Garvicin-SHAMU-LG6, A Novel Bacteriocin from Lactococcus garvieae That Exert Broad Antimicrobial Activity Against Drug-Resistant Pathogens

Garvicin-SHAMU-LG6, A Novel Bacteriocin from Lactococcus garvieae That Exert Broad Antimicrobial Activity Against Drug-Resistant Pathogens | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Garvicin-SHAMU-LG6, A Novel Bacteriocin from Lactococcus garvieae That Exert Broad Antimicrobial Activity Against Drug-Resistant Pathogens Shengnan Weng, Guiyun Leng, Ju Gao, Yawu Wang, Jie Yao, Xin Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3972345/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Accelerating growth and global expansion of antimicrobial resistance has deepened the demand for discovery of novel antimicrobial agents. Bacteriocins have attracted increasing interest because of their high efficiency, low toxicity and being not easy to cause drug resistance. This study aims to investigate a novel broad-spectrum bacteriocin, contributing to the pharmaceutical fields. Results From a human urine specimen, we isolated a strain thatproduced a novel broad-spectrum bacteriocin, which was identified as Lactococcus garvieae SHAMU-LG6. The bacteriocin, termed garvicin-SHAMU-LG6. The Oxford cup methoddemonstrated it could inhibit the growth of various clinically pathogenic and multidrug-resistant pathogens (MDRP). Whole genome sequencing analysis found a putative gene cluster, that shared 10% similarity with the most similar known bacteriocin cluster. In addition, the cell-free supernatant (CFS) of L. garvieae SHAMU-LG6 exerts antimicrobial activity against S. aureus by disrupting the integrity of bacterial cells. Furthermore, garvicin-SHAMU-LG6 was preliminary purified from the CFS of L. garvieae SHAMU-LG6. Its crude extraction showed good pH (pH 3 to 11) and heat stability (30℃ to 121℃) and resistance to the digestion of chymotrypsin, trypsin, proteinase K, and bromelain. Conclusions All these studies suggested that garvicin-SHAMU-LG6 has the potential to be used as a therapeutic drug against pathogenic bacteria as well as MDRP in the food and pharmaceutical fields. L. garvieae bacteriocin antimicrobial activity multidrug-resistant pathogens Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Antibiotics were once capable of treating most bacterial infections and hence, it can be argued that the identification and development of antibiotic therapy is one of the most significant scientific achievements of the twentieth century. However, extensive use of antibiotics has led to the emergence of antibiotic resistance and subsequent multidrug-resistant pathogens (MDRP), which diminished the effectiveness of existing antibiotics [ 1 ]. They are not only becoming increasingly problematic for human health and other fields crucial for human survival [ 2 ] but also increasing the global economic burden. According to a study led by Jim O'Neill, drug-resistant diseases cause more than 700,000 deaths annually [ 3 ]. Therefore, new antimicrobial drugs treating microbial infections are urgently needed. Recently, alternative treatments such as antibodies, probiotics, bacteriophages, and antimicrobial peptides (AMPs) are currently being investigated for the treatment of bacterial infections [ 4 ] AMPs from synthetic and natural sources are considered new-generation antibacterial agents. AMPs secreted by bacteria also known as bacteriocins, which can inhibit the growth of bacteria, fungi, parasites, or viruses [ 5 ]. Bacteriocins are generally considered as promising therapeutics against resistant microorganisms [ 6 ] and hence have drawn substantial attention. Bacteriocins are small, ribosomally synthesized AMPs or proteins produced by bacteria, which usually are extracellularly released and have a bactericidal or bacteriostatic effect (usually on species that are closely related to the producer strains) [ 7 ], but the cells that synthesize bacteriocins themselves have immunity to their products. Based on their structural and physico-chemical properties, the latest classification arranges bacteriocins into three major classes [ 8 , 9 ]. (Ⅰ) Class I bacteriocins are small post-translationally modified peptides (< 5 kDa, heat-stable), which contain wool sulfur amino acid and could be further subdivided into Class Ia (lantibiotics), Class Ib (labyrinthopeptins), and Class Ic (sanctibiotics). (Ⅱ) Class II bacteriocins are small (< 10 kDa), heat-stable, non-modified peptides described as unmodified bacteriocins, including Class IIa (pediocin-like bacteriocins), Class IIb (two-peptide bacteriocins), Class IIc (cyclic bacteriocins) and Class IId (linear, non-pediocin-like bacteriocins); (Ⅲ) Class III bacteriocins are larger peptides (> 10 kDa, heat-labile), which are classified into two subclasses: IIIa and IIIb. Bacteriocins have various modes of functions, including forming pores on target cell membranes, blocking metabolic pathways, inhibiting cell wall synthesis, and affecting functional gene expression, etc [ 10 ]. Generally, they exert antibacterial effects through one or more mechanisms. Bacteriocins have numerous properties as follows versus common antibiotics indicating that they are promising antimicrobial agents. (Ⅰ) They have stronger inhibitory effectory yet display no toxicity toward eukaryotic cells [ 11 ]. Generally, antibiotics need a micromolar level for their activity, but bacteriocins act specifically to target bacteria only at the nanomolar level [ 10 ] (Ⅱ) They show a lower propensity to develop resistance[ 2 ] and will not cause environmental pollution. On the one hand, bacteriocins act through quick pore formation on the target cell membrane at extremely low concentrations; on the other hand, they can be degraded easily in our bodies and other environments because of their proteinaceous nature [ 11 ]. (Ⅲ) They can be conveniently modified by genetic manipulation. (Ⅳ) They are usually stable to both pH and heat[ 12 ]. (Ⅴ) There are narrow- and broad-spectrum bacteriocins, which have strong antibacterial activity both in vivo and in vitro. The former has a high specific activity that can control targeted pathogens without negatively affecting commensal populations, the latter can be used to target infections of unknown etiology [ 13 ]. So far, research on bacteriocins has mostly focused on narrow-spectrum bacteriocins, and the mechanism of action of broad-spectrum bacteriocins with greater application value still needs to be elucidated. To date, the best-studied bacteriocins are from lactic acid bacteria (LAB) that are generally regarded as safe (GRAS) and possess qualified presumption of safety (QPS) status[ 8 ]. Their bacteriocins are regarded as safe by the USFDA (the U.S. Food and Drug Administration) [ 9 ] and have enormous application value in the food industry [ 14 ] and human health fields [ 5 ]. LAB includes various genera, such as Lactobacillus , Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Tetragenococcus, Vagococcus and Weissella [ 15 ]. Among them, Lactococcus genera bacteria are well-known bacteriocin producers. For example, the most representative Class-Ia bacteriocin, nisin [ 9 ], is produced by some Lactococcus spp. and Streptococcus spp. Lactococcus genera bacteria do not form spores, have no capsule, have no motility, and are facultative anaerobic Gram-positive cocci. Recently there are many bacteriocins produced by L. garvieae have been identified, such as Garvicin ML [ 11 ], Garvieacin Q [ 16 ], Garvicin KS [ 17 , 18 ]. Although many bacteriocins are being discovered continually, most of them are narrow-spectrum bacteriocins. So far, only two bacteriocins, both with wide antimicrobial spectra, nisin and pediocin PA-1, have been authorized as preservatives in the food industry [ 19 ]. Therefore, there is a need to identify bacteriocins with broad activity. In this study, we described here the screening assay, identification and rough purification of a novel broad-spectrum bacteriocin, garvicin-SHAMU-LG6, with potent activity against many important pathogens and MDRP. We measured the antimicrobial spectrum and mode of action of garvicin-SHAMU-LG6, and performed entire genome sequencing to identify the genes involved in bacteriocin synthesis. At the same time, the susceptibility to pH, temperature, and proteases was measured, and the mode of action of garvicin-SHAMU-LG6 was evaluated. The genome information of L. garvieae SHAMU-LG6 and the antibacterial properties of garvicin-SHAMU-LG6 provide the theoretical foundation for the potential use of a clinical therapeutic drug. MATERIALS AND METHODS Screening and identification of producers of broad‑spectrum antimicrobial bacteriocins To screen for novel broad-inhibition‑spectrum bacteriocins, we employed the Oxford cup diffusion method to test the antimicrobial activity of strains from clinical specimens in daily microbiological testing work. Finally, a higher activity strain was screened out. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) and MALDI Biotyper 3.1 software were used to preliminarily identify this strain . To further identify the species of strain, the 16S rRNA gene fragment of this strain was amplified using primers 27F and 1492R, and the PCR product was sequenced. The 16S rDNA gene sequence of this strain was blasted to obtain the closely related sequence using the Nucleotide Blast (Blast-n) algorithm program, the sequence was aligned with its closely related sequences and a phylogenetic tree was constructed using MEGA 11.0 software. Antibacterial activity assay The antibacterial activity of L. garvieae SHAMU‑LG6 against various indicator strains was determined by the Oxford cup (internal diameter of 6.0 mm) diffusion method. Briefly, each of the indicator strains was adjusted to the turbidity of 0.5 McFarlane (MCF), diluted tenfold, and subsequently spread on an LB agar plate. At the same time, the L. garvieae SHAMU‑LG6 solution was adjusted to the turbidity of 3.0 MCF. Subsequently, the Oxford cups were placed on the LB agar plate and pressed to the appropriate depth, followed by adding 30 μL L. garvieae SHAMU‑LG6 solution to the cups. Finally, removed the Oxford cups when the solution dried. The plates were incubated at 29°C for 14-18h, followed by the measurement of inhibition zone diameters to assess the antimicrobial activity. Time and growth-inhibition curves The growth kinetics of cell-free supernatant (CFS) against S. aureus strain ATCC25923 and S. aureus strain ATCC29213 were respectively determined to verify its antimicrobial activity. The specific method can be divided into two steps. In step 1, the L. garvieae strain SHAMU‑LG6 was cultured overnight in the liquid medium (LB, Luria broth) at 29°C with shaking at 100 rpm. After overnight incubation, cells were removed by centrifugation at 8000 × g for 10 min, and the suspension was filter sterilization with a membrane of pore size 0.22 μm, this was then referred to as CFS. In step 2, we selected S. aureus ATCC25923 and S. aureus ATCC29213 as the indicator strains and adjusted them to 0.5 MCF. The S. aureus cultures were respectively incubated with the CFS in a certain proportion (0.5%, 1.0%, and 1.5%), and then 200 μL of the mixtures were transferred to a 96-well microtitre plate. In this experiment, we set up four groups, including the experimental group above (CFS and S. aureus cultures, positive control (vancomycin, LB broth, and S. aureus cultures), negative control (LB broth and S. aureus cultures) and blank control (added only 200 μL LB broth). Finally, we used a SpectraMax i3x (Molecular Devices) microplate reader to continuously measure the growth of bacter (read mode: absorbance, read type: kinetics, wavelength: 600 nm, temperature: 35°C, shaking 10 s before read). At least three independent replicates of each data were obtained. Finally, arranged the data through software to respectively draw time and growth-inhibition curves of two S. aureus strains treated with CFS. Scanning electron microscopy S. aureus ATCC29213 was incubated overnight in 40ml LB broth, and diluted with LB broth to 0.5 MCF. The L. garvieae SHAMU‑LG6 was incubated at 29°C with shaking at 100 rpm for 24 h, followed by using the centrifugation method (10,000 rpm for 10 min) and filter sterilization with a membrane of pore size 0.22 μm to obtain the CFS. The S. aureus ATCC29213 solution was incubated with CFS at a ratio of 1% for 15 h at 4°C. Subsequently, cells were collected by centrifugation at 8000 rpm for 10 min, then washed with aquae sterilisata and fixed with 2.5% glutaraldehyde for 24h. The cells were dehydrated using gradient alcohol solutions (10 to 100%) for 20 min each, treated with isoamyl acetate for 20 min, dried by a vacuum freeze dryer for 12 h, and sputter-coated with gold. The morphological images of S. aureus ATCC29213 were observed and acquired with scanning electron microscopy (SEM). Genome sequencing and sequence analysis of L. garvieae SHAMU‑LG6 The genome of L. garvieae SHAMU‑LG6 was sequenced on an Illumina NovaSeq /Oxford Nanopore ONT platform by Shanghai Paisennuo Biotechnology Co. Ltd. The paired-end-read quality control was performed with FASTP. The third-generation sequencing data was assembled using Unicycle and Flye softwares to obtain contig sequences, then through pilon software to correct errors in the contig. Finally, the complete genome sequence of L. garvieae SHAMU‑LG6 was obtained by splicing. Compared all predicted protein coding genes with the proteins contained in various databases for functional annotation of protein-coding genes. The genome circle map was drawn by using cgview software. The bacteriocin biosynthetic gene clusters were predicted by AntiSMASH (https://antismash.secondarymetabolites.org). Functional analysis of proteins was performed using the BLASTP program. Rough extraction of garvicin-SHAMU-LG6 The L. garvieae SHAMU‑LG6 strain was inoculated in LB broth (400ml) for 24h at 29°C. The fermentation supernatant was centrifuged at 7450×g for 10min at 4°C, and then the supernatant was decanted and filtered twice with 0.22 µm filters, to obtain CFS. The garvicin-SHAMU-LG6 crude extract was obtained using the Amberlite XAD-16 N resin adsorption method. Briefly, the 400ml CFS was loaded onto a column containing 220g Amberlite XAD-16 N resin. Then, the resin was washed with 3 L of ultrapure water and 1 L of 30% (v/v) ethanol. The active substances were sequentially eluted with 800 ml of 50%, 60% 70%, 80%, and 90% (vol/vol) ethanol, followed by using a rotary evaporator to remove the ethanol. The eluates of each ethanol gradient were concentrated until completely dry, then respectively dissolved in 5mL of ultrapure water and transferred to an EP tube. Finally, filtered with 0.22 µm filters and tested their antimicrobial activity by the Oxford cup method described above. Sensitivity to pH, temperature, and proteases The effect of pH on the garvicin-SHAMU-LG6 was investigated by adjusting the pH of its crude extract (100 µl) from 3.0 to 11.0 with CH 3 COOH or NaOH solution, followed by incubation at 35℃ for 1h. The residual antimicrobial activity was tested after neutralizing the sample to pH 7.0. To research the thermal stability of garvicin-SHAMU-LG6, the crude extract (100 µl) was exposed to 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃ and 121℃ for 10 min, followed by measuring the residual antimicrobial activity. The effects of various proteases on garvicin-SHAMU-LG6 were also tested. The proteases included chymotrypsin, trypsin, proteinase K, and bromelain, and each protease solution (20 mg/ml) and crude extract (100 µl) was mixed and incubated at 35°C for 1 h. The residual antimicrobial activity was determined as well. The garvicin-SHAMU-LG6 sample used in this study was concentrated 80-fold in the original fermentation broth volume. The indicator strain was S. aureus and antimicrobial activity was determined with the Oxford cup diffusion method as described above . The untreated crude extract solution was used as a control. All experiments were performed in triplicate. RESULTS Screening and identification of a bacteriocin-producing strain, L. garvieae SHAMU‑LG6 We screened a strain with antagonistic activity against many Gram-positive cocci, Gram-positive bacilli, and Gram-negative bacilli from a urine specimen collected from an ordinary patient (non-urological infections) in urology. MALDITOF MS and MALDI Biotyper 3.1 software analysis preliminarily identified this strain was L. garvieae. The 16S rDNA gene sequence of this strain was deposited in the GenBank, accession number: OQ918061. To further determine the strain, a phylogenetic tree ( Fig 1 ) was generated to compare its 16S rDNA to its closely related sequences using MEGA 11.0. Two isolates having 16S rDNA sequence similarities of more than 97% can generally be considered as homology within the genus. Genomic analysis showed the 16S rDNA gene of the isolate shares 99.66% sequence similarity with that of Lactococcus garvieae. Thus, it was given the strain designation L. garvieae SHAMU-LG6, and preserved in China Center for Type Culture Collection (CCTCC) under no. M 2023482. Antibacterial spectrum of L. garvieae SHAMU‑LG6 When tested against a series of gram-negative and gram-positive bacteria by using the Oxford cup diffusion method, L. garvieae SHAMU‑LG6 exhibited a broad spectrum of antimicrobial activity. As shown by the results in Table 1 , L. garvieae SHAMU‑LG6 exerted wide antimicrobial activities against all of the gram-positive bacteria in this study, including Staphylococcus spp. , E. faecalis , E. faecium , K. kristinae , S. agalactiae , E. durans, L. innocua and D. hominus. Moreover, it also showed antimicrobial activity against most tested gram-negative bacteria. Further inspection of the table showed that L. garvieae SHAMU‑LG6 also had potent antibacterial activity against some common MDRP. These results demonstrated that L. garvieae SHAMU‑LG6 could inhibit the growth of a series of clinical pathogenic bacteria ( Fig 3 ) and some common MDRP ( Fig 2 ). Table 1 Antimicrobial spectrum of Lactococcus garvieae SHAMU‑LG6 Indicator strain a Number of strains Hypostatic rate Antibacterial activity b - + ++ +++ Gram-positive cocci Staphylococcus aureus (sensitive) 15 100.0% 0 0 6 9 Staphylococcus aureus (MRSA) 15 100.0% 0 0 10 5 Staphylococcus hominis 16 100.0% 0 2 1 13 Staphylococcus epidermidis 15 100.0% 0 1 1 13 Staphylococcus capitis 11 100.0% 0 0 0 11 Staphylococcus haemolyticus 8 87.5% 1 0 1 6 Staphylococcus saprophyticus 1 100.0% 0 0 1 0 Staphylococcus simulans 1 100.0% 0 0 1 0 Staphylococcus lugdunensis 3 100.0% 0 0 0 3 Staphylococcus petenkovii 1 100.0% 0 0 0 1 Enterococcus faecalis 15 100.0% 0 7 8 0 Enterococcus faecium 15 100.0% 0 1 10 4 Kocuria kristinae 1 100.0% 0 0 0 1 Streptococcus agalactiae 3 100.0% 0 0 2 1 Enterococcus durans 1 100.0% 0 0 0 1 Gram-positive bacilli Listeria innocua 1 100.0% 0 0 0 1 Dermabacter hominus 1 100.0% 0 0 0 1 Gram-negative bacilli Escherichia coli (sensitive) 17 0.0% 17 0 0 0 Escherichia coli (CRE) 15 20.0% 12 3 0 0 Escherichia coli (ESBLs) 17 70.6% 5 3 9 0 Acinetobacter baumannii (sensitive) 15 93.3% 1 6 9 0 Acinetobacter baumannii (MDR-AB) 17 100.0% 0 15 2 0 Klebsiellar pneumonia (sensitive) 15 100.0% 0 9 6 0 Klebsiellar pneumonia (CRE) 16 75.0% 4 10 2 0 Klebsiellar pneumonia (ESBLs) 15 86.7% 2 5 8 0 Klebsiella oxytoca (sensitive ) 2 50.0% 1 1 0 0 Klebsiella oxytoca (ESBLs) 2 100.0% 0 2 0 0 Pseudomonas. aeruginosa (sensitive ) 16 93.8% 1 3 12 0 Pseudomonas aeruginosa (MDR-PA) 15 100.0% 0 0 13 2 Pseudomonas montelli 1 100.0% 0 0 1 0 Pseudomonas putida 1 100.0% 0 0 0 1 Stenotrophomonas maltophilia 17 100.0% 0 0 15 2 Aeromonas caviae 6 100.0% 0 0 2 4 Aeromonas veronii 4 100.0% 0 0 3 1 Aeromonas hydrophila 2 100.0% 0 0 2 0 Klebsiella pneumoniae 2 100.0% 0 0 2 0 Pseudomonas aeruginosa 1 100.0% 0 0 0 1 Pseudomonas mosaica 1 100.0% 0 0 0 1 Enterobacter cloacae (sensitive) 13 35.7% 9 4 1 0 Enterobacter cloacae (CRE) 15 6.7% 14 1 0 0 Enterobacter asburiae 7 14.3% 6 0 1 0 Enterobacter bougainvilli 5 60.0% 2 1 2 0 Proteus vulgaris 2 100.0% 0 0 2 0 Proteus hauterii 4 100.0% 0 0 4 0 Proteus mirabilis 7 100.0% 0 0 6 1 Serratia marcescens 9 100.0% 0 4 5 0 Alcaligenes faecalis 1 100.0% 0 0 0 1 Achromobacter xylosoxidans 1 100.0% 0 0 0 1 Acinetobacter junii 4 100.0% 0 0 4 0 Acinetobacter Pittii 4 100.0% 0 1 3 0 Hospital Acinetobacter 3 100.0% 0 0 3 0 Citrobacter freudii (sensitive) 3 100.0% 0 3 0 0 Citrobacter freudii (CRE) 1 100.0% 0 0 1 0 Citrobacter Klebsiella 2 100.0% 0 0 2 0 Chryseobacterium indologenes 1 100.0% 0 0 1 0 Shewanella putrefaciens 1 100.0% 0 0 0 1 Vibrio parahaemolyticus 2 100.0% 0 0 2 0 Morganella morganii 5 100.0% 0 0 5 0 Moraxella osloensis 1 100.0% 0 0 1 0 a MRSA, Methicillin-resistant Staphylococcus aureus ; CRE, Carbapenem-resistant Enterobacteriaceae ; ESBLs, Extended-spectrum β-lactamases; sensitive, drug-sensitive bacteria. b "-" represents the inhibition zone diameter ≤ 6 mm (no inhibitory effect); "+" represents the inhibition zone diameter 6-10 mm (including 10 mm); "++" represents the inhibition zone diameter of 10-20 mm (including 20 mm); "+++" represents the inhibition zone diameter > 20 mm. L. garvieae SHAMU‑LG6 cell-free supernatant inhibits the growth of S. aureus To verify whether L. garvieae SHAMU‑LG6 inhibits the growth of other bacteria by secreting antibacterial active substances outside the cell. We selected S. aureus ATCC25923 and S. aureus ATCC29213 as models to measure the antibacterial activity of CFS. The experiment was divided into four groups, and each experiment was consistently repeated in triplicate. The optical density (OD 600 ) of each group was measured at set intervals by a SpectraMax i3x (Molecular Devices) microplate reader, and the mean value was calculated from the obtained data. The time and growth-inhibition curves of two S. aureus strains respectively treated with CFS were shown in Fig 4 . Neither of them multiply in wells with vancomycin and the blank control group, conversely, they multiply normally in negative controls containing LB broth. Compared with the control groups, in the experimental group adding the CFS, the growths of two S. aureus strains were all inhibited with the delayed logarithmic phase and stationary phase under CFS treatment. These results demonstrated that the CFS contained an active substance, which may be a bacteriocin and was preliminary named garvicin-SHAMU-LG6. Garvicin-SHAMU-LG6 exerts bactericidal activity by damaging the cell integrity To deduce the mode of action of garvicin-SHAMU-LG6, the cell morphological change of S.aureus ATCC29213 cells when incubated under CFS of L. garvieae SHAMU‑LG6 was observed and analyzed by scanning electron microscopy (SEM). SEM analysis demonstrated that the cell membrane of S. aureus ATCC 29213 cells became deformed and collapsed ( Fig 5 AB ) when incubated with CFS for15 h at 4°C, whereas the untreated control cells showed smooth and intact surfaces ( Fig 5 CD) . In conclusion, garvicin-SHAMU-LG6 may cause cell membrane damage, and disrupt the integrity of target cells, thereby exerting bactericidal activity on S.aureus . Putative biosynthetic gene cluster of garvicin-SHAMU-LG6 Further, the entire genome of the L. garvieae SHAMU-LG6 was sequenced and the genome was uploaded on antiSMASH to find the biosynthetic gene cluster of the garvicin-SHAMU-LG6. The complete genome of L. garvieae SHAMU-LG6 consists of a 2,033,399-base pair (bp) circular chromosome, a 39,838-bp circular plasmid, and a 2,235-bp circular plasmid, with a GC content of 38.27%, 35.58% and 33.29%, respectively. The complete chromosome genome information of the strain SHAMU-LG6 is shown in Fig 6 . The result of using AntiSMASH to search for secondary metabolites is shown in Table 2 . The putative biosynthetic gene cluster of garvicin-SHAMU-LG6 is illustrated in Fig 7 . Functional analysis of proteins encoded by these genes was performed using the BLASTP program. The gene cluster consisted of thirteen genes, including two genes (chr1825 and chr1827) encoding bacteriocin, one gene (chr1823) with biosynthetic-additional function, two genes (chr1821 and chr1832) possibly were transport-related genes, two genes (chr1824 and chr1830) may involved in immunity, one regulatory gene (chr1820), four genes (chr1822, chr1828, chr1831) possibly associated with gene transfer and rearrangement, one gene (chr1829) may be related to DNA segregation machinery, and one gene (chr1826) with unknown function. Moreover, using BLAST software, the gene encoding protein sequence was compared with the amino acid sequence in the virulence factor-related gene database, and no virulence factor-related genes were found in the genome. Table 2 Antismash analysis Seq ID a Type Start End Most similar known cluster Similarity chr RiPP-like 1,823,808 1,834,020 lactocin S RiPP: Lanthipeptide 10% a Sequence ID; b RiPP, other unspecified ribosomally synthesised and post-translationally modified peptide product. Table 3 Garvicin-SHAMU-LG6 biosynthetic related genes Gene No. of AA a Known protein with the highest homology b [microorganism] Proposed function chr1820 119 Metalloregulator ArsR/SmtB family transcription factor [Bacteria] (NCBI Reference Sequence: WP_003726380.1, identity: 100%) Gene regulation chr1821 705 Heavy metal translocating P-type ATPase [Lactobacillales] (NCBI Reference Sequence: WP_001291323.1, identity: 100%) Transport chr1822 144 IS6 family transposase [Lactococcus garvieae] (NCBI Reference Sequence: WP_285013987.1, 95.80%) Gene transfer and rearrangement chr1823 56 Cysteine peptidase family C39 domain-containing protein [Lactococcus] (NCBI Reference Sequence: WP_258272642.1, 100%) Biosynthetic-additional chr1824 104 Bacteriocin immunity protein [Lactococcus] (NCBI Reference Sequence: WP_032495339.1, 100%) Immunity chr1825 70 Garvicin Q family class ll bacteriocin [Lactococcus] (NCBI Reference Sequence: WP_165719065.1, 98.57%) Bacteriocin biosynthetic chr1826 99 Lactococcus garvieae strain Lg-Granada plasmid pLG50, complete sequence (GenBank:CP084378.1, 100%) Unknown function chr1827 61 Bacteriocin [Lactococcus] (NCBI Reference Sequence: WP_165719075.1, 98.36%) Bacteriocin biosynthetic chr1828 190 Recombinase family protein [Lactococcus] (NCBI Reference Sequence: WP-003134225.1, 100%) Gene transfer and rearrangement chr1829 255 ParA family protein [Lactococcus] (NCBI Reference Sequence: WP-003134226.1, 100%) DNA segregate chr1830 99 Lactococcus garvieae strain IPLA 31405 plasmid pLG42, complete sequence (GenBank: KM007160.1, 100%) Immunity chr1831 226 IS6-like element IS1216 family transposase [Lactococcus] (NCBI Reference Sequence: WP 162523730.1, 100%) Gene transfer and rearrangement chr1832 172 PTS sugar transporter subunit IIC [Lactococcus petauri] (GenBank: NHI79097.1, 100%) Transport a AA, amino acids b analyses were performed using the BLASTP program, and the proteins listed were the closest homologue to each gene in the putative biosynthetic gene cluster of Garvicin-SHAMU-LG6 in the NCBI and GenBank database. c Function annotation of predicted proteins encoded by genes in the putative bacteriocin biosyntheti gene cluster. Rough extraction of garvicin-SHAMU-LG6 The L. garvieae SHAMU-LG6 strain was cultured in LB broth for 24 h, and the active substance in the culture supernatants was rough extraction using Amberlite XAD-16 N resin. Using S. aureus as an indicator bacterium, the Oxford cup diffusion method mentioned above was used to verify the antibacterial activity of the collected liquids eluted with ethanol solutions of different concentrations [50%, 60% 70%, 80%, 90% (vol/vol) ethanol]. As shown by the results in Fig 8 , the fraction eluted with 70% and 80% (vol/vol) ethanol solutions had the best antibacterial effect. Therefore, we ultimately used 75% (vol/vol) ethanol as the elution solution, and the resulting supernatant was designated the crude extract, which was used for subsequent purification and study on physicochemical properties. Stability of garvicin-SHAMU-LG6 The susceptibility of garvicin-SHAMU-LG6 to pH, temperature, and proteases was investigated using the Oxford cup diffusion method. Untreated crude extract solution was taken as control. Garvicin-SHAMU-LG6 was found to be stable at high temperatures (up to 121℃) and in a wide range of pH (pH 3-11). In terms of thermostability, as presented in Fig 9 B, the antimicrobial activity of garvicin-SHAMU-LG6 slightly decreased with increasing temperature, but it could resist the temperature of 121℃ and retained most of its antimicrobial activity. Upon treatment in a range of pH, garvicin-SHAMU-LG6 showed its stability in the pH range of 3-11 ( Fig 9 A). Although the residual antimicrobial activity was greatly decreased under near-neutral conditions (ph 6.0 to 8.0), garvicin-SHAMU-LG6 exhibited superior activity under alkaline (ph 11.0) and acidic (ph 3.0) conditions. The antibacterial activity of garvicin-SHAMU-LG6 remained almost unchanged after treatment with four enzymes, as illustrated in Fig 9 C . Among them, garvicin-SHAMU-LG6 could retain its full antimicrobial activity after treatment with chymotrypsin and bromelain, majority of antimicrobial activity after treatment with trypsin and proteinase K. DISCUSSION The emergence of antibiotic resistance is a growing global public crisis. As more and more antibiotics become ineffective due to drug-resistant bacteria, it is imperative to focus on alternative therapies to conventional antibiotics[ 4 ]. In the past two decades, bacteriocins have shown their potential as promising alternative therapeutic [ 20 ]. The LAB is a well-known producer of bacteriocins and has great potential for application in food and pharmaceutical fields. Several reports have shown that over 230 bacteriocins from LAB have been isolated and reported, but only half of them were identified at the protein or DNA levels [ 21 ]. The most representative bacteriocin is the lactibiotic nisin, which is the first and most widely used bacteriocin in the food preservation and there have been no reports of widespread drug resistance [ 8 , 22 ]. L. garvieae is a LAB and so far, various bacteriocins have been found in different L. garvieae strains, including garviecin L1-5 [ 23 ], Garvicin ML [ 11 ], Garvieacin Q [ 16 ], garvicin A [ 24 ], Garvicin KS [ 17 , 18 ], Garvicins AG1, and Garvicins AG2 [ 25 ]. This study reports a novel bacteriocin, garvicin-SHAMU-LG6, that was produced by L. garvieae strain SHAMU-LG6 (CCTCC No: M 2023482), and whose wide antimicrobial activity against various pathogenic bacteria as well as MDRP, and high pH and thermal stability highlight its potential as a promising clinical therapeutic drug. The antimicrobial spectrum of one bacteriocin is vital for its application potential. Garviecin L1-5 is a small bacteriocin (about 2.5 kDa), produced by L. garvieae isolated from a raw cow’s milk sample. It was bactericidal against closely related species and strains of species from different genera, including Listeria and Clostridium spp [ 23 ]. The 60-amino-acid circular bacteriocin, garvicin ML (about 6 kDa), is produced by L. garvieae DCC43 isolated from mallard ducks and has a broad antimicrobial spectrum. It inhibits some strains of species from the Lactococcus , Lactobacillus , Pediococcus , Propionibacterium , Clostridium , Streptococcus,and Enterococcus spp. [ 11 ]. Garvieacin Q, a class II bacteriocin, consists of 50 amino acids (5.3 kDa) and is produced by strain BCC 43578, isolated from fermented pork sausage. It is especially active against Listeria monocytogenes ATCC 19115 and other L. garvieae strains [ 16 ]. Garvicin A is a class IId narrow-spectrum bacteriocin, with a mass of 4.7 kDa, produced by L. garvieae 21881 isolated in a human clinical case. It is active only against other strains of the same species [ 24 ]. Garvicin KS is a leaderless multipeptide bacteriocin, produced by L. garvieae KS1546 isolated from raw milk. Garvicin KS is composed of three similar peptides of 32 to 34 amino acids and shows wide antibacterial activity. Its inhibitory spectrum includes important pathogens belonging to the genera Staphylococcus, Bacillus, Listeria , and Enterococcus [ 17 , 18 ]. Garvicins AG1 and AG2 are two novel class IId bacteriocins of L. garvieae Lg-Granada isolated from the blood of a patient with endocarditis. They exhibit antimicrobial activity against other L. garvieae strains, and AG2 can also inhibit the growth of Listeria monocytogene s, Listeria ivanovii , and Enterococcus faecalis [ 25 ]. Apparently, that the majority of bacteriocins produced by L. garvieae are narrow-spectra bacteriocins. Herein, garvicin-SHAMU-LG6, the bacteriocin identified in the present study, has a more extensive antibacterial spectrum in comparison with the aforementioned bacteriocins. It exhibits antimicrobial activity against all of the gram-positive cocci in this study, two gram-positive bacilli strains, and many gram-negative bacilli. In addition, in terms of antibiotic resistance, we divided the indicator strains into drug-resistant and drug-sensitive groups. The results, as shown in Fig. 2 and Fig. 3 , indicate that it has good antagonistic activity against and many clinical pathogenic bacteria and common MDRP, such as MRSA, MDRAB, MDRPA, and some CRE. Therefore, the study of garvicin-SHAMU-LG6 may have important application value for the treatment of antibiotic-resistant bacteria. The growth kinetics of S. aureus strains treated with CFS indicated that the fermentation supernatant of strain L. garvieae SHAMU-LG6 contained an antimicrobial substance. Additionally, the whole-genome sequencing and DNA sequence by the antiSMASH database located the presence of a putative biosynthetic gene cluster which was identical 10% similarity with the lactocin S gene cluster (Fig. 7 ). This gene cluster contained genes coding for the bacteriocin, transporter-related proteins, proteins responsible for immunity, multiple transposase, and recombinase that probably relate to gene transfer and rearrangement and transcription factor. Sequence homology analysis showed that the similarity between this gene cluster and the known most similar gene cluster is only 10% (Table 2 ). Additionally, in terms of application security, no evidence of virulence-related genes was obtained. This further confirms that L. garvieae SHAMULG6 inhibits the growth of other bacteria by secreting a promising novel bacteriocin. Alvarez-Sieiro et al.[ 8 ] proposed dividing bacteriocins into three classes according to their biological activities and biosynthesis mechanisms. Most bacteriocins produced by LAB were Classes I and II small bacteriocins. Class I bacteriocins are heat-stable and smaller than 5 kDa peptides, which undergo post-translational modification; Class II includes heat-stable, smaller than 10 kDa, unmodified bacteriocins. Different classes of bacteriocins have different structures and mechanisms of action. Bacteriocins differ in the mode of action of antibiotics, most LAB bacteriocins, especially those inhibiting Gram-positive bacteria, exert their antimicrobial effects by targeting bacterial cell membranes and forming pores[ 13 ]. Furthermore, some bacteriocins exert antibacterial effects through various mechanisms. For example, nisins effectively belong to the Class I bacteriocins that contain lanthionine and possess a double mode of action[ 9 ]: (i) their N-terminal binds to lipid II and the C-terminal penetrates cytoplasmic membrane, resulting in pore formation and induces ion and ATP leakage. (ii) they inhibit cell wall synthesis by masking lipid II. In the present study, SEM analysis clearly showed that sensitive cells treated with CFS of L. garvieae SHAMU-LG6 exhibited significant deformation and collapse (Fig. 5 ). Moreover, L. garvieae SHAMU-LG6 exhibited antagonistic activity against both Gram-positive and Gram-negative bacteria. Therefore, we speculate that garvicin-SHAMU-LG6 targets universal molecules (such as the cell membrane, or intracellular target molecules) in bacterial cells, disrupting the integrity of bacterial cells through one or more modes of action, thereby killing the target bacteria. This hypothesis should be verified in further studies. Although bacteriocins have many advantages, antibacterial spectrum, purification, and yield have limited the application of bacteriocins. Breaking the bottleneck has become the focus of current research. In this study, we found that the garvicin-SHAMU-LG6 crude extract obtained by using Amberlite XAD-16 N resin to adsorb bacteriocin in the culture supernatants and eluting with 75% alcohol has the strongest activity. These results indicated the above-said method is capable of preliminary purification of garvicin-SHAMU-LG6 from fermentation supernatant. We will attempt to further purify garvicin-SHAMU-LG6 by using various chromatographic techniques for studying its structure and characteristics in future studies. The stability of garvicin-SHAMU-LG6 under acidic or basic and heat conditions was tested. Garvicin-SHAMU-LG6 was stable under acidic (PH 3) and basic conditions (PH 11) and exhibited a slight loss of activity under neutral conditions. Additionally, in terms of thermostability, upon autoclaving (121℃), garvicin-SHAMU-LG6 retained most of its antimicrobial activity. Many bacteriocins, including nisin, are readily inactivated under neutral and alkaline conditions. We also tested the susceptibility of garvicin-SHAMU-LG6 to four proteases. Although it showed resistance to the digestion of chymotrypsin, trypsin, proteinase K, and bromelain, we cannot completely deny that it is a protein or polypeptide. On the one hand, it may be because that garvicin-SHAMU-LG6 is a small molecule polypeptide with a simple structure and thus does not have the target of these enzymes. On the other hand, it might also be due to its special three-dimensional structure that blocks the recognition or combination of the protease and the target. For the example, the previous study indicated that the resistance of GarML to trypsin, pepsin, papain, and proteinase K, is not due to the absence of digestive sites, but to the inaccessibility of recognition sites [ 11 ]. These hypotheses should be verified in further studies. In conclusion, the extraordinary stability of garvicin-SHAMU-LG6 is another desirable property supporting its potential application value in food and pharmaceutical fields. As well, determining the biosafety of bacteriocin is vital for its development and application in medicine or food. Although no virulence factors have currently been found in its genome, hemolytic activity and cytotoxicity examination remain crucial. In general, more detailed studies may be carried out about the safety of garvicin-SHAMU-LG6 to evaluate the feasibility of its application as a promising clinical therapeutic drug. Conclusions The present study identified a broad-spectrum bacteriocin-producer, Lactococcus garvieae SHAMU-LG6, and preliminary purified its bacteriocin. Garvicin-SHAMU-LG6 showed good pH, heat, and protease stability, and it has wide antimicrobial activity against pathogenic bacteria as well as MDRP, likely by destroying their cell membrane integrity. These findings will provide the theoretical foundation for its potential application as a food preservative and clinical therapeutic drug. Declarations ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant number 82102460). Ethics approval and consent to participate This article does not contain any studies with human participants or animals performed by any of the authors. Consent for publication All authors agree to publish this review. Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests The authors declare no competing interests. Author contributions SW: Preparation, Investigation, Data curation,Writing - Original Draft. GL: Preparation, Methodology, Investigation. JG: Methodology, Investigation,Validation. YW: Validation, Data Curation. JY: Investigation, Validation. XL: Investigation, Validation. ZL: Investigation, Supervision. QZ: Investigation, Writing - review and editing. WT: Supervision, Funding acquisition, Writing - Review & Editing, Project administration. References Magana M, Pushpanathan M, Santos AL, Leanse L, Fernandez M, Ioannidis A, Giulianotti MA, Apidianakis Y, Bradfute S, Ferguson AL, et al. The value of antimicrobial peptides in the age of resistance. LANCET INFECT DIS. 2020;20(9):e216–30. Neves JV. Editorial for Special Issue Alternatives to Antibiotics: Bacteriocins and Antimicrobial Peptides. ANTIBIOTICS-BASEL 2022, 11(7). Liu S, Deng S, Liu H, Tang L, Wang M, Xin B, Li F. Four Novel Leaderless Bacteriocins, Bacin A1, A2, A3, and A4 Exhibit Potent Antimicrobial and Antibiofilm Activities against Methicillin-Resistant Staphylococcus aureus. MICROBIOL SPECTR. 2022;10(5):e94522. Ghosh C, Sarkar P, Issa R, Haldar J. Alternatives to Conventional Antibiotics in the Era of Antimicrobial Resistance. TRENDS MICROBIOL. 2019;27(4):323–38. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3972345","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":274159359,"identity":"53a66f86-fe54-4259-b19d-ea9bda3c5b92","order_by":0,"name":"Shengnan Weng","email":"","orcid":"","institution":"Second Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shengnan","middleName":"","lastName":"Weng","suffix":""},{"id":274159360,"identity":"183605ff-9c56-4481-9957-f41a5ac9b7c6","order_by":1,"name":"Guiyun Leng","email":"","orcid":"","institution":"Second Hospital of Anhui Medical 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09:31:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3972345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3972345/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51525187,"identity":"cc774d0b-38f0-43c4-90da-da83e6d9f078","added_by":"auto","created_at":"2024-02-23 05:21:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1323285,"visible":true,"origin":"","legend":"\u003cp\u003eThe phylogenetic tree of 16S rDNA gene sequences for isolated strain.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/bda7d2d3cc196d97e05eb2a3.png"},{"id":51524876,"identity":"f9e8697b-6669-45b2-be4b-f21210f25c5a","added_by":"auto","created_at":"2024-02-23 05:13:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2107677,"visible":true,"origin":"","legend":"\u003cp\u003eThe antimicrobial activities of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 against common MDRP.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/4e6ecf1eb02af0f980a53025.png"},{"id":51524875,"identity":"36f16489-66ee-45e7-979a-b2968d2abdb9","added_by":"auto","created_at":"2024-02-23 05:13:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2807658,"visible":true,"origin":"","legend":"\u003cp\u003eThe antimicrobial activities of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 against common drug-sensitive bacteria.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/8d18b91b9fafee30d006f7b7.png"},{"id":51524877,"identity":"b973c589-4191-4b29-b1a6-e6d0a9e6a456","added_by":"auto","created_at":"2024-02-23 05:13:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3764425,"visible":true,"origin":"","legend":"\u003cp\u003eThe time and growth-inhibition curves of two \u003cem\u003eS. aureus \u003c/em\u003estrains treated with CFS, control of BC (blank control), PC (positive control) and NC (negative control). “0.5%, 1% and 1.5%” represented proportion of \u003cem\u003eS. aureus\u003c/em\u003e solution add. The datas were represented as the mean values of three repeated experiments. (A) The indicator strain was S. aureus ATCC25923. (B) The indicator strain was S. aureus ATCC29213.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/9cf71981dd065b0eb8b5e3d2.png"},{"id":51524884,"identity":"cdfa6fc8-8444-4c1a-83ce-b2c82e3c9442","added_by":"auto","created_at":"2024-02-23 05:13:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53106988,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology observation of \u003cem\u003eS.aureus\u003c/em\u003e ATCC29213 cells after treated with CFS. (\u003cstrong\u003eAB\u003c/strong\u003e) The images of \u003cem\u003eS.aureus \u003c/em\u003ecells treated with CFS. The white arrows indicated the significant damage of \u003cem\u003eS.aureus\u003c/em\u003ecells. (\u003cstrong\u003eCD\u003c/strong\u003e) The images of untreated \u003cem\u003eS.aureus \u003c/em\u003ecells.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/19691c8efd36e8af80964982.png"},{"id":51525295,"identity":"24998c66-4779-4c43-ba16-ce734bddad82","added_by":"auto","created_at":"2024-02-23 05:29:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1589059,"visible":true,"origin":"","legend":"\u003cp\u003eCircular genome map of the chromosome of the L. garvieae SHAMU-LG6. From center to outside, ring 1 : scale; ring 2 :GC-skew; ring 3 : GC content; ring 4 and 7 : the COG to which each CDS belongs; ring 5 and 6: the positions of CDS, tRNA and rRNA on the genome.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/92598067677b120b7ab04439.png"},{"id":51524879,"identity":"d2d8271e-f964-4a05-af3c-e266d7de4516","added_by":"auto","created_at":"2024-02-23 05:13:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":470803,"visible":true,"origin":"","legend":"\u003cp\u003ePutative biosynthetic gene cluster of garvicin-SHAMU-LG6. Genes are colored according to their predicted functions. From left to right, genes are named in order from chr1820 to chr1832. Purplish red, regulatory gene; red, biosynthetic genses; green, immunity genes; grey, transport genes.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/de9c15dac75596e37a8dd3ee.png"},{"id":51524881,"identity":"ff7bc47c-b0b5-4304-91b9-a50018983694","added_by":"auto","created_at":"2024-02-23 05:13:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":10442077,"visible":true,"origin":"","legend":"\u003cp\u003eThe antibacterial activity of the fraction eluted with ethanol solutions of different concentrations.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/a91d2b22f853d9924f19eae7.png"},{"id":51524882,"identity":"060f0d18-4275-464d-b855-89ec4788613e","added_by":"auto","created_at":"2024-02-23 05:13:11","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":7205810,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of pH, temperature, and proteases on antimicrobial activity of garvicin-SHAMU-LG6. The indicator strain was \u003cem\u003eS. aureus\u003c/em\u003e and untreated crude extract (CE) solutions were used as the control. (A) Stability of garvicin-SHAMU-LG6 to PH. (B) Stability of garvicin-SHAMU-LG6 to temperature. (C) Stability of garvicin-SHAMU-LG6 to proteases.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/ca3f384610aa29b313a2f157.png"},{"id":51713076,"identity":"ea55e97c-6620-4cb1-8f36-319b9f9de4b4","added_by":"auto","created_at":"2024-02-27 19:22:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9919679,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3972345/v1/4a1005f7-c627-46a5-aa54-905cf70575da.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Garvicin-SHAMU-LG6, A Novel Bacteriocin from Lactococcus garvieae That Exert Broad Antimicrobial Activity Against Drug-Resistant Pathogens","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntibiotics were once capable of treating most bacterial infections and hence, it can be argued that the identification and development of antibiotic therapy is one of the most significant scientific achievements of the twentieth century. However, extensive use of antibiotics has led to the emergence of antibiotic resistance and subsequent multidrug-resistant pathogens (MDRP), which diminished the effectiveness of existing antibiotics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They are not only becoming increasingly problematic for human health and other fields crucial for human survival [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] but also increasing the global economic burden. According to a study led by Jim O'Neill, drug-resistant diseases cause more than 700,000 deaths annually [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, new antimicrobial drugs treating microbial infections are urgently needed. Recently, alternative treatments such as antibodies, probiotics, bacteriophages, and antimicrobial peptides (AMPs) are currently being investigated for the treatment of bacterial infections [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] AMPs from synthetic and natural sources are considered new-generation antibacterial agents. AMPs secreted by bacteria also known as bacteriocins, which can inhibit the growth of bacteria, fungi, parasites, or viruses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Bacteriocins are generally considered as promising therapeutics against resistant microorganisms [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and hence have drawn substantial attention.\u003c/p\u003e \u003cp\u003eBacteriocins are small, ribosomally synthesized AMPs or proteins produced by bacteria, which usually are extracellularly released and have a bactericidal or bacteriostatic effect (usually on species that are closely related to the producer strains) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], but the cells that synthesize bacteriocins themselves have immunity to their products. Based on their structural and physico-chemical properties, the latest classification arranges bacteriocins into three major classes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. (Ⅰ) Class I bacteriocins are small post-translationally modified peptides (\u0026lt;\u0026thinsp;5 kDa, heat-stable), which contain wool sulfur amino acid and could be further subdivided into Class Ia (lantibiotics), Class Ib (labyrinthopeptins), and Class Ic (sanctibiotics). (Ⅱ) Class II bacteriocins are small (\u0026lt;\u0026thinsp;10 kDa), heat-stable, non-modified peptides described as unmodified bacteriocins, including Class IIa (pediocin-like bacteriocins), Class IIb (two-peptide bacteriocins), Class IIc (cyclic bacteriocins) and Class IId (linear, non-pediocin-like bacteriocins); (Ⅲ) Class III bacteriocins are larger peptides (\u0026gt;\u0026thinsp;10 kDa, heat-labile), which are classified into two subclasses: IIIa and IIIb. Bacteriocins have various modes of functions, including forming pores on target cell membranes, blocking metabolic pathways, inhibiting cell wall synthesis, and affecting functional gene expression, etc [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Generally, they exert antibacterial effects through one or more mechanisms.\u003c/p\u003e \u003cp\u003eBacteriocins have numerous properties as follows versus common antibiotics indicating that they are promising antimicrobial agents. (Ⅰ) They have stronger inhibitory effectory yet display no toxicity toward eukaryotic cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Generally, antibiotics need a micromolar level for their activity, but bacteriocins act specifically to target bacteria only at the nanomolar level [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] (Ⅱ) They show a lower propensity to develop resistance[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and will not cause environmental pollution. On the one hand, bacteriocins act through quick pore formation on the target cell membrane at extremely low concentrations; on the other hand, they can be degraded easily in our bodies and other environments because of their proteinaceous nature [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. (Ⅲ) They can be conveniently modified by genetic manipulation. (Ⅳ) They are usually stable to both pH and heat[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. (Ⅴ) There are narrow- and broad-spectrum bacteriocins, which have strong antibacterial activity both in vivo and in vitro. The former has a high specific activity that can control targeted pathogens without negatively affecting commensal populations, the latter can be used to target infections of unknown etiology [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. So far, research on bacteriocins has mostly focused on narrow-spectrum bacteriocins, and the mechanism of action of broad-spectrum bacteriocins with greater application value still needs to be elucidated.\u003c/p\u003e \u003cp\u003eTo date, the best-studied bacteriocins are from lactic acid bacteria (LAB) that are generally regarded as safe (GRAS) and possess qualified presumption of safety (QPS) status[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Their bacteriocins are regarded as safe by the USFDA (the U.S. Food and Drug Administration) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and have enormous application value in the food industry [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and human health fields [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. LAB includes various genera, such as \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eLactococcus, Leuconostoc, Pediococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Tetragenococcus, Vagococcus\u003c/em\u003e and \u003cem\u003eWeissella\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among them, \u003cem\u003eLactococcus\u003c/em\u003e genera bacteria are well-known bacteriocin producers. For example, the most representative Class-Ia bacteriocin, nisin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], is produced by some \u003cem\u003eLactococcus\u003c/em\u003e spp. and \u003cem\u003eStreptococcus\u003c/em\u003e spp. \u003cem\u003eLactococcus\u003c/em\u003e genera bacteria do not form spores, have no capsule, have no motility, and are facultative anaerobic Gram-positive cocci. Recently there are many bacteriocins produced by \u003cem\u003eL. garvieae\u003c/em\u003e have been identified, such as Garvicin ML [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], Garvieacin Q [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], Garvicin KS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although many bacteriocins are being discovered continually, most of them are narrow-spectrum bacteriocins. So far, only two bacteriocins, both with wide antimicrobial spectra, nisin and pediocin PA-1, have been authorized as preservatives in the food industry [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, there is a need to identify bacteriocins with broad activity.\u003c/p\u003e \u003cp\u003eIn this study, we described here the screening assay, identification and rough purification of a novel broad-spectrum bacteriocin, garvicin-SHAMU-LG6, with potent activity against many important pathogens and MDRP. We measured the antimicrobial spectrum and mode of action of garvicin-SHAMU-LG6, and performed entire genome sequencing to identify the genes involved in bacteriocin synthesis. At the same time, the susceptibility to pH, temperature, and proteases was measured, and the mode of action of garvicin-SHAMU-LG6 was evaluated. The genome information of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU-LG6 and the antibacterial properties of garvicin-SHAMU-LG6 provide the theoretical foundation for the potential use of a clinical therapeutic drug.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eScreening and identification of producers of broad‑spectrum antimicrobial bacteriocins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo screen for novel broad-inhibition‑spectrum bacteriocins, we employed the Oxford cup diffusion method to test the antimicrobial activity of strains from clinical specimens in daily microbiological testing work. Finally, a higher activity strain was screened out. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) and\u0026nbsp;MALDI Biotyper 3.1 software were used to preliminarily identify this strain\u003cem\u003e.\u003c/em\u003e To further identify the species of strain, the 16S rRNA gene fragment of this strain was amplified using primers 27F and 1492R, and the PCR product was sequenced.\u0026nbsp;The 16S rDNA gene sequence of this strain was blasted to obtain the closely related sequence using the\u0026nbsp;Nucleotide Blast (Blast-n)\u0026nbsp;algorithm program, the sequence was aligned with its closely related sequences\u0026nbsp;and a phylogenetic tree was constructed using MEGA 11.0 software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial activity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibacterial activity of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 against various indicator strains was determined by the Oxford cup (internal diameter of 6.0\u0026thinsp;mm) diffusion method. Briefly, each of the indicator strains was adjusted to the turbidity of 0.5 McFarlane (MCF), diluted tenfold, and subsequently spread on an LB agar plate. At the same time, the \u003cem\u003eL. garvieae\u0026nbsp;\u003c/em\u003eSHAMU‑LG6 solution was adjusted to the turbidity of 3.0 MCF. Subsequently, the Oxford cups were placed on the LB agar plate and pressed to the appropriate depth, followed by adding 30 \u0026mu;L \u003cem\u003eL. garvieae\u0026nbsp;\u003c/em\u003eSHAMU‑LG6 solution to the cups. Finally, removed the Oxford cups when the solution dried. The plates were incubated at 29\u0026deg;C for 14-18h, followed by the measurement of inhibition zone diameters to assess the antimicrobial activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTime and growth-inhibition curves\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth kinetics of cell-free supernatant (CFS) against \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003estrain ATCC25923 and \u003cem\u003eS. aureus\u003c/em\u003e strain ATCC29213 were respectively determined to verify its antimicrobial activity. The specific method can be divided into two steps. In step 1, the \u003cem\u003eL. garvieae\u003c/em\u003e strain SHAMU‑LG6 was cultured overnight in the liquid medium (LB, Luria broth) at 29\u0026deg;C with shaking at 100 rpm.\u0026nbsp;After overnight incubation, cells were removed by centrifugation at 8000 \u0026times; g for 10 min, and the suspension was filter sterilization with a membrane of pore size 0.22 \u0026mu;m, this was then referred to as CFS. In step 2, we selected \u003cem\u003eS. aureus\u003c/em\u003e ATCC25923 and \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eATCC29213 as the indicator strains and adjusted them to 0.5 MCF. The \u003cem\u003eS. aureus\u003c/em\u003e cultures were respectively incubated with the CFS in a certain proportion (0.5%, 1.0%, and 1.5%), and then 200 \u0026mu;L of the mixtures were transferred to a 96-well microtitre plate. In this experiment, we set up four groups, including the experimental group above (CFS and \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003ecultures, positive control (vancomycin, LB broth, and \u003cem\u003eS. aureus\u003c/em\u003e cultures), negative control (LB broth and \u003cem\u003eS. aureus\u003c/em\u003e cultures) and blank control (added only 200 \u0026mu;L LB broth). Finally, we used a SpectraMax i3x (Molecular Devices) microplate reader to continuously measure the growth of bacter (read mode: absorbance, read type: kinetics, wavelength: 600 nm, temperature: 35\u0026deg;C, shaking 10 s before read). At least three independent replicates of each data were obtained. Finally, arranged the data through software to respectively draw time and growth-inhibition curves of two \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003estrains treated with CFS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eATCC29213 was incubated overnight in 40ml LB broth, and diluted with LB broth to 0.5 MCF. The \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 was incubated at 29\u0026deg;C with shaking at 100 rpm for 24 h, followed by\u0026nbsp;using the centrifugation method (10,000 rpm for 10 min) and filter sterilization with a membrane of pore size 0.22 \u0026mu;m to obtain the CFS. The \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eATCC29213\u003cem\u003e\u0026nbsp;\u003c/em\u003esolution was incubated with\u0026nbsp;CFS at\u0026nbsp;a ratio of 1% for 15 h at 4\u0026deg;C. Subsequently, cells were collected by centrifugation at 8000 rpm for 10 min, then washed with aquae sterilisata and fixed with 2.5% glutaraldehyde for 24h. The cells were dehydrated using gradient alcohol solutions (10 to 100%) for 20 min each, treated with isoamyl acetate for 20 min, dried by a vacuum freeze dryer for 12 h, and sputter-coated with gold. The morphological images of \u003cem\u003eS. aureus\u003c/em\u003e ATCC29213 were observed and acquired with scanning electron microscopy (SEM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequencing and sequence analysis of L. garvieae SHAMU‑LG6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome of\u0026nbsp;\u003cem\u003eL. garvieae\u0026nbsp;\u003c/em\u003eSHAMU‑LG6\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas sequenced on an Illumina NovaSeq /Oxford Nanopore ONT platform by Shanghai Paisennuo Biotechnology Co. Ltd. The paired-end-read quality control was performed with FASTP. The third-generation sequencing data was assembled using Unicycle and Flye softwares to obtain contig sequences, then through pilon software to correct errors in the contig. Finally, the complete genome sequence of\u0026nbsp;\u003cem\u003eL. garvieae\u0026nbsp;\u003c/em\u003eSHAMU‑LG6\u0026nbsp;was obtained by splicing. Compared all predicted protein coding genes with the proteins contained in various databases for functional annotation of protein-coding genes. The genome circle map was drawn by using cgview software. The bacteriocin biosynthetic gene clusters were predicted by AntiSMASH (https://antismash.secondarymetabolites.org). Functional analysis of proteins was performed using the BLASTP program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRough extraction of garvicin-SHAMU-LG6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 strain was inoculated in LB broth (400ml) for 24h at 29\u0026deg;C. The fermentation supernatant was centrifuged at 7450\u0026times;g for 10min at 4\u0026deg;C, and then the supernatant was decanted and filtered twice with 0.22 \u0026micro;m filters, to obtain CFS. The garvicin-SHAMU-LG6 crude extract was obtained using the Amberlite XAD-16 N resin\u0026nbsp;adsorption method. Briefly, the 400ml CFS was loaded onto a column containing 220g Amberlite XAD-16 N resin. Then, the resin was washed with 3 L of ultrapure water and 1 L of 30% (v/v) ethanol. The active substances were sequentially eluted with 800 ml of 50%, 60% 70%, 80%, and 90% (vol/vol) ethanol,\u0026nbsp;followed by using a rotary evaporator to remove the ethanol. The\u0026nbsp;eluates of each ethanol gradient were\u0026nbsp;concentrated\u0026nbsp;until completely dry, then respectively\u0026nbsp;dissolved in 5mL of\u0026nbsp;ultrapure water and transferred to an EP tube. Finally, filtered with 0.22 \u0026micro;m filters and tested their antimicrobial activity by the Oxford cup method described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSensitivity to pH, temperature, and proteases\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effect of pH on the\u0026nbsp;garvicin-SHAMU-LG6 was investigated by adjusting the pH of its\u0026nbsp;crude extract\u0026nbsp;(100 \u0026micro;l) from 3.0 to 11.0 with CH\u003csub\u003e3\u003c/sub\u003eCOOH or NaOH solution, followed by incubation at 35℃ for 1h. The residual antimicrobial activity was tested after neutralizing the sample to pH 7.0. To research the thermal stability of garvicin-SHAMU-LG6, the\u0026nbsp;crude extract\u0026nbsp;(100 \u0026micro;l) was exposed to 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃ and 121℃ for 10 min, followed by measuring the residual antimicrobial activity. The effects of various proteases on garvicin-SHAMU-LG6 were also tested. The proteases included chymotrypsin, trypsin, proteinase K, and bromelain, and each protease solution (20 mg/ml) and\u0026nbsp;crude extract\u0026nbsp;(100 \u0026micro;l) was mixed and incubated at 35\u0026deg;C for 1 h. The residual antimicrobial activity was determined as well. The garvicin-SHAMU-LG6 sample used in this study was concentrated 80-fold in the original fermentation broth volume. The indicator strain was \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eand antimicrobial activity was determined with the Oxford cup diffusion method as described above\u003cem\u003e.\u003c/em\u003e The untreated crude extract solution was used as a control. All experiments were performed in triplicate.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eScreening and identification of a bacteriocin-producing strain, \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe screened a strain with antagonistic activity against many Gram-positive cocci, Gram-positive bacilli, and Gram-negative bacilli from a urine specimen collected from an ordinary patient (non-urological infections) in urology. MALDITOF MS and MALDI Biotyper 3.1 software analysis preliminarily identified this strain was \u003cem\u003eL. garvieae.\u0026nbsp;\u003c/em\u003eThe 16S rDNA gene sequence of this strain was deposited in the GenBank, accession number: OQ918061. To further determine the strain, a phylogenetic tree (\u003cstrong\u003eFig 1\u003c/strong\u003e) was generated to compare its 16S rDNA to its closely related sequences using MEGA 11.0. Two isolates having 16S rDNA sequence similarities of more than 97% can generally be considered as homology within the genus. Genomic analysis showed the 16S rDNA gene of the isolate shares 99.66% sequence similarity with that of \u003cem\u003eLactococcus garvieae.\u0026nbsp;\u003c/em\u003eThus, it was given the strain designation \u003cem\u003eL. garvieae\u0026nbsp;\u003c/em\u003eSHAMU-LG6, and preserved in China Center for Type Culture Collection (CCTCC) under no. M 2023482.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibacterial spectrum of L. garvieae SHAMU‑LG6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen tested against a series of gram-negative and gram-positive bacteria by using the Oxford cup diffusion method, \u003cem\u003eL. garvieae\u0026nbsp;\u003c/em\u003eSHAMU‑LG6 exhibited a broad spectrum of antimicrobial activity. As shown by the results in \u003cstrong\u003eTable 1\u003c/strong\u003e, \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 exerted wide antimicrobial activities against all of the gram-positive bacteria in this study, including \u003cem\u003eStaphylococcus spp.\u003c/em\u003e, \u003cem\u003eE. faecalis\u003c/em\u003e, \u003cem\u003eE. faecium\u003c/em\u003e, \u003cem\u003eK. kristinae\u003c/em\u003e, \u003cem\u003eS. agalactiae\u003c/em\u003e, \u003cem\u003eE. durans,\u003c/em\u003e \u003cem\u003eL. innocua\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;D. hominus.\u003c/em\u003e Moreover, it also showed antimicrobial activity against most tested gram-negative bacteria. Further inspection of the table showed that \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 also had potent antibacterial activity against some common MDRP. These results demonstrated that \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 could inhibit the growth of a series of clinical pathogenic bacteria (\u003cstrong\u003eFig 3\u003c/strong\u003e) and some common MDRP \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFig 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAntimicrobial spectrum of \u003cem\u003eLactococcus garvieae\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eSHAMU‑LG6\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eIndicator strain\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of strains\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eHypostatic rate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.64788732394366%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAntibacterial activity\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"21.428571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.428571428571427%\" valign=\"top\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.142857142857146%\" valign=\"top\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eGram-positive cocci\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003e(sensitive)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u0026nbsp;\u003c/em\u003e(MRSA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus hominis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus capitis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus haemolyticus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e87.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus saprophyticus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus simulans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus lugdunensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStaphylococcus petenkovii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterococcus faecium\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eKocuria kristinae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStreptococcus agalactiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterococcus durans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eGram-positive bacilli\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eListeria innocua\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eDermabacter hominus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"7\"\u003e\n \u003cp\u003e\u003cstrong\u003eGram-negative bacilli\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003e(sensitive)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e0.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003e(CRE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e20.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003e(ESBLs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e70.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u0026nbsp;\u003c/em\u003e(sensitive)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e93.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter baumannii\u0026nbsp;\u003c/em\u003e(MDR-AB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003eKlebsiellar \u003cem\u003epneumonia\u0026nbsp;\u003c/em\u003e(sensitive)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiellar pneumonia\u0026nbsp;\u003c/em\u003e(CRE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e75.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003eKlebsiellar \u003cem\u003epneumonia\u0026nbsp;\u003c/em\u003e(ESBLs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e86.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003eKlebsiella oxytoca\u0026nbsp;(sensitive )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e50.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003eKlebsiella oxytoca\u0026nbsp;(ESBLs)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas. aeruginosa\u0026nbsp;\u003c/em\u003e(sensitive )\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e93.8%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u0026nbsp;\u003c/em\u003e(MDR-PA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas montelli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas putida\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eStenotrophomonas maltophilia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAeromonas caviae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAeromonas veronii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAeromonas hydrophila\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003ePseudomonas mosaica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter cloacae\u0026nbsp;\u003c/em\u003e(sensitive)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e35.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter cloacae\u0026nbsp;\u003c/em\u003e(CRE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e6.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter asburiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e14.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eEnterobacter bougainvilli\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e60.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eProteus vulgaris\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eProteus hauterii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eProteus mirabilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eSerratia marcescens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAlcaligenes faecalis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAchromobacter xylosoxidans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter junii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eAcinetobacter Pittii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eHospital Acinetobacter\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eCitrobacter freudii\u0026nbsp;\u003c/em\u003e(sensitive)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eCitrobacter freudii\u003c/em\u003e (CRE)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eCitrobacter Klebsiella\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eChryseobacterium indologenes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eShewanella putrefaciens\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eMorganella morganii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.32394366197183%\"\u003e\n \u003cp\u003e\u003cem\u003eMoraxella osloensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.246478873239436%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.781690140845072%\"\u003e\n \u003cp\u003e100.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.28169014084507%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.161971830985915%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.922535211267606%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eMRSA, Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e; CRE, Carbapenem-resistant \u003cem\u003eEnterobacteriaceae\u003c/em\u003e; ESBLs, Extended-spectrum \u0026beta;-lactamases; sensitive, drug-sensitive bacteria.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e\u0026quot;-\u0026quot; represents the inhibition zone diameter \u0026le; 6 mm (no inhibitory effect); \u0026quot;+\u0026quot; represents the inhibition zone diameter 6-10 mm (including 10 mm); \u0026quot;++\u0026quot; represents the inhibition zone diameter of 10-20 mm (including 20 mm); \u0026quot;+++\u0026quot; represents the inhibition zone diameter \u0026gt; 20 mm.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 cell-free supernatant inhibits the growth of \u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo verify whether \u003cem\u003eL. garvieae\u0026nbsp;\u003c/em\u003eSHAMU‑LG6 inhibits the growth of other bacteria by secreting antibacterial active substances outside the cell. We selected \u003cem\u003eS. aureus\u003c/em\u003e ATCC25923 and \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eATCC29213 as models to measure the antibacterial activity of CFS. The experiment was divided into four groups, and each experiment was consistently repeated in triplicate. The optical density (OD\u003csub\u003e600\u003c/sub\u003e) of each group was measured at set intervals by a SpectraMax i3x (Molecular Devices) microplate reader, and the mean value was calculated from the obtained data. The time and growth-inhibition curves of two \u003cem\u003eS. aureus strains\u003c/em\u003e respectively treated with CFS were shown in \u003cstrong\u003eFig 4\u003c/strong\u003e. Neither of them multiply in wells with vancomycin and the blank control group, conversely, they multiply normally in negative controls containing LB broth. Compared with the control groups, in the experimental group adding the CFS, the growths of two \u003cem\u003eS. aureus\u003c/em\u003e strains were all inhibited with the delayed logarithmic phase and stationary phase under CFS treatment. These results demonstrated that the CFS contained an active substance, which may be a bacteriocin and was preliminary named garvicin-SHAMU-LG6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGarvicin-SHAMU-LG6 exerts bactericidal activity by damaging the cell integrity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo deduce the mode of action of garvicin-SHAMU-LG6, the cell morphological change of \u003cem\u003eS.aureus\u003c/em\u003e ATCC29213 cells when incubated under CFS of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU‑LG6 was observed and analyzed by scanning electron microscopy (SEM). SEM analysis demonstrated that the cell membrane of \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003eATCC 29213 cells became deformed and collapsed (\u003cstrong\u003eFig 5\u003c/strong\u003e\u003cstrong\u003eAB\u003c/strong\u003e) when incubated with CFS for15 h at 4\u0026deg;C, whereas the untreated control cells showed smooth and intact surfaces \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFig 5\u003c/strong\u003e\u003cstrong\u003eCD)\u003c/strong\u003e. In conclusion, garvicin-SHAMU-LG6 may cause cell membrane damage, and disrupt the integrity of target cells, thereby exerting bactericidal activity on \u003cem\u003eS.aureus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003ePutative biosynthetic gene cluster of garvicin-SHAMU-LG6\u003c/p\u003e\n\u003cp\u003eFurther, the entire genome of the \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU-LG6 was sequenced and the genome was uploaded on antiSMASH to find the biosynthetic gene cluster of the garvicin-SHAMU-LG6. The complete genome of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU-LG6 consists of a 2,033,399-base pair (bp) circular chromosome, a 39,838-bp circular plasmid, and a 2,235-bp circular plasmid, with a GC content of 38.27%, 35.58% and 33.29%, respectively. The complete chromosome genome information of the strain SHAMU-LG6 is shown in\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFig 6\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe result of using AntiSMASH to search for secondary metabolites is shown in \u003cstrong\u003eTable 2\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e The putative biosynthetic gene cluster of garvicin-SHAMU-LG6 is illustrated in \u003cstrong\u003eFig 7\u003c/strong\u003e. Functional analysis of proteins encoded by these genes was performed using the BLASTP program.\u0026nbsp;The gene cluster consisted of thirteen genes, including two genes (chr1825 and chr1827) encoding bacteriocin, one gene (chr1823) with biosynthetic-additional function, two genes (chr1821 and chr1832) possibly were transport-related genes, two genes (chr1824 and chr1830) may involved in immunity, one regulatory gene (chr1820), four genes (chr1822, chr1828, chr1831) possibly associated with gene transfer and rearrangement, one gene (chr1829) may be related to DNA segregation machinery, and one gene (chr1826) with unknown function. Moreover, using BLAST software, the gene encoding protein sequence was compared with the amino acid sequence in the virulence factor-related gene database, and no virulence factor-related genes were found in the genome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e Antismash analysis\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"103%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.34020618556701%\" valign=\"top\"\u003e\n \u003cp\u003eSeq ID\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"top\"\u003e\n \u003cp\u003eType\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"top\"\u003e\n \u003cp\u003eStart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"top\"\u003e\n \u003cp\u003eEnd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.11340206185567%\" valign=\"top\"\u003e\n \u003cp\u003eMost similar known cluster\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"top\"\u003e\n \u003cp\u003eSimilarity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.34020618556701%\" valign=\"top\"\u003e\n \u003cp\u003echr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"top\"\u003e\n \u003cp\u003eRiPP-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"top\"\u003e\n \u003cp\u003e1,823,808\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.371134020618557%\" valign=\"top\"\u003e\n \u003cp\u003e1,834,020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.11340206185567%\" valign=\"top\"\u003e\n \u003cp\u003elactocin S RiPP: Lanthipeptide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\" valign=\"top\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eSequence ID;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eRiPP, other unspecified ribosomally synthesised and post-translationally modified peptide product.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e3\u003c/strong\u003e Garvicin-SHAMU-LG6 biosynthetic related genes\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"619\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\" valign=\"top\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\" valign=\"top\"\u003e\n \u003cp\u003eNo. of AA\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eKnown protein with the highest homology\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[microorganism]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\" valign=\"top\"\u003e\n \u003cp\u003eProposed function\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1820\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eMetalloregulator ArsR/SmtB family transcription factor [Bacteria]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP_003726380.1, identity: 100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eGene regulation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1821\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e705\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eHeavy metal translocating P-type ATPase [Lactobacillales]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP_001291323.1, identity: 100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eTransport\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1822\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eIS6 family transposase [Lactococcus garvieae]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP_285013987.1, 95.80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eGene transfer and rearrangement\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1823\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eCysteine peptidase family C39 domain-containing protein [Lactococcus]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP_258272642.1,\u0026nbsp;100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\" valign=\"top\"\u003e\n \u003cp\u003eBiosynthetic-additional\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1824\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eBacteriocin immunity protein [Lactococcus]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP_032495339.1, 100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eImmunity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1825\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eGarvicin Q family class ll bacteriocin [Lactococcus]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP_165719065.1, 98.57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\" valign=\"top\"\u003e\n \u003cp\u003eBacteriocin biosynthetic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1826\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eLactococcus garvieae strain Lg-Granada plasmid pLG50, complete sequence (GenBank:CP084378.1,\u0026nbsp;100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eUnknown function\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1827\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eBacteriocin [Lactococcus]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP_165719075.1,\u0026nbsp;98.36%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eBacteriocin biosynthetic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1828\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eRecombinase family protein [Lactococcus]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP-003134225.1,\u0026nbsp;100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eGene transfer and rearrangement\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1829\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eParA family protein [Lactococcus]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP-003134226.1,\u0026nbsp;100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eDNA segregate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1830\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eLactococcus garvieae strain IPLA 31405 plasmid pLG42, complete sequence (GenBank: KM007160.1, 100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eImmunity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1831\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003eIS6-like element IS1216 family transposase [Lactococcus]\u003c/p\u003e\n \u003cp\u003e(NCBI Reference Sequence: WP 162523730.1, 100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eGene transfer and rearrangement\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.723747980613894%\"\u003e\n \u003cp\u003echr1832\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.046849757673668%\"\u003e\n \u003cp\u003e172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"53.150242326332794%\" valign=\"top\"\u003e\n \u003cp\u003ePTS sugar transporter subunit IIC [Lactococcus petauri]\u003c/p\u003e\n \u003cp\u003e(GenBank: NHI79097.1, 100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.079159935379643%\"\u003e\n \u003cp\u003eTransport\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eAA, amino acids\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eanalyses were performed using the BLASTP program, and the proteins listed were the closest homologue to each gene in the putative biosynthetic gene cluster of Garvicin-SHAMU-LG6 in the NCBI and GenBank database.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003eFunction annotation of predicted proteins encoded by genes in the putative bacteriocin biosyntheti gene cluster.\u003c/p\u003e\n\u003cp\u003eRough extraction of garvicin-SHAMU-LG6\u003c/p\u003e\n\u003cp\u003eThe\u003cem\u003e\u0026nbsp;L. garvieae\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eSHAMU-LG6 strain was cultured in LB broth for 24 h, and the\u0026nbsp;active substance\u0026nbsp;in the culture supernatants was rough extraction using Amberlite XAD-16 N resin. Using\u003cem\u003e\u0026nbsp;S. aureus\u003c/em\u003e as an indicator bacterium, the Oxford cup diffusion method mentioned above was used to verify the antibacterial activity of the collected liquids eluted with ethanol solutions of different concentrations [50%, 60% 70%, 80%, 90% (vol/vol) ethanol]. As shown by the results in \u003cstrong\u003eFig 8\u003c/strong\u003e, the fraction eluted with 70% and 80% (vol/vol) ethanol solutions had the best antibacterial effect. Therefore, we ultimately used 75% (vol/vol) ethanol as the elution solution, and the resulting supernatant was designated the crude extract, which was used for subsequent purification and study on physicochemical properties.\u003c/p\u003e\n\u003cp\u003eStability of garvicin-SHAMU-LG6\u003c/p\u003e\n\u003cp\u003eThe susceptibility of garvicin-SHAMU-LG6 to pH, temperature, and proteases was investigated using the Oxford cup diffusion method. Untreated crude extract solution was taken as control. Garvicin-SHAMU-LG6 was found to be stable at high temperatures (up to 121℃) and in a wide range of pH (pH 3-11). In terms of thermostability, as presented in \u003cstrong\u003eFig 9\u003c/strong\u003e\u003cstrong\u003eB,\u003c/strong\u003e the antimicrobial activity of garvicin-SHAMU-LG6 slightly decreased with increasing temperature, but it could resist the temperature of 121℃ and retained most of its antimicrobial activity. Upon treatment in a range of pH, garvicin-SHAMU-LG6 showed its stability in the pH range of 3-11 (\u003cstrong\u003eFig 9\u003c/strong\u003e\u003cstrong\u003eA).\u0026nbsp;\u003c/strong\u003eAlthough the residual antimicrobial activity was greatly decreased\u0026nbsp;under near-neutral\u0026nbsp;conditions (ph 6.0 to 8.0),\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003egarvicin-SHAMU-LG6 exhibited superior activity under alkaline (ph 11.0) and acidic (ph 3.0) conditions. The antibacterial activity of garvicin-SHAMU-LG6 remained almost unchanged after treatment with four enzymes, as illustrated in \u003cstrong\u003eFig 9\u003c/strong\u003e\u003cstrong\u003eC\u003c/strong\u003e. Among them, garvicin-SHAMU-LG6 could retain its full antimicrobial activity after treatment with chymotrypsin and bromelain, majority of antimicrobial activity after treatment with trypsin and proteinase K.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe emergence of antibiotic resistance is a growing global public crisis. As more and more antibiotics become ineffective due to drug-resistant bacteria, it is imperative to focus on alternative therapies to conventional antibiotics[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the past two decades, bacteriocins have shown their potential as promising alternative therapeutic [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The LAB is a well-known producer of bacteriocins and has great potential for application in food and pharmaceutical fields. Several reports have shown that over 230 bacteriocins from LAB have been isolated and reported, but only half of them were identified at the protein or DNA levels [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The most representative bacteriocin is the lactibiotic nisin, which is the first and most widely used bacteriocin in the food preservation and there have been no reports of widespread drug resistance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. \u003cem\u003eL. garvieae\u003c/em\u003e is a LAB and so far, various bacteriocins have been found in different \u003cem\u003eL. garvieae\u003c/em\u003e strains, including garviecin L1-5 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], Garvicin ML [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], Garvieacin Q [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], garvicin A [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], Garvicin KS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Garvicins AG1, and Garvicins AG2 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This study reports a novel bacteriocin, garvicin-SHAMU-LG6, that was produced by \u003cem\u003eL. garvieae\u003c/em\u003e strain SHAMU-LG6 (CCTCC No: M 2023482), and whose wide antimicrobial activity against various pathogenic bacteria as well as MDRP, and high pH and thermal stability highlight its potential as a promising clinical therapeutic drug.\u003c/p\u003e \u003cp\u003eThe antimicrobial spectrum of one bacteriocin is vital for its application potential. Garviecin L1-5 is a small bacteriocin (about 2.5 kDa), produced by \u003cem\u003eL. garvieae\u003c/em\u003e isolated from a raw cow\u0026rsquo;s milk sample. It was bactericidal against closely related species and strains of species from different genera, including \u003cem\u003eListeria\u003c/em\u003e and \u003cem\u003eClostridium\u003c/em\u003e spp [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The 60-amino-acid circular bacteriocin, garvicin ML (about 6 kDa), is produced by \u003cem\u003eL. garvieae\u003c/em\u003e DCC43 isolated from mallard ducks and has a broad antimicrobial spectrum. It inhibits some strains of species from the \u003cem\u003eLactococcus\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003ePediococcus\u003c/em\u003e, \u003cem\u003ePropionibacterium\u003c/em\u003e, \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eStreptococcus,and Enterococcus\u003c/em\u003e spp. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Garvieacin Q, a class II bacteriocin, consists of 50 amino acids (5.3 kDa) and is produced by strain BCC 43578, isolated from fermented pork sausage. It is especially active against \u003cem\u003eListeria monocytogenes\u003c/em\u003e ATCC 19115 and other \u003cem\u003eL. garvieae\u003c/em\u003e strains [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Garvicin A is a class IId narrow-spectrum bacteriocin, with a mass of 4.7 kDa, produced by \u003cem\u003eL. garvieae\u003c/em\u003e 21881 isolated in a human clinical case. It is active only against other strains of the same species [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Garvicin KS is a leaderless multipeptide bacteriocin, produced by \u003cem\u003eL. garvieae\u003c/em\u003e KS1546 isolated from raw milk. Garvicin KS is composed of three similar peptides of 32 to 34 amino acids and shows wide antibacterial activity. Its inhibitory spectrum includes important pathogens belonging to the genera \u003cem\u003eStaphylococcus, Bacillus, Listeria\u003c/em\u003e, and \u003cem\u003eEnterococcus\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Garvicins AG1 and AG2 are two novel class IId bacteriocins of \u003cem\u003eL. garvieae\u003c/em\u003e Lg-Granada isolated from the blood of a patient with endocarditis. They exhibit antimicrobial activity against other \u003cem\u003eL. garvieae\u003c/em\u003e strains, and AG2 can also inhibit the growth of \u003cem\u003eListeria monocytogene\u003c/em\u003es, \u003cem\u003eListeria ivanovii\u003c/em\u003e, and \u003cem\u003eEnterococcus faecalis\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Apparently, that the majority of bacteriocins produced by \u003cem\u003eL. garvieae\u003c/em\u003e are narrow-spectra bacteriocins. Herein, garvicin-SHAMU-LG6, the bacteriocin identified in the present study, has a more extensive antibacterial spectrum in comparison with the aforementioned bacteriocins. It exhibits antimicrobial activity against all of the gram-positive cocci in this study, two gram-positive bacilli strains, and many gram-negative bacilli. In addition, in terms of antibiotic resistance, we divided the indicator strains into drug-resistant and drug-sensitive groups. The results, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, indicate that it has good antagonistic activity against and many clinical pathogenic bacteria and common MDRP, such as MRSA, MDRAB, MDRPA, and some CRE. Therefore, the study of garvicin-SHAMU-LG6 may have important application value for the treatment of antibiotic-resistant bacteria.\u003c/p\u003e \u003cp\u003eThe growth kinetics of \u003cem\u003eS. aureus\u003c/em\u003e strains treated with CFS indicated that the fermentation supernatant of strain \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU-LG6 contained an antimicrobial substance. Additionally, the whole-genome sequencing and DNA sequence by the antiSMASH database located the presence of a putative biosynthetic gene cluster which was identical 10% similarity with the lactocin S gene cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This gene cluster contained genes coding for the bacteriocin, transporter-related proteins, proteins responsible for immunity, multiple transposase, and recombinase that probably relate to gene transfer and rearrangement and transcription factor. Sequence homology analysis showed that the similarity between this gene cluster and the known most similar gene cluster is only 10% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, in terms of application security, no evidence of virulence-related genes was obtained. This further confirms that \u003cem\u003eL. garvieae\u003c/em\u003e SHAMULG6 inhibits the growth of other bacteria by secreting a promising novel bacteriocin.\u003c/p\u003e \u003cp\u003eAlvarez-Sieiro et al.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] proposed dividing bacteriocins into three classes according to their biological activities and biosynthesis mechanisms. Most bacteriocins produced by LAB were Classes I and II small bacteriocins. Class I bacteriocins are heat-stable and smaller than 5 kDa peptides, which undergo post-translational modification; Class II includes heat-stable, smaller than 10 kDa, unmodified bacteriocins. Different classes of bacteriocins have different structures and mechanisms of action. Bacteriocins differ in the mode of action of antibiotics, most LAB bacteriocins, especially those inhibiting Gram-positive bacteria, exert their antimicrobial effects by targeting bacterial cell membranes and forming pores[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, some bacteriocins exert antibacterial effects through various mechanisms. For example, nisins effectively belong to the Class I bacteriocins that contain lanthionine and possess a double mode of action[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]: (i) their N-terminal binds to lipid II and the C-terminal penetrates cytoplasmic membrane, resulting in pore formation and induces ion and ATP leakage. (ii) they inhibit cell wall synthesis by masking lipid II. In the present study, SEM analysis clearly showed that sensitive cells treated with CFS of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU-LG6 exhibited significant deformation and collapse (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Moreover, \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU-LG6 exhibited antagonistic activity against both Gram-positive and Gram-negative bacteria. Therefore, we speculate that garvicin-SHAMU-LG6 targets universal molecules (such as the cell membrane, or intracellular target molecules) in bacterial cells, disrupting the integrity of bacterial cells through one or more modes of action, thereby killing the target bacteria. This hypothesis should be verified in further studies.\u003c/p\u003e \u003cp\u003eAlthough bacteriocins have many advantages, antibacterial spectrum, purification, and yield have limited the application of bacteriocins. Breaking the bottleneck has become the focus of current research. In this study, we found that the garvicin-SHAMU-LG6 crude extract obtained by using Amberlite XAD-16 N resin to adsorb bacteriocin in the culture supernatants and eluting with 75% alcohol has the strongest activity. These results indicated the above-said method is capable of preliminary purification of garvicin-SHAMU-LG6 from fermentation supernatant. We will attempt to further purify garvicin-SHAMU-LG6 by using various chromatographic techniques for studying its structure and characteristics in future studies.\u003c/p\u003e \u003cp\u003eThe stability of garvicin-SHAMU-LG6 under acidic or basic and heat conditions was tested. Garvicin-SHAMU-LG6 was stable under acidic (PH 3) and basic conditions (PH 11) and exhibited a slight loss of activity under neutral conditions. Additionally, in terms of thermostability, upon autoclaving (121℃), garvicin-SHAMU-LG6 retained most of its antimicrobial activity. Many bacteriocins, including nisin, are readily inactivated under neutral and alkaline conditions. We also tested the susceptibility of garvicin-SHAMU-LG6 to four proteases. Although it showed resistance to the digestion of chymotrypsin, trypsin, proteinase K, and bromelain, we cannot completely deny that it is a protein or polypeptide. On the one hand, it may be because that garvicin-SHAMU-LG6 is a small molecule polypeptide with a simple structure and thus does not have the target of these enzymes. On the other hand, it might also be due to its special three-dimensional structure that blocks the recognition or combination of the protease and the target. For the example, the previous study indicated that the resistance of GarML to trypsin, pepsin, papain, and proteinase K, is not due to the absence of digestive sites, but to the inaccessibility of recognition sites [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These hypotheses should be verified in further studies. In conclusion, the extraordinary stability of garvicin-SHAMU-LG6 is another desirable property supporting its potential application value in food and pharmaceutical fields.\u003c/p\u003e \u003cp\u003eAs well, determining the biosafety of bacteriocin is vital for its development and application in medicine or food. Although no virulence factors have currently been found in its genome, hemolytic activity and cytotoxicity examination remain crucial. In general, more detailed studies may be carried out about the safety of garvicin-SHAMU-LG6 to evaluate the feasibility of its application as a promising clinical therapeutic drug.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study identified a broad-spectrum bacteriocin-producer, \u003cem\u003eLactococcus garvieae\u003c/em\u003e SHAMU-LG6, and preliminary purified its bacteriocin. Garvicin-SHAMU-LG6 showed good pH, heat, and protease stability, and it has wide antimicrobial activity against pathogenic bacteria as well as MDRP, likely by destroying their cell membrane integrity. These findings will provide the theoretical foundation for its potential application as a food preservative and clinical therapeutic drug.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant number 82102460).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish this review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSW: Preparation, Investigation, Data curation,Writing - Original Draft. GL: Preparation, Methodology, Investigation. JG: Methodology, Investigation,Validation. YW: Validation, Data Curation. JY: Investigation, Validation. XL: Investigation, Validation. ZL: Investigation, Supervision. QZ: Investigation, Writing - review and editing. WT: Supervision, Funding acquisition, Writing - Review \u0026amp; Editing, Project administration.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMagana M, Pushpanathan M, Santos AL, Leanse L, Fernandez M, Ioannidis A, Giulianotti MA, Apidianakis Y, Bradfute S, Ferguson AL, et al. The value of antimicrobial peptides in the age of resistance. LANCET INFECT DIS. 2020;20(9):e216\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeves JV. Editorial for Special Issue Alternatives to Antibiotics: Bacteriocins and Antimicrobial Peptides. ANTIBIOTICS-BASEL 2022, 11(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu S, Deng S, Liu H, Tang L, Wang M, Xin B, Li F. Four Novel Leaderless Bacteriocins, Bacin A1, A2, A3, and A4 Exhibit Potent Antimicrobial and Antibiofilm Activities against Methicillin-Resistant Staphylococcus aureus. MICROBIOL SPECTR. 2022;10(5):e94522.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhosh C, Sarkar P, Issa R, Haldar J. Alternatives to Conventional Antibiotics in the Era of Antimicrobial Resistance. TRENDS MICROBIOL. 2019;27(4):323\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHernandez-Gonzalez JC, Martinez-Tapia A, Lazcano-Hernandez G, Garcia-Perez BE, Castrejon-Jimenez NS. Bacteriocins from Lactic Acid Bacteria. A Powerful Alternative as Antimicrobials, Probiotics, and Immunomodulators in Veterinary Medicine. ANIMALS-BASEL 2021, 11(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGradisteanu PG, Popa LI, Marutescu L, Gheorghe I, Popa M, Czobor BI, Cristescu R, Chifiriuc MC. Bacteriocins in the Era of Antibiotic Resistance: Rising to the Challenge. PHARMACEUTICS 2021, 13(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshibashi N, Seto H, Koga S, Zendo T, Sonomoto K. Identification of Lactococcus-Specific Bacteriocins Produced by Lactococcal Isolates, and the Discovery of a Novel Bacteriocin, Lactococcin Z. PROBIOTICS ANTIMICRO. 2015;7(3):222\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlvarez-Sieiro P, Montalban-Lopez M, Mu D, Kuipers OP. Bacteriocins of lactic acid bacteria: extending the family. APPL MICROBIOL BIOT. 2016;100(7):2939\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDarbandi A, Asadi A, Mahdizade Ari M, Ohadi E, Talebi M, Halaj Zadeh M, Darb Emamie A, Ghanavati R, Kakanj M. Bacteriocins: Properties and potential use as antimicrobials. J CLIN LAB ANAL 2022, 36(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZendo T, Yoneyama F, Sonomoto K. Lactococcal membrane-permeabilizing antimicrobial peptides. APPL MICROBIOL BIOT. 2010;88(1):1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorrero J, Brede DA, Skaugen M, Diep DB, Herranz C, Nes IF, Cintas LM, Hern\u0026aacute;ndez PE. Characterization of Garvicin ML, a Novel Circular Bacteriocin Produced byLactococcus garvieae DCC43, Isolated from Mallard Ducks (Anas platyrhynchos). APPL ENVIRON MICROB. 2011;77(1):369\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXin B, Zheng J, Xu Z, Li C, Ruan L, Peng D, Sun M. Three Novel Lantibiotics, Ticins A1, A3, and A4, Have Extremely Stable Properties and Are Promising Food Biopreservatives. APPL ENVIRON MICROB. 2015;81(20):6964\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCotter PD, Ross RP, Hill C. Bacteriocins - a viable alternative to antibiotics? NAT REV MICROBIOL. 2013;11(2):95\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTavares LM, de Jesus L, Da ST, Barroso F, Batista VL, Coelho-Rocha ND, Azevedo V, Drumond MM, Mancha-Agresti P. Novel Strategies for Efficient Production and Delivery of Live Biotherapeutics and Biotechnological Uses of Lactococcus lactis: The Lactic Acid Bacterium Model. FRONT BIOENG BIOTECH. 2020;8:517166.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMokoena MP, Omatola CA, Olaniran AO. Applications of Lactic Acid Bacteria and Their Bacteriocins against Food Spoilage Microorganisms and Foodborne Pathogens. \u003cem\u003eMOLECULES\u003c/em\u003e 2021, 26(22).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTosukhowong A, Zendo T, Visessanguan W, Roytrakul S, Pumpuang L, Jaresitthikunchai J, Sonomoto K. Garvieacin Q, a Novel Class II Bacteriocin from Lactococcus garvieae BCC 43578. APPL ENVIRON MICROB. 2012;78(5):1619\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubey S, Diep DB, Evensen \u0026Oslash;, Munang Andu HM. Garvicin KS, a Broad-Spectrum Bacteriocin Protects Zebrafish Larvae against Lactococcus garvieae Infection. Int J Mol Sci. 2022;23(5):2833.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOvchinnikov KV, Chi H, Mehmeti I, Holo H, Nes IF, Diep DB. Novel Group of Leaderless Multipeptide Bacteriocins from Gram-Positive Bacteria. APPL ENVIRON MICROB. 2016;82(17):5216\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOvchinnikov KV, Chi H, Mehmeti I, Holo H, Nes IF, Diep DB. Novel Group of Leaderless Multipeptide Bacteriocins from Gram-Positive Bacteria. APPL ENVIRON MICROB. 2016;82(17):5216\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi GH, Holzapfel WH, Todorov SD. Diversity of the bacteriocins, their classification and potential applications in combat of antibiotic resistant and clinically relevant pathogens. CRIT REV MICROBIOL. 2023;49(5):578\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaskoniene V, Stankevicius M, Bimbiraite-Surviliene K, Naujokaityte G, Serniene L, Mulkyte K, Malakauskas M, Maruska A. Current state of purification, isolation and analysis of bacteriocins produced by lactic acid bacteria. APPL MICROBIOL BIOT. 2017;101(4):1323\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Xu H, Liu S, Song B, Liu H, Li F, Deng S, Wang G, Zeng H, Zeng X, et al. Toyoncin, a Novel Leaderless Bacteriocin That Is Produced by Bacillus toyonensis XIN-YC13 and Specifically Targets B. cereus and Listeria monocytogenes. APPL ENVIRON MICROB. 2021;87(12):e18521.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVillani F, Aponte M, Blaiotta G, Mauriello G, Pepe O, Moschetti G. Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk. J APPL MICROBIOL. 2001;90(3):430\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaldonado-Barrag\u0026iuml; NA, C\u0026iuml; Rdenas N, Mart\u0026iuml; Nez B, Ruiz-Barba JL, Fern\u0026iuml; Ndez-Garayz\u0026iuml; Bal JF, Rodr\u0026iuml; Guez JM, Gibello A. Garvicin A, a Novel Class IId Bacteriocin from Lactococcus garvieae That Inhibits Septum Formation in L. garvieae Strains. APPL ENVIRON MICROB. 2013;79(14):4336\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaldonado-Barragan A, Alegria-Carrasco E, Blanco M, Vela AI, Fernandez-Garayzabal JF, Rodriguez JM, Gibello A. Garvicins AG1 and AG2: Two Novel Class IId Bacteriocins of Lactococcus garvieae Lg-Granada. INT J MOL SCI 2022, 23(9).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"L. garvieae, bacteriocin, antimicrobial activity, multidrug-resistant pathogens","lastPublishedDoi":"10.21203/rs.3.rs-3972345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3972345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccelerating growth and global expansion of antimicrobial resistance has deepened the demand for discovery of novel antimicrobial agents. Bacteriocins have attracted increasing interest because of their high efficiency, low toxicity and being not easy to cause drug resistance. This study aims to investigate a novel broad-spectrum bacteriocin, contributing to the pharmaceutical fields.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom a human urine specimen, we isolated a strain thatproduced a novel broad-spectrum bacteriocin, which was identified as \u003cem\u003eLactococcus garvieae\u003c/em\u003e SHAMU-LG6. The bacteriocin, termed garvicin-SHAMU-LG6. The Oxford cup methoddemonstrated it could inhibit the growth of various clinically pathogenic and multidrug-resistant pathogens (MDRP). Whole genome sequencing analysis found a putative gene cluster, that shared 10% similarity with the most similar known bacteriocin cluster. In addition, the cell-free supernatant (CFS) of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU-LG6 exerts antimicrobial activity against \u003cem\u003eS. aureus \u003c/em\u003eby\u003cem\u003e \u003c/em\u003edisrupting the integrity of bacterial cells. Furthermore, garvicin-SHAMU-LG6 was preliminary purified from the CFS of \u003cem\u003eL. garvieae\u003c/em\u003e SHAMU-LG6. Its crude extraction showed good pH (pH 3 to 11) and heat stability (30℃ to 121℃) and resistance to the digestion of chymotrypsin, trypsin, proteinase K, and bromelain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll these studies suggested that garvicin-SHAMU-LG6 has the potential to be used as a therapeutic drug against pathogenic bacteria as well as MDRP in the food and pharmaceutical fields.\u003c/p\u003e","manuscriptTitle":"Garvicin-SHAMU-LG6, A Novel Bacteriocin from Lactococcus garvieae That Exert Broad Antimicrobial Activity Against Drug-Resistant Pathogens","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-23 05:13:06","doi":"10.21203/rs.3.rs-3972345/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9b2de238-2482-4450-a379-cb656ba7d1ed","owner":[],"postedDate":"February 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-27T19:14:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-23 05:13:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3972345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3972345","identity":"rs-3972345","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}