High antagonistic activity and antibiotic resistance of flavobacteria of polar microbial freshwater mats (King George Island, Antarctica)

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For the first time, polar-region flavobacterial strains have been investigated for their antagonistic activity and their antibiotic resistance. These strains were derived from microbial mats occurring in ephemeral freshwater ponds, i.e. ponds and streams of the periglacial zone of Ecology Glacier (King George Island, Maritime Antarctica). The study demonstrated the strains’ surprisingly high phylogenetic diversity, with 20 species among 50 isolates. Flavobacteria were characterised by four different patterns of antagonism and sensitivity: PRS, PR, SR and R, with ‘P’ representing the production of antimicrobial substances, ‘R’ – resistance, and ‘S’ – sensitivity to antimicrobials. Over 50% of strains produced substances inhibiting the growth of other isolates, with 40% being sensitive to such compounds. 68% of the isolates represented multidrug-resistant (MDR) strains. The antibiotic resistance index (ARI) demonstrated a significantly higher proportion of MDR strains and ARI ≥ 0.2 in stream mats (87%) as compared to the strains derived from pond mats (55%). A strong correlation was observed between the strains’ antagonistic potential and antibiotic resistance. Diverse chemoecological responses were found among the flavobacterial strains. An important role in these phenomena is accomplished by the “super bacteria” strains that effectively accumulate numerous traits associated with antagonistic potential and can be involved in the potential transfer of these traits. The individualisation of antagonistic interaction patterns and antibiotic resistance is one of the mechanisms that maintain mat homeostasis. Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences flavobacteria antagonistic activity antibiotic resistance microbial mats Antarctica Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Polar regions are characterised by extreme environmental conditions, including low temperatures, high UV intensity, limited nutrient availability and the pressure from frequent freezing and thawing (Králová et al., 2021 ). Due to the selectivity of these factors, only microorganisms with high adaptive capacities thrive in these environments. Flavobacteria are widely distributed in nature and characterised by high molecular, metabolic and physiological plasticity (Bernardet & Bowman, 2015 ; Králová, 2017 ). Currently (September 2024), over 400 species are classified in the genus Flavobacterium , including strains described as occurring in natural environments and relevant to veterinary medicine, agriculture, medicine and biotechnology (Parte et al., 2020 ). This taxon’s widespread occurrence, diversity and environmental potential have aroused strong interest among researchers in both cognitive and applied terms. Flavobacteria are among the most commonly isolated cold-adapted microorganisms of polar-region environments. These include organisms indigenous to these regions, e.g. F. antarcticum. F. glaciei, F. fryxellicola, F. degerlachei and F. sinopsychrotolerans (Yi et al., 2005 ; McCammon & Bowman, 2000 ; Van Trappen et al., 2004 ; 2005 ; Zhang et al., 2006 ; Xu et al., 2011 ; Králová et al., 2018 , 2021 ). The characteristic features of psychrotolerant and psychrophilic flavobacteria include an adaptive strategy for producing pigments (mainly carotenoids), proteins that neutralise reactive oxygen species as well as antifreeze and cold shock proteins (Liu et al., 2019 ). Flavobacteria colonise diverse ecosystems of Arctic and Antarctic polar environments, including glaciers, lakes, streams, soils and plant rhizosphere zones, as well as microbial mats (Van Trappen et al., 2004 ; Xin et al., 2009 ; Králová et al., 2017; 2021 ). Polar-region freshwater microbial mats contain a particularly diverse microbiota that mainly comprises Cyanobacteria, Chloroflexota, Pseudomonadota, Actinomycetota, Bacteroidota, Bacillota and Archaea (Prieto-Barajas et al., 2018 , Valdespino-Castillo et al. 2018 ). In the structure of cold-environment microbial mats flavobacteria are an abundant component (McCammon and Bowman, 2000 ; Králová et al., 2017). Conglomerates of diverse microbial groups usually comprise aggregated, self-sustaining, autonomous ecosystems. Their distinctive feature is the close link between the primary and secondary production processes and the regeneration of nutrients (Rochera et al., 2013 ; de los Ríos et al., 2015 ). The dominant portion of the total biomass of the microbiome of polar terrestrial ecosystems is contained within microbial mats indicating the crucial role they play in the functioning of these extreme environments (Quesada et al., 2009 ; Callejas et al., 2018 ). In heterogeneous microbial mat biocenotic systems, the essential factor that shapes the structure of the consortia is the intercellular interaction taking place at the species, functional group, and whole microbiota levels. Positive and negative interactions determine the dynamics of the colonised environment’s microbiota (Long et al., 2013 ). Antagonistic responses are among the most primeval and essential forms of interactions occurring between microorganisms that make up these consortia (Aguirre-von-Wobeser, 2015; Núñez-Montero & Barrientos, 2018 ). Aggregated microorganisms have developed mechanisms to actively compete through bacteriostatic or bactericidal interactions with rival microbial communities or species (Peterson et al., 2020 ). Bacteria, which have a very wide range of different compounds at their disposal, i.e. exotoxins, toxic enzymes, bacteriocins and antibiotics, use them in antagonistic intercellular interactions (Cotter et al., 2013 ; Mullis et al., 2019 ). The consequence of such interactions is the acquisition of resistance to these compounds. Forming an extended antibiotic resistome is particularly important for bacteria. Widespread antibiotic resistance in microbial mat microbiocenoses is the result of two mechanisms, i.e. natural evolutionary processes, which represent important adaptation of bacteria developing in heterogeneous microbiocenoses (Ambrožič Avguštin et al., 2019 ; Tallada et al., 2022 ; Depta & Niedźwiedzka-Rystwej, 2023 ), and induced resistance resulting from environmental contamination by commercial antibiotics used e.g. in medicine, agriculture and veterinary medicine (Hwengwere et al., 2022 ; Stanton et al., 2022 ). At the same time, diverse horizontal gene transfer (HGT) mechanisms lead to rapid and efficient development of drug resistance across the microbiota (Abe et al., 2020 ). These processes result in a pool of strains characterised by multi-antibiotic resistance, representing an important reservoir of resistance genes in the global microbiome (Van Goethem et al., 2018 ). Referring to these phenomena, contemporary research into microbial mats lacks reports describing antagonistic interactions between their biotic components and their antibiotic resistance. The novelty of the current study is that it describes these relationships within flavobacteria, which are ecologically important and abundant in natural environments, including polar environments. The current study contains a phylogenetic analysis of flavobacterial strains isolated from polar-region microbial mats that develop in small freshwater ponds and streams of the proglacial zone of the Ecology Glacier on King George Island, Antarctica (vicinity of Arctowski Polish Antarctic Station). It also presents the results of investigated antagonistic activity of flavobacterial isolates in the cross-inhibition reaction and their resistance to antibiotics. It was hypothesised that antagonism at the species level in aggregated microbial mat systems is a common phenomenon and plays a significant role in shaping bacterial communities. It was assumed that the antagonistic interactions are linked to the antibiotic resistance of strains, and the intensity of these interactions is determined by the nature of the aquatic environment in which the microbial mat is developing. Material and Methods Study site The Ecology Glacier is situated near the Arctowski Polish Antarctic Station at the western shore of Admiralty Bay, on King George Island, South Shetland Archipelago, Maritime Antarctica (Fig. 1). It is subjected to annual surface snow melt like other glaciers in the vicinity (Braun & Gossmann, 2002). The study was carried out in the vicinity of this glacier. Samples were taken during the Antarctic summer (March/April) of 2019 at twenty sites from streams (S) and ponds (P). Detailed description of research sites, environmental parameters and physicochemical conditions are presented in Zębek et al. (2021). Research material A collection of Flavobacterium spp. strains was obtained as part of a large study into the diversity of bacteria in microbial mats from the periglacial zone of the Ecology Glacier. The mats collected for research were also the subject of other analyses, including metagenomic and phycological analyses. The study showed that the structure of the microbial mats was characterised by the dominance of two main groups of photoautotrophs: cyanobacteria and diatoms (Zębek et al. 2021). An example of the macroscopic and microscopic (cross-section) structure of the microbial mat of the periglacial zone of Ecology Glacier is shown in Figure 2. Microbial mats sampling and isolation of bacterial strains Microbial mats were taken from mineral and organic substrates from proglacial streams and ponds. Using an aseptic technique, the samples were placed into sterile Petri dishes. Mat samples were collected twice using a flat metal mesh with an area of 5 cm 2 . A total of 20 microbial mats were collected. For bacterial cultivation, 1 g of each mat sample was suspended in 20 mL sterile saline (0.85 %) in 100 mL sterile flasks with glass beads and shaken gently on a universal shaker (type 327) (150 rpm, 20 min, 5 o C). Suspensions were then stored in the refrigerator (4 o C) for 10–20 min to allow larger particles to settle. A decimal dilution series of the supernatant to 10 -3 was prepared in sterile saline, and then 100 µL of suspension was inoculated on Petri plates with R2A medium in triplicates. The cultures were incubated at 7 o C for one month and inspected every third day for colony development and growth. This also enabled observing particular colony types from different sampling points and CFU growing rates . Pure cultures of different bacterial colonies were isolated after transport to Poland. 16S rRNA gene-sequencing-based taxonomic identification Bacterial DNA obtained from a single colony was isolated using the Genomic Mini AX Bacteria+ kit (A&A Biotechnology, Poland) with additional mechanical lysis of the sample in a FastPrep-24 device using zirconium beads. DNA concentration was measured using the fluorometric method on a Qubit 4 Fluorometer. Primers 27f (5'-GAG TTT GAT CCT GGC TCA G-3', Johnsen et al. 1999) and rp2 (5'-ACG GCT ACC TTG TTA CGA CTT-3', Weisburg et al. 1991) were used in the PCR reaction. DNA obtained from the amplification reaction was purified using the Clean-Up AX kit (A&A Biotechnology, Poland). PCR products, suspended in 10 mM Tris-HCl pH 8.0 buffer and diluted to a concentration of 50 ng/μL, were sent for sequencing to Macrogen Europe BV (The Netherlands). The current study examined the antibiotic resistance and antagonist interactions of 50 isolates belonging to the genus Flavobacterium , originating from microbial mats of freshwater ponds and streams. Antibiotic resistance According to Bauer et al. (1966), the disc diffusion method was applied using R2A as a replacement for Mueller-Hinton agar to determine the phenotypic antibiotic resistance of flavobacteria strains originating from microbial mats. The 25 antibiotics with different modes of action, belonging to 12 functional groups, were used (Antimicrobial Susceptibility Discs, Oxoid) (Table 1). The plates were incubated at 20 o C for 48-96 hours, depending on the growth rate of strains. Antibiotic resistance was considered to be the absence of a zone of inhibition of the strain’s growth around the antibiotic disk. The antibiotic resistance index (ARI) was determined according to Krumperman et al. (1983). Multi-drug resistance (MDR) was determined according to resistance to at least one of each mode of action of antibiotics used (Exner et al. 2017). Antagonistic interactions Taxonomically identified flavobacteria strains were screened for antimicrobial substance production using the spot-on-lawn method described by Prasad et al. (2011). Each strain was tested against the other strains for cross-inhibition. The cells were washed out from 1 mL of liquid culture of each strain by centrifuging three times (10 min/8,000 rpm), each time suspending the cell pellet in sterile physiological saline at 20°C. The cell pellet was then adjusted to a density of 0.5 McFarland and plated on the R2A medium, treating the inoculated strain as a indicator strain for examining the relationship with other strains. Following this, 10 μL of liquid culture of each of the remaining strains was applied pointwise. The culture was carried out at 20°C for seven days. After this time, the formation of a growth inhibition zone of the indicator strain around the spotted strains was considered an antagonistic effect. Statistics All data were statistically analysed using Statistica version 13.3 (StatSoft Inc.). The assessment of the significance of differences in the data obtained used a multivariate analysis of variance (ANOVA), and for data that failed to meet the normality test, a non-parametric Kruskal-Wallis test was applied. Linear regression analysis (LRA) was used to investigate correlational relationships. The antagonistic relationships associated with antibiotic resistance were illustrated in network graphs using the program Cytoscape 3.1.0 (Shannon et al. 2003), and the data were processed in the R Package's in-house scripts. Table 1. Characteristics of the antibiotics used in the study. Antibiotic Symbol Functional group Mode of action (inhibitors of) Disk potency (µg) Ampicillin AM β-lactams cell-wall synthesis 2 Carbenicillin PY β-lactams cell-wall synthesis 10 Cefixime CFM Modified β-lactams/3 rd generation cephalosporine cell-wall synthesis 100 Cefotaxime CTX Modified β-lactams/3 rd generation cephalosporine cell-wall synthesis 5 Ceftazidime CAZ Modified β-lactams/3 rd generation cephalosporine cell-wall synthesis 5 Imipenem IMP Modified β-lactams/Carbapenem cell-wall synthesis 2 Vancomycin VA Glycopeptide cell-wall synthesis/ RNA synthesis 30 Gentamicin CN Aminoglycosides protein synthesis 15 Kanamycin K Aminoglycosides protein synthesis 15 Streptomycin S Aminoglycosides protein synthesis 30 Chloramphenicol C Chloramphenicol/ Aminoglycosides protein synthesis 10 Tetracycline TE Tetracycline/Polyketide protein synthesis 1.25 Clarithromycin CLR Macrolide protein synthesis 10 Erythromycin E Macrolide protein synthesis 5 Clindamycin DA Lincosamide protein synthesis 15 Lincomycin L Lincosamide protein synthesis 30 Mupirocin MUP Monocarboxylic acid protein and RNA synthesis 5 Nitrofurantoin F Nitrofuran Inhibitor of folic acid synthesis DNA/RNA synthesis 5 Novobiocin NV Aminocoumarin DNA/RNA synthesis, 10 Ciprofloxacin CIP Quinolones DNA/RNA synthesis 30 Nalidixic acid NA Quinolones DNA/RNA synthesis 5 Metronidazole MET Metronidazole/Quinolones DNA/RNA synthesis 100 Rifampicin RA Rifamycin DNA/RNA synthesis 25 Trimethoprim TMP Trimethoprim RNA synthesis 5 Cotrimoxazole SXT Trimethoprim/Sulfamethoxazole RNA synthesis 5 Results Phylogenetic analysis Twenty species were identified among the 50 strains belonging to the genus Flavobacterium , derived from microbial mats of ponds and streams (Fig. 3, Table 2). Ten species were noted in pond and stream mats, with the more numerous species including F. aquidurense, F. hydatis, F. kayseriense, F. saccharophilum and F. xanthum . Five species originated from ponds with the F. antarcticum strains being the most abundant. Five species were isolated exclusively from stream mats, among which F. pectinovorum strains were the most abundant. Sequence data of studied flavobacteria strains have been deposited in the GenBank database, and the accession numbers are listed in Table 2. A phylogenetic tree based on 16S rRNA gene sequences comparing the isolated flavobacteria strains among their closest related species within the genus Flavobacterium is shown in Figure 3. Antibiotic resistance Antarctic flavobacterial strains isolated from microbial mats exhibited a broad spectrum of antibiotic resistance (Fig. 4). Among the isolates under study, 98% exhibited resistance to at least one of the 25 antibiotics applied. Nearly half (45%) of the strains were resistant to 1-5 antibiotics, 20% to 6-10 antibiotics, and 15% to 11-25 antibiotics. A significant percentage among the isolates under study (68%) were multi-drug resistant (MDR) strains, i.e. strains resistant to at least one antibiotic from each of the three functional groups, i.e. inhibiting cell wall synthesis (CW), protein synthesis (P), and nucleic acid synthesis (NA). MDR strains accounted for 65% and 70% of isolates in ponds and streams, respectively. 42% of them represented strains resistant to 10 or more antibiotics. Among the strains derived from pond mats, 80% were resistant to at least one antibiotic from the CW group and 65% to antibiotics from the P group. All strains were resistant to at least one antibiotic from the NA group. In the group of antibiotics inhibiting cell wall synthesis (CW), the largest pool was bacteria resistant to ß-lactams, i.e. ampicillin (40%) and carbenicillin (40%), as well as a 3rd generation cephalosporin, i.e. cefixime (45%). Only two strains derived from pond mats, F. antarcticum and F. kayseriense , were resistant to imipenem. In the group of antibiotics inhibiting protein synthesis (P), 40% were resistant to clindamycin, and 45% to lincomycin. Only one isolate, F. antarcticum , was resistant to tetracycline. In the group of antibiotics inhibiting nucleic acid synthesis (NA), all strains derived from pond mats exhibited resistance to at least one antibiotic. 70% of strains were resistant to trimethoprim, 55% to metronidazole, and 50% to mupirocin and ciprofloxacin. Three MDR strains derived from pond mats, i.e. F. degerlachei, F. hibernum and F. saccharophilum , were resistant to 16 of the 25 antibiotics applied. Two strains, i.e. F. glaciei and F. hydatis exhibited a very low resistance to only one antibiotic. Among the strains derived from stream mats, 87% were resistant to at least one antibiotic from the CW group, 77% to antibiotics from the P group, and 93% to antibiotics from the NA group. Most (87%) represented strains resistant to 6 or more antibiotics. Only 13% of strains derived from stream mats were resistant to 1-5 antibiotics. Table 2. 16S rRNA gene sequence affiliation to the closest phylogenetic neighbours, isolate origin and Gen Bank Accession Number of the flavobacteria isolated from an Antarctic pond and stream microbial mats. Isolate code Origin Gen Bank Accession Number Nearest taxonomic neighbour by BLAST Identity [%] P5 Pond OR739435 Flavobacterium antarcticum DSM 19726 98.76 P7 Pond OR739436 99.1 P8 Pond OR739437 99.03 P4 Pond OR739434 Flavobacterium degerlachei R-9106 99.46 P49 Pond OR739456 99.28 P40 Pond OR739449 Flavobacterium fryxellicola LMG 22022 99.87 P13 Pond OR739439 Flavobacterium glaciei 0499 99.48 P12 Pond OR739438 Flavobacterium sinopsychrotolerans 0533 99.79 P43 Pond OR739450 Flavobacterium aquidurense WB 1.1-56 99.02 P47 Pond OR739454 99.02 S79 Stream OR739477 99.33 S90 Stream OR739482 99.61 S68 Stream OR739469 Flavobacterium cupreum P2685 99.26 S71 Stream OR739472 98.88 P87 Pond OR739480 99.13 S55 Stream OR739459 Flavobacterium flabelliforme P4025 99.21 S58 Stream OR739462 99.35 P63 Pond OR739467 99.62 P62 Pond OR739466 Flavobacterium hibernum ATCC 51468 98.76 S81 Stream OR739479 99.22 P45 Pond OR739452 Flavobacterium hydatis DSM 2063 99.66 P48 Pond OR739455 99.68 S56 Stream OR739460 99.76 S57 Stream OR739461 99.68 S59 Stream OR739463 99.41 S34 Stream OR739445 Flavobacterium kayseriense F-49 99.89 S35 Stream OR739446 99.89 P44 Pond OR739451 100 S80 Stream OR739478 99.79 P50 Pond OR739457 Flavobacterium psychroterrae P3922 99.79 S54 Stream OR739458 99.76 P46 Pond OR739453 Flavobacterium saccharophilum NBRC 15944 99.76 S61 Stream OR739465 99.63 S65 Stream OR739468 99.67 S74 Stream OR739474 99.77 P14 Pond OR739440 Flavobacterium xanthum NBRC 14972 98.96 P15 Pond OR739441 100 S17 Stream OR739442 99.38 S39 Stream OR739448 100 S36 Stream OR739447 Flavobacterium alvei HR-AY 99.22 S73 Stream OR739471 Flavobacterium bizetiae CIP 105534 98.56 S72 Stream OR739473 99.02 S77 Stream OR739475 Flavobacterium chryseum P3160 99.69 S78 Stream OR739476 99.77 S19 Stream OR739443 Flavobacterium pectinovorum NBRC 15945 99.45 S20 Stream OR739444 99.37 S70 Stream OR739470 99.45 S91 Stream OR739483 Flavobacterium piscis 412r-09 99.18 S60 Stream OR739464 Flavobacterium sp. 95.44 S89 Stream OR739481 96.49 In the group of antibiotics inhibiting cell wall synthesis (CW), the largest pool consisted of bacteria resistant to 3rd generation cephalosporins, i.e. cefixime (83%), cefotaxime (73%) as well as ampicillin (70%) and vancomycin (63%). What should be emphasised is the lack of resistance of the analysed strains to imipenem. Nearly 80% of isolates were resistant to at least one antibiotic inhibiting protein synthesis (P), 57% were resistant to lincomycin, and 50% to chloramphenicol, erythromycin and clindamycin. The study found the lack of resistance to tetracycline and resistance of only two strains, i.e. F. hydatis and F. saccharophilum , to gentamycin. In the group of antibiotics inhibiting nucleic acid synthesis (NA), 93% of strains exhibited resistance to at least one antibiotic. 87% were resistant to metronidazole, 77% to trimethoprim, 73% to mupirocin, and 67% to nitrofurantoin. What is remarkable among the MDR strains is the broad resistance of F. piscis and F. flabelliforme to 19 and 18 antibiotics, respectively, and of three strains: F. aquidurense, F. hydatis and F. bizetiae , to 17 out of the 25 antibiotics applied. Two F. kayseriense isolates were resistant only to two antibiotics, i.e. ciprofloxacin and trimethoprim (belonging to the NA group). One strain, F. pectinovorum , exhibited no resistance to the antibiotics applied. The existence of individual antibiotic resistance patterns among flavobacterial strains, even among those of the same species, is noteworthy. Analysis of the antibiotic resistance index (ARI) showed a significantly higher proportion of multi-drug resistant strains (ARI ≥ 0.2) in stream mats (87%) compared to the strains isolated from pond mats (55%) (Fig. 4, Fig. 5). At the same time, the average ARI value for flavobacteria isolated from pond mats (0.27) was significantly higher, compared to the value for the strains isolated from stream mats (0.44). A statistically significantly higher resistance to antibiotics was demonstrated in strains derived from stream mats (n=50, p<0.01) (Fig. 5). In these strains, significantly higher resistance to antibiotics from the group inhibiting cell wall synthesis (CW) (p<0.001), and from the group inhibiting nucleic acid synthesis (NA) (p<0.05), was demonstrated. At the same time, no significant differences were noted in the resistance of the strains under study to antibiotics belonging to the group inhibiting protein synthesis (P). Antagonistic relations Antagonistic relations demonstrated among flavobacterial strains derived from polar-region microbial mats showed noticeable differences between strains isolated from stream mats and from ponds (Fig. 4). It was demonstrated that 50% of the flavobacteria derived from pond mats and 63% derived from stream mats produced compounds inhibiting the development of other isolates. Four groups of strains were distinguished among the possible types of interaction, with all types of interaction occurring among isolates from both pond mats and stream mats. The following strains were distinguished: PR – where the isolate produces antibacterial compounds but is also resistant to antibacterial compounds produced by other strains; PRS – where the isolate is an antagonist, i.e. produces antibacterial compounds, while being resistant and sensitive to one or more strains; SR – where the isolate produces no antibacterial compounds while being sensitive and resistant to antibacterial compounds produced by other strains; R – the isolate produces no antibacterial compounds but is resistant to the action of these compounds produced by other strains. Among the isolates derived from the environments under study, significant differences in antagonistic activity were demonstrated (Fig. 6). In the pond mats, the PR strains accounted for 25%, with the highest antagonistic activity exhibited by F. saccharophilum , which at the same time was resistant to the majority of the antibiotics applied. The remaining strains from this group exhibited low and moderate antibiotic resistance. In the stream mats, the PR strains were the dominant group (43%), with the most antagonistically active strains including F. flabelliforme , F. hydatis and F. bizetiae . These strains also exhibited a very high antibiotic resistance. Moderate and high antibiotic resistance characterised the remaining strains from this group. The PRS strains in pond and stream mats accounted for similar percentages, 25% and 20%, respectively. The PRS strains derived from pond mats were characterised by moderate antagonistic activity and antibiotic resistance. In contrast, Flavobacterium glaciei and F. hydatis inhibited the growth of only a few strains while being resistant to just one antibiotic. The PRS strains were characterised by higher antagonistic activity and a broader antibiotic resistance in stream mats. Flavobacterium saccharophilum and F. pectinovorum exhibited moderate antagonistic properties while being resistant to many antibiotics. The SR strains in ponds represented the least numerous group (15%) and were distinguished by a high resistance to antagonistic responses and a low antibiotic resistance (ARI<0.2). Only F. antarcticum , derived from this environment, was distinguished by higher sensitivity to antagonistic interactions than the remaining strains and it displayed resistance to one antibiotic from each of the antibiotic groups applied. Only two SR strains derived from stream mats showed a low resistance to antagonistic interactions and a low antibiotic resistance (ARI=0.08). The R strains in pond mats represented a dominant group (35%). F. degerlachei and F. hibernum were characterised by a lack of sensitivity to antibacterial compounds produced by other strains and the highest antibiotic resistance. All R strains from stream mats exhibited similar properties, yet it was the least numerous group isolated from this environment (13%). Antagonistic activity and antibiotic resistance interactions The results show a statistically significant correlation between the strains’ ability to produce antibacterial compounds and their resistance to antibiotics (p<0.05). In general, strains with high antagonistic activity were characterised by lower sensitivity to antagonistic interactions and higher antibiotic resistance (Fig. 7). Strains from both: pond mats and stream mats, exhibited a statistically significant correlation between the antagonistic potential of the strains, i.e. their production of antibacterial compounds, and their resistance to antibiotics: r=0.7 (p<0.05) and r=0.64 (p<0.01), respectively (Fig. 8a). These traits were exhibited by F. saccharophilum derived from pond mats, and F. piscis isolated from stream mats, which were characterised by the highest antagonism and antibiotic resistance values. Furthermore, among strains derived from both environments (ponds, streams), a positive correlation was noted between resistance to antagonistic interactions and their antibiotic resistance: r=0.52 (p<0.05) and r=0.49 (p<0.01), respectively. These properties were exhibited by F. hibernum and F. degerlachei derived from pond mats, and F. piscis derived from stream mats (Fig. 8b). Discussion The phylogenetic structure of the collection of flavobacterial strains isolated from the microbial mats of the periglacial zone of the Ecology Glacier on King George Island showed surprisingly high species heterogeneity, which corresponded, for the most part, to strains found in cold environments, including polar regions. The five species isolated from pond mats: F. antarcticum. F. glaciei, F. fryxellicola, F. degerlachei and F. sinopsychrotolerans were indigenous to Antarctic lakes (Yi et al., 2005 ; Zhang et al., 2006 ; Van Trappen et al., 2004 ; 2005 ; Xu et al., 2011 ; The species derived from stream mats, i.e. F. chryseum , F. pectinovorum , F. alvei, F. bizetiae and F. piscis , are described as occurring in various inland polar ponds, and their origin is also linked to cold-adapted fish pathogens (Králová et al., 2018 ; Serra, 2024; Lee & Jeon, 2018 ; Mühle et al., 2021 ; Zamora et al., 2014 ). Of the nine species found in both pond mats and stream mats, the following eight have so far been described in polar environments: F. flabelliforme, F. hibernum, F. hydatis, F. kayseriense, F. psychroterrae, F. saccharophilum, F. xanthum and F. cupreum (Králová et al., 2021 ; McCammon et al., 1998 ; Mantareva et al., 2022 ; Saticioglu et al., 2021 ; Králová et al., 2018 ; Asano et al., 1984 ; Matsui et al., 2017 ; Králová et al., 2018 ). One species, F. aquidurense , was also isolated from water of temperate zone streams (Cousin et al., 2007 ; Kim et al., 2014 ). This confirms the cosmopolitan nature of flavobacteria and their role as an integral component of the microbiocenoses of natural environments, including polar environments. The results highlight the importance of antagonistic interactions in forming the structure and functioning of the bacteriocenoses of polar microbial mats. In the available literature, few papers exist on interactions occurring in heterogeneous microbial consortia dependant on the same environmental resources. These relationships are often studied in the context of interactions with test microorganisms, e.g., human and animal pathogens or food contaminants (Kan et al., 2011 ). Chemoecological studies conducted to date have shown the existence of various types of interactions within heterogeneous microbial consortia: amensalism, commensalism and mutualism, which shape the structure of microbial communities (Long et al., 2013 ). Meanwhile, this phenomenon has been studied, to a limited extent, in primeval ecosystems such as polar-region microbial mats. Antagonism is an important survival strategy for aggregated bacteria, enabling them to gain an advantage over competing microorganisms (Tait et al., 2002; Lo Giudice et al., 2007 ; Mangano et al., 2009 ; Long et al., 2013 ). In the current study, over 50% of the strains exhibited the ability to produce substances that inhibit the growth other Flavobacterium spp. strains, and approximately 40% were sensitive to substances produced by microorganisms inhabiting the same niche. A similar, high antagonistic activity was demonstrated for the heterotrophic bacterial strains originating from saline lake microbial mats (60%), oceanic seston (53.3%), aggregated marine organic matter (54.1%), and the microbiome of Antarctic sponges (62.1%) (Long & Azam, 2001 ; Grossart et al., 2004 ; Mangano et al., 2009 ; Long et al., 2013 ). A considerably lower antagonistic activity was demonstrated in the bacteriocenoses of pelagic aquatic environments. Lo Giudice et al. ( 2007 ) identified only 15% of antagonistically active bacteria among strains isolated from the Ross Sea waters. A study by Long et al. (2001) demonstrated that the level of bacterial cell aggregation determines the production of antibacterial substances. Over 50% of bacteria aggregated on the seston surface revealed antagonistic activity, while only 5% of bacterioplankton had such an ability. The current study demonstrated that antagonistic interactions among microbial mat bacteria are complex. This was confirmed by the occurrence of four different patterns of antagonism and sensitivity interactions in the flavobacteria isolates under study: PRS, PR, SR and R. These intraspecies and interspecies properties maintain the structural and functional stability of the bacteriocenosis. This proves that different mechanisms can determine antagonism, which is usually an individual feature of the strain. It is noteworthy that all the flavobacterial strains isolated from microbial mats were resistant to at least one antibacterial agent, and more than half were producers of antibacterial substances. The individualisation of antagonistic properties of individual strains is a rather poorly understood phenomenon, which, however, appears to be of key importance in the formation of the structure of microbial consortia (Mangano et al., 2009 ; Prasad, 2011; Long et al., 2013 ). The clear difference in the phylogenetic structure and antagonistic activity of the flavobacteria of the microbial mats of ponds and watercourses, demonstrated in the current study, could be determined by the physicochemical and trophic properties of the aquatic environment. A crucial role in shaping the structure and activity of benthic consortia in such ponds is also played by the dynamics of water flow (Stanish et al., 2012 ; 2013; Kohler et al., 2015; Akulava et al., 2022 ). Ylla et al. ( 2013 ) emphasise the importance of organic matter availability and assimilability for developing bacterial groupings and their metabolic and physiological activity. The poorer trophic conditions found in streams are usually associated with a quantitative predominance of hardly decomposable plant polymers, mainly celluloses and hemicelluloses. In stagnant water bodies, the organic matter usually accumulates and “ages” gradually. Its structure simplifies, and readily available nutrient substrates are released (Ylla et al., 2013 ). The current study demonstrated that trophic constraints in the stream environment necessitate a high degree of competition between microbial mat bacteria. This is confirmed by the ability, shown for a significant proportion of the isolated flavobacterial strains, to produce antimicrobial substances, which effectively regulate the structure of the bacteriocenosis. This is expressed by the unexpectedly high species diversity of flavobacteria found in the mats under study, i.e. 20 species identified among 50 isolates. The current study suggests that impaired microbial development under suboptimal conditions of an extreme polar environment induces high susceptibility of the bacteriocenosis to even extremely low antagonistic interactions. A study by Grossart et al. ( 2004 ) points to the existing correlation between antagonistic activity and the ability of bacteriocenosis to degrade organic matter. The production of antibiotics, bacteriocins and other antimicrobial substances, even in subthreshold quantities, can ensure an advantage over other microorganisms that co-form the bacteriocenosis and inhibit colonisation of the niche by allochthonous microorganisms (Tait et al., 2002; Rypien et al., 2010 ). The high antibiotic resistance of flavobacteria strains observed in the current study, in which over 70% of isolates were MDR strains, was surprising but not unexpected. Broad antibiotic resistance is increasingly being identified in isolated strains and the metagenome of the microbiocenoses of various polar environments, indicating that this feature is common. The presence of multi-antibiotic-resistant bacteria was found, e.g. in different polar terrestrial environments (Jara et al., 2020 ; Marcoleta et al., 2022 ; Opazo-Capurro et al., 2020 ), marine bottom sediments (De Souza et al., 2006 ) and glacial ice (Brown & Balkwil, 2009). Numerous publications on polar areas point to the importance of the active soil layer as a reservoir of antibiotic resistance gene-harbouring bacteria (Králová et al., 2017; 2021 ; Van Goethem et al., 2018 ). Marcoleta et al. ( 2022 ), when investigating the resistome within the Antarctic Peninsula soils, demonstrated a very high frequency and diversity of antibiotic resistance genes. Microbial mats are a specific formation in which the interrelationships between the biotic components provide the basis for structural and functional homeostasis (Núñez-Montero & Barrientos, 2018 ; Ambrožič Avguštin et al., 2019 ). Antibiotic resistance in such aggregated microbiocenoses is a key phenomenon confirmed by the current study. Numerous researchers have noted the importance of microevolutionary processes in the development of natural antibiotic resistance in native environmental bacteria (Blair et al., 2015 ; Paun et al., 2021 ). The occurrence of antibiotic resistance in bacteria in areas remote from human activities may also be due to horizontal gene transfer from allochthonous bacteria introduced into the environment. Antibiotic resistance genes (ARGs), identified in polar-region bacteria, are perceived as a biotic contaminant and a significant ecological problem (Von Wintersdorff et al., 2016 ; Králová et al., 2021 ). Metagenomic research suggests that the Antarctic microbiota is the source of ancient antibiotic resistance genes, but, at the same time, multi-antibiotic-resistant strains are found in areas of high anthropogenic impact. King George Island is certainly one such region, which explains the resistance to antibiotics routinely used in modern therapy found in the current study. There are few reports on the resistome of Antarctic flavobacterial strains. As demonstrated by a study by Králová et al. ( 2021 ), two Antarctic species of F. flabelliforme and F. geliluteum , derived from the isolated environment of the James Ross Island, were phenotypically MDR and, at the same time, contained multiple resistance genes to a broader spectrum of antimicrobial drugs. The varying antibiotic resistance profiles among flavobacterial strains of the same species in this study indicate the acquired nature of these traits, influenced by external factors. The different antibiotic resistance in strains originating from two different environments, i.e. flowing and stagnant water, may be determined by the different dynamics of these waters. The current observations suggest that Antarctic bacteria from different ecosystems are genetically distinct due to the need to adapt to ecologically distinct habitats, which is also supported by studies conducted by other authors (Zhang et al., 2008 ). The narrower antibiotic resistance among flavobacterial strains derived from pond mats may be related to the greater stability of this environment and the availability of biogenic compounds and, consequently, to the lower competitiveness within the microbiocenosis. Evidence indicates that antibiotic resistance genes originate from multiple bacterial sources, showing that the genomes of all bacteria can be considered as one global set of genes, from which most, if not all, bacteria can draw in search of genes necessary for survival (Bennett, 2008 ). On the other hand, microbial mats can be important reservoirs of resistance to antimicrobial agents (Flores-Vargas et al., 2021 ). Summary This is the first study to demonstrate a relationship between antagonistic interactions with antibiotic resistance within flavobacteria, a component of polar-region microbial mats. The study revealed the complex nature of these interactions occurring at intra- and interspecies levels. It also demonstrated the distinctiveness of the relationships found among flavobacteria of the microbial mats of two types of water bodies, i.e. ponds and streams. The surprisingly high biodiversity of flavobacterial strains in the mat microbiocenosis is an example of the existing strong intra- and interspecies relationships that are key to maintaining structural and functional homeostasis. The results of the study demonstrate that there are individual patterns of antagonistic interactions and antibiotic resistance among the biotic components of mats. The nature of these patterns plays a crucial role in forming the structure and functions of microbial mats. In the mat microbiocenosis, strains of “superbacteria” are characterised by outstanding antagonistic potential and broad antibiotic resistance. As demonstrated by the study, environmental factors, mainly hydrological and trophic parameters of a pond, also play an important role in promoting specific patterns of intercellular interactions. The study also positively verified the hypothesis that antagonism at the species level in aggregated microbial mat systems is a common phenomenon and plays a significant role in shaping bacterial communities. Moreover, it was confirmed that antagonistic interactions are linked to the antibiotic resistance of strains, and the nature of the aquatic environment determines the intensity of these interactions. Although these findings on flavobacteria may not fully reflect all interactions within microbial mats, the obtained results can be used to develop a dynamic model of the formation of microbial mat communities. Declarations Competing Interests The authors declare no competing interests. Funding This work was supported by the financial support of University of Warmia and Mazury in Olsztyn (D. G., A.Ś. J. K.) and Institute of Biochemistry and Biophysics of the Polish Academy of Science in Warszawa (J. G., M.K.Z). Author Contribution Author Contribution: D.G., and A.Ś.; conceptualization: D.G., A.Ś., M.K.Z. and J. K.; sampling: D.G., A.Ś; and J.G. laboratory analysis: D.G., A.Ś, J.K.; data analysis and visualization: D.G., A.Ś and J.J.; manuscript preparation: D.G., A.Ś.; review and editing: D.G., A.Ś, J.G and M.K.Z. Acknowledgement We thank the Polish Antarctic Station Arctowski staff for the support in collecting samples from the periglacial zone of Ecology Glacier and for helping us in the expedition. Data Availability Sequence data of studied flavobacteria strains have been deposited in the GenBank NCBI database, under accession numbers OR739434–OR739483. 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H., Zhang, D. C., Liu, H. C., Zhang, J. L., Yu, Y., Xu, M. S., Zhou, P. J., Zhou Y. G. (2009). Flavobacterium tiangeerense sp. nov., a cold-living bacterium isolated from a glacier. International Journal of Systematic and Evolutionary Microbiology, 59(11), 2773-2777. https://doi.org/10.1099/ijs.0.007906-0 Xu M, Xin Y, Tian J, Dong K, Yu Y, Zhang J, Liu H, Zhou Y. (2011). Flavobacterium sinopsychrotolerans sp. nov., isolated from a glacier. Int J Syst Evol Microbiol ., 61(Pt 1):20-24. doi: 10.1099/ijs.0.014126-0. Yi H., Oh, H. M., Lee, J. H., Kim, S. J., & Chun, J. (2005). Flavobacterium antarcticum sp. nov., a novel psychrotolerant bacterium isolated from the Antarctic. International journal of systematic and evolutionary microbiology , 55(2), 637-641. Ylla, I., Peter, H., Romaní, A. M., & Tranvik, L. J. (2013). Different diversity–functioning relationship in lake and stream bacterial communities. FEMS microbiology ecology , 85(1), 95-103. Zamora, L., Vela, A. I., Sánchez-Porro, C., Palacios, M. A., Moore, E. R. B., Domínguez, L., Ventosa, A., Fernández-Garayzábal, J. F. (2014). Flavobacterium tructae sp. nov. and Flavobacterium piscis sp. nov., isolated from farmed rainbow trout (Oncorhynchus mykiss). Int J Syst Evol Microbiol ., 64(Pt 2):392-399. doi: 10.1099/ijs.0.056341-0 Zębek, E., Napórkowska-Krzebietke, A., Świątecki, A., Górniak, D. (2021). Biodiversity of periphytic assemblages in polar region: a case study of the vicinity of Arctowski Polish Antarctic Station (King George Island, Antarctica). Biodiversity and Conservation , 30, 2751–2771. https://doi.org/10.1007/s10531-021-02219-2 Zhang DC, Wang HX, Liu HC, Dong XZ, Zhou PJ. 2006. Flavobacterium glaciei sp. nov., a novel psychrophilic bacterium isolated from the China No.1 glacier. Int J Syst Evol Microbiol. 12:2921-2925. doi: 10.1099/ijs.0.64564-0. PMID: 17158999.10.1099/ijs.0.63423-0. PMID: 15774636. Zhang, X. F., Yao, T. D., Tian, L. D., Xu, S. J., An, L. Z. (2008) Phylogenetic and physiological diversity of bacteria isolated from Puruogangri ice core. Microb Ecol 55:476–488. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 19 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 15 Jan, 2025 Reviews received at journal 14 Jan, 2025 Reviews received at journal 30 Dec, 2024 Reviewers agreed at journal 20 Dec, 2024 Reviews received at journal 27 Nov, 2024 Reviewers agreed at journal 18 Nov, 2024 Reviewers agreed at journal 15 Nov, 2024 Reviewers agreed at journal 13 Nov, 2024 Reviewers invited by journal 13 Nov, 2024 Editor assigned by journal 13 Nov, 2024 Editor invited by journal 11 Nov, 2024 Submission checks completed at journal 08 Nov, 2024 First submitted to journal 23 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-5318460","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":379338883,"identity":"415e8056-f90a-41fc-abea-8e66da5506f8","order_by":0,"name":"Dorota Górniak","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYLCCBIYEOQiLDYjZidRijNDCTKQ9iQ1Ea5F373324EFNWvqGG9kJDB/KDjOYE9JieOa4uUHCsZzcDTdyNzDOOHeYwbKZkJYZaWwSCWwVuRtu525g5m07zGBwmCgt/yrSDUBa/hKjRV4CqCWxLScBrIWRGC0GPMeAWvrSDGfef7vhYM+5dB6CfpFvb2OT/PEtWZ7vzNmND36UWcuZszcQsOUAEgfE5jEgYAmDPIaRBLWMglEwCkbBiAMARmJDB+wo7ngAAAAASUVORK5CYII=","orcid":"","institution":"University of Warmia and Mazury in Olsztyn","correspondingAuthor":true,"prefix":"","firstName":"Dorota","middleName":"","lastName":"Górniak","suffix":""},{"id":379338885,"identity":"334ea839-10b2-4920-9b63-25cdf07fd01b","order_by":1,"name":"Aleksander Świątecki","email":"","orcid":"","institution":"University of Warmia and Mazury in Olsztyn","correspondingAuthor":false,"prefix":"","firstName":"Aleksander","middleName":"","lastName":"Świątecki","suffix":""},{"id":379338886,"identity":"bbab15d0-60db-436c-abdf-bf5900db008f","order_by":2,"name":"Jakub Kowalik","email":"","orcid":"","institution":"University of Warmia and Mazury in Olsztyn","correspondingAuthor":false,"prefix":"","firstName":"Jakub","middleName":"","lastName":"Kowalik","suffix":""},{"id":379338887,"identity":"e65d53c3-4391-4426-aa2f-626ac92df727","order_by":3,"name":"Jakub Grzesiak","email":"","orcid":"","institution":"Polish Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jakub","middleName":"","lastName":"Grzesiak","suffix":""},{"id":379338888,"identity":"66cc6002-709e-4866-a42f-ff1cc1f1d392","order_by":4,"name":"Jan Jastrzębski","email":"","orcid":"","institution":"University of Warmia and Mazury in Olsztyn","correspondingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Jastrzębski","suffix":""},{"id":379338889,"identity":"6111df06-6f57-45c6-92c1-5f342ff9f4d1","order_by":5,"name":"Marek K. Zdanowski","email":"","orcid":"","institution":"Polish Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Marek","middleName":"K.","lastName":"Zdanowski","suffix":""}],"badges":[],"createdAt":"2024-10-23 11:23:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5318460/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5318460/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-97205-x","type":"published","date":"2025-04-19T15:57:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69996686,"identity":"972b9b76-e832-4cac-9c58-701224a1c23f","added_by":"auto","created_at":"2024-11-27 10:28:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203794,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of the sampling area in the periglacial zone of Ecology Glacier, King George Island, Maritime Antarctica.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/9cf9291d310db96c839f22d7.png"},{"id":69995935,"identity":"bfeb0f50-a7d1-4b90-b62a-f4922d04d56b","added_by":"auto","created_at":"2024-11-27 10:20:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1026144,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic and microscopic (vertical cross section) structure of the microbial mats of the periglacial zone of Ecology Glacier; stream (\u003cstrong\u003ea, b\u003c/strong\u003e) and pond (\u003cstrong\u003ec, d\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/a7cd89622f6721d927d857a0.png"},{"id":69995936,"identity":"4fc33fd7-1625-4d74-b278-ff1dfe1dce90","added_by":"auto","created_at":"2024-11-27 10:20:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":323108,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on 16S rRNA gene sequences comparison of the isolated flavobacteria strains among their closest related species within the genus Flavobacterium. The evolutionary history was inferred using the maximum likelihood method based on the Tamura-Nei distance with the gamma model. Bootstrap probability values (percentages of 500 tree replications) greater than 50% are indicated at branch points. Bar, 0.01 substitutions per nucleotide position. Flavobacterium isolates are marked by a code number (see Table 2).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/155ba0a4291f66df6d1f48b6.png"},{"id":69996927,"identity":"0f4be955-b38c-4182-b3fe-153e871059b2","added_by":"auto","created_at":"2024-11-27 10:36:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":124302,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the antimicrobial resistance profiles (orange – resistant; grey – sensitive) and antagonistic activity data of flavobacteria isolates from the microbial mat of ponds and streams in the periglacial zone of Ecology Glacier. NAR – number of antibiotic resistance; ARI – antibiotic resistance index; I % – percentage of inhibited strains; R % – percentage of resistance to antagonistic interactions; ITP – interactive type profile: P – production of antibacterial compounds; R – resistance to antibacterial compounds; S – sensitivity to antibacterial compounds. Characteristics of antibiotics are listed in Table 1.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/25e5187e69478697ca7f7297.png"},{"id":69995929,"identity":"05fd31c4-b00f-4c8a-9337-3e682b165f23","added_by":"auto","created_at":"2024-11-27 10:20:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3687,"visible":true,"origin":"","legend":"\u003cp\u003eAntibiotic resistance index (ARI) of flavobacteria to a selected group of antibiotics: inhibitors of cell wall synthesis (CW), proteins (P) and nucleic acids (NA). ARI index with p\u0026lt;0.05 is indicated (*), and p\u0026lt;0.01 is indicated with (**).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/1ef975103aa2366ef2b63f07.png"},{"id":69996685,"identity":"63e645d2-65af-43c0-8889-813be0640ed7","added_by":"auto","created_at":"2024-11-27 10:28:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3887,"visible":true,"origin":"","legend":"\u003cp\u003eAntagonistic interactive types of flavobacteria strains isolated from pond and stream microbial mats; P – production of antibacterial compounds; R – resistance to antibacterial compounds; S – sensitivity to antibacterial compounds.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/0447f82dff89c203104af888.png"},{"id":69996687,"identity":"ad4bea7b-a75b-43c2-af17-28308e3cd8ec","added_by":"auto","created_at":"2024-11-27 10:28:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":422469,"visible":true,"origin":"","legend":"\u003cp\u003eNetwork analyses of antagonistic interactions and antibiotic resistance among flavobacteria isolated from microbial mats, (\u003cstrong\u003ea\u003c/strong\u003e) 20 x 20 array of tests among ponds isolates, (\u003cstrong\u003eb\u003c/strong\u003e) 30 x 30 array among streams isolates. Each node represents bacterial strains. Each line represents an antagonistic interaction from an active strain towards a sensitive strain. Strains with the same type of antagonistic activity (ITP – interactive type profile) have the same fill colour: PR – green, PRS – blue, SR – purple, and R – orange. Each line represents an antagonistic interaction from an active strain (red dot) towards a sensitive strain (arrow). The size of the red ring indicates the antibiotic resistance range. The species names of the strains are listed in Figure 4 and Table 2.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/6f6381a1e55ae94d00de13ee.png"},{"id":69995930,"identity":"5a124658-dc7d-4453-9522-d2222dec33fd","added_by":"auto","created_at":"2024-11-27 10:20:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":258830,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between antibiotic resistance index (ARI) and antagonistic activity (\u003cstrong\u003ea\u003c/strong\u003e) and resistance to antagonistic reactions (\u003cstrong\u003eb\u003c/strong\u003e) of flavobacteria strains.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/7dbc40013f8bb14132511e47.png"},{"id":81050954,"identity":"ae2b527f-aeae-4a0d-b82a-3c050a91fb64","added_by":"auto","created_at":"2025-04-21 16:08:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3435492,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5318460/v1/87ae3040-0592-4179-8491-77dea4fd012b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"High antagonistic activity and antibiotic resistance of flavobacteria of polar microbial freshwater mats (King George Island, Antarctica)","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolar regions are characterised by extreme environmental conditions, including low temperatures, high UV intensity, limited nutrient availability and the pressure from frequent freezing and thawing (Kr\u0026aacute;lov\u0026aacute; et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Due to the selectivity of these factors, only microorganisms with high adaptive capacities thrive in these environments. Flavobacteria are widely distributed in nature and characterised by high molecular, metabolic and physiological plasticity (Bernardet \u0026amp; Bowman, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kr\u0026aacute;lov\u0026aacute;, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Currently (September 2024), over 400 species are classified in the genus \u003cem\u003eFlavobacterium\u003c/em\u003e, including strains described as occurring in natural environments and relevant to veterinary medicine, agriculture, medicine and biotechnology (Parte et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This taxon\u0026rsquo;s widespread occurrence, diversity and environmental potential have aroused strong interest among researchers in both cognitive and applied terms. Flavobacteria are among the most commonly isolated cold-adapted microorganisms of polar-region environments. These include organisms indigenous to these regions, e.g. \u003cem\u003eF. antarcticum. F. glaciei, F. fryxellicola, F. degerlachei and F. sinopsychrotolerans\u003c/em\u003e (Yi et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; McCammon \u0026amp; Bowman, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Van Trappen et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kr\u0026aacute;lov\u0026aacute; et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The characteristic features of psychrotolerant and psychrophilic flavobacteria include an adaptive strategy for producing pigments (mainly carotenoids), proteins that neutralise reactive oxygen species as well as antifreeze and cold shock proteins (Liu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Flavobacteria colonise diverse ecosystems of Arctic and Antarctic polar environments, including glaciers, lakes, streams, soils and plant rhizosphere zones, as well as microbial mats (Van Trappen et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Xin et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Kr\u0026aacute;lov\u0026aacute; et al., 2017; \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Polar-region freshwater microbial mats contain a particularly diverse microbiota that mainly comprises Cyanobacteria, Chloroflexota, Pseudomonadota, Actinomycetota, Bacteroidota, Bacillota and Archaea (Prieto-Barajas et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Valdespino-Castillo et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the structure of cold-environment microbial mats flavobacteria are an abundant component (McCammon and Bowman, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Kr\u0026aacute;lov\u0026aacute; et al., 2017). Conglomerates of diverse microbial groups usually comprise aggregated, self-sustaining, autonomous ecosystems. Their distinctive feature is the close link between the primary and secondary production processes and the regeneration of nutrients (Rochera et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; de los R\u0026iacute;os et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The dominant portion of the total biomass of the microbiome of polar terrestrial ecosystems is contained within microbial mats indicating the crucial role they play in the functioning of these extreme environments (Quesada et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Callejas et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In heterogeneous microbial mat biocenotic systems, the essential factor that shapes the structure of the consortia is the intercellular interaction taking place at the species, functional group, and whole microbiota levels. Positive and negative interactions determine the dynamics of the colonised environment\u0026rsquo;s microbiota (Long et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Antagonistic responses are among the most primeval and essential forms of interactions occurring between microorganisms that make up these consortia (Aguirre-von-Wobeser, 2015; N\u0026uacute;\u0026ntilde;ez-Montero \u0026amp; Barrientos, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Aggregated microorganisms have developed mechanisms to actively compete through bacteriostatic or bactericidal interactions with rival microbial communities or species (Peterson et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Bacteria, which have a very wide range of different compounds at their disposal, i.e. exotoxins, toxic enzymes, bacteriocins and antibiotics, use them in antagonistic intercellular interactions (Cotter et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mullis et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The consequence of such interactions is the acquisition of resistance to these compounds. Forming an extended antibiotic resistome is particularly important for bacteria. Widespread antibiotic resistance in microbial mat microbiocenoses is the result of two mechanisms, i.e. natural evolutionary processes, which represent important adaptation of bacteria developing in heterogeneous microbiocenoses (Ambrožič Avguštin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tallada et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Depta \u0026amp; Niedźwiedzka-Rystwej, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and induced resistance resulting from environmental contamination by commercial antibiotics used e.g. in medicine, agriculture and veterinary medicine (Hwengwere et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Stanton et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). At the same time, diverse horizontal gene transfer (HGT) mechanisms lead to rapid and efficient development of drug resistance across the microbiota (Abe et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These processes result in a pool of strains characterised by multi-antibiotic resistance, representing an important reservoir of resistance genes in the global microbiome (Van Goethem et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Referring to these phenomena, contemporary research into microbial mats lacks reports describing antagonistic interactions between their biotic components and their antibiotic resistance. The novelty of the current study is that it describes these relationships within flavobacteria, which are ecologically important and abundant in natural environments, including polar environments.\u003c/p\u003e \u003cp\u003eThe current study contains a phylogenetic analysis of flavobacterial strains isolated\u003c/p\u003e \u003cp\u003efrom polar-region microbial mats that develop in small freshwater ponds and streams of the proglacial zone of the Ecology Glacier on King George Island, Antarctica (vicinity of Arctowski Polish Antarctic Station). It also presents the results of investigated antagonistic activity of flavobacterial isolates in the cross-inhibition reaction and their resistance to antibiotics. It was hypothesised that antagonism at the species level in aggregated microbial mat systems is a common phenomenon and plays a significant role in shaping bacterial communities. It was assumed that the antagonistic interactions are linked to the antibiotic resistance of strains, and the intensity of these interactions is determined by the nature of the aquatic environment in which the microbial mat is developing.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cem\u003eStudy site\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe Ecology Glacier is situated near the Arctowski Polish Antarctic Station at the western shore of Admiralty Bay, on King George Island, South Shetland Archipelago, Maritime Antarctica (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is subjected to annual surface snow melt like other glaciers in the vicinity (Braun \u0026amp; Gossmann, 2002). The study was carried out in the vicinity of this glacier. Samples were taken during the Antarctic summer (March/April) of 2019 at twenty sites from streams (S) and ponds (P). Detailed description of research sites, environmental parameters and physicochemical conditions are presented in Zębek et al. (2021).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eResearch material\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA collection of \u003cem\u003eFlavobacterium\u003c/em\u003e spp. \u0026nbsp;strains was obtained as part of a large study into the diversity of bacteria in microbial mats from the periglacial zone of the Ecology Glacier. The mats collected for research were also the subject of other analyses, including metagenomic and phycological analyses. The study showed that the structure of the microbial mats was characterised by the dominance of two main groups of photoautotrophs: cyanobacteria and diatoms (Zębek et al. 2021). An example of the macroscopic and microscopic (cross-section) structure of the microbial mat of the periglacial zone of Ecology Glacier is shown in Figure 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicrobial mats sampling and isolation of bacterial strains\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMicrobial mats were taken from mineral and organic substrates from proglacial streams and ponds. Using an aseptic technique, the samples were placed into sterile Petri dishes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMat samples were collected twice using a flat metal mesh with an area of 5 cm\u003csup\u003e2\u003c/sup\u003e. A total of 20 microbial mats were collected. For bacterial cultivation, 1 g of each mat sample was suspended in 20 mL sterile saline (0.85 %) in 100 mL sterile flasks with glass beads and shaken gently on a universal shaker (type 327) (150 rpm, 20 min, 5\u003csup\u003eo\u003c/sup\u003eC). Suspensions were then stored in the refrigerator (4\u003csup\u003eo\u003c/sup\u003eC) for 10\u0026ndash;20 min to allow larger particles to settle. A decimal dilution series of the supernatant to 10\u003csup\u003e-3\u003c/sup\u003e was prepared in sterile saline, and then 100 \u0026micro;L of suspension was inoculated on Petri plates with R2A medium in triplicates. The cultures were incubated at 7\u003csup\u003eo\u003c/sup\u003eC for one month and inspected every third day for colony development and growth. This also enabled observing particular colony types from different sampling points and CFU growing rates . Pure cultures of different bacterial colonies were isolated after transport to Poland.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e16S rRNA gene-sequencing-based taxonomic identification\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBacterial DNA obtained from a single colony was isolated using the Genomic Mini AX Bacteria+ kit (A\u0026amp;A Biotechnology, Poland) with additional mechanical lysis of the sample in a FastPrep-24 device using zirconium beads. DNA concentration was measured using the fluorometric method on a Qubit 4 Fluorometer. Primers \u003cstrong\u003e27f\u003c/strong\u003e (5\u0026apos;-GAG TTT GAT CCT GGC TCA G-3\u0026apos;, Johnsen et al. 1999) and \u003cstrong\u003erp2\u003c/strong\u003e (5\u0026apos;-ACG GCT ACC TTG TTA CGA CTT-3\u0026apos;, Weisburg et al. 1991) were used in the PCR reaction. DNA obtained from the amplification reaction was purified using the Clean-Up AX kit (A\u0026amp;A Biotechnology, Poland). PCR products, suspended in 10 mM Tris-HCl pH 8.0 buffer and diluted to a concentration of 50 ng/\u0026mu;L, were sent for sequencing to Macrogen Europe BV (The Netherlands).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe current study examined the antibiotic resistance and antagonist interactions of 50 isolates belonging to the genus \u003cem\u003eFlavobacterium\u003c/em\u003e, originating from microbial mats of freshwater ponds and streams.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAntibiotic resistance\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAccording to Bauer et al. (1966), the disc diffusion method was applied using R2A as a replacement for Mueller-Hinton agar to determine the phenotypic antibiotic resistance of flavobacteria strains originating from microbial mats.\u0026nbsp;The 25 antibiotics with different modes of action, belonging to 12 functional groups, were used (Antimicrobial Susceptibility Discs, Oxoid) (Table 1). The plates were incubated at 20\u003csup\u003eo\u003c/sup\u003eC for 48-96 hours, depending on the growth rate of strains. Antibiotic resistance was considered to be the absence of a zone of inhibition of the strain\u0026rsquo;s growth around the antibiotic disk. The antibiotic resistance index (ARI) was determined according to Krumperman et al. (1983). Multi-drug resistance (MDR) was determined according to resistance to at least one of each mode of action of antibiotics used (Exner et al. 2017).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAntagonistic interactions\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTaxonomically identified flavobacteria strains were screened for antimicrobial substance production using the spot-on-lawn method described by Prasad et al. (2011). Each strain was tested against the other strains for cross-inhibition. The cells were washed out from 1 mL of liquid culture of each strain by centrifuging three times (10 min/8,000 rpm), each time suspending the cell pellet in sterile physiological saline at 20\u0026deg;C. The cell pellet was then adjusted to a density of 0.5 McFarland and plated on the R2A medium, treating the inoculated strain as a indicator strain for examining the relationship with other strains. Following this, 10 \u0026mu;L of liquid culture of each of the remaining strains was applied pointwise. The culture was carried out at 20\u0026deg;C for seven days. After this time, the formation of a growth inhibition zone of the indicator strain around the spotted strains was considered an antagonistic effect.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data were statistically analysed using Statistica version 13.3 (StatSoft Inc.). The assessment of the significance of differences in the data obtained used a multivariate analysis of variance (ANOVA), and for data that failed to meet the normality test, a non-parametric Kruskal-Wallis test was applied. Linear regression analysis (LRA) was used to investigate correlational relationships. The antagonistic relationships associated with antibiotic resistance were illustrated in network graphs using the program Cytoscape 3.1.0 (Shannon et al. 2003), and the data were processed in the R Package\u0026apos;s in-house scripts.\u003c/p\u003e\n\u003cp\u003eTable 1. Characteristics of the antibiotics used in the study.\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"566\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eAntibiotic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eSymbol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eFunctional group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eMode of action\u003c/p\u003e\n \u003cp\u003e(inhibitors of)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003eDisk potency\u003c/p\u003e\n \u003cp\u003e(\u0026micro;g)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eAmpicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eAM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003e\u0026beta;-lactams\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003ecell-wall synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eCarbenicillin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003ePY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003e\u0026beta;-lactams\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003ecell-wall synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eCefixime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eCFM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eModified \u0026beta;-lactams/3\u003csup\u003erd\u003c/sup\u003e generation cephalosporine\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003ecell-wall synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eCefotaxime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eCTX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eModified \u0026beta;-lactams/3\u003csup\u003erd\u003c/sup\u003e generation cephalosporine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003ecell-wall synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eCeftazidime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eCAZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eModified \u0026beta;-lactams/3\u003csup\u003erd\u003c/sup\u003e generation cephalosporine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003ecell-wall synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eImipenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eIMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eModified \u0026beta;-lactams/Carbapenem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003ecell-wall synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eVancomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eVA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eGlycopeptide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003ecell-wall synthesis/\u003c/p\u003e\n \u003cp\u003eRNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eGentamicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eCN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eAminoglycosides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eKanamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eAminoglycosides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eStreptomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eAminoglycosides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eChloramphenicol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eChloramphenicol/ Aminoglycosides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eTetracycline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eTE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eTetracycline/Polyketide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eClarithromycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eCLR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eMacrolide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eErythromycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eMacrolide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eClindamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eLincosamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eLincomycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eLincosamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eMupirocin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eMUP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eMonocarboxylic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eprotein and RNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eNitrofurantoin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eNitrofuran Inhibitor of folic acid synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eDNA/RNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eNovobiocin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eNV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eAminocoumarin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eDNA/RNA synthesis,\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eCiprofloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eCIP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eQuinolones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eDNA/RNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eNalidixic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eQuinolones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eDNA/RNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eMetronidazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eMET\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eMetronidazole/Quinolones\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eDNA/RNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eRifampicin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eRA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eRifamycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eDNA/RNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eTrimethoprim\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eTMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eTrimethoprim\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eRNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.9647%;\"\u003e\n \u003cp\u003eCotrimoxazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.0707%;\"\u003e\n \u003cp\u003eSXT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 34.9823%;\"\u003e\n \u003cp\u003eTrimethoprim/Sulfamethoxazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.3746%;\"\u003e\n \u003cp\u003eRNA synthesis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.6078%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003ePhylogenetic analysis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTwenty species were identified among the 50 strains belonging to the genus \u003cem\u003eFlavobacterium\u003c/em\u003e, derived from microbial mats of ponds and streams (Fig. 3, Table 2). Ten species were noted in pond and stream mats, with the more numerous species including \u003cem\u003eF. aquidurense, F. hydatis, F. kayseriense, F. saccharophilum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;F. xanthum\u003c/em\u003e. Five species originated from ponds with the \u003cem\u003eF. antarcticum\u003c/em\u003e strains being the most abundant. Five species were isolated exclusively from stream mats, among which \u003cem\u003eF. pectinovorum\u003c/em\u003e strains were the most abundant. Sequence data of studied flavobacteria strains have been deposited in the GenBank database, and the accession numbers are listed in Table 2. A phylogenetic tree based on 16S rRNA gene sequences comparing the isolated flavobacteria strains among their closest related species within the genus \u003cem\u003eFlavobacterium\u003c/em\u003e is shown in Figure 3.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAntibiotic resistance\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAntarctic flavobacterial strains isolated from microbial mats exhibited a broad spectrum of antibiotic resistance (Fig. 4). Among the isolates under study, 98% exhibited resistance to at least one of the 25 antibiotics applied. Nearly half (45%) of the strains were resistant to 1-5 antibiotics, 20% to 6-10 antibiotics, and 15% to 11-25 antibiotics. A significant percentage among the isolates under study (68%) were multi-drug resistant (MDR) strains, i.e. strains resistant to at least one antibiotic from each of the three functional groups, i.e. inhibiting cell wall synthesis (CW), protein synthesis (P), and nucleic acid synthesis (NA). MDR strains accounted for 65% and 70% of isolates in ponds and streams, respectively. 42% of them represented strains resistant to 10 or more antibiotics. Among the strains derived from pond mats, 80% were resistant to at least one antibiotic from the CW group and 65% to antibiotics from the P group. All strains were resistant to at least one antibiotic from the NA group. In the group of antibiotics inhibiting cell wall synthesis (CW), the largest pool was bacteria resistant to \u0026szlig;-lactams, i.e. ampicillin (40%) and carbenicillin (40%), as well as a 3rd generation cephalosporin, i.e. cefixime (45%). Only two strains derived from pond mats, \u003cem\u003eF. antarcticum\u003c/em\u003e and \u003cem\u003eF. kayseriense\u003c/em\u003e, were resistant to imipenem. In the group of antibiotics inhibiting protein synthesis (P), 40% were resistant to clindamycin, and 45% to lincomycin. Only one isolate, \u003cem\u003eF. antarcticum\u003c/em\u003e, was resistant to tetracycline. In the group of antibiotics inhibiting nucleic acid synthesis (NA), all strains derived from pond mats exhibited resistance to at least one antibiotic. 70% of strains were resistant to trimethoprim, 55% to metronidazole, and 50% to mupirocin and ciprofloxacin. Three MDR strains derived from pond mats, i.e. \u003cem\u003eF. degerlachei, F. hibernum\u003c/em\u003e and \u003cem\u003eF. saccharophilum\u003c/em\u003e, were resistant to 16 of the 25 antibiotics applied. Two strains, i.e. \u003cem\u003eF. glaciei\u003c/em\u003e and \u003cem\u003eF. hydatis\u003c/em\u003e exhibited a very low resistance to only one antibiotic. Among the strains derived from stream mats, 87% were resistant to at least one antibiotic from the CW group, 77% to antibiotics from the P group, and 93% to antibiotics from the NA group. Most (87%) represented strains resistant to 6 or more antibiotics. Only 13% of strains derived from stream mats were resistant to 1-5 antibiotics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. 16S rRNA gene sequence affiliation to the closest phylogenetic neighbours, isolate origin and Gen Bank Accession Number of the flavobacteria isolated from an Antarctic pond and stream microbial mats.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"558\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eIsolate code\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eOrigin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eGen Bank Accession Number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 244px;\"\u003e\n \u003cp\u003eNearest taxonomic neighbour by BLAST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003eIdentity\u003c/p\u003e\n \u003cp\u003e[%]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739435\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium antarcticum\u003c/em\u003e DSM 19726\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e98.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739436\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739437\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739434\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium degerlachei\u003c/em\u003e R-9106\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739456\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.28\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium fryxellicola\u003c/em\u003e LMG 22022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739439\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium glaciei\u003c/em\u003e 0499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium sinopsychrotolerans\u003c/em\u003e 0533\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium aquidurense\u003c/em\u003e WB 1.1-56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739482\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739469\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium cupreum\u003c/em\u003e P2685\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739472\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e98.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739480\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739459\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium flabelliforme\u003c/em\u003e P4025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739466\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium hibernum\u003c/em\u003e ATCC 51468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e98.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739479\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739452\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium hydatis\u003c/em\u003e DSM 2063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739455\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739461\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739463\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739445\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium kayseriense\u003c/em\u003e F-49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739446\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.89\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739457\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium psychroterrae\u0026nbsp;\u003c/em\u003eP3922\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium saccharophilum\u003c/em\u003e NBRC 15944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739465\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739468\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739474\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium xanthum\u003c/em\u003e NBRC 14972\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e98.96\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eP15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003ePond\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739441\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739448\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739447\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium alvei\u0026nbsp;\u003c/em\u003eHR-AY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.22\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739471\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium bizetiae\u003c/em\u003e CIP 105534\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e98.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739473\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium chryseum\u003c/em\u003e P3160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739476\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739443\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium pectinovorum\u003c/em\u003e NBRC 15945\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium piscis\u003c/em\u003e 412r-09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e99.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739464\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 244px;\"\u003e\n \u003cp\u003e\u003cem\u003eFlavobacterium\u0026nbsp;\u003c/em\u003esp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e95.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003eS89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 50px;\"\u003e\n \u003cp\u003eStream\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003eOR739481\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e96.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIn the group of antibiotics inhibiting cell wall synthesis (CW), the largest pool consisted of bacteria resistant to 3rd generation cephalosporins, i.e. cefixime (83%), cefotaxime (73%) as well as ampicillin (70%) and vancomycin (63%). What should be emphasised is the lack of resistance of the analysed strains to imipenem. Nearly 80% of isolates were resistant to at least one antibiotic inhibiting protein synthesis (P), 57% were resistant to lincomycin, and 50% to chloramphenicol, erythromycin and clindamycin. The study found the lack of resistance to tetracycline and resistance of only two strains, i.e.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eF. hydatis\u003c/em\u003e and \u003cem\u003eF. saccharophilum\u003c/em\u003e, to gentamycin. In the group of antibiotics inhibiting nucleic acid synthesis (NA), 93% of strains exhibited resistance to at least one antibiotic. 87% were resistant to metronidazole, 77% to trimethoprim, 73% to mupirocin, and 67% to nitrofurantoin. What is remarkable among the MDR strains is the broad resistance of \u003cem\u003eF. piscis\u003c/em\u003e and \u003cem\u003eF. flabelliforme\u003c/em\u003e to 19 and 18 antibiotics, respectively, and of three strains: \u003cem\u003eF. aquidurense,\u003c/em\u003e \u003cem\u003eF. hydatis\u003c/em\u003e and \u003cem\u003eF. bizetiae\u003c/em\u003e, to 17 out of the 25 antibiotics applied. Two \u003cem\u003eF. kayseriense\u003c/em\u003e isolates were resistant only to two antibiotics, i.e. ciprofloxacin and trimethoprim (belonging to the NA group). One strain, \u003cem\u003eF. pectinovorum\u003c/em\u003e, exhibited no resistance to the antibiotics applied.\u0026nbsp;The existence of individual antibiotic resistance patterns among flavobacterial strains, even among those of the same species, is noteworthy.\u003c/p\u003e\n\u003cp\u003eAnalysis of the antibiotic resistance index (ARI) showed a significantly higher proportion of multi-drug resistant strains (ARI \u0026ge; 0.2) in stream mats (87%) compared to the strains isolated from pond mats (55%) (Fig. 4, Fig. 5). At the same time, the average ARI value for flavobacteria isolated from pond mats (0.27) was significantly higher, compared to the value for the strains isolated from stream mats (0.44). A statistically significantly higher resistance to antibiotics was demonstrated in strains derived from stream mats (n=50, p\u0026lt;0.01) (Fig. 5). In these strains, significantly higher resistance to antibiotics from the group inhibiting cell wall synthesis (CW) (p\u0026lt;0.001), and from the group inhibiting nucleic acid synthesis (NA) (p\u0026lt;0.05), was demonstrated. At the same time, no significant differences were noted in the resistance of the strains under study to antibiotics belonging to the group inhibiting protein synthesis (P).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAntagonistic relations\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAntagonistic relations demonstrated among flavobacterial strains derived from polar-region microbial mats showed noticeable differences between strains isolated from stream mats and from ponds (Fig. 4). It was demonstrated that 50% of the flavobacteria derived from pond mats and 63% derived from stream mats produced compounds inhibiting the development of other isolates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFour groups of strains were distinguished among the possible types of interaction, with all types of interaction occurring among isolates from both pond mats and stream mats. The following strains were distinguished: PR \u0026ndash; where the isolate produces antibacterial compounds but is also resistant to antibacterial compounds produced by other strains; PRS \u0026ndash; where the isolate is an antagonist, i.e. produces antibacterial compounds, while being resistant and sensitive to one or more strains; SR \u0026ndash; where the isolate produces no antibacterial compounds while being sensitive and resistant to antibacterial compounds produced by other strains; R \u0026ndash; the isolate produces no antibacterial compounds but is resistant to the action of these compounds produced by other strains.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the isolates derived from the environments under study, significant differences in antagonistic activity were demonstrated (Fig. 6). In the pond mats, the PR strains accounted for 25%, with the highest antagonistic activity exhibited by \u003cem\u003eF. saccharophilum\u003c/em\u003e, which at the same time was resistant to the majority of the antibiotics applied.\u003cem\u003e\u0026nbsp;\u003c/em\u003eThe remaining strains from this group exhibited low and moderate antibiotic resistance. In the stream mats, the PR strains were the dominant group (43%), with the most antagonistically active strains including \u003cem\u003eF. flabelliforme\u003c/em\u003e, \u003cem\u003eF. hydatis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eF. bizetiae\u003c/em\u003e. These strains also exhibited a very high antibiotic resistance. Moderate and high antibiotic resistance characterised the remaining strains from this group. The PRS strains in pond and stream mats accounted for similar percentages, 25% and 20%, respectively. The PRS strains derived from pond mats were characterised by moderate antagonistic activity and antibiotic resistance. In contrast, \u003cem\u003eFlavobacterium glaciei\u003c/em\u003e and \u003cem\u003eF. hydatis\u003c/em\u003e inhibited the growth of only a few strains while being resistant to just one antibiotic. The PRS strains were characterised by higher antagonistic activity and a broader antibiotic resistance in stream mats. \u003cem\u003eFlavobacterium saccharophilum\u003c/em\u003e and \u003cem\u003eF. pectinovorum\u003c/em\u003e exhibited moderate antagonistic properties while being resistant to many antibiotics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe SR strains in ponds represented the least numerous group (15%) and were distinguished by a high resistance to antagonistic responses and a low antibiotic resistance (ARI\u0026lt;0.2). Only \u003cem\u003eF. antarcticum\u003c/em\u003e, derived from this environment, was distinguished by higher sensitivity to antagonistic interactions than the remaining strains and it displayed resistance to one antibiotic from each of the antibiotic groups applied. Only two SR strains derived from stream mats showed a low resistance to antagonistic interactions and a low antibiotic resistance (ARI=0.08). The R strains in pond mats represented a dominant group (35%). \u003cem\u003eF. degerlachei\u003c/em\u003e and \u003cem\u003eF. hibernum\u003c/em\u003e were characterised by a lack of sensitivity to antibacterial compounds produced by other strains and the highest antibiotic resistance. All R strains from stream mats exhibited similar properties, yet it was the least numerous group isolated from this environment (13%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAntagonistic activity and antibiotic resistance interactions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe results show a statistically significant correlation between the strains\u0026rsquo; ability to produce antibacterial compounds and their resistance to antibiotics (p\u0026lt;0.05). In general, strains with high antagonistic activity were characterised by lower sensitivity to antagonistic interactions and higher antibiotic resistance (Fig. 7). Strains from both: pond mats and stream mats, exhibited a statistically significant correlation between the antagonistic potential of the strains, i.e. their production of antibacterial compounds, and their resistance to antibiotics: r=0.7 (p\u0026lt;0.05) and r=0.64 (p\u0026lt;0.01), respectively (Fig. 8a).\u003c/p\u003e\n\u003cp\u003eThese traits were exhibited by \u003cem\u003eF. saccharophilum\u003c/em\u003e derived from pond mats, and \u003cem\u003eF. piscis\u003c/em\u003e isolated from stream mats, which were characterised by the highest antagonism and antibiotic resistance values. Furthermore, among strains derived from both environments (ponds, streams), a positive correlation was noted between resistance to antagonistic interactions and their antibiotic resistance: r=0.52 (p\u0026lt;0.05) and r=0.49 (p\u0026lt;0.01), respectively. These properties were exhibited by \u003cem\u003eF. hibernum\u003c/em\u003e and \u003cem\u003eF. degerlachei\u003c/em\u003e derived from pond mats, and \u003cem\u003eF. piscis\u003c/em\u003e derived from stream mats (Fig. 8b).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe phylogenetic structure of the collection of flavobacterial strains isolated from the microbial mats of the periglacial zone of the Ecology Glacier on King George Island showed surprisingly high species heterogeneity, which corresponded, for the most part, to strains found in cold environments, including polar regions. The five species isolated from pond mats: \u003cem\u003eF. antarcticum. F. glaciei, F. fryxellicola, F. degerlachei\u003c/em\u003e and \u003cem\u003eF. sinopsychrotolerans\u003c/em\u003e were indigenous to Antarctic lakes (Yi et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Van Trappen et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; The species derived from stream mats, i.e. \u003cem\u003eF. chryseum\u003c/em\u003e, \u003cem\u003eF. pectinovorum\u003c/em\u003e, \u003cem\u003eF. alvei, F. bizetiae\u003c/em\u003e and \u003cem\u003eF. piscis\u003c/em\u003e, are described as occurring in various inland polar ponds, and their origin is also linked to cold-adapted fish pathogens (Kr\u0026aacute;lov\u0026aacute; et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Serra, 2024; Lee \u0026amp; Jeon, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; M\u0026uuml;hle et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zamora et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Of the nine species found in both pond mats and stream mats, the following eight have so far been described in polar environments: \u003cem\u003eF. flabelliforme, F. hibernum, F. hydatis, F. kayseriense, F. psychroterrae, F. saccharophilum, F. xanthum\u003c/em\u003e and \u003cem\u003eF. cupreum\u003c/em\u003e (Kr\u0026aacute;lov\u0026aacute; et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; McCammon et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Mantareva et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Saticioglu et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kr\u0026aacute;lov\u0026aacute; et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Asano et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Matsui et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kr\u0026aacute;lov\u0026aacute; et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). One species, \u003cem\u003eF. aquidurense\u003c/em\u003e, was also isolated from water of temperate zone streams (Cousin et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This confirms the cosmopolitan nature of flavobacteria and their role as an integral component of the microbiocenoses of natural environments, including polar environments.\u003c/p\u003e \u003cp\u003eThe results highlight the importance of antagonistic interactions in forming the structure and functioning of the bacteriocenoses of polar microbial mats. In the available literature, few papers exist on interactions occurring in heterogeneous microbial consortia dependant on the same environmental resources. These relationships are often studied in the context of interactions with test microorganisms, e.g., human and animal pathogens or food contaminants (Kan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Chemoecological studies conducted to date have shown the existence of various types of interactions within heterogeneous microbial consortia: amensalism, commensalism and mutualism, which shape the structure of microbial communities (Long et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Meanwhile, this phenomenon has been studied, to a limited extent, in primeval ecosystems such as polar-region microbial mats. Antagonism is an important survival strategy for aggregated bacteria, enabling them to gain an advantage over competing microorganisms (Tait et al., 2002; Lo Giudice et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mangano et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Long et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In the current study, over 50% of the strains exhibited the ability to produce substances that inhibit the growth other \u003cem\u003eFlavobacterium\u003c/em\u003e spp. strains, and approximately 40% were sensitive to substances produced by microorganisms inhabiting the same niche. A similar, high antagonistic activity was demonstrated for the heterotrophic bacterial strains originating from saline lake microbial mats (60%), oceanic seston (53.3%), aggregated marine organic matter (54.1%), and the microbiome of Antarctic sponges (62.1%) (Long \u0026amp; Azam, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Grossart et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Mangano et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Long et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). A considerably lower antagonistic activity was demonstrated in the bacteriocenoses of pelagic aquatic environments. Lo Giudice et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) identified only 15% of antagonistically active bacteria among strains isolated from the Ross Sea waters. A study by Long et al. (2001) demonstrated that the level of bacterial cell aggregation determines the production of antibacterial substances. Over 50% of bacteria aggregated on the seston surface revealed antagonistic activity, while only 5% of bacterioplankton had such an ability. The current study demonstrated that antagonistic interactions among microbial mat bacteria are complex. This was confirmed by the occurrence of four different patterns of antagonism and sensitivity interactions in the flavobacteria isolates under study: PRS, PR, SR and R. These intraspecies and interspecies properties maintain the structural and functional stability of the bacteriocenosis. This proves that different mechanisms can determine antagonism, which is usually an individual feature of the strain. It is noteworthy that all the flavobacterial strains isolated from microbial mats were resistant to at least one antibacterial agent, and more than half were producers of antibacterial substances. The individualisation of antagonistic properties of individual strains is a rather poorly understood phenomenon, which, however, appears to be of key importance in the formation of the structure of microbial consortia (Mangano et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Prasad, 2011; Long et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The clear difference in the phylogenetic structure and antagonistic activity of the flavobacteria of the microbial mats of ponds and watercourses, demonstrated in the current study, could be determined by the physicochemical and trophic properties of the aquatic environment. A crucial role in shaping the structure and activity of benthic consortia in such ponds is also played by the dynamics of water flow (Stanish et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; 2013; Kohler et al., 2015; Akulava et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Ylla et al. (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) emphasise the importance of organic matter availability and assimilability for developing bacterial groupings and their metabolic and physiological activity. The poorer trophic conditions found in streams are usually associated with a quantitative predominance of hardly decomposable plant polymers, mainly celluloses and hemicelluloses. In stagnant water bodies, the organic matter usually accumulates and \u0026ldquo;ages\u0026rdquo; gradually. Its structure simplifies, and readily available nutrient substrates are released (Ylla et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The current study demonstrated that trophic constraints in the stream environment necessitate a high degree of competition between microbial mat bacteria. This is confirmed by the ability, shown for a significant proportion of the isolated flavobacterial strains, to produce antimicrobial substances, which effectively regulate the structure of the bacteriocenosis. This is expressed by the unexpectedly high species diversity of flavobacteria found in the mats under study, i.e. 20 species identified among 50 isolates. The current study suggests that impaired microbial development under suboptimal conditions of an extreme polar environment induces high susceptibility of the bacteriocenosis to even extremely low antagonistic interactions. A study by Grossart et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) points to the existing correlation between antagonistic activity and the ability of bacteriocenosis to degrade organic matter. The production of antibiotics, bacteriocins and other antimicrobial substances, even in subthreshold quantities, can ensure an advantage over other microorganisms that co-form the bacteriocenosis and inhibit colonisation of the niche by allochthonous microorganisms (Tait et al., 2002; Rypien et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe high antibiotic resistance of flavobacteria strains observed in the current study, in which over 70% of isolates were MDR strains, was surprising but not unexpected. Broad antibiotic resistance is increasingly being identified in isolated strains and the metagenome of the microbiocenoses of various polar environments, indicating that this feature is common. The presence of multi-antibiotic-resistant bacteria was found, e.g. in different polar terrestrial environments (Jara et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Marcoleta et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Opazo-Capurro et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), marine bottom sediments (De Souza et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and glacial ice (Brown \u0026amp; Balkwil, 2009). Numerous publications on polar areas point to the importance of the active soil layer as a reservoir of antibiotic resistance gene-harbouring bacteria (Kr\u0026aacute;lov\u0026aacute; et al., 2017; \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Van Goethem et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Marcoleta et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), when investigating the resistome within the Antarctic Peninsula soils, demonstrated a very high frequency and diversity of antibiotic resistance genes. Microbial mats are a specific formation in which the interrelationships between the biotic components provide the basis for structural and functional homeostasis (N\u0026uacute;\u0026ntilde;ez-Montero \u0026amp; Barrientos, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ambrožič Avguštin et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Antibiotic resistance in such aggregated microbiocenoses is a key phenomenon confirmed by the current study. Numerous researchers have noted the importance of microevolutionary processes in the development of natural antibiotic resistance in native environmental bacteria (Blair et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Paun et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The occurrence of antibiotic resistance in bacteria in areas remote from human activities may also be due to horizontal gene transfer from allochthonous bacteria introduced into the environment. Antibiotic resistance genes (ARGs), identified in polar-region bacteria, are perceived as a biotic contaminant and a significant ecological problem (Von Wintersdorff et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kr\u0026aacute;lov\u0026aacute; et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Metagenomic research suggests that the Antarctic microbiota is the source of ancient antibiotic resistance genes, but, at the same time, multi-antibiotic-resistant strains are found in areas of high anthropogenic impact. King George Island is certainly one such region, which explains the resistance to antibiotics routinely used in modern therapy found in the current study. There are few reports on the resistome of Antarctic flavobacterial strains. As demonstrated by a study by Kr\u0026aacute;lov\u0026aacute; et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), two Antarctic species of \u003cem\u003eF. flabelliforme\u003c/em\u003e and \u003cem\u003eF. geliluteum\u003c/em\u003e, derived from the isolated environment of the James Ross Island, were phenotypically MDR and, at the same time, contained multiple resistance genes to a broader spectrum of antimicrobial drugs. The varying antibiotic resistance profiles among flavobacterial strains of the same species in this study indicate the acquired nature of these traits, influenced by external factors. The different antibiotic resistance in strains originating from two different environments, i.e. flowing and stagnant water, may be determined by the different dynamics of these waters. The current observations suggest that Antarctic bacteria from different ecosystems are genetically distinct due to the need to adapt to ecologically distinct habitats, which is also supported by studies conducted by other authors (Zhang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The narrower antibiotic resistance among flavobacterial strains derived from pond mats may be related to the greater stability of this environment and the availability of biogenic compounds and, consequently, to the lower competitiveness within the microbiocenosis. Evidence indicates that antibiotic resistance genes originate from multiple bacterial sources, showing that the genomes of all bacteria can be considered as one global set of genes, from which most, if not all, bacteria can draw in search of genes necessary for survival (Bennett, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). On the other hand, microbial mats can be important reservoirs of resistance to antimicrobial agents (Flores-Vargas et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSummary\u003c/h2\u003e \u003cp\u003eThis is the first study to demonstrate a relationship between antagonistic interactions with antibiotic resistance within flavobacteria, a component of polar-region microbial mats. The study revealed the complex nature of these interactions occurring at intra- and interspecies levels. It also demonstrated the distinctiveness of the relationships found among flavobacteria of the microbial mats of two types of water bodies, i.e. ponds and streams. The surprisingly high biodiversity of flavobacterial strains in the mat microbiocenosis is an example of the existing strong intra- and interspecies relationships that are key to maintaining structural and functional homeostasis. The results of the study demonstrate that there are individual patterns of antagonistic interactions and antibiotic resistance among the biotic components of mats. The nature of these patterns plays a crucial role in forming the structure and functions of microbial mats. In the mat microbiocenosis, strains of \u0026ldquo;superbacteria\u0026rdquo; are characterised by outstanding antagonistic potential and broad antibiotic resistance. As demonstrated by the study, environmental factors, mainly hydrological and trophic parameters of a pond, also play an important role in promoting specific patterns of intercellular interactions. The study also positively verified the hypothesis that antagonism at the species level in aggregated microbial mat systems is a common phenomenon and plays a significant role in shaping bacterial communities. Moreover, it was confirmed that antagonistic interactions are linked to the antibiotic resistance of strains, and the nature of the aquatic environment determines the intensity of these interactions. Although these findings on flavobacteria may not fully reflect all interactions within microbial mats, the obtained results can be used to develop a dynamic model of the formation of microbial mat communities.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eCompeting Interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the financial support of University of Warmia and Mazury in Olsztyn (D. G., A.Ś. J. K.) and Institute of Biochemistry and Biophysics of the Polish Academy of Science in Warszawa (J. G., M.K.Z).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor Contribution: D.G., and A.Ś.; conceptualization: D.G., A.Ś., M.K.Z. and J. K.; sampling: D.G., A.Ś; and J.G. laboratory analysis: D.G., A.Ś, J.K.; data analysis and visualization: D.G., A.Ś and J.J.; manuscript preparation: D.G., A.Ś.; review and editing: D.G., A.Ś, J.G and M.K.Z.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank the Polish Antarctic Station Arctowski staff for the support in collecting samples from the periglacial zone of Ecology Glacier and for helping us in the expedition.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSequence data of studied flavobacteria strains have been deposited in the GenBank NCBI database, under accession numbers OR739434\u0026ndash;OR739483. The datasets generated and analysed during the current study are available from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbe, K., Nomura, N., Suzuki, S. (2020). 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(2008) Phylogenetic and physiological diversity of bacteria isolated from Puruogangri ice core. \u003cem\u003eMicrob Ecol\u003c/em\u003e 55:476\u0026ndash;488.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"flavobacteria, antagonistic activity, antibiotic resistance, microbial mats, Antarctica","lastPublishedDoi":"10.21203/rs.3.rs-5318460/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5318460/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn polar-region environments, flavobacteria are an abundant component of freshwater microbial mats. For the first time, polar-region flavobacterial strains have been investigated for their antagonistic activity and their antibiotic resistance. These strains were derived from microbial mats occurring in ephemeral freshwater ponds, i.e. ponds and streams of the periglacial zone of Ecology Glacier (King George Island, Maritime Antarctica). The study demonstrated the strains\u0026rsquo; surprisingly high phylogenetic diversity, with 20 species among 50 isolates. Flavobacteria were characterised by four different patterns of antagonism and sensitivity: PRS, PR, SR and R, with \u0026lsquo;P\u0026rsquo; representing the production of antimicrobial substances, \u0026lsquo;R\u0026rsquo; \u0026ndash; resistance, and \u0026lsquo;S\u0026rsquo; \u0026ndash; sensitivity to antimicrobials. Over 50% of strains produced substances inhibiting the growth of other isolates, with 40% being sensitive to such compounds. 68% of the isolates represented multidrug-resistant (MDR) strains. The antibiotic resistance index (ARI) demonstrated a significantly higher proportion of MDR strains and ARI\u0026thinsp;\u0026ge;\u0026thinsp;0.2 in stream mats (87%) as compared to the strains derived from pond mats (55%). A strong correlation was observed between the strains\u0026rsquo; antagonistic potential and antibiotic resistance. Diverse chemoecological responses were found among the flavobacterial strains. An important role in these phenomena is accomplished by the \u0026ldquo;super bacteria\u0026rdquo; strains that effectively accumulate numerous traits associated with antagonistic potential and can be involved in the potential transfer of these traits. The individualisation of antagonistic interaction patterns and antibiotic resistance is one of the mechanisms that maintain mat homeostasis.\u003c/p\u003e","manuscriptTitle":"High antagonistic activity and antibiotic resistance of flavobacteria of polar microbial freshwater mats (King George Island, Antarctica)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-27 10:20:26","doi":"10.21203/rs.3.rs-5318460/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-15T12:31:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-14T14:28:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-30T14:53:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87007955112638155968266401840137211085","date":"2024-12-20T14:29:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-27T17:01:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334251604869361604517648643185517832955","date":"2024-11-18T08:48:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55023319036276828272051506428191362975","date":"2024-11-15T15:04:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173250676755976994397285512677542377383","date":"2024-11-13T08:29:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-13T08:17:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-13T08:14:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-11T14:11:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-08T13:04:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-23T11:09:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c5bd942-8e0e-401a-83ed-850337113426","owner":[],"postedDate":"November 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40390199,"name":"Earth and environmental sciences/Ecology"},{"id":40390200,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2025-04-21T16:03:04+00:00","versionOfRecord":{"articleIdentity":"rs-5318460","link":"https://doi.org/10.1038/s41598-025-97205-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-19 15:57:00","publishedOnDateReadable":"April 19th, 2025"},"versionCreatedAt":"2024-11-27 10:20:26","video":"","vorDoi":"10.1038/s41598-025-97205-x","vorDoiUrl":"https://doi.org/10.1038/s41598-025-97205-x","workflowStages":[]},"version":"v1","identity":"rs-5318460","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5318460","identity":"rs-5318460","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}