Bacteriophages isolate of stream water in Brazil lyses the superbugs Klebsiella spp

Bacteriophages isolate of stream water in Brazil lyses the superbugs Klebsiella spp | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bacteriophages isolate of stream water in Brazil lyses the superbugs Klebsiella spp Ingrid Emanoelly Oliveira Camilo, Marcely de Oliveira Peixoto, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7724689/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Klebsiella pneumoniae is a Gram-negative bacterium associated with high infection rates worldwide and is currently listed by the World Health Organization (WHO) as a critical priority pathogen for the developme 1 nt of new antimicrobials. The indiscriminate use of antibiotics has accelerated the emergence of multidrug-resistant strains, underscoring the urgent need for alternative therapeutic strategies such as phage therapy. In this study, two bacteriophages (vB_MC_KP1 and vB_MC_KP2) were isolated from stream water in Minas Gerais, Brazil, i a site receiving domestic sewage and used for irrigation and other human activities. Both phages specifically infected Klebsiella spp. and were characterized by in vitro assays assessing host range and physicochemical stability. They demonstrated high specificity, lytic activity against multiple Klebsiella species, and remarkable stability under a wide range of temperatures and pH values. These findings indicate that the isolated phages are environmentally persistent and hold potential for both therapeutic applications against multidrug-resistant K. pneumoniae and future use in environmental or food safety interventions. Further molecular characterization and in vivo studies are warranted to validate their application. Phage therapy Klebsiella pneumoniae phage stability Food microbiology water quality Figures Figure 1 Figure 2 Figure 3 Introduction In 2019, it was estimated that one in every eight deaths worldwide was caused by bacterial infections, with Klebsiella pneumoniae being included among the five pathogens responsible for more than 50% of deaths. Alone, it accounted for 790 000 deaths (Rocha and Andrade, 2022). K. pneumoniae is a Gram-negative, non-motile, facultative anaerobic enterobacterium in bacillus form. This microorganism is found in almost all environments such as nature, animals, poorly sanitized food, hospitals and humans. It is considered an opportunistic microorganism, affects immunocompromised individuals and pediatric patients, causing infections such as pneumonia, meningitis, hepatic abscesses, intestinal and urinary tract infections (Martin and Bachman, 2018). The indiscriminate use of antimicrobials contributes to the selection of drugs resistance genes among bacteria. The main mechanisms of bacterial resistance include alterations in membrane permeability, efflux proteins that pump out the antibiotics, mutation, production of enzymes that destroy antibiotics, among others. Also, the emergence of new genes through mutation may occur (Da Costa and Silva Junior, 2017). In recent years, antibiotic therapy options have become increasingly limited regarding bacteria such as K. pneumoniae . Different strains of this bacterium have become multidrug-resistant (MDR) due drugs misuse of antibiotics such as aminoglycosides, quinolones, polymyxins, beta-lactams, and tigecycline, and today it is considered a public health problem (De Souza, et al., 2019; Herridge et al., 2020). Different antibiotic resistance mechanisms can be found in K. pneumoniae strains. The resistance to β-lactam agents, associated with β-lactam ring hydrolysis by β-lactamases, is one of the main, which have a greater impact on infection treatment effectiveness (Herridge et al., (2020). Also, liposaccharide and polysaccharide capsules production, as well as biofilm synthesis, hinders antibiotics action and immune responses, besides fixing it on surfaces that can become areas of contagion. These characteristics make K. pneumoniae more virulent. Due to K. pneumoniae increasing antibiotics resistance, new drugs capable of inhibiting these microorganisms are extremely need (de Souza, et al., 2019). In addition to new antimicrobials, which have been the subject of extensive studies worldwide to combat multidrug-resistant bacteria, phage therapy also emerges as a possibility in the treatment of bacterial infections. Phages can infect and lyse specific bacteria, making the treatment more accurate and effective (Loganathan et al ., 2021). Félix d'Herelle described, in 1917, the bacteriophage as an obligatory intracellular organism, whereas in 1915, F.W. Twort observed lysis plaques but did not associate them with a virus (Summers, 2016). They are viruses that infect and parasitize only bacteria and can present cycles that determine the type of infection in the infected bacterium. Each phage is specific to a particular genus or bacteria species (Reina and Reina, 2018). Lytic phages can bind to bacterial surface, inject their genetic material into the bacterial cell's cytoplasm, and multiply within the infected cell. When viral particles reach their maximum volume within the bacterium and the cytoplasmic environment is conducive to lytic proteins activation, they rupture the bacterial wall, releasing new phages and triggering the lytic cycle (Loganathan et al., (2021). This type of phage is most recommended for use in phage therapy, since they cause lysis in the host cell at cycle end. Additionally, because they replicate exclusively in a specific bacterial type, the quantity of phages administered in treatment is exceedingly small (Tan et al., 2019). The first tests with phage therapy in humans were successful in clinical cases such as pediatric dysentery, cholera, and bubonic plague, which generated greater interest in the use of therapy by European countries such as France, Georgia, Russia, and Poland (Caflisch, Suh & Patel, 2019). Studies have demonstrated the success of using phages in vitro and in vivo in the treatment of birds and humans with Salmonella spp. Infections (Li et al., 2020), and against a wide range of bacteria (Loganathan et al. 2021). Phage therapy offers an advantage over antibiotic therapy since lytic phages are used, there is high specificity of phages for their hosts, their use depends on a single dose due to their high replication capacity, and they do not require large administrations (Chang et al., 2018). Environmental waters act as important reservoirs for multidrug-resistant bacteria and their bacteriophages. The detection of Klebsiella- specific phages in aquatic ecosystems directly or indirectly indicates the presence of their bacterial hosts, highlighting the role of streams receiving sewage or agricultural runoff as potential foci of persistence and dissemination of pathogenic strains. This fact is exemplified in the study by Wee and Yap (2022), who isolated Klebsiella pneumoniae from urban drain water. This isolated harbored genes for resistance to 10 different classes of antimicrobials and genes for resistance to metals. Untreated water used to irrigate vegetables consumed raw also represents a critical route for bacterial spread in the food chain. Contamination of resistant bacteria in food and packaging has generated growing concern in recent years, highlighting the importance of monitoring phages in aquatic environments related to agricultural and food production practices. List Shield is a product made from phages that infect Listeria monocytogenes that aims to control and eliminate contamination of foods such as lettuce, cheese, salmon, and frozen dishes (Perera et al . 2015). The present study isolated two bacteriophages called thereafter vB_MC_KP1 and vB_MC_KP2 from water samples of Corredor Stream in Mário Campos-MG, Brazil. This stream receives sewage from the surrounding population, which has been using it to irrigate vegetables consumed raw. The studied phages were able to lyse a multidrug-resistant strain of K. pneumoniae and other genotyped strains. They remained stable for infection under different physicochemical conditions regarding pH and temperature. The data obtained here suggests the potential use of these phages in phage therapy assays against K. pneumoniae . Materials and Methods 2.1 K. pneumoniae cultivation The target bacterium used in this study was Klebsiella pneumoniae (ATCC 700603), which produces beta-lactamase SHV-18, being used as a control for extended-spectrum beta-lactamase production (Rasheed et al., 2000). A simple streak of this bacterium was performed on nutrient agar (according to the manufacturer’s recommendation). The plate was then incubated at 37°C for 18 hours. After this period, a colony from the plate was transferred to a flask containing 15mL of nutrient broth, which was then incubated for 4 hours at 37°C. Subsequently, the cell concentration of the broth was adjusted using spectrophotometry to an optical density of 0.4 at 600 nm, and this concentration was used in all subsequent tests . All experiments in this study were done in triplicate. 2.2. Isolation of bacteriophages from environmental samples Water samples from Corredor Stream in Mário Campos, Brazil, were collected in sterile screw-capped bottles. The water was centrifuged for 30 minutes, and the supernatant filtered through a polyether sulfone membrane with a pore size of 0.22μm. In an Erlenmeyer flask, 10mL of Luria Bertani medium (Bertani, 1951), 30mL of the filtered sample, and 1mL of fresh bacterial culture were added. After18 hours of incubation, the culture was centrifuged for 15 minutes at 3500rpm. The supernatant underwent further filtration through a 22µm pore membrane, followed by serial dilution and plating for observation of lysis plaques using the overlay method. To phage isolation, four successive passages were performed on bacterial cells. To each passage, an isolated lysis plaque was removed using a pipette tip and transferred to a microtube containing 180μL of SM buffer (5.8 g NaCl, 2.0 g MgSO 4 ·7H2O, 50 ml 1 M Tris-HCl pH 7.4, 0,01% gelatin (v/w) in 1 liter of dH 2 O). From this first tube, serial dilutions were made by transferring 20μL from this tube to the next until reaching a dilution of 10 -9 . Various lysis plaques formed were then isolated and inoculated onto new Petri plates containing K. pneumoniae cultures. 2.3. Host Range Assay For the host range determination, six bacteria were used: Escherichia coli (ATCC 25922), Enterobacter hormaechei (ATCC 700323), Pseudomonas aeruginosa (ATCC 15442), Klebsiella pneumoniae (ATCC 700603), Klebsiella oxytoca (ATCC 13182), and a clinical sample of Klebsiella pneumoniae isolated from a lung infection in patient of Clinical Hospital of Belo Horizonte, Brazil, generously donated by Laboratory of Basic Virology of Federal University of Minas Gerais, Brazil. All these bacteria underwent the same procedure as K. pneumoniae (ATCC 700603) to achieve an optical density of 0.4, as described previously. After cultivation, each bacterium was plated in triplicate using the overlay method. 2.4. Testing viral stability at different pH levels and temperatures The bacteriophages suspension, with a viral title of 10 17 UFP/ml were exposed to different temperatures: 25°C, 37°C, and 60°C. The phages were serially diluted in SM buffer and exposed to each of the specified temperatures for 10 minutes. Then, the dilutions were plated using the overlay method. For the pH test, SM buffers were prepared at different pHs: 5.5, 6.5, 7.0, and 8.0. From each buffer, serial dilutions of each bacteriophage were made. The last 5 dilutions were plated using the overlay method on Petri dishes containing nutrient agar. After inoculation, the cultures were incubated at 37°C for 24 hours. In both tests, counting was performed on plates with a minimum of 30 and a maximum of 300 PFU. The results obtained from temperature and pH tests were calculated and expressed in plaque-forming units per milliliter (PFU/mL). Statistical tests were conducted to compare the data of each bacteriophage, confirm the normal distribution, and verify the confidence intervals using the GraphPad Prism. Results 3.1. Isolation of bacteriophages and host range test In the process of phage isolation, two distinct patterns of lysis plaques were observed (Figure 1). One phage showed a small lysis plaque, while the other a large lysis plaque. These two patterns indicated the presence of more than one bacteriophage in the collected water sample. The isolated phages were named vB_MC_KP1, and vB_MC_KP2. Both phages were capable of infecting and lysing K. pneumoniae (ATCC 700603), the hospital strain of K. pneumoniae , and K.oxytoca (ATCC 13182). Plates containing E. coli, P. aeruginosa, and E. hormaechei showed no lysis despite presence of bacterial growth (Table 1). Table 1 Host range of phages isolated from water samples of Corredor Stream: Bacterial strains tested VB_MC_KP1 VB_MC_KP2 K. pneumoniae K6 ( ATCC 700603) + + Clinical K. pneumoniae + + E. coli (ATCC 25922) - - P. aeruginosa ( ATCC 15442) - - E. hormaechei ( ATCC 700323) - - K. oxytoca ( ATCC 13182) + + Note: The intensity of lysis is represented as follows: "+" for clear lysis; "-" for no lysis. Source: authorship, 2024. 3.2 Bacteriophages stability The stability tests were performed using K. pneumoniae ( ATCC 70006) because it was as the bait strain for phage isolation. Lysis by vB-MC_KP1 was observed at all analyzed temperatures without statistical difference (Figure 2). In vB_MC_KP2, lysis was observed at 25°C and 37°C without statistical difference; no lysis halos were observed at 60°C (p<0,001). These results indicate vB_MC_KP2 is more sensible to high temperatures than vB_MC_KP1, as observed at 60°C. However, both isolates were stable and viable in room and body temperature. This finding is important for proposing future applications using these viruses. Both vB_MC_KP1 and vB_MC_KP2 remained stable and viable across all tested pH values (Figure 3). Phage vB_MC_KP1 showed its optimal pH at 6.5, exhibiting a statistically significant difference compared to the other evaluated pH levels (Figure 3A). For phage vB_MC_KP2 no statistical difference between the analyzed pH was observed (Figure 3B). This finding about infection of two superbugs and the ability to induce lysis in different temperature and pHs makes vB_MC_KP1 and vB_MC_KP2 promising candidates for molecular characterization and further exploration of biotechnological potential. Discussion Phages that lyse K. pneumoniae have been employed as a treatment with excellent results when used against multidrug-resistant strains. In 2018, phage therapy was applied as a treatment method in a victim of a bombing incident, in which wounds became infected by K. pneumoniae despite the use of antibiotics. As the infection persisted in the victim even after two years, bacteriophages combined with antibiotics were administered intravenously, and within weeks, the wounds, which extended to the femur, healed (Eskenazi et al. , 2022). In this study, the specificity and stability of two phages, vB_MC_KP1 and vB_MC_KP2, recently isolated from water samples from Corredor Stream in Mário Campos, Brazil, were analyzed. The water from this stream is used for irrigation of vegetables consumed raw, for animal husbandry, among other uses by the local population. The presence of these phages indicates the presence of Klebsiella in the water. In this case, the findings may demonstrate the risk of using untreated water, which can serve as a vehicle for dissemination of infection by multi-resistant bacteria. Rodrigues et al. (2025) evaluated the microbiological and physicochemical parameters of stream water and reported the detection of bacteriophages specific to Escherichia coli . The occurrence of these viruses can be attributed to fecal contamination of the water, combined with surface runoff resulting from rainfall, which promotes the transport of organic matter derived from animals inhabiting the surrounding area, thus serving as a potential source for the dissemination of both the phages and their bacterial hosts. Liu et al. (2024) also reported the isolation of a novel bacteriophage specific to Klebsiella quasi pneumoniae from karst waters impacted by industrial and agricultural activities. In agreement with their findings, the detection of environmental phages serves as indirect evidence that the bacterial host is, or has recently been, present in the aquatic ecosystem. This observation underscores the potential risk of pathogen dissemination through multiple routes, including the consumption of raw vegetables irrigated with contaminated water and the subsequent contamination of the local food chain. Moreover, environmental phages are not only indicators of the presence of their bacterial hosts, but may also participate in horizontal gene transfer, contributing to the environmental dissemination of antimicrobial resistance determinants. vB_MC_KP1 presents clear rounded lysis, while vB_MC_KP2 presents clear and slightly elongated lysis, indicating a lytic cycle, which means that the bacteriophages studied here are classified as virulent. Jurczak-Kurek et al. (2016) analyzed the lysis morphologies of 83 distinct phages and found that those with clear morphology would be lytic phages and those with halos around them would also be lytic. The host range results showed lysis in K. pneumoniae (ATCC 700603), in the clinical sample of K. pneumoniae , and in K. oxytoca ( ATCC 13182). The other enterobacteria studied were not infected by any of the phages, confirming the specificity of vB_MC_KP1 and vB_MC_KP2, which probably only bind to host receptors presented by bacteria of the Klebsiella genus. It is interesting to note that these phages can lyse different Klebsiella species, which suggests it may have a broad spectrum of action against several members of this genus specifically (Rohnelt, 2020). Unlike antibiotic therapy, phage therapy does not alter the individual's intestinal microbiota (Cully, 2019), thus avoiding dysbiosis and future secondary infections or autoimmune diseases [Cully, 2019; Drulis-Kawa, et al. , 2012). The results obtained in the present research were promising, as it suggests the possibility to phage therapies, they would remain stable and maintain their lytic activity in thermal variations; this also allows for various methods and forms of storage and transport of bacteriophages, as well as enabling an approach with phages for environmental and industrial control. Bacteriophages belonging to the former families Siphoviridae, Myoviridae, and Podoviridae were analyzed in culture with different strains of K. pneumoniae ( Zurabov and Zhilenkov, 2021). These phages were resistant, like vB_MC_KP1, to temperatures of 25°C and 60°C, but when subjected to temperatures above 65°C, they were found to be unable to infect the analyzed bacterial cells like observed to vB_MC_KP2. Other bacteriophage infecting Pseudomonas cichorii remained stable at temperatures between 5°C and 45°C, and after 60°C, the titer decreased drastically (Alves, 2021); these results resemble the pattern observed in vB_MC_KP2. Too, different bacteriophages (Φ i Lp 84 and Φ i Lp 1308) infecting Lactobacillus paracasei showed a better adsorption rate at 37°C with minimal efficiency of adsorption at 0°C and an increasing with the temperature up to 37°C. However, temperatures higher than 45°C had little effect on the adsorption of Φ i Lp 84, but it greatly affected that of Φ i Lp 1308. These phages exhibited a lower adsorption rate when subjected to temperatures above 60°C (Mercanti, Ackermann & Quiberoni, 2015). Since these phages are derived from complex samples, such as stream water, where the temperature can vary from 15°C to 26°C at certain times of the year, it is expected that they would show better viability at temperatures close to ambient. The same pattern was seen in phages isolated from sewage effluents in Pakistan, when tested at temperatures up to 50°C, and they withstand higher temperatures as in this work up to 60°C (Ullah et al., 2022). Rodrigues et al. (2025) observed that the pH of the stream water ranged from 6.2 to 8.9 between February and July. This condition may account for the stability of the bacteriophages studied here across a broad pH spectrum and suggests their potential persistence in the environment throughout this period. The fact that phages maintain their ability to form lysis plaques at different pHs suggests that, in cases of alkalosis, blood acidosis, or pH changes in the solution they are inserted into, they will remain viable and capable of lysing K. pneumoniae. Ni et al . (2021) stated that stability of phages can be observed in both basic and acidic pH, indicating that different phages exhibit different resistances to alkalinity or acidity (Tey et al., 2009). Results of bacteriophage tested in mice infected with K. pneumoniae (Anand et al., 2020) showed better activity when subjected to basic pH. Phages capable of lysing K. pneumoniae were stable at pHs ranging from 5.0 to 11.0 (Obradović et al., 2023). vB_MC_KP1 and vB_MC_KP2 follow the patterns found in other studies with different phages. Meanwhile, a diversity of phages tested for K. pneumoniae infection performs better activity when cultivated in pH 5 to 8; however, when incubated at pH 4, the viral titer decreased significantly (Kęsik-Szeloch et al., 2013). This reduction in activity may be observed in the performance of phage vB_MC_KP1 evaluated in this study, as at pH 6.5, it showed better performance with reduced efficacy at pH 5.5. Although the growth of the phage at pH 4.0 was not evaluated, the data suggest that the growth at this pH value could be even lower. Herridge et al . (2020) also highlighted the use of phages as an alternative therapy for patients infected with K. pneumoniae , P. aeruginosa , Staphylococcus aureus and E. coli . They demonstrated significant effect with oral administration of phages in humans, whose average body temperature ranges from 31.6°C to 37.2°C. The data found in this study may suggest that phages vB_MC_KP1 and vB_MC_KP2, which showed better survival rates at room temperature, could be used in future in vivo tests. Furthermore, these phages found in this study may serve as an alternative for the control of K. pneumoniae in water treatment systems. Other studies showed a significant reduction in K. pneumoniae MTCC109 (Anand et al., 2020) pulmonary burden in mice after a single intranasal administration of phages. According to Tan et al . (2019), hypervirulent lytic bacteriophages should be selected for K. pneumoniae treatment, as this bacterium presents resistance mechanisms such as biofilm formation. In addition to phage therapy, interests from the food industry are observed when discussing bacteriophages. Contaminated foods are responsible for causing illnesses in 600 million people annually (Al Sharif, 2021). In artisanal foods ready for consumption, 75 isolates of Klebsiella were found, with 52.69% being K. oxytoca and 23.31% K. pneumoniae (Crippa et al., 2023). In this context, vB_MC_KP1 and vB_MC_KP2 could be applied as a means of food control and quality assurance. Studies evaluated bacteriophage P100 applied in the food industry for the biocontrol of Listeria monocytogenes (Rossi and Almeida, 2010), concluded that phages could be used as biocontrol agents, thus preventing ingestion of contaminated foods and related health problems. Conclusions The viruses vB_MC_KP1 and vB_MC_KP2 represent promising candidates for the control of Klebsiella infections, exhibiting strong in vitro lytic activity against both K. pneumoniae and K. oxytoca strains. Beyond their medical potential, these phages could play a significant role in environmental and food safety contexts, such as reducing bacterial contamination in water sources, soil, and food products. Their capacity to specifically target and eliminate pathogenic bacteria highlights their potential for biotechnological applications aimed at minimizing the spread of Klebsiella in both ecological and alimentary settings. Further studies are warranted to fully explore their efficacy, stability, and practical implementation in diverse environmental and food-related scenarios. Declarations Competing Interests and Funding Statement: The authors declare that they have no financial or non-financial interests that could have appeared to influence the work reported in this paper. This study was supported by the Research Support Foundation of Minas Gerais State (FAPEMIG) through the Research Support Program of University of Minas Gerais State (PAPQ/UEMG). acknowledgements The authors would like to thank the Program for Financial Support for Research at the University of Minas Gerais State (PAPq - UEMG) for providing financial support to the researchers. References Al Sharif, P. (2021) Mundo tem 600 milhões de casos de doenças por alimentos contaminados todos os anos , ONU News . Available at: https://news.un.org/pt/story/2021/06/1752552 (Accessed: March 5, 2024). Alves, M. K. (2021) Isolamento e caracterização genômica de bacteriófago para biocontrole de Pseudomonas cichorri. Tese (Doutorado em Biotecnologia)-Universidade de Caxias do Sul, Caxias do Sul-Rio Grande do Sul. Anand, T., et al. 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(2019) “Characterization of Klebsiella pneumoniae ST11 isolates and their interactions with lytic phages,” Viruses , 11(11), p. 1080. doi: 10.3390/v11111080; PMID: 31752386; PMCID: PMC6893751. Tey, B. T. et al . (2009). “Production of fusion m13 phage bearing the di-sulphide constrained peptide sequence (CWSFFSNI-C) that interacts with hepatitis B core antigen,” African Journal of Biotechnology , 8(2), 268-273. Ullah, A. et al. (2022) “Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan,” Brazilian Journal of Biology , 82. doi: 10.1590/1519-6984.240943. Wee SK, Yap EPH. Draft Genome Sequence of Multidrug Resistant Klebsiella pneumoniae Strain C43 Isolated from Environmental Water Sample. Microbiol Resour Announc . 2022 Sep 15;11(9):e0025222. doi: 10.1128/mra.00252-22. Epub 2022 Aug 22. PMID: 35993705; PMCID: PMC9476979. Zurabov, F. and Zhilenkov, E. (2021) “Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation,” Virology journal , 18(1). doi: 10.1186/s12985-020-01485-w; PMID: 33407669; PMCID: PMC7789013. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7724689","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":528836158,"identity":"112be876-aad7-4a71-8310-d6808cd19d0b","order_by":0,"name":"Ingrid Emanoelly Oliveira Camilo","email":"data:image/png;base64,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","orcid":"","institution":"State University of Minas Gerais","correspondingAuthor":true,"prefix":"","firstName":"Ingrid","middleName":"Emanoelly Oliveira","lastName":"Camilo","suffix":""},{"id":528836159,"identity":"a0cf1e83-206b-47fc-98d3-fc17d48121e9","order_by":1,"name":"Marcely de Oliveira Peixoto","email":"","orcid":"","institution":"State University of Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Marcely","middleName":"de Oliveira","lastName":"Peixoto","suffix":""},{"id":528836160,"identity":"30b34906-025c-41eb-ac3d-260b4c28a764","order_by":2,"name":"Eduarda Geovana Coelho dos Santos","email":"","orcid":"","institution":"State University of Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Eduarda","middleName":"Geovana Coelho dos","lastName":"Santos","suffix":""},{"id":528836161,"identity":"b96b48e5-5467-4568-9ef4-77d18e7de546","order_by":3,"name":"Fernanda Prieto Bruckner","email":"","orcid":"","institution":"State University of Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Fernanda","middleName":"Prieto","lastName":"Bruckner","suffix":""},{"id":528836162,"identity":"15101968-d3dc-465c-9e37-972146cf5ce2","order_by":4,"name":"Marisa Cristina da Fonseca Casteluber","email":"","orcid":"","institution":"State University of Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Marisa","middleName":"Cristina da Fonseca","lastName":"Casteluber","suffix":""}],"badges":[],"createdAt":"2025-09-26 20:38:23","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7724689/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7724689/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93482134,"identity":"3c857440-cfaf-44a5-92c3-28773a6d26f6","added_by":"auto","created_at":"2025-10-14 10:18:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1550633,"visible":true,"origin":"","legend":"\u003cp\u003eLyses plaques of B_MC_KP1 and vB_MC_KP2 in \u003cem\u003eK. pneumoniae \u003c/em\u003e(ATCC 7000603). A- Lyses plaques observed prior to phages isolation. B- Separation of phages according to the morphology of lyses plaques formed.\u003c/p\u003e\n\u003cp\u003eSource: Authors’ private collection (June, 2024)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7724689/v1/b837d333900716b960877be9.png"},{"id":93482131,"identity":"c07f491d-cd6c-408d-b8d2-292264a40170","added_by":"auto","created_at":"2025-10-14 10:18:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":254313,"visible":true,"origin":"","legend":"\u003cp\u003eBacteriophage viability at different pH levels. A) Plate counting of vB_MC_KP1. B) Plate counting of vB_MC_KP2. The Y-axis indicates phage counting on a logarithmic scale. Error bars indicate standard deviation. The letters above the bars indicate statistical correspondence by ANOVA and Tukey test, with a p-value \u0026lt; 0,05.\u003c/p\u003e\n\u003cp\u003eSource: authorship, 2024.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7724689/v1/1c5052af35acb8c86a4cb6c0.png"},{"id":93482132,"identity":"f6080a59-edef-4c0a-a7fa-22dc8694f2e9","added_by":"auto","created_at":"2025-10-14 10:18:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":240160,"visible":true,"origin":"","legend":"\u003cp\u003eViability of bacteriophages at different temperatures in \u003cem\u003eK. pneumoniae \u003c/em\u003e(ATCC 700603). A) Plate counting of vB_MC_KP1. B) Plate counting of vB_MC_KP2. The Y-axisindicates phage counting on a logarithmic scale. Error bars indicate standard deviation. The statistical analysis was performed by ANOVA with a p-value \u0026lt; 0,05.\u003c/p\u003e\n\u003cp\u003eSource: authorship, 2024.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7724689/v1/5441d0215aac43efc59bbcc5.png"},{"id":93483153,"identity":"d6f3b6ac-cf29-42ed-b06c-07cc2b0d461f","added_by":"auto","created_at":"2025-10-14 10:26:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4110293,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7724689/v1/f737a229-d330-4d04-9f76-9e89f4314a3f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bacteriophages isolate of stream water in Brazil lyses the superbugs Klebsiella spp","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn 2019, it was estimated that one in every eight deaths worldwide was caused by bacterial infections, with \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e being included among the five pathogens responsible for more than 50% of deaths. Alone, it accounted for 790 000 deaths (Rocha and Andrade, 2022). \u003cem\u003eK. pneumoniae\u003c/em\u003e is a Gram-negative, non-motile, facultative anaerobic enterobacterium in bacillus form. This microorganism is found in almost all environments such as nature, animals, poorly sanitized food, hospitals and humans. It is considered an opportunistic microorganism, affects immunocompromised individuals and pediatric patients, causing infections such as pneumonia, meningitis, hepatic abscesses, intestinal and urinary tract infections (Martin and Bachman, 2018).\u003c/p\u003e\n\u003cp\u003eThe indiscriminate use of antimicrobials contributes to the selection of drugs resistance genes among bacteria. The main mechanisms of bacterial resistance include alterations in membrane permeability, efflux proteins that pump out the antibiotics, mutation, production of enzymes that destroy antibiotics, among others. \u0026nbsp;Also, the emergence of new genes through mutation may occur (Da Costa and Silva Junior, 2017).\u003c/p\u003e\n\u003cp\u003eIn recent years, antibiotic therapy options have become increasingly limited regarding bacteria such as \u003cem\u003eK. pneumoniae\u003c/em\u003e. Different strains of this bacterium have become multidrug-resistant (MDR) due drugs misuse of antibiotics such as aminoglycosides, quinolones, polymyxins, beta-lactams, and tigecycline, and today it is considered a public health problem (De Souza, et al., 2019; Herridge et al., 2020). Different antibiotic resistance mechanisms can be found in \u003cem\u003eK. pneumoniae\u003c/em\u003e strains. The resistance to \u0026beta;-lactam agents, associated with \u0026beta;-lactam ring hydrolysis by \u0026beta;-lactamases, is one of the main, which have a greater impact on infection treatment effectiveness (Herridge et al., (2020).\u003c/p\u003e\n\u003cp\u003eAlso, liposaccharide and polysaccharide capsules production, as well as biofilm synthesis, hinders antibiotics action and immune responses, besides fixing it on surfaces that can become areas of contagion. These characteristics make \u003cem\u003eK. pneumoniae\u003c/em\u003e more virulent. \u0026nbsp;Due to \u003cem\u003eK. pneumoniae\u003c/em\u003e increasing antibiotics resistance, new drugs capable of inhibiting these microorganisms are extremely need (de Souza, et al., 2019).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to new antimicrobials, which have been the subject of extensive studies worldwide to combat multidrug-resistant bacteria, phage therapy also emerges as a possibility in the treatment of bacterial infections. Phages can infect and lyse specific bacteria, making the treatment more accurate and effective (Loganathan et al\u003cem\u003e.,\u003c/em\u003e 2021).\u003c/p\u003e\n\u003cp\u003eF\u0026eacute;lix d\u0026apos;Herelle described, in 1917, the bacteriophage as an obligatory intracellular organism, whereas in 1915, F.W. Twort observed lysis plaques but did not associate them with a virus (Summers, 2016). They are viruses that infect and parasitize only bacteria and can present cycles that determine the type of infection in the infected bacterium. Each phage is specific to a particular genus or bacteria species (Reina and Reina, 2018).\u003c/p\u003e\n\u003cp\u003eLytic phages can bind to bacterial surface, inject their genetic material into the bacterial cell\u0026apos;s cytoplasm, and multiply within the infected cell. When viral particles reach their maximum volume within the bacterium and the cytoplasmic environment is conducive to lytic proteins activation, they rupture the bacterial wall, releasing new phages and triggering the lytic cycle (Loganathan et al., (2021). This type of phage is most recommended for use in phage therapy, since they cause lysis in the host cell at cycle end. Additionally, because they replicate exclusively in a specific bacterial type, the quantity of phages administered in treatment is exceedingly small (Tan et al., 2019).\u003c/p\u003e\n\u003cp\u003eThe first tests with phage therapy in humans were successful in clinical cases such as pediatric dysentery, cholera, and bubonic plague, which generated greater interest in the use of therapy by European countries such as France, Georgia, Russia, and Poland (Caflisch, Suh \u0026amp; Patel, 2019).\u003c/p\u003e\n\u003cp\u003eStudies have demonstrated the success of using phages in vitro and in vivo in the treatment of birds and humans with \u003cem\u003eSalmonella\u003c/em\u003e spp. Infections (Li et al., 2020), and against a wide range of bacteria (Loganathan et al. 2021). Phage therapy offers an advantage over antibiotic therapy since lytic phages are used, there is high specificity of phages for their hosts, their use depends on a single dose due to their high replication capacity, and they do not require large administrations (Chang et al., 2018).\u003c/p\u003e\n\u003cp\u003eEnvironmental waters act as important reservoirs for multidrug-resistant bacteria and their bacteriophages. The detection of\u003cem\u003e\u0026nbsp;Klebsiella-\u003c/em\u003especific phages in aquatic ecosystems directly or indirectly indicates the presence of their bacterial hosts, highlighting the role of streams receiving sewage or agricultural runoff as potential foci of persistence and dissemination of pathogenic strains. This fact is exemplified in the study by Wee and Yap (2022), who isolated \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e from urban drain water. This isolated harbored genes for resistance to 10 different classes of antimicrobials and genes for resistance to metals.\u003c/p\u003e\n\u003cp\u003eUntreated water used to irrigate vegetables consumed raw also represents a critical route for bacterial spread in the food chain. Contamination of resistant bacteria in food and packaging has generated growing concern in recent years, highlighting the importance of monitoring phages in aquatic environments related to agricultural and food production practices. List Shield is a product made from phages that infect \u003cem\u003eListeria monocytogenes\u003c/em\u003e that aims to control and eliminate contamination of foods such as lettuce, cheese, salmon, and frozen dishes (Perera \u003cem\u003eet al\u003c/em\u003e. 2015).\u003c/p\u003e\n\u003cp\u003eThe present study isolated two bacteriophages called thereafter vB_MC_KP1 and vB_MC_KP2 from water samples of Corredor Stream in M\u0026aacute;rio Campos-MG, Brazil. This stream receives sewage from the surrounding population, which has been using it to irrigate vegetables consumed raw. The studied phages were able to lyse a multidrug-resistant strain of \u003cem\u003eK. pneumoniae\u003c/em\u003e and other genotyped strains. They remained stable for infection under different physicochemical conditions regarding pH and temperature. The data obtained here suggests the potential use of these phages in phage therapy assays against \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1 K. pneumoniae\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003ecultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe target bacterium used in this study was \u003cem\u003eKlebsiella pneumoniae\u0026nbsp;\u003c/em\u003e(ATCC 700603), which produces beta-lactamase SHV-18, being used as a control for extended-spectrum beta-lactamase production (Rasheed et al., 2000). A simple streak of this bacterium was performed on nutrient agar (according to the manufacturer’s recommendation). The plate was then incubated at 37°C for 18 hours. After this period, a colony from the plate was transferred to a flask containing 15mL of nutrient broth, which was then incubated for 4 hours at 37°C. Subsequently, the cell concentration of the broth was adjusted using spectrophotometry to an optical density of 0.4 at 600 nm, and this concentration was used in all subsequent tests\u003cem\u003e.\u0026nbsp;\u003c/em\u003eAll experiments in this study were done in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2.2. Isolation of bacteriophages from environmental samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWater samples from Corredor Stream in Mário Campos, Brazil, were collected in sterile screw-capped bottles. The water was centrifuged for 30 minutes, and the supernatant filtered through a polyether sulfone membrane with a pore size of 0.22μm. In an Erlenmeyer flask, 10mL of Luria Bertani medium (Bertani, 1951), 30mL of the filtered sample, and 1mL of fresh bacterial culture were added.\u003c/p\u003e\n\u003cp\u003eAfter18 hours of incubation, the culture was centrifuged for 15 minutes at 3500rpm. The supernatant underwent further filtration through a 22µm pore membrane, followed by serial dilution and plating for observation of lysis plaques using the overlay method. To phage isolation, four successive passages were performed on bacterial cells. To each passage, an isolated lysis plaque was removed using a pipette tip and transferred to a microtube containing 180μL of SM buffer (5.8 g NaCl, 2.0 g MgSO\u003csub\u003e4\u003c/sub\u003e·7H2O, 50 ml 1 M Tris-HCl pH 7.4, 0,01% gelatin (v/w) in 1 liter of dH\u003csub\u003e2\u003c/sub\u003eO).\u003c/p\u003e\n\u003cp\u003eFrom this first tube, serial dilutions were made by transferring 20μL from this tube to the next until reaching a dilution of 10\u003csup\u003e-9\u003c/sup\u003e. Various lysis plaques formed were then isolated and inoculated onto new Petri plates containing \u003cem\u003eK. pneumoniae\u003c/em\u003e cultures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Host Range Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the host range determination, six bacteria were used: \u003cem\u003eEscherichia coli\u003c/em\u003e (ATCC 25922), \u003cem\u003eEnterobacter hormaechei\u003c/em\u003e (ATCC 700323), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (ATCC 15442), \u003cem\u003eKlebsiella pneumoniae\u0026nbsp;\u003c/em\u003e(ATCC 700603), \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e (ATCC 13182), and a clinical sample of \u003cem\u003eKlebsiella pneumoniae\u0026nbsp;\u003c/em\u003eisolated from a lung infection in patient of Clinical Hospital of Belo Horizonte, Brazil, generously donated by Laboratory of Basic Virology of Federal University of Minas Gerais, Brazil. All these bacteria underwent the same procedure as \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 700603) to achieve an optical density of 0.4, as described previously. After cultivation, each bacterium was plated in triplicate using the overlay method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Testing viral stability at different pH levels and temperatures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacteriophages suspension, with a viral title of 10\u003csup\u003e17\u003c/sup\u003e UFP/ml were exposed to different temperatures: 25°C, 37°C, and 60°C. The phages were serially diluted in SM buffer and exposed to each of the specified temperatures for 10 minutes. Then, the dilutions were plated using the overlay method. For the pH test, SM buffers were prepared at different pHs: 5.5, 6.5, 7.0, and 8.0. From each buffer, serial dilutions of each bacteriophage were made. The last 5 dilutions were plated using the overlay method on Petri dishes containing nutrient agar.\u003c/p\u003e\n\u003cp\u003eAfter inoculation, the cultures were incubated at 37°C for 24 hours. In both tests, counting was performed on plates with a minimum of 30 and a maximum of 300 PFU. The results obtained from temperature and pH tests were calculated and expressed in plaque-forming units per milliliter (PFU/mL). Statistical tests were conducted to compare the data of each bacteriophage, confirm the normal distribution, and verify the confidence intervals using the GraphPad Prism.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Isolation of bacteriophages and host range test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the process of phage isolation, two distinct patterns of lysis plaques were observed (Figure 1). One phage showed a small lysis plaque, while the other a large lysis plaque. These two patterns indicated the presence of more than one bacteriophage in the collected water sample. The isolated phages were named vB_MC_KP1, and vB_MC_KP2. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Both phages were capable of infecting and \u0026nbsp; lysing\u003cem\u003e\u0026nbsp;K. pneumoniae\u0026nbsp;\u003c/em\u003e(ATCC 700603), the hospital strain of \u003cem\u003eK. pneumoniae\u003c/em\u003e, and \u003cem\u003eK.oxytoca\u003c/em\u003e (ATCC 13182). Plates containing \u003cem\u003eE. coli, P. aeruginosa,\u003c/em\u003e and \u003cem\u003eE. hormaechei\u0026nbsp;\u003c/em\u003eshowed no lysis despite presence of bacterial growth (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 Host range of phages isolated from water samples of Corredor Stream:\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 39.9293%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBacterial strains tested\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.9117%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVB_MC_KP1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.159%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVB_MC_KP2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 39.9293%;\"\u003e\n \u003cp\u003e\u003cem\u003eK. pneumoniae K6 (\u003c/em\u003eATCC 700603)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.9117%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.159%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 39.9293%;\"\u003e\n \u003cp\u003eClinical\u003cem\u003e\u0026nbsp;K. pneumoniae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.9117%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.159%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 39.9293%;\"\u003e\n \u003cp\u003e\u003cem\u003eE. coli (ATCC\u003c/em\u003e 25922)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.9117%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.159%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 39.9293%;\"\u003e\n \u003cp\u003e\u003cem\u003eP. aeruginosa (\u003c/em\u003eATCC 15442)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.9117%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.159%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 39.9293%;\"\u003e\n \u003cp\u003e\u003cem\u003eE. hormaechei (\u003c/em\u003eATCC 700323)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.9117%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.159%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 39.9293%;\"\u003e\n \u003cp\u003e\u003cem\u003eK. oxytoca (\u003c/em\u003eATCC 13182)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24.9117%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.159%;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eNote: The intensity of lysis is represented as follows: \u0026quot;+\u0026quot; for clear lysis; \u0026quot;-\u0026quot; for no lysis.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSource: authorship, 2024.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Bacteriophages stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stability tests were performed using \u003cem\u003eK. pneumoniae (\u003c/em\u003eATCC 70006) because it was as the bait strain for phage isolation. Lysis by vB-MC_KP1 was observed at all analyzed temperatures without statistical difference (Figure 2). In vB_MC_KP2, lysis was observed at 25\u0026deg;C and 37\u0026deg;C without statistical difference; no lysis halos were observed at 60\u0026deg;C (p\u0026lt;0,001). \u0026nbsp; These results indicate vB_MC_KP2 is more sensible to high temperatures than vB_MC_KP1, as observed at 60\u0026deg;C. However, both isolates were stable and viable in room and body temperature. This finding is important for proposing future applications using these viruses.\u003c/p\u003e\n\u003cp\u003eBoth vB_MC_KP1 and vB_MC_KP2 remained stable and viable across all tested pH values (Figure 3). Phage vB_MC_KP1 showed its optimal pH at 6.5, exhibiting a statistically significant difference compared to the other evaluated pH levels (Figure 3A). For phage vB_MC_KP2 no statistical difference between the analyzed pH was observed (Figure 3B). This finding about infection of two superbugs and the ability to induce lysis in different temperature and pHs makes vB_MC_KP1 and vB_MC_KP2 promising candidates for molecular characterization and further exploration of biotechnological potential.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePhages that lyse \u003cem\u003eK. pneumoniae\u0026nbsp;\u003c/em\u003ehave been employed as a treatment with excellent results when used against multidrug-resistant strains. In 2018, phage therapy was applied as a treatment method in a victim of a bombing incident, in which wounds became infected by \u003cem\u003eK. pneumoniae\u0026nbsp;\u003c/em\u003edespite the use of antibiotics. As the infection persisted in the victim even after two years, bacteriophages combined with antibiotics were administered intravenously, and within weeks, the wounds, which extended to the femur, healed (Eskenazi et al.\u003cem\u003e,\u003c/em\u003e 2022).\u003c/p\u003e\n\u003cp\u003eIn this study, the specificity and stability of two phages, vB_MC_KP1 and vB_MC_KP2, recently isolated from water samples from Corredor Stream in M\u0026aacute;rio Campos, Brazil, were analyzed. The water from this stream is used for irrigation of vegetables consumed raw, for animal husbandry, among other uses by the local population. The presence of these phages indicates the presence of \u003cem\u003eKlebsiella\u003c/em\u003e in the water. In this case, the findings may demonstrate the risk of using untreated water, which can serve as a vehicle for dissemination of infection by multi-resistant bacteria.\u003c/p\u003e\n\u003cp\u003eRodrigues et al. (2025) evaluated the microbiological and physicochemical parameters of stream water and reported the detection of bacteriophages specific to \u003cem\u003eEscherichia coli\u003c/em\u003e. The occurrence of these viruses can be attributed to fecal contamination of the water, combined with surface runoff resulting from rainfall, which promotes the transport of organic matter derived from animals inhabiting the surrounding area, thus serving as a potential source for the dissemination of both the phages and their bacterial hosts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLiu et al. (2024) also reported the isolation of a novel bacteriophage specific to \u003cem\u003eKlebsiella quasi pneumoniae\u003c/em\u003e from karst waters impacted by industrial and agricultural activities. In agreement with their findings, the detection of environmental phages serves as indirect evidence that the bacterial host is, or has recently been, present in the aquatic ecosystem. This observation underscores the potential risk of pathogen dissemination through multiple routes, including the consumption of raw vegetables irrigated with contaminated water and the subsequent contamination of the local food chain.\u003c/p\u003e\n\u003cp\u003eMoreover, environmental phages are not only indicators of the presence of their bacterial hosts, but may also participate in horizontal gene transfer, contributing to the environmental dissemination of antimicrobial resistance determinants.\u003c/p\u003e\n\u003cp\u003evB_MC_KP1 presents clear rounded lysis, while vB_MC_KP2 presents clear and slightly elongated lysis, indicating a lytic cycle, which means that the bacteriophages studied here are classified as virulent.\u0026nbsp;Jurczak-Kurek et al. (2016)\u003csup\u003e\u0026nbsp;\u003c/sup\u003eanalyzed the lysis morphologies of 83 distinct phages and found that those with clear morphology would be lytic phages and those with halos around them would also be lytic.\u003c/p\u003e\n\u003cp\u003eThe host range results showed lysis in \u003cem\u003eK. pneumoniae\u003c/em\u003e (ATCC 700603), in the clinical sample of \u003cem\u003eK. pneumoniae\u003c/em\u003e, and in \u003cem\u003eK. oxytoca (\u003c/em\u003eATCC 13182). The other enterobacteria studied were not infected by any of the phages, confirming the specificity of vB_MC_KP1 and vB_MC_KP2, which probably only bind to host receptors presented by bacteria of the \u003cem\u003eKlebsiella\u0026nbsp;\u003c/em\u003egenus. It is interesting to note that these phages can lyse different \u003cem\u003eKlebsiella\u003c/em\u003e species, which suggests it may have a broad spectrum of action against several members of this genus specifically (Rohnelt, 2020).\u003c/p\u003e\n\u003cp\u003eUnlike antibiotic therapy, phage therapy does not alter the individual\u0026apos;s intestinal microbiota (Cully, 2019), thus avoiding dysbiosis and future secondary infections or autoimmune diseases [Cully, 2019; Drulis-Kawa, et al.\u003cem\u003e,\u0026nbsp;\u003c/em\u003e2012). The results obtained in the present research were promising, as it suggests the possibility to phage therapies, they would remain stable and maintain their lytic activity in thermal variations; this also allows for various methods and forms of storage and transport of bacteriophages, as well as enabling an approach with phages for environmental and industrial control.\u003c/p\u003e\n\u003cp\u003eBacteriophages belonging to the former families Siphoviridae, Myoviridae, and Podoviridae were analyzed in culture with different strains of \u003cem\u003eK. pneumoniae (\u003c/em\u003eZurabov and Zhilenkov, 2021). These phages were resistant, like vB_MC_KP1, to temperatures of 25\u0026deg;C and 60\u0026deg;C, but when subjected to temperatures above 65\u0026deg;C, they were found to be unable to infect the analyzed bacterial cells like observed to vB_MC_KP2.\u003c/p\u003e\n\u003cp\u003eOther bacteriophage infecting \u003cem\u003ePseudomonas cichorii\u003c/em\u003e remained stable at temperatures between 5\u0026deg;C and 45\u0026deg;C, and after 60\u0026deg;C, the titer decreased drastically (Alves, 2021); these results resemble the pattern observed in vB_MC_KP2. Too, different bacteriophages (\u0026Phi; i\u003cem\u003eLp\u003c/em\u003e84 and \u0026Phi; i\u003cem\u003eLp\u003c/em\u003e1308) infecting \u003cem\u003eLactobacillus paracasei\u003c/em\u003e showed a better adsorption rate at 37\u0026deg;C with minimal efficiency of adsorption at 0\u0026deg;C and an increasing with the temperature up to 37\u0026deg;C. However, temperatures higher than 45\u0026deg;C had little effect on the adsorption of \u0026Phi; i\u003cem\u003eLp\u003c/em\u003e84, but it greatly affected that of \u0026Phi; i\u003cem\u003eLp\u003c/em\u003e1308. These phages exhibited a lower adsorption rate when subjected to temperatures above 60\u0026deg;C (Mercanti, Ackermann \u0026amp; Quiberoni, 2015). Since these phages are derived from complex samples, such as stream water, where the temperature can vary from 15\u0026deg;C to 26\u0026deg;C at certain times of the year, it is expected that they would show better viability at temperatures close to ambient. The same pattern was seen in phages isolated from sewage effluents in Pakistan, when tested at temperatures up to 50\u0026deg;C, and they withstand higher temperatures as in this work up to 60\u0026deg;C (Ullah et al.,\u003cem\u003e\u0026nbsp;\u003c/em\u003e2022).\u003c/p\u003e\n\u003cp\u003eRodrigues et al. (2025) observed that the pH of the stream water ranged from 6.2 to 8.9 between February and July. This condition may account for the stability of the bacteriophages studied here across a broad pH spectrum and suggests their potential persistence in the environment throughout this period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe fact that phages maintain their ability to form lysis plaques at different pHs suggests that, in cases of alkalosis, blood acidosis, or pH changes in the solution they are inserted into, they will remain viable and capable of lysing \u003cem\u003eK. pneumoniae.\u003c/em\u003e Ni \u003cem\u003eet al\u003c/em\u003e. (2021) stated that stability of phages can be observed in both basic and acidic pH, indicating that different phages exhibit different resistances to alkalinity or acidity (Tey \u0026nbsp;et al., 2009). Results of bacteriophage tested in mice infected with \u003cem\u003eK. pneumoniae\u003c/em\u003e (Anand et al., 2020) showed better activity when subjected to basic pH. Phages capable of lysing \u003cem\u003eK.\u0026nbsp;\u003c/em\u003epneumoniae were stable at pHs ranging from 5.0 to 11.0 (Obradović et al., 2023). vB_MC_KP1 and vB_MC_KP2 follow the patterns found in other studies with different phages. Meanwhile, a diversity of phages tested for \u003cem\u003eK. pneumoniae\u003c/em\u003e infection performs better activity when cultivated in pH 5 to 8; however, when incubated at pH 4, the viral titer decreased significantly (Kęsik-Szeloch \u0026nbsp;et al., 2013). This reduction in activity may be observed in the performance of phage vB_MC_KP1 evaluated in this study, as at pH 6.5, it showed better performance with reduced efficacy at pH 5.5. Although the growth of the phage at pH 4.0 was not evaluated, the data suggest that the growth at this pH value could be even lower.\u003c/p\u003e\n\u003cp\u003eHerridge \u003cem\u003eet al\u003c/em\u003e. (2020) also highlighted the use of phages as an alternative therapy for patients infected with \u003cem\u003eK. pneumoniae\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. They demonstrated significant effect with oral administration of phages\u003cem\u003e\u0026nbsp;\u003c/em\u003ein humans, whose average body temperature ranges from 31.6\u0026deg;C to 37.2\u0026deg;C. \u0026nbsp;The data found in this study may suggest that phages vB_MC_KP1 and vB_MC_KP2, which showed better survival rates at room temperature, could be used in future \u003cem\u003ein vivo\u003c/em\u003e tests. Furthermore, these phages found in this study may serve as an alternative for the control of \u003cem\u003eK. pneumoniae\u003c/em\u003e in water treatment systems.\u003c/p\u003e\n\u003cp\u003eOther studies showed a significant reduction in \u003cem\u003eK. pneumoniae\u003c/em\u003e MTCC109 (Anand et al., 2020) pulmonary burden in mice after a single intranasal administration of phages. According to Tan et al\u003cem\u003e.\u003c/em\u003e (2019), hypervirulent lytic bacteriophages should be selected for \u003cem\u003eK. pneumoniae\u003c/em\u003e treatment, as this bacterium presents resistance mechanisms such as biofilm formation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to phage therapy, interests from the food industry are observed when discussing bacteriophages. Contaminated foods are responsible for causing illnesses in 600 million people annually (Al Sharif, 2021). In artisanal foods ready for consumption, 75 isolates of \u003cem\u003eKlebsiella\u003c/em\u003e were found, with 52.69% being \u003cem\u003eK. oxytoca\u0026nbsp;\u003c/em\u003eand 23.31% \u003cem\u003eK. pneumoniae\u0026nbsp;\u003c/em\u003e(Crippa et al., 2023). In this context, vB_MC_KP1 and vB_MC_KP2 could be applied as a means of food control and quality assurance. Studies evaluated bacteriophage P100 applied in the food industry for the biocontrol of \u003cem\u003eListeria monocytogenes\u0026nbsp;\u003c/em\u003e(Rossi and Almeida, 2010), concluded that phages could be used as biocontrol agents, thus preventing ingestion of contaminated foods and related health problems.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe viruses vB_MC_KP1 and vB_MC_KP2 represent promising candidates for the control of \u003cem\u003eKlebsiella\u003c/em\u003e infections, exhibiting strong in vitro lytic activity against both \u003cem\u003eK. pneumoniae\u003c/em\u003e and \u003cem\u003eK. oxytoca\u003c/em\u003e strains. Beyond their medical potential, these phages could play a significant role in environmental and food safety contexts, such as reducing bacterial contamination in water sources, soil, and food products. Their capacity to specifically target and eliminate pathogenic bacteria highlights their potential for biotechnological applications aimed at minimizing the spread of \u003cem\u003eKlebsiella\u003c/em\u003e in both ecological and alimentary settings. Further studies are warranted to fully explore their efficacy, stability, and practical implementation in diverse environmental and food-related scenarios.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests and Funding Statement:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no financial or non-financial interests that could have appeared to influence the work reported in this paper.\u003cbr\u003e\u0026nbsp;This study was supported by the Research Support Foundation of Minas Gerais State (FAPEMIG) through the Research Support Program of University of Minas Gerais State (PAPQ/UEMG).\u003c/p\u003e\n\u003cp\u003eacknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Program for Financial Support for Research at the University of Minas Gerais State (PAPq - UEMG) for providing financial support to the researchers.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl Sharif, P. (2021) \u003cem\u003eMundo tem 600 milh\u0026otilde;es de casos de doen\u0026ccedil;as por alimentos contaminados\u003cem\u003e todos os anos\u003c/em\u003e, \u003cem\u003eONU News\u003c/em\u003e. Available at: https://news.un.org/pt/story/2021/06/1752552 (Accessed: March 5, 2024).\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eAlves, M. K. (2021) \u003cem\u003eIsolamento e caracteriza\u0026ccedil;\u0026atilde;o gen\u0026ocirc;mica de bacteri\u0026oacute;fago para biocontrole de Pseudomonas cichorri.\u003c/em\u003e Tese (Doutorado em Biotecnologia)-Universidade de Caxias do Sul, Caxias do Sul-Rio Grande do Sul.\u003c/li\u003e\n\u003cli\u003eAnand, T., et al. (2020) \u0026ldquo;Phage therapy for treatment of virulent Klebsiella pneumoniae infection in a mouse model,\u0026rdquo; \u003cem\u003eJournal of global antimicrobial resistance\u003c/em\u003e, 21, pp. 34\u0026ndash;41. doi: 10.1016/j.jgar.2019.09.018; PMID: 31604128.\u003c/li\u003e\n\u003cli\u003eBertani, G. (1951) \u0026ldquo;Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli\u0026rdquo;. \u003cem\u003eJournal of Bacteriology, \u003c/em\u003e62(3), pp.293-300. doi: 10.1128/jb.62.3.293-300.1951; PMID: 14888646; PMCID: PMC386127.\u003c/li\u003e\n\u003cli\u003eCaflisch, K. M., Suh, G. A. and Patel, R. (2019) \u0026ldquo;Biological challenges of phage therapy and proposed solutions: a literature review,\u0026rdquo; \u003cem\u003eExpert review of anti-infective therapy\u003c/em\u003e, 17(12), pp. 1011\u0026ndash;1041. doi: 10.1080/14787210.2019.1694905; PMID: 31735090; PMCID: PMC6919273.\u003c/li\u003e\n\u003cli\u003eChang, R. Y. K., et al. \u003cem\u003e(\u003c/em\u003e2018) \u0026ldquo;Phage therapy for respiratory infections,\u0026rdquo; \u003cem\u003eAdvanced drug delivery reviews, \u003c/em\u003e133, pp. 76-86. doi: 10.1016/j.addr.2018.08.001.\u003c/li\u003e\n\u003cli\u003eCrippa, C., Pasquali, F., Rodrigues, C. \u003cem\u003eet al. 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A. \u003cem\u003eet al.\u003c/em\u003e (2019) \u003cem\u003eM\u0026eacute;todos Alternativos De Controle Microbiano\u003c/em\u003e. \u003cem\u003ePERSPECTIVA, Erechim\u003c/em\u003e. v. 43, n.163.\u003c/li\u003e\n\u003cli\u003eDrulis-Kawa, Z. \u003cem\u003eet al.\u003c/em\u003e (2012) \u0026ldquo;Learning from bacteriophages - advantages and limitations of phage and phage-encoded protein applications,\u0026rdquo; \u003cem\u003eCurrent protein \u0026amp; peptide science\u003c/em\u003e, 13(8), pp. 699\u0026ndash;722. doi: 10.2174/138920312804871193.\u003c/li\u003e\n\u003cli\u003eEskenazi, A. \u003cem\u003eet al.\u003c/em\u003e (2022) \u0026ldquo;Combination of pre-adapted bacteriophage therapy and antibiotics for treatment of fracture-related infection due to pandrug-resistant Klebsiella pneumoniae,\u0026rdquo; \u003cem\u003eNature communications\u003c/em\u003e, 13(1). doi: 10.1038/s41467-021-27656-z; PMID: 35042848; PMCID: PMC8766457\u003c/li\u003e\n\u003cli\u003eHerridge, W. 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M. and Bachman, M. A. (2018) \u0026ldquo;Colonization, Infection, and the Accessory Genome of Klebsiella pneumoniae,\u0026rdquo; \u003cem\u003eFrontiers in cellular and infection microbiology\u003c/em\u003e, 8. doi: 10.3389/fcimb.2018.00004.\u003c/li\u003e\n\u003cli\u003eMercanti, D. J., Ackermann, H.-W. and Quiberoni, A. (2015) \u0026ldquo;Characterization of two temperate Lactobacillus paracasei bacteriophages: Morphology, kinetics and adsorption,\u0026rdquo; \u003cem\u003eIntervirology\u003c/em\u003e, 58(1), pp. 49\u0026ndash;56. doi: 10.1159/000369207; PMID: 25591620.\u003c/li\u003e\n\u003cli\u003eNi, P. \u003cem\u003eet al.\u003c/em\u003e (2021) \u0026ldquo;Characterization of a Lytic Bacteriophage against Pseudomonas syringae pv. actinidiae and Its Endolysin,\u0026rdquo; \u003cem\u003eViruses\u003c/em\u003e, 13(4), p. 631. doi: 10.3390/v13040631.\u003c/li\u003e\n\u003cli\u003ePerera MN, Abuladze T, Li M, Woolston J, Sulakvelidze A. Bacteriophage cocktail significantly reduces or eliminates Listeria monocytogenes contamination on lettuce, apples, cheese, smoked salmon and frozen foods. \u003cem\u003eFood Microbiol.\u003c/em\u003e 2015 Dec;52:42-8. doi: 10.1016/j.fm.2015.06.006. Epub 2015 Jun 23. PMID: 26338115.\u003c/li\u003e\n\u003cli\u003eRasheed, J. K. \u003cem\u003eet al.\u003c/em\u003e (2000) \u0026ldquo;Characterization of the extended-spectrum \u0026beta;-lactamase reference strain, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e K6 (ATCC 700603), which produces the novel enzyme SHV-18,\u0026rdquo; \u003cem\u003eAntimicrobial agents and chemotherapy\u003c/em\u003e, 44(9), pp. 2382\u0026ndash;2388. doi: 10.1128/aac.44.9.2382-2388.2000\u003c/li\u003e\n\u003cli\u003eReina, J. and Reina, N. 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(2009). \u0026ldquo;Production of fusion m13 phage bearing the di-sulphide constrained peptide sequence (CWSFFSNI-C) that interacts with hepatitis B core antigen,\u0026rdquo; \u003cem\u003eAfrican Journal of Biotechnology\u003c/em\u003e, 8(2), 268-273.\u003c/li\u003e\n\u003cli\u003eUllah, A. \u003cem\u003eet al.\u003c/em\u003e (2022) \u0026ldquo;Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan,\u0026rdquo; \u003cem\u003eBrazilian Journal of Biology\u003c/em\u003e, 82. doi: 10.1590/1519-6984.240943.\u003c/li\u003e\n\u003cli\u003eWee SK, Yap EPH. Draft Genome Sequence of Multidrug Resistant Klebsiella pneumoniae Strain C43 Isolated from Environmental Water Sample. \u003cem\u003eMicrobiol Resour Announc\u003c/em\u003e. 2022 Sep 15;11(9):e0025222. doi: 10.1128/mra.00252-22. Epub 2022 Aug 22. PMID: 35993705; PMCID: PMC9476979.\u003c/li\u003e\n\u003cli\u003eZurabov, F. and Zhilenkov, E. (2021) \u0026ldquo;Characterization of four virulent Klebsiella pneumoniae bacteriophages, and evaluation of their potential use in complex phage preparation,\u0026rdquo; \u003cem\u003eVirology journal\u003c/em\u003e, 18(1). doi: 10.1186/s12985-020-01485-w; PMID: 33407669; PMCID: PMC7789013.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Phage therapy, Klebsiella pneumoniae, phage stability, Food microbiology, water quality","lastPublishedDoi":"10.21203/rs.3.rs-7724689/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7724689/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKlebsiella pneumoniae is a Gram-negative bacterium associated with high infection rates worldwide and is currently listed by the World Health Organization (WHO) as a critical priority pathogen for the developme\u003csup\u003e1\u003c/sup\u003e nt of new antimicrobials. The indiscriminate use of antibiotics has accelerated the emergence of multidrug-resistant strains, underscoring the urgent need for alternative therapeutic strategies such as phage therapy. In this study, two bacteriophages (vB_MC_KP1 and vB_MC_KP2) were isolated from stream water in Minas Gerais, Brazil, \u003csup\u003ei\u003c/sup\u003ea site receiving domestic sewage and used for irrigation and other human activities. Both phages specifically infected Klebsiella spp. and were characterized by in vitro assays assessing host range and physicochemical stability. They demonstrated high specificity, lytic activity against multiple Klebsiella species, and remarkable stability under a wide range of temperatures and pH values. These findings indicate that the isolated phages are environmentally persistent and hold potential for both therapeutic applications against multidrug-resistant K. pneumoniae and future use in environmental or food safety interventions. Further molecular characterization and in vivo studies are warranted to validate their application.\u003c/p\u003e","manuscriptTitle":"Bacteriophages isolate of stream water in Brazil lyses the superbugs Klebsiella spp","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-14 10:18:18","doi":"10.21203/rs.3.rs-7724689/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b82795b1-8b10-4c0c-8375-c833eca1487c","owner":[],"postedDate":"October 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-14T10:18:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-14 10:18:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7724689","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7724689","identity":"rs-7724689","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}