Genomic insight on Klebsiella variicola isolated from wastewater treatment plant has uncovered a novel bacteriophage.

Klebsiella variicola is considered an emerging pathogen, which may colonize a variety of hosts, including environmental sources. Klebsiella variicola investigated in this study was obtained from an influent wastewater treatment plant in the North-West Province, South Africa. Whole genome sequencing was conducted to unravel the genetic diversity and antibiotic resistance patterns of K. variicola. Whole genome core SNP phylogeny was employed on publicly available 170 genomes. Furthermore, capsule types and antibiotic resistance genes, particularly beta-lactamase and carbapenems genes were investigated from the compared genomes. A 38 099 bp bacteriophage was uncovered alongside with K. variicola genome. Whole genome sequencing revealed that the extended beta-lactamase blaLEN (75.3%) of the beta-lactamase is dominant among compared K. variicola strains. The identified IncF plasmid AA035 confers resistance genes of metal and heat element subtypes, i.e., silver, copper, and tellurium. The capsule type KL107-D1 is a predominant capsule type present in 88.2% of the compared K. variicola genomes. The phage was determined to be integrase-deficient consisting of a fosB gene associated with fosfomycin resistance and clusters with the Wbeta genus Bacillus phage group. In silico analysis showed that the phage genome interacts with B. cereus as opposed to K. variicola strain T2. The phage has anti-repressor proteins involved in the lysis-lysogeny decision. This phage will enhance our understanding of its impact on bacterial dissemination and how it may affect disease development and antibiotic resistance mechanisms in wastewater treatment plants. This study highlights the need for ongoing genomic epidemiological surveillance of environmental K. variicola isolates.

Zap the clap with DNA: a novel microbicide for preventing Neisseria gonorrhoeae infection.

Each year, Neisseria gonorrhoeae (Ngo) causes over 1.5 million new infections in the United States, and >87 million worldwide. The absence of a vaccine for preventing gonorrhea, the rapid emergence of multidrug-resistant and extremely drug-resistant Ngo strains, and the limited number of antibiotics available for treating gonorrhea underscore the importance of developing new modalities for addressing Ngo infection. Here, we describe DNA-based microbicides that kill Ngo but not commensals. Previously, we showed that Ngo is killed when it takes up differentially methylated DNA with homology to its genome. We exploited this Achilles heel to develop a new class of microbicides for preventing Ngo infection. These microbicides consist of DNA molecules with specific sequences and a methylation pattern different from Ngo DNA. These DNAs kill low-passage and antibiotic-resistant clinical isolates with high efficiency but leave commensals unharmed. Equally important, the DNAs are equally effective against Ngo whether they are in buffered media or personal lubricants. These findings illustrate the potential of this new class of practical, low-cost, self-administered DNA-based microbicides for preventing Ngo transmission during sexual intercourse.

Culture-independent detection of low-abundant Clostridioides difficile in environmental DNA via PCR.

Clostridioides difficile represents a major burden to public health. As a well-known nosocomial pathogen whose occurrence is highly associated with antibiotic treatment, most examined C. difficile strains originated from clinical specimen and were isolated under selective conditions employing antibiotics. This suggests a significant bias among analyzed C. difficile strains, which impedes a holistic view on this pathogen. In order to support extensive isolation of C. difficile strains from environmental samples, we designed a detection PCR that targets the hpdBCA-operon and thereby identifies low abundances of C. difficile in environmental samples. This operon encodes the 4-hydroxyphenylacetate decarboxylase, which catalyzes the production of the antimicrobial compound para-cresol. Amplicon-based analyses of diverse environmental samples demonstrated that the designed PCR is highly specific for C. difficile and successfully detected C. difficile despite its absence in general 16S rRNA gene-based detection strategies. Further analyses revealed the potential of the hpdBCA detection PCR sequence for initial phylogenetic classification, which allows assessment of C. difficile diversity in environmental samples via amplicon sequencing. Our findings furthermore showed that C. difficile strains isolated under antibiotic treatment from environmental samples were originally dominated by other strains according to PCR amplicon results. This provided evidence for selective cultivation of under-represented but antibiotic-resistant isolates. Thereby, we revealed a substantial bias in C. difficile isolation and research.IMPORTANCEClostridioides difficile is a main cause of diarrheic infections after antibiotic treatment with serious morbidity and mortality worldwide. Research on this pathogen and its virulence has focused on bacterial isolation from clinical specimens under antibiotic treatment, which implies a substantial bias in isolated strains. Comprehensive studies, however, require an unbiased strain collection, which is accomplished by isolation of C. difficile from diverse environmental samples and avoidance of antibiotic-based enrichment strategies. Thus, isolation can significantly benefit from our C. difficile-specific detection PCR, which rapidly verifies C. difficile presence in environmental samples and further allows estimation of the C. difficile diversity by using next-generation sequencing.

Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal.

BACKGROUND: Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae. MATERIALS AND METHODS: A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 µg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. RESULT: Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. CONCLUSION: Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Gene expression profile of Campylobacter jejuni in response to macrolide antibiotics.

Campylobacter jejuni is a foodborne pathogen that causes gastroenteritis in humans and has developed resistance to various antibiotics. The primary objective of this research was to examine the network of antibiotic resistance in C. jejuni. The study involved the wild and antibiotic-resistant strains placed in the presence and absence of antibiotics to review their gene expression profiles in response to ciprofloxacin via microarray. Differentially expressed genes (DEGs) analysis and Protein-Protein Interaction (PPI) Network studies were performed for these genes. The results showed that the resistance network of C. jejuni is modular, with different genes involved in bacterial motility, capsule synthesis, efflux, and amino acid and sugar synthesis. Antibiotic treatment resulted in the down-regulation of cluster genes related to translation, flagellum formation, and chemotaxis. In contrast, cluster genes involved in homeostasis, capsule formation, and cation efflux were up-regulated. The study also found that macrolide antibiotics inhibit the progression of C. jejuni infection by inactivating topoisomerase enzymes and increasing the activity of epimerase enzymes, trying to compensate for the effect of DNA twisting. Then, the bacterium limits the movement to conserve energy. Identifying the antibiotic resistance network in C. jejuni can aid in developing drugs to combat these bacteria. Genes involved in cell division, capsule formation, and substance transport may be potential targets for inhibitory drugs. Future research must be directed toward comprehending the underlying mechanisms contributing to the modularity of antibiotic resistance and developing strategies to disrupt and mitigate the growing threat of antibiotic resistance effectively.

Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus.

Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60% of its maximum activity observed within the broad pH range of 6.0-10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba2+, Co2+, and Cu2+ showed inhibitory effects on the activity of LysVPB, while Ca2+ can boost it to 126.8%. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions.

Photocatalysis Inhibits the Emergence of Multidrug-Resistant Bacteria in an Antibiotic-Resistant Bacterial Community in Aquatic Environments.

Bacterial antibiotic resistance has recently attracted increasing amounts of attention. Here, an artificially antibiotic-resistant bacterial community (ARBC) combined with five different constructed antibiotic-resistant bacteria (ARB) with single antibiotic resistance, namely, kanamycin (KAN), tetracycline (TET), cefotaxime (CTX), polymyxin B (PB), or gentamicin (GEM), was studied for the stress response to photocatalysis. With photocatalytic inactivation, the transfer and diffusion of antibiotic resistance genes (ARGs) in the ARBC decreased, and fewer multidrug-resistant bacteria (MDRB) emerged in aquatic environments. After several days of photocatalytic inactivation or Luria broth cultivation, >90% ARB were transformed to antibiotic-susceptible bacteria by discarding ARGs. Bacteria with double antibiotic resistance were the dominant species (99%) of residual ARB. The changes in ARG abundance varied, decreasing for the GEM and TET resistance genes and increasing for the KAN resistance genes. The change in the antibiotic resistance level was consistent with the change in ARG abundance. Correspondingly, point mutations occurred for the KAN, CTX and PB resistance genes after photocatalytic inactivation, which might be the reason why these genes persisted longer in the studied ARBC. In summary, photocatalytic inactivation could reduce the abundance of some ARGs and inhibit the emergence of MDRB as well as block ARG transfer in the bacterial community in aquatic environments. This work highlights the advantages of long-term photocatalytic inactivation for controlling antibiotic resistance and facilitates a better understanding of bacterial communities in real aquatic environments.

Deciphering Multidrug-Resistant Plasmids in Disinfection Residual Bacteria from a Wastewater Treatment Plant.

Current disinfection processes pose an emerging environmental risk due to the ineffective removal of antibiotic-resistant bacteria, especially disinfection residual bacteria (DRB) carrying multidrug-resistant plasmids (MRPs). However, the characteristics of DRB-carried MRPs are poorly understood. In this study, qPCR analysis reveals that the total absolute abundance of four plasmids in postdisinfection effluent decreases by 1.15 log units, while their relative abundance increases by 0.11 copies/cell compared to investigated wastewater treatment plant (WWTP) influent. We obtain three distinctive DRB-carried MRPs (pWWTP-01-03) from postdisinfection effluent, each carrying 9-11 antibiotic-resistant genes (ARGs). pWWTP-01 contains all 11 ARGs within an ∼25 Kbp chimeric genomic island showing strong patterns of recombination with MRPs from foodborne outbreaks and hospitals. Antibiotic-, disinfectant-, and heavy-metal-resistant genes on the same plasmid underscore the potential roles of disinfectants and heavy metals in the coselection of ARGs. Additionally, pWWTP-02 harbors an adhesin-type virulence operon, implying risks of both antibiotic resistance and pathogenicity upon entering environments. Furthermore, some MRPs from DRB are capable of transferring and could confer selective advantages to recipients under environmentally relevant antibiotic pressure. Overall, this study advances our understanding of DRB-carried MRPs and highlights the imminent need to monitor and control wastewater MRPs for environmental security.

One Health Threat of Treated Wastewater Discharge in Urban Ohio Rivers: Implications for Surface Water and Fish Gut Microbiome and Resistome.

Wastewater treatment plants (WWTPs) are thought to be a major disseminating source of antibiotic resistance (AR) to the environment, establishing a crucial connection between human and environmental resistome. The objectives of this study were to determine how wastewater effluents impact microbiome and resistome of freshwater and fish, and identify potential AR-carrying clinically relevant pathogens in these matrices. We analyzed wastewater influent and effluent from four WWTPs in three metropolitan areas of Ohio, USA via shotgun metagenomic sequencing. We also sequenced river water and fish guts from three reaches (upstream, at the WWTP outfall, and downstream). Notably, we observed a decline in microbiome diversity and AR gene abundance from wastewater to the receiving river. We also found significant differences by reach and trophic level (diet) in beta-diversity of the fish gut microbiomes. SourceTracker revealed that 0.443 and 0.248 more of the of the fish gut microbiome was sourced from wastewater effluent in fish from the outfall and downstream locations, respectively, compared to upstream fish. Additionally, AR bacteria of public health concern were annotated in effluent and river water samples, indicating potential concern for human exposure. In summary, our findings show the continued role of wastewater as a significant AR reservoir and underscores the considerable impact of wastewater discharge on aquatic wildlife, which highlights the One Health nature of this issue.

Evaluating the Impact of Cl2•- Generation on Antibiotic-Resistance Contamination Removal via UV/Peroxydisulfate.

The removal of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) using sulfate anion radical (SO4•-)-based advanced oxidation processes has gained considerable attention recently. However, immense uncertainties persist in technology transfer. Particularly, the impact of dichlorine radical (Cl2•-) generation during SO4•--mediated disinfection on ARB/ARGs removal remains unclear, despite the Cl2•- concentration reaching levels notably higher than those of SO4•- in certain SO4•--based procedures applied to secondary effluents, hospital wastewaters, and marine waters. The experimental results of this study reveal a detrimental effect on the disinfection efficiency of tetracycline-resistant Escherichia coli (Tc-ARB) during SO4•--mediated treatment owing to Cl2•- generation. Through a comparative investigation of the distinct inactivation mechanisms of Tc-ARB in the Cl2•-- and SO4•--mediated disinfection processes, encompassing various perspectives, we confirm that Cl2•- is less effective in inducing cellular structural damage, perturbing cellular metabolic activity, disrupting antioxidant enzyme system, damaging genetic material, and inducing the viable but nonculturable state. Consequently, this diminishes the disinfection efficiency of SO4•--mediated treatment owing to Cl2•- generation. Importantly, the results indicate that Cl2•- generation increases the potential risk associated with the dark reactivation of Tc-ARB and the vertical gene transfer process of tetracycline-resistant genes following SO4•--mediated disinfection. This study underscores the undesired role of Cl2•- for ARB/ARGs removal during the SO4•--mediated disinfection process.

High-Throughput Single-Cell Metabolic Labeling, Sorting, and Sequencing of Active Antibiotic-Resistant Bacteria in the Environment.

Active antibiotic-resistant bacteria (ARB) play a major role in spreading antimicrobial resistance (AMR) in the environment; however, they have remained largely unexplored. Herein, we coupled bio-orthogonal noncanonical amino acid tagging with high-throughput fluorescence-activated single-cell sorting (FACS) and sequencing to characterize the phenome and genome of active ARB in complex environmental matrices. Active ARB, conferring resistance to six antibiotics throughout wastewater treatment, were distinguished and quantified. The percentage and concentration of active ARB ranged from 0.28% to 45.3% and from 1.1 × 104 to 2.09 × 107 cells/mL, respectively. Notably, the final effluents retained up to 4.79 × 104 cells/mL of active ARB. Targeted FACS and genomic sequencing revealed a distinct taxonomic composition of active ARB compared with that of the overall population. The coexistence of antibiotic resistome and mobilome in active ARB was also identified, including three high-quality metagenomic assembly genomes assigned to pathogenic bacteria, highlighting the substantial health risks due to their activity, phenotypic resistance, mobility, and pathogenicity. This study advances our understanding of previously overlooked active ARB in the environment by linking their resistance phenotype to their genotype. This high-throughput method will enable efficient quantitative surveillance of active AMR, providing valuable insights into risk control and management.

Antibiotics and antibiotic resistance change bacterial community compositions in marine sediments.

Emerging contaminants, including antibiotics, antibiotic-resistant bacteria (ARB), and extracellular antibiotic resistance genes (eARGs), have been detected in large numbers in the aquatic environment. The effects of emerging contaminants on bacterial communities in marine sediments are not well studied. In this study, the effects of emerging contaminants (antibiotics, ARB, and eARGs) on the variations of bacterial populations in marine sediments of the Bohai Sea, Yellow Sea, East China Sea, and South China Sea were investigated. The results showed that the abundance of the host bacterial phylum Probacteria in the marine sediments of the Bohai Sea was the lowest among the four seas after exposure to different antibiotics, ARB, and eARGs. The inputs of exogenous antibiotics and resistance genes significantly affected the community function, resulting in significant differences in community abundance at the genus level. The abundance of Halomonas, Sulfitobacter, and Alcanivorax in the four sea areas displayed noteworthy differences in response to the addition of exogenous antibiotics and eARGs. These findings contribute to a more comprehensive understanding of the intricate interplay between emerging contaminants and the dynamics of bacterial communities in natural ecosystems.

Correlation between the development of phage resistance and the original antibiotic resistance of host bacteria under the co-exposure of antibiotic and bacteriophage.

Bacteriophages (phages) are viruses capable of regulating the proliferation of antibiotic resistant bacteria (ARB). However, phages that directly cause host lethality may quickly select for phage resistant bacteria, and the co-evolutionary trade-offs under varying environmental conditions, including the presence of antibiotics, remains unclear as to their impact on phage and antibiotic resistance. Here, we report the emergence of phage resistance in three distinct E. coli strains with varying resistance to ß-lactam antibiotics, treated with different ampicillin (AMP) concentrations. Hosts exhibiting stronger antibiotic resistance demonstrated a higher propensity to develop and maintain stable phage resistance. When exposed to polyvalent phage KNT-1, the growth of AMP-sensitive E. coli K12 was nearly suppressed within 18 h, while the exponential growth of AMP-resistant E. coli TEM and super-resistant E. coli NDM-1 was delayed by 12 h and 8 h, respectively. The mutation frequency and mutated colony count of E. coli NDM-1 were almost unaffected by co-existing AMP, whereas for E. coli TEM and K12, these metrics significantly decreased with increasing AMP concentration from 8 to 50 µg/mL, becoming unquantifiable at 100 µg/mL. Furthermore, the fitness costs of phage resistance mutation and its impact on initial antibiotic resistance in bacteria were further examined, through analyzing AMP susceptibility, biofilm formation and EPS secretion of the isolated phage resistant mutants. The results indicated that acquiring phage resistance could decrease antibiotic resistance, particularly for hosts lacking strong antibiotic resistance. The ability of mutants to form biofilm contributes to antibiotic resistance, but the correlation is not entirely positive, while the secretion of extracellular polymeric substance (EPS), especially the protein content, plays a crucial role in protecting the bacteria from both antibiotic and phage exposure. This study explores phage resistance development in hosts with different antibiotic resistance and helps to understand the limitations and possible solutions of phage-based technologies.

Combined applications of UV and chlorine on antibiotic resistance control: A critical review.

Environmental health problems caused by antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) have become a global concern. ARB and ARGs have been continuously detected in various water environments, which pose a new challenge for water quality safety assurance. Disinfection is a key water treatment process to eliminate pathogenic microorganisms in water, and combined chlorine and UV processes (the UV/Cl2 process, the UV-Cl2 process, and the Cl2-UV process) are considered potential disinfection methods to control antibiotic resistance. This review documented the efficacy and mechanism of combined UV and chlorine processes for the control of antibiotic resistance, as well as the effects of chlorine dose, solution pH, UV wavelength, and water matrix on the effectiveness of the processes. There are knowledge gaps in research on the combined chlorine and UV processes for antibiotic resistance control, in particular the UV-Cl2 process and the Cl2-UV process. In addition, changes in the structure of microbial communities and the distribution of ARGs, which are closely related to the spread of antibiotic resistance in the water, induced by combined processes were also addressed. Whether these changes could lead to the re-transmission of antibiotic resistance and harm human health may need to be further evaluated.

A systematic scoping review of antibiotic-resistance in drinking tap water.

Environmental matrices have been considered of paramount importance in the spread of antibiotic-resistance; however, the role of drinking waters is still underexplored. Therefore, a scoping review was performed using a systematic approach based on PRISMA guidelines, with the aim of identifying and characterizing antibiotic-resistance in tap water, specifically, water treated at a potabilization plant and provided for drinking use through a water distribution system. The review included 45 studies, the majority of which were conducted in upper-middle-income economies (42.2%), mainly from the Western Pacific region (26.7%), followed by Europe (24.4%). Most of the papers focused on detecting antibiotic-resistant bacteria (ARB), either alone (37.8%) or in combination with antibiotic-resistant genes (ARGs) (26.7%). Multidrug-resistance profile was often identified in heterotrophic bacteria, including various species of nontuberculous mycobacteria, Pseudomonas spp., and Aeromonas spp., which were especially resistant to penicillins, cephalosporins (including 3rd-generation), and also to macrolides (erythromycin) and tetracyclines. Resistance to a wide range of antibiotics was also prevalent in fecal bacteria, e.g., the Enterobacteriaceae family, with common resistance to (fluoro)quinolones and sulfonamide groups. ARGs were investigated either in bacterial strains isolated from tap waters or directly in water samples, and the most frequently detected ARGs belonged to ß-lactam, sulfonamide, and tetracycline types. Additionally, mobile genetic elements were found (i.e., int1 and tnpA). Sulfonamides and macrolides were the most frequently detected antibiotics across countries, although their concentrations were generally low (<10 ng/L) in Europe and the United States. From a health perspective, tap water hosted ARB of health concern based on the 2024 WHO bacterial priority pathogens list, mainly Enterobacteriaceae resistant to 3rd-generation cephalosporin and/or carbapenem. Despite the fact that tap water is treated to meet chemical and microbiological quality standards, current evidence suggests that it can harbor antibiotic-resistance determinants, thus supporting its potential role in environmental pathways contributing to antibiotic resistance.

Antibiotic residues in the cirata reservoir, Indonesia and their effect on ecology and the selection for antibiotic-resistant bacteria.

Antibiotic residues, their mixture toxicity, and the potential selection for antibiotic-resistant bacteria could pose a problem for water use and the ecosystem of reservoirs. This study aims to provide a comprehensive understanding of the occurrence, concentration, distribution, and ecological risks associated with various antibiotics in the Cirata reservoir, Indonesia. In our water and sediment samples, we detected 24 out of the 65 antibiotic residues analyzed, revealing a diverse range of antibiotic classes present. Notably, sulphonamides, diaminopyrimidine, and lincosamides were frequently found in the water, while the sediment predominantly contained tetracyclines and fluoroquinolones. Most antibiotic classes reached their highest concentrations in the water during the dry season. However, fluoroquinolones and tetracyclines showed their highest concentrations in the water during the wet season. Ecotoxicological risk assessments indicated that the impact of most antibiotic residues on aquatic organisms was negligible, except for fluoroquinolones. Looking at the impact on cyanobacteria, however, varying risks were indicated, ranging from medium to critical, with antibiotics like sulfamethoxazole, ciprofloxacin, norfloxacin, and lincomycin posing substantial threats. Among these, ciprofloxacin emerged as the antibiotic with the strongest risk. Furthermore, fluoroquinolones may have the potential to contribute to the selection of antibiotic-resistant bacteria. The presence of mixtures of antibiotic residues during the wet season significantly impacted species loss, with Potentially Affected Fraction of Species (msPAF) values exceeding 0.75 in almost 90% of locations. However, the impact of mixtures of antibiotic residues in sediment remained consistently low across all locations and seasons. Based on their occurrences and associated risks, 12 priority antibiotic residues were identified for monitoring in the reservoir and its tributaries. Moreover, the study suggests that river inflow serves as the most significant source of antibiotic residues in the reservoir. Further investigations into the relative share attribution of antibiotic sources in the reservoir is recommended to help identify effective interventions.

Pre-exposure to peracetic acid followed by UV treatment for deactivating vancomycin-resistant Enterococcus faecalis through intracellular attack.

Antimicrobial resistance (AMR) poses a global health threat to aquatic environments and its propagation is a hot topic. Therefore, deactivating antibiotic-resistant bacteria (ARB) and removing antibiotic resistance genes (ARGs) from water is crucial for controlling AMR transmission. Peracetic acid (PAA), which is known for its potent oxidizing properties and limited by-product formation, is emerging as a favorable disinfectant for water treatment. In this study, we aimed to assess the efficacy of pre-exposure to PAA followed by UV treatment (PAA-UV/PAA) compared with the simultaneous application of UV and PAA (UV/PAA). The focus was on deactivating vancomycin-resistant Enterococcus faecalis (VREfs), a typical ARB in water. Pre-exposure to PAA significantly enhanced the efficacy of subsequent UV/PAA treatment. At a UV fluence of 7.2 mJ cm-2, the PAA-UV/PAA method achieved a 6.21 log reduction in VREfs, surpassing the 1.29 log reduction observed with UV/PAA. Moreover, compared to UV/PAA, PAA-UV/PAA showed increased efficacy with longer pre-exposure times and higher PAA concentrations, maintaining superior performance across a broad pH range and in the presence of humic acid. Flow cytometry analysis indicated minimal cellular membrane damage using both methods. However, the assessments of superoxide dismutase (SOD) activity and adenosine triphosphate content revealed that PAA-UV/PAA induced greater oxidative stress under similar UV irradiation conditions, leading to slower bacterial regrowth. Specifically, SOD activity in PAA-UV/PAA surged to 3.06 times its baseline, exceeding the 1.73-fold increase under UV/PAA conditions. Additionally, pre-exposure to PAA amplified ARGs degradation and reduced resistance gene leakage, effectively mitigating the spread of AMR. Pre-exposure to 200 µM PAA for 10 and 20 min enhanced vanB gene removal efficiency by 0.14 log and 1.29 log, respectively. Our study provides a feasible approach for optimizing UV/PAA disinfection for efficient removal of ARB and ARGs.

Bacteria populating freshly appeared supraglacial lake possess metals and antibiotic-resistant genes.

Antibiotic resistance (AR) has been extensively studied in natural habitats and clinical applications. AR is mainly reported with the use and misuse of antibiotics; however, little is known about its presence in antibiotic-free remote supraglacial lake environments. This study evaluated bacterial strains isolated from supraglacial lake debris and meltwater in Dook Pal Glacier, northern Pakistan, for antibiotic-resistant genes (ARGs) and metal-tolerant genes (MTGs) using conventional PCR. Several distinct ARGs were reported in the bacterial strains isolated from lake debris (92.5%) and meltwater (100%). In lake debris, 57.5% of isolates harbored the blaTEM gene, whereas 58.3% of isolates in meltwater possessed blaTEM and qnrA each. Among the ARGs, qnrA was dominant in debris isolates (19%), whereas in meltwater isolates, qnrA (15.2%) and blaTEM (15.2%) were dominant. ARGs were widely distributed among the bacterial isolates and different bacteria shared similar types of ARGs. Relatively greater number of ARGs were reported in Gram-negative bacterial strains. In addition, 92.5% of bacterial isolates from lake debris and 83.3% of isolates from meltwater harbored MTGs. Gene copA was dominant in meltwater isolates (50%), whereas czcA was greater in debris bacterial isolates (45%). Among the MTGs, czcA (18.75%) was dominant in debris strains, whereas copA (26.0%) was greater in meltwater isolates. This presents the co-occurrence and co-selection of MTGs and ARGs in a freshly appeared supraglacial lake. The same ARGs and MTGs were present in different bacteria, exhibiting horizontal gene transfer (HGT). Both positive and negative correlations were determined between ARGs and MTGs. The research provides insights into the existence of MTGs and ARGs in bacterial strains isolated from remote supraglacial lake environments, signifying the need for a more detailed study of bacteria harboring ARGs and MTGs in supraglacial lakes.

Metagenomic analysis of hot spring soil for mining a novel thermostable enzybiotic.

The misuse and overuse of antibiotics have contributed to a rapid emergence of antibiotic-resistant bacterial pathogens. This global health threat underlines the urgent need for innovative and novel antimicrobials. Endolysins derived from bacteriophages or prophages constitute promising new antimicrobials (so-called enzybiotics), exhibiting the ability to break down bacterial peptidoglycan (PG). In the present work, metagenomic analysis of soil samples, collected from thermal springs, allowed the identification of a prophage-derived endolysin that belongs to the N-acetylmuramoyl-L-alanine amidase type 2 (NALAA-2) family and possesses a LysM (lysin motif) region as a cell wall binding domain (CWBD). The enzyme (Ami1) was cloned and expressed in Escherichia coli, and its bactericidal and lytic activity was characterized. The results indicate that Ami1 exhibits strong bactericidal and antimicrobial activity against a broad range of bacterial pathogens, as well as against isolated peptidoglycan (PG). Among the examined bacterial pathogens, Ami1 showed highest bactericidal activity against Staphylococcus aureus sand Staphylococcus epidermidis cells. Thermostability analysis revealed a melting temperature of 64.2 ± 0.6 °C. Overall, these findings support the potential that Ami1, as a broad spectrum antimicrobial agent, could be further assessed as enzybiotic for the effective treatment of bacterial infections. KEY POINTS: • Metagenomic analysis allowed the identification of a novel prophage endolysin • The endolysin belongs to type 2 amidase family with lysin motif region • The endolysin displays high thermostability and broad bactericidal spectrum.

Conformal immunomodulatory hydrogels for the treatment of otitis media.

Otitis media (OM), a condition stemming from the proliferation of various bacteria within the tympanic cavity (TC), is commonly addressed through the administration of ofloxacin (OFL), a fluoroquinolone antibiotic. Nevertheless, the escalating issue of antibiotic resistance and the challenge of drug leakage underscore the exploration of an alternative, more effective treatment modality in clinical practice. Here, we introduce a simple and easily implementable fluid-regulated strategy aimed at delivering immunomodulatory hydrogels into the TC, ensuring conformal contact with the irregular anatomical surfaces of the middle ear cavity to more effectively eliminate bacteria and treat OM. This innovative strategy exhibits expedited therapeutic process of antibiotic-resistant, acute and chronic OM rats, and significant reductions in the severity of tympanic membrane (TM) inflammation, residual bacteria within the TC (0.12 *105 CFU), and the thickness of TM/TC mucosa (17.63/32.43 µm), as compared to conventional OFL treatment (3.6, 0.76 *105 CFU, 48.70/151.26 µm). The broad-spectrum antibacterial and antibiofilm properties of this strategy against a spectrum of OM pathogens are demonstrated. The strategy is validated to bolster the host's innate immune response through the stimulation of antibacterial protein synthesis, macrophage proliferation and activation, thereby accelerating bacterial eradication and inflammation resolution within the TC. This facile, cost-effective and in vivo degradable technology exhibits promising prospects for future clinical implementation.