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.

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.

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.

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.

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.

Wild fish from a highly urbanized river (Orge, France) as vectors of culturable Enterobacterales resistant to antibiotics.

This study shows how wild fishes from urbanized rivers could be involved in the spread of antibiotic-resistant Enterobacterales. Antibiotic resistance profiles and molecular detection of clinical integron (IntI1) were carried out on 105 Enterobacterales isolated from 89 wildfish (skin or gut) belonging to 8 species. The proportion of isolates resistant to at least one antibiotic was independent of fish species and reached 28.3% within the Escherichia coli (E. coli) population and 84.7% in the non-E.coli Enterobacterales. Bacteria involved in nosocomial infections were isolated, such as E. coli, Klebsiella, and Enterobacter, as well as the environmental bacteria (Lelliottia, Butiauxella, and Kluyvera).

Cymbopogon citratus L. essential oil as a potential anti-biofilm agent active against antibiotic-resistant bacteria isolated from chronic rhinosinusitis patients.

Chronic rhinosinusitis (CRS) is long-term inflammation of the sinuses that can be caused by infection due to antibiotic-resistant bacteria. Biofilm developed by microbes is postulated to cause antibiotic treatment failure. Thus, the anti-biofilm activities of seven Thai herbal essential oils (EOs) against antibiotic-resistant bacteria isolated from CRS patients was investigated. Lemongrass (Cymbopogon citratus L.) EO showed the most effective antibiofilm activity against Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus epidermidis grown as biofilm. GC-MS analysis found that myrcene was the major bioactive compound. Pretreatment with lemongrass EO significantly inhibited biofilm formation of all bacterial strains in more than 50% of cases. Furthermore, confocal microscopy analysis revealed the biofilm-disrupting activity of lemongrass EO against the biofilm matrix of all these bacterial species and also increased P. aeruginosa swarming motility with no toxicity to human cells. These results suggest that lemongrass EO has promising clinical applications as an anti-biofilm agent for CRS patients.

Simultaneous effect of medicinal plants as natural photosensitizers and low-level laser on photodynamic inactivation.

Photodynamic inactivation (PDI) technology is a promising alternative to antibiotics. This technology is defined as the inhibition of bacterial growth with photosensitizers while irradiated with low-level laser light in the wavelength of 532 ± 2.08 nm. A challenging area in this field is selecting photosensitizers with antibacterial potential. In this paper, to enhance the antibacterial efficiency, the photosensitizers (the selected plant extracts) with a high absorption peak at the selected laser frequency, 532 nm, were prepared. Low-concentration ethanolic plant extracts of Hibiscus sabdariffa and Opuntia ficus-indica were found to exhibit significant antibacterial activity against, Acinetobacter baumannii ATCC 19606 and, Staphylococcus aureus ATCC 33591 as two important human pathogenic bacteria. The effectiveness of these natural photosensitizers was measured by determining their Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values and by performing a time-killing assay in the absence and the presence of laser irradiation. Our results showed that the combination of low-level laser irradiation and the selected photosensitizers had excellent potential for treating in vitro bacterial infections. Therefore, PDI technology has great potential as a viable alternative to traditional antibiotics for combating bacterial infections. This study presents a promising avenue for further exploration of PDI and the use of laser technology in medical science.

Risk of Illness with Salmonella Spp. and Antibiotic-Resistant Salmonella sp. due to Consumption of Lettuce Irrigated with Water from La Ramada Irrigation District.

Despite heavy contamination of the Bogotá River with domestic and industrial waste, it remains vital for various purposes, including agricultural use at La Ramada Irrigation District. There are important concerns regarding pathogen concentrations in irrigation water at La Ramada, including the presence of antibiotic-resistant Salmonella spp. This study aimed to estimate the risk of Salmonella-related illness from consuming lettuce irrigated with Bogotá River water at La Ramada. We collected lettuce samples from 4 different sites, all irrigated with water from La Ramada. The methodology involved a process to detach Salmonella spp. from lettuce leaves, quantification through plate counts on SS agar, and establishment of antibiotic-resistant bacteria concentrations through growth on media supplemented with ampicillin or ciprofloxacin. The results showed concentrations of Salmonella spp. of 103.59,102.66, and 104.56 CFU/g lettuce at sites 1, 2, and 3, respectively, and ampicillin-resistant Salmonella spp. of 101.93, 101.31, and 102.07 CFU/g lettuce at sites 1, 2, and 3, respectively. No colonies were obtained from lettuce samples collected from site 4. Notably, we detected no isolates resistant to ciprofloxacin at any of the sites. Salmonella spp. concentrations varied greatly among sampling sites. Salmonella spp. concentrations were used to predict the daily probability of illness, with a probability of 0.59 (0.33 to 0.78, CI 95%) for Salmonella spp. and 0.3 (0.03 to 0.53, CI 95%) for ampicillin-resistant Salmonella spp.

Wounds of Companion Animals as a Habitat of Antibiotic-Resistant Bacteria That Are Potentially Harmful to Humans-Phenotypic, Proteomic and Molecular Detection.

Skin wounds and their infections by antibiotic-resistant bacteria (ARB) are very common in small animals, posing the risk of acquiring ARB by pet owners or antibiotic resistance gene (ARG) transfer to the owners' microbiota. The aim of this study was to identify the most common pathogens infecting wounds of companion animals, assess their antibiotic resistance, and determine the ARGs using culture-based, molecular, and proteomic methods. A total of 136 bacterial strains were isolated from wound swabs. Their species was identified using chromogenic media, followed by MALDI-TOF spectrometry. Antibiotic resistance was tested using disc diffusion, and twelve ARGs were detected using PCRs. The dominant species included Staphylococcus pseudintermedius (9.56%), E. coli, and E. faecalis (both n = 11, 8.09%). Enterobacterales were mostly resistant to amoxicillin/clavulanic acid (68.3% strains), all Pseudomonas were resistant to ceftazidime, piperacillin/tazobactam, imipenem, and tylosin, Acinetobacter were mostly resistant to tylosin (55.5%), all Enterococcus were resistant to imipenem, and 39.2% of Staphylococci were resistant to clindamycin. Among ARGs, strA (streptomycin resistance), sul3 (sulfonamide resistance), and blaTEM, an extended-spectrum beta-lactamase determinant, were the most frequent. The risk of ARB and ARG transfer between animals and humans causes the need to search for new antimicrobial therapies in future veterinary medicine.

The stress response of tetracycline resistance genes and bacterial communities under the existence of microplastics in typical leachate biological treatment system.

Landfill leachate is an important source of microplastics (MPs) and antibiotic-resistance genes (ARGs). Here, in the presence of polystyrene MPs (PS-MPs) and polyethylene MPs (PE-MPs), the nitrogen and phosphorus removal effect and sludge structure performance were affected in an anaerobic-anoxic-aerobic system, a typical biological leachate treatment process. The abundance of tetracycline-resistance genes (tet genes) in biofilms on the two types of MP was significantly higher than that in the leachate and sludge, and the load on PE-MPs was higher than that on PS-MPs because of the porous structure of PE-MPs. Aging of the MPs increased their surface roughness and abundance of oxygen-containing functional groups and shaped the profile of ARGs in the MP biofilms. The biofilm biomass and growth rate on the two types of MP increased with the incubation time in the first 30 days, and was affected by environmental factors. Structural equation models and co-occurrence network analysis demonstrated that the MPs indirectly affected the spectrum of ARGs by affecting biofilm formation, and, to a lesser extent, had a direct impact on the selective enrichment of ARGs. We discuss the mechanisms of the relationships between MPs and ARGs in the leachate treatment system, which will have guiding significance for future research. Our data on the colonization of microorganisms and tet genes in MPs biofilms provide new evidence concerning the accumulation and transmission of these ARGs, and are important for understanding the mechanisms of MPs in spreading pollution.

Municipal wastewater treatment plant showing a potential reservoir for clinically relevant MDR bacterial strains co-occurrence of ESBL genes and integron-integrase genes.

Municipal wastewater treatment plants (MWWTPs) are a milieu for co-occurrence of multiple antibiotic resistance genes (ARGs). This facilitates mixing and genetic exchange; and promotes dissemination of multidrug resistance (MDR) to wastewater bacterial communities which is hazardous for the effluent receiving environment. This study investigated the co-occurrence of extended-spectrum beta-lactamase (ESBL) genes (blaTEM, blaCTX-M, blaSHV, blaOXA), and integron-integrase genes (intI1, intI2, intI3) in MDR bacteria isolated from the Bharwara MWWTP in Lucknow, India. Thirty-one MDR bacterial colonies resistant to three or more antibiotics were isolated from three treatment stages of this MWWTP. Six of these: Staphylococcus aureus, Serratia marcescens, Salmonella enterica, Shigella sonnei, Escherichia coli, and Bacillus sp. Had co-occurrence of ESBL and integron-integrase genes. These six isolates were examined for the occurrence of MDR efflux genes (qacA, acrB) and ARGs (aac(3)-1, qnrA1, tetA, vanA) and tested for resistance against 12 different antibiotics. The highest resistance was against penicillin-G (100%) and lowest for chloramphenicol (16.66%). Bacillus sp. Isolate BWKRC6 had the highest co-occurrence of antibiotic resistance-determining genes and was resistant to all the 12 antibiotics tested. The co-occurrence of ESBL, integron-integrase, antibiotic resistance-determining and MDR efflux genes in bacteria isolated from the Bharwara MWWTP indicates that the wastewaters of this treatment plant may have become a hotspot for MDR bacteria and may present human and environmental health hazards. Therefore, there is need for a rapid action to limit the spread of this threat. Public regulatory authorities must urgently implement measures to prevent MWWTPs becoming reservoirs for evolution of antibiotic resistance genes and development of antibiotic resistance.

Impact of PVC microplastics in photodynamic inactivation of Staphylococcus aureus and MRSA.

Photodynamic processes have found widespread application in therapies. These processes involve photosensitizers (PSs) that, when excited by specific light wavelengths and in the presence of molecular oxygen, generate reactive oxygen species (ROS), that target cells leading to inactivation. Photodynamic action has gained notable attention in environmental applications, particularly against pathogens and antibiotic-resistant bacteria (ARB) that pose a significant challenge to public health. However, environmental matrices frequently encompass additional contaminants and interferents, including microplastics (MPs), which are pollutants of current concern. Their presence in water and effluents has been extensively documented, highlighting their impact on conventional treatment methods, but this information remains scarce in the context of photodynamic inactivation (PDI) setups. Here, we described the effects of polyvinyl chloride (PVC) microparticles in PDI targeting Staphylococcus aureus and its methicillin-resistant strain (MRSA), using curcumin as a PS under blue light. The presence of PVC microparticles does not hinder ROS formation; however, depending on its concentration, it can impact bacterial inactivation. Our results underscore that PDI remains a potent method for reducing bacterial concentrations in water and wastewater containing ARB, even in highly contaminated scenarios with MPs.

Dynamics of antimicrobial resistance and susceptibility profile in full-scale hospital wastewater treatment plants.

Drug resistance has become a matter of great concern, with many bacteria now resist multiple antibiotics. This study depicts the occurrence of antibiotic-resistant bacteria (ARB) and resistance patterns in five full-scale hospital wastewater treatment plants (WWTPs). Samples of raw influent wastewater, as well as pre- and post-disinfected effluents, were monitored for targeted ARB and resistance genes in September 2022 and February 2023. Shifts in resistance profiles of Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii antimicrobial-resistant indicators in the treated effluent compared to that in the raw wastewater were also worked out. Ceftazidime (6.78 × 105 CFU/mL) and cefotaxime (6.14 × 105 CFU/mL) resistant species showed the highest concentrations followed by ciprofloxacin (6.29 × 104 CFU/mL), and gentamicin (4.88 × 104 CFU/mL), in raw influent respectively. WWTP-D employing a combination of biological treatment and coagulation/clarification for wastewater decontamination showed promising results for reducing ARB emissions from wastewater. Relationships between treated effluent quality parameters and ARB loadings showed that high BOD5 and nitrate levels were possibly contributing to the persistence and/or selection of ARBs in WWTPs. Furthermore, antimicrobial susceptibility tests of targeted species revealed dynamic shifts in resistance profiles through treatment processes, highlighting the potential for ARB and ARGs in hospital wastewater to persist or amplify during treatment.

Mechanism-guided strategies for combating antibiotic resistance.

Bacterial antibiotic resistance has been recognized as a global threat to public health. It challenges the antibiotics currently used in clinical practice and causes severe and often fatal infectious diseases. Fighting against antibiotic-resistant bacteria (ARB) is growing more urgent. While understanding the molecular mechanisms that underlie resistance is a prerequisite, several major mechanisms have been previously proposed including bacterial efflux systems, reduced cell membrane permeability, antibiotic inactivation by enzymes, target modification, and target protection. In this context, this review presents a panel of promising and potential strategies to combat antibiotic resistance/resistant bacteria. Different types of direct-acting and indirect resistance breakers, such as efflux pump inhibitors, antibiotic adjuvants, and oxidative treatments are discussed. In addition, the emerging multi-omics approaches for rapid resistance identification and promising alternatives to existing antibiotics are highlighted. Overall, this review suggests that continued effort and investment in research are required to develop new antibiotics and alternatives to existing antibiotics and translate them into environmental and clinical applications.

Distribution of bacterial community structures and spread of antibiotic resistome at industrially polluted sites of Mini River, Vadodara, Gujarat, India.

The influence of anthropogenic pollution on the distribution of bacterial diversity, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) was mapped at various geo-tagged sites of Mini River, Vadodara, Gujarat, India. The high-throughput 16S rRNA gene amplicon sequencing analysis revealed a higher relative abundance of Planctomycetota at the polluted sites, compared to the pristine site. Moreover, the relative abundance of Actinobacteriota increased, whereas Chloroflexi decreased in the water samples of polluted sites than the pristine site. The annotation of functional genes in the metagenome samples of Mini River sites indicated the presence of genes involved in the defence mechanisms against bacitracin, aminoglycosides, cephalosporins, chloramphenicol, streptogramin, streptomycin, methicillin, and colicin. The analysis of antibiotic resistome at the polluted sites of Mini River revealed the abundance of sulfonamide, beta-lactam, and aminoglycoside resistance. The presence of pathogens and ARB was significantly higher in water and sediment samples of polluted sites compared to the pristine site. The highest resistance of bacterial populations in the Mini River was recorded against sulfonamide (≥ 7.943 × 103 CFU/mL) and ampicillin (≥ 8.128 × 103 CFU/mL). The real-time PCR-based quantification of ARGs revealed the highest abundance of sulfonamide resistance genes sul1 and sul2 at the polluted sites of the Mini River. Additionally, the antimicrobial resistance genes aac(6')-Ib-Cr and blaTEM were also found abundantly at polluted sites of the Mini River. The findings provide insights into how anthropogenic pollution drives the ARG and ARB distribution in the riverine ecosystem, which may help with the development of antimicrobial resistance mitigation strategies.

Changes of composition and antibiotic resistance of fecal coliform bacteria in municipal wastewater treatment plant.

The dynamics of the composition and antibiotic resistance of the fecal coliform bacteria (FCB) in a typical wastewater treatment plant (WWTP) were investigated concerning the seasonal changes. Results showed that WWTP could remove the FCB concentration by 3∼5 logs within the effluent of 104∼105 CFU/L, but the antibiotic resistant rate of FCB species increased significantly after WWTP. The dominant FCB changed from Escherichia coli in the influent (∼73.0%) to Klebsiella pneumoniae in the effluent (∼53.3%) after WWTP, where the Escherichia coli was removed the most, while Klebsiella pneumoniae was the most persistent. The secondary tank removed the most of FCB (by 3∼4 logs) compared to other processes, but increased all the concerned antibiotic resistant rate. The potential super bugs of FCB community showing resistance to all the target antibiotics were selected in the biological treatment unit of WWTP. The FCB showed the highest multiple antibiotic resistance (92.9%) in total which even increased to 100% in the effluent. Klebsiella has the highest antibiotic resistant rate in FCB, with a multiple antibiotic resistance rate of 98.4%. These indicated that the Klebsiella pneumoniae not just Escherichia coli should be specially emphasized after WWTP concerning the health risk associated with FCB community.

Phenotypic Characterization and Comparative Genomic Analyses of Mycobacteriophage WIVsmall as A New Member Assigned to F1 Subcluster.

In this study, we conducted the morphological observation, biological and genomic characterization, evolutionary analysis, comparative genomics description, and proteome identification of a recently isolated mycobacteriophage, WIVsmall. Morphologically, WIVsmall is classified as a member of the Siphoviridae family, characterized by a flexible tail, measuring approximately 212 nm in length. The double-stranded phage genome DNA of WIVsmall spans 53,359 base pairs, and exhibits a G + C content of 61.01%. The genome of WIVsmall comprises 103 protein-coding genes, while no tRNA genes were detected. The genome annotation unveiled the presence of functional gene clusters responsible for mycobacteriophage assembly and maturation, replication, cell lysis, and functional protein synthesis. Based on the analysis of the phylogenetic tree, the genome of WIVsmall was classified as belonging to subgroup F1. A comparative genomics analysis indicated that the WIVsmall genome exhibited the highest similarity to the phage SG4, with a percentage of 64%. The single-step growth curve analysis of WIVsmall revealed a latent period of 120 min, and an outbreak period of 200 min.

Multiplex Microarrays in 96-Well Plates Photoactivated with 4-Azidotetrafluorobenzaldehyde for the Identification and Quantification of ß-Lactamase Genes and Their RNA Transcripts.

Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin-peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to ß-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.