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Multidrug-resistant P. aeruginosa infections pose a serious public health threat due to the rise in antimicrobial resistance. Phage therapy has emerged as a promising alternative. However, P. aeruginosa has evolved various mechanisms to thwart phage attacks, making it crucial to decipher these resistance mechanisms to develop effective therapeutic strategies. In this study, we conducted a forward-genetic screen of the P. aeruginosa PA14 non-redundant transposon library (PA14NR) to identify dominant-negative mutants displaying phage-resistant phenotypes. Our screening process revealed 78 mutants capable of thriving in the presence of phages, with 23 of them carrying insertions in genes associated with membrane composition. Six mutants exhibited total resistance to phage infection. Transposon insertions were found in genes known to be linked to phage-resistance such as galU and a glycosyl transferase gene, as well as novel genes such as mexB, lasB, and two hypothetical proteins. Functional experiments demonstrated that these genes played pivotal roles in phage adsorption and biofilm formation, indicating that altering the bacterial membrane composition commonly leads to phage resistance in P. aeruginosa. Importantly, these mutants displayed phenotypic trade-offs, as their resistance to phages inversely affected antibiotic resistance and hindered biofilm formation, shedding light on the complex interplay between phage susceptibility and bacterial fitness. This study highlights the potential of transposon mutant libraries and forward-genetic screens in identifying key genes involved in phage-host interactions and resistance mechanisms. These findings support the development of innovative strategies for combating antibiotic-resistant pathogens.

Sewage-based surveillance is widely employed to understand the occurrence and distribution of antimicrobial resistance (AMR) in urban community. However, there are limited studies which investigated the sewage of different sources within community. The present study used metagenomics to decipher the AMR profiles in five sources: local residence’s source, animal source, migrant workers’ source, clinical source , and urban wastewater treatment plant influent. A core resistome of ARGs was found across all samples, accounting for 81.4%-93.3% of the abundance of total resistome with only 17.3% diversity, irrespective of the sewage sources. Clinically relevant ARGs were identified in the core resistome across all wastewater sources. This included genes conferring resistance to beta-lactams as biomarkers of hospital sewage. The pet center wastewater showed a high abundance of genes encoding resistance to tetracycline, which is a commonly used veterinary antibiotic. The resistome profile of sewage from the migrant workers’ dormitories showed a slight variation to that of the local residential population, suggesting possible differences in the human gut resistome of the foreign/migrant population, with biomarkers of genes encoding resistance to fosfomycin, fosmidomycin, kasugamycin, MLS, and polymyxin. The co-localization of ARGs and plasmid, MGEs and integrative and conjugative elements (ICEs) could explain variations in the core resistome, presumably a result of high antibiotic selection pressure. Further analysis showed a specific host-associated resistance pattern, in which core hosts mediated the core resistome profile. The core BMRGs were also co-localized with MGEs/ICEs and carried by core potential bacterial hosts. Local healthy population carried the lowest ARG load (copy number discharged by each person per day) but contributed the highest ARG burden (copy number discharged by the population). This study elucidates population-based variations of a core resistome, and further provides important insights into source tracking and management of AMR in urban environments.

[This corrects the article DOI: 10.1371/journal.pone.0143123.].

Indicator organisms and antibiotic resistance were used as a proxy to measure microbial water quality of ballast tanks of ships, and surface waters in a tropical harbor. The survival of marine bacteria in ballast tanks appeared to diminish over longer water retention time, with a reduction of cell viability observed after a week based on heterotrophic plate counts. Pyrosequencing of 16S rRNA genes showed distinct differences in microbial composition of ballast and harbor waters. The harbor waters had a higher abundance of operational taxonomic units (OTUs) assigned to Cyanobacteria (Synechococcus spp.) and α-proteobacteria (SAR11 members), while marine hydrocarbon degraders such as γ-proteobacteria (Ocenspirillaes spp., Thiotrchales spp.) and Bacteroidetes (Flavobacteriales spp.) dominated the ballast water samples. Screening of indicator organisms found Escherichia coli (E. coli), Enterococcus and Pseudomonas aeruginosa (P. aeruginosa) in two or more of the ballast and harbor water samples tested. Vibrio spp. and Salmonella spp. were detected exclusively in harbor water samples. Using quantitative PCR (qPCR), we screened for 13 antibiotic resistant gene (ARG) targets and found higher abundances of sul1 (4.13-3.44 x 102 copies/mL), dfrA (0.77-1.80 x10 copies/mL) and cfr (2.00-5.21 copies/mL) genes compared to the other ARG targets selected for this survey. These genes encode for resistance to sulfonamides, trimethoprim and chloramphenicol-florfenicol antibiotics, which are also known to persist in sediments of aquaculture farms and coastal environments. Among the ARGs screened, we found significant correlations (P

Wet weather conditions have been associated with increased bacterial and viral counts in surface waters. Moreover, heavy rainfall and flooding were found to be the most common events preceding waterborne disease outbreaks associated with extreme weather conditions. This study aimed to examine the effect of rainfall on the quality of surface waters and to determine its suitability for primary contact recreation during wet weather conditions. A total of 228 catchment water samples were collected during wet and dry periods. Parameters that were found to increase with increasing rainfall were Escherichia coli, enterococci, somatic coliphages, and turbidity, whereas total dissolved solids were found to decrease. Positive correlations (p < 0.05) were observed between cumulative rainfall and geometric mean concentrations of E. coli, enterococci, somatic coliphages, and turbidity (r = 0.69-0.95), whereas a negative correlation was observed between cumulative rainfall and total dissolved solids (r = -0.58). In addition, a rapid decline in water quality was observed during heavy rainfall that resulted in failure to meet recreational water quality guidelines. In view of public health and safety, primary recreational activities in the water catchment may not be advisable during or immediately after a rainfall event due to poor water quality. [ABSTRACT FROM AUTHOR]

This study evaluated the geospatial distribution of fecal indicator bacteria (FIB) (i.e., Escherichia coli, Enterococcus spp.) and the alternative fecal indicator pepper mild mottle virus (PMMoV) in tropical freshwater environments under different land use patterns. Results show that the occurrence and concentration of microbial fecal indicators were higher for urban than for parkland-dominated areas, consistent with land use weightage. Significant positive correlations with traditional FIB indicate that PMMoV is a suitable indicator of fecal contamination in tropical catchments waters (0.549 ≤ rho ≤ 0.612; P < 0.01). PMMoV exhibited a strong significant correlation with land use weightage (rho = 0.728; P < 0.01) compared to traditional FIB (rho = 0.583; P < 0.01). In addition, chemical tracers were also added to evaluate the potential relationships with microbial fecal indicators. The relationships between diverse variables (e.g., environmental parameters, land use coverage, and chemical tracers) and the occurrence of FIB and PMMoV were evaluated. By using stepwise multiple linear regression (MLR), the empirical experimental models substantiate the impact of land use patterns and anthropogenic activities on microbial water quality, and the output results of the empirical models may be able to predict the sources and transportation of human fecal pollution or sewage contamination. In addition, the high correlation between PMMoV data obtained from quantitative real-time PCR (qPCR) and viral metagenomics data supports the possibility of using viral metagenomics to relatively quantify specific microbial indicators for monitoring microbial water quality (0.588 ≤ rho ≤ 0.879; P < 0.05). IMPORTANCE The results of this study may support the hypothesis of using PMMoV as an alternative indicator of human fecal contamination in tropical surface waters from the perspective of land use patterns. The predictive result of the occurrence of human fecal indicators with high accuracy may reflect the source and transportation of human fecal pollution, which are directly related to the risk to human health, and thereafter, steps can be taken to mitigate these risks. [ABSTRACT FROM AUTHOR]