Insights into effects of algae on decay and distribution of bacterial pathogens in recreational water: Implications for microbial risk management.

The decay and distribution of bacterial pathogens in water is an important information for the health risk assessment to guide water safety management, and suspended algae might affect bacterial pathogens in water. This study established microcosms to investigate the effects of algae-related factors on the representative indicators and opportunistic pathogen species in water. We found that suspended algae increased the persistence of targeted species by 1-2 orders of magnitude of concentrations compared to microcosms without algae; and the effect of algae on microbial survival was affected by water nutrient levels (i.e., carbon, nitrogen and phosphorus), as the increased microbial persistence were correlated to the increased algae concentrations with more nutrient supplies. Moreover, decay and distribution profiles of representative species were determined. The three opportunistic pathogen species (Pseudomonas aeruginosa, Aeromonas hydrophila and Staphylococcus aureus) showed lower decay rates (0.82-0.98/day, 0.76-0.98/day, 0.63-0.87/day) largely affected by algae-related factors, while the enteric species (Escherichia coli and Enterococcus faecalis) had higher decay rates (0.94-1.31/day, 0.89-1.21/day) with little association with algae, indicating the propensity for attachment to algae is an important parameter in microbial fate. Together results suggest suspended algae played an evident role in the decay and distribution of bacterial pathogens, providing important implications regarding microbial safety in recreational water.

Diversity and abundance of bacterial pathogens in urban rivers impacted by domestic sewage.

In developing countries, many urban rivers are suffering from heavy contamination by untreated sewage, which implies great microbial risks. However, information regarding the bacterial pathogen diversity and distribution in urban rivers is highly limited. In this study, 41 water samples of fifteen rivers and eight samples from two sewage treatment plants in Changzhou City of Yangtze River Delta were sampled. Next-generation sequencing and a self-built reference pathogen database were used to investigate the diversity of enteric and environmental pathogens. The results indicated that the studied urban rivers were harboring diverse potential pathogen species, which primarily included enteric pathogens in Arcobacter and Bacteroides, and environmental pathogens in Acinetobacter, Aeromonas and Pseudomonas. Quantification of twelve pathogens/indicators of interest by qPCR showed that Escherichia coli, Enterococcus faecalis, Campylobacter jejuni, Arcobacter cryaerophilus, Acinetobacter johnsonii, Acinetobacter lwoffii and Aeromonas spp. were abundant, with median values ranging from 3.30 to 5.85 log10 copies/100 mL, while Salmonella, Legionella pheumophila, Mycobacterium avium, Pseudomonas aeruginosa and Staphylococcus aureus were infrequently quantified. The pollution of nutrients and human intestinal microorganisms indicated by specific markers were found to be prevalent but with different levels in the rivers. The correlation analyses revealed that the diversity (p < 0.01) and concentrations (p < 0.05) of the enteric pathogens highly correlated to the human fecal marker abundances, which indicated that the enteric pathogens in the urban rivers were likely to have originated from domestic sewage. The environmental pathogens, which are different from the enteric ones, showed various distribution patterns, and some of them were more abundant in the rivers of rich nutrient. Our findings provide a comprehensive understanding of the bacterial pathogen distribution and influencing factors in urban rivers that are impacted by domestic sewage, thereby establishing the foundation for urban water management.

Occurrence of antibiotic resistance genes and bacterial pathogens in water and sediment in urban recreational water.

The emergence and prevalence of antibiotic resistance genes (ARGs) and pathogens in the environment are serious global health concern. However, information about the occurrence of ARGs and pathogens in recreational water is still limited. Accordingly, we investigated the occurrence of six ARGs and human pathogens in three recreational lakes, and the correlations between ARGs and one mobile genetic element (intI1) were analyzed. The quantitative PCR results showed that the concentration of ARGs ranged from 4.58 × 100 to 5.0 × 105 copies/mL in water and from 5.78 × 103 to 5.89 × 108 copies/g dry weight (dw) in sediment. Sul1 exhibited the highest level among the five quantifiable ARGs. The concentrations of sul1, bla-TEM, and tetX exhibited significant positive correlations with intI1 (p < 0.05), indicating that intI1 may be involved in their proliferation. The detection frequencies of ARGs ranged from 75%-100%, indicating the prevalence of these risks in this region. The concentration of Escherichia coli, Aeromonas spp., Mycobacterium avium, Pseudomonas aeruginosa, and Salmonella enterica ranged from 103 to 105 copies/100 mL in water and 104-106 copies/g dw in sediment. In total, 25% of the samples harbored all pathogen genes, indicating the prevalence of these pathogens in recreational lakes. Furthermore, the next-generation sequencing results showed that 68 genera of pathogens were present, among which Aeromonas, Mycobacterium, and Pseudomonas were the dominant ones in this region, posing a considerable potential health risk to public health. Overall, the widespread distribution of ARGs and pathogens underscores the need to better monitor and mitigate their propagation in recreational lakes and the associated risks to human health.

Diversity of potential antibiotic-resistant bacterial pathogens and the effect of suspended particles on the spread of antibiotic resistance in urban recreational water.

Evidence of the increasing incidence of antibiotic resistance in watersheds has attracted worldwide attention. Limited in formation is available on the occurrences of health-related antibiotic-resistant bacterial pathogens (ARBPs) in recreational waters. The effects of certain environmental factors (e.g., suspended particles) on the spread of resistance also has not been characterized to date. In this study, a combination of culture and molecular methods was employed to comprehensively investigate the patterns of microbial resistance to representative antibiotics in samples from three recreational lakes in Beijing. The antibiotic resistance index (ARI) based on the gradient concentration assay revealed that samples showed high resistance to penicillin-G, moderate resistance to ampicillin, vancomycin and erythromycin and low resistance to ceftriaxone, gentamycin, tetracycline and chloramphenicol. Antibiotic-resistant bacteria (ARB) were cultured and collected, and the diversity of potential ARBP species was further explored using next-generation sequencing (NGS). The results showed that most of the identified ARBPs were environmental opportunistic pathogens with emerging clinical concerns, e.g., the multidrug-resistant Acinetobacter junii. Furthermore, particle-attached (PA) fractions presented higher ARI values than free-floating (FL) fractions did, indicating that the PA fractions were more resistant to selected antibiotics. And the NGS results revealed that the PA fractions showed higher similarity in the screened ARB community compositions in comparison with the FL fractions, primarily due to a protective effect provided by the particles. Accordingly, ARBPs could persist for a longer time in protective particle matrices. However, quantification of antibiotic-resistant genes (ARGs) by qPCR showed no significant abundance differences between the two fractions. Overall, these findings suggest a potential health risk from the prevalence of ARBPs in recreational waters and provides a better understanding of the contribution of particles in the spread of antibiotic resistance in aquatic systems, with implications for the control of excessive suspended particles by water management.

Distribution comparison and risk assessment of free-floating and particle-attached bacterial pathogens in urban recreational water: Implications for water quality management.

The risk of pathogen exposure in recreational water is a concern worldwide. Moreover, suspended particles, as ideal shelters for pathogens, in these waters also need attention. However, the risk caused by the pathogen-particle attachment is largely unknown. Accordingly, water samples in three recreational lakes in Beijing were collected and separated into free-floating (FL, 0.22-5µm) and particle-attached (PA, >5µm) fractions. Next-generation sequencing (NGS) was employed to determine the diversity of genera containing pathogens, and quantitative PCR (qPCR) was used to assess the presence of genes from Escherichia coli (uidA), Salmonella enterica (invA), Aeromonas spp. (aerA), Mycobacterium avium (16S) and Pseudomonas aeruginosa (oaa). The NGS results showed stable pathogen genera composition distinctions between the PA and FL fractions. Some genera, such as Aeromonas and Mycobacterium, exhibited higher abundances in the PA fractions. qPCR revealed that most of the gene concentrations were higher within particles than were FL fractions. Some gene levels showed correlations with the particle concentrations and lake nutrient levels. Further quantitative microbial risk assessment (QMRA) of selected strains (S. enterica and M. avium) indicated a higher health risk during secondary contact activities in lakes with more nutrients and particles. We concluded that suspended particles (mainly composed of algae) in urban recreational water might influence the pathogen distribution and could serve as reservoirs for pathogen contamination, with important management implications.

Evaluation of bacterial pathogen diversity, abundance and health risks in urban recreational water by amplicon next-generation sequencing and quantitative PCR.

The microbial quality of urban recreational water is of great concern to public health. The monitoring of indicator organisms and several pathogens alone is not sufficient to accurately and comprehensively identify microbial risks. To assess the levels of bacterial pathogens and health risks in urban recreational water, we analyzed pathogen diversity and quantified four pathogens in 46 water samples collected from waterbodies in Beijing Olympic Forest Park in one year. The pathogen diversity revealed by 16S rRNA gene targeted next-generation sequencing (NGS) showed that 16 of 40 genera and 13 of 76 reference species were present. The most abundant species were Acinetobacter johnsonii, Mycobacterium avium and Aeromonas spp. Quantitative polymerase chain reaction (qPCR) of Escherichia coli (uidA), Aeromonas (aerA), M. avium (16S rRNA), Pseudomonas aeruginosa (oaa) and Salmonella (invA) showed that the aerA genes were the most abundant, occurring in all samples with concentrations of 104-6 genome copies/100mL, followed by oaa, invA and M. avium. In total, 34.8% of the samples harbored all genes, indicating the prevalence of these pathogens in this recreational waterbody. Based on the qPCR results, a quantitative microbial risk assessment (QMRA) showed that the annual infection risks of Salmonella, M. avium and P. aeruginosa in five activities were mostly greater than the U.S. EPA risk limit for recreational contacts, and children playing with water may be exposed to the greatest infection risk. Our findings provide a comprehensive understanding of bacterial pathogen diversity and pathogen abundance in urban recreational water by applying both NGS and qPCR.

Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium.

Study of enzymes in halophiles will help to understand the mechanism of aromatic hydrocarbons degradation in saline environment. In this study, two novel catechol 2,3-dioxygenases (C23O1 and C23O2) were cloned and overexpressed from a halophilic bacterial consortium enriched from an oil-contaminated saline soil. Phylogenetic analysis indicated that the novel C23Os and their relatives formed a new branch in subfamily I.2.A of extradiol dioxygenases and the sequence differences were further analyzed by amino acid sequence alignment. Two enzymes with the halotolerant feature were active over a range of 0-30% salinity and they performed more stable at high salinity than in the absence of salt. Surface electrostatic potential and amino acids composition calculation suggested high acidic residues content, accounting for their tolerance to high salinity. Moreover, two enzymes were further characterized. The enzymes activity both increased in the presence of Fe(3+), Fe(2+), Cu(2+) and Al(3+) and showed no significant inhibition by other tested metal ions. The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively. As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation.