Removal efficiency of antibiotic residues, antibiotic resistant bacteria, and genes across parallel secondary settling tank and membrane bioreactor treatment trains in a water reclamation plant.

Antimicrobial resistance is recognized as a potent threat to human health. Wastewater treatment facilities are viewed as hotspots for the spread of antimicrobial resistance. This study provides comprehensive data on the occurrences of 3 different antibiotic resistant opportunistic pathogens (with resistance to up to 5 antibiotics), 13 antibiotic resistant genes and intI1, and 22 different antimicrobial residues in a large water reclamation plant (176 million gallons per day) that runs a conventional Modified Ludzack-Ettinger (MLE) reactor followed by a secondary settling tank (SST) and membrane bioreactor (MBR) in parallel. All the antibiotic resistant bacteria and most of the antibiotic resistance genes were present in the raw influent, ranging from 2.5 × 102-3.7 × 106 CFU/mL and 1.2× 10-1-6.5 × 1010 GCN/mL, respectively. MBR outperformed the SST system in terms of ARB removal as the ARB targets were largely undetected in MBR effluent, with log removals ranging from 2.7 to 6.8, while SST only had log removals ranging from 0.27 to 4.6. Most of the ARG concentrations were found to have significantly higher in SST effluent than MBR permeate, and MBR had significantly higher removal efficiency for most targets (p < 0.05) except for sul1, sul2, blaOXA48, intI1 and 16S rRNA genes (p > 0.05). As for the antibiotic residues (AR), there was no significant removal from the start to the end of the treatment process, although MBR had higher removal efficiencies for azithromycin, chloramphenicol, erythromycin, erythromycin-H2O, lincomycin, sulfamethoxazole and triclosan, compared to the SST system. In conclusion, MBR outperformed SST in terms of ARB and ARGs removal. However low removal efficiencies of most AR targets were apparent.

Assessing the additional health burden of antibiotic resistant Enterobacteriaceae in surface waters through an integrated QMRA and DALY approach.

Antibiotic resistant Enterobacteriaceae pose a significant threat to public health. However, limited studies have evaluated the health risks associated with exposure to antibiotic-resistant bacteria (ARB), especially in natural environments. While quantitative microbial risk assessment (QMRA) assesses microbial risks in terms of the probability of infection, it does not account for the severity of health outcomes. In this study, a QMRA-DALY model was developed to integrate QMRA with health burden (disability-adjusted life years (DALY)) from infections caused by ARB. The model considers uncertainties in probability of infection and health burden assessment using Monte Carlo simulations. The study collected antimicrobial resistance (AMR) surveillance data from surface waters with different land uses. Results revealed water bodies with agricultural land use to be the main AMR hotspots, with the highest additional health burden observed in infections caused by meropenem-resistant E. coli (∆DALY = 0.0105 DALY/event) compared to antibiotic-susceptible E. coli. The estimated ∆DALY for antibiotic-resistant K. pneumoniae was lower than for antibiotic-resistant E. coli (highest ∆DALY = 0.00048 DALY/event). The study highlights the need for better evaluation of AMR associated health burden, and effective measures to mitigate the risks associated with antibiotic-resistant bacteria in natural environments.

A new modelling framework for assessing the relative burden of antimicrobial resistance in aquatic environments.

The infections caused by antibiotic resistant bacteria (ARB) can lead to higher medical costs, prolonged hospital stays, and increased mortality compared to bacteria that are susceptible to antibiotics. Challenges exist in quantifying the potential risk/burden associated with antimicrobial resistance (AMR) as there is a lack of dose-response models available for pathogens which are resistant to antibiotics, in addition to the fact that very little is known regarding the health risks posed by antibiotic resistant genes (ARG). In this paper, we proposed a new modelling framework to evaluate the relative burden of AMR in natural aquatic environments. With this framework, an AMR burden score for each sample was calculated based on burden coefficients assigned for each ARB and ARG, as well as weighted burdens for the separate ARBs and ARGs components. The method developed in this study was applied to assess the relative burden of AMR in local aquatic environments with different land uses at different seasons. The collected filed data were used to verify the applicability of the proposed relative burden assessment method. Through the established method, the spatial and temporal hotspots of AMR were identified, which could provide useful information to agencies for better control and management of AMR emergence in natural aquatic environments.

Metagenomic and Resistome Analysis of a Full-Scale Municipal Wastewater Treatment Plant in Singapore Containing Membrane Bioreactors.

Reclaimed water provides a water supply alternative to address problems of scarcity in urbanized cities with high living densities and limited natural water resources. In this study, wastewater metagenomes from 6 stages of a wastewater treatment plant (WWTP) integrating conventional and membrane bioreactor (MBR) treatment were evaluated for diversity of antibiotic resistance genes (ARGs) and bacteria, and relative abundance of class 1 integron integrases (intl1). ARGs confering resistance to 12 classes of antibiotics (ARG types) persisted through the treatment stages, which included genes that confer resistance to aminoglycoside [aadA, aph(6)-I, aph(3')-I, aac(6')-I, aac(6')-II, ant(2″)-I], beta-lactams [class A, class C, class D beta-lactamases (bla OXA)], chloramphenicol (acetyltransferase, exporters, floR, cmIA), fosmidomycin (rosAB), macrolide-lincosamide-streptogramin (macAB, ereA, ermFB), multidrug resistance (subunits of transporters), polymyxin (arnA), quinolone (qnrS), rifamycin (arr), sulfonamide (sul1, sul2), and tetracycline (tetM, tetG, tetE, tet36, tet39, tetR, tet43, tetQ, tetX). Although the ARG subtypes in sludge and MBR effluents reduced in diversity relative to the influent, clinically relevant beta lactamases (i.e., bla KPC, bla OXA) were detected, casting light on other potential point sources of ARG dissemination within the wastewater treatment process. To gain a deeper insight into the types of bacteria that may survive the MBR removal process, genome bins were recovered from metagenomic data of MBR effluents. A total of 101 close to complete draft genomes were assembled and annotated to reveal a variety of bacteria bearing metal resistance genes and ARGs in the MBR effluent. Three bins in particular were affiliated to Mycobacterium smegmatis, Acinetobacter Iwoffii, and Flavobacterium psychrophila, and carried aquired ARGs aac(2')-Ib, bla OXA-278, and tet36 respectively. In terms of indicator organisms, cumulative log removal values (LRV) of Escherichia coli, Enterococci, and P. aeruginosa from influent to conventional treated effluent was lower (0-2.4), compared to MBR effluent (5.3-7.4). We conclude that MBR is an effective treatment method for reducing fecal indicators and ARGs; however, incomplete removal of P. aeruginosa in MBR treated effluents (<8 MPN/100 mL) and the presence of ARGs and intl1 underscores the need to establish if further treatment should be applied prior to reuse.

Draft Genome Sequences of a Ceftazidime-Resistant Acinetobacter baumannii Donor and a Conjugal Escherichia coli Recipient with Acquired Resistance.

A ceftazidime-resistant Acinetobacter baumannii strain was isolated from hospital wastewater and used as the donor in a filter mating experiment with an Escherichia coli strain as the recipient. Recipient, donor, and transconjugant were sequenced, and both donor and transconjugant were found to harbor highly similar plasmid sequences, suggesting that plasmid transfer had occurred.

Draft Genome Sequences of Four Multidrug-Resistant Pseudomonas aeruginosa Isolates from Hospital Wastewater in Singapore.

Four multidrug-resistant Pseudomonas aeruginosa isolates were cultured from intensive care unit wastewater. All isolates exhibited resistance to carbapenem and extended-spectrum beta-lactam antibiotics. Genome characterization revealed the presence of beta-lactamase resistance genes (bla PAO and bla OXA), and three out of the four isolates carried the bla NDM-1 gene encoding resistance against carbapenems.

Microbial water quality and the detection of multidrug resistant E. coli and antibiotic resistance genes in aquaculture sites of Singapore.

Poor microbial water quality jeopardizes the health and safety of food produced by aquaculture farms. Three fish farms and transect sites in Singapore were assessed for microbial water quality and antimicrobial resistance determinants. Of the 33 multidrug resistant E. coli isolated from surface waters of the Johor Straits, 81.8% were ESBL producers. The relative abundance of sul1, qnrA and intI1 genes were higher in sediments than surface waters. Among the surface water samples, higher concentrations (10-1-101) of beta-lactamases (blaSHV, blaOXA, blaCTX-M) were detected in the transect sites. This study highlights a potential antimicrobial resistance transmission chain from environmental waters, to animal carriers and humans.

Occurrence and characteristics of extended-spectrum ß-lactamase- and carbapenemase- producing bacteria from hospital effluents in Singapore.

One of the most important resistance mechanisms in Gram-negative bacteria today is the production of enzymes causing resistance to cephalosporin and carbapenem antibiotics. The spread of extended-spectrum ß-lactamases (ESBL)- and carbapenemase- producing Gram-negative bacteria is an emerging global public health problem. The aim of the present study was to (i) assess the prevalence of carbapenem-resistant bacteria (CRB) and ESBL-producing strains in sewage effluents from two major hospitals in Singapore, (ii) characterize the isolated strains and (iii) identify some of the ESBL and carbapenemase genes responsible for the resistance. CHROMagar ESBL and KPC plates were used to rapidly screen for ESBL-producing bacteria and those expressing reduced susceptibility to carbapenems, respectively. The abundance of ESBL-producers and CRB in hospital wastewater ranged between 103 and 106CFU/mL. Out of the 66 isolates picked from ESBL and KPC plates, 95%, 82%, 82% and 76% were resistant to ceftriaxone, ceftazidime (3rd generation cephalosporin family), ertapenem and meropenem (carbapenem family), respectively. Among the resistant isolates, the most predominant taxa identified were Pseudomonas spp. (28.2%), Klebsiella spp. (28.2%), Enterobacter spp. (18.3%) and Citrobacter spp. (11.3%). PCR and sequencing analysis showed that the predominant ß-lactamase genes were blaSHV (41.1%) followed by blaNDM-1 (35.6%), blaCTX (35.6%) and blaKPC (28.8%). The results of this study show a high prevalence of bacteria resistant to modern extended-spectrum cephalosporins and carbapenems and the presence of ESBL- and carbapenemase producers in hospital effluents. These findings support the need to improve management of hospital wastewater in order to minimize the spread of antimicrobial resistant microorganisms from this source.

Corrigendum: Characterization of Metagenomes in Urban Aquatic Compartments Reveals High Prevalence of Clinically Relevant Antibiotic Resistance Genes in Wastewaters.

[This corrects the article on p. 2200 in vol. 8, PMID: 29201017.].

Characterization of Metagenomes in Urban Aquatic Compartments Reveals High Prevalence of Clinically Relevant Antibiotic Resistance Genes in Wastewaters.

The dissemination of antimicrobial resistance (AMR) is an escalating problem and a threat to public health. Comparative metagenomics was used to investigate the occurrence of antibiotic resistant genes (ARGs) in wastewater and urban surface water environments in Singapore. Hospital and municipal wastewater (n = 6) were found to have higher diversity and average abundance of ARGs (303 ARG subtypes, 197,816 x/Gb) compared to treated wastewater effluent (n = 2, 58 ARG subtypes, 2,692 x/Gb) and surface water (n = 5, 35 subtypes, 7,985 x/Gb). A cluster analysis showed that the taxonomic composition of wastewaters was highly similar and had a bacterial community composition enriched in gut bacteria (Bacteroides, Faecalibacterium, Bifidobacterium, Blautia, Roseburia, Ruminococcus), the Enterobacteriaceae group (Klebsiella, Aeromonas, Enterobacter) and opportunistic pathogens (Prevotella, Comamonas, Neisseria). Wastewater, treated effluents and surface waters had a shared resistome of 21 ARGs encoding multidrug resistant efflux pumps or resistance to aminoglycoside, macrolide-lincosamide-streptogramins (MLS), quinolones, sulfonamide, and tetracycline resistance which suggests that these genes are wide spread across different environments. Wastewater had a distinctively higher average abundance of clinically relevant, class A beta-lactamase resistant genes (i.e., blaKPC, blaCTX-M, blaSHV, blaTEM). The wastewaters from clinical isolation wards, in particular, had a exceedingly high levels of blaKPC-2 genes (142,200 x/Gb), encoding for carbapenem resistance. Assembled scaffolds (16 and 30 kbp) from isolation ward wastewater samples indicated this gene was located on a Tn3-based transposon (Tn4401), a mobilization element found in Klebsiella pneumonia plasmids. In the longer scaffold, transposable elements were flanked by a toxin-antitoxin (TA) system and other metal resistant genes that likely increase the persistence, fitness and propagation of the plasmid in the bacterial host under conditions of stress. A few bacterial species (Enterobacter cloacae, Klebsiella pneumoniae, Citrobacter freundii, Pseudomonas aeruginosa) that were cultured from the isolation ward wastewaters on CHROMagar media harbored the blaKPC-2 gene. This suggests that hospital wastewaters derived from clinical specialty wards are hotspots for the spread of AMR. Assembled scaffolds of other mobile genetic elements such as IncQ and IncF plasmids bearing quinolone resistance genes (qnrS1, qnrS2) and the class A beta-lactamase gene (blaTEM-1) were recovered in wastewater samples which may aid the transfer of AMR.

Composition of bacterial and archaeal communities in freshwater sediments with different contamination levels (Lake Geneva, Switzerland).

The aim of this study was to compare the composition of bacterial and archaeal communities in contaminated sediments (Vidy Bay) with uncontaminated sediments (Ouchy area) of Lake Geneva using 16S rRNA clone libraries. Sediments of both sites were analysed for physicochemical characteristics including porewater composition, organic carbon, and heavy metals. Results show high concentrations of contaminants in sediments from Vidy. Particularly, high contents of fresh organic matter and nutrients led to intense mineralisation, which was dominated by sulphate-reduction and methanogenesis. The bacterial diversity in Vidy sediments was significantly different from the communities in the uncontaminated sediments. Phylogenetic analysis revealed a large proportion of Betaproteobacteria clones in Vidy sediments related to Dechloromonas sp., a group of dechlorinating and contaminant degrading bacteria. Deltaproteobacteria, including clones related to sulphate-reducing bacteria and Fe(III)-reducing bacteria (Geobacter sp.) were also more abundant in the contaminated sediments. The archaeal communities consisted essentially of methanogenic Euryarchaeota, mainly found in the contaminated sediments rich in organic matter. Multiple factor analysis revealed that the microbial community composition and the environmental variables were correlated at the two sites, which suggests that in addition to environmental parameters, pollution may be one of the factors affecting microbial community structure.

Persistence and growth of faecal culturable bacterial indicators in water column and sediments of Vidy Bay, Lake Geneva, Switzerland.

The aims of this study was to investigate the persistence and the growth of culturable bacterial indicators (CBI) including total coliforms (TC) and faecal coliforms represented by Escherichia coli, enterococcus (ENT), and aerobic mesophilic bacteria (AMB) in the surface sediments and the water column of Vidy Bay (Lake Geneva, City of Lausanne, Switzerland). The study was carried out for 60 d using microcosms containing Sewage Treatment Plant (STP) effluent and nonsterile water without CBI, as well as contaminated and non-contaminated sediments. The effects of water temperature and of organic matter associated with sediments on the survival of CBI in the sediments and the water column were observed. The number of CBI colonies in the contaminated sediments of Vidy Bay and in the STP effluent was almost identical in the order of 10(5)-10(7), 10(4)-10(6), 10(3)-10(5), and 10(4)-10(7) CFU/100 g sediment or/100 mL water for TC, E. coli, ENT, and AMB respectively. A degradation of CBI was observed in the sediments where organic matter content was low and in the water column at a temperature of 10 degrees C after 5 d of experimentation. In addition, a growth of CBI was observed in the sediment which is rich in organic matter at 20 degrees C. The results of this study indicate: (1) the higher concentrations of the CBI observed in different points in the water column of Vidy Bay may not be explained only by the recent contribution of the three potential sources of the Bay contamination including STP and the Chamberonne and Flon Rivers, but also by the persistence, removal from sediment and multiplication of CBI in the sediment and water column; (2) the sediment of Vidy Bay constitute a reservoir of CBI and can even support their growth; and (3) the CBI not only survive in sediments, but also can be remobilized and increased in the water column, therefore, it become a permanent microbiological pollution in Vidy Bay.

Origin and spatial-temporal distribution of faecal bacteria in a bay of Lake Geneva, Switzerland.

The origin and distribution of microbial contamination in Lake Geneva's most polluted bay were assessed using faecal indicator bacteria (FIB). The lake is used as drinking water, for recreation and fishing. During 1 year, water samples were taken at 23 points in the bay and three contamination sources: a wastewater treatment plant (WWTP), a river and a storm water outlet. Analyses included Escherichia coli, enterococci (ENT), total coliforms (TC), and heterotrophic plate counts (HPC). E. coli input flux rates from the WWTP can reach 2.5 x 10(10) CFU/s; those from the river are one to three orders of magnitude lower. Different pathogenic Salmonella serotypes were identified in water from these sources. FIB levels in the bay are highly variable. Results demonstrate that (1) the WWTP outlet at 30 m depth impacts near-surface water quality during holomixis in winter; (2) when the lake is stratified, the effluent water is generally trapped below the thermocline; (3) during major floods, upwelling across the thermocline may occur; (4) the river permanently contributes to contamination, mainly near the river mouth and during floods, when the storm water outlet contributes additionally; (5) the lowest FIB levels in the near-surface water occur during low-flow periods in the bathing season.

Effects of a sewage treatment plant outlet pipe extension on the distribution of contaminants in the sediments of the Bay of Vidy, Lake Geneva, Switzerland.

In 2001, the municipality of Lausanne extended the outlet pipe of the sewage treatment plant into the Bay of Vidy (Lake Geneva, Switzerland) as a measure to reduce bacterial water pollution and sediment contamination close to the lake beaches. The aim of the present study was to assess the impact of this measure. Lake bottom sediments were collected and analyzed for grain size, organic matter, organic carbon, nitrogen, phosphorus, heavy metals and hydrophobic organic compounds to evaluate their concentration and spatial distribution. Our results demonstrate that, compared to results obtained before the outlet pipe prolongation, the surface area of highly contaminated sediments was reduced by one third after the pipe extension. However, contaminant concentrations are still high and the accumulation of pollutants may represent a significant source of toxicity for benthic organisms. One concludes that contaminant reduction at the source will be necessary for a further improvement.

Characterizing water circulation and contaminant transport in Lake Geneva using bacteriophage tracer experiments and limnological methods.

Multi-tracer tests with three types of marine bacteriophages (H4/4, H6/1, and H40/1), together with various limnological methods, including physicochemical depth profiling, surface drifters, deep current measurements, and fecal indicator bacteria analyses, have been applied to characterize water circulation and pathogen transport in the Bay of Vidy (Lake Geneva, Switzerland). The experimental program was carried out twice, first in November 2005, when the lake was stratified, and a second time during holomixis in February 2006. The bacteriophages were injected at three points at different depths, where contaminated waters enter the lake, including the outlet pipe of a wastewater treatment plant, a river, and a stormwater outlet. Thereafter, water samples were collected in the lake at 2 m depth during a 48 h sampling campaign. The results demonstrate that (i) contaminated river water spreads rapidly in the bay; (ii) a well-developed thermocline is highly effective in preventing contamination from the depth to rise up to the surface; (iii) rapid vertical mixing and pathogen transport occur under thermally homogeneous conditions; and (iv) repeated multi-tracertests with bacteriophages are a powerful technique to assess water circulation and contaminant transport in lakes where high dilution occurs.

Estimating the costs and health benefits of water and sanitation improvements at global level.

The aim of this study was to estimate the costs and the health benefits of the following interventions: increasing access to improved water supply and sanitation facilities, increasing access to in house piped water and sewerage connection, and providing household water treatment, in ten WHO sub-regions. The cost-effectiveness of each intervention was assessed in terms of US dollars per disability adjusted life year (DALY) averted. This analysis found that almost all interventions were cost-effective, especially in developing countries with high mortality rates. The estimated cost-effectiveness ratio (CER) varied between US$20 per DALY averted for disinfection at point of use to US$13,000 per DALY averted for improved water and sanitation facilities. While increasing access to piped water supply and sewage connections on plot was the intervention that had the largest health impact across all sub-regions, household water treatment was found to be the most cost-effective intervention. A policy shift to include better household water quality management to complement the continuing expansion of coverage and upgrading of services would appear to be a cost-effective health intervention in many developing countries.

Global cost-benefit analysis of water supply and sanitation interventions.

The aim of this study was to estimate the economic benefits and costs of a range of interventions to improve access to water supply and sanitation facilities in the developing world. Results are presented for eleven developing country WHO sub-regions as well as at the global level, in United States Dollars (US$) for the year 2000. Five different types of water supply and sanitation improvement were modelled: achieving the water millennium development goal of reducing by half in 2015 those without improved water supply in the year 1990; achieving the combined water supply and sanitation MDG; universal basic access to water supply and sanitation; universal basic access plus water purification at the point-of-use; and regulated piped water supply and sewer connection. Predicted reductions in the incidence of diarrhoeal disease were calculated based on the expected population receiving these interventions. The costs of the interventions included estimations of the full investment and annual running costs. The benefits of the interventions included time savings due to easier access, gain in productive time and reduced health care costs saved due to less illness, and prevented deaths. The results show that all water and sanitation improvements are cost-beneficial in all developing world sub-regions. In developing regions, the return on a US$1 investment was in the range US$5 to US$46, depending on the intervention. For the least developed regions, investing every US$1 to meet the combined water supply and sanitation MDG lead to a return of at least US$5 (AFR-D, AFR-E, SEAR-D) or US$12 (AMR-B; EMR-B; WPR-B). The main contributor to economic benefits was time savings associated with better access to water and sanitation services, contributing at least 80% to overall economic benefits. One-way sensitivity analysis showed that even under pessimistic data assumptions the potential economic benefits outweighed the costs in all developing world regions. Further country case-studies are recommended as a follow up to this global analysis.

Cost-effectiveness of water quality interventions for preventing diarrhoeal disease in developing countries.

Using effectiveness data from a recent systematic review and cost data from programme implementers and World Health Organization (WHO) databases, we conducted a cost-effectiveness analysis to compare non-piped in source- (dug well, borehole and communal stand post) and four types of household- (chlorination, filtration, solar disinfection, flocculation/disinfection) based interventions to improve the microbial quality of water for preventing diarrhoeal disease. Results are reported for two WHO epidemiological sub-regions, Afr-E (sub-Saharan African countries with very high adult and child mortality) and Sear-D (South East Asian countries with high adult and child mortality) at 50% intervention coverage. Measured against international benchmarks, source- and household-based interventions were generally cost effective or highly cost effective even before the estimated saving in health costs that would offset the cost of implementation. Household-based chlorination was the most cost-effective where resources are limited; household filtration yields additional health gains at higher budget levels. Flocculation/disinfection was strongly dominated by all other interventions; solar disinfection was weakly dominated by chlorination. In addition to cost-effectiveness, choices among water quality interventions must be guided by local conditions, user preferences, potential for cost recovery from beneficiaries and other factors.

Water, sanitation, and hygiene interventions to reduce diarrhoea in less developed countries: a systematic review and meta-analysis.

Many studies have reported the results of interventions to reduce illness through improvements in drinking water, sanitation facilities, and hygiene practices in less developed countries. There has, however, been no formal systematic review and meta-analysis comparing the evidence of the relative effectiveness of these interventions. We developed a comprehensive search strategy designed to identify all peer-reviewed articles, in any language, that presented water, sanitation, or hygiene interventions. We examined only those articles with specific measurement of diarrhoea morbidity as a health outcome in non-outbreak conditions. We screened the titles and, where necessary, the abstracts of 2120 publications. 46 studies were judged to contain relevant evidence and were reviewed in detail. Data were extracted from these studies and pooled by meta-analysis to provide summary estimates of the effectiveness of each type of intervention. All of the interventions studied were found to reduce significantly the risks of diarrhoeal illness. Most of the interventions had a similar degree of impact on diarrhoeal illness, with the relative risk estimates from the overall meta-analyses ranging between 0.63 and 0.75. The results generally agree with those from previous reviews, but water quality interventions (point-of-use water treatment) were found to be more effective than previously thought, and multiple interventions (consisting of combined water, sanitation, and hygiene measures) were not more effective than interventions with a single focus. There is some evidence of publication bias in the findings from the hygiene and water treatment interventions.