Sampling Considerations for Wastewater Surveillance of Antibiotic Resistance in Fecal Bacteria.

Wastewaters can be analyzed to generate population-level data for public health surveillance, such as antibiotic resistance monitoring. To provide representative data for the contributing population, bacterial isolates collected from wastewater should originate from different individuals and not be distorted by a selection pressure in the wastewater. Here we use Escherichia coli diversity as a proxy for representativeness when comparing grab and composite sampling at a major municipal wastewater treatment plant influent and an untreated hospital effluent in Gothenburg, Sweden. All municipal samples showed high E. coli diversity irrespective of the sampling method. In contrast, a marked increase in diversity was seen for composite compared to grab samples from the hospital effluent. Virtual resampling also showed the value of collecting fewer isolates on multiple occasions rather than many isolates from a single sample. Time-kill tests where individual E. coli strains were exposed to sterile-filtered hospital wastewater showed rapid killing of antibiotic-susceptible strains and significant selection of multi-resistant strains when incubated at 20 °C, an effect which could be avoided at 4 °C. In conclusion, depending on the wastewater collection site, both sampling method and collection/storage temperature could significantly impact the representativeness of the wastewater sample.

Surveillance of antibiotic resistant Escherichia coli in human populations through urban wastewater in ten European countries.

Antibiotic resistance surveillance data is lacking in many parts of the world, limiting effective therapy and management of resistance development. Analysis of urban wastewater, which contains bacteria from thousands of individuals, opens up possibilities to generate informative surveillance data in a standardized and resource-efficient way. Here, we evaluate the relationship between antibiotic resistance prevalence in E. coli from wastewater and clinical samples by studying countries with different resistance situations as assessed by traditional clinical surveillance. Composite, influent wastewater samples were collected over 24 h from treatment plants serving major cities in ten European countries. Using a broth screening method, resistance to six antibiotic classes was analyzed for 2507 E. coli isolates (n = 247-252 per country). Resistance prevalence in wastewater E. coli was compared to that in clinical E. coli reported by the European Antibiotic Resistance Surveillance Network. Resistance prevalence was lower in wastewater than clinical E. coli but followed similar geographic trends. Significant relationships were found for resistance to aminopenicillins (R2 = 0.72, p = 0.0019) and fluoroquinolones (R2 = 0.62, p = 0.0072), but not for aminoglycosides (R2 = 0.13, p = 0.31) and third-generation cephalosporins (R2 = 0.00, p = 0.99) where regression analyses were based on considerably fewer resistant isolates. When all four antibiotic classes were taken into account, the relationship was strong (R2 = 0.85, p < 0.0001). Carbapenem resistance was rare in both wastewater and clinical isolates. Wastewater monitoring shows promise as method for generating surveillance data reflecting the clinical prevalence of antibiotic resistant bacteria. Such data may become especially valuable in regions where clinical surveillance is currently limited.

A conceptual framework for the environmental surveillance of antibiotics and antibiotic resistance.

Environmental surveillance of antibiotics and antibiotic resistance could contribute toward the protection of human, animal and ecosystem health. However, justification for the choice of markers and sampling sites that informs about different risk scenarios is often lacking. Here, we define five fundamentally different objectives for surveillance of antibiotics and antibiotic resistance in the environment. The first objective is (1) to address the risk of transmission of already antibiotic-resistant bacteria to humans via environmental routes. The second is (2) to address the risk for accelerating the evolution of antibiotic resistance in pathogens through pollution with selective agents and bacteria of human or animal origin. The third objective is (3) to address the risks antibiotics pose for aquatic and terrestrial ecosystem health, including the effects on ecosystem functions and services. The two final objectives overlap with those of traditional clinical surveillance, namely, to identify (4) the population-level resistance prevalence and (5) population-level antibiotic use. The latter two environmental surveillance objectives have particular potential in countries where traditional clinical surveillance data and antibiotic consumption data are scarce or absent. For each objective, the levels of evidence provided by different phenotypic and genotypic microbial surveillance markers, as well as antibiotic residues, are discussed and evaluated on a conceptual level. Furthermore, sites where monitoring would be particularly informative are identified. The proposed framework could be one of the starting points for guiding environmental monitoring and surveillance of antibiotics and antibiotic resistance on various spatiotemporal scales, as well as for harmonizing such activities with existing human and animal surveillance systems.

Longitudinal study of ESBL Escherichia coli carriage on an organic broiler farm.

Objectives: To determine the molecular characteristics of ESBL-producing Escherichia coli (ESBL-E) collected during a longitudinal study on an organic broiler farm in order to investigate clonal expansion and horizontal gene transfer. Methods: Isolates were obtained from a longitudinal study performed previously on an organic broiler fattening farm. Samples from individually followed-up broilers, the broiler house, the transport van and persons that took the samples, taken at several timepoints (days 1, 3, 4, 7, 10, 42 and 70) within a production round and during the consecutive one (days 1, 2, 3 and 70), had been investigated for the occurrence of ESBL-E. In the current study, ESBL genes and MLST STs of these ESBL-E were determined. Plasmids were characterized and subtyped. Results: On arrival in round_1, ESBL-E of ST88 predominated, while on days 3, 4, 7 and 10 ST10 was most often found and at slaughter age ST155 and ST1551 prevailed. A shift in STs was also observed in round_2. None of the 35 individually selected broilers followed up in round_1 was positive for the same ESBL-E ST at all sampling times. All isolates carried CTX-M-1 group genes, confirmed as blaCTX-M-1 in 158 isolates. Further analysis of 36 isolates of different STs showed blaCTX-M-1 on IncI1/ST3 plasmids. Conclusions: The rapid dissemination of ESBL-E on this broiler farm was not due to the spread of one specific E. coli clone, but most likely the result of horizontal transfer of an IncI1/ST3 plasmid carrying blaCTX-M-1 resulting in a shift in the predominant ESBL-E population in broilers.

Transmission dynamics of extended-spectrum ß-lactamase and AmpC ß-lactamase-producing Escherichia coli in a broiler flock without antibiotic use.

Extended-spectrum and AmpC ß-lactamase-producing Escherichia coli (ESBL/AmpC-EC) are found throughout the broiler production pyramid. Transmission of resistance between broilers and humans could occur at any point, representing a potential public health issue. Insight in farm transmission dynamics could provide a basis for control, leading to fewer contaminated broilers. The aim was quantifying transmission rates and routes of ESBL/AmpC-EC, and specific phylogenetic groups, in an organic broiler flock without antibiotic use. In each of two consecutive production rounds, 80 randomly chosen broilers were followed individually. Cloacal swabs from these, 20 other randomly chosen broilers, and 11 environmental samples were taken at several moments from arrival till slaughter. ESBL/AmpC-EC were isolated by selective pre-enrichment, and ESBL/AmpC-genes and E. coli phylogenetic groups were determined. Transmission parameters (ß) were estimated using a Generalised Linear Model with a susceptible-infectious-susceptible model. Effect of direct broiler contact as compared to contact through the environment and previous carriage c.q. infectious status and their interaction were included as explanatory variables. Multiplying ß by the length of the infectious period gives the reproduction ratio (R). On day 1, prevalence was 28.8% (95%CI 19.2-40.0%) and 0.0% (95%CI 0.0-4.5%) among individually followed broilers, in round 1 and 2 respectively. In round 2, the environment was positive before arrival of day-old chicks. After 3 days, almost 100% of broilers and environmental samples were positive in both rounds. Most samples were positive for CTX-M-1 group genes, and A1 and B1 were predominant phylogenetic groups. From day 3 there was a shift towards more phylogenetic groups. R was 1.70 (95%CI 0.55-5.25) for total ESBL/AmpC-EC. Risk for broilers to become infectious was lower if previously infectious (ßpreviously infectious=0.02 vs. ßnot previously infectious=3.41; P<0.0001). For phylogenetic groups separately, R was 0.88 (95%CI 0.38-2.07), 0.51 (95%CI 0.27-0.98), 0.99 (95%CI 0.65-1.51) for A1, B1 and rest (i.e. A0, B2, D1, D2) groups, respectively. The interaction effect for A1 and B1 was reflected in the fact that when broilers were previous infectious, the environment was relatively more important for transmission of the A1 group, while this was direct contact between broilers for the B1 group. Positive day-old chicks and the environment both play a role in introduction and transmission of ESBL/AmpC-EC in flocks. These results suggest that, even without selective pressure from antibiotics, total ESBL/AmpC-EC persistence, and resulting endemic situation, seem to be caused by shifts in carriage of different phylogenetic groups. It implies that contaminated broilers enter the slaughterhouse.

Role of the Environment in the Transmission of Antimicrobial Resistance to Humans: A Review.

To establish a possible role for the natural environment in the transmission of clinically relevant AMR bacteria to humans, a literature review was conducted to systematically collect and categorize evidence for human exposure to extended-spectrum ß-lactamase-producing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus spp. in the environment. In total, 239 datasets adhered to inclusion criteria. AMR bacteria were detected at exposure-relevant sites (35/38), including recreational areas, drinking water, ambient air, and shellfish, and in fresh produce (8/16). More datasets were available for environmental compartments (139/157), including wildlife, water, soil, and air/dust. Quantitative data from exposure-relevant sites (6/35) and environmental compartments (11/139) were scarce. AMR bacteria were detected in the contamination sources (66/66) wastewater and manure, and molecular data supporting their transmission from wastewater to the environment (1/66) were found. The abundance of AMR bacteria at exposure-relevant sites suggests risk for human exposure. Of publications pertaining to both environmental and human isolates, however, only one compared isolates from samples that had a clear spatial and temporal relationship, and no direct evidence was found for transmission to humans through the environment. To what extent the environment, compared to the clinical and veterinary domains, contributes to human exposure needs to be quantified. AMR bacteria in the environment, including sites relevant for human exposure, originate from contamination sources. Intervention strategies targeted at these sources could therefore limit emission of AMR bacteria to the environment.

Methicillin-resistant Staphylococcus aureus and extended-spectrum and AmpC ß-lactamase-producing Escherichia coli in broilers and in people living and/or working on organic broiler farms.

The aim of this study was to estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum and AmpC ß-lactamase (ESBL/AmpC)-producing Escherichia coli among broilers, and humans living and/or working on organic broiler farms; further characterise isolates; and compare these results with those from conventional farms. In the Netherlands, only 9 certified organic broiler farms were present. On 8 of these farms, 60 throat swabs and 20 cloacal swabs were taken per farm for MRSA and ESBL/AmpC-E. coli detection, respectively, at an average age of both 34 (T1) and 68 (T2) days. Faecal swabs and questionnaires were returned by 27 out of 36 humans. For selected ESBL/AmpC-producing E. coli isolates, phylogenetic groups, ß-lactamase genes, plasmid families, and sequence types were determined. MRSA was not detected in broiler and human samples. ESBL/AmpC-producing E. coli were isolated from broilers on 7/8 farms at T1 and on all farms at T2. Furthermore, 3 farmers at T1, and 2 farmers and 1 family member at T2 were positive. Genes found in broilers and humans were almost exclusively blaCTX-M-1 and blaCMY-2. Given the high overall human ESBL/AmpC-prevalence (18.5%), which is similar to conventional farms, contact with live broilers is assumed a risk factor for carriage. Farm and sample-level prevalence at T1 are consistent with those from conventional farms. At T2, just before slaughter, sample-level prevalence of ESBL/AmpC-E. coli appears to have decreased (94.3% vs. 80%), which could have important consequences for contamination of retail meat.