Quantification of ß-lactamase producing bacteria in German surface waters with subsequent MALDI-TOF MS-based identification and ß-lactamase activity assay.

Environmental oligotrophic bacteria are suspected to be highly relevant carriers of antimicrobial resistance (AMR). However, there is a lack of validated methods for monitoring in the aquatic environment. Since extended-spectrum ß-lactamases (ESBLs) play a particularly important role in the clinical sector, a culturing method based on R2A-medium spiked with different combinations of ß-lactams was applied to quantify ß-lactamase-producing environmental bacteria from surface waters. In German surface water samples (n = 28), oligotrophic bacteria ranging from 4.0 × 103 to 1.7 × 104 CFU per 100 mL were detected on the nutrient-poor medium spiked with 3rd generation cephalosporins and carbapenems. These numbers were 3 log10 higher compared to ESBL-producing Enterobacteriales of clinical relevance from the same water samples. A MALDI-TOF MS identification of the isolates demonstrated, that the method leads to the isolation of environmentally relevant strains with Pseudomonas, Flavobacterium, and Janthinobacterium being predominant ß-lactam resistant genera. Subsequent micro-dilution antibiotic susceptibility tests (Micronaut-S test) confirmed the expression of ß-lactamases. The qPCR analysis of surface waters DNA extracts showed the presence of ß-lactamase genes (blaTEM, blaCMY-2, blaOXA-48, blaVIM-2, blaSHV, and blaNDM-1) at concentrations of 3.7 (±1.2) to 1.0 (±1.9) log10 gene copies per 100 mL. Overall, the results demonstrate a widespread distribution of cephalosporinase and carbapenemase enzymes in oligotrophic environmental bacteria that have to be considered as a reservoir of ARGs and contribute to the spread of antibiotic resistance.

Microbiological characterization of stormwater in a high-income neighborhood in Rio de Janeiro, Brazil.

Stormwater harvesting and reuse in the urban environment is emerging as an alternative water source, despite human pathogens in the stormwater may represent a hazard to public health. This study presents the results of 1-year monitoring to evaluate the quality of stormwater obtained in a high-income neighborhood in Rio de Janeiro for a set of microbiological parameters as total coliforms, Escherichia coli (E. coli), human adenovirus (HAdV), human JC polyomavirus (JCPyV), Group A rotavirus (RVA), and norovirus GI and GII. Forty-eight stormwater samples obtained from two multiplex units presented total coliforms and E. coli in 91.7% (n = 44) and 58.3% (n = 28) of samples, while HAdV and JCPyV were detected in 20.8% (n = 10) and 12.5% (n = 6), respectively. Viral quantification ranged from 103 to 104 genomic copies/liter (GC/L) for HAdV and from 101 to 104 GC/L for JCPyV. Neither RVA nor norovirus GI and GII was detected. Fifteen out of sixteen (93.8%) samples containing viruses were compliant as per fecal indicator bacteria (FIB) according to Brazilian standards for rainwater reuse and US EPA Guidelines for Water Reuse, suggesting that viruses monitoring should complement the study of bacterial indicators.

Optimization of sampling strategy to determine pathogen removal efficacy of activated sludge treatment plant.

Large-scale wastewater schemes rely on multi-barrier approach for the production of safe and sustainable recycled water. In multi-barrier wastewater reclamation systems, conventional activated sludge process (ASP) often constitutes a major initial treatment step. The main aim of this research was to determine most appropriate sampling approach to establish pathogen removal efficacy of ASP. The results suggest that ASP is capable of reducing human adenovirus (HAdV) and polyomavirus (HPyV) by up to 3 log10. The virus removal data suggests that HAdV removal is comparable to somatic bacteriophage belonging to Microviridae family. Due to the high removal of Escherichia coli (>3 log10) and very poor correlation with the enteric virus, it is not recommended that E. coli be used as a surrogate for enteric virus removal. The results also demonstrated no statistically significant differences (t test, P > 0.05) in calculated log removal values (LRVs) for HAdV, HPyV, and Microviridae from samples collected on hydraulic retention time (HRT) or simultaneous paired samples collected for influent and effluent. This indicates that a more practical approach of simultaneous sampling for influent and effluent could be used to determine pathogen removal efficiency of ASP. The results also suggest that a minimum of 10, preferably 20 samples, are required to fully capture variability in the removal of virus. In order to cover for the potential seasonal prevalence of viruses such as norovirus and rotavirus, sampling should be spread across all seasons.

Attachment and Detachment Behavior of Human Adenovirus and Surrogates in Fine Granular Limestone Aquifer Material.

The transport of human adenovirus, nanoparticles, and PRD1 and MS2 bacteriophages was tested in fine granular limestone aquifer material taken from a borehole at a managed aquifer recharge site in Adelaide, South Australia. Comparison of transport and removal of virus surrogates with the pathogenic virus is necessary to understand the differences between the virus and surrogate. Because experiments using pathogenic viruses cannot be done in the field, laboratory tests using flow-through soil columns were used. Results show that PRD1 is the most appropriate surrogate for adenovirus in an aquifer dominated by calcite material but not under high ionic strength or high pH conditions. It was also found that straining due to size and the charge of the colloid were not dominant removal mechanisms in this system. Implications of this study indicate that a certain surrogate may not represent a specific pathogen solely based on similar size, morphology, and/or surface charge. Moreover, if a particular surrogate is representative of a pathogen in one aquifer system, it may not be the most appropriate surrogate in another porous media system. This was apparent in the inferior performance of MS2 as a surrogate, which is commonly used in virus transport studies.

Sensitive detection of human adenovirus from small volume of primary wastewater samples by quantitative PCR.

An accurate quantitative detection of enteric viruses from the primary wastewater requires, sample concentration followed by extraction of nucleic acid with high purity. A highly efficient and sensitive method was developed for the concentration and quantitative detection of human adenovirus (HAdv) from wastewater samples. The two-step method which combines concentration of virus from 10 mL sample with centrifugal filters followed by extraction and purification of DNA with commercially available nucleic acid extraction kit resulted in high purity DNA for downstream quantitative PCR (qPCR). The results obtained on analytical sensitivities of five commercial nucleic acid extraction kits show that they differ in their ability for DNA yield and purity. Nevertheless, despite variable analytical sensitivities extracted nucleic acid was found to be relatively PCR inhibition free. The genomic copy numbers of HAdv detected from the same concentrated wastewater sample were significantly higher (P<0.01) when Qiagen Blood and Tissue kit (1.54×10(6) L(-1)) was used as compared to Mo-Bio PowerSoil kit (5.30×10(5) L(-1)) which suggests that the nucleic acid extraction kit can influence the sensitivity of qPCR assays. The method developed in this study is simple, rapid, sensitive, and can be applicable for the qPCR detection of adenovirus and other DNA virus in wastewater.