Novel clinically relevant antibiotic resistance genes associated with sewage sludge and industrial waste streams revealed by functional metagenomic screening.

A growing body of evidence indicates that anthropogenic activities can result in increased prevalence of antimicrobial resistance genes (ARGs) in bacteria in natural environments. Many environmental studies have used next-generation sequencing methods to sequence the metagenome. However, this approach is limited as it does not identify divergent uncharacterized genes or demonstrate activity. Characterization of ARGs in environmental metagenomes is important for understanding the evolution and dissemination of resistance, as there are several examples of clinically important resistance genes originating in environmental species. The current study employed a functional metagenomic approach to detect genes encoding resistance to extended spectrum ß-lactams (ESBLs) and carbapenems in sewage sludge, sludge amended soil, quaternary ammonium compound (QAC) impacted reed bed sediment and less impacted long term curated grassland soil. ESBL and carbapenemase genes were detected in sewage sludge, sludge amended soils and QAC impacted soil with varying degrees of homology to clinically important ß-lactamase genes. The flanking regions were sequenced to identify potential host background and genetic context. Novel ß-lactamase genes were found in Gram negative bacteria, with one gene adjacent to an insertion sequence ISPme1, suggesting a recent mobilization event and/ the potential for future transfer. Sewage sludge and quaternary ammonium compound (QAC) rich industrial effluent appear to disseminate and/or select for ESBL genes which were not detected in long term curated grassland soils. This work confirms the natural environment as a reservoir of novel and mobilizable resistance genes, which may pose a threat to human and animal health.

A coliform-targeted metagenomic method facilitating human exposure estimates to Escherichia coli-borne antibiotic resistance genes.

Antimicrobial resistance and the spread of antibiotic resistance genes (ARGs) pose a threat to human health. Community-acquired infections resistant to treatment with first-line antibiotics are increasing, and there are few studies investigating environmental exposures and transmission. Our objective is to develop a novel targeted metagenomic method to quantify the abundance and diversity of ARGs in a faecal indicator bacterium, and to estimate human exposure to resistant bacteria in a natural environment. Sequence data from Escherichia coli metagenomes from 13 bathing waters in England were analysed using the ARGs Online Analysis Pipeline to estimate the abundance and diversity of resistance determinants borne by this indicator bacterium. These data were averaged over the 13 sites and used along with data on the levels of E. coli in English bathing waters in 2016 and estimates of the volume of water that water users typically ingest in an average session of their chosen activityto quantify the numbers of ARGs that water users ingest. Escherichia coli in coastal bathing waters were found to harbour on average 1.24 ARGs per cell. Approximately 2.5 million water sports sessions occurred in England in 2016 that resulted in water users ingesting at least 100 E. coli-borne ARGs.

Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment.

OBJECTIVES: Multidrug-resistant Enterobacteriaceae pose a significant threat to public health. We aimed to study the impact of sewage treatment effluent on antibiotic resistance reservoirs in a river. METHODS: River sediment samples were taken from downstream and upstream of a waste water treatment plant (WWTP) in 2009 and 2011. Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae were enumerated. PCR-based techniques were used to elucidate mechanisms of resistance, with a new two-step PCR-based assay developed to investigate bla(CTX-M-15) mobilization. Conjugation experiments and incompatibility replicon typing were used to investigate plasmid ecology. RESULTS: We report the first examples of bla(CTX-M-15) in UK river sediment; the prevalence of bla(CTX-M-15) was dramatically increased downstream of the WWTP. Ten novel genetic contexts for this gene were identified, carried in pathogens such as Escherichia coli ST131 as well as indigenous aquatic bacteria such as Aeromonas media. The bla(CTX-M-15) -gene was readily transferable to other Gram-negative bacteria. We also report the first finding of an imipenem-resistant E. coli in a UK river. CONCLUSIONS: The high diversity and host range of novel genetic contexts proves that evolution of novel combinations of resistance genes is occurring at high frequency and has to date been significantly underestimated. We have identified a worrying reservoir of highly resistant enteric bacteria in the environment that poses a threat to human and animal health.

Functional metagenomic analysis reveals rivers are a reservoir for diverse antibiotic resistance genes.

The environment harbours a significant diversity of uncultured bacteria and a potential source of novel and extant resistance genes which may recombine with clinically important bacteria disseminated into environmental reservoirs. There is evidence that pollution can select for resistance due to the aggregation of adaptive genes on mobile elements. The aim of this study was to establish the impact of waste water treatment plant (WWTP) effluent disposal to a river by using culture independent methods to study diversity of resistance genes downstream of the WWTP in comparison to upstream. Metagenomic libraries were constructed in Escherichia coli and screened for phenotypic resistance to amikacin, gentamicin, neomycin, ampicillin and ciprofloxacin. Resistance genes were identified by using transposon mutagenesis. A significant increase downstream of the WWTP was observed in the number of phenotypic resistant clones recovered in metagenomic libraries. Common ß-lactamases such as blaTEM were recovered as well as a diverse range of acetyltransferases and unusual transporter genes, with evidence for newly emerging resistance mechanisms. The similarities of the predicted proteins to known sequences suggested origins of genes from a very diverse range of bacteria. The study suggests that waste water disposal increases the reservoir of resistance mechanisms in the environment either by addition of resistance genes or by input of agents selective for resistant phenotypes.

Integron prevalence and diversity in manured soil.

The levels of integron abundance and diversity in soil amended with pig slurry were studied. Real-time PCR illustrated a significant increase in class 1 integron prevalence after slurry application, with increased prevalence still evident at 10 months after application. Culture-dependent data revealed 10 genera, including putative human pathogens, carrying class 1 and 2 integrons.

Prevalence of sulfonamide resistance genes in bacterial isolates from manured agricultural soils and pig slurry in the United Kingdom.

The prevalences of three sulfonamide resistance genes, sul1, sul2, and sul3 and sulfachloropyridazine (SCP) resistance were determined in bacteria isolated from manured agricultural clay soils and slurry samples in the United Kingdom over a 2-year period. Slurry from tylosin-fed pigs amended with SCP and oxytetracycline was used for manuring. Isolates positive for sul genes were further screened for the presence of class 1 and 2 integrons. Phenotypic resistance to SCP was significantly higher in isolates from pig slurry and postapplication soil than in those from preapplication soil. Of 531 isolates, 23% carried sul1, 18% sul2, and 9% sul3 only. Two percent of isolates contained all three sul genes. Class 1 and class 2 integrons were identified in 5% and 11.7%, respectively, of sul-positive isolates. In previous reports, sul1 was linked to class 1 integrons, but in this study only 8% of sul1-positive isolates carried the intI1 gene. Sulfonamide-resistant pathogens, including Shigella flexneri, Aerococcus spp., and Acinetobacter baumannii, were identified in slurry-amended soil and soil leachate, suggesting a potential environmental reservoir. Sulfonamide resistance in Psychrobacter, Enterococcus, and Bacillus spp. is reported for the first time, and this study also provides the first description of the genotypes sul1, sul2, and sul3 outside the Enterobacteriaceae and in the soil environment.

Environmental monitoring of Mycobacterium bovis in badger feces and badger sett soil by real-time PCR, as confirmed by immunofluorescence, immunocapture, and cultivation.

Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger feces. Immunomagnetic capture, immunofluorescence, and selective culture confirmed species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle.

Incidence of class 1 integrons in a quaternary ammonium compound-polluted environment.

Samples of effluent and soil were collected from a reed bed system used to remediate liquid waste from a wool finishing mill with a high use of quaternary ammonium compounds (QACs) and were compared with samples of agricultural soils. Resistance quotients of aerobic gram-negative and gram-positive bacteria to ditallowdimethylammomium chloride (DTDMAC) and cetyltrimethylammonium bromide (CTAB) were established by plating onto nutrient agar containing 5 microg/ml or 50 microg/ml DTDMAC or CTAB. Approximately 500 isolates were obtained and screened for the presence of the intI1 (class 1 integrase), qacE (multidrug efflux), and qacE Delta1 (attenuated qacE) genes. QAC resistance was higher in isolates from reed bed samples, and class 1 integron incidence was significantly higher for populations that were preexposed to QACs. This is the first study to demonstrate that QAC selection in the natural environment has the potential to coselect for antibiotic resistance, as class 1 integrons are well-established vectors for cassette genes encoding antibiotic resistance.

Interactions between Salmonella typhimurium and Acanthamoeba polyphaga, and observation of a new mode of intracellular growth within contractile vacuoles.

Acanthamoeba polyphaga feeding on Salmonella typhimurium in a simple model biofilm were observed by light microscopy and a detailed record of interactions kept by digital image capture and image analysis. A strain of S. typhimurium SL1344 carrying a fis: gfp reporter construct (pPDT105) was used to assess intracellular growth in A. polyphaga on non-nutrient agar (NNA) plates. Invasion of the contractile vacuole (CV) was observed at a frequency of 1:100-1000 acanthamoebae at 35 degrees C. The salmonellae contained in CVs illustrated significant up-regulation of fis relative to extracellular bacteria, indicating that they were in the early stages of logarithmic growth, and reached numbers of 100-200 cells per vacuole after 4 days. This is the first report of this mode of intracellular growth. Up-regulation of fis was also observed in a proportion of S. typhimurium cells contained within food vacuoles. Filamentation of S. typhimurium and E. coli cells was frequently observed in coculture with A. polyphaga on NNA plates, with bacterial cells reaching lengths of up to 500 microm after 10 days' incubation at 35 degrees C. A. polyphaga was also seen to mediate bacterial translocation over the agar surface; egested salmonellae subsequently formed microcolonies along amoebal tracks. This illustrated intracellular survival of a fraction of the S. typhimurium population. These phenomena suggest that protozoa such as A. polyhaga may play an important role in the ecology of S. typhimurium in soil and aquatic environments.

An SEM study of adhesive disc skeletal structures isolated from trichodinids (Ciliophora: Peritrichida) of the genera Trichodina Ehrenberg, 1838 and Paratrichodina Lom, 1963.

Specimens of Trichodina domerguei Wallengren, 1897, T. intermedia (Lom, 1961) and Paratrichodina incissa (Lom, 1959) were sonicated to liberate skeletal components of the adhesive disc. This enabled SEM observation of the taxonomically important structures obscured in preparations of complete cells. A previously undescribed peg-like structure on the centrifugal surface of the central part of the denticles is revealed in T. domerguei. In P. incissa the ray apophysis and its supporting apophysis appear to be absent, providing an additional characteristic to discriminate it from species of the genus Trichondina Ehrenberg, 1838. From silver stained and SEM preparations of T. intermedia and P. incissa important differences in denticle blade form are apparent, underlining the value of observation of isolated skeletal structures by electron microscopy.

Ectoparasitic species of the genus Trichodina (Ciliophora: Peritrichida) parasitising British freshwater fish.

Seven species of the genus Trichodina Ehrenberg, 1838 were identified during a sampling programme of twenty freshwater fish species from approximately sixty sites in Scotland, England and Wales. Species found include: Trichodina acuta Lom, 1961 from Cyprinus carpio L., Carassius auratus L., Oncorhynchus mykiss (Walbaum), Salmo trutta L. and Phoxinus phoxinus L.; Trichodina domerguei Wallengren, 1897 from Gasterosteus aculeatus L.; Trichodina tenuidens Faure-Fremiet, 1944 from Gasterosteus aculeatus; Trichodina pediculus Ehrenberg, 1838 from Gasterosteus aculeatus; Trichodina modesta Lom, 1970 from Abramis brama L.; Trichodina nigra Lom, 1960 from Cyprinus carpio, Salmo trutta and Oncorhynchus mykiss; and Trichodina intermedia Lom, 1960 from Phoxinus. Morphological variation within and between host populations and host specificity of the Trichodina species recovered are described.