Carolin Rutgersson

Pyrosequencing of Antibiotic-Contaminated River Sediments Reveals High Levels of Resistance and Gene Transfer Elements

The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.

Fluoroquinolones and qnr Genes in Sediment, Water, Soil, and Human Fecal Flora in an Environment Polluted by Manufacturing Discharges

There is increasing concern that environmental antibiotic pollution promotes transfer of resistance genes to the human microbiota. Here, fluoroquinolone-polluted river sediment, well water, irrigated farmland, and human fecal flora of local villagers within a pharmaceutical industrial region in India were analyzed for quinolone resistance (qnr) genes by quantitative PCR. Similar samples from Indian villages farther away from industrial areas, as well as fecal samples from Swedish study participants and river sediment from Sweden, were included for comparison. Fluoroquinolones were detected by MS/MS in well water and soil from all villages located within three km from industrially polluted waterways. Quinolone resistance genes were detected in 42% of well water, 7% of soil samples and in 100% and 18% of Indian and Swedish river sediments, respectively. High antibiotic concentrations in Indian sediment coincided with high abundances of qnr, whereas lower fluoroquinolone levels in well water and soil did not. We could not find support for an enrichment of qnr in fecal samples from people living in the fluoroquinolone-contaminated villages. However, as qnr was detected in 91% of all Indian fecal samples (24% of the Swedish) it suggests that the spread of qnr between people is currently a dominating transmission route.

Pharmaceutical industry effluent diluted 1:500 affects global gene expression, cytochrome P450 1A activity, and plasma phosphate in fish

Patancheru, near Hyderabad, India, is a major production site for the global bulk drug market. Approximately 90 manufacturers send their wastewater to a common treatment plant in Patancheru. Extraordinary high levels of a wide range of pharmaceuticals have recently been demonstrated in the treated effluent. As little as 0.2% of this effluent can strongly reduce the growth rate of tadpoles, but the underlying mechanisms of toxicity are not known. To begin addressing how the effluent affects aquatic vertebrates, rainbow trout (Oncorhynchus mykiss) were exposed to 0.2% effluent for 5 d. Several physiological endpoints, together with effects on global hepatic gene expression patterns, were analyzed. The exposed fish showed both an induction of hepatic cytochrome P450 1A (CYP1A) gene expression, as well as enzyme activity. Clinical blood chemistry analyses revealed an increase in plasma phosphate levels, which in humans indicates impaired kidney function. Several oxidative stress-related genes were induced in the livers; however, no significant changes in antioxidant enzyme activities or in the hepatic glutathione levels were found. Furthermore, estrogen-regulated genes were slightly up-regulated following exposure, and moderate levels of estriol were detected in the effluent. The present study identifies changes in gene expression triggered by exposure to a high dilution of the effluent, supporting the hypothesis that these fish are responding to chemical exposure. The pattern of regulated genes may contribute to the identification of mechanisms of sublethal toxicity, as well as illuminate possible causative agents.

Comparison of six different sewage treatment processes-Reduction of estrogenic substances and effects on gene expression in exposed male fish

Treated sewage effluents often contain a mixture of estrogenic compounds in low concentrations. The total combined activity of these, however, may be sufficiently high to affect the reproduction of aquatic vertebrates. The introduction of advanced treatment technologies has been suggested as a way to remove micro-contaminants, including estrogenic substances. In this study, one municipal influent was treated with six different processes in parallel on a semi-large scale in order to assess their potential to reduce substances that could contribute to estrogenic effects in male fish. The effluent from a conventional, activated sludge treatment line was compared to a similarly treated effluent with a final sand-filtering step. The addition of ozonation (15 g O(3)/m(3)), a moving bed biofilm reactor (MBBR) or both in combination was also evaluated. There was also a separate treatment line that was based on a membrane bioreactor. A small battery of hepatic estrogen-responsive genes was measured in the exposed fish using quantitative PCR. Concentrations of steroid estrogens and estrogenic phenols in the effluents were measured by GC-ECNI-MS. The ozonated effluents were the only tested effluents for which all measured biological effects in exposed fish were removed. Chemical data suggested that the MBBR technology was equally effective in removing the analyzed estrogens; however, elevated expression of estrogen-responsive genes suggested that some estrogenic substances were still present in the effluent. The membrane bioreactor removed most of the measured estrogens and it reduced the induction of the estrogen-responsive genes. However, fish exposed to this effluent had significantly enlarged livers. Given that the same influent was treated in parallel with a broad set of technologies and that the chemical analyses were combined with an in vivo assessment of estrogenic responses, this study provides valuable input into the assessment of advanced treatment processes for removing estrogenic substances.

An assay for determining minimal concentrations of antibiotics that drive horizontal transfer of resistance.

Ability to understand the factors driving horizontal transfer of antibiotic resistance from unknown, harmless bacteria to pathogens is crucial in order to tackle the growing resistance problem. However, current methods to measure effects of stressors on horizontal gene transfer have limitations and often fall short, as the estimated endpoints can be a mix of both the number of transfer events and clonal growth of transconjugants. Our aim was therefore to achieve a proper strategy for assessing the minimal concentration of a stressor (exemplified by tetracycline) that drives horizontal transfer of antibiotic resistance from a complex community to a model pathogen. Conditions were optimized to improve a culture-based approach using the bacterial community of treated sewage effluent as donor, and fluorescent, traceable Escherichia coli as recipient. Reduced level of background resistance, differentiation of isolates as well as decreased risk for measuring effects of selection were achieved through the use of chromogenic medium, optimization of conjugation time as well as applying a different antibiotic for isolation of transconjugants than the one tested for its ability to drive transfer. Using this assay, we showed that a very low concentration of tetracycline, 10μg/L i.e. 150 times below the minimal inhibitory concentration of the recipient, promoted horizontal transfer of multiple antibiotic-resistance determinants. Higher concentrations favoured selection of a tetracycline-resistance phenotype along with a decline in the number of detectable transfer events. The described method can be used to evaluate different environmental conditions and factors that trigger horizontal dissemination of mobile resistance elements, eventually resulting in the formation of drug-resistant pathogens.

Minimal selective concentrations of tetracycline in complex aquatic bacterial biofilms

Selection pressure generated by antibiotics released into the environment could enrich for antibiotic resistance genes and antibiotic resistant bacteria, thereby increasing the risk for transmission to humans and animals. Tetracyclines comprise an antibiotic class of great importance to both human and animal health. Accordingly, residues of tetracycline are commonly detected in aquatic environments. To assess if tetracycline pollution in aquatic environments promotes development of resistance, we determined minimal selective concentrations (MSCs) in biofilms of complex aquatic bacterial communities using both phenotypic and genotypic assays. Tetracycline significantly increased the relative abundance of resistant bacteria at 10 μg/L, while specific tet genes (tetA and tetG) increased significantly at the lowest concentration tested (1 μg/L). Taxonomic composition of the biofilm communities was altered with increasing tetracycline concentrations. Metagenomic analysis revealed a concurrent increase of several tet genes and a range of other genes providing resistance to different classes of antibiotics (e.g. cmlA, floR, sul1, and mphA), indicating potential for co-selection. Consequently, MSCs for the tet genes of ≤ 1 μg/L suggests that current exposure levels in e.g. sewage treatment plants could be sufficient to promote resistance. The methodology used here to assess MSCs could be applied in risk assessment of other antibiotics as well.

Global hepatic gene expression in rainbow trout exposed to sewage effluents: A comparison of different sewage treatment technologies

Effluents from sewage treatment plants contain a mixture of micropollutants with the potential of harming aquatic organisms. Thus, addition of advanced treatment techniques to complement existing conventional methods has been proposed. Some of the advanced techniques could, however, potentially produce additional compounds affecting exposed organisms by unknown modes of action. In the present study the aim was to improve our understanding of how exposure to different sewage effluents affects fish. This was achieved by explorative microarray and quantitative PCR analyses of hepatic gene expression, as well as relative organ sizes of rainbow trout exposed to different sewage effluents (conventionally treated, granular activated carbon, ozonation (5 or 15 mg/L), 5 mg/L ozone plus a moving bed biofilm reactor, or UV-light treatment in combination with hydrogen peroxide). Exposure to the conventionally treated effluent caused a significant increase in liver and heart somatic indexes, an effect removed by all other treatments. Genes connected to xenobiotic metabolism, including cytochrome p450 1A, were differentially expressed in the fish exposed to the conventionally treated effluents, though only effluent treatment with granular activated carbon or ozone at 15 mg/L completely removed this response. The mRNA expression of heat shock protein 70 kDa was induced in all three groups exposed to ozone-treated effluents, suggesting some form of added stress in these fish. The induction of estrogen-responsive genes in the fish exposed to the conventionally treated effluent was effectively reduced by all investigated advanced treatment technologies, although the moving bed biofilm reactor was least efficient. Taken together, granular activated carbon showed the highest potential of reducing responses in fish induced by exposure to sewage effluents.

Oral exposure to industrial effluent with exceptionally high levels of drugs does not indicate acute toxic effects in rats

The Patancheru area near Hyderabad in India is recognized as a key link in the global supply chain for many bulk drugs. A central treatment plant receives wastewater from approximately 90 different manufacturers, and the resulting complex effluent has contaminated surface, ground, and drinking water in the region. Ecotoxicological testing of the effluent has shown adverse effects for several organisms, including aquatic vertebrates, at high dilutions. In addition, a recent study of microbial communities in river sediment indicated that the contamination of antibiotic substances might contribute to the emergence and spread of antibiotic resistance genes. In an attempt to start investigating how exposure to effluent-contaminated water may directly affect humans and other terrestrial vertebrates, rats were tube-fed effluent. Several pharmaceuticals present in the effluent could be detected in rat blood serum at low concentrations. However, results from exploratory microarray and quantitative polymerase chain reaction assays indicated no marked effects on hepatic gene transcription after 5 d of exposure. Clinical analysis of blood serum constituents, used as biomarkers for human disease did not reveal any significant changes, nor was there an effect on weight gain. The authors could not find evidence for any acute toxicity in the rat; however, the authors cannot rule out that [corrected] higher doses of effluent or a longer exposure time may still be associated with risks for terrestrial vertebrates.

Differential removal of human pathogenic viruses from sewage by conventional and ozone treatments

Abstract Sewage contains a mixed ecosystem of diverse sets of microorganisms, including human pathogenic viruses. Little is known about how conventional as well as advanced treatments of sewage, such as ozonation, reduce the environmental spread of viruses. Analyses for viruses were therefore conducted for three weeks in influent, after conventional treatment, after additional ozonation, and after passing an open dam system at a full-scale treatment plant in Knivsta, Sweden. Viruses were concentrated by adsorption to a positively charged filter, from which they were eluted and pelleted by ultracentrifugation, with a recovery of about 10%. Ion Torrent sequencing was used to analyze influent, leading to the identification of at least 327 viral species, most of which belonged to 25 families with some having unclear classification. Real-time PCR was used to test for 21 human-related viruses in inlet, conventionally treated, and ozone-treated sewage and outlet waters. The viruses identified in influent and further analyzed were adenovirus, norovirus, sapovirus, parechovirus, hepatitis E virus, astrovirus, pecovirus, picobirnavirus, parvovirus, and gokushovirus. Conventional treatment reduced viral concentrations by one to four log10, with the exception of adenovirus and parvovirus, for which the removal was less efficient. Ozone treatment led to a further reduction by one to two log10, but less for adenovirus. This study showed that the amount of all viruses was reduced by conventional sewage treatment. Further ozonation reduced the amounts of several viruses to undetectable levels, indicating that this is a promising technique for reducing the transmission of many pathogenic human viruses.