Björn Berglund

Occurrence and Abundance of Antibiotics and Resistance Genes in Rivers, Canal and near Drug Formulation Facilities – A Study in Pakistan

Antibiotic resistance (AR) is a global phenomenon that has severe epidemiological ramifications world-wide. It has been suggested that antibiotics that have been discharged into the natural aquatic environments after usage or manufacture can promote the occurrence of antibiotic resistance genes (ARG). These environmental ARGs could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. The aim of this study was to investigate the anthropogenic load of antibiotics in Northern Pakistan and study the occurrence of ARGs in selected samples from this region. 19 sampling sites were selected; including six rivers, one dam, one canal, one sewage drain and four drug formulation facilities. Our results show that five of the rivers have antibiotic levels comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics could be detected in the downstream river in close vicinity of the 10 million city Lahore, 1100, 1700 and 2700 ng L−1 for oxytetracycline, trimethoprim, and sulfamethoxazole respectively. Highest detected levels were at one of the drug formulation facilities, with the measured levels of 1100, 4100, 6200, 7300, 8000, 27000, 28000 and 49000 ng L−1 of erythromycin, lincomycin, ciprofloxacin, ofloxacin, levofloxacin, oxytetracycline, trimethoprim and sulfamethoxazole respectively. ARGs were also detected at the sites and the highest levels of ARGs detected, sulI and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. Highest levels of both antibiotics and ARGs were seen at a drug formulation facility, within an industrial estate with a low number of local residents and no hospitals in the vicinity, which indicates that the levels of ARGs at this site were associated with the environmental levels of antibiotics.

Environmental dissemination of antibiotic resistance genes and correlation to anthropogenic contamination with antibiotics

Antibiotic resistance is a growing problem which threatens modern healthcare globally. Resistance has traditionally been viewed as a clinical problem, but recently non-clinical environments have been highlighted as an important factor in the dissemination of antibiotic resistance genes (ARGs). Horizontal gene transfer (HGT) events are likely to be common in aquatic environments; integrons in particular are well suited for mediating environmental dissemination of ARGs. A growing body of evidence suggests that ARGs are ubiquitous in natural environments. Particularly, elevated levels of ARGs and integrons in aquatic environments are correlated to proximity to anthropogenic activities. The source of this increase is likely to be routine discharge of antibiotics and resistance genes, for example, via wastewater or run-off from livestock facilities and agriculture. While very high levels of antibiotic contamination are likely to select for resistant bacteria directly, the role of sub-inhibitory concentrations of antibiotics in environmental antibiotic resistance dissemination remains unclear. In vitro studies have shown that low levels of antibiotics can select for resistant mutants and also facilitate HGT, indicating the need for caution. Overall, it is becoming increasingly clear that the environment plays an important role in dissemination of antibiotic resistance; further studies are needed to elucidate key aspects of this process. Importantly, the levels of environmental antibiotic contamination at which resistant bacteria are selected for and HGT is facilitated at should be determined. This would enable better risk analyses and facilitate measures for preventing dissemination and development of antibiotic resistance in the environment.

Urban wastewater effluent increases antibiotic resistance gene concentrations in a receiving northern European river

Antibiotic-resistant bacteria are an emerging global problem that threatens to undermine important advances in modern medicine. The environment is likely to play an important role in the dissemination of antibiotic-resistance genes (ARGs) among both environmental and pathogenic bacteria. Wastewater treatment plants (WWTPs) accumulate both chemical and biological waste from the surrounding urban milieu and have therefore been viewed as potential hotspots for dissemination and development of antibiotic resistance. To assess the effect of wastewater effluent on a river that flows through a Swedish city, sediment and water samples were collected from Stångån River, both upstream and downstream of an adjacent WWTP over 3 mo. Seven ARGs and the integrase gene on class 1 integrons were quantified in the collected sediment using real-time polymerase chain reaction (PCR). Liquid chromatography-mass spectrometry was used to assess the abundance of 10 different antibiotics in the water phase of the samples. The results showed an increase in ARGs and integrons downstream of the WWTP. The measured concentrations of antibiotics were low in the water samples from the Stångån River, suggesting that selection for ARGs did not occur in the surface water. Instead, the downstream increase in ARGs is likely to be attributable to accumulation of genes present in the treated effluent discharged from the WWTP.

Efficient removal of antibiotics in surface-flow constructed wetlands, with no observed impact on antibiotic resistance genes

Recently, there have been growing concerns about pharmaceuticals including antibiotics as environmental contaminants. Antibiotics of concentrations commonly encountered in wastewater have been suggested to affect bacterial population dynamics and to promote dissemination of antibiotic resistance. Conventional wastewater treatment processes do not always adequately remove pharmaceuticals causing environmental dissemination of low levels of these compounds. Using constructed wetlands as an additional treatment step after sewage treatment plants have been proposed as a cheap alternative to increase reduction of wastewater contaminants, however this means that the natural microbial community of the wetlands becomes exposed to elevated levels of antibiotics. In this study, experimental surface-flow wetlands in Sweden were continuously exposed to antibiotics of concentrations commonly encountered in wastewater. The aim was to assess the antibiotic removal efficiency of constructed wetlands and to evaluate the impact of low levels of antibiotics on bacterial diversity, resistance development and expression in the wetland bacterial community. Antibiotic concentrations were measured using liquid chromatography-mass spectrometry and the effect on the bacterial diversity was assessed with 16S rRNA-based denaturing gradient gel electrophoresis. Real-time PCR was used to detect and quantify antibiotic resistance genes and integrons in the wetlands, during and after the exposure period. The results indicated that the antibiotic removal efficiency of constructed wetlands was comparable to conventional wastewater treatment schemes. Furthermore, short-term treatment of the constructed wetlands with environmentally relevant concentrations (i.e. 100-2000 ng×l(-1)) of antibiotics did not significantly affect resistance gene concentrations, suggesting that surface-flow constructed wetlands are well-suited for wastewater treatment purposes.

Abundance and dynamics of antibiotic resistance genes and integrons in lake sediment microcosms

Antibiotic resistance in bacteria causing disease is an ever growing threat to the world. Recently, environmental bacteria have become established as important both as sources of antibiotic resistance genes and in disseminating resistance genes. Low levels of antibiotics and other pharmaceuticals are regularly released into water environments via wastewater, and the concern is that such environmental contamination may serve to create hotspots for antibiotic resistance gene selection and dissemination. In this study, microcosms were created from water and sediments gathered from a lake in Sweden only lightly affected by human activities. The microcosms were exposed to a mixture of antibiotics of varying environmentally relevant concentrations (i.e., concentrations commonly encountered in wastewaters) in order to investigate the effect of low levels of antibiotics on antibiotic resistance gene abundances and dynamics in a previously uncontaminated environment. Antibiotic concentrations were measured using liquid chromatography-tandem mass spectrometry. Abundances of seven antibiotic resistance genes and the class 1 integron integrase gene, intI1, were quantified using real-time PCR. Resistance genes sulI and ermB were quantified in the microcosm sediments with mean abundances 5 and 15 gene copies/106 16S rRNA gene copies, respectively. Class 1 integrons were determined in the sediments with a mean concentration of 3.8×104 copies/106 16S rRNA gene copies. The antibiotic treatment had no observable effect on antibiotic resistance gene or integron abundances.

Occurrence of blaKPC-2, blaCTX-M and mcr-1 in Enterobacteriaceae from Well Water in rural China.

ABSTRACT We report on the coexistence of mcr-1 and blaCTX-M in multidrug-resistant, extended-spectrum β-lactamase-producing Escherichia coli belonging to the sequence type 10 complex isolated from well water in rural China. Raoultella ornithinolytica with blaKPC-2 was also detected in well water from the same area. This study shows that genes coding for resistance to last-resort antibiotics are present in wells in rural China, indicating a potential source of antibiotic resistance.

Prevalence of the mcr-1 colistin resistance gene in extended-spectrum β-lactamase-producing Escherichia coli from human faecal samples collected in 2012 in rural villages in Shandong Province, China

Since its initial discovery in China in 2015, the plasmid-mediated colistin resistance gene mcr-1 has been reported in Escherichia coli isolated from clinical samples, animals and meat worldwide. In this study, 706 extended-spectrum β-lactamase (ESBL)-producing E. coli from 411 persons were detected in a collection of faecal samples from 1000 rural residents in three counties in Shandong Province, China. These isolates were screened for mcr-1 and phenotypic colistin resistance. The gene was found in 3.5% of the isolates (from 4.9% of persons) from all three counties. All isolates with phenotypic colistin resistance carried mcr-1. These data indicate that commensal carriage of ESBL-producing E. coli with mcr-1 among persons in rural China was already present in 2012 and that mcr-1 was the most important colistin resistance mechanism. Interventions are necessary to minimise further dissemination of mcr-1, which would limit the future usefulness of colistin as a last-resort antibiotic.

Identical genotypes of community-associated MRSA (ST59) and livestock-associated MRSA (ST9) in humans and pigs in rural China

This study investigated the prevalence of MRSA in samples taken in households, with and without backyard pigs in villages in a rural area of Shandong Province, China. Community‐associated MRSA and livestock‐associated MRSA, belonging to ST59 and ST9, respectively, were identified in both humans and pigs. The genotypic and phenotypic comparison of isolates indicates that bidirectional transmission of MRSA has occurred between humans and pigs in the villages.

Presence of antibiotic residues in various environmental compartments of Shandong province in eastern China: Its potential for resistance development and ecological and human risk

OBJECTIVES To investigate the occurrence of antibiotic residues in different types of environmental samples including water samples in rural Shandong province, China. Further, to characterize the potential ecological risk for development of antibiotic resistance in the environment, and the potential direct human health risk of exposure to antibiotics via drinking water and vegetables. METHODS Environmental samples (n = 214) (river water, waste water, drinking water, sediments, manure, soil and edible parts of vegetables) were collected in twelve villages in Shandong province in eastern China. High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to determine the concentration of antibiotic residues. The ratio of the measured environmental concentrations (MEC) to the predicted no-effect concentrations (PNEC) was used to evaluate the ecological risk (risk quotient, RQ) for development of antibiotic resistance. The potential risks to human health through exposure to antibiotics in drinking water were assessed by comparing measured environmental concentrations (MEC) and predicted no-effect concentration in drinking water (PNECDW), and in vegetables by comparing estimated daily intake (EDI) to ADI. RESULTS Sulfapyridine, sulfamethoxazole, ciprofloxacin, enrofloxacin, levofloxacin, norfloxacin, chloramphenicol, florfenicol, doxycycline, and metronidazole were detected at concentrations ranging between 0.3 and 3.9 ng/L in river water, 1.3 and 12.5 ng/L in waste water, 0.5 and 21.4 ng/L in drinking water, 0.31 and 1.21 μg/kg in river sediment, 0.82 and 1.91 μg/kg in pig manure, 0.1 and 11.68 μg/kg in outlet sediment, 0.5 and 2.5 μg/kg in soil, and 6.3 and 27.2 μg/kg in vegetables. The RQs for resistance development were >1 for enrofloxacin, levofloxacin, and ranged between 0.1 and 1 for ciprofloxacin. MECs/PNECDW ratios were <1 from exposure to antibiotics through drinking water for both adults and children. EDI/ADI ratios were <0.1 from exposure to antibiotics by vegetable consumption. CONCLUSIONS Antibiotic pollutants were ubiquitous in various environmental compartments of Shandong province of China. Risk estimates indicated a potential for the measured levels of enrofloxacin, levofloxacin and ciprofloxacin in waste water to pose an ecological risk for resistance selection, and further studies are needed to validate this finding. The investigated antibiotics did not appear to pose an appreciable direct human health risk from environmental exposure through drinking water or vegetables consumption. However, they might still pose a risk for resistance development.

Occurrence and removal efficiency of parasitic protozoa in Swedish wastewater treatment plants

Giardia intestinalis, Cryptosporidium spp., Entamoeba histolytica and Dientamoeba fragilis are parasitic protozoa and causative agents of gastroenteritis in humans. G. intestinalis and Cryptosporidium spp. in particular are the most common protozoa associated with waterborne outbreaks in high-income countries. Surveillance of protozoan prevalence in wastewater and evaluation of wastewater treatment removal efficiencies of protozoan pathogens is therefore imperative for assessment of human health risk. In this study, influent and effluent wastewater samples from three wastewater treatment plants in Sweden were collected over nearly one year and assessed for prevalence of parasitic protozoa. Quantitative real-time PCR using primers specific for the selected protozoa Cryptosporidium spp., G. intestinalis, E. histolytica, Entamoeba dispar and D. fragilis was used for protozoan DNA detection and assessment of wastewater treatment removal efficiencies. Occurrence of G. intestinalis, E. dispar and D. fragilis DNA was assessed in both influent (44, 30 and 39 out of 51 samples respectively) and effluent wastewater (14, 9 and 33 out of 51 samples respectively) in all three wastewater treatment plants. Mean removal efficiencies of G. intestinalis, E. dispar and D. fragilis DNA quantities, based on all three wastewater treatment plants studied varied between 67 and 87%, 37-75% and 20-34% respectively. Neither E. histolytica nor Cryptosporidium spp. were detected in any samples. Overall, higher quantities of protozoan DNA were observed from February to June 2012. The high prevalence of protozoa in influent wastewater indicates the need for continued monitoring of these pathogens in wastewater-associated aquatic environments to minimise the potential risk for human infection.