Tracking spatio-temporal distribution and transmission of antibiotic resistance in aquatic environments by using ESBL-producing Escherichia coli as an indicator.

Wastewater treatment plants (WWTPs) play an important role in the production, and transmission of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) as a convergence for human, animal, and environmental wastewater. The aim of this study was to investigate the spatio-temporal variation and influencing factors of ARB in different functional areas of the urban WWTP and the connecting rivers for 1-year monitoring using extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-Ec) as an indicator bacteria, and to study the transmission patterns of ARB in the aquatic environment. The results showed that ESBL-Ec isolates were identified from the WWTP (n = 219), including influent (n = 53), anaerobiotic tank (n = 40), aerobiotic tank (n = 36), activated sludge tank (n = 31), sludge thickner tank (n = 30), effluent (n = 16), and mudcake storage area (n = 13). The dehydration process can significantly remove the ESBL-Ec isolates; however, ESBL-Ec was still detected in samples collected from the effluent of the WWTP (37.0%). The detection rate of ESBL-Ec was significantly different across seasons (P < 0.05), and ambient temperature was negatively correlated with the detection rate of ESBL-Ec (P < 0.05). Furthermore, a high prevalence of ESBL-Ec isolates (29/187, 15.5%) was detected in samples collected from the river system. These findings emphasize that the high majority of ESBL-Ec in aquatic environments is alarming because it poses a significant threat to public health. Clonal transmission of ESBL-Ec isolates between the WWTP and rivers based on the spatio-temporal scale was observed by pulsed-field gel electrophoresis analysis, ST38 and ST69 ESBL-Ec clone were selected as prioritized isolates for antibiotic resistance monitoring in the aquatic environment. Further phylogenetic analysis showed human-associated (feces, blood) E. coli was the main source contributing to the presence of antibiotic resistance in aquatic environments. Longitudinal and targeted monitoring of ESBL-Ec in WWTPs and the development of effective wastewater disinfection strategies before effluent discharge from WWTPs are urgently required, to prevent and control the spread of antibiotic resistance in the environment.

Persistent transmission of carbapenem-resistant, hypervirulent Klebsiella pneumoniae between a hospital and urban aquatic environments.

The increasing prevalence of infections caused by carbapenem-resistant hypervirulent Klebsiella pneumoniae strains (CR-hvKP) prompts the question of whether these strains also circulate outside of clinical settings. However, the environmental occurrence and dissemination of CR-hvKP are poorly studied. In the current study, we investigated the epidemiological characteristics, and dissemination dynamics of carbapenem-resistant K. pneumoniae (CRKP) isolated from a hospital, an urban wastewater treatment plant (WWTP), and adjacent rivers in Eastern China during one year of monitoring. A total of 101 CRKP were isolated, 54 were determined to be CR-hvKP harboring pLVPK-like virulence plasmids, which were isolated from the hospital (29 out of 51), WWTP (23 out of 46), and rivers (2 out of 4), respectively. The period with lowest detection rate of CR-hvKP in the WWTP, August, corresponded with the lowest detection rate at the hospital. Comparing the inlet and outlet of the WWTP, no significant reduction of the detection of CR-hvKP and relative abundance of carbapenem resistance genes was observed. The detection rate of CR-hvKP and the relative abundance of carbapenemase genes were significantly higher in the WWTP in colder months compared to warmer months. Clonal dissemination of CR-hvKP clones of ST11-KL64 between the hospital and the aquatic environment, as well as the horizontal spread of IncFII-IncR and IncC plasmids carrying carbapenemase genes, was observed. Furthermore, phylogenetic analysis showed that the ST11-KL64 CR-hvKP strain has spread nationally by interregional transmission. These results indicated transmission of CR-hvKP clones between hospital and urban aquatic environments, prompting the need for improved wastewater disinfection and epidemiological models to predict the public health hazard from prevalence data of CR-hvKP.

Genomic insight into transmission mechanisms of carbapenem-producing Citrobacter spp. isolates between the WWTP and connecting rivers.

Carbapenem-resistant Enterobacteriaceae (CRE) poses major health risks worldwide. Most studies have focused on carbapenem resistance in Klebsiella pneumoniae and Escherichia coli; however, the occurrence and transmission of carbapenem-resistant Citrobacter spp. (CRCS) are poorly understood. In this study, we investigated the occurrence and potential transmission patterns of CRCS in different functional areas of an urban wastewater treatment plant (WWTP) and connecting rivers during one-year monitoring in Shandong Province, China. In total, 14 CRCS were detected in 376 environmental samples, including those from the WWTP inlet (n = 7), WWTP anaerobic tank (n = 2), and rivers (n = 5). Citrobacter braakii (n = 6) was the dominant subtype among 14 CRCS isolates, followed by Citrobacter freundii (n = 5), Citrobacter sedlakii (n = 2), and Citrobacter werkmanii (n = 1). All CRCS were resistant to imipenem, meropenem, ampicillin, amoxicillin/clavulanic acid, cefotaxime, ceftazidime, trimethoprim/sulfamethoxazole, and ciprofloxacin. Plasmid analysis showed that the blaKPC-2 gene was located on IncN and IncFII (Yp) plasmids, whereas the blaNDM gene was located on IncX3 and IncN2 plasmids. Clonal transmission of CRCS harboring carbapenem genes occurred between the WWTP and connecting rivers on a temporal or spatial scale. High genomic relatedness of NDM-5-producing C. sedlakii was identified between river water and WWTP aerosol, suggesting a potential exposure risk of CRCS for workers and surrounding residents near the WWTP. Furthermore, NDM-5-producing C. sedlakii isolated from rivers was related to C. sedlakii isolated from soil and well water in different regions of China, indicating that NDM-5-producing C. sedlakii may be widespread in China. These findings indicate that rare healthcare-associated pathogens such as CRCS can contribute to widespread carbapenem production in the environment; thus, CRCS should be continuously monitored.

Occurrence, consumption level, fate and ecotoxicology risk of beta-agonist pharmaceuticals in a wastewater treatment plant in Eastern China.

Beta-agonist pharmaceuticals are widely used in humans and livestock for disease treatment, legal or illegal growth promotion in food animals, bodybuilding, weight loss, and sports doping. The occurrence of beta-agonists in wastewater treatment plants and their subsequent environmental impacts require greater attention. This study determined the levels of 12 beta-agonists in a wastewater treatment plant and evaluated their ecotoxicological risks as well as consumption levels and risks to human health. Among the 12 selected beta-agonists, all were detected in wastewater and 11 in sludge. In most cases, the concentrations of beta-agonists were higher in spring than in summer. Their total average daily mass loads per capita in the influent and effluent were 1.35 µg/d/p and 2.11 µg/d/p, respectively. The overall removal efficiencies of individual beta-agonists ranged from -295.3 to 71.2%. Ecotoxicological risk assessment revealed a low risk to daphnid and green algae from the levels of fenoterol and the mixture of 12 selected beta-agonists in the effluent. The daily consumption levels of individual beta-agonists per capita were 0.028-1.200 µg/d/p. Regular monitoring of beta-agonists in municipal sewage systems and their risk assessment based on toxicological data are urgently required in the future.

High prevalence of Escherichia coli co-harboring conjugative plasmids with colistin- and carbapenem resistance genes in a wastewater treatment plant in China.

Emergence and dissemination of resistance to last-resort antibiotics such as carbapenem and colistin is a growing, global health concern. Wastewater treatment plants (WWTPs) link human activities and the environment, can act as reservoirs and sources for emerging antibiotic resistance, and likely play a large role in antibiotic resistance transmission. The aim of this study was to investigate occurrence and characteristics of colistin- and carbapenem-resistant Escherichia coli (CCREC) in wastewater and sludge samples collected over a one-year period from different functional areas of an urban WWTP in Jinan city, Shandong, China. A total of 8 CCREC were isolated from 168 samples with selective agar and PCR, corresponding to high prevalence of 4.8%, co-harboring carbapenem resistance genes (blaNDM) and colistin resistance gene (mcr-1) and subsequently whole-genome sequenced. Additionally, all isolates were multidrug-resistant by antimicrobial susceptibility testing and carried a variety of antibiotic resistance genes. Two isolates carrying virulence genes associated with avian pathogenic E. coli were identified, one belonging to the high-risk clone O101:H9-ST167. Southern blotting was used to characterize CCREC isolates and plasmids carrying blaNDM-genes or mcr-1 could be transferred to a recipient strain E. coli J53 by in vitro conjugation assays. Resistance to other antibiotic classes were sporadically co-transferred to the transconjugant. Transposition of and mcr-1-carrying element from a transferable IncHI2-plasmid was observed among two CCREC clones isolated within 4 days of each other. The occurrence of multidrug-resistant CCREC capable of transferring their antibiotic resistance genotypes via conjugative plasmids is alarming. WWTPs bring bacteria from different sources together, providing opportunities for horizontal exchange of DNA among compatible hosts. Further dissemination of the colistin-, carbapenem-, or both colistin- and carbapenem resistant E. coli could lead to a serious threat to public health.

Seasonal distribution and dynamic evolution of antibiotics and evaluation of their resistance selection potential and ecotoxicological risk at a wastewater treatment plant in Jinan, China.

The seasonal distribution and dynamic evolution of antibiotics in wastewater from main treatment areas and in sludge and their resistance selection potential and ecotoxicological risk were studied at a municipal wastewater treatment plant in Jinan, East China. Ten antibiotics were selected, and all were detected in wastewater and sludge samples, with fluoroquinolones showing the highest detection concentrations and frequencies. Seasonal fluctuations in the antibiotic concentrations in the influent, effluent, and sludge were observed, with the highest values in winter in most cases. The dynamic evolution of antibiotics during the treatment process differed among the seasons. The antibiotic removal efficiencies were incomplete, ranging from - 40.47 to 100%. Mass balance analysis showed that sulfonamides, roxithromycin, and metronidazole were mainly removed through biological processing, whereas fluoroquinolones, doxycycline, and chloramphenicol were removed through sludge adsorption. Levofloxacin, as well as a mixture of the 10 antibiotics from the effluent, could pose a low ecotoxicological risk to Daphnia in the receiving waters. Additionally, levofloxacin and ciprofloxacin in the effluent and ciprofloxacin and metronidazole in the sludge may facilitate the selection of antibiotic-resistant bacteria in the environment.

The shared NDM-positive strains in the hospital and connecting aquatic environments.

The spread of antibiotic-resistant priority pathogens outside hospital settings is, both, a significant public health concern and an environmental problem. In recent years, New Delhi Metallo-ß-lactamase (NDM)-positive strains have caused nosocomial infections with high mortality and poor prognosis worldwide. Our study investigated the links of NDM-positive strains between the hospital and the connecting river system in Jinan city, Eastern China by using NDM-producing Escherichia coli (NDM-EC) as an indicator via whole genome sequencing. Thirteen NDM-EC isolates were detected from 187 river water and sediment samples, while 9 isolates were identified from patients at the local hospital. All NDM-EC isolates were resistant to imipenem, meropenem, cefotaxime, cefoxitin, ampicillin, tetracycline, fosfomycin, piperacillin-tazobactam. The blaNDM-5 (n = 20) and blaNDM-9 (n = 2) genes were identified, which were predominantly on IncX3 plasmids (n = 13), followed by IncFII plasmids (n = 5) and IncFIA plasmids (n = 2). Conjugation experiments showed that 21 isolates could transfer NDM-harboring plasmids. The well-conserved blaNDM-5 genetic environment (ISAba125-blaNDM-5/9-bleMBL-trpF-dsbD-IS26) of these plasmids suggested a common genetic origin. Nine sequence types (STs) were detected, including three international high-risk clones ST167 (n = 8), ST410 (n = 1), and ST617 (n = 1). Phylogenetic analysis showed ST167 E. coli from the river was genotypically related to clinical isolates recovered from patients. Furthermore, ST167 isolates showed high genetic similarities with other clinical strains from geographically distinct regions. The genetic concordance between isolates from different sampling sites in the same river (ST218 clone), and different rivers (ST448 clone) raises concerns regarding the rapid dissemination of NDM-EC in the aquatic environment. The emergence and spread of the clinically relevant NDM-positive strains, especially for E. coli ST167 clone, an international high-risk clone associated with multi-resistance and virulence capacity, within and between the hospital and aquatic environments were elucidated, highlighting the need for attention and action.

Dissemination of bla NDM-5 and mcr-8.1 in carbapenem-resistant Klebsiella pneumoniae and Klebsiella quasipneumoniae in an animal breeding area in Eastern China.

Animal farms have become one of the most important reservoirs of carbapenem-resistant Klebsiella spp. (CRK) owing to the wide usage of veterinary antibiotics. "One Health"-studies observing animals, the environment, and humans are necessary to understand the dissemination of CRK in animal breeding areas. Based on the concept of "One-Health," 263 samples of animal feces, wastewater, well water, and human feces from 60 livestock and poultry farms in Shandong province, China were screened for CRK. Five carbapenem-resistant Klebsiella pneumoniae (CRKP) and three carbapenem-resistant Klebsiella quasipneumoniae (CRKQ) strains were isolated from animal feces, human feces, and well water. The eight strains were characterized by antimicrobial susceptibility testing, plasmid conjugation assays, whole-genome sequencing, and bioinformatics analysis. All strains carried the carbapenemase-encoding gene bla NDM-5, which was flanked by the same core genetic structure (IS5-bla NDM-5-ble MBL-trpF-dsbD-IS26-ISKox3) and was located on highly related conjugative IncX3 plasmids. The colistin resistance gene mcr-8.1 was carried by three CRKP and located on self-transmissible IncFII(K)/IncFIA(HI1) and IncFII(pKP91)/IncFIA(HI1) plasmids. The genetic context of mcr-8.1 consisted of IS903-orf-mcr-8.1-copR-baeS-dgkA-orf-IS903 in three strains. Single nucleotide polymorphism (SNP) analysis confirmed the clonal spread of CRKP carrying-bla NDM-5 and mcr-8.1 between two human workers in the same chicken farm. Additionally, the SNP analysis showed clonal expansion of CRKP and CRKQ strains from well water in different farms, and the clonal CRKP was clonally related to isolates from animal farms and a wastewater treatment plant collected in other studies in the same province. These findings suggest that CRKP and CRKQ are capable of disseminating via horizontal gene transfer and clonal expansion and may pose a significant threat to public health unless preventative measures are taken.

Prioritized antibiotics screening based on comprehensive risk assessments and related management strategy in various animal farms.

Antibiotic pollution in the environment caused by animal breeding has become a serious issue. The persistent release of antibiotics with animal waste may lead to antibiotic resistances in the environment, which poses a threat to human health. This study tries to provides a practical method for screening prioritized antibiotics via a comprehensive risk assessment and determination of their major sources, and put forward corresponding regulatory measures for animal industries. We investigated the occurrence and distribution of 20 antibiotics belonging to eight classes, spanning the areas of animal feed, drinking water, and animal feces on 59 animal farms in Shandong Province, China. The results showed that antibiotic contamination was prevalent in different environmental mediums (feed, feces, and drinking water) on these farms. Tetracyclines typically exhibited higher concentrations than the other classes in all samples, and the majority of antibiotics had greater concentrations in cattle feces than in pig- or chicken feces. For the antibiotic ecological risks in feces, doxycycline, tetracycline, and enrofloxacin exhibited much more toxic effects on terrestrial organisms (e.g., wheat, cucumber, and rice). Ciprofloxacin, enrofloxacin, ofloxacin, and tetracycline levels in drinking water samples can lead to high risk of antibiotic resistance, while no antibiotic posed obvious risks to human health. Based on compressive risk assessments, 11 antibiotics were prioritized to control in the animal breeding environment. Based on the survey of feeds, drinking water and animal waste from the farm, roxithromycin in the feces mainly originated from the feeds, while most prioritized antibiotics, were from extra addition in the animal breeding process (including injection and other oral routes). The key point of local antibiotic management in animal farms should be adjusted from the feed factory to the extra addition of antibiotics in animal breeding processes.

Emergence of blaNDM-1, blaNDM-5, blaKPC-2 and blaIMP-4 carrying plasmids in Raoultella spp. in the environment.

To date, carbapenem-resistant Enterobacteriaceae have been found predominantly in clinical settings worldwide. Raoultella belongs to the Enterobacteriaceae family which can cause hospital-acquired infections, and carbapenem-resistant Raoultella spp. (CRR) is sporadically reported in the environment. We investigated the distribution and underlying resistance mechanisms of CRR in a wastewater treatment plant (WWTP) from eastern China between January 2018 and February 2019. A total of 17 CRR were isolated from 324 environmental samples, including Raoultella ornithinolytica (n = 15) and Raoultella planticola (n = 2). The detection of CRR was more frequent in the water inlet compared to anaerobic tank, aerobic tank, sludge thickener, activated sludge, mud cake storage area, and water outlet, and CRR was detected in mud cake stacking area. All CRR were resistant to imipenem, meropenem, ampicillin, piperacillin-tazobactam, cefotaxime, ceftazidime, trimethoprim-sulfamethoxazole and fosfomycin. Four different carbapenemase genes were identified, including blaKPC-2 (n = 13), blaNDM-1 (n = 8), blaNDM-5 (n = 1), blaIMP-4 (n = 1). Interestingly, isolated R. ornithinolytica from the WWTP were closely related to those reported from human samples in China. Plasmid analysis indicated that IncFII(Yp), IncP6, and IncU mediated blaKPC-2 spread, IncX3 and IncN2 mediated blaNDM spread in the environment. The core structure of the Tn3-ISKpn27-blaKPC-2-ISKpn6, ISAba125-blaNDM-bleMBL-trpF-dsbD were identified. The study provides evidence that Raoultella spp. may spread alarming carbapenem resistance in the environment and, therefore, the continuous surveillance for carbapenem resistance in the WWTP should be conducted, especially sludge.

Rapid Degradation of Chlortetracycline Using Hydrodynamic Cavitation with Hydrogen Peroxide.

Chlortetracycline (CTC), which has been frequently detected in surface water, is generated primarily by the discharge of high-concentration CTC wastewater from pharmaceutical and livestock plants. The development of effective CTC degradation technology is critical. In this study, the extent of CTC degradation at 80 mg/L was investigated by combining hydrodynamic cavitation (HC) and hydrogen peroxide (H2O2). The results indicate degradation ratios of 88.7% and 93.8% at 5 and 30 min, respectively. Furthermore, the possible mechanisms of CTC degradation were determined via HPLC-MS. The CTC degradation pathways include ring openings, C-N bond cleavage, demethylation, dehydroxylation, and desaturation in the sole system of HC, and a series of additional reactions, such as glycine conjugation and the cleavage of C-C double bonds, occurs in the binary system of HC + H2O2. Nevertheless, the treated water poses ecological risks and cannot be directly discharged into the environment. Therefore, HC + H2O2 treatment may be a rapid and effective primary method for the degradation of high-concentration CTC in pharmaceutical factories.

Clonal and plasmid-mediated dissemination of environmental carbapenem-resistant Enterobacteriaceae in large animal breeding areas in northern China.

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) constitute a major global health problem. The environment plays an important role in the dissemination of CRE, but large-scale studies on CRE in groundwater environments in animal breeding areas are scarce. The aim of this study was to investigate CRE occurrence and environmental transmission of carbapenem resistance genes in large animal breeding areas in northern China. In total, 280 well water and 102 animal feces samples in large animal breeding areas in six counties from the two provinces Inner Mongolia and Shandong in China, were screened for CRE. A total of 39 CRE were isolated and characterized with next-generation sequencing. 5.3% of well water samples were contaminated with CRE. The well water in chicken farms had the highest number of detections of CRE (15.9%). More than half of the isolates carried closely related, conjugative IncX3 plasmids with blaNDM-genes from multiple geographic areas, indicating that this kind of plasmid plays an important role in dissemination of carbapenem resistance determinants. The clonal expansion of various CRE isolates in well water and animal feces were demonstrated; clonally related CRE were isolated from different wells within the same county, from different counties in the same province, and even from different provinces. In addition to harboring various ARGs, two closely related K. pneumoniae belonging to ST11 isolated from well water carried genetic hypervirulence determinants on a virulence plasmid, highlighting the potential health risk posed by further dissemination of this strain. These findings suggest that groundwater may be an underappreciated reservoir and source of dissemination of CRE, from which resistance genes may disseminate among different bacterial strains and over large geographic distances. Further research and multi-sectorial monitoring, with a "One health" perspective, is urgently needed to investigate the need for interventions aimed at preventing CRE dissemination.

Emergence of IncX3 Plasmid-Harboring blaNDM-5 in a Citrobacter sedlakii Isolated from Outdoor Aerosol in Wastewater Treatment Plant.

A carbapenem-resistant Citrobacter sedlakii strain AA2CS carrying blaNDM-5 was detected in outdoor aerosols of a wastewater treatment plant (WWTP) in China and the whole genome was sequenced subsequently. AA2CS was captured in an aerobic tank with aerosol particles of sizes ranging from 4.7 to 7.0 µm. Besides blaNDM-5, AA2CS also harbored 21 other antibiotic resistance genes and displayed a high level of resistance to ampicillin, cefotaxime, ceftazidime, tetracycline, and meropenem. BlaNDM-5 was located on the IncX3 plasmid (pCSNDM-5) with an IS3000-IS5-blaNDM-5-bleMBL-trpF-dsbD-IS26 structure. pCSNDM-5 was highly homologous to other blaNDM-5-carrying IncX3 plasmids in China and can be transferred to the Escherichia coli recipient J53. To our knowledge, this is the first report of carbapenem-resistant Enterobacteriaceae in outdoor aerosols in WWTPs.

Dissemination of blaNDM-5 via IncX3 plasmids in carbapenem-resistant Enterobacteriaceae among humans and in the environment in an intensive vegetable cultivation area in eastern China.

The environment of a large-scale vegetable production area can be exposed to antibiotic residues and antibiotic-resistant bacteria (ARB) via animal manure and irrigation with contaminated water, which can facilitate the dissemination of ARB. However, the occurrence of ARB in plantation areas and their dissemination in this environment remain largely unexplored. In total, 382 samples including those from vegetable (n = 106), soil (n = 87), well water (n = 24), river water (n = 20), river sediments (n = 20), farmer feces (n = 58) and farmer hands (n = 67) were collected in 2019 from a large-scale cultivation area in Shandong, China. Selective agar plates were used to screen for carbapenem-resistant Enterobacteriaceae (CRE) and whole-genome sequencing and Southern blotting were used to characterise isolates and mobile genetic elements carrying carbapenem resistance determinants. A total of nine NDM-5-producing isolates of Escherichia coli, Klebsiella pneumoniae, and Citrobacter spp. were identified from environmental sources and human feces, all of which were multidrug-resistant. Single nucleotide polymorphism analysis suggested clonal transmission of carbapenem-resistant Citrobacter sedlakii within greenhouse soils in the area. Eight of the isolates carried closely related or identical IncX3 plasmids carrying blaNDM-5, which were shown to be conjugative via filter mating experiments, indicating the highly transmissible nature of this genetic element. Isolates of E. coli and Citrobacter freundii were detected in the feces of local farm workers and contained similar IncX3 plasmids with blaNDM-5 environmental isolates, suggesting a potential risk of CRE transfer from the work environment to the farm workers. Thus, further research is required to investigate the potential health risks associated with environmental exposure to CRE in vegetable cultivation areas.

Genetic characterization and virulence of a carbapenem-resistant Raoultella ornithinolytica isolated from well water carrying a novel megaplasmid containing blaNDM-1.

Infections caused by carbapenem-resistant Enterobacteriaceae are a growing concern worldwide. Raoultella ornithinolytica is a species in the Enterobacteriaceae family which can cause hospital-acquired infections and is sporadically reported as carbapenem-resistant from human and environmental sources. In this study, we firstly report on an NDM-1-producing R. ornithinolytica, Rao166, isolated from drinking water in an animal cultivation area in China. In addition to carbapenem-resistance, Rao166 was resistant to several other antibiotics including gentamicin, sulfamethoxazole-trimethoprim, tetracycline and fosfomycin. Rao166 carried a novel IncFIC-type megaplasmid, 382,325 bp in length (pRAO166a). A multidrug resistance region, 60,600 bp in length, was identified in the plasmid containing an aac(3)-IId-like gene, aac(6')-Ib-cr, blaDHA-1, blaTEM-1B, blaCTX-M-3, blaOXA-1, blaNDM-1, qnrB4, catB3, arr-3, sul1, and tet(D). Results from virulence assays implied that Rao166 has considerable pathogenic potential. Although pRAO166a was found to be non-transmissible, dissemination of the NDM-1 producing strain may occur from well water to humans or animals through cross-contamination during food preparation or directly via drinking water, and potentially lead to difficult-to-treat infections. Thus, contamination of well water by this carbapenem-resistant and presumptively virulent strain of R. ornithinolytica should be considered a potential public health risk.

Characterization of Chromosome-Mediated BlaOXA-894 in Shewanella xiamenensis Isolated from Pig Wastewater.

A new variant of the blaOXA-546 gene, namely blaOXA-894, was identified on the chromosome of Shewanella xiamenensis isolated from pig wastewater in rural China. OXA-894 differs from OXA-546 (A46V, I219del) and OXA-48 (T167I, I219del) with two amino acid substitutions, respectively. The isolate was resistant to ampicillin, aztreonam, imipenem, meropenem and fosfomycin. Carba NP test confirmed S. xiamenensis strain sx20 as a carbapenemase-producer. The blaOXA-894 gene was located between the gene encoding a LysR family transcriptional regulator and the C15 gene. Its gene environment was similar to other S. xiamenensis with chromosome-located blaOXA-48-like genes. The T24H and T94V amino acid substitutions of LuxS protein were predicted to be deleterious, which may affect the virulence phenotype. The occurrence and potential health risk of carbapenem-resistant S. xiamenensis in a water environment is of concern.

Genomic Analysis Of A KPC-2-Producing Klebsiella Pneumoniae ST11 Outbreak From A Teaching Hospital In Shandong Province, China.

PURPOSE: Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bacteria causes nosocomial infections worldwide. However, KPC-producing K. pneumoniae outbreak has never been reported in Shandong Province, China. The purpose of our study was to elucidate the epidemiological and drug resistance mechanisms of KPC-producing K. pneumoniae strains collected from a large teaching hospital in Shandong during the outbreak. Moreover, we attempted to characterize the genetic environment and phylogenetic analysis of bla KPC-2 in outbreak isolates. METHODS: We monitored a 64-day outbreak of infection in a general hospital in Shandong Province, and the bacteria causing the infection were all ST11-type K. pneumoniae. The genotype correlation of KPC-producing K. pneumoniae isolates was assessed by whole-genome sequencing (WGS) phylogenetic analysis. Subsequent studies included antibiotic susceptibility testing, multilocus sequence typing (MLST) and S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blot hybridization. RESULTS: From February 1, 2018 to April 5, 2018, 14 KPC-producing K. pneumoniae isolates from different wards were collected. All 14 isolates were resistant to carbapenems and carried the extended-spectrum ß-lactamase (ESBL) gene as well as fosA, and sul genes. Whole-genome analysis showed that all 14 the outbreak isolates were all ST11 type. The bla KPC-2 carrying plasmids were all belong to IncFIIK2 type, and the size ranged from 94 kb to 368 kb. CONCLUSION: As far as we know, this report first describes the genomics characterization of KPC-2-producing K. pneumoniae outbreak isolates from Shandong Province, China. In our study, these isolates appeared to be cloned, and ST11 K. pneumoniae was the major clone caused the outbreak. Therefore, routine surveillance of such strains in this region is urgently warranted.

Dissemination of extended-spectrum ß-lactamase-producing Escherichia coli carrying mcr-1 among multiple environmental sources in rural China and associated risk to human health.

Antibiotic resistance among gram-negative bacteria is increasingly becoming a problem of global concern. Particularly problematic is the emergence of resistance to last-resort antibiotics such as carbapenems and colistin. The increasing number of reports on the plasmid-mediated colistin resistance gene mcr-1 in isolates worldwide is raising concerns for the future usefulness of this class of antibiotics. Dissemination of mcr-1 is believed to have originated mainly from animal breeding, however, the role of the environment as a transmission source is not yet fully understood. In the current study, 89 extended-spectrum ß-lactamase-producing Escherichia coli isolated from 231 samples from different environmental sources in 12 villages in a rural area of Shandong, China, were screened for mcr-1. 17 (19.1%) mcr-1-positive isolates were found from different environmental sources, aggregated in 6 villages. Plasmids of three different Inc-groups carrying mcr-1 were confirmed, indicating that the widespread geographical distribution of mcr-1 in the local area is due to a number of different plasmids. Additionally, almost a third (29.4%) of the isolates carried virulence factors associated to intestinal pathogenic E. coli. These results illustrate the high complexity of the transmission patterns of mcr-1 among different environmental matrices on a local scale and the potential for the environment to facilitate dissemination and emergence of antibiotic-resistant and virulent strains of bacteria.

Evaluating Dissemination Mechanisms of Antibiotic-Resistant Bacteria in Rural Environments in China by Using CTX-M-Producing Escherichia coli as an Indicator.

It is becoming increasingly recognized that the environment plays an important role both in the emergence and in dissemination of antibiotic-resistant bacteria (ARB), Mechanisms and factors facilitating this development are, however, not yet well understood. The high detection rate of CTX-M genes in environmental sources provides an opportunity to explore this issue. In this study, 88 CTX-M-producing Escherichia coli were isolated from 30 pig feces samples from 30 pig farms and 201 environmental samples. CTX-M-producing E. coli was detected with the following frequencies in the different types of samples: pig feces, 73%; river water, 64%; river sediment, 52%; wastewater, 31%; drinking water, 23%; outlet sediment, 21%; soil, 17%; and vegetables, 4.4%. Dissemination of CTX-M-producing E. coli to different environmental matrices was evaluated by analyzing the genetic relatedness of isolates from different environmental sources, and putative transmission routes through bird feces, pig feces, drinking water, river sediment, river water, and wastewater were hypothesized. Dissemination through these routes is likely facilitated by anthropogenic activities and environmental factors. Wild birds as potential vectors for dissemination of CTX-M-producing E. coli have the capacity to spread ARB across long distances. Regional dissemination between different environmental matrices of CTX-M-producing E. coli increases the exposure risk of humans and animals in the area.

Characterization of Clinically Relevant Strains of Extended-Spectrum ß-Lactamase-Producing Klebsiella pneumoniae Occurring in Environmental Sources in a Rural Area of China by Using Whole-Genome Sequencing.

Klebsiella pneumoniae is a gram-negative, opportunistic pathogen, and a common cause of healthcare-associated infections such as pneumonia, septicemia, and urinary tract infection. The purpose of this study was to survey the occurrence of and characterize K. pneumoniae in different environmental sources in a rural area of Shandong province, China. Two hundred and thirty-one samples from different environmental sources in 12 villages were screened for extended-spectrum ß-lactamase-(ESBL)-producing K. pneumoniae, and 14 (6%) samples were positive. All isolates were multidrug-resistant and a few of them belonged to clinically relevant strains which are known to cause hospital outbreaks worldwide. Serotypes, virulence genes, serum survival, and phagocytosis survival were analyzed and the results showed the presence of virulence factors associated with highly virulent clones and a high degree of phagocytosis survivability, indicating the potential virulence of these isolates. These results emphasize the need for further studies designed to elucidate the role of the environment in transmission and dissemination of ESBL-producing K. pneumoniae and the potential risk posed to human and environmental health.