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Analysis of Wastewater Reveals the Spread of Diverse Extended-Spectrum β-Lactamase-Producing E. coli Strains in uMgungundlovu District, South Africa.

ABSTRACT: Wastewater treatment plants (WWTPs) are major reservoirs of antibiotic-resistant bacteria (ARB), favouring antibiotic resistance genes (ARGs) interchange among bacteria and they can provide valuable information on ARB circulating in a community. This study characterised extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli from the influent and effluent of four WWTPs in uMgungundlovu District, KwaZulu-Natal, South Africa. E. coli was enumerated using the membrane filtration method and confirmed using the API 20E test and real-time polymerase chain reaction. ESBL-producers were phenotypically identified by their susceptibility to the third-generation cephalosporins using the disc diffusion and the double-disc synergy methods against cefotaxime (30 µg) with and without 10 µg clavulanic acid. Genotypic verification was by PCR of the TEM, SHV, and CTX-M genes. The clonality of isolates was assessed by ERIC-PCR. The highest E. coli count ranged between 1.1 × 10⁵ (influent) and 4.3 × 10³ CFU/mL (effluent). Eighty pure isolates were randomly selected, ten from the influent and effluent of each of the four WWTP. ESBLs were phenotypically confirmed in 49% (n = 39) of the isolates, of which 77% (n = 30) were genotypically confirmed. Seventy-three percent of the total isolates were multidrug-resistant (MDR). Only two isolates were susceptible to all antibiotics. Overall, resistance to first and second-generation cephalosporins was higher than to third and fourth generation cephalosporins. Also, 15% of the isolates were resistant to carbapenems. The CTX-M-type ESBL (67%; n = 20) was the most common ESBL antibiotic resistance gene (ARG) followed by TEM (57%; n = 17) and SHV-types (27%; n = 8). Also, a substantial number of isolates simultaneously carried all three ESBL genes. ERIC-PCR revealed a high diversity of isolates. The diversity of the isolates observed in the influent samples suggest the potential circulation of different ESBL-producing strains within the studied district, requiring a more comprehensive epidemiological study to prevent the spread of ESBL-producing bacteria within impoverished communities.

SUBMITTER: Gumede SN

PROVIDER: S-EPMC8300763 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeThe isolation rate of extended spectrum β-lactamase (ESBL)-producing Escherichia coli is increasing, posing a challenge to clinical anti-infective therapy. This study aims to provide new insight into the genomic characteristics and antimicrobial resistance mechanisms of extended spectrum β-lactamase producing E. coli isolates recovered from a district hospital in China.MethodsA total of 36 ESBL-producing E. coli isolates were collected from body fluid samples from a Chinese district hospital. All isolates were subjected to whole genome sequencing to identify their antimicrobial resistance genes, virulence genes, serotypes, sequence types, and phylogenetic relationships by BacWGSTdb 2.0 webserver.ResultsAmong these isolates, all were resistant to cefazolin, cefotaxime, ceftriaxone, ampicillin, 24 (66.7%) were resistant to aztreonam, 16 (44.4%) were resistant to cefepime, and 15 were resistant (41.7%) to ceftazidime. The blaCTX-M gene was detected in all ESBL-producing E. coli isolates. Two isolates carrying two different types of blaCTX-M genes simultaneously. The carbapenem resistance gene blaKPC-2 was detected in one (2.8%) isolate. A total of 17 sequence types (STs) were found, with ST131 accounting for the majority (n =13; 36.1%). The most common serotype was O16:H5 associated with seven ST131 strains, followed by O25:H4/ST131 (n = 5) and O75:H5/ST1193 (n = 5). Evaluation of clonal relatedness revealed that all blaCTX-M gene-carrying E. coli had a difference of SNPs range from 7 to 79,198, which could be divided into four clusters. Only 7 SNPs could be found between EC266 and EC622, indicating that they are variants of the same clonal lineage.ConclusionThis study investigated the genomic characteristics of ESBL-producing E. coli isolates recovered from a district hospital in China. Continuous surveillance of ESBL-producing E. coli infections is imperative to create efficient strategies for controlling the transmission of these multi-drug resistant bacteria in clinical and community settings.

Project description:Escherichia coli significantly causes nosocomial infections and rampant spread of antimicrobial resistance (AMR). There is limited data on genomic characterization of extended-spectrum β-lactamase (ESBL)-producing E. coli from African clinical settings. This hospital-based longitudinal study unraveled the genetic resistance elements in ESBL E. coli isolates from Uganda and Tanzania using whole-genome sequencing (WGS). A total of 142 ESBL multi-drug resistant E. coli bacterial isolates from both Tanzania and Uganda were sequenced and out of these, 36/57 (63.1%) and 67/85 (78.8%) originated from Uganda and Tanzania respectively. Mutations in RarD, yaaA and ybgl conferring resistances to chloramphenicol, peroxidase and quinolones were observed from Ugandan and Tanzanian isolates. We reported very high frequencies for blaCTX-M-15 with 11/18(61.1%), and blaCTX-M-27 with 12/23 (52.1%), blaTEM-1B with 13/23 (56.5%) of isolates originating from Uganda and Tanzania respectively all conferring resistance to Beta-lactam-penicillin inhibitors. We observed chloramphenicol resistance-conferring gene mdfA in 21/23 (91.3%) of Tanzanian isolates. Extraintestinal E. coli sequence type (ST) 131 accounted for 5/59 (8.4%) of Tanzanian isolates while enterotoxigenic E. coli ST656 was reported in 9/34 (26.4%) of Ugandan isolates. Virulence factors originating from Shigella dysenteriae Sd197 (gspC, gspD, gspE, gspF, gspG, gspF, gspH, gspI), Yersinia pestis CO92 (irp1, ybtU, ybtX, iucA), Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (csgF and csgG), and Pseudomonas aeruginosa PAO1 (flhA, fliG, fliM) were identified in these isolates. Overall, this study highlights a concerning prevalence and diversity of AMR-conferring elements shaping the genomic structure of multi-drug resistant E. coli in clinical settings in East Africa. It underscores the urgent need to strengthen infection-prevention controls and advocate for the routine use of WGS in national AMR surveillance and monitoring programs.Availability of WGS analysis pipeline: the rMAP source codes, installation, and implementation manual can free be accessed via https://github.com/GunzIvan28/rMAP .

Project description:The wastewater of livestock slaughterhouses is considered a source of antimicrobial-resistant bacteria with clinical relevance and may thus be important for their dissemination into the environment. To get an overview of their occurrence and characteristics, we investigated process water (n = 50) from delivery and unclean areas as well as wastewater (n = 32) from the in-house wastewater treatment plants (WWTPs) of two German poultry slaughterhouses (slaughterhouses S1 and S2). The samples were screened for ESKAPE bacteria (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Escherichia coli Their antimicrobial resistance phenotypes and the presence of extended-spectrum-β-lactamase (ESBL), carbapenemase, and mobilizable colistin resistance genes were determined. Selected ESKAPE bacteria were epidemiologically classified using different molecular typing techniques. At least one of the target species was detected in 87.5% (n = 28/32) of the wastewater samples and 86.0% (n = 43/50) of the process water samples. The vast majority of the recovered isolates (94.9%, n = 448/472) was represented by E. coli (39.4%), the A. calcoaceticus-A. baumannii (ACB) complex (32.4%), S. aureus (12.3%), and K. pneumoniae (10.8%), which were widely distributed in the delivery and unclean areas of the individual slaughterhouses, including their wastewater effluents. Enterobacter spp., Enterococcus spp., and P. aeruginosa were less abundant and made up 5.1% of the isolates. Phenotypic and genotypic analyses revealed that the recovered isolates exhibited diverse resistance phenotypes and β-lactamase genes. In conclusion, wastewater effluents from the investigated poultry slaughterhouses exhibited clinically relevant bacteria (E. coli, methicillin-resistant S. aureus, K. pneumoniae, and species of the ACB and Enterobacter cloacae complexes) that contribute to the dissemination of clinically relevant resistances (i.e., blaCTX-M or blaSHV and mcr-1) in the environment.IMPORTANCE Bacteria from livestock may be opportunistic pathogens and carriers of clinically relevant resistance genes, as many antimicrobials are used in both veterinary and human medicine. They may be released into the environment from wastewater treatment plants (WWTPs), which are influenced by wastewater from slaughterhouses, thereby endangering public health. Moreover, process water that accumulates during the slaughtering of poultry is an important reservoir for livestock-associated multidrug-resistant bacteria and may serve as a vector of transmission to occupationally exposed slaughterhouse employees. Mitigation solutions aimed at the reduction of the bacterial discharge into the production water circuit as well as interventions against their further transmission and dissemination need to be elaborated. Furthermore, the efficacy of in-house WWTPs needs to be questioned. Reliable data on the occurrence and diversity of clinically relevant bacteria within the slaughtering production chain and in the WWTP effluents in Germany will help to assess their impact on public and environmental health.

Project description:The plasmid-mediated fosfomycin resistance gene fosA3 has been detected in Enterobacterales from various sources but has rarely been reported in vegetables. In this study, the aim was to investigate the prevalence of and, subsequently, to characterize fosA3-positive Enterobacterales isolates from retail vegetables. Seventeen (7.3%) fosA3-carrying strains were identified from 233 extended-spectrum-?-lactamase-producing Enterobacterales isolates from vegetables. All 17 isolates, including six Escherichia coli, seven Klebsiella pneumoniae, two Raoultella ornithinolytica, and two Citrobacter freundii isolates, carried bla CTX-M S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and hybridization confirmed that the fosA3 genes in 16 isolates were located on plasmids ranging in size from ?40?kb to ?250?kb, except one located on chromosome of C. freundii All the fosA3-carrying plasmids from 16 fosA3-positive isolates were successfully transferred into the recipient bacteria by transformation or conjugation. In agreement with data determined with isolates from food animals, the IncHI2/ST3 and IncN-F33:A-:B-/F33:A-:B plasmids were the main vectors of fosA3 in E. coli Additionally, F24:A-:B1, IncFIIK-IncR, IncFIIS, IncR, and two untypeable plasmids were found for the first time to be vectors for fosA3 in Enterobacterales The genetic contexts of fosA3 in 15 Enterobacterales isolates differed due to insertion and/or loss of molecular modules mediated by mobile elements. However, all fosA3 genes were flanked by IS26, as commonly observed in other fosA3-carrying plasmids. Here, we report a high rate of detection of fosA3 genes, mediated by multiple plasmid vectors, in ESBL-producing Enterobacterales from retail vegetables. FosA3-producing Enterobacterales could be transmitted to the human body by direct contact or consumption of vegetables, which might pose a potential threat to public health.IMPORTANCE This report provides important information on the transmission and epidemiology of fosA3 among Enterobacterales isolates from vegetables. The rate of occurrence of fosA3 in ESBL-producing Enterobacterales from retail vegetables is high, and fosA3 was found to be carried by diverse plasmids. Some novel genetic contexts of fosA3 and novel fosA3-carrying plasmids, including several plasmid types common in K. pneumoniae, were identified, increasing the number of known transfer vectors for the fosA3 gene and reflecting the complexity of fosA3 transmission in Enterobacterales The capture of fosA3 by the resident plasmid of K. pneumoniae will accelerate the spread of fosA3 in K. pneumoniae, one of the most pathogenic species in clinical medicine. Considering the clinical importance of fosfomycin, and the fact that vegetables are directly consumed, the fosfomycin resistance genes present a risk of transmission to the human body through the food chain and thus pose a threat to public health.

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Analysis of Wastewater Reveals the Spread of Diverse Extended-Spectrum β-Lactamase-Producing E. coli Strains in uMgungundlovu District, South Africa.

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