Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Jurong Lake, Singapore with Whole-Genome-Sequencing

Background: The fast-spreading of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli (ESBL-producing E. coli) and ESBL genes has become a big challenge to public health. The risk of spreading ESBL genes and pathogens in the environment and community has raised public health concern. The characterizing and whole-genome sequencing studies of ESBL-producing bacteria from reservoir water in Singapore is still limited. Materials and methods: The reservoir water sample was taken from two randomly selected sampling points of the Chinese Garden (Jurong river reservoir), which is a popular reservoir park in Singapore. The bacteria of the water sample were collected with 0.45 µm filter membranes and enriched before processing with ESBL-producing E. coli screening. The collected ESBL positive isolates were further characterized by both phenotypic tests including disc diffusion and microdilution Minimum Inhibitory Concentration (MIC) test, and also genotypic test as whole-genome sequencing analysis. Besides, to investigate the transferability of the resistance gene, a conjugation test was performed with the J53 E. coli strain as the gene receptor. Result: Nine ESBL-producing E. coli isolates were collected and confirmed as ESBL-producing with both phenotypic and genotypic tests. A potential pathogen as ST131 clade A isolate was identified, and all isolates were determined to harbor a blaCTX-M gene. Among them, strain J1E4 was resistant to polymyxin E and confirmed to harboring a conjugatable mcr-1 gene. Further genetic environment analysis has reflected a conversed gene cluster formed by insert sequence (IS), blaCTX-M-15, and WbuC family cupin-fold metalloprotein, which may potentially jump from the plasmids to the chromosome. Conclusion: The first time we reported the whole genome sequencing (WGS) data of ESBL-producing E. coli including potential pathogen (ST131) present in reservoir water in Singapore. The ESBL-producing E. coli from reservoir water also carrying conjugatable colistin resistance genes which may become a risk to human health

Characterization of extended-spectrum beta-lactamase-producing escherichia coli isolates from Jurong Lake, Singapore with whole-genome-sequencing

Background: The fast-spreading of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli (ESBL-producing E. coli) and ESBL genes has become a big challenge to public health. The risk of spreading ESBL genes and pathogens in the environment and community has raised public health concern. The characterizing and whole-genome sequencing studies of ESBL-producing bacteria from reservoir water in Singapore is still limited. Materials and methods: The reservoir water sample was taken from two randomly selected sampling points of the Chinese Garden (Jurong river reservoir), which is a popular reservoir park in Singapore. The bacteria of the water sample were collected with 0.45 µm filter membranes and enriched before processing with ESBL-producing E. coli screening. The collected ESBL positive isolates were further characterized by both phenotypic tests including disc diffusion and microdilution Minimum Inhibitory Concentration (MIC) test, and also genotypic test as whole-genome sequencing analysis. Besides, to investigate the transferability of the resistance gene, a conjugation test was performed with the J53 E. coli strain as the gene receptor. Result: Nine ESBL-producing E. coli isolates were collected and confirmed as ESBL-producing with both phenotypic and genotypic tests. A potential pathogen as ST131 clade A isolate was identified, and all isolates were determined to harbor a blaCTX-M gene. Among them, strain J1E4 was resistant to polymyxin E and confirmed to harboring a conjugatable mcr-1 gene. Further genetic environment analysis has reflected a conversed gene cluster formed by insert sequence (IS), blaCTX-M-15, and WbuC family cupin-fold metalloprotein, which may potentially jump from the plasmids to the chromosome. Conclusion: The first time we reported the whole genome sequencing (WGS) data of ESBL-producing E. coli including potential pathogen (ST131) present in reservoir water in Singapore. The ESBL-producing E. coli from reservoir water also carrying conjugatable colistin resistance genes which may become a risk to human health.Nanyang Technological UniversityPublished versionThis research was supported by Nanyang Technological University, Singapore

Conjugative IncX1 plasmid harboring colistin resistance gene mcr-5.1 in Escherichia coli isolated from chicken rice retailed in Singapore

Colistin is regarded as one of the last-resort antimicrobials for treatment of Gram-negative bacterial infections (1). Several cases of plasmid-borne colistin resistance genes mcr-1, mcr-3-like, and mcr-4.2 in clinical Enterobacteriaceae isolates, including Escherichia coli, have been reported in Singapore (2-4). However, the mcr-5 gene has not been reported in clinical isolates in Singapore. Previously, we reported the antimicrobial resistance (AMR) genotype and phenotype of E. coli SGEHI2010ENV103 isolates from ready-to-eat food in Singapore and documented the first isolate carrying mcr-5.1 in Singapore (5). In this study, we further analyzed this isolate. Here, we report the first complete nucleotide sequence of a transferable plasmid harboring mcr-5.1 in E. coli isolated from ready-to-eat chicken rice in Singapore. (Chicken rice is a common dish in Singapore which is composed of cooked chicken and seasoned rice, served with sauce and cucumber garnishes).Published versio

Phenotypic and genotypic characterization of antimicrobial resistant Escherichia coli isolated from ready-to-eat food in Singapore using disk diffusion, broth microdilution and whole genome sequencing methods

This study aimed to determine the antimicrobial resistance (AMR) profiles of Escherichia coli isolated from ready-to-eat (RTE) food sold in retail food premises in Singapore. In this study, a total of 99 E. coli isolates from poultry-based dishes (n = 77) and fish-based dishes (n = 22), obtained between 2009 and 2014, were included for disk diffusion testing. Of the 99 isolates, 24 (24.2%) were resistant to at least one antimicrobial agent. These isolates were then subjected to broth microdilution testing against 33 antimicrobial agents, including beta-lactams, aminoglycosides, tetracycline, fluoroquinolones and polymyxin E (also known as colistin) to determine the minimum inhibitory concentration (MIC) of isolates. Finally, whole genome sequence (WGS) was carried out on the strains in order to correlate resistant phenotypes to putative antimicrobial-related genes. Of the 24 isolates, 15 (62.5%) were found to be resistant to three or more classes of antimicrobials and thus were defined as multidrug resistant strains. Two isolates (8.3%) were confirmed as Extended-Spectrum Beta-Lactamase (ESBL)-producing E. coli by double-disk synergy test. Based on WGS data, online analysis tool ResFinder detected 7 classes of AMR genes and resistance-related chromosomal point mutations in 19 of the 24 E. coli isolates. Prediction of AMR using WGS data was evaluated for six antimicrobials including ampicillin, chloramphenicol, colistin, fluoroquinolones, tetracycline and trimethoprim. By analyzing the WGS contigs using BLASTn and KmerFinder, quinolone resistance genes, ESBL genes and transferable colistin resistance gene mcr-1 and mcr-5 were determined to be located on plasmids, which could pose a greater risk of AMR transfer among bacteria. Mutations were detected in four isolates within genes previously shown to confer resistance to quinolones (gyrA and parE) and tetracycline (rrsB). This study showed the presence of AMR E. coli isolates in RTE food, and raises a concern on the possible transmission of AMR bacteria from food to humans.Nanyang Technological UniversityNational Environmental Agency (NEA)This study was supported by the National Environment Agency (NEA) and Nanyang Technological University Research Initiative. Authors would like to thank Man Ling Chau and Ramona Alikiiteaga Gutiérrez for manuscript vetting, and thank Food Hygiene team members in the Environmental Health Institute of NEA for providing isolates and experimental guidance

Occurrence and antimicrobial resistance traits of Escherichia coli from wild birds and rodents in Singapore

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) poses a public health concern worldwide. Wild birds and rodents, due to their mobility, are potential vehicles for transmission of AMR bacteria to humans. Ninety-six wild birds’ faecal samples and 135 rodents’ droppings samples were collected and analysed in 2017. Forty-six E. coli isolates from wild birds and rodents were subjected to AMR phenotypic and genotypic characterisation. The proportion of E. coli isolates resistant to at least one of the antimicrobials tested from wild birds (80.8%) was significantly higher than that of isolates from rodents (40.0%). The proportion of E. coli isolates resistant to each antimicrobial class for wild birds was 3.8% to 73.1% and that for rodents was 5.0% to 35.0%. Six out of 26 E. coli isolates from wild birds (23.1%) and two out of 20 (10.0%) isolates from rodents were multi-drug resistant (MDR) strains. These MDR E. coli isolates were detected with various antimicrobial resistance genes such as blaTEM-1B and qnrS1 and could be considered as part of the environmental resistome. Findings in this study suggested that wild birds and rodents could play a role in disseminating antimicrobial resistant E. coli, and this underscores the necessity of environment management and close monitoring on AMR bacteria in wild birds and rodents to prevent spreading of resistant organisms to other wildlife animals and humans.Nanyang Technological UniversityNational Environmental Agency (NEA)Published versionThe authors thank the Rodent Control Unit, of the Central Regional Office, National Environment Agency for the collection of rodent droppings. In addition, the authors thank collaborators from the National Parks Board Singapore, and the Wildlife Reserves Singapore who provided the wild birds samples (carcasses). Lastly, the authors thank Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University for providing whole genome sequencing services