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.

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Objectives To determine the prevalence and genetic characteristics of ESBL-producing Escherichia coli in retail raw meats from Singapore markets. Methods A total of 634 raw meat (chicken, pork and beef) samples were collected from markets in Singapore during June 2017–October 2018. The samples were enriched overnight and then incubated on Brilliance™ ESBL Agar. Presumptive ESBL isolates were confirmed using the double-disc synergy test. Confirmed ESBL-producing E. coli were sent for WGS and bioinformatic analysis was performed. Results The prevalence of ESBL-producing E. coli in chicken, pork and beef meats was 51.2% (109/213), 26.9% (58/216) and 7.3% (15/205), respectively. A total of 225 ESBL-producing E. coli were isolated from 184 samples. β-Lactam resistance genes were detected in all isolates. After β-lactam resistance genes, the most common antimicrobial resistance genes detected were aminoglycoside resistance genes (92.4%). One hundred and seventy-two (76.4%), 102 (45.3%) and 52 (23.1%) isolates carried blaCTX-M genes, blaTEM genes and blaSHV genes, respectively. blaCTX-M-55 (57/225, 25.3%) and blaCTX-M-65 (40/225, 17.8%) were the most frequent ESBL genes. Colistin resistance genes (including mcr-1, mcr-3 and mcr-5) were found in 15.6% of all isolates. Conclusions This study indicates that ESBL-producing E. coli are widely found in retail raw meats, especially chicken, in Singapore. Occurrence of MDR (resistance to at least three classes of antimicrobial) and colistin resistance genes in retail raw meat suggests potential food safety and public health risks.

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.

C olistin is regarded as one of the last-resort antimicrobials for treatment of Gramnegative 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.) This E. coli isolate was obtained through the retail food surveillance program by the National Environmental Agency (NEA). It was isolated from retail chicken rice from a hawker center in Singapore in 2010. The MIC of colistin for this isolate was further determined to be 8 g/ml using broth dilution assay as described by CLSI (6). Doubledisk synergy testing was performed as previously described (5), and no extendedspectrum-␤-lactamase (ESBL) phenotype was detected. Conjugation experiments were performed using the filter mating method (7). Sodium azide-resistant E. coli strain J53 was the recipient strain, and transconjugants were selected using 4 g/ml colistin plus 200 g/ml sodium azide. PCR confirmed the presence of mcr-5 in transconjugants after 24 h of coculture (8), suggesting that the mcr-5.1 genes were able to be transferred to recipient E. coli strain J53. The transfer frequency determined was 10 Ϫ6. This suggests that this mcr-5.1-carrying plasmid might have the potential to transfer to other strains. The stability test for the plasmid carrying mcr-5.1 was performed as previously described (9). The plasmid was still stable after 20 successive days of subculture in LB broth (ϳ200 generations) without colistin selection, demonstrating the stability of the plasmid. The drug MICs of the transconjugant and E. coli J53 were determined using MicroScan Neg MIC panel type 44 (Beckman Coulter, Inc., Brea, CA, USA), in accordance with the manufacturer's instructions (data not shown). The colistin MIC of transconjugants was further determined by broth dilution assay, and the results showed an 8-fold increase (from 1 to 8 g/ml).