{'fraction_non_alphanumeric': 0.0380125284738041, 'fraction_numerical': 0.02035876993166287, 'mean_word_length': 4.865970772442589, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Prevalence Of mcr-1 Among Cefotaxime-Resistant Commensal Escherichia coli In Residents Of Vietnam

Purpose The dissemination of colistin-resistant bacteria harboring the colistin-resistance gene mcr-1 in developing countries has recently entered the spotlight as an emerging public health threat, which is attributed to the abuse of colistin use in these countries. However, the prevalence of these bacteria in developing countries has not been extensively investigated. Therefore, in the present study, we examined the prevalence of cefotaxime-resistant commensal Escherichia coli harboring mcr-1 among residents of a representative Vietnamese village and assessed the characteristics of these isolates. Materials and methods The stool samples, one stool sample per resident, of 612 residents were cultured on MacConkey agar with cefotaxime. Resulting E. coli-like colonies were isolated and examined further for the presence of colistin-resistant extended-spectrum β-lactamase (ESBL)-producing E. coli with mcr-1. Antibiotic susceptibility tests were performed, and clonal relationship among colistin-resistant isolates was assessed. Results Thirty-one of the 451 cefotaxime-resistant E. coli isolates were resistant to colistin and the majority possessed mcr-1, blaCTX-M, and/or blaTEM, except for two isolates that produced the AmpC β-lactamase. All mcr-1 ESBL-producing E. coli isolates were multidrug-resistant (5–11 antibiotics). The isolates contained various plasmid replicon types, including the most prevalent types IncHI2 (54.8%), IncFIB (48.4%), and IncN (41.9%). In addition, 83.9% of the mcr-1 ESBL-E. coli isolates possessed a transposon ISApl1-mcr-1 segment. Furthermore, 77.4% of the mcr-1 ESBL-E. coli isolates belonged to phylogenetic group A. Pulsed-field gel electrophoresis analysis indicated limited clonal expansion of a specific strain. Conclusion These results demonstrate the wide dissemination of colistin-resistant ESBL-E. coli harboring mcr-1 among commensal bacteria of rural residents in Vietnam, suggesting possible mobilization of the mcr-1 gene among ESBL-producing microbiota, which is a great public health concern.


Introduction
Colistin has been recognized as a last-resort antibiotic for the treatment of intractable infections involving multidrug-resistant (MDR) gram-negative bacteria, especially carbapenem-resistant MDR bacteria. 1 However, the increasing usage of colistin in food-producing animals has resulted in a rising prevalence of colistin-resistant bacteria. 2 Moreover, the mobile colistin-resistant gene mcr-1 was recently discovered in Escherichia coli isolates from animals and humans in China. 3 To date, MCR-1producing E. coli strains have been reported in many countries throughout Europe, Asia, and South and North America. [4][5][6] Therefore, the possible global dissemination of mcr-1-positive bacteria has become a serious public health concern.
Although the characteristics of animal and human E. coli isolates harboring mcr-1 have been examined in detail, 1,3,4,7,8 there is limited comprehensive information regarding the prevalence and characteristics of colistin-resistant bacteria harboring mcr-1 among human residents of developing countries. Because the human intestinal flora is an important reservoir of resistance genes, 9,10 the prevalence and characteristics of colistin-resistance in human commensal E. coli should be taken into account in prevention and control efforts. Therefore, the aim of this study was to explore the prevalence of mcr-1-positive E. coli in the feces of residents of a local community in Thai Binh province, Vietnam, which is known to be widely contaminated with extended-spectrum β-lactamase (ESBL)-producing bacteria, 11 and to investigate the molecular characteristics of these strains. These data should provide a useful resource for understanding current threats in the region to help initiate appropriate screening, treatment, and control.

Specimen Collection
The bacterial strains used in this study were originally isolated from residents for assessment of the dissemination of ESBL-producing bacteria in Nguyen Xa village, Thai Binh province, Vietnam, from September 2013 to February 2016. The village is a representative rural community in this province, with a reported population of 7730 residents in 2008 households at the time of the study. A total of 612 asymptomatic healthy resident volunteers who had not received any medical treatment for the last 3 months participated in the study. One stool specimen was obtained from each participant using a transport swab with Cary-Blair transport medium (Eiken Chemical, Tokyo, Japan). The study was approved by the ethics committees of Osaka University and Thai Binh University of Medicine and Pharmacy. All participants provided written informed consent and that this was conducted in accordance with the Declaration of Helsinki.

Detection Of ESBL-And AmpC-Producing E. coli
The presence of ESBLs was confirmed using the doubledisc synergy test using CTX and ceftazidime with and without clavulanic acid, as recommended by the Clinical and Laboratory Standards Institute (CLSI 2014, Wayne, PA, USA). Cefoxitin (FOX)-resistant strains were classified as putative AmpC-producing E. coli.
Genetic detection and genotyping of β-lactamases were performed by PCR using boiled bacterial suspensions in Tris-EDTA buffer as the template. PCR analysis was performed with universal primers specific to the TEM, SHV, CTX-M-1, CTX-M-2, CTX-M-9, and CTX-M-8/25 groups, as previously described. 12 The PCR products were visualized by 2% agarose gel electrophoresis and stained with GelRed nucleic acid (Biotium, Hayward, CA, USA). The sequence analysis of CTX-M genes was performed as previously described. 13

Phylogenetic Determination
Phylogenetic groups were determined by triplex PCR using a combination of two genes, chuA and yjaA, and the DNA fragment TSPE4.C2 as previously described. 14

mcr-1 Detection And Colistin MIC
The presence of the colistin resistance gene mcr-1 was detected by PCR with bacterial DNA and sequencing of the resulting products. 3 The colistin MIC of the mcr-1positive isolates was evaluated by ETEST® (bioMerieux) according to the manufacturer's protocol.

Pulsed-Field Gel Electrophoresis (PFGE) Analysis
XbaI-digested genomic DNA samples from the E. coli isolates were analyzed on a CHEF-DR III System (Bio-Rad, Hercules, CA, USA) following the detailed methods reported previously. 16

Prevalence Of Colistin-Resistant E. coli Harboring mcr-1 In Vietnamese Residents
The characteristics of the 612 participants are listed in Table 1. Bacterial growth on CTX-MacConkey was assessed for all participant stool specimens. One colony exhibiting E. coli characteristics from each participant specimen was obtained and subjected to further microbiological analysis. ESBL production was observed in 375 of the 451 E. coli isolates (83.1%). Moreover, 21 of the 451 isolates (4.7%) were AmpC producers.
A wide variety of plasmid replicon types were present in the mcr-1-harboring E. coli isolates. The most frequently detected replicon type was HI2 (54.8%), followed by FIB (48.4%) and N (41.9%), whereas B/C, FIC, HI1, U, X2, and X3 were not detected. Replicon types A/C, FIB, and P were detected in the E. coli isolates harboring mcr-1 as a single replicon type.
The presence of the transposon, ISApl1, a key component contributing to the mobilization of mcr-1, 21 in the mcr-1 E. coli isolates was also investigated. Because the PCR used in this study targeted only the ISApl1-mcr-1 segment, only the upstream portion of ISApl1 that is directly attached to mcr-1 was detected. The majority of mcr-1-harboring E. coli isolates (83.9%, 26/31) had the ISApl1-mcr-1 segment.

Discussion
This study revealed that the prevalence of colistin/CTXresistant commensal E. coli harboring mcr-1 among residents of a rural community in northern Vietnam is as high as 5.1% (31 positives/612 residents tested), although the  resistance genes highlights a high risk of extensively drugresistant bacteria circulating in the area. Dissemination studies conducted in other countries demonstrated the absence of mcr-1 isolates in healthy people of Switzerland 23 and the Netherlands. 24 In contrast, 4.9% of the Chinese resident samples 25 were found to be carriers of mcr-1-positive bacteria. Therefore, at the moment, the degree of the spread of E. coli harboring mcr-1 in healthy individuals has only been demonstrated in Asian countries, although there have been several reports of bacteria harboring mcr-1 in food and foodproducing animals worldwide. 26 Phylogenetic analysis revealed that the majority of the mcr-1-harboring E. coli isolates belonged to phylogenetic group A, with a few group B1, B2, and D isolates detected. These results are similar to the prevalence of phylogenetic groups in the normal human intestinal microbiota; A is the predominant group, followed by B2, whereas B1 and D are less common. 27 Thus, the frequency of mcr-1-harboring E. coli clone phylogenetic groups in the residents of this Vietnamese village may reflect the normal intestinal microbiota in humans.
Analysis of the antibiotic susceptibility of the mcr-1harboring E. coli isolates revealed that all isolates were MDR, ie, resistant to at least one antibiotic drug from three or more antibiotic classes. 28 However, resistance to FOX and CAZ was less common among these isolates. In addition, the overall antibiotic resistance profile of the mcr-1harboring E. coli isolates was similar to that reported from ESBL-producing E. coli isolated from food in Vietnam. 12 Therefore, it is possible that the mcr-1 gene may have been transmitted to ESBL-producing E. coli.
Indeed, nearly all of the mcr-1-harboring E. coli isolates were ESBL producers except for two isolates, which were class C β-lactamase producers, as determined by both phenotypic and genetic assays. The majority of these ESBL-mcr-1 isolates possessed CTX-M-9 group genes; the bla CTX-M-14 gene was identified in nearly one-third of the CTX-M-9 group and mcr-1 isolates tested. In contrast, a recent paper on MDR E. coli harboring mcr-1 reported that most isolates from pig were co-harboring CTX-M-1 group genes. 29 The difference between this previous study and our study may be due to the difference in specimen source.
Although it remains unclear whether the mcr-1 and CTX-M genes are located on the same plasmid, the present results demonstrate that ESBL-producing E. coli isolates with CTX-M-9 group genes are dominant among the colistin-resistant commensal E. coli in residents of the community. A previous study showed that mcr-1 and CTX-M-9 group genes are co-transferred by one or more plasmid types. 30 Moreover, the mcr-1-carrying plasmid identified in the first-ever discovered mcr-1 isolate is known to harbor the CTX-M-9 group, bla CTX-M-14 . 31 Plasmid type plays an important role in the spread of mcr-1, not only in the human intestinal microbiota but also in community settings. Therefore, characterization of the plasmids in mcr-1-harboring E. coli is important for understanding their dissemination. Replicon typing of the present isolates revealed a high diversity of plasmid backbones. The major replicon types of the isolates were HI2 (54.8%), FIB (48.4%), and N (41.9%), which have been reported as mcr-1-associated replicon types. 3,32,33 Replicon type I2, which is known as an mcr-1 disseminator, 34 was detected in 22.6% (7/31) of the isolates. Moreover, plasmid replicon type X4 was identified in one of our isolates, which was recently identified as a common mcr-1carrying plasmid replicon type in Enterobacteriaceae from food, animal, and human samples recovered in different countries. 5 However, since we assessed the replicon type of all plasmids in the isolates, the replicon types of the plasmid(s) harboring the mcr-1 gene remain unclear. In spite of this limitation, these results indicate that the diversity of plasmid types among mcr-1 isolates may contribute to the successful spread of the mcr-1 gene among different E. coli clones.
Besides class 1 integrons, 35 the transposon, ISApl1, is also known to be involved in horizontal gene transfer and maybe a key factor contributing to the widespread dissemination of the mcr-1 gene. 36 In this study, transposon ISApl1 with mcr-1 was detected in the majority of the isolates, except for six. As ISApl1 can be lost through improper recombination, 36 the ISApl1-mcr-1 segment may not have been detected in all of the mcr-1 harboring isolates. Zurfluh et al 33 reported that the mcr-1 gene may be mobilized independently. The possible occurrence of the mcr-1 gene or gene module mobilization among plasmids in the E. coli clones is also supported by the PFGE analysis, showing the limited clonal expansion of mcr-1harboring E. coli.
Overall, this study suggests that the wide dissemination of colistin-resistant ESBL-producing E. coli harboring mcr-1 in commensal bacteria in Vietnamese residents involves diverse clones with a variety of plasmid replicon types. Mobilization of the mcr-1 gene module among microbiota under certain circumstances such as exposure to colistin including suboptimal treatment during therapy is speculated as one of the likely causes of this dissemination. 37 Such wide dissemination of the mcr-1 gene in the commensal bacteria of healthy people constitutes a great public health concern in regard to the prevention, monitoring, and treatment of colistin-resistant bacteria.

Funding
This work was supported by grants from the Japan Agency for Medical Research and Development (AMED)/Japan International Cooperation Agency (JICA) as part of the Science and Technology Research Partnership for Sustainable Development (SATREPS), and Japan Society for Promotion of Science KAKENHI Grant Number 17H01687.

Disclosure
Dr Yoshimasa Yamamoto reports grants from Japan AMED/ JICA, and Japan Society for Promotion of Science, during the conduct of the study. The authors report no other conflicts of interest in this work.