Carriage of colistin-resistant, extended-spectrum β-lactamase-producing Escherichia coli harboring the mcr-1 resistance gene after short-term international travel to Vietnam

Introduction

With increasing globalization, people frequently travel to other countries. Under such circumstances, travelers can represent potential reservoirs for the dissemination of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae.¹

Our previous study revealed that veterinarians in Vietnam prefer to prescribe colistin-based drugs for use on chicken farms.² Although colistin is effective for treating multidrug-resistant (MDR) bacteria, including carbapenem-resistant bacteria, this has resulted in the widespread dissemination of colistin-resistant bacteria harboring plasmids encoding the colistin-resistant gene, mcr-1.³ Therefore, the prevalence of mcr-1 requires investigation. Additionally, a recent report showed that colistin-resistant Escherichia coli harboring mcr-1 had been isolated from food animals as well as from farmers and urban persons in Vietnam.^4,5 Haenni et al⁶ reported that the ESBL-related gene bla_CTX-M is encoded on mobile plasmids with mcr-1. Recently, transmission of colistin-resistant ESBL-producing E. coli by international travelers from China and India to developed countries has been reported.^7,8 Accordingly, we investigated whether travelers were bringing ESBL-producing E. coli harboring mcr-1 back to their home countries after short-term stays in developing countries, such as Vietnam.

Materials and methods

A prospective cohort study was approved by the ethics committees of Osaka University (no eki31) and Osaka Prefectural Institute of Public Health (no 1602-02-2). All participants provided written informed consent. For any participant younger than 18 years, the parents provided written informed consent. A total of 34 travel events from Japan to Vietnam made by 19 travelers (age: 12–61 years, males: 11, length of stay in Vietnam: 2–12 days) between June 2015 and August 2016 were investigated.

An ~0.1 g fecal specimen was collected from each traveler within 6 days before and up to 21 days after each travel event, and 10 mL of sterile phosphate-buffered saline was added to each sample and homogenized. A 100 μL serially diluted homogenized solution was inoculated on CHROMagar™ ECC (Chromagar, Paris, France) containing 1 µg/mL of cefotaxime (CTX) and cultured at 37°C for 24 h. Three to seven colonies exhibiting the characteristics of E. coli were collected from each sample plate.

For E. coli identification, biochemical tests were conducted, including assays in lysine indole motility medium (Nissui, Tokyo, Japan), cellobiose lactose indole β-glucuronidase medium (Kyokuto Pharmaceutical Industrial, Tokyo, Japan), triple sugar iron slants (Nissui), and API 20E (bioMérieux, Marcy l’Etoile, France).

Susceptibility to antibiotics (ampicillin, fosfomycin, cefoxitin, CTX, ceftazidime, meropenem, gentamycin, kanamycin, streptomycin, tetracycline, ciprofloxacin, chloramphenicol, nalidixic acid, and sulfamethoxazole–trimethoprim) was determined by disk diffusion assay. ESBL production was determined by the double-disk synergy test using ceftazidime and CTX with and without clavulanic acid. Both antibiotic susceptibility and ESBL production tests were conducted as recommended by the Clinical and Laboratory Standards Institute.⁹

Bacterial DNA was extracted by boiling the bacterial suspension in tris(hydroxymethyl)aminomethane–ethylenediaminetetraacetic acid buffer. Genotyping of ESBL-encoding genes and phylogenetic groupings were determined by multiplex polymerase chain reaction (PCR).¹⁰ The presence of the colistin-resistant gene mcr-1 was also confirmed by PCR.³ For both ESBL-encoding genes and mcr-1, the PCR products were identified by sequencing.^3,10 In mcr-1-positive strains, the colistin minimum inhibitory concentration (MIC) was evaluated by E-test^® (bioMérieux).

The phylogenetic grouping of identified E. coli isolates was also determined by triplex PCR using a combination of two genes (chuA and yjaA) and the DNA fragment TSPE4.C2, as previously described.¹¹

The clonal relationships of isolated E. coli strains were assessed by pulsed-field gel electrophoresis (PFGE), as previously described.¹²

The conjugation assay was performed as previously described.¹³

Statistical analysis was performed using Student’s t-test. The significance level was set at P<0.05.

Results

A total of 207 CTX-resistant E. coli strains (43 and 164 before and after travel, respectively) were isolated from the fecal specimens of travelers before and up to 2 weeks after traveling to Vietnam. Among these isolates, 175 strains were identified as ESBL-producing E. coli (Table 1).

Table 1 Detection of ESBL-producing Escherichia coli harboring mcr-1 before and after travel Notes: aSpecimens were collected up to 2 weeks after traveling. bExtended- spectrum β-lactamase-producing E. coli. cmcr-1-positive E. coli. dStrain name. *P<0.001. Abbreviation: ESBL, extended-spectrum β-lactamase.

A significant number of travel events (88.2%, 30/34) by the 19 travelers brought ESBL-producing E. coli back to Japan, while in 32.3% (11/34) of travel events, it was detected before traveling in seven travelers (Table 1). Among these, 42.9% of ESBL-producing E. coli isolates showed the same PFGE pattern as before traveling.

Genotyping of ESBL-producing isolates showed that bla_CTX-M-1 group/bla_TEM and bla_CTX-M-9 group were the most prevalent genotypes, while bla_CTX-M-2 and bla_CTX-M-8 groups and bla_SHV were not detected in travelers. Phylogenetic analysis of ESBL-producing E. coli isolates was also conducted. The most frequently detected phylogenetic group was D (41.9%, 62/142) followed by B2 (23.0%, 34/142), while B1 (11.5%, 17/142) and A (16.2%, 24/142) were the least common.

ESBL-producing E. coli isolates showed 100% resistance to ampicillin and CTX. In the case of quinolones, ESBL-producing E. coli isolates exhibited a rate of resistance of 76.1% (108/142) to nalidixic acid, and >50% of isolates were resistant to sulfamethoxazole–trimethoprim (66.9%, 95/142) and tetracycline (52.1%, 74/142).

Moreover, three events by three travelers brought back mcr-1. The three travelers with mcr-1, who visited Vietnam on business, did not carry mcr-1 before traveling. ESBL-producing E. coli harboring mcr-1 was identified by the co-occurrence of bla_CTX-M and mcr-1. Furthermore, the transmissibility of these mcr-1 genes was confirmed by conjugation assay.

Characteristics of these three ESBL-producing mcr-1- positive E. coli isolates are summarized in Table 2. Traveler A, a 38-year-old man, traveled to Nha Trang by the way of Hanoi for 4 days. One day after returning to Japan, a fecal sample was collected and investigated for the presence of ESBL-producing mcr-1-positive E. coli, and three ESBL-producing isolates with different phylogenetic patterns were identified. These belonged to the bla_CTX-M-9 group and the phylogenetic group A, the bla_CTX-M-1 group/bla_TEM and the phylogenetic group D, and the bla_CTX-M-14 genotype and the phylogenetic group B2, the latter of which harbored mcr-1. However, no ESBL-producing E. coli harboring mcr-1 was detected in a sample collected 17 days after returning to Japan. Traveler B traveled to Ho Chi Minh City for 12 days. Three days after returning to Japan, a fecal specimen was collected and four types of ESBL-producing E. coli were identified. These belonged to the bla_CTX-M-1 group and the phylogenetic group D, the bla_CTX-M-9 group and the phylogenetic group A, the bla_CTX-M-1 group/bla_TEM and the phylogenetic group D, and the bla_CTX-M-55/bla_TEM genotype and the phylogenetic group B2, the latter of which harbored mcr-1. Traveler C traveled to Can Tho by the way of Ho Chi Minh City for 5 days. Six days after returning to Japan, a fecal specimen was collected and four types of ESBL-producing E. coli were identified. These belonged to the bla_CTX-M-9 group and the phylogenetic group A, the bla_CTX-M-1 group and the phylogenetic group D, and the bla_CTX-M-14 genotype and the phylogenetic group D, the latter of which harbored mcr-1. In travelers B and C, no ESBL-producing E. coli harboring mcr-1 was detected 20 and 19 days, respectively, after returning to Japan.

Table 2 Characteristics of travelers carrying ESBL-producing Escherichia coli isolates harboring mcr-1 Note: aAntibiotic resistance was determined by the Clinical & Laboratory Standards Institute guidelines: AMP, CTX, NAL, CHL, SXT, TET, GEN, KAN, and STR. Abbreviations: AMP, ampicillin; CHL, chloramphenicol; CTX, cefotaxime; ESBL, extended-spectrum b-lactamase; GEN, gentamycin; KAN, kanamycin; MIC, minimum inhibitory concentration; NAL, nalidixic acid; STR, streptomycin; SXT, sulfa/trimethoprim; TET, tetracycline.

Discussion

Widespread dissemination of colistin-resistant MDR bacteria represents a worldwide threat owing to the limited number of effective antibiotics for treatment. In particular, the emergence of the mobile colistin-resistant gene mcr-1 suggests the possibility of the rapid dissemination of MDR bacteria with colistin resistance in a community because of the potential for horizontal gene transfer. In these circumstances, international travelers could act as carriers, transferring mcr-1-positive MDR bacteria to their home countries. Even if only a limited number of travelers carry mcr-1-positive bacteria, the possibility of genetic transfer and expansion of mcr-1 across borders seems to be likely. In this regard, there have been several reports of healthy travelers that carried ESBL-producing E. coli back to their home countries.^1,14 However, it is not clear whether a traveler on a short trip can carry mcr-1 back to his/her home country.

In our previous reports, a high prevalence of human ESBL carriage (51%–71%) was demonstrated among Southeast Asian countries, such as Vietnam, Thailand, and Laos.¹¹ In contrast, the dissemination of ESBL-producing E. coli is limited in Japan, where there is a low prevalence of human ESBL carriers.¹⁵ To date, only a limited number of bacterial isolates have been reported to harbor mcr-1 in E. coli isolates from healthy animals, but in sick swine, there were high mcr-1- positive bacteria detected in Japan.^16,17

It has been reported that in Hanoi, Vietnam, 37.5% of food animals are contaminated with E. coli harboring mcr-1.⁴ In this study, we revealed that 15.8% of the travelers assessed became the carriers of ESBL-producing E. coli harboring mcr-1 after a short stay in Vietnam. Even though the details of these carriage mechanisms are not clear, our results indicate that travelers may come to resemble Vietnamese residents, a percentage of whom are carriers of mcr-1-positive bacteria (unpublished data), during their stay in Vietnam. The three travelers who became mcr-1 carriers ate breakfast in four-star hotels every morning, and they frequented middle-class restaurants for lunch and dinner. They experienced upper-class conditions during their stays in Vietnam and after returning to Japan. Their genotyping results were similar to those of healthy Vietnamese residents in terms of bla_CTX-M genotypes, such as bla_CTX-M-1 and bla_CTX-M-9 groups.¹⁰ Conversely, an abundance of phylogenetic group D was observed in the isolates. However, the reason for this difference from healthy Vietnamese residents¹⁰ was not clear. It may be due to differences in intestinal flora between Japanese travelers and Vietnamese residents.

More than 40% of isolates from each traveler before and after travel showed the same PFGE patterns, suggesting low genetic diversity within each traveler. Further study regarding the clonal relationships with residents may provide valuable information on the carriage of MDR bacteria by travelers.

Since the intestinal flora in travelers can be changed by diet and environment factors during travel, it may be responsible for the carriage of ESBL-producing E. coli with mcr-1 after short-term travel.

Our follow-up study showed that the ESBL-producing E. coli harboring mcr-1 disappeared in the three travelers, who were not treated with any antibiotics, within 3 weeks of returning home. Therefore, the stability of the carriage of mcr-1-positive bacteria may be limited if there is no exposure to antibiotics.

The three ESBL-producing E. coli isolates harboring mcr-1 were further characterized, and the colistin MICs of the isolates were relatively low in comparison with those of isolates from food animals (4–8 µg/mL).⁴ Since antibiotic MICs can be increased by treatment with a high concentration of another antibiotic, the acquisition of high colistin MICs may be possible.

Conclusion

The findings in this study suggest that even a short-term international trip to a developing country may result in ESBL-producing mcr-1-positive E. coli being carried back to the travelers’ home countries. Although the dissemination of E. coli harboring mcr-1 is limited in developing countries, the number of international travelers continues to increase, thereby increasing the risk of spreading E. coli harboring mcr-1 into developed countries.

Acknowledgments

This work was supported by the Japan Agency for Medical Research and Development (AMED)/Japan International Cooperation Agency (JICA) as a part of the Science and Technology Research Partnership for Sustainable Development (SATREPS), and MEXT/JSPS KAKENHI Grant 17H01687.

Disclosure

The authors report no conflicts of interest in this work.

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