Antibiotic resistance is a global public health challenge of our time. Colistin (polymyxin E), first discovered in 1947, is a polycationic peptide antibiotic (Storm et al., 1977). It mainly targets the bacterial cell membrane and increases its permeability, which results in the leakage of cell contents, thereby ultimately causing cell death (Luo et al., 2020). Furthermore, it has broad-spectrum antimicrobial activities, and it is used to treat multidrug-resistant gram-negative infections (Poirel et al., 2017). Before 2015, studies showed that colistin resistance was regulated by mutation in chromosomal genes (Olaitan et al., 2014). However, in 2016, Liu et al. (2016) discovered the plasmid-mediated colistin resistance gene—mcr-1. Since then, the mcr-1 gene has been identified as part of several bacterial species in humans, animals, and the environment, thus, posing a great threat to treatment (Elbediwi et al., 2019).

The human gut is a reservoir of antimicrobial resistance genes. In the gut, antimicrobial resistance genes can spread through horizontal gene transfer (Rolain, 2013; McInnes et al., 2020), which plays an important role in transmitting drug-resistant bacteria. Previous studies have also reported that the prevalence of mcr-1-positive Enterobacteriaceae in fecal carriages was 0.4–15.0% in Asia (Chan et al., 2018; Shen et al., 2018; La et al., 2019; Wu et al., 2019), 0–0.35% in Europe (Terveer et al., 2017; Zurfluh et al., 2017b), and 38.3% in South America (Giani et al., 2018), showing great variability in different geographic areas and populations. In the aspects of risk factors, several literatures from China reported antibiotic use before admission, the consumption of meat and aquaculture products were linked to mcr-1-positive Enterobacteriaceae in fecal carriages (Wang et al., 2017b; Shen et al., 2018). Furthermore, traveling to Southeast Asia has also been associated with mcr-1-positive Escherichia coli (E. coli) in fecal carriages (Nakayama et al., 2018). Despite these results, most studies on the risk factors for mcr-1 Enterobacteriaceae in fecal carriages were from the adult population, and studies among children were scarce. Furthermore, E. coli is a common inhabitant of the human intestinal tract and has the highest mcr prevalence among all bacterial species (Elbediwi et al., 2019). Therefore, we conducted a prospective study to identify the prevalence and risk factors of mcr-1-positive E. coli in fecal carriages of community children in Southern Taiwan.

We collected 510 non-duplicated fecal specimens from patients in our pediatric ward. Among these specimens, 268 yielded 514 E. coli isolates. The characteristics of these 510 participants are demonstrated in Table 1. The participants’ ages ranged from 1 day to 17 years (mean age was 1.5 years), and males accounted for 58.6% (299/510). Hospitalization and antibiotic therapy in the last 3 months were recorded in 9.6% (49/510) and 7.8% (40/510) of participants, respectively. In addition, 7.5% (38/510) of the participants had traveled abroad in the last 12 months. Among them, 10 had been to China, 23 to Japan, 2 to Korea, 5 to Southeast Asian nations, and one to the United States. The prevalence of fecal carriages of mcr-1-positive E. coli was 2.4% (12/510). Looking further, we divided the study period into three segments. The prevalence was 1.6% (2/124) from April 2016 to March 2017, 2.5% (8/324) from April 2017 to March 2018, and 3.2% (2/62) from April 2018 to August 2019.

Subsequently, we compared the variables between individuals with and without fecal carriages of mcr-1-positive E. coli through univariate analysis. Individuals with fecal carriages of mcr-1-positive E. coli were predominantly male [Odds Ratio (OR) 8.02, 95% confidence interval (CI) 1.03–62.61] and people frequently ate chicken (OR 3.13, 95% CI 0.77–12.72), with a consumption of between 1 and 3 times per week. Another set of included individuals (OR 7.10, 95% CI 1.72–29.35) had a chicken consumption frequency of at least 3 times per week. Furthermore, antibiotic use in the past 3 months was borderline associated with mcr-1-positive E. coli carriage (OR 4.15, 95% CI 1.08–16.00) (Table 1). Through binary logistic regression analysis, chicken consumption with a higher frequency (least 3 times per week) was the only factor that had a significant association with fecal carriages of mcr-1-positive E. coli (adjust OR 6.60, 95% CI 1.58–27.62, p = 0.033) (Table 1).

The distribution of reasons for admission between participants with and without mcr-1-positive E. coli in fecal carriages are presented in Table 2, showing that individuals that were hospitalized due to infectious diseases had a higher proportion of mcr-1-positive E. coli in their fecal carriages than those hospitalized due to non-infectious diseases (3.6 and 0.9%, respectively, p = 0.041), and individuals hospitalized due to urinary tract infection (UTI) had the highest proportion (7.4%) of mcr-1-positive E. coli in their fecal carriages.

In Table 3, we compare the antibiotic susceptibility results between mcr-1-positive and non-mcr-1-positive E. coli isolates determined using Vitek. Results showed that mcr-1-positive E. coli isolates were more prone to be resistant to cefoxitin (50.0% vs. 9.0%, p = 0.001), minocycline (41.7% vs. 15.2%, p = 0.031), and sulfamethoxazole-trimethoprim (75.0% vs. 40.5%, p = 0.018) than non-mcr-1-positive E. coli isolates. Multidrug resistance was defined as the non-susceptibility to at least 1 antibiotic in 3 or more antimicrobial categories (Magiorakos et al., 2012). Multidrug resistance (75.0% vs. 39.5%, p = 0.031) was also more common in mcr-1-positive E. coli isolates. Non-mcr-1 -positive E. coli isolates were all susceptible to colistin. All E. coli isolates were susceptible to ertapenem, imipenem, amikacin, and tigecycline. Moreover, the MIC and molecular characteristics of mcr-1-positve E. coli isolates are shown in Table 4. Most mcr-1-positive E. coli isolates exhibited colistin MICs of 4–8 μg/mL. Two isolates possessed the ESBL-producing phenotype. One had bla_CTX–M and bla_TEM, and the other had bla_TEM. Among mcr-1-positive E. coli isolates, 10 (83.3%) belonged to ExPEC.

Sequence type (ST) 58 accounted for the highest proportion (n = 4, 33.3%). The genetic relatedness of mcr-1-positive E. coli in the study and those isolated from food animals reported in the literatures of Taiwan was evaluated using goeBURST’s algorithm (Figure 1; based on data from the MLST database)² (Kuo et al., 2016; Liu J. Y. et al., 2020). Results showed that ST10, ST101, and ST162 were detected in both our participants’ fecal samples and food animals, including chicken, pork, and beef, reported in the literatures of Taiwan (Kuo et al., 2016; Liu J. Y. et al., 2020).

The prevalence of mcr-1-positive E. coli in fecal carriages of patients included in the study was 2.4% (12/510), which was lower than what had been reported in children (4–38.3%) from either community or hospital settings in other countries, such as China, Bolivia and India (Bi et al., 2017; Hu et al., 2017; Giani et al., 2018; Purohit et al., 2019). However, the results were similar to other recent studies in Taiwan, including data from clinical isolates and commensal E. coli (Lee et al., 2019; Wu et al., 2019). These findings demonstrated that the prevalence of the mcr-1 gene was still low in Taiwan. However, an upward trend of the prevalence over time was found. Furthermore, in this study at the individual level, a significant predictor of fecal carriages in mcr-1-positive E. coli was chicken consumption. A study from China of approximately 90% adults also demonstrated that meat consumption, especially pork and mutton, was associated with mcr-1 prevalence in a provincial region’s level (Shen et al., 2018). Another epidemiological study from southern China using vegetarian and non-vegetarian classifications did not find diet as a risk factor for mcr-1 in fecal carriages (Wang et al., 2017b). Additionally, previous studies revealed a correlation of drug-resistance bacteria between contaminated food and the human gut. Sørensen et al. (2001) observed the same resistant strains in stool after ingestion of chickens containing those resistant bacteria. Donabedian et al. (2003) also observed related pulsed-field gel electrophoresis patterns of gentamicin-resistant isolates from human stools and meat. A recent review article indicated that foodborne transmission was a pathway of mcr-1-positive E. coli transmission (Elbediwi et al., 2019). Literature also revealed that chicken had a higher percentage of the mcr-1 gene than other meats (Kuo et al., 2016). Therefore, chicken consumption is a possible pathway to transmit mcr-1-positive E. coli to humans. Nevertheless, more studies should investigate the detailed mechanisms.

Traveling abroad was proposed as a risk factor for drug-resistant gene transmission (Hu et al., 2020). Limited studies on the relation between traveling abroad and fecal carriages of mcr-1-positive E. coli have been reported. von Wintersdorff et al. (2016) and Nakayama et al. (2018) demonstrated that traveling to Southeast Asia or southern Africa increases the risk to mcr-1-positive E. coli in fecal carriages. However, traveling abroad was not associated with mcr-1-positive E. coli in fecal carriages in this study. The period between the time of travel and fecal collection may affect the results. In those two studies, fecal samples were collected within no more than 3 weeks after travel events. In the study presented here, travel history was traced back to 12 months before the fecal sample collection. Previous studies revealed that the proportion of drug-resistant bacterial colonization declined as time after international travels increased. For example, only about 10% of fecal colonization of ESBL E. coli cases remained positive after 12 months follow-up (von Wintersdorff et al., 2016; Arcilla et al., 2017; OstholmBalkhed et al., 2018). Additionally, people in Taiwan have more opportunities of contacting people from China and Southeast Asia due to their geographic location, which may weaken the influence of traveling to these countries. Therefore, more studies should investigate the relationship between international travels and mcr-1-positive E. coli in fecal carriages.

A higher proportion of participants who were hospitalized due to UTI were positive for mcr-1-positive E. coli in the study. The association between UTI pathogens and the gut microbiome had been reported before (Moreno et al., 2008; Paalanne et al., 2018). Paalanne et al. (2018) revealed that the gut microbiome was associated with the risk of febrile UTI in children. However, we did not collect urine data from the participants in the study. The relevance of UTI pathogens and fecal carriages of mcr-1-positive E. coli thus needs further study. The four participants with UTI with mcr-1-positive E. coli isolates were under 5 years. Furthermore, two of them had a history of UTI and antibiotic exposure in the last 12 months. Except for the undefined one, the other three isolates belonged to ExPEC. Interestingly, the positive rate of mcr-1-positive E. coli presence in the fecal carriage of participants with acute gastroenteritis was low (1.7%, 1/58).

Additionally, variations were observed in genotypes among the 12 mcr-1-positive E. coli cases. ST58 was the most common type in the study. E. coli ST58 has been isolated from different samples, including humans, the environment, food animals, and wildlife (Fuentes-Castillo et al., 2021). Furthermore, mcr-1-positive E. coli ST58 was found in chicken, turkey, cattle, and the environment (Brennan et al., 2016; Donà et al., 2017; Zurfluh et al., 2017a; Sacramento et al., 2018; Zajac et al., 2019). Similarly, mcr-1-positive E. coli ST162 was also isolated from ducks, chickens, turkeys, and dogs (Lim et al., 2016; Gelbicova et al., 2019; Zhang et al., 2019; Liu Y. Y. et al., 2020). E. coli ST101 and ST10 were the widespread clones that harbored mcr-1 genes (Elbediwi et al., 2019). mcr-1-positive E. coli ST162, ST101, and ST10 have been reported in food animals and meats in Taiwan as well (Kuo et al., 2016; Liu J. Y. et al., 2020). Our preliminary data also showed that plasmid analysis from mcr-1-positive E. coli in children was similar to the plasmid sequence reported in the animals (unpublished data). Most E. coli ST108 have been isolated from chicken (Yang et al., 2014; Agabou et al., 2016; Pietsch et al., 2018). E. coli ST657 has been reported from rectal swabs of humans (Yang et al., 2014; Wang et al., 2017a). E. coli ST795 was found in retail vegetables and pigs (Hammerum et al., 2012; Luo et al., 2017; Xu et al., 2017). Additionally, E. coli ST-847 has been isolated from retail vegetables, pigs, cattle, and birds (Guenther et al., 2012; Leimbach et al., 2015; Freitag et al., 2018; Wang et al., 2020). These genotypes isolates, including ST-108, ST-657, ST-795, and ST-847, have been reported to carry different drug-resistant genes. This study is the first to reveal that these STs of E. coli carried the mcr-1 gene.

Some limitations exist in this study. First, the diet information relied on self-reports provided by the participants’ parents or caregivers. Recall bias is inevitable. Second, the number of participants with mcr-1-positive E. coli in fecal carriages was relatively small, which would not detect certain risk factors. Third, mcr-1 gene is detected mainly from Enterobacteriaceae. Although E. coli has the highest mcr prevalence among all bacterial species (Elbediwi et al., 2019), there are still some common bacterial species with mcr gene, such as Klebsiella pneumoniae, that have not been analyzed in the study. Furthermore, the study was limited to mcr-1 gene, which is the most widely disseminated mcr gene worldwide (Ling et al., 2020). However, other mcr variants may exist. Last, the diversity of STs of mcr-1-positive E. coli not only means clonal expansion but also the possibility of certain plasmid transmission in the community. In our preliminary result, some of the plasmid in the mcr-1 positive E. coli had the same sequence as the plasmid identified in food animals, as reported by studies published in Taiwan (unpublished data).

In conclusion, the prevalence of mcr-1-positive E. coli in fecal carriages is low among community children in Southern Taiwan who were included in this study. Nevertheless, we can found a gradual upward trend of the prevalence from 2016 to 2019. This phenomenon shows the need for continuous follow-up. A higher frequency of chicken consumption was also associated with fecal carriages of mcr-1-positive E. coli. However, the genetic relationship between our colonized isolates and isolates from chicken in Taiwan warrants further evaluation.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by VGHKS 16-CT2–04, VGHKS 18-CT3–11. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

P-CW, M-FC, and J-LW: conceptualization. P-CW, M-FC, W-YH, and C-HH: methodology. W-LC, M-FC, and C-HH: validation. P-CW, W-LC, and J-LW: formal analysis. P-CW and J-LW: investigation and writing—original draft preparation. W-YH and C-HH: data curation. J-LW and C-HH: writing—review and editing. C-HH: supervision. M-FC and J-LW: funding acquisition. All authors have read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.