The rapid rise of multidrug-resistant (MDR) bacterial infections is a major public health concern and a growing threat to the global health security. Unregulated use of broad spectrum antibiotics and widespread reservoirs of these pathogens are main contributors to this problem (Hilty et al., 2012). Broad spectrum antibiotics, in particular third generation cephalosporins (3GC), are among the most frequently prescribed drugs for the treatment of infections caused by Enterobacteriaceae (Pereira et al., 2004; World Health Organization [WHO], 2017). Failure of treatment with these antibiotics has increasingly been reported due to the emergence of extended spectrum beta-lactamases (ESBLs) during the last two decades (Pitout and Laupland, 2008). Several studies have suggested that children are more likely to be exposed to antibiotics both directly (Alexander et al., 2011; Saari et al., 2015) and indirectly, through exposure to antibiotics taken by their mothers (Verani et al., 2010; Macones et al., 2012; Ledger and Blaser, 2013; de Tejada, 2014). This direct and/or indirect consumption of antibiotics might thus affect infants’ intestinal microflora, including Escherichia coli, which is one of the first bacterial species that colonizes the infant gut (Hewitt and Rigby, 1976).

Antimicrobial resistance in commensal bacteria is worrisome due to its ability to spread to pathogens (Munk et al., 2018). Recent studies have showed that school children and children up to 2 years of age were colonized by E. coli resistant to broad spectrum antibiotics and ciprofloxacin, respectively (Gurnee et al., 2015; Ferjani et al., 2017). However, there is no information on the carriage rate and abundance of this antibiotic-resistant E. coli in relation to the total number of E. coli present in the gut. In addition, there is no data available on the carriage rate of MDR E. coli, including ESBL-producing E. coli, among infants under 1 year old. Therefore, this study evaluated the prevalence and rate of colonization of this organism during the early life of infants. We determined the prevalence, abundance, and carriage rate of 3GC-resistant (3GCr) E. coli, including pathogenic E. coli, by analyzing culturable E. coli in infant’s stool samples.

We found an extremely high prevalence of both 3GCr E. coli (82%) and ESBL-producing E. coli (74%) in stool samples of healthy infants living in rural areas of Bangladesh. Despite the relatively small sample size (n = 100) chosen based on logistic constraints, the high prevalence rates are likely representative of Bangladeshi infants under 1 year old in the study area within a margin of error of 10% (with 95% confidence interval). Even with the associated uncertainty, this is the highest prevalence of ESBL-producing E. coli in healthy human guts observed to date. For example, in a study in Tunisia, the prevalence of MDR E. coli was 6.6% in children aged between 6 and 12 years (Ferjani et al., 2017). The rates of 3GCr E. coli in healthy children at various ages was reported as 2.9% in Sweden, 2.7% in Portugal, and 10% in Senegal (Guimaraes et al., 2009; Kaarme et al., 2013). The fecal carriage rate of ESBL-producing Enterobacteriaceae in healthy children (0–59 months) was much higher (59%) in central Africa (Farra et al., 2016). None of these studies have reported the data on culturable 3GCr E. coli as a proportion of total culturable E. coli in stool samples. In our study we found that around one third of the total E. coli colonies obtained in culture were 3GCr, which is alarming.

The difference between 3GCr and 3GCs E. coli gives an indication of fitness costs for the maintenance of resistance within the gut microbial community. The proportion of 3GCr amongst the total E. coli (sum of 3GCr and 3GCs E. coli) was not significant in infants at different age groups, indicating that the competitiveness of resistant bacteria with normal residential flora within the gut is not influenced by age. There may be a low fitness cost associated with persistence and dissemination of resistance. Indeed, Cottell et al. (2012) suggest a low fitness cost associated with plasmid (pCT) carrying the resistance gene bla_CTX–M–14 for E. coli, as the E. coli were able to persist and disseminate readily even in the absence of selective pressure from antibiotics (Cottell et al., 2012). Our findings also displayed congruence with previous reports demonstrating that tetracycline- and ampicillin-resistant isolates persist continuously without any selective pressure of antibiotics in the gut of different age groups of children (Karami et al., 2006, 2008).

The implication of this high load of 3GCr E. coli is substantial in the context of child health safety. Antibiotic resistant E. coli and other common commensals of the gut including Klebsiella spp. and Acinetobacter spp. are amongst the leading causes of community-acquired serious infection in Southeast Asia. In a cross sectional study at five sites across Southeast Asia, Saha et al. (2018) found that only 17% of possible serious bacterial infections (pSBI) identified in young children were resistant to first line antibiotics. Resistant infections, as compared to susceptible infections, are linked with worse outcomes. For example, in Tanzania, children with septicemia caused by bacteria producing extended-spectrum beta-lactamases were almost twice as likely to die compared to non-ESBL infections (71% mortality rate vs. 39%) (Blomberg et al., 2007). In the present study, the observed high carriage rate and high relative proportion of culturable 3GCr E. coli may harbinger higher rates of 3GCr E. coli as a proportion of pSBI infections. Further research linking fecal carriage of resistant bacteria to risks of resistant infections is warranted.

Apart from increasing the risk of resistant infections, high carriage of 3GCr Enterobacteriaceae in the gut results in shedding through infant stool and thus contributes to an elevated risk of exposure to nearby people and animals. There is a common belief among illiterate or less literate mothers in the community that infant stool is not a health hazard or harmful, especially in comparison to adult stool (Yeager et al., 1999; Gil et al., 2004). According to a national survey during the period from 2012 to 2013 in Bangladesh, feces of about 61% children of age 0–2 years were disposed unsafely where the percentage was much higher in rural areas (67%) compared to urban areas (40%) (Bangladesh Bureau of Statistics [BBS] and United Nations Children’s Fund [UNICEF], 2014). Thus household members including mothers or caregivers are exposed to fecal MDR bacteria through unsafe disposal of infant feces. Similarly, improper disposal of infant’s stool in front yards or nearby ditches may contribute to transmission of these resistant bacteria to domestic and wild birds and/or other animals (Hasan et al., 2012).

The 3GCr E. coli isolates in this study were predominantly resistant to azithromycin and ciprofloxacin, among other antibiotics (Figure 1). Azithromycin is the first line of choice for treatment of shigellosis in children (Centers for Disease Control and Prevention [CDC], 2006) and a second line of choice for treatment of shigellosis in adults (World Health Organization [WHO], 2005). Although no infants were reported to be suffering from shigellosis during the study period, shigellosis has been identified as a major contributor to moderate-to-severe diarrheal disease in neighboring Mirzapur (Kotloff et al., 2013). Management of this infection might be complicated due to the high prevalence of azithromycin resistance in the study community. Among other antibiotics, ciprofloxacin resistance was found among 37% of 3GCr isolates, which is even higher than a previous report that showed resistance in 19% of E. coli obtained from healthy children (Gurnee et al., 2015). Interestingly, only 29% of E. coli isolates in this study were resistant to tetracycline, a first generation antibiotic which is less commonly used for the treatment of E. coli infections in the community (Calva et al., 1996; Domínguez et al., 2002). Tetracycline is not prescribed in children due to its effect on the growth of bones and teeth (Sánchez et al., 2004). It suggests that by reducing the use of antibiotics in humans and animals, it is possible to reduce the burden of resistant microorganisms and this can eventually restore the efficacy of the existing antibiotics even in a setting like Bangladesh where overuse of antibiotics and burden of AMR, both are way too high.

Our study demonstrated that infant’s guts serve as a reservoir of E. coli resistant to multiple antibiotics including 3GCr and fluoroquinolones, which are critical for the treatment of many infectious diseases in humans (World Health Organization [WHO], 2017). High rates of ESBL-producers among 3GCr isolates in the present study is alarming because patients with community acquired infections as well as their household members carrying ESBL-producing Enterobacteriaceae may spread resistance to other people in their community (Valverde et al., 2008). This can be explained by the overall high prevalence of ESBL E. coli infections in the community. A recent study in Bangladesh has reported that 34% of all clinical isolates of E. coli from patients with extra-intestinal infections were ESBL-producing (Khan et al., 2018).

The probable cause of colonization with ESBL-producing Enterobacteriaceae among pre-school children in Laos was reported due to misuse of antibiotics (Stoesser et al., 2015). In our study, we did not observe this: there was no significant association between colonization and reported previous use of antibiotic treatment among infants. Previous studies have suggested that acquisition of antimicrobial resistant bacteria or antimicrobial resistant genes in the infant gut might occur during and/or after the delivery (Zhang et al., 2011). Specifically, mothers’ flora during normal vaginal delivery or environmental flora during caesarian (C-section) delivery colonize the infant gut (Zhang et al., 2011). Therefore, AMR bacteria from the mother or hospital environment may contribute to infant carriage. For example, a study carried out in Tunisia showed that 20% of patients acquired 3GCr E. coli in their gut due to nosocomial infection (Maamar et al., 2016). In our study, we did not find any significant differences in the level of colonization with 3GCr E. coli between infants with normal vaginal delivery and infants delivered through C-section (Tables 1, 2). In Bangladesh, a recent study showed that delivery by C-section increased from 3.5 to 23% between 2004 and 2014 (Khan et al., 2017) and it is a common practice that prophylactic antibiotics are used before and after the surgery. During post-operative care mothers start to breastfeed the newborn while still on antibiotic treatment. This leads to transfer of antibiotics in its active form to infants and thus their gut microbiota may shift to survive in an antibiotic selective environment (Mathew, 2004). Further in Dhaka, Bangladesh, a significant proportion of newborns (98%) receive antibiotics (sulfonamides, fluoroquinolones, metronidazole, penicillins, etc) before 6 months of age (Rogawski et al., 2017), which renders the selective environment for antibiotic-resistant bacteria. Even in a very low concentration of antibiotics, fitness cost for microorganisms to become resistant is lower than becoming antibiotic susceptible (Sandegren, 2014). Surprisingly, in our study neither history of antibiotic use or previous record of hospitalization was associated with 3GCr colonization nor were gender, religion and feeding practices. The lack of association may be due to the high rate of colonization combined with relatively small sample size (n = 100). The low proportion of infants without 3GCr limits statistical analyses. Therefore, further studies, particularly focused on larger sample sizes, are needed to identify the causes of the high rate of antibiotic resistance carriage among infants under 1 year old in this setting.

The high rate of intestinal carriage with MDR microorganisms among infants in rural Bangladesh is a serious concern that can jeopardize the management of infectious diseases. In addition, shedding of high number of MDR microorganisms through infant feces increases the risk of widespread transmission of these microorganisms in the community and environment. This study raises important questions about how the acquisition of resistant microorganisms takes place in infants’ guts within the first 3 months of life, what are the major drivers of acquisition, and what are the implications on infant health and well-being. Future studies should explore the source of acquisition of resistance in infants, to understand whether such resistance is primarily acquired from the environment, vertically from the child’s mother, or through selective pressure from pediatric antibiotic use.

MAI and TJ conceived the development. MAI, TJ, MK, KL, TN-D, and MM designed the study and developed the protocol. MA, SR, and MBA contributed to the experiments, collection, and assembly of the data. MRI, MBA, and TJ contributed to the data entry and statistical analysis. MAI and MBA performed the first draft of the manuscript. TJ, MK, KL, TN-D, MM, MA, and MAI revised the manuscript and prepared the final draft 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.