Background
Multidrug-resistant (MDR) Gram-negative bacterial species are an established prominent international public health threat [
1]. MDR pathogens have an especially high prevalence in South Asia where bacterial infections are common and antibiotic use is widely unregulated [
2‐
4]. A lack of comprehensive surveillance programs may result in the unchecked spread of MDR Gram-negative bacterial species and their associated resistance mechanisms. Developing countries with an established national surveillance network, such as Nepal, do not have the resources to perform in-depth isolate characterization to comprehensively identify resistance mechanisms carried by MDR bacterial isolates [
5‐
7]. It is imperative that both archived and current bacterial isolates from underdeveloped regions undergo extensive MDR characterization to inform national strategies designed to halt the continuing spread of these dangerous pathogens.
Diarrheagenic
Escherichia coli is a ubiquitous presence in the developing world and is responsible for a range of enteric infections [
8‐
10]. Enterotoxigenic
E. coli (ETEC) is defined by production of 1–3 enterotoxins, and is one of the leading causes of both acute infant diarrhea and traveler’s diarrhea worldwide, including Nepal [
11‐
14]. ETEC attaches to and colonizes cells in the small intestine along the epithelial surface through the interaction of bacterial colonization factors (CFs) with the intestinal cell wall [
15,
16]. After the establishment of intestinal colonization, the bacterial cells produce heat-labile enterotoxins (LT) and/or heat-stable enterotoxins (STh or STp) that target vital cellular processes [
8,
17,
18]. The production of these enterotoxins most commonly results in abdominal cramping and watery diarrhea, with more severe cases also presenting with fever and nausea. ETEC infections are typically self-limiting with resolution of symptoms 3–7 days after initial onset and a recommended treatment strategy of rest and oral rehydration therapy [
8,
19]. In spite of ongoing efforts to develop an ETEC vaccine, no viable vaccine candidates have been produced thus far [
20,
21]. ETEC virulence factors, such as enterotoxins and CFs, are potential targets for vaccines or therapeutic candidates, and determination of regional prevalence rates and specific virulence factors may serve to inform the development of globally effective vaccines [
15,
18‐
21].
Severe ETEC cases presenting with cholera-like watery diarrhea that do not resolve through oral rehydration therapy may require treatment with antibiotics [
8,
10]. Initial treatment has historically been administration of first-line beta-lactams and quinolones. However, the evolution and global dissemination of multiple classes of antimicrobial resistance mechanisms has led to an increase of MDR ETEC infections that are insensitive to first-line antibiotics [
11,
22]. Beta-lactamases and Extended-spectrum β-lactamases (ESBLs) confer resistance to a number of antibiotic classes, such as penicillins, extended-spectrum cephalosporins, and monobactams [
23,
24]. Global dissemination of these mechanisms has occurred through clonal expansion and/or horizontal transfer of genetic resistance mechanisms between bacterial species [
24]. Bacterial pathogens carrying ESBL resistance mechanisms are commonly associated with, and detected in, hospital-acquired infections. However, these antibiotic resistance mechanisms have spread to community-acquired isolates, and are increasingly identified in acute diarrhea and other enteric infections [
12,
25‐
30]. ETEC infections are a large cause of acute diarrhea cases in Nepal, but prevalence rates of MDR ETEC isolates, both from a historic or current perspective, are unknown.
The current study determined antibiotic resistance and virulence factors of ETEC isolates previously identified from clinical acute diarrheal samples (263/265) or patient control samples (2/265) collected in Nepal between 2001 and 2016. The ETEC isolates were assayed to determine antimicrobial resistance profiles (including ESBL production) and virulence factor characterization, specifically enterotoxins and colonization factors. Antimicrobial resistance studies that encompass retrospective and current clinical isolates are vital to understanding the regional emergence of ESBL producing organisms, the existing treatment challenges, and the potential for ongoing dissemination. This study serves as a thorough investigation of antibiotic resistance in ETEC isolates from acute diarrheal samples in Nepal, and highlights the need for interventions that may address and stem the continual spread of MDR mechanisms in community settings.
Discussion
MDR Gram-negative bacterial pathogens are a global public health threat [
1,
6]. ESBL-producing bacterial strains were first detected in, and were mostly limited to, hospital-associated infections, but began being detected in community-associated infections beginning in the early 2000’s [
25,
27,
28,
34]. Initial reports of community-associated ESBL-producing
E. coli strains, including reports originating from Nepal, are mainly comprised of pathogens isolated from urinary tract or blood stream infections [
35‐
39]. However, community-associated infections isolated from enteric MDR pathogens are being increasingly reported in South and Southeast Asia [
8,
11,
12,
22,
26,
30].
ETEC is an enteric pathogen that results in both acute infant diarrhea and traveler’s diarrhea [
8,
10,
15]. ESBL-producing ETEC may result in treatment failures for infections that were previously easily treatable with first-line antibiotic administration. In the current study, only a single ESBL-positive ETEC isolate was identified from isolates archived in 2001–2009 from Nepal. However, over 30% of the ETEC isolates collected after 2013 phenotypically display ESBL production. This suggests that ESBL resistance mechanisms in Nepal have expanded relatively recently into community-associated bacterial pathogens, and represents a new threat to both the community as well as international travelers in the region. Notably, international travelers have been shown to be a potential vector for the spread of resistant bacterial pathogens through acquisition during travel, contributing to the global dissemination of MDR enteric pathogens [
40]. The combination of established community prevalence and additional spread by infected travelers may contribute to local and global dissemination of ESBL-positive ETEC isolates.
ETEC virulence factors, such as enterotoxins and CFs, are of particular interest as potential targets for infection intervention as vaccine or therapeutic targets [
15,
18,
21]. The expression of enterotoxins and CFs are essential for ETEC colonization of the small intestine and resulting pathogenesis. Blocking bacterial cell adhesion to the small intestine and/or neutralizing enterotoxins results in a limited ETEC infection and significantly reduces infection morbidity [
15,
18,
21]. The currently reported prevalence of the enterotoxins LT, STh, and STp in this study were comparative to previous studies reviewed and analyzed by Isidean, et al. [
18]. The most prevalent CFs in the current study were CS21, CS6, CS3, and CS2, while the most prevalent CFs in previously conducted studies reviewed by Isidean et al were CFA/1, CS6, and CS21 [
18]. Notably, CFA/1 was detected at a lower rate in the current study than previously reported (6.8 vs 20%), indicating CFA/1 may not be a priority target for ETEC interventions in South Asia [
18]. Overall, the current study suggests vaccines and therapeutics targeting the enterotoxins LT and STh, and the CF’s CS21, CS6, CS2, and CS3 would likely be most efficacious against ETEC infections in South Asia. This study demonstrated that these virulence factors are also prevalent in the ESBL-positive ETEC population, a group that is important to target for vaccine efficacy as MDR ETEC continues to emerge and spread throughout the region. It is important that ongoing studies characterizing virulence factors in MDR ETEC populations are performed to determine relevant intervention strategies against currently circulating strains.
Many ESBL genes have been reported as actively circulating in South Asia in hospital- and community-associated infections, including
blaCTX-M group 1 and group 9,
blaPER,
blaVEB,
blaGES, and
blaSHV [
41]. Within the gene family
blaCTX-M, blaCTX-M-15 and
blaCTX-M-14 are the most common type of ESBL found worldwide [
27,
42]. Aligning with the global findings of gene prevalence, previous studies conducted in Nepal have identified
blaCTX-M-15 as prevalent in hospital-associated
E. coli infections, while
blaTEM and
blaSHV were also detected at a lower frequencies [
43,
44]. In the current study,
blaCTX-M-15 was detected in the majority of ESBL-positive ETEC isolates. The genes
blaSHV-12,
blaCTX-M-14,
blaTEM-1, and
blaCMY-2 were also identified at lower rates, and multiple resistance genes were detected in 40% of the characterized isolates. Importantly, ESBL resistance mechanisms are often located on mobile plasmids [
42,
45,
46]. As such, enteric ETEC strains that are ESBL-positive may potentially serve as a community reservoir in Nepal for the dissemination of ESBL resistance mechanisms via clonal expansion and/or horizontal transfer to other bacterial species that do not result in enteric infections. A large percentage of ETEC isolates identified in the current study contain multiple resistance mechanisms, suggesting that horizontal transfer of ESBL genes is actively occurring within enteric bacterial pathogens. Interestingly, this study identified an ESBL-positive ETEC isolate from a patient control sample, absent of diarrheal symptoms, indicating the established presence of asymptomatic MDR pathogens in the community that may contribute to gene dissemination. Additional studies are necessary to observe the source and spread of ESBL resistance mechanisms in community-associated infections in Nepal.
Limitations of the current study include small sample sizes per year and the lack of clinical patient information. These limitations serve to highlight the need for additional long-term, comprehensive enteric pathogen surveillance to track ongoing MDR emergence and spread in community settings. ESBL-positive E. coli often have limited treatment options, such as carbapenem antibiotics. However, the efficacy of these treatment options are compromised by increased prevalence of carbapenemase resistance mechanisms, such as blaNDM-1, blaKPC, and blaIMP. No carbapenemase-producing ETEC were identified in the current study, but continued surveillance is imperative for tracking if, or more likely when, community-associated MDR ETEC isolates acquire carbapenem-resistance mechanisms in Nepal.
Acknowledgements
The authors would like to thank Dr. John Crawford, members of the AFRIMS Department of Enteric Diseases, Walter Reed/AFRIMS Research Unit Nepal, Bharatpur Hospital, Kanti Children’s Hospital, Sukraraj Tropical and Infectious Disease Hospital, and CIWEC Hospital and Travel Medicine Clinic for assistance, advice, and helpful discussion throughout the project and during manuscript preparation.
Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.