Recent Antimicrobial Resistance Situation and Mechanisms of Resistance to Key Antimicrobials in Enterotoxigenic Escherichia coli

Abstract

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in developing countries and is regularly imported into developed countries as a major cause of traveler’s diarrhea. ETEC is usually self-limiting and not necessarily treated with antimicrobials, although antimicrobial treatment is recommended in malnourished children, severe cases, and traveler’s diarrhea. However, resistant strains to representative therapeutic agents such as ciprofloxacin and azithromycin have been reported in recent years, and multidrug-resistant ETEC has also emerged. This review discusses the recent antimicrobial resistance surveillance in ETEC and the mechanisms of resistance to major antimicrobials.

1. INTRODUCTION

Enterotoxigenic Escherichia coli (ETEC) is a highly important bacteria as a cause of diarrhea in developing countries with poor sanitation. The main pathogens of acute infectious diarrhea, which account for about 20% of all child deaths in developing countries, are ETEC, rotaviruses, and Shigella spp.^1–3) Whereas Shigella spp. and rotaviruses are easily detected by standard assays, ETEC is more difficult to discriminate from other E. coli, particularly diarrheagenic E. coli, and the frequency and characteristics of ETEC on a global scale are poorly understood. ETEC is a frequent cause of diarrhea in children and adults living in developing countries and is a major cause of traveler’s diarrhea (TD) in travelers to these regions, so the organism is regularly imported into developed countries.^4–6)

ETEC is a type of E. coli that produces heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST) and a colonization factor for attachment to the small intestine.⁷⁾ There is a wide distribution of clinical ETEC isolates across E. coli species; however, there are several representative ETECs (O6, O8, O78, O128, and O153) with a global distribution.⁸⁾ ETEC infection has clinical symptoms ranging from mild diarrhea to severe syndromes such as the cholera-like diarrhea caused by ETEC is usually self-limiting and correction and maintenance of hydration are most important. Diarrhea in children can be caused not only by ETEC but also by other bacterial and viral pathogens and the insufficient clinical features to distinguish between them makes it difficult to study the effect of antimicrobials in children with ETEC disease, and antimicrobials are not routinely used to treat diarrhea in children. However, children with poor nutrition or other medical complications may have more severe symptoms and require the use of antimicrobials. Patients with persistent diarrhea due to ETEC may also benefit from antimicrobial therapy. Antimicrobials are certainly beneficial in the treatment of TD since ETEC is known to be the most frequent pathogen.

When ETEC was first identified, the organism was usually highly susceptible to all antimicrobials, including tetracyclines and trimethoprim-sulfamethoxazole (SXT).^5,9) However, over time, antimicrobial resistance (AMR) has emerged, and azithromycin and fluoroquinolones are now used as first-line drugs for ETEC infections.¹⁰⁾ However, in Bangladesh and India, as well as resistance to fluoroquinolones, high levels of AMR to several antimicrobials have been observed.^11,12) Azithromycin-resistant ETEC has also emerged in several countries.^11,13–16) Taken together, azithromycin and fluoroquinolones remain the most common drug of choice, although effective antimicrobial treatment for diarrhea caused by ETEC may differ slightly by region. The increase in AMR underlines the need for continuous surveillance of AMR, as it affects the drugs used to treat TD.

This review aims to provide information on recent research and AMR in ETEC. We surveyed the literature on resistance rates of ETEC to commonly used antimicrobials and resistance factor detection, as well as resistance factor analysis using whole-genome sequencing (WGS) of a large collection of globally representative ETEC isolates.

2. MATERIALS AND METHODS

2.1 De Novo Genome Assembly, and Resistance Gene Analyses

All ETEC raw sequence data were from the PRJEB1252, PRJEB2215, PRJEB2290, PRJEB2581, PRJEB2796, PRJEB2827, and PRJEB3144 project.

The raw sequences were data quality assessed and contig assembled by bactpia 3.0.0 with default settings.¹⁷⁾ The assembled genomes were identified for AMR genes using the ResFinder database¹⁸⁾ by ABRicate (https://github.com/phac-nml/staramr) and point mutations associated with AMR were identified using Pointfinder v050218¹⁹⁾ in STARAMR 0.7.2.²⁰⁾

3. MAJOR LINEAGE, TOXINS, AND VIRULENCE FACTORS IN ETEC

ETEC are strains of E. coli that acquire and maintain plasmids carrying toxins (LT and/or ST) and colonization factors (CFs). ETEC are found across phylogenetic groups A, B1, B2, D, and E (A and B1 being more common) and are widely distributed across E. coli species.^21,22) More than 100 O antigens have been reported to be associated with clinical ETEC isolates, with O6, O8, O78, O128, and O153 known as representative ETEC.⁸⁾ A recent comparative genomics study of ETEC using global ETEC collection between 1980 and 2011 identified 21 lineages (L1–L21) characterized by distinct profiles of enterotoxin and CF.²¹⁾

LT is biologically, structurally, and antigenically very similar to cholera toxin and has a similar mechanism of action. LT is an A1B5 toxin family protein²³⁾ in which the B subunit irreversibly binds to the GM1 ganglioside²⁴⁾ and the A subunit activates adenylate cyclase, resulting in increased cyclic AMP,²⁵⁾ stimulating salt and water into the intestinal lumen resulting in watery diarrhea. LT has two subtypes, LTI and LTII; the difference between LTI and LTII is based largely on the dissimilarity of their B subunits.²⁶⁾ LTI is divided into LTIh and LTIp, produced by humans and by porcine and human ETEC, respectively. LTII is antigenically distinct from LTI and has only 41% sequence identity with LTI, but has similar biological activity. LTII has been detected in humans, bovine, buffalo, porcine, and ostrich.²⁷⁾ In human-derived ETEC strains, LTI is mainly detected, and 28 variants have been reported.^28,29)

ST is a non-antigenic small peptide, classified as STa (STI) and STb (STII), with STa predominantly detected in humans.³⁰⁾ STa is further classified into 2 subtypes, STh detected in human ETEC and STp detected in porcine, bovine, and human ETEC. Increased cGMP activates cGMP-dependent protein kinase II, which, in turn, causes phosphorylation of cystic fibrosis transmembrane regulator (CFTR), leading to inhibition of Cl-secretion and NaCl uptake, followed by net water loss due to osmotic diarrhea.^31,32) STa has also been reported in 6 variants.²⁹⁾

CFs are classified as helical, fimbrial, fibrillar, or afimbrial proteins.³³⁾ CF settles the organism in the small intestine and expresses either LT and/or ST in close proximity to the intestinal epithelium. Due to the specificity of CFs, animal ETEC strains do not generally infect humans. Currently, more than 20 CF have been reported in human ETEC, with individual ETEC strains typically expressing 1 to 3 types of CF. However, CF was not detected in all ETEC, and approximately 30–50% of strains had no known CF. This may be due to either the absence of CFs, loss of CF due to strain subcultures, or the presence of unknown CFs.³³⁾

4. ANTIMICROBIAL TREATMENT FOR ETEC

TD is the most common disease in travelers, 80–90% of which is caused by bacterial pathogens (e.g., ETEC and enteroaggregative E. coli (EAEC), Campylobacter spp, Shigella spp., and non-typhoidal Salmonella spp.). ETEC is frequently detected in TD patients during or after visits to sub-Saharan Africa. ETEC is also a common cause of TD in Latin America and the Caribbean. In South and South-East Asian travelers, the prevalence of ETEC is second highest after Campylobacter.^4,6) TD is a self-limiting disease that often recovers within 5 days, but has a negative impact on travel plans and there is potential for long-term intestinal and extra-intestinal complications.

Antimicrobial therapy has been proven to be effective, significantly reducing associated symptoms and shortening the duration of morbidity.³⁴⁾ Currently, guidelines for TD treatment recommend antimicrobial therapy with azithromycin, fluoroquinolones, and rifaximin in moderate to severe cases, and fluoroquinolones are traditionally used as first-line antimicrobials.¹⁰⁾ Increased fluoroquinolone resistance in ETEC from various regions has been reported, and ETEC with low susceptibility to azithromycin have also been reported in travelers to Asia.^{11,12,35–37)} Multidrug resistance in Enterobacteriaceae acquiring ESBL (extended-spectrum β-lactamase) is also becoming increasingly common worldwide and ESBL-ETEC have been reported.³⁸⁾

5. AMR SURVEILLANCE AND RESISTANCE FACTOR IN ETEC

5.1 AMR Surveillance

Due to the widespread use of antimicrobial agents in diarrhea-endemic countries, ETEC has also seen an increase in multidrug resistance. However, reporting of antimicrobial susceptibility in ETEC is limited, having been studied in epidemiological surveys of TD patients traveling to or returning from the country where diarrhea is widespread. Resistance rates in ETEC vary from region to region.

Although fluoroquinolone and azithromycin are recommended for ETEC treatment, low-cost classical antimicrobials such as ampicillin, SXT, and tetracycline can be used in developing countries. As shown in Table 1,^{11,12,35–37,39–42)} β-lactam antimicrobial, ampicillin was frequently resistant in all reports, with resistance in 40%-90% of cases. Acquisition of ESBL in ETEC has also been reported, with a small number of reports of susceptibility testing for amoxicillin/clavulanic acid and third-/fourth-generation cephalosporin, but a low number (approx. 30%) have become resistant. Resistance to tetracyclines (30–90%) and SXT (25–65%) was also prevalent. Regarding quinolones including the first-line antimicrobial ciprofloxacin for the treatment of ETEC, resistance to nalidixic acid, the old quinolone, was observed in the majority of ETEC in Asia (70–90%). For ciprofloxacin, a certain number of resistant strains have been identified in India (70%) and Bangladesh (25%), and a few resistant strains have emerged in other regions (2–15%). For azithromycin, which is recommended for the treatment of TD in Southeast Asia where Ciprofloxacin-resistant Campylobacter are more frequent, resistant strains have also been identified in India (80%), Bangladesh (25%), and Nepal (10%).

Table 1. Antimicrobial Resistance Rates (%) of ETEC by Country

Country	China	China	Bangladesh	India	Nepal	Nepal	Spain	Ethiopia	Peru
Period	2012–2013	2012	2005–2009	2015–2019	2001–2016	2012–2014	2001–2004	2020–2021	2006–2008
No. of strains	318	123	903	153	265	60	108	39	205
Reference	35)	40)	12)	11)	36)	42)	37)	41)	39)
β-Lactam
Ampicillin	51.6	37.4	66.4	75.2	42.6	46.7	52.8	92.3	63.9
Amoxicillin /clavulanate	16.7	—	—	—	—	—	17.6	10.3	8.8
Ceftriaxone	—	17.9	12.6	31.4	—	—	—	—	—
Ceftazidime	9.7	—	—	—	—	—	—	15.4	1.5
Cefepime	19.2	—	—	24.2	—	—	—	2.6	—
Cefotaxime	21.1	—	—	—	—	—	—	15.4	—
Quinolone
Nalidixic acid	68.9	71.5	82.7	87.6	—	—	22.2	—	10.2
Norfloxacin	—	—	26.7	36.6	—	—	—	—	—
Ciprofloxacin	5.3	1.6	26.7	69.3	5.7	10.0	8.3	15.4	—
Ofloxacin	3.1	1.6	—	—	—	—	—	—	—
Macrolide
Erythromycin	—	—	97.0	98.0	—	—	—	—	—
Azithromycin	—	—	24.7	83.0	—	10.0	—	—	—
Aminoglycoside
Gentamicin	6.0	3.3	—	—	—	—	—	25.6	0.5
Streptomycin	92.5	13.0	48.1	99.3	—	—	—	—	—
Tetracycline
Tetracycline	28.6	35.0	42.3	32.0	27.9	26.7	59.3	92.3	37.1
Doxycycline	—	—	43.7	26.8	—	—	—	—	—
Phenicol
Chloramphenicol	3.8	—	—	6.5	—	—	30.6	23.1	6.8
Sulfamethoxazole-trimethoprim (SXT)
SXT	26.1	3.3	46.0	39.9	29.1	28.3	66.7	33.3	52.2
Sulfamethoxazole	27.7	55.3	—	—	—	—	—	—	—
Trimethoprim	38.1	—	—	—	—	—	—	—	—

ETEC, Enterotoxigenic Escherichia coli.

5.2 Resistance Factors

Resistance factors were conventionally detected by PCR methods, which could only detect selected resistance factors from the respective surveys, but recently the development of WGS has enabled comprehensive detection of resistance factors. The reports of resistance factor analysis using WGS in ETEC are limited,^16,43–46) and many reports have been based on PCR methods. Resistance factors for ETEC are described below, based on the analysis in the global ETEC collection used to determine the global lineage (Fig. 1) and the literature on ETEC resistance factor detection.

Fig. 1. Antimicrobial Resistance Factor Distribution on Global ETEC Collection

Genetic determinants for antimicrobial resistance (resistance genes and mutations in targets of antimicrobials) present in 362 genomes of global ETEC collection were identified by in silico analysis as described in “Materials and Methods”. ETEC, Enterotoxigenic Escherichia coli.

5.3 β-Lactams

β-Lactams inhibit bacterial cell wall biosynthesis. β-Lactams are the most widely used antimicrobials and the prevalence of β-lactam-resistant bacteria has increased greatly over the past few decades. In Gram-negative bacteria, the acquisition of β-lactamase, which degrades β-lactams, is the major resistance mechanism. ESBL confers resistance to many β-lactam antimicrobials, including penicillins, broad-spectrum cephalosporins, and monobactams, and is spread worldwide by clonal expansion and horizontal transfer between species.

The acquisition of ESBLs is progressing in ETEC as third-/fourth-generation cephalosporin resistance is often observed in surveillance.^{11,12,14,35,40,47)} In the global ETEC collection, blaTEM1-B was the most frequently identified β-lactamase, whereas other β-lactamases were rare. Reports on β-lactamase typing in ETEC are limited, but mainly blaCTX-M-15 is reported^{15,36,43,48,49)} and blaTEM is often reported,^{36,39,45,50–52)} suggesting that these 2 ESBLs are widespread in ETEC. Since the detection of blaCTX-M-15 was reported in more recent ETECs than the global ETEC collection, blaCTX-M-15 may be a recent spread.

5.4 Aminoglycoside

Aminoglycosides bind to the 30S ribosomal subunit and inhibit protein synthesis. Aminoglycoside modifying enzymes (AMEs) such as aminoglycoside acetyltransferases, aminoglycoside phosphotransferases, and aminoglycoside nucleotidyltransferases are most important in the aminoglycoside resistance mechanism.

AMEs also play an important role in ETEC, with aph(3ʺ)-Ib and aph(6)-Id being particularly frequent in the global ETEC collection, followed by ant(3ʺ)-Ia and ant(3ʺ)-Ib. Both aph(3ʺ)-Ib and aph(6)-Id coexist and spread widely with diverse transfer factors such as plasmids, ICE, and genomic islands⁵³⁾ and are also frequently found in E. coli.^54–58) Aminoglycoside is not a recommended treatment for ETEC in humans and information on gene detection and susceptibility testing is limited. However, AMEs are often reported in animal-derived ETECs.^59–61)

5.5 Quinolone

Quinolones inhibit DNA replication by targeting DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE). Ciprofloxacin is a standard treatment for TD. The main resistance mechanism to quinolones in ETEC, as in most bacteria, is mutations in the region between amino acids 67 and 106 of subunit A (GyrA, ParC), known as the QRDR (Quinolone Resistance Determining Region).^62,63)

There are many reports of quinolone resistance in ETEC, a single mutation in GyrA (S83L) is sufficient for the old quinolone nalidixic acid, while double mutations in GyrA (S83L + D87N) together with a single mutation in ParC (S80I) are required for the fluoroquinolones.^12,37,45) In global ETEC collections, GyrA mutations (S83L) were identified in small numbers and mutations in QRDR were relatively rare. Meanwhile, GyrA + ParC mutations are also often reported,^12,15,37) although they are often not investigated in surveillance, as the detection of mutations in QRDR requires sequencing. Nalidixic acid resistance is very common on surveillance in Asia,^12,35,40,47) and ETEC acquiring GyrA (S83L) is probably widespread. In addition to mutations in QRDR, transferable mechanisms of quinolone resistance (TMQR) are also rarely reported. Qnr, which encodes a target protection protein that protects gyrase from quinolone inhibition, and a mutant of the aminoglycoside acetyltransferase aac(6′)-Ib, which acetylates fluoroquinolones aac(6′)-Ib-cr have been detected, but both only confer low-level resistance, with mutations in QRDR being the more important resistance factor.^{15,43,45,46,50)}

5.6 Macrolide

Macrolides bind to the 50S ribosomal subunit and inhibit protein synthesis. Generally, macrolide antimicrobials have low membrane permeability, exhibit low activity against Enterobacteriaceae, and cannot be used for treatment, whereas azithromycin is a basic compound and has been used to treat diarrheal infections associated with a range of Enterobacteriaceae, including ETEC. Although azithromycin has no defined resistance breakpoint against E. coli, it is a representative antimicrobial for ETEC infections alongside ciprofloxacin.

Macrolide resistance is mediated by 3 mechanisms: enzymatic inactivation of macrolides, target alterations through modification or point mutations, and increased drug efflux.⁶⁴⁾ In global ETEC collections, detection of macrolide resistance genes was very rare. However, azithromycin resistance has been reported sporadically in ETEC in recent years,^{11,12,15,42,65)} mainly due to the acquisition of phosphotransferases mph(A).^16,39,44) Reports of macrolide efflux pumps such as msr(A), mef(A), and mef(B) and ribosomal RNA (rRNA) methylase, erm, were rare in ETEC.¹³⁾

5.7 Tetracycline

Tetracycline antimicrobials inhibit protein synthesis by preventing the binding of aminoacyl-tRNA to the ribosomal acceptor (A) site. ETECs were initially sensitive to tetracyclines, but resistance has increased over time. Resistance to tetracycline is caused by the acquisition of tetracycline resistance (tet) genes associated with efflux pumps, ribosome protection, and enzyme inactivation.⁶⁶⁾ Currently, 38 tet genes have been reported,⁶⁷⁾ with the efflux pump system being particularly important, and tet(A) and tet(B) are reported to be the most common tetracycline resistance determinants in E. coli in many countries.⁶⁸⁾

Acquisition of tet(A) or tet(B) is frequently observed in global ETEC collections and these acquisitions are the most common in ETEC and have been reported in surveillance in various regions.^{39,44–46,50)}

5.8 SXT

SXT, the combination of trimethoprim and sulfamethoxazole, inhibits bacterial folate synthesis. Sulfamethoxazole inhibits dihydropteroate synthase (DHPS), which catalyzes the formation of dihydropteroate from p-aminobenzoic acid, while trimethoprim inhibits dihydrofolate reductase (DHFR), which catalyzes the reduction of dihydrofolate to tetrahydrofolate. Sulfamethoxazole and trimethoprim resistance are mediated by the acquisition of sulfamethoxazole resistance genes (sul genes) and trimethoprim resistance genes (dfr genes), which are low-affinity DHPS and DHFR homologs, respectively. To date, only 4 sul genes have been reported,⁶⁹⁾ whereas more than 40 dfr genes have been reported.⁷⁰⁾

E. coli was widely susceptible to SXT in the 1970s, but high resistance has currently been reported in many regions of the world.^4,71,72) The sul and dfr genes were found at high frequencies in the global ETEC collection, with sul2 being the most frequent sul gene, followed by sul1, and dfrA8 being the most frequent dfr gene, followed by dfrA1, dfrA7, and dfrA15. Although sul2 was also reported with high frequency in surveillance reports,^{16,39,44–46)} dfr was reported in a variety of variants other than the variants observed in the global ETEC collection.^{16,39,44–46,50,52)}

5.9 Chloramphenicol

Chloramphenicol binds to the 50S ribosomal subunit and inhibits protein synthesis. Resistance to chloramphenicol is mainly enzymatic modification by the chloramphenicol acetyltransferase (cat). Acquisition of specific efflux systems for chloramphenicol such as cml and floR has also been reported.^73,74) In the global ETEC collection, catA1 was detected in a small number of cases. Although reports of resistance genes are not frequent due to the low frequency of chloramphenicol resistance in ETEC, catA is often reported,^39,50) and floR is rarely reported.³⁹⁾

6. SUMMARY AND PROSPECTS

ETEC is a major pathogen of TD and defense strategies include antimicrobial treatment as well as improved hygiene and the development of effective vaccines, but hygiene improvement is slow and vaccine development is underway.^75,76) Resistance to ciprofloxacin and azithromycin for the treatment of ETEC is gradually spreading and multidrug-resistant ETEC is emerging, which may limit therapeutic options in the future. This underlines the importance of continuous surveillance of AMR in ETEC.

With advances in next-generation sequencing technology, the cost of WGS of bacteria has decreased significantly and has been widely applied in epidemiological studies and the characterization of bacterial isolates for virulence and resistance gene identification. AMR surveillance by susceptibility testing is limited in elucidating the entire profile of AMR given different variations of antimicrobials in each institution due to technical and cost issues. However, WGS enables comprehensive resistance factor analysis and detailed comparisons between different time periods, regions, and institutions. Also, ETEC have acquired toxins and CF on plasmids, and plasmid incompatibility may limit resistant plasmids that can be acquired, which may bias the acquisition of resistance in ETEC. The development of long-read sequencing, such as Nanopore and PacBio, has made it possible to determine the complete genome sequence, distinguishing between genome and plasmid, which was not possible with short-read sequencing. The sequencing of resistance and virulence plasmids may clarify this question. In the future, these technologies could provide more detailed and rapid information necessary for infection prevention and control measures, such as the selection of vaccine antigens that can protect against circulating ETEC, monitoring against novel ETEC lineages, and the use of appropriate antimicrobials through resistance factor analysis.

Conflict of Interest

The authors declare no conflict of interest.

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