Current perspectivesin pathogenesis and antimicrobial resistance of enteroaggregative Escherichia coli

Microbial Pathogenesis 85 (2015) 44e49

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Review

Current perspectivesin pathogenesis and antimicrobial resistance of enteroaggregative Escherichia coli Haishen Kong a, Xiaoping Hong b, Xuefen Li a, * a

State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, 310003, PR China b Department of Laboratory Medicine, SGCC Occupational Disease Prevention and Treatment Hospital, Jiande, 311600, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 January 2015 Received in revised form 28 May 2015 Accepted 5 June 2015 Available online 6 June 2015

Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen that causes acute and persistent diarrhea in children and adults. While the pathogenic mechanisms of EAEC intestinal colonization have been uncovered (including bacterial adhesion, enterotoxin and cytotoxin secretion, and stimulation of mucosal inflammation), those of severe extraintestinal infections remain largely unknown. The recent emergence of multidrug resistant EAEC represents an alarming public health threat and clinical challenge, and research on the molecular mechanisms of resistance is urgently needed. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Escherichia coli Enteroaggregative Escherichia coli Pathogenicity Multidrug-resistant

1. Introduction Diarrheal disease remains a significant human health burden on the global scale. Developing countries not only account for the highest incidence rates, but diarrheal disease is the leading cause of death in many. Moreover, diarrhea is a common cause of malnutrition in infants and young children worldwide. China is no exception, and among the nation's incidences of infectious diseases, the most common are infections with enteric pathogens that promote malabsorption and cause diarrhea. Enteric infection with diarrheagenic Escherichia coli (DEC) is often sporadic and frequently represents domestic outbreaks of

Abbreviations: AA, aggregative adherence; AAF, aggregative adhesion fimbria; Caco-2, human colonic adenocarcinoma cells; DAEC, diffuse adhering E. coli; DEC, diarrheagenic E. Coli; E. coli, Escherichia coli; EAEC, enteroaggregative E. Coli; EAST1, EAEC heat-stable enterotoxin 1; EHEC, enterohaemorrhagic E. coli; EPEC, enteropathogenic E. coli; ESBLs, extended spectrum b-lactamases; ExPEC, extraintestinal pathogenic E. coli; ETEC, enterotoxigenic E. coli; FliC, flagellin; GM-CSF, granulocyte macrophage colony-stimulating factor; GRO, growth-related gene product; Hra1, heat-resistant agglutinin 1; HEp-2, human epithelial cell line; ICAM, intercellular adhesion molecule; IL, interleukin; INF-g, interferon-g; OMP, outer membrane protein; Pet, plasmid-encoded toxin; Pic, protease involved in colonization; PMQR, plasmid-mediated quinolone resistance; TLR, Toll-like receptor; TNF, tumor necrosis factor. * Corresponding author. E-mail address: [email protected] (X. Li). http://dx.doi.org/10.1016/j.micpath.2015.06.002 0882-4010/© 2015 Elsevier Ltd. All rights reserved.

diarrhea. The majority of infectious diarrheal disease research has focused on the enteroaggregative E. coli (EAEC) subgroup of DEC with main serotype O44, O42, O3 and O86. EAEC causes acute and persistent diarrhea [1e3] and represents a particular threat to travelers and immunocompromised individuals [4e6]. While many cases of EAEC manifest the clinical features of malnutrition and stunted growth (the latter being related to younger patients), asymptomatic carriers have also been reported [7]. For symptomatic cases of EAEC infection, the most common symptoms are gastrointestinal and can include all or a combination of the following: abdominal pain, nausea, vomiting, fever, watery stools, and/or blood/mucus in the stool. The first identification of EAEC occurred relatively recently. In 1987, Nataro et al. [8] described the unique aggregate- and bricklike bacterial formations formed in tissue and cell cultures of samples taken from a pediatric case of acute diarrhea in South America. The adhesive property shown by these bacterial isolates to the human epithelial cell line (HEp-2) led to the characterization of the aggregative-adherent (AA) phenotype. Since their discovery, EAEC sporadic outbreaks have been reported in multiple developed nations, including the United Kingdom, Switzerland, Japan and the United States. Epidemiologic studies of the cases in the United States have shown that the various EAEC strains are evenly distributed among age groups [9]. All strains of the EAEC subgroup of DEC pathogens are capable of

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dense adhesion to intestinal tissues, production of a variety of enterotoxins and cytotoxins, and stimulation of mucosal inflammatory activity [10e13]. However, the various EAEC strains have significant heterogeneity in their genetic composition, correlating with their pathogenic evolution [10,14e16]. Molecular evolution events that have been detected in the EAEC strains include gene gain and loss, as well as bacteriophage-, plasmid-, and transposonrelated modifications; in particular, genetic changes involving the pathogenicity islands have impacted virulence [17]. A typical example of genetic change leading to increased virulence potential is the emergence of a hybrid strain of EAEC and enterohemorrhagic E. coli (EHEC); the EAEC/EHEC hybrid expresses the shiga toxins (Stx1/2) and has an increased adsorption capacity, both of which support the higher mortality rate that has been seen in the cases of human infections detected to date [18,19].The sporadic outbreaks of EAEC/EHEC strains harboring stx2 that have occurred in Germany within the past few years highlight the eminent threat of these rare and under-studied strains [18e20]. EAEC strains are also capable of causing extraintestinal infections, including severe systemic infection. Furthermore, a variety of drug-resistant EAEC strains have already emerged, further occluding our understanding of their pathogenic mechanisms and complicating the clinical management of these cases. In this article, we provide a summary of the known features of EAEC and their underlying pathogenic mechanisms, with a particular focus on those related to drug resistance; this collective information is expected to stimulate and support future research to understand the epidemiology, control and treatment of this emerging threat to human health. 2. Pathogenic mechanisms of EAEC Pathogenesis studies have characterized the following three major features for EAEC: (1) adherence to the intestinal mucosa, (2) amplification of enterotoxins and cytotoxins, and (3) induction of mucosal inflammation. The major virulence factors of EAEC are presented in Table 1. 2.1. Intestinal adhesion Adhesion to the intestinal mucosa is the first step in establishment of EAEC enteric colonization. Two adhesive phenotypes have

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been described, according to the presence (fimbrial) or absence (afimbrial) of pili, which mediate the bacteria accumulating at the mucus layer of the intestine and biofilm formation [3]. Recent research has indicated that the adhesion property is also associated with expression and function of the aggregative adhesion fimbria (AAF) family member of the chaperone assembled fimbrial polyadhesins and the adhesion cell surface protein. As the major adhesion factor of EAEC, AAF mediates intestinal colonization by recognizing and binding to host proteins (receptors) in the extracellular matrix, such as fibronectin [21]. Four variants of AAF have been defined in EAEC to date: AAF/I, AAF/II, AAF/III and AAF/IV. All four are encoded on the pAA virulence plasmid (by the aggA, aafA, agg3 and hda genes, respectively). The four variants have been detected in various strains of EAEC and detailed studies of their mechanisms of action have determined that AAF/I mediates EAEC agglutination to human erythrocytes and AAF/II mediates adhesion to the intestinal mucosa, with both activated upon transcription of the aggR gene which encodes a transcriptional activator [22,23]. Comparative bacterial genome analysis showed that the EAEC AAF/III is also present in the diffuseadhering E. coli (DAEC) and that the agg and aaf adhesion operons are closely related among these bacteria species [24]. In addition, type 1 pili and the E. coli common pilus (ECP) have also been suggested as responsible for the establishment of the AA in some EAEC strains [25,26]. EAEC strains also express non-pilus adhesins, including outer membrane porin (OMP), dispersin, and Hda (a DnaA related protein involved in bacterial replication). The Hda adhesin was found in EAEC isolates from Denmark, and shown to be related to the aggregative adhesion property [27]. The OMPs are common nonstructural proteins in gram-negative bacteria and play important roles in establishment of bacterial colonization, as well as in aggregation and biofilm formation; in the EAECs, these proteins mediate adhesion and aggregation involving the hosts’ red blood cells [28]. Dispersin is a colonization factor related to the adhesion property and penetration function that supports dispersion of the bacteria into the intestinal mucosa [29]. Studies of the mechanisms underlying the latter property have shown that dispersin acts to neutralize the negative charge of EAEC that is mediated by the lipopolysaccharideon its surface, creating a positive charge that supports AAF binding to its host receptors and outward cell diffusion [29]. Dispersin and AAF remain attractive targets for novel

Table 1 Major virulence factors of enteroaggregative Escherichia coli (EAEC). Gene

Virulence factor

Function

Reference

aggR

AggR

[3,4]

aggA

AAF/I

aafA

AAF/II

agg3

AAF/III

had

AAF/IV type 1 pili

virulence regulator transcription activator human erythrocyte hemagglutination biofilm formation binding to extracellular matrix components adherence to the intestinal mucosa biofilm formation Bacterial aggregation aggregative adhesion to cultivated epithelial cells aggregative adhesion aggregative adhesion biofilm formation aggregative adhesion adhesion and aggregation involving host red blood cells promotion of EAEC dispersion across intestinal mucosa biofilm formation enterotoxic activity due to cAMP/cGMP activation serine protease autotransport autotransportin intestinal colonization pore-forming hemolysis, cytolysis

aap astA pet pic hlyE

ECP OMP Dispersin FIS, YafK, Hra1 EAST-1 Pet Pic HlyE

[22] [23]

[24] [27] [26] [25] [28] [29] [11,30,31] [32] [33] [34] [36]

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molecular therapies and/or vaccines; however, the antigenic heterogeneity of EAEC strains has proven an obstacle to the realization of this goal. EAEC are capable of forming biofilms on both biotic and abiotic surfaces and this property is meditated by AAF/I and II, type 1 fimbriae, FIS (a mediator of DNA replication in E. coli), YafK, and heat-resistant agglutinin 1(Hra1) [11,30,31]. Animal and in vitro experiments have shown that EAEC survive in the intestinal mucosa biofilm, which serves to block absorption of nutrients and leads to malnourishment in infected individuals, particularly children, and manifests the EAEC infection symptoms of mucous in stools and persistent diarrhea. Intriguingly, the isolates from the 2011 O104:H4 outbreak in Germany showed remarkably increased levels of AAF/I and biofilm formation, suggesting that the adherence and biofilm formation properties are key factors of high virulence [19]. 2.2. Toxin secretion When the O42 EAEC prototype strain's circular chromosome (5241977 bp) and single pAA plasmid (113346 bp) was sequenced and compared with other non-pathogenic E. coli strains revealed a plethora of virulence-related factors in the pathogenic strain [13]. Two genes in the 042 EAEC pAA plasmid were identified that encode toxins, namely the EAEC heat-stable enterotoxin 1 (EAST-1) gene which adjoins the plasmid-encoded toxin (Pet) genes [32]. The EAST-1 is a well-studied enterotoxin and known to cause watery diarrhea. Moreover, the theorized functional relation to virulence of the EAEC strains is supported by the fact that EAST-1 has also been found in other non-EAEC pathogenic E. coli strains, the enteropathogenic E. coli (EPEC)-related strains of non-EPEC serotypes [3]. Pet, on the other hand, is a serine protease cytopathic autotransporter, capable of functioning as both a heat-labile enterotoxin and cytotoxin. Studies of purified Pet have shown that its toxic effects on host cells include changes to cell morphology (such as cellular elongation, which can lead to cell lysis), cell mobility and contractility (such as functional loss of actin stress fibers), and cellular electrophysiological properties. These changes, individually or collectively, can culminate in loss of the mucosal tissue integrity and potentially life-threatening diarrhea [33]. Further studies of the EAEC circular chromosome have identified other toxin-encoding genes, such as that of the autotransporter protease involved in intestinal colonization (Pic), which is also a key toxin expressed by the pathogenic, diarrhea-causing, gram-negative bacteria Shigella flexneri. Studies in the latter have characterized Pic's role in hemagglutination and biofilm formation. Similar to Pet, Pic is immunogenic and induces intestinal hypersecretion, which is an important pathophysiological characteristic of EAECrelated diarrhea [34]. Also Pic mediate immune evasion by the direct cleavage of complement proteins [35]. As such, Pet and Pic are considered significant mediators of EAEC pathogenicity, playing roles in cytotoxicity, colonization and bacteria-mucus biofilm formation. In addition, the O42 EAEC prototype strain was found to chromosomally encode the gene for a hemolytic perforin, HlyE. Oligomerization of this toxin leads to pore-formation in host cells, resulting in cell death by cytolysis and pathological lesions in mucosal tissue [36]. 2.3. Induction of mucosal inflammation The pathogenic outcome of EAEC infections is also a product of inappropriate activation of the host inflammatory response. EAECinduced intestinal inflammation has been demonstrated in a mouse model and observed in human subjects [37]. Studies of the molecular mechanisms underlying this pathogenic process have

indicated that EAEC infection is associated with increases in host inflammatory markers, such as interleukins (ILs; including IL-1 receptor antagonist, IL-1b and IL-8), interferon (IFN)-g, lactoferr in and white blood cells count, in fecal occult blood [38,39]. In the normal inflammatory response, IL-8 activates neutrophils, which express adhesion molecules on their surface that function to restore and protect the integrity of the epithelial barrier of the intestine against abnormal fluid secretion and the resultant diarrhea. However, Steiner et al. [40] showed that EAEC O42 serotypes isolated from pediatric cases of persistent diarrhea stimulated overproduction of IL-8 from human colonic adenocarcinoma cells (Caco-2) intestinal epithelial cells; increased levels of IL-8 could cause overstimulation of neutrophils (and their subsequent production/secretion of pro-inflammatory chemokines), thereby perpetuating the inflammatory response to the detriment of intestinal tissue integrity. The authors also determined that the overproduction of IL-8 was mediated by a then unknown heat-stable, high molecular weight EAEC protein. Future investigations by Jiang et al. [38] showed that the increased levels of IL-8 observed in specimens from cases of traveler's diarrhea were associated with infection with EAEC strains carrying the aggR or aafA genes on the pAA plasmid. The O42 EAEC prototype strain has also been shown to upregulate cytokines in intestinal mucosal tissue during infection; these cytokines include IL-6, tumor necrosis factor (TNF)-a, growthrelated gene product (GRO)-a and GRO-g, intercellular adhesion molecule (ICAM)-1, granulocyte macrophage colony-stimulating factor (GM-CSF), and IL-1b. Upregulation of these cytokines has been attributed to host signaling pathways related to the Toll-like receptor (TLR)-5 upon recognition and interaction with the EAEC flagellin (Flic) surface protein [41]. The collective studies of EAEC effects on host expression of IL-8 have indicated that the pathogenic mechanism of this bacterial species involves a multitude of bacteria- and host-expressed factors [39]. 3. Parenteral EAEC infection In 1991, Denmark experienced the first recorded outbreak of parenteral EAEC infections with the uropathogenic multidrugresistant O78:H10 strains, which were identified as ST10 (phylogenetic group A) with a unique combination of extraintestinal pathogenic E. coli (ExPEC) and EAEC characteristics. Phylogenetic analysis and multilocus sequence typing of the O78:H10 strains from these outbreak isolates identified the ExPEC-relatedgenes of fyuA, traT, and iutA and the EAEC-related genes of sat, pic, aatA, aggR, aggA, ORF61, aaiC, aap, and ORF3. The fact that all strains examined had been isolated from the patients’ urine suggested pathogenicity involving the urethral microcosm [42]. Boll et al. [43] confirmed that the outbreak strainshad a significantly enhanced adhesion ability for human bladder epithelial cells and that this property was mediated by the EAEC-expressed AAFs. Moreover, the authors showed that these strains were able to form a biofilm on the abiotic surface of a urinary catheter, highlighting the increased resilience (survivability) and more robust threat of these newly emergent hybrid strains. Another important finding from the studies of these uropathogenic strains is that EAEC can readily evolve to cause severe extraintestinal infections, possibly leading to such life-threatening systemic events as sepsis. Indeed, Herzog et al. [44] recently reported a case of traveler's diarrhea in an immunocompromised patient, for whom culturable EAEC were detected in the feces, urine and blood and who necessitated urethral catheterization during the hospitalization; fortunately, although the patient advanced to sepsis, she responded to therapy with trimethoprimesulfamethoxazole, followed by meropenem.

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4. Antimicrobial resistance of EAEC Although most cases of diarrhea are self-limiting, the aggressive and chronic cases of infectious diarrhea often require therapeutic intervention with antimicrobial drugs to ensure resolution and halt progression to dehydration, hypovolemic shock and death. The most frequently used first-line antimicrobials are ampicillin, trimethoprim-sulfamethoxazole, tetracyclines and quinolones, due to their ready availability and inexpensive cost. The recent emergence of antimicrobial resistance and rapid dissemination of multidrug-resistant DEC strains has created a serious challenge to clinical management of infectious diarrhea, for both acute and persistent cases. The problem is even worse in economically underdeveloped countries, which lack the infrastructure and resources to provide alternative interventions. Gene screening of populations with confirmed intestinal pathogens may allow for identification and monitoring of the evolution of microbes with drug resistance. A long-term study in Nigeria [45] has already confirmed that drug-resistant E. coli strains rapidly increase with time. In that 12-year study (from 1986 to 1998) of 758 apparently healthy young adults (16e32 years-old) the rates of intestinal E. coli isolates with resistance to tetracycline, ampicillin, chloramphenicol or streptomycinincreased from 9e35% to 56e100%. This finding indicated that the presence of strains carrying antimicrobial-resistant genesis increasing in a healthy (nonsymptomatic) population. In addition, this finding may raise further alarm when it is considered in the context of population classification; for example, urban residents are more mobile (traveling for business and pleasure, both locally and internationally) and physically interact with a broader segment of society than their rural dwelling counterparts, representing a greater risk of encountering and distributing multidrug-resistant E. coli strains while appearing and feeling healthy [46]. In a very recent study of 140 pediatric diarrhea cases in Iran due to DEC infection, Haghi et al. [47] found extremely high prevalence of strains with robust resistance to erythromycin (100%), aztreonam (80.7%), amoxicillin (74.4%) and tetracycline (69.3%); in addition, the majority (86.4%)of the strains from these cases demonstrated the multidrug resistance phenotype. Similar studies from China [48] and Nicaragua [49] have shown the trend in emergence of multidrug-resistant strains. Chen et al. [48] investigated 347 DEC isolates from southeast China and found the multidrug resistance phenotype for 70.2%; in addition, 91.8% of these strains from Chinese patients were resistant to ampicillin, which was higher than the rate reported from the Nicaraguan study (67.7%) [49] and the Iranian study (62.0%) [50]. The Nicaraguan and Iranian studies also showed similar resistance rates for tetracycline and trimethoprim-sulfamethoxazole (>50%). Finally, the study from southeast China by Chen et al. [48] showed the effect of DEC serotype and genotype on resistance, demonstrating that O6, O25, O159, ST48, ST218, ST94 and ST1491 were sensitive to most antibiotics, while O45, O15, O1, O169, ST38, ST226, ST69, ST31, ST93, ST394 and ST648 were highly resistant to most antibiotics. Studies have uncovered varying degrees of antimicrobial resistance among the various DEC types. For example, the clinical EAEC isolates show a higher resistance in general to the most commonly used antibiotics compared to the other DECs [48,49,51]. In a Peruvian study of infectious diarrhea in infants, EAEC was found to be the most common DEC, and that it had significantly higher resistance to ampicillin, cotrimoxazole, tetracycline and nalidixic acid than the ETEC and EPEC isolates examined from the study population [7,52]. A study in India similarly showed that most of the EAEC isolates had resistance to many antibiotics, including the quinolones [53]. Studies of pediatric cases of infectious diarrhea in Iran [54,55] showed rates of EAEC resistance to ampicillin,

47

erythromycin, cephalothin and trimethoprim-sulfamethoxazole ranging from 70 to 100%, and slightly lower rates (42e57%) for resistance to nalidixic acid and ciprofloxacin; in addition, there was a remarkable (86.4%) rate of multidrug resistant isolates. EAEC resistance may be associated with a more robust ability of the bacteria to colonize the intestine. As described above, colonization involves aggregation and biofilm formation, features which may decrease the pathogen's physical exposure to antibiotics in the micro environment and increase the chance of acquiring resistance genes (via horizontal gene transfer) [7] or virulence plasmids (via conjugation), such as pCVD, which may possibly be further promoted by antibiotic stress conditions [56]. Research to understand the mechanisms underlying EAEC resistance has provided significant insights through studies of the ß-lactam-resistant phenotype. In general, EAEC produce extended spectrum b-lactamases (ESBLs), enzymes that inactivate cephalosporins and aztreonam via hydrolysis. Khoshvaght et al. [55] showed that among the ESBLsproducing EAEC strains examined the transposable blaTEM genes are the most common (78.9%) feature associated with this phenotype followed by blaCTX-M gene (63.1%), with 42.1% of the stains carrying both the blaTEM and blaCTX-M genes. Our previously mentioned study also examined ESBL-producing EAEC strains and showed that the major genes associated with this resistance phenotype encode CTX-M-14, CTX-M-55, CTX-M-15 and CTX-M-65 [48]. In another study from Peru examining the genetic mechanisms of quinolone resistance, point mutations in the gyrA and parC genes and overexpression of efflux pumps were shown to play an important role in diarrheogenic (DEC) and commensal (non-DEC) isolates [57]. In addition, an earlier study in Peru showed that the qnrB19 gene of the plasmid-mediated quinolone resistance (PMQR) genes contributes to the quinolone-resistant phenotype in EAEC [58]. 5. Conclusion and future perspective EAEC is frequently detected in the world's pediatric population with acute and chronic diarrhea and remains the major pathogenic agent of global traveler's diarrhea. While epidemiologic studies have been carried out to gain an understanding of the distribution and emergence of EAEC strains, especially those with antimicrobial-resistant phenotypes, the pathogenic mechanisms of EAEC have yet to be fully elucidated. Enhancing worldwide surveillance of EAEC infections, both symptomatic (diarrheal) and asymptomatic, will support studies of the pathogenic and resistance mechanisms and development of prevention and treatment programs. Conflicts of interest None of the authors has declared any conflict of interest which pertains to this manuscript. Acknowledgments This study was supported by grants from the National Key Basic Research Program (973) of China (2013CB531605) and the National Key Program for Infectious Diseases of China (2012ZX10004210). References [1] D.B. Huang, P.C. Okhuysen, Z.D. Jiang, H.L. DuPont, Enteroaggregative Escherichia coli: an emerging enteric pathogen, Am. J. Gastroenterol. 99 (2) (2004) 383e389. [2] I.N. Okeke, J.P. Nataro, Enteroaggregative Escherichia coli, Lancet Infect. Dis. 1 (5) (2001) 304e313. [3] F. Navarro-Garcia, W.P. Elias, Autotransporters and virulence of

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