CHARACTERIZATION OF ENTEROAGGREGATIVE ESCHERICHIA COLI AMONG DIARRHEAL CHILDRENIN WESTERN BRAZILIAN AMAZON

Caracterização de Escherichia coli enteroagregativa entre crianças com diarreia na Amazônia ocidental brasileira

Roger Lafontaine Mesquita TABORDA Luiz Antônio da SILVA Patricia Puccinelli ORLANDI Flávia Serrano BATISTA Renata Santos RODRIGUES Najla Benevides MATOS About the authors

ABSTRACT

BACKGROUND:

Enteroaggregative Escherichia coli (EAEC) is one of the main acute and chronic diarrhea causes both in children and adults, mainly in developing countries.

OBJECTIVE:

The aim of the present study is to characterize EAEC strains isolated from faecal samples and to identify genes potentially contributing to virulence, biofilm production and antimicrobial resistance in children admitted to a pediatric hospital in Porto Velho, Rondônia State.

METHODS:

The total of 1,625 E. coli specimens were isolated from 591 children in the age group 6 years or younger who were hospitalized in Cosme and Damião Children Hospital in Porto Velho, between February 2010 and February 2012, with acute gastroenteritis. Colonies suggestive of E. coli were subjected to polymerase chain reaction testing in order to identify the virulence factors. The in vitro adhesion assays using HEp-2 adherence were tests. Biofilm detection through spectrophotometry and antimicrobial susceptibility tests were conducted in the disk diffusion method.

RESULTS:

The mentioned study examined 591 stool samples from children with diarrhea. Diarrheogenic E. coli was found in 27.4% (162/591) of the children. EAEC was the diarreagenic E. coli most frequently associated with diarrhea 52.4% (85/162), which was followed by enteropathogenic E. coli 43.8% (71/162), enterotoxigenic E. coli 2.4% (4/162), and enterohemorrhagic E. coli 1.2% (2/162). The aggR gene was detected in 63.5% (54/85) of EAEC isolates; moreover, statistically significant correlation was observed among typical EAEC (aggR) and aatA (P<0.0001), irp2 (P=0.0357) and shf (P=0.0328). It was recorded that 69% (59/85) of the 85 analyzed EAEC strains were biofilm producers; 73% (43/59) of the biofilm producers carried the aggR gene versus 42.3% (11/26) of non-producers (P=0.0135). In addition, there was association between the aatA gene and biofilm production; 61% (36/59) of the samples presented producer strains, versus 19.2% (5/26) of non-producers (P<0.0004). Antibiotic sensitivity test evidenced that most EAEC were ampicillin 70.6% (60/85), sulfamethoxazole 60% (51/85), tetracycline 44.7% (38/85) and cefotaxime 22.4% (19/85) resistant.

CONCLUSION:

As far as it is known, the present study is pioneer in Northern Brazil to investigate EAEC virulence factors and to show the antimicrobial susceptibility of EAEC strains isolated from children with diarrhea.

HEADINGS:
Escherichia coli, classification; Infantile diarrhea; Child; Virulence, genetics

RESUMO

CONTEXTO:

A Escherichia coli enteroagregativa (EAEC) é um dos principais agentes causadores de diarreia aguda e crônica em crianças e adultos, principalmente em países em desenvolvimento.

OBJETIVO:

Caracterizar cepas de EAEC isoladas de amostras fecais e identificar genes que potencialmente contribuem para a virulência, produção de biofilme e resistência antimicrobiana em crianças internadas em um hospital pediátrico em Porto Velho, Rondônia.

MÉTODOS:

Um total de 1.625 cepas de E. coli foram isolados de 591 crianças com gastroenterite aguda na faixa etária de 6 anos que foram internadas no Hospital Infantil Cosme e Damião na cidade de Porto Velho, entre fevereiro de 2010 e fevereiro de 2012. Colônias sugestivas de E. coli foram submetidas a reação em cadeia da polimerase para identificação de fatores de virulência. O ensaio de adesão in vitro foi desenvolvido com célula HEp-2. A detecção de biofilme foi realizada através do teste de espectrofotometria e os testes de susceptibilidade aos antimicrobiana foram realizados através do método de difusão em disco.

RESULTADOS:

A E. coli diarreiogênica foi encontrada em 27,4% (162/591) das crianças e a EAEC foi a E. coli diarreiogênica mais frequentemente associada à diarreia com 52,4% (85/162), seguida pela E. coli enteropatogênica 43,8% (71/162), E. coli enterotoxigênica 2,4% (4/162) e E. coli enterohemorrágica 1,2% (2/162). O gene aggR foi detectado em 63,5% (54/85) dos isolados de EAEC com correlação estatisticamente significante entre esse gene com os genes aatA (P<0,0001), irp2 (P=0,0357) e shf (P=0,0328). Neste estudo 69% (59/85) das cepas de EAEC eram produtoras de biofilme, destas 73% (43/59) possuíam o gene aggR, ao passo que entre as não produtoras 42,3% (11/26) possuíam o gene (P=0,0135). Essa associação também foi observada com o gene aatA, presente em 61% (36/59) das cepas produtoras e em 19,2% (5/26) das não produtoras (P<0,0004). O teste de sensibilidade aos antibimicrobianos evidenciou que a maioria das EAEC eram resistentes a ampicilina 70,6% (60/85), ao sulfametoxazol 60% (51/85), a tetraciclina 44,7% (38/85) e a cefotaxima 22,4% (19/85).

CONCLUSÃO:

Este é o primeiro estudo no Norte do Brasil sobre a investigação dos fatores de virulência de EAEC mostrando a susceptibilidade antimicrobiana de cepas de EAEC isoladas de crianças com diarreia.

DESCRITORES:
Escherichia coli, classificação; Diarreia infantil; Criança; Virulência, genética

INTRODUCTION

According to the World Health Organization (WHO), diarrhea is the second leading morbidity and mortality cause among children in the age group 5 years or younger. Diarrhea resulted in 530,000 deaths in 2015, or in approximately 1,400 deaths per day, worldwide11. WHO. UNICEF. Diarrhoea remains a leading killer of young children, despite the availability of a simple treatment solution. Monitoring the Situation of Children and Women. 2017.. The main diarrheal-syndrome causes in children include inappropriate hygiene, insufficient water and food sanitation, besides lack of adequate health infrastructure22. Nweze EI. Aetiology of diarrhoea and virulence properties of diarrhoeagenic Escherichia coli among patients and healthy subjects in Southeast Nigeria. J Heal Popul Nutr. 2010;28:245-52.. Several viral, bacterial and parasitic agents are associated with diarrhea, among them bacteria belonging to species Escherichia coli (which is a natural species composing the intestinal flora). This species is strongly associated with diarrhea cases in children due to the acquisition of specific virulence factors contributing to its pathogenicity, the so-called diarreagenic E. coli (DEC)33. Beau De Rochars VEM, Alam MT, Telisma T, Masse R, Chavannes S, Anilis MG, et al. Spectrum of Outpatient Illness in a School-Based Cohort in Haiti, with a Focus on Diarrheal Pathogens. Am J Trop Med Hyg. 2015;92:752-7..

Diarreagenic E. coli is divided in six pathotypes depending on the pathogenicity mechanism, namely: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli, enteroaggrevative E. coli (EAEC), enterotoxigenic E. coli (ETEC) and diffusely adherent E. coli44. Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11:142-201.. The EAEC is associated with chronic and persistent diarrhea cases in developing countries, besides being one of the most relevant opportunistic pathogens affecting HIV patients. However, despite the several advances concerning the understanding about this bacterium species, its infection mechanism is not entirely understood because the heterogeneity of the pathogen impairs a proper diagnosis, as well as the understanding about its pathogenicity55. Jensen BH, Olsen KEP, Struve C, Krogfelt KA, Petersen AM. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli. Clin Microbiol Rev . 2014;27:614-30..

Previous studies have described the three-stage model applied to EAEC pathogenesis: 1- initial adherence to mucosal surface, 2- biofilm formation, and 3- inflammatory response induction and toxin release. Several virulence factors affect the infection process; however, they can be used as pathogenesis identification markers66. Navarro-Garcia F, Elias WP. Autotransporters and virulence of enteroaggregative E. coli. Gut Microbes. 2011;2:13-24..

The EAEC strains are defined in HEp-2 cells through their aggregative adherence (AA) or “stacked brick” phenotype, which is the gold standard method to identify such pathogen. However, the adherence test is not able to distinguish pathogenic from nonpathogenic EAEC strains. In addition, molecular biology assays based on virulence factor detection have been widely used to identify and characterize DEC isolates77. Zhang R, Gu D, Huang Y, Chan EW-C, Chen G-X, Chen S. Comparative genetic characterization of Enteroaggregative Escherichia coli strains recovered from clinical and non-clinical settings. Sci Rep. 2016;6(October 2015):24321.. The use of molecular techniques, and of epidemiological studies on this isolates, to investigate virulence markers in EAEC is important. Studies have been demonstrating that EAEC strains are heterogeneous and complex groups associated with many virulence factors. Different gene targets have been used to detect EAEC by using polymerase chain reaction (PCR). The knowledge about this genes and their function has been making the development of diagnostic methods and the understanding about the pathogenicity of EAEC possible88. Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent Advances in Understanding Enteric Pathogenic Escherichia coli. Clin Microbiol Rev . 2013;26:822-80..

Some studies adopted the PCR technique to conduct EAEC molecular identification of DEC categories or to assess the presence of specific virulence factors associated with genes such as aggregative adherence fimbriae (AAFs), transcriptional activator (aggR)99. Nataro JP, Yikang D, Yingkang D, Walker K. AggR, a transcriptional activator of aggregative adherence fimbria I expression in enteroaggregative Escherichia coli. J Bacteriol. 1994;176:4691-9., plasmid-encoded toxin (pet)1010. Navarro-Garcia F, Sears C, Eslava C, Cravioto A, Nataro JP. Cytoskeletal effects induced by Pet, the serine protease enterotoxin of enteroaggregative Escherichia coli. Infect Immun. 1999;67:2184-92., Shigella enterotoxin 1 (ShET1)1111. Henderson IR, Czeczulin J, Eslava C, Noriega F, Nataro JP. Characterization of Pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun . 1999;67:5587-96., EAEC heat-stable enterotoxin (EAST1)1212. Ménard L-P, Dubreuil JD. Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1): a new toxin with an old twist. Crit Rev Microbiol. 2002;28:43-60., mucinase activity (Pic)1111. Henderson IR, Czeczulin J, Eslava C, Noriega F, Nataro JP. Characterization of Pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli. Infect Immun . 1999;67:5587-96., secreted proteins (aap)1313. Nataro JP, Deng Y, Cookson S, Cravioto A, Savarino SJ, Guers LD, et al. Heterogeneity of enteroaggregative Escherichia coli virulence demonstrated in volunteers. J Infect Dis. 1995;171:465-8., dispersin transporter (aatA)1414. Nishi J, Sheikh J, Mizuguchi K, Luisi B, Burland V, Boutin A, et al. The Export of Coat Protein from Enteroaggregative Escherichia coli by a Specific ATP-binding Cassette Transporter System. J Biol Chem. 2003;278:45680-9., and yersiniabactin system (irp2)1515. Schubert S, Rakin A, Karch H, Carniel E, Heesemann J. Prevalence of the “high-pathogenicity island” of Yersinia species among Escherichia coli strains that are pathogenic to humans. Infect Immun . 1998;66:480-5. associated with genes in EAEC. The aggR gene is important for the pathogenesis and adherence properties of EAEC; moreover, the presence or absence of aggR is used to classify EAEC as typical or atypical, respectively.

The biofilm formation has been linked to several human diseases. Biofilms are highly organized communities of microorganisms structured within an array of exopolysaccharides (EPS)1616. Beloin C, Roux A, Ghigo JM. Escherichia coli biofilms. Curr Top Microbiol Immunol. 2008;322:249-89.. The bacterial arranged in biofilms tend to be more resistant to antimicrobial therapy, and the ability to biofilm formation in combination with the heterogeneity of virulence genes have been evidenced in EAEC strains1717. Mohamed JA, Huang DB, Jiang ZD, DuPont HL, Nataro JP, Belkind-Gerson J, et al. Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries. J Clin Microbiol. 2007;45:121-6..

In addition to mechanisms directly related to virulence, the antimicrobial resistance has been identified through clinical and non-clinical EAEC sources. The antimicrobial resistance has been seen as one of the most important factors to help assessing the impact this pathogen has on public health, and EAEC associated with multidrug resistant (MDR) was reported from different parts of the world1919. Raju B, Ballal M. Multidrug resistant enteroaggregative Escherichia coli diarrhoea in rural southern Indian population. Scand J Infect Dis . 2009:41:105-8.. MDR is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories1919. Raju B, Ballal M. Multidrug resistant enteroaggregative Escherichia coli diarrhoea in rural southern Indian population. Scand J Infect Dis . 2009:41:105-8..

Since EAEC is one of the main agents causing diarrhea, mainly in developing countries, it is worth conducting characterization and epidemiological surveillance studies, mainly in Northern Brazil, where public and environmental health is directly associated with poverty. Porto Velho is the capital of Rondônia State (Western Brazilian Amazon); the city is among the worst capitals when it comes to basic sanitation. Such poor sanitation condition has strong impact on local health indicators, which depict the epidemiological scenario in the region, as well as the high incidence of diarrheal diseases and infant mortality rates. Given the need of setting the profile of aetiological agents causing acute gastroenteritis, the aim of the present study was to characterize EAEC strains isolated from faecal samples and to identify genes potentially contributing to virulence, biofilm production and antimicrobial resistance in children admitted to a pediatric hospital in Porto Velho, Rondônia.

METHODS

Study site and patients

The total of 1,625 E. coli specimens were isolated from 591 children in the age group 6 years or younger who were hospitalized in Cosme and Damião Children Hospital in Porto Velho, between February 2010 and February 2012, with acute gastroenteritis. Cases were defined as acute gastroenteritis when patients presented liquid or semi-liquid stools, and three or more evacuations within 24-h periods. Sample collection was carried out three times a week, for two consecutive years. One fecal sample was collected from each child participating in the experiment. A sterile universal collector was used during the collection procedure. The samples were registered, labeled and stored at −80ºC. The experiment was approved by the Ethical Committee of Rondônia Tropical Medicine Research Centre (protocol N. 0113/2010).

Bacteriology

E. coli strains were selected from MacConkey, Salmonella-Shigella and xylose lysine deoxycholate selective agar provided by HiMedia U.S.A selective agar. All colonies were processed through routine microbiological and biochemical tests purchased at bioMérieux France (API20E system). Five colonies suggestive of E. coli were subjected to PCR testing in order to identify the virulence factors.

HEp-2 adherence test

All E. coli isolates were subjected to HEp-2 adherence tests2020. Cravioto A, Tello A, Navarro A, Ruiz J, Villafán H, Uribe F, et al. Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet (London, England). 1991;337:262-4.. The EAEC 042 was used as aggregative adherence positive control.

Analysis of E. coli virulence factors through multiplex polymerase chain reaction (PCR)

The EAEC virulence factors aggR, astA, and pic were identified through PCR by using specific primers as previously described by Müller et al.1818. Müller D, Greune L, Heusipp G, Karch H, Fruth A, Tschäpe H, et al. Identification of unconventional intestinal pathogenic Escherichia coli isolates expressing intermediate virulence factor profiles by using a novel single-step multiplex PCR. Appl Environ Microbiol. 2007;73:3380-90.. The aatA, shet1A, shf, irp2, and pet virulence factors were identified according to the method described by Mohamed et al.1717. Mohamed JA, Huang DB, Jiang ZD, DuPont HL, Nataro JP, Belkind-Gerson J, et al. Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries. J Clin Microbiol. 2007;45:121-6..The EAEC 042 was used as positive control. Non-pathogenic E. coli strain HB101 was used as negative control, as well as to monitor PCR contamination.

Biofilm detection through spectrophotometry

The 96-well polystyrene microtiter plates were used to detect biofilms on polystyrene, according to previously described procedures2121. Stepanović S, Vuković D, Hola V, Di Bonaventura G, Djukić S, Cirković I, et al. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS. 2007;115:891-9.. The following strains were used to assure the quality of the biofilm assay: Pseudomonas aeruginosa PAOI, EAEC 042 - which is a strong biofilm producer, and the non-pathogenic E. coli strain HB101 (negative control).

Antimicrobial sensitivity test

Antimicrobial susceptibility tests were conducted in Mueller-Hinton agar (HiMedia U.S.A) through the disk diffusion method, according to Clinical and Laboratory Standards Institute guidelines2222. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard - Ninth Edition. vol. 32. 2012.. Gentamicin (GEN 10 µg), imipenem (IMP 10 µg), piperacillin/tazobactam (TZP 10 µg), tetracycline (TET 30 µg), trimethoprim/sulfamethoxazole (SXT 25 µg), amoxicillin/clavulanic acid (AMC 30 µg), amikacin (AMI 30 µg), ampicillin (AMP 10 µg), cefotaxime (CTX 30 µg), and ceftazidime (CFZ 30 µg) antibiotic disks (Sensifar-cefar®, Brazil) were used. The E. coli strain ATCC 25922 was used for quality control in all tests.

Statistical analysis

Data were analyzed through Fisher’s exact test conducted in GraphPad Prism 5.0. Results were significant at P-values < 0.05.

RESULTS

Pathogens associated with diarrhea

Pathogenic agent isolates analyzed in the study were taken from a study conducted from February 2010 to February 2012 in Porto Velho, Rondonia, Brazil. The mentioned study examined 591 stool samples from children with diarrhea. Diarrheogenic E. coli was found in 27.4% (162/591) of the children. EAEC was the DEC most frequently associated with diarrhea - 52.4% (85/162), which was followed by EPEC 43.8% (71/162), ETEC 2.4% (4/162), and EHEC 1.2% (2/162). Among other enteropathogenic bacteria, Salmonella sp was found in 7.1% (42/591) cases and Shigella species were recorded in 2.1% (13/591) of the cases.

The mean age of the patients was 17.2 months; nonetheless, this pathotype incidence was higher in children under 2 years old, although there was not statistically significant difference.

The total of 85 EAEC isolates (all of them presenting aggregative adherence pattern) from 51 patients were identified through cell assays in order to characterize cellular adhesion. All EAEC strains were subjected to the PCR technique in order to find the virulence factors. Infection caused by EAEC was detected in both male 60.8% (31/51) and female 39.2% (20/51) patients. Typical EAEC (aggR-positive) was found in 63,5 % (54/85) and atypical EAEC (aggR-negative) in 36.5% (31/85) of the strains (Table 1).

TABLE 1
Distribution of epidemiological factors among children infected by EAEC.

All children participating in the study presented typical enteropathogen infection symptoms including diarrhea, vomiting and fever. Bloody diarrhea was recorded in 23.5% (12/51) of the cases and EAEC was the only pathogen detected in 58.3% (7/12) of these cases.

Prevalence of virulence genes

One or more virulence marker genes were detected in all EAEC isolates. The irp2 gene was the most commonly identified one, since it was recorded in 75.3% (64/85) of the isolates; it was followed by astA, aggR, and aatA, which were found in 64.7%, (55/85), 63.5% (54/85), and 48.2% (41/85) of the isolates, respectively. In addition, many other genes were involved in EAEC pathogenesis, including set1A, afa1, shf, pic, and pet, these genes were more often found in typical EAEC strains than in atypical EAEC strains. There was statistically significant correlation between typical EAEC and the presence of aatA (P<0.0001), irp2 (P=0.0357) or shf (P=0.0328) genes (Table 2).

TABLE 2
Incidence of virulence genes in typical and atypical ente­roaggrevative E. coli strains.

Association with biofilm production

Of the 85 analyzed EAEC strains, 69.4% (59/85) were biofilm producers; therefore, there was statistically significant relation between biofilm production and the presence of aggR gene (Table 2). It is worth highlighting that 73% (43/59) of the biofilm producers had the aggR, whereas this gene frequency in non-producer strains was 42.3% (11/26; P=0.0135 [OR=3.665; IC95%=1.393 to 9.639]). Similarly, there was association between the presence of aatA gene and biofilm producers (61% [36/59]), versus 19.2% (5/26) association in non-producers; P=0.0004 [OR=6.574; IC95% 2.173 to 19.89]. No correlation was observed between biofilm production and the presence of other virulence genes such as set1A, afa1, irp2, pic, astA, pet, and shf (Figure 1).

FIGURE 1
Biofilm formation and presence of virulence genes in EAEC isolates among diarrheal children. a shigella enterotoxin 1; b enteroaggregative heat-stable enterotoxin 1; c aggregative adherence; factor; d dispersin transporter; e transcriptional activator; f plasmid encoded toxins; g yersiniabactin biosynthesis; h cryptic open reading frame; i mucinase activity genes. P values represent correlation between biofilm production and virulence genes in EAEC: * P=0.0004; **P=0.0135 (Fisher’s exact test).

Antibiotic sensitivity in EAEC isolates

All 85 EAEC isolates were tested against 10 different antibiotics; the susceptibility profiles are shown in Table 3. At least one strain resistant to each of the tested antibiotics was identified. Results evidenced that most EAEC isolates were resistant to AMP 70.6% (60/85), SXT 60% (51/85), TET 44.7% (38/85), and CTX 22.3% (19/85). Multidrug resistance (MDR) was detected in 36.5% (31/85) of the tested strains. The MDR strains were resistant to antibiotics belonging to the most common chemotherapy classes, including AMP 96.8% (30/31), SXT 96.8% (30/31), TET 61.3% (19/31), CTX 54.8% (17/31).

TABLE 3
Antimicrobial profiles of EAEC: enteroaggrevative E. coli isolates.

DISCUSSION

Studies about the interaction and importance of different pathogenic E. coli strains associated with acute diarrhea in Brazil have highlighted EPEC as the major etiologic agent of infant diarrhea2323. Hu J, Torres AG. Enteropathogenic Escherichia coli: Foe or innocent bystander? Clin Microbiol Infect. 2015;21:729-34.,2424. Blanco Crivelli X, Bonino MP, Von Wernich Castillo P, Navarro A, Degregorio O, Bentancor A. Detection and Characterization of Enteropathogenic and Shiga Toxin-Producing Escherichia coli Strains in Rattus spp. from Buenos Aires. Front Microbiol. 2018;9(1).. However, studies have been reporting EAEC as an emerging enteric pathogen that causes persistent diarrhea and malnutrition in children living in developed countries2525. Kaur P, Chakraborti A, Asea A. Enteroaggregative Escherichia coli: An Emerging Enteric Food Borne Pathogen. Interdiscip Perspect Infect Dis. 2010;2010:254159..

With regard to the current study, 8.6% (51/591) of childhood gastrointestinal singular infection or co-infection by other enteropathogens could be attributed to EAEC. Studies conducted abroad reported EAEC infection prevalence from 2% to 24%2626. Ifeanyi CI feanyichukwu C, Ikeneche NF lorence, Bassey BE nya, Al-Gallas N, Ben Aissa R, Boudabous A. Diarrheagenic Escherichia coli pathotypes isolated from children with diarrhea in the Federal Capital Territory Abuja, Nigeria. J Infect Dev Ctries. 2015;9:165-74.,2727. Patzi-Vargas S, Zaidi MB, Perez-Martinez I, León-Cen M, Michel-Ayala A, Chaussabel D, et al. Diarrheagenic Escherichia coli carrying supplementary virulence genes are an important cause of moderate to severe diarrhoeal disease in Mexico. PLoS Negl Trop Dis. 2015;9:e0003510.. Previous studies carried out in Brazil showed that 0.5% to 41% of acute gastroenteritis cases requiring hospitalization were caused by this pathotype2828. Orlandi PP, Magalhães GF, Matos NB, Silva T, Penatti M, Nogueira PA, et al. Etiology of diarrheal infections in children of Porto Velho (Rondonia, Western Amazon region, Brazil). Brazilian J Med Biol Res. 2006;39:507-17.

29. Lima IFN, Boisen N, Da Silva Quetz J, Havt A, De Carvalho EB, Soares AM, et al. Prevalence of enteroaggregative Escherichia coli and its virulence-related genes in a case-control study among children from north-eastern Brazil. J Med Microbiol. 2013;62:683-93.
-3030. Assis FEA, Wolf S, Surek M, De Toni F, Souza EM, Pedrosa FO, et al. Impact of Aeromonas and diarrheagenic Escherichia coli screening in patients with diarrhea in Paraná, southern Brazil. J Infect Dev Ctries . 2014;8:3-8.. The high incidence of diarrhea cases in developing countries can be attributed to standard fecal-oral contamination routes resulting from deficient sanitary infrastructure, low education level, nutritional deficiency and inappropriate personal and food-related hygiene practices. Such incidence results in high economic burden for national public health systems3131. Breurec S, Vanel N, Bata P, Chartier L, Farra A, Favennec L, et al. Etiology and Epidemiology of Diarrhea in Hospitalized Children from Low Income Country: A Matched Case-Control Study in Central African Republic. PLoS Negl Trop Dis . 2016;10:e0004283.,3232. Giles-Vernick T, Bainilago L, Fofana M, Bata P, Vray M. Home Care of Children With Diarrhea in Bangui’s Therapeutic Landscape (Central African Republic). Qual Health Res. 2016;26:164-75.. It is worth emphasizing that patients in the current study presented critical social patterns related to low per capita income and untreated water intake.

Diarrheagenic E. coli infections in the current study most affected children in the age group 2 years or younger. Besides diarrhea, the major clinical symptoms associated with DEC among the assessed children were vomiting and fever. This outcome is consistent with reports from India3333. Dutta S, Guin S, Ghosh S, Pazhani GP, Rajendran K, Bhattacharya MK, et al. Trends in the prevalence of diarrheagenic Escherichia coli among hospitalized diarrheal patients in Kolkata, India. PLoS One. 2013;8:e56068., Tanzania3434. Moyo SJ, Maselle SY, Matee MI, Langeland N, Mylvaganam H. Identification of diarrheagenic Escherichia coli isolated from infants and children in Dar es Salaam, Tanzania. BMC Infect Dis. 2007;7:92., Libya3535. Ali MMM, Mohamed ZK, Klena JD, Ahmed SF, Moussa TAA, Ghenghesh KS. Molecular Characterization of Diarrheagenic Escherichia coli from Libya. Am J Trop Med Hyg . 2012;86:866-71., Cambodia3636. Meng CY, Smith BL, Bodhidatta L, Richard S a, Vansith K, Thy B, et al. Etiology of diarrhea in young children and patterns of antibiotic resistance in Cambodia. Pediatr Infect Dis J. 2011;30:331-5., Panama3737. Jimenez Gutierrez E, Pineda V, Calzada JE, Guerrant RL, Lima Neto JB, Pinkerton RC, et al. Enteric Parasites and Enteroaggregative Escherichia coli in Children from Canazas County, Veraguas Province, Panama. Am J Trop Med Hyg . 2014;91:267-72., and Israel3838. Tobias J, Kassem E, Rubinstein U, Bialik A, Vutukuru S-R, Navaro A, et al. Involvement of main diarrheagenic Escherichia coli, with emphasis on enteroaggregative E. coli, in severe non-epidemic pediatric diarrhea in a high-income country. BMC Infect Dis . 2015;15:1-7..

There are just few reports about bloody diarrhea cases associated with EAEC and they rarely concern children55. Jensen BH, Olsen KEP, Struve C, Krogfelt KA, Petersen AM. Epidemiology and clinical manifestations of enteroaggregative Escherichia coli. Clin Microbiol Rev . 2014;27:614-30.,3333. Dutta S, Guin S, Ghosh S, Pazhani GP, Rajendran K, Bhattacharya MK, et al. Trends in the prevalence of diarrheagenic Escherichia coli among hospitalized diarrheal patients in Kolkata, India. PLoS One. 2013;8:e56068.. With regard to the current study, bloody diarrhea was observed in 23.5% (12/51) of the assessed cases and EAEC was identified as the only pathogen in 58.3% (7/12) of them; however, other studies have reported conflicting results. A study conducted in Nigeria did not identify bloody stool as symptom of childhood EAEC infection2626. Ifeanyi CI feanyichukwu C, Ikeneche NF lorence, Bassey BE nya, Al-Gallas N, Ben Aissa R, Boudabous A. Diarrheagenic Escherichia coli pathotypes isolated from children with diarrhea in the Federal Capital Territory Abuja, Nigeria. J Infect Dev Ctries. 2015;9:165-74.. The prevalent virulence genes in the herein analyzed isolates were irp2, in 75.3% (64/85) of the cases; astA, in 64.7% (55/85); and aggR, in 63.5% (54/85). Previous studies have reported irp2 genes in EAEC samples but it was rarely found in EPEC, enteroinvasive E. coli and ETEC, besides being absent in EHEC, Shigella and Salmonella enterica1717. Mohamed JA, Huang DB, Jiang ZD, DuPont HL, Nataro JP, Belkind-Gerson J, et al. Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries. J Clin Microbiol. 2007;45:121-6.,3939. Czeczulin JR, Whittam TS, Henderson IR, Navarro-Garcia F, Nataro JP. Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli. Infect Immun . 1999;67:2692-9.. The irp2 gene is part of the High-pathogenicity island found in the chromosome of Yersinia species (Y. pestis, Y. pseudotuberculosis sorotype O1 and Y.enterocolitica biotype 1B) involved in iron uptake, which is mediated by the siderophore yersiniabactin and found in EAEC 042.

Elias et al., 2002, used the EAEC probe and found that irp2 was the second most prevalent marker (91.4%) among the 70 EAEC probe + strains4040. Elias W, Barros S, Moreira C, Trabulsi L, Gomes T. Enteroaggregative Escherichia coli Strains among Classical Enteropathogenic Escherichia coli O Serogroups. J Cinical Microbiol. 2002;40:3540-2.. There was statistically significant association between the typical EAEC aatA (P<0.0001) and irp2 (P=0.0357) genes. Patzi-Vargas conducted a study about DEC carrying supplementary virulence genes; he showed that the aatA genes are significantly more common in EAEC isolates than in non-DEC strains2525. Kaur P, Chakraborti A, Asea A. Enteroaggregative Escherichia coli: An Emerging Enteric Food Borne Pathogen. Interdiscip Perspect Infect Dis. 2010;2010:254159.. Tokuda et al4141. Tokuda K, Nishi J, Imuta N, Fujiyama R, Kamenosono A, Manago K, et al. Characterization of typical and atypical enteroaggregative escherichia coli in Kagoshima, Japan: biofilm formation and acid resistance. Microbiol Immunol 2010;54(6):320-9. showed that aatA and irp2 genes are more prevalent in typical EAEC isolates than in atypical ones. Results in the present study evidenced a whole variety of virulence genes combinations in typical EAECs, fact that was not so common in atypical EAECs.

Some researchers have assumed that several virulence gene combinations can be directly associated with diarrhea or with strain virulence3535. Ali MMM, Mohamed ZK, Klena JD, Ahmed SF, Moussa TAA, Ghenghesh KS. Molecular Characterization of Diarrheagenic Escherichia coli from Libya. Am J Trop Med Hyg . 2012;86:866-71.,4242. Cennimo D, Abbas A, Huang DB, Chiang T. The prevalence and virulence characteristics of enteroaggregative Escherichia coli at an urgentcare clinic in the USA: A case-control study. J Med Microbiol . 2009;58:403-7..

Pathogenic implications of aggR gene have been assessed in a number of studies worldwide1717. Mohamed JA, Huang DB, Jiang ZD, DuPont HL, Nataro JP, Belkind-Gerson J, et al. Association of putative enteroaggregative Escherichia coli virulence genes and biofilm production in isolates from travelers to developing countries. J Clin Microbiol. 2007;45:121-6.,4141. Tokuda K, Nishi J, Imuta N, Fujiyama R, Kamenosono A, Manago K, et al. Characterization of typical and atypical enteroaggregative escherichia coli in Kagoshima, Japan: biofilm formation and acid resistance. Microbiol Immunol 2010;54(6):320-9.. Interestingly, current results showed that children over one month old presented greater risk of being infected by typical than by atypical EAEC (P=0.0155). These findings suggest that children have greater probability of being exposed to the most virulent strains as they age.

Andrade and collaborators showed that EAEC strains found in small intestine and colon mucosa produce large amounts of biofilm and cause changes in the epithelia, fact that possibly explains the long duration of diarrheal episodes4343. Andrade JAB de, Freymüller E, Fagundes-Neto U. Adherence of enteroaggregative Escherichia coli to the ileal and colonic mucosa: an in vitro study utilizing the scanning electron microscopy. Arq Gastroenterol. 2011;48:199-204.. Biofilm production, adhesiveness and other EAEC virulence factors are directly associated with certain genes, including the regulatory gene aggR. The aggR is one of the most studied genes since it controls the expression of other genes related to EAEC pathogenesis. The presence of aggR in the present study was indeed statistically correlated with biofilm production (P=0.0135); this finding corroborates findings reported in studies conducted in Mexico and Mongolia, which have also indicated that the presence of aggR gene is associated with biofilm formation4444. Sarantuya J, Nishi J, Wakimoto N, Erdene S, Nataro JP, Sheikh J, et al. Typical enteroaggregative Escherichia coli is the most prevalent pathotype among E. coli strains causing diarrhea in Mongolian children. J Clin Microbiol . 2004;42:133-9.,4545. Huang DB, Mohamed J a, Nataro JP, DuPont HL, Jiang Z-D, Okhuysen PC. Virulence characteristics and the molecular epidemiology of enteroaggregative Escherichia coli isolates from travellers to developing countries. J Med Microbiol . 2007;56:1386-92.. Although it was possible recording significant association between the presence of aatA gene and biofilm production (P=0.0004), the shf gene and biofilm production were not correlated to each other. This results were different from those evidenced in a previous study that correlated shf with biofilm formation, even when it was associated with aggR4646. Fujiyama R, Nishi J, Imuta N, Tokuda K, Manago K, Kawano Y. The shf gene of a Shigella flexneri homologue on the virulent plasmid pAA2 of enteroaggregative Escherichia coli 042 is required for firm biofilm formation. Curr Microbiol. 2008;56:474-80..

Antimicrobial resistance, in EAEC populations, mainly MDR, has been reported in several studies4747. Regua-Mangia AH, Bezerra RMP, Esparis CM, Teixeira LM. Escherichia coli enteroagregativa (EAEC): Filotipagem e resistência a antimicrobianos em um enteropatógeno emergente. Rev Patol Trop. 2009;38:27-34.,4848. Ali MM, Ahmed SF, Klena JD, Mohamed ZK, Moussa TA, Ghenghesh KS. Enteroaggregative Escherichia coli in diarrheic children in Egypt: Molecular characterization and antimicrobial susceptibility. J Infect Dev Ctries . 2014;8:589.. Results in the present study evidenced greater EAEC resistance to AMP, SXT, TET and CTX than to other antibiotics. The resistance to these antibiotics was expected since they are the low-cost chemotherapeutic drugs of choice in many studies4949. Kong H, Hong X, Li X. Current perspectivesin pathogenesis and antimicrobial resistance of enteroaggregative Escherichia coli. Microb Pathog. 2015;85:44-9.. Antibiotics, mainly β-lactams, are often used to treat DEC infections, mainly in persistent diarrhea cases when the infectious agent is not identified, although these infections are self-limiting and only require oral-rehydration therapy. Most of the herein tested EAECs showed MDR phenotype, and this result is consistent with other studies. The most common MDR phenotypes recorded in the current study included resistance to β-lactams, TET, aminoglycosides, SXT, and GEN. There was no association between antimicrobial resistance and biofilm production, although this correlation was previously described by other researchers5050. Høiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents. 2010;35:322-32..

As far as it is known, the present study is pioneer in showing the pathogenic potential and heterogeneity of virulence EAEC genes and co-infections caused by other enteric pathogens in children with gastroenteritis living in Rondônia. Data presented in the current study may contribute to the better understanding about the role played by EAEC in children with diarrheal illnesses living in Porto Velho. The results may help developing strategic plans to control antimicrobial resistance in these poor regions.

ACKNOWLEDGEMENTS

The authors thank all the children and their parents who enrolled in this study. The Rosimeire Cristina Dalla Marta, and Nucia Cristiane da Silva Lima for helping in the laboratory analysis, and Paulo Afonso Nogueira for statistical analyses. We thank the Cosme and Damião Children Hospital for pacient recruitment. This research was supported by grants from Instituto de Pesquisa em Patologias Tropicais (IPEPATRO), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Saúde - Programa Pesquisa para o SUS (PPSUS), and Fundação de Amparo à Pesquisa do Estado de Rondônia (FAPERO).

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    Andrade JAB de, Freymüller E, Fagundes-Neto U. Adherence of enteroaggregative Escherichia coli to the ileal and colonic mucosa: an in vitro study utilizing the scanning electron microscopy. Arq Gastroenterol. 2011;48:199-204.
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  • Disclosure of funding: no funding received

Publication Dates

  • Publication in this collection
    Oct-Dec 2018

History

  • Received
    17 July 2018
  • Accepted
    12 Oct 2018
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