Emerging Problems in Infectious Diseases Molecular characterization of virulence factors in diarrhoeagenic Escherichia coli isolates from children in Nairobi , Kenya

Introduction: Among the bacterial causes, diarrheagenic Escherichia coli (DEC) is the most important etiologic agent of childhood diarrhoea and represents a major public health problem in developing countries. New evidence suggests that major differences in virulence among groups of DEC pathotypes may be related to the presence of specific pathogenicity islands (PAIs). Methodology: Multiplex and conventional PCR assays were used to identify the DEC pathotypes and PAIs respectively from 207 E. coli isolates. Results: The predominant DEC pathotype isolated was EPEC 19.3% (40/207), followed by ETEC 7.25% (15/207), EAEC 3.86% (8/207), STEC 0.97% (2/207) and EIEC 0.48% (1/207). The PAIs detected were enteropathogenic secreted protein C (EspC) 12.2% (8/66), locus of enterocyte effacement (LEE) 62.1% (41/66), and high pathogenicity island (HPI) 57.6% (38/66). Six percent (4/66) expressed only fyuA gene, 12.2% (8/66) irp2 only, and 39.4% (26/66) expressed both fyuA and irp2 genes. SHI-2 39.4% (26/66), she 6% (4/66) and O island 33.3% (22/66), 19.8% (13/66) expressed only efa/lifA gene, 7.6% (5/66) pagC gene only and 6.1% (4/66) expressed both efa/lifA and pagC genes. Toxigenic invasion A (TIA) PAI was not detected. Conclusion: This study revealed that in addition to eaeA, stx, aat, einv, st and lt virulence genes exhibited in the different DEC pathotypes there are numerous PAIs in the DEC pathotypes. The PAIs can increase gene mobility within various motile elements, which has implications for the spread of virulence factors from DEC to commensal E. coli.


Introduction
Diarrhoea is one of the leading causes of morbidity and mortality among children under five years of age in the developing world [1].Currently diarrhoea has been reported to account for 1.6 to 2.5 million deaths annually; Diarrhoea in most developing countries still remains one of the principal causes of morbidity in children with each child reported to experience an average of three episodes of diarrhoea per year [2,3].Diarrhoeal diseases in Kenya are among the five main causes of mortality in children younger than five years.Bacterial diarrhoea has been reported to account for up to 30% of all cases of infantile diarrhoea and as the most common cause of travellers' diarrhoea (TD) [4].The spread of different pathogenic E. coli is a major concern in developing countries where it is enhanced by factors such as harsh climatic conditions, poor sanitation, malnutrition and immunosuppression related to HIV and AIDS [5].Evidence from studies indicates DEC is a potential public health risk, with EHEC O157: H7 causing life-threatening sequelae, including hemolytic uremic-syndrome (HUS) and thrombocytopenic purpura (TTP), which causes kidney failure, hemolytic anemia, and thrombocytopenia [6].New evidence suggests that major differences in virulence between groups of DEC pathotypes might be related to the presence of specific pathogenicity islands (PAIs) [7].The PAIs increase gene mobility within various mobile elements such as plasmids, and from chromosomal location to mobile elements, which has implications for the spread of virulence factors from DEC to commensal E. coli; thus the PAIs play a significant role in pathogenesis of DEC and increased virulence in disease presentation.This study was conducted to establish the prevalence of DEC pathotypes causing diarrhoea among children in Mbagathi District Hospital and to identify pathogenicity islands in different DEC pathotypes.

Study design
This was a laboratory based study on E. coli isolates collected between January 2005 and May 2008, from children treated at the Mbagathi District Hospital, Nairobi, Kenya.Mbagathi District Hospital serves mainly a population of medium to low socioeconomic status.The E. coli isolates were collected from children younger than five years of age admitted with diarrhoea (defined as three or more loose movements in the previous 24 hours) before being treated with antibiotics.The study was ethically approved by the Kenya Medical Research Institute (KEMRI), Kenya Ethical Review Committee N0.1419.

Bacterial isolates
Two hundred and seven (207) E. coli isolates were randomly selected from archived E. coli isolates processed by previously described methods [8,9] and revived by inoculation on Brain Heart Infusion broth.The isolates were incubated at 37°C for 18 to 24 hours, then sub cultured onto lactose MacConkey and sorbitol MacConkey Agar and incubated at 37 °C for 18 to 24 hours.Morphological characteristics on these media were used to re-confirm E. coli isolates.

PCR detection of diarrhoeagenic E. coli pathotypes and PAIs
Bacterial colonies from MacConkey (MAC) and sorbital MacConkey (SMAC) plates were inoculated into 5 ml phosphate-buffered saline (PBS) tube to a density of MacFarland 4 (10 9 5X10 9 bacterial/ml).The 5 ml bacterial suspension was boiled for 20 minutes and then centrifuged at 2,500 X g for 10 minutes to pellet cell debris.The supernatant was used for PCR assays.

Preparation of DNA template and primers
The DNA templates were subjected to multiplex PCR for the detection of the virulence markers shown in Table 1.Positive controls containing template DNA of the following reference strains were used in every amplification round: EHEC ATCC 43890, EHEC ATCC 43887, EPEC ATCC 43887, ETEC ATCC 35401, EIEC ATCC 43893, EAEC 97R and E. coli ATCC 11775 (negative control without virulence genes).Conventional PCR assay was used for detection of pathogenicity islands (PAIs) in the diarrhoeagenic E. coli isolates.The following distinct genes in particular islands were targeted she PAI (pic gene), HPI PAI (irp2 and fyu A gene), LEE PAI (eae gene), TIA PAI (tia gene), SHI-2 PAI (iutA gene), EspC PAI (espC gene) and O-island PAI (efa/lifA gene and pagC gene).Primers used in this study are shown in Table 2.

PCR conditions
Each PCR test was performed in 0.5 ml microcentrifuge tubes using a 25-μl-reaction mixture containing: 2 μl of 10 mM mix deoxynucleotide triphosphate (dNTP); 2.5 μl of MgCl 2 (25mM); 2.5 μl 10X buffer solution; 1.25 μl of PCR primer (forward and reverse primer each) with concentration of pmol/ml (Bioserve Biotechnologies, Laurel, MD, USA); 0.3 μl of Taq polymerase (Applied Biosystems, Roche Molecular, Inc, and Branchbury, New Jersey, USA); and 2 μl of DNA template and water to the final volume of 25 μl.PCR amplifications were performed on a PTC-200 thermal cycler (MJ Research Inc, Watertown, Massachusetts, USA).A multiplex PCR cycle was used as previously described [10] in amplification of DEC pathogenic genes.PCR conditions for the detection of PAIs were used as earlier described for she PAI (pic gene) [11], HPI PAI (irp2 and fyu A gene) [12], LEE PAI (eae gene) [13], TIA PAI (tia gene) [14], SHI-2 PAI (iutA gene) [13], EspC PAI (espC gene) [15] and O-island PAI (efa/lifA gene and pagC gene) [16].The amplified PCR products were separated by electrophoresis in 2% agarose gels stained with ethidium bromide in Tris Borate (TBE) buffer at 100 V for one and half hours.The DNA in the gel was visualized on a UV transilluminator and photographed under ultraviolet light using a blackand-white instant Polaroid camera.A molecular size marker (100-bp DNA ladder; Promega, Madison, Wisconsin, USA) was included in each agarose gel run to estimate the size of the amplicons.

Statistical analysis
Data was entered using excel (Microsoft, Redmond, WA, USA)) and checked for consistency and integrity.Statistical analysis was performed with Statistical Package for Social Scientists (SPSS) version 11.5 (IBM Chicago, IL, USA); categorical variables were analysed using frequency distributions to establish prevalence of pathotype and pathogenicity islands.

Pathogenicity islands detected in DEC
The pathogenicity islands are shown in Table 3. TIA PAI was not detected in any of the DEC pathotypes.

Discussion
Diarrhoeagenic Escherichia coli is a potential public health risk in children in developing countries, causing persistent diarrhoea.DEC pathotypes such as EHEC O157: H7 and ETEC have been reported to cause severe forms of diarrhoea in children.In this study EPEC was the predominant DEC pathotype followed by ETEC, EAEC, STEC and EIEC.The study further established that DEC pathotypes carried a number of PAIs with the huge majority of the pathogenic isolates possessing at least two PAIs systems.According to Schmidt and Hensel, genomic plasticity in bacteria is largely enhanced by horizontal gene transfer where large DNA fragments containing virulence-associated genes, or PAIs, can be exchanged between different bacterial species, and their acquisition can generate new pathogenic variants [7].The majority (71%) of the DEC pathotypes possessed at least HPI and SHI-2 PAIs iron-uptake systems, a feature which may contribute to higher pathogenicity and facilitate colonization of the host [17,18,19].The ability to acquire iron is crucial for bacteria for growth and during infection for their ability to form biofilms, slime-encased colonies of microbes that cause many chronic infections such as those that occur in wounds, on medical devices, and in the lungs of people with cystic fibrosis [20].Other researchers have also observed the presence of the HPI in genomes of E. coli as seen from a study by Xu et al. [21] which isolated HPI-harboring E. coli strains from patients in all age groups and was most frequently detected in association with diarrhoea in children under 10 years of age.This observation shows HPI could play a major role in the pathogenesis of diarrhoea.
Detection of the pic gene in this study significantly shows acquisition of virulence factors, which aid in intestinal colonization of the host and is vital for successful infection; however, the low isolation of the pic gene in this study may indicate that the she island could be one of but not a great contributor to diarrhoeal pathogenesis in the DEC pathotypes.Presence of the pic gene, which is used as a marker for she PAI, may indicate conferment of set1A and set1B encoding the two subunits of shET1 enterotoxin and sigA that encodes a cytopathic, autotransported protease.This process may aid in further pathogenesis of diarrhoea to the DEC pathotypes [11,22,].
The eaeA gene, which is a marker for LEE PAI, was detected in all EPEC pathotypes and only one of the STEC pathotypes.This is in agreement with findings by Morabito et al. [23], who showed that LEE PAI is mainly found in EPEC and STEC pathotypes.The eaeA gene, which is necessary for attaching and effacing activity, encodes a 94 to 97-KDa outer membrane protein termed intimin [24].The LEE PAI plays a major role in the colonization process, particularly in STEC, which is reported to contribute to its higher virulence and development of hemolytic uremic syndrome (HUS) in patients [25].
EspC gene was detected only in the EPEC pathotype.This result concurs with observations by Navarro-García et al. [15] who determined that it mainly confers virulence properties to EPEC.The presence of EspC PAI shows significance of acquisition of other virulent PAIs in aiding pathogenesis of diarrhoeal illness.
This study also established that among the STEC strains one isolate contained both efa/lifA and pagC genes, while another contained the efa/lifA gene.efa/lifA and pagC genes were also detected in the other DEC pathotypes, however, at low rates.Studies by Morabito et al. [23] on efa/lifA virulence gene show that it is involved in the capability of EHEC to adhere to cells and in the repression of the host lymphocyte activation response of EPEC.pagC gene virulence is indicated as a putative adhesion and hemeagglutinin [23,26].Detection of other virulence markers in the O-island of EHEC apart from LEE and stx/vt may help to explain the increase in pathogenicity of EHEC strains.Wickham et al. [27] proposed that the additive effect of efa/lifA and pagC contributes significantly to causing HUS while the pagC locus may contribute to pathogenicity in more virulent STEC.In this study the tia gene was not detected although other investigations show that it aids Enterotoxigenic Escherichia coli (ETEC) in adherence and invasion of epithelial cells originating from the human ileum or colon, and also may aid in maximum secretion of the LT enterotoxin.This function may add pathogenicity to the ETEC virulent strains [7,28].

Conclusion and recommendations
Results from this study showed that the DEC pathotypes carried a number of PAIs with the vast majority of the pathogenic isolates possessing at least two PAIs systems.Acquisition of the virulence gene to all the DEC pathotypes through horizontal gene transfer was observed by detection of PAIs documented from Yersinia spp.(HPI PAI), Shigella spp.(she and SHI-2 PAI) and EHEC (O-islands PAI).This feature may contribute to higher pathogenicity.Moreover, the virulence associated traits may also contribute to the persistence in the intestinal micro flora as well as increase in their pathogenic capability in the urinary tract.
Based on the results of this study, the authors make the following recommendations for further studies: 1) to confirm the role of the PAIs in severity of diarrhoeal disease presentation in Kenya; 2) to target the presence of other genes in the PAIs investigated in this study; 3) to investigate other PAIs and genes which can enhance persistence of diarrhoea; and 4) to initiate a correlation study of the different PAIs that exist and their virulence presentation in diarrhoea.

Table 1 .
Sequences for DEC Multiplex PCR primers and their respective product size

Table 2 .
Sequences for PAIs PCR primers and their respective product size