- Volume 67, Issue 9, 2018

Nearly all bacterial species express two or more chaperonin genes. Recent data indicate that type I chaperonins may be key players in bacterial infections. This is partly due to the well-known contribution of chaperonins in cellular proteostasis, the latter being compromised during bacterial host infection. In addition to their protein-folding activity, it has been revealed that certain chaperonins also exhibit moonlighting functions that can contribute in different ways to bacterial pathogenicity. Examples range from inducing adhesion molecules in Chlamydophila pneumoniae to supporting intracellular survival in Mycobacterium tuberculosis and Leishmania donovani, to inducing cytokines in Helicobacter pylori to promoting antimicrobial resistance in Escherichia coli, amongst others. This article provides a thorough reviews of our current understanding of the different mechanisms involving type I chaperonins during bacteria–host interactions, and suggests new areas to be explored and the potential of finding new targets for fighting bacterial infections.

Purpose. This study was undertaken to evaluate the efficiency of the pyrosequencing (PSQ) assay for the rapid detection of resistance to rifampicin (RIF), fluoroquinolones (FQs) and second-line injectables (SLIs) such as capreomycin (CAP) and kanamycin (KAN) in Mycobacterium tuberculosis (Mtb) clinical isolates.

Methodology. Pyrosequencing is a simple and accurate short read DNA sequencing method for genome analysis. DNA extraction from Mtb clinical isolates was performed using Tris-HCl buffer and chloroform. The rpoB (RIF), gyrA (FQs) and rrs (aminoglycosides) genes were amplified, followed by sequencing using the PyroMark Q24 ID system. The PSQ results were compared with the results from the conventional drug susceptibility testing performed in the laboratory.

Results. The sensitivity of the PSQ assay for the detection of resistance to RIF, FQ, CAP and KAN was 100 %, 100 %, 40 % and 50 %, respectively. The specificity of the PSQ assay was 100 %.

Conclusion. The PSQ assay is a rapid and effective method for detecting drug resistance mutations from Mtb clinical isolates in a short period of time.

The in vitro activity of anti-pseudomonal β-lactams in combination with avibactam was evaluated against 54 multidrug-resistant non-fermenting Gram-negative bacilli isolated from cystic fibrosis patients. Avibactam increased and/or restored the antibacterial activities of ceftazidime and aztreonam against Pseudomonas aeruginosa and Stenotrophomonas maltophilia, respectively. No β-lactam–avibactam combination was active against Achromobacter xylosoxidans.

An NDM-5-producing Klebsiella pneumoniae ST147 strain was isolated from a Japanese patient who had not travelled abroad in at least 5 years. Whole-genome sequencing revealed a genomic rearrangement in an FII plasmid harbouring bla _NDM-5 due to the replicative transposition of IS26. A hypothetical structure was proposed for its ancestral plasmid, and comparative genomic analysis of the plasmid suggested the dissemination of structurally similar plasmids harbouring bla _NDM-5 in Asian and Middle Eastern countries.

Purpose. Biofilms comprise bacterial populations enclosed in a matrix that attaches to surfaces such as medical stents. We characterized the biofilm components in occluding pancreatic stents and investigated potential factors for the formation of the biofilms.

Methodology. The clinical details of 24 patients (M : F, 15 : 9) undergoing pancreatic stent retrieval were noted and the retrieved stents were processed for the quantification of biofilm proteins and polysaccharides and the molecular identification of bacteria.

Results. The patients’ ages ranged from 16 to 62 years. The underlying indications for stent insertion were bile duct stone prophylaxis against post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (n=7; 29.1 %) and pancreatic ductal leaks (n=17; 70.9 %). The retrieved stent sizes were 5 Fr (n=5; 20.8 %) and 7 Fr (n=19; 79.2 %), with a mean insertion duration of 103 days. The polybacteria detected by PCR in 95.8 % of the stents were Pseudomonas (n=8), Staphylococcus (n=8), Serratia (n=5), Aeromonas (n=4), Proteus (n=4), Klebsiella (n=4), Escherichia coli (n=4), Enterococcus (n=4), Streptococcus (n=4), Citrobacter (n=3), Bacillus (n=2), Enterobacter (n=1), Vibrio (n=1) and Clostridium (n=1). Several other organisms were identified by sequencing. The mean protein concentration was 0.585±0.29 mg ml−1 and the mean polysaccharide concentration was 0.054±0.03 mg ml−1. No significant differences were observed in the quantity of proteins and polysaccharides (P=0.933) for various factors, namely gender, presence of cholangitis, indications for stenting, stent sizes and duration of indwelling stents. Age was found to be a significant (P=0.013) factor for protein deposition for those aged >50 years.

Conclusion. The majority of the pancreatic stents grew polymicro-organisms, and those from patients aged >50 years showed significant deposition of protein, which is a key element in biofilm formation. Understanding the constituents of the biofilms in pancreatic stents could be very useful in developing future strategies for the prevention of biofilm formation.

Purpose. Respiratory tract infections are a major cause of global morbidity and mortality. Pneumonia is the ninth leading cause of mortality in Sri Lanka. Atypical pathogens cause about one-fifth of community-acquired pneumonia, while Mycoplasma pneumoniae accounts for about 50 %. This study aimed to determine the seroprevalence of M. pneumoniae respiratory tract infections in Sri Lanka while attempting to understand the relationships between the serology and PCR.

Methodology. Paired sera from 418 adult patients (pneumonia, n=97; bronchitis, n=183; pharyngitis, n=138) and 87 healthy controls were studied. IgM, IgG and IgA antibodies were tested by M. pneumoniae enzyme-linked immunosorbent assay (ELISA). Positive IgM and or IgG seroconversion was considered to be seropositive. M. pneumoniae DNA were tested by PCR in age and gender-matched seropositives and seronegatives.

Results. M. pneumoniae IgG was in 14.4 % (14/97), 6.0 % (11/183) and 1.5 % (2/138) of pneumonia, bronchitis and pharyngitis patients, respectively, whilst IgM was in 6.2 % (6/97), 1.1 % (2/183) and 0 % (0/138), respectively. Amongst the pneumonia seropositives, 64.7 % (11/17) showed IgG alone, 17.5 % (3/17) showed IgM alone and 17.5 % (3/17) showed IgM and IgG. Amongst the bronchitis seropositives, 84.6 % (11/13) had IgG alone and 15.4 % (2/13) had IgM alone. In the pharyngitis seropositives, only IgG was detected 100 % (2/2). M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. In pneumonia or bronchitis patients, specific DNA was in 77.8 % (7/10) and 50 % (6/12) of patients, respectively. M. pneumoniae DNA was not found in pharyngitis patients. Of the seropositive PCR-negative pneumonia patients, 66.7 % (2/3) showed IgG alone and 33.3 % (1/3)showed IgM alone. In bronchitis patients, 83.3 % (5/6) showed IgG alone and 16.7 % (1/6) showed IgM alone. Of the seronegative PCR-positive patients, 16.7 % (2/12) had pneumonia and 18.2 % (2/11) had bronchitis.

Conclusion.The serological evidence for M. pneumoniae infection in Sri Lanka comprised the following prevalences: 17.5 % (17/97), 7.1 % (13/183) and 1.4 % (2/138) in adults with pneumonia, bronchitis or pharyngitis, respectively. M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. IgG was predominant in PCR positives and negatives.

Purpose. Although Mycobacterium fortuitum (M. fortuitum) is not an organism rarely isolated from respiratory samples, its clinical importance is still not fully understood, which therefore prompted our current study.

Methodology. We evaluated respiratory samples from 6800 patients with suspected tuberculosis from May 2014 to May 2016, for the detection of M. fortuitum using phenotypic and genotyping methods.

Results/Key findings. Of the 40 patients with M. fortuitum lung disease, 35 had two or more positive culture results. The mean age of these 35 patients was 50.7±18.4 years, and 20 (57.1 %) were men. Sputum (68.6 %), haemoptysis (51.4 %), cough (45.7 %) and gastroesophageal disease (22.9 %) were the major presenting symptoms. Cystic fibrosis, other bacterial lung diseases and lung cancer were the main underlying pulmonary diseases. Five patients (12.5 %) were human immunodeficiency virus (HIV) positive. The most common chest X-ray findings were reticulonodular opacities (53.3 %). Multivariate logistic regression analysis revealed that cigarette smoking history (OR 0.334, 95 % CI 0.125–0.843, P=0.048) and underlying lung disease (OR 0.393, 95 % CI 0.216–0.588, P=0.023) were significant predictors for positive M. fortuitum infection.

Conclusion. These results demonstrated the high frequency of M. fortuitum in respiratory samples and that this bacterium causes transient infection or colonization in patients with underlying pulmonary conditions, such as cystic fibrosis and cigarette smoking-induced. Additionally, it appears that infection with M. fortuitum is particularly common and may be important in patients with HIV.

Leptospirosis is an endemic infectious disease causing considerable morbidity and mortality in Sri Lanka; however, reports on the isolation of Leptospira from infected patients in Sri Lanka have been largely unavailable since the 1970s. Two isolates were obtained and characterized from 100 blood cultures from leptospirosis-suspected patients. Phylogenic analysis of partial flaB gene sequences identified the isolates as Leptospira interrogans. The patient serum samples from which Leptospira was isolated reacted with the Leptospira serogroups Sejroe and Canicola at a titre of 1 : 200. Exposure to domestic sewage and gutters filled with muddy water was suspected to be the source of infection in these two culture-positive patients. This study reports the successful isolation of pathogenic Leptospira from two patients in Western Province, Sri Lanka.

To improve time to identification of pathogens and detection of resistance genes, we evaluated the BioFire FilmArray Blood Culture Identification Panel (BCID) as compared to: (1) direct MALDI-TOF MS (DM) and (2) standardized culture-based identification (ID) with antibiotic susceptibility testing (AST). BCID gave an accurate identification in 102/112 (91 %) of cases (102/103 for on-panel organisms). DM gave an accurate identification in 91/112 (81 %) of cases, with 13/91 (14 %) requiring repeat testing from the residual pellet. The mean time to an identification result was 2.4 and 2.9 h for BCID and DM, respectively. Standardized ID and AST results were available at a mean time of 26.5 and 33 h, respectively. There were 44 BCID/DM results that had an antimicrobial treatment change made based on rapid identification and resistant gene detection of pathogens. Both BCID and DM are accurate and rapid methods for the identification of new positive blood culture pathogens.

Purpose. Both Shigella and enteroinvasive Escherichia coli (EIEC) can cause enterocolitis, but they have a distinct epidemiology and public health relevance. Current culture-independent testing (CIT) methods to identify Shigella in faecal samples rely on the ipaH gene as the target, which is also found in EIEC genomes. The aim of this study was to design an assay that can identify EIEC in cultures from CIT ipaH-positive samples.

Methodology. Shigella and EIEC genomes were screened to find unique regions present in EIEC genomes using a comparative genomics approach and differentiating genetic loci that are suitable PCR targets were identified. The primers for these loci were designed and tested in 6501 and 104 genomes of Shigella and EIEC, respectively.

Results. An assay with two sets of multiplex PCR reactions that differentiates Shigella and EIEC based on the presence/absence of at least two out of six loci was developed and evaluated. The majority of Shigella genomes lacked all six loci, while at least two loci were present in most EIEC genomes. This assay successfully differentiated clinical EIEC from Shigella with a limit of detection of 105 cells ml−1. The sensitivity and specificity were over 95 and 99%, respectively. The assay can further subtype EIEC genomes into their genetic lineages.

Conclusion. This new highly specific assay can assist in the identification of EIEC in ipaH PCR-positive samples and augment the public health laboratory surveillance of EIEC and shigellosis.

Purpose. Bacterial vaginosis (BV) is a common clinical condition characterized by odorous vaginal discharge, vaginal itching and/or burning. BV can occur when vaginal lactobacilli are depleted and replaced by diverse anaerobic bacteria. We evaluated a commercial multiplex PCR (ATRiDA) for the diagnosis of BV.

Methods. Cervicovaginal samples were included from women reporting urogenital symptoms and from women notified for sexually transmitted infections (STI) – who were not (necessarily) symptomatic. Clinical BV diagnoses were obtained from electronic patient files. The ATRiDA test measures the loads of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus species in relation to overall bacterial load. The ATRiDA test outcome was compared to the clinical BV diagnosis and to vaginal microbiota composition, determined by 16SrRNA gene sequencing.

Results. We included samples from 185 women reporting urogenital symptoms, of whom 81 had BV and 93 women who were notified for an STI, of whom 16 had BV. Overall, compared to the clinical BV diagnosis, the ATRiDA test demonstrated high sensitivity (96.9 %) and moderate specificity (70.2 %). The negative predictive value was high (>97.3). The positive predictive value differed by study group and was highest in women reporting urogenital symptoms (78.2 %). Sequencing showed that 54 % of women who had an ATRiDA BV-positive test outcome, but who were not clinically diagnosed with BV, had diverse anaerobic vaginal microbiota (asymptomatic vaginal dysbiosis).

Conclusion. The ATRiDA test is a sensitive method for the detection of BV but, given the high occurrence of asymptomatic vaginal dysbiosis, a positive test outcome should be interpreted together with clinical symptoms.

Purpose. The taxonomy of Aeromonas keeps expanding and their identification remains problematic due to their phenotypic and genotypic heterogeneity. In this study, we aimed to develop a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism assay targeting the rpoD gene to enable the differentiation of aeromonads into 27 distinct species using microfluidic capillary electrophoresis.

Methodology. A pair of degenerate primers (Aero F: 5′-YGARATCGAYATCGCCAARCGB-3′ and Aero R: 5′-GRCCDATGCTCATRCGRCGGTT-3′) was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966T, A. caviae ATCC15468T, A. veronii ATCC9071T, A. media DSM4881T, A. allosaccharophila DSM11576T, A. dhakensis DSM17689T, A. enteropelogens DSM7312T, A. jandaei DSM7311T, A. rivuli DSM22539T, A. salmonicida ATCC33658T, A. taiwanensis DSM24096T and A. sanarelli DSM24094T) were randomly selected from the 27 Aeromonas species for experimental validation.

Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement.

Conclusion. This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.

Purpose. Human papillomavirus (HPV) infection in women is known to promote the development of cervical neoplasia. Specific HPV genotypes are more highly associated with disease, and therefore detection and genotyping of HPV infection is critical for preventing and effectively treating cervical cancer. Consequently, various assays using diverse technologies have been developed to detect HPV genotype. Recently the OmniPlex-HPV and GeneFinder HPV methods, based on PCR and Luminex xMAP liquid bead microarray technologies, were developed for the detection of 40 and 32 HPV genotypes, respectively. The purpose of this study was to compare the clinical performance of OmniPlex-HPV and GeneFinder HPV.

Methodology. The study included 300 cytology-confirmed cervical swab specimens. In cases where there was a discrepancy between the two assay results, type-specific direct sequencing was performed.

Result. We found a high overall agreement between OmniPlex-HPV and GeneFinder HPV for detecting the presence or absence of high-risk HPV (HR HPV) (90.7 %, κ=0.810). However, OmniPlex-HPV showed greater sensitivity than GeneFinder HPV in the identification of multiple genotype-infected samples. Specifically, diagnostic sensitivities for HR HPV positivity in high-grade squamous intra-epithelial lesions (HSIL) were 100.0 % for OmniPlex-HPV and 96.8 % for GeneFinder HPV.

Conclusions. Our results suggest that OmniPlex-HPV and GeneFinder HPV are highly comparable for the detection and genotyping of HPV, but OmniPlex-HPV displays greater accuracy in cases of multiple HPV infection.

Objective. The development of an accurate, sensitive, specific, rapid, reproducible, stable-at-room-temperature and cost-effective diagnostic kit, and a low-cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries.

Methods. We developed the ‘Chlamy and Ness CT/NG kit’ based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi-centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)-based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 ˚C and the inter-day reproducibility of results were also analysed.

Results. The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the ‘inter-rater’ κ=0.976 for Chlamydia and κ=0.943 for Neisseria.

Conclusion. Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource-limited settings of developing countries.

Purpose. Bloodstream infections remain an important cause of morbidity and mortality. Rapid diagnosis can reduce the time from empiric antimicrobial therapy to targeted therapy and improve patient outcomes.

Methodology. The fully automated Unyvero Blood Culture (BCU) Application (Curetis GmbH) can identify a broad panel of pathogens (36 analytes covering over 50 pathogens) and 16 antibiotic resistance gene markers simultaneously in about 5 h. The assay was evaluated in three clinical laboratories in comparison to routine microbiological procedures.

Results. A total of 207 blood cultures were included in the study, and 90.5 % of the species identified by culture were covered by the Unyvero BCU panel with an overall sensitivity of 96.8 % and specificity of 99.8 %. The time to result was reduced on average by about 34 h. The assay accurately identified 95 % of the species, including 158/164 monomicrobial and 7/9 polymicrobial cultures. The Unyvero BCU Cartridge detected a large number of resistance markers including mecA (n=57), aac(6′)aph(2′′) (n=40), one vanB resistance gene, and six instances of bla _CTX-M.

Conclusion. The Unyvero BCU Application provided fast, reliable results, while significantly improving turnaround time in blood culture diagnostics.

Introduction. Although hepatitis E virus (HEV) is mainly transmitted via the faecal–oral route, the rate of HEV transmission via blood donation is on the rise. However, the seroprevalence of HEV among blood donors is not well established and is thought to be affected by the type of diagnostic assay used. We aimed to evaluate performance and correlation among widely used commercial diagnostic assays for the seroprevalence assessment of HEV-IgM/IgG among blood donors.

Methodology. A total of 1049 blood donor samples were tested for HEV IgG and IgM using different enzyme immunoassays (Wantai, Eruoimmune, MP diagnostics, Mikrogen immunoblot, HEV-IgM rapid test). The performance of each assay was evaluated according to our established silver standard value based on three or more IgG concordant assay results.

Results. HEV seroprevalence varied considerably using these assays, ranging from 10.1 % (Euroimmune-ELISA) to 18.0 % (Wanti-ELISA) for HEV-IgG, and from 0.2 % (Wanti-ELISA) to 2.6 % (MP Rapid test) for HEV-IgM. A total of 155 of 216 (71.6%) samples tested positive for HEV-IgG by three or more concordant assays. On the other hand, IgM assays showed poor agreement as only 7.6 % (4/52) of the specimens were positive according to three or more concordant assay test results. All HEV-IgG assays revealed high sensitivity and specificity (ranging 96.5–100 %),and excellent Kappa concordance (0.88–0.95), except for Euroimmun ELISA (sensitivity=61.5 %, kappa=0.63). MP ELISA showed the highest levels of sensitivity (100 %) and specificity (98.5 %).

Conclusions. Due to discrepancies in the performance of various IgG and IgM assays, seroprevalence studies should be based on furher confirmatory testing for decisive conclusions to be reached.

Purpose. To develop a fast and inexpensive genotyping assay to identify the Mycobacterium tuberculosis complex (MTC) species most prevalent in human tuberculosis (TB), based on the thermal denaturation profiles of PCR products from mycobacterial 16S rDNA and three MTC genomic regions of difference (RD).

Methodology. Genotypes were determined by the presence and thermal denaturation profiles of the amplicons generated in the ‘preliminary’ PCR mixture (16S rDNA), followed by those of the simultaneous D1 (RD9+, RD1−) and D2 (RD4+, RD4−) PCR mixtures. The 16S rDNA profile identifies the genus Mycobacterium; the absence of any additional RD profile identifies Mycobacterium non-tuberculous (MNT) strains; additional RD4+ and RD9+ profiles without RD1− identify M. tuberculosis; an additional RD4+ profile per se identifies M. africanum; an additional RD4− profile per se identifies Mycobaterium bovis; additional RD1− and RD4− profiles identify M. bovis BCG.

Results. Genotypes of a panel with 44 mycobacterial strains coincided in 16 MB and five non-MTC strains; in the remaining 23 MTC strains, 17 MTB and five MA concordant genotypes and one discordant MB genotype were resolved. The genotypes of 13 human and bovine MTC isolates coincided in all four MB and eight of the nine MTB isolates.

Conclusion. Sensitivity, specificity and positive and negative predictive values of the method are 100 % for the genus Mycobacterium, which resolves MB, MTB and MA genotypes. Species/genotype agreement is 97.7 % for the panel and 92.3 % for the MTC isolates. This method may be advantageously used to identify the most prevalent MTC species in humans.

The effect of storage time and temperature on the recovery of pathogen DNA from polytetrafluorethylene (PTFE) was investigated. PTFE impression membranes were inoculated with Pseudomonas aeruginosa, Herpes Simplex Virus-1 (HSV-1) or Acanthamoeba and stored at −70 °C, −20 °C, +4 °C or +35 °C. PCR was performed on days 0, 1, 2, 3, 7 and months 1, 3 and 10 post-inoculation. We found no reduction in the DNA recovery of any of the studied microorganisms for the first 3 days of storage up to +35 °C. For HSV-1 and P. aeruginosa, storage for 3 months at +35 °C was associated with a significant reduction in DNA recovery (P<0.001), but not at +4 °C, −20 °C or −70 °C for 1 month for P. aeruginosa and for 10 months for HSV-1. Acanthamoeba DNA recovery was not affected by any storage parameters (P=0.203). These results will inform the investigation of microbial keratitis where access to microbiological testing is not readily available.

Purpose. Two natural epidemic biotypes of Vibrio cholerae O1, classical and El Tor, exhibit different patterns of sensitivity against the antimicrobial peptide polymyxin B. This difference in sensitivity has been one of the major markers in biotype classification system for several decades. A recent report regarding the emergence of polymyxin B-sensitive El Tor V. cholerae O1 in Kolkata has motivated us to track the spread of the strains containing this important trait, along with Haitian-like genetic content, in different parts of India.

Methodology. We have collected 260 clinical V. cholerae O1 strains from 12 states in India and screened them for polymyxin B susceptibility. Genetic characterization was also performed to study the tcpA, ctxB and rtxA genotypes by allele-specific polymerase chain reaction (PCR) and nucleotide sequencing.

Results. Interestingly, 88.85 % of the isolates were found to be sensitive to polymyxin B. All of the states, with the exception of Assam, had polymyxin B-sensitive V. cholerae strains and complete replacement with this strain was found in eight of the states. However, from 2016 onwards, all the strains tested showed sensitivity to polymyxin B. Allele-specific PCR and sequencing confirmed that all strains possessed Haitian-like genetic traits.

Conclusion. Polymyxin B-sensitive strains have begun to spread throughout India and may lead to the revision of the biotype classification. The dissemination of these new variant strains needs to be carefully monitored in different endemic populations through active holistic surveillance to understand their clinical and epidemiological consequences.

Purpose. Entero-aggregative Escherichia coli (EAEC) is one of the main causes of diarrhoea worldwide. Several virulence factors have been identified in EAEC. This study was conducted to investigate the distribution of virulence factor genes in EAEC strains isolated in Iran from children with diarrhoea, as well as the genetic similarity of these isolates.

Methodology. A total of 37 EAEC isolates were tested for the presence of 11 virulence genes by PCR, and the genetic relatedness of these strains was further determined by multilocus variable-number tandem-repeat analysis (MLVA).

Results. All EAEC isolates were typical EAEC. pic, set1A and set1B were the most prevalent genes, detected in 54.1 % of the isolates, followed by sat (43.2 %), astA (32.4 %), pet (24.3 %), agg4A (24.3 %), sepA (18.9 %), agg3A (13.5 %), sigA (8.1 %), aggA (8.1 %) and aafA (5.4 %). Using MLVA, the 37 isolates were divided into 32 types and classified into five clonal complexes.

Conclusion. This study showed that EAEC is a heterogeneous group of E. coli possessing a broad range of virulence factors. There was no notable association between MLVA patterns and virulence profiles.