Molecular study on virulence and resistance genes of ST131 clone (uropathogenic/enteropathogenic Escherichia coli) hybrids in children.

Aim: To analyze ST131 clones and other characteristics in uropathogenic and atypical enteropathogenic Escherichia coli hybrids. Methods: Samples were collected from children with urinary tract infections and underwent testing for antimicrobial susceptibility, multidrug resistance and extended-spectrum ß-lactamases, in vitro biofilm formation and virulence, resistance genes, hybrid pathotypes and ST131 clones. Results: E. coli isolates showed high levels of antibiotic resistance, extended-spectrum ß-lactamase production, virulence genes, multidrug resistance and biofilm formation. Four (5.0%) isolates were identified as uropathogenic/atypical enteropathogenic E. coli hybrids, all of which belonged to the high-risk ST131 clone. Conclusion: Our results provide promising insights about hybrid isolates and should be addressed to improve prevention measures for hybrid pathotypes.

The genotypic and phenotypic characteristics contributing to high virulence and antibiotics resistance in Escherichia coli O25-B2-ST131 in comparison to non- O25-B2-ST131.

BACKGROUND: Escherichia coli serogroup O25b-sequence type 131 (E. coli O25-B2-ST131) is considered as multidrug-resistant and hypervirulent organism. There is lack of data about involvement of this pathogen in the children's infection. In this study, the prevalence, and clonality, virulence capacity, and antibiotic resistance phenotype and genotype of E. coli O25-B2-ST131 compared with non-O25-B2-ST131 isolates were investigated in children with urinary tract infection in Tehran, Iran. METHODS: The E. coli isolates from urine samples were identified using conventional microbiological methods. Characterization of E. coli O25-B2-ST131 clone, antibiotic susceptibility, biofilm formation, ESBLs phenotype and genotype, serum resistance, hemolysis, hydrophobicity, and formation of curli fimbriae were done using conventional microbiological and molecular methods. Clonality of the isolates was done by rep-PCR typing. RESULTS: Among 120 E. coli isolates, the highest and lowest antibiotic resistance was detected against ampicillin (92, 76.6%) and imipenem 5, (4.1%), respectively. Sixty-eight (56.6%) isolates were ESBL-producing and 58 (48.3%) isolates were considered as multi-drug resistance (MDR). The prevalence of ESBL-producing and MDR isolates in O25-B2-ST131 strains was higher compared with the non-O25-B2-ST131 strains (p value < 0.05). O25-B2-ST131 strains showed significant correlation with serum resistance and biofilm formation. Amongst the resistance and virulence genes, the prevalence of iucD, kpsMTII, cnf1, vat, blaCTX-M-15, and blaSHV were significantly higher among O25-B2-ST131 isolates in comparison with non-O25-B2-ST131 isolates (p value < 0.05). Considering a ≥ 80% homology cut-off, fifteen different clusters of the isolates were shown with the same rep-PCR pattern. CONCLUSIONS: Our results confirmed the involvement of MDR-ESBLs producing E. coli strain O25-B2-ST131 in the occurrence of UTIs among children. Source tracking and control measures seem to be necessary for containment of the spread of hypervirulent and resistance variants in children.

Evaluation of the frequency of Escherichia coli pathogroups in Brassica oleracea cultivars.

Background and Objectives: Pathogenic diseases resulting from microbial contamination of food have been widely distributed in many parts of the world. Among these, Escherichia coli is one of the most important foodborne pathogenic bacteria. Diarrhea is one of the major causes of children's death in developing countries, with approximately 2 million deaths annually. The current study aimed to determine the frequency of diarrheagenic E. coli pathotypes such as Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), Enteroaggregative E. coli (EAEC), and Shiga toxin-producing E. coli (STEC) in Brassica oleracea cultivars in order to provide information on the assessment of diarrheagenic E. coli pathogenesis risk. Materials and Methods: 100 samples of vegetables were collected in Tehran, including cabbage, cauliflower, broccoli and Brussels sprouts. After homogenizing samples, enrichment was done in the EC broth medium. Five colonies of pure culture were used for DNA extraction. Pathotypes were identified by PCR using virulence genes. Results: The results showed that the prevalence of diarrheagenic E. coli strains was 7%. The EPEC prevalence was 3%, All EPEC isolates were atypical. The ETEC frequency was 3%, And the EAEC prevalence was 1%. Conclusion: These findings indicated that Brassica oleracea cultivars could be consideredas a source of contamination with diarrhea-causing E. coli strains.

Involvement of Pseudomonas aeruginosa in the occurrence of community and hospital acquired diarrhea, and its virulence diversity among the stool and the environmental samples.

Transmission of Pseudomonas aeruginosa along the food chain could cause gastrointestinal infections. To show this involvement, the prevalence, putative virulence genotype, and antibiotic resistance phenotype of P. aeruginosa isolates from stool of 1482 patients with community and hospital acquired diarrhea were compared with 87 isolates from the environmental samples. The results showed infection with P. aeruginosa in 3.4% of the cases, while 57.4% of vegetable samples were contaminated. Significantly higher frequency of lasB (98%), aprA (98%), exoY (98%), and exoS (90%), but lower rate of exoT (39.2%), was detected among the stool isolates. Multi-drug resistance (MDR) phenotype was detected in 25.5% and 4% of the stool and vegetable isolates, respectively. A higher rate of studied virulence genes was detected among the MDR strains vs non-MDR strains. These results indicate P. aeruginosa as a causative agent of diarrhea either among the hospitalized patients and those with community-acquired diarrhea.

A comparison of antibiotic disks from different sources on Quicolor and Mueller-Hinton agar media in evaluation of antibacterial susceptibility testing.

BACKGROUND AND OBJECTIVES: Antibacterial susceptibility testing of clinical bacterial isolates through disk diffusion method plays a major role in antibacterial treatment. One of the main factors affecting the result of these tests is the type, structure and quality of the disks. The main objective of this study was to compare the agreement of antibiotic disks originated from three companies on Quicolor and Mueller-Hinton agar. MATERIALS AND METHODS: Quicolor and Mueller-Hinton agar media were used in disk diffusion method. Seventy clinical isolates from Enterobacteriaceae family (21 Klebsiella spp., 36 Escherichia coli, 1 Enterobacter spp. and 12 Shigella spp.) were investigated in the study. After obtaining data, the results were interpreted as resistant, sensitive or intermediate. Kappa coefficient measured the agreement of two media. Coefficient of variation (CV) was also calculated for antibiotic disks. RESULTS: The kappa agreement values for three types of antibiotic disks on Quicolor and Mueller-Hinton agar plates were good or excellent for all the examined antibiotics. CV values were also very satisfactory in the majority of cases. CONCLUSION: Antibiotic disks from three manufacturers can successfully be used on both Quicolor and Mueller-Hinton agar plates.

Isolation of Enteropathogenic Escherichia coli from lettuce samples in Tehran.

AIM: The purpose of this study was to find the isolation rate of enteropathogenic Escherichia coli (EPEC) from lettuce samples collected in Tehran. BACKGROUND: During the last decade, the prevalence of infectious diarrheal diseases due to consumption of contaminated food especially raw vegetable has been increasingly reported. Enteropathogenic Escherichia coli strains are an important group of diarrheagenic E. coli that can cause infant diarrhea especially in the developing world. MATERIAL AND METHODS: One hundred lettuce samples collected in Tehran were transported to the laboratory, homogenized by a stomacher in EC broth containing cefixime, and cultured on MacConkey agar plates. Bacterial DNA was extracted by boiling method and PCR was performed using three pairs of primers targeting stx 1, stx 2 and eaeA genes. RESULTS: Screening of 100 lettuce samples by PCR showed four samples were positive for the presence of EPEC. CONCLUSION: This study suggests contamination of the lettuce by the EPEC and its possible role as the source of infection in this region.

Shiga toxin-producing Escherichia coli isolated from lettuce samples in tehran, iran.

BACKGROUND: During the last decade, the prevalence of foodborne diseases due to contaminated food as well as the outbreaks of diseases due to Shiga toxin-producing Escherichia coli (STEC) strains has increased. OBJECTIVES: The aim of this study was to evaluate the prevalence and antibiotic resistance pattern of STEC strains in lettuce samples. Since lettuce is used as a raw vegetable in salads, the rates of infections caused by this vegetable are high. MATERIALS AND METHODS: A total of 100 samples collected from Tehran, Iran, were transported to the laboratory, homogenized by a stomacher in E. coli broth containing cefixime, and cultured on MacConkey agar medium. Their DNA was extracted by boiling method and polymerase chain reaction (PCR) was performed, using five primers targeting the stx1, stx2, fliCh7, rbfO157, and eaeA genes. Susceptibility testing against ampicillin, imipenem, cephalosporin, tetracycline, aminoglycosides, chloramphenicol and quinolones was performed using disk diffusion method. RESULTS: Eight samples were positive for presence of STEC strains, three contained stx1, five contained stx2, and one sample was positive for presence of both rbfO157 and fliCh7. They were susceptible to all the antibiotics except for ampicillin and tetracycline. CONCLUSIONS: This study indicated the contamination of lettuce by STEC strains and its possible role as the source of infection. Resistance to both tetracycline and ampicillin may be considered as an emergency alarm for a multidrug resistance of STEC strains.

Isolation of Shiga toxin-producing Escherichia coli from raw milk in Kermanshah, Iran.

BACKGROUND AND OBJECTIVES: Infectious diarrhoeal diseases are great problem throughout the world and are responsible for considerable morbidity and mortality. Shiga toxin-producing Escherichia coli (STEC) is a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (HC) or the hemolytic uremic syndrome (HUS), which is the main cause of acute renal failure in children. Food-borne outbreaks associated with Shiga toxin-producing Escherichia coli have been well documented worldwide. The aim of this study was to investigate the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains in raw milk samples. MATERIALS AND METHODS: Raw milk samples collected from various cow farms in Kermanshah, Iran during June - September 2009 were investigated for STEC using PCR targeting stx1 and stx2 and then eaeA. RESULTS: Of 206 samples, 36 (17.47%) were contaminated with STEC. STEC isolates harbored 56.41% and 43.59% stx 2 and stx 1 gene respectively. In antibiotic resistance test, all strains were sensitive to ceftazidime, cefepime, gentamicin, imipenem and ciprofloxacin. 23.08% of isolates were resistat to tetracycline, and 38.5% of them showed intermediate sensitvity to cephalothin. CONCLUSIONS: The high presence of STEC in raw milk confirms the important role of raw milk as putative vehicle of infection to human. Moreover, this study suggests that the development of antibiotic resistant STEC must be a major concern in Iran and more studies are needed to identify the prevalence of STEC in other food samples.

Evaluation of a liquid-phase colorimetric method for rapid antibacterial susceptibility testing.

BACKGROUND AND OBJECTIVE: Early determination of antibacterial susceptibility increases the success of therapy, decreases unnecessary use of antibacterial agents and side-effects, and lowers the overall healthcare cost. MATERIALS AND METHODS: A rapid colorimetric method for antibacterial susceptibility testing named Qui-Sensitest (Inovative Biotechnology Organization, Istanbul, Turkey) was evaluated in this study. RESULTS: Qui-Sensitest proved to be a reliable rapid test for determining antibacterial susceptibility having an overall agreement of 97.6% with Kirby Bauer disk diffusion test results for enteric bacteria with 0.4% of major discrepancies and 2.0% of minor discrepancy. CONCLUSION: Since the test makes the results available in 3.5-6 hours, it can provide the means for choosing the right treatment regimen the same day the infectious agent is isolated.

Ribotyping of EPEC isolates from diarrheal and asymptomatic patients in Iran.

AIM: We intended to find out the diversity of EPEC isolates among asymptomatic or diarrheal children in Iran using ribotyping. BACKGROUND: Enteropathogenic Escherichia coli (EPEC) is responsible for gastroenteritis especially in young children. PATIENTS AND METHODS: A total of 39 EPEC collected strains were serotyped and the presence of virulence genes as well as EAF plasmid among the strains was studied. Adherence assay was also performed. Clonal diversity of the isolates was investigated using ribotyping. RESULTS: Of 39 studied strains of E. coli, 6 serogroups of EPEC were represented. The presence of the stx gene was ascertained in 7 isolates and the eaeA, eaeB and bfpA genes were harbored by 5, 3 and 1 strains, respectively. Ribotyping yielded 9 different clusters. CONCLUSION: According to our results there was not a significant correlation between the results of serotyping and those of ribotyping. However, different serotypes of E. coli may belong to the same ribotype clusters and vice versa.

Prevalence and antimicrobial resistance of diarrheagenic Escherichia coli and Shigella species associated with acute diarrhea in Tehran, Iran.

A study was performed to determine the prevalence and antimicrobial resistance of Shigella species and diarrheagenic Escherichia coli isolates cultured from patients with acute diarrhea in Tehran, Iran. Between May 2003 and May 2005, 1120 diarrheal specimens were collected and assayed for bacterial enteropathogens by conventional and molecular methods. Etiological agents were isolated from 564 (50.3%) specimens, and included 305 (54%) E coli, 157 (27.8%) Shigella species, and 102 (18%) from other genera of bacteria. The predominant E coli was Shiga toxin-producing E coli (105 isolates [34.5%]) and the predominant Shigella serotype was Shigella sonnei (88 isolates [56.1%]). A high rate of antibiotic resistance was observed among E coli, with 40 of 53 (75.5%) Shiga toxin-producing E coli isolates resistant to amoxicillin and tetra-cycline, and eight (5.2%) E coli isolates resistant to more than six antibiotics. Most Shigella isolates were resistant to tetracycline (95%) and trimethoprim-sulfamethoxazole (91.7%), with greatest antibiotic resistance observed among S sonnei (53 of 88 [60.2%] isolates). Antibiotic resistance is widespread in diarrheagenic E coli and Shigella in children with acute diarrhea in Tehran, Iran; hence, updated strategies for appropriate use of antimicrobial agents in Iran are needed.

[Serotype distribution and antimicrobial resistance rates of Shigella spp. isolates in Tehran, Iran]. / Tahran'da izole edilen Shigella türlerinin serotip dagilimi ve antimikrobiyal direnç oranlari.

In this study, antimicrobial resistance patterns and serotype distributions of Shigella spp. isolated from pediatric and adult patients with diarrhea, inhabiting in Tehran, were investigated. Stool specimens of 1350 patients with diarrhea who were admitted to the seven different hospitals in Tehran from November 2003 till March 2005 were taken into the study. Antibacterial susceptibility patterns of Shigella spp. isolates were determined by standard disk diffusion method. Overall isolation rate of Shigella spp. was found as 11.5 percent. S. sonnei was the most frequent species (55.1%) followed by S. flexneri (30.8%), S. boydii (9.6%) and S. dysenteria (4.5%). Resistance rates to ampicillin (81.4%), trimethoprim-sulphamethoxazole (93.6%), chloramphenicol (28.2%) and tetracycline (98.7%) were high, whereas low resistance rates to cefixime (5.1%) and nalidixic acid (2.6%) were detected. All isolates were found susceptible to ceftriaxone and ciprofloxacine. These data may help physicians for choosing appropriate empirical chemotherapy although subsequent antibacterial susceptibility testing is always recommended.

Molecular epidemiology of Escherichia coli diarrhoea in children in Tehran.

From July to December 2003, four categories of diarrhoeagenic Escherichia coli were investigated in Tehranian children with acute diarrhoea. Stool specimens of children under 5 years of age with diarrhoea (n=200) and matched controls (n=200) without diarrhoea were studied for the presence of entero-aggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC) and Shiga toxin-producing (STEC) E. coli by PCR identification of six different genes of diarrhoeagenic E. coli. STEC isolates were typed by O157 and H7 antisera. EAEC was the most prevalent category and was found in 24% of patients with diarrhoea and 8% of controls (p<0.0001). ETEC was isolated in 15.5% of patients with diarrhoea but not in any controls ( p<0.0001), STEC in 15% of patients and 2% of controls (p<0.0001) and EPEC in 6% of patients and 5% of controls. Of 30 STEC isolates from patients with diarrhoea, seven were O157:H7 and 23 were non-O157:H7.

[The prevalence of nasopharyngeal Neisseria meningitidis carriage, serogroup distribution, and antibiotic resistance among healthy children in Cankaya municipality schools of Ankara province]. / Ankara ili Cankaya ilçesi okullarindaki saglikli cocuklarda nazofaringeal Neisseria meningitidis tasiyicilik prevalansi, serogrup dagilimi ve antibiyotik direnci.

Meningococci responsible for significant morbidity and mortality rates in children are found in the oropharynx and nasopharynx and communicated with droplets. In this study, the prevalence of nasopharyngeal Neisseria meningitidis carriage, serogroup distribution and antibiotic resistance were determined among healthy children in Cankaya municipality of Ankara province. The study involved 1155 students aged 7-19 years. Systematic sampling method was used for sample selection. To isolate N. meningitidis, modified Thayer-Martin medium was used. The antibiotic susceptibilities of N. meningitidis isolates were determined by agar dilution method for penicillin, sulfadiazine, rifampicin, and azithromycin. N. meningitidis carriage prevalence was found as 10.4% with serogroup B being the most predominant (47.5%). The prevalence of N. meningitidis carriage was found to be closely associated with living conditions however, tonsillectomy, tonsillar hypertrophy, passive or active smoking did not affect the rate of carriage. Overcrowded life style, use of old-fashioned stoves for heating, and living in shanty housing were determined as risk factors increasing N. meningitidis carriage. None of the strains showed beta-lactamase activity, and five strains (4.2%) had decreased sensitivity to penicillin. The resistance against sulfadiazine was 54.4%, while it was 26.9% against azithromycin. No rifampicin-resistant strain were detected. It can be concluded that the prevalence of meningococcal carriage in this study was similar to that of other European countries. Rifampicin should be the first drug of choice both for the treatment of meningococcal carriers and for the prophylaxis of the subjects who had been in contact with patients with meningococcal infection.

Prevalence and pattern of antimicrobial resistance of Shigella species among patients with acute diarrhoea in Karaj, Tehran, Iran.

The study was carried out to determine the prevalence and pattern of antimicrobial resistance of Shigella species among patients with acute diarrhoea in Karaj, Tehran, Iran. The study included all acute diarrhoea patients who visited the hospitals and treatment centres of Karaj during November 2001-October 2002. Of 734 stool samples collected from patients with acute diarrhoea and analyzed for Shigella spp., 123 (16.8%) yielded Shigella spp. (7.5% Shigella flexneri, 5.2% S. sonnei, 2.6% S. dysenteriae, and 1.5% S. boydii). Of the Shigella isolates, 90.8% were resistant to one or more antimicrobial agent(s), and 87.8% were multidrug resistant. The most common resistance was to tetracycline (73.5%), trimethoprim-sulphamethoxazole (70.4%), and amoxicillin-clavulanic acid (50.0%). Resistance to cefixime, ciprofloxacin, ceftriaxone, and nalidixic acid was observed in 6.1%, 3.1%, 2.0%, and 1.0% of the isolates respectively. These findings suggest that Shigella spp. may be an important aetiological agent of diarrhoea with a high rate of drug resistance in this region, which requires further study.