Molecular typing of cytotoxin-producing Klebsiella oxytoca isolates by 16S-23S internal transcribed spacer PCR.

Cytotoxin is one of the important pathogenic factors, which plays a role in the virulence of Klebsiella oxytoca. The aim of this study was to investigate molecular typing of clinical isolates of the cytotoxin-producing K. oxytoca using internal transcribed spacer (ITS) PCR. A total of 75 isolates of K. oxytoca were isolated from clinical samples; they were verified as K. oxytoca by standard microbiological tests and PCR. Production of toxin determines the cytotoxic effects on HEp-2 cells. The genetic diversity of isolates of the cytotoxin-producing K. oxytoca were defined by ITS-PCR. Of all the isolates investigated, five K. oxytoca strains isolated from stool cultures, two strains from blood samples, one strain from a wound and one strain isolated from urine had cytotoxic effects on HEp-2 cells. The ITS-PCR patterns showed genetic diversity among cytotoxin-producing isolates. The ITS-PCR method had good discriminatory power; performance of this method and interpretation of the results were easy and repeatable. Five genetic diversity patterns were identified by ITS-PCR.

PFGE genotyping and molecular characterization of Campylobacter spp. isolated from chicken meat.

A total of 70 samples were collected from chicken meat obtained from 10 markets in Tehran, Iran from which 39 Campylobacter coli were isolated. Among 10 antibiotics used, maximum resistance was seen to trimethoprim-sulphamethoxazole (SXT) (97.36%), nalidixic acid (94.8%), ciprofloxacin (87.7%), streptomycin (89.72%), and tetracycline (97.4%). No resistance was to gentamycin was observed. None of the Campylobacter strains under study harbored integron, suggesting the involvement of other resistance mechanisms in emergence of multi drug resistance (MDR) phenotype among the isolates. Two major types (A and B) and 15 subtypes (A1-A8 and B1-B7) were identified. Pulsed-field gel electrophoresis (PFGE) analysis demonstrated a high degree of homogeneity while the majority of the isolates shared identical or very similar PFGE genotypes. Isolates with identical genotypes differed in their resistance profile, although all of them assigned to MDR phenotype. To our knowledge, this is the first molecular survey from Iran characterizing Campylobacter isolates from poultry, which adds to our knowledge the epidemiological linkage of Campylobacter isolates with MDR properties from different sources and emphasizes the need for cautious use of antimicrobials in different fields of food production chain.

Molecular characterization of Salmonella enterica serotype Enteritidis isolates from food and human samples by serotyping, antimicrobial resistance, plasmid profiling, (GTG)5-PCR and ERIC-PCR.

In recent years, Salmonella enterica serovar Enteritidis has been a primary cause of human salmonellosis in many countries. The major objective of this study was to investigate genetic diversity among Salmonella Enteritidis strains from different origins (food and human) by Enterobacterial Repetitive Intergenic Consensus (ERIC) -PCR, as well as to assess their plasmid profiling and antimicrobial resistance. A total of 30 Salmonella Enteritidis isolates, 15 from food samples (chicken, lamb, beef and duck meats) and 15 from clinical samples were collected in Tehran. Identification of isolates as Salmonella was confirmed by using conventional standard biochemical and serological tests. Multiplex-PCR was used for serotyping of isolates to identify Salmonella Enteritidis. Antimicrobial susceptibility testing to 16 agents founds drug resistance patterns among Salmonella Enteritidis isolates. No resistance was observed to cephalexin, ceftriaxone, ceftazidime and cefotaxime, ciprofloxacin, imipenem or meropenem, chloramphenicol and gentamicin. The highest resistance (96.7%) was observed to nitrofurantoin. Seven plasmid profiles (P1-P7) were detected, and a 68-kb plasmid was found in all isolates. Two different primers; ERIC and (GTG)5 were used for genotyping, which each produced four profiles. The majority of clinical and food isolates fell into two separate common types (CTs) with a similar percentage of 95% by ERIC-PCR. Using primer (GTG)5, 29 isolates incorporated in three CTs with 70% of isolates showing a single banding pattern. Limited genetic diversity among human and food isolates of Salmonella Enteritidis may indicate that contaminated foods were possibly the source of human salmonellosis. These results confirmed that ERIC-PCR genotyping has limited discriminatory power for Salmonella Enteritidis of different origin.

Multi-locus variable number tandem repeat analysis of Vibrio cholerae isolates from 2012 to 2013 cholera outbreaks in Iran.

Cholera remains to be an international threat, with high rates of illness and death. In 2012 and 2013, two cholera outbreak happened in Iran, affecting lots of people. Vibrio cholerae O1 was confirmed as the etiological agent. Source identification and controlling the spread of the cholera disease are two critical approaches in cholera outbreaks. In this study, thirty V. cholerae O1 isolates were selected and has been evaluated for antimicrobial resistant as well as molecular typing by multilocus variable-number tandem-repeat analysis (MLVA) method. Twenty-nine (97%) isolates were sero-grouped as El Tor (one isolate was classical) and 100% were related to Inaba serotype. All of the isolates were susceptible to ciprofloxacin, chloramphenicol, ampicillin and gentamicin. On the other hand, 60% of the isolates were MDR (resistant to 3 or more classes). There were three resistance patterns. The most prevalent pattern was resistance to streptomycin, erythromycin, trimethoprim-sulfamethoxazole, and tetracycline (ST-SXT-E-T) which was seen in 50% of isolates. Using MLVA method 14 MLVA types were identified. MLVA type 2 (5-7-7-16-15) accounted for 43% of isolates. Isolates with the same genotype often did not have the same antibiogram. Overall, the data indicate that the Iranian V. cholerae were MDR and clonaly related. Furthermore, the results of this study shows that MLVA can be used as useful method for V. cholerae genotyping in epidemiological investigations.

Molecular characterization of Vibrio cholerae isolates from Iran 2012 and 2013 outbreaks.

UNLABELLED: The aim of this study was to assess the genetic diversity of Vibrio cholerae isolated from 2012 and 2013 outbreaks in Iran, with regard to their virulence properties. A total of 20 V. cholerae strains were collected from Sistan-Baluchestan province of Iran during 2012 and 2013 outbreaks. Hybridization assays showed the presence of ctx, zot, ace and rstC genes related to CTX and RS1 phages in all of the isolates. PCR assay indicated the concomitant presence of ORFs within RTX (1448, 1451) and TLC (1465, 1469) elements within the genome of the isolates. ERIC-PCR analysis showed four homogeneous profiles among which strains from 2013 outbreak and 72·7% of 2012 outbreak uniformly showed a common ERIC-PCR fingerprint. Ribotyping assay showed a single dominant profile (ribotype A) among 77·7 and 72·7% of isolates recovered from 2013 and 2012 outbreaks respectively. In conclusion, this study reports high degree of homogeneity among isolates from 2012 and 2013 outbreaks in Iran and emphasizes on the primary application of ERIC-PCR to generate fingerprints and differentiate between V. cholerae isolates of clinical origin in a timely manner for epidemiological investigations and source tracking purposes, although ribotyping method was proved to be more discriminatory. SIGNIFICANCE AND IMPACT OF THE STUDY: The clonality of Vibrio cholerae isolates recovered from patients with Afghan nationality during 2012 and 2013 outbreaks in Iran emphasizes on the need for monitoring Iran boundaries. This highlights the demand for a simple, reproducible and time-saving typing method for rapid and reliable assessment of clonal correlation of isolates in outbreaks. In this regard, ERIC-PCR produced results comparable with those obtained by PFGE and ribotyping which is of great significance in public health and source tracking purposes.

Emergence of Vibrio cholerae O1 classical biotype in 2012 in Iran.

UNLABELLED: Cholera outbreaks annually occur in many parts of Iran. The aim of this study was to investigate the biotype and genotype diversity of V. cholerae isolates from recent outbreak (2012) in Iran and to characterize the ctxB allelic sequence of isolates. The ctxB sequence of all isolates was analyzed and compared with the reference ctxB sequences for El Tor and classical biotypes in GenBank database. The PFGE genotype specification of isolates was determined and genetic relatedness among isolates and also with those previously reported from Iran was assessed. Ten out of eleven isolates were identified as El Tor biotype and one single isolate belonged to classical biotype. All isolates except three possessed tcpA, ctxA, ctxB and wbeT genes. All the ctxB(+) isolates in this study (classical and El Tor biotypes) possessed the ctxB sequence of classical biotype allele providing evidences of El Tor variants. Eight out of 11 isolates (73%) showed identical pulsotypes (P1). Each of the remaining three isolates showed distinct pulsotypes (P2-P4) with more than three band differences. Pulsotype P2 was corresponded to an isolate (9%) with classical biotype. The result demonstrated diversity in virulence gene content of strains with identical PFGE patterns and also provided evidences of the import of a V. cholerae strain with classical biotype from out of our country as no classical biotype strain has been previously (1998-2011) reported from Iran. SIGNIFICANCE AND IMPACT OF THE STUDY: Emergence of V. cholerae with Classical biotype after at least one decade in Iran is alarming due to fear of expansion of V. cholerae strains with high virulence potential and signifies the need to monitor and analyze all new cases in countries with cholera outbreaks or even sporadic cases as this could conceivably affect the neighbouring countries and may expose the world to the seventh pandemic with Classical biotype strains.

Comparison of two recombinant systems for expression of cholera toxin B subunit from Vibrio cholerae.

PURPOSE: The aim of this study was to assess the production of recombinant cholera toxin B subunit (rCTB) protein in two different expression systems (pAE_ctxB and pQE_ctxB constructs) in Escherichia coli BL21 (DE3). MATERIALS AND METHODS: The ctxB fragment was amplified from Vibrio cholerae O 1 ATCC14035 and cloned in pGETM-T easy vector after which it was transformed to E. coli Top 10F' and grown on LB-ampicillin agar medium. Sequence analysis confirmed the complete ctxB gene sequence in the construct which was further subcloned to pQE-30 vector. The construct was subsequently transformed to E. coli M15 (pREP4). The recombinant pAE_ctxB and pQE_ctxB were transformed to competent E. coli BL21 (DE3) cells to express CTB protein. RESULT: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the maximum expression of rCTB in both systems at 5 h after induction and western blot analysis confirmed the presence of recombinant CTB in blotting membranes. CONCLUSION: Expression of rCTB in pAE_ctxB construct was more efficient (15-fold) than pQE_ctxB, and it seems that Lac UV5 in E. coli BL21 (DE3) is more compatible with the former construct. This expression system can be used to produce recombinant CTB in high yield which may enable us to study the oral tolerance or mucosal adjuvant properties of rCTB using animal models.

Investigation into immunological responses against a native recombinant CTB whole-cell Vibrio cholerae vaccine in a rabbit model.

AIM: The aim of this study was to express and purify the recombinant CTB (rCTB) protein from Vibrio cholerae and investigate the biological and immunological characteristics of purified protein in rabbit animal model and in combination with Iranian inactivated V. cholerae whole cells as a domestic recombinant WC-CTB vaccine. METHODS AND RESULTS: Expressed 6XHis-tagged rCTB was properly purified, and its identity was confirmed by Western blotting using cholera toxin-specific antibody. Concentration of purified protein was assessed to be 700 mg l(-1) . GM(1) -ELISA assay showed that purified rCTB pentamer was functionally active and able to bind GM(1) in a dose-dependent manner. Recombinant CTB was inoculated into rabbits through intestinal rout alone and in combination with inactivated whole-cell V. cholerae strains (WC). The anti-CTB IgG titre showed that serum IgG responses were significantly increased in groups immunized with rCTB mixed with inactivated WC in comparison with control group. Furthermore, rCTB without V. cholerae WC also stimulated the IgG responses when inoculated into rabbit intestine. Challenge experiments of immunized rabbits showed an adequate protection against V. cholerae strains. CONCLUSIONS: Recombinant CTB alone and in combination with inactivated Iranian strains was protective against live toxigenic V. cholerae strains, made it a potential candidate for an indigenous vaccine. SIGNIFICANCE AND IMPACT OF THE STUDY: It was proved that rCTB produced in this system can be used as a potent immunogenic protein to stimulate the immunity against V. cholerae strains and can be used for developing a native vaccine composed of our local strains with their own surface structures and antigenic determinants against cholera.

Emergence of Pseudomonas aeruginosa cross-infection in children with cystic fibrosis attending an Iranian referral pediatric center.

BACKGROUND AND OBJECTIVES: This study was carried out with the objective of determining the genomic variability of P. aeruginosa strains isolated from patients suffering from cystic fibrosis or from environmental cultures collected from different locations in the unit they admitted. MATERIALS AND METHODS: A total of 57 clinical and environmental P. aeruginosa isolates were genotyped by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR), and antimicrobial susceptibility testing was performed using the Clinical and Laboratory Standards Institute method. RESULTS: One predominant ERIC profile (type A) was identified in 46 strains (81% of all typed isolates) which was responsible for thirty-nine of 44 clinical isolates (89%) and 7 of 13 environmental isolates (54%). All clinical isolates were susceptible to piperacillin-tazobactam, ceftazidime and cefepime followed by ticarcillin, aztreonam, amikacin and tobramycin (96.5%). CONCLUSIONS: In our country CF patients are not segregated from other patients, and transmission of bacteria between these patients and other patients might occur in the wards via personal contact or contaminated environment. Future evaluation for policy of patient segregation is necessary and the elimination of contaminated sources and control of environmental spread and recurrent contamination risk is needed.

Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance.

PURPOSE: The aim of this study was to understand the epidemiological linkage of clinical and environmental isolates of Vibrio cholerae and to determine their genotypes and virulence genes content. MATERIALS AND METHODS: A total of 60 V. cholerae strains obtained from clinical specimens (n = 40) and surface waters (n = 20) were subjected to genotyping using PFGE and determination of their virulence-associated gene clusters. RESULT: PCR analysis showed the presence of chromosomally located hly and RTX genetic elements in 100% and 90% of the environmental isolates, respectively. The phage-mediated genetic elements such as CTX, TLC and VPI were detected in 5% of the environmental isolates suggesting that the environmental isolates cannot acquire certain mobile gene clusters. A total of 4 and 18 pulsotypes were obtained among the clinical and environmental V. cholerae isolates, respectively. Non-pathogenic environmentally isolated V. cholerae constituted a distinct cluster with one single non-O1, non-O139 strain (EP6) carrying the virulence genes similar to the epidemic strains. This may suggest the possible potential of conversion of non-pathogenic to a pathogenic environmental strain. CONCLUSIONS: The emergence of a single environmental isolate in our study containing the pathogenicity genes amongst the diverse non-pathogenic environmental isolates needs to be further studied in the context of V. cholerae pathogenicity sero-coversion.

Distribution of class 1 integrons among enteropathogenic Escherichia coli.

The aim of this study was to investigate the incidence of and resistance gene content of class 1 integrons among enteropathogenic Escherichia coli (EPEC) and non-EPEC and to investigate intraspecies genetic diversity of EPEC strains isolated from children with diarrhea in Iran. Twenty-eight EPEC and 16 non-EPEC strains isolated from children with diarrhea were tested for the presence of a class 1 integron associated integrase gene (int1). Sequence analysis was performed to identify the resistance gene content of integrons. Genetic diversity and cluster analysis of EPEC isolates were also investigated using enterobacterial repetitive intergenic concensus-polymerase chain reaction (ERIC-PCR) fingerprinting. Twenty-three (82%) EPEC isolates and 11 (68.7%) non-EPEC isolates harbored the int1 gene specific to the conserved integrase region of class 1 integrons. Sequence analysis revealed the dominance of dfrA and aadA gene cassettes among the isolates of both groups. ERIC-PCR fingerprinting of EPEC isolates revealed a high diversity among these isolates. The widespread distribution of 2 resistance gene families (dfrA and aadA) among both groups of EPEC and non-EPEC isolates indicates the significance of integrons in antibiotic resistance transfer among these bacteria. Furthermore, clonal diversity of EPEC isolates harbouring a class 1 integron also suggests the circulation of these mobile elements among a diverse population of EPEC in this country.

Molecular diversity of CTX prophage in Vibrio cholerae.

AIMS: The objective of this study was to investigate the molecular diversity of CTX genetic element within toxigenic Vibrio cholerae genomes and to determine the genetic diversity of V. cholerae population collected in a 6-year period (2004-2009) in Iran. METHODS AND RESULTS: The results of mismatch amplification mutation assay (MAMA)-PCR and sequencing showed cytosine nucleotide in positions 203 and 115 in all 50 El Tor V. cholerae strains, which is the same as classical ctxB sequence. One strain yielded amplicons with both El Tor and classical biotype primers in MAMA-PCR indicative of presence of two copies of CTX phages with different genotypes (rstR(ET) ctxB(class) and rstR(ET) ctxB(ET)) integrated within the genome of this isolate, which suggested the integration of two different CTX phages at different occasions or point mutation in one copy of CTX. Sequencing and PCR analysis indicated the presence of hybrid CTX genotype (rstR(ET) ctx(class)) in 70.6% of the isolates; however, only El Tor RS1 phage has been integrated in flanking to the CTX phages with different genotypes. CONCLUSIONS: Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and ribosomal gene spacer-PCR (RS-PCR) showed a relatively homogenous population in different years. Our findings indicate that sequence analysis of RS and ctxB regions has more discriminative power than restriction-based methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigating the molecular diversity of CTX prophage among V. cholerae strains helps to establish a new valuable database of genetic information about isolates, which is of great importance for epidemiologic studies in Iran and other countries encountering cholera epidemics.

A rapid and reliable species-specific identification of clinical and environmental isolates of Vibrio cholerae using a three-test procedure and recA polymerase chain reaction.

PURPOSE: Vibrio cholerae, the cause of cholera, is one of the leading causes of morbidity and mortality in many developing countries. Most laboratories initially rely on biochemical tests for a presumptive identification of these strains, followed by a polymerase chain reaction (PCR)-based method to confirm their identification. The aim of this study is to establish a rapid and reliable identification scheme for V. cholerae using a minimal, but highly specific number of biochemical tests and a PCR assay. MATERIALS AND METHODS: We developed a species-specific PCR to identify V. cholerae, using a housekeeping gene recA, and used that to evaluate the sensitivity and specificity of 12 biochemical tests commonly used for screening and / or presumptive identification of V. cholerae in the clinical and environmental samples. RESULTS: Here we introduced a combination of three biochemical tests, namely, sucrose fermentation, oxidase test, and growth in trypton broth containing 0% NaCl, as also the PCR of the recA gene, for rapid identification of V. cholerae isolates, with 100% sensitivity and specificity. The established method accurately identified a collection of 47 V. cholerae strains isolated from the clinical cases (n = 26) and surface waters (n = 21), while none of the 32 control strains belonging to different species were positive in this assay. CONCLUSION: The triple-test procedure introduced here is a simple and useful assay which can be adopted in cholera surveillance programs for efficient monitoring of V. cholerae in surface water and fecal samples.

wbeT sequence typing and IS1004 profiling of Vibrio cholerae isolates.

AIMS: To investigate the molecular basis for serotype variation in Vibrio cholerae O1 and the genetic relatedness amongst different serotypes isolated from 2004 to 2008 in Iran. METHODS AND RESULTS: Despite the presence of all three serotypes of V.cholerae O1 (Ogawa, Inaba and Hikojima) in Iran in the last decade, the Inaba strains have been the dominated serotype. Sequence analysis of wbeT determined only a single substitution of G for A at position 295 in all Inaba strains resulting in a replacement of serine to proline. No difference was found in the copy numbers and profile of IS1004 between the classical and El Tor V. cholerae O1 strains, supporting the clonality amongst the isolates obtained over 5 years in Iran. In addition, Southern blots of HpaII-digested chromosomal DNAs of our Ogawa and Inaba isolates showed the presence of an incomplete copy of IS1004 for all isolates. CONCLUSIONS: IS1004 profiling can be a reliable method for analysis of clonal dissemination of V. cholerae. The results indicated that specific point mutation at a particular position within the wbeT of V. cholerae O1 strains in Iran may occur which, in turn, may result in serotype switching. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the molecular basis for serotype conversion of V. cholerae and their genetic relatedness could give insights for the incoming cholera epidemic prediction and control.

Characterization of pathogenicity island prophage in clinical and environmental strains of Vibrio cholerae.

In this study 86 isolates of Vibrio cholerae were analysed for their adhesive properties and the presence of pathogenicity island genes. With the exception of three isolates, all of the other clinical isolates (92.5%) contained an intact TCP (toxin-co-regulated pilus) gene cluster. In contrast, 95% of all environmental non-O1-non-O139 isolates were negative for the TCP gene cluster. The majority of clinical isolates (82.5%) possessed the complete vibrio pathogenicity island (VPI) gene cluster and had a similar RFLP pattern, while only a single environmental strain possessed an almost complete VPI cluster (lacking 0.4 kb in the tcpA and toxT region). The result showed that the isolates with tcpA(+)/toxT(+) had a strong attachment for HT-29 and Vero cells, whereas isolates with tcpA(+)/toxT(-) or tcpA(-)/toxT(-) genomic characteristics showed no autoagglutination and weak attachment for the cell lines. Two environmental strains (tcpA(-)/toxT(-)) showed strong adhesive properties to the cell lines, indicating that non-fimbrial adhesive factors are involved in the environmental V. cholerae strains in the absence of TCP.

Comparative sequence analysis of recA gene among Vibrio cholerae isolates from Iran with globally reported sequences.

AIMS: To study the genetic relatedness between V. cholerae isolates from Iran and other countries based on housekeeping gene recA sequence analysis. METHODS AND RESULTS: A 995-bp region of the recA gene from 24 V. cholerae isolates obtained from human and surface water origins in Iran over a 5-year period was sequenced and compared with the sequence data from the isolates belonging to other places. Cluster analysis of the constructed dendrogram based on recA sequence divergence for our clinical isolates showed one sequence type (ST), whereas environmental isolates revealed eight STs. Interestingly, one of our environmental isolates was intermixed with clinical isolates in the largest cluster containing the epidemic strains. Our 24 isolates plus 198 global isolates available in the GenBank showed 77 sequence types (STs) with at least one nucleotide difference. CONCLUSIONS: Our result suggested that recA sequencing is a reliable analysis method for understanding the relatedness of the local isolates with the isolates obtained elsewhere. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the genetic relatedness between V. cholerae isolates could give insights into the health care system for better control and prevention of the cholera.

A molecular survey on virulence associated genotypes of non-O1 non-O139 Vibrio cholerae in aquatic environment of Tehran, Iran.

Thirty-seven Vibrio cholerae strains were isolated from surface water sources at 5 different locations in Tehran, Iran during 2006 and were identified as non-O1 and non-O139 isolates. PCR for SXT element and class 1 integron was positive for 19% and 5.4% of isolates, respectively. PCR for virulence associated-genes within the vibrio pathogenicity island (VPI) gene cluster showed the presence of LJ, int and RJ in 8, 59 and 30% of the isolates, respectively. None of the V. cholerae isolates contained the toxin encoding genes (ace, zot, ctx) in the CTX genetic element. Biochemical fingerprinting using PhPlate system (PhP-RV) was able to type all strains and resulted in 8 common types (containing 78% of the isolates) and 8 single types (22%). Out of 37 isolates, only 26 isolates were typeable with pulsed-field gel electrophoresis (PFGE) producing banding patterns. The results presented in this study showed no genotyping correlation between the V. cholerae isolated from surface water and the clinical setting which had been reported previously by this laboratory. Furthermore, combination of PFGE and PhP-RV methods was proved beneficial for non-typeable V. cholerae isolates.

Genomic organisation of the CTX element among toxigenic Vibrio cholerae isolates.

The composition and gene arrangement of the CTX genetic element were compared in 36 Vibrio cholerae isolates obtained during 2004-2006 from Iran. Long-PCR amplification of the CTX genetic element, using primers targeting ig1 and attB2, revealed three PCR products of c. 6.9, 5.6 and 2.6 kb, respectively. Southern blot hybridisation revealed that 30%, 17% and 53% of the isolates had one, two and three copies of the zot gene, respectively. PCR analysis of internal regions showed that isolates with three copies of the CTX genetic element carried one complete (6.9 kb) and two truncated CTX elements (each of 5.6 kb). In contrast, isolates with one or two copies of CTX carried the complete 6.9-kb CTX element. Pulsed-field gel electrophoresis revealed two pulsotypes among the isolates, with 75% of the isolates belonging to pulsotype 2. The pulsotype 2 isolates had varying CTX genomic arrangements, whereas the pulsotype 1 isolates had a homogeneous CTX arrangement. Thus, variations in the content, arrangement and copy number of the CTX genetic element may occur in isolates belonging to the same clone.

Dissemination of a single Vibrio cholerae clone in cholera outbreaks during 2005 in Iran.

In this study, 50 Vibrio cholerae O1 serotype Inaba isolates were collected during several cholera outbreaks throughout Iran during the summer of 2005. The results of antibiotic susceptibility testing showed that 86, 84, 84 and 82 % of the isolates were resistant to streptomycin, chloramphenicol, co-trimoxazole and tetracycline, respectively. The strains were genotyped using randomly amplified polymorphic DNA (RAPD), PFGE and ribotyping techniques. PCR showed that 100, 98 and 98 % carried the ctx, zot and ace genes, respectively. Biochemical fingerprinting of the isolates using the PhenePlate (PhP) system showed a low diversity index level (0.755), suggesting that the strains were highly homogeneous. Among the strains, 100 and 96 % showed an identical ribotype and PFGE patterns, respectively. The two isolates showing different PFGE patterns also exhibited discrete PhP types. RAPD was able to discriminate the isolates into six distinct groups, suggesting some genetic dissimilarity was present among the strains. These ribotyping, PFGE and PhP techniques revealed the clonal dissemination of a single V. cholerae strain throughout Iran in 2005.