The potential role of Bifidobacterium spp. as a preventive and therapeutic agent in controlling inflammation via affecting inflammatory signalling pathways.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease with relapses and periods of remission. Forasmuch as, dysregulation of the immune system is one of the triggers of IBD, taking probiotics as one of the immunomodulators in the gut, could help to control inflammation and IBD via influencing signalling pathways. Here, we aimed to investigate the efficacy of five selected Bifidobacterium strains in modulating JAK/STAT and NF-kB inflammatory signalling pathways via using the in vitro assay. A quantitative real-time polymerase chain reaction assay was used to analyse the expression of JAK/STAT and inflammatory genes followed by potential probiotic treatments before, after and simultaneously with the inflammation induction (sonicated pathogen). The production of IL-6 and IL-1ß after probiotic treatment was evaluated. Probiotic treatment resulted in the downregulation of TIRAP, IRAK4, NEMO and RIP genes in the NF-kB pathway, as well as JAK genes compared to sonicate-treated cells. The expression of STAT genes was different after our selected Bifidobacterium strains treatment. The production of IL-6 and IL-1ß decreased after probiotic treatment. These strains of Bifidobacterium spp. showed anti-inflammatory effects on HT-29 cells via modulation of JAK/STAT and NF-kB signalling pathways. The use of Bifidobacterium spp. could be considered as a suitable preventive and complementary treatment for patients with inflammatory bowel disease.

Rise of antibiotic resistance in clinical enterococcal isolates during 2001-2016 in Iran: a review.

INTRODUCTION: The clinical significance of enterococci is mostly related to its antibiotic resistance which contributes to colonization and infection, in particular amongst the hospitalized patients. The present review has examined the literature to provide a comprehensive data on enterococci antibiotic resistance during the last 20 years in Iran. METHODS: Search engines such as Google Scholar and PubMed were used to identify all Persian and English-language articles investigating enterococci in Iran from 1996 to 2017. The search terms were "enterococci", "enterococcal", "enterococcus", "Iran", "bacterial resistance", "antibiotic resistance" and "resistance". RESULTS: Decrease in the resistance trend against ampicillin, gentamycin and ciprofloxacin was observed over a period of 15 years (2001 to 2016) in Iran. During a 10 years period from 2001 to 2015, the rate of resistance among Enterococcus faecalis species was less than Enterococcus faecium. The resistancerate, however, was considerably increased for both species during this period. The mean resistance rates for vancomycin, gentamicin, ciprofloxacin, erythromycin, nitrofurantoin, chloramphenicol, trimethoprim-sulfametoxazol, imipenem and teicoplanin were higher among complicated cases (patients with underlying debilitating disorders) compared to general cases (hospitalized or outpatients with no specific underlying disorder). CONCLUSIONS: E. faecalis and E. faecium showed a rise in the mean resistance against all the antibiotics during a 10-year period from 2010 to 2015. With the exception of penicillin and ampicillin, resistance to all antibiotics was higher amongst complicated cases compared to general patients.

Evaluation of potential probiotic characteristics and antibacterial effects of strains of Pediococcus species isolated from broiler chickens.

Pediococcus spp. were isolated from poultry rectum, faeces and food as good probiotic candidates in order to select strains to be used as probiotic in poultry feed. A total of 168 lactic acid bacteria were isolated and 51 isolates including 31 Lactobacillus spp. and 20 Pediococcus spp. were able to survive in low pH and bile salt concentration. The Pediococcus spp. were identified and their ability to form biofilm, adhesion to Caco-2 cells and antimicrobial activities against enteric pathogenic bacteria were determined. The results showed the presence of two strains, Pediococcus acidilactici P17 and P19 in rectal swab samples from 21-d old chickens with significant antibacterial activities against Salmonella enteritidis and Escherichia coli. The results suggest that only a few isolates of Pediococcus with potential probiotic activities are present in the poultry industry.

Virulence and antimicrobial resistance of Enterococcus faecium isolated from water samples.

UNLABELLED: The aim of this study was to determine the incidence of Enterococcus species and six virulence factors of Enterococcus faecium which were isolated from surface water and wells. Fifteen different water samples, which were used for drinking as well as agricultural irrigation, were collected from nine private wells and surface water from six rivers located at the east of Tehran. The Ent. faecium isolates were tested for their resistance to 10 antibiotics and their virulence factors were detected using multiplex PCR for esp, acm, gelE, asa1, cylA and hyl genes. The most predominant species in 315 isolates were Ent. faecium (n = 118) followed by Enterococcus galinarom (n = 110), Enterococcus mundeti (n = 18), Enterococcus hirea (n = 37) and Enterococcus casselifelavus (n = 32). The resistance rates were observed in 41·5, 27·1, 12·7, 6·8 and 1·7% isolates for tetracycline, erythromycin, ampicillin, ciprofloxacin and chloramphenicol respectively. None of the Ent. faecium isolates were resistant to vancomycin, teicoplanin, linezolid, gentamicin and quinuspristin-dalfopristin. Virulence determinant was found in 84·7, 33·9, 16·1 and 2·5% of isolates for acm, asa1, esp, cylA respectively. None of the isolates carried hyl and gelE gene. The presence of virulence factors and antibiotic resistance indicated that water might be an important source of dissemination of virulent enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of drinking or recreational water by human or animal faecal waste is a major public health threat. In this study, we determine the incidence of Enterococcus species and six virulence factors of Enterococcus faecium which were isolated from surface water and wells. Results from this study suggest that the presence of Ent. faecium in natural and well waters was found to be significant in rural areas of Tehran. Resistant to erythromycin among Ent. faecium was relatively high and the incidence of acm and asa1 among our isolates was common overall.

Molecular analysis of pbp2b in Streptococcus pneumonia isolated from clinical and normal flora samples.

Streptococcus pneumoniae is an important bacterial pathogen responsible for respiratory infections, bacteraemia, and meningitis remains an important cause of disease and mortality in infants and younger children around the world, with penicillin being considered the drug of choice for the treatment of infections. However, penicillin-resistant S. pneumonia is now becoming endemic worldwide. In this study, a total of 80 pneumococcal isolates were collected from different clinical sources as well as normal flora. These isolates were subjected to antimicrobial susceptibility testing and MIC determination. The penicillin-binding proteins, pbp2b, were amplified by PCR, and they were sequenced. The genetic relationship of the penicillin-resistant isolates was performed by BOX PCR. Overall, 36 pneumococcal (45 %) isolates were found to be resistant to penicillin with different MICs. The majority of them (80 %) were intermediately resistant with MIC of 0.12-1 µg/ml, whereas 20 % of isolates were penicillin resistant with MICs of >2 µg/ml. The results identified seven groups which were based on the amino acid substitutions of pbp2b. Sequencing analysis revealed that the most prevalent mutation was the substitution of Adenine for Thymine at the position 445 which is next to the second PBP2b-conserved motif (SSN). This study indicates that resistance to penicillin appears to be dependent on specific mutations in pbp2b, and the substitution in S620 → T near to the third PBP2b-conserved motif appears to be important in developing highly antibiotic-resistant isolates. Moreover, there was a positive correlation between the mutations in pbp2b gene and MIC.

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.

Characterisation of uropathogenic Escherichia coli from children with urinary tract infection in different countries.

Uropathogenic Escherichia coli (UPEC) carry many virulence factors, including those involved in long-term survival in the urinary tract. However, their prevalence and role among UPEC causing urinary tract infection (UTI) in children is not well studied. To further understand the virulence characteristics of these bacteria, we investigated the prevalence of antibiotic resistance, antigen 43 genes, curli and cellulose among UPEC in children from different countries. Isolates (n = 337) from five countries were tested for antibiotic susceptibility, phylogenetic groups, prevalence of flu, fluA(CFT073), fluB(CFT073), curli and cellulose. High prevalence of multidrug resistance and extended spectrum beta lactamase production was found among Iranian and Vietnamese isolates. Resistance was associated with phylogenetic group D while group B2 was associated with fluA(CFT073) and fluB(CFT073). Fewer Iranian isolates carried fluA(CFT073), curli and cellulose. fluB(CFT073) was most prevalent among Slovak isolates. Ampicillin and amoxicillin/clavulanic acid resistance was prevalent among fluA(CFT073)- and fluB(CFT073)-positive Australian, Iranian and Swedish isolates. Lack of curli and cellulose was associated with resistance among Vietnamese isolates. We conclude that major differences exist in the prevalence of antibiotic resistance among UPEC from different countries. Associations observed between resistance and virulence factors may, in different ways, promote the long-term survival of UPEC in the urinary tract.

Prevalence of virulence factors and antibiotic resistance in vancomycin-resistant Enterococcus faecium isolated from sewage and clinical samples in Iran.

PURPOSE: The purpose of the present study was to perform a molecular epidemiological survey by investigating the antibiotic resistance and the presence of known virulence factors in Enterococcus faecium isolates in Iran. The data collected from this study would allow us to control the spread and develop strategies for treatment of the enterococcal infections. MATERIALS AND METHODS: In this study, 156 vancomycin-sensitive E. faecium (VSEF; 58) and vancomycin-resistant E. faecium (VREF; 98) samples were isolated from clinical specimen and sewage treatment plants (STPs). These isolates were screened for the presence of genes encoding for aggregation substance (asa1), cytolysin (cyl), enterococcal surface protein (esp), gelatinase (gelE) and hyaluronidase (hyl) by polymerase chain reaction (PCR). RESULTS: Although significantly different, the results showed the presence of hyl and esp genes in both clinical (41 and 75%, respectively) and sewage (3.2 and 41%, respectively) isolates. Sensitivity of all isolates to seven antibiotics was examined. The results of the clinical isolates showed that the majority of esp positive isolates were also resistant to vancomycin, ciprofloxacin and erythromycin. Furthermore, cyl, gelE and asa1 were not found in either clinical or STP isolates. Finally, we determined the distinct types of isolates using Pulse Field Gel Electrophoresis (PFGE), which confirmed that most of the isolates were clonally unrelated. CONCLUSION: Our results demonstrated that higher number of the clinical E. faecium isolates carried virulence genes than the isolates from STP. Finally, the lack of the genes in clinical and STP isolates confirmed that these genes do not transfer horizontally.

Clonal groups of high-level gentamicin-resistant Enterococcus faecium isolated from municipal wastewater and clinical samples in Tehran, Iran.

AIMS: Clonality among high-level gentamicin-resistant Enterococcus faecium (HLGR-EF) isolates obtained from clinical and sewage treatment plants (STP) were investigated using PhePlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE). METHODS AND RESULTS: During 1 year study (September 2005-2006), a total of 106 HLGR-EF isolates were collected from clinical (n = 48) and STP (n = 58) samples in Tehran, Iran. Biochemical fingerprinting of these isolates using the PhP showed the presence of 21 PhP types (diversity index, Di = 0.97) among the clinical and 21 PhP types (Di = 0.91) among the STP isolates. Representative isolates of each PhP type (n = 42) were further characterized by the ribotyping method. Sixteen ribotypes were identified among the isolates with five types shared between the clinical and STP isolates. PFGE recognized 24 clonal types among these isolates with three pulsotypes shared between the clinical and STP isolates. Combination of the two techniques (PFGE and ribotyping) resulted in 24 (Di = 0.96) and 16 (Di = 0.93) types among the strains isolated from clinical and STP samples, respectively. CONCLUSIONS: We concluded that the combination of PhP typing, ribotyping and PFGE could be extremely discriminatory when examining HLGR-EF isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The emergence of highly diverse HLGR-EF population in Iran is of serious concern especially because of their multi-resistances.

The homogeneity of vanB gene cluster among enterococcal isolates in Iran.

AIMS: This study aimed to analyse the diversity of the vanB gene cluster in enterococcal species isolated from sewage treatment plants (STP) in Tehran, Iran. METHODS AND RESULTS: The enterococcal isolates were collected from three sewage treatment plants in Tehran, Iran, during 2005. A total of 203 enterococcal isolates, collected over six rounds of sampling from three STPs were tested for the presence of vanB gene. Long-PCR showed that amongst the isolates, three Enterococcus faecium, one Enterococcus gallinarum and one Enterococcus casseliflavus harboured the complete vanB gene cluster. Restriction fragment length polymorphism (RFLP) of the vanB1 gene cluster (5900 bp) from the isolates showed an identical pattern to a standard strain of Enterococcus faecalis (V583). None of the isolates were able to transfer the vanB gene in conjugation experiments. Different pulsed-field gel electrophoresis patterns were obtained for the three E. faecium isolates with vanB gene clusters. CONCLUSIONS: Our results indicated that the dissemination of vanB is not widespread in Tehran. Although only a few vanB positive isolates were detected, vanB was found in several enterococcal species. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the lack of information on vanB resistance genes and their diversity in Iran, knowledge of the global dissemination of vanB genes in Enterococcus spp. is noteworthy.

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.

Molecular structure and transferability of Tn1546-like elements in Enterococcus faecium isolates from clinical, sewage, and surface water samples in Iran.

The molecular structure and transferability of Tn1546 in 143 vancomycin-resistant Enterococcus faecium (VREF) isolates obtained from patients (n = 49), surface water (n = 28), and urban and hospital sewage (n = 66) in Tehran, Iran, were investigated. Molecular characterization of Tn1546 elements in vanA VREF was performed using a combination of restriction fragment length polymorphism analysis and DNA sequencing of the internal PCR fragments of vanA transposons. Long-PCR amplification showed that the molecular size of Tn1546 elements varied from 10.8 to 12.8 kb. The molecular analysis of Tn1546 showed that 45 isolates (31.5%) harbored a deletion/mutation upstream from nucleotide 170. No horizontal transfer of Tn1546 was observed following filter-mating conjugation with these isolates. Nevertheless, the rates of transferability for other isolates were 10(-5) to 10(-6) per donor. Insertion sequences IS1216V and IS1542 were present in 103 (72%) and 138 (96.5%) of the isolates, respectively. The molecular analysis of Tn1546 elements resulted in three genomic organizations. The genomic organization lineage 1 was dominated by the isolates from clinical samples (3.4%), lineage 2 was dominated mostly by sewage isolates (24.5%), and lineage 3 contained isolates obtained from all sources (72.1%). The genetic diversity determined using pulsed-field gel electrophoresis (PFGE) revealed a single E. faecium clone, designated 44, which was common to the samples obtained from clinical specimens and hospital and municipal sewage. Furthermore, the results suggest that lineage 3 Tn1546 was highly disseminated among our enterococcal isolates in different PFGE patterns.

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.