Genetic relatedness of the Escherichia coli fecal population and strains causing urinary tract infection in the same host.

It is common knowledge that fecal microbiota is a primary source of Escherichia coli causing urinary tract infections (UTIs) via the fecal-perineal-urethral route. But, it is still unknown whether E. coli UTI is mainly caused by dominant fecal E. coli isolates (prevalence hypothesis) or the isolates that possess more virulence factors (special pathogenicity hypothesis). In the present study, the urine E. coli isolates of 30 women with UTI were compared with the fecal E. coli isolates of the same patients and healthy control individuals according to the phylogenetic group, virulence genotype, and antibiotic susceptibility pattern. The genetic relatedness of the isolates was specified and compared by pulsed-field gel electrophoresis (PFGE). PFGE analysis showed that most patients (73.3%) had distinct urine isolates which were not similar to any of their fecal isolates. Based on the phylogenetic analysis, most of the urine and fecal isolates of healthy women were assigned to phylogenetic group B2, followed by D. The distribution of phylogenetic groups was significantly different between the urine and the fecal isolates of patients (p < 0.05). The prevalence of fimH and ompT among urine isolates was significantly more than that among fecal isolates. The level of multidrug resistance was higher among urine isolates. Although more in-depth researches are required, the present study could be supported by pathogenicity hypothesis. Furthermore, concerning the antibiotic resistance pattern among uropathogenic E. coli should be highly considered.

In-silico design, expression, and purification of novel chimeric Escherichia coli O157:H7 OmpA fused to LTB protein in Escherichia coli.

E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-ß-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression and purification of OmpA-LTB divalent under the above-mentioned conditions.

Genotyping of the Helicobacter pylori cagA Gene Isolated From Gastric Biopsies in Shiraz, Southern Iran: A PCR-RFLP and Sequence Analysis Approach.

BACKGROUND: Cytotoxin-associated gene A (cagA) is an important virulence factor in the pathogenesis of Helicobacter pylori. OBJECTIVES: The aim of this study was to genotype the H. pylori cagA gene isolated from antral biopsies of patients with stomach symptoms, using a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. PATIENTS AND METHODS: A total of 161 gastric biopsies were collected from patients with stomach symptoms. After isolation of H. pylori from the biopsy culture, the cagA gene was assessed using PCR. The PCR products were then digested by the HinfI restriction endonuclease enzyme. A sample of each genotype was also subjected to direct sequencing for further analysis. RESULTS: From 161 antral biopsies, 61 (37.9%) were positive for H. pylori in culture. Overall, 24 cagA-positives were detected in the isolates. RFLP indicated three different genotypes (I, II, and III) of cagA with a frequency of 62.5%, 25%, and 12.5% among the isolates, respectively. Genotypes I and II of cagA were predominant in patients who had gastritis. However, genotype III was found in three patients with duodenitis and duodenal ulcers. Alignment of the nucleotide sequences of the three isolated genotypes, with H. pylori 26695 as a reference strain, revealed 12 inserted nucleotides in genotype III. When the sequence of genotype III was aligned with 15 additional H. pylori strains available in GenBank, the same inserted nucleotides were detected in six of them. CONCLUSIONS: Using the PCR-RFLP method, three distinctive H. pylori cagA genotypes were detected in antral biopsies. Genotype I, which was predominant among the isolates, was significantly associated with gastritis. However, the data showed that cagA genotype III may play a role in duodenitis and duodenal ulcers in patients infected with H. pylori.

Molecular Characterization of Streptococcus agalactiae Isolates From Pregnant and Non-Pregnant Women at Yazd University Hospital, Iran.

BACKGROUND: Streptococcus agalactiae (Group B streptococcus, GBS) that colonize the vaginas of pregnant women may occasionally cause neonatal infections. It is one of the most common causes of sepsis and meningitis in neonates and of invasive diseases in pregnant women. It can also cause infectious disease among immunocompromised individuals. The distribution of capsular serotypes and genotypes varies over time and by geographic era. The serotyping and genotyping data of GBS in Iranian pregnant and non-pregnant women seems very limited. OBJECTIVES: The aim of this study was to investigate the GBS molecular capsular serotype and genotype distribution of pregnant and non-pregnant carrier women at Yazd university hospital, in Iran. . PATIENTS AND METHODS: In this cross-sectional study, a total of 100 GBS strains isolated from 237 pregnant and 413 non-pregnant women were investigated for molecular capsular serotypes and surface protein genes using the multiplex PCR assay. The Chi-square method was used for statistical analysis. RESULTS: Out of 650 samples, 100 (15.4%) were identified as GBS, with a predominance of capsular serotypes III (50%) [III-1 (49), III-3 (1)], followed by II (25%), Ia (12%), V (11%), and Ib (2%), which was similar with another study conducted in Tehran, Iran, but they had no serotype Ia in their report. The surface protein antigen genes distribution was rib (53%), epsilon (38%), alp2/3 (6%), and alpha-c (3%). CONCLUSIONS: The determination of serotype and surface proteins of GBS strains distribution would be relevant for the future possible formulation of a GBS vaccine.

Characterization of carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex isolates from nosocomial bloodstream infections in southern Iran.

Acinetobacter baumannii is an important opportunistic bacterial pathogen responsible for serious infections in hospitalized patients. From a total of 78 consecutive non-repetitive Acinetobacter spp. isolates from patients with blood infections, 61 were carbapenem resistant, which were positive for blaOXA-51-like (96.7%), blaOXA-23-like (77 %), blaOXA-58-like (8.1%) and blaOXA-40-like genes (32.8%) by multiplex PCR. The isolates were identified as A. baumannii (n = 59) and Acinetobacter nosocomialis (n = 2). Also, we found a case of Acinetobacter junii, causing bacteraemia, that possessed the IMP gene. High levels of resistance were observed to fluoroquinolones, aminoglycosides, tigecycline and to the beta-lactam antibiotics, including piperacillin/tazobactam and ampicillin/sulbactam. ISAba1 was present in 96.7% of all Acinetobacter calcoaceticus-baumannii complex (Acb) isolates. Also, 33 (54.1%) and 23 (37.7%) isolates harboured ISAba1 upstream of blaOXA-23-like and blaOXA-51-like genes, respectively, though this was not observed in A. nosocomialis isolates. No relationship was observed between the presence of ISAba1 upstream of oxacillinase genes and the level of carbapenem resistance in all Acb isolates. Only two genes encoding metallo-beta-lactamase (VIM, SPM) were detected in all Acb isolates. This suggests that carbapenem resistance in blood-isolate Acb is mostly due to the presence of acquired carbapenemases. This is the first report from Iran on the identification of A. nosocomialis isolates that possess multiple oxacillinase genes and lack upstream ISAba1.

Virulence Characteristics and Antibiotic Resistance Patterns among Various Phylogenetic Groups of Uropathogenic Escherichia coli Isolates.

The aim of this study was to determine the resistance patterns of uropathogenic Escherichia coli (UPEC) isolates and to investigate the frequency of several virulence genes, including fimH, papA, hlyD, cnf-1, sitA, and tsh, among various phylogenetic groups of UPEC isolates. A total of 85 E. coli isolates were recovered from urine samples from outpatients with a clinical diagnosis of uncomplicated urinary tract infections. A molecular approach to examine the antimicrobial resistance patterns was employed using PCR and the disc diffusion method. The detected frequencies of the virulence factor genes determined using PCR were: fimH (34.1%), papA (9.4%), hlyD (21.2%), cnf-1 (3.5%), sitA (15.3%), and tsh (27.1%). These results revealed that the isolates were resistant to trimethoprim-sulfamethoxazole (SXT) (74.1%), cefotaxime (CTX) (68.2%), and amoxicillin-clavulanic acid (AMC) (94.1%), and they were relatively less resistant to N (56.5%). According to these results, further investigation is needed to determine exactly whether or not SXT, CTX, and AMC are appropriate antibiotics for the treatment of UPEC infections in southern Iran. Although these results demonstrate that fimH is the most frequent virulence gene among UPEC isolates, the high prevalence of isolates that do not encode fimH (75.9%) and the relatively low frequency of isolates that carry other virulence genes require further investigation to clarify the role of the other potential virulence factors in the pathogenesis of these isolates.

Association of Three Plasmid-Encoded spv Genes Among Different Salmonella Serotypes Isolated from Different Origins.

The virulence plasmid associated Salmonella plasmid virulence (spv) locus is strongly concomitant with strains that cause non typhoid bacteremia. The spv region contains three genes required for the virulence, the positive transcriptional regulator spvR and two structural genes spvB and spvC. The purpose of this study was to investigate the presence of these three genes among salmonella serotypes isolated from different sources. A collection of 60 salmonella serotypes from different sources were used. Polymerase chain reaction was carried out for the presence of these genes using specific primers. The prevalence of spvB, spvC, and spvR genes were 26 (43.3 %), 44 (73.3 %), and 28 (46.6 %), respectively. The findings revealed that the distribution of these genes was dissimilar among these serotypes. Many of the human pathogenic salmonella strains which can be transmitted by animals may have these genes and can be very injurious for public health.

Detection of virulence genes (bvfA, virB and ure) in Brucella melitensis isolated from aborted fetuses of sheep and goats.

BACKGROUND AND OBJECTIVES: Brucella, causative of brucellosis, has some potential virulence factors involved in Brucella replication and its strategies to circumvent the immune response. One of them is the virB gene that encodes the type IV secretion system proteins (T4SS) involved in intracellular replication of organism. Brucella virulence factor A (bvfA), and urease (ure) has also been described as being implicated in survival, and virulence in the hosts. The aim of this study was to investigate the B. melitensis virulence factor genes among Brucella isolated from aborted fetuses of sheep and goats in Fars province, southern Iran. MATERIALS AND METHODS: A total of 42 isolates of B. melitensis isolated from aborted fetuses between 2005-2011 in Fars province of Iran was used in this study. PCR assay was performed in order to detect the virB, bvfA, and ure genes using specific primers. RESULTS AND CONCLUSIONS: The frequency of bvfA, virB, and ure genes was 78.50%, 73.80%, and 88.09% among all isolates respectively. The results of the present study showed that most Brucella isolates from this region have virulence factors genes (virB, bvfA, ure) in their genome, and most B. melitensis had ure genes that has been hypothesized to play a role in the pathogenesis of disease.