Differentiation of Brucella abortus and B. melitensis biovars using PCR-RFLP and REP-PCR.

Brucellosis is one of the most common zoonotic diseases of animal and human beings. This study aimed to differentiate the Brucella spp. and determines the patterns of biovars by using repetitive element palindromic (REP)-PCR and PCR restriction fragment length polymorphism (RFLP) methods. A total of 100 blood specimens suspected of harbouring brucellosis were collected. Conventional culture methods and multiplex PCR were used for the detection of Brucella genus and species; and REP-PCR was used for Brucella spp. differentiation and polymorphisms sequence analysis. In addition, to identify the biovar patterns of REP-PCR, PCR-RFLP was used. Eighty-three samples were identified as harbouring Brucella spp. by the implementation of multiplex PCR, 72 of which were detected as Brucella melitensis and 11 as B. abortus. Also, through analysing the results of PCR-RFLP, it was found that of 72 B. melitensis samples, 69 were B. melitensis biovar 1 and three species were from other biovars. In addition, the obtained patterns for all of the B. abortus samples were from biovars 3, 5, 6 and 9. This study also optimized a test for the detection of Brucella biovar with the REP-PCR method such that Brucella spp. and biovars could be separated in the shortest possible time.

Frequency of bacteria causing urinary tract infections and their antimicrobial resistance patterns among pediatric patients in Western Iran from 2007-2009.

Urinary Tract infections ( UTIs) are among the most common infections in infants and neonates. The aim of the current study was to evaluate the frequency of bacteria causing UTI and their relevant drug resistance patterns among infants and neonates hospitalized in Ilam province, Western Iran during 2007-2009. A total of 220 cases of UTI were enrolled in this cross-sectional retrospective study. A standard checklist was used for demographic and clinical data to be collected from their health records. Data was then analyzed using SPSS version 16.0. More than two-thirds (64.8%) of the cases were female. E. coli (44.5%), Klebsiella spp., (18.6%), Enterobacter spp., (15%) and Staphylococcus spp. (12.7%) were the most common microorganisms isolated from UTIs, respectively. High rates of resistance to tetracycline, ampicillin, and nalidixic acid were observed among these isolates. Similar to other studies, E. coli was the most common bacteria causing UTI and showed a high rate of resistance against most of the antimicrobial agents. Determining the antimicrobial sensitivity can be helpful for physicians in choosing an appropriate treatment for patients suffering from UTI, and also to reduce the complications related to serious UTI.

Development of a multilocus sequence typing scheme for intestinal spirochaetes within the genus Brachyspira.

The purpose of this study was to evaluate a multilocus sequence typing (MLST) scheme for intestinal spirochaetes of the genus Brachyspira. Eight loci mainly coding for enzymes previously used in multilocus enzyme electrophoresis analysis of Brachyspira species were examined in 66 Brachyspira field isolates and type/reference strains. The isolates and strains were recovered from pigs, birds, dogs and a mouse and originated from seven European countries, the USA and Canada. Forty-six isolates represented recognized Brachyspira species and 20 represented provisionally designated species or isolates that have not been classified. Only two loci gave PCR products for all 66 strains and isolates, but amplicons for seven loci were obtained for 44 of the isolates. Sequences for each locus had a DNA allelic variation of 30-47 and an amino acid allelic variation of 14-47 that gave rise to the same number of sequence and amino acid types (58) for the strains and isolates studied. A population snapshot based on sequence and amino acid types showed a close phylogenetic relationship amongst the porcine isolates from the same geographical regions, and indicated a close evolutionary relationship between isolates recovered from pigs and mallards. A general concordance was obtained between the MLST groupings and classifications based on culture and biochemical tests, 16S rDNA sequence analysis and random amplified polymorphic DNA analysis. This is a first step towards establishing an MLST system for use in identifying Brachyspira species and determining relationships between individual strains and species in the genus.

Evaluation of selective and nonselective media for isolation of Helicobacter pylori from gastric biopsy specimens.

The aim of the present study was to compare six media, three selective and three nonselective media, to determine the best combination of media for the primary isolation of Helicobacter pylori. Over a period of 8 months, mucosal antral biopsy specimens were obtained from 97 dyspeptic patients undergoing endoscopy. Biopsy samples were plated in parallel on all six media. Egg yolk emulsion agar (EYE), Skirrow's medium and modified Thayer-Martin medium were used as selective media; modified chocolate agar (MCHOC), Triptycase Soy Agar (TSA) and brain heart infusion agar were used as nonselective media. Overall, by using these six media, H. pylori were recovered from biopsy specimens from 48 of 97 patients, yielding an isolation rate of 49%. Comparison of all possible combinations of the six media showed that the highest rate of isolation of H. pylori was 100% (48 of 48) with EYE-MCHOC, followed by 97% (47 of 48) when EYE-SK was used. Conversely, it was found that none of the media used alone yielded a 100% rate of recovery (the maximum recovery rate was 92%, which was achieved with EYE). These results indicate that the association of EYE and MCHOC yielded the maximum recovery of H. pylori from gastric biopsy specimens. Therefore, the use of selective and nonselective media in parallel offers optimal recovery rates with only a slight increase in costs.

Evaluation of different primer sets for the rapid diagnosis of tuberculosis.

Pleural fluid samples from patients with exudative effusion who were diagnosed with tuberculous pleuritis are examined using a new designed primer set based on IS1081 gene (IS1081-PCR) and rpoB-PCR. The PCR results are compared with the results of the sample cultures, using Loewenstein-Jensen (LJ) medium and Ziehl-Neelsen (ZN) staining. Of 78 cases that were confirmed as tuberculous pleuritis by histopathology, supported by sputum culture, biochemical markers (adenosine deaminase, gamma interferon and tumor necrosis factor), radiographic and clinical data, 61 (78.2%) were positive by IS1081-PCR, 43 (55.1%) by rpoB-PCR, 17 (21.7%) by culture and 3 (3.8%) by ZN stain. When IS1081-PCR test results were compared with the confirmed culture, the sensitivity, specificity, positive predictive value and negative predictive value for the IS1081-PCR were 94.1, 55.7, 37.2 and 97.1%, respectively. The corresponding values for the rpoB-PCR were 94.1, 26.2, 26.2 and 94.1%, respectively. When tests results were compare with the confirmed radiographic, histopathology, biochemical markers and clinical diagnosis of tuberculous pleuritis, the IS1081-PCR assay is more sensitive, specific and reliable than both rpoB-PCR assay and culture.