Prevalence and molecular characterization of extendedspectrum -lactamase-producing Klebsiella pneumoniae Method

Specified number of K pneumoniae non-duplicate isolates will be collected from hospitalized patients. Susceptibility testing will be performed by the disc diffusion method according to Clinical and Laboratory Standards Institute recommendations. The minimum inhibitory concentration (MIC) will be determined by an E-strip test as described by the manufacturer. A laboratory control strain,Escherichia coli ATCC 25922, will be used in the sensitivity test and in the MIC determination. Phenotypic detection of ESBL will be carried out by two different methods: 1) the combined disc method using discs containing cefotaxime and ceftazidime with and without clavulanate with the ESBL phenotype defined as an increase of 5 mm in the zone around the disc containing clavulanate compared to the zone of corresponding discs without clavulanate; and 2) using the E-test ESBL-strip with a ceftazidime gradient at one end and a ceftazidime plus clavulanic acid gradient at the other end. ESBL was detected if the ratio of the MIC of ceftazidime to the MIC of ceftazidime plus clavulanic acid was 8.

PCR methods will be used to detect blaTEM , blaSHV , and blaCTX-M using the primers. A further group-specific CTX-M PCR will be performed to differentiate between the CTX-M-1, -2, -8 and -9 groups of enzymes using primers.

or Molecular characterization of CTX-M -lactamases among Klebsiella pneumoniae isolated from patients at tertiary care hospital.

Investigation of genetic heterogeneity in Mycobacterium tuberculosis isolates from tuberculosis patients using DNA fingerprinting
Materials and methods

A total of 180 isolates derived from all new-diagnosed TB patients referred to the center, were analyzed The patients were 133 men and 47 women and their age ranged from 20 to 60 years with a mean of 34.6. The isolates were identified as MTB by acid fast staining of direct smears prepared from sputum samples, culture in Lowenstein Jensen (LJ) medium and subsequently tested biochemically, using the niacin accumulation test, the nitrate reduction test, and heat-labile catalase test.[13] Chromosomal DNA was extracted from growth harvested from the surface of LJ medium by the simple boiling method. In short, few colonies were removed and suspended in 500 ml of sterile double distilled water in a microfuge tube and was boiled for 10 min. After centrifugation at 12 000 g for 3 min in a microcentrifuge, 10 ml of supernatant was used for the PCR.[12] An IS 6110 -based PCR approach was performed with a single primer, 5-GAGTCTCCGGACTCACCGG3, targeted at the inverted repeat sequence of the IS 6110 insertion.[14] Reaction volumes were 25 ml and contained 1 PCR buffer, 1.5 mmol MgCl2, 0.2 mmol of each deoxynucleotide triphosphate, 25 p mol of primer, 1 unit of Taq polymerase and 5 ml of DNA template primer. The reaction conditions were as follows. An initial denaturation at 95C for 120 s; 1 cycle of 95C for 20 s, 45C for 360 s and 72C for 120 s; 30 cycles of 95C for 20 s, 62C for 30 s and 72C for 180 s; and a final extension at 72C for 10 min.[14] The PCR products were loaded on an 1.5% (w/vol.) agarose gel with 0.5 mg/ml of ethidium bromide and were analyzed by gel electrophoresis. Hae III restriction enzyme was used in this study and the experiment was carried out as described by other investigators.[15] In brief, 10 ml of PCR product was added to 6 ml of sterile distilled water, 2 ml of restriction enzyme Hae III (Cinnagen Co., Tehran, Iran), and 2 ml of corresponding buffer. The mixture was incubated for 1-2 h at 37C in a water bath. The results were analyzed on a 2% (w/vol.) agarose gel with 0.5 mg/ml of ethidium bromide. Gels were photographed and the digestion bands were measured. The base pair size of each DNA fragment was determined by comparing the migration distance of the bands with the molecular markers visually.

The prevalence of virulence genes of E. coli strains isolated from patients of different age group with urinary tract infection
Introduction E. coli accounts for as much as 90% of urinary tract infections (UTI) and displays variable virulence properties. Several genes encode urovirulent factors such as hemolysin (hly gene), cytotoxic nercotizing factor type 1 (cnf-1-1 gene), pyelonephritis associated pili (pap genes) and S-family adhesions (sfa gene). Some genes, such as the hly pap or p fimbriae and cnf-1 1 play an important role in the pathogenesis of E. coli strains. In the present study, we evaluated the prevalence of the virulence genes, and their correlation with clinical data in uropathogenic E. coli strains isolated from patients with UTI. Meterial and methods Diagnosis of UTI was established by clinical symptoms and laboratory investigations (colony count of more than 10 5 colony forming unit/mL in meadstream sampling, 10 4 cfu/mL in urine sampling by catheterization and any count in suprapubic sampling). None of the patients was immune compromised. Acute pyelonephritis was clinically defined as fever (temperature > 38.5C), lumbar tenderness, and sepsis in neonate. Cystitis was considered when dysuria and pyuria were present without fever. Data including age, sex, previous history of infection, recent antibiotic usage and history of hospitalization during the previous 28 days were collected on all the patients with community acquired UTI in questionnaire forms. The exclusion criterion was nosocomial infection, which was defined as infection noted 48 h after admission or within four weeks after a previous discharge. Isolation of Bacteria E. coli strains were isolated from urine samples and identified using standard methods. Urine culture was considered positive if at least 10 5 colony forming unit of E. coli per mL of cleanvoided urine were grown in the culture media. The strains were then stored and sub cultured for further analysis as previously described in literature. DNA extraction E. coli isolates were grown in Luria Bertani broth at 37C overnight. Bacteria were then pelleted from broth, resuspended in sterile distilled water and boiled at 95C for ten min. After centrifugation, the supernatants were stored as DNA template at -20C until they were used in the PCR. PCR assay Detection of pap, sfa, cnf-1, and hly genes was performed by amplifying the genes by PCR. The

primers sequences were previously reported in the literature, and obtained from TIB MOLBIOL Syntheselabor GmbH (Berlin, Germany). Descriptions and sequences of the PCR primers used in this study are shown in. Other enzymes and chemicals were provided by Cinnagen Chemical Company (Tehran, Iran). Amplification was performed in a thermal cycler (Eppendorf, Germany) according to the methods described by Yamamoto et al.Expected sizes of the amplicons were ascertained by electrophoresis in 1.5 % agarose gel with an appropriate molecular size marker (100-bp DNA ladder, MBI, Fermentas, Lithuania). Statistical Analysis

Statistical analysis was performed using SPSS software for Windows, version 11.5 (SPSS). Descriptive analyses were performed for parametric and non parametric variables. Student 't' test, Chi square, and logistic regression were done for evaluation of variables correlation. P value less than 0.05 was considered as significant. ( study was done by Shohreh Farshad1, Fatemeh Emamghorashi2 ,Iran )

Epidemiology and molecular typing of Candida isolates

his study, spread over a span of 2years describes Candida infections in burn patients of an Indian hospital. A total of 220 burn patients were monitored and Candida could be isolated from 138 patients. A total of 228 different Candida species were obtained from various body locations of these patients. Species identification revealed that Candida albicans was the most predominant (45) followed by Candida tropicalis(33), Candida glabrata (13.5), C. parapsilosis (4), C. krusei (2.75) and C. kefyr (1.75). DNA fingerprinting of all C. albicans isolates was done by using CARE-2 probe