Iranian Journal of Microbiology
Volume:4 Issue: 2, Jun 2012

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5’-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was < 1000 copies/ml for HIV-1 and < 500copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Urinary tract infection (UTI) is one of the most common infections in the world. The majority of UTI caused by Uropathogenic Escherichia coli (UPEC) strains. FimH and FliC are the most important virulence factors of UPEC. To date, any ideal vaccine against UTI has not been approved for human use and we need to test new target to develop an ideal vaccine against UTI. In this study, we constructed fusion FimH/FliC of UPEC as a novel vaccine candidate against UTI. PCR amplification of fimH and fliC genes of the UPEC isolates was performed by specific primers designed for this purpose. Construction of fimH/fliC hybrid gene was performed by overlap PCR. The fimH, fliC and fimH/fliC were cloned in pET28a vector. The confirmation of expression of the proteins was done by SDS-PAGE and Western blot. The fliC and fimH genes were amplified in all of the UPEC isolates tested. The fimH showed significant homology with the sequences in GenBank. We generated a fusion consisting of the fimH gene linked to the N-terminal end of fliC. Sequencing of the fusion fimH/fliC showed that fusion was constructed precisely. SDS-PAGE and western blot confirmed the expression of the proteins in optimized condition. Urinary tract infection is a huge burden on healthcare system in many of the countries. UPEC isolated in around 80% of UTI cases. Antibiotic therapy resulted in the emergence of antibiotic resistance in UPEC strains. This is the major cause for an increasing requirement to vaccine to prevent UTI. This work describes for the first time the construction of a novel fusion protein from Iranian UPEC isolate. Further immunological studies are required for evaluation of this protein as a novel and safe vaccine candidate against UTI caused by UPEC.

Shiga toxin-producing Escherichia coli (STEC) have emerged as human pathogens and contamination of foods of animal origin has been a major public health concern. The aim of the present study was to determine the dissemination of STEC in healthy and diarrheic calves in Urmia region which is located in West Azerbaijan province, Iran. In the current study, a total of 124 Escherichia coli isolates from clinically healthy (n=73) and diarrheic calves (51) belonging to 6 different farms located in West Azerbaijan province, Iran, were screened by the polymerase chain reaction (PCR) assay for the presence of virulence genes characteristic for STEC, that is, Shiga-toxin producing gene(s) (stx1, stx2), intimin (eaeA) and enterohaemolysin (hlyA). STEC strains were recovered from 21.92% (16/73) in healthy calves, and 19.6% (10/51) in diarrheic calves. Overall, PCR results showed that 6 (23.1%) isolates carried stx1 gene, 7 (26.92%) possessed stx2 gene while 13 isolates (50%) gave positive amplicon both for stx1 and stx2 genes. All stx positive strains were assayed further to detect eaeA and hlyA sequences. 7 out of the 26 (26.92%) Shiga toxin gene positive isolates were positive for the eaeA gene, and 15 (57.69%) were positive for the hlyA gene. Both virulence genes (eaeA and hlyA) in the same isolate were observed in 5 (19.23%) of the stx+ isolates. In total, diverse virulence gene profiles were detected, from which strains with the genetic profile stx1 stx2 hlyA was the most prevalent. In addition, eaeA gene was more evident in isolates from diarrheic calves than in healthy calves. As a result, calves seem to be the group with higher rate of STEC excretion within the herds and aspects of calf management may represent specific control points for reducing STEC spread within dairy units. The results also appear to indicate that STEC E. coli strains are a normal part of intestinal bacterial populations in calves.

Streptococcosis/lactococcosis is the cause of high morbidity and mortality in aquaculture sector and to date a number of species of Streptococcus and Lactococcus genera including S. iniae, S. agalactiae, S. dysagalactiae, S. parauberis, S. feacalis, L. garvieae and L. lactis have been discriminated as the cause of disease in aquatic animals. Despite the use of diagnostic molecular methods for each of these bacterial species, no data is available on a suitable, rapid and simple simultaneous detection tool for these pathogens. This paper describes a simultaneous detection method which is PCR based on a reverse line blot (RLB) for rapid detection and differentiation of four species of genera of Streptococcus and Lactococcus genera consisting of S. iniae, S. agalactiae, S. parauberis and L. garvieae the most important agents of the disease in fish. A reverse line blot (RLB) assay was developed for the simultaneously identification of four species of Streptococcus/lactococcusconsisting of S. iniae, S. parauberis, S. agalactiaeand Lactococcusgarvieae. The assay employs one set of primer pair for specific amplification of the 16S rRNA gene. These were designed based on the nucleotide sequences of 16S rRNA gene sharing a homology region with Streptococcus spp. and Lactococcus spp. DNA was extracted from the pure bacterial colonies and amplified. A membrane was prepared with specific oligonucleotide for each bacterial species. PCR products were then hybridized to a membrane.Results and The amplification resulted in PCR product of 241 bp in length. No cross-reactions were observed between any of the tested bacterial species, and mixed DNAs from these four bacterial species were correctly identified. This RLB method is a suitable technique for a simultaneous detection of these species of bacterial fish pathogens that are some of the main causes of streptococcal/lactococcal infections in both freshwater and marine aquatic animals, and so we recommend its use for integrated epidemiological monitoring of streptococcosis/lactococcis in aquaculture industry.

Background/ Although habitual consumption of xylitol reduces cariogenic streptococci levels, its effect on beneficial oral streptococci is less clear. The main aim of the study is to investigate the effect of short-term xylitol consumption on the oral beneficial streptococci level of saliva, Streptococcus sanguinis and S. mitis. Twenty four volunteers with a median age of 23.7 years (range: 20-28) harboring Streptococcus mutans, S. sobrinus, S. sanguinis and S. mitis participated in the randomized, double-blind, cross-over study. The experimental chewing gum (1.5 g/pellet) contained 70% xylitol w/w while the control gum contained 63% sorbitol w/w. Saliva samples were collected before and after two three-week test periods with a four-week washout interval. Colony-forming units (CFU)/ml were enumerated for the estimation of S. mutans levels on Mitis Salivarius-Mutans valinomycin (MS-MUTV), S. sobrinus on Mitis Salivarius- Sobrinus (MS-SOB), S. sanguinis on Modified Medium 10 -Sucrose (MM10-S) and S. mitis on Mitis Salivarius Agar with Tellurite (MSAT) media. The S. mutans and S. sobrinus counts of the saliva samples decreased significantly (p=0.01 and p=0.011, respectively) in the xylitol gum group but not in the sorbitol gum group. The salivary S. sanguinis and S. mitis counts did not decrease in both xylitol and sorbitol gum groups. Based on the findings of this study, xylitol consumption reduced S. mutans and S. sobrinus counts in saliva but appeared not to effect numbers of S. sanguinis and S. mitis in saliva. So, habitual consumption of xylitol reduces cariogenic streptococci levels without any effect on beneficial sterptococci for the oral cavity.

Staphylococcus aureus is an important agent in hospital and community-associated infections, causing high morbidity and mortality. Introduction of the new antimicrobial classes for this pathogen is usually followed by the emergence of resistant strains through multiple mechanisms. For instance, resistance to clindamycin (CLI), can be constitutive or inducible. Inducible clindamycin resistance which may lead to treatment failure can simply be identified by performing D-test. The aim of this study was to determine the prevalence of inducible clindamycin resistance among Staphylococcus aureus isolates by D-test method. This was a cross-sectional study conducted on 254 non-duplicated S. aureus isolates in Imam Reza hospital of Mashhad during 2010. Susceptibility to oxacillin, cefoxitin, erythromycin and clindamycin was performed by disk agar diffusion method according to CLSI guidelines and D-shaped clindamycin susceptibility patterns where considered as D-test positive (D+). Of 211 S. aureus isolates 88 (37.82%) were methicillin resistant. It was found that of 88 MRSA isolates, 78 (88.63%) were erythromycin (ERY) resistant and 46 (52.27%) were CLI resistant. ERY and CLI resistance in MSSA strains was 21.95% and 11.96% respectively. Inducible clindamycin resistance was detected in 18 (20.45%) MRSA isolates. 47(53.40%) of MRSA isolates and 9 (7.32%) of MSSA showed constitutive MLSB phenotype. In conclusion, we found a high prevalence of inducible clindamycin resistance phenotype in our region. We recommend that whenever clindamycin is intended for S. aureus infections, D-test should be performed to facilitate the optimal treatment of patients.

Butyric acid has many applications in chemical, food and pharmaceutical industries. Applications of butyric acid are as an additive to food, flavorings, varnishes, perfumes, pharmaceuticals and disinfectants. Butyric acid concentrations have positive impact on the quality control of milk, yogurt and other probiotic dairy products. The present investigation was undertaken to determine and compare the concentrations of butyric acid (C4) in the ordinary and probiotic yogurt samples by GC method. Probiotic yogurt samples were prepared under laboratory scale conditions using two different commercial starters ABY1 and 211, while ordinary yogurt samples lacked the probiotic starter cultures. All samples were analyzed in duplicate, for C4 concentrations by gas chromatography after day 1, 2, 10 and 20 of production, during storage at 4ºC. The results were analyzed using ANOVA and Duncan test. The level of the mentioned fatty acid in ABY1 yogurt sample was significantly higher (0.2%) than in 211 samples (0.17%). These values were significantly lower in ordinary yogurt samples and only 0.07% was recorded in these samples on first day of storage which decreased gradually during storage. The level of reduction in the yogurt samples tested during different time intervals was not similar in all the examined samples, and some showed enhanced reduction than other samples. Compared to ordinary yogurt samples, probiotic yogurt samples used in study showed higher levels of butyric acid with increased shelf life.

Cystic fibrosis (CF) is an autosomal recessive genetic disease. Infections in these patients are mostly caused by three bacteria: Staphylococcus aureus, Haemophilus influenza and particularly Pseudomonas aeruginosa. Carbapenems including antibiotics are used to combat infections with Pseudomonas aeruginosa. In recent years, carbapenems resistant strains of P. aeruginosa isolated from clinical specimens are being reported. Decrease in drug penetration and production of metalobeta lactamase (MBLS) have been proposed as mechanisms of resistance. In this descriptive study, the population under investigation was 27 patients suffering from CF in Alzahra hospital of Isfahan. Clinical specimens were taken by deep swabbing from throat and data from every patient was recorded in a questionnaire. The specimens were cultured and isolated organisms were identified as P. aeruginosa using standard tests. Kirby-Bauer disk diffusion method was used to determine the bacterial drug resistance pattern. Strains of P. aeruginosa were checked for production of MBLS using disk impregnated with IPM-EDTA and PCR targeting of bla VIM. Among the 27 patients, 7 (26%) had P. aeruginosa infection. In total, 11 P. aeruginosa isolates were taken. All isolates were susceptible to imipenem, ticarcillin, ciprofloxacin and piperacillin. The lowest scale of susceptibility belonged to ceftazidime (72.2%) followed by tobramycin (45.4%). None of the strains were positive for the blaVIM gene. Isolates of P. aeruginosa from CF patients in Isfahan were susceptible to antibiotics during the study period.

Staphylococcus aureus produces a particular morphological variant called small colony variant(SCV) which is responsible for persistent subclinical infections in predisposed individuals and is usually resistant to aminoglycosides and cell wall active antibiotics. Infections by SCV of S. aureus is an upcoming problem due to difficulty in laboratory diagnosis and resistance to antimicrobial chemotherapy. We here report a case of infective endocarditis caused by SCV of Staphylococcus aureus in a pediatric patient.

1. Iranian Journal of Microbiology (2012) Mar; 4(1): 44-66.In the article titled: Development of a new DNA extraction protocol for PFGE typing of Mycobacterium tuberculosis complex.The correct names of authors are as follows:Ghodousi A, Vatani S, Darban-Sarokhalil D, Omrani M, Fooladi A, Khosaravi A, Feizabadi MM2. Iranian Journal of Microbiology (2012) Mar; 4(1): 40-43.In the article titled: Oral chromoblastomycosis: a case report.The correct names of authors are: Fatemi MJ, Bateni H3. Iranian Journal of Microbiology (2011) Sep; 3(3): 112-7.In the article titled: Antimicrobial resistance profile and presence of class I integrongs among Salmonella enterica serovars isolated from human clinical specimens in Tehran, Iran.The correct names of authors are: Firoozeh F, Shahcheraghi F, Zahraei Salehi T, Karimi V, Aslani MM.4. Iranian Journal of Microbiology (2010) Sep; 2(3): 165-7.In the article titled: Catalase-negative Staphylococcus aureus isolated from a diabetic foot ulcer.The correct names of authors are: Dezfulian A, Salehian M, Amini V, Dabiri H, Azimirad M, Aslani MM, Zali M, Fazel I.