Iranian Journal of Microbiology
Volume:7 Issue: 4, Aug 2015

Loop-mediated isothermal amplification is a novel nucleic acid amplification assay providing as a simple diagnostic tool for rapid identification of microbial diseases in developing countries. In this study, a LAMP assay was established for Yersinia enterocolitica, a leading cause of acute enterocolitis in young children. LAMP assay was established with four primers targeting a specific locus for the detection of Y. enterocolitica. The assay was conducted at 65°C in thermo block for 90min. The sensitivity of LAMP was evaluated in com- parison to conventional PCR using pTZ57R containing the target locus. Finally, specificity was assessed using DNA from common enteropathogenic bacteria. Results showed that the sensitivity of LAMP assay was 44-copy number, which was 10-fold higher than that of PCR. No cross-reactivity was observed when testing against other enteropathogenic pathogens. This study showed that LAMP assay is an alternative molecular diagnostic tool for infections with Y. enteroco- litica. In addition, this method may be useful in diagnosis at field or in laboratories without PCR machine.

Shiga toxin-producing Escherichia coli (STEC) have emerged as human pathogens and con- tamination via animal origin has been a major public health concern. We compared the distribution of phylogenetic groups and prevalence of stx gene variants among the pathogenic strains of Escherichia coli isolated from feces of diarrheatic calves in Tehran suburb farms. In this study we screened 140 diarrheatic calves (1-15 days old) for E. coli strains during a 3 months period of time. The isolated strains were grouped into different phylotypes according to the presence of chuA, yjaA and TSPE4.C2 genes. Then, the prevalence of stx gene subtypes was evaluated in the B phylotypes. From diarrheatic calves, 51 bacterial isolates were biochemically identified as E. coli and 31 isolates out of 51 were considered B phylotype using DNA Microarray technology. Of these isolates, 20 contained stx a and stx b and one harbored all mentioned variants of stx genes except stx b. This study showed that in Tehran suburb, the B phylotype of E. coli is prevalent as a causative agent of diarrhea in calves and the prevalence of stx gene subtypes is dominant in comparison with other subtypes. Considering the possibility that these stx genes can be spread to other strains, bovine E. coli strains are an important source of stx genes for other strains and further study and surveillance seems to be required for the exact identification of virulence profile of E. coli phylotypes in different hosts.

Enteroinvasive Escherichia coli (EIEC) is one the cause of acute diarrhea and bacillary dysentery in developing countries. Routine diagnostic microbiology tests are not capable to distinguish EIEC from other pathogenic or non-pathogenic E. coli. PCR, targeting ipaH, virF, virB and other virulence genes, is a diagnostic method for detecting E. coli pathotypes. Using PCR, we identified EIEC by PCR targeting ipaH and virF genes among E.coli isolates from patients with diarrhea at the selected hospitals in Tehran. Isolates of E. coli were cultured from 140 specimens of patients with diarrhea using culture and IMViC test. DNA was extracted using commercial kits and and tested for uidA, ipaH and virF genes by PCR. Totally, 140 E. coli isolates were confirmed by IMViC tests and PCR targeting uidA gene. Of 140 E. coli isolates, 5 (3.6%) were positive for the ipaH gene, 4 (2.9%) contained virF gene and 4 (2.9%) were positive for both ipaH and virF genes. These results indicated that EIEC is a considerable acute diarrheagenic pathogen in adults and infants. More- over, virF gene is suggested for evaluation of invasiveness of EIEC.

Salmonella is an important food-borne pathogen in humans. Strains of Salmonella spp. That producing extended-spectrum β-Lactamases have become a concern in medicine regarding both antimicrobial treatment and infection control program. The objective of this study was to describe the antibiotic susceptibility, ESBL production and determining the prevalence of the blaCTX-M-1 group among clinical isolates of Salmonella spp. A total of 110 Salmonella isolates collected from four Tehran hospitals during May 2012 and April 2013. The specific monovalan Salmonella antisera were used for serogrouping of Salmonella isolates. Antibacterial susceptibility was determined by disk diffusion and ESBL phenotype was confirmed by combination disk method. The blaCTX-M-1 group was identified by PCR with specific primers. The transferability of the blaCTX-1 group was tested by conjugation with broth matting method. The prevalence of Salmonella serogroups consist of 56.4% serogroup D, 13.6 % serogroup C, 10 % serogroup B, and 1.8 % serogroup A and 18.2% other serogroups. Maximal resistance in Salmonella isolates was noticed against trimethoprim-sulfamethoxazole (63.6%) and nalidixic-acid (47/3%). All isolates were susceptible to imipenem and ciprofloxacin. Four isolates (3.6%) showed ESBLs phenotype. All Salmonella spp. that produce ESBls have blaCTX-1 genes group. A conjugative plasmid containing blaCTX-1 group was found in one Salmonella isolate. This study demonstrates the predominant presence of the gene encoding CTX-M-1 group among ESBLs producing of Salmonella spp. They can transmit to bacteria of this genus or even other genera of enteric bacteria.

Methicillin resistance Staphylococcus aureus (MRSA) and coagulase negative staphylococci (MRCoNS) have recognized as the major cause of nosocomial infections that threat the burn patient’s life. The aims of this study were to determine the frequency of MRSA and MRCoNS and their antibiotic resistance patterns among burn patients in a burn center in Ahvaz, Iran. A total of 340 clinical specimens: (80%) wound and (20%) blood were obtained from patients in Taleghani burn hospital during February 2013-2014. Staphylococci species identification and antibiogram were performed by standard procedures using disk diffusion method. The Methicillin resistance strains were detected by Etest and PCR using mecA specific primers. Out of 30.2% (103) isolates that were recognized as staphylococci, 82 % (84) and 18% (19) were identified as S. aureus and coagulase negative staphylococci (CoNS) respectively. Resistance to methicillin was detected in 60% and 63% of the S. aureus and CoNS isolates respectively. Seven different antimicrobial resistance patterns observed among methicillin resistant staphylococci. The MRSA and MRCoNS strains showed closed resistance phenotypes. All the methicillin resistant isolates showed a high rate resistance to the other studied antibiotics in comparison to methicilin sensitive isolates. Vancomy- cin and imipenem showed the greatest effect against methicillin resistant isolates. During 8 years in the studied burn hospital, no significant changes in the methicillin resistance staphylococci frequency were detected. The presence of multi resistant MRSA and MRCoNS strains is cause of concern in burn hospitals. Vancomycin remains as a drug of choice for methicillin resistance staphylococci infections.

Iranian chronic HBV carrier’s population has shown a unique pattern of genotype D distri- bution all around the country. The aim of this study was to explore more details of evolutionary history of carriers based on structural surface proteins from different provinces. Sera obtained from 360 isolates from 12 Different regions of country were used for amplificationand sequencing of surface proteins. A detailed mutational analysis was undertaken. The total ratio for Missense/Silent nucleotide substitutions was 0.96. Sistan and Kermanshah showed the lowest rate of evolution between provinces (P = 0.055). On the other hand, Khorasan Razavi and Khoozestan contained the highest ratio (P = 0.055). The rest of regions were laid between these two extremes. Azarbayjan and Guilan showed the highest proportion of immune epitope distribution (91.3% and 96%, respectively). Conversely, Sistan and Tehran harbored the least percentage (66.6% and 68.8%, respectively). Kermanshah province contained only 5.2%, whereas Isfahan had 54.5% of B cell epitope distribution. In terms of T helper epitopes, all provinces showed a somehow homogeneity: 22.58% (Fars) to 46.6% (Khuz- estan). On the other hand, distribution of substitutions within the CTL epitopes showed a wide range of variation between 6.6% (Khuzestan) and 63% (Kermanshah). Further to low selection pressure found in Iranian population, the variations between different regions designate random genetic drift within the surface proteins. These finding would have some applications in terms of specific antiviral regimen, design of more efficient vaccine and public health issues.

Hepatitis C virus (HCV) chronically infects around 200 million people worldwide and fre- quently causes liver cirrhosis and hepatocellular carcinoma. Rapid detection of this virus results in decreasing the distance between infection and initiation the anti-viral treatment, and may prevent most of the undesirable consequences. The new detected HCV protein «Core+1» made from the ribosomal frame shift in Core region is an important candidate for diagnostic tools. This study was conducted to design a recombinant Bacmid plasmid expressing the HCV 1a Core+1 sequence in the Baculovirus expression system for further diagnostic applications. The HCV Core +1 gene was amplified by PCR using the pcDNA-HAF recombinant vector that contained the Core+1 sequence from HCV genotype 1a as a template, and the specific primers with 2 restriction sites for Nco I and Xba I restriction enzymes. The PCR product was cloned in XbaI/NcoI restriction sites of the linearized pFastBac-HTB vector and evaluated by using those restriction enzymes and sequencing. Then the recombinant pFastBac-HTB vector was transformed in DH10Bac and the result was screened and confirmed by X-Gal discrimination and PCR. The HCV 1a Core+1 was successfully amplified and the PCR product was confirmed by using the related restriction enzymes and sequencing. Cloning of pFastBac vector with the purified PCR product of HCV Core+1 was confirmed. Finally, the recombinant Bacmid was successfully transformed in DH10Bac. The recombinant Bac-Core+1 expression vector is considered as an important tool to transfect the sf9 cell line and expression the Core+1 protein.