[Expression of leoA gene of Helicobacter pylori in CHO animal cells by RT-PCR method]

Background and Objective: Helicobacter pylori infection is one of the most common chronic bacterial infections all over the world, particularly in the developing countries. LeoA gene plays an important role in pathogenesis, and the main role of this gene is to increase the bacterial toxin secretion. This study was conducted to isolate and clone the leoA gene in a pEGFP-C2 expression vector and evaluate its expression in eukaryotic system

Methods: In this laboratory study, the leoA gene was amplified from the standard strain of Helicobacter pylori genome [ATCC 43504] by PCR method. It was then inserted into the pTZ vector by cloning T/A. Sub cloning of this gene was performed in a pEGFP-C2 expression vector with a ligase enzyme. The final structure of pEGFP-C2-leoA was transformed by electroporation in CHO [Chinese hamster ovary] cells and the expression of the leoA gene was evaluated by SDS-PAGE and RT-PCR

Results: The results of PCR indicated that the 1758 bp fragment was amplified from the leoA gene. Cloning of this gene was performed successfully in pTZ and pEGFP-C2 vectors, respectively. The enzyme digestion with two KpnI and SacII enzymes, as well as sequencing, confirmed the accuracy of gene cloning. The observation of the protein product of the leoA gene in CHO cells indicated the successful expression of the LeoA gene in the eukaryotic system of Helicobacter pylori

Conclusion: The final construct of pEGFP-C2-leoA had a successful expression of the leoA gene in animal cells

Is there a correlation between helicobacter pylori and enterohepatic helicobacter species and gallstone cholecystitis?

Background: Cholecystitis is a common surgical condition. Recently, several authors have reported that DNA of bile tolerant Helicobacter spp. has been found in the human bile colonizing the biliary tract. The aim of this study was to evaluate the association between the presence of Helicobacter spp. and gallstone cholecystitis

Methods: In this case-control study, gallstones, bile, and gallbladder mucosa were collected from 25 patients without gallstone disease, 24 with acute cholecystitis, and 28 with chronic cholecystitis. The presence of Helicobacter pylori [H. pylori], Helicobacter bilis [H. bilis], Helicobacter hepaticus [H. hepaticus], and Helicobacter pullorum [H. pullorum] were investigated by polymerase chain reaction [PCR] using species-specific primers

Results: In this study, 77 subjects with acute and chronic cholecystitis and control groups with a mean age of 46.85 +/- 14.53 years, including 58 [67.25%] women and 19 [32.75%] men were included. DNA of 10 Helicobacter spp. was detected in the bile of the patients with cholecystitis including eight H. pylori and two H. bilis. However, we could not detect H. hepaticus and H. pullorum DNA in the samples. Moreover, there was an association between H. pylori and acute cholecystitis [p = 0.048], which was found to be stronger in 31-40-year-olds group [p = 0.003]

Conclusion: We found an association between the presence of H. pylori DNA and acute gallstone cholecystitis. There is not statistically significant correlation between three enterohepatic Helicobacter spp. [H. bilis, H. hepaticus, and H. pullorum] and cholelithiasis. Given the low sample size of the patients, more studies are required to clear the clinical role of Helicobacter spp. in the gallstone disease and cholecystitis

High frequency of class 2 and 3 integrons related to drug-resistance in clinical isolates of diarrheagenic E. coli in Iran

Background: Integrons are mobile genetic elements able to obtain the antibiotic resistance gene cassettes. The prevalence of integrons in the Enterobacteriaceae family has been varied and played an important role in the development of the drug resistant bacteria. The present study aimed to investigate the contribution of class 2 and 3 integrons in drug resistant Diarrheagenic Escherichia coli strains

Materials and Methods: The 164 Diarrheagenic E. coli collected from feces samples of children in the Yasuj -Iran and all isolates were identified by standard biochemical tests. The antimicrobial susceptibility for 14 antibiotics, which are used conventionally was determined by disk diffusion. The presence of class 2 and 3 integrons in all isolates was investigated by PCR

Results: Of 164 E. coli isolates from children, 80.49% carried class 2 integron and the length of the amplicons ranged from 800 bp to 2 kb. Class 3 integrons were identified among 24 E. coli isolates. All the E.coli isolates were susceptible to imipenem and the greatest resistance was correspondent to nalidixic acid. A significant correlation was revealed between Class 2 integron and resistance to kanamycin, amikacin, gentamicin, ceftazidime, chloramphenicol and cephalexin. The presence of class 3 integron was significantly associated with resistance to ampicillin, gentamicin, streptomycin, kanamycin, tetracycline and trimetoprime-sulfametoxazol

Conclusion: The results indicated that integrons are widespread in Diarrheagenic E. coli and its carriage contributed significantly to the emergence of resistance among Diarrheagenic E. coli. However, factors leading to the wide spread of integrons are still to be determined

Isolation and cloning of Helicobacter Pylori ureE gene into pIRES2-DSRed expression vector to generate a gene vaccine

Background and Aim: As one of the factors of gastric ulcers and cancer, Helicobacter pylori can live in the acidic environment of stomach for many years due to having urease enzyme. This enzyme requires Ni2+ and a group of auxiliary proteins such as ureE for its catalytic activity. Urease is not only a requisite factor to colonize the Helicobacter pylori but it is also pathogenic with different mechanisms. Regarding the high prevalence of these bacteria finding a way to prevent infection with them is necessary. The present research aimed at homogenizing . cloning of Helicobacter Pylori ureE gene into pIRES2-DS Red expression vector in order to create a DNA [gene] vaccine

Materials and Methods: In this experimental study, the ureE gene fragment was amplified through PCR method and it was cloned using T/A cloning in the pTZ vector. Sub-cloning of the gene was done in pIRES2-DS Red vector using T4-ligase enzyme and it was transformed into E. coli TOP10F strain; and, then, amplified. The gene construct, which is a DNA vaccine candidate, was transferred to CHO cells using electroporation method to investigate the gene expression in eukaryotic systems. UreE gene expression was assessed in eukaryotic cells by means of SDS-PAGE

Results: UreE gene cloning was confirmed in two vectors including pTZ as a replicative vector and pIRES2- DS Red expression vector by PCR, enzyme digestion, and sequencing methods. SDS-PAGE results confirmed the successful expression of the ureE gene in the eukaryotic system of CHO cells

Conclusion: The recombinant pIRES2-DSRed-ureE construct is capable of successfully generating of polypeptides derived from Helicobacter Pylori ureE gene expression in bestial cells. Given that the protein product of the ureE gene is one of the most important proteins of the mentioned bacterium, . the created recombinant DNA in this research can be used as a DNA vaccine candidate against Helicobacter pylori in future

Application of multiplex PCR for Rapid and sensitive detection of human papillomaviruses in cervical cancer

Objectives: Reffering to an increase in cervical cancer in the recent years, rapid, sensitive and economical detection of human papillomaviruses [HPVs] as causative agents of cervical cancer is important. The traditional methods for the detection of HPVs in cervical cancer, such as pap smear, suffer from limitation and PCR has a potential to overcome the limitaitons. The purpose of present research work was to identify the five important strains of HPV [16, 18, 31, 33 and 45] simultaneously by Multiplex PCR application

Methods: Study was done on 100 cervical lesions of women. DNA was extracted from specimens by a genomic DNA purification kit. A 5-plex PCR was developed for the simultaneous detection of major HPV. Five pair of new primers was designed for detection of HPV 16, 18, 31, 33 and 45 by Multiplex PCR

Results: Among the 100 evaluated samples, 82 were found positive to HPVs. In the meantime the highest rate of infection was for HPV 16. Also 30 of HPV positive samples had infections with two or more HPV types

Conclusion: Multiplex PCR assay used in present study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of vaginal samples

[Prevalence and antibiotic susceptibility patterns of Staphylococcus aureus isolated from cockroaches from Chaharmahal va Bakhtiari hospitals]

Background: Hospital infection is usually taken into consideration as a serious public health problem. A set of factors might give rise to the occurrence of these infections, including the presence of insects as the carriers of multidrug-resistant bacterial species. The present study, then, aimed to evaluate the contamination of cockroaches' methicillin-resistant Staphylococcus aureus [MRSA] and its susceptibility pattern

Materials and methods: In this descriptive study, over 100 cockroaches were collected and the bacterial culture of the outer and inner body was performed. MRSA was confirmed on the basis of standard methods and PCR [polymerase chain reaction]. Disk diffusion method was also used to specify resistance patterns according to CLSI [Clinical and Laboratory Standards Institute] guidelines

Results: Of 100 samples, 44 [44%] sample were contaminated to S. aureus, and also 8 [18.18%] sample of 44 samples to MRSA. The overall resistance of isolated MRSA strains to antimicrobial agents turned out to be 8 [100%] for methicillin, 7 [87.5%] for cefixime and 6 [75%] for vancomycin

Conclusion: Cockroaches can transmit potential pathogenic bacteria therefore their presence in hospitals should be regarded as a sanitation challenge. Then, it is recommended to arrange plans to fight these pests in hospitals

Deletion of Salmonella enterica serovar typhimurium sipC gene

Objective: To construct a novel plasmid as Salmonella enterica serovar typhimurium (S. typhimurium) sipC gene knockouts candidate. Methods: In this research, 5' upstream and 3' downstream regions of S. typhimurium sipC gene and kanamycin gene were PCR amplified. Each of these DNA fragment was cloned into pGEM T-easy vector. The construct was confirmed by PCR and restriction digest. Results: PCR amplified 320, 206 and 835 bp DNA fragments were subcloned into pET-32 vector resulting with a plasmid called pET-32-sipC up-kan- sip C down. Conclusions: The new plasmid (pET-32-sipC up-kan- sip C down) is useful for genetic engineering and for future manipulation of S. typhimurium sipC gene.

Prevalence of Helicobacter pylori vacuolating cytotoxin A gene as a predictive marker for different gastrodoudenal diseases

Helicobacter pylori [H. pylori] has several virulence factors such as vacA and cagA genes. Mosaicism in vacA alleles with two distinct families of vacA signal sequences [s1, s2] and two distinct families of middle region alleles [m1, m2] is reported. The aim of this study was determination of H. pylori vacA allelic types in Chaharmahal and Bakhtiyari province, Iran and their correlation with six different gastroduodenal diseases. This cross-sectional descriptive study was performed on 200 antral gastric biopsy specimens that were obtained from patients undergoing upper gastrointestinal tract endoscopy in Hajar Hospital of Shahrekord. Initially, H. pylori strains were identified by rapid urease test [RUT] and ureC primers, and thereafter, we used seven specific primers for detection of vacA genotypes. Statistical analyses were used to find their relationship to gastric disorders. The frequency of the vacA alleles, s1a/m1a, s1a/m1b, s1a/m2, s1b/m1a, s1b/m1b, s1b/m2, s1c/m1a, s1c/m1b, s1c/m2, s2/m1a, s2/m1b and s2/m2 were respectively, 27[16.46%], 8[4.87%], 45[28.43%], 7[4.26%], 5[3.04%], 10[6.09%], 12[7.31%], 4[2.43%], 18[10.97%], 6[3.65%], 0 and 22[13.41%]. s1a/m2 strains were the most prevalent strains in this region and there was a considerable relationship between s1a/m1a, s1a/m2, s2/m2 and s1c/m1a with some gastric disorders. As the findings are different from other regions of the world, extended molecular epidemiologic investigations are recommended in other cities of Iran

[Compression of three methods of polymerase chain reaction, culture and rapid urease test in diagnosis of Helicobacter pylori in gastric biopsy specimen]

Helicobactr pylori [H.pylori] infection is related to chronic gastritis, peptic ulcer and duodenal ulcer. Thus, identification and treatment of the infection has a considerable importance. The aim of this study was to compare three methods of polymerase chain reaction [PCR], culture and rapid urease test [RUT] in identification of Helicobactr pylori in gastric biopsy specimens. 263 patients [157 woman and 106 man] who suffering from digestive complaints and referred to the endoscopy department of Hajar Hospital in Sharkord [2007, Iran] were participated in the study. Three gastric biopsy samples were collected from each patient. Samples were examined by standard RUT and culture methods for diagnosis of H.pylori. PCR was used for diagnosis of ureC gene. Out of 263 patients, 36.49%, 9.50%, 13.30%, 9.48% and 5.50% had criteria gastritis, gastric ulcer, duodenal ulcer, esophagitis and gastric cancer, respectively. H.pylori infection was diagnosed in 54.37%, 31.94% and 84.79% of the patients by RUT, culture and PCR method, respectively. Findings of this study indicated that PCR has a greater sensitivity rather than rapid urease test and culture methods for diagnosis of H.pylori

[Evaluation of metronidazole resistance Helicobacter pylori strains isolated from Shahrekord]

Metronidazole is one of the most important antibiotics in Helicobacter pylori [H.pylori] eradication regimens. This study was designed to determine metronidazole resistance H. pylori isolates from patients referred to Hajar hospital, Shahrekord, Iran. In this cross-sectional study, 263 patients who referred to endoscopy department of Hajar hospital, Shahrekord in 2007 were randomly selected. Gastric biopsy samples were cultured on selective Brucella agar containing 10% blood and incubated under microerophilic condition at 37°C between 3 and 7 days. Gram stain, urease, catalase and oxidase tests were used to detect H. Pylori isolates, and PCR [polymerase chain reaction] to determine ureC gene. H.pylori indentified. Standard CLSI [Clinical and Laboratory Standard Institute] was used to evaluate metronidazole resistance. Rapid urease test [RUT], culture and PCR detect H. pylori infection in 54.37%, 84% and 84.79%, respectively. Of isolated strains, 49 [58.33%] metronidazole resistance and 7 [8.33%] semisensitive were recognized. Of 49 metronidazole resistant patients, 32.65%, 20.40%, 12.24%, 6.10%, 4.80% and 24.48% had gastritis, gastric ulcer, duodenal ulcer, esophagitis, gastric cancer and normal endoscopy, respectively. Fisher exact test shows that there are relation between metronidazole resistance and women. Regarding high metronidazole resistance among studied patients, alternative antibiotics with less resistance is recommended in Helicobacter pylori [H.pylori] eradication regimens