Jundishapur Journal of Microbiology
Volume:9 Issue: 2, Feb 2016

Shiga toxins (Stxs, also referred to as verotoxins) are a family of bacterial protein toxins generated by Stx producing-Escherichia coli (STEC), such as E. coli serotype O157:H7.
The aim of this study was to investigate the effect of recombinant and native Shiga toxin (Stx) in induction of pro- and anti-apoptosis factors and stimulation of immune response to HeLa and THP-1 cells.
The HeLa and THP-1 cells were used to study the effect of native and recombinant Shiga toxin. For this purpose, 106 cells were cultured overnight in six-well plates and different concentrations of Stx were added to each well. The cells were then collected after 24 hours of incubation. Total RNA and protein was extracted. Firstly, the total RNA was used in reverse transcription-polymerase chain reaction (RT-PCR) for detection of interleukin (IL)-1α, IL-1β, IL-8, tumor necrosis factor (TNF)-α, B-cell lymphoma (Bcl)-2 and Bcl-xl transcript. Protein expression of pro- and anti-apoptotic factors was also confirmed by western blot analysis.
The IL-1α and IL-8 were increased by recombinant and native Stx. Interleukin-1β was detected in THP-1, while TNF-α was detected HeLa cells. Furthermore, Bcl-2 and Bcl-xl expression was observed in HeLa cells. However, expression of Bak was reduced by recombinant Stx and native toxin at the protein level, while Bcl-xl expression was increased.
These results suggest that toxins induce inflammatory responses, particularly through expression of chemokine. Recombinant Stx and native toxin induced apoptosis by balancing between different pro- and anti-apoptotic Bcl-2 family-factors in epithelial cells. In this study, for the first time, recombinant and native Stx induction of apoptotic factors and stimulation of immune response to HeLa and THP-1 cells were compared.

Acute diarrheal disease and urinary tract infection are leading causes of childhood morbidity and mortality in the developing world. Diarrheagenic Escherichia coli (DEC) has been identified as a major etiologic agent of diarrhea worldwide, and urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is one of the most common bacterial infections among human beings. Quick and precise detection of these bacteria help provide more effective intervention and management of infection.
In this study we present a precise and sensitive typing and phylogenetic study of UPEC and DEC using multiplex PCR in order to simplify and improve the intervention and management of diarrheal and UT infections.
In total, 100 urinary tract infection samples (UTI) and 200 specimens from children with diarrhea, which had been diagnosed with E. coli as the underlying agent by differential diagnosis using MacConkey’s agar and biochemical study, were submitted for molecular detection. Pathotyping of E. coli pathotypes causing urinary tract infection and diarrhea were examined using a two set multiplex PCR, targeting six specific genes. Phylogenetic typing was done by targeting three genes, including ChuA, YjaA and TspE4C2.
Overall, 88% of DEC and 54% of UTI isolates were positive for one or more of the six genes encoding virulence factors. Prevalence of the genes encoding virulence factors for DEC were 62%, 25%, 24%, 13%, 7% and 5% for ST (ETEC), LT (ETEC), aggR (EAggEC), daaD (DAEC), invE (EIEC) and eae (EPEC), respectively; whereas, the prevalence rates for the UTI samples were 23%, 14%, 6%, 6% and 4% for aggR (EAggEC), LT (ETEC), daaD (DAEC), invE (EIEC) and ST (ETEC), respectively. No coding virulence factors were detected for eae (EPEC). Group B2 was the most prevalent phylogroup and ST was the most frequently detected pathotype in all phylogroups.
ETEC and EAggEC were the most detected E. coli among stool and UTI samples, emphasizing the need to dedicate more health care attention to this group. In addition, our phylogenetic study may be helpful in figuring out the infection origin and for epidemiological studies. Nonetheless, more research studies with larger sample sizes are suggested for confirming our results.

Campylobacteriosis is a zoonotic infectious disease caused by Campylobacter jejuni and C. coli. The cadF gene is considered as a genus-specific gene while other genes are mainly used for discrimination at the species level.
This study aimed to analyze the cadF gene and to develop a duplex PCR assay for simultaneous detection of C. coli and C. jejuni, the two commonly encountered species.
In silico analysis of the cadF gene was carried out by several software and available online tools. A duplex PCR optimized with specific primers was used for detection and differentiation of both species. To evaluate specificity and sensitivity of the test, a panel of different Campylobacter spp. together with several intestinal bacterial pathogens was tested. The limit of detection (LOD) of method was determined using serial dilutions of standard genomes.
The analysis of the full size cadF gene indicated variations in this gene, which can be used to differentiate C. jejuni and C. coli. The duplex PCR designed in this study showed that it could simultaneously detect and differentiate both C. jejuni and C. coli with product sizes of 737 bp and 461 bp, respectively. This assay, with 100% specificity and sensitivity, had a limit of detection (LOD) of about 14 and 0.7 µg/mL for C. jejuni and C. coli, respectively.
In silico analysis of the cadF full-gene showed variations between the two species that can be used as a molecular target for differentiating C. jejuni and C. coli in a single-step duplex-PCR assay with high specificity and sensitivity.

Candidiasis is one of the most prevalent and important opportunistic fungal infections of the oral cavity caused by Candida yeast species like Candida albicans, C. glabrata, and C. krusei. In addition, several bacteria can cause oral infections. The inhibition of microbial biofilm is the best way to prevent oral infections.
The aim of the present study is to evaluate the antifungal, antimicrobial, and anti-biofilm properties of ginger (Zingiber officinale) extract against Candida species and some bacterial pathogens and the extract’s effects on biofilm formation.
Ginger ethanolic extract as a potential mouthwash was used to evaluate its effect against fungi and bacteria using the microdilution method, and biofilm was evaluated using the crystal violet staining method and dead/alive staining. MTT assay was used to evaluate the possible cytotoxicity effects of the extract.
The minimum inhibitory concentrations (MICs) of ginger extract for evaluated strains were 40, 40, 20, 20, 20, 20, 10, and 5 mg/mL for Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Bacillus cereus, Acinetobacter baumannii, C. albicans, and C. krusei, respectively. Ginger extract successfully inhibited biofilm formation by A. baumannii, B. cereus, C. krusei, and C. albicans. MTT assay revealed no significant reduction in cell viability after 24 hours. The minimum inhibitory biofilm concentrations (MIBCs) of ginger extract for fungi strains (C. krusei and C. albicans) were greater than those of fluconazole and nystatin (P = 0.000)..
The findings of the present study indicate that ginger extract has good antifungal and antibiofilm formation by fungi against C. albicans and C. Krusei. Concentrations between 0.625 mg/mL and 5 mg/mL had the highest antibiofilm and antifungal effects. Perhaps, the use of herbal extracts such as ginger represents a new era for antimicrobial therapy after developing antibiotic resistance in microbes.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are among the most important health issues in Turkey. Human immunodeficiency virus (HIV) infections are less frequently observed in the country. The individuals who had blood transfusions, patients undergoing hemodialysis, and intravenous drug addicted individuals, people who had tattoos/piercings, communal living environments, contamination of a family member, and prisoners are the main risk groups.
The current study aimed to discuss the prevalence and the genotypes of hepatitis and HIV infections among a specific group, namely individuals incarcerated in prisons.
Patients and Two-hundred and sixty-six prisoners sentenced for crimes such as robbery, sexual assault, assault substance abuse or selling drugs in the Kahramanmaras closed prison were recruited for the study. Demographic data and the presence of hepatitis B, hepatitis C and HIV were investigated in the study subjects.
Out of the 266 cases included in the study, 89.5% were male, 10.5% were female and the mean age was 31.21 ± 8.99 years. Risk factors were detected in 27.4% of the subjects. Out of the 73 subjects, among whom the risk factors were detected, 20.3% had intravenous substance use, 3.8% had a history of operation/transfusion, 1.9% had a history of indentation and 1.5% had unprotected sexual contact. The rate of hepatitis B surface antigen (HBsAg) positivity was 2.6%, the ratio of anti-HBs positive subjects was 35.0% and immunity was achieved with vaccination in 43% of the subjects. Anti-HCV was positive in 17.7% of the prisoners and the genotype 3 and genotype 1 were 68.1% (n = 32) and 2.1% (n = 1), respectively.
Continued substance abuse among most of the drug addicted individuals in prisons, common use of injection materials, tattoos and other circumstances that cause blood contact increase the risk of blood-borne infections.

Toxoplasma gondii is an obligate intracellular protozoan parasite that exists worldwide. Various techniques have been developed for T. gondii detection.

The aim of this study was the detection of T. gondii in diabetic patients with RE and B1 genes and the comparison of these two genes for diagnosis using the nested-PCR assay method.
Patients and

DNA samples from 205 diabetic patients who had been referred to the diabetes center of Ali Asghar hospital in Zahedan, Iran, were collected and analyzed using the nested-PCR assay method. Toxoplasma antibody data gathered using the enzyme-linked immunosorbent assay (ELISA) method from a previous study was used to group patients. The data were analyzed using SPSS 18. The chi-square test was used for comparison.

Of the diabetic patients selected, the following results were obtained: 53 (IgG, IgM); 20 (IgG-, IgM); 72 (IgG, IgM-); and 60 (IgG-, IgM-). The nested-PCR detected the following: in the acute group, 21/53 (39.63%), 30/53 (56.60%) (IgM, IgG); in the chronic group, 40/72 (55.56%), 51/72 (70.83%), (IgG, IgM-); in the false positive group, 18/20 (90%), 17/20 (85%) (IgM, IgG-); and sero-negative samples of 38/60 (63.33%) and 60/ 41 (77.35%) for RE and B1 genes, respectively. The prevalence of toxoplasmosis showed positive in patients with diabetes in the B1 gene 139 (67.8%) and RE gene 117 (57.1%).

Our study demonstrated that the B1 gene, more so than the RE gene, showed positive samples and can be used to detect toxoplasmosis, although the B1 gene, in comparison to the RE gene, did not show any superiority of molecular diagnosing capability. Results also showed that toxoplasma molecular detection methods can be used instead of routine serological detection methods in a clinical laboratory testing.

Even without treatment, most acute hepatitis E virus (HEV) infected patients resolve HEV but sometimes the disease leads to acute liver failure, chronic infection, or extrahepatic symptoms. The mechanisms of HEV pathogenesis appear to be substantially immune mediated. However, the immune responses to HEV are not precisely identified.
This study aimed to evaluate the Th1/Th2 ratio by investigating serum soluble markers from Th1 and Th2 cells in acute HEV infected patients.
Patients and This case-control study included 35 acute HEV infected patients and 35 age and gender matched anti-HEV negative healthy controls. The serum levels of Interferon (IFN)-γ, IL-4, soluble CD26 (sCD26) and sCD30 were determined by the enzyme-linked immunosorbent assay.
The results showed a significant difference in IFN-γ and sCD26 (P Acute HEV infection shows a pattern of Th1-type immune response, and the direct significant positive correlation between the serum level of sCD26 and IFN-γ in acute HEV infected patients, suggests that the trend of sCD26 levels is a valuable marker for predicting hepatic inflammation in hepatitis E.

Acute otitis externa, an inflammatory condition of the external auditory canal, is a common clinical problem in general medicine.
This study aimed to determine the etiology of otitis externa in patients from the Mazandaran province, north of Iran, which has a humid climate, as humidity can affect the prevalence of pathogenic microorganisms.
Patients and This cross-sectional study involved 116 patients with otitis externa. Two sets of samples were collected from their ears; one set was used for slide preparations, and the other for microbial culturing. After culturing, the microorganisms were identified by conventional methods.
Patients between 35 and 44 years of age were most frequently affected (25.00%) by otitis externa (average age, 43.87 ± 18.08 years). Moreover, women (54.31%) were more frequently affected than men (45.69%). Upon direct investigation, Gram-positive bacilli were the most commonly identified microorganisms (22.41%). Furthermore, Bacillus spp. and coagulase-negative staphylococci (22.41% and 19.83%, respectively), were the organisms most frequently identified from cultures of otitis externa samples.
Direct examination and culture showed that a mixed infection of fungi and bacteria is the most common cause of otitis externa. The present study revealed that Bacilli spp. were the most abundant bacteria isolated from patients with otitis externa. Thus, it is recommended that both organisms should be considered as etiologic agents in protocols for treatment of otitis externa.

Cell-mediated immunity (CMI) by CD4 Th (T helper)-type cells is the predominant host defense mechanism against Oral Candidiasis (OC) in HIV-infected individuals. Weakened CMI and depletion of CD4 T cells are the main factor contributing to the output of OC in HIV-positive individuals. The cytokines produced by Th1, Th2 and Th17 cells play a role in mediating an increased susceptibility to OC during HIV infection.
The present study investigated plasma concentration of IFN-γ, IL-4, IL-6 and IL-17 in HIV-1 patients suffering from OC.
Patients and In total, 98 samples in four groups (HIV-positive and HIV-negative persons with and without OC) were obtained from the oral cavities and cultured on Sabouraud’s dextrose agar and CHROMagar. Also blood samples were obtained to assess plasma level of IFN-γ, IL-4, IL-6 and IL-17 using ELISA technique.
There was a statistically significant difference in the plasma concentration of IFN-γ, IL-6 and IL-17 but not about IL-4. Our findings suggest a significant interaction between fungal infection and HIV on expression of assessed cytokines.
Fungal infection and HIV alone and together could seriously alter immune system function as assessed by measuring the levels of the plasma cytokines. Therefore, these results provide important new information relative to the putative immune-based factors associated with resistance and/or susceptibility to OC in HIV-positive persons.

Helicobacter pylori is curved Gram negative and microaerophilic bacilli that have infected half of the world’s population. It is recognized as the causative agent of duodenal ulcer, gastritis peptic ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma and is associated with gastric adenocarcinoma. Resistance to clarithromycin is related to point mutations in 23SrRNA gene on nt 2143 and 2144, when A turns to G, and A2143G is the most important type. These mutations lead to reduced affinity of antibiotics to their ribosomal target and are considered as the main cause of treatment failure.
The aim of this study was to determine the frequency of A2143G point mutation in 23SrRNA of H. pylori strains isolated from gastric biopsies of patients in Rasht, north of Iran, by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
Patients and A descriptive study was performed on 89 H. pylori strains, which were isolated from gastric biopsies of patients with gastric disorders such as gastritis, peptic ulcer, duodenal ulcer, non-ulcer dyspepsia and gastric adenocarcinoma. Isolated strains were tested for clarithromycin resistance using as breakpoint a minimum inhibitory concentration (MIC) of ≥ 1 mg/L by the E-test. The presence of H. pylori DNA was confirmed by amplifying the ureC (glmM) gene by PCR. Also, point mutation on 23SrRNA gene (A2142G and A2143G) was detected by PCR-RFLP using MboII and BsaI restriction endonucleases in all extracted DNA.
Of the 89 H. pylori isolates, eighty-four were susceptible to clarithromycin, while five (5.6%) were resistant. All DNA samples of resistant strains, which were treated with BsaI had A2143G mutation. There was no point mutation in the sensitive strains of H. pylori. Also, we detected no mutation on nt A2142G of resistant strains.
In the present study, the frequency of clarithromycin resistance was lower than the other studies conducted in Iran. Resistance frequency in samples isolated from gastric ulcer was higher than other gastric disorders. Women and patients aged more than 60 years old showed the most resistance frequency in this study. All resistant strains had the A2143G genotype.

PCR has been used for confirmation of leishmaniasis in epidemiological studies, but complexity of DNA extraction and PCR approach has confined its routine use in developing countries.
In this study, recent epidemiological situation of cutaneous leishmaniasis (CL) in two hyper-endemic metropolises of Shiraz and Isfahan in Iran was studied using DNA extraction by commercial FTA cards and kinetoplastid DNA (kDNA)-PCR amplification for detection/identification of Leishmania directly from stained skin scraping imprints.
Patients and Fifty four and 30 samples were collected from clinically diagnosed CL patients referred to clinical laboratories of leishmaniasis control centers in Isfahan and Shiraz cities, respectively. The samples were examined by direct microscopy and then scrapings of the stained smears were applied to FTA cards and used directly as DNA source in a nested-PCR to amplify kDNA to detect and identify Leishmania species.
Fifty four of 84 (64.2%) slides obtained from patients had positive results microscopically, while 79/84 (94%) of slides had positive results by FTA card-nested-PCR. PCR and microscopy showed a sensitivity of 96.4% and 64.2% and specificity of 100% and 100%, respectively. Interestingly, Leishmania major as causative agent of zoonotic CL was identified in 100% and 90.7% of CL cases from Isfahan and Shiraz cities, respectively, but L. tropica was detected from only 9.3% of cases from Shiraz city. All cases from central regions of Shiraz were L. tropica and no CL case was found in Isfahan central areas.
Filter paper-based DNA extraction can facilitate routine use of PCR for diagnosis of CL in research and diagnostic laboratories in Iran and countries with similar conditions. Epidemiologic changes including dominancy of L. major in suburbs of Shiraz and Isfahan metropolises where anthroponotic CL caused by L. tropica had been established, showed necessity of precise studies on CL epidemiology in old urban and newly added districts in the suburbs.

Emergence of resistance to respective antifungal drugs is a primary concern for the treatment of candidiasis. Hence, determining antifungal susceptibility of the isolated yeasts is of special importance for effective therapy. For this purpose, the clinical laboratory standard institute (CLSI) has introduced a broth microdilution method to determine minimum inhibitory concentration (MIC). However, the so-called “Trailing effect” phenomenon might sometimes pose ambiguity in the interpretation of the results.
The present study aimed to determine the in vitro susceptibility of clinical isolates of Candida against azoles and the frequency of the Trailing effect.
A total of 193 Candida isolates were prospectively collected and identified through the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Using a broth microdilution test, according to the guidelines of CLSI M27-A3, antifungal susceptibilities of the isolated yeasts against Fluconazole (FLU), Itraconazole (ITR), Ketoconazole (KET) and Voriconazole (VOR) were assessed. Moreover, trailing growth was determined when a susceptible MIC was incubated for 24 hours, and turned into a resistant one after 48 hours of incubation.
Among the tested antifungal drugs in this study, the highest rate of resistance was observed against ITR (28.5%) followed by VOR (26.4%), FLU (20.8%) and KET (1.5%). The trailing effect was induced in 27 isolates (14.0%) by VOR, in 26 isolates (13.5%) by ITR, in 24 isolates (12.4%) by FLU, and in 19 isolates (9.8%) by KET.
The monitoring of antifungal susceptibilities of Candida species isolated from clinical sources is highly recommended for the efficient management of patients. Moreover, the trailing effect should be taken into consideration once the interpretation of the results is intended.

Streptococcus agalactiae (Group B streptococcus, GBS) that colonize the vaginas of pregnant women may occasionally cause neonatal infections. It is one of the most common causes of sepsis and meningitis in neonates and of invasive diseases in pregnant women. It can also cause infectious disease among immunocompromised individuals. The distribution of capsular serotypes and genotypes varies over time and by geographic era. The serotyping and genotyping data of GBS in Iranian pregnant and non-pregnant women seems very limited.
The aim of this study was to investigate the GBS ýmolecular capsular serotype ýand genotype distribution of pregnant and non-pregnant carrier ýwomen at Yazd university hospital, in Iran.ý
Patients and In this cross-sectional study, a total of 100 GBS strains isolated from 237 pregnant and 413 non-pregnant women were investigated for molecular capsular serotypes and surface protein genes using the multiplex PCR assay. The Chi-square method was used for statistical analysis.
Out of 650 samples, 100 (15.4%) were identified as GBS, with a predominance of capsular serotypes III (50%) [III-1 (49), III-3 (1)], followed by II (25%), Ia (12%), V (11%), and Ib (2%), which was similar with another study conducted in Tehran, Iran, but they had no serotype Ia in their report. The surface protein antigen genes distribution was rib (53%), epsilon (38%), alp2/3 (6%), and alpha-c (3%).
The determination of serotype and surface proteins of GBS strains distribution would ýbe ýrelevant ýfor the future possible formulation of a GBS vaccine.