Jundishapur Journal of Microbiology
Volume:6 Issue: 2, Apr 2013

Although infection with Helicobacter pylori is a global health problem, its prevalence is different among countries. Serologic tests for the diagnosis of H. pylori infection are limited by low accuracy rates and lack of validation. Recent studies indicate that the stool antigen test has an acceptable level of accuracy. Our objective was to investigate the prevalence of H. pylori infection in Khuzestan province, south-west of Iran by stool antigen test and to evaluate possible risk factors for the H. pylori infection. Patients and In this study, from September to October 2009, 861 healthy individuals aged 0- years were sampled by cluster sampling from 4.5 million inhabitants of the province. Infection with H. pylori was evaluated by detection of H. pylori antigens in stool (HpSA). Epidemiologic data of each subject were determined by filling up a questionnaire. The overall HpSA positivity was (53.5%, CI 95% = 50.2-56.84%) and the mean age of infected cases was greater than that of non-infected ones (29.2 yrs vs. 24.5 yrs) (P < 0.001). HpSA positivity increased with age. The peak prevalence was reached in the 41–50 years age group (66%). No association was detected between H. pylori positivity and gender. Additionally, there was no significant relationship between H. pylori infection and previous hospital admission, gastrointestinal symptoms, and living area (P > 0.05). H. pylori infection is highly prevalent (57%) in Khuzestan province, south-west of Iran. Low educational levels, low socioeconomic jobs, and increasing age were related to high prevalence of H. pylori infection in this area.

Proteases are often used as catalysts for peptide synthesis. To shift the thermodynamic equilibrium in favor of the peptide synthesis, reaction media should contain organic solvent. However, known enzymes are usually inactivated by adding organic solvents to reaction media. In this study, we reported a bacterium isolated from soil of Ahvaz, Iran contaminated by crude oil producing an organic solventstable protease, and the effect of organic solvents on proteolytic activity was investigated. Isolated bacterium was cultured in mineral salt medium containing glucose and peptone. After 48 hours incubation at 35 °C and 130 rpm, culture media were centrifuged and resulted supernatant filtered using 0.22 μm nitrocellulose membrane filter. Proteolytic activity of supernatant was determined by Keay and Wildi method (1970) by using casein as substrate. The effects of different concentrations of various organic solvents including acetone, ethanol, pentanol, cyclohexane, benzene, n-hexane, and n-decane and, also, the effects of temperature and pH on protease stability and activity were examined. According to 16SrDNA sequencing, strain ISA9 was identified as a new strain of Bacillus licheniformis. This strain was able to produce an extracellular organic solvent- tolerant protease. After 30 minutes incubation at 37 °C, caseinolytic activity of crude protease was increased in 25 and 50% of acetone, ethanol, benzene, cyclohexane, and hexane compared to non-solvent control. The enzyme was also activated 1.64, 1.23, and 1.17 times by 75% (v/v) of benzene, decane, and hexane, respectively. The protease was active in a broad range of pH (from 6 to 10) with the optimum pH 10. The optimum temperature for the activity of this protease was 70 °C and the enzyme remained active after incubation at 30-50 °C for 30 minutes. In this study, we isolated B. licheniformis producing an organic solvent-stable protease from oil-contaminated soil. The protease was stable and active in various organic solvents. By purification, the protease could be used as a biocatalyst for organic solvent-based enzymatic synthesis.

The 30 kDa major secretory protein of Mycobacterium tuberculosis (antigen 85B) is a primary vaccine candidate. This secreted antigen induces a protective immune response and stimulates the production of IFN-γ in animal models. The aim of this study was cloning and expression of Ag 85B of M. tuberculosis in Escherichia coli. To produce recombinant Ag85B, the fbpB gene was amplified by PCR method. Then inserted into the pET101/D vector and transported into E. coli strain TOPO10. Plasmid containing pET101/D: Ag85B was transformed into competence E.coli BL21 (DE3). The transformed E.coli strain BL21 was effectively expressed recombinant Ag85B. The expressed fusion protein was found almost entirely in the insoluble form. Followed by sonication to disrupt the cells, Solution of the cell debris was centrifuged and after use of Ni–NTA column and 6 molar urea and 6 M guanidine-HCl solutions recombinant protein was purified. These results could serve as a basis for further studies in endemic regions of tuberculosis on the usefulness of this gene and its expression product in the development of subunit vaccine and DNA vaccine against tuberculosis.

Genetic manipulation of chloroplast in higher plants offers a number of unique prerogatives, including; undesirable of pleiotropic genome and gene silencing effects and also use as an important agronomic trait for producing essential biomaterials and industrial enzymes. In order to manipulate chloroplast genome, specific vectors are required. These vectors can be transformed and expressed in Escherichia coli due to the same evolutionary origin of bacteria and chloroplasts. The aim of the present study was to construct chloroplast vector specified for spinach and assessing the chloroplast regulatory elements in a prokaryotic expression host, E. coli. Flanking sequences (INSL+INSR) were isolated by PCR from the spinach chloroplast genome and blunt-end ligated into the PvuII site of pUC19 vector to form an intermediate vector, pUCINS. Then the selectable marker cassette (including aadA gene, Prrn promoter and rbcL terminator) was isolated via PCR and blunt-end cloned into the unique PvuII site of pUCINS to make the final chloroplast vector, named pCSI. The constructed vector transformed to E.coli strain DH5α and several procedures such as colony PCR, digestion and sequencing were assigned to confirm the consequence of the construct. The appearance of bacterial colonies on the plate containing different concentrations of streptomycin indicated the strength of resistance to streptomycin which showed the bacterial cells capability to express aadA gene under the controls of chloroplast regulatory elements.

Fungal vaginitis originates from yeasts that are active in the mucosa of the women‘s genital tract. The main yeast that causes fungal vaginitis is Candida albicans. The current study aimed to detect frequency of yeasts mainly C. albicans in vaginal specimens of women from Tabriz, Iran. For the above purpose, the sensitivity and specifity of traditional laboratory assays were compared with those of molecular method (PCR) by universal and species primers to detect C. albicans in vaginal samples.Patients and In this study, 250 vaginal specimens were collected from women in Tabriz, East Azerbaijan province, Iran during 2009- 2010. Samples were examined to identify C. albicans by germ-tube test, chlamydoconidium formation test, preparation of wet smear using potassium hydroxide, and Polymerase Chain Reaction (PCR). 162 yeast species from 250 specimens were isolated in Sabroud Dextrose Agar (SDA) Medium. 106 (65.4%) of them were germ tube formation positive, 86 (53%) chlamydoconidium formation positive and 101(62.4%) were PCR positive. Yeast cells and mycelia were detected in the isolates on direct microscopic examination. C. albicans accounted for 66.04% of cases and 34% were non-C. albicans species. In conclusion, PCR may be the best method to detect Candida species.

The importance of preserving and maintaining printed materials is crucial for the libraries. Fungi play the main role in destroying wood and paper. This research aimed to study and identify threatening fungal agents of library resources in Isfahan University of Medical Sciences. This is a descriptive analytical study. 126 samples were collected and examined for the presence of fungi. An open plate method was used to scan airborne fungal contents and triplicate samples were collected at four different locations in the morning, at noon, and in the evening. The fungal culture media were incubated at 25-30 ºC until growth appeared and then the fungi colonies were identified by routine mycological laboratory methods. 1265 colonies of fungi belonging to 26 genera were identified in the air and different surfaces of books (references and circulation departments) and also surfaces of shelves in libraries. Cladosporium sp., Penicillium sp., Aspergillus sp. and Alternaria sp. were the most common isolated fungi in libraries of Isfahan University of medical sciences. We suggest training librarians as one of the most important steps in libraries to preserve library materials because having no knowledge about threatening factors and the way to fight with them are the main reasons of most frequent damages to library resources. Using new methods and technologies of preserving and maintenance of materials should be a priority in library managers’ planning.

Visceral leishmaniasis (Kala-azar) is one of the most serious tropical diseases, and it can lead to death. The kinetoplastid membrane protein-11 (KMP-11) is highly conserved in all stages of the Leishmania life cycle. In the present study, the KMP-11 gene was extracted from Leishmania infantum and then, cloned and expressed in an expression vector.The main objective of this study was to provide an introduction to DNA vaccine production against visceral leishmaniasis. DNA was extracted from L. infantum (MHOM/TN/80/IPI1), and amplified by PCR and a specific primer. Afterwards the purified PCR products were successfully ligated into the pTZ57R/T plasmid vector. The pcDNA3 plasmid was used as an expression vector and the KMP-11 gene subcloned into this plasmid. The pTKMP-11 plasmid was digested by restriction enzymes to confirm cloning of this gene in the pTZ57R/T plasmid. In the last step, the subcloned gene was expressed in a eukaryotic cell. The KMP-11 gene was successfully subcloned in pcDNA3 as a eukaryotic expression vector. Recombinant KMP-11 protein was confirmed by the reverse transcriptase polymerase chain reaction (RT-PCR) method. The results of the present study will increase our knowledge about molecular cloning and expression of the L. infantum KMP-11 gene, and this may be used as an effective target for controlling visceral leishmaniasis.

Antibiotic resistance in Pseudomonas aeruginosa, as one of the most important pathogens commonly implicated in nosocomial infections, has been increased in recent years, moreover the presence of integrons and the associated resistance gene cassettes is well established. The aim of the present study was to ascertain the presence and spread of class 1 integrons amongst environmental isolates of P. aeruginosa from Intensive Care Unit (ICU) as well as its association with drug resistance. This cross-sectional study was performed on 33 P. aeruginosa, isolated from different places and devices used in ICU at Shahid Beheshti Hospital in Babol, north of Iran, from 2008 to 2009. Antibiotic susceptibility profiles and minimum inhibitory concentration against 12 antibacterial agents were performed by micro dilution and disk diffusion methods. The detection of class 1 integron was performed by the PCR method. The demographic and microbiological data between the integron positive and negative isolates were compared with SPSS software. Thirteen of 33 (39.4%) of P. aeruginosa had intl gene, among which 24.2% were characterized as multidrug-resistant P. aeruginosa (MDRPA) on the other hand, 15.2% showed intermediate or complete sensitivity. No significant differences were seen between the presence of integron gene and resistance to the antibiotics except for ofloxacin. Most resistance was observed in cefepime (100%) and the lowest to ofloxacin and ciprofloxacin (42.5%). The result of this study showed a high prevalence of class 1 integron gene in most P. aeruginosa strains isolated from different parts of the environment and equipment used in ICU. The role of these transferable genetic agents has been proven in the creation of resistance. So, the environmental bacteria represent a reservoir for dissemination of clinically relevant multidrug-resistant antibiotics and should be taken under control to reduce the appearance or distribution of these antibiotic resistant agents.

Staphylococcus aureus is associated with different infections ranging from skin and soft tissue infections to endocarditis and fatal pneumonia. S. aureus is still the most common bacterial species isolated from inpatient specimens and the second most common from outpatient specimens. Today, methicillin resistant S. aureus (MRSA) isolates are present in the hospitals of most countries and are often resistant to several antibiotics. This study was conducted from 2007 to 2011 to detect prevalence and antibiotic resistance patterns among MRSA and methicillin sensitive S. aureus (MSSA) isolated from hospitals in Tehran, Iran. Totally 726 isolates of S. aureus were collected from three referral hospitals in Tehran. All isolates were identified at the species level by standard biochemical tests. Susceptibility to eighteen antibiotics was determined by disc diffusion method. Then oxacillin and vancomycin minimum inhibitory concentration (MIC) of resistant isolates was also determined using Etest. mecA gene was detected using specific primers. A total of 216 (30%) strains were found to be MRSA isolates. The highest antibiotic resistance was to penicillin, clindamycin, tobramycin and tetracycline respectively. Ninety three and 61% of MRSA and MSSA isolates were multidrug resistant (MDR) respectively. However, no strain was resistant to vancomycin, synercid, linezolid and chloramphenicol. Sixty nine percent of MRSA isolates showed high level of resistance to oxacillin (MIC ≥ 256 μg/mL). mecA gene was detected among all MRSA isolates. Although the frequency of MRSA isolates in the current study was low, resistance to other antibiotics was high and most of the isolates were found to be MDR. Regular surveillance of hospital-associated infections and monitoring of their antibiotic sensitivity patterns are required to reduce MRSA prevalence. High frequency of MDR isolates of S. aureus could be considered as an urgent warning for public health.

Because of great concerns about mortality and morbidity of infection in febrile neutropenic patients, the appropriate empirical antibiotic should be started immediately. Although there are established guidelines for the use of empirical therapy, local microbiological pattern and antibiotic susceptibility should be considered. The current study aimed to identify the etiological pathogens in febrile neutropenic cancer patients in Isfahan, Iran.Patients and This single-centre population-based study was conducted on 81 febrile neutropenic patients referring to Sayed-Al- Shohada hospital, the only referral malignant care center in Isfahan, Iran. Demographic data, duration and kind of malignancy, duration from last chemotherapy, duration of fever, and also physical exam were recorded for each patient. Moreover, procalcitonin, CRP, ESR, white blood cells, hemoglobin, platelet, and absolute neutrophil count were measured. BACTEC and E-test were used for blood culturing and determining the antibiotic susceptibility. Out of 81 participants, 28.4% had positive blood cultures which mostly consisted of Gram positive microorganisms. In addition, Staphylococcus epidermidis was the most isolated Gram positive bacteria (39.1%). ESR, CRP, and procalcitonin were significantly higher in patients with positive blood culture. The risk of infection increased with raise in duration of hospital stay, catheterization, increased pulse rate, increased oral temperature, low level of oxygen saturation, decreased systolic blood pressure, and low absolute neutrophile count. However, in this case, no relationship was found among the patients’ age, diastolic blood pressure, respiratory rate, duration of diagnosis, duration from last chemotherapy, duration of fever, white blood cells, hemoglobin, platelet, and finally the type of malignancies. It was concluded that Gram-positive bacteria were more prevalent as a cause of infection in the patients with malignancy and the most common pathogen was S. epidermidis. Larger studies are needed to determine the bacterial susceptibility of this center.

Human enteroviruses are members of Picornaviridae family; they are non-enveloped, icosahedral viruses with positive RNA as genome. Echovirus 30 is an important member of enteroviruses that is recognized in outbreaks of enterovirus meningitis. The aim of this study was to determine relative frequency of echovirus 30 as an important agent of aseptic meningitis among children referred to Aboozar hospital, Ahvaz, Iran.Patients and 34 cerebrospinal fluid samples from patients with enterovirus aseptic meningitis, negative bacterial culture, WBC (white blood cell) count > 5x106/mm3, and aseptic meningitis symptoms were entered in the study. These samples were collected in a year between May 2010 and May 2011. RNA of enteroviruses were extracted and investigated for echovirus 30 infection with RT-PCR (reverse transcription polymerase chain reaction) test. Samples also were cultured in RD (rhabdomyosarcoma) cell and positive results were approved by RT-PCR test with enterovirus specific primers. To recognize PCR-product, 440 bp RT-PCR product was sequenced and phylogenic tree was drawn based on Neighbor Joining method with 1000 replication bootstrap. Echovirus 30 infection was not detected in any case. Just one CSF sample grew in RD cell culture. This sample was approved by RT-PCR and sequencing. Positive sample was recognized as coxsackie virus B3. There was no echovirus 30 in Ahvaz because of diverse nature of enteroviruses and several serotypes with various distribution patterns in different geographical regions, and the fact that echovirus 30 is mostly detected in outbreaks rather than endemism. Coxsackie virus B3 was responsible for aseptic meningitis of a child in this study. Based on another study conducted in Tehran, it seems that Coxsackie B viruses are among current agents causing enterovirus aseptic meningitis in Iran. Of course we need to conduct more studies in Ahvaz and other parts of the country to approve this hypothesis.

Pseudomonas aeruginosais an opportunistic pathogen which causes severe, acute and chronic nosocomial infections. These infections are difficult to eradicate since the organisms are usually multidrug-resistant. Carbapenems are considered as the most effective drugs against these isolates. However, recent emergence of carbapenem-resistant P. aeruginosa has become a major healthcare problem. The present study was conducted to determine the antibiotic susceptibility of P. aeruginosa burn isolates to 13 antibiotics including imipemen and meropenem. One hundred and thirty three P. aeruginosa burn isolates were collected from Shahid Motahari Burn Hospital between July and December 2011. The majority of the isolates were from wounds (88.7%), followed by 5.26% from blood, 4.15% from subclavian catheters and 1.5% from urine. The antibiotic susceptibility profiles were studied by the agar disc diffusion. The results showed 99.2% resistance to carbenicillin, 98.4% to ticarcillin, 96.2% to ciprofloxacin, 95.4% to co-trimoxazole, 94.7% to imipenem and meropenem, 93.9% to piperacillin, 93.2% to azetronam, 92.4% to tobramycin, 91.7% to cefepime, 89.4% to amikacin and ceftazidime, and finally 87.2% to piperacillin-tazobactam. Overall, 100% of the isolates showed multidrug-resistance (resistance to ≥ 3 classes of antibiotics) including theimipenem- resistant isolates. The high rate of multidrug-resistance is alarming and it is crucial to screen for carbapenem resistance prior to - antibiotic therapy.

Bacterium-bacterium antagonistic interactions could be important in the ecology of marine bacteria. Antimicrobial properties of microorganisms are exploited in various fields of human activities. Antagonism of heterotrophic bacteria from different marine environments of tropical and temperate zones was examined. Bacteria were isolated from biofilm samples, tissues of hydrobionts and sea water. Isolates were characterized by phenotypic and 16S rRNA phylogenetic analyses. Agar diffusion assay was applied to investigate inhibitory interactions. 5 type strains and 21 strains of marine origin were used as test cultures. 68.97% of isolates from temperate zone and 56.76% of tropical zone showed antimicrobial activity. The most active strains belonged to genera Pseudomonas and Pseudoalteromonas. Bacterial interspecies growth inhibition is widely distributed in marine environments. Marine bacteria, especially Vibrio spp., may be good probiotics which are active against pathogenic bacteria.

Beta-lactam resistance in Gram-negative bacteria, especially Escherichia coli, is a main clinical problem. It is often caused by the coproduction of β-lactamases, particularly extended-spectrum β-lactamases (ESBLs) and AmpC enzymes. It may lead to a problem for diagnosis via recommended phenotypic tests by the Clinical and Laboratory Standards Institute (CLSI). Conversely, β lactamase genes have several subfamilies; therefore, using designed primers with high ability could be valuable to detect all of them. This investigation focused on evaluating the prevalence of extended-spectrum β-lactamases using disk diffusion method and confirmatory test (Combined Disk), as well as PCR for complete detection of these genes (SHV- and AmpC (CITM, FOX)- type β-lactamase genes). 500 clinical samples were collected from different hospitals of Tehran, Iran and 200 E. coli isolates were detected by standard biochemical tests such as IMVIC. These specimens were isolated from urine, stool, blood, and wound. Subsequently, the isolates were screened by disk diffusion method and combined disk assay for β-lactamase production. Resistant isolates were evaluated by PCR for molecular assessing. In disk agar diffusion test, 128(64%) E. coli isolates resistant to ceftazidime (55.5%) and cefotaxime (64%) were selected and followed by combined disk (ceftazidime, cefotaxime, and clavulanic acid) assay. In combined disk assay, among 128 screened isolates, 115 (89.8%) and 13 (10.2%) isolates were detected as ESBLs and AmpC producers, respectively. PCR performed on all 128 selected isolates via phenotypic tests and the results showed that among 115 and 13 isolates, 7 (6.1%) and 13(100%) cases contained blaSHV and blaCITM, respectively. Fox gene was not detected in any sample. Our findings indicate that the prevalence of ESBLs in Tehran is rising. According to CLSI, isolates showing negative confirmatory test are potentially considered as producers of AmpC (the result of CITM PCR was 100% positive). On the other hand, co-production of ESBLs and AmpC may lead to ESBLs false negative. Thus, development of diagnosis methods for complete detection of β-lactamase enzymes is important for resistance control and the treatment with high achievement.

Hospital-acquired infections caused by multi-drug resistant Acinetobacter spp. are often extremely difficult to treatand this has proved to be a serious problem worldwide. The aim of this study was to determine the incidence rates and distribution patterns of multi-drug resistant (MDR) Acinetobacter spp. strains and the occurrence of Acinetobacter-derived cephalosporinase (ADC-7) and OXA-type carbapenemases (OXAsetC genes) in clinical specimens in the Beheshti Teaching Hospital in Kashan, Iran. This descriptive study was carried out on sixty isolates of Acinetobacter spp. and clinical samples collected from patients. The level of antibiotic resistance was determined by the disc diffusion method and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) procedure. Polymerase chain reaction (PCR) amplification of the genetic determinants of resistance was also determined. The resistance rates were; amikacin (80%), tobramycin (68.3%), ceftazidime (60%), ciprofloxacin (55%), piperacillin/tazobactam (51.7%), doxycycline (50%), SXT/TMP (sulfamethoxazole / trimethoprim)(48.3%), levofloxacin 43.3%), gentamicin (40%), imipenem (25%), and sulbactam/ampicillin (20%). The frequency of MDR Acinetobacter spp. strains isolated was found to be 56.7%. These isolates were most sensitive to imipenem followed by ampicillin/sulbactam and gentamicin. The prevalence of genes for ADC-7 and OXAsetC in the Acinetobacter spp. was; 34 (56.7%) and 32 (53.3%), respectively. The positive percentages of MDR isolates for ADC-7 were 82.4% and for OXAsetC they were 73.5%. Our phenotypic analysis demonstrated that Acinetobacter spp. isolates were resistant to most clinically significant antibiotic classes. This is the first report concerning Acinetobacter-derived cephalosporinase, blaADC, enzymes in Acinetobacter spp. isolates from Iran.

Nosocomial fungal infections could arise from independent exposure to airborne spores of filamentous fungi existing in the hospital environment. The present study aimed to determine the mycoflora of indoor and outdoor environments of five major hospitals in Khorramabad, Iran. Sampling of air was done from indoor and outdoor environments of wards, surroundings and green space of hospitals by settle plate method. To obtain the sample from surfaces, pre-moistened swabs with cotton-tipped sticks were applied on different surfaces (floor, the walls, windows, beds, trolleys, laryngoscope and angiography devices). Culture plates of air and surfaces on Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) were incubated in the dark at 28 ºC and examined daily for fungal colonies for two to three weeks. Fungal isolates were identified by a combination of their macroscopic and microscopic criteria after purification on isolation culture media. A total of 707 fungal colonies including, Penicillium (29.14%), Cladosporium (24.04%), Aspergillus (20.65%), Fusarium (9.05%), Alternaria (3.96%), Rhodotorula (1.69%), Cryptococcus neoformans (0.7%) and other fungi (10.77%) were isolated. All the examined high-risk parts of the hospitals were found to be contaminated by various fungi. Aspergillus was the most prominent genus in Intensive Care Unit (ICU) and surgery, Cladosporium in Critical Care Unit (CCU), emergency and thalassemia, and Penicillium in orthopedic, emergency and neonatal sections. Among pathogenic yeasts, C. neoformans was isolated from ICU, surgery and orthopedic sections. The dimorphic fungal pathogen, Sporothrix schenckii, was reported from CCU. Theisolated fungi specially the genera Aspergillus and Penicillium are potential threats for immunocompromised patients in the hospitals.

Human immunodeficiency virus (HIV) testing strategy for tuberculosis (TB) patients in some high HIV incidence countries has been recommended based on interaction between HIV and TB.3 The present study aimed to determine the prevalence of HIV infection among newly detected pulmonary TB patients.Patients and In a cross sectional study, 104 cases including 95 non injecting drug users (IDU) and nine IDU, from 2009 to 2011 in Ahvaz capital of Khuzestan province in the south west of Iran, underwent HIV testing as early as being diagnosed as pulmonary TB. Inclusion criteria for HIV positivity was two EIA-HIV positive tests and confirmatory western blots. SPSS-16 software was employed to analyze data, including demographical characteristics, epidemiological and laboratory findings. Overall HIV prevalence among pulmonary TB patients was 7.6%. Comparing HIV prevalence between non IDU pulmonary TB and IDU pulmonary TB showed that IDU cases were significantly at the risk of HIV infection [2.1% vs. 66.6%,odds ratio (95%CI): 1.6,0.31-12.97, P < 0.0001]. In the region under study HIV prevalence in newly detected pulmonary TB patients (except in IDU patients) was low compared with HIV high prevalence region. Therefore, routine HIV testing may not be required.