Iranian Journal of Microbiology
Volume:8 Issue: 4, Aug 2016

The microbiological monitoring of the water used for haemodialysis is important especially for Legionella and non-fermentative bacteria since patients with end stage renal disease (ESRD) are suffering from deteriorated function of immune system.
A total 50 water and dialysate samples were weekly collected over a period of 10 weeks from 5 sites. Total and faecal coliforms were determined by utilizing the most probable number (MPN) method. For isolation of Legionella, water samples were inoculated on a BCYE medium. DNA extraction was performed and was used to amplify 16S rRNA gene of Legionella species. Airborne bacteria were sampled using a single stage Andersen air sampler.
Out of total 50 water samples, 24 samples had bacterial contamination. The highest rate of Legionella contamination was observed in the storage tank (67 cfu/ml). Legionella was not isolated from the dialysate effluent samples. The highest rate of total bacterial count was related to the dialysate effluent and the maximum total count of coliforms was related to the reverse osmosis. The isolated bacteria were Gram-negative bacilli (mostly Pseudomonas isolates), Gram-positive cocci (mostly Micrococcus spp.) and Gram-positive bacilli (mostly Bacillus spp.). Six samples were contaminated with coliforms. No faecal coliform was isolated from the samples.
These results indicated that dialysis machine is an important source of contaminations such as Staphylococcus, Pseudomonas and Legionella. Therefore an efficient prevention program is needed to eliminate bacterial contamination of dialysis water system. Moreover, in haemodialysis centres, periodic surveillance programs for microbiological qualification can lead to a better planning for disinfection of haemodialysis water systems.

Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene.
Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b() expression vector and transformed into E. coli Rosetta (DE3) host strain.
The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector.
We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development.

Cholera is an endemic diarrheal disease in Iran, caused by Vibrio Cholerae. The epidemiology, transmission route, environmental determinants and antimicrobial resistant pattern of cholera have been changed during recent years. In this study the epidemiology and antimicrobial resistance of cholera in Iran during 2013 outbreak was investigated.
A retrospective, cross-sectional study was carried out using cholera national surveillance system collected data in 2013. Bacterial identification and antimicrobial susceptibility testing were done on 60 Vibrio cholerae isolates, serotype Inaba.
During July to November 2013, 256 confirmed cholera cases were diagnosed by stool culture. Two hundred and eleven out of 256 (83%) cases were imported from Afghanistan and Pakistan. The prevalent age group was 16-30 years old, 90% were male, 98.8% affected by Inaba serotype and case fatality rate was 2.7%. The results of antimicrobial susceptibility testing on 60 V. cholerae, serotype Inaba showed that all isolates were resistant to nalidixic acid, tetracyclin and trimethoprim- sulfamethoxazole and intermediate resistance to erythromycin but sensitive to ciprofloxacin, cefixime and ampicillin.
Migrants from neighboring countries played a key role in cholera outbreak in Iran during 2013. The results of antimicrobial susceptibility testing on 60 V. cholerae, serotype Inaba showed an increasing resistance rate in comparison with previous years.

Tuberculosis (TB) is a major problem in the world. Treatment and control of TB needs detection of the Mycobacterium tuberculosis (MT) in the proper samples. While smear doesn’t have enough sensitivity, culture and PCR are expensive, time consuming and unavailable in many centers. Recent development of a rapid TB antigen detection test (PrTBK) at Pasteur Institute of Iran could give a simple way for diagnosis of TB in about two hours. In this test the antigen-antibody complex will change color when gold conjugated mouse anti- rabbit antibody detects specific MT cell wall antigen in suspected samples.
We evaluated the diagnostic accuracy of PrTBK for diagnosis of pulmonary TB in comparison with smear, culture and PCR techniques in 56 consecutive samples (47 BAL and 13 sputum samples) obtained from patients with clinical suspicion of active TB.
Twentynine patients (52%) were female and seven patients were HIV positive. PrTBK was positive in 17 culture positive and 4 culture negative samples (100% sensitivity, 89% specificity and 92% accuracy in comparison with culture method). In two out of four patients with negative culture who were positive for PrTBK, PCR and anti-tuberculosis drugs trial therapy responses were in favor of tuberculosis. If we take this finding into account, the accuracy of PrTBK will rise.
High sensitivity and accuracy of PrTBK test enable us to initiate treatment on the basis of this convenient and rapid test.

Accurate designation of antimicrobial susceptibility pattern of the infecting microorganisms is an important crucial factor in making appropriate therapeutic decisions. Macrolide, lincosamide and streptogramin B antibiotics are in a family, reserved as an alternative approach in treatment of resistant Gram positive cocci. Amongst them, clindamycin has been considered as the preferred agent due to its excellent pharmacokinetic properties. The inducible resistance to clindamycin in Gram positive staphylococci and streptococci cannot be recognized by routine broth or agar based susceptibility tests and D-zone testing is necessary. This study is conducted to evaluate the frequency of inducible clindamycin resistance in Gram positive cocci.
Using traditional culture methods, 487 isolates of staphylococcus and β-hemolytic streptococcus were evaluated. If they were resistant to erythromycin and sensitive to clindamycin in primary antibiotic susceptibility testing by Kirby-Bauer method, they were subjected to D-zone testing to detect possible inducible clindamycin resistance.
Thirty three out of 172 isolates of Staphylococcus aureus and 50 out of 277 isolates of coagulase-negative staphylococci (CoNS) were subjected for D-zone testing. Among them 13/33 and 28/50 showed inducible clindamycin resistance, respectively. There was no significant difference in inducible clindamycin resistance regarding to methicillin susceptibility pattern. Positive D-test was observed in 17.39 and 13.33% of Group B streptococci and Streptococcus spp., respectively.
Considerable number of isolates showed inducible clindamycin resistance in our study which falsely would be reported susceptible if D-zone testing was not performed. Thus, performing D-Zone testing is necessary to avoid misleading results which may cause treatment failure.

Pseudomonas aeruginosa (PA) is one of the most important causes of nosocomial infections and has an intrinsic resistance to many antibiotics. Among all the resistance-nodulation-division (RND) pumps of P. aeruginosa, MexAB-OprM is the first efflux pump found to target multiple classes of antibiotics. This study was aimed to evaluate the expression level of genes expressing MexAB-OprM in clinical isolates of P. aeruginosa.
In this study, 45 P. aeruginosa strains were isolated from patients admitted to Children's Medical Center Hospital, an Iranian referral hospital. Disk diffusion and Minimum Inhibitory Concentration (MIC) methods were used for determination of the patterns of resistance to antibiotics. Real-time PCR was used to investigate the expression level of genes of MexAB-OprM efflux pump.
Among 45 resistant PA isolates, the frequency of genes overexpression was as follows: MexA (n=25, 55.5%), MexB (n=24, 53.3%) and OprM (n=16, 35.5%). In addition, in 28 strains (62%) overexpression was observed in one of the studied three genes of MexAB-OprM efflux pump.
In our study 28 isolates (62%) had increased expression level of efflux pumps genes, MexAB-OprM. Although the efflux pumps play important roles in increasing the resistance towards different antibiotics but the role of other agents and mechanisms in evolution of resistance should not be ignored. Since the concomitant overproduction of other Mex efflux systems might have additive effects on antibiotic resistance, the co-expressing of a multicomponent efflux pump is recommended. On the other hand, the concomitant overproduction of two Mex pumps might have additive effects on resistance to antibiotic. Therefore co-expressing of Mex efflux systems is recommended.

Biomaterials are widely used in medical devices such as urinary catheters. One of the main problems associated with long term using of the urinary catheters is biofilm formation on their surfaces. Many techniques have been presented to reduce the biofilm formation. One of the most revolutionary techniques allowing such surface fictionalization is plasma surface modification.
In this study, a glow discharge plasma (GDP) effect on Escherichia coli biofilm formation on the surface of urinary catheter in the pressure of 1.6 × 10-1 Torr of nitrogen, discharge voltage about 1.2 kV and current of 150 mA for 20 minutes has been investigated. Crystal violet binding assay and sonication method were performed in order to evaluate the amount of biofilm formation on tested biomaterials.
Characterization of modified surfaces by Attenuated Total Reflectance Fourier Transform Infrared Spectrometry (ATR-FTIR) and atomic force microscopy (AFM) revealed a noticeable change in hydrophobicity and roughness of catheter surfaces achieved by nitrogen plasma. The results of crystal violet binding assay and sonication method showed that the amount of biofilm formation on modified surface was about 86% less than the pristine sample.
Plasma surface modification can reduce the risk of infections in patients with long-term use of urinary catheters.

Acinetobacter spp. are important causes of nosocomial infections. They possess various antibiotic resistance mechanisms including extended spectrum beta lactamases (ESBLs). The aim of this study was to determine antibiotic resistance profile of Acinetobacter clinical isolates especially among ESBL-producing strains and to investigate the antimicrobial effects of oleo-gum-resin extract and essential oil of Ferula gummosa Boiss.
120 Acinetobacter strains were isolated from various clinical samples of hospitalized patients in Baqiyatallah hospital, Tehran during 2011-2012. Antibiotic susceptibility test was performed on the isolates using disk diffusion method. To detect and confirm the ESBL-positive isolates, phenotypic and genotypic tests were performed. Three types of F. gummosa oleo-gum-resin extracts and essential oils were prepared and the bioactive components of F. gummosa Boiss extracts were determined by GC-Mass chromatography. F. gummosa antimicrobial activity was evaluated against standard strain of Acinetobacter baumannii (ATCC19606) as well as Acinetobacter clinical isolates using well and disk diffusion methods. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution method.
46 isolates were resistant to all tested antibiotics. All clinical isolates were resistant to cefotaxime. 12.94% of the isolates were phenotypically ESBL-producing among which 94.2% carried ESBL genes (blaPER-1, blaOXA-4 and blaCTX-M) detected by PCR. Oleo-gum-resin of F. gummosa had significant antibacterial activity and alcoholic essential oil had higher inhibitory effect on Acinetobacter strains (MIC of 18.75 mg/ml).
Ferula gummosa extract contained components with well-known antimicrobial effects.

Lactic acid bacteria (LAB) play important roles in processing of Sayur Asin (spontaneously fermented mustard). Unfortunately, information about LAB in Indonesian Sayur Asin, prepared by traditional manufactures which is important as baseline data for maintenance of food quality and safety, is unclear. The aim of this study was to describe the diversity and distribution of culturable lactic acid bacteria in Sayur Asin of Indonesia.
Four Sayur Asin samples (fermentation liquor and fermented mustard) were collected at harvesting times (3-7 days after fermentation) from two traditional manufactures in Tulung Agung (TA) and Kediri (KDR), East Java provinces, Indonesia. LAB strains were isolated by using MRS agar method supplemented with 1% CaCO3 and characterized morphologically. Identification of the strains was performed basedon 16S rDNA analysis and the phylogenetic tree was drawn to understand the phylogenetic relationship of the collected strains.
Different profiles were detected in total count of the plates, salinity and pH of fermenting liquor of Sayur Asin in TA and KDR provinces. A total of 172 LAB isolates were successfully isolated and identified based on their 16S rDNA sequences. Phylogenetic analysis of 27 representative LAB strains from Sayur Asin showed that these strains belonged to 5 distinct species namely Lactobacilus farciminis (N=32), L. fermentum (N=4), L. namurensis (N=15), L. plantarum (N=118) and L. parafarraginis (N=1). Strains D5-S-2013 and B4-S-2013 showed a close phylogenetic relationship with L. composti and L. paralimentarius, respectively where as the sequence had slightly lower similarity of lower than 99%, suggesting that they may be classified into novel species and need further investigation due to exhibition of significant differences in their nucleotide sequences. Lactobacillus plantarum was found being dominant in all sayur asin samples.
Lactobacilli were recognized as the major group of lactic acid bacteria in Sayur Asin including 5 known and 2 novel candidate species. The distribution of LAB species was associated with the manufactures where Sayur Asin is produced.