Iranian Journal of Microbiology
Volume:9 Issue: 1, Feb 2017

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). Fosfomycin is a borad-spectrum antibiotic with inhibitory effects on biofilm production and β-Chloro-L-alanine (β-CLA) is an amino acid analog. The aim of this study was to determine effect of the combination of fosfomycin and β-CLA on biofilm production by MRSA isolates. Also,the clonal relatedness of the isolates was evaluated.
To determine the ability of biofilm production by 42 MRSA isolates, microtiter plate method was used. Antibacterial activities of fosfomycin and β-CLA were investigated by determining MICs and MBCs. Antibiofilm activities were measured in the presence of sub-MIC concentrations of fosfomycin, β-CLA or a combination of both. RAPDPCR was used for investigating the clonal relationship between isolates by the two specific primers.
21.4% of isolates were strong and 5% were moderate biofilm producers. The effect of fosfomycin plus β-CLA treatment on biofilm production was significantly different from non-treated, fosfomycin and β-CLA groups (p=0.00, 0.004 and 0.000 respectively). RAPD-PCR analysis revealed that the RAPD1 primer had more discriminatory power. The Sizes of RAPD-PCR bands ranged from 150 bp to 1500 bp and the number of bands varied from 1 to 13.
Clonal relatedness of isolates showed that the majority of biofilm producing isolates had identical pattern and only three isolates showed more than 80% similarity. The combination of fosfomycin and β-CLA could be introduced as an excellent mixture for eradication of MRSA biofilms in vitro.

Endophytic actinobacteria colonize inside the plant tissues without causing damages to the host plant. Since these microorganisms colonize in the different parts of plants and can stop plant disease, they have been applied as biological agents for controlling human diseases. The aim of this study was molecular identification and measuring the antimicrobial activity of endophytic Actinomycetes isolated from medicinal plants of Iran.
The total of 23 medicinal plant samples were collected, sterilized, and crushed. Small pieces of the crushed samples were then cultured directly on three selective media. Grown colonies were identified by 16S rRNA gene sequencing method. Each isolate was cultured in TSB medium and then antimicrobial compound was extracted using ethyl acetate and tested against the target bacteria.
Sixteen out of 23 bacterial isolates (69%) exhibited antimicrobial activity against the selected pathogenic bacteria, such as Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae, Citrobacter freundii, Proteus mirabilis, Shigella flexneri and Escherichia coli.
Our Study showed a high phylogenetic diversity and the potent antibiotic activity of endophytic bacteria in medicinal plants of Iran.

The selection of alternative treatment options with antibiotic combinations may be used for successful managing of multidrug-resistant Acinetobacter baumannii. The aim of this study was to determine the synergistic effects of ampicillin-sulbactam combined with either levofloxacin or tigecycline against MDR A. baumannii.
A total 124 of A.baumannii isolates collected from clinical samples of hospitalized patients which assessed for antibiotic susceptibility using disk diffusion method. E-test was used on 10 MDR A. baumannii isolates to determine the minimum inhibitory concentration (MIC) of ampicillin-sulbactam, levofloxacin and tigecycline. Any synergistic effects were evaluated at their own MIC using E-test assay at 37°C for 24 hours. Synergy was defined as a fractional inhibitory concentration index (FICI) of ≤0.5.
Levofloxacin plus ampicillin-sulbactam combination was found to have synergistic effects (FIC index: ≤0.5) in 90% of the isolates, but there was no synergistic effect for ampicillin-sulbactam/tigecycline and tigecycline/ levofloxacin combination. The antagonist effect in 50% of isolates (FIC index: >2) showed in combination of levofloxacin/tigecycline.
The emergence of multidrug A. baumannii isolates requires evaluating by combination therapy. The combination of levofloxacin plus a bactericidal antibiotic such as ampicillin-sulbactam is recommended. Results should be confirmed by clinical studies.

This study was designed to evaluate the activity of Quercus infectoria galls extract (QIFGE) on virulence factor production and inhibition of quorum sensing (QS) in Pseudomonas aeruginosa.
Minimum inhibitory concentration (MIC) of QIFGE against 5 strains of P. aeruginosa was determined. The extract at sub-MIC was used to determine biofilm formation, level of protease LasA, LasB, swarming and twitching motility and QS using Chromobacterium violaceum CV026 as a biosensor. Effect of the extract on expression levels of lasR gene was determined by real time PCR.
QIFGE inhibited the QS and all other tested virulence factors compared with the control grown in the absence of the extract (P=0.001). Real time PCR showed 2 to 8-fold reduction in lasR gene expression in presence of the extracts compared with the control. QIFGE significantly inhibited the virulence factor production, had inhibitory effect on QS, and resulted in the lower expression of lasR gene.
QIFGE showed novel inhibitory effect against QS related virulence factor production, which was unrelated to antimicrobial effect. The extract can down regulate the production of virulence factor and should be evaluated as a candidate for alternative treatment of pseudomonad infections in future.

Diarrhea is one of the most prevalent diseases in the world, specially in developing countries. One of the most important causative agents of bacterial diarrhea is diarrheagenic Escherichia coli (DEC) which causes gastroenteritis and this group involving enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterohemoragic E. coli (EHEC), enteroinvasive E. coli (EIEC), diffusely adherence E. coli (DAEC). The aim of this study was to identify different E. coli pathotypes in north and north-west of Iran, among the clinical isolates.
In this study for identification of E. coli, 170 fecal samples were cultured on MacConkey agar and identified by biochemical tests. Samples with E. coli characteristics were selected (145 samples) and their genomes were purified by phenol-chloroform method. After extraction of genomes, lt and sta genes identified by PCR for ETEC, eae gene for atypical and eae and bfp for typical EPEC, AA region for EAEC, stx1 and stx2 and eae genes for EHEC (stx1 or stx2 or both for STEC) and invE for EIEC.
Finally 10 samples identified as ETEC (%5.88), 18 (%10.58) EPEC, 6 (%3.52) EHEC and 12 (7.05%) samples were STEC. None of the samples were positive for EAEC and EIEC.
The results obtained in this study showed that ETEC, EPEC, EHEC and STEC are prevalent bacterial agents in north and north-west of Iran. Complementary studies to identify these pathotypes in other seasons can help to adopt necessary policies against outbreaks in Iran.

For further confirmation of the previous in-house real-time PCR and CYP 141 target as a rapid and cheap diagnostic technique and a new target for direct detection of Mycobacterium tuberculosis, we evaluated and compared the results of smear, culture and real-time PCR in sputa that were collected from 2 health centers. Moreover we tried to evaluate the diagnostic accuracy of phenotypical methods for detection of tuberculosis that have been applied in two health centers of Iran.
Thirty two sputa (including 15 smear positive and 17 smear negative) and 53 Sputa (29 smear and culture positive and 24 smear and culture negative specimens) were collected from tuberculosis suspected patients from health center No. 1 and 2 respectively. A Taqman probe was used for direct detection of M. tuberculosis using the specific primers.
Because of the results, data of health center No. 1 was not reliable. The average number of bacteria that was detected in health center No. 2 by real time PCR was 1.2E퍍7.3퍎; 1.4E퍎1.29퍎 and 8.27E퍎1.07퍎 for one to three plus smear result groups, respectively. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of real-time PCR were 96.5% (28/29), 95.8% (23/24), (96.6%) and (96%), respectively.
Compared with the results of previous studies and being a good correlation between real-time PCR and phenotypic methods, emphasize that CYP141 is a good target for quantification of M. tuberculosis in sputa and real-time PCR can be a good method for evaluation of smear microscopy. Moreover, further surveillance is needed to evaluate the phenotypical methods and final decisions that are taken in health centers of Iran that can be observed and evaluated by the cheap molecular methods like in-house methods.

The linear epitopes of gp46-I, gp46-II, gp21 and p19 are used in diagnosis of HTLV-I/-II infections. The aims of this study was to obtain high-level expression and purification of recombinant antigen (RA) containing these epitopes. Large-scale preparation of such antigen probably worths for diagnostic purpose.
The synthetic DNA encoding RA was synthesized and over-expressed as soluble in Escherichia coli BL21 (DE3) cells. Expression and distribution of the His-GST-RA protein were evaluated using SDS-PAGE. The soluble RA was purified utilizing Ni-NTA agarose beads under native conditions and was concentrated by ultra filtration. Using 20 sera specimens from HTLV infected patients, the antigenicity of the purified protein was confirmed in ELISA and western blotting analysis.
SDS-PAGE revealed that the purified protein was more than 90% pure. The final yield was approximately 25 mg per liter of culture medium. ELISA results showed that RA could specifically bind to anti-HTLV-I/-II antibodies in infected sera.
RA could be a candidate for HTLV-I/-II screening and the strategy presented in this study could be used for easy production of this diagnostic protein.

Pathogen reduction technologies are among methods to eliminate transfusion transmitted infections. Mirasol method using riboflavin in combination with ultraviolet rays is one of them. The aims of this study were to investigate the effectiveness of Mirasol method to inactivate some model pathogens as well as examination of the sensitivity of plasma proteins after treatment.
Riboflavin in 50μM concentration and ultraviolet (365 nm) in three different energy doses (3.6, 7.2, and 10.8 j/cm2) were employed to inactivate model pathogens. Four standard viruses were used in this study including Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus1 (HSV-1), Bovine Viral Diarrhea Virus (BVDV) and Polio Virus. 50% Tissue Culture Infectious Dose (TCID50) and Reed–Muench Methods were used to estimate viruses’ titers. E. coli and Staphylococcus aureus were used as bacterial models. Four plasma proteins including factor V, VIII, fibrinogen and antithromin were used to determine their sensitivity to pathogen inactivation treatment.
The most pathogen reduction titre was determined for 15 minutes irradiation period equal to 10.8 J/cm2 that is corresponding to Log 6.10 for BVDV, Log 6.09 for HSV-1, Log 6.62 for VSV and Log 3.36 for Polio. Bacterial reduction titer was Log 6.94 for E. coli and Log 7.00 for S. aureus. Indicator proteins for plasma activity were determined to be 75% for factor V, 88% for factor VIII, 52% for fibrinogen and 94% for antithrombin.
Results showed that the employed method can inactivate most of the pathogens in fresh frozen plasma. The acceptable activities of selected plasma proteins remained after treatment.