Jundishapur Journal of Microbiology
Volume:7 Issue: 2, Feb 2014

Secondary metabolite production from wild strains is very low for economical purpose therefore certain strain improvement strategies are required to achieve hundred times greater yield of metabolites. Most important strain improvement techniques include physical and chemical mutagenesis. Broad spectrum mutagenesis through UV irradiation is the most important and convenient physical method.. The present study was conducted for enhanced production of avermectin B1b from Streptomyces avermitilis 41445 by mutagenesis using ultraviolet (UV) radiation, ethidium bromide (EB), and ethyl methanesulfonate (EMS) as mutagens.. S. avermitilis DSM 41445 maintained on yeast extract malt extract glucose medium (YMG) was used as inoculum for SM2 fermentation medium. Spores of S. avermitilis DSM 41445 were exposed to UV radiation for physical broad spectrum mutagenesis and to EMS and EB for chemical mutagenesis. For each mutagen, the lethality rate and mutation rate were calculated along with positive mutation rate.. Avermectin B1b-hyper-producing mutant, produced using these three different methods, was selected according to the HPLC results. The mutant obtained after 45 minutes of UV radiation to the spores of S. avermitilis 41445, was found to be the best mutant for the enhanced production of avermectin B1b component (254.14 mg/L). Other avermectin B1b-hyper-producing mutants, were obtained from EMS (1 µL/mL) and EB (30 µL/mL) treatments, and yielded 202.63 mg/L and 199.30 mg/L of B1b, respectively.. The hereditary stability analysis of the UV mentioning 45 minutes revealed the UV exposure time for mutants and 3 represented the colony taken from the plate irradiated for 45 minutes mutant showed that the production of avermectin B1b remained constant and no reverse mutation occurred after 15 generations..

Some genetic and phenotypic variables are associated among distinct microbial populations.. The associations between multi-drug resistance (MDR) phenotypes, prevalence of antibiotic resistance integrons (ARIs), blaSHV, blaTEM and blaCTX-M gene carriage and genetic fingerprints of random amplified polymorphic DNA (RAPD), confirmed by pulsed field gel electrophoresis (PFGE), were investigated among extended-spectrum β-lactamases (ESBL)-producing nosocomial isolates of Klebsiella pneumoniae.. Susceptibility of 35 ESBL-producing K. pneumoniae nosocomial isolates to 22 antimicrobial agents was determined. Integron carriage was detected using specific primers for intI1, intI2 and intI3 genes by PCR.. All isolates were resistant to piperacillin and susceptible to imipenem. MDR phenotype was observed in 91.4% of the isolates. Class 1 integrons were detected in 21 (60%) and class 2 integrons in 3 (8.57%) of the isolates. Two of the isolates carried both classes and none harbored class 3 integrons. Significant correlations were observed between resistance to aminoglycosides, fluoroquinolones and sulfonamides, and between genotype groups with carriage of ARIs, MDR phenotype and blaSHV gene carriage. ARI carriage was also significantly associated with MDR phenotype.. Our findings suggest the possible co-carriage of some blaSHV genes and ARIs on the same plasmids harboring the MDR genes. Possible role of integrons in dissemination of ESBL-encoding blaSHV genes among ESBL-producing K. pneumoniae nosocomial isolates may be inferred..

Extended spectrum β-lactamases (ESBLs) and AmpC β-lactamases enzyme are major sources of resistance to β-lactam antibiotics especially in Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae. Increasing frequency of the co-existence of ESBLs with AmpC-β-lactamases in bacteria is a serious threat for treating bacterial infections..

The aim of this study was to determine the presence of AmpC and CTX-M types of β-lactamases in clinical isolates of E. coli and K. pneumoniae producing ESBLs..

Resistance to different antibiotics was determined using the standard disk diffusion method. ESBLs, MBLs and AmpC-β-lactamases were detected by the combination double disk test (CDDT) method and polymerase chain reaction (PCR) was used to determine blaCTX-M genes in the ESBLs and AmpC positive isolates..

The prevalence of ESBLs and AmpC-β-lactamase producer isolates was 181 (43.8%) and 133 (37.2%), respectively. The prevalence of blaCTX-M among isolates was 61 (14.7%)..

Outbreak of isolates co-expressing AmpC-β-lactamases and ESBLs can cause serious problems in the future, regarding the treatment of infections caused by these common enteric pathogens..

Extensive quantities of keratinic by-products are disposed annually by animal-processing industry, causing a mounting ecological problem due to extreme resilience of these materials to enzymatic breakdown. There is a growing trend to apply cheap and environment-friendly methods to recycle keratinic wastes. Soil bacteria of profound keratinolytic potential, especially spore-forming rods from the genus Bacillus, play a significant role in keratinase-mediated biodegradation of keratins, therefore could be effective in hastening their biodegradation. Keratin hydrolysis in microbial cultures is one of the most promising techniques not only to utilize this protein but also to obtain valuable by products.. The study was undertaken to investigate the biodegradation process of various keratinic materials by two Bacillus strains.. Two keratinolytic strains, Bacillus cereus and B. polymyxa, were subject to cultures in the presence of several keratinic appendages, like chicken feathers, barbs and rachea of ostrich feathers, pig bristle, lamb wool, human hair and stratum corneum of epidermis, as main nutrient sources. Bacterial ability to decompose these waste materials was evaluated, at the background of keratinase and protease biosynthesis, in brief four-day cultures. Keratinolytic activity was measured on soluble keratin preparation and proteases were assayed on casein. Additionally, amounts of liberated proteins, amino acids and thiols were evaluated. Residual keratin weight was tested afterwards.. Both tested strains proved to be more adapted for fast biodegradation of feather β-keratins than hair-type α-keratins. B. cereus revealed its significant proteolytic potential, especially on whole chicken feathers (230 PU) and stratum corneum (180 PU), but also on separated barbs and rachea, which appeared to be moderate protease inducers. Keratinolytic activity of B. cereus was comparable on most substrates and maximum level obtained was 11 KU. B. polymyxa was found to be a better producer of keratinases, up to 32 KU on chicken feathers and 14 KU on both fractions of ostrich feathers. Its proteolytic activity was mostly revealed on stratum corneum and human hair. Stratum corneum was extensively degraded by both bacterial strains up to 99% - 87%, chicken feathers 47-56%, ostrich barbs and rachea, 28% and 35% at maximum, respectively. Keratin fibres of structures like human hair, lamb wool and pig bristle remained highly resilient to this short microbiological treatment, however certain extent of keratinase induction was also observed.. The obtained results prove that keratinolytic potential of both tested bacterial strains could be applied mainly in biodegradation of feathers, however, B. cereus and B. polymyxa differed in terms of keratinase and protease production on each of the substrates. Biodegradation of highly resilient structures like hair or pig bristle requires further analysis of process conditions..

Tuberculosis remains a global epidemic, especially in developing countries, including Iran. Rapid diagnosis of active Mycobacterium tuberculosis infection plays a critical role in controlling the spread of tuberculosis. Conventional methods may take up to several weeks or longer to produce results. In addition to multiplicity of steps involved in conventional detection, including isolation, identification and drug susceptibility testing, the slow growth rate of M. tuberculosis is also responsible for this lengthy time.. The aim of this study was to compare the polymerase chain reaction (PCR) and culture methods for the detection of M. tuberculosis in different clinical specimens.. This study was performed on different samples (urine, gastric aspirate, bronchoalveolar lavage, pleural fluid, cerebrospinal fluid, ascetic fluid and joint fluid specimens) of tuberculosis suspected patients. M. tuberculosis DNA was extracted directly from different samples using two different protocols. Next, PCR was performed using three sets of specific primers to detect members of Mycobacterium genus, M. tuberculosis complex and non-tuberculosis Mycobacteria. The results were then compared with that of the culture method, which is considered as the gold standard method.. The concordance rate between the three sets of primers was calculated and IS6110/buffer PCR method showed good agreement with the LJ culture method (κ = 0.627, P < 0.0001). The sensitivity of IS6110/buffer PCR was 58.33%, with specificity of 77.78%; the positive and negative predictive values were 100% and 78.26%, respectively. Buffer method for DNA extraction was proved to give a higher accuracy to PCR in comparison with the boiling method.. PCR method is a valuable, cost-effective and alternative tool for quick diagnosis of active tuberculosis in different clinical specimens..

Sewage treatment plants are considered to be the hotspots for antibiotic resistance transfer among bacterial species. Many fecal bacteria including Enterococci circulate and are exposed to antibiotic residues in this environment. Being as one of the most common cause of nosocomial infections, special concerns have risen worldwide about the rate and characteristics of Enterococci (especially, isolates with high resistance against glycopeptides) which are available in raw sewages.. Study on the vancomycin Resistant E. faecium diversity in Tehran sewage by plasmid profile, biochemical fingerprinting and antibiotic resistance. Forty isolates recovered from an urban sewage treatment plant were studied during 2009- 2010. The antibiotic resistance of isolates against 7 antibiotics was examined by disk diffusion method. Extraction of plasmid DNA was performed and identification of van genotype (vanA and vanB) was done by PCR. Biochemical fingerprinting was done by the use of Phene-Plate system (PhP).. All isolates were found to be resistant to erythromycin, ampicillin and ciprofloxacin. The PCR analyses showed that all E. faecium isolates harbored vanA gene and 5 (13%) isolates harbored vanA and vanB concomitantly. By plasmid profiling the VRE isolates differentiated into 11 types. PhP showed that VRE isolates were grouped into 23 biochemical types.. The combination of plasmid profiling and PhP techniques revealed the presence of diverse population of VRE in sewage treatment plant in Tehran. Furthermore, the results showed that the PhP technique is a reliable method in determining the VRE clonal diversity..

The Thiol-specific antioxidant (TSA) is an antigen of Leishmania major which is believed to be the most promising molecule as a vaccine candidate against leishmaniasis.. In this study, we investigated the protective efficacy of TSA-based DNA vaccine against L. major infection.. Recombinant plasmid construction TSA (pcTSA) was prepared and transfected into eukaryotic cells and expression was confirmed with western blot and RT-PCR. The mice were assigned to six different groups and DNA immunization was performed with 100 µg intramuscular recombinant plasmid with a two-week interval. Cytokines and lymphocyte proliferation assay, antibody responses and determination of parasite burden were performed following immunization and the challenging infection with L. major.. The antibody and IFN-γ titers were higher in pcTSA + AlPO4 group the immunized mice with pcTSA alone, but there was no statistically significant difference between the two groups. Additionally the IL-4 titer was not statistically different between the groups following immunization and challenge. After infection with L. major promastigotes, the immunized mice with pcTSA and the one immunized with both pcTSA + AlPO4 presented a considerable reduction in diameter of lesion but there was no statistical difference between the two groups. The immunized mice had significantly lower parasite loads. No significant differences were observed between the two vaccinated groups. However the highest reduction in parasite burden was observed in the group immunized with pcDNA + AlPO4. No significant differences were observed in survival rate of the immunized mice after the challenge with L. major.. In conclusion, TSA-based DNA vaccine induced Th1 platform immune response and aluminum phosphate could improve the efficacy of these vaccines with induction of humoral and cellular immune responses against L. major infection. There were no significant differences observed between pcTSA and pcTSA + AlPO4 groups.

Acinetobacter baumannii plays an important role in some types of nosocomial infections as an opportunist microorganism which increases levels of resistance to antibacterial drugs and disinfectants.. The aim of this study was to determine the resistance and sensitivity of A. baumannii to different antibiotics and evaluate the minimal inhibitory concentration (MIC) for Ciprofloxacin and Tetracycline; in addition to Surfanios, Citron and Aniosyme DD1 disinfectants, and also to detect the presence of gyrA, parC and tetB gene bands in the isolates.. In this study, 65 A. baumannii isolates were collected from the hospitalized patients in NIOC hospital (National Iranian Oil Company hospital) of Tehran, Iran during 2010-2011. The pattern of sensitivity to antibiotics was determined using CSLI disk diffusion and MIC methods. Furthermore, resistance of isolates to the common disinfectants (Surfanios Citron and Aniosyme DD1) was determined in different hospital wards. Presence of gyrA, parC and tetB gene bands was also detected by PCR method.. Frequency of Acinetobacter resistance to Amikacin, Ciprofloxacin, co-Trimoxazole, Ceftazidime and Ceftriaxone was 100% in the isolates reviewed in this study. The frequency of resistance to Gentamicin and Tetracycline were 86.1% in the isolates. The MIC of Ciprofloxacin in all (100%) of isolates was 32-64 μg/mL which showed the resistance to Ciprofloxacin In 86.1% of cases the Gentamicin and Tetracycline MIC were ≥ 16 μg/mL and in 13.9% of isolates the Gentamicin and Tetracycline MIC were 4μg/mL, these results showed the resistance and sensitivity to the Gentamicin and Tetracycline, respectively. Additionally, all (100%) of the A. baumannii isolates were resistant to disinfectant concentrations, which were used with the methods recommended by manufacturers (0.5%). In 100% of the isolates parC and gyrA genes bands were detected, and tetB gene was also detected in 86.1% of Tetracycline resistant isolates.. Due to the high resistance of A. baumannii isolates to most antibiotics in our study and also its high resistance to the common disinfectants usually used in hospitals, it seems that more attentions should be paid for applying disinfectants. Since most of the isolates were collected from tracheal and sputum samples (46%), it seems that respiratory tract is the most t prevalent site of infection among Acinetobacter infections. Therefore, disinfecting the respiratory tract related equipment and instruments by using proper disinfectants seems to be an appropriate way to prevent these infections..

One of the most important problems in production of recombinant protein is to attain over-expression of the target gene and high cell density. In such conditions, the secondary metabolites of bacteria become toxic for the medium and cause cells to die. One of these aforementioned metabolites is acetate, which enormously accumulated in the medium, so that both cell and protein yields are affected.. To overcome this problem, several strategies applied. In this research we used antisense RNA strategy, where the transcription of phosphotransacetylase (PTA) and acetate kinase (ACK), two acetate pathway key enzymes, could be controlled, which led to reduced acetate production.. In order to achieve this, recombinant plasmid harboring antisense sequences targeting both of pta and ackA was assembled, after transfecting to the cells, its effects on the cell growth and acetate accumulation in the minimal media was assessed and compared with the control, the plasmid without antisense cassette, in presence and absence of IPTG in Escherichia coli BL21 (DE3).. It was observed that the mentioned strategy partially affect the growth and amount of excreted acetate in comparison with the control. In addition it was found that high down-regulation of the acetate production pathway reduces the growth rate of E. coli BL21 (DE3).. The study principally proved the importance of this strategy in acetate excretion control..

Nowadays natural products such as pure compounds and plant extract scan provide unlimited opportunities for new antiviral drugs. Newcastle disease virus (NDV) is one of the most important viral diseases in poultry industry. Vaccination could provide protection against NDV outbreaks, but it is not sufficient because infections by NDVs have remained frequent around the world.. The current research aimed to study Achillea millefolium and Thymus vulgaris antiviral activity against Newcastle disease virus (NDV).. The antiviral activity of the plants was measured by the reduction assay of viral titer, and explained by inhibition percentage (IP).. Inhibition percentage was determined as 10 1.75, which indicated the ability of the extracts to reduce the viral potency by more than 56 folds.. Both plants were found effective against Newcastle disease virus..

Biofilm formation is a major pathogenic factor in different bacteria such as Pseudomonas aeruginosa. A number of studies have reported that bacterial biofilms show different levels of antibiotic resistance. In order to re-sensitize the bacterial biofilms to antibiotics, biofilms should be dispersed.. In this study, the effect of n-butanolic Cyclamen coum extract in combination with ciprofloxacin was examined on one, three and five day old P. aeruginosa biofilms. The synergistic effect of n-butanolic C. coum extract and ciprofloxacin towards dispersing pre-established P. aeruginosa biofilms was also studied.. The ability of biofilm formation by six different P. aeruginosa strains was confirmed by microtiter plate method and PCR assay for the cupA gene. The extraction of C. coum tubers was achieved by fractionation method using different solvents. The minimum inhibitory concentration (MIC) of n-butanolic C. coum extract and ciprofloxacin against planktonic cells was evaluated using agar well diffusion and microdilution methods. The microdilution chequerboard method was used to determine the fractional biofilm eradication concentration index (FBCI), when the combination of n-butanolic C. coum extract and ciprofloxacin were used against P. aeruginosa biofilms.. The ability of biofilm formation by P. aeruginosa strains was quantitatively confirmed. The PCR method confirmed the existence of cup A gene (172 bp) in all studied strains. Saponin content of the n-butanolic C. coum extract was 156 µg/mL. The extract revealed antibacterial activity against planktonic cells of P. aeruginosa strains. The results showed that one and three day old biofilms are affected by either ciprofloxacin or n-butanolic C. coum extract. However, n-butanolic C. coum extract in combination with ciprofloxacin was significantly more effective against P. aeruginosa biofilms.. Using n-butanolic C. coum extract in combination with ciprofloxacin offers a novel strategy to control biofilm-based infections caused by P. aeruginosa..