Molecular detection of virulence factors and biofilm formation in Pseudomonas aeruginosa obtained from different clinical specimens in Bandar Abbas.

BACKGROUND AND OBJECTIVES: Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen. The presence of several virulence factors such as exotoxin and exoenzyme genes and biofilm may contribute to its pathogenicity. The purpose of this study was to investigate the presence of toxA, exoU and exoS, the determination of biofilm production and antimicrobial susceptibility patterns among clinical isolates of P. aeruginosa. MATERIALS AND METHODS: In this study, 75 isolates of P. aeruginosa were recovered from various clinical specimens. Antimicrobial susceptibility pattern of isolates were identified. Virulence genes toxA, exoU and exoS were determined using PCR. The ability of biofilm production was assessed. RESULTS: Antimicrobial susceptibility test showed that 12 strains were resistant to more than 8 antibiotics (17.14%). The most effective antibiotic was colistin as 98.6% of isolates were sensitive. The frequencies of exoU and exoS genes were detected as 36.6% and 55.7%, respectively. In addition, 98.6% of the isolates were biofilm producers. Exotoxin A was detected in sixty-eight isolates (95.7%). CONCLUSION: The findings of this study showed that, the presence of P. aeruginosa exotoxin and exoenzyme genes, particularly, the exoU gene is the most common virulence factors in the bacterial isolates from urine samples. Biofilm is a serious challenge in the treatment of P. aeruginosa infection.

Marine microbial L-asparaginase: Biochemistry, molecular approaches and applications in tumor therapy and in food industry.

The marine environment is a rich source of biological and chemical diversity. It covers more than 70% of the Earth's surface and features a wide diversity of habitats, often displaying extreme conditions, where marine organisms thrive, offering a vast pool for microorganisms and enzymes. Given the dissimilarity between marine and terrestrial habitats, enzymes and microorganisms, either novel or with different and appealing features as compared to terrestrial counterparts, may be identified and isolated. L-asparaginase (E.C. 3.5.1.1), is among the relevant enzymes that can be obtained from marine sources. This amidohydrolase acts on L-asparagine and produce L-aspartate and ammonia, accordingly it has an acknowledged chemotherapeutic application, namely in acute lymphoblastic leukemia. Moreover, L-asparaginase is also of interest in the food industry as it prevents acrylamide formation. Terrestrial organisms have been largely tapped for L-asparaginases, but most failed to comply with criteria for practical applications, whereas marine sources have only been marginally screened. This work provides an overview on the relevant features of this enzyme and the framework for its application, with a clear emphasis on the use of L-asparaginase from marine sources. The review envisages to highlight the unique properties of marine L-asparaginases that could make them good candidates for medical applications and industries, especially in food safety.

Identification of Carbapenem-Resistant Klebsiella pneumoniae with Emphasis on New Delhi Metallo-Beta-Lactamase-1 (blaNDM-1) in Bandar Abbas, South of Iran.

BACKGROUND AND OBJECTIVE: The spread of carbapenem-resistant Klebsiella pneumoniae especially blaNDM-1-carrying isolates is a great concern worldwide. In this study we describe the molecular basis of carbapenem-resistant K. pneumoniae in three teaching hospitals at Bandar Abbas, south of Iran. MATERIALS AND METHODS: A total of 170 nonduplicate clinical isolates of K. pneumoniae were investigated. Antimicrobial susceptibility test was performed by disc diffusion method. PCR was carried out for detection of carbapenemase (blaKPC, blaIMP, blaVIM, blaNDM, blaSPM, blaOXA-48, and blaOXA-181) and extended-spectrum ß-lactamase (blaCTX-M, blaSHV, blaTEM, blaVEB, blaGES, and blaPER). Clonal relatedness of blaNDM-1-positive isolates was evaluated by multilocus sequence typing (MLST). RESULTS: Tigecycline was the most effective antimicrobial agent with 96.5% susceptibility. In addition, 6.5% of the isolates were carbapenem resistant. BlaNDM-1 was identified in four isolates (isolate A-D) and all of them were multidrug-resistant. MLST revealed that blaNDM-1-positive isolates were clonally related and belonged to two distinct clonal complexes, including sequence type (ST) 13 and ST 392. In addition to blaNDM-1, isolate A coharbored blaSHV-11, blaCTX-M-15, and blaTEM-1, isolate B harbored blaSHV-11 and blaCTX-M-15, and isolates C and D contained both blaSHV-1 and blaCTX-M-15. CONCLUSION: Our results indicate that NDM-1-producing K. pneumoniae ST 13 and ST 392 are disseminated in our region. Moreover, one of our major concerns is that these isolates may be more prevalent in the near future. Tracking and urgent intervention is necessary for control and prevention of these resistant isolates.

MIRU-VNTR analysis of Mycobacterium tuberculosis from Tehran, Sistan-Baluchestan, Kermanshah and Hormozgan during 2014 and 2015.

Standard 15-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis (MTB) is a valuable instrumentation for TB control. Our knowledge about the genetic diversity of MTB and population structure of MTB circulating in Iran is limited. During 2014-2015, 98 MTB isolates were collected from the TB centers of four provinces of Iran. Isolates were genetically characterized using 15-locus based MIRU-VNTR typing. Ninety-five distinct mycobacterial interspersed repetitive unit MIRU-VNTR patterns were found among 98 isolates. 5 (5.1%) isolates grouped into 2 clusters and 93 (94.89%) isolates had a unique pattern. The HGDI was as high as 0.99 and 10 of loci were designated as highly discriminative. Clusters belonged to Tehran only. This indicates these patterns are rotating in Tehran. Unique patterns suggest that distribution of samples in each province and population differs. HGDI is higher than previous studies for MIRU-VNTR typing in Iran. We suggest MIRU-15 because it is a valid epidemiological background for clustering defined. Limited data is available on the genetic diversity and transmission dynamics of MTB in Iran. To examine the genetic diversity and transmission dynamics of MTB strains we genotyped a collection of isolates from four different parts of Iran. The method of 15-loci MIRU-VNTR demonstrated high discriminatory power and may be applied as a first-line genotyping instrumentation in investigating the molecular epidemiology of M. tuberculosis in Iran.

Purification, Characterization and Comparison between Two New L-asparaginases from Bacillus PG03 and Bacillus PG04.

BACKGROUND: L-asparaginase has been used as a chemotherapeutic agent in treatment of lymphoblastic leukemia. In the present investigation, Bacillus sp. PG03 and Bacillus sp. PG04 were studied. METHODS: L- asparaginases were produced using different culture media and were purified using ion exchange chromatography. RESULTS: Maximum productivity was obtained when asparagine was used as the nitrogen source at pH 7 and 48 h after cultivation. New intracellular L-asparaginases showed an apparent molecular weight of 25 kDa and 30 kDa by SDS-PAGE respectively. These enzymes were active in a wide pH range (3-9) with maximum activity at pH 6 for Bacillus PG03 and pH 7 for Bacillus PG04 L-asparaginase. Bacillus PG03 enzyme was optimally active at 37 ˚C and Bacillus PG04 maximum activity was observed at 40˚C. Kinetic parameters km and Vmax of both enzymes were studied using L-asparagine as the substrate. Thermal inactivation studies of Bacillus PG03 and Bacillus PG04 L-asparaginase exhibited t1/2 of 69.3 min and 34.6 min in 37 ˚C respectively. Also T50 and ∆G of inactivation were measured for both enzymes. CONCLUSION: The results revealed that both enzymes had appropriate characteristics and thus could be a potential candidate for medical applications.

Genotyping of Mycobacterium tuberculosis Isolates from Hormozgan Province of Iran Based on 15-Locus MIRU-VNTR and Spoligotyping.

Background. Considering that Hormozgan province in Iran (southern part of Iran on the Persian Gulf) is among the areas with high prevalence of MDR-MTB and attracts so many sailors and tourists, genetic diversity of MTB isolates circulating in this part of Iran was evaluated. Pattern of TB transmission was also examined. Methods and Material. A total of 38 isolates of MTB were cultured from TB patients from Hormozgan province of Iran and standard MIRU-VNTR typing and spoligotyping were applied to genotype these isolates. Drug susceptibility testing was performed using proportion method. Results. There were 28 VNTR profiles comprising 5 clusters and 23 unique isolates compared to 21 spoligotyping profiles, which contained 9 clusters and 12 unique isolates. Latin American-Mediterranean (n = 9, 23.6%) was found to be the most predominant lineage. MIRU-VNTR analysis, with an HGDI of 0.975, was more discriminating than spoligotyping, which had an HGDI of 0.955. The estimated proportion of TB cases due to recent transmission was 26.3% and 44.7% by MIRU-VNTR and spoligotyping, respectively. The rates of monodrug resistance and MDR were 15.8% and 7.9%, respectively. Two of 3 MDR strains were found to be related to MIRU-VNTR and belonged to the same spoligotyping cluster characterized with T1/SIT53 genotype. Conclusions. The high genetic diversity among MTB isolates suggests that transmission occurred from different sources to this area. Reactivation of a priori, latent MTB infection was found to contribute mainly to TB cases in this geographic region.

In Vitro Activity of Tigecycline and Colistin against clinical isolates of Acinetobacter baumannii in Hospitals in Tehran and Bandar-Abbas, Iran.

BACKGROUND: The Acinetobacter species, particularly A. baumannii, has emerged as one of the main causes of nosocomial infections in recent years. The high prevalence of drug resistance in A. baumannii limits the therapeutic options for treating infections caused by these bacteria. The objective of this study was to determine the in vitro activity of Tigecycline and Colistin against clinical isolates of A. baumannii in Tehran and Bandar Abbas, Iran. METHODS: This study was conducted from March 2009 to November 2010 at three hospitals in Tehran and Bandar Abbas, Iran, using 165 Acinetobacter species isolated from clinical specimens. All isolates were subjected to PCR to detect bla OXA-51-like genes that are unique to Acinetobacter baumannii. Isolates that gave a band for the bla OXA-51-like genes were identified as A. baumannii. Anti-microbial susceptibility tests were performed for Tigecycline, Colistin, and other antibiotics. RESULTS: Sensitivity rates to Colistin and Polymyxin-B were 100%. Resistance rates for Tigecycline were 4.2% in Tehran and 8.8% in Bandar-Abbas according to Jones criteria, whereas, according to U.S. FDA criteria, the resistance rates were 20.8% and 17.6%, respectively. CONCLUSIONS: New alternative drugs are needed for the treatment of drug resistant A. baumannii. Although Colistin appears to be a good choice, adverse reactions have limited its usage. Tigecycline is effective against A. baumannii isolates, and it shows promise for solving the problem.

Spoligotyping and drug resistance patterns of Mycobacterium tuberculosis isolates from five provinces of Iran.

Tuberculosis (TB) persists as a public health problem in Iran. Characterization of Mycobacterium tuberculosis isolates circulating in this area will contribute to understand and control the spread of the strains. The aims of this study were to understand the genetic diversity and drug susceptibility of M. tuberculosis isolates circulating in Iran and to analyze the relationship between genotype and drug resistance. A total of 291 M. tuberculosis isolates collected from TB patients were genotyped by spoligotyping. Drug susceptibility testing was performed using proportion method. Spoligotyping resulted in 75 distinct patterns. 86.2% of isolates were grouped in 35 clusters while the remaining isolates were unique. Ural was found to be the most predominant lineage (34.3%) followed by Central Asian strain (CAS) (24%), T (18.2%), Manu2 (7.5%) and Latin American-Mediterranean (LAM) (6.1%). The five largest clusters were Ural/Spoligotype International Type (SIT)127 (15.8%), CAS1/SIT26 (9.2%), T1/SIT53 (6.1%), T1/SIT284 (5.4%), and CAS1/SIT25 (4.4%). About 5% of isolates had multidrug resistance (MDR) and 10% had other resistance. MDR was significantly associated with Beijing strains, but not with Ural family. This study highlights dominance of Ural, CAS, and T families in Iran. Biogeographic specificity of CAS and T families to border provinces of Iran including Sistan-Baluchestan and Kermanshah, respectively, suggested that this family strains might be transmitted from these regions to other provinces of the country.