Genomic analysis of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex by isoenzyme analysis and pulsed-field gel electrophoresis.

Initially, multilocus enzyme electrophoresis was used to examine genetic relationships among 63 isolates of Mycobacterium bovis and 13 other members of the M. tuberculosis complex. The isolates were divided into five electrophoretic types, with a mean genetic diversity of 0.1. The strains were genetically homogenous, indicating that members of the complex were closely related. This supported the suggestion that they should be considered as subspecies of a single species. Pulsed-field gel electrophoresis (PFGE) was then used to differentiate these isolates, as well as 59 additional isolates of M. bovis from different parts of the world. PFGE differentiated these strains into 63 patterns (53 patterns for M. bovis). Isolates of M. bovis from Western Australia (n = 46) were more homogenous than isolates from other regions. Eight strains were identified in that state, and one predominantly bovine strain was isolated from two human beings and a feral pig. Although M. bovis isolates from different parts of the world had distinct DNA patterns, some were very similar. PFGE is a highly discriminatory technique for epidemiological studies of bovine tuberculosis. For example, it allowed differentiation between isolates of M. bovis cultured from animals in separate outbreaks of tuberculosis, it suggested the transmission of infection between certain properties, and it demonstrated the existence of multiple infections with different strains at certain farms

The inhibitory effect of a Lactobacillus acidophilus derived biosurfactant on Serratia marcescens biofilm formation

Objective(s): Serratia marcescens is one of the nosocomial pathogen. The ability to form biofilm is an important feature in the pathogenesis of S. marcescens. The aim of this study was to determine the anti�adhesive properties of a biosurfactant isolated from Lactobacillus acidophilus ATCC 4356, on S. marcescens strains. Materials and Methods: Lactobacillus acidophilus ATCC 4356, was selected as a probiotic strain to produce biosurfactant. Anti�adhesive activities was determined by pre�coating and co� incubating methods in 96�well culture plates. Results: The FTIR analysis of derived biosurfactant revealed the composition as protein component. Because of the release of such biosurfactants, L. acidophilus was able to interfere with the adhesion and biofilm formation of the S. marcescens strains. In co� incubation method this biosurfactant in 2.5 mg/ml concentration showed anti�adhesive activity against all tested strains of S. marcescens (P<0.05). Conclusion: Our results show anti�adhesive properties of L. acidophilus biosurfactant will be useful against microorganisms responsible for infections in the urinary, vaginal and gastrointestinal tracts, as well as skin, making it a suitable alternative to conventional antibiotics. © 2015, Mashhad University of Medical Sciences. All rights reserved

Single tube real time PCR for detection of Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila from clinical samples of CAP

We designed a multiplex real time PCR for rapid, sensitive and specific detection of Streptococcus pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae. The study cases consisted of 129 patients with community acquired pneumonia (CAP). Bacteriological techniques were implemented for detection of the cultivable organisms. DNA were extracted from sputa, throat swabs, bronchoalveolar lavages and tracheal aspirates and used as templates in real time PCR. The primers and probes were designed for cbpA (S. pneumoniae), p1adhesin (M. pneumoniae), mip (L. pneumophila) and ompA (C. pneumoniae). After optimization of real time PCR for every organism, the experiments were continued in multiplex in a single tube. Of 129 CAP specimens, the positive cultures included 14 (10.85) for S. pneumoniae, 9 (6.98) for L. pneumophila and 3 (2.33) for M. pneumoniae. Four specimens (3.10) were positive for C. pneumoniae by real time PCR. The sensitivity of our real time PCR was 100 for all selected bacteria. The specificity of the test was 98.26, 98.34, 100 and 100 for S. pneumoniae, L. pneumophila, M. pneumoniae and C. pneumoniae, respectively. This is the first report on the use of multiplex real time PCR for detection of CAP patients in the Middle East. The method covers more than 90 of the bacterial pathogens causing CAP. © 2012 Akadémiai Kiadó, Budapest

Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis using TaqMan Allelic Discrimination

Objectives: Multidrug-resistant tuberculosis (MDR-TB) is a global problem that many countries are challenged with. Rapid and accurate detection of MDR-TB is critical for appropriate treatment and controlling of TB. The aim of the present study was to evaluate the TaqMan allelic discrimination without minor groove binder (MGB) as a rapid, efficient, and low-cost method for detection of drug resistant strains of Mycobacterium tuberculosis. Methods: A total of 112 M. tuberculosis isolates from cases with diagnosed TB were subjected to drug susceptibility testing (DST), using the proportion method. Resistant isolates were tested for characterization of mutations in the rpoB and KatG genes by TaqMan genotyping. Results: Of 112 M. tuberculosis isolates for which DST was performed, three, one, and two isolates were MDR, rifampin (RIF) resistant, and isoniazid (INH) resistant, respectively. According to the threshold cycle (Ct) and curve pattern of mutants, TaqMan probes detect all of the mutations in the analyzed genes (katG 315, AGC�ACC, rpoB 531, TCG�TTG, and rpoB 531, TCG�TGG). Conclusion: The present study suggests that drug-resistant strains of M. tuberculosis can be detected by pattern's curve or Ct with TaqMan probes without MGB in real-time polymerase chain reaction (PCR). © 2016

Molecular detection of simultaneous occurrence of antibiotic- and heavy metal-resistance in Klebsiella Pneumoniae isolated from urinary tract infection

Background: Microorganisms resistant to both antibiotics and metals have been isolated from nosocomial and urinary tract infection. Most heavy metal-resistant hospital infections including Klebsiella pneumoniae (K. pneumoniae) harbor plasmids with different molecular sizes. The aim of this study was the molecular detection of simultaneous occurrence of antibiotic and heavy metal resistance in Klebsiella isolated from urinary tract infection (UTI). Methods: Overall, 144 K. pneumonia strains were isolated from UTI samples in the laboratories of hospitals and clinics. Primary selection of β-lactam-resistant strains was conducted using combined disk and double-disk (DD) synergy methods according to the guidelines of Clinical and Laboratory Standards Institute (CLSI). Polymerase chain reaction (PCR) was also performed to detect TEM-1 and SHV-1 genes in resistant strains. Minimal inhibitory concentrations (MICs) of the heavy metals were determined for Hg2+, Cu2+, Pb2+, and Cd2+. Findings: Among the 61.81 antibiotic-resistant strains, 42.69 were β-lactamase producers. After performing PCR, from 38 positive extended spectrum beta lactamase (ESBL) strains, 28.94 of Klebsiella strains harbored SHV gene and; harbored TEM gene. The highest resistance was to Hg2+ (35 mg/L), Cu2+ (650 mg/L), Pb2+ (350 mg/L), and Cd2+ (200 mg/L). A significant difference was observed between antibiotic-resistant and heavy metals-resistant strains (P = 0.012). Conclusion: Plasmid-carrying isolates that showed resistant to heavy metals were highly resistant to antibiotics. The results showed that it is possible for the plasmids which carry genes for resistance to antibiotics and heavy metals to be exchanged by bacterial strains

Molecular identification and antibiotic resistance pattern of actinomycetes isolates among immunocompromised patients in Iran, emerging of new infections

Recent advancements in DNA-based approaches have led to the identification of uncommon and rare bacterial pathogens. In this study, by utilizing a DNA-based approach, a total of 1043 clinical specimens were processed for the identification of actinobacteria targeting the 16S rRNA and gyrB genes. Drug susceptibility testing was also conducted using micro-broth dilution and PCR. Two isolates of Nocardia flavorosea and Rhodococcus erythropolis were reported for the first time in Iran. Also, Nocardiopsis dassonvillei, Streptomyces olivaceus, and Streptomyces griseus were reported for the first time in Asia. Infections caused by Nocardia caishijiensis and Prauserella muralis have also been reported in this study. The first Asian case of pulmonary infection caused by Nocardia ignorata and the first global case of brain abscess caused by Nocardia ninae and Nocardia neocaledoniensis have been reported in this study. Overall 30 isolates belonging to 6 genera (Nocardia, Streptomyces, Rodoccoccus, Nocardiopsis, Rothia, and Prauserella) were detected in 30 patients. All 30 isolates were susceptible to amikacin and linezolid. Three isolates including Nocardia otitidiscaviarum (n = 2) and Nocardia flavorosea (n = 1) were resistant to trimethoprim-sulfamethoxazole which were the first trimethoprim-sulfamethoxazole resistant clinical actinomycetes in Iran. Isolation of rare species of actinomycetes particularly Nocardia spp. requires urgent action before they spread clinically particularly among immunocompromised patients. © 2021, The Author(s)

Antagonistic activities of some probiotic lactobacilli culture supernatant on serratia marcescens swarming motility and antibiotic resistance

Background and Objectives: Serratia marcescens, a potentially pathogenic bacterium, benefits from its swarming motility and resistance to antibiotic as two important virulence factors. Inappropriate use of antibiotics often results in drug resistance phenomenon in bacterial population. Use of probiotic bacteria has been recommended as partial replacement. In this study, we investigated the effects of some lactobacilli culture supernatant on swarming, motility and antibiotic resistance of S. marcescens. Materials and Methods: Antimicrobial activity of lactobacilli supernatant and susceptibility testing carried out on S. marcescens isolates. Pretreatment effect of lactobacilli culture supernatant on antibiotic - resistance pattern in S. marcescens was determined by comparison of the MIC of bacteria before and after the treatment. Results: Our results showed that pretreatment with L. acidophilus ATCC 4356 supernatant can affect the resistance of Serratia strains against ceftriaxone, but it had no effect on the resistance to other antibiotics. Furthermore, culture supernatant of lactobacilli with concentrations greater than 2, had an effect on the swarming ability of S. marcescens ATCC 13880 and inhibited it. Conclusion: Probiotic bacteria and their metabolites have the ability to inhibit virulence factors such as antibiotic resistance and swarming motility and can be used as alternatives to antibiotics. © 2018, Tehran University of Medical Science. All rights reserved

A bioassay-guided fractionation scheme for characterization of new antibacterial compounds from Prosopis cineraria aerial parts

Background and Objectives:Due to the importance of finding of new antibacterial agents, the antibacterial properties of Prosopis cinerariaaerial parts were investigated using a bioassay guided fractionation scheme. Materials and Methods:The organic extract was prepared via maceration in methanol, followed by the fractionation using n-hexane and ethyl acetate. The MICs of fractions were determined against some human pathogenic bacteria using broth micro-dilution assay. The primary characterization and identification of bioactive substance(s) was based on a bio-autograph- ical method using HPTLC and flash chromatography in parallel with agar overlay assays. Finally the exact mass of effective compound(s) was determined by LC-MS. Results:The best antibacterial activities were related to the ethyl acetate fraction. The effective antibacterial compound of the plant were 2 substances with molecular weight of 348 and 184 Dalton that inhibited the growth of assessed Gram positive bacteria with MIC values lower than 125 to 62.5 µg/ml synergistically. Conclusion: Further analysis using nuclear magnetic resonance could reveal the exact structure of these two antibacterial substances. These 2 effective antibacterial compounds could be applied as lead compound for synthesis of new antibacterial agents. © 2016, Tehran University of Medical Science. All Rights reserved

Differentiation of Australian isolates of Mycobacterium paratuberculosis using pulsed-field gel electrophoresis

Objectives To examine strain variation amongst Australian isolates of Mycobacterium paratuberculosis. Design Pulsed field gel electrophoresis was optimised for differentiation of M paratuberculosis strains, and this typing technique was then applied to a collection of Australian isolates. Procedure DNAs from 35 Australian isolates of M para-tuberculosis and a UK reference strain were digested with one or other of three restriction endonucleases. The banding patterns obtained after pulsed field gel electrophoresis of the DNA fragments were compared. Results The Australian isolates were divided into two groups on the basis of their DNA banding pattern. Both were different from the UK reference strain. Seven isolates from cattle in Victoria and the Northern Territory had the same pattern as five isolates from alpacas in Victoria and Western Australia. Another 20 isolates from cattle in Victoria, Western Australia and the Northern Territory had the same pattern as isolates from two sheep and a goat in New South Wales. Conclusion Pulsed field gel electrophoresis was a useful tool for strain typing of M paratuberculosis, and could be used to study the transmission of strains in Australia