Differentiation of Brucella abortus and B. melitensis biovars using PCR-RFLP and REP-PCR.

Brucellosis is one of the most common zoonotic diseases of animal and human beings. This study aimed to differentiate the Brucella spp. and determines the patterns of biovars by using repetitive element palindromic (REP)-PCR and PCR restriction fragment length polymorphism (RFLP) methods. A total of 100 blood specimens suspected of harbouring brucellosis were collected. Conventional culture methods and multiplex PCR were used for the detection of Brucella genus and species; and REP-PCR was used for Brucella spp. differentiation and polymorphisms sequence analysis. In addition, to identify the biovar patterns of REP-PCR, PCR-RFLP was used. Eighty-three samples were identified as harbouring Brucella spp. by the implementation of multiplex PCR, 72 of which were detected as Brucella melitensis and 11 as B. abortus. Also, through analysing the results of PCR-RFLP, it was found that of 72 B. melitensis samples, 69 were B. melitensis biovar 1 and three species were from other biovars. In addition, the obtained patterns for all of the B. abortus samples were from biovars 3, 5, 6 and 9. This study also optimized a test for the detection of Brucella biovar with the REP-PCR method such that Brucella spp. and biovars could be separated in the shortest possible time.

Comparison of in vitro antibacterial activities of two cationic peptides CM15 and CM11 against five pathogenic bacteria: Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Acinetobacter baumannii, and Escherichia coli.

In recent years, the widespread use of antibiotics has caused many bacterial pathogens resistance to conventional antibiotics. Therefore, generation of new antibiotics to control and reduce the effects of these pathogens is urgently needed. Antimicrobial peptides and proteins are important members of the host defense system in eukaryotes. These peptides are potent, broad-spectrum antibiotics that demonstrate potential as novel and alternative therapeutic agents for the treatment of drug-resistant infections. Accordingly, we evaluated two hybrid peptides CM11 (WKLFKKILKVL-NH2) and CM15 (KWKLFKKIGAVLKVL-NH2) on five important pathogenic bacteria. These peptides are short cecropin-melittin hybrid peptides obtained through a sequence combination approach, which are highly effective to inhibit the growth of important pathogenic bacteria. The activity of these two cationic peptides (CM11 and CM15) in different concentrations (2-64 mg/L) was investigated against standard and clinical isolates of important hospital infection bacteria by measuring MIC, MBC, and bactericidal assay. These peptides demonstrated the same ranges of inhibitory values: The organisms in early 24 h were more susceptible to polycationic peptides (MIC: 8 mg/L and MBC 32 mg/L), but after 48 h the MIC and MBC remained constant for the CM11 peptide. Bactericidal assay showed that all bacteria strains did not have any growth in agar plates after 40 min. The result showed that these two peptides are more effective than other peptides.

Diversity and frequency of Nocardia spp. in the soil of Isfahan province, Iran.

OBJECTIVE: To isolate and identify Nocardia spp. from soil in different regions of Isfahan province in the center of Iran. METHODS: This study was conducted in 32 districts (16 cities and 16 villages) in Isfahan province during two years. A total of 800 soil samples from these regions were studied by using kanamycin. The isolated Nocardia species were examined by gram and acid-fast staining and were identified biochemically and morphologically. The frequency and distribution of Nocardia spp. were determined in relation to different factors such as soil pH and temperate climate. RESULTS: From 153 (19.1%) Nocardia isolates identified, Nocardia asteroids (N. asteroids) complex (45.5%) and Nocardia brasiliensis (N. brasiliensis) (24.7%) were the most frequently isolated species, followed by Nocardia otitidiscaviarum (2.2%), Nocardiopsis dassonvillei, Actinomadura actinomadura (each 1.7%) and Nocardia transvalensis (1.1%) and also unknown spp. (23.0%). In this study, most species (54.4%) of Nocardia, especially N. asteroides complex were isolated from soils with pH: 7.01-8, whereas in pH: 8.01-9 more N. brasiliensis was isolated. The most Nocardia spp. was detected from regions with semi-nomadic and temperate climate (41.1%). CONCLUSIONS: N. asteroids complex is more prevalent in Isfahan province and soil can be a potential source of nocardiosis infections. It is to be considering that climate and soil pH are involved in the frequency and diversity of aerobic Actinomycetes.

Simultaneous and rapid differential diagnosis of Mycoplasma genitalium and Ureaplasma urealyticum based on a polymerase chain reaction-restriction fragment length polymorphism.

OBJECTIVES: The aim of this investigation was to simultaneously detect and differentiate Mycoplasma genitalium and Ureaplasma urealyticum in female patients suffering from genital complications by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). MATERIALS AND METHODS: Genital swabs were taken from 210 patients. They were transported to the laboratory in phosphate-buffered saline. For PCR, samples were analysed with genus-specific MyUu-R and MyUu-F primers. This primer set, which was originally designed in our laboratory, amplified a 465 bp fragment (M. genitalium) and a 559 bp fragment (U. urealyticum). Samples containing a band of the expected sizes for the Mycoplasma strains were subjected to digestion with a restriction endonuclease enzyme of TaqI and Cac8I. RESULTS: Of the 210 samples, a total of 100 (47.6%) samples were found to be positive for Mycoplasmas (seven M. genitalium isolates, 3.3%; and 89 U. urealyticum isolates, 42.4%), and coinfections with both species were detected in four samples (1.9%). The PCR-RFLP results showed that M. genitalium and U. urealyticum are different by enzyme patterns. CONCLUSION: PCR-RFLP offers a rapid and easily applicable protocol to simultaneous detection and differentiation of M. genitalium and U. urealyticum from clinical samples when specific primers and restriction enzymes are used.