Differentiation of Staphylococcus aureus and Staphylococcus epidermidis by PCR for the fibrinogen binding protein gene.

Mastitis is one of the most common and burdensome diseases afflicting dairy animals. Among other causes of mastitis, staphylococci are frequently associated with clinical and subclinical mastitis. Although Staphylococcus aureus is the predominant species involved, Staphylococcus epidermidis and other coagulase-negative staphylococci are increasingly being isolated from cases of bovine mastitis. Although Staph. aureus and Staph. epidermidis can be easily differentiated based on their biochemical properties, such phenotypic identification is time consuming and laborious. This study aimed to rapidly identify Staph. aureus and Staph. epidermidis. Accordingly, a multiplex PCR was developed and we found that a single gene encoding the adhesin fibrinogen binding protein could be used to identify and differentiate the two species. Consequently, a multiplex reaction combining a triplex PCR for Staph. aureus and a duplex PCR for Staph. epidermidis was standardized, first using bacterial cultures and then with pasteurized milk spiked with live organisms or DNA extracted from the organisms. The test could specifically detect Staph. aureus and Staph. epidermidis even in the presence of a dozen other organisms. The limit of detection for detecting Staph. aureus and Staph. epidermidis separately was 10 to 100 cfu/mL for simplex PCR and 10(4)cfu/mL for multiplex PCR. Conversely, the limit was 10(6)cfu/mL by multiplex PCR for simultaneous detection of both the organisms when spiked into culture medium or pasteurized milk. Overnight enrichment enhanced the assay sensitivity 100-fold. The assay had a high diagnostic sensitivity and specificity. The application of the test was verified on 602 field isolates of staphylococci that had been characterized earlier by phenotypic methods. Importantly, 25 coagulase-negative isolates were identified as Staph. aureus by the multiplex PCR. The test could be adapted for use in clinical diagnostic laboratories.

Antifungal Activity of Leaves of Mangroves Plant Acanthus licifolius Against Aspergillus fumigatus.

The antifungal activity of chloroform extract of leaves of Acanthus ilicifolius was evaluated in Aspergillus fumigatus infected mice. Swiss albino mice (60) were divided into five groups. All the groups were immunosuppressed with cyclophosphamide and cortisone acetate couple of days prior to intranasal inoculation with Aspergillus fumigatus conidia (10(6)) in all the groups, except the first. Treatment was initiated at 24 h of fungal inoculation and continued up to day 14, and included amphotericin B (1 mg/kg orally) for group III and extract of Acanthus ilicifolius at 250 mg and 500 mg/kg for group IV and V, respectively. Groups I and II received sterile water orally for the same period. From each group, three mice were sacrificed after 1 h and the remaining mice on the 14(th) day of inoculation. One hour post-inoculation lung colony forming unit count confirmed the delivery of conidia into the lungs. Colony forming unit count, intensity of gross necropsy changes and histopathological changes were highest in group II. It improved in group III and also in groups IV and V in dose-dependent manner. Lesions were absent in the noninfected group. Lesions included maximum granulomatous inflammation of lung, multifocal diffused necrotic granulomas on kidney and moderate microgranulomas on liver. From this study, it was concluded that chloroform extract of Acanthus ilicifolius contains active principles that are absorbed after oral administration to produce systemic effects when given at 500 mg/kg dose.

Molecular detection of meat animal species targeting MT 12S rRNA gene.

The efficacy of PCR-RFLP analysis of mt 12S rRNA gene in identification of animal species from meat samples of known and unknown origin and adulterated meat samples was evaluated. In PCR, all the samples generated an amplicon of 456 bp. Restriction enzyme digestion of the PCR product with AluI, HhaI, BspTI and ApoI revealed characteristic RFLP patterns. Of the samples of unknown origin few were identified as cattle, few as buffalo and some were admixtures of two, suggesting adulteration. The RFLP pattern of one did not match any of species included in the study, which on sequencing was confirmed as camel meat. Application of this technique on adulterated meat samples could detect both animal species in proportion of 50:50 and 75:25 (except in case of goat+cattle). The technique however could not detect any of the two species when proportion of mixture was 90:10 (except in case of cattle+buffalo).

Detection of Mycobacterium avium & M. tuberculosis from human sputum cultures by PCR-RFLP analysis of hsp65 gene & pncA PCR.

BACKGROUND & OBJECTIVE: Identification of mycobacteria by conventional methods is slow, labour intensive and may at times fail to produce precise results. Molecular techniques developed in the recent past, overcome these disadvantages facilitating rapid identification of most species. We undertook this study to characterize mycobacteria isolated from sputa of human patients suspected to have tuberculosis by conventional methods and later, by polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) of hsp65 gene and pncA PCR. METHODS: Twenty two mycobacteria isolated from 30 sputum samples were identified based on growth rate, pigmentation, cultural and biochemical properties and subjected to PRA of hsp65 gene involving amplification of hsp65 gene and digestion of the product with BstEII and HaeIII in separate reactions and analysis of digests by 3 per cent agarose gel electrophoresis. The mycobacteria were simultaneously evaluated by M. tuberculosis-specific and M. bovis-specific pncA PCR assays in separate reactions. RESULTS: With the conventional biochemical tests, the 22 sputum culture isolates were identified as M. tuberculosis (19) and M. avium complex (MAC) (3). PCR of hsp65 gene yielded 439 bp product in all the mycobacteria tested. The RFLP patterns of three MAC isolates with BstEII and HaeIII were identical to reference M. avium strain with two fragments in each of the digest. M. intracellulare reference strain showed a distinct pattern with 3 fragments each in both enzyme digests. The PRA of hsp65 confirmed MAC isolates as M. avium. M. tuberculosis isolates including H37Rv and M. bovis strains could not be discriminated by PRA of hsp65. The two pncA PCR assays (M. bovis-specific and M. tuberculosis-specific) detected specifically the respective organisms with an amplification product of 185 bp. The MAC strains yielded no amplification product in both the pncA PCR assays. INTERPRETATION & CONCLUSION: PRA profiles of hsp65 could differentiate MAC isolates into M. avium and M. intracellulare but could not distinguish between M. tuberculosis and M. bovis. pncA PCR assays were found specific in detecting the respective mycobacterial species. The study confirms utility of pncA PCR assays in differential identification of M. tuberculosis and M. bovis and that of PRA of hsp65 in the identification of M. avium.