Development of ISSR-derived SCAR marker-targeted PCR for identification of Aspergillus section Flavi members.

UNLABELLED: Aspergillus section Flavi is a heterogeneous fungal cluster including some of the most economically important Aspergillus species. The section is comprised of toxigenic and nontoxigenic aspergilli that are phenotypically undistinguishable. The aim of this study was to develop a genetic marker specific to Aspergillus section Flavi on the whole. Based on inter-simple sequence repeat (ISSR) fingerprinting profiles of major Aspergillus section Flavi members, a sequence-characterized amplified region (SCAR) marker was identified. Primers were designed in the conserved regions of the SCAR marker and were utilized in a PCR for concurrent identification of major members of the section. The detection level of the SCAR-PCR was found to be 0·1 ng purified DNA, and when applied to 45 naturally contaminated food samples, 28 samples were found infected with Aspergillus section Flavi members. The present SCAR-PCR is rapid and less cumbersome unlike conventional identification techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of Aspergillus section Flavi members is important owing to their impact on human health and economy. The ISSR-based SCAR-PCR developed in this study is superior over the other existing Aspergillus section Flavi detection systems due to its simplicity and minimal requirement of sample handling. This PCR could be a supplementary strategy to time-consuming and rather ambiguous conventional polyphasic detection techniques and a reliable tool for high-throughput sample analysis.

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications.

AIM: To evaluate an inter-generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. METHODS AND RESULTS: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap-extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r-αCS specifically detected 42-kDa and 33-kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild-type toxins in vitro. CONCLUSIONS: The r-αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild-type toxins in vitro. SIGNIFICANCE OF THE STUDY: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co-infections like gangrenous ischaemia, gangrenous mastitis, etc.

Molecular characterization of lactic acid bacteria recovered from natural fermentation of beet root and carrot Kanji.

The lactic acid bacteria (LAB) play an important role in the fermentation of vegetables to improve nutritive value, palatability, acceptability, microbial quality and shelf life of the fermented produce. The LAB associated with beetroot and carrot fermentation were identified and characterized using different molecular tools. Amplified ribosomal DNA restriction analysis (ARDRA) provided similar DNA profile for the 16 LAB strains isolated from beetroot and carrot fermentation while repetitive extragenic palindromic PCR (rep-PCR) genotyping could differentiate the LAB strains into eight genotypes. Thirteen strains represented by five genotypes could be clustered in five distinct groups while three LAB strains exhibiting distinct genotypes remained ungrouped. These genotypes could be identified to be belonging to L. plantarum group by 16S rDNA sequencing. The recAnested multiplex PCR employing species-specific primers for the L. plantarum group members identified the LAB strains of six genotypes to be L. paraplantarum and the other two genotypes to be L. pentosus. Three genotypes of L. paraplantarum were consistently found on the third and sixth day of beetroot fermentation whereas a distinct genotype of L. paraplantarum and L. pentosus appeared predominant on the tenth day. From carrot Kanji two distinct genotypes of L. paraplantarum and one genotype of L. pentosus were identified. REP-PCR DNA fingerprinting coupled with 16S rDNA sequencing and recA-nested multiplex PCR could clearly identify as well as differentiate the diverse L. plantarum group strains involved in the fermentation.

Antimicrobial susceptibility and molecular characterization of Vibrio cholerae from cholera outbreaks in Chennai.

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002-2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002-2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.