Detection of toxigenic strains of Aeromonas species in foods by a multiplex PCR assay

Abstract

Aeromonas hydrophila and other aeromonads are ubiquitous organisms found in meat, vegetables, drinking water and various other food items. They cause diarrhea and extra-intestinal infections in normal and immunocompromised patients. The aim of the study was to develop a multiplex PCR assay for the detection of virulenceassociated genes of Aeromonas including hemolysin (hlyA), aerolysin (aerA), glycerophospholipid-cholesterol acyl transferase (GCAT) alongwith a 16S rRNA gene. Internal amplifi cation control (IAC), which was coamplifi ed with aerA primers, was also included in this study. The results showed that all cultures of Aeromonas were accurately identifi ed by the assay without showing non-specifi city. A. hydrophila could be detected at a range of 10-50 CFU ml -1 from experimentally spiked fi sh, chicken and milk samples following overnight enrichment in alkaline peptone water supplemented with 10 μg/ml ampicillin (APW-A) by this multiplex PCR (mPCR). When evaluated on a total of 74 naturally occurring food samples, four samples were identifi ed to contain Aeromonas by mPCR. All these results were compared to the conventional culture, isolation and biochemical identifi cation procedures. The high throughput and cost-effective mPCR method developed in this study could provide a powerful tool for detection of pathogenic Aeromonas spp. from food and environmental samples and in addition, the method has advantages in terms of specifi city, sensitivity and ease of use compared to other reported PCR methods and DNA hybridization assays.

Detection of hemolytic strains of Aeromonas hydrophila and A. sobria along with other Aeromonas spp. from fish and fishery products by multiplex PCR

“…hydrophila. On the other hand the MPCR assay developed by Balakrishna et al (2010) could detect 50 CFU/g of A. hydrophila in fish samples based on aerolysin and hemolysin genes with overnight incubation in alkaline peptone water (APW-A) broth and the detection limit of the assay was 5 pg with pure genomic DNA. As the amplification of bacterial DNA directly from food complexes by PCR often give poor yield, enrichment of the samples prior to PCR has been recommended by many workers (Merino et al 1995).…”

“…2). The multiplex PCR assays developed by Nam and Joh (2007) and Balakrishna et al (2010) were specific for Aeromonas , as they did not amplify the selected virulent genes in E . coli, Vibrio cholerae, Salmonella typhimurium , Staphylococcus aureus and Proteus vulgaris.…”

Prevalence of antimicrobial resistant Aeromonas in chicken and fish washings

“…Since, laboratory procedures for isolation and identification of aeromonads are laborious and time consuming, several virulence associated genes have been targeted for detection of potentially pathogenic aeromonads by PCR including hemolysin, cytolysin, aerolysin genes (Wang et al, 2003;Aslani and Hamzeh, 2004;Balakrishna et al, 2010). Earliest report on development of PCR assay to detect aerolysin gene in A. hydrophila is from Pollard et al (1990) with a detection limit of 1 ng of total nucleic acid.…”

Prevalence of Hemolytic and Enterotoxigenic Aeromonas Spp. in Healthy and Diseased Freshwater Food Fishes as Assessed by Multiplex PCR

“…Yogananth et al (2009) found that 50% of the Aeromonas isolated from fish samples collected from local market in Chennai, India were positive for A. hydrophila. Balakrishna et al (2010) detected A. hydrophila from 8.5% of raw fish samples procured from different location in Mysore, India using a MPCR assay targeting virulence associated genes of Aeromonas including hlyA, aerA, GCAT, along with 16S rRNA gene. However, Bin Kingombe et al (2004) found that 59% of raw food samples were contaminated with Aeromonas harbouring virulent genes and the contamination of raw foods with Aeromonas was proportionally high in raw shrimps (56%) and fish (75%).…”