Aims
To develop a pcsB‐based Loop‐mediated isothermal amplification (LAMP) method for the detection of Streptococcus agalactiae (GBS) in milk, tilapia and vaginal swabs.
Methods and Results
The sensitivity of the LAMP method using real‐time turbidity monitoring was 1 pg of template within 1 h at 64°C, 100‐fold higher than conventional PCR. The sensitivity of visual detection dropped an order of magnitude using SYBR Green I or hydroxynaphthol blue. The validity of the visual LAMP assay was assessed by the detection of GBS in 180 vaginal swabs from one hospital, 14 brain tissues samples of diseased tilapias from two fishponds and fresh milk of 67 dairy cattle from one farm. In total, 17 samples (4 vaginal swabs, 13 tilapia brain tissues but no milk sample) tested positive for GBS. Subsequent bacterial identification confirmed the specificity and reliability of the LAMP method. Molecular serotyping and multilocus sequence typing demonstrated that all 13 tilapia GBS isolates were identical (serotype Ia, ST7), whereas the four human GBS isolates were more diverse and could be classified into two serotypes (Ia, III) and four sequence types (ST19, ST23, ST24, ST862). Virulence gene testing showed that only the bac, rib and lmb genes were not present in all isolates. Antimicrobial susceptibility profiles of the isolates were basically consistent with their genotypes, except for sulphonamide and fluoroquinolone.
Conclusions
We developed a reliable pcsB‐based LAMP assay for GBS detection. Our results demonstrated that the prevalence of GBS was 92·9% among diseased tilapia, 2·2% among female patients and 0% on a dairy farm in Hainan.
Significance and Impact of the Study
The pcsB‐based LAMP method is suitable for GBS detection and contains great potential of application in dairy industry, aquiculture and clinical.
Twenty-five strains of ciprofloxacin-resistant lactic acid bacteria (LAB; 14 Lactobacillus spp. and 11 Streptococcus thermophiles spp.) isolated from commercial yogurts in China were analyzed in this study. For each of these strains, amino acid changes associated with quinolone resistance-determining regions (QRDRs) were investigated using PCR-based detection methods. The same methodology was used to identify the presence of plasmid-mediated quinolone resistance (PMQR) genes in LAB. Sequencing analyses and an efflux pump inhibition test (using reserpine) were also performed as part of the analysis. Our results showed that typical mutations corresponding to quinolone resistance were found in the QRDRs of LAB strains. Detected mutations included Ser80Leu in parC, and Ser83Leu and Glu87Asp in gyrA. In addition, a Tyr74Phe substitution in parC, which had not previously been reported to be associated with quinolone resistance, was observed in two Lactobacillus delbrueckii subsp. bulgaricus strains. For each of the tested strains, the presence of the efflux pump inhibitor, reserpine, resulted in a two-to eightfold reduction in the minimum inhibitory concentrations (MICs) of ciprofloxacin. However, PMQR genes were not observed in any of the strains analyzed. Our results suggest that mutations in the QRDRs or efflux pump could be involved in ciprofloxacin resistance, and that a combination of these mechanisms may lead to increased ciprofloxacin resistance in LAB strains.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.