Isolation and Characterization of Staphylococcus aureus from Raw Cow's Milk and Investigating the Effect of Bifidobacterium bifidum Probiotic Cell Free Supernatant on Their Enterotoxins Genes Expression

Abstract

The present reserach aimed to detect and isolate the genes involved in the staphylococcal enterotoxins (SEs) production in strains isolated from unprocessed cow's milk and to examine the impact of Bifidobacterium bifidum probiotic cell-free supernatant (CFS) on their expression. Standard techniques were used for isolation and identification of Staphylococci strains in unprocessed milk. The PCR was used to identify strains carrying enterotoxin genes. The B. bifidum CFS was applied to strains containing the target genes, and the genes expression levels were quantified using Real-time PCR. Using 16SrDNA sequencing, the phylogenic relationship of the isolated strains was determined. Analysis revealed that bacteria such as Staphylococcus species were found in the 72% of the samples. The PCR test showed the presence of various SE superantigens, including SEA (16.7%), SEC (11.7%), SED (8.3%), SEE (6.7%), and SEB (1.7%) in isolated strains. The B. bifidum CFS had obvious antimicrobial activity against strains 24, 51, 54, and 35 of Staphylococcus species, and the minimum inhibitory concentration and minimum bactericidal concentration values for these strains treated with B. bifidum CFS were in the range of 31.25-125 μg/ml. Strains 51 and 24 were clustered with S.aureus ATCC 25923, and strains 54 and 35 were clustered with S.aureus ATCC 12600, respectively. The RT-PCR exhibited that probiotics CFS suppressed the expression of SEA, SEB, SEC, and SEE genes (P<0.05). The average fold change for SEA, SEB, SEC, and SED genes was -1.681, -1.28, -1.52, and -0.84, respectively. The research demonstrated that probiotic bacteria can lower enterotoxin production by downregulating the expression of SEs genes.

Keywords: Enterotoxin; Bifidobacterium bifidum; Staphylococcus aureus.

Figure 1

Agarose gel electrophoresis of PCR products of random samples. The M Lane: 100bp ladder, +: positive control, -: negative control

Figure 2

Quantitative RT-PCR demonstrating fold changes of SEA and SEB genes in CFS-treated strains. Strain numbers were expressed within the columns. Data represent the averages from three independent experiments. Error bars represent the standard deviations from the means. *: P < 0.05; **: P < 0.01, ***: P < 0.001 versus control group.

Figure 3

Quantitative real-time PCR showing fold changes of SEC, SED, and SEE genes in CFS-treated strains. Strain numbers were expressed within the columns. Data represent the averages from three independent experiments. Error bars represent the standard deviations from the means. *: P < 0.05; **: P < 0.01, ***: P < 0.001 versus control group.

Figure 4

Phylogenetic relationship of strains based on 16S rDNA sequences of the four isolates, outgroup, and type strains