Identification of enterococci by MALDI-TOF-MS & 16S rRNA sequencing isolated from squeezed cheeses and evaluation of antibiotic susceptibility and antibacterial activity

Objective: This study aims to identify enterococci isolated from squeezed cheeses by MALDI-TOF-MS and 16S rRNA sequence analysis and to evaluate antibiotic resistance and antibacterial effects of these isolates against some food pathogens. Methods: Identification of 84 Enterococcus isolates obtained from squeezed cheese was carried out using MALDI-TOF-MS and 16S rRNA gene sequencing. The isolates were tested for resistance to 14 different antibiotics by the disc diffusion method. The antimicrobial effects of isolates against various food pathogens were determined by the agar spot test. SPSS 22.0.0 software (SPSS Inc., Chicago, USA) was used for the assessment of the correlation between variance analysis (ANOVA, F test) and antibacterial results. Results: As a result of sequence analysis; 33 (39.3%) were described to E. faecalis, 29 (34.5%) to E. faecium, 14 (16.7%) to E. durans, 4 (4.8%) to E. gallinarum, 3 (3.5%) to E. casselifavus and 1 (1.2%) to E. thailandicus. Eighty-one isolates gave the same identification result as the MALDI-TOF-MS method (96.43%). The difference between two identification methods has not been found to be statistically significant (p>0.05); however, MALDI-TOF-MS has some advantages over 16S rRNA sequencing, such as being less labor-intensive, more economical and faster. In total, 83.3% of the strains exhibited multidrug-resistant phenotypes. A high incidence of resistance was detected for nalidixic acid, oxacillin, and streptomycin. E. faecalis isolates were found to show lower sensitivity to antibiotics tested than E. faecium (p <0.05). The anti-listerial effect of E. faecalis was determined among all enterococcal isolates (p <0.05). Along with this, a strong correlation was found between L. innocua and L. monocytogenes inhibition. The results of antibacterial activity revealed that enterococci are more effective for the inhibition of Gram-positive food pathogens. Conclusion: The correlation between the results from the two identification methods showed that MALDI-TOF-MS is a fast, economical, robust, and reliable method for the characterization of enterococci. When the results were examined in terms of food safety, it was observed that squeezed cheese produced from raw milk without using starter culture was reservoirs of Enterecoccus spp. with multiple antibiotic resistance. Enterococci, which does not carry multiple antibiotic resistance, can be used in starter culture combinations and in hurdle technology to prevent the growth of Grampositive food pathogens; however, to this end, virulence determinants must be determined to achieve these goals.