The Characterization and Beta-Lactam Resistance of Staphylococcal Community Recovered from Raw Bovine Milk

Abstract

Staphylococci is an opportunistic bacterial population that is permanent in the normal flora of milk and poses a serious threat to animal and human health with some virulence factors and antibiotic-resistance genes. This study was aimed at identifying staphylococcal species isolated from raw milk and to determine hemolysis, biofilm, coagulase activities, and beta-lactam resistance. The raw milk samples were collected from the Düzce (Türkiye) region, and the study data represent a first for this region. The characterization of the bacteria was performed with MALDI-TOF MS and 16S rRNA sequence analysis. The presence of coa, icaB, blaZ, and mecA was investigated with PCR. A nitrocefin chromogenic assay was used for beta-lactamase screening. In this context, 84 staphylococci were isolated from 10 different species, and the dominant species was determined as S. aureus (32.14%). Although 32.14% of all staphylococci were positive for beta hemolysis, the icaB gene was found in 57.14%, coa in 46.42%, mecA in 15.47%, and blaZ in 8.33%. As a result, Staphylococcus spp. strains that were isolated from raw milk in this study contained some virulence factors at a high level, but also contained a relatively low level of beta-lactam resistance genes. However, considering the animal-environment-human interaction, it is considered that the current situation must be monitored constantly in terms of resistance concerns. It must not be forgotten that the development of resistance is in constant change among bacteria.

Keywords: beta-lactamase; biofilm; blaZ; coagulase; mecA; staphylococci; virulence.

Figure 1

Staphylococcus species isolated from raw milk.

Figure 2

Dispersion of MALDI-TOF MS Scores.

Figure 3

Staphylococcus spp. on CRA: (A) Strong slime-forming strain. (B) Non-producing slime. (C) Weak slime-forming strains.

Figure 4

The images of disk diffusion assay on S. aureus.

Figure 5

Chromogenic disk assay: (A,C) Beta-lactamase positive strains. (B,D) Beta-lactamase negative strains.

Figure 6

PCR results: (A) PCR bands for icaB (880 bp), 1,2: Positive sample, 3: Negative sample. (B) PCR bands for blaZ (421 bp) and coa (variable), 1: Positive control for blaZ, 2,3: Positive samples for blaZ, which is from the S. aureus strains, 4,5: Positive result for coa is almost 850 bp, 6,7: Negative samples, 8: Positive result for coa is almost 550 bp. (C) PCR bands for mecA (583 bp), 1,3: Positive samples for mecA, which is from the S. epidermidis strains, 2: Negative sample. M: Marker, 100–1000 bp DNA ladder is used.

Figure 7

Distribution of virulence genes within the species: This table does not give the general distribution of virulence genes. In this table, information is given about how many of a species detected in the study have which gene. For example, while all S. epidermidis strains were icaB positive, more than 30% of them were positive for coa, mecA, and blaZ. At least six different species in the study contain one or more virulence genes. In the remaining four species, the virulence genes investigated in this study could not be detected.