Characterization and molecular epidemiology of Staphylococcus aureus strains resistant to beta-lactams isolated from the milk of cows diagnosed with subclinical mastitis

Abstract

Background and aim: The term ESKAPE, recognized by the WHO, is an acronym, which refers to the pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., which is extremely virulent and multidrug-resistant. Although the term is used to designate nosocomial pathogens, in a milking environment, strains of Methicillin-resistant S. aureus have been isolated from cattle diagnosed with clinical and subclinical mastitis. Resistant strains may be involved in the transfer of genes conferring resistance to beta-lactam antimicrobials among the species of microorganisms related to mastitis etiology. This study aimed to trace the phenotypic and genotypic profiles of susceptibility to beta-lactams in S. aureus isolated from milk of cattle diagnosed with subclinical mastitis obtained from different rural properties located in the North of Minas Gerais State, Brazil.

Materials and methods: Sixteen microorganisms previously identified as S. aureus isolated from milk of cattle diagnosed with subclinical mastitis were submitted to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF), mass spectrometry, and polymerase chain reaction (PCR) analysis for microbial species confirmation. The S. aureus beta-lactams antimicrobial phenotypic resistance profile was investigated by disk diffusion method. PCR methods were also performed to investigate the S. aureus genotypic beta-lactams resistance profile. For this purpose, bla _Z, mec A, mec _ALGA251, bla _Oxa23, and bla _KPC genes were screened among S. aureus isolates. The genetic diversity of S. aureus by fingerprint random amplified polymorphic DNA (RAPD)-PCR was also performed in this study.

Results: All isolates showed phenotypic resistance to at least three beta-lactams, among which was meropenem. None of the isolates tested positive for the genes mec _ALGA251, bla _Oxa23, and bla _KPC; however, the presence of the genes bla _Z and mecA was detected among the isolates. The fingerprint analysis divided isolates into two distinct groups and 15 different subgroups. Despite the presence of clonality among the isolates, the PCR-RAPD analysis unveiled a heterogeneous profile with genetic diversity among the S. aureus isolates.

Conclusion: In this study, we identified beta-lactams resistant S. aureus strains isolated from the milk of cows diagnosed with subclinical mastitis. The S. aureus beta-lactams resistance was investigated using a phenotypic and genotypic approach. We believe that molecular epidemiology, improved knowledge, and genetic basis of resistance to beta-lactams might assist in asserting guidelines for better management practices of dealing with subclinical mastitis and mapping of origin of resistant pathogens in the studied Brazilian area.

Keywords: Staphylococcus aureus; beta-lactams; genetic diversity; infection; resistance genes.

Figure-1

Polymerase chain reaction (PCR) optimization with positive controls for universal bacteria 16S rDNA, bla_OXA23,bla_KPC,bla_Z, mecA, and mec_ALG250 genes _. M: Molecular weight marker Mid-Range (Cellco Biotecnologia), Line 1: PCR Staphylococcus aureus strain ATCC 43300 for detection of universal bacteria 16S rDNA gene (~370pb). Line 2: PCR nosocomial Klebsiella pneumoniae isolate for detection of bla_KPC gene (~876pb). Line 3: PCR Acinetobacter baumannii isolate for detection of bla_OXA23 gene (~1057pb) Line 4: PCR S. aureus strain ATCC 43300 for detection of bla_Z gene (~377pb). Line 5: PCR S. aureus strain ATCC 43300 for detection of mecA gene (533pb ). Line 6: PCR S. aureus strain ATCC 43300 for detection of gene mec_ALGA251 (~356pb). Electrophoresis on 1.5% agarose gel.

Figure-2

Random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and dendrogram of genetic relationship of the Staphylococcus isolated from milk of bovines diagnosed with subclinical mastitis. Panel A: Electrophoresis on 1.8% agarose gel containing RAPD-PCR amplicons of five isolates of Staphylococcus aureus. M: Molecular weight marker Mid-Range (Cellco Biotecnologia), Line 1 to 5: RAPD-PCR of S. aureus FL2, FL3, MU1, TR3, and SL2 isolates. Panel B: Dendrogram of genetic relationship of the Staphylococcus aureus (n=16) isolated from milk of bovines diagnosed with subclinical mastitis. Dendrogram of genetic relationship of the Staphylococcus aureus (n=16) isolated from milk of bovines diagnosed with subclinical mastitis. The 16 isolates were divided into two groups (I and II) according to the degree of similarity based on the Euclidean distance calculation. The RAPD-PCR profile of standard strain of S. aureus ATCC 43300 was used for the dendrogram generation (corresponding to 17).