Molecular typing and antimicrobial resistance profiling of 33 mastitis-related Staphylococcus aureus isolates from cows in the Comarca Lagunera region of Mexico

Abstract

Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphylococcus aureus. Little is known about the S. aureus lineages causing mastitis in Mexican cattle. The aim of this study was to type S. aureus isolates causing mastitis in cows from the Comarca Lagunera region in Mexico in 2015-2016. Multi-locus variable number tandem repeat fingerprinting (MLVF) of 33 S. aureus isolates obtained from 210 milk samples revealed the MLVF clusters A (n = 1), B (n = 26), C (n = 5) and D (n = 1). Spa-typing showed that clusters A and B represent the spa-type t224, cluster C includes spa-types t3196 and t416, and cluster D represents spa-type t114. The different spa-types were mirrored by the masses of protein A bands as detected by Western blotting. Antimicrobial susceptibility testing showed that one isolate was susceptible to all antimicrobials tested, whereas all other strains were resistant only to benzylpenicillin. These findings show that only four S. aureus lineages, susceptible to most antimicrobials, were responsible for causing mastitis at the time of sampling. Lastly, many isolates carried the same small plasmid, designated pSAM1. The high prevalence of pSAM1 amongst the antimicrobial-susceptible isolates suggests an association with bovine colonization or mastitis rather than antimicrobial resistance.

Figure 1

Map of Mexico indicating the Comarca Lagunera region from which the mastitis isolates were collected. The Comarca Lagunera region is marked with blue and pink dots. This region includes 5 municipalities of the Coahuila state (blue dots) and 11 municipalities of the Durango state (pink dots). The map was created from Google Maps (Map data Copyright 2020 Google) and edited in Microsoft PowerPoint 2016.

Figure 2

MLVF dendrogram of 33 S. aureus isolates from cows with mastitis. PCR fragments of seven VNTRs of S. aureus were separated on a Bioanalyzer 2100 with microfluidic DNA 7500 chips. The dendrogram was constructed in silico from CSV files of the Bioanalyzer runs using GelCompar II software (Applied Maths; https://www.applied-maths.com/gelcompar-ii). The PCR fragments (clfA [1014-bp], clfB [850-bp], sdrD [735-bp] sdrC [651-bp] sdrE [603-bp], spa [195-bp] sspA [110-bp]) obtained for the control DNA of S. aureus USA300 are indicated. For the generation of the dendrogram, a cut-off value of 81% was used, yielding a concordance of 0.980. The respective MLVF clusters (A-D), the respective spa-types, and the presence of the nuc gene and plasmid pSAM1 are specified. The Figure was created with Microsoft PowerPoint 2016.

Figure 3

Schematic representation of the polymorphic VNTRs associated with identified spa-types. The order of identified VNTRs of the identified spa-types t224, t416, t3196 and t114 as detected in the present study isolates is schematically represented. The Figure was created with Microsoft PowerPoint 2016.

Figure 4

Western blotting analysis to detect the presence of SdrE and protein A. Proteins in the cellular fractions of S. aureus strains RF122 and Newman, and the mastitis isolates F-1, B-1, G-1 and E-3 were separated by LDS-PAGE and transferred to a nitrocellulose membrane. To visualize the presence of SdrE and protein A, the membrane was incubated with SdrE-specific rabbit antibodies (1:5000) and, subsequently, with an IRDye800CW-labelled goat anti-rabbit IgG secondary antibody (1:10,000). Note that protein A is detected by its capacity to bind rabbit IgG. The sizes of the marker proteins are indicated in kDa at the left side of the membrane. On the right side, the position of SdrE- and protein A-specific protein bands is marked. The complete original Western blot is presented as Supplemental Fig. S2. The Figure was created with Microsoft PowerPoint 2016.

Figure 5

Schematic representation of open reading frames on plasmid pSAM1. The relative positions of open reading frames (repL, 2, 3, 4, 5 and 6) are marked by arrows. Predicted stem-loop structures are indicated by lollypops. Regions of sequence identity between different plasmids are indicated by red lines, in which mutations (*) and insertions (∆) are marked. The names of the homologous plasmids are indicated on the left. The Figure was created with Microsoft PowerPoint 2016.