Antimicrobial resistance of Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus canis in quarter milk samples from Bavaria, Southern Germany, between 2012 and 2022.

The objective of this study was to analyze the in vitro antimicrobial resistance (AMR) of Streptococcus (Sc.) dysgalactiae, Sc. agalactiae, and Sc. canis over a 10-year period from 2012 to 2022 against the most commonly used antimicrobial agents. For this purpose, all quarter milk samples (QMS) submitted to the milk laboratory of the Bavarian Animal Health Service (TGD) were analyzed. Each QMS was tested using the California Mastitis Test (CMT) and categorized as negative (N), subclinical (S), or clinical (C) mastitis if the milk character was abnormal. Samples with Sc. dysgalactiae, Sc. agalactiae, or Sc. canis were included and a subset of isolates were further tested for in vitro antimicrobial resistance by breakpoint analysis with broth microdilution. Sc. dysgalactiae (61%, n = 65,750) was the most abundant pathogen among those 3 species, followed by Sc. agalactiae (28%, n = 30,486), and Sc. canis (11%, n = 11,336). All 3 species showed the highest resistance to the same 4 antimicrobial agents: erythromycin, marbofloxacin, pirlimycin, and cefalexin/kanamycin with varying degrees of resistance. Throughout the study period, Sc. dysgalactiae, Sc. agalactiae, and Sc. canis were largely susceptible to the remaining antimicrobial agents tested (penicillin, amoxicillin-clavulanate, oxacillin, cefazolin, cefoperazone, cefquinome). Only less than 14% of isolates of Sc. dysgalactiae and Sc. canis were resistant against any of the antimicrobials tested. Sc. agalactiae was the species with the highest percentage of resistant isolates. While the percentage of resistant isolates from Sc. canis and Sc. dysgalactiae decreased, the percentage of resistant Sc. agalactiae isolates increased since 2017. In summary, most isolates were not resistant to the most commonly used antimicrobial agents for mastitis therapy, including ß-lactam antibiotics and penicillin should remain the first-choice therapy against streptococcal mastitis.

Changes in antimicrobial resistance of Staphylococcus aureus in bovine quarter milk samples from Southern Germany between 2012 and 2022.

The objective of this study was to describe the in vitro resistance of Staphylococcus (S.) aureus from bovine quarter milk samples obtained by the udder health laboratory of the Bavarian Animal Health Services between 2012 and 2022. All S. aureus samples were tested for ß-lactamase production and only forwarded to further microbroth susceptibility testing either if the ß-lactamase result was positive or upon explicit request by the submitter. The growth of most S. aureus isolates was inhibited at the lowest evaluated minimum inhibitory concentration (MIC) of tested antimicrobials, with the MIC50 and MIC90 mostly beneath the respective breakpoint. On average, about a quarter (24%, n = 5,718) of tested isolates was resistant to erythromycin. However, the prevalence of resistant isolates dropped from 53% (n = 1,018) in 2012 to 8% (n = 113) in 2022. The second highest prevalence of in vitro resistance was to penicillin (17%, of all isolates tested for ß-lactamase production, n = 28,069). Less than 14% of isolates were resistant to the remaining assessed antimicrobial agents (cefoperazone, pirlimycin, kanamycin-cefalexin, marbofloxacin, amoxicillin-clavulanate, cefquinome, or cefazolin, respectively). Over the years, 4% (n = 959) of the S. aureus isolates selected for microbroth susceptibility testing (and 0.8% (n = 1,392) of all submitted S. aureus isolates) were methicillin-resistant S. aureus (MRSA), and 5% (n = 1,162) of S. aureus isolates were multidrug-resistant. However, there was an overall trend toward fewer resistant isolates. These findings are consistent with those of several European monitoring programs that reported a slight decrease of AMR of bovine S. aureus in countries where antibiotic use in veterinary medicine was reduced. Notably, isolates of clinical mastitis cases were consistently less likely to express in vitro resistance than isolates obtained from milk of healthy cows or subclinical mastitis cases. In conclusion, antimicrobial resistance (AMR) of S. aureus was decreasing and penicillin should remain the first-choice antimicrobial in the attempt of treating S. aureus intramammary infections in Bavaria.

In vitro antimicrobial resistance profiles of Streptococcus uberis, Lactococcus spp., and Enterococcus spp. from quarter milk samples of cows between 2015 and 2019 in Southern Germany.

The objective was to describe and compare antimicrobial resistance patters of esculin-hydrolyzing streptococci and streptococcal-like organisms (Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Lactococcus garvieae, Lactococcus lactis) from routine diagnostic samples of the udder health laboratory of the Bavarian Animal Health Services between 2015 and 2019. All routine diagnostic samples of the udder health laboratory of the Bavarian Animal Health Services, that were tested with a standard microbroth dilution, were eligible to be included in this retrospective case series. A California Mastitis Test result was available for all samples. Most Strep. uberis and L. lactis were susceptible to all antibiotics tested. Enterococcus faecium had consistently the highest minimum inhibitory concentration required to inhibit the growth of 90% of tested isolates. The resistance patterns of Lactococcus garvieae were positioned between enterococci and L. lactis. The minimum inhibitory concentration for various antibiotics and pathogens tended to decrease over the 5-yr period. Regardless of the pathogen, isolates of clinical cases were less likely to express in vitro resistance than isolates of healthy or subclinical cases. Streptococcus uberis or L. lactis showed hardly any in vitro resistance to tested antibiotic groups. Penicillin should remain the first-choice antimicrobial for the therapy of Strep. uberis and Lactococcus spp. However, a success of any antimicrobial treatment of enterococcal infections seems questionable.

Multiple cases of methicillin-resistant CC130 Staphylococcus aureus harboring mecC in milk and swab samples from a Bavarian dairy herd.

The discovery of a new mecA homolog, mecC, necessitates a modification of diagnostic procedures for the identification of methicillin-resistant Staphylococcus aureus (MRSA), as most assays used for the genotypic and phenotypic mecA detection cannot currently recognize mecC. Although the prevalence, distribution, and importance of mecC are not yet completely understood, an exchange of mecC-MRSA between humans and animals seems possible. All previously reported observations of mecC-positive strains have been sporadic. To the best of our knowledge, this is the first report about multiple cases of mecC-positive Staph. aureus in 1 dairy herd. Clonal complex 130 Staph. aureus harboring mecC were found in milk samples from 16 of 56 lactating cows kept in a herd in Bavaria, Germany. Almost all quarter milk samples positive for mecC-MRSA had the lowest possible California Mastitis Test score; composite somatic cell counts obtained from monthly milk recordings showed a mean of 51,600 cells/mL in mecC-MRSA affected cows. Additionally, mecC-positive clonal complex 130 Staph. aureus were detected in swab samples from the mammary skin and a teat lesion of 1 cow from this herd. This report suggests that mecC-carrying strains are able to spread among livestock, and that they have the ability to cause multiple cases in single herds. Therefore, future studies targeting MRSA in dairy cows need to consider mecC.

Hot topic: Bovine milk samples yielding negative or nonspecific results in bacterial culturing--the possible role of PCR-single strand conformation polymorphism in mastitis diagnosis.

A large proportion of mastitis milk samples yield negative or nonspecific results (i.e., no mastitis pathogen can be identified) in bacterial culturing. Therefore, the culture-independent PCR-single strand conformation polymorphism method was applied to the investigation of bovine mastitis milk samples. In addition to the known mastitis pathogens, the method was suitable for the detection of fastidious bacteria such as Mycoplasma spp., which are often missed by conventional culturing methods. The detection of Helcococcus ovis in 4 samples might indicate an involvement of this species in pathogenesis of bovine mastitis. In conclusion, PCR-single-strand conformation polymorphism is a promising tool for gaining new insights into the bacteriological etiology of mastitis.

Molecular characterization of phenotypically CAMP-negative Streptococcus agalactiae isolated from bovine mastitis.

In the present study three phenotypically CAMP-negative Streptococcus agalactiae, isolated from three cows with mastitis, were characterized by molecular analysis. An identification of the S. agalactiae was performed by conventional methods and by PCR amplification of species specific parts of the 16S rRNA gene and the 16S-23S rDNA intergenic spacer region. In addition all three phenotypically CAMP-negative isolates harboured a normal sized CAMP-factor encoding cfb gene indicating a reduced expression of CAMP-factor or a gene defect elsewhere along the pathway of expression. The clonal identity of the three isolates could be demonstrated by macrorestriction analysis of their chromosomal DNA.