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.

Identification of streptococci isolated from various sources by determination of cfb gene and other CAMP-factor genes.

In the present study, the CAMP-factor (cfb) gene of streptococci of serological group B (Streptococcus agalactiae) and the CAMP-factor (cfu) gene of S. uberis could be amplified by polymerase chain reaction. A cfb specific amplicon could be observed for all 128 phenotypically CAMP-positive S. agalactiae, for the phenotypically CAMP-negative S. agalactiae strain 74-360, and for 2 S. difficile reference strains. A cfu specific amplicon could be observed for all 7 phenotypically CAMP-positive S. uberis. Four S. agalactiae strains isolated from 4 cows with mastitis appeared to be phenotypically CAMP-negative and negative in the cfb gene PCR. The CAMP-positive and CAMP-negative isolates, including both S. difficile, could be identified as S. agalactiae by amplification of a S. agalactiae specific part of the V2 region of the 16S rRNA and a species-specific part of the 16S-23S rRNA intergenic spacer region. Amplification of an internal fragment of the cfb gene with a reduced annealing temperature yielded positive reactions not only for CAMP-positive S. agalactiae, but also for phenotypically CAMP-positive S. pyogenes (n = 4), S. canis (n = 28), and S. uberis (n = 7), indicating a close relation of the CAMP genes of these 4 species. The relation could be further demonstrated by sequencing the internal fragment of the CAMP-factor (cfg) gene of S. canis and comparing the sequence with those of S. agalactiae, S. pyogenes, and S. uberis.

[Examination of a novel wax teat bougie for treatment of wounds in the papillary lactiferous sinus of dairy cows with special regard to endoscopic documentation of mucosal reactions compared with conventional teat bougies and a teat cannula]. / Prüfung eines neuartigen Strichkanalstabes zur Behandlung von Strichkanalwunden unter besonderer Berücksichtigung der endoskopisch dokumentierten Schleimhautverträglichkeit im Vergleich zu konventionellen Zitzenstiften und Verweilkanülen.

The present study on a novel wax-teat bougie, developed by Dr. T. GEISHAUSER for treatment of wounds in the papillary lactiferous sinus of dairy cows, has been performed in two phases of investigation. In a first step, fourteen healthy dairy cows (Deutsch Schwarzbunt) in state of early lactation were dried off temporarily for five days, after checking milk samples of all quarters for six following-on days on their bacteriological status, number of somatic cells, conductivity and results of the California-Mastitis-Test (CMT). The drying off was performed on two Quarters with similar cell-count in milk samples each. In one teat of the selected pairs off udder quarters, the wax-bougie was applied (group I), the other corresponding udder quarters were used as a control (group II). After five days of drying off, lactation was started again after removing the bougies in treated quarters. For another five days milk samples out of the quarters in groups I and II were checked in the laboratory tests named above and mean values and increase differences of results were compared. Comparison of increase differences with t-test showed no statistically significant difference between drying off procedures with the wax-bougie and without any treatment in the parameters named above. Courses of counts of somatic cells, conductivity and CMT showed no significant differences between drying off probands by using the novel wax-bougie and drying off without further treatment. In the second phase of the study, fifteen lactating, healthy dairy cows as probands have been split in five groups. Three cows (twelve udder quarters) were dried off for five days by using the novel teat bougie (group D), in three groups (A,B,C) two different types of conventional teat bougies and one type of cannula were used, and, as a control three cows (group E) were dried off without any treatment. Before drying off the udder quarters and after starting lactation again, the condition of the mucosa in the teat papillary lactiferous sinus has been documented by means of endoscopy and the bacteriological status of milk samples has been determined. As a result all udder quarters dried off temporarily showed endoscopically detectable inflammatory-proliferating reactions in the mucosa of the papillary lactiferous sinus, whereas in cows treated with the novel wax-bougie (group D), as well as in the cows of the control group (E), there were no inflammatory reactions detectable at all. These different reactions, basing on the Fisher-test, have to be considered statistically significant (p < 0.001).

[Occurrence of Prototheca mastitis in dairy farms in Hesse]. / Vorkommen von Protothekenmastitiden in hessischen Milcherzeugerbetrieben.

During January 1994 and August 1996 from dairy farms in Hessia a total of 305,609 milk samples were investigated. Prototheca sp. as etiological agent of a mastitis was isolated from milk samples of seven dairy herds. According to our experiences and to several reports from various countries dealing with Prototheca infections in dairy herds, mastitis control programs should include Prototheca algae as potential pathogens. Mastitis due to this organism usually occurs in different semeiologies, one with clinical symptoms, and the other, more common type, as subclinical mastitis. In both cases, Prototheca organisms use to persist in the tissue of the mammary gland also during the dry period and antimicrobial treatment proves to be ineffective. Considering the wide distribution of these algae as saprophytes in the environment and in feces of several domestic animals, predisposing factors like a humid aerobic milieu and unsanitary milking conditions are necessary for Prototheca infections becoming manifest in the udder of dairy cows. Control measures should preferably stress the identification and removal of infected animals, in particular when the disease is sporadic in the herd. Due to the more questionable occurrence of spontaneous healing and the lack of an efficient drug, slaughtering of infected cows appears as a suitable method to eliminate the disease from the herd. Additionally, improvement of the hygiene status concerning feeding and milking management within a herd is as essential as in the control of other opportunistic udder pathogens.

[Endoscopic assessment of the mucosal tolerance of a new papillary duct bougie compared with conventional mammary cannulas]. / Endoskopische Prüfung der Schleimhautver-träglichkeit eines neuartigen Strichkanalstabes im Vergleich zu konventionellen Zitzenstiften und einer Verweilkanüle.

In this study on healthy, milking cows three per group a temporary drying off for five days, using two types of conventional teat bougies (group A, B), one type of a teat canula (group C) and one type of a novel wax-bougie, developed by Dr. Th. Geishauser (group D) was performed. As a control, three cows were dried off without using any teat bougies (group E). Before drying off the udder quarters, the condition of the mucosa in the teat papillary lactiferous sinus was documented by means of endoscopy. After five days, lactation was started and endoscopy was performed again. Endoscopical pictures and video tapes were evaluated for existence of inflammatory-proliferating reactions on the mucosa of the papillary lactiferous sinus. Comparison of the five groups with statistical methods (Fisher test) showed significant (P < 0.001) effects: all udder quarters, dried off temporarily by using conventional teat bougies and teat canulas (groups A, B, C) showed more or less severe alterations in the mucosa of the papillary lactiferous sinus, whereas in cows, treated with the novel wax bougie (group D) as well as in cows of the control group (E) there were no alterations detectable at all.