Estimation of epidemiological cut-off values for eight antibiotics used for treatment of bovine mastitis caused by Streptococcus uberis and Streptococcus dysgalactiae subsp. dysgalactiae.

Interpretive criteria for antimicrobial susceptibility testing are lacking for most antimicrobials used for bovine streptococcal mastitis. The objectives of this study were to determine (tentative) epidemiological cut-off ((T)ECOFF) values for clinically relevant antibiotics used for treatment of bovine mastitis, and to estimate the proportion of acquired resistance (non-wild-types) in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus uberis. A total of 255 S. uberis and 231 S. dysgalactiae subsp. dysgalactiae isolates were obtained in Denmark and Norway from bovine mastitis. The isolates were tested for susceptibility to 10 antibiotics using broth microdilution. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard operating procedure, additional published MIC distributions were included for the estimation of ECOFFs for cloxacillin, cephapirin, lincomycin and tylosin, and TECOFFs for amoxicillin, benzylpenicillin, cephapirin and oxytetracycline. The proportion of non-wild-type (NWT) isolates for the beta-lactams was significantly higher in the Danish S. uberis (45-55%) compared to the Norwegian isolates (10-13%). For oxytetracycline, the proportion of NWT was significantly higher in the Danish isolates, both for S. uberis (28% vs. 3%) and S. dysgalactiae (22% vs. 0%). A bridging study testing in parallel MICs in a subset of isolates (n = 83) with the CLSI-specified and the EUCAST-specified broths showed excellent correlation between the MICs obtained with the two methods. The new ECOFFs and TECOFFs proposed in this study can be used for surveillance of antimicrobial resistance, and - for antimicrobials licensed for streptococcal bovine mastitis - as surrogate clinical breakpoints for predicting their clinical efficacy for this indication.

Comparative Genomics of Staphylococcus rostri, an Undescribed Bacterium Isolated from Dairy Mastitis.

This study characterizes 81 S. rostri isolates from bovine mastitis (of which 80 were subclinical). The isolates were first identified as S. microti by MALDI-TOF MS, but later whole genome sequencing analysis allowed reclassification as S. rostri. The isolates were derived from 52 cows and nine dairy herds in Denmark. To describe the pathogenicity of S. rostri, we used whole genome sequencing to infer the distribution of genes associated with virulence, antibiotic resistance, and mobile genetic elements. Also, we performed a core-genome phylogeny analysis to study the genetic relatedness among the isolates. All 81 isolates expressed the same virulence profile comprising two putative virulence genes, clpP and clpC. Three isolates carried a resistance gene encoding streptomycin (str) or lincomycin (lnuA) resistance. The distribution of plasmids suggested the detected antibiotic resistance genes to be plasmid-mediated. Phages were abundant among the isolates, and the single isolate from clinical mastitis acquired a phage disparate from the rest, which potentially could be involved with virulence in S. rostri. The core genome phylogeny revealed a strong genetic intra-herd conservation, which indicates the source of introduction being herd-specific and might further imply the ability of S. rostri to adapt to the bovine niche and spread from cow-to-cow in a contagious manner. With this study, we aim to acquaint clinicians and professionals with the existence of S. rostri which might have been overlooked so far.

First Finding of a Methicillin-Resistant Staphylococcus aureus (MRSA) t304/ST6 from Bovine Clinical Mastitis.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) comprises a global threat to humans and animals. Here, we report and characterize the MRSA t304/ST6 variant which, to our knowledge, represents the first case found in bovine clinical mastitis. In general, the MRSA t304/ST6 variant is rarely described in livestock, contrary to humans where it is widely recognized. Phenotypic and genotypic resistance profiling showed that the bovine-MRSA t304/ST6 isolate expressed low susceptibility toward cefoxitin (MICcefoxitin = 16 µg/mL) and carried the mecA resistance gene in the SCCmec IVa. The bovine-MRSA t304/ST6 isolate carried a plasmid similar to that which has been frequently observed among human-MRSA t304/ST6 isolates in Denmark (GenBank accession no. NZ_CP047022). In addition, a Staphylococcus prophage 3 (ϕSA3) was detected, encoding an immune evasion cluster (IEC) of putative virulence genes associated with human host-specificity (sea, sak, and scn). Taken together, these findings suggest that the MRSA t304/ST6 found in this study represents a recent host-jump event, with human to cow transmission. This study emphasizes the importance of and the need for performance of antimicrobial resistance surveillance among bovine mastitis pathogens, including S. aureus and MRSA.

Microbiological Diagnoses on Clinical Mastitis-Comparison between Diagnoses Made in Veterinary Clinics versus in Laboratory Applying MALDI-TOF MS.

The present study compares the diagnoses on clinical bovine mastitis made in veterinary clinics using conventional diagnostic methods with diagnoses on the same samples made by a veterinary reference laboratory using MALDI-TOF MS as diagnostics. The study enables targeted and evidence-based consulting on prudent mastitis diagnostics and related antibiotic usage. In total, 492 samples from clinical mastitis were included. When applying MALDI-TOF MS as gold standard, only 90 out of 492 diagnoses made in veterinary clinics, equal to 18%, were correct. Four main findings were important: (1) the veterinary clinics overlooked contamination in mastitis samples; (2) the veterinary clinics only assigned 2 fully correct diagnoses out of 119 samples with mixed growth cultures; (3) the veterinary clinics made close to half of their diagnoses on pure culture erroneously; (4) the veterinary clinics applied a limited number of the relevant pathogen identifications on pure culture samples. Altogether, the present study shows that a large part of Danish clinical mastitis cases are misdiagnosed. Lack of correct diagnoses and diagnostic quality control may lead to the choice of wrong treatment and thus hamper prudent use of antibiotics. Hence, the present study warns a risk of overuse of antibiotics in Denmark. Consequently, the present study calls for training of veterinary clinics in diagnostics of mastitis pathogens and national guidelines on quality assurance of mastitis diagnostics.

In vitro Antimicrobial Resistance of Causative Agents to Clinical Mastitis in Danish Dairy Cows.

This study was undertaken to investigate the antimicrobial resistance patterns of major causative agents to clinical mastitis in Danish dairy cows collected in 2016 to provide data on the current resistance patterns. Such data may subsequently serve as basis for a guideline for prudent use of antimicrobial agents in mastitis treatment. In addition, this study serves as a baseline for future comparison. The minimum inhibitory concentrations in Escherichia coli (n = 62), Klebsiella pneumoniae (n = 18), Staphylococcus aureus (n = 63), coagulase-negative Staphylococci (CNS) (n = 49), Streptococcus uberis (n = 61), Streptococcus dysgalactiae (n = 33), and Streptococcus agalactiae (n = 13) were determined to antimicrobial agents representing most classes relevant for treatment. The occurrence of resistance in the 299 bacterial isolates in total was evaluated using Clinical and Laboratory Standards Institute clinical breakpoints or in-house breakpoint values. For E. coli, low resistance levels were detected, 11.3% being resistant to ampicillin while resistance to other compounds was lower or zero. In contrast, K. pneumoniae revealed frequent ampicillin resistance (83.3%), but was susceptible to most other antimicrobial agents tested. Staphylococci were susceptible to the majority of antimicrobial agents tested, only 17.7% of the S. aureus isolates and 22.4% of the CNS being resistant to penicillin. Species distribution of the CNS isolates revealed that Staphylococcus simulans, Staphylococcus chromogenes, and Staphylococcus epidermidis were the most prevalent species. One S. aureus and one S. chromogenes isolate was found to be cefoxitin resistant and confirmed as methicillin resistant by polymerase chain reaction detection of the mecA gene, showing that methicillin resistance in staphylococci is present. All species of streptococci were susceptible to penicillin. No other critical resistance was found in any species, and resistance was in general low to all clinically relevant compounds. We emphasize the need for continuous surveillance of antibiotic resistance in major mastitis pathogens and the need for harmonization of methods and interpretations.

Staphylococcus aureus infected embolic stroke upregulates Orm1 and Cxcl2 in a rat model of septic stroke pathology.

OBJECTIVE: Ischaemic brain lesions and brain abscesses are frequent in both human and animal cases of septic embolic stroke. However, existing models of brain infection do not reflect central aspects of septic embolic stroke. Our aim was to compare septic and non-septic embolic stroke in order to identify gene expressions, inflammatory mediators and brain damage in a rat model. METHODS: We created precisely located focal brain infarcts in a rat model of Staphylococcus aureus infected embolic stroke. To cause septic embolic stroke we used a fibrin-rich embolus with bacteria, while every rat in the control group received a non-infected embolus. 64 rats were randomized to receive sham-surgery, sterile embolic stroke or septic embolic stroke. All groups were compared for brain pathology, mortality, gene expressions and inflammatory mediators using histology and reverse transcription quantitative real-time PCR. RESULTS: Although infarct volumes did not differ, septic embolic stroke caused higher mortality than sterile embolic stroke (p=  0.002). Brain abscesses were observed only in the septic group. Approximately 400-500 fold increases were observed for Orm1 and Cxcl2 respectively (1.00E-08 < p < 1.92E-07) in the septic group compared to the sterile group, and these were the most dramatically regulated genes in septic embolic stroke compared to sterile embolic stroke. CONCLUSIONS: Septic embolic stroke caused brain abscesses, increased mortality and upregulated Orm1 and Cxcl2 gene expressions compared to non-infected embolic stroke. The dramatic Orm1 increase observed in the septic group is unprecedented and suggests a significant biological role of Orm1 during septic neuroinflammation.