Treatment of mild to moderate clinical bovine mastitis caused by gram-positive bacteria: A noninferiority randomized trial of local penicillin treatment alone or combined with systemic treatment.

Bovine mastitis is one of the most important diseases in modern dairy farming, as it leads to reduced welfare and milk production and increased need for antibiotic use. Clinical mastitis in Denmark is most often treated with a combination of local and systemic treatment with penicillin. The objective of this randomized clinical trial was to assess whether worse results could be expected with local intramammary treatment with penicillin compared with a combination of local and systemic treatment with penicillin in terms of the bacteriological cure of mild and moderate clinical mastitis cases caused by gram-positive bacteria. We carried out a noninferiority trial with a noninferiority margin set to a relative reduction in bacteriological cure of 15% between these 2 treatment groups to assess the effect of reducing the total antibiotic use by a factor of 16 for each treated case. Clinical mastitis cases from 12 Danish dairy farms were considered for enrollment. On-farm selection of gram-positive cases was carried out by the farm personnel within the first 24 h after a clinical mastitis case was detected. A single farm used bacterial culture results from the on-farm veterinarian, whereas the other 11 farms were provided with an on-farm test to distinguish gram-positive bacteria from gram-negative or samples without bacterial growth. Cases with suspected gram-positive bacteria were allocated to a treatment group: either local or combination. Bacteriological cure was assessed based on the bacterial species identified in the milk sample from the clinical mastitis case and 2 follow-up samples collected approximately 2 and 3 wk after ended treatment. Identification of bacteria was carried out using MALDI-TOF on bacterial culture growth. Noninferiority was assessed using unadjusted cure rates and adjusted cure rates from a multivariable mixed logistic regression model. Of the 1,972 clinical mastitis cases registered, 345 (18%) met all criteria for inclusion (full data). The data set was further reduced to 265 cases for the multivariable analysis to include only complete registrations. Streptococcus uberis was the most commonly isolated pathogen. Noninferiority was demonstrated for both unadjusted and adjusted cure rates. The unadjusted cure rates were 76.8% and 83.1% for the local and combined treatments, respectively (full data). The pathogen and somatic cell count before the clinical case had an effect on the efficacy of treatment; thus efficient treatment protocols should be herd- and case-specific. The effect of pathogen and somatic cell count on treatment efficacy was similar irrespective of the treatment protocol. We conclude that bacteriological cure of local penicillin treatment for mild and moderate clinical mastitis cases was noninferior to the combination of local and systemic treatment using a 15% noninferiority margin. This suggests that a potential 16-fold reduction in antimicrobial use per mastitis treatment can be achieved with no adverse effect on cure rate.

Genotyping and Antimicrobial Susceptibility Profiling of Streptococcus uberis Isolated from a Clinical Bovine Mastitis Outbreak in a Dairy Farm.

Streptococcus uberis, an environmental pathogen responsible also for contagious transmission, has been increasingly implicated in clinical mastitis (CM) cases in Europe. We described a 4-month epidemiological investigation of Strep. uberis CM cases in an Italian dairy farm. We determined molecular characteristics and phenotypic antimicrobial resistance of 71 Strep. uberis isolates from dairy cows with CM. Genotypic variability was investigated via multiplex PCR of housekeeping and virulence genes, and by RAPD-PCR typing. Antimicrobial susceptibility was assessed for 14 antimicrobials by MIC assay. All the isolates carried the 11 genes investigated. At 90% similarity, two distinct clusters, grouping 69 of the 71 isolates, were detected in the dendrogram derived from the primer ERIC1. The predominant cluster I could be separated into two subclusters, containing 38 and 14 isolates, respectively. Strep. uberis strains belonging to the same RAPD pattern differed in their resistance profiles. Most (97.2%) of them were resistant to at least one of the drugs tested, but only 25.4% showed a multidrug resistance phenotype. The highest resistance rate was observed for lincomycin (93%), followed by tetracycline (85.9%). This study confirmed a low prevalence of ß-lactam resistance in Strep. uberis, with only one isolate showing resistance to six antimicrobial classes, including cephalosporins.

Genomic investigation of Staphylococcus aureus isolates from bulk tank milk and dairy cows with clinical mastitis.

Staphylococcus aureus is one of the most common pathogens that cause mastitis in dairy cows. Various subtypes, virulence genes and mobile genetic elements have been associated with isolates from bulk tank milk and clinical mastitis. So far, no Danish cattle associated S. aureus isolates have been whole-genome sequenced and further analyzed. Thus, the main objective was to investigate the population structure and genomic content of isolates from bulk tank milk and clinical mastitis, using whole-genome sequencing. This may reveal the origin of strains that cause clinical mastitis. S. aureus isolates from bulk tank milk (n = 94) and clinical mastitis (n = 63) were collected from 91 and 24 different farms, respectively and whole-genome sequenced. The genomic content was analyzed and a phylogenetic tree based on single nucleotide polymorphisms was constructed. In general, the isolates from both bulk tank milk and clinical mastitis were of similar genetic background. This suggests that dairy cows are natural carriers of the S. aureus subtypes that cause clinical mastitis if the right conditions are present and that a broad range of subtypes cause mastitis. A phylogenetic cluster that mostly consisted of ST151 isolates carried three mobile genetic elements that were primarily found in this group. The prevalence of resistance genes was generally low. However, the first ST398 methicillin resistant S. aureus isolate from a Danish dairy cow with clinical mastitis was detected.

Brain microabscesses in a porcine model of Staphylococcus aureus sepsis.

BACKGROUND: Sepsis caused by Staphylococcus aureus often leads to brain microabscesses in humans. Animal models of haematogenous brain abscesses would be useful to study this condition in detail. Recently, we developed a model of S. aureus sepsis in pigs and here we report that brain microabscesses develop in pigs with such induced S. aureus sepsis.Twelve pigs were divided into three groups. Nine pigs received an intravenous inoculation of S. aureus once at time 0 h (group 1) or twice at time 0 h and 12 h (groups 2 and 3). In each group the fourth pig served as control. The pigs were euthanized at time 12 h (Group 1), 24 h (Group 2) and 48 h (Group 3) after the first inoculation. The brains were collected and examined histopathologically. RESULTS: All inoculated pigs developed sepsis and seven out of nine pigs developed brain microabscesses. The microabscesses contained S. aureus and were located in the prosencephalon and mesencephalon. Chorioditis and meningitis occurred from 12 h after inoculation. CONCLUSIONS: Pigs with experimental S. aureus sepsis often develop brain microabscesses. The porcine brain pathology mirrors the findings in human sepsis patients. We therefore suggest the pig as a useful animal model of the development of brain microabscesses caused by S. aureus sepsis.

Intravenous inoculation of Staphylococcus aureus in pigs induces severe sepsis as indicated by increased hypercoagulability and hepatic dysfunction.

Nine pigs were inoculated intravenously once or twice with 10(8) Staphylococcus aureus per kilogram body weight and sacrificed 12, 24 and 48 h after inoculation. Three sham-infected pigs served as controls. Blood samples were taken for bacteriology, haematology and clinical chemistry. A necropsy was carried out and tissue samples were collected for bacteriology and histology. The onset of clinical disease was seen at 7-8 h after inoculation. The blood bacterial counts remained low throughout the study. All infected pigs developed sepsis characterized by fever, neutrophilia, increased levels of C-reactive protein (CRP) and IL-6, and decreased levels of serum iron. The CRP and IL-6 levels peaked at 36 h, whereas IL-1beta and tumour necrosis factor-alpha showed no obvious changes. Thromboelastography showed increasing hypercoagulability from 12 h and onwards, whereas the platelet numbers declined slightly throughout the experiment. The levels of serum aspartate aminotransferase and bilirubin were elevated at 24 and 36 h. In conclusion, sepsis and severe sepsis were induced as evidenced by dysfunction of the blood clotting system and the liver.