Bovine mastitis bacteria resolved by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method to identify the most common pathogenic bacteria in humans and animals. The goals of this study were to amend a commercial database with additional species, evaluate the amended database for identification of bacterial genera and species causing bovine mastitis, and describe the plethora of species involved. In total, 500 udder pathogenic isolates were subjected to MALDI-TOF MS using bacterial or fungal colony material; 93.5% could be identified to the species level, and 6.5% were identified only to the genus level. Isolates identified to the genus level required further identification to the species level by conventional methods or 16S rDNA sequencing. Mass spectra from verified species were used to expand the MALDI-TOF MS database to improve future identification ability. A total of 24 genera and 61 species were identified in this study. Identified isolates were mainly staphylococci, streptococci, Enterobacteriaceae, and coryneforme bacteria. In conclusion, MALDI-TOF MS is a powerful, rapid, and reliable technique to identify the most common microorganisms causing bovine mastitis, and the database can be continuously expanded and improved with additional species.

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.

Typeability of MALDI-TOF assay for identification of non-aureus staphylococci associated with bovine intramammary infections and teat apex colonization.

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), a culture-dependent assay, has recently been implemented for routine identification of non-aureus staphylococci (NAS) species from milk, but the assay has never been investigated for NAS from nonmilk or environmental samples. The objective of this study was to evaluate the typeability of the MALDI-TOF assay for the identification and differentiation of bovine-associated NAS species on aseptically collected quarter milk and teat skin samples in dairy herds. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabs and foremilk samples from right hind and left front quarters. Teat skin swabs and milk samples were collected aseptically for preliminary identification using bacterial culture on chromogenic and calf blood agars. Colonies from milk and teat skin samples with suspicion of having NAS were identified to species-level by MALDI-TOF assay. Out of 511 isolates from 284 quarters (142 cows), 78% (n = 399) were identified by MALDI-TOF. The percentage of correctly identified NAS from milk (91%, 105/115) using MALDI-TOF was higher than the percentage from teat skin (68%, 268/396). Out of the identified isolates, 93% (n = 373) were successfully identified as NAS, whereas the remaining 26 (7%) were shown to be other bacterial species. Out of 26 NAS isolates, 1 originated from milk (Corynebacterium stationis), whereas 25 originated from teat skin representing Aerococcus viridans (n = 7), Bacillus pumilus (n = 13), Enterococcus saccharolyticus (n = 1), Clostridium septicum (n = 1), Corynebacterium stationis (n = 2), and Corynebacterium casei (n = 1). The MALDI-TOF identified 85 (98/115) and 62% (245/396) of the isolates in the first test. Isolates that were not identified to species-level at first test were subjected to a second test, and 47 (8/17) and 32% (48/151) from milk and teat skin, respectively, were identified. After 2 rounds of MALDI-TOF, 22% (n = 112) of the isolates were not identified, representing 103 from teat skin and 9 from milk. Eighteen isolates without identification by MALDI-TOF were successfully identified to species-level using sequencing, where 16 were correctly identified as NAS, whereas the other 2 were Corynebacterium stationis. In conclusion, MALDI-TOF is a reliable assay for identification and typeability of NAS species from aseptically collected quarter milk samples. The assay may be used for identification of NAS species from teat skin swabs. However, confirmation using nucleic acid-based tools is vital for accurate species identification of some species and strains.

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.

Microbiological quality of mink feed raw materials and feed production area.

BACKGROUND: The quality of mink feed and raw ingredients affect health and growth. The objectives of this study were to examine the microbiological quality of ready-to-eat mink feed and its raw ingredients, screen the plant part of the feed for mycotoxins, and determine the hygiene of the production environment in the feed processing facilities. The results of the study are important for identification of critical steps in the feed production and for formulation of recommendations for improvements of production processes to obtain better quality feed. Feed and swab samples were taken at three Danish mink feed producers October 2016 and May 2017, respectively. Viable counts, detection of methicillin-resistant Staphylococcus aureus (MRSA), influenza virus and filamentous fungi were performed together with qualitative chemical analyses for bioactive fungal metabolites and mycotoxins. Swab samples were analyzed for total viable counts. RESULTS: Viable counts varied between 7.2 × 102 and 9.3 × 107 cfu/g in raw ingredients and between 107 and 109 cfu/cm2 on different surfaces at the feed production facilities. A pork meat product, pork haemoglobin, pork liver and a poultry mix was found positive for MRSA, while monophasic Salmonella [4,5,12:i:-] was detected in a pork meat product. Neither MRSA nor Salmonella was detected in any ready-to-eat feed. Influenza A virus was not detected in any sample. Filamentous fungi were detected in all analysed samples of ready-to-eat feed while dihydro-demethyl-sterigmatocystin was found in almost 50% of all ready-to-eat feed samples and in 80% of the sugar beet pulp. Fumonisins and other Fusarium toxins were found especially in corn gluten meal and extruded barley and wheat. CONCLUSIONS: Mink feed contained a cocktail of mycotoxins and bacteria, which may not per se cause clinical disease, but may affect organ function and animal performance and well-being.

Arcanobacterium phocae infection in mink (Neovison vison), seals (Phoca vitulina, Halichoerus grypus) and otters (Lutra lutra).

BACKGROUND: Infectious skin disorders are not uncommon in mink. Such disorders are important as they have a negative impact on animal health and welfare as well as on the quality and value of the fur. This study presents the isolation of Arcanobacterium phocae from mink with severe skin lesions and other pathological conditions, and from wild seals and otters. RESULTS: In 2015, A. phocae was isolated for the first time in Denmark from outbreaks of dermatitis in mink farms. The outbreaks affected at least 12 farms. Originating from these 12 farms, 23 animals cultured positive for A. phocae. The main clinical findings were necrotizing pododermatitis or dermatitis located to other body sites, such as the lumbar and cervical regions. A. phocae could be isolated from skin lesions and in nine animals also from liver, spleen and lung, indicating a systemic spread. The bacterium was also, for the first time in Denmark, detected in dead seals (n = 9) (lungs, throat or wounds) and otters (n = 2) (throat and foot). CONCLUSIONS: An infectious skin disorder in mink associated with A. phocae has started to occur in Danish farmed mink. The origin of the infection has not been identified and it is still not clear what the pathogenesis or the port of entry for A. phocae infections are.

Identification of pathogenic microorganisms directly from positive blood vials by matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is a promising and fast method for identifying fungi and bacteria directly from positive blood cultures. Various pre-treatment methods for MALDI-TOF MS identification have been reported for this purpose. In-house results for identification of bacterial colonies by MALDI-TOF MS using a cut-off score of 1.5 did not reduce the diagnostic accuracy compared with the recommended cut-off score of 1.8. A 3-month consecutive study of positive blood cultures was carried out in our laboratory to evaluate whether the Sepsityper™ Kit (Bruker Daltonics) with Biotyper 2.0 software could be used as a fast diagnostic tool for bacteria and fungi and whether a 1.5 cut-off score could improve species identification compared with the recommended score of 1.8. Two hundred and fifty-six positive blood vials from 210 patients and 19 blood vials spiked with fungi were examined. Using the cut-off score of 1.8, 81% Gram-negative bacteria were identified to the species level compared to 84% using a cut-off score of 1.5. For Gram-positive bacteria 44% were identified to the species level with a cut-off of 1.8 compared to 55% with the value of 1.5. The overall identification rate was 63% (cut-off 1.5) and 54% (cut-off 1.8). Seventy-seven per cent of fungal species were identified with both log scores. MALDI-TOF MS was in this study found to be a powerful tool in fast diagnosis of Gram-negative bacteria and fungi and to a lesser degree of Gram positives. Using 1.5 as cut-off score increased the diagnosis for both Gram-positives and -negatives bacteria.