Genetic and Phenotypic Characterization of Subclinical Mastitis-Causing Multidrug-Resistant Staphylococcus aureus.

The core objective of this study was to genetically and phenotypically characterize subclinical mastitis-causing multidrug-resistant Staphylococcus aureus (MDRSA). In addition, risk factors associated with subclinical mastitis caused by MDRSA were investigated. Bacterial cultures were performed on 2120 mammary quarters, 40 swabs of milk utensils, 5 bulk tank milk samples, and 11 nostril and 11 hand swabs from milkers from five dairy farms. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was conducted for S. aureus identification. Antimicrobial resistance was screened phenotypically using the disk diffusion test in all S. aureus isolates. A biofilm formation assay; detection of genes associated with beta-lactam resistance, efflux pump, and biofilm formation; and pulsed-field gel electrophoresis (PFGE) were performed in all MDRSA isolates. Multi-locus sequence typing (MLST) was carried out in cefoxitin-resistant MDRSA isolates. A total of 188 S. aureus isolates from milk as well as two from milking utensils and one from bulk tank milk were identified. Most of the isolates (92.7%; 177 of 191) showed beta-lactam resistance, and 7% (14 of 191) were MDRSA. Interestingly, 36% (5 of 14) of MDRSA isolates were cefoxitin-resistant, but none carried mecA or mecC genes. Based on PFGE results, it was observed that S. aureus strains were more likely to be unique to a specific herd. Two clonal complexes were identified, CC97 (ST126; commonly livestock-associated) and CC1 (ST7440; usually community-associated). To the best of our knowledge, this is the first report of ST7440 isolated from bovine mastitis in Brazil. The risk factor results underscored the importance of considering parity, stage of lactation, SCC, milk production, and herd size when studying the risk of subclinical mastitis and antimicrobial resistance in S. aureus. Thus, to implement effective strategies to prevent subclinical mastitis in dairy herds and to minimize MDRSA spread, it is important to understand MDRSA strains' distribution and their antimicrobial resistance profile.

Virulence and antimicrobial resistance genes profiles of spa type t605 methicillin-susceptible Staphylococcus aureus isolated from subclinical bovine mastitis.

AIMS: Staphylococcus aureus is one of the most common pathogens associated with mastitis in dairy herds worldwide. This study evaluated the profile of virulence and antimicrobial resistance genes of spa type t605 methicillin-susceptible Staphylococcus aureus isolated from subclinical bovine mastitis in São Paulo, Brazil. METHODS AND RESULTS: A total of 57 S. aureus strains were screened by conventional PCR (Polymerase Chain Reaction) for 49 virulence genes. The most prevalent virulence genes detected were icaD (94.7%), fib (93%), fnbA (82.5%), clfA (80.7%), bap (78.9%), clfB (73.7%), icaA (66.7%), see (64.9%), and sed (61.4%). The blaZ (94.7%), aac6'aph2' (15.8%), and ant4 (12.3%) genes were the most common antimicrobial resistance genes; however, mecA and mecC genes were not found. All methicillin-susceptible S. aureus (MSSA) strains were characterized through spa and agr typing. The spa type t605 was found in all isolates. By agr typing, the most prevalent were type II (56.1%). Antimicrobial resistance was determined by the disk diffusion method, and 93% showed resistance to at least one antibiotic. Penicillin resistance was the most prevalent (87.7%), followed by tetracycline (12.3%), oxacillin (10.5%), and gentamicin (10.5%) resistance. CONCLUSION: Our study confirmed the spa type t605 as endemic, carrying a wide variety of virulence factors and high-level penicillin resistance. The profile seems to be associated with the colonization of MSSA and its persistence in subclinical mastitis.

Molecular characterization of persistent subclinical mastitis-causing Staphylococcus aureus from dairy farms.

The study aimed to evaluate the genetic diversity of Staphylococcus aureus causing subclinical mastitis (SM) isolated from dairy cows and to assess the effect of the infection status (transient vs. persistent) on the milk and component yield. A total of six dairy farms in São Paulo state were used for the selection of cows with SM caused by S. aureus. S. aureus strains (n = 56) obtained from three biweekly aseptic mammary quarter milk samplings (n = 1140 from 95 cows) were subjected to MALDI-TOF MS analysis for species confirmation and further PFGE analysis. Intramammary infections (IMI) caused by S. aureus were categorized as transient (T: when only one out of 3 milk samplings had positive isolation of any pulsotype) or persistent (P: when two (P2) or three (P3) milk samplings had positive isolation of identical pulsotype over the consecutive episodes of SM. The SmaI macrorestriction fragment profiles of 56 S. aureus isolates showed a dominant S. aureus clonal pattern (PFGE type A; n = 50; 89.3%) within and among the herds. The SM-causing S. aureus represented a reduction of quarter milk yield of 26.2% in transient and 54.8% in persistent cases as well as a reduction of total solid yield of 38.1% and 49.4%, respectively, when compared with the healthy control quarters. Overall, the greater chance of S. aureus to be persistent is when a dominant clonal pattern is present in the herd which consequently may be associated with the cause of accentuated milk loss.

Antimicrobial activities of polyhexamethylene biguanide against biofilm-producing Prototheca bovis causing bovine mastitis.

Prototheca spp. is a frequent cause of bovine mastitis and is highly resistant to commonly used disinfectants. This study aimed to: (1) evaluate the antimicrobial activity of polyhexamethylene biguanide (PHMB) against mastitis-causing Prototheca spp., and (2) evaluate the biofilm production ability of Prototheca spp. A total of 85 Prototheca bovis and 2 Prototheca blaschkeae isolates from bovine mastitis cases were submitted to biofilm production assays and antimicrobial susceptibility tests against PHMB and disinfectants commonly used in dairy herds (chlorhexidine digluconate, povidone-iodine, sodium dichloroisocyanurate, and sodium hypochlorite). The minimal inhibitory concentration (MIC) and minimal algicidal concentration (MAC) were determined by microdilution assays. We observed that PHMB (MIC90: ≥2 µg/mL and MAC90: ≥4 µg/mL) and chlorhexidine gluconate (MIC90 and MAC90: ≥2 µg/mL) presented the highest antimicrobial activity against P. bovis isolates, followed by sodium dichloroisocyanurate (MIC90 and MAC90: ≥1,400 µg/mL), sodium hypochlorite (MIC90 and MAC90: ≥2,800 µg/mL), and povidone-iodine (MIC90 and MAC90: ≥3,200 µg/mL). Concerning P. blaschkeae isolates, PHMB (MIC and MAC ≥1 µg/mL) and chlorhexidine gluconate (MIC and MAC ≥1 µg/mL) were the disinfectants that presented the lowest concentration values required to inhibit the isolates. Regarding biofilms formation, 63.5% (n = 54/85) of the P. bovis isolates were classified as strong, 28.3% (n = 24/85) moderate, and 8.2% (n = 7/85) weak biofilm producers. In contrast, the P. blaschkeae isolates were classified as weak and moderate biofilm producers. These findings suggest that PHMB has the potential to be used for teat and milking-equipment disinfection for the prevention of mastitis-causing Prototheca spp. in dairy herds.

Chronic subclinical mastitis reduces milk and components yield at the cow level.

We evaluated the effects of chronic subclinical mastitis (CSM) caused by different types of pathogens on milk yield and milk components at the cow level. A total of 388 Holstein cows had milk yield measured and were milk sampled three times at intervals of two weeks for determination of SCC and milk composition, and microbiological culture was performed. Cows were considered healthy if all three samples of SCC were ≤200 000 cells/ml and were culture-negative at the third milk sampling. Cows with one result of SCC > 200 000 cells/ml were considered to suffer non-chronic subclinical mastitis whereas cows with at least 2 out of 3 results of SCC > 200 000 cells/ml had CSM. These latter cows were further sorted according to culture results into chronic negative-culture or chronic positive-culture. This resulted in four udder health statuses: healthy, non-chronic, chronicNC or chronicPC. The milk and components yields were evaluated according to the udder health status and by pathogen using a linear mixed effects model. A total of 134 out of 388 cows (34.5%) were chronicPC, 57 cows (14.7%) were chronicNC, 78 cows (20.1%) were non-chronic and 119 cows (30.7%) were considered healthy, which resulted in a grand total of 1164 cow records included in the statistical model. The healthy cows produced more milk than each of the other groups (+2.1 to +5.7 kg/cow/day) and produced higher milk component yields than the chronicPC cows. The healthy cows produced more milk than cows with chronicPC caused by minor (+5.2 kg/cow/day) and major pathogens (+7.1 kg/cow/day) and losses varied from 5.8 to 11.8 kg/cow/day depending on the pathogen causing chronicPC mastitis. Chronic positive-culture cows had a reduction of at least 24.5% of milk yield and 22.4% of total solids yield.

Subclinical intramammary infection does not affect bovine milk ethanol stability / Infecção intramamária subclínica não afeta a estabilidade do leite bovino

The present study hypothesized that intramammary infection (IMI) might reduce milk ethanol stability (MES), mainly when IMI is caused by major pathogens. Thus, this study evaluated the effect of IMI on bovine MES using a natural exposure experimental design. Ninety-four lactating cows from five dairy herds were selected once they were determined to have an IMI, based on milk bacteriological culturing with positive isolation and somatic cell count (SCC) > 200×103 cells/mL in two out of three composite milk samples collected during three consecutive weeks. After selection, cows were sampled a second time (within two weeks) for evaluation at mammary quarter level (n = 326): milk yield (kg/quarter/day), MES, composition (fat, protein, lactose, casein, total solids and solids-non-fat), and bacteriologic culture. The effect of subclinical mastitis on MES was tested by two models: 1) comparison of healthy vs. infected quarters; and 2) comparison of contralateral mammary quarter within cow. The only milk composition variable associated with MES was lactose (r = 0.18; P < 0.01). Subclinical IMI did not affect MES when the comparison was performed using both models (1 and 2). Likewise, MES did not change when infected quarters were sorted into two groups of pathogens (major, minor and infrequent; and contagious, environmental, minor and infrequent) and compared with healthy mammary quarters. Considering the results of both models, subclinical IMI did not affect MES of dairy cows.(AU)

Neste trabalho investigou-se a hipótese de que a infecção intramamária (IIM) poderia reduzir a estabilidade do leite ao etanol (ELA), principalmente quando a IIM é causada por agentes primários. Assim, em um experimento de exposição natural, foi avaliado o efeito da IIM sobre a ELA em bovinos. Noventa e quatro vacas em lactação de cinco rebanhos leiteiros foram selecionadas por apresentar IIM, segundo resultados de cultura bacteriológica de amostras compostas de leite (isolamento positivo) e contagem de células somáticas (CCS) > 200×103 células/mL em pelo menos duas de três coletas semanais consecutivas. Após essa seleção, as vacas foram amostradas pela segunda vez (dentro de duas semanas) para avaliação da IIM em amostras de leite coletadas por quarto mamário (n = 326): produção de leite (kg/quarto/dia), ELA, composição (gordura, proteína, lactose, caseína, sólidos totais e sólidos não gordurosos) e cultura bacteriológica. O efeito da mastite subclínica sobre a ELA foi testada por dois modelos: 1) comparação de quarto sadio versus infectado; e 2) comparação de quartos mamários contralaterais. A única variável de composição do leite associada à ELA foi a lactose (r = 0,18; P < 0,01). A IIM subclínica não afetou a ELA quando a comparação foi realizada utilizando-se os dois modelos (1 e 2); bem como a ELA não foi alterada quando os quartos infectados foram classificados em grupos de agentes patogênicos (primários, secundários e infrequentes; ou contagiosos, ambientais, secundários e infrequentes) e comparados com os quartos mamários sadios. Os resultados obtidos com os dois modelos empregados demonstraram que a IIM subclínica não afetou a ELA de vacas leiteiras.(AU)

Effect of somatic cell count on composition and hygiene indicators of bulk tank milk / Efeito da contagem de células somáticas sobre a composição e indicadores de higiene do leite de tanque

This study evaluated the effect of somatic cell count (SCC) on composition and hygienic quality of dairy herd bulk tank milk ­ specifically, the effect of SCC of bulk tank of dairy herds on composition (fat, protein, total solids, nonfat dry solids) and on total bacterial count (TBC), psychrotrophic count (PC) and coliform count (CC) were evaluated. A total of 230 dairy herds located south of Minas Gerais and west of São Paulo were selected based on SCC geometric mean obtained from five monthly analyses preceding the start of the sampling. The dairy farms were classified according to SCC in three groups: low (< 250,000 cells/mL, n = 84), medium (> 250,000 and < 750,000 cells/mL, n = 79) and high SCC (> 750,000 cells/mL, n = 67). After herd selection, bulk tank milk samples were collected every 14 days for three months totaling 1380 samples, which were subjected to analysis of composition, TBC, PC, and CC. A decrease of TBC and CC was observed in herds with low SCC; however, herds with medium and high SCC had an increase in fat, crude protein, and total solids contents. A medium correlation was observed between TBC and PC (r = 0.6215), and also between PC and CC (r = 0.3692). Based on hygiene indicators and milk composition, a low and negative correlation between TBC and fat (r = -0.0585), PC and fat (r = -0.0585), and PC and total solids (r = -0.0662) was observed. Dairy herds with SCC < 250,000 cells/mL had higher bulk tank milk hygienic quality; however, considering the composition, herds with higher SCC produced higher milk fat and protein concentration.(AU)

Este trabalho avaliou o efeito da contagem de células somáticas (CCS) na composição e na qualidade higiênica do leite de tanque de rebanhos leiteiros. Especificamente, foram avaliados o efeito da CCS do tanque de rebanhos leiteiros na composição (gordura, proteína, sólidos totais, extrato seco desengordurado) e nas contagens bacteriana total (CBT), de psicrotróficos (CP) e de coliformes (CC). Um total de 230 rebanhos leiteiros localizados no sul de Minas Gerais e oeste de São Paulo foram selecionados com base na média geométrica da CCS obtida de cinco análises mensais anteriores ao início das coletas das amostras. As fazendas foram classificadas de acordo com a CCS em três grupos: baixa (< 250.000 células/mL, n = 84), média (> 250.000 e < 750.000 células/mL, n = 79) e alta CCS (> 750.000 células/mL, n = 67). Após a seleção dos rebanhos, amostras de leite do tanque foram coletadas a cada catorze dias durante três meses, totalizando 1.380 amostras, as quais foram submetidas às análises de composição, CBT, CP e CC. Uma redução da CBT e da CC foi observada em rebanho com baixa CCS; entretanto, rebanhos com média e alta CCS tiveram aumento nos teores de gordura, proteína bruta e sólidos totais. Uma média correlação foi observada entre CBT e CP (r = 0,6215) e também entre CP e CC (r = 0,3692). Com base nos indicadores de higiene e na composição do leite, foi observada uma correlação baixa e negativa entre CBT e gordura (r = -0,0585), CP e gordura (r = -0,0585) e CP e sólidos totais (r = -0,0662). Os rebanhos leiteiros com CCS < 250.000 células/mL apresentaram maior qualidade higiênica do leite de tanque; entretanto, considerando a composição, rebanhos com maior CCS tiveram maiores concentrações de gordura e proteína.(AU)

Using milk leukocyte differentials for diagnosis of subclinical bovine mastitis.

This research study aimed to evaluate the use of the milk leukocyte differential (MLD) to: (a) identify quarter milks that are culture-positive; and (b) characterize the milk leukocyte responses to specific groups of pathogens causing subclinical mastitis. The MLD measures the absolute number and relative percentage of inflammatory cells in milk samples. Using the MLD in two dairy herds (170 and 172 lactating cows, respectively), we studied all lactating cows with a most recent monthly Dairy Herd Improvement Association somatic cell count (SCC) >200 × 103 cells/ml. Quarter milk samples from 78 cows meeting study criteria were analysed by MLD and aseptically collected milk samples were subjected to microbiological culture (MC). Based upon automated instrument evaluation of the number and percentage of inflammatory cells in milk, samples were designated as either MLD-positive or - negative for subclinicial mastitis. Positive MC were obtained from 102/156 (65·4%) of MLD-positive milk samples, and 28/135 (20·7%) of MLD-negative milk samples were MC-positive. When MC was considered the gold standard for mastitis diagnosis, the calculated diagnostic Se of the MLD was 78·5% (IC(95%) = 70·4 to 85·2%) and the Sp was 66·5% (IC(95%) = 58·6 to 73·7%). [corrected]. Quarter milks positive on MC had higher absolute numbers of neutrophils, lymphocytes and macrophages, with higher neutrophils% and lymphocytes% but lower macrophages%. The Log10 (N/L) ratios were the most useful ratio to differentiate specific subclinical mastitis quarters from healthy quarters. Use of the MLD on cows with monthly composite SCC > 200 × 103 cells/ml for screening at quarter level identified quarters more likely to be culture-positive. In conclusion, the MLD can provide an analysis of mammary quarter status more detailed than provided by SCC alone; however, the MLD response to subclinical mastitis was not found useful to specifically identify the causative pathogen.

Non-culture-based identification of mastitis-causing bacteria by MALDI-TOF mass spectrometry.

The purpose of this study was to evaluate the detection limit of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct identification, without previous microbiological culture, of bovine mastitis-causing bacteria from milk samples. Milk samples (n = 15) were experimentally contaminated with Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Escherichia coli to have bacterial counts ranging from 103 to 109 cfu/mL. These contaminated milk samples were subjected to a preparation protocol for bacterial ribosomal protein extraction using the MALDI Sepsityper kit (Bruker Daltonik, Bremen, Germany), which allowed MALDI-TOF MS coupled with Biotyper software (Bruker Daltonik) to identify bacterial fingerprints based on intact ribosomal proteins. The ability of MALDI-TOF MS to correctly identify bacterial strains from experimentally contaminated milk (without previous microbiological culture) depended on the bacterial count of the samples and on the species of the bacteria evaluated. Adequate identification at the bacterial species level (score ≥2.0) directly from milk samples required bacterial counts in the following ranges: ≥106 cfu/mL of Staph. aureus, ≥107 cfu/mL of E. coli, and ≥108 cfu/mL of Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis. We concluded that direct identification of mastitis-causing pathogens is possible for Staph. aureus, E. coli, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis, but correct identification depended on the bacterial count in the milk samples.

Effects of bovine subclinical mastitis caused by Corynebacterium spp. on somatic cell count, milk yield and composition by comparing contralateral quarters.

Subclinical mastitis caused by Corynebacterium spp. (as a group and at the species level) was investigated by evaluating contralateral (healthy and infected) mammary quarters for somatic cell count (SCC), milk yield and composition. Selection of cows with subclinical mastitis caused by Corynebacterium spp. was performed by microbiological culture of composite samples collected from 1242 dairy cows from 21 dairy herds. For each of the selected cows, milk yield was measured and milk samples were collected at the mammary quarter level (i.e., 1140 mammary samples collected from 285 cows) for analysis of milk composition and SCC. The identification of Corynebacterium spp. isolates was performed by 16S rRNA gene sequencing. One hundred and eighty Corynebacterium spp. isolates were identified, of which 167 (92.77%) were C.bovis and eight (4.44%) non-C.bovis; for five of the Corynebacterium spp. isolates (2.77%), sequencing of 16S rRNA genes did not allow identification at the species level. Mammary quarters infected with Corynebacterium spp. as a group had a higher geometric mean SCC (197,900 cells/mL) than healthy contralateral mammary quarters (85,800 cells/mL). Species of Corynebacterium non-C.bovis were infrequently isolated and did not change SCC, milk yield or milk solid contents when evaluated at the contralateral quarter level. Although C.bovis infection showed no effect on milk yield, fat, protein, casein or total solids in milk, it increased SCC and decreased lactose and milk solids non-fat content.

Detection and Enumeration of Streptococcus agalactiae from Bovine Milk Samples by Real-Time Polymerase Chain Reaction.

The aim of this study was to evaluate the use of real-time polymerase chain reaction (qPCR) combined with DNA extraction directly from composite milk and bulk tank samples for detection and enumeration of Streptococcus agalactiae (SAG) causing subclinical mastitis. Dilutions of sterile reconstituted skim milk inoculated with SAG ATCC 13813 were used to establish a standard curve (cfu/mL) for the qPCR assay targeting SAG. The analytical sensitivity and repeatability of the qPCR assay were determined. Bulk tank (BTM; n = 38) and composite milk samples (CM; n = 26) collected from lactating cows with positive isolation of SAG were submitted to the qPCR protocol and SAG plate counting, with results from both methods compared. Amplification of DNA was not possible in two out of 64 samples, indicating that qPCR was able to detect SAG in 96 and 97% of BTM and CM samples, respectively. The inter-assay coefficient of variation was <5%, showing that the technique had adequate repeatability. The qPCR protocol can be a high-throughput and rapid diagnostic assay to accurately detect SAG from BTM and CM samples compared with conventional microbiological culture method. However, the evaluated qPCR protocol is not accurate for enumerating SAG in milk samples, probably due to quantification of DNA of non-viable cells.

Biofilm-producing ability and efficiency of sanitizing agents against Prototheca zopfii isolates from bovine subclinical mastitis.

The objectives of the present study were to evaluate (1) the capacity of the microalga Prototheca zopfii isolated from subclinical bovine mastitis cases to form biofilms; and (2) the resistance of these isolates to sanitizing agents. Ten isolates of P. zopfii from cows with subclinical mastitis (somatic cell count>200×10(3) cells/mL), distributed in 5 dairy farms, were evaluated for their capacity to form biofilms in polystyrene microplate assays and stainless steel coupons, at 25°C and 37°C±1°C. Prototheca zopfii were isolated from milk samples via microbiological culture and analyzed by 18S rRNA gene sequencing. Biofilm formation on the coupons was observed by scanning electron microscopy. The resistance to sanitizing agents was assessed using the biofilm-forming P. zopfii isolates in stainless steel coupon assays, which were subjected to 3 sanitizers: peracetic acid, sodium hypochlorite, and iodine solution. To evaluate resistance to the sanitizers, the minimum inhibitory concentration (MIC) technique was performed using decreasing concentrations of the sanitizing agents (20, 10, 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, 0.039, and 0.019 g/L). After inoculating the isolates, all concentrations were evaluated at 3 distinct incubation periods (24, 48, and 72 h) to assess the effect of incubation time on the MIC. Using the polystyrene microplate assays, 1 isolate showed weak biofilm production, 5 moderate, and 4 strong, when incubated at 25°C±1. For isolates incubated at 37°C±1, 6 showed weak biofilm production and 4 moderate. All P. zopfii isolates (n=10) had the capacity to form biofilms on stainless steel coupons. The longer the incubation period of the P. zopfii isolates at different dilutions, the greater the concentrations of sanitizer needed to prevent growth of the microalgae under the tested conditions. We detected a significant effect of sanitizer and time of incubation (24, 48, and 72 h) on MIC values against P. zopfii isolates. The isolates were sensitive in vitro to peracetic acid (MIC90≥0.019 g/L), sodium hypochlorite (MIC90≥0.312 g/L), and iodine solution (MIC90≥0.625 g/L), after 24 h of incubation (where MIC90=concentration needed to inhibit 90% of isolates). Of the tested sanitizers, peracetic acid had the greatest efficiency against P. zopfii. We conclude that P. zopfii isolates are capable of biofilm production, which may contribute to their persistence in a milking and dairy environment.

Identification of Corynebacterium spp. isolated from bovine intramammary infections by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Identification of coagulase-negative staphylococci from bovine intramammary infection by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Coagulase-negative staphylococci (CoNS) are among the main pathogens causing bovine intramammary infection (IMI) in many countries. However, one of the limitations related to the specific diagnosis of CoNS is the lack of an accurate, rapid, and convenient method that can differentiate the bacterial species comprising this group. The aim of this study was to evaluate the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to accurately identify CoNS species in dairy cow IMI. In addition, the study aimed to determine the frequency of CoNS species causing bovine IMI. A total of 108 bacterial isolates were diagnosed as CoNS by microbiological cultures from two milk samples collected from 21 dairy herds; the first sample was collected at the cow level (i.e., 1,242 composite samples from all quarters), while the second sample was collected at the mammary quarter level (i.e., 1,140 mammary samples collected from 285 cows). After CoNS isolation was confirmed by microbiological culture for both samples, all CoNS isolates (n=108) were genotypically differentiated by PCR restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and subjected to the MALDI-TOF MS identification procedure. MALDI-TOF MS correctly identified 103 (95.4%) of the CoNS isolates identified by PCR-RFLP at the species level. Eleven CoNS species isolated from bovine IMI were identified by PCR-RFLP, and the most prevalent species was Staphylococcus chromogenes (n=80; 74.1%). In conclusion, MALDI-TOF MS may be a reliable alternative method for differentiating CoNS species causing bovine IMI.