Biofilm Formation and Antimicrobial Resistance of Staphylococcus aureus and Streptococcus uberis Isolates from Bovine Mastitis.

This study aimed to assess (a) the biofilm producer ability and antimicrobial resistance profiles of Staphylococcus (Staph.) aureus and Streptococcus (Strep.) uberis isolated from cows with clinical mastitis (CM) and subclinical mastitis (SCM), and (b) the association between biofilm producer ability and antimicrobial resistance. We isolated a total of 197 Staph. aureus strains (SCM = 111, CM = 86) and 119 Strep. uberis strains (SCM = 15, CM = 104) from milk samples obtained from 316 cows distributed in 24 dairy herds. Biofilm-forming ability was assessed using the microplate method, while antimicrobial susceptibility was determined using the disk diffusion method against 13 antimicrobials. Among the isolates examined, 57.3% of Staph. aureus and 53.8% of Strep. uberis exhibited the ability to produce biofilm, which was categorized as strong, moderate, or weak. In terms of antimicrobial susceptibility, Staph. aureus isolates displayed resistance to penicillin (92.9%), ampicillin (50.8%), and tetracycline (52.7%). Conversely, Strep. uberis isolates exhibited resistance to penicillin (80.6%), oxacillin (80.6%), and tetracycline (37.8%). However, no significant correlation was found between antimicrobial resistance patterns and biofilm formation ability among the isolates.

Comparison of Minimum Inhibitory Concentrations of Selected Antimicrobials for Non-Aureus Staphylococci, Enterococci, Lactococci, and Streptococci Isolated from Milk Samples of Cows with Clinical Mastitis.

The objective of this study was to compare the minimum inhibitory concentrations of antimicrobials included in a commercial broth microdilution panel among Gram-positive pathogens that caused non-severe clinical mastitis on three Michigan dairy farms. Duplicate quarter milk samples were collected from eligible quarters of cows enrolled in a randomized clinical trial, cultured in a university laboratory, and identified using MALDI-TOF. Etiologies were grouped by genus as Enterococcus species (n = 11), Lactococcus species (n = 44), non-aureus Staphylococcus species (n = 39), or Streptococcus species (n = 25). Minimum inhibitory concentrations (MICs) were determined using the mastitis panel of a commercially available broth microdilution test. In vitro susceptibility was determined using approved guidelines and included breakpoints for mastitis pathogens, or when not available, breakpoints from other species. Most isolates were inhibited at or below breakpoints that demonstrated in vitro susceptibility. The proportions of susceptible isolates varied among pathogens for pirlimycin, penicillin, and tetracycline. The greatest proportion of resistance was observed for pirlimycin, tetracycline, and sulfadimethoxine. Survival analysis was performed to evaluate differences in MICs among pathogen groups. MIC values varied among pathogens for ceftiofur, cephalothin, erythromycin, penicillin, pirlimycin, and tetracycline. However, nearly all isolates were susceptible to ceftiofur and cephalothin, indicating that pathogen differences in MIC are not likely clinically relevant, as these are the two most commonly administered mastitis treatments in the United States. While differences in vitro susceptibility were observed for some antimicrobials, susceptibility was high to cephalosporin-based IMM treatments that are most commonly used and did not vary among pathogens.

Apparent prevalence and selected risk factors of methicillin-resistant Staphylococcus aureus and non-aureus staphylococci and mammaliicocci in bulk tank milk of dairy herds in Indiana, Ohio, and Michigan.

The purpose of this study was to determine the apparent prevalence and risk factors of methicillin-resistant Staphylococcus aureus and non-aureus staphylococci and mammaliicocci (NASM) in bulk tank milk (BTM) obtained from 300 dairy farms that belong to a cooperative collecting milk from Indiana, Michigan, and Ohio. Dairy field personnel recorded information about selected farm level risk factors and collected and froze BTM samples (n = 300) that were sent to Michigan State University researchers. Milk samples were thawed at room temperature and pre-enriched by adding 1 to 4 mL of Mueller-Hinton broth supplemented with 6.5% NaCl and incubated at 37°C for 24 h. Subsequently, 10 µL was plated on mannitol salt agar and Mueller-Hinton agar supplemented with 2.5% NaCl containing 2 mg/L oxacillin and 20 mg/L aztreonam. Colonies that grew on the selective media were subcultured on blood agar and identified using MALDI-TOF mass spectrometry. Phenotypic methicillin resistance was tested using cefoxitin disk diffusion. Conventional PCR was used to detect mecA and mecC in phenotypically resistant isolates. Of 550 isolates that were obtained from mannitol salt agar plates and 10 isolates from Mueller-Hinton agar plates, 16 species of NASM accounted for 84% of staphylococci, while S. aureus accounted for the remaining 16%. Among S. aureus, 4 isolates from 4 farms (1.3%) demonstrated phenotypic resistance to methicillin resistance but none carried mecA or mecC genes. Among NASM, 45 isolates from 40 farms (13.3%) demonstrated phenotypic resistance to methicillin. However, only 13 NASM isolates (7 Mammaliicoccus sciuri, 2 Staphylococcus haemolyticus, 1 Mammaliicoccus fleuretti, 1 Staphylococcus epidermidis, 1 Staphylococcus saprophyticus, and 1 Staphylococcus hyicus) from 13 farms were positive for mecA, whereas all were negative for mecC. Thus, the prevalence of mecA-positive NASM in BTM was 4.3%. Based on molecular results, this study demonstrated a low prevalence of methicillin resistance NASM from BTM samples collected from farms in the Upper Midwest. Dairy farms that contained ≤200 lactating cows and had swine located on the farm had a higher prevalence of methicillin-resistant NASM than smaller farms that did not contain swine.

MALDI-TOF bacterial subtyping for rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis.

AIMS: This study aimed to evaluate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial subtyping for the rapid detection of biomarkers in Staphylococcus aureus from subclinical bovine mastitis. METHODS AND RESULTS: A total of 229 S. aureus isolates were obtained from milk samples collected from cows with subclinical mastitis using microbiological culture. Staphylococcus aureus isolates were also submitted to PCR analysis targeting the mecA and mecC genes, which are indicative of methicillin resistance. Confirmation of the species was achieved through MALDI-TOF MS analysis. To analyze antimicrobial resistance patterns, the MALDI BioTyper Compass Explorer and ClinProTools Bruker software were employed, and dendrograms were generated using Bionumerics software. CONCLUSIONS: MALDI-TOF MS successfully identified S. aureus at the species level, but no methicillin resistance was observed. Moreover, spectral typing displayed limited similarity when compared to pulsed-field gel electrophoresis (PFGE).

Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

Incidence and Treatments of Bovine Mastitis and Other Diseases on 37 Dairy Farms in Wisconsin.

The aim of this research was to describe the incidence and treatments of mastitis and other common bovine diseases using one year of retrospective observational data (n = 50,329 cow-lactations) obtained from herd management software of 37 large dairy farms in Wisconsin. Incidence rate (IR) was defined as the number of first cases of each disease divided by the number of lactations per farm. Clinical mastitis (CM) remains the most diagnosed disease of dairy cows. Across all herds, the mean IR (cases per 100 cow-lactations) was 24.4 for clinical mastitis, 14.5 for foot disorders (FD), 11.2 for metritis (ME), 8.6 for ketosis (KE), 7.4 for retained fetal membranes (RFM), 4.5 for diarrhea (DI), 3.1 for displaced abomasum (DA), 2.9 for pneumonia (PN) and 1.9 for milk fever (MF). More than 30% of cows that had first cases of CM, DA, RFM, DI, and FD did not receive antibiotics. Of those treated, more than 50% of cows diagnosed with PN, ME and CM received ceftiofur as a treatment. The IR of mastitis and most other diseases was greater in older cows (parity ≥ 3) during the first 100 days of lactation and these cows were more likely to receive antibiotic treatments (as compared to younger cows diagnosed in later lactation). Cows of first and second parities in early lactation were more likely to remain in the herd after diagnosis of disease, as compared to older cows and cows in later stages of lactation. Most older cows diagnosed with CM in later lactation were culled before completion of the lactation. These results provide baseline data for disease incidence in dairy cows on modern U.S. dairy farms and reinforce the role of mastitis as an important cause of dairy cow morbidity.

The Impact of Selective Dry Cow Therapy Adopted in a Brazilian Farm on Bacterial Diversity and the Abundance of Quarter Milk.

We aimed to evaluate the impact of selective dry cow therapy (SDCT) (protocol 1: antimicrobial combined with internal teat sealant (ITS); vs. protocol 2: ITS alone) on bacterial diversity and the abundance of quarter milk. Eighty high production cows (parity ≤ 3 and an average milk yield of 36.5 kg/cow/day) from the largest Brazilian dairy herd available were randomly selected; milk quarter samples were collected for microbiological culture (MC) on the day of drying-off (n = 313) and on day 7 post-calving (n = 313). Based on the results of the MC before and after calving, 240 quarters out of 313 were considered healthy, 38 were cured, 29 showed new infections and 6 had persistent infections. Mammary quarters were randomly selected based on intramammary information status and SDCT protocols for bacterial diversity analyses. The bacterial diversity was similar when comparing both healthy and cured quarters submitted to both drying-off protocols. Despite healthy cows that were treated at dry-off using only teat sealant showing no alteration in the alpha and beta bacterial diversity, they did show a higher abundance of bacterial groups that may be beneficial to or commensals of the mammary gland, which implies that antibiotic therapy should be reserved for mammary quarters with a history of mastitis.

MALDI-TOF MS identification of Prototheca algae associated with bovine mastitis.

We evaluated the use of MALDI-TOF MS for the identification of 3 major, dairy-associated Prototheca species, namely, Prototheca bovis (formerly P. zopfii genotype 2), P. blaschkeae, and P. ciferrii (formerly P. zopfii genotype 1). The MALDI-TOF MS spectra established for those species were introduced into the reference spectra library of the Bruker Biotyper MALDI-TOF MS analysis software. Next, 31 Prototheca isolates from Holstein cows with mastitis, from herds located in the midwestern area of São Paulo State, Brazil, were subjected to MALDI-TOF MS profiling. MALDI-TOF MS allowed identification of 22 of 27 P. bovis and 3 of 4 P. blaschkeae isolates with scores >2.0, with 5 of 27 P. bovis and 1 of 4 P. blaschkeae isolates identified only to the genus level. With our extended algae database, MALDI-TOF MS can contribute to quick and effective speciation of Prototheca from mastitis cases.

Association between antimicrobial use and antimicrobial resistance of Streptococcus uberis causing clinical mastitis.

It is unknown whether overuse of antimicrobials against clinical mastitis (CM) from Streptococcus uberis is associated with increased antimicrobial resistance (AMR). Therefore, the aim of this study was to evaluate the association between antimicrobial use (AMU) and AMR in relation to the Strep. uberis causing CM in dairy herds. A total of 83 Strep. uberis isolates were selected from a collection created during a previous study evaluating the epidemiology of CM in dairy herds (n = 17) of southeastern Brazil. For each case of CM identified on farm, the following information was recorded: cow's identification number, affected mammary quarter, date of CM diagnosis, antimicrobial commercial names, number of administrations, and descriptions of protocol changes during the treatment. Streptococcus uberis isolates were confirmed by conventional culture, MALDI-TOF mass spectrometry, and quantitative multiplex PCR analyses. Thus, a total of 8 antimicrobials commonly used for CM treatment were evaluated for antimicrobial activity against Strep. uberis isolates. The minimum inhibitory levels of antimicrobials were determined at the lowest concentrations able to inhibit 50 and 90%, respectively, of Strep. uberis isolates. Data related to the antibiotics used for treatment of CM was used to calculate the frequency of administered antimicrobials as the number of defined daily doses (DDD). The highest frequencies of resistant Strep. uberis were observed for erythromycin (80.7% resistant, R), tetracycline (R = 59%), and penicillin G (R = 57.8%), whereas against ceftiofur only 10.8% of Strep. uberis isolates were resistant, and only 1.2% of the Strep. uberis isolates were resistant to enrofloxacin. Regarding the evaluation of resistance for antimicrobial classes, the highest frequency was observed for macrolides (R = 80.7%; 19.3% susceptible, S). Additionally, a frequency of 18.7% of Strep. uberis isolates were resistant to cephalosporins (S = 81.3%), respectively. Further, 94% of Strep. uberis isolates were multiresistant; all these isolates presented resistance to at least 3 different antimicrobial classes. The overall monthly average of antimicrobial treatment incidence (ATI) among the 17 herds enrolled in the study was 23.7 DDD per 1,000 lactating dairy cows [standard deviation (SD) = 13.9], ranging from 5.0 to 55.4 DDD per 1,000 cows in lactation-day. Cephalosporins and penicillins were the most commonly used antimicrobial classes among the evaluated herds (n = 16; 94.1%), followed by tetracyclines (n = 15 herds; 88.2%), fluoroquinolones (n = 14; 82.3%), and sulfonamides (n = 14; 82.3%). The tetracycline class had the highest ATI mean (5.0 DDD per 1,000 lactating cow-days, SD = 5.8), followed by fluoroquinolones (4.7 DDD per 1,000 lactating cow-days, SD = 6.0) and cephalosporins (3.8 DDD per 1,000 lactating cow-days, SD = 6.0). The overall use of antimicrobials was associated with the resistance of Strep. uberis to the antimicrobial tetracycline.

Herd-level associations between somatic cell counts and economic performance indicators in Brazilian dairy herds.

The aims of the present study were to provide a portrait of the techno-economic status of dairy herds in Minas Gerais, Brazil, particularly with respect to bulk-tank somatic cell count (BTSCC) data, and to examine the herd-level associations of BTSCC with various economic performance indicators (EPI). Data from 543 herds, 1,052 herd-year records in total, spread over 3 years (2015-2017), from the South and Southwest mesoregions of Minas Gerais State were provided by the Brazilian Support Agency to Micro and Small Companies Division Minas Gerais (SEBRAE). Herds had an average of 82 lactating cows per herd, milk yield of 17 L/cow per day, and availability of financial information via routine monthly economic surveys. The EPI data (revenue, gross margin, GM; net margin, NM; profit; break-even point; and operational profitability) of each herd was measured monthly by SEBRAE personnel, and herd-year averages of all variables were computed. Bulk-tank data (SCC, total bacterial count, content of crude protein and fat) taken by producers or dairy processors were recorded by SEBRAE personal; and corresponding herd-year averages were calculated and included in the SEBRAE database. There were 209 selected herds, which passed all edit checks, and which had data for all 3 years. The EPI (all expressed on a per-cow basis, $/cow per year) were analyzed, including the effects of region, year, log (ln) BTSCC, production level, and herd size, together with the random effect of herd nested within region. A high proportion of herds (94.6%) presented data records (herd-years) with an average BTSCC > 200 × 103 cells/mL: 37.8% of herd-year records had BTSCC between >200 and ≤400, 14.5% with BTSCC between >400 and ≤500, 25% with BTSCC between >500 and ≤750, and 17.3% with BTSCC >750. For each unit increase in ln BTSCC, revenue declined by $228.5/cow per year, GM by $155.6/cow per year, and profit by $138.6/cow per year. Herds with cows of lower production (<14 kg/d) presented lower GM ($286.8/cow per year) compared with herds containing cows producing ≥14 kg/d (≥14 and <19 kg/d = $446.5, and ≥19 kg/d = $601.9). The small-scale milk producers (<39 lactating cows) presented lower revenue ($1,914.9/cow per year) and GM ($274.5/cow per year) and consequently a negative profit (-$224.1/cow per year) compared with other herd size categories (≥39 lactating cows). The reduction in milk yield was 641 L/cow per lactation for each unit increase in ln BTSCC; this represented 9.4% of the milk yield per lactation, assuming an average milk production of 6,843.3 L/cow per lactation of cows from herds that had BTSCC ≤ 200 × 103 cells/mL. Consequently, we found a negative association of BTSCC with profit; profit declining from $227.0 to -53.1/cow per year when the BTSCC increased from 100 to 750 × 103 cell/mL. In short, the lower the BTSCC, the greater the revenue, GM and NM, profit, and operational profitability of the herds. The reduction of milk yield was the main factor associated with higher BTSCC.

Short communication: Association between the accessory gene regulator (agr) group and the severity of bovine mastitis caused by Staphylococcus aureus.

Staphylococcus aureus can elicit mild to more severe degrees of mastitis in cattle, depending on the response of the host's immune system and the virulence factors of the specific isolate. Several virulence factors are controlled by a global regulatory system, designated accessory gene regulator (agr). Thus, the objective was to examine associations between different capsular and agr types and the severity of bovine mastitis caused by S. aureus. All isolates were obtained from bovine subclinical (n = 50), mild clinical (n = 73), and moderate clinical mastitis cases (n = 28). Isolates containing the agrI gene and lacking the agr locus (agr-) were more prevalent among subclinical than clinical mastitis cases, whereas isolates containing the agrII and agrIII genes were more prevalent among clinical mastitis cases. The capsular types 5 (cap5) and 8 (cap8) were found in 42 and 44%, respectively, of the isolates obtained from subclinical cases and in 38.6 and 58.4%, respectively, of those isolated from clinical mastitis cases. Capsular type was not associated with type of mastitis (subclinical, mild clinical, or moderate clinical). We found a strong association between agr type and type of mastitis, suggesting that knowledge of S. aureus genetic profiles could be an additional tool to control this disease.

First investigation of Staphylococcus argenteus in a Brazilian collections of S. aureus isolated from bovine mastitis.

BACKGROUND: Staphylococcus argenteus is a new specie positive coagulase staphylococci. We investigate the presence of S. argenteus in isolates previously classified as S. aureus, obtained from the milk of cows with mastitis in Brazil. RESULTS: Among 856 S. aureus tested in chocolate agar, tryptone soya agar and salt egg yolk agar, white or colorless colonies were observed in 185 (21.6%) isolates. Regarding the ctrOPQMN operon, 111 (60%) presented the complete cluster. Despite some missing genes in this cluster, the remaining strains (74) were confirmed as S. aureus using the nrps gene. CONCLUSIONS: As far as we know, this is the first review of S. aureus collection in Brazil and S. argenteus does not appear to be a significant problem in Brazilian herds.

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

ABSTRACT The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Association of herd-level risk factors and incidence rate of clinical mastitis in 20 Brazilian dairy herds.

The objectives of this study were to characterize the pathogen frequency and severity of clinical mastitis (CM) in 20 dairy herds of southeastern Brazil; and to determine the incidence rate of clinical mastitis (IRCM; overall and based on specific-pathogen groups) based on quarter time at risk and its association with risk factors at the herd-level. Data were recorded in each herd for a period of 8 to 15 months. The association between herd-level risk factors and IRCM were determined by two groups of mixed regression models: one based on the overall IRCM, and five based on the following specific-pathogen groups: contagious, other Gram-positive, Gram-negative, other, and negative culture. The following herd-level risk factors were evaluated: herd size, housing system, average daily milk yield per cow, bulk milk somatic cell count (BMSCC), and bulk milk total bacterial count (BMTBC). A total of 5957 quarter-cases of CM were recorded from 2637 cows, but only 4212 cases had milk samples collected for culture. The most frequently isolated pathogens were Escherichia coli (6.6% of total cultures), Streptococcus uberis (6.1%), and Streptococcus agalactiae (5.9%). The majority of CM cases were mild (60.3%), while 34.1% were moderate and 5.6% severe. The frequency of severe CM cases was lower for those with a Gram-positive result (4.6%) compared to a Gram-negative result (11.4%). Overall, monthly mean IRCM was 9.7 cases per 10,000 quarter-days at risk (QDAR). Herds with a geometric mean BMSCC ≥ 601 × 103 cell/mL had higher overall IRCM (16/10,000 QDAR) than those with BMSCC ≤ 600 × 103 cell/mL (≤7.7/10,000 QDAR). When the specific-pathogen groups were evaluated, for contagious pathogens, variables housing (free-stalls or compost-bedded pack barns), BMSCC (≥601 × 103 cells/mL), and average daily milk yield per cow (21 and 25 Kg/d) presented the highest IRCM. Furthermore, in Gram-negative group, herds with BMTBC ≥ 31 × 103 cfu/mL had higher IRCM compared with herds with BMTBC ≤ 30 × 103 cfu/mL. Although environmental pathogens were the most common cause of CM in this study, contagious pathogens (e.g., Strep. agalactiae and Staph. aureus) are still a concern in dairy herds of Brazil. Additionally, as there were some herd-level risk factors associated with the IRCM, there may be opportunity for management strategies aiming to improve the control of CM in dairy herds.

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk.

The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n=120) were skimmed by centrifugation (10,000×g/10min) and pre-incubated at 37°C for 12h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Rapid identification of bovine mastitis pathogens by MALDI-TOF Mass Spectrometry / Rápida identificação de agentes causadores de mastite por espectrometria de massas MALDI-TOF

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to be an alternative method for identification of bacteria via their protein profile spectra, being able to identify bacteria at the genus, species and even at subspecies level. With the aim of large-scale identification of pathogens causing mastitis by this platform, a total of 305 isolates of bacteria identified from cows with subclinical mastitis were analyzed by conventional microbiological culture (MC) as well as by MALDI-TOF MS coupled with Biotyper data processing. Approximately 89% of the identifications performed by MALDI-TOF MS were consistent with results obtained by MC. From the remaining isolates (11%), 6.3% of isolates were classified as misidentified (discordance for both genus and species level), and 4.7% showed identification agreement at the genus level but not at the species level, being classified as unidentified at species level. The disagreement results were mostly associated with identification of Streptococcus and Enterococcus species probably due to the narrow phenotypic similarity between these two genera. These disagreement results suggest that biochemical assays might be prone to identification errors and, MALDI-TOF MS therefore may be an alternative to overcome incorrect species-specific identification. Standard microbiological methods for bovine mastitis diagnosis are time consuming, laborious and prone to errors for some bacteria genera. In our study, we showed that MALDI-TOF MS coupled with Biotyper may be an alternative method for large-scale identification of bacteria isolated from milk samples compared to classical microbiological routine protocols.(AU)

A espectrometria de massas (MALDI-TOF MS) tem mostrado ser um método alternativo para a identificação de bactérias, sendo capaz de identificar as bactérias causadoras de mastite em gênero, espécie ou até mesmo subespécie. Com o objetivo de identificar os patógenos causadores de mastite em grande-escala por esta plataforma, um total de 305 isolados bacterianos oriundos de vacas com mastite subclínica foram analisados pela cultura microbiológica convencional (CM) e pela MALDI-TOF MS acoplada ao software Biotyper. Aproximadamente 89% das identificações realizadas pela MALDI-TOF MS foram consistentes com os resultados obtidos pela CM. Do restante de isolados bacterianos (11%), 6,3% foram classificados como identificação errônea (discordância de gênero e espécie), e 4,7% apresentaram concordância de gênero, mas discordância da espécie. Os resultados que apresentaram divergência estavam mais associados com a identificação das espécies de Streptococcus spp. e Enterococcus spp. devido à similaridade fenotípica entre os dois gêneros. Estes resultados divergentes sugerem que os ensaios bioquímicos podem ser propensos a erros de identificação, por isso a MALDI-TOF MS pode ser considerada um método alternativo para superar os erros de identificação da CM. A cultura microbiológica padrão e os ensaios bioquímicos utilizados na identificação de agentes causadores de mastite são demorados, trabalhosos e propensos a erros quando utilizados na identificação em nível de espécie. No presente estudo, demonstramos que a MALDI-TOF MS acoplada ao software Biotyper pode ser considerada um método alternativo de identificação de bactérias causadoras de mastite em grande-escala quando comparado com a cultura microbiológica convencional.(AU)

Milk losses associated with somatic cell counts by parity and stage of lactation.

The reduction of milk production caused by subclinical mastitis in dairy cows was evaluated through the regression of test-day milk yield on log-transformed somatic cell counts (LnSCC). Official test-day records (n = 1,688,054) of Holstein cows (n = 87,695) were obtained from 719 herds from January 2010 to December 2015. Editing was performed to ensure both reliability and consistency for the statistical analysis, and the final data set comprised 232,937 test-day records from 31,692 Holstein cows in 243 herds. A segmented regression was fitted to estimate the cutoff point in the LnSCC scale where milk yield started to be affected by mastitis. The statistical model used to explain daily milk yield included the effect of herd as a random effect and days in milk and LnSCC as fixed effects regressions, and analyses were performed by parity and stage of lactation. The cutoff point where milk yield starts to be affected by changes in LnSCC was estimated to be around 2.52 (the average of all estimates of approximately 12,400 cells/mL) for Holsteins cows from Brazilian herds. For first-lactation cows, milk losses per unit increase of LnSCC had estimates around 0.68 kg/d in the beginning of the lactation [5 to 19 d in milk (DIM)], 0.55 kg/d in mid-lactation (110 to 124 DIM), and 0.97 kg/d at the end of the lactation (289 to 304 DIM). For second-lactation cows, milk losses per unit increase of LnSCC had estimates around 1.47 kg/d in the beginning of the lactation (5 to 19 DIM), 1.09 kg/d in mid-lactation (110 to 124 DIM), and 2.45 kg/d at the end of the lactation (289 to 304 DIM). For third-lactation cows, milk losses per unit increase of LnSCC had estimates around 2.22 kg/d in the beginning of the lactation (5 to 19 DIM), 1.13 kg/d in mid-lactation (140 to 154 DIM), and 2.65 kg/d at the end of the lactation (289 to 304 DIM). Daily milk losses caused by increased LnSCC were dependent on parity and stage of lactation, and these factors should be considered when estimating losses associated with subclinical mastitis.

Risk factors associated with the antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis / Fatores de risco associados com a resistência antimicrobiana de Staphylococcus aureus isolados da mastite bovina

The objective of this study was to evaluate herd management practices and mastitis treatment procedures as risk factors associated with Staphylococcus aureus antimicrobial resistance. For this study, 13 herds were selected to participate in the study to evaluate the association between their management practices and mastitis treatment procedures and in vitro antimicrobial susceptibility. A total of 1069 composite milk samples were collected aseptically from the selected cows in four different periods over two years. The samples were used for microbiological culturing of S. aureus isolates and evaluation of their antimicrobial susceptibility. A total of 756 samples (70.7%) were culture-positive, and S. aureus comprised 27.77% (n=210) of the isolates. The S. aureus isolates were tested using the disk-diffusion susceptibility assay with the following antimicrobials: ampicillin 10mg; clindamycin 2μg; penicillin 1mg; ceftiofur 30μg; gentamicin 10mg; sulfa-trimethoprim 25μg; enrofloxacin 5μg; sulfonamide 300μg; tetracycline 30μg; oxacillin 1mg; cephalothin 30μg and erythromycin 5μg. The variables that were significantly associated with S. aureus resistance were as follows: the treatment of clinical mastitis for ampicillin (OR=2.18), dry cow treatment for enrofloxacin (OR=2.11) and not sending milk samples for microbiological culture and susceptibility tests, for ampicillin (OR=2.57) and penicillin (OR=4.69). In conclusion, the identification of risk factors for S. aureus resistance against various mastitis antimicrobials is an important information that may help in practical recommendations for prudent use of antimicrobial in milk production.

Objetivou-se com este estudo avaliar os fatores de risco associados às práticas de manejo e tratamento de mastite e a resistência aos antimicrobianos de Staphylococcus aureus isolados de vacas com mastite. Foram selecionados para o presente estudo 13 rebanhos localizados na região de Pirassununga/SP. Foi aplicado um questionário contendo informações para o levantamento de fatores de risco relacionados à resistência aos antimicrobianos e às práticas de manejo e tratamento de mastite. Após a seleção dos rebanhos e aplicação dos questionários, foram utilizados 210 isolados de S. aureus de amostras compostas de leite coletadas durante 24 meses, em quatro períodos, para realização dos testes de resistência. Os antimicrobianos testados foram: ampicilina 10µg, clindamicina 2µg, penicilina 1µg, eftiofour 30µg, gentamicina 10µg, sulfatrimetropin 25µg, enrofloxacina 5µg, sulfonamida 300µg, tetraciclina 30µg, oxacilina 1µg, cefalotina 30µg e eritromicina 5µg. As variáveis que foram significativamente associadas à resistência de S. aureus foram: o tratamento da mastite clínica para ampicilina (OR = 2,18), o tratamento da vaca seca para enrofloxacina (OR=2,11), e o não envio de amostras de leite para a cultura microbiológica e testes de sensibilidade, para ampicilina (OR=2,57) e penicilina (OR=4,69). Em conclusão, a identificação dos fatores de risco para a resistência S. aureus frente aos principais agentes antimicrobianos, utilizados para tratamento da mastite, pode auxiliar o estabelecimento do uso prudente de antimicrobianos na produção de leite.