Molecular characterization of non-aureus Staphylococcus spp. from heifer intramammary infections and body sites

The purpose of this study was to investigate non-aureus Staphylococcus spp. intramammary infections (IMI) in periparturient heifers and determine the relationship of precalving body site isolation with precalving IMI and postcalving IMI using molecular speciation and strain-typing methods. Primiparous heifers were enrolled at approximately 14 d before expected calving date. Precalving mammary quarter secretions and body site swabbing samples (teat skin, inguinal skin, muzzle, and perineum) were collected. Postcalving, mammary quarter milk samples were collected for culture and somatic cell counting. Precalving body site samples were cultured, and up to 10 staphylococcal colonies were saved for characterization. Staphylococcal isolates were speciated using matrix-assisted laser/desorption ionization time-of-flight mass spectrometry or sequencing of rpoB or tuf. Pulsed-field gel electrophoresis was used to strain type a subset of isolates. Overall, Staphylococcus chromogenes, Staphylococcus agnetis, and Staphylococcus simulans were the most common species identified in precalving mammary secretions, whereas S. chromogenes, Staphylococcus xylosus, and S. agnetis were the most common species found in postcalving milk samples. The most common species identified from body site samples were S. chromogenes, S. xylosus, and Staphylococcus haemolyticus. Mammary quarters that had a precalving mammary secretion that was culture positive for S. agnetis, S. chromogenes, or Staphylococcus devriesei had increased odds of having an IMI with the same species postcalving. A S. chromogenes IMI postcalving was associated with higher somatic cell count when compared with postcalving culture-negative quarters. Among heifers identified with a non-aureus Staphylococcus spp. IMI either precalving or postcalving, heifers that had S. agnetis or S. chromogenes isolated from their teat skin had increased odds of having the same species found in their precalving mammary secretions, and heifers with S. chromogenes, S. simulans, and S. xylosus isolated from their teat skin precalving were at increased odds of having an IMI with the same species postcalving. Overall, 44% of all heifers with a S. chromogenes IMI around the time of parturition had the same strain isolated from a body site. Based on pulsed-field gel electrophoresis, a high level of strain diversity was found.     

Non-aureus staphylococci in fecal samples of dairy cows: First report and phenotypic and genotypic characterization

The aims of this study were to determine whether non-aureus staphylococci (NAS) are present in rectal feces of healthy dairy cows, and if so, to delineate species to which they belong and to study several phenotypic and genotypic traits as a first step toward determining the potential impact of fecal shedding of NAS on bovine udder health. Fecal samples were aseptically collected from the rectum of 25 randomly selected clinically healthy dairy cows in a commercial dairy herd using an automated milking system. Fecal NAS were isolated and then identified at the species level using transfer RNA-intergenic spacer PCR and sequencing of the 16S rRNA housekeeping gene. Strain typing was performed using random amplification of polymorphic DNA (RAPD)-PCR. The antimicrobial resistance profiles, biofilm formation, and growth and inhibitory characteristics of all NAS isolates were evaluated. Half of the cows were shedding NAS, resulting in 31 NAS isolates belonging to 11 different species. The most prevalent species were Staphylococcus rostri (23%, n = 7), Staphylococcus cohnii (16%, n = 5), and Staphylococcus haemolyticus (13%, n = 4) with all Staphylococcus agnetis, Staphylococcus chromogenes, and Staph. rostri isolates belonging to the same strain according to RAPD banding patterns. Acquired antimicrobial resistance was observed in 28 of the 31 NAS isolates, mainly due to β-lactamase production. Most of the isolates (84%, n = 27) had a weak biofilm-forming potential, but only 2 contained the bap gene. The ica and aap genes were not detected in any of the isolates. In vitro growth of Staphylococcus aureus and Streptococcus dysgalactiae was inhibited by Staph. agnetis isolates, and Staph. chromogenes isolates were able to inhibit the growth of Strep. dysgalactiae and Streptococcus uberis. All fecal isolates were able to grow when oxygen and iron were limitedly available, mimicking the growth conditions in the mammary gland.     

Strain diversity and infection durations of Staphylococcus spp. and Streptococcus spp. causing intramammary infections in dairy cows

To effectively prevent and control bovine mastitis, farmers and their advisors need to take infection pathways and durations into account. Still, studies exploring both aspects through molecular epidemiology with sampling of entire dairy cow herds over longer periods are scarce. Therefore, quarter foremilk samples were collected at 14-d intervals from all lactating dairy cows (n = 263) over 18 wk in one commercial dairy herd. Quarters were considered infected with Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae when ≥100 cfu/mL of the respective pathogen was detected, or with Staphylococcus epidermidis or Staphylococcus haemolyticus when ≥500 cfu/mL of the respective pathogen was detected. All isolates of the mentioned species underwent randomly amplified polymorphic DNA (RAPD)-PCR to explore strain diversity and to distinguish ongoing from new infections. Survival analysis was used to estimate infection durations. Five different strains of Staph. aureus were isolated, and the most prevalent strain caused more than 80% of all Staph. aureus infections (n = 46). In contrast, 46 Staph. epidermidis and 69 Staph. haemolyticus strains were isolated, and none of these caused infections in more than 2 different quarters. The 3 most dominant strains of Strep. dysgalactiae (7 strains) and Strep. uberis (18 strains) caused 81% of 33 and 49% of 37 infections in total, respectively. The estimated median infection duration for Staph. aureus was 80 d, and that for Staph. epidermidis and Staph. haemolyticus was 28 and 22 d, respectively. The probability of remaining infected with Strep. dysgalactiae or Strep. uberis for more than 84 and 70 d was 58.7 and 53.5%, respectively. Staphylococcus epidermidis and Staph. haemolyticus were not transmitted contagiously and the average infection durations were short, which brings into question whether antimicrobial treatment of intramammary infections with these organisms is justified. In contrast, infections with the other 3 pathogens lasted longer and largely originated from contagious transmission.     

Characterization of coagulase-negative staphylococcus species from cows’ milk and environment based on bap,icaA,and mecA genes and phenotypic susceptibility to antimicrobials and teat dips

The aim of this study was to investigate whether the main coagulase-negative staphylococci (CNS) species involved in bovine intramammary infections (IMI) possess specific characteristics that promote colonization of the udder. Virulence markers associated with biofilm formation, antimicrobial resistance, and biocide tolerance were compared between typically contagious CNS species (Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus simulans) and those rarely causing IMI (Staphylococcus sciuri, Staphylococcus equorum, and others) to find possible associations with pathogenicity. Coagulase-negative staphylococci isolates (n = 366) belonging to 22 different species were analyzed by PCR for the presence of the biofilm-associated genes bap and icaA, and the methicillin resistance gene mecA. A selection of 82 isolates was additionally tested for their susceptibility to 5 antibiotics and 2 commercial teat dip products. Minimum inhibitory concentrations of antimicrobials were determined by Etest (AB bioMérieux, Marcy l’Etoile, France), and a microdilution method was optimized to determine minimum biocidal concentrations of teat dips. The bap, icaA, and mecA genes were detected significantly more in isolates from CNS species typically living in the cows’ environment than in isolates from IMI-causing species. Antimicrobial resistance was mainly against erythromycin (23%) or oxacillin (16%), and was detected more often in the environmental species. The isolates least susceptible to the teat dips belonged to the IMI-causing species Staph. chromogenes and Staph. simulans. We concluded that carriage of biofilm genes and antimicrobial resistance were not associated with the ability to colonize the mammary gland because free-living CNS species constituted a more significant reservoir of biofilm and resistance determinants than did IMI-causing species. In contrast, increased tolerance to biocides may favor the establishment of bovine IMI by some CNS species.     

Short communication: Diversity of species and transmission of antimicrobial resistance among Staphylococcus spp. isolated from goat milk

The increasing production of goat milk and its derivatives is affected by the occurrence of intramammary infections, which are highly associated with the presence of Staphylococcus species, including some with zoonotic potential. Staphylococci in general can exchange mobile genetic elements, a process that may be facilitated by the isolate's capacity of forming biofilms. In this study we identified, to the species level, Staphylococcus isolated from goat milk samples by MALDI-TOF and confirmed the identification by sequencing housekeeping genes (rrs and tuf). Eight species were identified, more than half being either Staphylococcus epidermidis or Staphylococcus lugdunensis. The isolates were shown by pulsed-field gel electrophoresis to be genetically diverse between the studied herds. Resistance to ampicillin and penicillin was widespread, and 2 Staph. epidermidis isolates contained the methicillin-resistance gene mecA. Most of the isolates that were resistant to at least 1 of the 13 antimicrobials tested harbored plasmids, one of which was demonstrated to be conjugative, being transferred from a Staph. epidermidis to a Staphylococcus aureus strain. Biofilm formation was observed in almost every isolate, which may contribute to their capacity of exchanging antimicrobial resistance genes in addition to acting as a physical barrier to the access of drugs. Our results showed that antimicrobial resistance among goat staphylococci may be emerging in a process facilitated by the exchange of mobile genetic elements between the bacteria and the establishment of biofilms, which calls for careful monitoring and more effective control therapies.     

Prevalence of non-aureus staphylococci species causing intramammary infections in Canadian dairy herds

Non-aureus staphylococci (NAS), the microorganisms most frequently isolated from bovine milk worldwide, are a heterogeneous group of numerous species. To establish their importance as a group, the distribution of individual species needs to be determined. In the present study, NAS intramammary infection (IMI) was defined as a milk sample containing ≥1,000 cfu/mL in pure or mixed culture that was obtained from a cohort of cows assembled by the Canadian Bovine Mastitis Research Network. Overall, 6,213 (6.3%) of 98,233 quarter-milk samples from 5,149 cows and 20,305 udder quarters were associated with an NAS IMI. Of the 6,213 phenotypically identified NAS isolates, 5,509 (89%) were stored by the Canadian Bovine Mastitis Research Network Mastitis Pathogen Collection and characterized using partial sequencing of the rpoB housekeeping gene, confirming 5,434 isolates as NAS. Prevalence of each NAS species IMI was estimated using Bayesian models, with presence of a specific NAS species as the outcome. Overall quarter-level NAS IMI prevalence was 26%. The most prevalent species causing IMI were Staphylococcus chromogenes (13%), Staphylococcus simulans (4%), Staphylococcus haemolyticus (3%), Staphylococcus xylosus (2%), and Staphylococcus epidermidis (1%). The prevalence of NAS IMI as a group was highest in first-parity heifers and was evenly distributed throughout cows in parities ≥2. The IMI prevalence of some species such as S. chromogenes, S. simulans, and S. epidermidis differed among parities. Overall prevalence of NAS IMI was 35% at calving, decreased over the next 10 d, and then gradually increased until the end of lactation. The prevalence of S. chromogenes, Staphylococcus gallinarum, Staphylococcus cohnii, and Staphylococcus capitis was highest at calving, whereas the prevalence of S. chromogenes, S. haemolyticus, S. xylosus, and S. cohnii increased during lactation. Although the overall prevalence of NAS IMI was similar across barn types, the prevalence of S. simulans, S. xylosus, S. cohnii, Staphylococcus saprophyticus, S. capitis, and Staphylococcus arlettae IMI was higher in tiestall barns; the prevalence of S. epidermidis IMI was lowest; and the prevalence of S. chromogenes and Staphylococcus sciuri IMI was highest in bedded-pack barns. Staphylococcus simulans, S. epidermidis, S. xylosus, and S. cohnii IMI were more prevalent in herds with intermediate to high bulk milk somatic cell count (BMSCC) and S. haemolyticus IMI was more prevalent in herds with high BMSCC, whereas other common NAS species IMI were equally prevalent in all 3 BMSCC categories. Distribution of NAS species IMI differed among the 4 regions of Canada. In conclusion, distribution differed considerably among NAS species IMI; therefore, accurate identification (species level) is essential for studying NAS epidemiology.     

Quarter- and cow-level risk factors for intramammary infection with coagulase-negative staphylococci species in Swiss dairy cows

Bacteriological status, evaluation of udder symmetry, udder hygiene, and teat end scores of 92 dairy cows were assessed on 3 Swiss dairy farms in a longitudinal 1-yr study to determine risk factors for intramammary infection (IMI) with coagulase-negative staphylococci (CNS) species. Farm visits were performed monthly including sterile quarter milk sampling and udder evaluation of all lactating cows. Milk samples were evaluated for the presence of staphylococci using selective agar plates. Species identification was performed using MALDI-TOF mass spectrometry. Intramammary infection was defined as milk samples having ≥100 cfu per mL of milk according to culture results. Overall, 3,151 quarter samples were included in the statistical analysis. Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus xylosus, and a Staphylococcus warneri-like species were the 4 most prevalent CNS species found. Hierarchical multivariable logistic regression models were built to evaluate risk factors for species-specific CNS IMI. Risk factors for Staph. chromogenes IMI were presence in herd B, the period from June 2014 to August 2014 and December 2014 to February 2015, and presence of udder edema. For Staph. haemolyticus, the relevant risk factor included coinfection with Staph. xylosus coinfection with other than the above-mentioned CNS species (“others”) and the period from June 2014 to November 2014. Coinfection with Staph. haemolyticus and “others,” the periods from June 2014 to August 2014 and December 2014 to February 2015, early phase of lactation (1–60 d in milk), and belonging to herd B were significantly associated with Staph. xylosus IMI. Mid and late lactation, coinfection with Staph. xylosus, and the period September 2014 to May 2015 were identified as significant risk factors for Staph. warneri-like IMI. For Staph. chromogenes, 60.6 and 26% of the variance was observed at the quarter and cow level, respectively, whereas for the other investigated species the highest variance was observed at the sample level. The predominant species within herds differed and was most pronounced for the Staph. warneri-like species.     

Antimicrobial resistance and virulence characteristics in 3 collections of staphylococci from bovine milk samples

Mastitis is a prevalent disease in dairy cattle, and staphylococci are among the most common causative pathogens. Staphylococci can express resistance to a range of antimicrobials, of which methicillin resistance is of particular public health concern. Additionally, Staphylococcus aureus carries a variety of virulence factors, although less is understood about the virulence of non-aureus staphylococci (NAS). The aim of our study was to identify and characterize 3 collections of staphylococcal isolates from bovine milk samples regarding antimicrobial resistance, with emphasis on methicillin resistance, and their carriage of virulence genes typically displayed by Staph. aureus. A total of 272 staphylococcal isolates collected in Norway and Belgium in 2016 were included, distributed as follows: group 1, Norway, 100 isolates; group 2, Flanders, Belgium, 64 isolates; group 3, Wallonia, Belgium, 108 isolates. Species identification was performed by use of MALDI-TOF mass spectrometry. Phenotypic resistance was determined via disk diffusion, and PCR was used for detection of methicillin resistance genes, mecA and mecC, and virulence genes. Antimicrobial resistance was common in Staphylococcus epidermidis and Staphylococcus haemolyticus from all different groups, with resistance to trimethoprim-sulfonamide frequently occurring in Staph. epidermidis and Staph. haemolyticus as well as in Staph. aureus. Resistance to penicillin was most frequently observed in group 1. Ten Belgian isolates (1 from group 2, 9 from group 3) carried the methicillin resistance determinant mecA: 5 Staph. aureus from 2 different farms and 5 NAS from 3 different farms. Almost all Staph. aureus isolates were positive for at least 3 of the screened virulence genes, whereas, in total, only 8 NAS isolates harbored any of the same genes. Our study contributes to the continuous need for knowledge regarding staphylococci from food-producing animals as a basis for better understanding of occurrence of resistance and virulence traits in these bacteria.     

Distribution of non-aureus staphylococci species in udder quarters with low and high somatic cell count,and clinical mastitis

The effect of non-aureus staphylococci (NAS) in bovine mammary health is controversial. Overall, NAS intramammary infections (IMI) increase somatic cell count (SCC), with an effect categorized as mild, mostly causing subclinical or mild to moderate clinical mastitis. However, based on recent studies, specific NAS may affect the udder more severely. Some of these apparent discrepancies could be attributed to the large number of species that compose the NAS group. The objectives of this study were to determine (1) the SCC of quarters infected by individual NAS species compared with NAS as a group, culture-negative, and major pathogen-infected quarters; (2) the distribution of NAS species isolated from quarters with low SCC (<200,000 cells/mL) and high SCC (≥200,000 cells/mL), and clinical mastitis; and (3) the prevalence of NAS species across quarters with low and high SCC. A total of 5,507 NAS isolates, 3,561 from low SCC quarters, 1,873 from high SCC quarters, and 73 from clinical mastitis cases, were obtained from the National Cohort of Dairy Farms of the Canadian Bovine Mastitis Research Network. Of quarters with low SCC, high SCC, or clinical mastitis, 7.6, 18.5, and 4.3% were NAS positive, respectively. The effect of NAS IMI on SCC was estimated using mixed-effect linear regression; prevalence of NAS IMI was estimated using Bayesian analyses. Mean SCC of NAS-positive quarters was 70,000 cells/mL, which was higher than culture-negative quarters (32,000 cells/mL) and lower than major pathogen-positive quarters (129,000 to 183,000 cells/mL). Compared with other NAS species, SCC was highest in quarters positive for Staphylococcus capitis, Staphylococcus gallinarum, Staphylococcus hyicus, Staphylococcus agnetis, or Staphylococcus simulans. In NAS-positive quarters, Staphylococcus xylosus (12.6%), Staphylococcus cohnii (3.1%), and Staphylococcus equorum (0.6%) were more frequently isolated from quarters with low SCC than other NAS species, whereas Staphylococcus sciuri (14%) was most frequently isolated from clinical mastitis cases. Finally, in NAS-positive quarters, Staphylococcus chromogenes, S. simulans, Staphylococcus epidermidis, and Staphylococcus haemolyticus were isolated with similar frequency from among low SCC and high SCC quarters and clinical mastitis cases. Staphylococcus chromogenes, S. simulans, S. xylosus, S. haemolyticus, S. epidermidis, S. agnetis, Staphylococcus arlettae, S. capitis, S. gallinarum, S. sciuri, and Staphylococcus warneri were more prevalent in high than in low SCC quarters. Because the NAS are a large, heterogeneous group, considering them as a single group rather than at the species, or even subspecies level, has undoubtedly contributed to apparent discrepancies among studies as to their distribution and importance in IMI and mastitis.     

Some coagulase-negative Staphylococcus species affect udder health more than others

A longitudinal study in 3 dairy herds was conducted to profile the distribution of coagulase-negative Staphylococcus (CNS) species causing bovine intramammary infection (IMI) using molecular identification and to gain more insight in the pathogenic potential of CNS as a group and of the most prevalent species causing IMI. Monthly milk samples from 25 cows in each herd as well as samples from clinical mastitis were collected over a 13-mo period. Coagulase-negative staphylococci were identified to the species level using transfer-RNA intergenic spacer PCR. The distribution of CNS causing IMI was highly herd-dependent, but overall, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus simulans were the most prevalent. No CNS species were found to cause clinical mastitis. The effect of the most prevalent species on the quarter milk somatic cell count (SCC) was analyzed using a linear mixed model, showing that Staph. chromogenes, Staph. simulans, and Staph. xylosus induced an increase in the SCC that is comparable with that of Staphylococcus aureus. Almost all CNS species were able to cause persistent IMI, with Staph. chromogenes causing the most persistent infections. In conclusion, accurate species identification cannot be ignored when studying the effect of CNS on udder health, as the effect on SCC differs between species and species distribution is herd-specific. Staphylococcus chromogenes, Staph. simulans, and Staph. xylosus seem to be the more important species and deserve special attention in further studies. Reasons for herd dependency and possible cow- and quarter-level risk factors should be examined in detail for the different species, eventually leading to cost-benefit analyses for management changes and, if needed, treatment recommendations.     

Short communication: Prevalence of methicillin resistance in coagulase-negative staphylococci and Staphylococcus aureus isolated from bulk milk on organic and conventional dairy farms in the United States

The objective of this study was to evaluate the presence of methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus spp. in bulk tank milk samples from 288 organic and conventional dairy farms located in New York, Wisconsin, and Oregon from March 2009 to May 2011. Due to recent publications reporting the presence mecC (a mecA homolog not detected by traditional mecA-based PCR methods), a combination of genotypic and phenotypic approaches was used to enhance the recovery of methicillin-resistant organisms from bulk tank milk. In total, 13 isolates were identified as methicillin resistant: Staph. aureus (n = 1), Staphylococcus sciuri (n = 5), Staphylococcus chromogenes (n = 2), Staphylococcus saprophyticus (n = 3), Staphylococcus agnetis (n = 1), and Macrococcus caseolyticus (n = 1). The single methicillin-resistant Staph. aureus isolate was identified from an organic farm in New York, for an observed 0.3% prevalence at the farm level. The methicillin-resistant coagulase-negative staphylococci prevalence was 2% in the organic population and 5% in the conventional population. We did not identify mecC in any of the isolates from our population. Of interest was the relatively high number of methicillin-resistant Staph. sciuri recovered, as the number of isolates from our study was considerably higher than those recovered from other recent studies that also assessed milk samples. Our research suggests that the presence of a potential methicillin-resistant Staphylococcus reservoir in milk, and likely the dairy farm population in the United States, is independent of the organic or conventional production system.     

Antimicrobial resistance and genetic characterization of coagulase-negative staphylococci from bovine mastitis milk samples in Korea

Coagulase-negative staphylococci (CNS) are one of the most common bovine mastitis pathogens found worldwide. In this study, we investigated the prevalence and distribution of CNS species in mastitis milk samples and further characterized the methicillin-resistant (MR) CNS. A total of 311 CNS were isolated from 3,692 quarter milk samples from 1,373 dairy cattle at 81 farms between 2013 and 2017. Further evaluation of the CNS isolates revealed 14 CNS species among the samples and 3 predominant species—namely, Staphylococcus chromogenes, Staphylococcus simulans, and Staphylococcus epidermidis. Resistance was higher in S. epidermidis than in other CNS species except for resistance against oxacillin in Staphylococcus sciuri. Resistance to β-lactams was the most common in all CNS species (8.4% in ampicillin, 21.2% in oxacillin, and 13.5% in penicillin). Conversely, only minimal resistance to cephalothin, ceftiofur, and pirlimycin/novobiocin was found. Twenty-one isolates from 4 species were mecA-carrying MRCNS strains, including 18 S. epidermidis and 1 each of S. sciuri, Staphylococcus equorum, and Staphylococcus hominis. The majority of the mecA-carrying MRCNS isolates were produced in the biofilm. Furthermore, multidrug-resistant sequence type 179 isolate produced the strongest biofilm. Seven genotypes were detected in the 18 MR S. epidermidis strains, the most predominant of which persisted on a farm for 2 yr. Our findings for the antimicrobial susceptibility profiles and genotypic characterization of the MRCNS isolates could provide valuable information for controlling the spread of resistance and the selection of appropriate antimicrobial therapies for mastitis in the future. Further, strategic antibiotic use for mastitis treatment and hygienic management practices aimed at the prevention of the growth of resistant bacteria are urgently needed on dairy farms.     

Antibacterial potential of Enterococcus faecium strains isolated from ewes’ milk and cheese

The study was conducted to evaluate the antibacterial activity of three bacteriocin-producing Enterococcus faecium strains (TW15, TW20 and TW22) isolated from ewes’ milk and cheese sampled in the Patagonian region of Argentina. The strains were tested against spoilage and pathogens microorganisms showing antimicrobial activity towards 4 strains of Listeria monocytogenes, one strain of Listeria innocua and 2 strains of Staphylococcus aureus. E. faecium TW15, E. faecium TW20 and E. faecium TW22 were sensitive to vancomycin. Furthermore, investigation of virulence factors revealed the absence of the genes encoding them. The bacteriocin-like substances (BLISs) produced by the 3 strains were thermostable, pH resistant and can be expressed even in the presence of NaCl (3.0 g/100 g). Moreover, they prove to have a bactericidal mode of action. Results from physicochemical and biochemical characteristics of BLIS produced by these E. faecium strains make them potential candidates to aid in preservation of foods.     

Antimicrobial activity of crude extracts from actinomycetes against mastitis pathogens

The emergence of antimicrobial resistance to commonly used antibiotics has necessitated the development of new antimicrobial products. Crude extracts produced by actinomycetes contain antimicrobial metabolites that can inhibit bacterial growth. The objective of our study was to evaluate the antimicrobial activity of crude extracts (Caat1–54 and CaatP5–8) produced by actinomycetes against isolates of Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, and Streptococcus uberis, which were obtained from the milk of cows affected by mastitis in 23 dairy herds. Twenty isolates of each bacterial species were used to define minimum inhibitory concentrations (MIC) of both crude extracts and ceftiofur (positive control). The MIC₅₀ and MIC₉₀ were defined at the concentration required to inhibit the growth of 50 and 90% of bacterial isolates tested, respectively. The MIC results were evaluated by survival analysis. Staphylococcus aureus isolates presented MIC₉₀ of Caat 1–54 ≥6.25 µg/mL, ceftiofur ≥12.5 µg/mL, and Caat P5–8 ≥100 µg/mL. Streptococcus uberis presented MIC₉₀ of ceftiofur ≥0.39 µg/mL, Caat 1–54 ≥50 µg/mL, and Caat P5–8 ≥100 µg/mL. Staphylococcus chromogenes isolated from subclinical mastitis presented MIC₉₀ of Caat 1–54 ≥0.78 µg/mL and ceftiofur and Caat P5–8 of ≥6.25 and ≥100 µg/mL, respectively. Streptococcus dysgalactiae isolated from clinical mastitis presented similar MIC₉₀ values between antimicrobials tested (ceftiofur, Caat 1–54, and Caat P-58), but these values (≥100 µg/mL) were higher than the values obtained from other pathogens evaluated in the present study. Our results indicate that Caat 1–54 and Caat P5–8 crude extracts present in vitro antimicrobial activity against isolates of Staph. aureus, Staph. chromogenes, Strep. dysgalactiae, and Strep. uberis isolated from clinical and subclinical mastitis.     

Distribution of coagulase-negative Staphylococcus species from milk and environment of dairy cows differs between herds

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant pathogens causing intramammary infections (IMI) in dairy cows. The cows’ environment is thought to be a possible source for CNS mastitis and this was investigated in the present paper. A longitudinal field study was carried out in 6 well-managed dairy herds to determine the distribution and epidemiology of various CNS species isolated from milk, causing IMI and living freely in the cows’ environment, respectively. In each herd, quarter milk samples from a cohort of 10 lactating cows and environmental samples from stall air, slatted floor, sawdust from cubicles, and sawdust stock were collected monthly (n = 13). Isolates from quarter milk samples (n = 134) and the environment (n = 637) were identified to species level using amplified fragment length polymorphism (AFLP) genotyping. Staphylococcus chromogenes, S. haemolyticus, S. epidermidis, and S. simulans accounted for 81.3% of all CNS milk isolates. Quarters were considered infected with CNS (positive IMI status) only when 2 out of 3 consecutive milk samples yielded the same CNS AFLP type. The species causing IMI were S. chromogenes (n = 35 samples with positive IMI status), S. haemolyticus (n = 29), S. simulans (n = 14), and S. epidermidis (n = 6). The observed persistent IMI cases (n = 17) had a mean duration of 149.4 d (range 63.0 to 329.8 d). The CNS species predominating in the environment were S. equorum, S. sciuri, S. haemolyticus, and S. fleurettii. Herd-to-herd differences in distribution of CNS species were observed in both milk and the environment, suggesting that herd-level factors are involved in the establishment of particular species in a dairy herd. Primary reservoirs of the species causing IMI varied. Staphylococcus chromogenes and S. epidermidis were rarely found in the environment, indicating that other reservoirs were more important in their epidemiology. For S. haemolyticus and S. simulans, the environment was found as a reservoir, suggesting that IMI with these species were possibly environmental in origin.     

Technical note: Antimicrobial susceptibility of Portuguese isolates of Staphylococcus aureus and Staphylococcus epidermidis in subclinical bovine mastitis

To evaluate the antimicrobial resistance traits of staphylococci responsible for subclinical bovine mastitis in Portugal, the minimum inhibitory concentrations (MIC) of 7 antimicrobial agents, frequently administered for mastitis treatment, were determined for 30 Staphylococcus aureus and 31 Staphylococcus epidermidis field isolates. β-Lactamase production was detected through the use of nitrocefin-impregnated discs. The MIC that inhibited 90% of the isolates tested (MIC₉₀) of penicillin, oxacillin, cefazolin, gentamicin, sulfamethoxazole/trimethoprim, oxytetracycline, and enrofloxacin were, respectively, 4, 0.5, 1, 1, 0.25, 0.25, and 0.06 μg/mL for Staph. aureus and ≥64, 8, 1, 32, ≥64, ≥64, and 0.06 μg/mL for Staph. epidermidis. All Staph. aureus isolates showed susceptibility to oxacillin, cefazolin, gentamicin, sulphamethoxazole/trimethoprim, and enrofloxacin. β-Lactamase production was detected in 20 of these isolates (66.7%), all of which were resistant to penicillin. Of the 31 Staph. epidermidis tested, 24 (77.4%) were β-lactamase positive. All isolates were susceptible to both cefazolin and enrofloxacin. Nine Staph. epidermidis isolates were resistant to oxacillin, with MIC values ranging from 4 to 8 μg/mL. The MIC values of 5 antimicrobial agents tested were higher than those reported in other countries. Enrofloxacin was the only exception, showing lower MIC values compared with other reports. Overall, the antimicrobial agents tested in our study, with the exception of penicillin, were active against the 61 isolates studied.     

Short communication: β-Lactam resistance and vancomycin heteroresistance in Staphylococcus spp. isolated from bovine subclinical mastitis

The use of antimicrobial agents has led to the emergence of resistant bacterial strains over a relatively short period. Furthermore, Staphylococcus spp. can produce β-lactamase, which explains the survival of these strains in a focus of infection despite the use of a β-lactam antibiotic. The aim of this study was to evaluate the resistance of Staphylococcus spp. isolated from bovine subclinical mastitis to oxacillin and vancomycin (by minimum inhibitory concentration) and to detect vancomycin heteroresistance by a screening method. We also evaluated β-lactamase production and resistance due to hyperproduction of this enzyme and investigated the mecA and mecC genes and performed staphylococcal cassette chromosome mec typing. For this purpose, 181 Staphylococcus spp. isolated from mastitis subclinical bovine were analyzed. Using the phenotypic method, 33 (18.2%) of Staphylococcus spp. were resistant to oxacillin. In contrast, all isolates were susceptible to vancomycin, and heteroresistance was detected by the screening method in 13 isolates. Production of β-lactamase was observed in 174 (96%) of the Staphylococcus spp. isolates. The mecA gene was detected in 8 isolates, all of them belonging to the species Staphylococcus epidermidis, and staphylococcal cassette chromosome mec typing revealed the presence of type I and type IV isolates.     

Antibacterial activity and cholesterol assimilation of lactic acid bacteria isolated from traditional Iranian dairy products

In this study, lactic acid bacteria were isolated from ewe milk, traditional yoghurt and sour buttermilk samples collected from different areas of Azarbayjan-e-sharqi in Iran. All the isolates were screened for their ability to produce bacteriocin like inhibitory substances (BLIS) by studying their inhibitory action against pathogens like Listeria monocytogenes, Salmonella enteritidis and Staphylococcus aureus, after eliminating the effect of organic acids and hydrogen peroxide. According to results, four of the isolates identified as Lactobacillus brevis, Lactobacillus pentosus, Pedoicoccus acidilactici and Lactobacillus paracasei were unaffected by the action of pH neutralization and hydrogen peroxide and showed inhibitory action against the tested pathogens. The inhibitory activities demonstrated by these isolates were completely inhibited in the presence of proteolytic enzymes.     

Molecular epidemiology and distribution of antimicrobial resistance genes of Staphylococcus species isolated from Chinese dairy cows with clinical mastitis

Staphylococcus species, categorized into Staphylococcus aureus and non-aureus staphylococci (NAS), are frequent causes of mastitis in dairy cattle around the world. Current treatments using antimicrobials are under increasing scrutiny due to rising prevalence of multi-drug resistance in S. aureus. Objectives of this study were to determine: (1) genetic diversity of Staphylococcus species isolated from clinical mastitis in cows from large Chinese dairy farms; and (2) prevalence and distribution of antimicrobial resistance genes (ARG) in these isolates. Staphylococcus aureus (n = 96) were isolated from 26 herds located in 12 provinces of China, whereas NAS (n = 112) were isolated from 59 herds located in 18 provinces of China. The NAS were identified at the species level using a partial 16S rRNA sequencing method, whereas random amplification of polymorphic DNA (RAPD) PCR was done to determine genetic relationships of isolates. Finally, PCR was used to detect resistance and biofilm formation genes. Staphylococcus chromogenes (33%) was the most common NAS species, followed by Staphylococcus sciuri (17%) and Staphylococcus epidermidis (8%). Staphylococcus aureus was grouped in 12 genotypes, of which 2 types represented 56% of isolates. Staphylococcus chromogenes (n = 37) clustered into 8 RAPD types, with 2 prevalent types containing 73% of isolates. The most prevalent ARG in S. aureus isolates was blaZ (95%), followed by tetM (33%), tetK (31%), ermT (26%), and aacA-aphD (23%). The mecA and vanA were detected in 16 and 4% of isolates, respectively. In NAS, blaZ (100%), mecA (73%), tetK (79%), tetM (96%), mphC (63%), and msrA (54%) were frequently detected. Antimicrobial resistance genes mecA, tetK, tetL, tetM, dfrG, ermB, msrA, mphC, aadD, and aphA3 were more commonly detected in NAS than in S. aureus. Biofilm formation genes (icaA and icaD) were frequently detected in staphylococci isolated from bovine clinical mastitis. The existence of predominant RAPD types in S. aureus and S. chromogenes isolates across Chinese dairy farms indicated that specific genotypes had disseminated within herds and become more udder-adapted. High prevalence of ARG, especially in NAS, highlighted the risk of selection of multi-drug resistant staphylococci with potential as a reservoir of ARG.     

Bovine-associated staphylococci and mammaliicocci trigger T-lymphocyte proliferative response and cytokine production differently

We examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B- and T-lymphocyte proliferation and interleukin (IL)-17A and interferon (IFN)-γ production by peripheral blood mononuclear cells in nulliparous, primiparous, and multiparous dairy cows. Flow cytometry was used to measure lymphocyte proliferation with the Ki67 antibody, and specific monoclonal antibodies were used to identify CD3, CD4, and CD8 T lymphocyte and CD21 B lymphocyte populations. The supernatant of the peripheral blood mononuclear cell culture was used to measure IL-17A and IFN-γ production. Two distinct, inactivated strains of bovine-associated Staphylococcus aureus [one causing a persistent intramammary infection (IMI) and the other from the nose], 2 inactivated Staphylococcus chromogenes strains [one causing an IMI and the other from a teat apex), as well as an inactivated Mammaliicoccus fleurettii strain originating from sawdust from a dairy farm, and the mitogens concanavalin A and phytohemagglutinin M-form (both specifically to measure lymphocyte proliferation) were studied. In contrast to the “commensal” Staph. aureus strain originating from the nose, the Staph. aureus strain causing a persistent IMI triggered proliferation of CD4⁺ and CD8⁺ subpopulations of T lymphocytes. The M. fleurettii strain and the 2 Staph. chromogenes strains had no effect on T- or B-cell proliferation. Furthermore, both Staph. aureus and Staph. chromogenes strains causing persistent IMI significantly increased IL-17A and IFN-γ production by peripheral blood mononuclear cells. Overall, multiparous cows tended to have a higher B-lymphocyte and a lower T-lymphocyte proliferative response than primiparous and nulliparous cows. Peripheral blood mononuclear cells of multiparous cows also produced significantly more IL-17A and IFN-γ. In contrast to concanavalin A, phytohemagglutinin M-form selectively stimulated T-cell proliferation.