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Coagulase-negative staphylococci (CNS) are abundantly present in the dairy farm environment and on bovine skin and mucosae. They are also the most prevalent bacteria causing bovine intramammary infections (IMI). Reservoirs and transmission routes of CNS are not yet fully unraveled. The objectives of this study were to explore the distribution of CNS in parlor-related extramammary niches and to compare it to the distributions of CNS causing IMI in those herds. Niches that were targeted in this study were cows' teat apices, milking machine unit liners, and milker's skin or gloves. Each of the three herds had its own CNS microbiota in those niches. The most prevalent species in the parlor-related extramammary niches were Staphylococcus cohnii, S. fleurettii, and S. equorum in the first, second, and third herd, respectively, whereas S. haemolyticus and S. sciuri were found in all herds. S. cohnii and S. fleurettii, as well as S. haemolyticus, which was present in each herd, were also frequently found in milk samples. By contrast, S. chromogenes, S. simulans, and S. xylosus favored the mammary gland, whereas S. equorum was more common in the parlor-associated niches. Within each herd, species distribution was similar between teat apices and milking machine unit liners. In conclusion, some of the extramammary niches related to the milking process might act as infection sources for IMI-causing CNS. This study provides further evidence that the group of CNS species is comprised of environmental, opportunistic and host-adapted species which differ in ecology.

Although many studies report coagulase-negative staphylococci (CNS) as the predominant cause of subclinical bovine mastitis, their epidemiology is poorly understood. In the current study, the genetic diversity within four CNS species frequently associated with bovine intramammary infections, Staphylococcus haemolyticus, S. simulans, S. chromogenes, and S. epidermidis, was determined. For epidemiological purposes, CNS genotypes recovered from bovine milk collected on six Flemish dairy farms were compared with those from the farm environment, and their distribution within the farms was investigated. Genetic diversity was assessed by two molecular typing techniques, amplification fragment length polymorphism (AFLP) and random amplification of polymorphic DNA (RAPD) analysis. Subtyping revealed the highest genetic heterogeneity among S. haemolyticus isolates. A large variety of genotypes was found among environmental isolates, of which several could be linked with intramammary infection, indicating that the environment could act as a potential source for infection. For S. simulans, various genotypes were found in the environment, but a link with IMI was less obvious. For S. epidermidis and S. chromogenes, genetic heterogeneity was limited and the sporadic isolates from environment displayed largely the same genotypes as those from milk. The higher clonality of the S. epidermidis and S. chromogenes isolates from milk suggests that specific genotypes probably disseminate within herds and are more udder-adapted. Environmental sources and cow-to-cow transmission both seem to be involved in the epidemiology of CNS, although their relative importance might substantially vary between species.

Coagulase-negative staphylococci (CNS) are a major cause of intramammary infections (IMI) in dairy cows and they colonize the teat skin. Staphylococcus haemolyticus, one of the more common CNS, has been identified as a highly versatile opportunistic species. The aim of the present study was to gain better insight into the adaptation of S. haemolyticus subtypes to the udder ecosystem with respect to IMI development. During a longitudinal observational study conducted over 13 mo on 6 Flemish dairy herds, S. haemolyticus isolates were recovered from milk and teat apices. A total of 44 S. haemolyticus isolates originating from milk (24 isolates) and teat apices (20 isolates) of 6 selected udder quarters were singled out and analyzed using a combined methodology of (GTG)5-PCR and amplified fragment length polymorphism (AFLP) fingerprinting to determine intraspecies differences. Combining both fingerprinting methods, 4 S. haemolyticus subtypes were obtained (I to IV). Subtypes I, II, and IV were recovered from both milk and teat apex samples and were found to be associated with persisting IMI. Subtype III, not apparently related to IMI, was isolated solely from teat apices and not from milk. In general, S. haemolyticus subtypes found in milk from infected quarters could be recovered from the corresponding teat apices, although the latter could be colonized with up to 3 different subtypes. Comparing subtypes from milk and teat apices indicates that the IMI-causing agent likely originates from the teat skin.

Swab samples (n=72) obtained from the teat apex of lactating dairy cows without visual signs of inflammation (n=18) were gathered on 2 well-managed Flemish dairy herds (herds 1 and 2) during the same month to assess the bacterial diversity of teat apices before milking. A combination of both culture-dependent [plating and (GTG)(5)-PCR fingerprinting of the colonies] and culture-independent [denaturing gradient gel electrophoresis (PCR-DGGE)] techniques indicated that the teat apices contain a wide diversity of bacterial genera. Despite a low bacterial load, 20 bacterial genera of 3 phyla (Actinobacteria, Firmicutes, and Proteobacteria) were present. The most prevalent bacteria were the coagulase-negative staphylococci (CNS), encompassing a total of 15 species, which were identified to the species level using a combination of (GTG)(5)-PCR fingerprinting, gene sequencing (16S ribosomal RNA and rpoB genes), and a novel PCR-DGGE technique based on the tuf-PCR amplicon. Overall bacterial diversity did not differ significantly between the herds or between noninfected and subclinically infected quarters in herd 1. In herd 1, borderline significant lower CNS species diversity was found on teat apices of noninfected quarters compared with subclinically infected quarters. The most prevalent CNS species were Staphylococcus haemolyticus and Staphylococcus equorum in both herds and Staphylococcus carnosus in herd 2.

Thirteen Gram-positive-staining coagulase-variable staphylococci were isolated from subclinical and mild clinical mastitic bovine milk (n = 12) and a teat apex (n = 1). The results of sequence analysis of the 16S rRNA gene and two housekeeping genes, rpoB and tuf, and DNA fingerprinting with amplified fragment length polymorphism (AFLP) analysis showed that the isolates formed a separate branch within the genus Staphylococcus. The phylogenetically most closely related species were Staphylococcus hyicus and Staphylococcus chromogenes. DNA–DNA hybridization with S. hyicus DSM 20459T and S. chromogenes DSM 20674T confirmed that the isolates belonged to a separate species. The predominant fatty acids were i-C15 : 0, ai-C15 : 0, i-C17 : 0 and C20 : 0 and the peptidoglycan type was A3α l-Lys–Gly5. Based on the results of genotypic and phenotypic analyses, it is proposed that the thirteen isolates represent a novel species, for which the name Staphylococcus agnetis sp. nov. is proposed. Strain 6-4T ( = DSM 23656T = CCUG 59809T) is the type strain.

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 withthese species were possibly environmental in origin.

A longitudinal study was carried out to detect intramammary infections caused by Klebsiella pneumoniae and to identify potential sources of this bacterial species in the environment of the cows. The study was performed in 6 well-managed Belgian dairy herds from May 2008 to May 2009. Monthly (n = 13), unused and used sawdust bedding samples as well as individual quarter milk and feces samples were collected from 10 randomly selected cohort cows in each herd. Cases of clinical mastitis of all lactating cows in the 6 herds were also sampled (n = 64). From the 3,518 collected samples, 153 K. pneumoniae isolates were obtained, of which 2 originated from milk (clinical mastitis cases). In feces (n = 728), used bedding (n = 73), and unused bedding (n = 73), respectively, 125 (17.2%), 20 (27.4%), and 6 (8.2%) isolates were found. The isolates were fingerprinted by means of pulsed field gel electrophoresis. In total, 109 different pulsotypes were differentiated, indicating a high degree of genetic diversity within the isolates. All isolates from unused bedding belonged to pulsotypes other than those from the other sources, suggesting that sources other than unused sawdust may introduce K. pneumoniae into the herd. Only 2 pulsotypes contained isolates originating from different sources. Pulsotype 10 was found in milk and used bedding and pulsotype 21 was found in feces and used bedding. The 2 milk isolates originated from 2 cows in the same herd but they belonged to a different pulsotype. The results indicate that K. pneumoniae can be prevalent in the environment without causing significant mastitis problems. Most cows were shedding K. pneumoniae in feces, substantiating findings under very different conditions (i.e., American dairy herds). Contamination of used bedding in the cubicles with K. pneumoniae from feces was confirmed, whereas unused bedding was not an important source of K. pneumoniae for the environment of the cows.

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 bioMerieux, 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.