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.
CashionP.,
Holder-FranklinM. A.,
McCullyJ.,
FranklinM.1977; A rapid method for the base ratio determination of bacterial DNA. Anal Biochem 81:461–466 [View Article][PubMed]
DevrieseL. A.,
van de KerckhoveA.1979; A comparison of methods and the validity of deoxyribonuclease tests for the characterization of staphylococci isolated from animals. J Appl Bacteriol 46:385–393 [View Article][PubMed]
FreneyJ.,
KloosW. E.,
HajekV.,
WebsterJ. A.,
BesM.,
BrunY.,
Vernozy-RozandC.
other authors 1999; Recommended minimal standards for description of new staphylococcal species. Subcommittee on the taxonomy of staphylococci and streptococci of the International Committee on Systematic Bacteriology. Int J Syst Bacteriol 49:489–502 [View Article][PubMed]
GohS. H.,
SantucciZ.,
KloosW. E.,
FaltynM.,
GeorgeC. G.,
DriedgerD.,
HemmingsenS. M.1997; Identification of Staphylococcus species and subspecies by the chaperonin 60 gene identification method and reverse checkerboard hybridization. J Clin Microbiol 35:3116–3121[PubMed]
GötzF.,
BannermanT.,
SchleiferK.-H.2006; The genera Staphylococcus and Macrococcus
. In The Prokaryotes vol. 4 Bacteria: Firmicutes, Cyanobacteria; pp. 5–75 Edited by
DworkinM.,
FalkowS.,
RosenbergE.,
SchleiferK.-H.,
StackebrandtE.
New York: Springer; [CrossRef]
JoussonO.,
Di BelloD.,
VanniM.,
CardiniG.,
SoldaniG.,
PrettiC.,
IntorreL.2007; Genotypic versus phenotypic identification of staphylococcal species of canine origin with special reference to Staphylococcus schleiferi subsp. coagulans
. Vet Microbiol 123:238–244 [View Article][PubMed]
KoivulaM.,
PitkäläA.,
PyöräläS.,
MäntysaariE. A.2007; Distribution of bacteria and seasonal and regional effects in a new database for mastitis pathogens in Finland. Acta Agric Scand A 57:89–96 [View Article]
KuykendallL. D.,
RoyM. A.,
O'NeillJ. J.,
DevineT. E.1988; Fatty acids, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradorhizobium japonicum
. Int J Syst Bacteriol 38:358–361 [View Article]
MesbahM.,
PremachandranU.,
WhitmanW.1989; Precise measurement of the G+C content of deoxyribonucleic acid by high performance liquid chromatography. Int J Syst Bacteriol 39:159–167 [View Article]
PiepersS. L.,
De MeulemeesterL.,
de KruifA.,
OpsomerG.,
BarkemaH. W.,
De VliegherS.2007; Prevalence and distribution of mastitis pathogens in subclinically infected dairy cows in Flanders, Belgium. J Dairy Res 74:478–483 [View Article][PubMed]
PiessensV.,
SupréK.,
HeyndrickxM.,
HaesebrouckF.,
De VliegherS.,
Van CoillieE.2010; Validation of amplified fragment length polymorphism genotyping for species identification of bovine associated coagulase-negative staphylococci. J Microbiol Methods 80:287–294 [View Article][PubMed]
SampimonO. C.,
ZadoksR. N.,
De VliegherS.,
SupréK.,
HaesebrouckF.,
BarkemaH. W.,
SolJ.,
LamT. J. G. M.2009; Performance of API Staph ID 32 and Staph-Zym for identification of coagulase-negative staphylococci isolated from bovine milk samples. Vet Microbiol 136:300–305 [View Article][PubMed]
SupréK.,
De VliegherS.,
SampimonO. C.,
ZadoksR. N.,
VaneechoutteM.,
BaeleM.,
De GraefE.,
PiepersS.,
HaesebrouckF.2009; Technical note: use of transfer RNA-intergenic spacer PCR combined with capillary electrophoresis to identify coagulase-negative Staphylococcus species originating from bovine milk and teat apices. J Dairy Sci 92:3204–3210 [View Article][PubMed]
TamaokaJ.,
KomagataK.1984; Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25:125–128 [View Article]
Taponen, S. S. (2008).Bovine mastitis caused by coagulase-negative staphylococci. PhD thesis, University of Helsinki, Finland.
TaponenS.,
SimojokiH.,
HaveriM.,
LarsenH. D.,
PyöräläS.2006; Clinical characteristics and persistence of bovine mastitis caused by different species of coagulase-negative staphylococci identified with API or AFLP. Vet Microbiol 115:199–207 [View Article][PubMed]
TaponenS.,
KoortJ.,
BjörkrothJ.,
SaloniemiH.,
PyöräläS.2007; Bovine intramammary infections caused by coagulase-negative staphylococci may persist throughout lactation according to amplified fragment length polymorphism-based analysis. J Dairy Sci 90:3301–3307 [View Article][PubMed]
TaponenS.,
BjörkrothJ.,
PyöräläS.2008; Coagulase-negative staphylococci isolated from bovine extramammary sites and intramammary infections in a single dairy herd. J Dairy Res 75:422–429 [View Article][PubMed]
WayneL. G.,
BrennerD. J.,
ColwellR. R.,
GrimontP. A. D.,
KandlerO.,
KrichevskyM. I.,
MooreL. H.,
MooreW. E. C.,
MurrayR. G. E. et al.1987; International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37:463–464 [View Article]