Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis

Abstract

-Unnerstad, et al.. Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis. Veterinary Microbiology, Elsevier, 2009, 134 (3-4) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Page 1 with results obtained by tuf gene sequencing showed that Staph-Zym™ correctly identified 29 CNS reference strains to species level more often than bovine milk CNS isolates (83% and 30 61%, respectively). In addition, tests supplementary to the Staph-Zym™ were frequently 31 needed in both groups of isolates (50% of reference strains and 33% of milk isolates) to 32 obtain an identification of the strain. It is notable that Staph-Zym™ judged two isolates as 33 CNS, although they belonged to other species, could not give a species name in 11% of the 34 bovine CNS isolates, and gave 28% of the isolates an incorrect species name. The present 35 study indicates that the studied phenotypic methods are unreliable for identification of CNS 36 from bovine intra-mammary infections. 37 38

Differentiation of lactic acid bacteria based on RFLP analysis of the tuf gene

“…In fact, recently diverged species such as L. plantarum, L. paraplantarum, L. pentosus or L. casei, L. rhamnosus, and L. zeae could not be clearly differentiated by 16S rRNA (6,7). Recently, the tuf gene has been successfully employed to differentiate closely-related bacterial strains such as nontuberculous mycobacteria (25), coagulase-negative staphylococci (26), and Yersinia species (27). Therefore, the feasibility and suitability of the tuf gene for identifying and differentiating many closely-related Lactobacillus spp.…”

Identification of Coagulase-Negative Staphylococci from Bovine Intramammary Infection by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…This gene has well-conserved DNA sequences within a given species but with sufficient sequence variations to allow species-specific identification (33). A number of PCR amplicon sequencing-based methods targeting the 16S rRNA, sodA, and tuf genes have been reported for identification of CoNS (9,16,34). However, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in the GenBank database, especially for identification of closely related Staphylococcus species (35).…”

“…According to Capurro et al (9), 16 CoNS species have been isolated from milk samples collected from dairy cows with mastitis. Staphylococcus chromogenes was the CoNS species most frequently isolated (74.07%) from dairy cows with IMI in this study.…”

“…Sixteen CoNS species have been previously isolated from cows with clinical and subclinical mastitis (9). Cows with mastitis caused by S. chromogenes, S. simulans, and S. xylosus had somatic cell counts (SCC) similar to those observed in cows with mastitis caused by S. aureus (10).…”

tuf Gene Sequence Analysis Has Greater Discriminatory Power than 16S rRNA Sequence Analysis in Identification of Clinical Isolates of Coagulase-Negative Staphylococci

“…PCR amplifications were conducted in a total volume of 25 l containing 2.5 mM deoxynucleoside triphosphates (dNTPs), 10 pmol of each PCR primer, 0.6 U Taq polymerase, 2.5 l of 10ϫ PCR buffer with 15 mM MgCl 2 (Takara Bio, Inc., Shiga, Japan), and 2.5 l of the template. In-house primers were designed using LightCycler Probe Design software (version 2.0) (Roche, Penzberg, Germany); published studies were used as a reference (7,10,15). 16S rRNA was amplified with primers MSQ-F (5Ј-TGA AGAGTTTGATCATGGCTCAG-3Ј) and MSQ-R (5Ј-ACCGCGGCTGCTGG CAC-3Ј), and the tuf gene was amplified with primers TUF-F (5Ј-GCCAGTTG AGGACGTATTCT-3Ј) and TUF-R (5Ј-CCATTTCAGTACCTTCTGGTAA-3Ј).…”