Comparison of phenotypic and genotypic methods for the species

Veterinary Microbiology 147 (2011) 142–148

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Research article

Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections Joo Youn Park a, Lawrence K. Fox a,*, Keun Seok Seo b, Mark A. McGuire c, Yong Ho Park d, Fred R. Rurangirwa e, William M. Sischo a, Gregory A. Bohach b a

Department of Veterinary Clinical Science, Washington State University, Pullman, WA 99164-6610, USA Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844, USA Department of Animal and Veterinary Science, University of Idaho, Moscow, ID 83844, USA d Department of Microbiology, College of Veterinary Medicine and the BK21 Program for Veterinary Science, Seoul National University, Seoul 151-742, South Korea e Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 September 2009 Received in revised form 21 June 2010 Accepted 23 June 2010

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCRRFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0% (200/ 263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0% (255/263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Coagulase-negative staphylococci PCR-RFLP gap gene

1. Introduction Coagulase-negative staphylococci (CNS) are a heterogeneous group of bacteria that are the most prevalent cause of bovine mastitis (Fox, 2009; Hogan et al., 1999; Pitkala et al., 2004; Pol and Ruegg, 2007). A variety of methods have been used to phenotypically identify CNS, including a conventional identification scheme (Kloos and

* Corresponding author. Tel.: +1 509 335 0786; fax: +1 509 335 0880. E-mail address: [email protected] (L.K. Fox). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.06.020

Schleifer, 1975) and commercial biochemical kits (Capurro et al., 2009; Sampimon et al., 2009; Taponen et al., 2008). One commercially available identification system is the API STAPH ID 20 which is currently recommended for the species identification of CNS isolated from bovine IMI by the National Mastitis Council (NMC) (Hogan et al., 1999). A drawback of the commercially available identification systems is that they were not designed to identify CNS isolates from veterinary specimens thus the database of phenotypic characteristics from veterinary CNS isolates is limited (Hogan et al., 1999; Matthews et al., 1990).

J.Y. Park et al. / Veterinary Microbiology 147 (2011) 142–148

A number of DNA sequence-based molecular methods have been developed for the identification of Staphylococcus spp. (Zadoks and Watts, 2009). Partial 16S rRNA gene sequence analysis has emerged as a preferred technique and is widely accepted as a standard method for the identification of Staphylococcus spp. as well as other microorganisms (Becker et al., 2004; Clarridge, 2004). This approach may not be practical for routine identification in clinical diagnostics because it is time-consuming, labor intensive, and relatively expensive compared to currently recommended methods. Therefore, there is a need for simple and reliable molecular tests for CNS identification. In this study we evaluated the validity of API STAPH ID 20 and gap PCR-RFLP analysis for the species identification of CNS isolates from bovine IMI at the species and subspecies levels by comparison with partial 16S rRNA gene sequence analysis as the definitive test. 2. Materials and methods 2.1. Control strains Sixteen reference and type (T) strains used in this study as control strains were purchased from the American Type Culture Collection (ATCC) and included the following: Staphylococcus aureus ATCC 29740, Staphylococcus capitis subsp. capitis ATCC 35661, Staphylococcus caprae ATCC 35538T, Staphylococcus chromogenes ATCC 43764T, Staphylococcus epidermidis ATCC 12228, Staphylococcus haemolyticus ATCC 29970T, Staphylococcus hyicus ATCC 11249T, Staphylococcus hominis subsp. hominis ATCC 25615, Staphylococcus intermedius ATCC 29663T, Staphylococcus saprophyticus ATCC 49453, Staphylococcus sciuri subsp. sciuri ATCC 29060, Staphylococcus sciuri subsp. carnaticus ATCC 700058T, Staphylococcus simulans ATCC 11631, Staphylococcus succinus ATCC 700337T, Staphylococcus warneri ATCC 49454, and Staphylococcus xylosus ATCC 29971T. 2.2. Isolation of bacterial strains and growth conditions Two hundred sixty-three CNS were isolated from milk samples collected in duplicate from mammary quarters with bovine IMI in dairies in Idaho and Washington. A mammary quarter was considered infected when the number of colony-forming unit/ml of milk was greater than 200 on sheep blood agar from duplicate milk samples (National Mastitis Council, 1987). All isolates were presumptively identified as CNS by primary phenotypic identification methods based on colony morphology, catalase, coagulase tube test, and Gram staining. Strains presumptively identified as CNS were subcultured to obtain single colonies in pure culture. Colonies were then transferred to Todd Hewitt broth (THB, Becton Dickinson Diagnostic Systems, Sparks, MD), cultured at 37 8C for 18 h, and stored in 20% glycerol solution at 80 8C until use. 2.3. Species identification by partial 16S rRNA and rpoB gene sequencing Chromosomal DNA was purified as previously described (Pitcher et al., 1989). Briefly, bacteria were

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cultured in 5 ml of THB at 37 8C for 18 h and then harvested by centrifugation at 12,000 g for 5 min. The cell pellet was resuspended in TE buffer (50 mM Tris–HCl, pH 7.0, 10 mM EDTA), digested by treatment with 10 ml of lysostaphin (1 mg/ml, Sigma–Aldrich Co., St. Louis, MO) and 100 ml of lysozyme (100 mg/ml, Sigma–Aldrich Co.) at 37 8C for 1 h. Cell lysates were treated with 500 ml of lysis buffer L6 (5 M guanidine thiocyanate, 5 mM EDTA, 0.5% (v/ v) sarkosyl) followed by 250 ml of ammonium acetate solution (7.5 M, Sigma–Aldrich Co.). After mixing with an equal volume of chloroform:isoamyl alcohol (24:1), the sample was centrifuged at 18,000 g for 10 min. The upper aqueous phase containing DNA was collected into a fresh microtube and precipitated with ethanol. The concentration of DNA was adjusted to 100 ng/ml by the addition of deionized water to achieve an OD = 2.0 at 260 nm (Nanodrop Technologies, Wilmington, DE). Partial 16S rRNA gene sequencing was performed for all control strains and 263 CNS isolates from bovine IMI as described previously (Becker et al., 2004). To amplify the relevant portion of the 16S rRNA gene, the broad-range primers SSU-bact-27f (50 -AGA GTT TGA TCM TGG CTC AG30 ) and SSU-bact-907r (50 -CCG TCA ATT CMT TTR AGT TT30 ) were used. The amplification products were purified with PCR purification kits (QIAGEN Inc., Valencia, CA). Sequencing reactions were carried out with the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) using SSU-bact-519r (50 -GWA TTA CCG CGG CKG CTG-30 ) primer. Partial 16S rRNA gene sequences from control strains and CNS isolates from bovine IMI were aligned and compared with sequences available in GenBank using multiple sequence alignment CLUSTALW program (Thompson et al., 1994). As previously reported, the partial 16S rRNA gene sequences of S. capitis and S. caprae were indistinguishable (Takahashi et al., 1999). Therefore, partial RNA polymerase B (rpoB) gene sequencing (Mellmann et al., 2006) was performed to differentiate these two species only when control strains or IMI isolates initially appeared to be S. capitis or S. caprae by partial 16S rRNA gene sequence analysis. The relevant portion of the rpoB gene (899 bp) was amplified using primers Staph rpoB 1418f (50 -CAA TTC ATG GAC CAA GC-30 ) and Staph rpoB 3554r (50 -CCG TCC CAA GTC ATG AAA C-30 ). Sequencing was performed as described above using primer Staph rpoB 1876r (50 -GAG TCA TCI TTY TCT AAG AAT GG-30 ). Partial rpoB gene sequences were analyzed by comparison with those available in GenBank (Mellmann et al., 2006). 2.4. Species identification by the gap PCR-RFLP All control strains and 263 CNS isolates from bovine IMI were cultured on Todd Hewitt agar (THA, Becton Dickinson Diagnostic Systems) plates at 37 8C for approximately 16 h. A single colony was suspended in 50 ml of PCR reaction buffer consisting of 10 mM Tris–HCl (pH 8.3), 2 mM MgCl2, and 50 mM KCl and incubated at 95 8C for 15 min to extract chromosomal DNA. Amplification of the relevant gap gene fragment was performed as described by Yugueros et al. (2000). Briefly, the PCR amplification was carried out with a PCR kit (Applied Biosystems, Foster City, CA) using 2 ml of extracted chromosomal DNA, 0.3 mM of GF-1 (50 -ATG GTT

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TTG GTA GAA TTG GTC GTT TA-30 ) and GR-2 (50 -GAC ATT TCG TTA TCA TAC CAA GCT G-30 ) primers. The thermal cycle for amplification was an initial denaturation at 95 8C for 10 min, followed by 40 cycles of denaturation at 95 8C for 20 s, annealing at 55 8C for 30 s, and extension at 72 8C for 40 s. The amplification product (approximately 931 bp) was digested with 10 U of AluI (Invitrogen Co., Carlsbad, CA) for 4 h at 37 8C. The resulting DNA fragments were analyzed in 2% agarose (Fisher Scientific Inc., Houston, TX) or MetaPhor agarose (Lonza Rockland, Inc., Rockland, ME) gel electrophoresis in 0.5 Tris–borate–EDTA (TBE) buffer. To differentiate S. chromogenes from S. intermedius, PCR products amplified from control strains and/or IMI isolates were digested with 10 U of RsaI (Invitrogen Co.) at 37 8C for 4 h. For an unambiguous species identification of S. caprae, S. saprophyticus, S. xylosus, S. sciuri subsp. sciuri, and S. sciuri subsp. carnaticus, PCR products amplified from control strains and/or IMI isolates were digested with 10 U of TaqI (New England Biolabs, Ipswich, MA) at 65 8C for 4 h. The partial gap gene sequences were not available in GenBank for S. succinus and S. sciuri subsp. carnaticus. Therefore, to determine the partial gap gene sequences, in those species that were used in our control strain comparisons, the gap gene amplicons were cloned using TOPO TA cloning kit (Invitrogen Co.) according to manufacturer’s instructions. Sequencing was performed as described above using M13F and M13R primers. 2.5. Species identification by API STAPH ID 20 The API STAPH ID 20 (bioMe´rieux, Inc., Durham, NC) miniaturized biochemical system for identification of Staphylococcus spp. was used as recommended by the manufacturers. Briefly, bacterial suspension was adjusted to a turbidity equivalent to 0.5 McFarland by spectrophotometer. One hundred thirty-five microliters of bacterial suspension were added to each microtube on API STAPH ID 20 test strip and the mixture was incubated at 37 8C for 18–24 h. A 7-digit numeric profile was generated by interpreting the biochemical reactions in each test strip following the API STAPH ID 20 instructions. The ApiwebTM (bioMe´rieux, Inc.) database was consulted and using the numeric profile and ApiwebTM (bioMe´rieux, Inc.) software a species was assigned to each bacterial isolate tested. A classification of ‘‘high probability’’ was assigned when the percentage identification accuracy (% ID), a probability of how closely the API numeric profile corresponded to that of the species relative to all other species in the API database, was greater than or equal to 80. A result was classified as ‘‘low probability’’ when the % ID was below 80. The % ID of 80 as a threshold differentiating between a high probability of correct species identification from a lesser probability was selected based on the recommendation of the ApiwebTM software system. The species of an isolate was designated by the highest % ID among the species listed in ApiwebTM software when multiple species were offered for a particular profile. Additionally, as recommended by ApiwebTM software, the b-glucuronidase test (b-glu) (Dahlen and Linde, 1973) was performed for all S. hyicus isolates and its control strain to differentiate them from S. chromogenes.

2.6. Nucleotide sequence accession numbers The partial gap gene sequences for S. sciuri subsp. sciuri control strain, S. sciuri subsp. carnaticus, S. hyicus and S. succinus control strains and isolates from bovine IMI determined in this study were deposited in GenBank under accession numbers FJ578000–FJ578006, respectively. 3. Results 3.1. Species identification by partial 16S rRNA and rpoB gene sequencing All ATCC control strains were correctly identified at species level using partial 16S rRNA gene sequence analysis except S. capitis subsp. capitis ATCC 35661 and S. caprae ATCC 35538T. The partial 16S rRNA gene sequences obtained from the 263 CNS IMI isolates showed high sequence similarity (>99%) with those from control strains and sequences available in GenBank, and resulted in identification of 11 different CNS species (Table 1). The majority of CNS isolates from bovine IMI were identified as S. chromogenes (72.2%), with S. xylosus (9.1%), and S. haemolyticus (6.1%) second and third most prevalent, respectively (Table 1). Similar to a previous report demonstrating indistinguishable partial 16S rRNA gene sequences of S. capitis or S. caprae (Takahashi et al., 1999), the partial 16S rRNA gene sequences of S. capitis or S. caprae control strains and IMI isolates (n = 4) were indistinguishable (>99.0% sequence similarity). As demonstrated by Mellmann et al. (2006), the identities of S. capitis and S. caprae control strains as well as IMI isolates were successfully identified at species level by partial rpoB gene sequencing, showing that one isolate was S. capitis and three were S. caprae. 3.2. Species identification by the gap PCR-RFLP The partial gap gene (approximately 931 bp) was successfully amplified by PCR from all ATCC control strains and all CNS IMI isolates. Most control strains showed distinctive gap PCR-RFLP patterns using AluI (Fig. 1A). Most gap PCR-RFLP patterns using AluI were determined by major restriction fragments in the size range between 100 bp and 400 bp. Compared with species identification results obtained by partial 16S rRNA gene sequence analysis, 97.0% (255/263) of CNS IMI isolates were correctly identified at species level by the gap PCR-RFLP (Table 1). However, the gap PCR-RFLP pattern of isolates classified by the 16S rRNA gene sequencing analysis as S. hyicus (four bands: 324 bp, 246 bp, 225 bp, and 69 bp) (n = 8) (Fig. 1B) differed from that of the S. hyicus ATCC 11249T control strain (five bands: 327 bp, 225 bp, 153 bp, 135 bp, and 69 bp) (Fig. 1A). S. caprae, S. saprophyticus and S. xylosus were not easily differentiated by the gap PCR-RFLP using AluI (Fig. 1A). Their patterns were composed of four major restriction fragments. However, the second restriction fragment (243 bp) is only 6 bp longer in S. saprophyticus than in S. caprae. The first two restriction fragments (300 bp and 219 bp) appear shorter in S. xylosus than in S. caprae


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Table 1 Speciation results of 263 CNS isolates from bovine intramammary infections using partial 16S rRNA gene sequence, gap PCR-RFLP analyses and API STAPH ID 20. Speciesa

S. S. S. S.

Partial 16S rRNA gene sequencing No. of isolates (%)

chromogenes xylosus haemolyticus sciuri subsp. carnaticus S. hyicus S. simulans S. caprae S. epidermidis S. succinus S. capitis S. hominis

190 24 16 9

Total

263 (100.0)

8 7 3 2 2 1 1

(72.2) (9.1) (6.1) (3.4) (3.0) (2.7) (1.1) (0.8) (0.8) (0.4) (0.4)

gap PCR-RFLP No. of isolates Matchedd

API STAPH ID 20 Not matchede

High probabilityb (% ID 80) No. of isolates

Low probabilityc (% ID < 80) No. of isolates

Matchedf

Not matchedg

Matched

Not matched

190 24 16 9

0 0 0 0

113 22 0 0

36 0 6 0

38 2 2 7

3 0 8 2

0 7 3 2 2 1 1

8 0 0 0 0 0 0

8 2 2 2 0 1 0

0 4 0 0 2 0 0

0 0 1 0 0 0 0

0 1 0 0 0 0 1

255

8

150

48

50

15

a

The species was determined by partial 16S rRNA gene sequence analysis except S. caprae and S. capitis which were determined by partial rpoB gene sequence analysis. b A result was classified as ‘‘high probability’’ when the percentage identification accuracy (% ID) was greater than 80. c A result was classified as ‘‘low probability’’ when % ID was below 80. The species was determined on the basis of the highest % ID provided by ApiwebTM software. d The speciation results obtained by the gap PCR-RFLP matched those obtained by partial 16S rRNA gene sequence analysis. e The speciation results obtained by the gap PCR-RFLP did not match those obtained by partial 16S rRNA gene sequence analysis. f The speciation results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis. g The speciation results obtained by API STAPH ID 20 did not match those obtained by partial 16S rRNA gene sequence analysis.

(327 bp and 237 bp) and S. saprophyticus (327 bp and 243 bp). To more clearly differentiate them, the gap gene PCR products from those control strains were digested by TaqI, showing distinctive and reproducible gap PCR-RFLP patterns (Fig. 2). All S. caprae, S. saprophyticus and S. xylosus IMI isolates showed the identical gap PCR-RFLP patterns using TaqI, as those observed with corresponding control strains. Similarly, S. chromogenes and S. intermedius showed very similar gap PCR-RFLP patterns using AluI (Fig. 1A). Both patterns consisted of three restriction fragments. The size of the first restriction fragment (327 bp) is the same in both species. However, the second (294 bp) and third restriction fragments (237 bp) appear slightly larger in S. intermedius than in S. chromogenes. To overcome the difficulty in differentiating these two species by AluI digestion, the gap genes from S. chromogenes and S. intermedius control strains were amplified and digested with RsaI. As shown in Fig. 2, their PCR-RFLP patterns by RsaI were clearly distinctive and reproducible. All S. chromogenes IMI isolates showed an identical gap PCR-RFLP pattern as that observed with the control strain. The partial gap gene sequences of some ATCC control strains had not been determined at the time of the study. The partial gap gene sequences of S. sciuri subsp. sciuri ATCC 29060 and S. sciuri subsp. carnaticus ATCC 700058T were determined and deposited in GenBank (accession numbers FJ578000 and FJ578001, respectively). It revealed that the partial gap gene sequence similarity between these two subspecies was 99%, resulting in an identical gap PCR-RFLP pattern using AluI (Fig. 1A). To differentiate these two subspecies, the gap gene PCR products were digested

with TaqI. As shown in Fig. 2, S. sciuri subsp. sciuri ATCC 29060 and S. sciuri subsp. carnaticus ATCC 700058T showed distinctive and reproducible gap PCR-RFLP patterns by TaqI digestion, indicating differentiation was achieved at the subspecies level. All S. sciuri subsp. carnaticus IMI isolates showed the same gap PCR-RFLP pattern as that observed with the control strain (see Table 1). 3.3. Species identification by API STAPH ID 20 Of the 263 CNS IMI isolates, the species identification results of 200 isolates (76%, 200/263) obtained by API STAPH ID 20 matched those obtained by the gold standard, partial 16S rRNA gene sequence analysis. Of 200 CNS IMI isolates found to be matched with partial 16S rRNA gene sequence analysis, 150 and 50 isolates were correctly identified at species level by API STAPH ID 20 with a high probability (% ID 80) or a low probability (% ID < 80), respectively (Table 1). Of 63 CNS IMI isolates that did not match the partial 16S rRNA gene sequence analysis, 48 and 15 CNS IMI isolates were misidentified as other Staphylococcus spp. with a high (% ID 80) or a low probability (% ID < 80), respectively (Table 1). Although some CNS isolates had a low probability of correct species identification, API STAPH ID 20 successfully matched 79.5% (151/ 190) of S. chromogenes, 7 of 9 of S. sciuri subsp. carnaticus, and all S. xylosus, S. hyicus, S. caprae, S. epidermidis, and S. capitis when compared to species identification by partial 16S rRNA gene sequence analysis (Table 1). By contrast, 2 of 16 S. haemolyticus, 2 of 7 S. simulans and none of S. succinus and S. hominis were correctly identified at species level by API STAPH ID 20 (Table 1).

[(Fig._1)TD$IG]

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[(Fig._2)TD$IG]


Fig. 2. The gap PCR-RFLP of control strains using TaqI or RsaI. The gap gene PCR products were digested with TaqI or RsaI and analyzed by agarose gel electrophoresis. Lane M, 50 bp DNA ladder.

Fig. 1. The gap PCR-RFLP of control strains (A) and CNS isolates from bovine IMI (B). The gap gene PCR products (approximately 931 bp) were digested with AluI and analyzed by agarose gel electrophoresis. Lane M, 50 bp DNA ladder.

4. Discussion Despite the high prevalence of CNS in bovine IMI, rapid and reliable species identification methods have not been established. Currently, API STAPH ID 20, a commercially available identification system, has been recommended by the NMC (Hogan et al., 1999). Its accuracy in the identification of veterinary isolates has not been fully established. The purpose of this study was to compare this phenotypic method with a proposed genotypic method, the gap PCR-RFLP, for species identification of CNS from bovine IMI. Partial 16S rRNA gene sequence analysis was performed as the ‘‘gold standard’’ for definitive identification. Partial 16S rRNA gene sequence analysis has served well as a gold standard method for the identification of a variety of microorganisms including Staphylococcus spp. due to large reference databases (Becker et al., 2004; Clarridge, 2004), although it was not fully sufficient to identify some Staphylococcus spp. at species level as described in others (Takahashi et al., 1999) and our results (e.g. S. caprae and S. capitis).

When analyzed by API STAPH ID 20, only 76% (200/263) of the CNS IMI isolates matched identification by partial 16S rRNA gene sequence analysis. Of the 200 isolates that matched the gold standard, 50 isolates appeared to have a low probability of identification (% ID < 80) (Table 1). The species of these 50 isolates were determined by the highest % ID provided by the ApiwebTM source information. The difficulty in species identification of CNS using API STAPH ID 20 rests with the ambiguity of likely identification. In this study, 75.3% (198/263) of CNS IMI isolates were classified by API STAPH ID 20 with a high probability (% ID 80), yet only 57% (150/263) of isolates matched the gold standard. A recent test of the (Sampimon et al., 2009) API STAPH ID 32 indicated only 41% of mastitis isolates were correctly identified, lower than reported herein. It should be noted that Sampimon et al. (2009) used a more stringent value, an identification threshold of >90% probability, for species identification. Additionally, the system they tested used almost 50% more biochemical tests to identify CNS at species level. Fifty of 65 isolates noted to have a low probability (% ID < 80) of identification in the current study could be considered to be correctly identified when compared to the gold standard. Thus, it could be argued that these 50 isolates were correctly, but not unambiguously, identified by API STAPH ID 20 system. Problems in identification appeared to be due to pheno-


typic variations in several key biochemical tests. For example, S. chromogenes appeared to be misidentified as other Staphylococcus spp. with a high probability mainly due to the variations in maltose fermentation test which API STAPH ID 20 system uses to differentiate S. chromogenes from S. hyicus. A recommended complimentary test, b-glu, was performed for the S. hyicus control strain and isolates, to discriminate them from S. chromogenes. Contrary to the API database, all S. hyicus isolates (n = 8) and the control strain were negative for b-glu. Thus the findings of this study and those of Sampimon et al. (2009) indicate that the API STAPH ID systems, both 20 and 32, are not well suited to identify CNS from cows with IMI. We have demonstrated the utility of the gap PCR-RFLP for identification of CNS isolates from bovine IMI. Recently, the partial gap gene sequences of 27 Staphylococcus spp. were determined (Ghebremedhin et al., 2008; Layer et al., 2007; Yugueros et al., 2000, 2001). However, partial gap gene sequences of some CNS species commonly isolated from bovine IMI were lacking. Partial gap gene sequences of S. hyicus, S. sciuri subsp. carnaticus, and S. succinus isolates from bovine IMI were determined, permitting us to report the gap PCR-RFLP patterns for those species or subspecies. Most CNS species from bovine IMI were correctly identified by the gap PCR-RFLP using AluI with exception of S. hyicus. The S. hyicus isolates from bovine IMI showed a unique gap PCR-RFLP pattern differing from that of the type strain, S. hyicus ATCC 11249T, and other staphylococcal species. Analysis of partial gap gene sequences showed a sequence similarity of 96.2% between control strain and S. hyicus IMI isolates. Partial gap gene sequences of S. hyicus isolates from bovine IMI were well conserved (>99% sequence similarity). Compared to the S. hyicus control strain, partial gap gene sequence of S. hyicus IMI isolates revealed two additional AluI sites beginning at positions 312 and 339 and one AluI restriction site (positioned at 449 bp) that was absent, explaining the unique gap PCR-RFLP pattern. Similarly, heterogeneous gap PCR-RFLP patterns within the species were reported in S. epidermidis, S. hominis, and S. simulans (Yugueros et al., 2000). Variant gap PCR-RFLP patterns in these species were not detected in the current study. Although the effectiveness of CNS species identification by the gap PCR-RFLP using AluI was demonstrated by other studies (Yugueros et al., 2000, 2001), we noticed that some gap PCR-RFLP patterns were difficult to interpret. The gap PCR-RFLP patterns of S. sciuri subsp. sciuri and carnaticus were indistinguishable and those of S. chromogenes and S. intermedius were difficult to differentiate. The gap PCRRFLP patterns of S. caprae, S. saprophyticus and S. xylosus were also not easily differentiated. However, these difficulties were overcome by the gap PCR-RFLP using TaqI or RsaI to easily and unambiguously differentiate these species or subspecies. This study demonstrated gap PCR-RFLP is a more accurate and rapid tool for the identification of CNS isolates from bovine IMI as compared to the current NMC (Hogan et al., 1999) recommended procedure, API STAPH ID 20. For practical application we demonstrated that the gap PCR-RFLP using TaqI or RsaI can be used for some CNS species which showed ambiguous gap PCR-RFLP patterns

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using AluI. The advantages of gap PCR-RFLP for the species identification of CNS isolates from bovine IMI in mastitis diagnostics are accuracy, ease of use and cost. We estimate that the cost of materials for gap PCR-RFLP is lower than the API STAPH ID 20, and its agreement with the gold standard better is better. Therefore the gap PCR-RFLP is considered a better test for species identification of CNS from bovine IMI.

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