identification of staphylococcus species bovine the api...

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1983, p. 1212-12190095-1137/83/11 1212-08$02.00/0Copyright ©C 1983, American Society for Microbiology

Vol. 18, No. 5

Identification of Staphylococcus Species of Bovine Origin withthe API Staph-Ident Systemt

BRUCE E. LANGLOIS,* ROBERT J. HARMON, AND KATHERINE AKERSDepartment of Animal Sciences, University of Kentucky, Lexington, Kentucky 40546-0215

Received 27 June 1983/Accepted 22 August 1983

The API Staph-Ident system was evaluated as a means for identifying thespecies of bovine strains of staphylococci routinely isolated from quarter-milksamples. The species identity of 314 of 581 (54%) isolates of staphylococci wascorrectly determined by this method. The API Staph-Ident system was moreaccurate in correctly identifying Staphylococcus alureus (93.9%) than in correctlyidentifying non-S. auireus species (41.8%). False identifications of Staphylococcusepidermidis and Staphylococcus hominis were the main reasons for the incorrectidentifications of the non-S. auireus species.

Bovine mastitis has been estimated to cost theU.S. dairy industry more than 2 billion dollars ayear (11). Staphylococcuis auireus can be a majorcause of mastitis in many dairy herds; however,control programs are available which can beused to reduce the incidence of mastitis causedby S. aureus. A recent study (12) indicated thatS. aureus mastitis was reduced from 16 to 5% bythe use of an effective control program; howev-er, more than 23% of the milk samples examinedcontained pathogens which were classified asStaphylococcus epidermidis.The latest edition of Bergey's Manlual of De-

terminativ,e Bacteriology (2) lists S. aulreuls, S.epidermidis, and Staphylococcus saprophyticusas the only species in the genus Staphylococcus.Recently, several new species have been de-scribed for this genus and new descriptions havebeen given for S. epidermidis and S. saprophyti-cus (4-6, 10, 14, 15, 22). An increasing numberof reports have described the incidence of coag-ulase-negative staphylococci in infections (1, 3,7-9, 20, 23). Studies in our laboratory haveshown elevated somatic cell counts in milk sam-ples containing coagulase-positive and coagu-lase-negative staphylococci species that couldnot be classified according to the descriptionsgiven for the three species listed in Bergey'sManual or by the identification scheme of Kloosand Schleifer (13) for human staphylococci iso-lates.

Miniaturized biochemical test systems havebeen developed for the rapid identification ofseveral groups of microorganisms. A 24-h sys-tem utilizing 20 biochemical tests was developed

t Published with the approval of the Director of the Ken-tucky Agricultural Experiment Station as Journal paper no.83-5-100.

for staphylococci by API System, S. A. Monta-lieu-Vercieu, France (3). In late 1981, AnalytabProducts, Plainview, N.Y., introduced the APIStaph-Ident system which permits the determi-nation of 10 biochemical characteristics afterincubation for 5 h. The system is capable ofdistinguishing all of the human staphylococcispecies described by Kloos and Schleifer (13)plus two species of veterinary interest, Staphy-lococcuis hyicus and Staphylococcus interme-diiis. Between 80 and 96% agreement has beenshown between the Staph-Ident system and thebiochemical identification scheme of Kloos andSchleifer (13) for clinical staphylococci isolates(1, 7, 8, 17). The purpose of this investigationwas to evaluate the API Staph-Ident system foruse in the identification to species level of staph-ylococcal isolates from bovine milk samples.

MATERIALS AND METHODSBacterial isolates. During a 20-month period, 581

gram-positive catalase-positive cocci of bovine originwere obtained by our laboratory from several sources.A total of 25 isolates were obtained by swabbing thedistal end of the streak canal of 14 cows in theUniversity of Kentucky dairy herd during their dryperiod. The remaining isolates were obtained from thefollowing sources: 96 were activated from freeze-driedcultures of isolates obtained from quarter foremilksamples from the University of Kentucky dairy herd in1969 (25, 26), 383 were from quarter foremilk samplesobtained from the University of Kentucky dairy herdduring the 20-month period of this study, 32 were fromtwo private dairy herds in Kentucky with a highincidence of mastitis (18, herd A; 14, herd B), 37 werefrom J. W. Pankey, North Louisiana Hill Farm Ex-periment Station, Homer, La., and 9 were from K. L.Smith, Department of Dairy Science, Ohio Agricultur-al Research and Development Center, Wooster, Ohio.Upon receipt at our laboratory, all milk samples orcultures were streaked on sheep blood esculin agar

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STAPH-IDENT IDENTIFICATION 1213

(BEA [21]), and the plates were incubated at 35°C for24 to 48 h. A single well-isolated colony was picked tobrain heart infusion (BHI) broth, incubated at 35°C for18 h, and stored at 2°C until activated for the APIStaph-Ident system and biochemical tests. In addition,each isolate was streaked onto a BHI agar slant andincubated at 35°C for 18 h, and the growth was washedoff with sterile 10o skim milk. Skim milk (1 ml) was

placed into a sterile Cryotube (GIBCO Laboratories)and stored at -80°C.API Staph-Ident system. Cultures were transferred

up to three times in BHI broth before they werestreaked onto BEA. The inocula used to inoculate thestrips were prepared from growth on BEA. Recom-mended procedures of the manufacturer were followedfor the preparation of strips and inoculum and for theinoculation, incubation, and reading of the strips.Borderline color reactions that were occasionally ob-tained with all tests and more often with mannose(MNE) and trehalose (TRE) were considered to benegative in determining the four-digit profile for spe-cies identification according to the API Staph-Identprofile register. When a code profile was not found inthe profile register, the species identity of the isolatewas determined by consulting the API computer cen-ter. Since several species were usually given for a

profile, the species with the highest probability ofoccurring for the profile was used to identify theisolate, unless additional tests were recommended forthe identification of a species.

Conventional biochemical tests. Inocula for test andtest cultures were prepared from the BHI broth usedto streak the BEA plate or by activation of the culturesstored at -80°C. The frozen cultures were activatedby transferring three times in BHI broth before beingused to inoculate test media. The broth cultures were

used to determine the following biochemical charac-teristics: aerobic acid production from arabinose; dex-trose; fructose; galactose; lactose; maltose; mannitol;MNE; melezitose; ribose; salicin; sucrose; TRE; tur-anose; xylitol and xylose (13); anaerobic utilization ofmannitol (9); coagulase activity with citrated EDTArabbit plasma (24); production of heat-sensitive (19)and heat-stable (18, 19) nuclease; lysostaphin suscepti-bility (19); type of growth in thioglycolate medium (9);alkaline phosphatase activity with p-nitrophenyl phos-phate (0.495 mg/ml; Sigma Chemical Co.); growth onTrypticase soy agar containing 7.5, 10, and 15% NaCl(16); nitrate reduction (24), and production of acetyl-methylcarbinol (24). Hemolysis, esculin hydrolysis,and pigmentation were determined by examination ofgrowth on BEA plates. When necessary to separatespecies, novobiocin susceptibility was determined byusing 5-,ug novobiocin sensitivity disks on inoculatedMueller-Hinton agar. Isolates giving zone sizes equalto or less than 11 mm were considered to be novobio-cin resistant. Isolates were identified according to amodification of the scheme of Kloos and Schleifer (13)which permitted the identification of S. hyicus and S.intermedius as well as the human staphylococcal spe-cies.

RESULTSAssuming that the identifications obtained by

conventional biochemical tests by the methods

of Kloos and Schleifer (13) were correct, then314 of 581 bovine staphylococci isolates (54%)were correctly identified by the API Staph-Identsystem (Table 1). The accuracy among speciesvaried from 0% for S. capitis to 100% for S.hyicus and S. simulans for isolates identified bythe API Staph-Ident system and confirmed byconventional biochemical tests. False identifica-tion or failure to identify a species by the APIStaph-Ident system generally was due to falsenegative reactions.Of 131 S. aureus isolates, 123 (93.9%) were

correctly identified by the API Staph-Ident sys-tem. A positive P-galactosidase reaction result-ed in eight S. aureus isolates being incorrectlyidentifed as S. xylosus. These eight isolates werecoagulase positive and negative for xylose utili-zation, and they were obtained from cows in thesame herd.Approximately 29% of the isolates tested were

identified as S. epidermidis by the API Staph-Ident system compared with 4.3% by conven-tional biochemical tests. The API Staph-Identsystem identified 7.1% of the isolates as S.hyicus compared with 39.4% by the biochemicaltests. The API system is unable to differentiatethe subspecies within S. hyicus. Therefore, thevalue in Table 1 includes both S. hyicus and S.hyicus subsp. chromogenes.The biggest problem encountered in this study

was the false identification of S. hyicus and S.hyicus subsp. chromogenes as S. epidermidis.Only 24 of 171 S. epidermidis isolates (14%)were correctly identified by the API Staph-Ident

TABLE 1. Identification of staphylococci of bovineorigin with the API Staph-Ident system and with

conventional biochemical tests

APINo. identifiedStaphylococcus N No. (%) by bio-

species identified correctly chemical testidniididentified'

S. aureus 124 123 (99.2) 131S. capitis 11 0 (0) 0S. epidermidis 171 24 (14.0) 25S. haemolyticus 40 3 (7.5) 6S. hominis 45 5 (11.1) 41S. hyicusb 41 41 (100) 229S. saprophyticus 2 1 (50.0) 1S. sciuri 20 17 (85.0) 17S. simulans 54 54 (100) 66S. warnerib 41 24 (58.5) 37S. xylosus 32 22 (68.8) 23Unknown 5

Total 581 314 (54.0) 581a Species identity was determined by conventional

biochemical tests according to Kloos and Schleifer(13).

b Includes subspecies.

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1214 LANGLOIS, HARMON, AND AKERS

TABLE 2. Frequency of API Staph-Ident four-digit profiles among Staphylococcus species of bovine origin

Correctly identifieda Incorrectly identifiedaStaphylococcus No. of

species isolates API % With API % With Correct speciesprofile profile profile profile

S. aureus 124 7740 64.5 3500 0.8 S. hyicus

S. capitis

S. epidermidis

S. haemolyticus

S. hominis

S. hyicus

S. saprophyticus

S. sciuri

S. simulans

S. warneri

770057005740670047003700

16.18.97.30.80.80.8

6.43.5

171 30403000310070402140

40 44400440

45 2440200020402400

41 3540154075402540

2 240120 5710

47104610

54 24612061204160616461242160416441

25 664064406650

0040

02403040

1.8 34401.8 70400.6 3140

30001040714070001140

5.0 44402.5 4640

0440006144000640

4660

1440004100600460

4.4 20402.22.2 24402.2 2000

04002400

68.314.612.24.9

50.060.015.010.031.529.613.09.33.71.91.91.9

12.012.04.0

260146504510

644066407440

27.327.318.218.29.1

55.00.614.64.73.52.92.31.20.60.6

25.019.67.57.57.55.02.52.52.52.52.52.52.52.5

62.24.415.62.22.22.2

50.010.05.0

28.016.08.0

S. hominisS. simulansS. haemolyticusS. hyicusS. warneriS. hyicusUnknownS. hyicusS. hyicusS. hyicusS. hyicusS. hyicusS. hyicusS. hyicusS. hyicusS. hominisS. warneri

S. hominisS. hominisS. simulansS. hominisS. hominisS. warneri

S. warneriUnknownS. hyicusS. simulansS. simulansS. simulansS. hyicusS. simulansS. hyicusS. epidermidisS. haemolyticusS. hyicus

S. xylosusS. warneriUnknown

S. hominisS. hominisS. hyicus

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TABLE 2-ContinuedCorrectly identified' Incorrectly identifieda

Staphylococcus No. of Corc spcespecies isolates API % With API % With Correct speciesprofile profile profile profile

6040 4.0 4500 4.0 S. hominis4240 4.0 6400 4.0 S. hominis

4200 4.0 UnknownS. warneri subsp. 1 16 6660 43.8 6660 6.3 S. hominis

6060 25.02620 12.52420 6.32020 6.3

S. xylosus 32 6021 9.4 7701 21.9 S. aureus6421 6.3 7601 3.1 S. aureus6721 6.3 6421 3.1 S. simulans7721 6.3 6711 3.1 Unknown7731 6.34721 3.16301 3.16321 3.16461 3.17001 3.17021 3.17421 3.17431 3.17521 3.17621 3.16731 3.1

a Species identity was determined by conventional biochemical tests according to the scheme of Kloos andSchleifer (13).

system. Of the 147 isolates incorrectly identifiedas S. epidermidis, 146 were identified as S.hyicus or its subspecies by biochemical tests.False identifications were due mainly to nega-tive MNE and TRE utilizations by S. hyicus andits subspecies that resulted in code profiles forS. epidermidis. Most of the false identificationsobtained were for pigmented isolates that wereidentified as S. hyicus subsp. chromogenes bybiochemical tests.A total of 36 S. hyicus were incorrectly identi-

fied as S. hominis owing to low percentages ofpositive reactions for alkaline phosphatase,MNE utilization, and TRE utilization. A nega-tive urease reaction or a positive P-glucosidasereaction resulted in 37 isolates being incorrectlyidentified as S. haemolyticus, rather than as S.hominis, S. warneri, and S. simulans.The API four-digit profiles obtained for each

species are shown in Table 2. A total of 89different profiles were obtained in this study, ofwhich 79.8% were unique for a single speciesand 16.9% overlapped two species. Approxi-mately 2% of the profiles overlapped three spe-cies and 1.1% of the profiles overlapped fourspecies.Approximately 78% of the isolates (451) yield-

ed profiles that were included in the profileregister of the manufacturer. The remaining 130

isolates were identified by the API computercenter. Unless additional tests were recom-mended for the identification of a species, thespecies with the highest probability of occurringfor a profile was used to identify the isolate.Percentages of species requiring the use of theAPI computer center for their identificationwere: S. warneri subsp. 1 (100%), S. warneri(96%), 5. haemolyticus (45%), 5. sciuri (25%),S. hominis (22.2%), S. epidermidis (18.7%), S.hyicus (17.1%), S. simulans (16.7%), and S.aureus (1.6%).The number of API four-digit profiles ob-

tained from each species ranged from 2 for S.capitis and S. saprophyticus to 19 for S. xylosus.The number of profiles that gave correct speciesidentification ranged from 0 for S. capitis to 16for S. xylosus.The S. aureus isolates that were correctly

identified had seven different code profiles, with7740 (64.5%), 7700 (16.1%), 5700 (8.9%), and5740 (7.3%) accounting for all but three isolates.One S. aureus isolate with profile 3500 wasidentified by biochemical tests as a coagulase-negative S. hyicus. The 11 isolates with APIcode profiles for S. capitis (0040 and 0240) wereidentified as S. hominis, S. simulans, S. haemo-lyticus, S. hyicus, and S. warneri. Of the 147 S.epidermidis that were incorrectly identified,

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63.2% had the same API code profiles (3000,3040, and 7040) as isolates correctly identified asS. epidermidis. Isolates with API code profiles(0440 and 4440) were identified as S. haemolyti-cus and S. hominis. Isolates incorrectly identi-fied as S. haemolyticus were identified as S.hominis (4440, 4640, 0440, 0640, and 4400), S.warneri (4640, 0640, and 4660), S. simulans(0061, 0041, 0060, and 0460), and S. hyicus(1440). All API code profiles correctly identifiedas S. hominis also had isolates that were identi-fied as S. hyicus (2040, 2440, and 2400), S.simulans (2040), and S. epidermidis (2000).The identification of S. warneri (96%) and S.

warneri subsp. 1 (100%) was based on assistancefrom the API computer center. API code pro-files 6440 and 6640 were identified biochemicallyas S. warneri and S. hominis, whereas 6660 wasidentified as S. warneri subsp. 1 and S. hominis.API code profiles 7440, 6400, and 4500 werefound to be S. hyicus and S. hominis rather thanS. warneri. S. simulans was found to have thesame API code profile as S. xylosus (6421). S.xylosus with API code profiles 7601 and 7701were found to be coagulase positive and nega-tive for xylose utilization. These isolates wereidentified as S. aureus.A summary of the percentages of isolates in

each species, as determined by conventionalbiochemical tests, giving positive results foreach of the biochemical tests in the API Staph-Ident strip and the additional test for coagulaseactivity is given in Table 3. The API strip isunable to differentiate the subspecies within S.hyicus. For comparison purposes, we dividedthe isolates identified as S. hyicus into those thatwere nonpigmented (S. hyicus) and pigmented(S. hyicus subsp. chromogenes). Based on ourresults, the percentages of positive reaction forthe biochemical tests in the API strip differbetween S. hyicus and S. hyicus subsp. chromo-genes, and values tended to be similar for S.epidermidis and S. hyicus subsp. chromogenes.S. warneri and S. warneri subsp. 1 are combinedin the table. The main difference between thetwo is that 100% of the isolates identified as S.warneri were negative for ,3-glucuronidase,whereas 100% of the isolates identified as S.warneri subsp. 1 were positive. Percentages ofpositive reactions obtained for the biochemicaltests in the API strips tended to be similar to thevalues reported by Kloos and Wolfshohl (17)and the manufacturer of the API Staph-Identsystem for S. aureus, S. epidermidis, S. sciuri,S. warneri, and S. xylosus. Some minor differ-ences obtained were negative results for alkalinephosphatase (S. simulans and S. xylosus), MNEutilization (S. haemolyticus and S. simulans),and ,-glucuronidase (S. haemolyticus and S.hyicus) and higher positive results for urea utili-

zation (S. hyicus), 0-glucosidase (S. hyicus andS. simulans), MNE utilization (S. hominis), andarginine utilization (S. hominis and S. warneri).Lower positive results were obtained for ureautilization (S. hominis), ,B-glucosidase (S. hyi-cus), MNE utilization (S. hyicus), and TREutilization (S. haemolyticus, S. hyicus, and S.simulans). Some of the differences obtained maybe due to the small number of isolates in somespecies.

DISCUSSIONA simple, rapid, and accurate method for

determining the species identities of bovine iso-lates of staphylococci would have value in iden-tifying species associated with varying degreesof clinical severity of bovine mastitis and withelevated somatic cell counts. Such a methodwould permit the veterinarian to rapidly deter-mine the Staphylococcus species responsible formastitis rather than having to submit samples toan animal diagnostic laboratory for analysis.The purpose of this study was to determine

the accuracy of a rapid, miniaturized biochemi-cal system (API Staph-Ident) that was designedfor use with human clinical isolates as a meansfor identifying the species of bovine isolates ofstaphylococci.The accuracy that we obtained in correctly

identifying bovine isolates as S. aureus (93.9%)by the API Staph-Ident system was similar tothe values reported in studies with human iso-lates (1, 7, 8, 17). However, owing to our lowaccuracy in correctly identifying non-S. aureusspecies (41.8%), the overall accuracy of 54%that we obtained for the 581 bovine isolatestested was much lower than the 80 to 96%reported by other researchers for human isolates(1, 7, 8, 17). The proportion of particular speciescomprising the isolates being tested will affectthe percentage agreement between the APIStaph-Ident system and conventional biochemi-cal tests. Our highest agreements were obtainedfor sources that gave a majority of isolates thatwere identified as S. aureus. The lowest agree-ments were obtained for sources that gave avariety of non-S. aureus species (especially S.hyicus subsp. chromogenes).

False identifications of the non-S. aureus spe-cies by the API system were due to incompletecolor changes, especially with MNE and TRE.After 5 h of incubation, the color of some sugarswas red-orange or orange, and slight colorchanges were observed for alkaline phospha-tase, P-glucosidase, and P-glucuronidase. Ac-cording to the directions of the manufacturer forthe interpretation of results, these color changesare to be considered negative. Unlike the resultsof Doern et al. (7), incubation for an additional19 h did not result in positive color changes. In

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fact, many times the color of a sugar changedback to the original red color. Our overall accu-racy would have approached the values reportedin other studies if we had considered some of theincomplete color changes positive rather thannegative. This would have been especially truefor the incomplete color changes obtained forMNE and TRE by S. hyicus and its subspecies.Our study indicated that unless positive reac-tions are definite for the biochemical tests of theAPI Staph-Ident system, problems will be en-countered in the proper identification of somespecies (S. epidermidis, S. hyicus, S. hyicussubsp. chromogenes, S. haemolyticus, and S.hominis) of bovine origin. Based on our experi-ence with bovine isolates, some type of colorchart would be helpful in standardizing the test,especially in deciding when to record an incom-plete color change positive. The color chart maybe more useful with nonhuman isolates, sincereactions of nonhuman isolates appear morevariable. The color change for a reaction to beconsidered positive for the API Staph-Ident sys-tem may depend on the source of the isolate.The reason for the large number of false

identifications of non-S. aureus species by theAPI Staph-Ident system is unknown. One prob-lem may be the use of a system designed for therapid identification of human isolates to identifybovine isolates. Differences in biochemical char-acteristics between human and bovine isolatesmay result in different rates of substrate utiliza-tion after 5 h of incubation. As a result of thesedifferences, code profiles are obtained that cor-rectly identify human isolates but incorrectlyidentify bovine isolates.Methods currently being used to control mas-

titis may affect some of the biochemical charac-teristics used for the identification of staphylo-cocci. Weckback and Langlois (26) foundsignificant differences between some biochemi-cal characteristics of isolates from teats routine-ly dipped after each milking with an iodophorteat dip and those not dipped. Biochemical char-acteristics normally associated with staphylo-cocci and used in their identification were affect-ed by the teat dip. They did not indicate whetherthe differences were due to the effect of the teatdip on the organisms or to the selection ofpopulations in the dipped teats that differedbiochemically from the nondipped teats.

Like our study, other studies (7, 8, 17, 23) alsoused the biochemical identification scheme ofKloos and Schleifer (13) for human isolates toevaluate the accuracy of the API Staph-Identsystem. Except for Kloos and Wolfshohl (17),other researchers (7, 8, 23) used human isolatesto evaluate the API Staph-Ident system. There-fore, the other studies did not encounter falseidentification of S. hyicus and S. hyicus subsp.

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chromogenes as S. epidermidis. This is notsurprising since both S. hyicus and S. hyicussubsp. chromogenes are considered to be pri-marily associated with animals (6). Both specieshave been isolated from cows with mastitis (6).The percentages of positive reactions for thebiochemical tests in the API Staph-Ident striptended to be similar for S. epidermidis and S.hyicus subsp. chromogenes (Table 3). The big-gest differences were for TRE and arginine utili-zation. Since S. hyicus subsp. chromogenes iscommon to bovine milk and gives reactionssimilar to S. epidermidis on the API strip, it isnot surprising that false identifications wereobtained. To prevent false identifications of bo-vine isolates, additional tests (DNase, Voges-Proskauer, maltose utilization, ribose utiliza-tion) are necessary to correctly identify S.hyicus and its subspecies. Our percent agree-ment between the API Staph-Ident system andconventional biochemical tests was markedlyimproved when we used one or more of thesetests with the API Staph-Ident system.The code profiles obtained were similar to

those reported by others (7, 17). The majority ofS. aureus isolates yielded a 7740 profile (64.5%).The most common profile for S. epidermidis was3040 (64%); however, based on biochemicaltests, 89.5% of the isolates with a 3040 profilewere identified as S. hyicus. Likewise, the 7040profile observed most frequently for S. epider-midis by Doern et al. (7) generally was found inour study to be S. hyicus. Other code profilesobtained were similar to those reported byKloos and Wolfshohl (17).The results of this study indicated that S.

aureus of bovine origin could be accuratelyidentified by the API Staph-Ident system. How-ever, only 41.8% of the non-S. aureus specieswere correctly identified. Since several non-S.aureus species have been associated with masti-tis and elevated somatic cell counts, the APIStaph-Ident system as it is currently manufac-tured or used for clinical human isolates appearsto be of limited value in determining the identityof non-S. aureus species of bovine origin.

LITERATURE CITED

1. Aldridge, K. E., C. W. Stratton, L. S. Patterson, M. E.Evans, and R. L. Hodges. 1983. Comparison of the Staph-Ident system with a conventional method for speciesidentification of urine and blood isolates of coagulase-negative staphylococci. J. Clin. Microbiol. 17:516-520.

2. Baird-Parker, A. C. 1974. Gram-positive cocci. Famili I.Micrococcaceae, p. 478-490. In R. E. Buchanan andN. E. Gibbon (ed.), Bergey's manual of determinativebacteriology, 8th ed. The Williams & Wilkins Co., Balti-more.

3. Brun, Y., J. Fleurette, and F. Orey. 1978. Micromethodfor biochemical identification of coagulase-negative staph-ylococci. J. Clin. Microbiol. 8:503-508.

4. Devriese, L. A. 1977. Isolation and identification of Staph-vlococcus hyicuis. Am. J. Vet. Res. 38:787-792.

5. Devriese, L. A., and J. Derycke. 1979. Staphylococcushyicus in cattle. Res. Vet. Sci. 26:356-358.

6. Devriese, L. A., V. Hajek, P. Oeding, S. A. Meyer, andK. H. Schleifer. 1978. Staphylococcus hyicuis (Sompo-linsky, 1953) comb. nov. and Staphylococcus hyicussubsp. chromogenes subsp. nov. Int. J. Syst. Bacteriol.28:482-490.

7. Doern, G. V., J. E. Earls, P. A. Jeznach, and D. S.Parker. 1983. Species identification and biotyping ofstaphylococci by the API Staph-Ident system. J. Clin.Microbiol. 17:260-263.

8. Gemmel, C. G., and J. E. Dawson. 1982. Identification ofcoagulase-negative staphylococci with API Staph system.J. Clin. Microbiol. 16:874-877.

9. Gunn, B. A., F. L. Singleton, E. R. Peepler, R. R. Col-well, J. K. Keiser, and C. 0. Kapfer. 1981. Comparison ofmethods for identifying Staphylococcus and Micrococcusspp. J. Clin. Microbiol. 14:195-200.

10. Hajek, V. 1976. Staphylococcus intermedius, a new spe-cies isolated from animals. Int. J. Syst. Bacteriol. 26:401-408.

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