characterization of staphylococcit - applied and environmental

12
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1983, p. 649-660 0099-2240/83/090649-12$02.00/0 Copyright 0 1983, American Society for Microbiology Vol. 46, No. 3 Characterization of Staphylococcit DALLAS G. HOOVER,1 SITA R. TATINI,l* AND JOANN B. MALTAIS2 Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota 551081 and Minneapolis Center for Microbiological Investigations, Food and Drug Administration, Minneapolis, Minnesota 554012 Received 16 February 1983/Accepted 3 June 1983 A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separat- ed these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotox- ins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxono- my, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods. Since the publication of the latest edition of Bergey's Manual in 1974 (4), many new species of staphylococci have been proposed. Coagu- lase-positive staphylococci are divided into three species: Staphylococcus aureus, Staphylo- coccus intermedius (21), and Staphylococcus hyicus (13). The coagulase-negative staphylo- cocci, Staphylococcus epidermidis and Staphy- lococcus saprophyticus, have been redefined into as many as 11 species (25, 27). However, this extensive restructuring of coagulase-nega- tive staphylococci has recently been criticized (19). Creation of new coagulase-positive species, which share phenotypic characteristics long as- sociated with S. aureus, is of concern for the practical use of these features in evaluating t Paper no. 13324 on the Scientific Journal Series, Minneso- ta Agricultural Experiment Station, St. Paul, Minn. staphylococcal food-borne hazards. Based upon the nomenclature of Bergey's Manual, S. aureus is considered the only enterotoxigenic species of Staphylococcus. With the more recently pro- posed nomenclatures, S. intermedius and S. hyicus subsp. hyicus join S. aureus as coagulase- positive species. Both S. intermedius (22) and S. hyicus subsp. hyicus (12, 36) have been shown to be pathogenic. Phenotypically, S. intermedius should not be overlooked as a possible entero- toxigenic species because, as defined by Hdjek (21), over 90% of S. intermedius strains coagu- late rabbit plasma (as does S. aureus). However, should S. hyicus subsp. hyicus contain entero- toxigenic strains, the chance of misdiagnosis is much greater. The population of S. hyicus subsp. hyicus is defined by Devriese et al. (13) to be 11 to 89%o coagulase positive and has been shown to demonstrate "irregular, often nega- tive, but sometimes delayed positive results" with the coagulase test (38). This group should 649 Downloaded from https://journals.asm.org/journal/aem on 16 February 2022 by 177.39.118.204.

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Page 1: Characterization of Staphylococcit - Applied and Environmental

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1983, p. 649-6600099-2240/83/090649-12$02.00/0Copyright 0 1983, American Society for Microbiology

Vol. 46, No. 3

Characterization of StaphylococcitDALLAS G. HOOVER,1 SITA R. TATINI,l* AND JOANN B. MALTAIS2

Department ofFood Science and Nutrition, University of Minnesota, St. Paul, Minnesota 551081 andMinneapolis Center for Microbiological Investigations, Food and Drug Administration, Minneapolis,

Minnesota 554012

Received 16 February 1983/Accepted 3 June 1983

A total of 158 Staphylococcus strains from various sources were characterizedby biochemical, physiological, and morphological tests. Numerical taxonomy wasapplied by using these features. Taxonomic analysis was done with programs rununder the MVS-TSO system of the IBM 370 complex and PDP-10 system of theNational Institutes of Health. DNA-DNA hybridization with nitrocellulose filterswas done to compare selected atypical cultures with American Type CultureCollection reference strains. We found that the use of the nomenclature ofBergey's Manual (8th edition) to identify these strains by species was notadequate. DNA homology values supported the formation of Staphylococcushyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcusepidermidis, and Staphylococcus saprophyticus. The three tests that best separat-ed these strains into four species were (i) tube coagulase (6-h or 24-h porcineplasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobicallyfrom ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin.Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gavetypical enterotoxigenic responses in monkey-feeding tests but were negative forenterotoxins A through E, suggesting the presence of one or more new enterotox-ins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM)could not be classified by either DNA-DNA hybridization or numerical taxono-my, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay.DNA homology showed that strain FRI-698M was more closely related to S.epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggestthe occurrence of coagulase-negative enterotoxigenic strains that are not S.aureus; nonetheless, a positive tube coagulase test and heat-stable DNase testshould together be useful for routine screening of most potentially enterotoxigenicstaphylococci in foods.

Since the publication of the latest edition ofBergey's Manual in 1974 (4), many new speciesof staphylococci have been proposed. Coagu-lase-positive staphylococci are divided intothree species: Staphylococcus aureus, Staphylo-coccus intermedius (21), and Staphylococcushyicus (13). The coagulase-negative staphylo-cocci, Staphylococcus epidermidis and Staphy-lococcus saprophyticus, have been redefinedinto as many as 11 species (25, 27). However,this extensive restructuring of coagulase-nega-tive staphylococci has recently been criticized(19).

Creation of new coagulase-positive species,which share phenotypic characteristics long as-sociated with S. aureus, is of concern for thepractical use of these features in evaluating

t Paper no. 13324 on the Scientific Journal Series, Minneso-ta Agricultural Experiment Station, St. Paul, Minn.

staphylococcal food-borne hazards. Based uponthe nomenclature ofBergey's Manual, S. aureusis considered the only enterotoxigenic species ofStaphylococcus. With the more recently pro-posed nomenclatures, S. intermedius and S.hyicus subsp. hyicus join S. aureus as coagulase-positive species. Both S. intermedius (22) and S.hyicus subsp. hyicus (12, 36) have been shown tobe pathogenic. Phenotypically, S. intermediusshould not be overlooked as a possible entero-toxigenic species because, as defined by Hdjek(21), over 90% of S. intermedius strains coagu-late rabbit plasma (as does S. aureus). However,should S. hyicus subsp. hyicus contain entero-toxigenic strains, the chance of misdiagnosis ismuch greater. The population of S. hyicussubsp. hyicus is defined by Devriese et al. (13) tobe 11 to 89%o coagulase positive and has beenshown to demonstrate "irregular, often nega-tive, but sometimes delayed positive results"with the coagulase test (38). This group should

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650 HOOVER, TATINI, AND MALTAIS

concern food microbiologists because S. hyicussubsp. hyicus is closely associated with suchmeat animals as cattle, pigs, and poultry andcould possibly be found in foods derived fromthese animals. Presently, the frequency of en-terotoxin production in S. hyicus subsp. hyicushas not been established.The purpose of this polyphasic study was to

examine and classify S. hyicus subsp. hyicusstrains and other cultures atypical to the Staphy-lococcus species descriptions of Bergey's Man-ual. Through the application of numerical taxon-omy and DNA-DNA hybridization, groups ofstrains were formed and compared with thenomenclature of Bergey's Manual and with therecently defined schema. Enterotoxin produc-tion was examined to evaluate the hazard poten-tial of these strains in foods.

MATERIALS AND METHODS

Cultures. The staphylococcal cultures used in thisstudy were obtained from investigators who isolatedthese strains from various sources, such as humans,poultry, pigs, cattle, and food. Most of the strainsexamined were from L. A. Devriese of the Faculty ofVeterinary Medicine, Casinoplein, Ghent, Belgium

(13). Other contributors were R. W. Bennett of theFood and Drug Administration, Washington, D.C.; L.Hoffner and M. Lund of the Department of LaboratoryMedicine and F. Bates of the Department of Veteri-nary Medicine, University of Minnesota, Minneapolis,Minn.; and W. Sperber and D. Pusch of the PillsburyCo., Minneapolis, Minn. (Table 1).

Cultures were preserved on porcelain beads con-tained in screw-capped test tubes filled with silica geland stored at -10°C. The strains were revived byplacement of a bead into 5 ml of brain heart infusionbroth (BBL Microbiology Systems, Cockeysville,Md.) with overnight incubation at 37°C.

Biochemical, physiological, and morphological tests.Colony morphology and pigmentation were observedon P agar (26) and Baird-Parker agar (BBL). Egg yolkreactions (clearing of agar and surface precipitation)and tellurite reduction were determined on Baird-Parker agar.

Catalase production was determined by direct appli-cation of 3% H202 to colonies on P agar. The benzi-dine test was done by the method of Deibel and Evans(10). Acetoin production was tested by the procedureof Coblenz (8). The method of Pennock and Huddy(32) was used to detect phosphatase. Nitrate reductionwas tested as outlined in the Compendium ofMethodsfor the Microbiological Evaluation of Foods (37).Oxygen requirement was tested in the semisolid thio-glycolate medium of Evans and Kloos (16).

TABLE 1. Culture designation and source for each cluster from the dendrogram

Cluster no. cultures Culture designationsa Source of isolation

1 37 VII53, V1182, C32, C25, ATCC 12600, Culture numbers preceded by VII areJAL, VA506, ATCC 13679, 800A, from L. A. Devriese (13) and isolatedVII90, VII65, 800C, 799, H(Str), from poultry. C32, C25 and C22 are52A5M, 52A5G, Z88, 326, 243, ATCC from L. Hoffner and are isolated from10832, VII23, VII47, VII29, VII89, human clinical patients (blood orV1148, V1118, VII87, VII84, 800B, urine). JAL, 799, 800A, B, C, 326,M47, M86, VII75, VII25, VI143, 243, Z88, M47, M86, M130 are foodVII13, C22, M130 isolates from R. W. Bennett. Others

are from unknown sources.

2 20 A4, Sato 12, Sato 13, A383, A332, K46, All cultures are from L. A. Devriese;K128, K127, Sato 16, Sato 17, Sato cultures Sato, K and VII are from14, ATCC 11249, VII115, VII131, poultry, and A series are from cattle.VII128, A35I, V1I108, VII28, VlIIll,VII76

3 2 VM10 and VA350 VM10 is from clinical lesions (turkey),F. Bates; VA350 is from L. A. Dev-riese, pigs.

Single culture 1 VA517 L. A. Devriese, pigs.

4 43 VA348, K57, VA351, VA349, VA361, Cultures ML55 and CS1 are from humanVA356, VA354, VA346, VA501, blood or urine and are from M. Lund.VA502, VA347, VA362, VA507, All other cultures are from L. A. Dev-VA508, VA353, VA516, VA360, riese; VA cultures are from pigs, andVA519, VA512, VA364, VA515, K24, K and VII are from poultry.VA504, VA505, A201-1, VA518,VA520, VA503, VA358, VA359,VA363, VA510, VA513, VA514,VA357, ML55, VII26, VII27, VII113,VII63, VII52, CS1

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VOL. 46, 1983 CHARACTERIZATION OF STAPHYLOCOCCI 651

TABLE 1-Continued

Cluster no. No. of Culture designationsa Source of isolation

5 3 VII93, FDA1, FDA2 V1193 is a poultry isolate from L. A.Devriese. FDA1 and 2 are food iso-lates from R. W. Bennett.

6 4 V11127, FB1, V11134, B10 V11127 and 134 are poultry strains fromL. A. Devriese. FB1 is an animal clin-ical from F. Bates. B10 is from humanblood from L. Hoffner.

7 3 VII66, V1186, A3 V1166 and 86 are from L. A. Devrieseand are poultry isolates.

8 2 476, R97 Food isolates from R. W. Bennett.

9 2 T26, Bi T26 is a food isolate from W. Sperber;Bi is a human blood isolate from L.Hoffner.

10 14 A17, Al, DPi, B12, C13, ML2, M22, Cultures A, B, and C are from humanATCC 14990, B2, A21, A39, VM5, blood or urine and are from L.FDA575 Hoffner. ML2 is from human urine

and from M. Lund. VM5 is an animalisolate from F. Bates. FDA575 andM22 food isolates from R. W. Ben-nett.

11 12 A15, FB3, B3, 134, 8048, ATCC 15305, Cultures A and B are human blood iso-90-2, FDA577, 90-1, ML56, ML57, lates from L. Hoffner. ML culturesML66 are human blood or urine isolates

from M. Lund; 90-1, 90-2, andFDA577 are food isolates from R. W.Bennett.

12 3 S20, FRI698M Food isolates: S20 was from R. W. Ben-nett and 698M from M. S. Bergdoll.

13 4 A12, ML26, A12-6, B9 Human clinical isolates from L. Hoffnerand M. Lund.

14 2 VM11, A18 Animal and human clinical lesions, fromF. Bates and L. Hoffner.

15 3 Armour, ARM, T3 Food isolates; T3 is from W. Sperber,and others are from W. McCall.

Single culture 1 Ti Food isolate from W. Sperber.

Single culture 1 D143 Food isolates from R. W. Bennett.

16 2 S53, S52 Food isolates from R. W. Bennett.

17 4 ST29, Z162, ST121, 5 Food isolates.a Cultures re-ad from left to right within each cluster correspond to numbers shown from top to bottom in the

dendrogram (Fig. 1). For example, in cluster 1 the first culture is 13 or VII53, and the last culture is 117 or M130;in cluster 2, the first culture is 75 or A4, and the last culture is 18 or VII76; etc. The last four cultures indendrogram are ST29, Z162, ST121, and 5, which are streptococci.

Hemolysins were detected by using plates with Tolerance to NaCl concentrations of 5, 7.5, 10, andblood agar base containing sheep and rabbit blood. 15% was estimated by streak culture growth on P agarStreak cultures were examined after 24 h of incuba- after incubation for 48 h at 37°C.tion, as described by Elek and Levy (15). Aerobic carbohydrate utilization was tested as de-

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652 HOOVER, TATINI, AND MALTAIS

scribed by Kloos et al. (26). The carbohydrates testedwere xylitol, xylose, maltose, mannose, mannitol,sucrose, ribose, rhamnose, trehalose, lactose, fruc-tose, glucose, galactose, and arabinose.Anaerobic mannitol fermentation was done in test

tubes containing purple broth base (Difco Labora-tories, Detroit, Mich.) with 1% agar and 1% mannitoladded. These were stab inoculated and sealed with alayer of Vaspar. The tubes were examined at 48, 72,and 96 h of incubation at 37°C.The coagulase tube test was done by mixing 0.2 ml

of overnight culture growth in brain heart infusionbroth at 37°C with 0.5 ml of coagulase plasma in asterile test tube (10 by 75 mm). The tubes werecovered, incubated at 37°C, and examined for clotformation after 2, 4, 6, and 24 h. Three plasmascontaining EDTA were used: Difco rabbit plasma,BBL rabbit plasma, and noncommercial porcine plas-ma obtained from the George H. Hormel Co., Austin,Minn.Thermonuclease production was detected by the

DNA agar diffusion assay with toluidine blue 0 dye asdescribed by Kamman and Tatini (24).

Lysostaphin and novobiocin (Sigma Chemical Co.,St. Louis, Mo.) susceptibilities were tested on inocu-lated overlay plates as described by Schleifer andKloos (34). Trypticase soy broth (BBL) with 0.5%yeast extract (Difco) and agar were used in the over-lay. Lysostaphin sensitivity was tested at 200 ,ug/ml,and novobiocin sensitivity was tested at 0.6 and 2.0,ug/ml.For protein A analysis, cells were grown in 500 ml of

brain heart infusion broth with shaking for 24 h at37°C, collected, and suspended in 30 ml of TSC buffer(0.05 M Tris-hydrochloride, 0.145 M NaCl, 0.015 Msodium citrate [pH 7.4]). Protein A was isolated by themethod of Sjoquist et al. (35), except that after over-night dialysis, the supernatant was concentrated withpolyethylene glycol 20,000. Low-speed centrifugationremoved particulate matter from the remaining fluid (1to 2 ml). Protein A in these concentrated preparationswas measured against human immunoglobulin G (Cap-pel Laboratories, Downingtown, Pa.) in a microslidegel double diffusion assay for staphylococcal entero-toxins developed by Casman et al. (7). A 750 jig/mlsolution of immunoglobulin G was used versus 100 ,gof purified protein A (Pharmacia Fine Chemicals,Piscataway, N.J.) per ml as the reference. The mini-mum amount of protein A detectable was approxi-mately 25 ,g/ml. Immunodiffusion was done in 1%agarose (electrophoresis grade; ICN Pharmaceuticals,Inc., Irvine, Calif.) containing 0.01 M Tris-acetic acid,pH 8.0. The reaction was positive if a line of identitywas observed between the test material and purifiedprotein A after 3 days of incubation at room tempera-ture.

Numerical taxonomy. Taxonomic analysis was donewith programs run under the MVS-TSO system of theIBM 370 complex of the National Institutes of Health,Bethesda, Md., and the PDP-10 system. Data wereedited with the program EDITMAT as described byWalczak and Krichevsy (40). The resulting data setcontained 162 strains and 48 of the original 61 features.Cluster analysis was performed with a modified ver-sion outlined by Walczak and Krichevsky (41) of theTAXAN 5 program developed by Sneath and Sackin.under the auspices of the Medical Research Council of

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Great Britain. The simple matching coefficient andsorting via unweighted average linkage were used.Feature frequency tables were generated by computerand used to select the most discriminating features.

Enterotoxdgenicity. Enterotoxin detection for typesA through E was by the microslide gel double diffusionassay of Casman et al. (7). Supernatant from a 72-hculture grown at 37°C with vigorous shaking in 4% NZ-Amine NAK (Humko-Sheffield Chemical Co., Mem-phis, Tenn.) plus 0.2% yeast extract was used to checkstrains for enterotoxin production.Monkey bioassay of culture filtrates was done with

cynomologus monkeys (Macacafascilicularis) weigh-ing 1.85 to 5.5 kg as described by Adesiyun and Tatini(1). Monkeys were first examined for response speci-ficity to enterotoxin A and to enterotoxin A inactivat-ed with antiserum A. Emesis in at least two of sixmonkeys within 5 h of feeding was considered apositive response.

Genetic parameters. To obtain high-molecular-weight unlabeled DNA for the DNA-DNA hybridiza-tion studies, cells were grown overnight at 37°C withshaking in 300 ml of Trypticase soy broth supplement-ed with 1% glycine (TSBG). Cells were pelleted bycentrifugation (10,000 x g, 15 min) and washed in TSCbuffer. The pellet was suspended in 30 ml of TSC towhich 50 U of lysostaphin was added. The cell suspen-sion was incubated for 2 h at 37°C to cause cell lysis,and the cell lysate was used for purification ofDNA bythe method of Marmur (30).The concentration and purity of the DNA were

measured spectrophotometrically. The DNA prepara-tion was considered sufficiently pure when the ratio ofabsorbance at 260 nm to absorbance at 280 nm wasgreater than 1.8 and the ratio of absorbance at 260 nmto absorbance at 230 nm was greater than 2.0. Ifpreparations exhibited lower ratios, chloroform-isoa-myl alcohol extractions and ethanol precipitationswere repeated.For the incorporation of radioactive label into DNA,

0.6 mCi of [6-3H]thymidine (New England NuclearCorp., Boston, Mass.) and 6 ,g of 5'-fluorodeoxyuri-dine (Sigma) were added to 50 ml ofTSBG 30 min afterthe addition of a 1% inoculum. The cells were harvest-ed after 18 h of growth and were washed in TSC buffer.The cells were suspended in 5 ml of TSC and lysedwith 50 U of lysostaphin. The labeled DNA wasisolated as described above for the unlabeled DNA.DNA preparations were stored at 4°C over a few dropsof chloroform. The DNA content was determinedspectrophotometrically by assuming that 1 opticaldensity unit at 260 nm corresponded to 50 ,g of DNA.Before hybridization, the necessary amount of labeledDNA was diluted with hybridization buffer andsheared by two 45-s bursts from a Branson sonifier at1.1 A.DNA reassociation was done based on the estab-

lished technique first described by Gillespie and Spie-gelmann (18) with modifications by Denhardt (11) andby Meyer and Schleifer (31). Unlabeled DNA fornitrocellulose filter attachment was alkali denaturedby the technique of Baker (5). Nonspecific binding wasminimized by the use of the preincubation medium ofJeffries and Flavell (23). The reassociation reactionwas allowed to proceed at 60°C for 40 h withoutagitation. The restrictive hybridization buffer was 4xSSC (SSC is 0.15 M NaCl plus 0.015 sodium citrate)

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CHARACTERIZATION OF STAPHYLOCOCCI 653

plus 20% formamide, pH 7.0. All reactions were run intriplicate. The amount of hybridized DNA was mea-sured as described by Meyer and Schleifer (31).DNA homology was expressed as the percentage

ratio between 3H-labeled DNA which was hybridizedwith heterologous unlabeled filter-fixed DNA and theamount of DNA which hybridized with the unlabeledDNA of the same strain as the labeled strain. Percent-age homologies were averaged. Each hybridizationwas repeated at least once. When counts appeareddisparate, additional hybridizations were done, anddata were selected by determining the confidenceinterval estimate of the mean.The guanine and cytosine (G+C) content of DNA

was determined spectrophotometrically by the methodof Ulitzur (39) with DNA from S. epidermidis ATCC14990 as the standard.

RESULTSExcept for the four non-staphylococcal con-

trol strains, all the cultures included in this studywere staphylococci on the basis of Gram stain(gram-positive cocci), cell morphology (1,m indiameter, irregular clusters), catalase and benzi-dine tests (both positive), and sensitivity tolysostaphin.

Numerical taxonomy. The dendrogram (Fig. 1)was developed from a similarity triangle basedon calculated similarity levels constructed fromthe biochemical, physiological, and morphologi-cal tests. The dendrogram shows the taxonomicorder of the strains as well as cluster linkagelevels. Sixteen clusters were formed from thedendrogram by using a similarity level of 80%.The strains were placed in descending order ofsimilarity. The four non-staphylococcal culturesappear at the end of the dendrogram with anintercluster similarity of less than 62%. Thesecultures were classified as streptococci becausethey were gram-positive cocci with negativecatalase and bendizine tests. These were used totest the effectiveness of clustering of unrelatedorganisms. This indicates that the numericaltaxonomy analysis was successful in discrimi-nating at the genus level since 70% relatednesshas generally been suggested as a suitable cut-offlevel in establishing genus borders (6). Basedupon the characteristics chosen for this study,clusters which included only staphylococcalspecies showed intracluster similarities of 83 to89% and intercluster similarities above 50%.This supports the credibility of our analysis atthe species level (high intracluster similaritywith a low standard deviation of c0.6%) and theplacement of the strains in the same genus. Thisis consistent with the levels of similarity basedupon the characteristics chosen for study.

Five major clusters were identified which con-tained 10 or more strains (Fig. 1). There were 11smaller clusters of two to four strains and 3clusters of only a single strain. The major clus-

ters 1, 10, and 11 represented members of thethree species S. aureus, S. epidermidis, and S.saprophyticus, respectively. This is partiallybased on the inclusion of the respective typestrains from the American Type Culture Collec-tion (Rockville, Md.), 12600, 14990, and 15305,in the respective clusters (Table 2).The two remaining major clusters (2 and 4)

primarily contained cultures not classifiable tothe species level on the basis of the nomencla-ture of Bergey's Manual (4). These clusterscontained strains that represented the newlyproposed species, S. hyicus subsp. hyicus. (TheAmerican Type Culture Collection type strainfor S. hyicus subsp. hyicus, 11249, was in cluster2.) Clusters 2, 3, and 4 were combined in Table 2and redesignated cluster 2. Although clusters 2,3, and 4 represented the majority of the S. hyicussubsp. hyicus cultures in this study, they had anintercluster similarity of 77.1%. These clusterswere combined into one cluster because themain diagnostic features, as defined by Devrieseet al. (13), would group them together phenotyp-ically in S. hyicus subsp. hyicus. It should benoted, however, that the primary phenotypicdifferences between clusters 2 and 4 were thatstrains of cluster 2 were mostly isolated frompoultry and cases of bovine mastitis, were coag-ulase positive (95%; 3+/4+ clot within 6 h), andwere negative for protein A (95%), whereasstrains of cluster 4 were mostly isolated frompigs, were coagulase negative (95%), and werepositive for protein A (81%).The percentages of strains in the four groups

positive for various characteristics were deter-mined (Table 2). These features were presentedin this key because of their common use in theidentification of staphylococci or their rankingby the group partitioning index (mean deviationof feature frequencies x 100). The group parti-tioning index varies between 0 and 50, with 50representing the most effective score and 0 theleast effective score in the separation of groupsof taxonomically related strains.The interpretation used for scoring positive

coagulase reactions was that defined by Ber-gey's Manual (4); any degree of clotting in 24 hat 370C. Most food laboratories use a 3+ or 4+clot within 6 h of incubation as the basis for apositive coagulase test. Under this latter criteri-on with rabbit plasma (Difco), cluster 1 strainswere 100% coagulase positive, cluster 2 strainswere 26% coagulase positive, and cluster 10 and11 strains were 100% coagulase negative. Over-all, the rabbit coagulase plasmas (Difco andBBL) were more sensitive than the noncommer-cial porcine plasma for detecting the coagulasereaction. Because the porcine plasma had alower tendency to clot, 1+ clots were lessfrequently formed than with use of the rabbit

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654 HOOVER, TATINI, AND MALTAIS

r---

Cluster 1

Cluster 2 ------

Cluster 3----_-. _,2

Cluster 4-

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I 75 70 65 60 55 50

ClusterClusterClusterClusterClusterCluster

ClusterClusterClusterClusterClusterCluster

167

8.>9g - -

1-2 _ ----113-----12- - >-v:::

16 - -. - '100 a' 0*o 60o 5 r0 6 60 9 so

FIG. 1. Dendrogram constructed from phenetic data.

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CHARACTERIZATION OF STAPHYLOCOCCI 655

TABLE 2. Phenotypic profiles of strains of major clusters

% of strains positivePhenotype Rank GPIa Cluster 1: Cluster 2: Cluster 10: Cluster 11:

S. aureus S. hyicus' S. epidermidis S. saprophyticus

CoagulasePorcine, 24 hPorcine, 6 hAcetoin

Clearing of egg yolk on Baird-Parker agar

Aerobic acid from mannitol

Thermonuclease

Coagulase (Difco, 24 h)

Aerobic acid from ribose

Protein A

1 45.3 100.03 40.6 100.02 40.6 100.0

82.865.60.0

4 40.6 92.0 87.0

5 37.5 92.0 14.0

6 35.9 46.0 100.0

7 35.9 100.0 50.8

8 35.9 53.0 98.0

10 35.9 95.8 54.8

Aerobic acid from naltose 11 34.4 91.7 0.0 92.9

Aerobic acid from trehalose

Coagulase (BBL, 24 h)

Growth in the presence of 2 g ofnovobiocin per ml

Precipitate on Baird-Parker agar

Black colonies on Baird-Parker agar

Coagulase (BBL, 6 h)

12 33.6 100.0 81.3

13 32.8 100.0 23.1

18 31.3 89.0 100.0

19 31.3 92.0 48.0

17 31.3 100.0 18.0

21 30.4 100.0 20.0

Anaerobic acid from mannitol 23 28.1 78.0 0.0 0.0

Coagulase (Difco, 6 h) 27 25.75 100.0 35.4

a GPI, Group partitioning index; 0 to 50 scale.b Cluster 2 consisted of cultures from clusters 2, 3,

plasmas. These 1 + clots, which are difficult todetermine, caused false-positive readings andthus inappropriately indicated that strains ofclusters 10 and 11 were 15.4 and 25.0o coagu-lase positive with BBL rabbit plasma at 6 h(Table 2). However, use of rabbit plasma (BBLor Difco) with the more stringent interpretation(3+/4+ clot, 6 h) showed clusters 10 and 11 asentirely coagulase negative but with a lowergroup partitioning index (data not shown).Only 46% of the cultures in cluster 1 (S.

aureus) were positive for thermonuclease pro-duction (Table 2). This was caused by the ratherlarge number of thermonuclease-negative, coag-ulase-positive atypical strains that were includedin this study and placed in cluster 1 by numericaltaxonomy. However, it was found that all thecultures in cluster 1 were actually very weaklypositive for thermonuclease production. A 100-

4 of the dendrogram (Fig. 1).

fold concentration of the assay supernatant gavea positive test result with the DNA agar diffusiontest (24).DNA homology and polyphasic comparisons.

The DNA homology values of individual strainsselected from the numerical taxonomy clustersand the American Type Culture Collection typestrains for the four species classified in thisstudy by numerical taxonomy were determined(Table 3). Labeled DNA was obtained from thetype strains, 12600, 14990, and 15305, as well as

two enterotoxigenic S. aureus cultures, 800Cand JAL, and two S. hyicus subsp. hyicusstrains, VII131 and A4, which were isolatedfrom different sources.

Generally, there was good agreement betweenclusters formed by numerical taxonomy andDNA homology values; however, there were

notable exceptions. Low homology values were

0.00.0

100.0

7.0

14.0

0.0

7.7

0.0

0.0

0.00.0

41.7

0.0

92.0

0.0

0.0

0.0

0.0

100.0

0.0

7.7

100.0

7.0

31.0

15.4

91.7

16.7

17.0

0.0

67.0

25.0

0.0

38.5 25.0

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656 HOOVER, TATINI, AND MALTAIS

found among cluster 1 (S. aureus) strains. Ther-monuclease-negative cultures VII13 and VII18,which were originally described as S. aureus,avian biotype 2 (14), displayed very low homolo-gy with the labeled DNA from the three S.aureus cultures, 12600, 800C, and JAL. Partlybased on this relationship, 30% was used as theDNA homology value to differentiate at thespecies level under the hybridization conditionsused in this study.Another exception to the agreement between

numerical taxonomy and DNA-DNA hybridiza-tion was strain FDA2. FDA2, a coagulase-nega-tive isolate from food, had relatively high homol-ogy values to the S. aureus strains of cluster 1but was placed in cluster 5.DNA homology values supported the defini-

tion of S. hyicus subsp. hyicus (13) but could notdistinguish S. hyicus subsp. hyicus clusters 2and 4, which were formed by numerical taxono-my. DNA homology values of S. hyicus subsp.hyicus strains were higher among strains isolat-ed from the same ecological source. NeitherDNA-DNA hybridization nor numerical taxono-my could distinguish those S. hyicus subsp.hyicus strains that gave a positive emetic re-sponse in the monkey bioassay. In this study, nostrains of S. hyicus subsp. hyicus were foundthat produced enterotoxins A through E, butfour strains (VII76, VII113, VII131, and VA519)did produce untyped, biologically active entero-toxin(s) as detected by the monkey bioassay.These four cultures were thermonuclease posi-tive and were also coagulase-negative when in-terpreted with the criterion of a 3+/4+ clotwithin 6 h with rabbit plasma. With any degreeof clotting (1 + to 4+) and 24 h of incubation, theresults from multiple testings were: (i) BBLrabbit plasma, all negative except V11113 (1+clots); (ii) Difco rabbit plasma, VII76 (1 + clot),VII113 (1 + clot), VI1131 (2+ and 4+ clots) werepositive, and VA519 was negative. Clearly,these strains were not strong producers of coag-ulase and probably would be missed in screeningstaphylococcal strains by the tube coagulasetest. Also, these strains did not exhibit typical S.aureus colony appearance on Baird-Parker agarand did not reduce tellurite strongly to form jet-black colonies, with strain VII76 showing nei-ther lipase nor protease reaction.

Strains ARM and D143 were originally isolat-ed from food. Both were coagulase negative andthermonuclease positive. Neither hybridizedsignificantly with any other strain. More impor-tant, D143 gave a positive enterotoxin responsein the monkey bioassay. The two strains did notproduce enterotoxins A through E.

Strain FRI-698M was coagulase negative andthermonuclease negative but produced entero-toxin D. It did not hybridize with any S. aureus

strain that was tested (Table 2). Instead, a 46%homology was shown with the type strain for S.epidermidis, 14990 (standard deviation, + 2.9%),and a 34% homology was shown with the typestrain for S. saprophyticus, 15305 (standard de-viation, ± 10.3%). Of the 26 strains examined inthis hybridization study, FRI-698M was the onlyculture that displayed a greater than 30% homol-ogy with two type strains. This may be ex-plained by the standard deviation of the mean;for FRI-698M with 15305, it was ±10.3%. Withsuch an interval, the homology range wouldextend below the 30% species boundary.

DISCUSSIONIn this study, strains other than American

Type Culture Collection type strains and non-staphylQcoccal control strains were selected topresent a cross section of staphylococci thatwould present difficulties in routine identifica-tion. This included not only isolates that wereatypical in matching modal strain features of thetaxonomic key of Bergey's Manual but alsostrains weakly positive or variable for suchdiagnostic tests as coagulase and thermonu-clease production (3, 13). The strains displayed ahigh degree of heterogeneity. Five major clus-ters of strains were formed by numerical taxono-my at an 80% similarity. Strains that fell outsidethese five major clusters represented 22% of thetotal number of cultures, indicative of the phe-notypic heterogeneity of the staphylococci inthis study.The best diagnostic features found to differen-

tiate the four species groups classified in thisstudy (Table 2) were the coagulase test (porcineplasma, 24 h of incubation) to separate clusters 1and 2 from clusters 10 and 11, acetoin produc-tion to separate cluster 2 from clusters 1 and 10,and aerobic acid from trehalose to separatecluster 1 from cluster 10 and cluster 10 fromcluster 11.Data from cell wall protein A measurement of

S. hyicus subsp. hyicus agreed with recent workby Phillips and Kloos (33), who suggested theuse of protein A as a possible marker to distin-guish ecotypes of S. hyicus. We found protein Aprimarily associated with S. hyicus subsp. hyi-cus strains from pigs and not from isolates fromcattle or poultry. There was no significant corre-lation between coagulase-positive S. hyicussubsp. hyicus strains and strains that were posi-tive for protein A.Except for a higher percentage of strains

utilizing mannitol aerobically, the feature fre-quencies shown in Table 2 for S. hyicus subsp.hyicus (cluster 2) fit the nomenclature describedby Devriese et al. (13). The DNA homologyvalues for the S. hyicus subsp. hyicus strains

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CHARACTERIZATION OF STAPHYLOCOCCI 657

(Table 3) were in agreement with homology datapublished by Devriese et al. (13) and by Phillipsand Kloos (33). It supported the subspeciesdesignation distinct from S. aureus, S. epidermi-dis, and S. saprophyticus. There was a distinctgenetic relatedness among ecotypes of S. hyicussubsp. hyicus. Strains from poultry showedhigher intragroup homologies with labeled DNAfrom strain V11131 (poultry) than with strain A4(bovine mastitis). Numerical taxonomy did notdifferentiate the ecotypes of S. hyicus subsp.hyicus as DNA-DNA hybridization did. StrainsVII131 and 11249 were placed in the samecluster at an 88% similarity by numerical taxon-omy, whereas 11249 had only a 26% DNAhomology with V11131. Devriese et al. (13) andPhillips and Kloos (33) found such homologydifferences among S. hyicus subsp. hyicus cul-tures, enough to suggest other possible subspe-cies of S. hyicus. Recent indications (27) are thatfurther subspecies will be defined in S. hyicus.From a practical standpoint, further classifica-

tion to the subspecies level may not be neces-sary. Very low homology values were found incluster 1 (S. aureus), the most homogeneouscluster formed by numerical taxonomy (Table3). Strains VII13 and VII18 (both thermonu-clease negative) were previously classified as S.aureus, avian biotype 2 (14). These strainsshowed low homology with labeled DNA fromthe three S. aureus cultures. Such lower valuesmay be acceptable in a functioning species defi-nition which includes different ecotypes.The biological activity of the enterotoxin(s)

from S. hyicus subsp. hyicus strains did notappear to be as great as the activity of enterotox-in produced by S. aureus. These S. hyicussubsp. hyicus strains were positive in the catinjection bioassay (data not shown) and themonkey-feeding bioassay with cynamologusmonkeys, but the strains were negative whentested with rhesus monkeys. We suggest thatcynamologus monkeys may be more sensitive toenterotoxins than rhesus monkeys. If this is thecase, the potential of food poisoning to humansfrom enterotoxigenic strains of S. hyicus subsp.hyicus may be low.

Strain FDA2 was placed in cluster 5 of thedendrogram. Although DNA homology indicat-ed that FDA2 was a S. aureus culture, it dis-played a phenotypic similarity of only 67% withcluster 1 (S. aureus). This strain may have lostseveral characteristics during storage since itsoriginal isolation (28), as reflected in phenotypicclustering. However, such a change would notresult in an extensive change in genetic makeupas reflected by DNA homology (6). Thus, ho-mology data indicate that FDA2 was a coagu-lase-negative variety of S. aureus. Gramoli andWilkinson (20) stated that the thermonuclease

test was useful in detecting coagulase-negativeS. aureus, and this would be true in detecting S.aureus isolates such as FDA2, but other testswould be necessary for definite classification byspecies.

Strains ARM and D143 were not similar to anydefined species group or type strain. CultureD143 was pigmented and thermonuclease posi-tive, but otherwise it had no phenotypic resem-blance with S. aureus. D143 was negative forenterotoxins A through E but gave an emeticresponse in the monkey bioassay. D143 is notgrouped with any other strain in the dendro-gram, and DNA-DNA hybridization showed nosignificant homology with any other culture.Phenotypically, this culture would be classifiedas S. hyicus subsp. chromogenes.

Strain ARM was also a coagulase-negative,thermonuclease-positive staphylococcus isolat-ed from food. This strain was a subculture ofArmour, an isolate studied by Gramoli and Wil-kinson (20). These authors found Armour un-identifiable. In this study, ARM could not beidentified by numerical taxonomy or DNA-DNAhybridization. Based on key characteristics,ARM would be identified as an isolate of S.hyicus subsp. hyicus. The genetic data of ARMsuggest that another species or subspecies otherthan S. hyicus. subsp. hyicus may be necessaryto describe varieties of Staphylococcus that arecoagulase negative, thermonuclease positiveand, as shown with D143, enterotoxigenic.

Strain FRI-698 was examined by Lotter andGenigeorgis (29) is their study of coagulase-negative variants of S. aureus. The isolate theyused was coagulase-negative and positive fordelta hemolysin, egg yolk reaction, thermonu-clease, penicillinase, phosphatase, acetoin, pro-tein A, acid from maltose, and production ofenterotoxin D and pigment. The culture used inthe present study was originally received fromthe Food Research Institute, University of Wis-consin, Madison, Wis.; however, the culturewill be designated FRI-698M since it appears tohave lost many of the characteristics associatedwith FRI-698. Strain FRI-698M was coagulasenegative and unable to produce hemolysin, ther-monuclease, pigment, phosphatase, acetoin, orprotein A or to reduce tellurite. FRI-698M couldutilize maltose, was resistant to penicillin, andproduced enterotoxin D. This phenotypic differ-ence was probably due to a spontaneous muta-tion caused by extended storage on desiccatedbeads, as suggested by Lotter and Genigeorgis(29) after observation of stored strains in theirstudy. Such mutations should not affect DNAhomology values. The data from Table 3 suggestthat FRI-698M was an atypical enterotoxigenicvariant of S. epidermidis that originally carriedmany phenotypic features associated with S.

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Page 10: Characterization of Staphylococcit - Applied and Environmental

658 HOOVER, TATINI, AND MALTAIS

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CHARACTERIZATION OF STAPHYLOCOCCI 659

aureus. Unfortunately, no data were recordedfrom strain FRI-698M when it was first receivedin our laboratory, and Lotter and Genigeorgisdid not identify FRI-698 by DNA-DNA hybrid-ization.

Specific strains in this study indicated that thegenus Staphylococcus may need to be expandedto include more species; however, the excep-tional nature of these strains may make anincrease unnecessary. More coagulase-negative,thermonuclease-positive strains will have to beexamined to estimate the frequency and entero-toxigenicity of such staphylococcal strains (9),especially from animal sources.Although the strains of S. hyicus subsp. hyi-

cus were negative for enterotoxins A through E,four cultures gave a typical response for staphy-lococcal enterotoxins in monkey-feeding tests.This suggests that enterotoxigenicity is a proper-ty found in S. hyicus subsp. hyicus, regardless ofthe association of a strain with a positive tubecoagulase test. Other examples of enterotox-igenic non-S. aureus strains were D143 and FRI-698M.

Because of the lack of direct assay for staphy-lococcal enterotoxins that is rapid, simple, andeconomical, indirect tests continue to be used toestimate staphylococcal hazard potential. Al-though no indirect test or tests would be 100%reliable in detecting enterotoxigenic staphylo-cocci (e.g., strain FRI-698M), the combined useof the coagulase test and the thermonucleasetest (2, 17) appear to be reliable and practical forthe screening of potentially hazardous staphylo-cocci, including enterotoxigenic varieties notdefined as S. aureus.

ACKNOWLEDGMENTS

We thank J. L. Smith and P. M. Walsh for their criticalreading of this manuscript. The authors also appreciate theassistance of A. A. Adesiyun for conducting the monkeybioassays for new enterotoxins.

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1. Adesiyun, A. A., and S. R. Tatini. 1981. Suitability ofcynomologous monkeys (Macacafascicularis) for staphy-lococcal enterotoxin bioassay. J. Food Safety 3:193-198.

2. Baer, E. F., R. J. H. Gray, and D. S. Orth. 1976. Methodsfor the isolation and enumeration of Staphylococcus aure-us, p. 374-386. In M. L. Speck (ed.), Compendium ofmethods for the microbiological examination of foods.American Public Health Association, Washington, D.C.

3. Baird-Parker, A. C. 1965. The classification of staphylo-cocci and micrococci from world-wide sources. J. Gen.Microbiol. 38:363-387.

4. Baird-Parker, A. C. 1974. Genus II. Staphylococcus Ro-senbach 1884, 18 nom. cons. opin. 17 Jud. Comm. 1958,153, p. 483-489. In R. E. Buchanan and N. E. Gibbons(ed.), Bergey's manual of determinative bacteriology, 8thed. The Williams & Wilkins Co., Baltimore.

5. Baker, R. F. 1977. Binding of DNA to cellulose nitratefilters under denaturing conditions. Anal. Biochem.78:569-571.

6. Brenner, D. J. 1980. Taxonomy, classification and no-menclature of bacteria, p. 1-6. In E. H. Lennette, A.Balows, W. J. Hausler, Jr., and J. P. Truant (ed.), Manualof clinical microbiology, 3rd ed. American Society forMicrobiology, Washington, D.C.

7. Casman, E. P., R. W. Bennett, A. E. Dorsey, and J. E.Stone. 1969. The microslide gel double diffusion test forthe detection and assay of staphylococcal enterotoxins.Health Lab. Sci. 6:185-198.

8. Coblenz, L. M. 1943. Rapid detection of the production ofacetylmethylcarbinol. Am. J. Public Health 33:815-817.

9. Danielsson, M. L., and B. Hellberg. 1977. The biochemicalactivity of enterotoxin and non-enterotoxin producingstaphylococci. Acta. Vet. Scand. 18:266-273.

10. Delbel, R. H., and J. B. Evans. 1960. Modified benzidinetest for the detection of cytochrome-containing respira-tory systems in microorganisms. J. Bacteriol. 79:356-360.

11. Denhardt, D. T. 1966. A membrane filter technique for thedetection of complementary DNA. Biochem. Biophys.Res. Commun. 23:641-646.

12. Devrlese, L. A., and J. Derycke. 1979. Staphylococcushyicus in cattle. Res. Vet. Sci. 26:787-792.

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

14. Devriese, L. A., and P. Oeding. 1975. Coagulase and heat-resistant nuclease-producing Staphylococcus epidermidisstrains from animals. J. Appl. Bacteriol. 39:197-207.

15. Elek, S. D., and E. Levy. 1950. Distribution of hemolysinsin pathogenic and nonpathogenic staphylococci. J. Pathol.Bacteriol. 62:541-553.

16. Evans, J. B., and W. E. Kloos. 1972. Use of shake cul-tures in a semisolid thioglycolate medium for differentiat-ing staphylococci from micrococci. Appl. Microbiol.23:326-331.

17. Food and Drug Administration. 1976. FDA bacteriologicalanalytical manual for foods. Association of Official Ana-lytical Chemists, Washington, D.C.

18. Gillespie, D., and S. Spiegelnann. 1965. A quantitativeassay for DNA-RNA hybrids with DNA immobilized on amembrane. J. Mol. Biol. 12:829-842.

19. Goodfellow, M., M. Modarski, A. Tkacz, K. Szyba, and G.Pulverer. 1980. Polynucleotide sequence divergenceamong some coagulase-negative staphylococci. Zentralbl.Bakteriol. Abt. 1 Orig. Reihe A 246:10-22.

20. Gramoli, J. L., and B. J. Wilkinson. 1978. Characteriza-tion and identification of coagulase-negative, heat-stabledeoxyribonuclease-positive staphylococci. J. Gen. Micro-biol. 105:275-286.

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

22. Hijek, V., and E. Marsalek. 1971. The differentiation ofpathogenic staphylococci and a suggestion for their taxo-nomic classification. Zentralbl. Bakteriol. Parasitenkd.Infektionskr. Hyg. Abt. 1 Orig. Reihe A 217:176-182.

23. Jeffries, A. J., and R. A. Flavell. 1977. Physical maparound rabbit beta-globulin gene. Cell 12:429-439.

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25. Kloos, W. E., and K. H. Schleifer. 1981. The genus Staph-ylococcus, p. 1548-1569. In M. P. Starr, H. Stolp, H. G.Truper, A. Balows, and H. G. Schlegel (ed.), The pro-karyotes, vol. 2. Springer-Verlag, New York.

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1971. Metachromatic agar-diffusion methods for detectingstaphylococcal nuclease activity. Appl. Microbiol.21:585-587.

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