COMPARATIVE STUDY OF GAFFKYA HOMARI, AEROCOCCUS VIRIDANS,TETRAD-FORMING COCCI FROM MEAT CURING BRINES, AND

THE GENUS PEDIOCOCCUS'

R. H. DEIBEL AND C. F. NIVEN, JR.Division of Bacteriology, American Meat Institute Foundation, The University of Chicago,

Chicago, Illinois

Received for publication May 20, 1959

Snieszko and Taylor (1947), and Hitchner andSnieszko (1947) described gram-positive, tetrad-forming micrococci associated with a septicemicdisease of lobsters. These organisms grew poorlyon laboratory media unless a fermentable sugarwas added. They were further characterized bytheir ability to ferment mannitol and their in-ability to reduce nitrate or to hydrolyze gelatin.Because of their morphology and pathogenicityfor the lobster, the authors suggested a new spe-cies, Gaffkya homari.

In 1951, Dr. W. S. Sturges, Jr., of The CudahyPacking Company Laboratory, Omaha, Ne-braska, submitted to this laboratory two strainsof cocci which had been isolated from cured meatproducts. These microorganisms were charac-terized by small colony formation on ordinarylaboratory agar media, tendency to occur astetrads, and ability to produce a greenish dis-coloration of cured meat products. Aaronson(1956) also obtained two cultures of these tetrad-forming cocci from Dr. Sturges and noted thatthey appeared to be identical to G. homari withrespect to morphology, minimal nutritional re-quirements, and biochemical characteristics.

Williams et al. (1953) attempted to enumeratethe oral streptococei from the air of occupiedrooms by the use of a selective culture mediumcontaining potassium tellurite and crystal violet.They noted the frequent occurrence of a uniquecoccus that had biochemical characteristics simi-lar to the enterococci. But, no tendency towardchain formation could be demonstrated. Thesemicroorganisms were catalase-negative, grew inthe presence of 40 per cent bile, and producedstrong greening on blood agar. In view of theseunique features, the authors proposed a new genusand species, Aerococcus viridans.The present report describes a group of tetrad-

' Journal paper no. 178, American Meat Insti-tute Foundation.

forming, gram-positive cocci that have beenfound to comprise a minor proportion of thenormal flora of meat curing brines. These micro-organisms appear to be identical, or very similar,to Aerococcus viridans, Gaffkya homari, and thecultures of Dr. Sturges. Since all of these micro-organisms have several characteristics commonto the genus Pediococcus, it is proposed that theybe included in this genus.

MATERIALS AND METHODS

Used ham curing pickle samples were obtainedfrom a number of meat packing companies andplated quantitatively on APT agar (Evans andNiven, 1951). After incubation for 3 days at 30 C,individual colonies were picked, streaked on APTagar, and reisolated. One tetrad-forming coccusfrom each packing plant was saved for furtherstudy. Although these organisms did not repre-sent the predominant morphological type in thesebrines, some were found in most of the brinesamples examined. Similar organisms also wereisolated from the skin of pig carcasses that hadbeen chilled overnight after slaughter, and fromoutbreaks of bacterial greening of bacon. Sixstrains from curing brines, 1 from a hog carcass,and the 2 from bacon were subjected to a detailedtaxonomic study.For comparative purposes, 6 cultures of A.

viridans were obtained from Dr. R. E. 0.Williams. The 2 strains from Dr. Sturges and 2 G.homari strains from Dr. Aaronson also were in-cluded in the study. The G. homari strains (ATCC10400 and RT-4) were the same as those em-ployed in the comparative study by Aaronson(1956).The laboratory methods employed were similar

to those described in the previous publications(Deibel and Niven, 1958, 1959). Nitrate reduc-tion was tested in the semi-solid indole-nitritemedium (BBL) supplemented with 0.5 per cent

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TABLE 1Physiological characteristics of tetrad-forming cocci from meat brines and cured meat products, Aerococcus

viridans, and Gaffkya homari

Relation to oxygen..............Catalase.........................Greening on blood agar..........Nitrate reduced.................Final pH, glucose broth.........CO2 from glucose................Litmus milk.....................

Polysaccharide from sucrose.Growth:10% NaCl broth...............10 C..........................40 C..........................45 C...........................40% bile agar.................

Hydrolysis:Sodium hippurate.............Esculin........................Arginine.......................Starch.........................Gelatin. ......................

Fermentation:Xylose ........................Arabinose .....................Galactose .....................Lactose .......................Trehalose .....................Cellobiose .....................Melibiose......................Raffinose ......................Inulin .........................Glycerol.......................Mannitol ......................Sorbitol .......................Inositol .......................Salicin ........................

Tetracocci (Meat Tetracocci (Sturges) Aerococcus viridans Gaffkya homariBrines etc.) (2 strains) (6 strains) (2 strains)(9 strins)

Facultative

4.7-5.0

2 acid; 7 nochange

+++

4+;5

7+; 2-6+; 3-2+; 7-

+4+; 5-7+; 2-

4+; 5-6+; 3-2+; 7-3+; 6-+

1+; 8-7+; 2-

Facultative

+

5.0

1 acid; 1 nochange

1+; 1-

1+; 1-1+; 1-

1+; 1-

+1+; 1-

Facultative4+; 2-+

4.8-5.1

4 acid; 2 nochange

++

2+; 4-+

+4+; 2-1+; 5-

4+; 2-4+; 2-++

5+; 1-4+; 2-2+; 4-4+; 2-1+; 5-5+; 1-5+; 1-4+; 2-

4+; 2-

Facultative1+; 1-+

4.7-4.9

1 acid; 1 nochange

1+; 1-

1+; 1-1+; 1-+

1+; 1-1+; 1-1+; 1-1+; 1-1+; 1-1+; 1-1+; 1-

All cultures fermented glucose, mannose, fructose, maltose, and sucrose. None fermented rhamnose,melezitose, or dulcitol.

yeast extract. Nitrite was detected using thesulfanilic acid-a-naphthylamine reagent. Cata-lase production was determined in APT brothand on an agar medium containing only 0.05 percent glucose (Felton et al., 1953). Growth in rela-tion to oxygen was determined in agar shake cul-tures using trypticase soy broth (BBL) plus 1.5per cent agar.

RESULTS

Table 1 summarizes the physiological char-acteristics of the 9 tetracocci from meat curingpickles, bacon, and hog carcass; the 2 tetracoccifrom Dr. Sturges; 6 strains of A. viridans; and 2strains of G. homari. Although some variationswere noted in the characteristics among the in-dividual strains, no single test served to differ-

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PEDIOCOCCUS HOMARI NOV. COMB.

4' ~ z -, X, 4 *

.sse as t b*=~~.

p. ' , p.

;, *.

5~~~%!~~~~ ., Sivt -~~~~~~ f

*~4 1640

U ofU. A

a IJ*f

_ - ^ *

1* 0~~~~.9 .9~~I

I

I

Figure 1. A comparison of the morphology and cell size of Pediococcus cerevisiae (left) and Pediococcushomari (right). Stained with crystal violet and magnified 730 X.

entiate one group from another. The characteris-tics of the 2 G. homari strains as reported byAaronson were largely confirmed, although somedeviations were noted. In contrast to Aaronson'sresults, strain RT-4 failed to ferment lactose ormannitol.

Morphology. Regardless of source, stained prep-arations of all cultures grown in APT brothshowed numerous tetrad formations, some pairs,and many larger clusters. All strains were gram-positive and were similar in appearance. Thetendency to form tetrads was diminished whenthese organisms were grown in or on other mediawhich yielded reduced growth. Therefore, smearswere routinely prepared from APT broth culturesafter 20 to 24 hr at 30 C. As demonstrated in fig-ure 1, Pediococcus cerevisiae produces similargroupings of cells, but the individual cells gen-erally are smaller when cultured in APT brothor similar media.

Catalase production. Regardless of source, allcultures either failed to produce detectable cata-lase or showed only weak catalase activity. In-deed, all the tetracocci isolated in the presentstudy failed to produce catalase even when cul-tured on the low-glucose agar of Felton et at.(1953). However, of the 6 strains of A. viridans, 4cultures (strains 7764, 7765, 7766, and 7794)

showed weak catalase activity on this agar me-dium. Both strains from Dr. Sturges were weaklycatalase-positive, even in broth culture. One ofthe G. homari strains (ATCC 10400) was weaklypositive on the low-glucose agar. The inability todemonstrate catalase or the finding of weak cata-lase activity parallels closely the distribution andactivity of this enzyme in the genus Pediococcus.

Cultural characteristics. When cultured in abroth medium, most of the strains produced uni-form turbidity, the cells tended to settle ratherquickly to form a packed sediment. The sedimentcan be resuspended uniformly with ease. Some ofthe cultures of A. viridans produced a more gran-ular type of growth that was difficult to resus-pend uniformly. None of the cultures producedany evidence of pellicle formation. In general,broth cultures appeared very similar to lacticacid bacteria.

After 48 hr at 30 C, surface colonies on agarmedia were 2 mm or less in diameter. The col-onies were greyish white and compact, and hadcharacteristics similar to P. cerevisiae or otherlactic acid bacteria.

Relation to oxygen. In suitable agar mediasuch as the trypticase soy medium, all cultureswere facultative with respect to oxygen. In otheragar media most cultures exhibited superior

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DEIBEL AND NIVEN

growth just beneath the surface of the agar. How-ever, in no instance did any of the cultures growextensively on the surface of the agar shake cul-ture as would be expected for a micrococcus or astaphylococcus.

Glucose fermentation. All strains from all sourcesproved to be homofermentative and achieved afinal pH value of 4.7 to 5.1. The lactic acid pro-duced by 5 strains isolated in this study, and thetype culture A. viridans (NCTC 8251), was theL( +)-isomer. In contrast to these results, P.cerevisiae cultures tested under the same condi-tions produced a final pH value between 3.7 and4.0 and accumulated inactive lactic acid as thepredominant fermentation product.

Mlliscellaneous tests. All cultures were relativelytolerant to salt and had a rather low temperaturerange for growth. The temperature limits ofgrowth of some of the tetracocci isolated in thisstudy have been shown to depend on the amountof salt in the medium (Deibel, Goldman andNiven, unpublished data).

In contrast with P. cerevisiae (Felton andNiven, 1953), none of the tetracocci required thecitrovorum factor (Leucovorin) when tested in afolic acid-free basal medium. The growth of eachof the tetracoccus strains was inhibited in anagar medium containing 0.02 per cent sodiumazide, except in those areas having a heavy in-oculum. P. cerevisiae did not evidence a similarinhibition of growth in this medium.

Characteristically, all strains produced astrong greening reaction when individual col-onies were examined in sheep blood agar pourplates. In contrast, the strains of P. cerevisiaetested produced either a very weak greening re-action or no evident change on blood agar. Asopposed to P. cerevisiae, all of the tetracocci, re-gardless of source, were capable of producing agreen discoloration when streaked upon the cutsurface of cured meats and incubated for 24 hrat 30 C.

Other minor differences were noted betweenthe tetracoccus collection and P. cerevisiae. Forexample, the majority of the tetracocci hydro-lyzed sodium hippurate, whereas P. cerevisiaecultures tested in this laboratory, as well as thosereported by Jensen and Seeley (1954), failed toattack this substrate. Also, the majority of thetetracocei fermented mannitol whereas P. cere-visiae does not (Pederson, 1949; Williams et al.,1953).

DISCUSSION

From the results, it seems reasonable to groupthe tetracocci from meat sources, Aerococcusviridans and Gaffkya homari, into one species.Although variations existed among strains, nosingle characteristic served to separate one groupof microorganisms from another. Furthermore,these microorganisms have characteristics verysimilar to the genus Pediococcus. This genus, ofwhich only two species are established, is char-acterized as being catalase-negative or weaklycatalase-positive, facultative or microaerophilic,homofermentative, nonproteolytic, incapable ofreducing nitrate, producing poor growth on thesurface of agar media, and having a tendency toform tetrads. All the cultures here studied havethese characters. Therefore, it is logical to con-clude that they belong to the genus Pediococcus.

Williams et al. (1953) recognized the similarityof A. viridans and the genus Pediococcus butdifferentiated the two groups of microorganismson the basis of morphology, relation to oxygen,final pH value, mannitol fermentation, and tol-erance to crystal violet and potassium tellurite.Our study has demonstrated that the morphol-ogy, and relation to oxygen of these groups, arenot markedly different, and the other characteris-tics would not appear to justify differentiationon a generic basis.

In selecting a species name for the tetracoceihere studied, it would be desirable to keep inmind the apparent ubiquity of these organismsand to recognize the inherent weakness of em-ploying parasitism as a criterion of species de-lineation. Nevertheless, on the basis of priorityas prescribed by the International Code of Nomen-clature of Bacteria and Viruses (1958), it becomesnecessary to classify the tetracocei as Pediococcushomari nov. comb. if it is accepted that all ofthem belong to one species within the genusPediococcus. Rule 23 of the Bacteriological Codestates that "a legitimate name or epithet mustnot be rejected merely because it is inappropriate,or disagreeable, or because another is preferableor better known, or because it has lost its originalmeaning." Indeed, it would be interesting to testthe pathogenicity of representative cultures ofA. viridans and the meat tetracocci for the lobster.The proposed classification places these micro-organisms appropriately among the lactic acidbacteria (family Lactobacillaceae, tribe Strepto-cocceae).

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PEDIOCOCCUS HOMARI NOV. COMB.

There are other tetrad-forming cocci that havebeen described and which may be related to P.homari. Hornsey and Mallows (1955) observedthe occurrence of a catalase-negative Micrococcusspecies in beef curing brines. Although the pub-lished description does not permit detailed com-parison, there is suggestive evidence that theseorganisms are similar or identical to P. homari.

Orla-Jensen (1919) described a catalase-nega-tive tetracoccus from anchovy pickle that pos-sessed a weak fermentative capacity and pro-duced dextro-rotatory lactic acid from glucosefermentation. Mees (1934) confirmed thesefindings and proposed the name Tetracoccushalophilus, since the growth of this species wasstimulated by sodium chloride.

Since completion of this study, attention hasbeen called to the description of a coccus fromsoy sauce mash and soy-koji (Sakaguchi, 1958)which appears to be identical or very similar tothe microorganisms described here. They existedas tetrads, were microaerophilic, salt tolerant,catalase-negative cultures which had a rather lowoptimal temperature for growth. They wereweakly fermenting, homofermentative micro-organisms which produced either L(+) or inac-tive lactic acid from glucose. They failed to reducenitrate, and the majority of the cultures fer-mented mannitol. The author proposed the namePediococcus soyae for this group of microorgan-isms. It would be interesting to determinewhether they are identical to the microorganismsincluded in the present study.Although P. homari has characteristics much

in common with the other Pediococcus species,it may be differentiated in that P. homari isrelatively tolerant to salt, produces dextro-rota-tory lactic acid from glucose, ferments sugarswith a higher limiting pH value, produces stronggreening on blood agar, generally ferments man-nitol, and evidences a marked inhibition whengrown on an agar medium containing 0.02 percent sodium azide. In contrast with P. cerevisiae,P. homari does not require the citrovorum factorfor growth, and the individual cells of P. homaritend to be larger than those of P. cerevisiae.Aaronson (1956) reviewed the genus Gaffkya,

and suggested that it may have to be dismantled.In view of the fact that the two species of thisgenus could well be distributed into other genera,it would be desirable to give serious considera-tion to abandoning the genus Gaffkya.

The partial overlapping of some physiologicalcharacteristics of P. homari with the enterococcimerits comment. When grown under somewhatunfavorable conditions the cellular morphologyof P. homari may be confused with that of theenterococci. Furthermore, catalase may be absentor undetected in P. homari, the degree of salttolerance is similar, and the strong greening re-action in blood agar is very similar to that pro-duced by Streptococcus faecium. Consequently,these tests fail to differentiate these two groupsof organisms.

ACKNOWLEDGMENTS

The authors wish to express their gratitude toDr. W. S. Sturges, Jr., Dr. R. E. 0. Williams,and Dr. S. Aaronson for kindly providing thecultures included in this study.

SUMMARY

A comparative study was made on a collectionof gram-positive, tetrad-forming cocci from curedmeat and meat curing brines, Gaffkya homari,and Aerococcus viridans. These cultures weresufficiently similar in their morphological andphysiological characteristics to justify placingthem into a single species. Because of their closerelationship to the genus Pediococcus, it is pro-posed that they be placed into the species Pedio-coccus homari nov. comb.

In addition to characteristic morphologicalfeatures, P. homari is a facultative, salt tolerant,homofermentative bacterium that produces littleor no catalase, forms dextro-rotatory lactic acidfrom sugar fermentation, and fails to reduce ni-trate, or liquefy gelatin. It differs from Pedio-coccus cerevisiae in that it has a rather high limit-ing pH, produces strong greening on blood agar,generally ferments mannitol, is less tolerant tosodium azide, and does not require the citro-vorum factor for growth.

REFERENCESAARONSON, S. 1956 A biochemical-taxonomic

study of a marine micrococcus, Gaffkyahomari, and a terrestrial counterpart. J.Gen. Microbiol., 15, 478-484.

DEIBEL, R. H. AND NIVEN, C. F., JR. 1958Microbiology of meat curing. I. The occur-rence and significance of a motile Lactobacillusin ham curing brines. Appl. Microbiol., 6,323-327.

DEIBEL, R. H. AND NIVEN, C. F., JR. 1959

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EVANS, J. B. AND NIVEN, C. F., JR. 1951 Nutri-tion of the heterofermentative lactobacillithat cause greening of cured meat. J. Bac-teriol., 62, 599-604.

FELTON, E. A. AND NIVEN, C. F., JR. 1953 Theidentity of "Leuconostoc citrovorum, strain8081." J. Bacteriol., 65, 482-483.

FELTON, E. A., EVANS, J. B., AND NIVEN, C. F.,JR. 1953 Production of catalase by thepediococci. J. Bacteriol., 65, 481-482.

HITCHNER, E. R. AND SNIESZKO, S. F. 1947 Astudy of a microorganism causing a bacterialdisease of lobster. J. Bacteriol., 54, 48.

HORNSEY, H. C. AND MALLOWS, J. H. 1955 Beefcuring brines. II. Bacterial and chemicalchanges occurring in a long-life, acid brine.J. Sci. Food Agr., 6, 705-712.

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JENSEN, E. M. AND SEELEY, H. W. 1954 Thenutrition and physiology of the genusPediococcus. J. Bacteriol., 67, 484-488.

MEES, R. H. 1934 Onderzoekingen over deBiersarcina. Diss. aus dem Lab. A. J. Kluy-ver, Delft.

ORLA-JENSEN, S. 1919 The lactic acid bacteriaM6m. acad. roy. sci. Danemark, Sect. sci.,8 s6r., 5, 81-197.

PEDERSON, C. S. 1949 The genus Pediococcus.Bacteriol. Rev., 13, 225-232.

SAKAGUCHI, K. 1958 Studies on the activitiesof bacteria in soy sauce brewing. III. Taxo-nomic studies on Pediococcus soyae nov.sp., the soy sauce lactic acid bacteria. Bull.Agr. Chem. Soc. Japan, 22, 353-362.

SNIESZKO, S. F. AND TAYLOR, C. C. 1947 Abacterial disease of the lobster (Homarusamericanus). Science, 105, 500.

WILLIAMS, R. E. O., HIRCH, A., AND COWAN, S. T.1953 Aerococcus, a new bacterial genus. J.Gen. Microbiol., 8, 475-480.

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