Vol. 13, No. 5JOURNAL OF CLINICAL MICROBIOLOGY, May 1981, p. 919-9330095-1 137/81/050919-15$02.00/0

Kluyvera, a New (Redefined) Genus in the FamilyEnterobacteriaceae: Identification of Kluyvera ascorbata sp.nov. and Kluyvera cryocrescens sp. nov. in Clinical Specimens

J. J. FARMER III,`* G. R. FANNING,2 G. P. HUNTLEY-CARTER,1 BARRY HOLMES,3F. W. HICKMAN,' C. RICHARD,4 AND DON J. BRENNER'

Enteric Section, Centers for Disease Control, Atlanta, Georgia 30333'; Department of Biochemistry, WalterReed Army Institute of Research, Washington, DC 200122; Computer Identification Laboratory, National

Collection of Type Cultures, Central Public Health Laboratory, London, England3; and Service desEnterobacteries, Institut Pasteur, Paris, France4

Received 12 January 1981/Accepted 23 January 1981

Kluyvera is proposed as a new genus for the group of organisms formerly knownas Enteric Group 8 (synonym = API group 1). Strains of Kluyvera share theproperties of most members of the family Enterobacteriaceae: they are gram-negative rods, motile with peritrichous flagella, catalase positive, and oxidasenegative; they grow on MacConkey agar, ferment D-glucose with the productionof acid and gas, and are susceptible to many antibiotics. Strains are usually indolepositive, methyl red positive, Voges-Proskauer negative, citrate positive, H2S(triple sugar iron) negative, urea negative, phenylalanine deaminase negative,lysine decarboxylase positive, arginine dihydrolase negative, and ornithine decar-boxylase positive. Kluyvera strains ferment many of the sugars and polyhydroxylalcohols used in identification. By deoxyribonucleic acid-deoxyribonucleic acidhybridization, strains of Kluyvera were divided into three groups. Kluyveraascorbata is proposed as the type species for the genus. Most strains of K.ascorbata have been isolated from clinical specimens. K. cryocrescens is proposedas the second species. It was occasionally isolated from clinical specimens, but itwas isolated more commonly from the environment. Kluyvera species group 3was heterogeneous, but was distinct from the two named species by deoxyribo-nucleic acid hybridization. This group was rare, so no species names will beproposed at this time. K. ascorbata can be differentiated from K. cryocrescens byits positive ascorbate test, inability to grow at 5°C in a refrigerator, and smallerzones of inhibition around carbenicillin and cephalothin disks. The test normallyused for identification does not clearly differentiate these two species. Kluyveraspecies are probably infrequent opportunistic pathogens. The most commonsource is sputum, where they are probably not clinically significant. Five strainshave been from blood cultures. More information is needed about the incidenceand clinical significance of the genus Kluyvera.

Since 1965 we have received over 100 strainsof a gram-negative, oxidase-negative, fermenta-tive bacterium which does not fit any of thedefined species in the family Enterobacteria-ceae. Most of these strains were from clinicalspecimens, and originally they were reportedsimply as "unidentified." However, the biochem-ical reactions of the new group were tabulatedin 1977, and it was given the vernacular name"Enteric Group 8" (7, 21). A synonym of EntericGroup 8 is "API group 1," used by AnalytabProducts, Plainview, N.Y. Since 1977 we havereported cultures as Enteric Group 8 and re-quested that other biochemically similar strainsbe sent for further study. In about 1978, one of

us (B.H.) noticed that the biochemical reactionsof Enteric Group 8 were almost identical to agroup of organisms that had been discovered inJapan (2), given the name Kluyvera (4, 5), andlater moved to the genus Escherichia (3).

In 1956 to 1957 Asai and co-workers (4, 5) inJapan proposed the genus Kluyvera for a groupof polarly flagellated bacteria which producedlarge amounts of a-ketoglutaric acid (2) duringthe fermentation of glucose. Five strains wereoriginally studied (2): one from soil and fourfrom sewage. Two species names were proposed(4), Kluyvera citrophila and Kluyvera noncitro-phila, which were based on the difference be-tween the two species in utilizing citric acid as

919

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

920 FARMER ET AL.

the sole source of carbon and energy. Asai andco-workers state (based on their interpretationof the Kluyver and van Niel paper [17]) that thegenus was named (4, 5) to honor A. J. Kluyverwho, with C. B. van Niel, in 1936 postulated (17)that there may be a group of polarly flagellatedorganisms in the tribe Pseudomonadeae whichhave a mixed-acid type of fermentation similarto the genus Escherichia (which was called Bac-terium in the paper). If such a group were to bediscovered, it could be a separate genus, whichwould differentiate it from the genus Aero-monas, which has a butylene glycol-fermenta-tive pathway rather than a mixed-acid pathway(17). Asai et al. (4) thought that they had dis-covered this postulated group of polarly flagel-lated organisms in 1956 to 1957 and named thegroup Kluyvera in honor of A. J. Kluyver for hisprediction and many contributions to microbialmetabolism and physiology. The genus Kluy-vera was classified in the tribe Pseudomona-deae, which at that time included nonfermen-tative genera, but also included the fermentersof the genus Aeromonas. Today, the familyPseudomonadaceae is restricted to bacteriawhich do not ferment glucose.

In 1962, Asai et al. (3) confirmed the obser-vations (unpublished data) of J. M. Shewan andRudolph Hugh (3) that all five of their Kluyverastrains actually had peritrichous rather thanpolar flagella, so they proposed that the twospecies K. citrophila and K. noncitrophila betransferred to the genus Escherichia in the fam-ily Enterobacteriaceae. Thus, the genus Kluy-vera was abolished in 1962 by its original pro-posers, although reports in the literature stilirefer to Kluyvera (6, 16, 19, 20, 22, 25, 26).Kluyvera lost all standing in nomenclature whenthe genus name and the two species names didnot appear on the "Approved Lists of BacterialNames" compiled and edited by Skerman et al.(23). Since 1 January 1980, none of the Kluyveranames has had standing in nomenclature.The purpose of this study was to better define

the group of organisms that we have been re-

porting as Enteric Group 8 and to compare our

strains with strains of Kluyvera. On the basis ofdeoxyribonucleic acid (DNA)-DNA hybridiza-tion, biochemical reactions, and antibiotic sus-

ceptibility, we propose Kluyvera as a new genusin the family Enterobacteriaceae with two spe-

cies, K. ascorbata and K. cryocrescens. Theremainder of this paper discusses the identifi-cation of these organisms which occur in clinicalspecimens.

MATERIALS AND METHODSGeneral. Unless exceptions are given, the following

statements hold throughout this paper: all experi-

ments were done in the Enteric Section, Centers forDisease Control; the temperature of incubation was 36+ 1 C; water refers to glass-distilled water; commercialmedia were used whenever possible (the terms "fromindividual ingredients" or "was made with" appear ifa commercial medium was not used); media weresterilized in an autoclave at 121°C for 15 min; opticaldensity was measured in a Bausch & Lomb Spectronic20 spectrophotometer at 650 nm in disposable glasstubes (13 by 100 mm); filter sterilization was througha 0.22-im nitrocellulose filter; refrigeration was at atemperature of 5 ± 1°C; all results are based oncultures picked twice, each time from a single isolatedcolony; names of enzymes are put in quotation marksunless actual enzyme assays were done; and the term"antibiotic" refers to true antibiotics and to syntheticantimicrobial agents. In most instances organic chem-icals were from Sigma Chemical Co., St. Louis, Mo.,and inorganic chemicals were Baker Analyzed Reagentgrade and were from J. T. Baker Chemical Co., Phil-lipsburg, N.J.Nomenclature. The names used are from the "Ap-

proved Lists of Bacterial Names" (23). In addition, weuse Citrobacter amalonaticus as a synonym of Levi-nea amalonatica. The genus Kluyvera is proposed inthis paper as a new (redefined) genus in the familyEnterobacteriaceae. The genus Kluyvera has two newnamed species, K. ascorbata and K. cryocrescens, anda third unnamed group, Kluytera species group 3 (seeResults).

Strains. As of 1 November 1980 we had identified144 strains as Kluyvera species. For this study 100strains were examined (in 1978 to 1980) in more detail:78 strains of K. ascorbata, 17 strains of K. cryocres-cens, and 5 strains of Kluyvera species group 3. Theirsources are given in Table 1. Several strains, includingthe five original isolates of Asai and co-workers, areimportant for historical or nomenclatural reasons, somore information about them is given in Table 2.

Stock cultures. A single colony was picked from a24-h-old Trypticase soy agar (BBL Microbiology Sys-tems, Cockeysville, Md.) plate, which had beenstreaked to give good isolated colonies. This step wasdone at least twice. The streak plates were then keptfor 14 to 28 days at room temperature before theywere discarded. Stock cultures were stored on Tryp-ticase soy agar (BBL) or blood agar base (Difco Lab-oratories, Detroit, Mich.) in sealed tubes (in the darkat room temperature) and have remained viable for 1to 10 years without transfer.DNA-DNA hybridization. The genetic related-

ness of strains was determined by DNA-DNA hybrid-ization on hydroxyapatite with 12P-labeled DNA.Reassociation was often done at both 60 and 75°C,but, unless otherwise indicated, all data refer to reas-sociations done at 60°C. The methods used have beendescribed by Brenner et al. (8, 24), and the originalpapers should be consulted for technical details toolengthy to repeat here. Closely or highly related strains(that is, belonging to the same species) usually have arelatedness (based on relative binding of the DNAs)of 70% or more (occasionally down to 60%) whenreassociation is done at 60°C (and relatedness of 50%or more when reassociation is done at 75°C). Species

J. CLIN. MICROBIOL.

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

IDENTIFICATION OF KLUYVERA 921

TABLE 1. Sources of 100 Kluyvera strains

K. ascorbata K. cryocrescens Kluyvera species group 3Source (78 strains) (17 strains) (5 strains)

Human clinical specimens:Sputum 28 2 2Urine 1i 2Stool 8Throat 5 1 2Blood 1 1Other, or not given 8 1 1

Water 1Sewage 1 3Soil 1Milk 1Hospital sink 1Cow 1Unknown 13 5

TABLE 2. Important strains of Kluyvera

Species Previously CDC ATCC Sourcedesignated number numberKluyvera ascorbata Enteric Group 8 0648-74 33433 Human, sputum, North CarolinaKluyvera cryocrescens Enteric Group 8 2065-78 33435 Kitchen food, Persian GulfKluyvera ascorbata K. citrophila 0408-78a 14236 Sewage (Asai's designation = 6, Beta)Kluyvera cryocrescens K. citrophila 0409-78a 14237 Soil (Asai's designation = 84C, Alpha)Kluyvera cryocrescens K. citrophila 0410-78a 14238 Sewage (Asai's designation = il, Gamma)Kluyvera cryocrescens K. noncitrophila 0411-78a 14239 Sewage (Asai's designation = 4)Kluyvera cryocrescens K. noncitrophila 0412-78a 14240 Sewage (Asai's designation = 10)Kluyvera ascorbata Enteric Group 8 2221-78 33434 Human ?, ?, New York

aThese cultures were studied twice at CDC. Numbers were assigned in 1961 also: 0408-78 = 2567-61, 0409-78 =2568-61, 0410-78 = 2569-61, 0411-78 = 2570-61, 0412-78 = 2571-61.

are considered to belong in the same genus if they aremore closely related to each other than to species inother genera and if they are similar in their phenotypicproperties.G+C content of DNA. The moles percent of gua-

nine plus cytosine (G+C) was determined by the buoy-ant density method (cesium chloride centrifugation)in the laboratory of Manley Mandel, M.D. AndersonHospital, Houston, Tex., from unsheared DNA pre-pared by A. G. Steigerwalt. Values were determinedtwice in the presence of reference DNAs from Bacillussubtilis bacteriophage 2C (density, 1.742 g/cm3). Be-cause of the variables in the technique, the results ofG+C content are sometimes rounded to two significantfigures.

Biochemical tests used for identification. Bio-chemical tests were basically done according to themethods of Edwards and Ewing (10); modificationsand more complete documentations have been givenin considerable detail in more recent publications (12-14). The biochemical reactions of the 100 Kluyverastrains were done twice, first by the standard tubemethod (10) as they arrived at the Enteric Sectionduring the period 1969 to 1980. They were then allretested in 1979 to 1980 in glass tubes (100 by 13 mm)by using the 72-prong multi-inoculator shown in Fig.

1. This later testing was done with the same mediaand was essentially the same as the standard testingin the Enteric Section; however, the advantage wasthat all strains could be compared at the same pointin time. Two racks, or a total of 144 tubes, were usedfor each test so that all 100 strains could be studied atone time.The following tests were done by C. Richard at the

Institut Pasteur. "Beta-xylosidase" was tested by themethod of Brisou et al. (9) which depends on theformation of yellow p-nitrophenol from colorlessp-nitrophenyl-,B-D-xylopyranoside. "Beta-glucuroni-dase" was tested in a similar manner by the method ofKilian and Bulow (15), but with p-nitrophenyl-,f-D-glucuronide as the colorless substrate. "Tetrathionatereductase" was tested by the method of LeMinor andPichinoty (18) and is based on acid production in apeptone medium from tetrathionate, which is reducedto thiosulfate with the production of HW."Ascorbate" test. The ascorbate test is a new test

which was devised to differentiate the two namedKluyvera species. Ascorbate medium 7795 (a numberis given to all media used in the Enteric Section)contains 10 g of peptone, 3 g of meat extract, 5 g ofNaCl, 10 g of L-ascorbic acid-sodium salt, and 0.004 gof bromothylmol blue (Difco furnished the first three

VOL. 13, 1981

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

922 FARMER ET AL.

__

FIG. 1. A 72-prong multi-inoculator (fabricated in the Centers for Disease Control machine shop), polypro-pylene 72-hole test tube rack (Scientific Products, McGraw Park, Ill.), polypropylene instrument tray (Bel-ArtProducts, Pequannock, N.J.) used as a top to prevent airborne contamination of tubes, and polypropyleneautoclave bag (York Scientific Inc, Ogdensburg, N. Y.) used to cover racks during incubation to preventcontamination.

ingredients as "Enteric Fermentation Base" no. 836-01, which can be purchased by anyone, even though itdoes not appear in the Difco catalog). All ingredientsare dissolved in 900 ml of water, and 0.1 N NaOH isadded until pH 7.5 is reached (dark blue in color).Water is added to make the final volume 1,000 ml. Themedium is then dispensed into 16- by 125-mm tubes(10 ml each), and a Durham tube is added. A plasticcap is added, and the medium is autoclaved. As soonas the tubes are removed from the autoclave, 4 ml ofsterile mineral oil is aseptically added to each tube.The medium is stored immediately in the refrigeratorand discarded if the pH drops to below 6.8 (green incolor), or 0.1 N NaOH can be added to change thecolor back to light blue (pH 7.4). The ascorbate test isinoculated with about 104 to 105 organisms (for exam-ple, 0.001 ml of a 24-h-old heart infusion broth culture)under the mineral oil. A positive reaction is the pro-duction of acid (pH to below 6.0; color change fromblue to yellow) and usually gas (for those strains whichproduce gas during fermentation). Strains which arenegative usually lower the pH to around 6.5 to 6.8(green to green-yellow) but do not produce gas. Acontrol tube of uninoculated ascorbate medium mustbe included in the incubator since the pH of uninocu-lated medium drops. The drop in pH of the uninocu-lated medium is much faster at 36°C than in therefrigerator.Glucose fermentation at 5°C. Peptone water

with Andrade indicator is satisfactory for this test, orthe ascorbate medium referred to above can be used(except 10 g of D-glucose is substituted for the 10 g ofL-ascorbic acid-sodium salt, the final pH is adjusted to7.6 before autoclaving, and the mineral oil overlay isomitted). The tube is inoculated with about 104 to 105organisms and "incubated" in a refrigerator main-tained at 5 + 1°C. The tubes are observed for 3 weeksfor the production of acid. A positive test is growth

and glucose fermentation to a pH of <6.0 within 21days.

Susceptibility to antibiotics-disk method.The standardized single-disk method (Kirby-Bauer)was used (13), and the zones of complete inhibitionaround the antibiotic disks (BBL) were measured.Table 3 gives the antibiotic disks used, potencies, andabbreviations. Conversion of the zone sizes into "sus-ceptible" (or "sensitive"), "intermediate," and "resist-ant" was based on the zone size interpretative chartsupplied with the disks. In addition, the zone of "anyinhibition" was measured around the carbenicillin disk(100 ,ug) and cephalothin disk (30 gg). This was definedto be twice the distance from the center of the disk tothe area of complete growth with no inhibition. Colo-nies in the zone were ignored in measuring this zoneof any inhibition which is different from the usualmeasurement which considers these resistant colonies.This zone of any inhibition was found useful in differ-entiating the two Kluyvera species.

Susceptibility to antibiotics-minimum inhib-itory concentrations in Mueller-Hinton broth.The Sensititre-Gram Negative Plate (made by SewardLaboratory, London, England; purchased from GIBCODiagnostics, Lawrence, Mass.) was used according tothe manufacturer's instructions. Strains were pickedfrom 24-h Trypticase soy agar plates and inoculated(about 106 ceils) into 0.5 ml of brain heart infusionbroth. They were grown for 4 to 6 h (to about 109 cellsper ml), and a 0.001-ml loopful was transferred to 10ml of Mueller-Hinton broth (GIBCO) to give about105 bacteria per ml. Then 0.05 ml of this dilutedsuspension was added with an eight-channel dispensor(GIBCO) to each of 96 wells of a disposable plasticplate containing the dehydrated antibiotics (13). Afterovernight incubation the wells were observed for tur-bidity.Requirements for vitamins or amino acids. To

J. CLIN. MICROBIOL.

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

IDENTIFICATION OF KLUYVERA 923

TABLE 3. Disk-diffusion, antibiotic susceptibility of Kluyvera

K. ascorbata (73 strains) K. cryocrescens (16 strains)Antibiotic Stnar tadrnMean Standard % susceptible Mean Standard % susceptibledeviation deviation

Colistin (10)a 12.2b 0.9 99 12.5a 0.6 100Nalidixic acid (30) 19.5 3.1 63 22.1 2.5 94Sulfadiazine (250) 18.9 3.5 78 22.4 3.9 94Gentamicin (10) 19.1 1.5 100 20.2 2.6 100Streptomycin (10) 15.0 1.9 65 16.1 2.5 69Kanamycin (30) ' 19.2 1.4 95 20.3 2.5 94Tetracycline (30) 19.9 2.5 85 22.0 1.9 100Chloramphenicol (30) 24.2 3.2 97 25.5 5.7 94Penicillin G (10 U) 6.3 1.2 0 6.9 2.3 0Ampicillin (10) 9.3 3.1 7 11.4 3.7 25Carbenicillin (100) 12.0 3.8 4 16.9 5.2 13Cephalothin (30) 12.9 2.8 6 15.8 3.1 31

aFigures in parentheses are potencies of the disks in micrograms (or units if the number is followed by a U).bThe mean value of the zones of complete inhibition

avoid any contamination by organic matter, only newglassware was used. Growth on D-glucose as the solesource of carbon and energy was determined in Cen-ters for Disease Control medium 7727H, which con-tained 3 g of Na2HP04, 2 g of KH2PO4, 0.5 g of NH4C1,0.5 g of (NH4)2S04, 0.05 g of MgSO4.7H20, 0.01 g ofFeSO4.7H20, 0.01 g of CaCl2.2H20, 0.1 g of ethylene-diaminetetracetic acid-disodium salt, 0.004 g of brom-othymol blue, and 1 g of D-glucose, all in 1,000 ml ofwater. The final pH was 6.8. This medium was filtersterilized, and 4 ml was dispensed into sterile dispos-able borosilicate glass tubes (100 by 13 mm). Eachtube was inoculated with 0.001 ml (72-prong inocula-tor) of a 24-h heart infusion broth culture. Serialtransfers (0.001 ml into 4 ml of fresh medium) weremade at 1- to 3-day intervals for seven transfers (es-timated to be about 70 cell divisions since the originalinoculum) to eliminate any nutrients carried over fromthe original heart infusion broth inoculum. If a culturegrew and fermented glucose for all seven transfers, weconsidered it able to grow on glucose as the sole sourceof carbon and energy. A vitamin requirement wasestablished if the culture would not grow for seventransfers in medium 7727H, but would grow throughseven transfers (0.001 ml into 4 ml of fresh mediumeach time) of medium 7727H supplemented with thefollowing vitamins: 0.813 mg of thiamine, pyridoxine,pyridoxamine dihydrochloride, pyridoxal hydrochlo-ride, pantothenic acid (hemi-calcium salt), riboflavin,nicotinamide, niacin, p-aminobenzoic acid, folic acid,lipoic acid, and glutathione per liter; and 0.0813 mgeach of biotin, cyanocobalamine (vitamin B12), andmenadione per liter.

Lactose fermentation, the "coliform" concept,and the "fecal coliform" concept. These tests weredone according to instructions in the 14th edition ofStandard Methods for the Examination of Waterand Wastewater (1), except that pure cultures wereused. Each tube was inoculated with 103 to 104 orga-nisms grown in brain heart infusion broth (exceptbrilliant green bile broth and EC medium, which were

inoculated with 0.001 ml of a 48-h-old culture in lauryltryptose broth as required in Standard Methods). Theinsert tubes were observed for gas information; thetubes were gently shaken, and the presence or absenceof gas bubbles was noted.

RESULTSNomenclature. On 1 January, 1980, the fol-

lowing names lost standing in nomenclature: thegenus Kluyvera Asai, Okumura, and Tsunoda1957; the species Escherichia ("Kluyvera") ci-trophila (Asai, Okumura, and Tsunoda 1957)Asai, Iizuka, and Komagata 1962; and the speciesEscherichia ("Kluyvera") noncitrophila (Asai,Okumura, and Tsunoda 1957) Asai, lizuka, andKomagata 1962. The species definitions even-tually proposed in our study did not correlatewith the ability to utilize citric acid as postulatedby Asai and co-workers (4, 5) so we thought itunwise to "revive" the names "citrophila" and"noncitrophila" (see reference 23, p. 230). Toavoid confusion we felt it best to propose thegenus name Kluyvera, but without either speciesname used previously. Enteric Group 8 and APIgroup 1 are synonyms of Kluyvera. When wemade a preliminary proposal on Kluyvera (G. R.Fanning, J. J. Farmer III, J. N. Parker, G. P.Huntley-Carter, and D. J. Brenner, Abstr. Annu.Meet. Am. Soc. Microbiol. 1979, I30, p. 100), wewere sure that the two species names wouldappear on the "Approved Lists of BacterialNames" (23) since they were validly publishedand were represented by type strains. They werenot, however, included on the Approved Lists.In this article, we propose Kluyvera gen. nov. asa new genus in the family Enterobacteriaceaewith two species, K. ascorbata sp. nov. (pronun-ciation = ah skohr bah' tah; holotype strain =

VOL. 13, 1981

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

924 FARMER ET AL.

American Type Culture Collection [ATCC]33433) and K. cryocrescens sp. nov. (pronuncia-tion = cry oh kress' enz; holotype strain = ATCC33435). Kluyvera is a Latinized form of Kluyver,after the late A. J. Kluyver, who many considerto be the father of microbial physiology. Thegenus name is treated as a modern (neo) Latinfeminine noun. The species name "ascorbata"is derived from the modern chemical term"ascorbate" designating the salt of ascorbic acid."Ascorbate" is derived from the modern Latinword "ascorbic," which is derived from "a"(Greek, for "negative") and "scorbutus" (Latin,for "scurvy"). The intended meaning is "theascorbate Kluyvera," or less literally, "the Kluy-vera which has a positive ascorbate test." Thespecies name "cryocrescens" is derived from"kryos" (Greek noun, for "cold") and from "cres-cens" (Latin feminine participle, for "growing").The intended meaning is "the Kluyvera whichgrows in the cold," because K. cryocrescens cangrow and ferment glucose at 5°C. Both speciesnames are treated as modern Latin feminineadjectives which agree in gender with the femi-nine generic name Kluyvera. The type speciesfor the genus Kluyvera is K. ascorbata. Severalcultures of Kluyvera did not fit K. ascorbata orK. cryocrescens. We propose the vernacularname Kluyvera species group 3 for them. Noscientific name(s) will be proposed until morestrains become available.DNA-DNA hybridization. Table 4 and Fig.

K. SPECIES 3

K. ASCORBATA E

K. CRYOCRESCENS

2 show that Kluyvera is made up of three hy-bridization groups. Strain 408-78 was more than80% related to 26 strains and was 69 and 77%related to 2 other strains. This group of 29 strainswhich was highly related to strain 408-78 wasinitially called "Kluyvera hybridization group 1"and was eventually named K. ascorbata. Strain409-78 was related by 75% or more to nine otherstrains. This group of 10 strains was initiallycalled "Kluyvera hybridization group 2" andevantually was named K. cryocrescens. (Thename Enteric Group 12 has been used to reportthese cultures.) Five other strains were 60 to68% related to both K. ascorbata and K. cry-ocrescens. These probably represent one ormore additional species of Kluyvera. The ver-nacular name Kluyvera species group 3 will beused until more strains become available forstudy. (The names Enteric Group 36 and EntericGroup 37 have been used to report these cul-tures.) Figure 2 shows that all three Kluyveragroups were much more closely related to eachother than to other genera of Enterobacteria-ceae. This closer genetic relatedness and strikingphenotype similarity argue that Kluyvera is adistinct genus in the family Enterobacteriaceae.The closest relatives of Kluyvera species werespecies of the genera Klebsiella, Enterobacter,and Citrobacter.G+C content of DNA. The moles percent of

G+C were as follows: K. ascorbata ATCC 33433= 56.1 (buoyant densities, 1.715 and 1.715 g/

EEIPROTEUS

E[EDWARDSIELLA

EMORGANELLA

EEE- ERWINIA

E SALMONELLA

IJ KLEBSIELLA

m t *0 est* .e ENTEROBACTER

ErE. CITROBACTER

.E* *ESCHERICHIA-SHIGELLA

E1 HAFNIA

E--EJ YERSINIA

E PROVIDENCIA

100 90 80 0 6o 5 40 30 20 10 O% RELATEDNESS

FIc. 2. Relatedness by DNA-DNA hybridization of Kluyvera to other genera in the familyy Enterobacteri-aceae. The other strains were compared to strain ATCC 14236 of K. ascorbata labeled with 32P. Percentrelatedness is based on the percent DNA reassociation at 60°C. The spatial location on the undesignatedvertical axis is arbitrary.

J. CLIN. MICROBIOL.

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

IDENTIFICATION OF KLUYVERA 925

TABLE 4. Identification of 44 Kluyvera strains by DNA-DNA hybridization

% Relatedness of test strain to reference strain:

Test strain K. ascorbata 0408-78 K. cryocrescens 0409-78

600C Divergence 75°C 600C Divergence 75°C

K. ascorbata

K. cryocrescens

K. species group 3

looa100949493939292929191919090909090909089898989898784847769

Oa looa1 952 861 882 861 852 861 841 861 881 881 891 832 951 891 901 951 891 851 861 850 851 841 911 881 825 807 626 65

62 12 4160 10 4469 il 4665 il 4361 il 3968 il 4765 12 4465 11 4366 11 4462 11 42

68 9 5366 11 4963 10 4562 11 4562 10 43

65 il 4569 12 4472 12 3865 12 3671 12 4766 12 3568 13 4166 il 4666 11 4568 il 4567 11 4063 11 4366 il 4467 12 4263 12 4064 11 4569 il 4670 10 5064 12 4465 12 4565 1 1 4360 12 4463 il 4467 12 4563 11 4663 il 4266 10 4966 il 4455 il 38

100969488868080807875

0 1000 1031 930 871 888 698 649 647 618 63

64 il 4367 12 4361 il 4060 il 4961 11 42

aThe first value is the % relatedness of the reference strain to the test strain when the reassociation is done at

600C. The second value is the percent divergence (synonym = ATm) which is a measurement of the relatednessof the DNA nucleotide sequences of the pair of strains. The larger the value of the divergence, the less related thestrains are (fox each percent of unpaired bases, divergence will increase by about 1°C). The third value.is the %relatedness of the reference strain to the test strain when the reassociation is done at 75°C.

cm3), K. ascorbata 1058-74 = 56.6 (buoyant den-sities, 1.715 and 1.716 g/cm3), K. cryocrescensATCC 14237 = 55.1 (buoyant densities, 1.714

and 1.714 g/cm3) and K. cryocrescens 0546-78= 55.1 (buoyant densities, 1.714 and 1.714 g/cm3).

0408-781322-752589-700972-781402-742221-781058-740412-752439-760842-743214-752070-780573-740648-744417-740204-750055-762892-763198-773858-694450-740720-761432-761153-770762-751220-732064-782066-780556-74

0409-78331 7-752065-780412-780411-781396-730410-780546-782063-781734-74

2774-702065-762891-764246-743108-76

VOL. 13, 1981

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

TABLE 5. Biochemical reactions for 100 strains of Kluyvera

Cumulative % positive at: Reactions for type strains:Test K. ascorbata K. cryocrescens

24h 48h 7d ATCC 33433 ATCC 33435

Indole production 83 89 ND +a +Methyl red 100 100 ND + +Voges-Proskauer 0 0 ND - -Citrate (Simmons') utilization 93 97 97 + +H2 S production (TSIb or PIAb) 0 0 0Urea-Christensen's O O O -Phenylalanine "deaminase" 0 ND NDLysine "decarboxylase" - Moeller's 87 87 87 +Arginine "dihydrolase" - Moeller's 0 0 2Ornithine "decarboxylase" - Moeller's 100 100 100 + +Motility 94 97 97 + +Gelatin liquefaction at 22°C 0 0 0KCN, growth in 93 95 96 + +Malonate utilization 3 94 94 +2 +

D-Glucose-acid production 100 100 100 + +D-Glucose-gas production 81 83 83 + +Acid production from:

Lactose 96 98 99 + +Sucrose 99 99 99 + +D-Mannitol 99 99 99 + +Dulcitol 27 29 29 - -Salicin 100 100 100 + +Adonitol 0 0 0 - -i-(myo) Inositol 0 0 0 - -D-Sorbitol 41 41 41L-Arabinose 99 100 100 + +Raffinose 98 98 98 + +L-Rhamnose 100 100 100 + +Maltose 100 100 100 + +D-Xylose 97 98 98 + +Trehalose 100 100 100 + +Cellobiose 92 100 100 + +a-Methyl-D-glucoside 97 98 98 + +Erythritol 0 0 0Melibiose 97 98 98 + +D-Arabitol 0 0 0Glycerol 13 15 95 +7 +7D-Mannose 100 100 100 + +D-Galactose 100 100 100 + +

Mucate - acid production 95 96 99 + +Tartrate Jordan's 0 0 0Esculin hydrolysis 100 100 100 + +Acetate utilization 19 69 100 +3 +3

Citrate - Christensen's 94 95 98 + +Lipase (corn oil) O O O"Deoxyribonuclease" - 25°C or 36°C O O O°Oxidase - Kovacs' O ND NDN03-. N02 100 ND ND + +ONPG test 100 100 100 + +Pectate hydrolysis 0 0 0Pigment production O oC ocTyrosine clearing 0 0 0

GSymbols: ND = Not Done, + = Positive at 24h,-= Negative at end of the incubation period, +2 = positive atday 2 (the superscript gives the day the reaction became positive).bAbbreviations: TSI = Triple Sugar Iron Agar, PIA = Peptone Iron Agar, ONPG = O-Nitrophenyl-p-D-galactoside.c4% of the strains produced red-blue crystals in and around the colonies with prolonged incubation.

926 FARMER ET AL. J. CLIN. MICROBIOL.

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

IDENTIFICATION OF KLUYVERA 927

Biochemical reactions. Table 5 gives thebiochemical reactions of Kluyvera in the testsoften used in American clinical laboratories foridentification. The strains were very uniform intheir reactions and were distinct from any otherdescribed species of Enterobacteriaceae, whichwas the original reason we reported them as

unidentified and later defined Enteric Group 8.The biochemical reactions for the type strainsof K. ascorbata (ATCC 33433) and K. cryocres-cens (ATCC 33435) are also given in Table 5.Description ofKluyvera. A brief description

of the genus Kluyvera would include: gram-neg-ative rods, motile with peritrichous flagella(however, arrangement of flagella may be inter-preted as polar; see reference 3), oxidase nega-

tive, catalase positive, nitrate reduced to nitrite,D-glucose and other carbohydrates fermentedwith the production of acid and usually gas,large amounts of a-ketoglutaric acid producedduring fermentation (2), G+C content of DNAof 55 to 57 mol% (buoyant density), and isolatedfrom soil, water, sewage, food and human clinicalspecimens. A further description of the genusand its two species is given in the tables, figures,and text. The type species is K. ascorbata.Differentiation ofK. ascorbata andK cry-

ocrescens. None of the 50 tests routinely usedfor identifying members of the family Entero-bacteriaceae (12-14) correlated exactly with thetwo distinct groups obvious after DNA-DNAhybridization. Other tests were therefore tried.Two of these, the ascorbate test and glucosefermentation at 5°C, correlated almost exactlywith the hybridization groups (Table 6). Otherbiochemical tests which correlated somewhatwith the hybridization data are also listed.Strains which were indole negative or lysinenegative or which did not produce gas were morelikely to be K. cryocrescens than K. ascorbata.A total of 45% of the K. ascorbata strains fer-mented dulcitol, but none of K. cryocrescensstrains did.Differentiation of Kluyvera from other,

similar members of the family Enterobac-teriaceae. Table 7 gives the tests (results fromtests done at the Institut Pasteur) which shouldprove useful in differentiating Kluyvera speciesfrom other indole-positive, Voges-Proskauer-negative species in the family. Kluyvera is usu-

ally positive in Moeller's lysine "decarboxylase"and is raffinose positive. Few other indole-posi-tive groups share these properties. The test forfB-xylosidase, which is often used in France, mayalso prove useful in identifying Kluyvera species.Biogroups. Although the biochemical reac-

tions of Kluyvera were fairly uniform, there wassome variation among the strains (Table 5). Thisvariation allows the definition of biogroups,which are defined and summarized in Tables 8and 9 (gas- refers to gas production from D-

glucose). The biogroups should be helpful inidentifying newly isolated strains and can beused as markers to study the ecology and epi-demiology of Kluyvera.Production of blue and red crystals. As

previously mentioned, streak plates on Trypti-case soy agar were held at room temperature forseveral weeks before they were discarded. It wasnoted that occasional colonies of four strainsturned blue-purple with time; typical coloniesare shown in Fig. 3. With magnification (X10 tox60 with a dissecting microscope), a mixture oftiny red and blue crystals was noted in andaround the colonies which appeared blue. Wepropose the name "kluyveramycin" for this mix-ture of blue and red crystals in the hope that itmay have a use in microbiology or medicine.Strain 2221-78 of K. ascorbata was a strongproducer of kluyveramycin and it was depositedin the ATCC as no. 33434.Antimicrobial susceptibility. Kluyvera

was generally susceptible to antibiotics. Table 3gives the mean zones of inhibition and theirstandard deviations for both species. Early inthe study it was apparent that K. cryocrescensoften had large zones of inhibition (often withcolonies in the zones) around the disks for car-

TABLE 6. Differentiation ofK. ascorbata and K. cryocrescens with biochemical tests

Test K. ascorbataa K. cryocrescens a

Ascorbate test +b 97%b _ 09D-Glucose-acid (5°C, 21 d) 3% + 100%Lysine "decarboxylase" - Moeller's + 100% V 50%0D.Glucose-gas G 86% V 50%Dulcitol-acid V 45% - 0%

aBased on the 29 strains of K. ascorbata and 10 strains of K. cryocrescens definitively identified by DNA-DNAhybridization.bThe percent positive at 48h, 36°C unless otherwise stated, + = 90-100% positive, ®) = 75-89% positive, V26-74% positive, ® = 11-25% positive, - = 0-10% positive.

VOL. 13, 1981

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

TABLE 7. Differentiation of indole+, Voges-Proskauer- species ofEnterobacteriaceae isolatedin clinical microbiology laboratories

Reaction for:

~~~~~~~~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~.

Test

Indole production +a + + G + +Citrate-Simmons' - + + + +Lysine "decarboxylase" - Moeller's G - - -_Arginine "dihydrolase" - Moeller's - - -Ornithine "decarboxylase" - Moeller's V + + +KCN-growthin - - + + G VMalonate - + - + G VYellowpigment - - - - G G"frXylosidase" - - - + V V"p-Glucuronidase" G - - - - -"Tetrathionate reductase" - + - t9 VD-Glucose-gas production G + + G +Acid production from:

Lactose + - - + VAdonitol - + - - QL-Sorbose Q - +D-Sorbitol + + + V V VDulcitol V V - V V VSucrose V V - +Salicin V - - + V VRaffinose Q _ _ +Melibiose G - - + G V

a+ = 90-100% positive, G = 75-89% positive, V = 26-74% positive,-= 11-25% positive,Q)= 0-10% positive.Data are based on reactions which occur within 48 hours (Institute Pasteur's data).

TABLE 8. Biogroups of Kluyvera ascorbata

Number of Fermentation of: Other phenotypic characteristicsBiogroup strains Dulcitol D-Sorbitol which differ from the wild type

1 37 -a2 18 + +3 5 +4 4 + + Indole b5 2 - - Gag6 2 - - Lysine77 2 + + Gas , KCN8 1 - - Mucate9 t - - o-methyl-D-glucoside10 1 - - Citrate, inotility~l l I - - Raffinose~, melibiose~l 2 I - - Indole , lysine , motility13 1 + + Gas-, malonate-14 l + + Indole, gas, lactose-15 1 _ + Indole6, gas-

aSymbols: + = positive within 2 days, - = negative at 2 days.bNegative reactions are for the end of the incubation period.

928 FARMER ET AL. J. CLIN. MICROBIOL.

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

IDENTIFICATION OF KLUYVERA 929

TABLE 9. Biogroups of Kluyvera cryocrescens

Number of Reactions For: Other phenotypic characteristicsBiogroup strains Lysine D-Sorbitol Gas which differ from the wild type

1 3 a ++2 2 - - -

3 2 - - +4 1 + + + Citrate-, KCN-b5 i + + + Motility-, KCN-6 1 - - - Indo!e, D-Xylose7 1 - Citrate, ONPG8 1 + + -9 1 + _ _ Indole, D-Mannitol, a-methyl-D-glucoside-10 1 - + -i1 1 - + +1 2 1 - - + Malonate1 3 1 - - + Indole , D-Xylose

aSymbols: + = positive within 2 days, - = negative at 2 daysbNegative reactions are for the end of the incubation period

" §< .: x^a sE Dss

.;.Xe. X-X°B. k i ilg 11 b: ` E 1E | . . .. .....| i * i ......| | F P ' ' . e:. , . j ,e ,., : `,,*' : : ,: a! .... ' j.'FIG. 3. Kluyveramysin crystals in and around the colonies ofK ascorbate ATCC 33434.

benicillin and cephalothin. Before the ascorbatetest and glucose fermentation at 5°C were de-veloped, these large zones on the antibiogramplate were the only way to differentiate the twospecies. Figure 5 shows the zones of completeinhibition (see Materials and Methods for thedefinition of this term) against carbenicillin andcephalothin. Two distinct groups are formed

when these zones are plotted (Fig. 4), and only2 of the 39 strains gave results which did notagree with their species assignment based onDNA-DNA hybridization. The difference inthese zone sizes (Fig. 4) provides an additionalway to differentiate K. ascorbata and K. cry-ocrescens. Table 10 gives the minimum inhibi-tory concentrations (broth dilution) of Kluyvera

VOL. 13, 1981

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

930 FARMER ET AL.

K. cryocrescens required one or more vitamins.Coliform concept. In the test for lactose

fermentation used for identification (enteric fer-mentation base with Andrade indicator and 1%filter-sterilized lactose), 90% of the Kluyverastrains fermented lactose with the production ofacid and gas. At 36°C, 89% of the strains alsoproduced gas from lactose in the liquid mediaused in coliform analysis. In the confirmatorytest, 87% produced gas in brilliant green bilelactose broth. At 45°C in EC medium, however,none of the strains formed gas. Thus, Kluyveracan be considered as "coliform," but not a "fecalcoliform."

6 0 15 20ZONE (mm) CARBENICILLIN

25 30

FIG. 4. Differentiation ofK ascorbata and K cry-ocrescens based on the zones of complete inhibitionaround carbenicillin (100-gg disk) and cephalothin(30-lig disk).

FIG. 5. Zones of inhibition around cephalothin(30-pg, disk, right) and carbenicillin (100-gg disk,left). Top strain is K cryocrescens ATCC 33435; bot-tom strain is K ascorbata 1402- 74.

in the Sensititre system. There was little if anydifference among the three Kluyvera groups, sothey were combined for this summary.Requirement for vitamins or amino

acids. Twenty-seven of the 29 strains of K.ascorbata grew through seven transfers on glu-cose as the sole source of carbon, as did 5 of 10strains of K. cryocrescens and 5 of 5 strains ofKluyvera species group 3. All of the strains grewthrough seven transfers when trace amounts of15 vitamins were added to the glucose-basal saltsmedium. Thus, it appears that none of the Kluy-vera strains required an amino acid, but thattwo strains of K. ascorbata and five strains of

DISCUSSIONThe genus Kluyvera has had a turbulent his-

tory since it was first proposed by Asai and hisco-workers in 1956. Few workers were familiarwith the genus, because it had not been includedin Bergey's Manual of Determinative Bacteri-ology or in other standard reference books. Someinvestigators have tested the Kluyvera strains,but not accepted them as new species. The fivestrains deposited in the ATCC (ATCC 14236 to14240) were examined by Ewing (10, p. 278) whoconcluded, "One of these (84C) proved to be a

strain of C. freundii (indole produced, hydrogensulfide not produced)." No comment was madeabout the identity or taxonomic position of theother four strains (10). To complicate mattersfurther, the original proposers of the genustransferred the two named species to the genusEscherichia (3). However, reports in the litera-ture continue to use the name Kluyvera (6, 16,17, 19, 20, 22, 25, 26), which is why we thoughtit best to repropose it rather than coin a new

genus name. We were fortunate that two of us

(J.J.F. and B.H.) were doing a collaborativeproject on the identification of new groups ofEnterobacteriaceae. The relationship betweenEnteric Group 8 and Kluyvera was discoveredbecause the computer programs for identifica-tion at the Computer Identification Laboratoryat Colindale contained Kluyvera as a namedgroup. Otherwise the identity of Enteric Group8 and Kluyvera might have gone undetected.Almost ail of the Kluyvera strains were sentbecause the original laboratory observed thattheir isolate did not fit any of the existing speciesin the family Enterobacteriaceae (7, 21). Severalof the strains were sent as "E. coli citrate posi-tive??"; others were referred as "Enterobacter?"or "Citrobacter?" Our first impression afterstudying the biochemical reactions of the new

group was that it was a Citrobacter-Enterobac-ter "intermediate" (7). All these vernacular(common) names can now be dropped in favor

K.

30-

25-zmb-

o

120a-

E

z

10

K.

J. CLIN. MICROBIOL.

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

IDENTIFICATION OF KLUYVERA 931

TABLE 10. Minimum inhibitory concentrations for 9 antibioticsa

Cumulative percent ot strains inhibited atantibiotic concentration (pig/ml) of:

Antibiotic.25 .5 1 2 4 8 16 32 64 128 256 512

Ampicillin 4 1 2 31 50 8,bCarbenicillin 4 12 15 31 42 77 81Cephalothin 4 12 35 58 81 96Amikacin 12 69 92 100Gentamicin 50 96 100Kanamycin 23 81 96 100Tobramycin 35 92 96 100Chloramphenicol 4 19 62 92 96 96 96Tetracycline 4 15 50 81 88 88 96

aBased on 10 strains each of K. ascorbata and K. cryocrescens and 6 of Kluyvera species group 3.bjf the last value is not 100, the test system did not include any higher concentrations of antibiotic.

of the scientific name Kluyvera.Our data indicate that Kluyvera is a new

genus in the family Enterobacteriaceae. Bio-chemically, Kluyvera is distinct from all othernamed genera, and it is also distinct by DNA-DNA hybridization (Fig. 2). Our concept of agenus is a group of distinct species which aremore closely related to each other by DNAhybridization than they are to other species orgenera. A new genus should also be phenotypi-cally distinct from other genera, and ail speciesin the genus should be similar enough so that agenus definition can be logically formed. Ideally,the species should be phenotypically distinctfrom each other, so that species definitions canbe easily formed. Kluyvera is a good genus basedon the first criterion, but the two named speciesare phenotypically very similar.The two species of Kluyvera were initially

very hard to differentiate. None of the 47 bio-chemical tests routinely done in the EntericSection at the Centers for Disease Controlclearly differentiated the two groups which wereformed by DNA hybridization. For over a yearthe only tests that showed a good correlationwere the zones of complete inhibition aroundthe antibiotic disks carbenicillin and cephalo-thin. K. cryocrescens had large zones. often withresistant colonies, but K. ascorbata had muchsmaller zones. Finally, two characteristics, theascorbate test and glucose fermentation at 50C,were found which correlated almost exactly withthe DNA hybridization data. Clinical laborato-ries should be able to recognize a strain of Kluy-vera because of its differences from the othernamed groups in Enterobacteriaceae. Theascorbate test is recommended for those whowish to differentiate the two Kluyvera species.Others may wish to report "Kluyvera species,"which would include all three groups. We en-

courage laboratories to identify Kluyvera to thespecies level so that more can be learned aboutthe ecology, epidemiology, and role in humandiseases of K. ascorbata, K. cryocrescens, andKluyvera species group 3. We have now reported11 strains as Kluyvera species group 3 becausethey were either different from both named spe-cies by DNA hybridization or different in theirphenotypic reactions. These strains probablyrepresent additional species of Kluyvera, butmore study is required before a scientificname(s) can be assigned.Concerning the clinical significance of Kluy-

vera, Schwach (21) reported three isolates ofEnteric Group 8, all from upper respiratory tractspecimens, which we subsequently received andidentified (phenotypically) as K. ascorbata.Since the strains were in mixed cultures, and notdetected in subsequent culture, she concludedthat they were probably not clinically signifi-cant. Braunstein and co-workers (7) recentlyreported on two cultures that we had identifiedas Enteric Group 8 (both are K. ascorbata). Oneof these was from the sputum of a 6-year-oldboy with pulmonary tuberculosis. This isolatewas not considered clinically significant. A sec-ond isolate was from gall bladder drainage fluidof a 63-year-old woman with acute pancreatitis.On the basis of chart review, this isolate wasconsidered clinically significant. We are aware ofno other published reports assessing the clinicalsignificance of Enteric Group 8 or Kluyvera. Ofour 144 Kluyvera strains, none have been fromspinal fluid, but 5 strains have been from blood:3 strains (2 from France) of K. ascorbata, 1strain of K. cryocrescens (from a 3-month-oldchild, at autopsy), and 1 strain of Kluyveraspecies group 3. No other information was in-cluded to assist in the evaluation of the fiveisolates. These five blood isolates and the report

VOL. 13, 1981

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

932 FARMER ET AL.

of Braunstein and co-workers (7) suggest thatKluyvera is more than a benign saprophyte.Most new species of Enterobacteriaceae have atleast attained the status of "infrequent oppor-tunistic pathogen." On the basis of presentknowledge this status also seems appropriate forKluyvera. The respiratory tract was the mostcommon source for Kluyvera, but there is nostrong evidence that it is clinically significant.(One isolate of K. ascorbata was, however, froma lung at autopsy.) The respiratory tract (partic-ularly sputum) is notoriously difficult to evalu-ate for clinical significance except in carefullydesigned prospective studies which include pa-tients' antibody responses. The urinary tract wasthe next most common source, but there was nomention of more than 100,000 organisms per mlof urine or the presence of leukocytes or eryth-rocytes. Feces was a common site of isolation,and the documented presence of Kluyvera infood (16; Table 1) is an obvious source of theseisolates. Since fecal cultures are usually takenbecause the patient has diarrhea, the role ofKluyvera as a possible cause should be investi-gated prospectively. Further study is needed tofully understand the ecology and epidemiologyof Kluyvera and to define its role in disease. Wehope that this report will stimulate others toidentify strains of Kluyvera and determine theirsource and significance.

ACKNOWLEDGMENTS

We thank Manley Mandel for doing the G+C determina-tions, A. G. Steigerwalt for preparing the DNA for thesedeterminations, Martin Busse for Kluyvera cultures isolatedfrom food (16) and water (26), T. Schwach for more informa-tion on her three case reports (21), Karla Tomfohrde forfurnishing Kluyvera cultures referred to Analytab Productsfor identification, John Holt and Warren Armstrong for sug-gesting possible names for the Kluyvera species, KathleenAnn Kelley, Emory University, for helping with the idiosyn-crasies of Greek and Latin grammar and for writing the sectionon the origins of the species names "ascorbata" and "cryocres-cens," and Robert Gherna for furnishing the ATCC strains.

LITERATURE CITED

1. American Public Health Association. 1975. Standardmethods for the examination of water and wastewater,14th ed. American Public Health Association, Inc., NewYork.

2. Asai, T., K. Aida, Z. Sugisaki, and N. Yakeishi. 1955.On a-ketoglutaric acid fermentation. J. Gen. Appl. Mi-crobiol. 1:308-346.

3. Asai, T., H. Iizuka, and K. Komagata. 1962. The flag-ellation of genus Kluyvera. J. Gen. Appl. Microbiol. 8:187-191.

4. Asai, T., S. Okumura, and T. Tsunoda. 1956. On a newgenus, Kluyvera. Proc. Jpn. Acad. 32:488-493.

5. Asai, T., S. Okumura, and T. Tsunoda. 1957. On theclassification of the a-ketoglutaric acid accumulatingbacteria in aerobic fermentation. J. Gen. Appl. Micro-

biol. 3:13-33.6. Beck, E., J. Wieczorek, and W. Reinecke. 1980. Puri-

fication and properties of hamamelosekinase. Eur. J.Biochem. 107:485-489.

7. Braunstein, H., M. Tomasulo, S. Scott, and M. P.Chadwick. 1980. A biotype of Enterobacteriaceae in-termediate between Citrobacter and Enterobacter. Am.J. Clin. Pathol. 73:114-116.

8. Brenner, D. J., J. J. Farmer III, G. R. Fanning, A. G.Steigerwalt, P. Klykken, H. G. Wathen, F. W.Hickman, and W. H. Ewing. 1978. Deoxyribonucleicacid relatedness of Proteus and Providencia species.Int. J. Syst. Bacteriol. 28:269-282.

9. Brisou, B., C. Richard, and A. Lenriot. 1972. Intérêttaxonomique de la recherche de la betaxylosidase chezles Enterobacteriaceae. Ann. Institut Pasteur (Paris)123:341-347.

10. Edwards, P. R., and W. H. Ewing. 1972. Identificationof Enterobacteriaceae, 3rd ed. Burgess Publishing Co.,Minneapolis.

11. Ewing, W. H., and M. A. Fife. 1971. Biochemical char-acterization of Enterobacter agglomerans. Center forDisease Control, Atlanta.

12. Farmer, J. J., III. 1978. Standardization and automationin biotyping, p. 68-97. In A. Balows, and H. D. Isenberg(ed.), Biotyping in the clinical microbiology laboratory.Charles C Thomas, Publisher, Springfield, 111.

13. Farmer, J. J., III, M. A. Asbury, F. W. Hickman, D.J. Brenner, and the Enterobacteriaceae StudyGroup. 1980. Enterobacter sakazakii: a new species of"Enterobacteriaceae" isolated from clinical specimens.Int. J. Syst. Bacteriol. 30:569-584.

14. Hickman, F. W., and J. J. Farmer III. 1978. Salmonellatyphi: identification, antibiograms, serology, and bacte-riophage typing. Am. J. Med. Technol. 44:1149-1159.

15. Kilian, M., and P. Bulow. 1979. Rapid identification ofEnterobacteriaceae. Acta Pathol. Microbiol. Scand.Sect. B 87:271-276.

16. Kleeberger, A. 1979. Untersuchungen zur Taxonomievon Enterobakterien und Pseudomonaden aus Hack-fleisch. Arch. Lebensmittelhyg. 30:130-137.

17. Kluyver, A. J., and C. B. van Niel. 1936. Prospects fora natural system of classification of bacteria. Zentrabl.Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 2 94:369-403.

18. LeMinor, L., and F. Pichinoty. 1963. Recherche de latétrathionate-réductase chez les bactéries Gram nega-tives anaérobies facultatives (Enterobacteriaceae,Aeromonas et Pasteurella). Méthode et valeur diag-nostique. Ann. Institut Pasteur (Paris) 104:384-393.

19. Nakazawa, H., H. Enei, S. Okumura, and H. Yamada.1972. Synthesis of L-tryptophane from pyruvate andindole. Agric. Biol. Chem. 36(Suppl. 13):2523-2528.

20. Nara, T., R. Okachi, and M. Misawa. 1971. Enzymaticsynthesis of D(-)-alpha-aminobenzylpenicillin by Kluy-vera citrophila. J. Antibiot. 24:321-323.

21. Schwach, T. 1979. Case report. Clin. Microbiol. Newsl.1(no. 16):4-5.

22. Serizawa, N., N. Nakagawa, G. Kamimura, T. Miyad-era, and M. Arai. 1979. Stereo-specific synthesis of 3-trifluromethylcephalosporin derivative by microbialacylase. J. Antibiot. 32:1016-1018.

23. Skerman, V. B. D., V. McGowan, and P. H. A. Sneath.1980. Approved lists of bacterial names. Int. J. Syst.Bacteriol. 30:225-420.

24. Steigerwalt, A. G., G. R. Fanning, M. A. Fife-Asbury,and D. J. Brenner. 1976. DNA relatedness amongspecies of Enterobacter and Serratia. Can. J. Microbiol.22:121-137.

J. CLIN. MICROBIOL.

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from

IDENTIFICATION OF KLUYVERA 933

25. Thanbichler, A., and E. Beck. 1974. Catabolism ofhamamelose. The anaerobic dissimilation of D-hama-melose by Kluyvera citrophila 627. Eur. J. Biochem.50:191-196.

26. Thurner, K., and M. Busse. 1978. Numerisch taxonom-ische Untersuchungen an Enterobakterien aus Ober-flâchenwasser. Zentralbl. Bakteriol. Parasitenkd. Infek-tionskr. Hyg. Abt. 1 Orig. Reihe B 167:262-271.

VOL. 13, 1981

on August 13, 2020 by guest

http://jcm.asm

.org/D

ownloaded from