THE CLASSIFICATION OF ORGANISMS TERMEDLEPTOTRICHIA (LEPTOTHRIX) BUCCALIS

III. GROWTH AND BIOCHEMICAL CHARACTERISTICS OF Bacterionema matruchotii'

MARION N. GILMOUR AND PATRICIA H. BECK

Eastman Dental Dispensary, Rochester, New York

Because of the confusion which has existed inthe classification of the large gram-positive oralfilamentous organism characteristically having afilament attached to a bacillus-like body (11), acomprehensive description of those traits usefulfor identification is essential. Some of these traitshave been reported by the following authors:Kligler (16), for anaerobic isolates which werenamed Leptothrix buccalis; Mendel (18), for anaerobically isolated organism which he termedCladothrix matruchoti; Bulleid (7), for anaerobicisolates subsequently cultured aerobically whichhe designated Leptothrix buccalis; Bibby (3), andBibby and Berry (4), for anaerobically isolatedorganisms which were termed Leptotrichiabuccalis; Bartels (1), for an anaerobically iso-lated filamentous organism; Morris (19) foranaerobic isolates which he believed were Lepto-trichia; Beck and Gilmour (2), for aerobic pourplate isolates of a filamentous organism; Davisand Baird-Parker (9), for the aerobically isolatedorganism which they termed Leptotrichia dentium;Richardson and Schmidt (22), for their aerobicpour plate isolates; and Gilmour et al. (11), in aliterature review of previously described organ-isms which they termed Bacterionema matruchotii.As shown in tables 1 and 2, the various descrip-tions differ in fermentation patterns, gaseousgrowth requirements, and the occurrence ofbranching. These discrepancies may be at-tributable to the varying cultural conditions em-ployed, to culture instability, or to a variety ofbiochemically different organisms having similarcellular morphologies. To test these possibilities,the physiological characteristics and environ-mental effects on morphology have been studiedin 55 strains of Bacterionema matruchotii.

1 This investigation was supported by a researchgrant, D-370, from the National Institute of Den-tal Research, U. S. Public Health Service. Reportsof parts of this work were presented at the meet-ings of the International Association for DentalResearch, San Francisco, California, March 19,1959, and Chicago, Illinois, March 17, 1960.

MATERIALS AND METHODS

Cultures and growth conditions. The isolationand morphology of the 55 strains studied has beenpreviously described (10). All cultures were main-tained on aerobically incubated slants of brainheart infusion agar (Difco) supplemented with0.2 per cent yeast extract, and transferred at 3-week intervals. For comparison purposes, testswere also made on Nocardia asteroides strains343 (isolated from a human subject) and ATCC9970, Nocardia polychromogenes ATCC 3409,Nocardia corallina ATCC 4273, Nocardia maduraeATCC 6245, and Nocardia brasiliensis 9961 (iso-lated from a human subject), all of which werekindly supplied by Dr. A. Howell, Jr. Nocardiastocks were transferred as above, but incubatedat room temperature.Media employed included brain heart infusion

(brain heart infusion broth (Difco), 37 g; yeastextract, 2 g; distilled water, 1000 ml; pH 7.4),half strength brain heart infusion (as above, butwith 18.5 g brain heart infusion broth), and nu-trient medium (nutrient broth, Difco, 8 g; yeastextract, 2 g; distilled water, 1000 ml; pH 7.4).Two per cent agar was added for solidification,and 5 per cent citrated sheep blood or sheep serumwas included when required.

Because most of the strains exhibited granulargrowth, inocula for the majority of tests werelightly homogenized buffer or broth suspensionsof log phase cells adjusted to standard opticaldensities ranging from scale readings of 50 to 70on a Klett-Summerson colorimeter, with filter54 and the menstruum as blank.

Cultures were incubated at 37 C aerobically,or anaerobically using the Pine and Howell (21)technique or anaerobic jars (McIntosh andFildes, or Brewer's). The latter were evacuatedand filled three times with 95 per cent H2 and 5per cent C02, and then attached to an electriccircuit for 1 hr. Cultures of N. asteroides wereused to detect deficient anaerobiosis. All tests onNocardia cultures were incubated aerobically.

152

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

CLASSIFICATION OF ORGANISMS TERMED LEPTOTRICHIA. III

TABLE 1Fermentative characteristics of organisms morphologically similar to Bacterionema matruchotii, reported

by other authors

Author and Number of Strains Tested

Carbohydrate Morris (17)KliglerMendelBulleid_____BibbyBartels__Davis and RichardsoneKligler Mende Bulleid Bibby Barte Baird-Parker and Schmidt

(15) (3) (10) (53) (1) Type Type 2 Type Type (?) (12)

Glucose...... + + slow+ + + - slow + + + + +Fructose..... + + +Salicin_.-.-- + and - +

strainsSucrose. + + - 98% + + - slow+ + + + +Mannose + +Maltose...... + slow+ 88% + + - + + + +Raffinose + - - - - - + and - + and -

strains strainsMannitol.... - - - - slow+ + +Lactose . - + - - + + -ArabinoseXylose - - - +Galactose.... slow + - + and -

strainsRhamnose.. .

Melibiose....Cellobiose ....

Trehalose ....Melizitose....

Inulin ....... +Dextrin ...... + + +Sorbitol.Dulcitol. -Inositol.

Gaseous conditions for growth. To determine thepreferential gaseous environment, shake tubescontaining 15 ml brain heart infusion and 2.5 percent agar were inoculated with 0.1 ml fromaerobic broth cultures of either Bacterionemamatruchotii or Nocardia species and observeddaily for a 2-week period.

Aerobically and anaerobically grown cell sus-pensions of all strains were each also tested forgrowth under both aerobic and anaerobic (jars)conditions. Suspensions of approximately 7 X104 cells per ml were streaked onto duplicateplates using either a standard loop for inoculationand streaking, or 0.05 ml of a 1:10 dilution and aglass rod. Seventeen or more strains were testedin duplicate broth tubes each containing 5 ml ofmedium and inoculated with 0.05 ml of the abovesuspension. The Nocardia species were similarlytested on plates. Media employed were brain

heart infusion agar with and without blood, andbrain heart infusions and nutrient broths withand without serum. Observations and Gramstains were made after 1 or 2 weeks of incubation,or both.To ascertain that anaerobic growth could not

be attributed to large cell masses, small inoculaof aerobically and anaerobically grown cellsfrom each of eight strains were tested for aerobicand anaerobic growth. Each strain was incubatedaerobically and anaerobically in brain heart in-fusion broth supplemented with 5 per cent sheepserum until a faintly turbid suspension (scalereading approximately 5 to 10 on the Klett-Summerson colorimeter) was obtained after theclumps had settled out. Aliquots of each super-natant were removed, counted in a hemocytome-ter, and diluted to 20 cells per ml in each of the 3media employed: (a) brain heart infusion broth;

1961] 153

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

GO

00

±Q+*S,

+ + Q+ +

._+++ II I+ + I+Q<+

> + +a)

C4 o <O Q+

Ou0Z- l + 00+

V-~~ ~ ~ ~ ~~~~I

a ¢

++

tji e + a_, )

X ~~~~~*

*

.Ca

b)05--

.

0 c.... .4

.0 0 05)Uc.. .

Ca

t =; C; >: Ca $-

154

0

a)

011

0r-

a)

00505

-4Ov

ma ¢*

'n

4)

o

0

¢

0

4-0;

*4-0

4-

*C

4-

0Sc.' 4-:

¢

r0

0*g

qo

4-a)

4-9

a)

b0ew:

._

Un

~04U

Cd

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

CLASSIFICATION OF ORGANISMS TERMED LEPTOTRICHIA. III

(b) brain heart infusion broth supplemented with5 per cent sheep serum; and (c) brain heart in-fusion broth supplemented with laked blood (5ml sheep blood in 10 ml distilled water added to100 ml medium). Eighty 1-ml aliquots of eachof the resulting 6 dilutions from each strain were

dispensed into 80 Kahn tubes, one-half of whichwere incubated aerobically, the other halfanaerobically. Following 7 days of incubation,the numbers of tubes showing growth were re-

corded.pH limitations on growth initiation. Fifteen

strains were tested on brain heart infusion brothsand agars adjusted at 0.5 pH unit intervals inthe range pH 4.5 to 9.0. Broth tubes and duplicateplates were each inoculated with 0.1 ml buffersuspensions of aerobically grown cells. All tubesand one set of plates were incubated aerobically,while the other set of plates was incubated an-

aerobically in an atmosphere of H2. Observationswere made daily over a 1-week incubation periodand Gram stains were prepared from all cultureswith growth.

Fermentation tests. Aerobically incubated testswere performed on all stains shortly after purifica-tion and, using stock cultures which were trans-ferred every 3 weeks during the interval, again

after 1 year. Tests were also repeated twice on 11strains incubated anaerobically and on the 5Nocardia cultures.Aqueous 20 per cent solutions of each of the

carbohydrates at pH 6.5 were sterilized and addedaseptically at a final concentration of 1 per centto the basal media. Sucrose, fructose, lactose,arabinose, galactose, xylose, mannose, and mal-tose were sterilized with filtration; mannitol,dulcitol, inulin, raffinose, salicin, glycerol, rham-nose, glucose, trehalose, cellobiose, melibiose,sorbitol, and a-methyl-glucoside, by steamingfor 20 min on each of 3 successive days. The basalmedium for aerobic tests was nutrient mediumbroth with 0.001 per cent final concentration ofbromothymol blue. Because this medium did notsupport anaerobic growth, nutrient medium serum

broth was used for the anaerobically incubatedtests, and aerobically incubated controls were

performed to ascertain that the fermentationpattern was not affected by the change ofmedium.

Aerobically incubated tests were performedin Durham tubes inoculated with 0.1-ml aliquotsof aerobically grown cell suspensions. Inoculated

and uninoculated control tubes were observeddaily for color change, and after 2 weeks of incu-bation the final pH was determined. Followinginoculation with anaerobically grown cells, anaer-obic tests were incubated for 2 weeks and the pHin both inoculated tubes and uninoculated con-trols of each sugar was measured with a pH meter.The rates of pH change with aerobic and ana-

erobic growth were determined with 2 diffuselygrowing strains in nutrient medium serum brothswith and without 1 per cent glucose. Each strainwas tested by inoculating 30 tubes of each me-dium with 0.1-ml aliquots of an aerobically growncell suspension giving a scale reading of 50 on theKlett-Summerson calorimeter. One half of theinoculated tubes and an equal number of unin-oculated control tubes were incubated aero-bically; the other half and their controls,anaerobically in jars. Following 2 hr incubation,and thereafter at 24-hr intervals over a period of14 days, pH determinations were made on onetube of each of the inoculated and uninoculatedmedia from each incubation condition, and thegrowth was estimated by visual comparison.

Additional biochemical tests. All tests were madeon each strain simultaneously with its fermenta-tion tests and using the same inoculum. Unlessotherwise specified, the basal media employedwere half strength brain heart infusion broth foraerobically incubated tests, and the same mediumsupplemented with serum for the 11 strains testedanaerobically. Serum was added to all media em-ployed for anaerobic cultures. The majority ofmethods used are described in the Manual ofmicrobiological methods (24) and the Manual ofmethods for pure culture study of bacteria (23) ofthe Society of American Bacteriologists. Esculinhydrolysis was tested in broth supplemented with0.5 per cent esculin and 0.5 per cent ferric am-monium citrate, and in streak plates containing2.5 per cent esculin, 2.5 per cent yeast extract, 2per cent agar, and 0.05 per cent ferric ammoniumcitrate (G. Knaysi, unpublished data); daily ob-servations were made over a 2-week incubationperiod. Hippurate hydrolysis was determined bymixing equal volumes of 50 per cent H2SO4 andthe supernatant from a 2-week culture grown innutrient medium broth containing 1 per centsodium hippurate. Starch hydrolysis was testedby flooding 5-day cultures on 0.5 per cent starchplates with Lugol's iodine. Gelatin liquefactionwas observed in chilled 2-week cultures in 12 per

19611 155

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

GILMOUR AND BECK

cent gelatin. Ammonia production was deter-mined by nesslerization of 1-week broth culturescontaining 1 per cent arginine hydrochloride.Tests for indole production were made withKovac's reagent on 2-week cultures; for nitratereduction to nitrite on 1-week cultures; and foracetoin production from glucose in 2-week 2 percent glucose cultures employing the method asoriginally described (8) and 0.3 per cent creatinein 40 per cent KOH. CO2 production was testedin Durham tubes using 1 per cent glucose nu-trient medium broth, in 2 per cent glucose agarshake cultures, and in Eldredge tubes using 25strains grown aerobically in 2 per cent glucosenutrient medium broth. The production ofcatalase was assessed by suspending an aero-bically grown colony in 3 per cent H202 and ob-serving at 32 X magnification.

Antibiotic sensitivities. Tests were made on 9strains with aerobic incubation, on an additional5 strains with aerobic and anaerobic incubationperformed in parallel, and on 5 Nocardia species.Following uniform inoculation of brain heart in-fusion serum agar plates with suspensions ofaerobically or anaerobically grown cells for therespective incubation condition, a Sensi-disc(BBL) was placed on each plate. Sensi-discs ofeach antibiotic were from a single series batchincluding high and low concentrations: penicillinand bacitracin, 2 and 10 units; dihydrostrepto-mycin, 10 and 50 ,g; triple sulfonamide (sulfadia-zine, sulfamethazine, and sulfamerazine), 0.25and 1.0 mg; erythromycin and carbomycin, 2 and15 Mg; chlortetracycline, oxytetracycline, tetra-cycline, neomycin, chloramphenicol, and poly-myxin B, 5 and 30 Mg. After 5 days of incubation,the diameter of the zone without growth, in-cluding the disc, was measured.

RESULTS

Gaseous conditions for growth. Of the 34 strainstested in agar shake cultures, 30 grew throughoutthe tube, 2 did not grow at the top, and 2 did notgrow at the bottom. The latter 4 strains did, how-ever, subculture on both aerobically and anaero-bically incubated streak plates. The majority ofthe first group had most abundant growth ex-tending from X in. below the surface halfwayto the bottom of the tube. Two of such strainsexhibited a ring of heavy growth approximately/% in. below the medium surface. In contrast,growth of the Nocardia species was restricted tothe top 14 in. of medium.

TABLE 3Growth of Bacterionema matruchotii and Nocardia

spp. under aerobic and strictly anaerobicconditions*

Ratio + Cultures Ratio + CulturesB. mtatruchoiii Nocardia spp.

Medium Anaero- Aerobic Anaero-Aerobic bcbicincuba- incAba- incuba- incuba-to tintion tiont

Brain heart infu-sion agar + 0.2%yeast extract ..... 55/55 55/55 5/5 0

Blood agar........ t 55/55 5/5 0Brain heart infu-

sion broth +0.2% yeast ex-tract ............. 22/22 16/22 5/5 0

Brain heart infu-sion broth + 5%sheep serum ..... 17/17 17/17 - -

Nutrient broth +0.2% yeast ex-tract ............. 17/17 2/17 - -

Nutrient broth +0.2% yeast ex-tract + 5% sheepserum ............ 17/17 16/17 - -

* From aerobic inocula.t All cultures grew when

bated aerobically.T- = Not tested.

subsequently incu-

The results with aerobically and anaerobicallyincubated broth and agar cultures of Bac-terionema matruchotii are compatible with thosegiven above, providing that nutritional require-ments, particularly in broths, were met (tables 3and 4). Successful growth from anaerobicallygrown small inocula (20 cells per ml) was particu-larly dependent on the medium (table 4), andrelatively few anaerobically grown cells of thetwo strains which grew only near the surface inshake cultures were able to reproduce (see strainWHB-9A, table 4). It should be noted thatgrowth rates in non-serum or non-blood con-taining media were greater aerobically thananaerobically, whereas when serum or laked bloodwas added, anaerobic and aerobic growth ratesof some strains were comparable as judged byvisual comparison. Although Bacterionema matru-chotii cultures were able to reproduce underaerobic or anaerobic conditions, the five Nocardiaspecies grew only aerobically (table 3).

156 [VOL. 25

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

CLASSIFICATION OF ORGANISMS TERMED LEPTOTRICHIA. III

TABLE 4Effect of media and aerobic and anaerobic incubation on growth of small inocula of Bacterionema

matruchotii

Subcultures % Subcultures Yielding GrowthtInoculum*

Incubation Mediumt No. 2A31 No. 12 No. 7 No. WHB9A No. 30

Aerobic Aerobic 0 100 95 100 100 100Blood 100 100 100 100 100Serum 100 100 100 100 100

Anaerobic 0 100 95 95 90 100Blood 100 97.5 100 92.5 100Serum 100 95 90 95 100

Anaerobic Aerobic 0 77.5 87.5 95 30 100Blood 100 100 100 20 100Serum 100 100 100 52.5 100

Anaerobic 0 97.5 15 10 5 25Blood 100 100 100 10 100Serum 97.5 80 100 32.5 32.5

* Grown on brain heart infusion medium supplemented with 5 per cent sheep serum.t Brain heart infusion with no additions, or supplemented with 5 per cent final concentration sheep

serum or laked blood, (5 ml. sterile sheep blood + 10 ml distilled water added to 100 ml medium).t Each figure represents tests of growth in 40 tubes.

Cell morphologies in aerobic and anaerobiccultures were consistently different in the fol-lowing respects. (a) Branching occurred veryrarely in anaerobically grown cultures, whereasit was a common phenomenon in aerobic cultures.(b) The frequency of occurrence of long fila-mentous cells was greatly increased in anaerobiccultures as compared to aerobic cultures. (c) Cellsize (both diameter and length) was larger inanaerobic cultures than in aerobic cultures.pH limitations on growth initiation. As shown

in table 5, the optimal pH range for initiation ofgrowth on both solid and fluid media was 7.0 to7.5. In general, growth was not initiated at a pHof 9.0, or at a pH of 5.5 or lower. Branchingcells were consistently found more frequently incultures grown at pH 6.0 than in cultures grownat higher pH values.

Fermentation tests. With a few exceptions, the55 aerobically tested strains and the 11 anaero-bically tested strains were remarkably similarand constant in their sugar fermentation patterns.One strain differed from all others in beinga "slow" fermenter of all fermentable substrates(i.e., requiring 1 to 2 weeks to give an acid re-action). All strains consistently fermented glu-

TABLE 5Effect of pH on growth initiation of Bacterionema

matruchotii in broth and agar media

Ratio Cultures with Growth

pH of Medium AgarBroth

. v(aerobic)Aerobic Anaerobic

4.5 0/15 *5.0 0/15 0/15 0/155.5 0/15 1/15 0/156.0 11/15 9/15 9/156.5 15/15 13/15 15/157.0 15/15 15/15 15/157.5 15/15 15/15 15/158.0 12/15 13/15 15/158.5 7/15 1/15 4/159.0 0/15

*- = Not tested.

cose, fructose, sucrose, and maltose to a finalpH of 4.7 to 5.2. Including 3 "slow" fermenters,mannose was fermented by 54 and 10 of the re-spective aerobically and anaerobically testedstrains, the final pH being 5.0 to 5.5. Fifty-one

1571961]

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

GILMOUR AND BECK

of the aerobically tested and 9 of the an-aerobically tested strains fermented salicin toyield a final pH of 5.3 to 5.4. However, salicinfermentation was not a reliable characteristic; 15strains were "slow" fermenters and 3 strainschanged from negative to fast fermenters betweenthe two test times. Raffinose was slowly fer-mented by 19 of the 55 aerobically and 5 of the 11anaerobically tested strains. One strain changedfrom raffinose i to raffinose + between the twotest times. Inocula from late positive tubes didnot cause a rapid fermentation when sub-sequently inoculated into fresh raffinose tubes,indicating that the "slow" fermentation wasnot attributable to a major population shift toraffinose positive cells. Mannitol was fermentedby 7 of the aerobically tested strains and 2 ofthose tested anaerobically, yielding a final pH of5.0 to 5.3, and one strain changed from negativeto positive. Trehalose was fermented by 3 ofthe aerobically tested strains, and by none ofthose tested anaerobically. None of the strainsfermented lactose, arabinose, xylose, galactose,a-methyl-glucoside, rhamnose, melibiose, cello-biose, inulin, glycerol, sorbitol, or dulcitol.None of the 5 Nocardia species tested under

these conditions produced acid or gas from anyof the carbohydrates tested. Final pH values wereeither the same as the uninoculated control tubes,or higher.Although the incubation condition (i.e., aerobic

or anaerobic) did not affect the sugar fermenta-tion pattern, it appeared to influence the final pHvalues obtained. In the absence of a fermentablesubstrate, the final pH increased as comparedto the uninoculated control tubes by an averageof 0.5 unit with aerobic incubation, but exhibitedlittle change anaerobically. In the presence of afermentable substrate, the final pH as comparedto the controls decreased an average of 2.6 unitswith aerobiosis, but only 1.6 units with anaerobio-sis. These differences in pH decrease could beattributable, for the majority of strains tested,to the slower growth rates found with anaerobio-sis than with aerobiosis.However, to determine if the gaseous growth

environment per se could affect the final pHvalues attained, the rates of pH changes weremeasured with growth conditions and strainssuch that aerobic and anaerobic growth rateswere comparable. The results of the rates of pHchanges given in figure 1 are from a typical ex-

I5.55.0- A

4.5- .

4.013.510

O 156789001 1 31C-).

INCUBATION TIME (DAYS)

Figure 1. Effect of aerobic and anaerobic growthof Bacterionema matruchotii on pH change in thepresence of the fermentable sugar, glucose. (a)Absolute pH measurements of inoculated tubesand their uninoculated controls. (b) pH changescalculated from the difference of pH between un-inoculated controls and inoculated experimentaltubes. A A aerobic growth; ------ aerobicuninoculated control; O0 o anaerobic growth;o-----0 anaerobic uninoculated control.

periment in which the stationary growth phasewas initiated after 5 or 6 days. In the presence ofthe fermentable substrate glucose there was agreater initial rate of pH decrease and a lowerfinal pH with anaerobic incubation than withaerobiosis (see figure la). When corrected for pHchanges in the anaerobically incubated uninocu-lated control tubes, the rates of pH decrease dueto fermentation differed aerobically and anaero-bically until at the initiation of stationary phasethe net pH change was similar in both incubationconditions (figure lb). Subsequent to this period,further acid production occurred aerobically toyield a net pH decrease greater aerobically thananaerobically. It is probable that the lowpH found in the anaerobic cultures preventedfurther acid production; cells from 14-day cul-tures did not stain well, and yielded few positivesubcultures as compared to cells from 14-dayaerobically incubated cultures. In the absence ofglucose and under aerobic conditions there was asteady slow increase in pH until a plateau regionwas established at pH 8.3, whereas under anaero-bic conditions no pH change occurred.

158 [VOL. 25

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

CLASSIFICATION OF ORGANISMS TERMED LEPTOTRICHIA. III

Additional tests. No changes were noted be-tween the two testing times in the followingtraits. Esculin was hydrolyzed by 48 of the aero-bically tested strains and 11 of those testedanaerobically. All strains hydrolyzed sodiumhippurate and starch. None liquefied gelatin,produced indole from tryptophan, or producedammonia from arginine. All reduced nitrate tonitrite. All strains produced acetoin from glucose,but not enouggh to give a positive reaction by theoriginal test. The 25 aerobically tested strainswere found to produce CO2 by Eldredge tubetechnique but not by the Durham tube or agarshake culture methods. All strains were catalasepositive.

Antibiotic sensitivities. Among the 14 strainstested with aerobic incubation, one was insen-sitive to all antibiotics tested, and all of theremaining 13 were sensitive to penicillin, dihy-drostreptomycin, erythromycin, carbomycin,neomycin, oxytetracycline, tetracycline, and baci-tracin. The 13 were sensitive to high concentra-tions of chloramphenicol and chlortetracyclinebut 2 were insensitive to the low concentrationof chloramphenicol and 5 to the low concentra-tion of chlortetracycline. Twelve were sensitiveto the high concentrations of polymyxin andtriple sulfonamide, and 4 to the low concentra-tions of each antibiotic; the strains insensitive toeach compound were not necessarily the same.Zones without growth for the respective high andlow concentrations ranged from 4.5 to 6.0 cmand 1.0 to 5.5 cm for the first group ofcompounds; 2.5 to 5.0 cm and 0.0 to 3.0 cm forchloramphenicol and triple sulfonamide; and0.0 to 3.0 cm and 0.0 to 2.0 cm for chlortetra-cycline and polymyxin. Comparison of the 5strains tested both aerobically and anaerobicallyshowed that the above sensitivity patterns wereessentially the same for both incubation condi-tions, although more strains were sensitive tochloramphenicol and triple sulfonamide andfewer to polymyxin when incubated anaero-bically than aerobically. Since the pH of themedium after anaerobic incubation was 6.6 ascompared to pH 6.9 after aerobic incubation,these differences might be attributable to differ-ing rates of antibiotic diffusion.The 5 Nocardia species tested were less sensi-

tive to the antibiotics. N. brasiliensis was in-sensitive to all the antibiotics. Among the 4remaining cultures, 1 was sensitive to penicillin,

3 to streptomycin, 3 to oxytetracycline, 3 totetracycline, 1 to triple sulfonamide, and 2 topolymyxin B. With the exceptions of N. poly-chromogenes on dihydrostreptomycin, neomycin,oxytetracycline, and triple sulfonamide, and N.asteroides on dihydrostreptomycin, the inhibitionzones were 3 cm or smaller in diameter.

DISCUSSION

Although one strain was characteristically a"slow" fermenter, and a second was resistant toall antibiotics tested, the 55 strains of Bac-terionema matruchotii studied herein comprise areasonably homogeneous group similar to manypreviously reported isolates (see tables 1 and 2).The finding that at least two growth conditions,pH and gaseous environment, govern the fre-quency of branching in this organism reconcilesthe data presented here with those descriptionsreporting the rareness of branching (3, 16, 19)and with those reporting its regular occurrencein this microorganism (1, 7, 9, 14, 18, 22). Itseems, therefore, that the controversy overwhether or not this organism branches arose be-cause of differences in the growth conditions em-ployed in the earlier studies. The ability of ourstrains to grow both aerobically and anaero-bically, (although growth rates were generallygreater with aerobiosis than with anaerobiosis)also differs from the aerobic (9, 14, 18) or morestrictly anaerobic (3, 16, 19) habits previouslyattributed to similar organisms. This discrep-ancy may be attributable to two factors: char-acterization as aerobic could result from failureto make experimental allowances for the mediarequirements and slower growth rates reportedhere for anaerobic growth; characterization asmore strictly anaerobic could result from anaero-bic isolates being more strictly anaerobic thanthe aerobic pour plate isolates studied herein.There are, therefore, probably three classes ofisolates: facultative aerobes, similar to thosedescribed here and by others (7, 9, 18, 22), facul-tative anaerobes as described by Kligler (16),Bibby (3), and Morris (19), and strict anaerobes(19). The aerobic and anaerobic pH limitationson growth are similar, and are the same as thosepreviously given for both anaerobic (3) and aero-bic (22) isolates. Additionally, biochemical char-acteristics, including sugar fermentation patterns,are similar to the majority of earlier descriptions(1, 3, 4, 9, 16, 22). Comparison with Bibby's

15919611

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

GILMOUR AND BECK

anaerobic isolates (3), the largest number ofstrains previously studied, shows differences onlyin the percentages of maltose-, raffinose-, andstarch-positive strains (respectively, 88, 100, and19 per cent as compared to 100, 35, and 100 percent reported here). There is no real discrepancyin the results for CO2 production, since assess-ment with the more sensitive Eldredge tube tech-nique yields positive results whereas Durhamtubes used here and by others give negative re-sults. There are, however, apparent radical differ-ences from the essentially asacharolytic strainsdescribed by Bulleid (7) and the 4 anaerobiccatalase-negative groups noted by Morris (19).Although the use of aerobiosis or anaerobiosisduring either isolation or testing cannot solelyaccount for these differences, it is possible thatthe asacharolytic strains are actually "slow"fermenters, since relatively short test incubationtimes were employed by both authors. Thus, themajority of isolated strains are both morphologi-cally and biochemically similar, the biochemicalcharacteristics being relatively stable despitelong culture periods and changes in media orgaseous environment during testing. However,as indicated by Morris' (19) work, other probablyrarer morphologically similar but biochemicallydifferent forms exist.

Because of its ability to branch, its reproduc-tion by fragmentation (12), its sensitivity to acidand antibiotics, and its size variations, this micro-organism has characteristics of both the higherfungi and the bacteria. Gilmour et al. (11) havetherefore classified this type of organism in thefamily Actinomycetaceae, but because of differ-ences from the two existing genera, Nocardia andActinomyces, have recommended that a newgenus, Bacterionema, be formed. The type speciessuggested, Bacterionema matruchotii, comprisesorganisms identical to those described here. Fur-ther differences from the former two genera arereported in this paper. Bacterionema matruchotiidiffers markedly from the Nocardia species testedin its gaseous growth characteristics, fermenta-tive capacities, and antibiotic sensitivities (seealso (17) and (25)). It differs in gaseous growthcharacteristics, fermentative pattern, reactionsin additional tests, and inability to grow onthe Howell and Pine (13) casitone medium forActinomyces (Gilmour, unpublished data), fromboth the anaerobic Actinomyces and the facul-tative oral form termed Actinomyces naeslundii

by Thompson and Lovestedt (26) and also de-scribed by Naeslund (20), Bibby and Knighton(5), Bowen (6), and Howell et al. (15).

SUMMARY

A number of characteristics of 55 strains ofBacterionema matruchotii have been studied.Fifty-three of the strains form a homogeneousgroup. They are facultative aerobes; initiategrowth optimally in the pH range 7.0 to 7.5;ferment glucose, fructose, sucrose, mannose, andmaltose to yield a final pH in the range 4.7 to5.2 in a nutrient broth yeast extract medium;hydrolyze starch and sodium hippurate; reducenitrate to nitrite; produce acetoin and CO2 fromglucose; are catalase positive and sensitive tomany antibiotics. Two strains differed from theothers: one required 1 to 2 weeks to give an acidreaction on fermentable substrates, and one wasinsensitive to all antibiotics tested. With the ex-ceptions of reactions to salicin, raffinose, andmannitol, all strains were stable in all character-istics tested. Morphologically and biochemicallythese strains resemble organisms described inthe majority of previous studies (1, 3, 4, 7, 9, 14,16, 18), but differ from the four anaerobic typesreported by Morris (19).

ACKNOWLEDGMENT

The authors wish to express their appreciationto Mrs. Ilona Cselenyi for her technical assistancein this study.

REFERENCES

1. BARTELS, H. A. 1943 A filamentous micro-organism isolated from stained teeth. J.Dental Research, 22, 97-102.

2. BECK, P. H. AND GILMOUR, M. N. 1959 Con-tinued studies on an oral gram-positivefilamentous organism. J. Dental Research,38, 657 (Abst.).

3. BIBBY, B. G. 1935 A study of the filamen-tous bacteria of the mouth. Ph.D. Thesis,University of Rochester, Rochester, N. Y.

4. BIBBY, B. G. AND BERRY, G. P. 1939 Acultural study of filamentous bacteria ob-tained from the human mouth. J. Bac-teriol., 38, 263-274.

5.'BIBBY, B. G. AND KNIGHTON, H. T. 1941Actinomyces of the human mouth. J.Infectious Diseases, 69, 148-154.

6. BOWEN, W. H. B. 1959 Oral Actinomycesand their possible role in calculus formation.

160 [VOL. 25

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from

CLASSIFICATION OF ORGANISMS TERMED LEPTOTRICHIA. III

M.A. Thesis, University, of RochesterRochester, N Y.

7. BULLEID, A. 1924 An experimental studyof Leptothrix buccalis. Guy's Hosp. Repts.,74, 444-458.

8. CLARK, W. M. AND LUBS, H. A. 1915 Thedifferentiation of bacteria of the Colon-Aerogenes family by the use of indicators.J. Infectious Diseases, 17, 160-173.

9. DAVIS, G. H. G. AND BAIRD-PARKER, A. C.1959 Leptotrichia buccalis. Brit. DentalJ., 106, 70-73.

10. GILMOUR, M. N. AND HUNTER, P. H. 1958Isolation of an oral filamentous microor-ganism. J. Bacteriol., 76, 294-300.

11. GILMOUR, M. N., HOWELL, A., JR., AND BIBBY,B. G. 1961 The classification of organ-isms termed Leptotrichia (Leptothrix) buc-calis. I. Review of the literature and pro-posed separation into Leptotrichia buccalisTrevisan, 1879 and Bacterionema gen. nov.,B. matruchotii (Mendel, 1919) comb. nov.Bacteriol. Rev., 25, 131-141.

12. GILMOUR, M. N. 1961 The classification oforganisms termed Leptotrichia (Leptothrix)buccalis. II. Reproduction of Bacterionemamatruchotii, Bacteriol. Rev. 25, 142-151.

13. HOWELL, A. JR., AND PINE, L. 1956 Studieson the growth of species of Actinomyces. I.Cultivation in a synthetic medium withstarch. J. Bacteriol., 71, 47-53.

14. HOWELL, A. JR., AND ROGOSA, M. 1958 Iso-lation of Leptotrichia buccalis. J. Bac-teriol., 76, 330-331.

15. HOWELL, A. JR., MURPHY, W. C., PAUL, F.,AND STEPHAN, R. M. 1959 Oral strains ofActinomyces. J. Bacteriol., 78, 82-95.

16. KLIGLER, I. J. 1915 A biochemical studyand differentiation of oral bacteria withspecial reference to dental caries. J. AlliedDental Soc., 10, 282-330.

17. McLUNG, N. 1954 The utilization of carboncompounds by Nocardia species. J. Bac-teriol., 68, 231-236.

18. MENDEL, J. 1919 Cladothrix et infectiond'origine dentaire. Compt. rend., 82,583-586.

19. MORRIS, E. 0. 1954 The bacteriology of theoral cavity. V. Corynebacterium and gram-positive filamentous organisms. Brit. Den-tal J., 97, 29-36.

20. NAESLUND, C. 1925 Studies of Actinomycesfrom the oral cavity. Acta Pathol. Micro-biol. Scand., 2, 110-140.

21. PINE, L. AND HOWELL, A. JR., 1956 Com-parison of physiological and biochemicalcharacters of Actinomyces sp. with those ofLactobacillus bifidus. J. Gen. Microbiol.,15, 428-445.

22. RICHARDSON, R. L. AND SCHMIDT, J. 1959An oral filamentous microorganism: Culturalcharacteristics and microbial relationshipsaffecting growth. J. Dental Research,38, 1016-1027.

23. Society of American Bacteriologists, Com-mittee on Bacteriological Technic 1946Manual of methods for pure culture studyof bacteria. Biotech Publications, Geneva,New York.

24. Society of American Bacteriologists, Com-mittee on Bacteriological Technic 1957Manual of microbiological methods. Mc-Graw-Hill Book Co., Inc., New York.

25. STRAUSS, R. E., KLIGMAN, A. M., AND PILLS-BURY, D. M. 1951 The chemotherapyof actinomycosis an I nocardiosis. Am.Rev. Tuberc., 63, 441-448.

26. THOMPSON, L. AND LOVESTEDT, B. A. 1951An Actinomyces-like organism obtainedfrom the human mouth. Proc. Staff Meet-ings, Mayo Clinic, 26, 169-175.

19611 161

on January 30, 2020 by guesthttp://m

mbr.asm

.org/D

ownloaded from