APPLED MICROBIOLOGY, July 1969, p. 114-121Copyright © 1969 American Society for Microbiology

Predominant Catalase-negative Soil Bacteria1II. Occurrence and Characterization of Actinomyces humiferus, sp. N.

WILLIAM E. GLEDHILL2 AND L. E. CASIDA, JR.Department of Microbiology, The Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication 27 March 1969

A microorganism resembling an Actinomyces species was found to be a numer-ically predominant inhabitant of various organically rich soils. This organism formsa hyphal-like structure with true branching that fragments into gram-positivediphtheroid and coccoid elements. Its cells ferment carbohydrates and containboth lysine and ornithine as the major basic amino acids of the cell wall. It is cata-lase-negative, microaerophilic to aerobic, and sensitive to lysozyme, and it is de-pendent on an organic nitrogen source and incubation at 30 C for optimum growth.Based on these characteristics, a new species, Actinomyces humiferus, is proposed.The ecological and medical implications of a large soil population of this micro-organism are discussed.

The genus Actinomyces includes those micro-organisms which are facultative to anaerobic,require CO2 for growth, ferment carbohydrates,grow best at 37 C, have cell walls containing lysineas a major basic amino acid, and form a myceliumwith true branching that fragments into diph-theroid and coccoid elements (24). These micro-organisms occur among the normal bacterialflora of the oral cavity, and they have beenassociated with various pathological conditionsin warm-blooded animals (2, 12, 13, 17, 18, 23,26). In contrast, their presence in soil and similarnatural sources has only rarely been noted (3, 19,20, 29).By employing a modified dilution-frequency

isolation procedure (6), we have now demon-strated that an Actinomyces-like organism con-stitutes a numerically dominant population ofcertain soils. Although this organism possessescertain major characteristics in common withthose presently ascribed to Actinomyces species,it differs significantly in other important aspects.The present communication, therefore, describesvarious microbiological properties of our isolates,and recommends that they be designated as a newspecies, Actinomyces humiferus.

MATERIALS AND METHODS

Except where noted, the methods employed in thisinvestigation were those used previously (15). The 26

'This research was authorized for publication as paper no.3564 in the journal series of the Pennsylvania Agricultural Ex-periment Station on March 17, 1969.

2Present address: Microbiological Research, Tenneco Chemi-cals, Inc., Piscataway, N. J. 08854

soils (15) were collected from many locations through-out the United States, and they ranged from organi-cally rich to barren desert soils with pH values from3.8 to 9.1, moisture contents from 0.7 to 51.9%, andorganic matter contents from 0.6 to 25.2%. Themodified dilution-frequency isolation technique ofCasida (6) was employed for isolation of the Actino-myces-like organism described in this study.

Acid products resulting from glucose fermentationwere analyzed by gas chromatography. For thisdetermination, the volatile and nonvolatile acids wereextracted from the medium (21); then the volatile acidswere regenerated from their sodium salts by adding4% aqueous phosphoric acid containing 5% formicacid (7). Separation and identification of the volatilefermentation acids were accomplished by direct injec-tion of aqueous solutions into a model 5300 Barber-Coleman gas chromatograph with a hydrogen flameionization detecter and containing a stainless-steelcolumn [6 feet by 1/8 inch outside diameter (183 by0.32 cm)] packed with Chromosorb 102 (AppliedScience Laboratories, State College, Pa.). Methylesters of nonvolatile acids (1) were chromatographedon a stainless-steel column [6 feet by 0.25 inch outsidediameter (183 by 0.32 cm)] containing 1O0% diethyleneglycol adipate and 2% phosphoric acid on Chromo-sorb W (Applied Science Laboratory). All sampleswere chromatographed isothermally at 160 C withnitrogen as the carrier gas (50 ml per min).

Lactic acid was also determined colorimetrically(22) after extraction from the medium.

RESULTS

Occurrence. An Actinomyces-like microorgan-ism was isolated regularly from soils with organicmatter contents of 6.5% or greater and pH valuesranging from 6.4 to 7.9. Forty-one strains wereisolated for the present study. These strains

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Celular opology. During the first 24 hr ofgrowth on agar and liquid media, most of thecas became flaentous with true branching(Fig. 1), and they often displayed swollen terminalregions. For most strains, frmentation to diph-theroid and coccoid cells of varied size and shape(Fig. 2) occurred between 24 and 48 hr. Thisfamentation prevented the formation of anextensive hyphal structure, although occasionalrough colony variants formed a more extensivemycehium; in this instance, frm tation was notinitiated until later in their growth cycle. Culturesof physiologically older cells (48 hr), similar tothose in Fig. 2, divided in the diphtheroid andcoccoid states without mycelial development.However, when these cells were placed in freshmedalium, hyphal formation was initiated by thedevelopment of "buds" at 1 or more points onthe parent coccoid or diphtheroid cell (Fig. 3).Cells ined gram-positive throughout theirdevelopment cycle.CooMymoog. Growth on Heart Infusion

FwG. 1. Branched mycelium observed at 12 hr of Agar (24 Kr) appeared as branched, filamentous,incubation. Phase contrast. X 1,630. "spider-like" microcolonies (Fig. 4), which are

typical of members of the genus Actinomyces (5,18). At 48 hr, as frmentation of older segmentsof the hyphal structure ensued, the coloniesformed a dense central region (Fig. 5). Maturecolonies (7 to 10 days) of most strains when

_examined by transmitted light were small (lessthan 1 mm), opaque, entire, and convex, and

FIG. 2. Fragmentation of 36-hr-ol mYCelium toyield coccoid and diphtheroid cells. Phase contrast.x 1,630.

represented a numrially predominant segmentof the bacterial flora, since they were recoveredfrom those soil dilutions 1 and 2 logarithmic unitsbeyond the dilution range for platable micro-oranisms (6). Attempts to isolate this ofrom a wide variety of soil types by the use Of FmG. 3. Growth initiation by the formation of "buds"standard plating techniques were unsuccessful. at 8 hr of incubation. Phase contrast. X 1,630.

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GLEDHILL AND CASIDA

Pigment production has not been observed foreither smooth or rough strains.Oxygen requirements and growth characteriza-

tion. The soil isolates were microaerophilic toaerobic. Growth in thioglycollate broth was re-stricted primarily to a distinct band of finelygranular or flocculent consistency at or just belowthe interface between the oxidized and reducedzones, with comparatively less growth in theoxidized region. Growth in Heart Infusion Brothcontaining 0.08% agar also occurred mainly as adiffuse band 0.5 cm below the surface of themedium, with decreasing turbidity at higher andlower regions of the tube. In Heart InfusionAgar shake tubes, maximum growth occurredin the upper 1 cm of agar, with isolated lightpatches of growth in lower portions of thetube. A white granular-to-flaky sediment ofgrowth without turbidity was produced in HeartInfusion Broth. Growth was not observed un-der anaerobic conditions, and the developmentof all strains either was not affected or was in-hibited by the presence of an increased CC2

FiG. 4. "Spider-like" microcolony at 24 hr of incu- tension.bation. X 650. The pH range for optimum growth in media

no. 1 and 2 was 7.2 to 7.5. The optimum growthtemperature for all isolates was approximately30 C, and this value did not change during con-tinued cultivation on laboratory media. At initialisolation from soil, all strains were unable to growat 37 C. However, after prolonged laboratorycultivation a few isolates did display feeblegrowth at this temperature, although successivetransfers at this temperature resulted in death ofthe cultures.

Biochemical characteristics. As may be seen inTables 1 and 2, these soil organisms can becharacterized as catalase-negative, nonacid-fast,susceptible to lysis by lysozyme, not reducing

FiG. 5. Microcolony with dense central region at48 hr of incubation. X 650.

they displayed a dark central area (Fig. 6).Rough-colony variants observed occasionallywere irregular and heaped, and they were smallerand more convex than the smooth colonies.Further cultivation of these variants was possible,but they tended to revert to the smooth form. FIG. 6. Mature colony at 8 days. X 6.

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CATALASE-NEGATIVE SOIL BACTERIA

TABLE 1. Biochemical properties of soil isolates'

Property Positive strains Property Poitive strains

Catalase activity............O0

Benzidine test.............0

Oxidase test ..................................0

Nitrate reduction............0

Hydrolysis ofStarch 100

Gelatin .................................. 44bTributyrin............0

Casein ..................................... 85Esculin............ 89

Decomposition ofTyrosine............0

Xanthine ...................................0

Urea............0

Methyl red test.................100

Voges-Proskauer test............... 15

Lysozyme sensitivity............... 93

Utilization ofAcetate...............0

Propionate......0...

Gluconate...... 26

Oxalate......0

Citrate....... 0

Lactate

Succinate......0

Fumarate................................. 100

Pyruvate.............100

a-Ketoglutarate............ 97

Litmus milklAcid and reduction.................. .. 82

No reaction................ ....... .. 18

Acid fast... 0

Production ofNH, from peptone

NH, from arginine.

Indole ...................................0..

H2S.67

Glucose fermentation 100

Acid fromGlycerol................ .. 74

Arabinose.. 96

Ribose......... 11Xylose.................... 89

Fructose 100

Galactose ...................... 93

Glucose.100Mannose ...... ............. 96Rhamnose ....................96

Salicin................... 78

Adonitol ....................0...

Dulcitol......0...........

Inositol.......................7

Mannitol......93

Sorbitol.4Cellobiose.67Gentiobiose......... 85Lactose.........5......... 59Maltose.................96

Melibiose............... 93

Sucrose.100Trehalose........... 70Turanose....... .......82

Melezitose.............. ..96

Raffinose.. 89

Stachyose 96

Dextrin 100

Inulin..4........... ....4Starch.100

Growth in presence of1% bile.33

3% bile

4% NaCI .......................52

a Results based on 27 strains.b These strains showed only a very narrow zone of hydrolysis.

TABLE 2. Various properties of soil isolates

Glucose fermentation products' Major cell wal componentsb DNA base inpotione

Acid Total Amino adds Amino sugas | SD5 | CsaI Per centacids density GC'S

Lactic 91-97 Alanine Glucosamine Rhamnnose 1.7315 73.0Succinic <5 Glutamic acid Muramic acidAcetic <5 LysinePropionic 0 Ornithine

Aspartic acid

aAverage of 12 strains.b Determinations based on 14 strains.c Data from one representative strain.d Moles per cent guanine plus cytosine of the total bases.

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GLEDHILL AND CASIDA

nitrate, not producing indole, able to utilizecertain organic acids for growth, and fermentinga broad spectrum of carbohydrates without gasformation. Lactic acid, their major end productof glucose fermentation, usually was produced inquantities just great enough to provide a positivemethyl red test.Based on the benzidine test (Table 1), iron

porphorin compounds, such as the cytochromes,were absent or present only to a minor extent.Gelatin either was not attacked or was onlyweakly hydrolyzed. In contrast, casein was readilydecomposed, and it markedly stimulated thegrowth of most of the isolates. Many of thestrains produced H2S, but this product could onlybe detected by suspending lead acetate paperstrips above growth on Brain Heart InfusionAgar slopes supplemented with 0.1% cysteine.

Cell wall composition (Table 2) was similar toother Actinomyces species (4, 8, 10) in that lysineand ornithine were the only basic amino acidsdetected. Rhamnose was the predominant cellwall sugar in all strains, although a few of theisolates also displayed trace amounts of glucoseand fucose.The deoxyribonucleic acid base composition

(Table 2), characterized by a high per cent guanineplus cytosine, lies within the 64 to 80% range ofvalues reported for other members of the orderActinomycetales (27, 28).

Nutritional requirements. The soil isolates andcultures of established Actinomyces species didnot grow on media lacking organic nitrogen.Also, little if any growth occurred in certainchemically defined media or media containingsimple peptones; these media were Conn'sglycerol asparagine, Sabouraud dextrose, Czapek-Dox, and nutrient broths. The addition of soilextract to medium BAVT (15) slightly stimulatedthe growth of 21 of the 27 strains studied.

Pathogenicity. The soil isolates did not induceexperimental infections in mice as have beendescribed for various Actinomyces species (5,24). Examination of 14 mice sacrificed 3 weeksafter intraperitoneal injection of washed salinecell suspensions revealed no significant pathology.

Serology. The five strains examined by thefluorescent antibody approach displayed no sero-logical cross-reactivity with specific antisera toaccepted Actinomyces and Rothia species. How-ever, two of the isolates did show a slight positive,but apparently nonspecific, reaction with Bacter-lonema antiserum.Taxonomy. From the results presented above,

it is evident that our soil isolates display majorcharacteristics in common with the genus Actino-myces. These characteristics are morphology,nutrition, cell wall composition, catalase reaction,

and carbohydrate fermentation. However, ourisolates differ from the described species of thisgenus as regards oxygen requirements, optimumgrowth temperature, and lysozyme sensitivity.Therefore, pending final consideration by theSubcommittee on Microaerophilic Actinomycetes,a new species, Actinomyces humiferus, is proposedfor these organisms.Actinomyces humiferus, sp. n. Gledhill and

Casida, 1969. L. noun humus soil; L. verb feroto bear; M.L. adj. humiferus soil-borne.

Microcolonies on agar and initial growth inliquid media composed of branched, often fila-mentous cells eventually fragmenting into coccoidand diphtheroid elements. Mature colonies small,opaque, smooth, entire, convex, with a darkcentral region. Rough-colony variation occasion-ally observed. Pigmentation not evident. Gram-positive, nonacid-fast, no aerial mycelium orspores, nonmotile. Growth in broth containing0.08% agar primarily as a diffuse band justbeneath the surface of the medium. Organicnitrogen required for growth. Catalase-negative;microaerophilic to aerobic, poor or no growthanaerobically. Oxidase-negative, benzidine-nega-tive. Growth not stimulated by increased C02tension. Temperature optimum at approximately30 C, poor or no growth at 37 C. Sugars fermentedwith lactic acid as a major product; no gas forma-tion. Cell walls contain lysine, ornithine, alanine,glutamic acid, aspartic acid, glucosamine, mu-ramic acid, and rhamnose as major components.Cells sensitive to lysis by lysozyme.

Casein, esculin, and starch hydrolyzed; gelatinweakly hydrolyzed but not liquified. Litmus milkacidified and reduced. Nitrates not reduced tonitrities. Indole not produced from tryptophan;ammonia not produced from peptone or arginine.Methyl red test positive; Voges-Proskauer testnegative. Xanthine, tyrosine, and urea not de-composed. Hydrogen sulfide produced. Nogrowth in the presence of 4% NaCl. Utilizationof pyruvate, fumarate, a-ketoglutarate, and glu-conate. Acid from arabinose, xylose, fructose,galactose, glucose, mannose, rhamnose, salicin,mannitol, cellobiose, gentiobiose, maltose, meli-biose, sucrose, turanose, melezitose, raffinose,stachyose, dextrin, and starch. No acid fromribose, adonitol, dulcitol, inositol, sorbitol, andinulin. Variable acid production from glycerol,lactose, and trehalose.

Habitat. Soil. The type culture was deposited atthe American Type Culture Collection as ATCC25174 (our isolate number PSU-16S).

DISCUSSIONThis study has demonstrated the occurrence of

an Actinomyces-like microorganism as a numer-

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TABLE 3. Selected characteristics of Actinomyces and Rothia species"

Paperty

HabitatCatalae activityRequirement forOxygeneOrganic nitrogenCarbon dioxide

Lysozyme sensi-tivityb

Nitrate reductionHydrolysis ofCaseinStarch

Gelatin liquefac-tion

Methyl red testVoges-Proskauer

testProduction of

IndoleHydrogen sulfide

Growth tempera-ture optimum

Cell wall rhamnose'Glucose fermenta-

tionAcid fromFructoseGlucoseLactoseSucroseGlycerolMannitolXyloseCellobioseMelezitoseRhamnoseInositolStarch

Ac-tixo--'(skumif-2517S

A. bevis13683

Soil Animals Animals Animals Animals

+

+

+8

+

+

+0

+

ii

-b

+

37 C

++

ii

+

-b

or -

37 C

+

+

+

+

b

or -

+7

+

ii

+

+

NDd

37 C

R. deuio-ariosa17931

Animals Animals Animals+

iv

+

ND

+

ND

37 C

+

+

ND

v

+

+

ND

+

+7

+

37+

+

+

vi

+7

+

+

+

& Data, unless otherwise stated, are compiled from ref. 2, 9, 11, 13, 14, 16, 17. Numbers identifyingspecies are American Type Culture Collection numbers.bData olWain from the present investigation.

(i) Microaerophilic to aerobic, (ii) microaerophilic to anaerobic, (iii) facultative, (iv) facultative toaerobic, (v) anaerobic, (vi) aerobic.

d Not determined.* Present as a major component.

ically predominant inhabitant of certain soils. Themajor properties of this organism, such as mor-phology, catalase reaction, organic nitrogen re-quirement, cell wall composition, and carbohy-drate fermentation, are in agreement with thosecommonly associated with the genus Actinomyces.However, at the species level our isolates are

rncroaerophilic to aerobic, inhibited or at leastnot stimulated by increased CO, tension, and

sensitive to lysis by lysozyme. Also, their optimumgrowth teprtre is approximately 30 C, andthey are able to hydrolyze casein. Since thesecharacteristics differ significantly from those ofpreviously described Actinomyces species (Table3), it is proposed that a new species, Actinomyceshuniferus, be created to encompass the propertiesof these soil isolates.Of the various unique properties displayed by

VOL 18, 1969 119

A.israds, A.uaas- A.odoxio- A.,iscosus A. aiksoxii10048 lundsi 12104 lyticas 17929 15987 15423

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120 GLEDHILL AND CASIDA

A. humiferus, lysozyme sensitivity is of primarysignificance. Of the strains examined, 93% weresusceptible to lysozyme lysis during growth, aphenomenon that we have not been able todemonstrate for other Actinomyces or Rothiaspecies. Also, protoplasts could be prepared byadding lysozyme to actively growing or earlystationary phase cells in an osmotically protectivemedium. Thus, although the general chemicalcomposition of A. hwniferus, as determined afteracid hydrolysis, is similar to that for otherActinomyces species, its lysozyme sensitivity in-dicates a fundamental difference in cell wallstructure.

A. humiferus displays more characteristics incommon with A. eriksonii (Table 3) than withother Actinomyces species. However, major dif-ferences are that A. eriksonii is an obligateanaerobe, its cell walls contain galactose as themajor sugar (13), and it does not ferment rham-nose.The generally aerobic nature of A. humiferus

is shared by the genus Rothia. This similaritycould prove important from a phylogeneticstandpoint if it is considered that A. humiferusmight represent a transitional group between theanaerobic, catalase-negative Actinomyces speciesand the aerobic, catalase-positive species ofRothia. In addition to its catalase reaction, Rothiadiffers from A. hwniferus by its ability to reducenitrate, inability to grow on organic acids (11),lysozyme insensitivity, proteolytic nature, cellwall composition, and fermentative ability.The occurrence of an Actinomyces species as a

major population of certain soils is of bothecological and medical importance. Since thisspecies is a numerically dominant soil inhabitant,it must possess certain unique capabilities whichallow population maintenance at a high level. Onemight postulate either dormancy of the cells insoil, or the presence in soil of a food source ofunique composition: one that is not readilydecomposed by other soil microflora and is con-stantly present in sufficient quantities to sustainthis large population. Since A. humiferus is pro-teolytic (at least for casein) and has only beenfound in soils with relatively high organic con-tents, humiclike soil material might offer anattractive possibility for a natural nutrient source.Detailed ecological studies must be initiated toprovide answers pertaining to the nutrient sourceand also to establish if these organisms are asfunctionally important in soil as their numbersmight suggest.

Further study of the relation of A. humiferus tomedically important Actinomyces species couldeventually lead to a clarification of the epidemi-ology of the diseases caused by the latter organ-

APPL. MIcRoBioL.

isms. It is hoped that with the development anduse of additional suitable isolation techniques,such as that employed in this study, other soilActinomyces species can be isolated which will beof a more anaerobic nature than A. humiferus andbetter able to adapt to growth at 37 C. Theconcept of soil as a natural reservoir for infectiousActinomyces species does not seem so remote whenone considers the greater prevalence of Actino-myces infections in grazing as opposed to carniv-orous animals and rural as opposed to urbanpopulations (30).

ACKNOWLEDGMENTS

The immunological relationships of our isolates to variousgenera of the Actinomycetaceae were determined by J. M. Slackand M. A. Gerencser (Medical Center, University of West Vir-ginia). Deoxyribonucleic acid base compositions were determinedby M. Mandel (M. D. Anderson Tumor Clinic, University ofTexas).

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