APPLIED MICROBIOLOGY, Jan. 1968, p. 33-38 Vol. 16, No. 1

Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Morphological and Biochemical Differentiation ofAchromobacter and M/oraxella (DeBord's

Tribe Mimeae)1GERALD L. GILARDI

Microbiology Department, Hospital for Joint Diseases and Medical Center, New York, New York 10035

Received for publication 12 July 1967

To determine the most useful laboratory tests for the differentiation of Achromo-bacter anitratus, Achromobacter lwoffii, and Moraxella duplex (DeBord's tribeMimeae), 157 strains of these bacteria, isolated from clinical specimens, were ex-amined for their morphological and biochemical characteristics. There were sev-eral differences between these nonfermentative, gram-negative diplococci: Morax-ella was nonglucolytic in either infusion base or synthetic base, oxidase-positive,and sensitive to penicillin, whereas Achromobacter produced variable carbohy-drate activity, and was oxidase-negative and resistant to penicillin. A. anitratuswas distinguished from A. lwoffii in that the former utilized infusion media con-taining either glucose or 10% lactose, whereas the latter did not. Both species uti-lized the same carbohydrates in a chemically defined medium, although the latteracted more sluggishly.

Various bacteria, previously considered to besoil or water saprophytes harmless to humans,have been found recently to be pathogens orpotential pathogens. One such group of bacteriais DeBord's (11, 12) tribe Mimeae, comprised ofMima polymorpha var. oxidans, Mima poly-morpha, and Herellea vaginicola. [These termshave appeared frequently in the literature buttheir dubious status has also been reported(32).] Other names used to describe these bac-teria include Bacterium anitratum (36), Diplo-coccus mucosus (27), Neisseria winogradskyi (26),B5W (38), Moraxella (28), Achromobacter (5),Acinetobacter (6), and Cytophaga (25). Recentinvestigations (10, 19, 20, 32) have suggested thatthe correct designations for the bacteria referredto by DeBord are, respectively, Moraxella duplex,Achromobacter lwoffii, and Achromobacter ani-tratus. The discrepancy in the nomenclature isdue, in part, to the general characteristics of thisgroup. Morphologically these gram-negativebacteria occur frequently as diplococci resem-bling Neisseria; biochemically they resemble thePseudomonas-Alcaligenes group in that theyeither attack glucose oxidatively or apparentlydo not utilize it at all. Although many reports

IPreliminary identification of the strains studiedwas made by the bacteriology laboratory of the Hos-pital for Joint Diseases and Medical Center, underthe direction of Jessie Maple.

have appeared in the literature concerning thisgroup of bacteria, descriptions of these organ-isms, in standard books of microbiology, arescant or missing altogether. Laboratory techni-cians may be unaware of the characteristics ofthis group, with the result that the bacillus isignored or misidentified when recovered frompathological materials.The present study was undertaken to correct

the general lack of knowledge concerningAchromobacter and Moraxella, and to deal withthe diagnostic problems associated with thesebacilli. The morphological and biochemicalcharacteristics of all strains of these bacteria,isolated from human sources, were examinedto devise laboratory tests which can be used toidentify these bacteria.

MATERIALS AND MErHODSCultures. From May 1965 to June 1967, 157 strains

of Achromobacter and Moraxella were isolated, fromhuman sources, in the Microbiology Department ofthe Hospital for Joint Diseases and Medical Center.Of these strains, 59 were isolated from variouswounds, 48 from urines, 14 from sputa, 7 from dia-betic ulcers, 6 from various abscesses, 3 each fromthroats, trachael secretions, and osteomyelitis, 2each from urethra, skin grafts, burn ulcers, and dia-betic gangrene, and 1 each from vagina, feces, esopha-geal washing, blood, ear, and lung at autopsy.

Morphological studies. Smears from TrypticaseSoy Agar (TSA; BBL) cultures, incubated for 24 hr

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at 37 C, were stained by Gram's method and thenobserved for microscopic morphology. Isolatedcolonies on TSA plates, containing 5% defibrinatedrabbit blood and incubated for 24 hr at 37 C, wereexamined for colonial morphology. Growth charac-teristics were observed on Kligler Iron Agar (KIA;Difco), Sellers' Medium (BBL), Salmonella ShigellaAgar (SS Agar; Difco), and MacConkey Agar(Difco). TSA slants were used for oxygen-require-ment studies and for growth-temperature studies at4 and 42 C. Motility was determined by microscopicexamination of a hanging drop of a Trypticase SoyBroth (BBL) culture incubated for 24 hr at 37 C.

Biochemical and sensitivity studies. Cultures wereincubated for 3 weeks at 37 C before tests were dis-carded as negative. The tests and media employedincluded: acid production from 1% glucose, fructose,galactose, mannose, rhamnose, arabinose, xylose,sucrose, maltose, lactose, mannitol, dulcitol, salicin,and dextrin (Purple Broth Base, Difco; basal saltsmedium); acid production from 10% lactose (PurpleAgar Base, Difco); acid production from 1% glucoseand lactose (OF Basal Medium, Difco); acid produc-tion from glucose, fluorescein production, nitrogengas production, and anaerobic growth in the presenceof nitrate (Sellers' Medium); hydrogen sulfide pro-duction (KIA); decarboxylase activity (DecarboxylaseBase M0ller, Difco); citrate utilization (Simmons'Citrate Agar, Difco); nitrate reduction (TrypticaseNitrate Broth, BBL); indole production (TryptoneBroth, BBL); urease activity (Christensen's UreaAgar, Difco); gelatinase activity (Nutrient Gelatin,Difco); and oxidase reaction (Oxidase Discs, Difco).The carbohydrates (10% solutions) were sterilized

at 10 psi for 10 min and were added aseptically topreviously heat-sterilized Purple Broth Base andbasal salts medium to give a final concentration of1%. Utilization of 10% lactose was studied by asep-tically adding this substance to previously sterilizedPurple Agar Base at 45 C. The basal salts mediumcontained, per 1,000 ml: (NH4)2SO4, 2.0 g; MgSO4,0.2 g; K2HPO4, 0.2 g; NaCI, 0.2 g; CaCl2, 0.1 g; andaqueous bromocresol purple, 0.015 g. Decarboxylaseactivity was determined according to the method ofMoller (29), with the exception that no paraffin oilseal was used. The OF Basal Medium studies were per-formed according to the technique of Hugh andLeifson (21); however, only tubes of basal mediumplus carbohydrate (without paraffin oil seals) wereused. Sellers' Medium (37) was used in accordancewith a previously described technique. Other mediawere prepared and tests were performed by use of themethods described by Edwards and Ewing (13), andantibiotic sensitivity was studied by the disc method(BBL Sensi-disc, low concentration). The criterionfor sensitivity was the appearance of a zone of in-hibition, regardless of size, around the disc after 18 hrof incubation at 37 C.

RESULTS

Morphological characteristics. In liquid media,strains of Achromobacter and Moraxella werepredominantly gram-negative diplococci, with

some strains demonstrating diplobacilli or bacilli;on solid media, diplococci predominated. Somedegree of pleomorphism was evident in liquidmedia. Colonies on blood-agar were p:edomi-nantly circular, convex, entire, smooth, opaque,grayish-white to cream in color, glistening, andbutyrous. Some Achromobacter colonies weremucoid and stringy to the touch. Colonies of A.anitratus and A. iwoffii were 1 to 3 mm in diam-eter, whereas colonies of M. duplex were charac-teristically smaller (0.5 to 1.5 mm in diameter).Good growth was obtained on all media at 37 C,but only 38 strains of Achromobacter and 2strains of Moraxella grew on SS Agar. Nogrowth occurred at 4 C, but the majority ofAchromobacter strains and all of the Moraxellastrains grew at 42 C. Twenty-one strains of Achro-mobacter, but none of the strains of Moraxella,produced ,B-hemolysis on blood-agar plates.Strains of M. duplex characteristically grewslowly in comparison to Achromobacter. None ofthe Achromobacter or Moraxelia strains grewanaerobically. The biochemical results are indi-cated in Tables 1 and 2 and the results of sensi-tivity studies in Table 3.

DIscUSSION

In the identification of Achromobacter andMoraxella, irregular results were obtained fromcarbohydrate studies with infusion base media.Aiken, Ward, and King (1) reported that theyobtained more satisfactory utilization of carbo-hydrates by use of Herellea-like bacteria in achemically defined medium devised by Elrodand Braun (15). In the present studies, oxidationreactions in the synthetic medium of Elrod andBraun compared favorably with the oxidationreactions obtained by Aiken, Ward, and King.M. duplex failed to utilize any of the carbohy-drates tested, in either the infusion or the syn-thetic base. In the infusion base, A. anitratusconsistently produced acid from glucose, galac-tose, and xylose, but attacked fructose, mannose,and arabinose irregularly. In the synthetic base,A. anitratus consistently produced acid fromglucose, fructose, galactose, mannose, arabinose,xylose, and lactose, but produced acid fromrhamnose and maltose only irregularly. Acid wasproduced from rhamnose, maltose, and lactosein the synthetic base, but these substances werenot oxidized at all in the infusion base. A. lwofflidid not attack any carbohydrates in the infusionbase but oxidized carbohydrates in the syntheticbase in a pattern identical to that of A. anitratus,although the former acted more sluggishly. Theadvantage of using a synthetic carbohydrate baseis that the small amount of acidity produced by

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TABLE 1. Characteristics of Achromobacter and Moraxella isolated from clinical sources-

Test or substrate A. anitratus (121 strains) A. Iwoffii (30 strains) M. duplex (6 strains)

KIA ...ALK (121) ALK (30) ALK (6)Hydrogen sulfide......... + (92) - (29) + (17) - (13) - (6)Urea................... + (90) - (31) + (2) - (28) - (6)Citrate................... + (108) - (13) + (24) - (6) - (6)Indole .. .- (121) - (30) - (6)Motility - (121) - (30) - (6)Growth on MacConkey + (121) + (30) + (6)Growth on SS.......... + (29) - (92) + (9) - (21) + (2) - (4)Oxidase - (121) - (30) + (6)Nitrate to nitrite - (121) - (30) + (2) - (4)Hemolysis................ + (10) - (111) + (11) - (19) - (6)Anaerobic growth - (121) - (30) - (6)Gelatin - (121) + (11) - (19) + (2) - (4)Growth at 4 C - (121) - (30) - (6)Growth at 42C .......... + (115) - (6) + (23) - (7) + (6)SM Butt .. .NC (121) NC (30) NC (6)

Slant ...ALK (121) ALK (30) ALK (6)Glucose oxidation...... + (121) - (30) - (6)Fluorescence - (121) - (30) - (6)Nitrogen gas - (121) - (30) - (6)

Glucose (OFBM). + (121) - (30) - (6)Lactose (OFBM) ......... + (121) - (30) - (6)Lactose, 10% (PAB) ...... + (121) - (30) - (6)Ornithine - (121) - (30) - (6)Arginine.........- (121) - (30) - (6)Lysine .. .- (121) - (30) - (6)

- KIA = Kligler Iron Agar; SS = Salmonella Shigella Agar; SM = Sellers' Medium; OFBM = Oxi-dation Fermentation Basal Medium; PAB = Purple Agar Base; ALK = alkaline reaction; NC = nochange in indicator; + = positive test result; - = negative test result. Urease activity and hydrogensulfide production were detected after 48 hr of incubation with some strains; all other reactions weredetected after 24 hr of incubation.

TABLE 2. Oxidation of carbohydrates, in infusion and synthetic bases, by Achromobacter andMoraxella-

Infusion base Synthetic base

Carbohydrates A. anitratus A. iwoffii M. duplex A. anitratus A. lwofii M. duplex

A NC A NC A NC A NC A NC A NC

Glucose ............. 121 0 0 30 0 6 121 0 23 7 0 6Fructose...... 8 113 0 30 0 6 121 0 23 7 0 6Galactose ........... 119 2 0 30 0 6 121 0 26 4 0 6Mannose ............ 92 29 0 30 0 6 121 0 15 15 0 6Rhamnose........... 0 121 0 30 0 6 93 28 11 19 0 6Arabinose ........... 92 29 0 30 0 6 121 0 17 13 0 6Xylose .............. 118 3 0 30 0 6 121 0 22 8 0 6Sucrose .............. 0 121 0 30 0 6 0 121 0 30 0 6Maltose ............. 0 121 0 30 0 6 79 42 12 18 0 6Lactose .............. 0 121 0 30 0 6 121 0 15 15 0 6Mannitol.....0...O 121 0 30 0 6 0 121 0 30 0 6Dulcitol ............. 0 121 0 30 0 6 0 121 0 30 0 6Salicin .............. 0 121 0 30 0 6 0 121 0 30 0 6Dextrin .............. 0 121 0 30 0 6 0 121 0 30 0 6

a A = acid; NC = no change in indicator. Acid production was detected after 24 to 48 hr of incubationwith the majority of the strains but after 7 to 14 days of incubation with a few strains.

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TABLE 3. Sensitivity of Achromobacter andMoraxella to antibiotics as determined

by the disc methoda

A.. A. M.ani- lofidpeAntibacterial agent Disc concn tratus W(O30I duplext(ra21s) strains) strains)

Penicillin......... 2 units 0 3 100Novobiocin...... 5.g 10 17 100Erythromycin..... 2 lAg 19 24 100Lincomycin....... 2 Mg 0 0 50Ampicillin........ 2 Ag 37 40 100Tetracycline ...... 5 jSg 48 51 100Chloramphenicol.. 5 Ag 5 52 100Streptomycin..... 2 Ag 71 41 100Cephalothin ...... 30 ,ug 6 41 100Neomycin ........ 5 Mlg 100 100 100Kanamycin....... 5 Ag 100 100 100Polymyxin........ 50 units 100 100 100Colistin .......... 2 /g 100 100 100Furazolidine ...... 100 ,g 23 57 100Nitrofurantoin.... 100 gg 5 21 100Nitrofurazone .... 100 Ag 94 73 100Methenaminemandelate ...... 300 Ag 92 98 100

Triple sulfa....... 250 ,g 62 45 100

a Percentage sensitive.

oxidative metabolism is not neutralized by thealkaline substances produced from organicprotein media. It is evident that the use of thesynthetic base is particularly valuable in differen-tiating A. lwoffii from M. duplex.

Various media have been developed recentlyto aid in the identification of the nonfermenting,gram-negative bacilli. One such medium, de-veloped by Sellers (37), demonstrates anaerobicgrowth in the presence of nitrate, oxidation ofglucose in the presence of a high peptone con-centration, nitrate reduction, and fluoresceinproduction. Of the strains discussed here, onlyA. anitratus was capable of producing acid fromglucose. Although A. Iwoffli has been shown tooxidize glucose in a synthetic base, glucose oxida-tion was not evident in Sellers' peptone medium.None of the species was able to grow anaerobi-cally, even in the presence of nitrate. Magnesiumsulfate and mannitol were incorporated into themedium to stimulate fluorescein production, butthe strains produced neither fluorescein nor anyother pigment. Owing to the characteristic failureto reduce nitrates, Schaub and Hauber (36) sug-

gested the epithet "anitratus" for this group.Chilton and Fulton (8) were among the first

to observe that paracolons fermented 10% lactosein agar slants within 24 hr of incubation. Using amodification of Chilton and Fulton's medium,Aiken, Ward, and King (1) demonstrated that

all Herellea-like strains utilized 10% lactose-agarslants and that a few Mima-like strains alsoproduced acid. Other investigators (14, 31) havealso reported B. anitratum to be capable ofproducing acid in 10% but not in 1% lactose.In the present studies, all strains of A. anitratusoxidized 10% lactose, but A. Iwoffii and M. duplexdid not.Some reports did not distinguish between

oxidation or fermentation of carbohydrates byAchromobacter, whereas other reports incor-rectly described the metabolic process as fer-mentation. Hugh and Leifson (21) developed anoxidation-fermentation medium which aids indistinguishing between the two metabolic proc-esses. They classified the metabolic pattern ex-hibited by B. anitratum as an oxidative metabo-lism. Elston and Hoffman (16) reported that A.anitratus, but not Mima or Moraxella, oxidizedglucose and lactose in this medium. In the presentstudies, the oxidative type of metabolism wasdemonstrated for A. anitratus, whereas A. lwoffiand Moraxella demonstrated a nonoxidativemetabolism.

M0ller (29) was probably the first to develop apractical amino acid decarboxylase test for theidentification of bacteria. This test has beenused primarily in the identification of the Entero-bacteriaceae, and only a few reports discuss itsapplication to the identification of nonfermenta-tive bacilli. Ballard, Griffith, and Controni (2)performed decarboxylase studies with Herelleaand found it capable of decarboxylating lysine,arginine, and ornithine, but, on further examina-tion, Controni, Stewart, and Jones (9) failed todetect any evidence of a lysine decarboxylatingenzyme in Herellea. In the present studies, noneof the strains of Achromobacter or Moraxella wascapable of decarboxylating arginine, lysine, orornithine. The failure to detect decarboxylaseactivity may be due to the lack of acidificationof the medium for optimal decarboxylase activity.There is general agreement among investiga-

tors on the majority of the characteristics ofAchromobacter and Moraxella, but there hasbeen some, perhaps doubtful, variation regard-ing a few important characteristics. Some of thesecharacteristics also differ from results of thepresent investigation.

Reduction of nitrate to nitrite by Moraxellawas a variable reaction in the present studies.In DeBord's (12) original description of M.polymorpha var. oxidans, there was no reductionof nitrate. Beer (3) and Elston and Hoffman(16) found this to be a variable characteristic,and Piechaud (33) and Ballard, Griffith, andControni (2) found that all strains they studiedreduced nitrate. In the present study, none of the

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strains of A. anitratus reduced nitrate, but thereduction of nitrate to nitrite, and of nitrite tosome unidentified product has been demon-strated in B. anitratum by Jyssum and Joner (22).Achromobacter and Moraxella are described

as nonmotile, but Nelson and Shelton (30) andPike, Schulze, and McCullough (35) reportedobserving motile strains of Moraxella. Lautrop(25) was able to demonstrate a gliding motilityin B. anitratum and this was confirmed by Hal-vorsen (18). By examining bacteria in an oilchamber, Piechaud (34) demonstrated glidingmotility in Moraxella species as well as in B.anitratum.

In the present study, none of the strains of A.anitratus produced acid from maltose, lactose,sucrose, or rhamnose in infusion media, but acidproduction from these carbohydrates has beenreported (3, 7, 24). The strains of A. Iwoffiistudied in this investigation produced acid fromcarbohydrates in synthetic base only, but A.Iwoffii has been observed to produce acid fromcarbohydrates in infusion base (7, 23).None of the bacteria in the present study grew

under anaerobic conditions, but both A. anitratus(23) and A. iwoffii (14, 39) have been reported togrow anaerobically as well as aerobically.

Other reports indicate that A. Iwoffii (3, 4, 30)and Moraxella (3, 4) are uniformly incapable ofgrowing at 44 C, but I found that all strains ofM. duplex and the majority of the strains of A.anitratus grew at 42 C.

Evaluation of the present studies shows thatAchromobacter and Moraxella are typicallyanaerogenic, aerobic, gram-negative, nonmotile,nonpigmented diplococci or diplobacilli, whichneither produce indole nor demonstrate decar-boxylase activity. Moraxella is distinguishedfrom Achromobacter in that it is citrate-, hydro-gen sulfide-, and urease-negative; it is nonglu-colytic in either infusion or synthetic base; itdoes not hemolyze blood-agar and is oxidase-positive; it may reduce nitrate to nitrite; and itproduces relatively smaller colonies on agarmedia. Achromobacter produces variable citrateutilization, hydrogen sulfide production, ureaseactivity, carbohydrate activity, and hemolysis ofblood-agar; is oxidase- and nitrate-negative;and produces relatively larger colonies on agarmedia. A. anitratus is distinguished from A.lwoffii in that the former produces acid fromglucose in Sellers' Medium, Purple Broth Base,and OF Basal Medium, as well as from 10%lactose in Purple Agar Base, whereas the latterdoes not. Both species attack carbohydrates in achemically defined medium, although A. Iwoffliacts more sluggishly. Achromobacter is highlysusceptible to neomycin, kanamycin, polymyxin,

colistin, nitrofurazone, and methenamine man-delate, and moderately susceptible to streptomy-cin and triple sulfa. A. Iwoffii is slightly moresusceptible than A. anitratus, but both speciesappear to have a similar pattern. M. duplex issusceptible to all agents tested except lincomycin.M. duplex is distinguished from Achromobacterin that the former is characteristically susceptibleto penicillin, whereas the latter is not.

Since A. Iwoffii produced acid from carbohy-drates in the synthetic medium in a pattern simi-lar to that of A. anitratus, and because bothforms are oxidase-negative and resistant to peni-cillin, it appears that these groups represent twoclosely related species within the same genus.In view of the gradation of characteristics withinthe two forms, it does not seem advisable to splitthem into several species. Since M. duplex is non-glucolytic, oxidase-positive, and susceptible topenicillin, it appears that this microorganism isnot related to A. Iwoffii and A. anitratus. In arecent review (17) of the nomenclature of thetribe Mimeae, it was suggested that the correctbinomial designations for the bacteria describedby DeBord are M. duplex for M. polymorpha var.oxidans, A. Iwoffii for M. polymorpha, and A.anitratus for H. vaginicola.

It is concluded that the criteria for differen-tiating these bacteria are the oxidase reaction,susceptibility to penicillin, utilization of glucosein infusion and synthetic media, and the utiliza-tion of 10% lactose.

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