Simple Genetic Transformation Assay for RapidDiagnosis

APPLIED MICROBIOLOGY, Jan. 1974, p. 16-24Copyright 0 1974 American Society for Microbiology

Vol. 27, No. 1Printed in U-SA.

Simple Genetic Transformation Assay for Rapid Diagnosis ofMoraxella osloensis

ELLIOT JUNI

Department of Microbiology, The University of Michigan, Ann Arbor, Michigan 48104

Received for publication 15 August 1973

A genetic transformation assay for unequivocal identification of strains ofMoraxella osloensis is described. In this assay a stable tryptophan auxotroph istransformed to prototrophy by deoxyribonucleic acid (DNA) samples from otherstrains of M. osloensis but not by DNA samples from unrelated bacteria. The testis simple to perform and definitive results can be obtained in less than 24 h. Theprocedure, which is suitable for routine diagnosis in a clinical laboratory,involves a rapid method for preparation of crude transforming DNA from smallquantities of bacterial cells and permits simultaneous examination of largenumbers of isolated cultures. The assay was shown to correctly identify 27 strainspreviously classified as M. osloensis. Forty-five other gram-negative, oxidase-positive, nonmotile coccobacilli, which might be confused with M. osloensisunless subject to more extensive testing, were shown to be unrelated geneticallyto M. osloensis. The transformation assay clearly distinguishes M. osloensis fromAcinetobacter. Although most strains of M. osloensis are nonfastidious, beingable to grow in a mineral medium supplemented with a single organic carbonsource, one of the strains tested was only able to grow on fairly complex mediaand could not be transformed to grow on simple media. Inability to alkalizeSimmons citrate agar was shown not to be characteristic of all strains of M.osloensis.

Several reports have appeared in the litera-ture implicating strains of Moraxella as thecausative agents of disease in man (5, 14, 16, 19,20, 21, 22, 33, 43, 45, 46, 48) and in animals (13,44). Taxonomic (4, 24) and genetic studies (9) ofaerobic gram-negative, oxidase-positive, andnonmotile coccobacilli, classified as Moraxella,have served to emphasize the existence of sev-eral distinct groups of these organisms whichappear to be more or less distantly related toeach other. To better establish the possible roleof moraxellas in disease it is essential to haverapid and positive methods for identification ofthese bacteria. It has been shown, however, thatit is frequently difficult to distinguish variousMoraxella strains from each other only byexamination of their phenotypic properties (12).

In 1962 Bovre and Henriksen (11) reportedthat many moraxellas are competent for genetictransformation of streptomycin resistancemarkers. As a result of quantitative interstraintransformation investigations Bovre (7, 8) wasable to demonstrate that strains formerly classi-fied as Moraxella nonliquefaciens could be di-vided into at least two distinct and genetically

compatible groups. Members of each groupshowed high ratios of inter- to intrastrain fre-quencies of transformation to streptomycin re-sistance. By contrast, very low transformationratios were observed when strains from each ofthe groups were compared (8). Although moststrains of M. nonliquefaciens have complexnutritional requirements, there is a geneticallydistinct group of Moraxella which can grow in amineral medium containing a single organiccarbon source. It has been suggested thatstrains in this group be classified as Moraxellaosloensis (12).

In a recent study of Acinetobacter it wasshown that deoxyribonucleic acid (DNA) sam-ples from all acinetobacters could transformauxotrophs of a genetically competent strain toprototrophy (29). It is possible to use thisprocedure as a routine diagnostic test for posi-tive identification of newly isolated strains ofAcinetobacter (29). The present investigationdescribes a similar simple diagnostic assay forrapid and unequivocal identification of strainsof Moraxella osloensis suitable for use in aclinical laboratory.

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RAPID DIAGNOSIS OF M. OSLOENSIS

MATERIALS AND METHODS

Bacterial strains. The strains of M. osloensis usedare listed in Table 1. Each strain bears the designa-tion which the culture or DNA sample had whenreceived.Growth media. Heart infusion agar (Difco) was

used for routine cultivation of all strains studied. Oneliter of lactic acid-mineral liquid medium was pre-pared by adding the following chemicals, one at atime, to 800 ml of distilled water until completelydissolved: lactic acid (reagent grade, supplied com-mercially as approximately 85%), 5 ml; KH2PO4, 1.5g; Na2HPO4, 13.5 g (or Na2HPO4.7H3O, 25.5 g);MgSO4, 0.1 g (or MgSO4.7H20, 0.2 g); NH4Cl, 2 g;CaCl2, 1 ml of a 1% solution; and FeSO4 7H20, 0.5 mlof a freshly prepared 0.1%7c solution. The final volumewas adjusted to 1 liter with distilled water (finalpH, 6.65) and sterilized by autoclaving for 20 min.Lactic acid-mineral agar plates were prepared bypouring a volume of lactic acid-mineral liquid me-dium (medium at room temperature) into an equalvolume of recently melted (90 to 100 C) sterile 3%agar, mixing, and pouring 15 to 20 ml per plate. Thesalts mixture used in this lactic acid-mineral mediumis the S-2 medium of Monod and Wollman (39).Preparation of the complete mineral medium, asdescribed above, avoids the precipitation of salts thatusually occurs when mixing the components of S-2medium. After drying in the inverted position allplates are stored in double plastic bags either at roomtemperature or in a refrigerator (5 C).

Preparation of crude transforming DNA. Asmall amount of bacterial cell paste on a bacteriologi-cal loop, from growth on any suitably plated medium,is carefully placed into 0.5 ml of a lysing solutionconsisting of sterile 0.05% sodium dodecyl sulfate instandard saline citrate solution (0.15 M sodium chlo-ride, 0.015 M Na3 citrate) contained in a screw-cappedtube (13 by 100 mm), and the cells are suspendeduniformly with the aid of an orbital mixer. Careshould be taken to avoid placing cell paste on the sideof the tube where it cannot come into contact with thedetergent solution. The suspended cells are thenheated in a deep 60 C water bath for 15 to 60 min, aprocedure which sterilizes the contents of the tube bycausing cell lysis and the release of intracellular DNA.DNA solutions prepared this way can be storedindefinitely in the refrigerator, if desired, providedthat the caps are screwed on tightly. If the lysingsolution, or DNA preparation, is permitted to evapo-rate before use, the concentration of sodium dodecylsulfate will increase to such an extent that therecipient cells to be transformed will be killed duringthe transformation assay. The use of screw-cappedtubes for containing the lysing solution facilitatespreparation of large numbers of such tubes at onetime and the tightly capped tubes may be storedindefinitely at room temperature.Auxotroph used in the transformation assay.

Auxotrophs of M. osloensis (strain 23) were preparedby mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine according to the procedure of Adel-

berg et al. (1). Strain trpE55, a mutant lacking afunctional anthranilate synthetase and requiringeither anthranilate or tryptophan for growth in lacticacid-mineral medium, was selected as the test orga-nism in the transformation assay since it is relativelystable and reverts spontaneously to prototrophy onlyrarely.

Transformation assay. A grid of squares ismarked on the bottom of a heart infusion platecontaining as many as 36 squares, as required. A smallamount of cell paste of auxotroph trpE55, grownovernight on a heart infusion plate at 34 to 35 C, isplaced in the center of one of the squares. Thequantity of cell paste used is not critical, an amountjust visible to the naked eye being sufficient. A sterileloopful (2 mm diameter loop) of crude DNA to betested is used to suspend the cells previously placedon the plate and the DNA-cell mixture is spread in acircular area somewhat smaller than the confines ofthe marked square. A second loopful ofDNA is spreadover the area of another square to serve as a sterilitycontrol; no growth should be visible in this squareafter incubation. A small amount of cell paste oftrpE55 is spread over the surface of a third square, thesubsequent growth in this square being used to checkthe stability of the auxotroph. Several DNA samplesmay be tested on the same plate, each sample beingmixed with trpE55 on a separate square, as describedabove. For each DNA sample a DNA sterility squareis also prepared. Only a single square with trpE55alone (non-DNA-treated control) need be made perplate. After incubation at 34 to 35 C for 2 to 24 h (seeResults for a discussion of incubation time) a generousportion of each growth area is streaked on a pie-shaped sector of a lactic acid-mineral agar plate. Asmany as eight sectors can be used conveniently perplate. One sector of each plate should be streakedwith trpE55 non-DNA-treated control cells. Thestreaked plate is incubated for 15 to 48 h at 34 to 35 C.After incubation the streaked areas are observed forcolonies derived from cells of trpE55 that were trans-formed to prototrophy during growth in the presenceof DNA on the heart infusion plate. The absence ofprototrophic transformant colonies after 48 h of incu-bation indicates that the organism being tested is nota strain of M. osloensis.

Since M. osloensis grows relatively slowly, observa-tion of transformant colonies at 15 h, or sooner, mayrequire use of a low-power dissecting microscope.After 24 h or more of incubation at 34 to 35 Cprototrophic transformant colonies will be clearlyvisible to the naked eye. The streak of non-DNA-treated trpE55 control cells should show no proto-trophic colonies. An extremely rare occasional colonyon the control sector is the result of spontaneousreversion of trpE55 to prototrophy.

Bacteriological tests. The oxidase test was carriedout by using the method of Kovacs (30). Ability toalkalize citrate medium was determined by streakingcultures on Simmons citrate agar (Difco) plates andincubating at 34 to 35 C. Each culture was streaked sothat both isolated colonies and massive growth couldbe observed on the same plate.

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TABLE 1. Strains tested

Organism Strain [ Received from | Isolated from

1. Mima Z42. M. polymorpha3. M. osloensis4. M. osloensis5. M. osloensis6. M. osloensis7. M. polymorpha var. oxidans8. M. osloensis9. M. osloensis

10. M. osloensis11. M. nonliquefaciens12. M. osloensis13. M. polymorpha var. oxidans14. M.osloensis15. M. osloensis16. M. osloensis17. M. osloensis18. M. osloensis19. M. osloensis20. M. osloensis21. M. osloensis22. M. osloensis23. M. nonliquefaciens

24. M. nonliquefaciens

25. M. osloensis26. M. osloensis27. M. osloensis

28. M. osloensis

29. Achromobacter sp.30. Achromobacter sp.31. Achromobacter sp.32. Achromobacter sp.33. Neisseria catarrhalis

34. Acinetobacter lwoffi35. Acinetobacter Iwoffi36. Mima polymorpha var. oxidans37. Mima polymorpha var. oxidans38. Mima polymorpha var. oxidans39. Mima polymorpha var. oxidans40. Micrococcus cryophilus41. Flavobacterium meningosepticum

42. Micrococcus cryophilus43. Moraxella nonliquefaciens44. Moraxella nonliquefaciens45. Mima polymorpha var. oxidans46. Moraxella nonliquefaciens

47. Moraxella phenylpyruvica

a DNA sample from M. Mandel.

a

RH486C572A608813482928375B9400B9762B9777CDC 9870a9893ATCC 10973aATCC 15276ATCC 17974ATCC 19954aATCC 19955aATCC 19956aATCC 19957aATCC 19958aATCC 19959ATCC 19960aATCC 19961 (19116/

51)aATCC 19962

ATCC 19963ATCC 19964ATCC 19965

ATCC 19976 (typestrain)

MJT F/4/11/5MJT F5/158MJT F5/199AMJT F5/21141

632633A4435A6571(1)A8620A9198(2)ATCC 12226ATCC 13253 as strainRH540

ATCC 15174ATCC 17953ATCC 17955ATCC 17960ATCC 19975 (Neotype

strain)ATCC 23333 (Neotype

strain)

M. MandelR. HughR. E. WeaverR. E. WeaverR. E. WeaverR. E. WeaverW. B. CherryR. E. WeaverR. E. WeaverR. E. WeaverM. MandelR. E. WeaverW. B. CherryATCCATCCM. MandelATCC

ATCC

ATCC

B. W. Catlin

B. W. Catlin

ATCCATCCATCC

S.D. Henriksen, K. Bovre,and R. E. Weaver

M. J. ThornleyM. J. ThornleyM. J. ThornleyM. J. ThornleyStock culture collection,

Dept. of Microbiology,Univ. of Michigan

G. L. GilardiG. L. GilardiR. E. WeaverR. E. WeaverR. E. WeaverR. E. WeaverATCCR. Hugh

ATCCE. J. OrdalE. J. OrdalATCCS. D. Henriksen and K.Bovre

S. D. Henriksen and K.Bo5vre

Blood

Blood

Nose

Spinal fluidGanglion of childUrineUrineMeningitis case'I

Patient with gon-orrhea-likesyndrome

Ulcer that devel-oped after veinstripping

Cerebrospinalfluid

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RAPID DIAGNOSIS OF M. OSLOENSIS

RESULTSAnalysis of DNA samples by the transfor-

mation assay. DNA samples from all 47 cul-tures listed in Materials and Methods weretested for ability to transform trpE55, a trypto-phan auxotroph of M. osloensis, strain 23, toprototrophy. Figure 1 shows the growth oftrpE55 after mixing with DNA samples fromeach of five different strains of M. osloensis, aswell as growth of trpE55 in the absence of addedDNA. Cell paste from each growth area wasstreaked heavily on a sector of a lactic acid-min-eral agar plate. Growth of cells of trpE55 thatwere transformed to prototrophy may be seen inFig. 2. It is evident that DNA from each of thefive strains of M. osloensis readily transformedtrpE55 to prototrophy. Sector A of Fig. 2, whichcontains a streaking of non-DNA-treatedtrpE55 (grown on square A, Fig. 1), shows no FIG 2. Interstrain transformation of trpE55. Cellprttrpi coois onl th amun of cell FG .Itrtantasomto ft .Clprototrophic colonies, only the amount of cell paste from the growth areas of the plate shown in Fig.

paste originally streaked on this sector being 1 were spread on sectors of a lactic acid-mineral agarvisible. Similar results were obtained with DNA plate and incubated for 48 h at 34 to 35 C. The letterssamples from strains 1 to 28, all of which had in this figure correspond to the similarly letteredbeen previously identified as strains of M. growth areas of Fig. 1. The growth observed is that ofosloensis. prototrophically transformed cells of trpE55 which areDNA samples from strains 29 to 47 all failed now able to synthesize their own tryptophan and are

to give even a single prototrophic transformant capable of growing on this simple medium. Only theoriginal amount of cell paste of trpE55 streaked insector A (non-DNA-treated control) is visible, thisauxotroph being unable to grow in the absence oftryptophan.

colony when used to treat trpE55 in the mannerdescribed above. All of these strains are gram-negative, oxidase-positive coccobacilli that canbe readily confused with M. osloensis unlesssubjected to further testing. In addition tostrains 29-47, a series of 27 oxidase-positiveMoraxella-like organisms were also examined.These bacteria were selected for study by theSubcommittee on the Taxonomy of Moraxellaand Allied Bacteria of the International Com-mittee on Nomenclature of Bacteria becausetheir taxonomic position and relationship toMoraxella have not yet been determined. Un-like authentic Moraxella species, most of theseorganisms form acid aerobically from sugars

FIG. 1. Growth of mixtures of trpE55 and DNA on and some of them also fail to grow at 35 C. DNAheart infusion agar for 21 h at 34 to 35 C. The growth samples from all these 27 strains failed toareas contained: A, no DNA; B, DNA from strain 6; transform trpE55 to prototrophy. DNA samplesC, DNA from strain 9; D, DNA from strain 14; E, from a variety of oxidase-negative acinetobac-DNA from strain 19; and F, DNA from strain 26. Each ters likewise failed to transform trpE55 toDNA sample used was also spread in the square prototrophy. All the M. osloensis strains exam-immediately to the right of the respective trpE55- proto the M.osloeni e xam-DNA mixture to verify that the DNA preparation was ined in the study of Baumann et al. (4) andsterile. The marks in the central portion of the growth seven of the 10 strains tested by B ivre(8) areareas were made when cell paste was removed with a included in the series of strains analyzed in theloop and spread on sectors of the lactic acid-mineral transformation assay described above.agar plate shown in Fig. 2. Optimum conditions for the transforma-

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tion assay. In the first step of the transforma-tion assay, cell paste from growth on anysuitable medium of the organism to be tested, isused for the preparation of crude transformingDNA. The amount of cell paste taken is notcritical and even the cell material in part of asingle small colony is sufficient for this purpose,as much as possible of the colony being trans-ferred with a loop to the lysing solution. Thetime of heating can vary considerably and maybe extended for several hours with no damage tothe DNA. Although 60 C is the recommendedtemperature for heating, temperatures from 55to 70 C may be used since even the highestvalue is below the melting temperature for DNAfrom M. osloensis, which has a DNA composi-tion of 43 to 43.5 mole % guanine plus cytosine(35). Cells incubated in the lysing solution arekilled within a few minutes, even at roomtemperature. Heating this cell suspension, how-ever, does accelerate lysis and release of intra-cellular DNA. When crude DNA is mixed withcells of trpE55 on a plate, as in the transforma-tion assay described above, detergent (sodiumdodecyl sulfate) in the DNA solution is ab-sorbed into the agar rapidly enough so that onlya few of the recipient cells are killed. The largemolecules of DNA are retained on the agarsurface until they are subsequently taken up bythe competent cells.The next step in the transformation assay

involves growth of trpE55 in the presence ofDNA. During growth on heart infusion agar thecells pass through a competency phase whereDNA is taken up. If the DNA used is derivedfrom a strain of M. osloensis it can recombinewith the chromosomal DNA of the recipienttrpE55 cells, some of which will be transformedto prototrophy. Since the interval of compe-tency is not known for growth of a competentstrain on semisolid media, a test was performedwhere trpE55 was mixed with DNA from M.osloensis, strain 19, and incubated at 34 to 35 Cfor various periods of time before the cellpaste-DNA mixture was streaked on sectors of alactic acid-mineral agar plate. The results ofthis study are shown in Fig. 3. It can be seenthat prototrophic transformant colonies ap-peared even when the cell paste was streaked onlactic acid-mineral agar immediately after mix-ing with DNA. The number of transformantcolonies increased significantly, however, whenincubation of the cell paste-DNA mixture wasextended to at least 2.5 h before streaking on theminimal agar plate (Fig. 3B). Although incuba-tion with DNA prior to streaking was onlycontinued for 12.5 h in the test illustrated inFig. 3, it has been shown that incubation

FIG. 3. Effect of time of incubation of trpE55 withDNA from another strain of M. osloensis prior tostreaking on lactic acid-mineral agar. DNA fromstrain 19 was mixed with trpE55 on squares of a heartinfusion agar plate at different times and incubated at34 to 35 C. The DNA-cell paste mixtures were thenstreaked on sectors of a lactic acid-mineral agar plateand incubated for 48 h. The colonies seen on this plateare derived from prototrophically transformed cells oftrpE55. The times of incubation of the DNA-trpE55mixtures on the original heart infusion agar plate (notshown) were: A, Oh; B, 2.5 h; C, 5 h; D, 7.5 h; E, 10 h;and F, 12.5 h.

periods as long as 24 to 48 h also result inmaximum numbers of transformant colonies.Because it is desirable in clinical procedures toperform the entire transformation assay in asshort a time period as possible, it is clear thatthe step in which incubation of cell paste oftrpE55 with DNA occurs can be extremelyshort. It is recommended that this incubationtime be at least 2 to 5 h in order to assurereasonably large numbers of prototrophic trans-formants for DNA samples from strains of M.osloensis.The assay step requiring the longest period of

time is the one in which prototrophic transform-ant cells of trpE55 grow to form visible colonieson sectors of the lactic acid-mineral agar plate(Fig. 2). Since M. osloensis has a longer genera-tion time than organisms such as Acinetobacteror Escherichia, it is important that the test beperformed in such a way as to permit a max-imum time for growth. If assays are startedearly in the morning the lactic acid-mineralagar plate can be streaked within a few hours.This will make it possible to look for proto-trophic transformant colonies by the next morn-ing. Early recognition of prototrophic colonies is

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greatly facilitated by the use of a low-powerdissecting microscope. The inclusion on thelactic acid-mineral agar plate of a sectorstreaked with trpE55 which has not beentreated with DNA (non-DNA-treated control,Fig. 2A) is most useful for comparative purposeswhen looking for such early transformant colo-nies. Furthermore, the non-DNA-treatedtrpE55 control serves to insure that the sampleof trpE55 used in the test has not revertedgrossly to prototrophy. This control also showsthat trpE55 is not contaminated with otherbacteria capable of growing on the lactic acid-mineral agar plate.An incubation temperature of 34 to 35 C is

suggested for growth of trpE55 on heart infusionand lactic acid-mineral agar. Although M.osloensis does grow well at 37 C, it is similar toAcinetobacter in that the optimal temperaturefor growth is slightly below body temperature.Plates may also be incubated at room tempera-ture. In this case, however, growth will besomewhat slower than is obtained at the opti-mal temperature. Although transformant colo-nies are visible to the naked eye after 24 h ofincubation, the plates used in Fig. 2 and 3 wereincubated for a total of 48 h for photographicpurposes.Mutants suitable for the transformation

assay. trpE55 was chosen as a test organismfor the transformation assay because of itsinherent stability and also because of the highefficiency with which it is transformed to proto-trophy by DNA samples from other strains ofM. osloensis. The ability of heterologous M.osloensis DNA samples to transform auxotrophsof strain 23 requiring either leucine, arginine, orhistidine for growth in lactic acid-mineral me-dium has also been demonstrated.Growth factor and carbon source re-

quirements ofM. osloensis strains. It has beenreported that strains of M. osloensis are allcapable of growing in a simple acetate-mineralmedium (4, 12) and should thus show no re-quirement for growth factors. All the strains ofM. osloensis examined in the present study,with one exception, have indeed been shown tobe able to grow in simple mineral media supple-mented with acetic acid. The one exceptionalstrain (strain 2) will only grow on complexmedia such as heart infusion or antibioticmedium 3 (Difco) agar. Virtually no growth ofthis strain takes place on nutrient agar (Difco)or on lactic acid-mineral agar supplementedwith vitamin-free casein hydrolysate. Strain 2,received from R. Hugh, was described as beingidentical with ATCC 10973. A culture of ATCC10973 (strain 13), received from W. B. Cherry,

was able to grow on an acetate-mineral mediumand appears to be identical with the same straintested by Baumann et al. (4). It seemed possiblethat strain 2 might be a spontaneous auxo-trophic mutant of strain 13. Since strain 2 iscompetent for genetic transformation, as shownby its ability to be transformed to streptomycinresistance using DNA samples from strep-tomycin-resistant mutants derived from severalstrains of M. osloensis, an attempt was made totransform strain 2 to prototrophy with DNAfrom strain 13. This experiment was not suc-cessful, however, not even a single prototrophictransformant of strain 2 being obtained. Fur-thermore, attempts to isolate a spontaneousprototrophic revertant of strain 2 were alsounsuccessful.

It is generally considered that all strains ofM.osloensis either grow slowly or not at all onSimmons citrate agar (12). In all cases reportedno strain ofM. osloensis has been observed to beable to alkalize this medium. All strains of M.osloensis examined in the present report werestreaked on Simmons citrate agar since it wasreasoned that even strains that grow slowly onthis medium might give some evidence of alkali-zation. The results listed in Table 2 show thatmost of the strains tested do not grow onSimmons citrate agar. Several strains appear togrow slowly on this medium with two strainsshowing weak alkalization after prolonged incu-bation. By contrast, strain 23 grows well on thisplate giving strong alkalization in less than 24 h.

DISCUSSIONThe suitability of genetic transformation as a

means of establishing taxonomic relationshipsamong various strains of Moraxella was firstreported in 1962 by Bovre and Henriksen (11).Using interstrain transformation of strep-tomycin-resistance markers Bovre (9) was ableto show that there appear to be several distinctgroups of Moraxella, members of a particulargroup showing high ratios of inter- to intraspe-cies transformation (range, 0.3 to 1.0), thecorresponding ratios for transformation betweenMoraxella strains from different groups beingconsiderably lower (usually less than 10-4).

In a transformation study of various strainsoriginally designated M. nonliquefaciens, hightransformation ratios (0.34 to 0.99) were foundfor 20 of the 22 strains investigated, ratios lessthan 2 x 10-5 being obtained for interstraintransformation between the remaining twostrains and representatives of the major class oforganisms (7). One of these unusual strains(strain 19116/51) was considered to possiblyrepresent a new taxonomic group (7). In 1965

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Bovre (8) showed that nine other independentlyisolated strains belong to the "19116/51" groupsince there were high ratios of inter- to intraspe-cies transformation of streptomycin resistance(range, 0.32 to 1.0) among the various membersof this group. On the basis of these studies,Bbvre and Henriksen (12) proposed that strainsbelonging to the "19116/51" group be desig-nated M. osloensis. This suggestion necessi-tated a new, more restrictive, definition of theM. nonliquefaciens group (12). Unfortunately,the phenotypic properties used to distinguishstrains of M. osloensis from strains of M.nonliquefaciens frequently overlap making itnecessary to rely on the results of transforma-tion studies for definitive diagnosis (12). Inter-strain transformation of organisms now knownto be M. osloensis has also been shown by Catlinand Cunningham (15).Both M. osloensis and M. nonliquefaciens are

aerobic, gram-negative, oxidase-positive, non-motile, nonsporeforming coccobacilli which donot produce acid from hexoses. Unlike M.nonliquefaciens, M. osloensis can grow in Hughand Leifson's medium (8, 12). Strains of M.osloensis are also somewhat more stable toheating than are strains of M. nonliquefaciens(12). The DNA base compositions of strains ofM. nonliquefaciens range from 40 to 42 mole %guanine plus cytosine, whereas the correspond-ing range for strains of M. osloensis is 43 to 43.5mole % guanine plus cytosine (12). It is quiteclear that phenotypic characteristics alone can-not be used with certainty to identify strains ofM. osloensis. Although strains of M. osloensisare said to grow on Simmons citrate mediumwithout alkalization (12), the results of thepresent study (Table 2) reveal that only somestrains are able to grow on this medium, a few ofthese giving rise to an alkaline reaction. It hasbeen reported recently that only a maximum of10% of M. osloensis strains grow in citratemedia when several passages are required (H.Lautrop, personal communication cited in ref.10).

TABLE 2. Growth of strains of M. osloensis onSimmons citrate agar

Type of growth Strain no.

No growth ........ 3, 6, 7, 8, 9, 14, 15, 17, 19,21, 24, 28

Poor growth ........ 4, 5, 10, 12a, 13, 25°, 26, 27Good growth ........ 23C

0 Alkalization after 3 to 4 days.a

Alkalization after 10 days.c Alkalization within 24 h.

The finding that one strain of M. osloensis(strain 2) is not, unlike the other strains stud-ied, able to grow in simple mineral mediasupplemented with a single carbon sourceserves to emphasize the fact that nonfastidious-ness cannot be taken as an absolute criterion fordiagnosis of strains of M. osloensis. Further-more, a new species, M. urethralis, has beendescribed (32) which grows on a simple acetate-or hydroxy butyrate-mineral medium and is notgenetically related to M. osloensis. Strain 45,originally described as Mima polymorpha var.oxidans, has been classified as M. urethralis(32). It is now clear that organisms formerlydescribed as Mima polymorpha var. oxidans (2,17, 23, 42) may, upon genetic analysis, prove tobe strains of either M. osloensis, M. non-liquefaciens, or M. urethralis.To date, streptomycin resistance has been the

principal marker used in transformation studiesof Moraxella strains. In order to perform trans-formation with this marker it is first necessaryto isolate a streptomycin-resistant mutant ofthe strain under study and then prepare DNAfrom this mutant for transformation of anothercompetent streptomycin-sensitive strain (6).Furthermore, the methods used for isolation oftransforming DNA are quite time consumingand require rather large quantities of cells (6).The fact that most strains of M. osloensis growin a simple mineral medium supplemented witha single carbon source, such as acetic or lacticacid, makes it possible to isolate auxotrophicmutants and use these as markers for transfor-mation studies. The development of a simpleand rapid procedure for the preparation of crudeand sterile transforming DNA samples from alarge number of bacterial strains has alreadyproven useful in the development of a transfor-mation assay for Acinetobacter strains (29).

In the present study this transformationassay has been modified for use in the routinediagnosis of strains of M. osloensis. It is gener-ally acknowledged that genetic interaction, asevidenced by ready interstrain transformation,is among the best means of establishing taxo-nomic relatedness (28, 34, 36). This is particu-larly true when the characteristics transformedare nonribosomal auxotrophic markers. In orderto transform a competent auxotroph to proto-trophy with DNA from another strain it isessential that the base sequence of donor DNAin the region of the marker be nearly identicalwith that of recipient chromosomal DNA in thesame region in order for recombination to takeplace (9, 25). Organisms that are geneticallyunrelated must have considerably different

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RAPID DIAGNOSIS OF M. OSLOENSIS

DNA base sequences in the corresponding re-gions of their respective chromosomes since ithas been well documented that in such casesthere can be virtually no DNA-DNA hybridiza-tion (27, 28, 34, 36-38). For example, there isalmost no homology between DNA species fromM. osloensis and Acinetobacter (26). It has beenshown, however, that ribosomal ribonucleicacid (rRNA)-DNA hybridization can occurusing ribosomal RNA species from strains un-related to those used as the source of DNA (26,40, 47). This finding has led to the conclusionthat the base sequences of ribosomal RNAspecies must be highly conserved in a widevariety of living forms, possibly because anyextensive compositional changes in ribosomalcomponents may result in poorly functioningribosomes (18, 47). Although there is little or nomeasurable homology between DNA samplesfrom M. osloensis and Acinetobacter, there is,nevertheless, good intergeneric rRNA-DNA ho-mology for these two organisms (26).Streptomycin resistance has been shown to

result from mutational alteration of a ribosomalprotein (41). Since the amino acid sequences ofribosomal proteins are also more conserved thanthe amino acid sequences of other proteins (18),the use of the streptomycin resistance marker ininterstrain crosses may reveal distant relation-ships which might not otherwise be evident ifless conserved genes, as represented by auxo-trophic markers, were used in such crosses. Thefact that DNA samples from all 27 strains of M.osloensis were able to readily transform fourdifferent unlinked auxotrophic markers ofstrain 23 in the present study must be taken asstrong evidence for the close genetic relatednessof all these strains. It will be of interest tocontinue testing DNA samples from new iso-lates of M. osloensis for ability to transformseveral auxotrophic markers of strain 23 todetermine whether strains have evolved in na-ture having extensive chromosomal alterationssuch that interstrain recombination of certainmarkers has become quantitatively less effi-cient. Such evolutionary changes have beenobserved in a study of several strains ofAcinetobacter (E. Juni, unpublished data).

All strains of M. osloensis analyzed in thisstudy were obtained from human materials.The natural sources for these organisms appearto be the genitourinary tract, spinal fluid,blood, the pleural cavity, and more rarely thenose and respiratory tract (12). Oxidase-posi-tive moraxellas have never been isolated fromsoil or water (3). Relatively few reports haveappeared specifically implicating M. osloensis

in human disease. In all probability this is aresult of difficulties frequently encountered inidentification of M. osloensis in clinical labora-tories. One report recently appeared in which anauthenticated strain of M. osloensis was shownto be the causative agent of septic arthritis andvaginal discharge in a young girl (20). It is alsopossible that other infections caused by orga-nisms described as Mima polymorpha var.oxidans may in fact be cases where M. osloenisis the infectious agent (5, 14, 19, 21, 22, 31, 33,43, 45). Introduction of the transformation assayas a routine diagnostic procedure for unequivo-cal identification of strains of M. osloensis, asdescribed in this report, should help in assessingthe distribution and clinical significance of thisorganism.

ACKNOWLEDGMENTS

The following individuals very generously supplied cul-tures used in the present study: K. Bovre, B. W. Catlin, W. B.Cherry, G. L. Gilardi, S. D. Henriksen, R. Hugh, M. J.Thornley, and R. E. Weaver. A few DNA samples from somestrains of Moraxella osloensis were supplied by M. Mandel.

This investigation was supported by Public Health Servicegrant AI-10107 from the National Institute of Allergy andInfectious Diseases.

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