Characteristics and biotypes of Pasteurella multocida isolated from humans

0095-1137/81/030566-06$02.00/0

Characteristics

and

Biotypes

of Pasteurella

multocida

Isolated

from

Humans

THOMAS R. OBERHOFER

Microbiology Section, Department of Pathology, Madigan ArmyMedical Center, Tacoma, Washington 98431

Fifty-two isolates ofPasteurella (48 strains of Pasteurella multocida and 4

strainsofatypical Pasteurella) wereidentifiedby conventional andcommercial

testsystems.Ail strains fermentedglucose, sucrose, andfructoseinpurple broth

base (Difco Laboratories) with bromocresol purple as indicator, although the

atypicalPasteurellaproducedfermentationreactions thatwerebarely percepti-ble.Elevendifferentbiotypeswereidentifiedbyfermentationreactions in maltose,

mannitol, xylose, sorbitol, and trehalose media. There was a correlation of

biotypesto catbites,with 61% ofcatbite isolatesfalling into biotype A and B. A

correlation of biotype and dog bite isolateswasnot seen.The choice ofmedium

used for fermentation tests was critical as evidenced by the inabiity of the

organisms to grow in a second commercially purchased preparation ofpurple

brothbase. Thereliabilityof commercialtestsystemsinidentifyingPasteurella

was81% forOxi/Ferm(Roche Diagnostics,Div.Hoffmann-LaRoche,Inc.,Nutley,

N.J.), 68% for API (Analytab Products, Plainview, N.Y.), and 11%for Minitek

(BBLMicrobiologySystems, Cockeysville, Md.).

Recently therehas beenarenewed interest in the bacteriology ofanimal bites (9) and in the

aerobicbacterialfloraof oral and nasal fluids of

dogs and cats (1, 17). The organisms most

fre-quently isolatedfrom the oralcavityand

gingiva

ofdogsincludethreeunclassifiedgroupsof

aero-bic

gram-negative

bacteria, IIj, EF-4, and M-5,

(17), and Pasteurella multocida (1,17),

al-though thehigh incidence of the alphanumeri-cally designated bacteria has been discounted

(9). The principal agent isolated from the dog

bite seems to be P. multocida since the few studies on the

quantitation

of flora have not conclusively shown an

etiological

role of the

otherorganismspresent (1, 17).

Physiological and biochemical differences

havebeendocumentedamongstrains of P.

mul-tocida recovered from various animal

species

(10-12, 18). Isolates initially categorizedon the

basis offermentative reactions in various

car-bohydrates were also obtained from many

dif-ferent animal hosts, especially fowl (4, 5).

Al-though much is known about the biochemical and morphological characteristics of P.

multo-cida,includingthe fact that therecanbe

consid-erable variation in fermentation results with

these organisms, new and additional data

per-tainingtoclinical isolates from human infections

arestilllacking.

Few attempts havebeen madetobiotype

Pas-teurella isolated from infections of humans(21).

Itis the purpose of this report to describe the

biochemical properties of human isolates, to

present the biotypesobtained, and to focus

at-tention on the methods used to identify and

biotype the

organisms.

Itis also the purpose to determine and present any relationship between

the biotypes and the original source of human

infections.

MATERIALS AND METHODS

Organisms. Fifty-twoisolatesofPasteurella were

recoveredin the past 4 yearsfrom lesions from infected

humanpatients. The specimens were collected from

thewound areas onswabsor byaspiration and sent

for culturetotheMicrobiology Laboratory,Madigan

Army Medical Center. Almost all specimenswerethe

result of animal bites. A comprehensive survey of bacterial florawas notconducted in most cases,and anaerobic analysiswasperformed only with specimens identified as "wounds". Each specimen for aerobic study was streaked for isolation and morphological

studies to 5% sheep blood agar, chocolate agar, MacConkey agar, andthioglycolatebroth.

Ail

cultures

except thioglycolate broth were incubated overnight

at35°Cin anatmosphereof5%C02.

Biochemicaltests.Growthandcolony character-istics on theplatedmedia wereexaminedafter 24 and 48 h ofincubation. Blood agarplates served as an indicator of hemolytic activity and as a source of

inoculumforbiochemicaltests.The inoculum forall

tests consisted of one droporloopful ofa 4- to 6-h

cultureinTrypticase soy broth(TSB;BBL

Microbi-ology Systems,Cockeysville, Md.). Conventional bio-chemical tests were performed by the methods of Edwards andEwing (6).Theindoletestwasperformed

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using both peptone broth and TSB. Carbohydrate

fermentation tests were performed with

laboratory-preparedpurple broth base(PBB)containing bromo-cresol purple asindicator, which wasobtained from

twocommercialsources (DifcoLaboratories, Detroit,

Mich.; BBL Microbiology Systems). Filter-sterilized carbohydrate solutionswereaddedtothe sterile PBB (pH 6.8) to give afinal concentration of 1%.

Repro-ducibilityof fermentationtests was determined with

different preparations of the Difco PBB containing

carbohydrates.Thephysiologicaltests wereincubated

at35C in ambient air for5daysandexamineddaily

for reactions.

Theproductionof nitrite and indolewasdetermined

after 24 h ofincubation, with negative cultures retested after an additional 24 h of incubation. Indole tests

negativeat48hwerefurthertestedbyxylene

extrac-tionat 4 or 5days (14, 21). Motilitywasdetermined by use of both semisolid agar and theslidemethod after24and48hof incubationat35°C.Abilitytogrow

at42°Cwasdeterminedby growthonTrypticasesoy agarslants(15; BBLMicrobiologySystems).Both the spot indoletest (20) and the spotoxidasetestusing

1% tetramethyl paraphenylenediamine

dihydrochlo-ride were performed using growth taken from the

blood agarplateat24h.Tests fordeoxyribonuclease,

starchhydrolysis, esculin hydrolysis, and f-D-galac-tosidaseweredescribedpreviously (14).

The API system (Analytab Products, Plainview, N.Y.), theOxi/Fermsystem(RocheDiagnostics,Div. Hoffmann-LaRoche,Inc.,Nutley, N.J.),andthe Min-itek system (BBL Microbiology Systems) wereused

accordingtothemanufacturers' directions.

Antimicrobial susceptibility.The agar disk

dif-fusionsusceptibility test wasperformed accordingto

accepted techniques (13) using Mueller-Hinton agar

supplemented with 5%sheepblood.

RESULTS

Isolates ofP. multocida consisted of small,

coccoid,gram-negativebacil withanoccasional

tendencytoformfilaments.Thegram

morphol-ogy of

atypical

Pasteurella taken from blood

agar showed

staining

of

varying

intensity and exhibited both

large

and small coccobacilli. Col-onies on blood agar were 0.5 to 1.0 mm in

di-ameterand smoothwithanentire

edge,

increas-ing in size to 1.0 to 2.0 mm with continued incubationat

35°C.

Greening

onbloodagar was

generally

absent at 24 h but present at 48 h.

Growth onbloodpresenteda

distinctly

charac-teristic

odor,

although

the odor wasnot a

con-stantfeature until48h ofincubation.

The biochemical reactions of48strainsof P.

multocida and4strains ofatypical Pasteurella

are shown in Table 1. Indole production was

inconsistent in peptone broth because of poor

growth in this medium. Growth in TSB was

improved

although

incubationbeyond48hand

xylene extraction were still necessary in a few

instances. The spot indole tests wererapid,

in-tense, andeffective. Sevenisolates of P.

multo-TABLE 1. Biochemical characteristics of 52 human isolates ofPasteurella'

Testorsubstrate

Hemolysis Oxidase Catalase Motility Indole Peptone broth TSBc Spottested Methylred Voges-Proskauer Citrate Phenylalanine deaminase Urease Nitratereduction Gelatinase Lysinedecarboxylase Argininedihydrolase Ornithinedecarboxylase Starch hydrolysis Esculin hydrolysis Deoxyribonuclease ,B-D-galactosidase Malonate MacConkey (growth) Litmus milkchange 420C growth Acid from: Glucose Lactose Sucrose Maltose Mannitol Xylose Fructose Salicin Dulcitol Inositol Adonitol Arabinose Raffinose Rhamnose Sorbitol Trehalose Cellobiose P.multocida (48strains) +(+) % o o 48> 100 48 100 o o

42(1) 90 27(3) 97 31 100 o o o o o o o o o o

45(3) 100

o o

o o

o o

39(2) 85

o o o o o o o o o o o o o o 18 100 48 100 o o 48 100 10(1) 23

35 73 34 71 48 100 o o o o o o o o o o o o o o 29 60 33 69 o o Atypical Pasteurella (4strains) + (+) % o 0 4b 100 4 100 o O

1(3) 100

2 50 4 100 o 0 o o o o o o o o 4 100 o o o o o o 1 25 o o o o o o o o o o o o o o NI' NT 4b 100 o o

2(2) 100

o o o o o o 4" 100 o o o o o o o o o o o o o o o o 4b 100 o o

aSymbols:+, number positive within 2days; (+) number positive within 3 to 5days; %, percent positive after 5 days.

bWeakreactions.

CThirty-oneisolatestestedby the spot test and in Trypti-casesoy broth.

dEighteenisolatestested at

420C.

'NT, Not tested.

cida andthree isolates ofthe atypical

Pasteu-rella failed todecarboxylate ornithine,with the

negative tests welldistributed amongthevarious

biotypes.

Small amounts of acid with no gas were

formed in theglucose, sucrose,andfructose

me-dia (Table 1). Although fermentation was

prompt (18 to 24 h),the color reactioninPBB

causedby P. multocida was notintense yellow,

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568 OBERHOFER

as seen with enteric bacteria, but a subdued

yellow. Fermentation of maltose, mannitol,

xy-lose,sorbitol, andtrehalosebyP.multocidawas

avariable characteristic andprovided the basis

forbiotype formation.Incontrast,fermentative

abilitiesoftheatypical Pasteurellawereabsent

within 24h,althoughgrowth inPBBwas

abun-dant. Theseorganisms manifested acidity only

by discolorationof theindicatorafter 2or more

days of incubation.

Table 2 presents the 11 biotypes of

Pasteu-rella derived from the 52human isolates. The

four atypical Pasteurella comprise biotype K.

Otherthan 13 strains(25%) belongingtobiotype

B,there isanapparentevendistributionacross

fivebiotypes (A, D,E, F,andJ)of P.multocida,

accounting for 54% of the strains tested. The reproducibility in PBB of 124 negative and 63

positive fermentation reactionswith11isolates

was 100%.

Table 3 presents the source of isolation of

each biotype of Pasteurella. Ofthe 52strains,

42 were clearly related to animal bites or

scratches,and it islikelythat thewound isolates

were actually the result of animal bites or

scratchesaswell. Therewas an evendistribution

between dog and cat bites. Of the 18 cat bite

isolates, 11fellinto biotypes AandB, and the

cat-relatedstrains accounted for themajor

pro-portion of thestrains withinbiotypesAand B.

Table 4 shows the fermentative abilities of

isolates recoveredfrom humansas aresultof cat

anddogbites and of isolatesreported byother

investigators. The current data show that with

theexceptionof maltosefermentation,the

cat-associated isolates had a greaterpropensityto

fermentthe listed carbohydrates than did the

dog-associated isolates. The human isolates

re-ported by Heddleston (10) failed to ferment

maltose and demonstrated a limited ability to

fermenttrehalose. Therewasamarkedtendency

of thedog isolates in both the Heddleston (10)

and Smith (18) studies to ferment maltose in

contrast to the human and cat isolates.

Con-versely,the cat andcat-associated strainswere

moreactiveagainstmannitol.

Table5shows the results ofthree commercial

test systems used for the identification of P.

multocida. The Minitek, the API, and the

Oxi/

Fermsystemsidentified11,68,and81%,

respec-tively, of the isolates tested. Reproducibility of

the APIprofile (14 strains tested)wasonly64%.

Reproducibility of Minitek and Oxi/Ferm

re-sultswasnotattempted.

The results of in vitro susceptibility testsof

32strains of P. multocidato 17 antibiotics are

given in Table 6. Although interpretive

stand-ardsarenotavailable for Pasteurella, it is clear

thatpenicillin and thepenicillin derivatives,

tet-racycline, chloramphenicol, and the

cephalospo-rins, were highly effective in inhibiting the

growth of P. multocida. The results also indicate

variable susceptibility to the aminoglycosides,

andtotalresistance tovancomycin and

clinda-mycin is indicated by failuretoproduceany zone

ofinhibition inmostinstances.

DISCUSSION

The organisms known asP. multocida are a

collection of closely related bacteria with phe-notypic similarities, but possessing

characteris-ticswhichalso pointtostrain differences.

Bio-typesare easilyselected duetothe fermentation

ofmaltose, mannitol, and xylose (21), and

sor-bitolandtrehalose.Thesebiotypes, however, do

notaccountfor other obvious strain variations,

which include, in part: (i) failure to

decarbox-ylate ornithine, (ii) reluctance ofsomestrainsto

growin nitrate brotheven though testing

posi-tive for nitrite, and (iii) reluctance to grow in

peptonebroth due eithertolow saltcontentor

lack ofenrichment, undoubtedly accounting for

negative indole testsin this medium. None of

these aberrantgrowthpatternscanbeascribed

to any biotype. For example, organisms were

encountered thatgrewwell inpeptonebrothor

nitrate broth but failed to decarboxylate

orni-thine.

Theorganisms tentativelyclassifiedas

atypi-calPasteurellaspecies consisted of four isolates

whichproduced anacid slant and butt intriple

sugar iron agarwithin 24 h of incubation, but

TABLE 2. Biotypesof52humansstrainsof Pasteurella

Result for biotype (no. of strains) Carbohydrate

A(6) B(13) C(1) D(6) E(7) F(4) G(1) H(3) I(2) J(5) Kb (4)

Maltose + - + - - - + + - -

-Mannitol + + + + + - - - + -

-Xylose + + + + + - +

Sorbitol + + + - + - _ _ +

Trehalose + + - + - + + + - - +(w)

a+,Positivereaction;-,negative reaction;w,weak reaction.

bBiotyperepresentedby the fourstrainsofatypicalPasteurella.

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BIOTYPES OF PASTEURELLA 569

producedaweak acid

change

inPBBwith brom-ocresol purple indicator

only

after 3 or more

days of incubation. This

differing

fermentation pattern, although similarto

biotype

J,

setthem apart from typical P. multocida isolates. An associationwith thenegative ornithinetestswas

suggested

although

the number of strains tested

was toosmall forfirmspeculation.

Phenol red (10, 11, 16) and Andrade

(16,

18,

21) indicators have been used

successfully

to

characterize

Pasteurella,

andbromocresol pur-ple hasproven tobeasensitive and useful

indi-catorforuseinfermentation studies. The growth

media used for

carbohydrate

fermentationtests

and the choice of indicator are critical when testing PasteurellaandActinobacillus

(16).

Sig-nificantly,

carbohydrate

reactionsare

probably

oneof themost

likely

sourcesoferroneous iden-tifications, and routine

quality

controlmeasures

using anavid

fennenter

may not detect

slight

variances in

fermentative

abilities of the more

exacting organisms.

Thiswasdramatized when

twocommercial

products

wereused for fermen-tation tests.

Eight

strains ofP. multocidawere

tested in

parallel

using

PBB

(Difco

and

BBL)

containinga

full

complement

of

carbohydrates.

Whereas Difco PBB

supported good growth

of P.multocida with

subsequent overnight

fermen-tation and indicator

change,

twodifferent lots of

TABLE 3. Source of Pasteurellaisolates according

tobiotype

No. of strains of biotype Source

A B C D E F G H I J K

Dog bite 1 2 0 2 2 3 0 3 0 3 3

Catbite 4 7 0 2 3 1 1 0 0 0 0

Catscratch 1 1 0 1 1 0 0 0 0 0 0

Animal bite 0 1 0 0 0 0 0 O O O 0

Wound 0 2 1 0 1 0 0 0 0 0 O

Abscess 0 0 0 0 0 0 0 0 1 0 1

Unknown O O O 1 0 0 0 0 1 2 0

the BBL

product

failed to support

adequate

growth

after 3

days

of

incubation,

witha

resul-tantlack offermentation reactions. The

discrep-ancy in

growth

patternbetween the two

com-mercial media may be accounted for by the

Gelysate

in the BBL product as opposed to

proteose-peptone no. 3 in the Difco

product.

Reproducibility

of fermentation test results in

DifcoPBBwas100%.

The biochemical identification of P.

multo-cida is system dependent as wellas media

de-pendent. When usingtheAPIsystem,failureto code wasprincipally a result ofaninability to

attackthe carbohydrate substrates inthe

sys-tem. The applicability of the API system for

biotyping

wasnotfavorable, basedonthe

find-ings

thatonly64% of theprofilescould be

repro-duced. The misidentificationwith the Minitek

system or the failure to code in the system

resulted fromaninactive ornithine

decarboxyl-ase, in additionto the inabilitytoferment

key

carbohydrates.The misidentifications with the Oxi/Fermsystemsimply resulted from the

fail-ure of the manufacturer to allow for dextrose fermentationin theprofile code.

Reproducibility

studieswerenotattemptedwith theMinitekor

Oxi/Fermsystemsbecauseofeconomy.

Dogsaretheanimalsmostfrequentlyinvolved

in bite episodes, although cat bites result in

secondary infections threetimesasoften (7,12).

It is of interest thatmorethanhalf of the

Pas-teurelladescribedinthisreportwererecovered

from lesionsresulting fromcatbitesorscratches.

Smith(18) attemptedtoexplainwhy local infec-tions due to P. multocida are more common

after cat bites than dog bites by showing the

high mouse pathogenicity ofcat strains as

op-posedtodog strains.

Itis alsoaccepted thatmostvictimsofanimal

bites are children or young adults. Complete

clinicaldatawereobtainedonthe last 35victims

TABLE 4. Physiological characteristicsof P.multocidareported byvariousinvestigators %Positiveinpresent %Pitv e<dbyH eb % Positivereportedby

study % PositivereportedbyHeddleStonb% Mithb Substrateor test

Dog bite Cat bite Human iso- Dog iso- Catiso- Dog iso- Cat

iso-(16) (22) lates (33) lates (13) lates (22) lates (39) lates (31)

Maltose 25 27 0 54 5 44 29

Mannitol 44 91 100 31 100 15 77

Xylose 44 95 91 100 59 23 55

Sorbitol 31 77 94 32 82 18 55

Trehalose 69 82 24 69 32 67 42

Ornithine 81 86 NTC NT NT NT NT

Indole(peptone) 87 91 94 100 100 NT NT

aFigures in

parentheses

arenumbers of strains tested.

bHeddleston,

reference

10;Smith,

reference

18.

CNT,

Not tested. VOL.13,1981

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TABLE 5. Resultsofcommercial test systems to

identifyP. multocida

Discrepancysource Test sys- No. No.

identi-tem tested fied(%) No.with No. misi-nocode identified

API 31 21(68) 6 4

Minitek 18 2(11) 12 4

Oxi/Ferm 31 25(81) 0 6

TABLE 6. SusceptibilityofP.multocidato 17

antibiotiCsa

Zonesize No. No. % Sus- (mm) Antibiotic suscep-

cepti-tested tibleb ble' Me-Range dian

Penicillin 32 32 100 23-40 28 Ampicillin 32 32 100 21-37 28 Carbenicillin 32 32 100 27-35 32 Methicillin 32 32 100 19-29 20 Tetracycline 32 32 100 22-38 25 Chlorampheni- 32 32 100 22-41 30

col

Cephalothin 32 32 100 24-35 28

Cefoxitin 18 18 100 23-40 27

Erythromycin 32 23(9) 100 15-25 18

Colistin 32 32 100 11-16 13

Kanamycin 32 15(16) 97 11-29 17 Gentamicin 32 21(7) 88 12-25 16 Tobramycin 29 19(8) 93 10-29 18 Neomycin 30 14(11) 83 6-22 16 Streptomycin 30 15(8) 77 10-26 15

Vancomycin 31 0 0 6-9 6

Clindamycin 32 0 0 6-13 6

'Disk diffusion methodusingMueller-Hintonagar with 5%

sheep blood.

bFigures inparenthesesarethenumbergiving intermediate susceptibility values usingzonestandardsgiveninNational Committee forClinicalLaboratory Standards1979.

'Percentsusceptible includes thoseinintermediate cate-gory.

of animal bitesshowingthat,of15personsbitten

by dogs, 11 (73%) were under 15 years of age,

with 4 of the 11 dog bites resulting in facial

lacerations inchildren under 2 years of age. In

contrast,only5of17persons(28%)sufferingcat

bitesorscratcheswereunder the age of15years,

andeightofthevictims

(44%)

were overthe age

of 45years.A definitecorrelation between

bio-typesanddog-relatedstrainswas not seen.

How-ever, there was anassociation between biotype

and catisolates, with 13of22 (59%) of the

cat-related strains being biotype A or B. These

results support the view of Talbot and Sneath

(19) that cat strains (unlike dog strains), when

examined by computer, form a characteristic

andcomparatively homogeneousgroup.

A marked degree ofspecificity by P.

multo-cida for different host species of animals has

been reported (2), suggestingthat biochemical

characteristics may vary according to animal

origin. Moreover, the fermentation of maltose

by P. multocida is stated to be a characteristic of strains isolated from dogs and cats (2). Hed-dleston (10) demonstrated that strains of P. mul-tocida isolated from dogs were more apt to ferment maltose, whereas those from cats and humans rarely did. Furthermore, a series of 30 isolates from humans failed to ferment maltose, and only 1.9% of 1,088 isolates from birds and mammals other than humans fermented this substrate (11). The data from this study are not in accordance with those from other reports

since 11human isolates (21%), of which 7 were

recovered from wounds inflicted by cats and 4

were recovered from wounds inflicted by dogs,

fermented maltose. This maltose-positive

char-acteristic wasstable upon repeat testing.

Hed-dleston and Wessman (11) also suggested that all strains of human origin fermented mannitol, whereas the results of this study and others (8,

12, 18,21) are atvariance with these findings.

The differences in strains from animal

cul-turesand human cultures with respect to

man-nitol fermentation are difficult to assess at this time. Smith (18) reported that the majority of his 39 canine strains were mannitol negative (85%) and sorbitol negative (82%). Frederiksen (8) also noted the propensity for canine strains

to be negative for mannitol and sorbitol and

suggested that his mannitol- and

sorbitol-nega-tivestrains(75%) representedadistinctbiotype,

which he referred to as "dog type." Of the 19

known dog-associated strainslisted inTable 3,

63% were mannitolnegative and 74% were

sor-bitolnegative, butonly47%(biotypesF, H,and J) were mannitol and sorbitol negative. Of

inter-est is that only 9% of22 known cat-associated

strains were mannitol negative and 23% were

sorbitol negative, and only one isolate (4%) was

negativefor both characteristics. Thesefindings

confirm Smith's observations that incontrast to

thedog type ("canine type" of Carter[3]),most

catstrainsfermented mannitol (77%) and

sorbi-tol (55%). Carter (3) recommended that strains

recovered from catspossessing positive

charac-teristics formannitol, sorbitol, andglycerol

fer-mentation bereferredtoas"felinebiotype."

The datapertaining to the true incidence of

P. multocida ininfected woundsareconflicting

(1, 9, 17). Itwasbeyond the scope of thisstudy

toconsider the incidence and relevance of

Pas-teurella in infected and noninfected animal bites

of humans. Until differences in fermentative

properties ofP.multocidacanbe assayed with

greater reliability, the true relationship of

bio-type to animal host species and to

non-bite-related infections cannot be accurately

deter-mined.

CLIN. MICROBIOL.

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ACKNOWLEDGMENT

Administrative supportwasreceived from theDepartment of ClinicalInvestigation,Madigan ArmyMedicalCenter, Ta-coma, Wash.

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