Evaluation of methods to detect oxidase activity in the genus Pasteurella

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 0095-1137/80/08-0220/06$02.00/0

Evaluation of Methods

to Detect Oxidase

Activity

in

the

Genus

Pasteurella

JOSEPH L. GADBERRY,t* KARL CLEMMONS,AND KIMBERLY DRUMM

Department of Microbiology, Miami University, Oxford, Ohio 45056

Several oxidasereagentsand commercial productswere evaluatedastotheir

efficacy in detecting oxidase activity in species of the genus Pasteurella.

Rec-ommendationsaremadeconcerning the reagent of choice fordeterminingoxidase

activity in thegenusPasteurella. Recommendations are made also concerning

theuseof commercialproductsand theirefficacyindetectingoxidaseactivity in

thisgenus.

In 1965 Smith and Thal (21) made a

taxo-nomic study of the genus Pasteurella. They

observed two distinct groups in this genus on

the basisof the oxidasereactionsaswellasother

biochemical differences. Group I was oxidase

positive and included P. multocida, P.

pneu-motropica, P.

haemolytica,

and P. ureae.Their

groupIIwasoxidasenegative andincluded

Pas-teurella"X"(Yersinia

enterocolitica),

P.pestis, andP.

pseudotuberculosis. They proposed

that

the members ofthegenusPasteurella ingroup

Il be placed in a newgenus, Yersinia, as

pro-posedbyvanLoghem(25). In addition to Smith

andThal,otherinvestigatorshave also studied

the oxidase activity ofPasteurella. Steel (22)

reportedthat 19 of 26 P.multocida strainswere

oxidase positiveand 6 of 6 strains of P.

haemo-lyticawere

positive.

Hisdataagreedwiththose

of Smith and Thal for theirgroup II Pasteurella

species.Steel usedfilterpaperimpregnatedwith 1%

tetramethyl-p-phenylenediamine

dihydro-chloride (TPD) todetect oxidaseactivity.

Hen-ricksen and Jyssum (14) tested a0.5% aqueous

solution of TPD as an oxidase reagent. They observed thatall 14 strains ofP. multocida, 6

strains of P. haemolytica, and 1 strain of P.

pneumotropicawereoxidasepositivewith TPD

reagent. Using the

dimethyl-p-phenylenedia-mine dihydrochloride reagent, most of these

strainsgavenegativereactions.However, several

clinicalmicrobiology manuals which are

availa-ble for laboratory use indicate that dimethyl

salts also may be used asacceptable reagents for

thedetection ofoxidase activity in bacteria (1,

10, 19, 24). The results in this study definitely

prove this not to be the case for the genus

Pasteurella.

t Present address: Department of Microbiology, Kansas CityCollegeofOsteopathic Medicine,Kansas City, MO 64124.

MATERIALS AND METHODS

Organismsand oxidase reagents. Themajority

of thePasteurella and Yersinia isolates used in this study were ofbovine, porcine,fowl, and human origin. They were obtained fromhospital and veterinary lab-oratories and from several investigators engaged in

Pasteurellaresearch. Isolates of P.multocida,P.

hae-molytica, P. pneumotropica, P. ureae, Y.pestis, Y. pseudotuberculosis, and Y. enterocoliticaweregrown

inTrypticase soybroth(BBLMicrobiology Systems, Cockeysville, Md.) for24h at37°C.Thecultureswere

mixed with aVortex mixer, and the cell suspensions were thenadjustedto areading of 95% transmittance

on aColemanJuniorII spectrophotometer model 6120 (Coleman Instruments Corp., Oak Brook, Ill.). After adjusting the cell suspensions, dilutions of 104 were made in sterile 0.85% saline. One-tenth-milliliter

amountsof the finaldilutions were plated on tryptose blood agarplates. The inoculum was spread across the plates with aglass rod dipped in 95% ethyl alcohol and flamed between each plating. After 24 to 26 and 48 to

50h ofincubation at 37°C, several drops of the re-spective oxidase reagents were applied to the isolated colonies. Aeromonashydrophilaand Staphylococcus

aureuswerechosen as the positive and negative con-trol organisms, respectively, for all the experiments. All oxidase tests were made in triplicate.

All oxidase reagents were used as aqueous solutions of0.5, 1, and 2% with and without 1% a-naphthol prepared in 95% ethyl alcohol (8, 9, 13). a-Naphthol wasused in a 1:1 ratio with the oxidase reagents. Gaby

andHadley observed thata-naphthol with the oxidase reagent in a 1:1 ratio was a satisfactory tool in the detection of oxidase activity in Pseudomonas

aerugi-nosa (13). Therefore, a-naphthol was used in this study to determine if it would make the respective

oxidase reagents more sensitive in the detection of oxidaseactivity in the genus Pasteurella. The phen-ylenediamine salts (Eastman Organic Chemicals, Rochester, N.Y.) used as oxidase reagents were: TPD, dimethyl-p-phenylenediamine monohydrochloride (DPH), dimethyl-p-phenylenediamine oxalate, and di-methyl-p-phenylenediamine sulfate. The reagents were stored at 4°C for 1 week and then discarded.

Strains were recorded as oxidase positive if the colonies developed a blue,pink, or rose color within 5

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DETECTION OF OXIDASE

min. Any colony not changing color in 5 min was recorded as oxidase negative.

Tosimulate a clinical laboratory situation, 33 ad-ditional isolates of P. multocida were plated on 5% tryptose blood agarplates and streaked for isolated colonies. The plates were incubated for 24 h at 37°C. One percent DPH and TPD with and without a-naph-tholwerethereagents used to detect oxidase activity. Tests were done in triplicate on different days to ensurereproducibility.

Commercial oxidase tests. The efficacies of com-mercially available prepared disks impregnated with oxidase reagents were also observed. These products wereused to detect oxidase activity in P. multocida AUltestsweredone as recommended by the

manufac-turers.Thecommercial products used were Patho Tec CytoO strips(General Diagnostics, Warner-Lambert Co., Morris Plains, N.J.), Taxo N disks (BBL), and oxidasedifferentiation disks (Difco Laboratories, De-troit,Mich.).Apositive reaction was a color change at the site of application of the inoculum. No color changewasrecorded as an oxidase-negative reaction. Theinoculumwasapplied to the disks with a platinum loop.

RESULTS

Twenty-four-hour coloniesofall fourspecies of Pasteurella showed poor oxidase activity when concentrations of 0.5 to 2% of the

di-methyl-p-phenylenediamine

salts (dimethyl-p-phenylenediamine sulfate,DPH, and dimethyl-p-phenylenediamine oxalate) without a-naph-thol were used (Table 1). Colonies growing for

48h showed a considerable increase in oxidase

activitywhen1% andparticularly 2%

concentra-tionsof the dimethyl-p-phenylenediamine salts

wereused.DPH wasgenerallythe leastreactive

of the threesaltsexcept in the case ofP. mul-tocida.

There was aconsiderableincrease inactivity

at 24h when TPDwithout

a-naphthol

wasused

todetect oxidase.Again,anincubationperiod of

48hgenerallyincreased oxidaseactivityto100% forallfourspecies, using allthree concentrations

ofTPD. However, earlier detection of oxidase

activity was

possible,

at 24 h, for all species if

theconcentration ofTPDwasincreased to2%

(Table

1).

Whena-naphtholwasaddedtothe

dimethyl-p-phenylenediamine

salts in a 1:1

'ratio,

there wasno

appreciable

increasein oxidase detection

at 24 h over that obtained by using the salts

without a-naphtholatall threeconcentrations,

except for P. ureae. Nosignificant increases in

oxidase production were seen at48 hwhen

a-naphthol wasadded tothe

dimethyl-p-phenyl-enediaminereagents for P.

haemolytica

and P.

pneumotropica (Table 1).Increased oxidase ac-tivitywasseenfor P. multocidaat48hwhen 1

and 2%concentrationswereused

(Table

1).

The addition ofa-naphthol to TPD in a 1:1

ratio at 24 hshowed anappreciable increase in

oxidase activity when compared with the

di-methyl-p-phenylenediamine salts. However,

when thedetectionsofoxidase activity by using

TPD with and without a-naphthol were

com-pared at 24h, nonoticeableincrease in activity

wasseen for any of the four species. In general,

48-h colonies of thefour species ofPasteurella showed nogreateroxidase activity than the 24-hcultures, especiallyat 1 and 2%concentrations.

A.hydrophiliawasoxidasepositive with each

of the dimethyl-p-phenylenediamine salts and

TPD at 24 and48 hwith and without

a-naph-thol. The oxidase-negative control, S. aureus,

showednooxidase activity with any of the four

reagents after 24 and 48 h of incubation with

andwithouta-naphthol.

Ail

33 isolates (100%) of P.multocida which

were plated on 5%tryptose blood agar plates, in

thesimulated clinical laboratorysituation, were

oxidase positivewith 1% TPD withand without

a-naphthol

after 24 h of incubation (Table 2).

Only24.2and42.4%of the P. multocida isolates

were oxidase positive with 1% DPH without

a-naphthol and with a-naphthol, respectively, after 24 h ofincubation (Table 2). It was

also

observed that viable P.multocidacould be

re-coveredfor up to 1 h aftercontinuousexposure

toaqueous1%TPD.

Commercial oxidase testing

procedures

were

based on the methods of Kovacs (18). These

werecompared with the previous techniquesto

determine which methodwas mostsuitable for

detecting oxidase activity in the genus

Pasteu-rella.Ofthe 33isolates of P.multocida,Taxo N

disks detected oxidase activity in 88% of the

isolates, oxidase differentiation disks detected

oxidaseactivityin63% of theisolates,and Patho

Tec Cyto O strips detected oxidase activity in

lessthan 3% of the isolates (Table 3). Some of

the isolatesgaveweakly

positive

and

question-able resultswith Patho Tec Cyto O strips and

were positive with one or both of the other

commercial products. Aeromonas spp. gave a

strongoxidase reaction with the three

commer-cialproducts, and S.aureus wasoxidase

negative

witheach oftheseproducts.

DISCUSSION

The results of these oxidase studies indicate

thatthe most

satisfactory

reagent for

detecting

oxidase activity ofall

species

within the genus

Pasteurellawas a1 or2%concentrationof TPD

without

a-naphthol.

Itwasalso shown that the

optimalincubation

period

for

detecting

oxidase

activity in the genusPasteurellawas24h.

Nei-theranincrease in incubation ofculturesto 48

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TABLE 1. Detectionof oxidaseactivityin the genus Pasteurella withdifferentoxidasereagentsafter24 and 48 hofincubation

No. of

strains Reagent' Concn(%) tested

26 DPS

DPH

DPO

TPD

3 DPS

DPH

DPO

TPD

7 DPS

DPH

DPO

TPD

7 DPS

DPH

0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0

% of strainsoxidasepositive Noa-naphthol a-Naphthol

24h 48h 24h 48h

4 46 4 27

8 50 19 77

23 85 42 96

8 31 35 58

27 58 42 85

27 81 65 100

0 38 8 62

4 73 12 88

8 73 23 85

65 92 84 96

96 100 100 100

100 96 100 100

33 0 33 67

0 100 33 100

0 100 33 100

0 33 33 67

0 33 33 33

0 67 33 67

0 67 0 33

0 100 33 100

0 100 100 100

67 100 100 100

100 100 100 100

100 100 100 100

o o o o

0 57 14 29

0 57 0 57

0 14 14 29

14 43 14 29

14 43 14 43

0 14 14 0

0 29 14 0

0 43 14 14

86 100 29 57

100 100 71 86

100 100 86 71

14 57 86 100

14 100 86 86

29 100 86 86

29 43 86 86

0 43 100 100

14 57 100 86

Organism

P.multocida

P.pneumotropica

P.haemolytica

P.ureae

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TABLE1-Continued

% of strains oxidase positive No. of

Organism strains Reagent' Concn (%) Noa-naphthol a-Naphthol tested

24h 48h 24h 48h

DPO 0.5 29 86 71 86

1.0 29 100 71 86

2.0 43 100 100 86

TPD 0.5 100 100 86 100

1.0 100 100 100 100

2.0 100 100 100 100

DPS, Dimethyl-p-phenylenediamine sulfate; DPO, dimethyl-p-phenylenediamine oxalate.

TABLE 2. Simulated clinicalevaluation on 1% TPD and DPH ofdetection of oxidase activity in 33 P.

multocida strains after 24 h of incubation

%of strains oxidase positive Reagent

No a-naphthol a-Naphthol

DPH 24.2 42.4

TPD 100 100

TABLE 3. Efficacy of commercial products in the

detectionof oxidaseactivity in33P.multocida

strains

% of strains Commercialproduct oxidase

posi-tive Taxo N disk ... 88 Oxidasedifferentiation disk ... 63

PathoTecCytoOstrip ... <3

h nor the addition of a-naphthol significantly

increased the sensitivity ofTPD to detect

oxi-dase-producingisolatesofPasteurella.

Further-more, this combination produced false-positive

reactions withinthe genus Yersinia, especially

when the incubation time of these organisms

wasextendedto48 h (Table 4).

Ingeneral, the detection of oxidase activityin

thegenusPasteurella bythe

dimethyl-p-phen-ylenediamine salts was not as consistent as it

was whenTPD was used. Although there was

someincrease insensitivitywhen cultureswere

testedforoxidase activitywith the

dimethyl-p-phenylenediaminesaltsaftera48-hgrowth

pe-riod and when a-naphthol was added to these

reagents, the sensitivity of these reagents was notasgreatasthat of TPD.Itwasreported by

Henricksen and Jyssum (14) that most strains

of P. multocida were oxidase negative when

tested with 1% DPH after 24 h, which agrees

with the results of thisstudy.

.TPDisa moresensitivereagentfordetecting

colorchange during the oxidase test. The color

is usuallyseen within 1 to 2 min afterapplication

ofthereagent. Steel (22)found that when

posi-tive reaction to the development of color was

limitedto within 60s,only 47% of his P.

multo-cida isolateswerepositive with TPD. The

pos-itivereactionsaregenerallyaveryintense blue,

which

facilitates

readingthereactions.The oxi-dase-positivereactionswith dimethyl-p-phenyl-enediamine oxalate and dimethyl-p-phenylene-diamine sulfatewerenot aseasily read,sincethe maximum intensity was onlya rose color. The

positive colonies turned brown to black when

DPH was used. The length of timefor a color

changetooccurvaried from1to 5 min whenthe

dimethyl-p-phenylenediamine

salts were used.

It wasofinterest, however, that when

a-naph-thol was used with each of these reagents, a

colorchangewasdetectedmore quickly.Itwas

often noticed that thecenterof the colonygave a more intense positive reaction than did the peripherywhen any of the reagents wereused. Thismaybe duetotheareaof heaviestgrowth,

assuggested by Henricksen and Jyssum (14). Since SmithandThal (21) suggested thatthe

oxidase test be used as a major criterion to

separate the genusPasteurella intotwogroups,

Yersinia andPasteurella, isolates of Yersinia

wereused in thisstudytocomparetheiroxidase

activity withthatofPasteurella. The studies of Smith and Thal and those of Henricksen and Jyssum indicatedthatall membersof thegenus

Pasteurella were oxidase

positive

when 0.5%

TPDwasusedasthe oxidasereagent.Our

stud-iesindicated that0.5% TPDwas notas

satisfac-tory indetectingoxidaseactivityin P.multocida

in 24 h asitwasfor P.

pneumotropica,

P.ureae,

andP. haemolytica.

Tryptoseagar with 5%sheep

erythrocytes

was

used asthe medium of choice for this

study,

as

Pasteurella grewmore

rapidly

onthis medium.

Blood agar also is the mediumrecommendedto

isolatethe

organisms

in theclinical

laboratory

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TABLE 4. Detectionof oxidaseactivityin the genus Yersinia withdifferentoxidasereagentsafter24 and 48 hofincubation

% of strains oxidasepositive No.of

Organism strains Reagenta Concn Noa-naphthol a-Naphthol tested

24h 48h 24h 48h

Y.pestis 3 DPS 0.5 0 0 0 0

1.0 0 0

2.0 0 0

DPH 0.5 0 0

1.0 0 0

2.0 0 0

DPO 0.5 0 0

1.0 0 0

2.0 0 0

4 TPD 0.5 0 0

1.0 0 0

2.0 0 0

o o

o o

o o

o

o

o o

o o

o o

o o

o o

o o

o o

Y.pseudotuberculosis 7 DPS 0.5 0 0

1.0 0 0

2.0 0 0

DPH 0.5 0 14

1.0 0 0

2.0 0 0

DPO 0.5 0 O

1.0 0 0

2.0 0 0

o o

o o

o o

o o

o o

o o

TPD 0.5 0 14 0 29

1.0 0 14 14 43

2.0 0 29 29 43

Y. enterocolitica 6 DPS 0.5 0 0

1.0 0 0

2.0 0 0

DPH 0.5 0 0

1.0 0 0

2.0 0 0

DPO 0.5 0 0

1.0 0 0

2.0 0 0

o o

o o

o o

0 17

0 17

0 33

TPD 0.5 0 100 100 83

1.0 0 83 83 100

2.0 0 83 100 100

'DPS,Dimethyl-p-phenylenediaminesulfate; DPO, dimethyl-p-phenylenediamine oxalate.

and,therefore, would be themediummost

com-monly used for the oxidase test.

Results of the evaluation of the three

com-mercial oxidase tests indicate the efficacy of

thesetests tobe approximatelyequivalenttothe

aqueousdimethyl solutions discussedabove.

P. multocida is animportant pathogen.

Hu-man infections caused by this bacterium have

beenrelatedtoboth animal andnonanimal

con-tact (2-7, 11, 12, 15-17, 20, 23). P.multocida is

oftenmisidentified in the clinical laboratory as

a result offalse-negative oxidase tests. Culture

andcorrect identificationareimperative if

ade-quate therapeutic treatmentistobe instituted.

0 29

0 14

0 29

0 33

0 67

0 67

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Basedontheresults ofourstudies, it is

recom-mended that freshaqueous1% TPD without

a-naphthol be thereagentofchoice for the

detec-tion ofoxidaseactivity in thegenusPasteurella.

Theoptimal conditions were foundto be

incu-bationat37°C for 24 hon5%sheep bloodagar.

These conditions eliminated much ofthe

prob-ability of Pasteurella giving false-negative

re-actions and of other microorganisms showing

false-positive oxidase reactions. It is also

rec-ommended that the three commercialtests

eval-uated not be used in the detection ofoxidase

activity in P. multocida,as noneof the

commer-cial tests consistently detected oxidase activity in P. multocida.

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