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DIAG. MICROBIOL.INFECT. DIS. 1990;13:205-208

BACTERIOLOGY

Rapid Identification of Branhamella catarrhalis A Comparison of Five Rapid Methods Marie Louie, Emma G. Ongsansoy, and Kevin R. Forward

Five methods for the rapid identification and differentiation of Branhamella catarrhalis from other Neisseria species in 86 respiratory specimens were compared. These tests included the 4-methylumbelliferyl butyrate (MUB), API quadFerm, B.CAT.Confirm, Gonochek II, and the Tributyrin disc. All five tests reliably and accurately identified 31 B. catarrhalis

isolates. However, the MUB test was the least expensive, least labor intensive, and did not require overnight purity plates for performance. The MUB test provided same-day identification of B. catarrhalis isolates from the initial primary isolation culture.

INTRODUCTION

for B. catarrhalis. We conducted a comparison of five rapid methods that might be used for the identification of B. catarrhalis from respiratory specimens.

Branhamella catarrhalis, once considered a nonpathogenic respiratory tract commensal, is now recognized to play an important role in the pathogenesis of acute sinusitis, otitis media, acute bronchitis, and acute exacerbations of chronic obstructive lung disease (Hager et al., 1987). Empiric antibiotic therapy for these infections often includes use of aminopenicillins. Because B. catarrhalis is frequently resistant to this class of antimicrobial agent, this organism should be identified promptly and accurately from clinical specimens. Although the Gram stain appearance of clinical specimens and colony morphology may be helpful in identifying B. catarrhalis, nonpathogenic Neisseria species frequently recovered from the respiratory tract may be mistaken From the Department of MedicalMicrobiology,St. Boniface General Hospital and Universityof Manitoba, Winnipeg, Manitoba, Canada. This paper was presented, in part, at the Twenty-ninthInterscience Conferenceon AntimicrobialAgents and Chemotherapy, Houston, Texas, September 1989. Address reprint requests to: Dr. KevinR. Forward, Department of Microbiology,McKenzieBuilding, Room31513, 5788 UniversityAvenue, Halifax,Nova Scotia, Canada B3H 1V8. Received May 2, 1989; revised and accepted January 11, 1990. © 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-889319013.50

MATERIALS A N D M E T H O D S St. Boniface General Hospital is an 801-bed pediatric and adult tertiary care hospital. Respiratory tract specimens submitted to the microbiology laboratory of St. Boniface General Hospital between June and October 1988 were evaluated, and specimens with colonies resembling B. catarrhalis were analyzed. Oxidase-positive, catalase-positive Gram-negative diplococci were identified by using non-growth-dependent rapid carbohydrate utilization tests (RCUTs) (Lennette et al., 1985) and the methyl green DNA medium for the detection of DNase (McFaddin, 1985). These two tests differentiated B. catarrhalis from other Neisseria species. We tested 86 strains of B. catarrhalis and other Neisseria species isolated from respiratory specimens. Of these, 31 were B. catarrhalis, 11 were Neisseria meningitidis, 1 was N. gonorrhoeae, and 43 were other Neisseria species. Well-characterized laboratory strains of B. catarrhalis, N. meningitidis, N. gonorrhoeae, and N. lactamica were used as control strains. Staphylococcus aureus ATCC 29213, N. lactamica ATCC 23970, and B. catarrhalis were used as controls in the

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methyl green DNase test. All isolates were initially tested from primary chocolate or sheep blood agar plates with the MUB reagent (described below). Isolates were stocked at - 7 0 ° C in skim milk and were passaged once before repeating the MUB test in conjunction with the other rapid tests. Prior to the performance of each of the five rapid identification tests, the stocked isolates were subcultured onto chocolate agar a n d incubated at 37°C for 18 hr. We c o m p a r e d the API q u a d F e r m (Analytab Products, NY), B.CAT.Confirm (Scott Laboratories Inc., Carson, CA), G o n o c h e k II (DuPont C o m p a n y , San Mateo, CA), and Tributyrin disc (Pro-Lab Inc., Scarborough, Ontario). Tests were p e r f o r m e d according to the manufacturers' instructions by using pure growth from overnight subcultures. The 4-methylumbelliferyl butyrate (MUB) reagent (Sigma C h e m ical Co., St. Louis, MO) was p r e p a r e d as described by V a n e e c h o u e t t e et al. (1988). A stock solution of the substrate was m a d e by dissolving 100 mg of MUB in 10 ml of dimethyl sulfoxide (Sigma) and 100 ixl of Triton X-100 (Sigma). This stock solution was diluted 1:10 in 0.1 M citrate buffer (pH 5.0), aliquoted, and stored at - 7 0 ° C for u p to 1 m o n t h .

RESULTS W h e n p e r f o r m e d from a purity plate, all five tests correctly differentiated B. catarrhalis from other Neisseria species, a n d there were no false-positive reactions. Primary isolates of clinical specimens tested directly with the MUB reagent identified 28 of 31 B. catarrhalis isolates. U p o n repeat testing from a p u r e culture, all were positive a n d there w e r e no falsely positive reactions. Both B.CAT.Confirm and Tribu-

TABLE 1.

tyrin discs m a y be suitable for testing directly from primary isolates, a l t h o u g h t h e y require a larger ino c u l u m than the MUB test. We did not use the B.CAT.Confirm and Tributyrin discs on initial plates, as there were seldom e n o u g h colonies to p e r f o r m more than the MUB test, G r a m stain, oxidase test, and catalase test from p r i m a r y cultures. Table 1 shows the cost and other factors that m a y influence the choice of each test. The MUB test was the least expensive b o t h in terms of direct cost of reagents and technologist's time required for test performance.

DISCUSSION Reported f r e q u e n c y of B. catarrhalis from clinical respiratory specimens is b e t w e e n 1.3% and 2.0%, alt h o u g h it m a y be u n d e r d i a g n o s e d d u e to confusion with n o n p a t h o g e n i c Neisseria species (Christensen et al., 1986; DiGiovanni et al., 1987). The increased incidence of B. catarrhalis infections and the emergence of resistance to empiric antibiotics enforce the importance of p r o m p t recognition so that appropriate t h e r a p y can be instituted. Minimal criteria for the diagnosis of B. catarrhalis have been detailed by D o e r n and Morse (1980). Failure to p r o d u c e acid forms carbohydrates, and reduction of nitrate and nitrite tests are time c o n s u m ing and m a y delay reporting. More recently, n o n g r o w t h - d e p e n d e n t RCUTs have b e e n employed. The p r o d u c t i o n of DNase has also b e e n u s e d as a s u p p l e m e n t a r y confirmation test for the identification of B. catarrhalis ( A h m a d et al., 1987). O n occasion, Neisseria caviae will p r o d u c e DNase, but this species is n e v e r f o u n d in m a n ( S o t o - H e r n a n d e z

Test Requirements and Costs

Costs and Requirements Cost/testb Indirect costs Purity plate Test tubes Saline/PBS Sticks, loops, swabs Pipettes Incubator (37°C) Fluorescent light Inoculum size Test incubation (min) Tech time (rain)

RCUT 1.32 + + + + + + Heavy 240 11.0

DNase a Media per Test (purchased)

MUB Reagent

B.Cat. Confirm

0.17

0.03

1.35

-+ + + Moderate Overnight 1.5

+ + + Light 5 1.0

-+ + + Moderate 60 3.0

Tributyrin Disc Test 0.48 -+ + + + + + Heavy 240 5.5

~DNase media with methyl green (prepared Media Laboratory, Richmond, British Columbia). bCosts in Canadian dollars.

API quadFerrn 2.42

Gonochek II 2.74

+ + + + + +

+ + + + +

Heavy 120 15.0

Heavy 30 5.0

B. catarrhalis by Five Methods

et al., 1988). Our laboratory routinely uses a combination of RCUT and DNase testing in differentiating B. catarrhalis from other Neisseria species. Although less laborious than the test outlined by Doern and Morse (1980), overnight pure cultures after primary isolation are still required for the RCUT. As a result, there is a delay in reporting B. catarrhalis isolates for at least 48 hr after inoculation of the original clinical specimens. Each of the five tests used in this study reliably identified B. catarrhalis. When the instructions by the manufacturers were followed, interpretation of reactions in each test was not difficult. The B.CAT.Confirm is designed as a confirmatory test requiring prior presumptive identification for B. catarrhalis. Our evaluation suggests that this is unnecessary because B. CAT. Confirm can be used alone to test oxidase-positive, Gram-negative diplococci resembling B. catarrhalis from respiratory specimens. However, its use is limited in that most cases require overnight pure cultures to obtain a sufficient inoculum. The API quadFerm system is used as a rapid screening test and includes a DNase test to differentiate asaccharolytic B. catarrhalis strains from asaccharolytic Neisseria species, such as Neisseria cinerea (Janda et al., 1987). Again, limitation of its usefulness is the need for a heavy inoculum from an overnight purity plate. The API quadFerm system is technically tedious and costs severalfold more than our laboratory method for B. catarrhalis identification (Table 1). Branhamella catarrhalis is able to hydrolyze tributyrin in contrast to other Neisseria species isolated from humans (Riou and Guibourdench, 1986). By using both tributyrin hydrolysis and nitrate reduction, differentiation between B. catarrhalis and Neisseria species can be achieved reliably, despite equivocal carbohydrate reactions (Christensen et al., 1986). Tributyrin hydrolysis, along with Gram strain and catalase and oxidase testing, allows for same-day identification of B. catarrhalis (Richards, 1988). Occasionally, false-negative tributyrin reactions have been reported, and such isolates may require further conventional biochemical testing. Despite its sensitivity and specificity, use of the Tributyrin disc often delays reporting because it requires a heavy suspension for inoculation. Gonochek II is a rapid chromogenic substrate uti-

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lization test used for the rapid identification of N. gonorrhoeae and N. meningitidis from primary cultures on selective media Branhamella catarrhalis can occa-

sionally grow on selective media (Janda et al., 1987), and Gonochek II can differentiate it from other Neisseria species (Dillon et al., 1988). Although Gonochek II is intended for use on isolates from selective media, we have shown, under the conditions tested, that it can reliably identify B. catarrhalis from primary nonselective isolation plates if sufficient colonies are present for a heavy suspension. Because a heavier inoculum is required for the performance of the Tributyrin Disc, Gonochek II, B.CAT.Confirm, and API quadFerm tests, use of the MUB reagent is preferred. Care should be exercised in performance of the test, as failure to use at least two large colonies may result in falsely negative MUB reactions. When the rapid 5-minute MUB reaction is negative and when colony morphology and Gram stain appearance from the clinical specimens suggests the presence of B. catarrhalis, the test should probably be repeated from a pure growth. Most studies evaluating various rapid methods have compared testing among subcultures of stocked isolates. Our study attempted the testing of all isolates from the initial primary isolation plates with the MUB reagent. Our results demonstrate no falsepositive reactions with the rapid 5-minute MUB test when an adequate inoculum was used. Early firstday presumptive identification of B. catarrhalis can be confirmed quickly by the MUB test, requiring no further overnight purity plates or other confirmatory tests such as RCUT or DNase detection. Suspicious colonies that test negative from the primary plates can be subcultured and retested with the MUB test in 18 hr. The MUB test has the advantage of ease in performance, light inoculation, immediate results, and minimal costs. Unfortunately, use of a MUB test alone would not allow for the recognition of occasional N. meningitidis and N. gonorrhoeae isolates that may be clinically significant. When colonies are carefully selected by morphology, Gram stain, and oxidase and catalase testing, false-positive MUB reactions of other Neisseria strains wil be detected infrequently (Vaneechouette et al., 1988). The costs and delay in reporting B. catarrhalis isolates with API quadFerm, B. CAT. Confirm, Gonochek II, and Tributyrin discs make these tests less desirable.

REFERENCES Ahmad F, Young H, Mcleod DT, Croughan MH, Calder MA (1987) Characterization of Branhamella catarrhalis and differentiation from Neisseria species in a diagnostic laboratory. J Clin Pathol 40:1369. Christensen JJ, Gadeberg O, Bruun B (1986) Branhamella

catarrhalis: significance in pulmonary infections and bacteriological features. Acta Pathol Microbiol Immunol Scand Sect B 94:89

DiGiovanni C, Riley TV, Hoyne GF, Yeo R, Cooksey P (1987) Respiratory tract infections due to Branhamella

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catarrhalis: epidemiological data from Western Australia. Epidemiol Inf 99:445. Dillon JR, Carballo M, Pauze M (1988) Evaluation of eight methods for identification of pathogenic Neisseria species: Neisseria-Kwik, RIM-N, Gonobio-Test, Minitek, Gonocek II, GonoGen, Phadebact Monoclonal GC OMNI Test, and Syva Microtrak Test. J Clin Microbiol 26:493. Doern GV, Morse SA (1980) Branhamella (Neisseria) catarrhalis: Criteria for laboratory identification. J Clin Microbiol 11:193. Hager H, Verghese A, Alvarez S, Berk SL (1987) Branhamella catarrhalis respiratory infections. Rev Infect Dis 9:1140. Janda WM, Zigler KL, Bradna JJ (1987) API quadFerm with rapid DNAse for identification of Neisseria species and Branhamella catarrhalis. J Clin Microbiol 25:203. Lennette EH, Balows A, Hausler WJ, Shadomy JH (1985) Neisseria and Branhamella. In Manual of Clinical Microbiology, 4th ed. Eds., JA Morello, WM Janda, and M

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Bohnhoff. Washington, DC: American Society for Microbiology, pp 176-192. MacFaddin JF (1985) Media for Medical Bacteria. In Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, vol 1. Eds., J Butler and J Seitz. Baltimore, MD: Williams and Wilkins, pp 275-284. Richards J (1988) Evaluation of a rapid method for identifying Branhamella catarrhalis. J Clin Pathol 41:462. Riou JY, Guibourdench M (1986) Branhamella catarrhalis: new methods of bacterial diagnosis. Drugs 31(suppl 3):1. Soto-Hernandez JL, Nunley D, Holtsclaw-Berk S, Berk SL (1988) Selective medium with DNAse test agar and a modified toluidine blue O technique for primary isolation of Branhamella catarrhalis in sputum. J Clin Microbiol 26:405. Vaneechouette M, Verschraegen G, Claeys G, Flamen P (1988) Rapid identification of Branhamella catarrhalis with 4-methylumbelliferyl butyrate. J Clin Microbiol 26:1227.

Rapid identification of Branhamella catarrhalis. A comparison of five rapid methods.

Five methods for the rapid identification and differentiation of Branhamella catarrhalis from other Neisseria species in 86 respiratory specimens were...
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