Modification of the phosphoketolase assay for rapid identification of bifidobacteria

MicrobiologicalJournal of Microbiological Methods 40 (2000) 221224JournalofM/locate/jmicmethModication of the phosphoketolase assay for rapid identication ofbidobacteriaJ.I. Orban, J.A. Patterson*Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-1151, USAReceived 13 October 1999; received in revised form 4 February 2000; accepted 6 February 2000AbstractThe phosphoketolase assay is commonly used as a denitive criterion for identication of bidobacteria. A limitation ofthe assay is the time-consuming process of cell disruption, either by use of the French Pressure Cell or by sonication. Wehave replaced the time consuming cell disruption process with a more rapid cell membrane disruption process by pretreatingcells with the detergent hexadecyltrimethylammonium bromide (cetrimonium bromide, CTAB). The effect of nopretreatment, sonication or the addition of CTAB (0.45 mg/ml) on color development in the phosphoketolase assay wastested using pure cultures of bidobacteria and lactobacilli. No phosphoketolase activity was observed with bidobacterialcultures without cell disruption or with lactobicilli that had undergone cell disruption. All bidobacterial cultures gave asimilar color formation whether sonication or CTAB addition was used to disrupt cells. Use of CTAB to disrupt cellmembranes is an effective alternative to the time consuming traditional cell disruption procedures and increases the numberof cultures that can be simultaneously assayed and presumptively identied using the phosphoketolase assay. 2000Elsevier Science B.V. All rights reserved.Keywords: Bidobacteria; Bidobacterium; cell disruption; CTAB; identication; phosphoketolase1. IntroductionThe identication of a bacterial isolate as amember of the genus Bidobacterium is difcult andlabor intensive. The most reliable non-molecular testfor identication of bidobacteria is fructose-6-phos-phate phosphoketolase activity (Scardovi andTrovatelli, 1965, 1969). Since the fructose-6-phos-phate phosphoketolase enzyme is intracellular, har-*Corresponding author. Tel.: 1 1-764-494-4826; fax: 1 1-764-494-9346.E-mail address: (J.A. Patterson)vested cells are rst disrupted by sonication torelease cellular extracts for the assay. Cultures areidentied as belonging to the genus Bidobacteriumby visual observation of the formation of a reddishviolet color.The most time consuming aspect of the assay iscell disruption, whether by sonication or use of aFrench pressure cell. The equipment is expensiveand cell disruption requires 5 to 10 min for eachculture tested. Thus, only a small number of samplescan be processed at a given time. The presentprocedure describes a modication of the phos-phoketolase test for identication of bidobacteria.The modied procedure does not require expensive0167-7012/00/$ see front matter 2000 Elsevier Science B.V. All rights reserved.PII: S0167-7012( 00 )00133-0222 J.I. Orban, J.A. Patterson / Journal of Microbiological Methods 40 (2000) 221 224cell disruption equipment and allows one to assaymore cultures within a given amount of time.2. Materials and methods2.1. ProcedureThe reagents and procedure are as described byScardovi (1981) for detecting fructose-6-phosphatephosphoketolase activity, except that CTAB wasused for cell disruption. CTAB or cetrimoniumbromide (hexadecyltrimethylammonium bromide) isa cationic detergent used as an antiseptic or cleaningagent. It is freely soluble in alcohol and also solublein water at 1:10 ratio (CTAB:water) (Stecher et al.,1976). It has also been used to disrupt cells forwhole cell enzyme assays (Patterson and Hespell,1985). All reagents were obtained from Sigma (St.Louis, MO) and were made up in distilled waterunless indicated otherwise. Trichloroacetic acid(TCA) and fructose-6-phosphate solutions weremade up fresh daily.2.2. Bacterial culturesBacterial cultures tested included strains ofbidobacteria and lactobacilli (obtained from Dr.Peter Muriano, Colorado State University, Stillwater,CO, Table 1), and 18 fecal isolates from humansubjects. Fecal cultures had been isolated fromanaerobic agar plates selective for bidobacteria(Munoa and Pares, 1988). Cultures were maintainedon Reinforced Clostridial broth (Difco Laboratories,Detroit, MI) and were grown overnight at 378C in 10to 20 ml of broth.2.3. Cell preparationCells were washed twice (10 000 3 g, 48C, 15min) with phosphate buffer (KH2PO4, 0.05 M, andcysteine ? HCl, 500 mg / liter, mixed 1:1 (V/ V), ad-justed to pH 6.5 with fresh NaOH) and resuspendedin 1.0 ml of phosphate buffer. Washed bacterial cellseither underwent no pretreatment, sonication for 6min in ice, or were incubated with CTAB for 5 minprior to the assay. CTAB was added in gradedamounts of 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 ml(450 mg / ml stock solution) to determine the level ofCTAB that would be effective for cell disruption.2.4. Phosphoketolase assay:After pretreatment, 0.25 ml of a solution con-taining sodium uoride (NaF, 3 mg / ml) and potas-sium or sodium iodoacetate (5 mg / ml) in H2O wasadded. To that, 0.25 ml sodium fructose-6-phosphate(80 mg / ml in H2O) was added, the solution wasTable 1Comparison of cetrimonium bromide (CTAB) or sonication for cell disruption in the phosphoketolase assay for identication of culturesbelonging to the genus BidobacteriumBacterial cultures Cell disruption method and color formationaATCCb CTAB (4 ml) Sonication (6 min)B.B.B.B.B.B.B.L.L.L.adolescentisadolescentisbidumbidumbreveinfantislongumacidophiluscaseifermentum15703157041186315696157001569715707C7464617501111111111111111111111111111111111Human fecal isolates (18) c cabcColor intensity 5 none, 1 5 light, 1 1 5 dark, 1 1 1 5 very dark.ATCC 5 American Type Culture Collection, Manassas, VA.Human fecal isolates; one negative out of 18 samples presumptively identied as belonging to the genus Bidobacterium.J.I. Orban, J.A. Patterson / Journal of Microbiological Methods 40 (2000) 221 224 223vortexed and then incubated at 378C for 30 min.Following incubation, 1.5 ml of hydroxylamine ? HCl(13 g / 100 ml) was added, the mixture was vortexedand allowed to incubate at room temperature for 10min. One milliliter of TCA (15%, W/ V), 1.0 ml of4N HCl and 1.0 ml of ferric chloride (FeCl ? 6H2O,5% W/ V in 0.1 N HCl) were added, tubes werevortexed and color formation was recorded eitherusing a qualitative scale or spectophotometrically at505 nm. For spectrophotometric determinations, thestopped reaction mixture was centrifuged (10 000 3g, 48C, 15 min) and the supernatant was measuredusing a Bausch and Lomb Spectronic 70 spec-trophotometer. Test tubes containing reagents with-out cells added, or cells plus all reagents, exceptfructose-6-phosphate, were used as blanks.The reaction mix without phosphoketolase activitydevelops a light yellow color. A reddishviolet colordevelops immediately with the addition of ferricchloride if the culture contains phosphoketolaseactivity. The intensity of the color formed may varydepending on the bidobacterial strain used, or ongrowth characteristics of the strain. For strains thatproduce a light color, the color was visually moreevident after standing for a while to allow debris tosettle. In some cases, a slight color may develop incontrols without addition of CTAB. We assume thiswas due to slight cell lysis during cell washing andpelleting of more fragile cultures.3. Results and discussionResults of the present study indicate that CTABcan be effectively used for cell disruption in thephosphoketolase test for identication of culturesbelonging to the genus Bidobacterium. The red-dishviolet color was formed by bidobacterial cellsdisrupted by sonication or treated with CTAB, butnot by lactobacilli cells, irrespective of cell disrup-tion procedure (Table 1). Out of the 18 human fecalisolates obtained from bidobacterial selectivemedia, only one gave no color formation in thephosphoketolase assay.Several bidobacterial cultures were used to de-termine the minimum concentration of CTAB re-quired to maximize color formation. Color formationwas measured spectrophotometrically and occurredat the lowest level of CTAB addition (45 mg);however, 180 mg (0.4 ml of stock solution) of CTABwas found to be the optimum concentration for avariety of cultures (Fig. 1). Color intensity wasfound to vary among bidobacterial strains. Whetherthis was due to different amounts of cells or differentFig. 1. Inuence of increasing concentrations of cetrimonium bromide (CTAB) stock solution on color formation by different strains ofbidobacteria in the phosphoketolase assay. Bidobacterium bidum , B. adolescentis 15704 j , B. breve m, B. adolescentis 15703 3 , B.infantis *, B. longum d, Lactobacillus casei 4646 s .224 J.I. Orban, J.A. Patterson / Journal of Microbiological Methods 40 (2000) 221 224amounts of phosphoketolase produced per cell wasnot determined. However, all bidobacterial cultureswith an initial culture OD (660 nm) above 1.0 gave adenitive color reaction. With the use of CTAB, itwas possible to test up to 20 samples within 1.5 hwhereas the same procedure using a sonicator wouldtake 3.3 h. Thus, a larger numbers of samples caneasily be assayed at a single time.CTAB was shown to be an effective alternative forcell disruption in the phosphoketolase assay foridentication of cultures belonging to the genusBidobacterium. Use of CTAB was less time-con-suming than sonication, thus removing the limitationon using the phosphoketolase assay as a massscreening method for identifying bidobacteria inunknown cultures.AcknowledgementsFunding for this project was provided by Larex, StPaul, MN. Pure cultures of bidobacteria and lac-tobacilli were provided by Dr. Peter Muriano,Colorado State University, Stillwater, CO. We thankPat Jaynes for her help in maintaining cultures andmedia preparation.ReferencesMunoa, F.J., Pares, R., 1988. Selective medium for isol