Whole-cell bacterial peroxidase test with isoproterenol as the hydrogen donor

Q ~I_SEVIER

Ann. Inst. Pasteur/Microbiol.

Pari:~ 1987

1987, 138, 407-414

WHOLE-CELL BACTERIAL PEROXIDASE TEST WITH ISOPROTERENOL AS THE HYDROGEN DONOR S. Giunta (1) (,), G. T,.wchetti Q), L. Galeazzi (i), G. Grilli (l), G. Groppa (1) and R. RoccPetti (2) (0 Sezione di Microbiologia, Laboratorio Clinico, Istituto di Ricovero e Cura a Carattere Scientifico INRCA~ 60100 Ancona (Italy), and (2) Laboratorio di Analisi Chimica Strumentale, lstituto ITIS, V. Volterra, Torrette di Ancona, Ancona (Italy)

SUMMARY The ~-adrenergic compound isoproterenol was used as oxidizable reagent in a whole-cell assay for the detection of bacterial peroxidase activities. Isoproterenol has been shown to constitute a useful reagent for detecting peroxidase activities in enzymatic tests, utilizing standard purified enzymes, and in the microbiological application proposed. The procedure developed is simple and rapid to perform. In contrast to currently used whole-cell tests for bacterial peroxidases, the assay described here does not need preliminary permeabilization; moreover, the compound utilized does not have related toxicological problems. Therefore, the isoproterenol assay may represent a low-cost safe additional peroxidase test in clinical bacteriology. KEY-WORDS: Peroxidase, Isoproterenol; Bacterial whole cell, Benzidine. INTRODUCTION Bacterial defence against oxygen toxicity necessitates the scavenging of both O~ and H202. This is accomplished by superoxide dismutases, which scavenge OF, and by catalases and peroxidases which scavenge H2Oz [5]. The detection and study of bacterial peroxidase activity is useful in general as well as in clinical microbiology for many purposes: metabolism, taxonomy, classification and identification. In the fields of microbiology previously mentioned, benzidine and its derivatives have been the most widely used hydrogen donors for detecting Submitted April 15, 1987, accepted July 21, 1987. (*) To whomcorrespondence should be sent.

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bacterial peroxidase activities [2, 6]. The utilization of benzidine or, more recently, of o-dianisidine, as chromogens for peroxidase investigations, encompasses bo~h fundamental studies, utilizing purified bacterial enzymes, and whole-cell peroxidase assays currently used in clinical microbiology. These whole-cell tests have a serious drawback • the need for bacterial permeabilization, since the peroxidase assay using o-dianisidine does not show any colour formation with many whole (untreated) cells [6]. Many attempts have been made to make the bacterial suspensions more permeable, but the permeabiF:zation procedures often caused interference in the subsequent detection of enzymatic activity [6]. Very recently, we have developed a new test for the detection of microbial peroxidase activities utilizing the phenothiazine d ~u~ chlorpromazine as oxidizable reagent [4]. In that assay, chlorpromazine acted both as cell permeabilizer and reagent for the detection and quantitative estima-. tion of bacterial peroxidase and peroxidase-like activities [4]. The utilization of a drug currently used in therapy for detecting microbial peroxidase activities prompted us to search for other pharmacc,logical compounds potentially useful for this purpose. Taking into account the fact that cathecol [1], and some of its derivatives were previously known to constitute donor reagents for peroxidases, screening among this class of compounds was carried out in search of potential hydrogen donors for peroxidase reactions. In this paper, we describe the utilization of the well known [3-adrenergic compound, isoproterenol, as reagent for whole-cell microbial peroxidase test.

MATERIALS AND METHODS

Microorganisms° The microbial strains tested were from specimens submitted to our clinical laboratory, and from collection cultures of the American-Type Culture collection (ATCC) Rockville, MD (table I). The bacterial suspension utilized in the whole-cell peroxidase determinations were prepared from colonies harvested from MuellerHinton agar (BBL) after 18-24-h incubation. Ceils could either be added directly to tt~e reaction mixlmre by a loop or a swab, oi introduced in a saline suspension. The latter system was preferred because it provided standardized inocula which could be turbidimetricallycontrolled. Inocula giving absorbances of about 1.5 at 540 nm were used in order to avoid false-negative reactions due to weak colour formation at lower inocula concentrations. Aliquots of the same suspensions were sonicated for data comparison with disrupted cells.

Reagents. (-)-Isoproterenol hydrochloride (Sigma Chemical Company, St. Louis, MO) 5 mg/ml in double-distilled water was used. This solution must be stored in the dark at 4°C and may be used for several days after filter sterilization. Hydrogen peroxide 40 % (~armil:a!ia-Carlo Erba, Montedison, Milan, Italy) was used. Benz~dinefor the reference memod [3] was from Merck AG (Darmstadt, W. Germany). Purified pe~'oxidase, catalase and guaiacol were from Sigma.

ISOPROTERENOL BACTERIAL PEROXIDASE

T~ST

409

Whole-cell procedure. A 0.1-ml aliquot o f bacterial suspension was added to 1 ml of isoproterenol solution. The mixture was allowed to stand 10 min at room temperature; then the reaction was started by the addition of 50 ~1 of 0.4 % H202. The colour development was monitored for the following 20-30 min. This procedure gives an easy, clear-cut evaluation of the whole-cell bacterial peroxidase activity. To more understand the isoproterenol-peroxidase reaction, we also performed spectrophotometric studies utilizing pure peroxidase. The absorbance was monitored at 470-490 nm, the range at which the oxidized product(s) present maximal absorption. The spectrophotometric determinations were performed with a ¢~Uvikon 860 r~ spectrophotometer (Kontron, Milan, Italy).

TABLE I. - - Whole-cell peroxidase lest, Peroxidase test results Strain

Isoproterenol test

Conventional benzidine test

Group A streptococcus

Streptococcus pyogenes ATCC 19615 Streptococcus faeca!is ATCC 19433 Staphylococcus aureus ~cherichia coil P/~teus mirabilis Klebsiella spp. Citrobacter freundii Pseudomonas aeruginosa

u

m

2+ 2+ 1+ 3+ 3+ 4+

1+ 3+ 1+ 2+ 3+ 3+

Reactions were semi-quantitativelyrated - to 5 + (whiteto dark green for the benzidine method, and white to dark orange-brownfor the isoproterencl method).

TABLE II. - - Absorbance results obtained with intact cells (IC) and with cells disrupted by mild sonication (DC). Strain

Optical density

S. faecalis

IC DC

0.026 0.019

S. aureus

IC DC

0.974 1.061

E. coil

IC DC

1.162 1.787

P. aeruginosa

IC DC

1.610 1.647

Absorbances were recorded at the end of the reaction after filtration of the mixtures.

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Determination of the K m. The determination of the K m values were carried out, using the double reciprocal LineweaveroBurk plot under the following assay conditions : phosphate buffer 0.05 M pH 7, H202 0.05 mM peroxidase 5 ~.g/ml, lambda 470 nm, temperature 20°C, isoproterenol (0.05, 0.10, 0.20, and 0.30 mM).

RESULTS

Isoproterenol was found to be a useful reagent for detecting bacterial peroxidase activity, in whole-cell assays. The test gave a clear-cut semi-quantitative evaluation of peroxidase activities. The color development was easily detected within the first few minutes after the addition of hydrogen peroxide° The reaction with pure enzyme showed a pH optimum in the range of 7-10. We chose to carry out the test at near neutral as we were working with living cells and because isoproterenol undergoes oxidation on exposure to alkaline conditions [7]. At the low concentration of H 2 0 2 used, practically no catalase activity was detected (absence of bubble formation). However, when present, the catalase activity did not interfere with the isoproterenol test.

li

,k

m

FIG. 1. - - Results o f the whole-cell isoproierenol test performed on six different bacterial strains. From left to right : S. faecalis, S. aureus, E. coil, S. epidermidis, K. oxytoca and C: freunati. Each reacted differentially to the assay.

I S O P R O T E R E N O L B A C T E R I A L P E R O X I D A S E TEST

411

Figure I .~;t,~owsthe reaction colour change of six different bacterial :~trains which reacted differentially in the isop~oterenol assay. Table I shows the results of t~e whole-ceil peroxidase test performed on different bacteria. The isoproterenol method was compared with the benzidine method: benzidine ( 1 % solution in methanol) 0.05 ml; EDTA 0.1 M 0.2 ml; cell suspension 0.1 ml; HzO2 4 °7o 0.1 ml. The results of semi-quantitative evaluation of the whole-cell tests were read with the naked eye and the colour formation expressed as degree of reaction, with clear positivity equal to 5 + and clear negativity equal to - . To investigate whether the differences in the isoproterenol assay observed between species could be due to differences in permeation of the compound, we performed parallel controls (in 4 of the strains tested) with cells disrupted by sonication, comparing the quantitative results obtained by recording the absorbances at the end of the reaction, immediately after filtration of the mixtuies. Table II reports these results, and shows that disrupted eels show slightly higher absorbances, but they maintain the same pattern as intact cells as coneerns the degree of reaction. Therefore, differences in the permeation of the compound which may be present, do not appear to be critical in the interpretation of the test. Figure 2 shows that catalase d~d not carry out the same reaction as peroxidase. Bacterial cells showed their greatest activity in the isoproterenolperoxidase test when grown on agar, whereas broth cultures gave low or negative results. The value of Km in the horseradish-peroxidase-catalysed oxidation of isoproterenol was 6.5 mM~ a value much greater than the 1.4 mM obtained with guaiacol. However, although some other classical reagents were better hydrogen donors in tests with pure enzymes, for an assay with intact cells, isoproterenol has the important advantages of avoiding permeabilization procedures, and above all, of not being toxic (unlike the majority of commonly used reagents).

DISCUSSION

Isoproterenol (3-4-dihydroxyphenyl-[3-etharrol-isopropylamine) (fig. 4) is a synthetically obtained compound currently used in medica! practice. This drug is the most potent of the sympathomimetic amines that act almost exclusively on [3 receptors [7]. It belongs to a class of compounds which have been previously shown to act as hydrogen donors in peroxidase reactions [1]. Peroxidases are enzymes with quite a narrow specificity for the hydrogen acceptor; however, the specificity of these enzymes ,_,.~_, ~'--- the hydrogen donor is low, and a number of compounds are active [1]. The donor may be an amine, a phenol or a leuko-dye; consequently, the list of potential donor compounds is wide. Unfortunately, very often these oxidizable reagents include many toxic chemicals, such as the aforementioned

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S. O I U N T A A N D COLL.

~b:~.

0.5@0 O.D.

i1

0.250-

/ 0.000 o.o

/

~.o

~.o

23.0

30.0 min

TIME

FJ6. 2. -- Reactio,,s o f purified peroxidase and catalase iu the isoproterenol assay. The curves clearly and quantitatively demonstrate that catalase 500 U / m l (curve 2) dit not carry out the same reaction as peroxidase 0.04 U / m l (curve 1).

OH H

H3 CH

I

OH

"'CH 2

NH--CH

I

CH 3

FIG. 3. - - Isoproterenol (3-4-dihydroxyphenyl-~-ethanoMsopropylamine).

ISOPROTERENOL BACTERIAL PEROXIDASE TEST

413

benzidine and phenol. The use of benzidine and its derivatives as chromogens has been questioned because they are hazardous carcinogens. As concerns bacterial peroxidase detection, benzJdine has been widely used as reagent, while a currently utilized assay is the whole-cell peroxidase test using o-dianisidine. The latter, however, has two serious drawbacks • a perweabilization requiremeat, and its inhibition in the presence of strong catalase activity [6]. Earlier reports from this laboratory showed that currently used pharmacological cerr.pounds can be both good hydrogen donors in peroxidase reactions and useful reagents for the detection of bacterial peroxidase activity [4]. The present report confirms the advantages of this approach; indeed, isoproterenol acts as an efficient oxidizable reagent in the whole-cell detection of bacterial peroxidases. The isoproterenol-peroxidase test gives a distinct colour reaction which may be easily read by the naked eye for semi-quantitative evaluation. The assay never gave false-positive reactions with streptococci, which are known to be benzidine-negative [3]. CataIase a,.tivity, when present, did not cause interference of any kind. Interestingly, and differently from the currently used tests, the i~oproterenol assay did not require preliminary permeabilization of the bacterial strains tested. The compound is a white powder readily soluble in water, non-carcinogenic [7] and does not pose other relevant toxicological problems. Thus, this new approach to the detection of bacterial peroxidase activities combines good sensitivity, specificity and stable colour formation with safety in handling.

R1ESUMI~ DOSAGE DE LA PEROXIDASE ASSOCII~E A DI: S CELLULES BACTIER~ENNES ENTII~RES A L'AIDE DE L'ISOPROTI~RI~NGLCOMME DONNEUR D'HYDROGI~NE

L'isoprot6r6nol, compos6 [3-adr6nergique, a 6t6 utilis6 en tant que substrat oxydable pour le dosage des activit6s peroxydasiques bact6riennes sur cellules enti&es. L'isoprot6r6nol s'est r6v616 i!tre un excellent r6actif pour le dosage des activit6s peroxidasiques bact6rienne:3, aussi bien dans les tests enzymatiques qui utilisent des enzymes purifi6s standards qt~e dans l'utilisation de cellules bact6riennes enti&es propos6e ici. La proc6dure st:;vie est simple et d'ex6cution rapide. Contrairement aux autres techniques actuellement en usage pour !e dosage des peroxidases bact6riennes sur cellules enti6res, l'essai d6crit dans le pr6sent rapport ne n6cessite aucune perm6abilisation pr61iminaire. En outre, le compos6 employ6 ne pr6sente pas de propri6t6s toxiques importantes. Par cons6quent, rutilisation de l'isoprot6r6nol repr6sente une technique suppl6mentaire pour le dosage des peroxidases bact6riennes caract6ris6e par son innocuit6 et son bas prix. MOTS-CLr~S: Peroxydase, Isoprot~r6nol; Cellules bact6riennes enti&es, Benzidine.

414

S. G I U N T A A N D C O L L . ACKNOWLEDGMENTS

This work was supported by a grant from the Italian Ministero delia Sanith, Ricerca Finalizzata Farmacologia Geriatrica, sottoprogetto FG4, Direttore Dr G. Groppa.

REFERENCES [1] BARMAN, T.E., Peroxidase, in ~tEnzyme Handbook~> (Barman, T.E.) (pp. 234-235). Springer-Verlag, Berlin, 1985. [2] CLAIRBORNE,A. • FRtDOVlCH, I., Purification of the o-dJanisidine peroxidase from Escherichia coli B. ~L biol. Chem., 1979, 254, 4245-4252. [3] DE1BEL, R.H. & SEELEY, H.W., Streptococcaceae, in ~Bergey's Manual of Determinative Bacteriology, (Buchanan, R.E. & Gibbon, N.E.) (p. 491). William & Wilkins Co., Baltimore, 1974. [4] GALEAZZI,L., TURCHETTI,G., GRILLI,G., GROPPA,G. & GIUNTA,S., Chlorpromazine as permeabilizer and reagent for detection of microbial peroxidase and peroxidase-!ike activities. App. environm. MicrobioL, 1986, 52, 1433-1435. [5] HASSA~, H.M. & FRIDOVICtt, I., Regulation of the synthesis of catalase and peroxidase in Escherichia coil J. biol. Chem., 1978, 253, 6445-6450. [6] PINE, L., HOFFMAN,G.B., MALCOM, G.B., BENSON, R.F. & GORMAN,G.W., Whole-cell peroxidase tests for identification of Legionella pneumophila. J. clin. MicrobioL, 1984, 19, 286-290. [7] WEINER,N., Isoproterenol, in ~ The Pharmacological Basis of Therapeutics, (Goodman, L., Gilman, A., Rail, T.W., Murad, F.) (pp. 160-161). MacMillan Co., London, 1985.