Journal of Applied Bacteriology 1992, 73, 14-22

Co-existence of P-lactamase and penicillin acylase in bacteria ; detection and quantitative determination of enzyme activities W.L. Baker Swinburne Institute of Technology, John Street, Hawthorn 3122, Victoria, Australia 4057/11/91: accepted 18 January 1992 W . L . B A K E R . 1992. Twenty-six bacteria were examined for the presence of penicillin acylase and 8-lactamase. A copper reducing assay, which was sensitive in the analytical range 2-20 pg/ml, was used for determination of penicilloates and a fluorescamine assay was used to determine 6-aminopenicillanic acid concentrations when both substances were produced by the action of the enzymes on a single substrate. Seventeen bacteria contained 8-lactamases, six contained penicillin acylases and four contained both enzymes. Two bacteria contained a T y p e 1 penicillin acylase and four bacteria contained a T y p e I1 enzyme. No ampicillin acylases were detected. All 8-lactamases were constitutive enzymes in those organisms where both enzymes co-existed. Bacillus subtilis and B . cereus produced inducible and extracellular 8-lactamases. Acinetobacter calcoaceticus ATCC 2 1288 produced a constitutive 8-lactamase which was detected extracellularly.

INTRODUCTION

Penicillin acylases (EC3.5.1.11) may interfere with certain methods for detecting b-lactamases (b-amidohydrolasesEC3.5.2.6) (Sykes & Matthew 1979). The problem of distinguishing between the two enzymes has often depended on the methods and criteria used for detecting penicillin acylases. Ideally the product of each enzyme reaction should be differentiated and estimated by chemical methods but there are not many convenient sensitive chemical methods which differentiate 6-aminopenicillanic acid (6-APA) from penicilloates. The acylase has usually been detected by phenylacetylation of 6-APA (Cole & Sutherland 1966) or colorimetry (Nara et af. 1971) after chromatographic separation from the parent penicillin but there could be problems with potential pathogens. 8Lactamases may be detected by several methods (Ayliffe 1964) but biological indicators (Cole & Sutherland 1966) or one of several variations of an iodometric assay (Perret 1954; Sykes & Nordstrom 1972) are reliable and commonly used. Other techniques which differentiate between the two enzymes in bacteria (Pruess & Johnson 1965; Arcos et af. 1968) are less convenient for routine laboratory use and also present a problem with potential pathogens. In the present work the problem associated with the coexistence of penicillin acylase and b-lactamase in bacteria has been re-examined by quantitative methods which differentiate between penicillin and 6-APA substrates of the Correspondence to : W.L. Baker, Swinburne Institute of Technology, John Street, Hawthorn 3/22, Victoria, Australia.

enzymes and the product penicilloates and penicic acid (6APOA). A combination of biological and chemical procedures, including copper reduction, has been used for detection and estimation of b-lactamase activity. Fluorimetry has been used for the estimation of 6-APA, formed by the action of penicillin acylases on penicillins, or its disappearance due to broad spectrum b-lactamases. A major aim was to establish unequivocal criteria for the presence of both enzymes in bacteria. The investigation included a consideration of the presence of broad-spectrum b-lactamases, which convert 6-APA to penicic acid (6-APOA), inducibility and extracellular nature of the enzymes, and the very low enzyme activity found in certain bacteria (Sykes & Smith 1979).

MATERIALS AND METHODS Chemicals

Penicillins came from the sources previously described (Baker 1983a). 6-APA (96% pure) was obtained from Aldrich Chemicals (Milwaukee, WI) and bicinchoninic acid (BCA) from Pierce Chemicals (Rockford, IL). Crystalline lysozyme was purchased from C.F. Boehringer (Mannheim, Germany). P-Lactamase (‘Labpenase’) came from Commonwealth Serum Laboratories, Parkville, Australia. Nitrocefin (chromogenic cephalosporin 87/3 12, O’Callaghan et af. 1972) was kindly donated by Glaxo Research Ltd (Green ford, Middlesex, UK) and solutions were prepared

8-LACTAMASE AND PENICILLIN ACYLASE IN BACTERIA 15

and used according to the manufacturer’s instructions. All other chemicals were analytical grade. instrumental

pH measurements were made with a Metrohm AG396 pH meter. Absorbance measurements were made with a Varian-Techtron 635 recording spectrophotometer. Fluorescence readings were made on a Shimadzu SP500 spectrophotofluorimeter, exciting at 390 nm, and reading emission at 480 nm. Growth was measured with an EEL nephelometer using the standard provided by the manufacturer for 100% turbidity. Bacterial cells were disrupted when necessary at 0°C by sonication at 75 W for two intervals of 3 min each from a Branson sonic probe. Phase contrast microscopy indicated a 75-80% cell breakage. Control enzymes

The crude penicillin acylase preparation, obtained from Escherichia coli ATCC 9637 as previously described (Baker 1983a), was used as a control. This preparation did not affect the colour of Nitrocefin. ‘Labpenase’ was used as a 8-lactamase control. Analytical methods

6-APA was estimated by the reaction with fluorescamine after dilution of solutions with 0.5 mol/l acetate buffer pH 4 (Baker 1983a). Penicilloates were estimated with a BCA colour reagent consisting of 2% BCA and 1% sodium potassium tartrate in 0-05 mol/l phosphate buffer, pH 7. Penicilloate solution (1 ml) in buffer was mixed with 2 ml of colour reagent and equilibrated at 37°C for 10 min. Copper sulphate (0.1 ml of 20 mmol/l in water) was added, solutions mixed and the reaction allowed to proceed for 15 min exactly before termination of colour development with 0.1 ml of 0.2 mol/l EDTA in buffer. Colour was read at 562 nm. Detection and estimatlon of fl-iactamases

Uninhibited growth on nutrient agar plates at 37°C for 16 h in the presence of discs containing benzylpenicillin (5 pg), phenoxymethylpenicillin (5 pg), ampicillin (5 pg) and 6-APA (5 and 100 pg) was presumptive evidence for /?lactamase and was supported by a ‘Nitrocefin’ spot test (Sykes & Matthew 1979) where possible. Quantitative estimations were made on cell suspensions prepared by washing 16 h growth from nutrient agar plates with 3 ml of 0.05 mol/l phosphate buffer pH 7. The turbidity of a diluted sample of these suspensions was meas-

ured nephelometrically and on the basis of this reading the concentrated suspension was adjusted to give the equivalent of a nephelometer reading of 500. Bacterial suspension (0.4 ml) was mixed with 1.6 ml of 6.25 mmol/l penicillin solution and incubated at 37°C with shaking. Samples (0.0250.1 ml) were taken as required, added to 0.1 ml of cold 8% trichloracetic acid and immediately diluted to 1 ml with 0-2 mol/l phosphate buffer p H 7. Penicilloate formed was estimated as above. Cell and penicillin controls were included with each set of incubations and the amount of penicilloate formed was determined by a calculation (Baker 1989) which allowed for copper-catalysed hydrolysis of the intact penicillin. The micro-iodomemc procedure (Sykes & Nordstrom 1972) was also used for analysis. The bacterial suspensions were also examined for penicillin acylase activity.

Attempts were made to induce /?-lactamases by adding 20 ml of a 16 h culture of organism in nutrient broth to 100 ml of nutrient broth in Erlenmeyer flasks. Flasks were shaken at 200 rev/min and 37°C in a Gallenkamp orbital incubator for 2 h to establish growth and methicillin added to a final concentration of 1.2 pmol/l. The shaking was continued and samples taken to follow growth and enzyme activity. Samples were maintained at 4°C until analysis. Enzyme was considered inducible when the ratio of activity to growth was considerably greater than 1. With inducible enzymes dilutions were required to obtain accurate estimations of enzyme activity. Detection and estimation of penicillin acyiase activity

Induction medium at pH 7 (Baker 1983b), solidified with 2% agar, was seeded with a 16 h culture of organism and incubated for 3 d at 24°C. Growth was slow but this temperature is considered most satisfactory for enzyme production (Kaufmann & Bauer 1964). Organisms were removed from the surface of the agar with 3 ml of 0.05 mol/l phosphate buffer pH 7.8 and adjusted to a nephelometer reading of 500. Bacterial suspension (0.4 ml) was mixed with 1.6 ml of buffer or benzylpenicillin (41.25 mmol/l or phenoxymethylpenicillin (2 1 mmol/l) or ampicillin (12.25 mmol/l) or 6-APA (6.25 mmol/l). Tubes were shaken at 37°C and samples (0.1 ml) taken at specific times, vigorously mixed with 0.1 ml of 1 : 1000 cetyltrimethyl ammonium bromide (CTAB) solution, left for 5 min and diluted with 0.5 mol/l acetate buffer pH 4. One ml samples of the diluted solution were treated with fluorescamine (Baker 1983a, 1984) and the fluorescence of 6-APA formed estimated against a 6-APA standard curve after removal of cell debris by centrifugation. The 6-APA substrate was used to detect p-lactamases which act on 6-APA. These cell suspensions were also tested for /?-lactamase activity.

16 W . L . B A K E R

15246 since a sonic extract of this organism readily changed colour. Changes in colour with sonic extracts of the two strains of E. coli were still incomplete and the pink component sedimented on centrifugation, indicating low enzyme activity and tight binding of Nitrocefin to the enzyme.

RESULTS Bicinchonlnlc acid and penlcilloates

Penicilloates reduced the Cu(I1)-BCA complex at pH 7 and 37°C (Fig. 1). The absorbance of the BCA-Cu(1) complex obeyed Beer's Law at penicilloate concentrations to 40 pg/ml and above but a working range of 2-20 pg/ml penicilloate was used in most experiments. EDTA disrupted the Cu(I1) complex but did not seem to affect the Cu(1) complex which was stable in colour. BCA was preferred to neocuproine (2 a-dimethyl-1 ,10-phenanthroline) as a copper reagent (Cohenford ef al. 1988) because the colour change was visible (green to violet) especially when plactamase activity in organisms was low. At pH 7 protein did not interfere significantly and treatment with chlorhexidine or CTAB (1/2000), borate (10 mmol/l) and formaldehyde (5%) did not affect colour development. Formaldehyde did not inhibit B-lactamase activity in many instances.

Estimation of piactamase activity

Bicinchoninic acid analysis was used to obtain quantitative results concerning p-lactamase activity in several organisms not exposed to methicillin induction (Table 1). While there was considerable variation in activity towards the penicillins used the most satisfactory substrates were benzylpenicillin and ampicillin. Some organisms hydrolysed Cephalexin and Bacillus subtilis, B . cereus, Acinetobacter calcoaceticus and Klebsiella aerogenes hydrolysed 6-APA as well as benzylpenicillin and are therefore classed as broad spectrum enzymes (Smith & Hamilton-Miller 1963). Estimation of fl-iactamase and penicillin acylase activity in one organism

Screening organisms for piactamase activity

Growth patterns suggested the possible presence of plactamase in all Gram-negative organisms (13/13) and 4/13 Gram-positive organisms. The response to 6-APA was variable. Inhibitory zones indicated the antibiotic activity of 6-APA whereas lack of zones could indicate either lack of activity or the presence of a B-lactamase in the organism which hydrolysed 6-APA. Quantitative results using penicillin and 6-APA substrates and BCA and fluorescamine analysis distinguished between these two options. Such results indicated that Sarcina lutea had no B-lactamase and 6-APA had no antibiotic activity against this organism but did inhibit the growth of four other organisms. B-Lactamase presence was generally confirmed with Nitrocefin but the colour change of this reagent was slow and incomplete (orange to pink) over 6 h with Alcaligenes faecalis ATCC 15246, E. coli ATCC 9637 and E. coli ATCC 11105. This was due to lack of permeability in Ah. faecalis ATCC

0'5r(a '

The lower activity of control enzymes in a mixture compared with the activity of the individual enzymes (Table 2) represents competition for a single substrate. This competition presumably occurs within organisms although factors such as accessibility, permeability and enzyme levels must be considered in vivo. T o ensure estimatable levels of penicillin acylase activity within 6 h in the presence of 8-lactamase it was necessary to grow the organism on the medium containing phenylacetic acid. The p-lactamase of Alc. faecalis ATCC 15246 was constitutive and more active than the penicillin acylase (Fig. 2) of this organism. The substrate level used for this procedure was barely sufficient to detect the penicillin acylase in Ac. calcoaceticus ATCC 21288 but higher levels of substrate gave very high blank readings in the B-lactamase copper assay and

P

0.4 W 0 C

0.3

Fig. 1 (a) Standard curve of

;

n a 0.2 0.I

0

10

20

Concentration of benzylpenicilloate (pg/mll

0

10

20

30

Time (mid

40

50

benzylpenicilloic acid in the concentration range 2-20 pg/ml(O) and benzylpenicillin under the same conditions (.).(b) Development of colour with time for 20 pg/ml benzylpenicilloate (0) and benzylpenicillin ( 0 )EDTA . (20 pmol) added where indicated by arrow

/?-LACTAMASE AND PENICILLIN ACYLASE IN BACTERIA 17

Table 1 Quantitative estimation of p-lactamases in some bacteria*

Enzyme activity (pmol penicillin destroyed/ml/6 h ) Organism

PenG

PenVK

Meth

Amp

Ceph

1. Escherichia coli K12 2. Bacillus subtilis 3. B. cereus 4. Staphylococcus aureus 5. Klebsiella aerogenes 6. Serratia marcescens 7. Erwinia carotovora 8. Proteus vulgaris 9. E. coli A T C C 11105 10. E. coli A T C C 9637

1.04 2.08 1.18 0.44 1.86 1.56 0.95 0.74 0.39 0.24

0.80 0.87 0.75 0.14 0.84 0.43 0.34 0.14 0.15 0.32

0 0 0 0 0.17 0 0-07 0 0.27 0

1-29 2-30 1.84 1.72 1.93 0.96 2.15 2-69 1.44 0.55

0-12 0 0 0 2.54 0.17 0 1.79 0 0

3.04

3.10

0.24

2.12

2.69

1.25

1.03

0

0

0

1 1. Alcalagenes faecalis ATCC 15246 12. Acinetobacter calcoaceticus ATCC 21288

* Cells incubated with 5 pmol respective penicillin for 6 h at 37°C. Duplicate samples (0.1 ml) were taken and analysed for penicilloate by bicinchoninic acid (BCA) method as under Materials and Methods. PenG, Benzylpenicillin ; PenVK, phenoxymethylpenicillin potassium salt; Meth, methicillin; Amp, ampicillin; Ceph, cephalexin. Table 2 Co-existence of penicillin acylase and j-lactamase in micro-organisms*

Product estimated (pmol/ml solution) Incubation time (h) 1

6

6

Growth medium

System, substrate + enzyme or bacteria

Control enzymes

+ /l-lactamase control + penicillin acylase control + both enzymes + Escherichia coli ATCC 9637

A

B

24

A

24

B

+ Alraligmcs faccaiis ATCC 15246 + Acinctobactcr calcoaccricus ATCC 21288t + E. coli ATCC 9637 + E. coli ATCC 11105

+ Alc. faecalis ATCC 15246 + AC.calcoaccticus ATCC 21288t + E. coli ATCC 9637

+ E. coli ATCC 11105 + Air. faecalis ATCC 15246 + Ac. calcoaceticus ATCC 21288t

+ E. coli ATCC 9637 + E. coli ATCC 11105 + Alc. faccalis ATCC 15246

+ Ac. calcoaccricus ATCC 21288t

BPOA or PVKOA

CAPA

1.31 0.62

-

0.05

0.03 0-22 0

0-69 0.67

1.91 1.20

4.62 2.96

0.113 0.08 2.37 1.74

0-18

0.67 0.67 1.12 2.02

0.04 0.11 0.92 0.18

0.27 0.23 4.1 2.70

5.0 4.8 1.2 0.17

1.20

* Growth medium A was nutrient agar and medium B was phenylacetic acid agar as described under Materials and Methods. After washing from the agar the cells were incubated in 5 mmol/l benzylpenicillin solution at pH 7 and analyses conducted as described under Materials and Methods. t Phenoxymethylpenicillin solution 5 mrnol/l was substituted for benzylpenicillin as substrate. BPOA, Penicilloate of penicillin G; PVKOA, penicilloate of phenoxymethylpenicillin potassium salt; 6-APA, 6-aminopenicillanic acid.

18 W . L . B A K E R

sensitivity to the Cu(I1) reduction procedure was used to confirm the presence of the enzyme in these organisms. With this technique 52 and 84 nmol of benzylpenicillin was destroyed in 1 ml of respective cell suspension in 6 h and 72 and 92 nmol was destroyed by sonic extracts of the same cell suspensions. Penicillin acylase type specificity

0

I

2

3

4

5

Higher levels of penicillin were required to confirm the presence of penicillin acylase in Ac. calcoaceticus ATCC 21288 which showed a typical Type I acylase pattern (Table 3). Nocardia glomerula ATCC 2 1022 also possessed a Type I enzyme but the other four ATCC organisms produced a Type I1 acylase. No other organism tested produced a penicillin- or an ampicillin-specific (Nara et al. 1971) acylase. Acylase activity in Alc. faecalis ATCC 15245 was always comparatively low due to the destruction of substrate by the 8-lactamase in this organism. Disappearance of fluorescence upon formation of penicic acid from 6-APA confirmed the presence of broad spectrum /3-lactamases in B. subtilis, B. cereus, Kl. aerogenes and Ac. calcoaceticus.

6

Time ( h )

Fig. 2 Formation of benzylpenicilloate (@) and

6-aminopenicillanic acid (6-APA) ( 0 )from benzylpenicillin by cell suspension of Alcaligenesfaecalis ATCC 15246. Cells were grown on phenylacetic acid induction medium (Baker 1983b) as described under Materials and Methods. Samples (25 pl) were taken at times indicated

were undesirable. The /3-lactamase activity by these two organisms was confirmed by the macro-iodometric procedure (Perret 1954). /3-Lactamase activities of E. coli ATCC 9637 and E. coli ATCC 11105 were very low and the micro-iodometric assay (Sykes & Nordstrom 1972) which is of a similar order of

inducible fl-iactamases

Methicillin had different effects on the growth rate of liquid cultures of B. subtilis and B. cereus (Fig. 3). ConseTable 3 Quantitative estimation of

Activity (pmol/ml/6 h)

penicillin acylase activity*

6-APA formed from Organism

PenVK

PenG

Amp

6-APA used

Bacillus megaterium ATCC 14945 Escherichia coli ATCC 9637 E. coli ATCC 11105 Aicaiigenes faecalis ATCC 15246 Nocardia glomerula ATCC 21022 Acinetobacter calcoaceticus ATCC 2 1288 Kiebsieila aerogenes B . subtilis B. cereus

0 0.67 0.6 1 0.09 2.64 4.4 0 0 0

0.86 4.6 3.9 0.77 0.52 0.42 0 0 0

0.30 0.44 0.27 0.64 0 0 0 0 0

0 0 0 0 0 1.94 2.1 0.92 0.52

* Cells grown on agar medium containing phenylacetic acid and collected and standardized as described under Materials and Methods. Cells were suspended in solutions of penicillins to give the following final concentrations : phenoxymethylpenicillin 16.7 mmol/l; benzylpenicillin, 33 mmol/l; ampicillin 10 mmol/l and 6-APA, 5 mmol/l. Samples were taken after 6 h incubation at 37"C, diluted with 0.5 mmol/l acetate buffer pH 4 and the amount of 6-APA formed or remaining estimated with fluorescamine as described under Materials and Methods. 6-APA, 6-aminopenicillanic acid ; PenG, benzylpenicillin; Amp, ampicillin ; PenVK, phenoxymethylpenicillin potassium salt.

b-LACTAMASE AND PENICILLIN ACYLASE I N BACTERIA 19

701

60

n 0

I

2

I

I

1

4

3

4

5

6

Time (h)

Flg. 4 Formation of benzylpenicilloic acid and penicic acid from

benzylpenicillin and 6-aminopenicillanic acid (6-APA) by cell suspension of Klebsiella aerogenes. Benzylpenicilloate (m) and penicic acid ( 0 )measured by copper reduction. Penicic acid ( 0 ) measured by disappearance of fluorescence of 6-APA with time. Samples were taken and treated at the time indicated as described under Materials and Methods

Fig. 3 Development of 8-lactamase activity with growth of Bacillus subtilis and B. cereus. Enzyme activity ( 0 )and growth

( 0 )of B . subtilis and enzyme activity (m) and growth ( 0 )of B . cereus

quently the ratios of enzyme activity to growth in the interval between 2 and 10 h was considered the best criterion for inducibility. The effects in the presence and absence of inducing agent (Table 4) show a 17-fold increase in growth of B. subtilis and a 64-fold increase in rate of enzyme activity in the presence of methicillin. Over the same interval B. cereus growth increased 20-fold and enzyme activity increased 640-fold. Enzyme activity increased broadly commensurate with growth rate in the absence of inducing agent..

Extracellular broad-spectrum &lactamases

The broad-spectrum /?-lactamase of KI. aerogenes was a non-inducible enzyme and longer incubation times were required to obtain estimations of activity than were required with the enzymes of B. subtilis and B. cereus (Table 5). All the broad-spectrum enzymes which were examined hydrolysed benzylpenicillin more rapidly than 6-APA. T h e rate of hydrolysis of each substrate by the /?lactamase in K1. aerogenes is shown in Fig. 4. The super-

Table 4 Induced and non-induced

Ratio 10 h/2 h

8-lactamases* Organism Bacillus subtilis

Inducer (1.2 pmol))

Sample time

Nil

2 10

10 62

0.04 0.28

2 10

9 153

0.75 47.8

2 10

15 59

0.17 0.29

2 10

5.6 112

0.065 41.6

2 10

24 156

0.193 0.674

1 6.5

1 3.5

2

32 131

0.321 0.619

1 4.1

1

Methicillin Bacillus cereus

Nil Methicillin

Acinetobacter calcoaceticus

Nil

ATCC 21288

Methicillin

10

Nephelometer reading

Enzyme activity (pmol/ml/h)

Growth

1 6.2 1 17 1 3.93 1 20

Enzyme activity 1 7 1 63.8 1

1.71 1 640

1.93

* Enzyme activity was determined with benzylpeniallin as substrate as described under Materials and Methods.

20 W . L . B A K E R

Organism

Reaction time (h)

Bacillus subtilis

0.17

Preparation

PenG

6-APA

Broth culture Supernatant fluid

2.47 2.92 0.90

1.38 1.2 0.15

4.04

4.38 1.69

2.22 0.93 0-65

cells

2.57 0.1 1 1.72

1.94 0.36 1.20

Broth culture Supernatant fluid cells

4.8 2.8 4.16

3.52 1.12 2.8

cells

Bacillus cereus

0.1

Broth culture Supernatant fluid cells

Klebsiella aerogenes

Acinetobacter calcoaceticus ATCC 21288

6

6

Broth culture Supernatant fluid

Cells were grown in nutrient broth with shaking and inducing agent as described under Materials and Methods and for Table 3. A sample of the original broth culture, a sample of the supernatant fluid obtained by centrifugation at SO00 rev/min for 20 min and a sample of cells, resuspended in the same volume as the broth culture, were mixed with benzylpenicillin or 6-APA at a final concentration of 5 mmol/l and incubated for the times indicated. 6-APA, 6-aminopenicillanic acid ;PenG, benzylpenicillin.

natant fluid of the growth medium of Kl. aerogenes showed little enzyme activity in comparison with that of the other three broad-spectrum organisms. DISCUSSION

Under the conditions of enzyme analysis the copper reduction procedure is specific for penicilloates and fluorescamine, used at pH 4, is specific for 6-APA (Baker 1983a). The protocol used (Fig. 5) gives unequivocal evidence of the coexistence of penicillin acylase and 8-lactamase in bacteria. Where 8-lactamase activity was low the microiodometric procedure (Sykes & Nordstrom 1972) was preferred to colorimetric substrates (O’Callaghan et al. 1972; Schindler & Huber 1980) for confirmation. These synthetic substrates may not be universally hydrolysed by 8-lactamases (Sykes & Matthew 1979). Many organisms can be processed simultaneously and a wide range of substrates can be used with relative convenience. Data are obtained rapidly and simply and modifications permit the constitutive, inducible, intra- or extracellular nature of the enzymes and their substrate profiles to be determined. There is scope for examination of fastidious organisms. Unlike chromatograhic techniques for 8-lactamases and penicillin acylases (Cole & Sutherland

1966; Nara et al. 1971) standard aseptic precautions give relative safety against pathogens. Safety is not ensured by the very sensitive dansylation technique (Chen 1986) where there is also potential for degradation of penicillins during derivatization. I n the present procedure penicillins, rather than artificial substrates (Holt & Stewart 1964; Kutzbach & Rauenbusch 1974) are always used for both enzymes. Twenty-six organisms were examined in this study. Seventeen bacteria contained 8-lactamases, six contained penicillin acylases and four contained both enzymes. All penicillin acylases were induced by phenylacetic acid. Two organisms produced a Type I penicillin acylase and four organisms a Type I1 penicillin acylase. No specific ampicillin acylases were detected. 8-Lactamases were constitutive in those organisms where both enzymes co-existed but their metabolic profiles varied. T h e enzymes in E. coli ATCC 9637 and E. coli ATCC 11105 hydrolysed benzylpenicillin at a very slow rate. T h e enzymes in A c . calcoaceticus and Alc. faecalis were more active against benzylpenicillin. Acinetobacter calcoaceticus had a Type I penicillin acylase and a broad-spectrum blactamase which was not reported in an earlier study (Nara et al. 1971). T h e Kl. aerogenes broad-spectrum 8-lactamase was constitutive and intracellular. T h e enzymes of the Bacillus genus were extracellular and inducible. T h e poten-

8-LACTAMASE AND PENICILLIN ACYLASE IN BACTERIA 21

I

I

8-Loctomose ond penicillin ocylose co-existence

Nutrient plates

PenVK

directly with

6-APA Others

ontibiotic discs susoension

Ouontitotive PenVK T I ocylose

1

I LLz-l

Ouontitotive PenG Amp PenVK Ceph Meth 6-APA Others

Techniques

I Fluorescomine

I Fig. 5 Protocol for detection and

estimation of B-lactamases and penicillin acylases in the one organism

I

Mocroiodometric Microiodometric CU(n) reduction

I +

I.Test qrowth vs enzyme activity for inducible enzymes. 2.Test culture medium, supernatant fluid and centrifuged cells for narrow spectrum and brood spectrum enzymes.

I

&a-Loctomoses

Fluorescomine for brood sDectrum 8-loctomoses

tial broad-spectrum /?-lactamases explain the requirement for high penicillin levels when determining penicillin acylase type activity and specificity.

REFERENCES ARCOS,J . M . , R U I Z , M . C . & M U G I C AJ, . D . (1968) BLactamase and penicillin acylase coexistence in Escherichia coli. Journal of Bacteriology 96, 1870-1872. AYLIFFE, G . A .J . (1963) Ampicillin inactivation and sensitivity of coliform bacilli. Journal of General Microbiology 36, 339-348. B A K E RW, . L . (1983a) Application of the fluorescamine reaction with 6-aminopenicillanic acid to estimation and detection of penicillin acylase activity. Antimicrobial Agents and Chemotherapy 23, 26-30. BAKER,W . L . (1983b) Activity of penicillin acylase producing bacteria against a-aminobenzylpenicillins.Antonie van Leeuwenhoek Journal of Microbiology and Serology 49, 551-558. BAKER, W . L . (1984) A sensitive procedure for screening microorganisms for the presence of penicillin amidase. Australian Journal of Biological Sciences 37, 257-265. B A K E RW, . L . (1989) Spectrophotometric determination of penicilloates in penicillins. Analyst 114, 1137-1 139.

Q3-Loctomoses Mocroiodometric Microiodometric Cu(ll) reduction 8.Penicillin o c y w Fluorescomine

CHEN, K .C . S . (1986) Two-dimensional thin-layer chromatography for simultaneous detection of bacterial B-lactam acylases and /3-lactamases. Antimicrobial Agents and Chemotherapy 30, 536541.

COHENFORD, M . A . , A B R A H A MJ ,. & MEDEIROS, A.E. (1988) A colorimetric procedure for measuring B-lactamase activity. Analytical Biochemistry 168, 252-258. COLE,M . & SUTHERLAND, R. (1966) The role of penicillin acylase in the resistance of gram negative bacteria to penicillins. Journal of General Microbiology 42, 345-356. HOLT,R.J. & STEWART, G . T . (1964) Production of amidase and B-lactamase by bacteria. Journal of General Microbiology 36,203-213.

K A U F M A NW N ,. & BAUER,K . (1964) Variety of substrates for a bacterial benzylpenicillin splitting enzyme. Nature (London) 203, 520.

KUTZBACH, C. & RAUENBUSCH, E . (1974) Preparation and general properties of crystalline penicillinacylase from Escherichia coli ATCC 11105. Zeitschrijl f u r Physiologische Chemie 354, 45-53.

N A R AT., , MISAWA,M . , O K A C H IR, . & YAMAMOTO, M. (1971) Enzymatic synthesis of D( -)-a-aminobenzylpenicillin. Part 1. Selection of penicillin acylase producing bacteria. Agricultural and Biological Chemistry 35, 16761682. O'CALLAGHAN, C . H . , MORRIS,A., K I R B Y , S . M . &

22 W . L . BAKER

S H I N G L E RA., (1972) Novel method for detection of /?lactamases using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy 1, 283-288. PERRETT, C. J . (1954) Iodometric assay of penicillinase. Nature (London) 174, 1012-1013. PRUESS,D . L . & J O H N S O N , M . J . (1965) Enzymatic deacylation of S’5 benzylpenicillin. Journal of Bacteriology 90, 380383. S C H I N D L EP. R , & HUBER,G . (1980) Use of PADAC, a novel chromogenic /?-lactamase substrate, for the detection of /?lactamase producing organisms and assay of /?-lactamase inhibitors/inactivators. In Enzyme Inhibitors, ed. Brodbeck, U., pp. 169-176. Weinheim: Verlag Chemie. S M I T H J.T. , & HAMILTON-MILLER, J . M . T . (1963) Dif-

ferences between penicillinases from Gram-positive and Gramnegative sources. Nature (London) 197,976978. S Y K E SR.B. , & MATTHEW, M . (1979) Detection, assay and immunology of /?-Lactamases. In /?-Lactamases, eds HamiltonMiller, J.M.T. & Smith, J.T. Ch.2, pp. 1749. London: Academic Press. S Y K E S ,R.B. 8z NORDSTROM, K. (1972) Microiodometric determination of /?-lactamase activity. Antimicrobial Agents and Chemotherapy 1,94-99. SYKES,R.B. & S M I T H J.T. , (1979) Biochemical aspects of /?-lactamase from Gram negative organisms. In /?-Lactamases, eds Hamilton-Miller, J.M.T. & Smith, J.T. Ch. 14, pp. 369401. London : Academic Press.