ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1986, p. 104-106 Vol. 29, No.

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0066-4804/86/010104-03$02.00/0
Copyright C) 1986, American Society for Microbiology

Synergistic Effects of Dicloxacillin or Clavulanic Acid in

Combination with Penicillin G or Cephalothin against
Yersinia Enterocolitica
MARIA JIMENEZ-VALERA,* ALFONSO RUIZ-BRAVO, AND ALBERTO RAMOS-CORMENZANA
Departamento de Microbiologia, Facultad de Farmacia, Universidad de Granada, Granada, Spain
Received 1 April 1985/Accepted 8 October 1985

Cultures of Yersinia enterocolitica grown at 22°C produced beta-lactamases, whereas cultures grown at 37°C
produced these enzymes much Iess effectively. Both dicloxacillin and clavulanic acid inhibited the beta-
lactamase activity of bacterial crude extracts and potentiated the activity of penicillin G or cephalothin against
14 Y. enterocolitica strains. It appeared that the beta-lactamase activity present in Y. enterocolitica cells grown
at 3rC was great enough to play a role in bacterial resistance to beta-lactam antibiotics, since combining
penicillin G or cephalothin with clavulanic acid or dicloxacillin resulted in synergistic activity against cultures
grown at 37°C that was equal to or greater than the activity against cultures grown at 22°C.

The production of beta-lactamase is recognized as one of
the main mechanisms of bacterial resistance to beta-lactam
antibiotics (8, 15). The approaches used to solve this resist-
ance problem have involved the development of stable
penicillins and cephalosporins and a search for beta-
lactamase inhibitors (3). Numerous compounds have been
included on the list of beta-lactamase inhibitors, and some of
these have shown potential clinical usefulness based on their
synergistic effects when they are combined with beta-
lactamase-labile antibiotics (3, 8). This is the case with
clavulanic acid (13, 16, 17) and isoxazolyl penicillins (1, 12,
18).
Yersinia enterocolitica is an enteropathogenic organism
(2) that produces intracellular beta-lactamases (4, 6), which
are readily detected in cultures grown at 22°C but almost
imperceptible in bacteria grown at 37°C (10). The simulta-
neous presence of two chromosome-mediated beta-
lactamases, called beta-lactamases A and B, is a common
property of strains belonging to serotypes 0:3 and 0:9 (4, 5).
In this report, we describe the potentiation of penicillin G
and cephalothin activities when these antibiotics were com-
bined with dicloxacillin or clavulanic acid against 14 Y.
enterocolitica strains. Our aim was to assess the role of
beta-lactamase production at 22 and 37°C in the resistance of
Y. enterocolitica to beta-lactam antibiotics.
MATERIALS AND METHODS
Bacterial and culture conditions. Fourteen strains of Y.
enterocolitica were used in this study. Seven of these strains
were serotype 0:3, five were serotype 0:9, one was serotype
Nitrocefin was provided by Glaxo Laboratories, Greenford,
Middlesex, England.
Nitrocefin assay. Bacterial cells were suspended in 0.85%
saline, and a Nitrocefin (cephalosporin 87/312) assay was
performed as described by Kammer et al. (9). Briefly, 50 p.1
of a Nitrocefin solution (5 mg of Nitrocefin in a solution
containing 0.5 ml of dimethyl sulfoxide and 9.5 ml of 0.1 M
phosphate buffer, pH 7.0) was added to 50 p.l of a bacterial
suspension. Development of a red color within 30 min
indicated the presence of beta-lactamases.
Assay for beta-lactamase inhibition. Bacteria were grown
at 37 and 22°C, and crude cell extracts were obtained by
sonic treatment of cells for 3 min in an ice bath. Beta-
lactamase activity was determined by using the iodometric
assay described by Sargent (19), except that the phosphate
buffer was adjusted to pH 5.6 when penicillin G was used as
the substrate and to pH 8.0 when the substrate was cepha-
lothin (11). The assay was performed in the presence of
several concentrations of clavulanic acid or dicloxacillin,
which were added to the substrate solution just before
incubation with the crude extract. Blank tests for iodine
reagent absorbance and for spontaneous substrate hydroly-
sis were included in order to calculate beta-lactamase activ-
ity by the method of Sawai et al. (20). Finally, the levels of
beta-lactamase inhibition were calculated as percentages by
using the following formula (after arithmetic simplification of
common factors): percentage of beta-lactamase inhibition =
(activity without inhibitor - activity in the presence of
inhibitor)/(activity without inhibitor) x 100 = KJ(enzyme +
inhibitor) K&(enzyme)]/[K&(substrate) - Kj(enzyme)] x
-

0:4,33, and one was serotype 0:6,31. Twelve were of human
origin and two were isolated from water. Bacterial inocula
were prepared from cultures grown on Trypticase soy agar
(BBL Microbiology Systems, Cockeysville, Md.) at either 37
or 22°C for 24 h.
Antimicrobial agents. Clavulanic acid (sodium salt) was
provided lyophilized by Beecham Pharmaceuticals Research
Div., Betchworth, England. Dicloxacillin was also provided
by Beecham Laboratories. Penicillin G was obtained from
Antibioticos S.A., Leon, Spain. Cephalothin power was
supplied by Lilly Research Laboratories, Indianapolis, Ind.
*
Corresponding author.
104
100, where K&(enzyme + inhibitor) was the absorbance (in
Klett units) after iodine consumption by enzymatic hydroly-
sis of the substrate in the presence of inhibitor, K&(enzyme)
was the amount of iodine consumption by enzymatic hydro-
lysis without inhibitor, and K&(substrate) was the amount of
iodine consumption by spontaneous hydrolysis of the sub-
strate.
Determination of MIC. Logarithmic-phase cells grown in
Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.)
at 37 or 22°C were diluted in Mueller-Hinton broth to provide
inocula having approximately 2 x 106 CFU/ml (confirmed by
culturing appropriate dilutions of inocula on Trypticase soy
agar). Serial twofold dilutions of the antibiotics were pre-
VOL. 29, 1986 ANTIBIOTIC SYNERGISM AGAINST Y. ENTEROCOLITICA 105-

TABLE 1. Inhibition of beta-lactamases from Y. enterocolitha IP134 by dicloxacillin and clavulanic acid
% of inhibition of beta-lactamase activity on
(.g/mI)
Inhlbltor
Inhibitor Concn (,ug/ml) Temp of bacterial
~~~~~growth
(°C)
the following substratesa
Penicillin G Cephalothin
Dicloxacillin 0.5 22 27 ± 9.4b 29 ± 8.2
5 22 47 ± 10.5 69 ± 8.5
50 22 >95 >95
0.5 37 60 ± 8.8 61 ± 4.5
5 37 >95 >95
Clavulanic acid 10 22 90 ± 6.4 84 ± 5.7
1 37 70 ± 8.4 69 ± 10.6
10 37 >95 >95
a
The following sustrate concentrations were used: penicillin G, 2.0 mg/ml; cephalothin, 2.4 mg/ml. At 22°C the beta-lactamase activities were as follows (in
nanomoles of substrate hydrolyzed per minute per milliliter): penicillin G, 47 ± 1.5; cephalothin, 74 ± 1.5. At 37°C the beta-lactamase activities were as follows
(in nanomoles of substrate hydrolyzed per minute per milliliter): penicillin G, 17 ± 2.0; cephalothin, 23 ± 4.2.
The values are arithmetic means ± standard deviations from at least four determinations.
b

pared in Mueller-Hinton broth and distributed in glass tubes. drug combinations which included clavulanic acid was
Each tube received 1.0 ml of an antibiotic dilution and 1.0 ml higher at 37°C than at 22°C; in contrast, the level of synergy
of an inoculum. Triplicate tubes were incubated at 37 or 22°C of drug combinations which included dicloxacillin was influ-
for 24 h. The MIC was the lowest antibiotic concentration enced little or was not influenced by the temperature.
which gave complete inhibition of visible growth. To study
synergy in combinations of penicillin G or cephalothin with DISCUSSION
dicloxacillin or clavulanic acid, the two antibiotics in each
combination were mixed to provide concentrations that were The results of this study indicate that dicloxacillin and
two times the MIC of each antibiotic alone, and the mixture clavulanic acid potentiated the activity of beta-lactamase-
was serially diluted twofold. Bacterial inocula (1.0 ml) were labile antibiotics against Y. enterocolitica strains. The ability
added to equal volumes of antibiotic mixture dilutions. MIC of dicloxacillin and clavulanic acid to protect penicillin G or
determinations were performed after 24 h at 37 or 22°C. An cephalothin against Y. enterocolitica beta-lactamases argues
antibiotic combination was considered synergistic when the for beta-lactamase inhibition as a major mechanism of these
MIC of each drug in the combination decreased at least synergisms. It is very interesting that the synergy against
fourfold (7). cultures grown at 37 was equal to or greater than the synergy
against cultures grown at 22°C. Our results suggest that the
RESULTS beta-lactamase activity detected by the Nitrocefin assay in
Beta-lactamase detection and inhibition. Previously, we whole cells grown at 37°C was great enough to play a role in
reported that Y. enterocolitica produces beta-lactamase at resistance to beta-lactam antibiotics. Minute amounts of
22°C, whereas beta-lactamase activity is not detected when strategically placed beta-lactamase may be enough to hydro-
cultures are grown at 37°C (10, 11). We used the very lyze the entering beta-lactam antibiotic and to protect bac-
sensitive Nitrocefin test for beta-lactamase detection in teria (14). The small quantity of beta-lactamase produced at
cultures of strains grown at both temperatures. Beta- 37°C should be inhibited more easily than the large amount
lactamase activity was always found; the appearance of the of enzyme produced at 22°C; this may explain the fact that
red color was immediate in cultures grown at 22°C, whereas the level of synergy was higher at 37°C than at 22°C for some
the red color appeared 5 to 10 min later in cultures grown at combinations.
37°C (data not shown). Y. enterocolitica produces beta-lactamases A and B,
To check the inhibition of Y. enterocolitica beta which have different substrate profiles and different pH
lactamases by dicloxacillin and clavulanic acid, we investi- optima (5, 11). Cloxacillin completely inhibits the activity of
gated the effect of both agents on protecting penicillin G and beta-lactamase B but partially inhibits the activity of beta-
cephalothin from hydrolysis by bacterial crude extracts.
Table 1 shows that the beta-lactamase activities of crude
extracts from Y. enterocolitica IP134 were inhibited by TABLE 2. Cumulative MICs of beta-lactam agents and
appropriate concentrations of clavulanic acid and dicloxacil- combinations for 14 Y. enterocolitica strains at 22 and 37°C
lin. Furthermore, our data show that beta-lactamase activity Range of MICs (,ug/ml)"
was present in crude sonic extracts of cultures grown at Antibiotic(s)
22°C 37°C
37°C, as determined by the iodometric assay, whereas there
was no activity when whole cells grown at the same temper- Penicillin G 512-2,048 256-2,048
ature were tested (11). Cephalothin 256-1,024 128-1,024
MICs. To investigate the true role of beta-lactamase Dicloxacillin 512-2,048 256-2,048
production in resistance of Y. enterocolitica to beta-lactam Clavulanic acid 32-256 32-64
Penicillin G + dicloxacillin 32-128/32-64b 8-128/16-64
antibiotics, we studied the interactions of penicillin G and Penicillin G + clavulanic acid 32-512/2-64 4-64/0.5-2
cephalothin with dicloxacillin and clavulanic acid against Cephalothin + dicloxacillin 16-64/32-128 4-64/8-128
cultures of 14 strains at 37 and 22°C (Table 2). When Cephalothin + clavulanic acid 32-256/4-32 2-64/0.5-4
combinations of penicillin G or cephalothin with the beta-
lactamase inhibitors were studied, synergism was found at Mean values.
bRange of MICs of the beta-lactamase-labile antibiotic in the
37 and 22°C (the MIC of each drug in the combination was combination/range of MICs of the beta-lactamase-inhibiting agent in the
reduced by 25%). As a general rule, the level of synergy of combination.
106 JIMENEZ-VALERA ET AL.
lactamase A (4); it may be assumed that beta-lactamase
inhibition due to dicloxacillin has a similar pattern. There is
currently no information available about the effect of clavu-
lanic acid on either enzyme. The influence of temperature on
the synergy between beta-lactamase-labile antibiotics and
clavulanic acid may be due to some hypothetical tempera-
ture-dependent change in the ratio of the beta-lactamases A
and B produced by Y. enterocolitica. However, alternative
explanations should be considered; for example, it is possi-
ble that clavulanic acid penetrated Y. enterocolitica more
easily when cells were grown at 37°C than when they were
grown at 22°C, whereas this mechanism might be negligible
for the larger dicloxacillin molecule.
LITERATURE CITED
1. Acred, P., and R. Sutherland. 1967. Antibacterial activities of
combinations of ampicillin and cloxacillin, p. 53-58. Antimi-
crob. Agents Chemother. 1966.
2. Bottone, E. J. 1977. Yersinia enterocolitica: a panoramic view of
a charismatic microorganism. Crit. Rev. Microbiol. 5:211-241.
3. Cole, M. 1979. Inhibition of beta-lactamases, p. 205-290. In J.
M. T. Hamilton-Miller and J. T. Smith (ed.), Beta lactamases.
Academic Press, Inc., London.
4. Cornelis, G. 1975. Distribution of beta-lactamases A and B in
some groups of Yersinia enterocolitica and their role in resist-
ance. J. Gen. Microbiol. 91:391-402.
5. Cornelis, G., and E. P. Abraham. 1975. Beta-lactamase from
Yersinia enterocolitica. J. Gen. Microbiol. 87:273-284.
6. Cornelis, G., G. Wauters, and M. Vanderhaehe. 1973. Presence
de beta-lactamase chez Yersinia enterocolitica. Ann. Microbiol.
(Paris) 124B:139-152.
7. Hallander, H. O., K. Dornbusch, L. Gezelius, K. Jacobson, and
I. Karlsson. 1982. Synergism between aminoglycosides and
cephalosporins with antipseudomonal activity: interaction index
and killing curve method. Antimicrob. Agents Chemother.
22:743-752.
8. Hamilton-Miller, J. M. T. 1977. Inhibition of beta-lactamase: a
ANTIMICROB. AGENTS CHEMOTHER.
continuig story. J. Antimicrob. Chemother. 3:195-196.
9. Kammer, R. B., D. A. Preston, J. R. Turner, and L. C. Hawley.
1975. Rapid detection of ampicillin-resistant Haemophilus influ-
enzae and their susceptibility to sixteen antibiotics. Antimicrob.
Agents Chemother. 8:91-94.
10. Kouwatli, K., V. Bejar, A. Ruiz-Bravo, and A. Ramos-
Cormenzana. 1979. Sensitivity of Yersinia enterocolitica to
several antimicrobial agents. Microbios Lett. 11:137-142.
11. Kouwatli, K., V. Bejar, A. Ruiz-Bravo, and A. Ramos.
Cormenzana. 1981. Effect of temperature on sensitivity of
Yersinia enterocolitica to beta-lactam-type agents. Microbios
Lett. 16:55-58.
12. Maddocks, J. L., and J. R. May. 1969. Indirect pathogenicity of
penicillinase-producing enterobacteria in chronic bronchial in-
fections. Lancet i:793-795.
13. Miller, J. M., C. N. Baker, and C. Thornsberry. 1978. Inhibition
of beta-lactamase in Neisseria gonorrhoeae by sodium clavulan-
ate. Antimicrob. Agents Chemother. 14:794-796.
14. Neu, H. C. 1974. The role of beta-lactamases in the resistance of
gram-negative bacteria to penicillin and cephalosporin deriva-
tives. Infect. Dis. Rev. 11:133-149.
15. Ogawara, H. 1981. Antibiotic resistance in pathogenic and
producing bacteria, with special reference to beta-lactam anti-
biotics. Microbiol. Rev. 45:591-619.
16. Paisley, J. W., and J. A. Washington. 1978. Combined activity of
clavulanic acid and ticarcillin against ticarcillin-resistant, gram-
negative bacilli. Antimicrob. Agents Chemother. 14:224-227.
17. Reading, C., and M. Cole. 1977. Clavulanic acid: a beta-
lactamase-inhibiting beta-lactam from Streptomyces
clavuligerus. Antimicrob. Agents Chemother. 11:852-857.
18. Renzini, G., G. Ravagnan, and B. Oliva. 1973. The microbiolog-
ical properties of a dicloxacillin-hetacillin combination. Chemo-
therapy (Basel) 19:367-378.
19. Sargent, M. G. 1968. Rapid fixed-time assay for penicillinase. J.
Bacteriol. 95:1493-1494.
20. Sawai, T., I. Takahashi, and S. Yamagishi. 1978. lodometric
assay method for beta-lactamase with various beta-lactam anti-
biotics as substrates. Antimicrob. Agents Chemother.
13:910-913.