a Department of Microbiology, The Prince of Wales Hospital, Randwick, NSW 2031, Australia; b WHO Yersinia Collaborating Centre, Institut Pasteur, 75724 Paris Cedex 15, France
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Abstract |
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Introduction |
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Matthew and colleagues subsequently examined the ß-lactamases of 37 strains of Y. enterocolitica of various serological and biochemical groups by means of isoelectric focusing.4 They found that strains of serotypes O:1, O:2, O:3 and O:9 produced both types of ß-lactamase, enzyme A with a pI of 8.7 and enzyme B with pIs of 5.3 and 5.7. Strains of serotype O:5,27 produced only enzyme B and strains of other serotypes produced what appeared to be different types of ß-lactamase. Subsequently, different patterns of susceptibility to ß-lactam antibiotics observed with strains of biotypes 1A, 3 and 4 isolated in Australia were explained by differences in the distribution of enzyme A, enzyme B and enzyme B-like which also was a group of cephalosporinases produced by strains of the non-pathogenic biotype 1A.5,6 The properties of the ß-lactamases of the enzyme B-like group were similar to those of enzyme B but their pIs were different, ranging from 6.2 to 7.3.6 In a study of Y. enterocolitica bio-serotype 4/O:3 isolated in various parts of the world, strains of phage type VIII and IXa were found to be homogeneous in the expression of ß-lactamase, with all strains producing both enzyme A and enzyme B. Also, while Y. enterocolitica 4/O:3/IXb strains isolated in Canada formed two subgroups, one produced only one type of ß-lactamase, enzyme A and the other produced both enzyme A and enzyme B. Isolates of Y. enterocolitica 4/O:3/IXb from Australia and New Zealand produced only enzyme A.7 In a study using molecular techniques to explain the heterogeneity of expression of ß-lactamase activity in Y. enterocolitica, de la Prieta and colleagues8 reported that, while the genes encoding the two types of ß-lactamase were present in all strains examined, they were not always active; strains of serotype O:5b or O:5,27 expressed only the cephalosporinase and another strain was ß-lactamase-negative.
Molecular techniques have provided interesting data, but a complete picture of the ß-lactamase activity of Y. enterocolitica, which would be useful for both epidemiological and therapeutic reasons, would require in-depth examination of a large number of isolates of various serotypes and biotypes collected from different parts of the world. This was the reason for the present study, which also included strains with atypical biochemical reactions. The susceptibility to 16 ß-lactam antibiotics was determined and the characteristics of ß-lactamase production of each of the 145 isolates were examined in detail. Characterization included isoelectric focusing of the crude enzyme preparations, response to the presence of two potent ß-lactamase inhibitors and the presence or absence of ß-lactamase induction.
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Materials and methods |
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One hundred and forty-five isolates of Y. enterocolitica of different biotypes/serotypes and from various parts of the world were examined (Table I). Five strains each of biotype 1A, biotype 2 (previously classified as biotype 3) and atypical biotype 3 and 48 strains of biotype 4 were among the strains examined in previous studies.57,9 Identification and biotyping were performed as described previously.5 With strains of biotypes 2 and 3, indole production was also tested in a more sensitive medium containing 1% tryptone and 3% l-tryptophan in peptone broth as described by Iteman et al.10 Each of the 145 strains was serotyped and phage typed at the WHO Collaborating Centre for Yersinia, Institut Pasteur, Paris. All cultures were incubated at 28°C in air.
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Amoxycillin, carbenicillin, clavulanic acid and ticarcillin (Beecham Research Pharmaceuticals, Melbourne, Australia), aztreonam and cefepime (Bristol Myers Squibb Pharmaceuticals, Melbourne, Australia), cefoxitin and imipenem (Merck, Sharp & Dohme, Sydney, Australia), meropenem and cefotetan (ICI Pharmaceuticals, Sydney, Australia), ceftazidime (Glaxo, Victoria, Australia), cefotaxime, cefpirome (Hoechst Marion Roussel, Sydney, Australia) and ceftriaxone (Roche, Sydney, Australia) were kindly supplied by the manufacturers or their agents. Nitrocefin (BR 63) was purchased from Oxoid (Basingstoke, UK) and ampicillin from Commonwealth Serum Laboratories (Sydney, Australia). All working solutions were freshly prepared before use.
Isoelectric focusing of ß-lactamases
A crude enzyme extract of each of the 145 isolates of Y. enterocolitica was prepared by sonication and isoelectric focusing was performed on Ampholine PAG plates (pH range 3.59.5; Linbrook International, Pharmacia Biotech, Sweden) as described previously.6 ß-Lactamase bands were visualized by overlaying the gels with a Whatman filter paper soaked in nitrocefin 1000 mg/L. The selective inhibitory effect of clavulanic acid and aztreonam on the ß-lactamase bands was also demonstrated by overlaying the gels with Whatman filter paper soaked in 40 µM clavulanate or 20 µM aztreonam for 2 min before the application of nitrocefin.
Assay of ß-lactamase activity and measurement of selective inhibition of ß-lactamase activity
Crude enzyme extracts of the 145 isolates of Y. enterocolitica were assayed for ß-lactamase activity and protein content as described previously.6 The selective inhibitory effect of 20 µM clavulanate and 10 µM aztreonam on the ß-lactamase activity of crude enzyme extracts was also measured and calculated as described previously.6
ß-Lactamase induction in broth culture
With the exception of the five strains of biotype 5, all isolates of Y. enterocolitica in this study were examined for the induction of ß-lactamase in tryptone soya broth using imipenem 0.5 mg/L as inducer at 28°C in a shaking water bath as described.11 With biotype 5, the induction conditions were modified to accommodate the lower imipenem MIC and the slower growth of these strains. Broth cultures of strains of biotype 5 were incubated in the presence of imipenem 0.25 mg/L for 180 min instead of imipenem 0.50 mg/L for 150 min.
Detection of ß-lactamase induction by a disc diffusion method
All isolates were examined for the presence of ß-lactamase induction by a disc diffusion method as described previously.12
Detection of enzyme A by a disc diffusion method
Enzyme A production by all isolates of biotypes 2 and 3 was investigated by a disc diffusion test as described previously.13 A modified enzyme A detection test using Isosensitest agar instead of Sensitest agar was performed in parallel.
Determination of antibiotic susceptibility
The MICs of 16 antibiotics were determined on all 145 isolates of Y. enterocolitica by an agar dilution technique on Sensitest agar (Oxoid) as described previously.5
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Results |
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Table I summarizes the characteristics of 145 Y. enterocolitica strains examined in the study according to their biotype, serotype, phage type and country of isolation. Seven of 14 strains of biotype/serotype 2/O:5,27 gave a negative indole reaction in peptone water and a positive reaction in the more sensitive medium containing 1% tryptone and 3% l-tryptophan. Of these, five were previously classified as belonging to biotype 3 according to the revised biogrouping scheme of Yersinia enterocolitica.5,14 A number of Y. enterocolitica isolates from Japan showed atypical biochemical reactions. Strains of atypical biotype 3 were VogesProskauer and l-sorbose-negative and strains of atypical biotype 4 were VogesProskauer and maltose-negative. Bio-serotype 4/O:3, phage type IXb strains were originally isolated only in Canada and Australia but are now found in France and in the USA. Likewise, European phage type VIII and South African phage type IXa are found in Australia; these strains have probably been disseminated by international travellers (Table I
).7,15
Isoelectric focusing
Table II shows the cumulative results of isoelectric focusing on PAG plates (pH range 3.59.5) of the enzyme extracts of the 145 strains of Y. enterocolitica examined in the present study. With the exception of one 4/O:3/IXb strain, all Y. enterocolitica examined showed the presence of either one or several ß-lactamase bands after application of nitrocefin.
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Assay of ß-lactamase activity and measurement of selective inhibition of ß-lactamase activity
All except one strain of Y. enterocolitica were found to produce ß-lactamase when nitrocefin was used as the substrate to test crude enzyme extracts. The ß-lactamase activity ranged from 0.06 to 0.74 µmol of nitrocefin hydrolysed/ min/mg protein. The results of selective inhibition on the ß-lactamase activity of crude enzyme preparations by clavulanate and aztreonam are summarized in Table II. With the majority of strains, ß-lactamase activity was inhibited partially by clavulanate and aztreonam. The ß-lactamase activity of all 14 2/O:5,27 strains and that of the two 4/O:3/VIII strains isolated in New Caledonia was completely inhibited by 10 µM aztreonam but not affected at all by 20 µM clavulanate and the reverse results were recorded with 21 4/O:3/IXb strains.
Induction of ß-lactamase in broth culture and detection of ß-lactamase induction by a disc diffusion method
The 21 4/O:3/IXb strains that lacked enzyme B and one lacking both enzyme A and enzyme B, together with all five strains of biotype 5, failed to show induction of ß-lactamase activity. Enhancement of ß-lactamase activity in the presence of imipenem in broth culture was demonstrated with the remaining strains of Y. enterocolitica. Table II summarizes the results of ß-lactamase induction in tryptone soya broth at 28°C by imipenem performed on the 145 strains of Y. enterocolitica. The corresponding results of the detection of ß-lactamase induction by a disc diffusion technique are also shown in Table II
. A characteristic flattened zone of inhibition of a cefotaxime 5 µg disc adjacent to an imipenem 10 µg disc demonstrated the presence of cephalosporinase induction of cells in contact with imipenem.
Detection of enzyme A by a disc diffusion method
A characteristic additional zone of inhibition between a ticarcillin 75 µg disc and a co-amoxiclav 3 µg disc was observed when enzyme A was present. In enzyme A detection tests performed on Sensitest or Isosensitest agar on strains of biotypes 2 and 3, with the exception of the 14 O:5,27 strains, all strains tested showed the characteristic additional zone of inhibition indicative of synergy between the ticarcillin 75 µg and co-amoxiclav 3 µg discs. In contrast, no synergy between the two antibiotic discs was observed with the O:5,27 strains and the two unusual 4/O:3/VIII strains isolated in New Caledonia. These strains produced a large zone of inhibition around the ticarcillin 75 µg discs, demonstrating the absence of the ticarcillin-hydrolysing enzyme A.
Antibiotic susceptibility testing
All 145 isolates were uniformly susceptible to the newer ß-lactam antibiotics in vitro. The MICs of these agents were (mg/L): aztreonam, 1; cefotetan,
1; cefpirome,
0.03; ceftriaxone,
0.06; imipenem,
0.25; meropenem,
0.03. With all strains, ampicillin was generally two-fold more potent than amoxycillin and ticarcillin was two-fold more active than carbenicillin. Table III
shows that, with the exception of ticarcillinclavulanate, to which all strains tested were uniformly susceptible, the susceptibility to ticarcillin, cefoxitin and amoxycillin in the presence of clavulanate 2 mg/L was specific to each biotype or subtype of Y. enterocolitica.
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Discussion |
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The complete inhibition of ß-lactamase activity by clavulanate and the presence of the ticarcillin hydrolysing enzyme A alone, observed in 21 of the 28 strains of Y. enterocolitica 4/O:3/IXb, explained the resistance to ticarcillin and the unusually low MIC of amoxycillin (0.5 mg/L) in the presence of clavulanate 2 mg/L (Tables II and III). This was also the MIC of amoxycillin alone observed with the unusual 4/O:3/IXb strain isolated in Australia, the only ß-lactamase-negative Y. enterocolitica strain described to date. This strain was probably a mutant that lacked the enzyme A of the typical Australian 4/O:3/IXb/A strain. These observations add strength to the argument that the sole mechanism of resistance to ß-lactams in Y. enterocolitica is the expression of ß-lactamase.
The present study confirmed the uniform susceptibility to ticarcillin and the absence of enzyme A in the 14 strains of serotype O:5,27 (Table III). Previously, strains of serotype O:5,27 were reported as belonging to either biotype 2 or biotype 3, depending on the results of an indole production test.19,20 When the 14 strains of serotype O:5,27 used in the present study were tested for indole production, seven of them gave a negative reaction in peptone water and a positive reaction in a tryptophan-enriched medium. Of these seven strains, five were previously classified as belonging to biotype 3 on the basis of the indole test performed in peptone water.5 As the indole reaction test is the only biochemical test that differentiates biotype 2 from biotype 3, according to the revised biogrouping scheme proposed by Wauters et al.,14 the use of the tryptophan-enriched medium for this test was necessary to achieve a clear result. However, a consistent and more readily observed characteristic of Y. enterocolitica O:5,27 strains was the susceptibility to carbenicillin and ticarcillin as a result of the lack of enzyme A as demonstrated in the enzyme A detection test.46,13,21 A database for the antimicrobial susceptibility of Y. enterocolitica recently compiled by Stock & Wiedemann22 also showed that strains classified as biotype 3 fell into two groups: ticarcillinsusceptible (ticarcillin MIC 14 mg/L) and ticarcillinresistant (MIC 64256 mg/L). It is probable that the susceptible strains corresponded to those lacking enzyme A, i.e. serotype O:5,27, which required a tryptophan-enriched medium to yield a positive indole reaction and to be clearly identified as biotype 2. The resistant strains, i.e. those that produced both enzymes A and B, would be biotype 3 strains.
The presence of the B-like enzyme in biotype 1A strains has been reported previously.6 In the present study, an additional type of ß-lactamase, an A-like enzyme with pIs of 8.18.3, was found to be specific to strains of biotype 1B. Whereas the B-like enzyme closely resembled enzyme B in activity, the A-like enzyme was not as efficient at hydrolysing carbenicillin and ampicillin as enzyme A. This explained the borderline susceptibility to carbenicillin and ampicillin of the five strains of biotype 1B examined in the present study and was consistent with the reported susceptibility of Y. enterocolitica 1B/O:8 strains in earlier studies.20,23
The variable inducibility of the cephalosporinase enzyme B in broth culture was unusual and unexpected. Unlike other members of the Enterobacteriaceae, such as Enterobacter cloacae and Citrobacter freundii, in which induction of chromosomal cephalosporinases appeared to be uniformly distributed, induction in Y. enterocolitica was extremely variable and appeared to be specific to each biotype. For example, induction could not be achieved with the five strains of biotype 5 that produced a low basal level of enzyme B and, in the case of biotype 4, induction was specific to the phage type. On the other hand, enzyme B in biotype 1B and the B-like enzyme in biotypes 1A were both highly inducible.
In the present study, detection of ß-lactamase induction by a disc diffusion method using a cefotaxime 5 µg disc and an imipenem 10 µg disc was not sensitive enough to detect ß-lactamase induction in strains with a low ß-lactamase activity. Furthermore, induction was not demonstrated clearly in disc diffusion tests with biotype 1B strains, although there was a 28- to 36-fold increase in ß-lactamase activity when compared with non-induced cells. This apparent paradox, where the disc diffusion test failed to detect a substantial increase, was explained by the very low basal ß-lactamase activity, so that, even after induction, the activity was in the order of 1.852.87 µmol nitrocefin hydrolysed/min/mg protein. This contrasted with the clear demonstration of ß-lactamase induction by disc diffusion with strains of biotypes 1A, 2 and 3, where the ß-lactamase activity of the induced cells was high (6.522.4 µmol nitrocefin hydrolysed/min/mg protein). The low sensitivity of disc diffusion methods in detecting ß-lactamase induction may explain the apparent lack of inducible ß-lactamase in two yersinia septicaemia isolates reported in an earlier study where a disc method of induction was used.24
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Notes |
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References |
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Received 7 February 2000; returned 23 May 2000; revised 5 July 2000; accepted 11 July 2000