1 Antibiotic Resistance Monitoring & Reference Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT; 2 Barts & The London School of Medicine & Dentistry, Turner Street, London E1 2AD, UK
Received 23 July 2002; returned 4 November 2002; revised 22 November 2002; accepted 26 November 2002
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Abstract |
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Keywords: piperacillin/tazobactam, Klebsiella, extended-spectrum ß-lactamases
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Introduction |
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What is surprising is that the MIC distributions of piperacillin/tazobactam for ESBL-producing Klebsiella spp. are strongly bimodal (see Results for examples), with values clustered either around 4 + 4 to 8 + 4 mg/L or 512 + 4 mg/L.2,3 Even when ESBL-producing isolates belong to a single outbreak strain, it is common to find that some representatives are susceptible to piperacillin + tazobactam, whereas others are highly resistant. Such stratification has not been reported with piperacillin/tazobactam for producers of other ß-lactamases, nor for other inhibitor combinations against ESBL producers. Skewed unimodal MIC distributions were found when tazobactam, clavulanate and sulbactam combinations were tested against Escherichia coli isolates with various amounts of TEM-1 enzyme;4 moreover, ESBL producers are consistently susceptible to the cephalosporin/clavulanate combinations used in ESBL detection tests.5
To investigate why bimodal MIC distributions arise for ESBL producers with piperacillin + tazobactam 4 mg/L, we tested piperacillin in combination with different concentrations of tazobactam or, as a control, with clavulanate. We also compared the ß-lactamase expression, kinetics and outer membrane profiles of piperacillin/tazobactam-susceptible and -resistant representatives of ESBL-producing outbreak strains of Klebsiella pneumoniae.
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Materials and methods |
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ESBL-producing isolates were collected in two surveys of klebsiellae from European ICUs, undertaken in 1994 and 1997/8.2,3 The 1994 survey included 220 ESBL producers from 27 hospitals, the 1997/8 survey included 161 producers from 16 hospitals. Species identification was with the API20E system (bioMérieux, La Balme les Grottes, France), and strains were typed by capsular serotyping and by pulsed-field gel electrophoresis (PFGE) of genomic DNA digested with XbaI. The methods of DNA digestion and PFGE were as described previously6 for the isolates collected in 1994; the method of Gautom7 was used for the isolates collected in 1997/8 as it was more convenient than the earlier method and gave comparable results. MICs of antibiotics were determined on Iso-Sensitest agar (Oxoid, Basingstoke, UK), as previously described.2,3 Piperacillin and tazobactam were from Wyeth (Taplow, UK), lithium clavulanate from SmithKline Beecham (Welwyn Garden City, UK) and cefotaxime from Sigma (Poole, UK).
Isolates were identified as ESBL producers on the basis of ceftazidime/ceftazidime + clavulanate MIC ratios of 16. ß-Lactamase profiles were examined by isoelectric focusing (IEF) of extracts prepared by the sonication (1994) or repeated freezing and thawing (1997/8) of cells harvested from nutrient agar plates;8 blaTEM and blaSHV genes were sought with the primers and conditions described previously.9 In a few cases, the PCR products of amplification of blaSHV were sequenced by chain termination, using the method and conditions described elsewhere;9 otherwise the PCR products of blaSHV genes were profiled by single-stranded conformational polymorphism (SSCP), as described previously.6
Quantification of ß-lactamase activity
Cultures were grown overnight, with shaking, in 10 mL amounts of nutrient broth at 37°C, then diluted 10-fold into fresh identical broth at 37°C. After incubation for a further 4 h, these cultures were harvested at 5000g and 37°C. The cells were washed in 10 mL of 0.1 M phosphate buffer pH 7.0, resuspended in 1.5 mL of the same buffer, chilled on ice and disrupted by sonication. The resulting extracts were used in spectrophotometric assays at 37°C and pH 7.0, with 0.1 mM cefotaxime or 0.2 mM piperacillin as substrates; assay wavelengths were 255 and 235 nm, respectively.
To assay inhibition of ß-lactamase activity, sonicates (250100 µL) were mixed with various concentrations of tazobactam, from 0.01 µM to 0.1 mM, in 1 mL volumes of 0.1 M phosphate buffer. After incubation for 10 min at 37°C, 100 µL amounts of 2 mM piperacillin were added, giving a final piperacillin concentration of 0.182 mM. Piperacillin hydrolysis was then measured by spectrophotometry at 235 nm and the I50 was defined as the tazobactam concentration reducing the piperacillin hydrolysis rate by 50%. Specific activity was defined as nmol ß-lactam hydrolysed/min per mg cell protein, with protein measured by the Lowry method10 or with the Bradford Reagent (Sigma). In either case, bovine serum albumin was used for the standards.
Extraction and electrophoresis of outer membrane proteins
Outer membrane proteins were extracted as the Sarkosyl-insoluble fraction from exponential phase cells that had been grown in Nutrient Broth No. 2 (Oxoid).8 These preparations were suspended in distilled water and adjusted to a protein concentration of 1 mg/mL, as assayed by the Lowry method.10 Protein profiles were then obtained by SDSPAGE, using the methods described by Livermore & Williams.8
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Results |
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Susceptibility tests with piperacillin in the presence of various concentrations of tazobactam or clavulanate were carried out for the 220 ESBL-producing Klebsiella spp. from the 1994 survey and for the 161 from the 1997/8 collection. All except six of the isolates were resistant to piperacillin 16 mg/L and the great majority (95.4%) required piperacillin MICs 128 mg/L. MIC distributions of piperacillin + tazobactam 4 mg/L were bimodal for both collections (Figure 1a), with peaks at 4 + 4 to 8 + 4 mg/L and at
1024 + 4 mg/L, and with the trough at MICs of 32 + 4 to 256 + 4 mg/L. These distributions have been published previously2,3 but are reproduced here to illustrate the phenomenon under investigation.
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Piperacillin/tazobactam resistance in relation to strain structure
The 220 ESBL producers collected in 1994 comprised 85 strains as defined by serotyping and PFGE, together with seven isolates for which satisfactory PFGE profiles could not be obtained.6 Fully 61% of these 220 isolates belonged to 16 outbreak strains, each with three or more representatives, whereas 39% were sporadics, with only one or two representatives. Of the 16 outbreak strains, eight exclusively comprised isolates susceptible to piperacillin + tazobactam at the breakpoint of 16 + 4 mg/L, whereas three comprised only resistant organisms and five included representatives of both phenotypes. Similar patterns were seen among the isolates collected in 1997/8. Among the 161 ESBL producers, 115 (71%) belonged to 17 outbreak strains, each with three or more representatives, whereas the remaining 46 (29%) belonged to 35 sporadic strains. Among the outbreak strains from 1997/8, four solely comprised organisms susceptible to piperacillin + tazobactam 4 mg/L, six had only resistant organisms and seven included both resistant and susceptible organisms. In those instances where outbreak strains included both susceptible and resistant isolates (Table 1), the MIC distributions of piperacillin + tazobactam 4 mg/L were extremely wide and, so far as could be judged (allowing for the small numbers of isolates per strain), recapitulated the bimodal pattern seen for the collections as a whole.
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Among the 220 ESBL producers collected in 1994, multiple ß-lactamases were found by IEF in 27/154 (17.5%) of those susceptible to piperacillin + tazobactam (16 + 4 mg/L) compared with 29 of the 76 (38.2%) that were resistant (2 = 16.29, P < 0.001). If the division was drawn at 64 + 4 mg/L, corresponding to the natural trough in the bimodal distribution rather than the breakpoint (Figure 1a) then the proportions of susceptible and resistant isolates with multiple ß-lactamases were 31/167 (18.6%) and 25/63 (39.7%), respectively (
2 = 15.6, P < 0.001). These analyses indicated an association between production of multiple ß-lactamases and resistance to piperacillin + tazobactam 4 mg/L, but this conclusion was not supported by results from the 1997/8 collection, when multiple ß-lactamases were found in 43 of the 66 isolates susceptible at breakpoint and in 72 of the 95 found resistant (
2 = 2.01; P > 0.05). Similarly, multiple ß-lactamases were observed in 51 of the 77 isolates from the 1997/8 collection that were susceptible to piperacillin + tazobactam at the natural trough of
64 + 4 mg/L, compared with 65 of 84 that were resistant (
2 = 2.47, P > 0.05). A complicating factor was that a greater proportion of isolates had multiple enzymes in the 1997/8 study, and that more had TEM enzymes (G. S. Babini, L. M. C. Hall, M. Yuan & D. M. Livermore, unpublished data).
Basis of variation in susceptibility to piperacillin/tazobactam within strains
Additional studies sought explanations for variation in susceptibility to piperacillin/tazobactam within strains. Investigation centred on the 52 representatives of the K25/PN1 strain from the 1994 survey (Table 1) and on six pairs of isolates from the 1997/8 study. One of these latter pairs (pair 1) also comprised organisms belonging to the K25/PN1 strain, which continued to be isolated at two centres in the more recent survey.
Members of the K25/PN1 strain were variously collected in Bordeaux, Clermont-Ferrand and Ghent during the 1994 survey, and were consistently found to carry a ß-lactamase gene with the PCR-SSCP profile of blaSHV-4, also to have the pI 7.8 band characteristic of this enzyme. This ß-lactamase identification was confirmed by gene sequencing for one representative, and is consistent with many other reports for this epidemic strain, which is long-established and widely distributed in France.6,11,12 Among the 20 representatives from Clermont-Ferrand, 15 were susceptible to piperacillin + tazobactam at 16 + 4 mg/L, whereas five had a low-level resistance, with MICs of 3264 + 4 mg/L. PCR indicated that these latter five organisms had blaTEM genes as well as blaSHV, whereas blaTEM was not detected in the 15 susceptible representatives. Of the 20 K25/PN1 isolates from Bordeaux, 18 were susceptible to piperacillin + tazobactam 16 + 4 mg/L, whereas two were resistant. Among the 12 representatives from Ghent, eight were susceptible and four were highly resistant, with MICs 512 + 4 mg/L. None of the isolates from Ghent or Bordeaux gave a PCR product with primers to blaTEM and none had other ß-lactamases besides SHV-4 (pI 7.8), as revealed by IEF.
ß-Lactamase quantification was undertaken on 10 representatives of the K25/PN1 strain that lacked blaTEM; three each were from Clermont-Ferrand and Bordeaux, and four from Ghent. These organisms were selected as ranging widely in their susceptibility to piperacillin + tazobactam 4 mg/L (Table 2); one was exceptional in retaining borderline susceptibility to unprotected piperacillin (MIC 16 mg/L), whereas the others were highly resistant. SHV-4 ß-lactamase activities ranged between 21 and 97 nmol cefotaxime hydrolysed/min per mg protein, and a relationship was evident between specific activity and the MIC of piperacillin/tazobactam (Table 2). Nevertheless, this relationship was imperfect and the two most resistant isolates (nos 619 and 613, MICs 1024 + 4 mg/L) had lower ß-lactamase-specific activities than no. 266, which was much less resistant (MIC 64 + 4 mg/L).
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The organisms of pair 1 showed the characteristic outer membrane profile of the K. pneumoniae K25/PN1 strain to which they belonged, with the two major bands separated only very narrowly (Figure 3, cf. Figure 2). Isolates belonging to the remaining five pairs showed the more typical profiles for the species, with major bands at 43 and 39 kDa. Only minor variations in profile were seen within the pairs and none of these was consistently associated with the differences in resistance phenotype.
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Discussion |
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The most plausible explanation of a bimodal distribution is that it is a titration effect, reflecting the steady-state proportion of enzyme that can be kept in an inactive form by the amount of inhibitor available. Aside from the inhibitor concentration itself, factors likely to co-determine the amount of enzyme that can be titrated into an inactive form include the permeability of the strain, the amount of ß-lactamase produced, the rate of enzyme re-synthesis, and the rate of enzyme regeneration by breakdown of inhibitor complexes.
Further investigations studied why some ESBL producers were more resistant to piperacillin/tazobactam than others. The results indicated mechanisms for individual groups of strains, but not a global explanation. Thus, electrofocusing results for isolates from the 1994 survey suggested that the more resistant organisms more often had multiple ß-lactamases. Likewise, some (but not all) of the more resistant members of the K25/PN1 strain had TEM as well as SHV-4 ß-lactamases. These data were in keeping with those of Rice et al.,16 who associated resistance to piperacillin/tazobactam with the simultaneous expression of TEM and SHV enzymes, but were not supported by the 1997/8 survey, where the prevalence of multiple enzyme types appeared to be as high among the susceptible as the resistant isolates. It should perhaps be added that production of multiple TEM and SHV enzymes does not guarantee resistance to piperacillin/tazobactam; up to five TEM and SHV variants were found in most of a series of K. pneumoniae isolates from Durban, South Africa, yet these were consistently susceptible to piperacillin + tazobactam.9
Variation in quantity of a single ß-lactamase is a further and obvious explanation for variation in susceptibility to inhibitor combinations, since it is evidently easier to inhibit a small amount of enzyme activity than a large amount.1 Studies on representatives of the K25/PN1 strain (Table 2) suggested such a pattern, with the MIC of piperacillin + tazobactam 4 mg/L broadly relating to specific activity against cefotaxime. Nevertheless, the variation in piperacillin + tazobactam MIC was 500-fold, whereas that in ß-lactamase specific activity was only five-fold, suggesting that some other factor(s) must also be implicated. Moreover, no clear relationship between ß-lactamase-specific activity and piperacillin/tazobactam MIC was seen for a further six pairs of ESBL-producing isolates from the 1997/8 collection. Thus, although some relationships existed between the amount and numbers of ß-lactamase(s) and the level of piperacillin + tazobactam resistance for individual strains, these relationships were neither consistent nor universal. Relationships between porin loss and piperacillin/tazobactam resistance were not found, although they have been demonstrated in individual K. pneumoniae isolates by others.17
There remains, critically, the question of whether piperacillin/tazobactam should be used in infections caused by ESBL producers. On the one hand, many producers are susceptible in vitro2,3 and the combination has been successfully used in experimental infections caused by susceptible producers.1820 On the other hand, some ESBL producers are resistant, and failure of empirical piperacillin + tazobactam has been reported in some infections caused by ESBL producers, which were subsequently shown to express in vitro resistance.21 In these circumstances the best advice is to be guided by susceptibility results for the individual isolates and to consider piperacillin/tazobactam as a therapeutic option when ESBL producers are demonstrably susceptible, but to be cautious with empirical use in settings where ESBL producers are prevalent, even when these belong to outbreak strains that are generally susceptible.
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Acknowledgements |
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Footnotes |
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Present address. Vascular Biology Group, University Department of Obstetrics and Gynaecology, Glasgow Royal Infirmary, 10 Alexandra Parade, Glasgow G31 2ER, UK.
¶ Corresponding author. Tel: +44-20-8200-4400; Fax: +44-20-8358-3292; E-mail: Dlivermore{at}phls.nhs.uk
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