JAC Advance Access originally published online on January 3, 2008
Journal of Antimicrobial Chemotherapy 2008 61(2):309-314; doi:10.1093/jac/dkm494
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Original research |
Role of β-lactamase inhibitors in enterobacterial isolates producing extended-spectrum β-lactamases
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
* Corresponding author. Tel: +91-9415820675; E-mail: mr_senbhu{at}yahoo.com
Received 3 September 2007; returned 8 October 2007; revised 22 November 2007; accepted 23 November 2007
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Abstract |
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Objectives: To determine the in vitro activity of β-lactamase inhibitors (clavulanic acid and sulbactam) in combination with third-generation cephalosporins and monobactam against extended-spectrum β-lactamase (ESBL)-producing members of the Enterobacteriaceae family.
Methods: A total of 361 ESBL-producing enterobacterial isolates obtained from patients of a university hospital were screened for the status of co-production of AmpC β-lactamase. These strains were further subjected to an MIC study using third-generation cephalosporins and monobactam, and reductions were observed after combining with β-lactamase inhibitors at a fixed concentration of 4 mg/L.
Results: Most of the isolates showed 8-fold reduction with sulbactam when combined with ceftriaxone, cefpodoxime and cefotaxime but not with ceftazidime and aztreonam, whereas clavulanic acid showed the same result with all the cephalosporins tested. Further, both the inhibitors showed greater reduced MIC when combined with aztreonam.
Conclusions: As the ability of clavulanic acid to induce AmpC production may interfere with ESBL detection, sulbactam is likely to be preferred over clavulanic acid after standardization of an appropriate concentration for ESBL detection in the scenario of increased prevalence of AmpC producers. Greater in vitro activity of these inhibitors when combined with aztreonam further indicates the need of studies to evaluate these combination antimicrobials in clinical settings as they can play a significant role for clinicians as viable alternatives to treat infections caused by such organisms.
Keywords: ESBLs , OXA-10 , aztreonam
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Introduction |
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The production of β-lactamase remains the major mechanism of resistance in Gram-negative bacilli to β-lactam antibiotics. In recent years, extended-spectrum β-lactamases (ESBLs) have become progressively widespread due to extensive use of third-generation cephalosporins in hospital settings. ESBLs have the ability to hydrolyse β-lactam antibiotics containing an oxyimino group, namely cefotaxime, ceftazidime, ceftriaxone, cefpodoxime and aztreonam, but they are usually inactivated by β-lactamase inhibitors.1
Carbapenems and cephamycins have the most consistent activity against ESBL producers. The former are regarded as the drugs of choice against these isolates. But, recently, combined cephamycin and carbapenem resistance has been observed in ESBL-producing organisms.2,3 Thus, these inhibitors should be investigated as a therapeutic option by combining them with respective cephalosporins/monobactam; in addition they can also play a leading role in laboratory detection of ESBLs. Clavulanic acid, which is recommended by the CLSI,4 is a suboptimal inhibitor of ESBLs for those isolates that produce inducible AmpC β-lactamases. Induction of these enzymes may not only obscure the recognition of the ESBL status, but it may also affect adversely the treatment of clinical conditions caused by such strains. Sulbactam is unlikely to cause this problem and could be a better alternative to clavulanic acid for detection of ESBLs. Although there are a number of combination antimicrobials utilizing the three β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) that are in clinical use, to date there is no consensus agreement on the concentration of inhibitors to be used with the companion drug and it clearly differs in the recommendations of the CLSI, German (Deutsche Industrie Norm—Medizinsche Mikrobiologie) and British (BSAC) guidelines.4–6
Thus, the objective was to study the in vitro activity of clavulanic acid and sulbactam in combinations with third-generation cephalosporins and monobactam against ESBL-producing Escherichia coli, Klebsiella spp. and Proteus mirabilis.
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Materials and methods |
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Strains
A total number of 361 consecutive, non-duplicate ESBL-producing enterobacterial isolates from different clinical specimens of patients admitted in different wards of the Sir Sunderlal Hospital, Banaras Hindu University, Varanasi, India, were collected. The strains were E. coli (n = 280), Klebsiella pneumoniae (n = 63), Klebsiella oxytoca (n = 8) and P. mirabilis (n = 10). The ESBL status of these strains was established by combined disc diffusion and MIC reduction method as per CLSI recommendations.4
PCR detection of β-lactamase genes
For partial gene PCR amplification, primers specific for blaTEM,7 blaSHV8, blaCTX-M-1,-2,-99, blaOXA10 and blaOXA27 (Table 1), respectively, were used for reaction with bacterial DNA as a template. Each single reaction mixture contained 1 µg of DNA, 15 pmol of each primer, 10 mM dNTPs, 1 U of Taq DNA polymerase (Genei, Bangalore, India) and 25 mM MgCl2 supplied by the manufacturer of the enzyme. A Biometra Thermal cycler was used and the reactions were run under the following conditions: initial denaturation at 94°C for 5 min; 40 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; and a final elongation at 72°C for 7 min.
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Typing by random amplification of polymorphic DNA (RAPD) analysis
All the isolates were typed by RAPD using primer 7 as described previously.10
Antimicrobial susceptibility testing
Antimicrobial susceptibility was performed by the Kirby–Bauer disc diffusion method for various antibiotics, namely: amikacin (30 µg), cefepime (30 µg), cefoxitin (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), imipenem (10 µg), netilmicin (30 µg) and piperacillin (100 µg) (Hi-Media, Mumbai, India). Activities of different β-lactam/β-lactamase inhibitors were also determined using amoxicillin/clavulanic acid (20/10 µg), ticarcillin/clavulanic acid (75/10 µg), ampicillin/sulbactam (20/10 µg) (Hi-Media), cefoperazone/sulbactam (75/30 µg) (Pfizer, Mumbai, India), ceftriaxone/sulbactam (30/15 µg) (Shreya Life Sciences, Gujarat, India) and piperacillin/tazobactam (100/10 µg) (Wyeth, Mumbai, India). K. pneumoniae ATCC 700603 was used as a positive control, whereas E. coli ATCC 25922 and E. coli ATCC 35218 were used as negative controls.
A test isolate (with a turbidity equivalent to that of a 0.5 McFarland standard) was spread over a Mueller–Hinton agar (Hi-Media) plate, and cefotaxime (30 µg) and cefoxitin (30 µg) (Hi-Media) discs were placed 20 mm apart from centre to centre. Isolates showing a blunting of the cefotaxime zone of inhibition adjacent to the cefoxitin disc were confirmed as positive for AmpC β-lactamase production. E. coli ATCC 25922 was used as a negative control.
All of the isolates were subjected to an MIC study (agar dilution) using cefotaxime, ceftazidime, ceftriaxone (Hi-Media), cefpodoxime and aztreonam (Ranbaxy, Gurgaon, India) alone and in combination with clavulanic acid (Ranbaxy) and sulbactam (Aurobindo pharma, Hyderabad, India). The inoculum size for the study was 1 x 106 cfu/mL. Since the purpose of this study was to compare the ability of each inhibitor to protect its companion drug, a fixed single inhibitor concentration of 4 mg/L was used, which allowed direct comparisons to be made.
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Results |
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Of the 361 enterobacterial isolates that were confirmed on the basis of phenotypic methods to be ESBL-positive, the majority of them harboured the CTX-M (n = 272) gene followed by SHV (n = 56), TEM (n = 29) and OXA-10 (n = 1). Although the presence of multiple β-lactamase genes was found in 50 isolates, a significant number of isolates, which included all of the P. mirabilis, did not show any amplification with the primers used in the present study (Table 2).
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After performing RAPD, there were 27 types of E. coli arranged in 17 clusters, 8 types of K. pneumoniae grouped in 6 clusters, and 3 types of K. oxytoca and 5 types of P. mirabilis grouped in 3 clusters each.
On observing the antimicrobial susceptibility of these strains, they showed 100% in vitro susceptibility towards imipenem and piperacillin/tazobactam followed by ceftriaxone/sulbactam (85%), cefoperazone/sulbactam (81%) and amikacin (75%) (Table 3). Depending on their antibiogram, these strains were found to be of nine subtypes (Table 4). CTX-M was found to be uniformly distributed among all of the subtypes.
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A total of 113 isolates showed co-production of AmpC β-lactamase, which was most frequently observed in K. pneumoniae (n = 27, 43%) followed by E. coli (n = 81, 29%). Three isolates of K. oxytoca and two P. mirabilis were also found to be co-producers. Among the AmpC β-lactamase co-producing strains, sulbactam was superior in detecting ESBL production over clavulanic acid for 17 isolates [E. coli (n = 13) and K. pneumoniae (n = 4)] when combined with cefotaxime and 18 isolates each when combined with cefpodoxime [E. coli (n = 16) and K. pneumoniae (n = 2)] and with ceftriaxone [E. coli (n = 15), K. pneumoniae (n = 2) and K. oxytoca (n = 1)], respectively.
Most of the isolates had 8-fold reduction when clavulanic acid was combined with all of the test drugs, whereas with sulbactam this was observed when combined with ceftriaxone, cefpodoxime and cefotaxime, but not when combined with ceftazidime and aztreonam (Table 5). Further, when the MIC50 and MIC90 values of the test drugs alone were compared with those for combinations with clavulanic acid and sulbactam, the efficacy of different β-lactamase inhibitors with their companion drugs became evident. In general, high MICs of the individual test drugs were observed, but there was a significant reduction in MICs of these drugs on addition of inhibitors. Furthermore, both of the inhibitors (i.e. clavulanic acid and sulbactam) showed greater reduction of MIC with aztreonam, well below its susceptibility range, implicating their therapeutic value (Table 6).
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8-fold against ESBL-positive organisms
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Discussion |
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In the current study, ESBL genes of various groups were found and the absence of an amplicon in some isolates could be due to the presence of some other type of gene that could not be targeted by our primers. Isolates harbouring such a diverse range of ESBL genes was previously reported from a similar hospital-based study.11 To our knowledge, this is the first report on the detection of the OXA-10 gene in E. Coli from India.
Although different opinions exist with regard to the use of a single inhibitor concentration (2 mg/L),12 and different concentrations for different inhibitors for finding 8-fold MIC reductions with their corresponding drugs,13 we have used 4 mg/L of both the inhibitors as CLSI recommends this concentration for clavulanic acid to detect 8-fold reduction in MIC when combined with cephalosporins and monobactam than when tested alone in ESBL producers. However, the proportion of MIC reduction by clavulanic acid and sulbactam was not uniform, which might be due to a difference in binding affinity of enzymes for each inhibitor or enzymatic action (hydrolysis rate) that varies with inhibitor type.
The very low level of susceptibility to cefoxitin may be due to a high prevalence of isolates containing AmpC type β-lactamases. Although on weight basis clavulanic acid is more potent than sulbactam, its ability to induce AmpC production may interfere with ESBL detection.14 Thus, sulbactam, which also showed good activity, is likely to be preferred over clavulanic acid after standardization of an appropriate concentration for ESBL detection in the scenario of increased prevalence of AmpC producers.
In the present in vitro study, a combination of antimicrobials with β-lactamase inhibitors was found to be effective against ESBL producers as observed previously.15 Piperacillin/tazobactam was found to be effective against all of the test isolates on the basis of in vitro disc diffusion testing. Furthermore, the combinations of aztreonam/clavulanic acid and aztreonam/sulbactam were found to be active against all of the test isolates and activity was below the MIC breakpoint of aztreonam alone, suggesting further studies are needed to evaluate these combination antimicrobials in clinical settings as they can play a significant role for clinicians as viable alternatives to treat infections caused by these organisms.
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Funding |
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The current study was financially supported by the University Grants Commission, India and the Department of Microbiology, IMS, BHU, Varanasi.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We would like to acknowledge the Head, Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi and the University Grants Commission, India, for providing financial support to carry out the study.
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References |
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Chmelnitsky I, Carmeli Y, Leavitt A, et al. CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum β-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel. Antimicrob Agents Chemother (2005) 49:4745–50.
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Thomson KS, Sanders CC, Moland ES. Use of microdilution panels with and without β-lactamase inhibitors as a phenotypic test for β-lactamase production among Escherichia coli, Klebsiella spp. Enterobacter spp. Citrobacter freundii, and Serratia marcescens. Antimicrob Agents Chemother (1999) 43:1393–400.
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