July-SeptemberIndian Journal of 2007 Medical Microbiology, (2007) 25 (3):203-8 203 Original Article SENSITIVITY PATTERN OF GRAM NEGATIVE BACILLI TO THREE β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS USING THE AUTOMATED API SYSTEM K Anuradha, VV Sailaja, P Umabala, T Satheesh, *V Lakshmi Abstract Purpose: To evaluate the spectrum of activity of three β-lactamase inhibitors such as amoxicillin/ clavulanic acid, ticarcillin/ clavulanic acid and piperacillin/ tazobactam in comparison to cephalosporins against gram negative bacilli. Methods: Gram-negative bacilli isolated from the clinical specimens received in the laboratory were included in the study. Using the API system (bioMérieux) during a one-year period, a total of 1,252 Enterobacteriaceae and 385 non-fermenters were evaluated. Results: The percentage resistance of the Enterobacteriaceae isolates was 82.92% to amoxicillin/ clavulanic acid, 58.22% to ticarcillin/clavulanic acid and 22.44% to piperacillin/tazobactam respectively. Pseudomonas aeruginosa showed resistance of 96% to ticarcillin/ clavulanic acid and 61% to piperacillin/ tazobactam and Acinetobacter baumannii showed 49% resistance to ticarcillin/ clavulanic acid and 77% resistance to piperacillin/ tazobactam respectively. The isolates exhibited high resistance to all the generations of cephalosporins and the other groups of antibiotics except carbapenems. Conclusions: Piperacillin/tazobactam was found to be the most active combination of the three against Enterobacteriaceae and Pseudomonas spp. and ticarcillin/clavulanic acid against Acinetobacter spp. and Stenotrophomonas maltophilia. Key words: API, β-lactamase inhibitors, gram negative bacteria, sensitivity An extensive use of β-lactam antibiotics in hospitals and emerging nosocomial pathogens such as Acinetobacter spp. community has created major resistance problems leading to and Stenotrophomonas maltophilia.5 increased morbidity, mortality and health-care costs.1 Of the several mechanisms of resistance, the most widespread and This study compared the spectrum of activity of β-lactam/ most important is the destruction of the β-lactam ring, which β-lactamase inhibitors to that of cephalosporins against gram- is mediated by β-lactamases.2 Extended spectrum β-lactamases negative bacilli. (ESBLs) are of greater concern because they are capable of Materials and Methods hydrolyzing several groups of β-lactam antibiotics, notably third and fourth generation cephalosporins and extended The in vitro activity of three β-lactamase inhibitors in spectrum penicillins such as piperacillin. There are 255 known comparison with cephalosporins was evaluated against clinical β-lactamases to date and the continued use of β-lactams may isolates from patients admitted in our institute using the API select for newer variants.3 system. Amoxycillin/clavulanic acid (AUG), ticarcillin/ (www.medknow.com).clavulanic acid (TIM) and piperacillin/tazobactam (TZP) and The use of β-lactamase inhibitors in combination with all the four generations of cephalosporins were included in β-lactam antibiotics is currently the most successful strategy the study. to combat this specific resistance mechanism. These β- lactamase inhibitorsThis are PDFthoughta site tois behosted available “suicide inhibitors” by forMedknow freeA totaldownload ofPublications 1252 Enterobacteriaceae from isolates and 385 that form stable complexes between the bacterial β-lactamase isolates from non-fermenters were tested for AUG, TIM and and the β-lactamase inhibitor in a multi-step chemical TZP and TIM and TZP respectively. Tables 1 and 2 show all 4 reaction. Their broad spectrum of activity originates from the isolates tested and the antibiotics used. the ability of respective inhibitors to inactivate a wide range of β-lactamases produced by gram-positive, gram-negative, All the isolates were identified and their minimum anaerobic and even acid-fast pathogens. β-lactam/ β-lactamase inhibitory concentrations (MICs) were determined by API inhibitor combinations are particularly useful against mixed system (bioMérieux) using the relevant panels. The inoculum infections and play a useful role in treating various multi was prepared as per the standard protocol given in the resistant pathogens such as ESBLs. These agents are gaining instruction manual of the API system. Quality control was importance in the treatment of various multi-resistant and performed by using Escherichia coli strain ATCC 25922 for Enterobacteriaceae and Pseudomonas aeruginosa strain ATCC *Corresponding author (email: <[email protected]>) 27853 for non-fermenters, with each new batch of strips. Department of Microbiology, Nizam’s Institute of Medical Sciences, Hyderabad - 500 082, Andhra Pradesh, India Results Received: 29-09-04 Accepted: 12-12-05 Among the Enterobacteriaceae, the overall resistance www.ijmm.org 204 Indian Journal of Medical Microbiology vol. 25, No. 3 to amoxicillin/clavulanic acid, ticarcillin/ clavulanic acid Discussion and piperacillin/tazobactam was 82.92, 58.22 and 22.44% respectively (Table 3) and 82-65% resistance to cephalosporins Penicillin, the Þ rst of the β-lactam antibiotics, was Þ rst from Þ rst to fourth generations. introduced into medical practice in the 1940s. Since then, a large number of different β-lactams, including penicillins, Table 4 shows the MICs of different β-lactam/β-beta- cephalosporins, monobactams and carbapenems have been lactamase organisms to Enterobacteriaceae group of inhibitors developed, all of which are structurally related through and Tables 5-10 show the MICs against cephalosporins. Table the presence of a core β-lactam ring.2 The most common 11 shows the MICs of different β-lactam/β-beta-lactamase mechanism of resistance to beta-lactam antibiotics is inhibitors and cephalosporins to various non-fermenters. the production of β-lactamase, which destroys β-lactam antibiotics before they reach the bacterial target.6 A highly effective and proven approach for tackling β-lactamase Table 1: Isolates of Enterobacteriaceae group and non- mediated resistance to β-lactams is the use of the β-lactam/ fermenters β-lactamase inhibitor combinations.7 In recent years, the use Enterobacteriaceae Non-fermenters of these combinations has been proven to be a useful and an Species No. of isolates Species No. of isolates effective strategy to improve upon the therapeutic value of β-lactam antibiotics.8 Several factors inß uence the activity E. coli 769 Acinetobacter 106 baumannii and pharmacodynamics of these combinations, including K. pneumoniae 236 Acinetobacter 12 potency of these agents, pharmacokinetics of the inhibitor, lwofÞ i type and quality of β-lactamase produced by the target Enterobacter 101 Pseudomonas 195 bacterium and potential for the inhibitor to induce expression cloacae aeruginosa of chromosomal cephalosporinases in the target bacterium.9 Other 56 Other 46 The overall antibacterial spectrum of these drug combinations Enterobacter spp. Pseudomonas spp. depends on the intrinsic activity of the β-lactam as well as the Proteus mirabilis 36 Stenotrophomonas 26 characteristics of the individual inhibitor towards different maltophilia β-lactamases.5 The differences among these β-lactam/ β- Proteus vulgaris 2 _ _ Providencia rettgeri 6 _ _ lactamase inhibitors such as spectrum of activity, need to be Serratia marcescens 39 _ _ considered in choosing an agent for a speciÞ c case. Other Serratia spp. 7 _ _ Currently, there are three commercially available Total 1252 Total 385 β-lactamase inhibitors-clavulanic acid, sulbactam and Table 2: Antibiotics with concentrations (μg/L) tested against Enterobacteriaceae group and non-fermenters β-Lactamase inhibitors Cephalosporins Group AUG TIM TZP 1 G CEPH CEF CF/CTX CB FEP CPO Enterobacteriaceae + + (www.medknow.com).+ + + + + + + n=1252 4/2 16/2 8/4 8 8 4 4 4 4 Non-fermenters _ + + _ _ _ + + _ n= 385 This PDFa site is 16/2-hosted available 16/4 by - forMedknow free download Publications from4-32 4-32 64/2 64/4 + - Tested against, - - Not tested, API strips used for antimicrobial sensitivity-For Enterobacteriaceae -rapid ID 32 E For Non-fermenters - ATB PSE, AUG - Amoxicillin/Clavulanic acid, TIM - Ticarcillin/Clavulanic acid, TZP - Piperacillin/Tazobactum, 1 G Ceph - First generation cephalosporin, CEF - Cefuroxime, CF/CTX - Cefotaxime/Ceftriaxone, CB - Ceftazidime, FEP - Cefepime, CPO - Cefpirome Table 3: Resistance pattern of Enterobacteriaceae and non-fermenters to β-lactamase inhibitors and cephalosporins Group AUG TIM TZP 1 G CEPH CEF CF/CTX CB FEP CPO Enterobacteriaceae n =1252 82.92 58.22 22.44 82.18 75.07 66.61 66.77 64.77 65.17 Non-fermenters n= 385 __ 49.61 48.83 __ __ __ 63.11 47.01 __ AUG - Amoxicillin/Clavulanic acid, TIM - Ticarcillin/Clavulanic acid, TZP - Piperacillin/Tazobactum, 1 G Ceph - First generation cephalosporin, CEF - Cefuroxime, CF/CTX - Cefotaxime/Ceftriaxone, CB - Ceftazidime, FEP - Cefepime, CPO - Cefpirome www.ijmm.org July-September 2007 Anuradha et al - Testing of GNB against β-lactamase Inhibitors using API System 205 Table 4: Sensitivity pattern of organisms of Enterobacteriaceae group to β-lactamase inhibitors Organism No. of AUG TIM TZP isolates S I R S I R S I R (%) (%) (%) (%) (%) (%) (%) (%) (%) E. coli 88 2 679 244 52 473 586 50 133 769 (11) (1) (88) (32) (7) (62) (76) (7) (17) K. pneumoniae 80 0 156 95 12 129 156 10 70 236 (34) (0) (66) (40) (5) (55) (66) (4) (30) Enterobacter 0 5 96 26 11 64 50 5 46 cloacae 101 (0) (5) (95) (26) (11) (63) (50) (5) (45) Other 8 1 47 25 2 29 36 1 19 Enterobacter spp. 56 (14) (2) (84) (44) (4) (52) (64) (2) (34) Proteus 29 0 7 33 3 0 35 1 0 mirabilis 36 (83) (0) (17) (91) (9) (0) (97) (3) (0) Proteus 0 0 2 2 0 0 2 0 0 vulgaris 2 (0) (0) (100) (100) (0) (0) (100) (0) (0) Providencia 0 0 6 5 0 1 5 0 1 rettgeri 6 (0) (0) (100) (83) (0) (17) (83) (0) (17) Serratia 0 0 39 9 3 27 17 12 10 marcescens 39 (0) (0) (100) (23) (8) (69) (44) (931) (26) Other 0 0 7 2 1 4 4 1 2 Serratia spp. 7 (0) (0) (100) (29) (14) (57) (57) (14) (29) S - Sensitive, I - Intermediate, R - Resistant, AUG - Amoxicillin/Clavulanic acid, TIM - Ticarcillin/Clavulanic acid, TZP - Piperacillin/ Tazobactum Table 5: Sensitivity pattern of E.