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1 July-September Indian Journal of 2007 Medical Microbiology, (2007) 25 (3):203-8 Original Article 203 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 community has created major resistance problems leading to increased morbidity, mortality and health-care costs. 1 Of the several mechanisms of resistance, the most widespread and most important is the destruction of the β-lactam ring, which is mediated by β-lactamases. 2 Extended spectrum β-lactamases (ESBLs) are of greater concern because they are capable of hydrolyzing several groups of β-lactam antibiotics, notably third and fourth generation cephalosporins and extended spectrum penicillins such as piperacillin. There are 255 known β-lactamases to date and the continued use of β-lactams may select for newer variants. 3 The use of β-lactamase inhibitors in combination with β-lactam antibiotics is currently the most successful strategy to combat this specific resistance mechanism. These β- lactamase inhibitors are thought to be suicide inhibitors that form stable complexes between the bacterial β-lactamase and the β-lactamase inhibitor in a multi-step chemical reaction. 4 Their broad spectrum of activity originates from the ability of respective inhibitors to inactivate a wide range of β-lactamases produced by gram-positive, gram-negative, anaerobic and even acid-fast pathogens. β-lactam/ β-lactamase inhibitor combinations are particularly useful against mixed infections and play a useful role in treating various multi resistant pathogens such as ESBLs. These agents are gaining importance in the treatment of various multi-resistant and *Corresponding author ( <lgorthi@hotmail.com>) Department of Microbiology, Nizam s Institute of Medical Sciences, Hyderabad , Andhra Pradesh, India Received: Accepted: emerging nosocomial pathogens such as Acinetobacter spp. and Stenotrophomonas maltophilia. 5 This study compared the spectrum of activity of β-lactam/ β-lactamase inhibitors to that of cephalosporins against gramnegative bacilli. Materials and Methods The in vitro activity of three β-lactamase inhibitors in comparison with cephalosporins was evaluated against clinical isolates from patients admitted in our institute using the API system. Amoxycillin/clavulanic acid (AUG), ticarcillin/ clavulanic acid (TIM) and piperacillin/tazobactam (TZP) and all the four generations of cephalosporins were included in the study. A total of 1252 Enterobacteriaceae isolates and 385 isolates from non-fermenters were tested for AUG, TIM and TZP and TIM and TZP respectively. Tables 1 and 2 show all the isolates tested and the antibiotics used. All the isolates were identified and their minimum inhibitory concentrations (MICs) were determined by API system (biomérieux) using the relevant panels. The inoculum was prepared as per the standard protocol given in the instruction manual of the API system. Quality control was performed by using Escherichia coli strain ATCC for Enterobacteriaceae and Pseudomonas aeruginosa strain ATCC for non-fermenters, with each new batch of strips. Results Among the Enterobacteriaceae, the overall resistance

2 204 Indian Journal of Medical Microbiology vol. 25, No. 3 to amoxicillin/clavulanic acid, ticarcillin/ clavulanic acid and piperacillin/tazobactam was 82.92, and 22.44% respectively (Table 3) and 82-65% resistance to cephalosporins from Þrst to fourth generations. Table 4 shows the MICs of different β-lactam/β-betalactamase organisms to Enterobacteriaceae group of inhibitors and Tables 5-10 show the MICs against cephalosporins. Table 11 shows the MICs of different β-lactam/β-beta-lactamase inhibitors and cephalosporins to various non-fermenters. Table 1: Isolates of Enterobacteriaceae group and nonfermenters Enterobacteriaceae Non-fermenters Species No. of isolates Species No. of isolates E. coli 769 Acinetobacter 106 baumannii K. pneumoniae 236 Acinetobacter 12 lwofþ i Enterobacter 101 Pseudomonas 195 cloacae aeruginosa Other 56 Other 46 Enterobacter spp. Pseudomonas spp. Proteus mirabilis 36 Stenotrophomonas 26 maltophilia Proteus vulgaris 2 Providencia rettgeri 6 Serratia marcescens 39 Other Serratia spp. 7 Total 1252 Total 385 Discussion Penicillin, the Þrst of the β-lactam antibiotics, was Þrst introduced into medical practice in the 1940s. Since then, a large number of different β-lactams, including penicillins, cephalosporins, monobactams and carbapenems have been developed, all of which are structurally related through the presence of a core β-lactam ring. 2 The most common mechanism of resistance to beta-lactam antibiotics is 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 mediated resistance to β-lactams is the use of the β-lactam/ β-lactamase inhibitor combinations. 7 In recent years, the use of these combinations has been proven to be a useful and an effective strategy to improve upon the therapeutic value of β-lactam antibiotics. 8 Several factors inßuence the activity and pharmacodynamics of these combinations, including potency of these agents, pharmacokinetics of the inhibitor, type and quality of β-lactamase produced by the target bacterium and potential for the inhibitor to induce expression of chromosomal cephalosporinases in the target bacterium. 9 The overall antibacterial spectrum of these drug combinations depends on the intrinsic activity of the β-lactam as well as the characteristics of the individual inhibitor towards different β-lactamases. 5 The differences among these β-lactam/ β- lactamase inhibitors such as spectrum of activity, need to be considered in choosing an agent for a speciþc case. Currently, there are three commercially available 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 n=1252 4/2 16/2 8/ Non-fermenters _ + + _ + + _ n= /2-16/ /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 = Non-fermenters n= 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

3 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 (11) (1) (88) (32) (7) (62) (76) (7) (17) K. pneumoniae (34) (0) (66) (40) (5) (55) (66) (4) (30) Enterobacter cloacae 101 (0) (5) (95) (26) (11) (63) (50) (5) (45) Other Enterobacter spp. 56 (14) (2) (84) (44) (4) (52) (64) (2) (34) Proteus mirabilis 36 (83) (0) (17) (91) (9) (0) (97) (3) (0) Proteus vulgaris 2 (0) (0) (100) (100) (0) (0) (100) (0) (0) Providencia rettgeri 6 (0) (0) (100) (83) (0) (17) (83) (0) (17) Serratia marcescens 39 (0) (0) (100) (23) (8) (69) (44) (931) (26) Other 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. coli to cephalosporins (n=769) 1 G Ceph (15) (0) (85) Cefuroxime (21) (1) (78) Cf/Ctx (33) (0) (67) Ceftazidime (30) (4) (66) Cefepime (30) (3) (67) Cefpirome (33) (0) (67) Table 6: Sensitivity pattern of Klebsiella pneumoniae to cephalosporins (n=236) 1 G Ceph (35) (0) (65) Cefuroxime (37) (1) (63) Cf/Ctx (38) (0) (62) Ceftazidime (32) (5) (63) Cefepime (33) (6) (61) Cefpirome (38) (0) (62) tazobactam. 7 These inhibitors are available in the combinations of amoxycillin/clavulanic acid (augmentin), ampicillin / sulbactum and piperacillin/tazobactam (tazact), ticarcillin/ clavulanic acid (timentin) and cefoperazone/sulbactam (magnex). Tazobactam in combination with piperacillin has an excellent clinical efþcacy in various infections, caused by class A, D and C β-lactamase producing bacteria, including ESBL producers. 10 The available API panels allowed MIC determinations of amoxycillin/clavulanic acid, piperacillin/ tazobactam and ticarcillin/clavulanic acid. In the present study, 71.06% of the isolates tested. were susceptible to piperacillin/tazobactam, 35.06% to ticarcillin/ clavulanic acid and 16.38% to amoxicillin/clavulanic acid among Enterobacteriaceae. In another similar study, 91.7% of Enterobacteriaceae isolates were susceptible to piperacillin/

4 206 Indian Journal of Medical Microbiology vol. 25, No. 3 Table 7: Sensitivity pattern of Enterobacter cloacae to cephalosporins (n=101) 1 G Ceph (0) (0) (100) Cefuroxime (13) (1) (86) Cf/Ctx (23) (3) (74) Ceftazidime (21) (0) (79) Cefepime (25) (2) (73) Cefpirome (28) (0) (72) Table 8: Sensitivity pattern of other Enterobacter spp. to cephalosporins (n=56) 1 G Ceph (5) (0) (95) Cefuroxime (26) (2) (72) Cf/Ctx (31) (0) (69) Ceftazidime (32) (0) (68) Cefepime (50) (7) (43) Cefpirome (57) (2) (41) Table 11: Sensitivity pattern of non-fermenters to β-lactamases and cephalosporins Organism No. of TIM TZP CB FEP isolates S I R S I R S I R S I R (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) Acinetobacter baumannii 106 (18) (36) (46) (12) (15) (73) (4) (16) (80) (6) (60) (34) Acinetobacter lwofþ i 12 (75) (25) (0) (92) (8) (0) (17) (25) (58) (42) (0) (58) Pseudomonas aeruginosa 195 (8) (43) (49) (50) (19) (31) (26) (24) (50) (13) (41) (46) Other Pseudomonas spp. 46 (20) (13) (67) (24) (20) (56) (13) (17) (70) (7) (33) (60) Stenotrophom onas maltophilia 26 (19) (23) (58) (0) (8) (92) (4) (19) (77) (0) (23) (77) AUG - Amoxicillin/Clavulanic acid, TIM - Ticarcillin/Clavulanic acid, TZP - Piperacillin/Tazobactum Table 9: Sensitivity pattern of Proteus mirabilis to cephalosporins (n=36) 1 G Ceph (60) (0) (40) Cefuroxime (60) (0) (40) Cf/Ctx (60) (0) (40) Ceftazidime (53) (3) (44) Cefepime (59) (3) (38) Cefpirome (62) (0) (38) Table 10: Sensitivity pattern of Serratia marcescens to cephalosporins (n=39) 1 G Ceph (0) (0) (100) Cefuroxime (0) (0) (100) Cf/Ctx (20) (0) (80) Ceftazidime (15) (0) (85) Cefepime (15) (0) (85) Cefpirome (20) (0) (80)

5 July-September 2007 Anuradha et al - Testing of GNB against β-lactamase Inhibitors using API System 207 Table 12: Comparison of other studies with the present study Studies (Ref. No.) Pseudomonas aeruginosa Acinetobacter baumannii Stenotrophamonas maltophilia TZP TIM TZP TIM AMP TZP TIM Sader et al _ Ellen et al Bonfoglio et al _ Gobernado et al _ _ Miller et al Present study _ 0 19 All Þgures represent % sensitivity, TZP - Piperacillin/Tazobactum, TIM - Ticarcillin/Clavulanic acid tazobactam while ticarcillin/clavulanic acid was active against 85.8% isolates. 11 Another study showed susceptibility of >74% against piperacillin/tazobactam, >69% against ticarcillin/ clavulanic acid and >34% susceptibility against ampicillin/ sulbactam. 12 As observed by other studies, 11,12 piperacillin/ tazobactam was documented as the most active β-lactam/βlactamase inhibitor combination against Enterobacteriaceae in our study. In the context of the non-fermenter isolates, piperacillin/ tazobactam was found to be the most active combination against P. aeruginosa and ticarcillin/clavulanic acid against A. baumannii and S. maltophilia similar to the above studies (Table 12). Although all these study results indicated the same rank orders of activity, difference in the susceptibility rates was observed. The reasons could possibly be contributed to the hospital organisms sampled, test methods, sites of infection and the study time interval. 12 We found lower susceptibility rates to all the three β-lactam/β-lactamases compared to other studies. Referral and a tertiary care hospital status of our hospital and prior treatment with multiple antibiotics may account for high resistance among the isolates from this hospital. In the treatment of P. aeruginosa infections, the potential for clavulanic acid to induce expression of chromosomal cephalosporinase and antagonize antibacterial activity of ticarcillin is a concern, especially in patients who lack protective host defences. These are not concerns with piperacillin/tazobactam. 9 Tazobactam seems to be the most promising β-lactamase inhibitor, which has, unlike clavulanic acid and sulbactam, its own antibiotic activity. 15 Multiple antibiotic resistance is becoming increasingly prevalent in the opportunistic pathogen A. baumannii. Ticarcillin/clavulanic acid was found to be the most effective compared to piperacillin/tazobactam against A. baumannii. Though sulbactam has the highest intrinsic activity, compared to the other inhibitors, against A. baumannii 9,16,12 ampicillin/sulbactam was not tested in our study as it was not included in the susceptibility strip used for testing nonfermenters of the API. The majority of clinical isolates of the recently emerging nosocomial pathogen S. maltophilia, are resistant to multiple antibiotics. Ticarcillin/clavulanic acid combination is one of the few agents that have greater activity against this pathogen than the other β-lactam/β-lactamase inhibitor combinations. 7 The same pattern was observed in the present study. The high resistance exhibited by all isolates included in this study to all generation of cephalosporins as compared to β-lactam/β-lactamase inhibitors may be due to an increased use of cephalosporins in our hospital. Under a selective pressure induced by the extensive use of the cephalosporins, especially the third generation, ESBL producers appear and spread within the hospital. β-lactam/β-lactamase inhibitors can be useful alternatives to conventional two-three drug regimens in mixed infections, such as foot infections in patients with diabetes mellitus and hospital-acquired intra-abdominal infections. 17 Their substitution in place of cephalosporins appears to reduce emergence of the ESBL producing pathogens. Similarly their use may also curtail the emergence of other resistant pathogens such as Clostridium difficile and vancomycin resistant Enterococci. These are generally well-tolerated and their oral forms provide effective outpatient therapy against many commonly encountered infections. 5 They could even be more cost-effective than conventional combination therapies. 5 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. The isolates exhibited high resistance to other groups of antibiotics except carbapenems. References Maiti SN, Phillips OA, Micetich RG, Livermore DM. Betalactamase inhibitors: Agents to overcome bacterial resistance. Curr Med Chem 1998;5: Williams JD. Beta-lactamases and Beta-lactamase inhibitors. Int J Antimicrob Agents 1999;12:S3-7. Kotra LP, Mobashery S. Mechanistic and clinical aspects of beta-

6 208 Indian Journal of Medical Microbiology vol. 25, No lactam antibiotics and Beta-lactamases. Arch Immunol Ther Exp (Warsz) 1999;47: Rotschafer JC, Ostergaard BE. Combination beta-lactam and beta-lactamase-inhibitor products: Antimicrobial activity and efficiency of enzyme inhibition. Am J Health Syst Pharm 1995;52:S clavulanic acid and ampicillin/sulbactam. Braz J Infect Dis 2000;4:22-8. Baron EJ, Jones RN. National survey of the in vitro spectrum of piperacillin-tazobactam tested against more than 40,000 aerobic clinical isolates from 236 medical centers. Diagn Microbiol Infect Dis 1995;21: Lee N, Yuen KY, Kumana CR. Clinical role of beta-lactam/ betalactamase inhibitor combinations. Drugs 2003;63: Sandanayaka VP, Prashad AS. Resistance to beta-lactam antibiotics: Structure and mechanism based design of betalactamase inhibitors. Curr Med Chem 2002;9: Miller LA, Ratnam K, Payne DJ. Beta-lactamase-inhibitor Combinations in the 21 st century: Current agents and new developments. Curr Opin Pharmacol 2001;1: de la Pena A, Derendorf H. Pharmacokinetic properties of betalactamase inhibitors. Int J Clin Pharmacol Ther 1999;37: Lister PD. Beta-lactamase inhibitor combinations with extendedspectrum penicillins: Factors inßuencing antibacterial activity against Enterobacteriaceae and Pseudomonas aeruginosa. Pharmacotherapy 2000;20:213S-8S. Niki Y. Fundamental and clinical studies on beta-lactamase inhibitors. Nippon Rinsho 2001;59: Sader HS, Tosin I, Sejas L, Miranda E. Comparative evaluation of the in vitro activity of three combinations of beta-lactams with beta-lactamase inhibitors: Piperacillin/tazobactam, ticarcillin/ Author Help: Reference checking facility BonÞglio G, Laksai Y, Franceschini N, Perilli M, Segatore B, Bianc Stefani S, et al. In vitro activity of piperacillin-tazobactam against 615 Pseudomonas aeruginosa strains isolated in intensive care units. Chemotherapy 1998;44: Gobernado M, Bouza E, Perea E, Alvarez-Bravo J, García Rodríguez JA. A national Multicenter study of the in-vitro activity of piperacillin-tazobactam. The Spanish piperacillin-tazobactam group. Rev Esp Quimioter 1998;11: Blahova J, Hupkova M, Kremery V Sr. The effectiveness of so-called potentiated penicillins (augmentin and tazobactam) in vitro. Cas Lek Cesk 1995;134: Suh B, Shapiro T, Jones R, Satishchandran V, Truant AL. In vitro activity of beta-lactamase inhibitors against clinical isolates of Acinetobacter species. Diagn Microbiol Infect Dis 1995;21: Hart SM, Bailey EM. A practical look at the clinical usefulness of the beta-lactam/beta-lactamase inhibitor combinations. Ann Pharmacother 1996;30: Source of Support: Nil, Conßict of Interest: None declared. The manuscript system ( allows the authors to check and verify the accuracy and style of references. The tool checks the references with PubMed as per a predefined style. Authors are encouraged to use this facility before submitting articles to the journal. The style as well as bibliographic elements should be 100% accurate to get the references verified from the system. A single spelling error or addition of issue number / month of publication will lead to error to verifying the reference. Example of a correct style Sheahan P, O leary G, Lee G, Fitzgibbon J. Cystic cervical metastases: Incidence and diagnosis using fine needle aspiration biopsy. Otolaryngol Head Neck Surg 2002;127: Only the references from journals indexed in PubMed would be checked. Enter each reference in new line, without a serial number. Add up to a maximum 15 reference at time. If the reference is correct for its bibliographic elements and punctuations, it will be shown as CORRECT and a link to the correct article in PubMed will be given. If any of the bibliographic elements are missing, incorrect or extra (such as issue number), it will be shown as INCORRECT and link to possible articles in PubMed will be given.