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 / , / clavulanic acid and / in comparison to 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. 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 except . 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 , 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 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 strain ATCC 25922 for Enterobacteriaceae and strain ATCC *Corresponding author (email: ) 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 , 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, 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, 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- 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, 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 from 4-32 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 , CEF - , CF/CTX - /, CB - , FEP - , CPO -

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

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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. coli to cephalosporins Table 6: Sensitivity pattern of to (n=769) cephalosporins (n=236) Cephalo Sensitive Intermediate Resistant Cephalo Sensitive Intermediate Resistant sporins (%) (%) (%) sporins (%) (%) (%) 1 G Ceph 114 0 655 1 G Ceph 83 0 153 (15) (0) (85) (35) (0) (65) Cefuroxime 166 5 598 Cefuroxime 86 1 149 (21) (1) (www.medknow.com).(78) (37) (1) (63) Cf/Ctx 252 1 516 Cf/Ctx 89 0 147 (33) (0) (67) (38) (0) (62) Ceftazidime This233 PDF a site is hosted 29available by507 forMedknow freeCeftazidime download Publications 75 from 12 149 (30) (4) (66) (32) (5) (63) Cefepime 235 22 512 Cefepime 78 14 144 (30) (3) (67) (33) (6) (61) Cefpirome 251 1 517 Cefpirome 89 0 147 (33) (0) (67) (38) (0) (62) tazobactam.7 These inhibitors are available in the combinations determinations of amoxycillin/clavulanic acid, piperacillin/ of amoxycillin/clavulanic acid (augmentin), / tazobactam and ticarcillin/clavulanic acid. sulbactum and piperacillin/tazobactam (tazact), ticarcillin/ clavulanic acid (timentin) and /sulbactam In the present study, 71.06% of the isolates tested. were (magnex). Tazobactam in combination with piperacillin has susceptible to piperacillin/tazobactam, 35.06% to ticarcillin/ an excellent clinical efÞ cacy in various infections, caused by clavulanic acid and 16.38% to amoxicillin/clavulanic acid class A, D and C β-lactamase producing bacteria, including among Enterobacteriaceae. In another similar study, 91.7% of ESBL producers.10 The available API panels allowed MIC Enterobacteriaceae isolates were susceptible to piperacillin/

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Table 7: Sensitivity pattern of Enterobacter cloacae to Table 9: Sensitivity pattern of Proteus mirabilis to cephalosporins (n=101) cephalosporins (n=36) Cephalo Sensitive Intermediate Resistant Cephalo Sensitive Intermediate Resistant sporins (%) (%) (%) sporins (%) (%) (%) 1 G Ceph 0 0 101 1 G Ceph 22 0 14 (0) (0) (100) (60) (0) (40) Cefuroxime 13 1 87 Cefuroxime 22 0 14 (13) (1) (86) (60) (0) (40) Cf/Ctx 24 2 75 Cf/Ctx 22 0 14 (23) (3) (74) (60) (0) (40) Ceftazidime 21 0 80 Ceftazidime 18 1 17 (21) (0) (79) (53) (3) (44) Cefepime 25 2 74 Cefepime 21 1 14 (25) (2) (73) (59) (3) (38) Cefpirome 28 0 73 Cefpirome 23 0 13 (28) (0) (72) (62) (0) (38)

Table 8: Sensitivity pattern of other Enterobacter spp. to Table 10: Sensitivity pattern of Serratia marcescens to cephalosporins (n=56) cephalosporins (n=39) Cephalo Sensitive Intermediate Resistant Cephalo Sensitive Intermediate Resistant sporins (%) (%) (%) sporins (%) (%) (%) 1 G Ceph 3 0 53 1 G Ceph 0 0 39 (5) (0) (95) (0) (0) (100) Cefuroxime 15 1 40 Cefuroxime 0 0 39 (26) (2) (72) (0) (0) (100) Cf/Ctx 17 0 39 Cf/Ctx 8 0 31 (31) (0) (69) (20) (0) (80) Ceftazidime 18 0 38 Ceftazidime 6 0 33 (32) (0) (68) (15) (0) (85) Cefepime 28 4 24 Cefepime 6 0 33 (50) (7) (43) (15) (0) (85) Cefpirome 32 1 23 Cefpirome 8 0 31 (57) (2) (41) (20) (0) (80) (www.medknow.com).

Table 11: Sensitivity pattern of non-fermenters to β-lactamases and cephalosporins Organism This No. of PDFa site TIMis hosted available by forMedknow TZP free download Publications CB from FEP isolates S I R S I R S I R S I R (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) Acinetobacter 19 38 49 13 16 77 4 17 85 6 64 36 baumannii 106 (18) (36) (46) (12) (15) (73) (4) (16) (80) (6) (60) (34) Acinetobacter 9 3 0 11 1 0 2 3 7 5 0 7 lwofÞ i 12 (75) (25) (0) (92) (8) (0) (17) (25) (58) (42) (0) (58) Pseudomonas 16 83 96 98 36 61 50 46 99 25 80 90 aeruginosa 195 (8) (43) (49) (50) (19) (31) (26) (24) (50) (13) (41) (46) Other 9 6 31 11 9 26 6 8 32 3 15 28 Pseudomonas spp. 46 (20) (13) (67) (24) (20) (56) (13) (17) (70) (7) (33) (60) Stenotrophom 5 6 15 0 2 24 1 5 20 0 6 20 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

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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.11 61.5 56.4 _ _ 87 100 _ Ellen et al.12 91 78 57 _ _ _ _ Bonfoglio et al.13 92.4 69 _ _ _ _ _ Gobernado et al.14 92 _ 42 _ _ 61 _ Miller et al.7 92 87 66 73 79 _ _ Present study 50 8 12 18 _ 0 19 All Þ gures represent % sensitivity, TZP - Piperacillin/Tazobactum, TIM - Ticarcillin/Clavulanic acid tazobactam while ticarcillin/clavulanic acid was active against The majority of clinical isolates of the recently emerging 85.8% isolates.11 Another study showed susceptibility of >74% nosocomial pathogen S. maltophilia, are resistant to multiple against piperacillin/tazobactam, >69% against ticarcillin/ antibiotics. Ticarcillin/clavulanic acid combination is one of clavulanic acid and >34% susceptibility against ampicillin/ the few agents that have greater activity against this pathogen sulbactam.12 As observed by other studies,11,12 piperacillin/ than the other β-lactam/β-lactamase inhibitor combinations.7 tazobactam was documented as the most active β-lactam/β- The same pattern was observed in the present study. lactamase inhibitor combination against Enterobacteriaceae in our study. The high resistance exhibited by all isolates included in this study to all generation of cephalosporins as compared to In the context of the non-fermenter isolates, piperacillin/ β-lactam/β-lactamase inhibitors may be due to an increased tazobactam was found to be the most active combination use of cephalosporins in our hospital. Under a selective against P. aeruginosa and ticarcillin/clavulanic acid against A. pressure induced by the extensive use of the cephalosporins, baumannii and S. maltophilia similar to the above studies11-14 especially the third generation, ESBL producers appear and (Table 12). spread within the hospital.

Although all these study results indicated the same rank β-lactam/β-lactamase inhibitors can be useful alternatives orders of activity, difference in the susceptibility rates was to conventional two-three drug regimens in mixed infections, observed. The reasons could possibly be contributed to the such as foot infections in patients with diabetes mellitus hospital organisms sampled, test methods, sites of infection and and hospital-acquired intra-abdominal infections.17 Their the study time interval.12 We found lower susceptibility rates to substitution in place of cephalosporins appears to reduce all the three β-lactam/β-lactamases compared to other studies. emergence of the ESBL producing pathogens. Similarly their Referral and a tertiary care hospital status of our hospital and use may also curtail the emergence of other resistant pathogens prior treatment with multiple antibiotics may account for high such as Clostridium difficile and resistant resistance among the isolates from this hospital. Enterococci. These are generally well-tolerated and their (www.medknow.com).oral forms provide effective outpatient therapy against many In the treatment of P. aeruginosa infections, the potential commonly encountered infections.5 They could even be more for clavulanic acid to induce expression of chromosomal cost-effective than conventional combination therapies.5 cephalosporinase and antagonizea site antibacterial hosted activity by Medknow of Publications ticarcillin is a Thisconcern, PDF especially is inavailable patients who lackfor freePiperacillin/tazobactam download from was found to be the most active protective host defences. These are not concerns with combination of the three against Enterobacteriaceae and piperacillin/tazobactam.9 Tazobactam seems to be the most Pseudomonas spp. and ticarcillin/clavulanic acid against promising β-lactamase inhibitor, which has, unlike clavulanic Acinetobacter spp. and Stenotrophomonas maltophilia. The acid and sulbactam, its own activity.15 isolates exhibited high resistance to other groups of antibiotics except carbapenems. Multiple antibiotic resistance is becoming increasingly prevalent in the opportunistic pathogen A. baumannii. References Ticarcillin/clavulanic acid was found to be the most effective compared to piperacillin/tazobactam against A. baumannii. 1. Maiti SN, Phillips OA, Micetich RG, Livermore DM. Beta- lactamase inhibitors: Agents to overcome bacterial resistance. Though sulbactam has the highest intrinsic activity, Curr Med Chem 1998;5:441-56. compared to the other inhibitors, against A. baumannii9,16,12 2. Williams JD. Beta-lactamases and Beta-lactamase inhibitors. Int ampicillin/sulbactam was not tested in our study as it was J Antimicrob Agents 1999;12:S3-7. not included in the susceptibility strip used for testing non- fermenters of the API. 3. Kotra LP, Mobashery S. Mechanistic and clinical aspects of beta-

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lactam antibiotics and Beta-lactamases. Arch Immunol Ther Exp clavulanic acid and ampicillin/sulbactam. Braz J Infect Dis (Warsz) 1999;47:211-6. 2000;4:22-8.

4. Rotschafer JC, Ostergaard BE. Combination beta-lactam and 12. Baron EJ, Jones RN. National survey of the in vitro spectrum of beta-lactamase-inhibitor products: Antimicrobial activity and piperacillin-tazobactam tested against more than 40,000 aerobic efficiency of inhibition. Am J Health Syst Pharm clinical isolates from 236 medical centers. Diagn Microbiol Infect 1995;52:S15-22. Dis 1995;21:141-51.

5. Lee N, Yuen KY, Kumana CR. Clinical role of beta-lactam/ beta- 13. BonÞ glio G, Laksai Y, Franceschini N, Perilli M, Segatore B, lactamase inhibitor combinations. Drugs 2003;63:1511-24. Bianc Stefani S, et al. In vitro activity of piperacillin-tazobactam against 615 Pseudomonas aeruginosa strains isolated in intensive 6. Sandanayaka VP, Prashad AS. Resistance to beta-lactam care units. Chemotherapy 1998;44:305-12. antibiotics: Structure and mechanism based design of beta- lactamase inhibitors. Curr Med Chem 2002;9:1145-65 14. Gobernado M, Bouza E, Perea E, Alvarez-Bravo J, García Rodríguez JA. A national Multicenter study of the in-vitro activity 7. Miller LA, Ratnam K, Payne DJ. Beta-lactamase-inhibitor of piperacillin-tazobactam. The Spanish piperacillin-tazobactam Combinations in the 21st century: Current agents and new group. Rev Esp Quimioter 1998;11:139-46. developments. Curr Opin Pharmacol 2001;1:451-8. 15. Blahova J, Hupkova M, Kremery V Sr. The effectiveness of 8. de la Pena A, Derendorf H. Pharmacokinetic properties of beta- so-called potentiated penicillins (augmentin and tazobactam) in lactamase inhibitors. Int J Clin Pharmacol Ther 1999;37:63-75. vitro. Cas Lek Cesk 1995;134:558-61.

9. Lister PD. Beta-lactamase inhibitor combinations with extended- 16. Suh B, Shapiro T, Jones R, Satishchandran V, Truant AL. In vitro spectrum penicillins: Factors inß uencing antibacterial activity activity of beta-lactamase inhibitors against clinical isolates of against Enterobacteriaceae and Pseudomonas aeruginosa. Acinetobacter species. Diagn Microbiol Infect Dis 1995;21:111-4. Pharmacotherapy 2000;20:213S-8S. 17. Hart SM, Bailey EM. A practical look at the clinical usefulness 10. Niki Y. Fundamental and clinical studies on beta-lactamase of the beta-lactam/beta-lactamase inhibitor combinations. Ann inhibitors. Nippon Rinsho 2001;59:771-6. Pharmacother 1996;30:1130-40. 11. Sader HS, Tosin I, Sejas L, Miranda E. Comparative evaluation of the in vitro activity of three combinations of beta-lactams with Source of Support: Nil, Conß ict of Interest: None declared. beta-lactamase inhibitors: Piperacillin/tazobactam, ticarcillin/

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