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Year : 2007  |  Volume : 25  |  Issue : 3  |  Page : 203-208

Sensitivity pattern of gram negative bacilli to three β-lactam/β-lactamase inhibitor combinations using the automated API system

Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad - 500 082, Andhra Pradesh, India

Date of Submission29-Sep-2004
Date of Acceptance12-Dec-2005

Correspondence Address:
V Lakshmi
Department of Microbiology, Nizam's Institute of Medical Sciences, Hyderabad - 500 082, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0255-0857.34759

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 ~ 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 (bioMιrieux) 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 .

Keywords: API, β-lactamase inhibitors, gram negative bacteria, sensitivity

How to cite this article:
Anuradha K, Sailaja V V, Umabala P, Satheesh T, Lakshmi V. Sensitivity pattern of gram negative bacilli to three β-lactam/β-lactamase inhibitor combinations using the automated API system. Indian J Med Microbiol 2007;25:203-8

How to cite this URL:
Anuradha K, Sailaja V V, Umabala P, Satheesh T, Lakshmi V. Sensitivity pattern of gram negative bacilli to three β-lactam/β-lactamase inhibitor combinations using the automated API system. Indian J Med Microbiol [serial online] 2007 [cited 2021 Mar 8];25:203-8. Available from:

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 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 gram-negative bacilli.

 ~ Materials and Methods Top

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. [Table - 1],[Table - 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 (bioMιrieux) 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 More Details coli strain ATCC 25922 for Enterobacteriaceae and Pseudomonas aeruginosa strain ATCC 27853 for non-fermenters, with each new batch of strips.

 ~ Results Top

Among the Enterobacteriaceae , the overall resistance to amoxicillin/clavulanic acid, ticarcillin/ clavulanic acid and piperacillin/tazobactam was 82.92, 58.22 and 22.44% respectively [Table - 3] and 82-65% resistance to cephalosporins from first to fourth generations.

[Table - 4] shows the MICs of different β -lactam/β -beta-lactamase organisms to Enterobacteriaceae group of inhibitors and [Table - 5],[Table - 6],[Table - 7],[Table - 8],[Table - 9],[Table - 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.

 ~ Discussion Top

Penicillin, the first of the β -lactam antibiotics, was first 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 influence the activity and pharmacodynamics of these combinations, including potency of these agents, pharmacokinetics of the inhibitor, type and quality of b-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 b-lactam/ β -lactamase inhibitors such as spectrum of activity, need to be considered in choosing an agent for a specific case.

Currently, there are three commercially available β -lactamase inhibitors-clavulanic acid, sulbactam and 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 efficacy 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/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/β -lactamase 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 [11],[12],[13],[14] [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 non-fermenters 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 Top

1.Maiti SN, Phillips OA, Micetich RG, Livermore DM. Beta-lactamase inhibitors: Agents to overcome bacterial resistance. Curr Med Chem 1998;5:441-56.  Back to cited text no. 1  [PUBMED]  
2.Williams JD. Beta-lactamases and Beta-lactamase inhibitors. Int J Antimicrob Agents 1999;12:S3-7.  Back to cited text no. 2    
3.Kotra LP, Mobashery S. Mechanistic and clinical aspects of beta-lactam antibiotics and Beta-lactamases. Arch Immunol Ther Exp (Warsz) 1999;47:211-6.  Back to cited text no. 3  [PUBMED]  
4.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 :S15-22.  Back to cited text no. 4  [PUBMED]  
5.Lee N, Yuen KY, Kumana CR. Clinical role of beta-lactam/ beta-lactamase inhibitor combinations. Drugs 2003;63:1511-24.   Back to cited text no. 5  [PUBMED]  
6.Sandanayaka VP, Prashad AS. Resistance to beta-lactam antibiotics: Structure and mechanism based design of beta-lactamase inhibitors. Curr Med Chem 2002;9:1145-65  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Miller LA, Ratnam K, Payne DJ. Beta-lactamase-inhibitor Combinations in the 21 st century: Current agents and new developments. Curr Opin Pharmacol 2001;1:451-8.  Back to cited text no. 7  [PUBMED]  [FULLTEXT] la Pena A, Derendorf H. Pharmacokinetic properties of beta-lactamase inhibitors. Int J Clin Pharmacol Ther 1999;37:63-75.  Back to cited text no. 8  [PUBMED]  
9.Lister PD. Beta-lactamase inhibitor combinations with extended-spectrum penicillins: Factors influencing antibacterial activity against Enterobacteriaceae and Pseudomonas aeruginosa. Pharmacotherapy 2000;20:213S-8S.  Back to cited text no. 9  [PUBMED]  
10.Niki Y. Fundamental and clinical studies on beta-lactamase inhibitors. Nippon Rinsho 2001;59:771-6.  Back to cited text no. 10    
11.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/clavulanic acid and ampicillin/sulbactam. Braz J Infect Dis 2000;4:22-8.  Back to cited text no. 11  [PUBMED]  
12.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;21:141-51.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]
13.Bonfiglio 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;44:305-12.  Back to cited text no. 13    
14.Gobernado M, Bouza E, Perea E, Alvarez-Bravo J, Garcνa Rodrνguez JA. A national Multicenter study of the in-vitro activity of piperacillin-tazobactam. The Spanish piperacillin-tazobactam group . Rev Esp Quimioter 1998;11:139-46.  Back to cited text no. 14    
15.Blahova J, Hupkova M, Kremery V Sr. The effectiveness of so-called potentiated penicillins (augmentin and tazobactam) in vitro. Cas Lek Cesk 1995;134:558-61.  Back to cited text no. 15    
16.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;21:111-4.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.Hart SM, Bailey EM. A practical look at the clinical usefulness of the beta-lactam/beta-lactamase inhibitor combinations. Ann Pharmacother 1996;30:1130-40.  Back to cited text no. 17  [PUBMED]  


  [Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6], [Table - 7], [Table - 8], [Table - 9], [Table - 10], [Table - 11], [Table - 12]

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