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ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 51  |  Issue : 2  |  Page : 222-224
Detection of extended-spectrum β-lactamase in Pseudomonas aeruginosa

Department of Microbiology, Pt. B. D. Sharma Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India

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Purpose: The present study was designed to detect the extended-spectrum β-lactamase (ESBL) production in Pseudomonas aeruginosa and to evaluate the susceptibility pattern. Materials and Methods: One hundred forty-eight isolates of P. aeruginosa were analyzed for the presence of ESBL enzyme by double disc synergy test. Antibiotic sensitivity pattern of ESBL-positive P. aeruginosa was determined. Results: Of the 148 isolates tested, 30 (20.27%) were found to be positive. Maximum ESBL production was found in sputum and tracheostomy swabs (28.57%), followed by pus (24.13%), urine (19.04%), cerebrospinal fluid (CSF) and other sterile body fluids (15.38%) and blood (7.14%). All the ESBL-producing P. aeruginosa isolates were multi-drug-resistant. Isolates were 100% sensitive to imipenem. Ofloxacin was the second most (70%) effective drug. Conclusion: From this study, we conclude the presence of ESBL-positive P. aeruginosa in our hospital. This has important implications as carbapenems remain the only choice of treatment for infections caused by these organisms. The control measures include judicious use of antibiotics and implementation of appropriate infection control measures to control the spread of these strains in the hospital

Keywords: β-lactams, Pseudomonas aeruginosa, third-generation cephalospronis

How to cite this article:
Aggarwal R, Chaudhary U, Bala K. Detection of extended-spectrum β-lactamase in Pseudomonas aeruginosa. Indian J Pathol Microbiol 2008;51:222-4

How to cite this URL:
Aggarwal R, Chaudhary U, Bala K. Detection of extended-spectrum β-lactamase in Pseudomonas aeruginosa. Indian J Pathol Microbiol [serial online] 2008 [cited 2023 Jun 1];51:222-4. Available from:

   Introduction Top
 Pseudomonas aeruginosa Scientific Name Search i> is an opportunistic pathogen with innate resistance to many antibiotics and disinfectants. P. aeruginosa is physiologically versatile and flourishes as a saprophyte in multiple environments, including sinks, drains, respirators, humidifiers and disinfectant solutions. Infections due to P. aeruginosa are seldom encountered in healthy adults; but in the last two decades, the organism has become increasingly recognized as the etiological agent in a variety of serious infections in hospitalized patients, especially those with impaired immune defenses. [1]

In addition to its innate resistance, acquired additional resistance due to plasmids is also a problem in P. aeruginosa . Plasmid-mediated resistance involving modifying enzymes is particularly associated with indiscriminate antibiotic use and with sites where high levels of antibiotics are achieved. [1]

Pseudomonads are more versatile than Enterobacteriaceae in acquiring drug resistance by various mechanisms. ESBL production is one of them. ESBL enzyme-encoding genes SHV-2a and TEM-42 have been found in P. aeruginosa, suggesting that ESBL reservoir is not restricted to Enterobacteriaceae family. [2],[3] However, the occurrence and detection of ESBL in P. aeruginosa are undefined in India. Clinical Laboratory Standards Institute (CLSI) guidelines do not describe any method to detect ESBL production in P. aeruginosa. [4]

Hence the present study was conducted with an objective to detect ESBL production in P. aeruginosa isolates from various clinical samples and to know the antibiotic sensitivity of these positive isolates.

   Materials And Methods Top

One hundred forty-eight clinical isolates of P. aeruginosa, inclusive of 28 isolates from blood, 58 isolates from pus, 21 isolates from urine, 13 isolates from cerebrospinal fluid and other sterile fluids and 28 isolates from sputum and tracheotomy swabs, were obtained from patients. P aeruginosa isolates that were obtained as a pure and predominant growth from the clinical specimens were only considered for the present study. The organisms were identified based on the colony morphology and biochemical reactions. [5] The sensitivity of the isolates to the third-generation cephalosporins (ceftazidime, cefotaxime, ceftriaxone, Third generation cephalosporins (3GC), 30 µg each) was determined by disc diffusion method using P. aeruginosa ATCC 27853 as control strain. Results were interpreted according to the CLSI guidelines, which suggest a diameter of inhibition zone ≥22 mm for ceftazidime, ≥27 mm for cefotaxime and ≥25 mm for ceftriaxone as susceptible. [6]

ESBL production in all the isolates was detected by double disc synergy test as described by Jarlier. [7] Synergy was determined between a disc of amoxyclav (20 µg amoxycillin and 10 µg clavulanic acid) and a 30-µg disc of each 3GC test antibiotic placed 15 mm apart on a lawn culture of the isolate under test on Mueller Hinton agar (MHA, Hi-Media). The test organism was considered to produce ESBL if the zone size around the antibiotic disc increased towards the amoxyclav disc. This criterion also fulfills the CLSI guidelines. [4] This increase occurs because the clavulanic acid present in the amoxyclav disc inactivates the ESBL produced by the test organism.

Antimicrobial susceptibility test of ESBL-producing P. aeruginosa was done by disc diffusion method using P. aeruginosa ATCC 27853 as control strain. Results were interpreted according to CLSI guidelines. [6] Commercially available (Hi-Media) 6-mm antibiotic discs were used on Mueller Hinton agar (MHA).

   Results Top

Out of 148 P. aeruginosa isolates, 30 (20.27%) were found to be positive for ESBL production [Table 1]. ESBL production was found in both sensitive and resistant groups. Of the 116 (78.38%) 3GC-resistant isolates, 25 (21.55%) produced ESBL; whereas of the 32 (21.62%) 3GC-sensitive isolates, 5 (15.62%) were found to be producers of ESBL enzyme [Table 2]. This resistance to 3GC was found to coexist with resistance to other antibiotics.

All the ESBL-positive P. aeruginosa were multi-drug resistant (drug resistance to more than three drugs). Maximum sensitivity (100%) was seen with imipenem; followed by ofloxacin, which showed good sensitivity (70%). The least effective drugs were cephalothin, cefamandole, azlocillin, ticarcillin, ticarcillin/clavulanate [Table 3].

   Discussion Top

Our study reported 20.27% ESBL production among P. aeruginosa isolates. Maximum ESBL production was found in sputum and tracheostomy wound swabs (28.57%), followed by pus (24.13%), urine (19.04%), CSF and other sterile body fluids (15.38%) and blood (7.14%). ESBL-mediated resistance to 3GC in P. aeruginosa as reported by Uma et al . (77.30%) [4] and Mathur et al . (64%) [9] is much higher than that reported in the present study. A study conducted by Jacobson et al. [9] depicted a very low rate, viz., 7.7%, of ESBL production in P. aeruginosa.

In our study, varying range of resistance, viz., 40% to 100%, for β-lactams has been observed. This resistance is due to the hydrolysis of β-lactam ring of β-lactam antibiotics by the action of β-lactamase enzymes. Other mechanisms of drug resistance to β-lactam group of antibiotics are loss of outer membrane protein, production of class C AmpC β-lactamases and altered target sites. These ESBL enzymes are inhibited by β-lactamase inhibitors, viz., clavulanic acid and sulbactam. Hence the use of β-lactam/β-lactamase inhibitor combination may be an alternative to 3GC, but the effect of this combination varies depending on the subtype of ESBL present. [10] Some TEM-derived β-lactamases are resistant to β-lactamase inhibitors. Also, there is limited clinical experience with the use β-lactam/β-lactamase inhibitors in treating serious infections with ESBL-producing organisms. Because of these variables, these agents should not be considered as first line of therapy. Among the combination group, 96.66% of resistance was seen to ticarcillin/clavulanate and 63.33% of resistance rate was observed to ampicillin/sulbactam. This shows that these combination drugs are unreliable for therapeutic purposes.

As already mentioned, ESBL-producing bacteria are frequently resistant to many other classes of antibiotics, including aminoglycosides and fluoroquinolones. This is due to the coexistence of genes encoding drug resistance to other antibiotics on the plasmids which encode ESBL. [10] This fact has also been observed in our study.

In our study, imipenem was the only drug found unaffected by the action of these enzymes. This finding is in concordance with the studies conducted by other authors, who also reported a rate of 100% sensitivity to imipenem. [11],[12],[13],[14],[15] These carbapenem agents may be of benefit in the treatment of ESBL infection; however, indiscriminate use of these agents may promote increased resistance to carbapenems.

One of the potential limitations of this study is that molecular epidemiologic analysis and characterization of ESBL types was not carried out. Our study reports the presence of ESBL enzyme in P. aeruginosa; our data suggests that the gene for this enzyme has spread from Enterobacteriaceae to non- Enterobacteriaceae . To combat this problem, efforts should be made to isolate and characterize plasmids and to screen ESBLs responsible for resistance in multi-drug-resistant P. aeruginosa strains in India; and a nationwide antibiotic policy should be instituted to minimize the emergence of resistance

   References Top

1.Shahid M, Malik A, Sheeba. Multidrug resistant Pseudomonas aeruginosa strains harbouring R-plasmids and Amp C β-lactamases isolated from hospitalized burn patients in tertiary care hospital of North India. FEMS Lett 2003;228:181-6.  Back to cited text no. 1    
2.Naas T, Phiippon L, Poirel L, Ronco E, Nordmann P. An SHV-derived extended-spectrum β-lactamase in Pseudomonas aeruginosa . Antimicrob Agents Chemother 1999;43:1281-4.  Back to cited text no. 2    
3.Mugnier P, Dubrous P, Casin I, Arlet G, Collatz E. A TEM-derived extended-spectrum β-lactamase in Pseudomonas aeruginosa . Antimicrob Agents Chemother 1996;40:2488-93.  Back to cited text no. 3    
4.Clinical laboratory standards institute. Performance standards for antimicrobial susceptibility testing. Sixteenth international supplement. CLSI document M100 - S 16, Wayne PA: 2007.  Back to cited text no. 4    
5.Colle JG, Miles RS, Wan B. Tests for the identification of bacteria. In : Collee JG, Fraser AG, Marmion BP, Simmons A, Editors. Mackie and McCartney Practical Medical Microbiology, 14th ed. Edinburg: Churchill Livingstone; 1996. p. 131-50.  Back to cited text no. 5    
6.National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disk susceptibility tests, 7 th ed. Approved standard. NCCLS document M2-A7, Vol. 20 No. 1; Wayne PA: January 2000.  Back to cited text no. 6    
7.Jarlier V, Nicolas MH, Fourneir G. Extended spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae : Hospital prevalence and susceptibility patterns. Rev Infect Dis 1998;10:867-78.  Back to cited text no. 7    
8.Mathur P, Kapil A, Das B, Dhawan B. Prevalence of extended spectrum β-lactamase producing gram negative bacteria in a tertiary care hospital. Indian J Med Res 2002;115:153-7.  Back to cited text no. 8    
9.Jacobson KL, Cohen SH, Inciardi JF, King JH, Lippert WE, Iglesias T, et al . The relationship between antecedent antibiotic use and resistance to extended spectrum cephalosporins in Group I β-lactamase producing organisms. Clin Infect Dis 1995;21:1107-13.  Back to cited text no. 9    
10.Nathisuwan S, Burgess DS, Lewis JS 2 nd . Extended spectrum β-lactamases: Epidemiology, detection and treatment. Pharmacotherapy 2001;21:920-8.  Back to cited text no. 10    
11.Thomson KS, Sanders CC. Detection of extended spectrum β-lactamases in members of family Enterobacteriaceae : Comparison of the double disk and three dimensional tests. Antimicrob Agents Chemother 1992;36:1877-82.  Back to cited text no. 11    
12.Pena C, Pujol M, Ardanuy C. Epidemiology and successful control of a large outbreak due to Klebsiella pneumoniae producing extended spectrum β-lactamases. Antimicrob Agents Chemother 1998;42:53-8.  Back to cited text no. 12    
13.Winokur PL, Canton R, Casellas JM, Legakis N. Variations in the prevalence of strains expressing an extended spectrum β-lactamase phenotype and characterisation of isolates from Europe, the Americans and the Western Pacific region. Clin Infect Dis 2001;32:594-603.  Back to cited text no. 13    
14.Abigail S, Mathai E, Jesudason MV, John TJ. Ceftazidime resistance among Klebsiella pneumoniae in South India. Indian J Med Res 1995;102:53-5.  Back to cited text no. 14    
15.Subha A, Ananthan S. Extended spectrum β-lactamase mediated resistance to third generation cephalosporins among Klebsiella pneumoniae in Chennai. Indian J Med Microbiol 2002;20:92-5.  Back to cited text no. 15    

Correspondence Address:
Ritu Aggarwal
H. No. 717, Sec. 1 HUDA, Rohtak - 124 001, Haryana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.41693

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  [Table 1], [Table 2], [Table 3]

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