Abstract
Resistance to broad-spectrum beta-lactams, mediated by extended-spectrum beta-lactamase enzymes (ESBL), is an increasing problem worldwide. The present study was undertaken to determine the incidence of ESBL-production among the clinical isolates of Pseudomonas aeruginosa and their susceptibility to selected antimicrobials. A total of one eighty-seven clinical specimens were tested for the presence of ESBL production using the double-disc synergy test. Of these, 25.13% (n = 47) isolates of P. aeruginosa were observed as ESBL positive. The maximum number of ESBL-producing strains were found in sputum (41.67%; n = 24) followed by pus (28.36%; n = 19), cerebrospinal fluid and other body fluids (21.74%; n = 5), urine (20.45%; n = 9) and blood (13.79%; n = 4). ESBL producing isolates exhibited co-resistance to an array of antibiotics tested. Imipenem and meropenem can be suggested as the drugs of choice in our study.
Keywords: Extended-spectrum beta-lactamases, Double-disc synergy test, Pseudomonas aeruginosa, Imipenem, Meropenem
1. Introduction
The worldwide emergence of multi-drug resistant bacterial strains in hospitals and community continues to be a problem of due scientific concern, especially infections caused by Pseudomonas species and Pseudomonas aeruginosa in particular. P. aeruginosa is an opportunistic pathogen with inherent resistance to many antibiotics and disinfectants including anti-pseudomonal Penicillins, Ceftazidime, Carbapenems, Aminoglycosides and Ciprofloxacin (Dundar and Otkun, 2010).
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 an aetiological agent in a variety of serious infections in hospitalized patients, especially those with impaired immune defenses (Shahid et al., 2003).
Extended-spectrum beta-lactamases (ESBLs) have emerged as an important cause of resistance in Gram-negative bacteria. Beta-lactam antibiotics are among the safest and most frequently prescribed antimicrobial agents all over the world in treating Gram positive and Gram negative infections (Bradford, 2001). Production of beta-lactamases is the most common mechanism of bacterial resistance to these antibiotics. These enzymes are numerous and are plasmid mediated, capable of hydrolysing and inactivating a wide variety of beta-lactam antibiotics. In addition, ESBL producing organisms exhibit co-resistance to many other classes of antibiotics resulting in the limitation of therapeutic options. For this reason, ESBL–mediated infections have been increasingly reported worldwide (Khanfar et al., 2009).
The aim of the present study was to determine the incidence of ESBL-production among the clinical isolates of P. aeruginosa and their susceptibility to antimicrobials.
2. Methods
2.1. Bacterial isolates
One hundred eighty-seven isolates of P. aeruginosa were recovered from various clinical specimens. These included 44 isolates from urine, 29 isolates from blood, 67 isolates from pus, 24 isolates from sputum and 23 isolates from cerebrospinal fluid and other body fluids. All the specimens were quickly sent to the laboratory to be processed. Standard methods for isolation and identification of these bacteria were used (Cowan and Steel, 1970).
2.2. Antimicrobial susceptibility testing and ESBL detection
Antimicrobial susceptibility testing of the isolates was performed by the standard disc diffusion method as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2008). P. aeruginosa ATCC 27853 was used as a control strain. ESBL production was detected by double disc synergy test as described by Jarlier et al. (1998). Synergy was determined between a disc of amoxyclav (20 μg amoxycillin and 10 μg clavulanic acid) and a 30 μg disc of each third generation cephalosporin test antibiotic placed 15 mm apart on a lawn culture of the isolate under test on Mueller Hinton agar plates. The test organism was considered to produce ESBL if the zone size around the antibiotic disc increased towards the amoxyclav disc. This increase occurs as the clavulanic acid present in the amoxyclav disc inactivates the ESBL produced by the test organism.
3. Results
A total of 187 bacterial isolates were analysed from various clinical specimens. Of these, 25.13% (n = 47) P. aeruginosa strains were found to be ESBL positive. The highest number of ESBL-producers were isolated from sputum (41.67%) followed by pus (28.36%), cerebrospinal fluid and other body fluids (21.74%), urine (20.45%) and blood (13.79%) (Table 1). Antibiotic susceptibility tests were performed for ESBL producing isolates. Imipenem and meropenem were found to be the most effective antibiotics against the ESBL-producing P. aeruginosa isolates. Zero percent resistance was found against both imipenem and meropenem. Among the third-generation cephalosporins, the highest resistance was found against ceftazidime, which was 91.49% (n = 43). The percent resistance to selected antimicrobials exclusively among the ESBL-producing isolates has been shown in Table 2.
Table 1.
ESBL-producing P. aeruginosa strains isolated from various samples.
| S. No | Specimen type | No. of P. aeruginosa strains(n = 187) | ESBL positive P. aeruginosa strains (n; %) (n = 47) |
|---|---|---|---|
| 1. | Urine | 44 | 09 (20.45%) |
| 2. | Blood | 29 | 04 (13.79%) |
| 3. | Pus | 67 | 19 (28.36%) |
| 4. | Sputum | 24 | 10 (41.67%) |
| 5. | Cerebrospinal fluid body fluid and other | 23 | 05 (21.74%) |
Table 2.
Percent resistance to the selected antimicrobial agents among the ESBL-positive P. aeruginosa isolates (n = 47).
| S. No. | Antibiotics | S | R | %R |
|---|---|---|---|---|
| 1 | Ampicillin | 00 | 47 | 100.00 |
| 2 | Aztreonam | 11 | 28 | 59.57 |
| 3 | Amikacin | 07 | 40 | 85.11 |
| 4 | Cefotaxime | 10 | 37 | 78.72 |
| 5 | Ceftazidime | 04 | 43 | 91.49 |
| 6 | Cefazolin | 13 | 34 | 72.34 |
| 7 | Cefepime | 16 | 31 | 65.96 |
| 8 | Cefoperazone | 15 | 32 | 68.09 |
| 9 | Ceftriaxone | 08 | 39 | 82.98 |
| 10 | Ciprofloxacin | 12 | 35 | 74.47 |
| 11 | Piperacillin | 18 | 29 | 61.70 |
| 12 | Tigecycline | 14 | 33 | 70.21 |
| 13 | Gentamicin | 02 | 45 | 95.74 |
| 14 | Imipenem | 47 | 00 | 00.00 |
| 15 | Meropenem | 47 | 00 | 00.00 |
S = Sensitive, R = Resistant.
4. Discussion
P. aeruginosa is a leading cause of nosocomial infections, including pneumonia, urinary tract infections, and bacteremia. The infections can be particularly severe in patients with impaired immune systems, such as neutropenic or cancer patients (Pagani et al., 2004). Infections caused by P. aeruginosa are difficult to treat as the majority of isolates show varying degrees of inherent resistance. Acquired resistance is also reported by the production of plasmid mediated AmpC beta (beta)-lactamase, ESBL and metallo beta-lactamase enzymes (Manchanda and Singh, 2008).
In the present study we observed that 25.13% (n = 47) P. aeruginosa were ESBL producers. The frequency of ESBL producing isolates was highest in sputum (41.67%) followed by pus (28.36%), cerebrospinal fluid and other body fluids (21.74%), urine (20.45%) and blood (13.79%). This is in harmony with the findings of Aggarwal et al. (2008).
The ESBL producing P. aeruginosa isolates exhibited co-resistance against most of the antibiotics tested. This is consistent with most of the recent findings (Bandekar et al., 2011; Begum et al., 2013). All ESBL producing P. aeruginosa isolates were sensitive to imipenem and meropenem. This is in harmony with the findings of Okesola and Oni (2012).
The introduction of carbapenems into clinical practice represented a great advance for the treatment of serious bacterial infections caused by beta-lactam resistant bacteria. Due to their broad spectrum of activity and stability to hydrolysis by most beta lactamases, carbapenems have been the drug of choice for treatment of infections caused by penicillin or cephalosporin-resistant Gram-negative bacilli especially, ESBL producing Gram-negative infections (Mendiratta et al., 2005).
5. Conclusion
The present study determines the prevalence of ESBL producing P. aeruginosa with limited susceptibility to antimicrobials in hospital environment. In order to combat these problems proper antibiotic policies should be formulated. Further, it was observed that all the ESBL-producing isolates were susceptible to imipenem and meropenem. This brings due relief as these are the drugs of choice in the treatment of Pseudomonas infection.
Acknowledgment
The financial support provided by the Department of Science & Technology (DST), New Delhi, India, Grant No. IF130056 to S Shaikh is deeply acknowledged.
Footnotes
Peer review under responsibility of King Saud University.
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