Prevalence and Antimicrobial Susceptibility Pattern of Extended Spectrum Beta-Lactamse Producing Escherichia coli and Klebsiella pneumoniae Isolated at a Tertiary Care Institute in North West Region of Rajasthan, India

Resistant bacteria are emerging world wide as a threat to the favourable outcome of common infections in the community and hospital settings. β-lactamases production by several gram negative and gram positive organisms is perhaps the most important single mechanism of resistance to penicillins and cephalosporins. In the past it was believed that cephalosporins were relatively immune to attack by β-lactamases, but latter on Klebsiella spp. was found to be resistant to cephalosporin. The mechanism of this resistance was production of extended spectrum β-lactamases (ESBLs) (Chaudhary et al., 2004).


Introduction
Resistant bacteria are emerging world wide as a threat to the favourable outcome of common infections in the community and hospital settings. β-lactamases production by several gram negative and gram positive organisms is perhaps the most important single mechanism of resistance to penicillins and cephalosporins. In the past it was believed that cephalosporins were relatively immune to attack by β-lactamases, but latter on Klebsiella spp. was found to be resistant to cephalosporin. The mechanism of this resistance was production of extended spectrum β-lactamases (ESBLs) (Chaudhary et al., 2004). specifically designed to be resistant to the hydrolytic action of β-lactamases. However, with each new class that has been used to treat patients, new β-lactamases emerged that caused resistance to that class of the drug. Presumably, the selective pressure of the use and overuse of new antibiotics in the treatment of patients has selected for new variants of β-lactamase. One of these new classes was the oxyimino-cephalosporins, which became widely used for the treatment of serious infections due to gram-negative bacteria in the 1980s. Not surprisingly, resistance to these expanded-spectrum βlactam antibiotics due to β-lactamases emerged quickly (Bradford, 2003).
Extended spectrum beta-lactamases (ESBLs) represent a major group of of βlactamases currently being identified world wide in large numbers, most commonly produced by Klebsiella pneumoniae and Escherichia coli but also occur in other gram negative bacteria (Agrawal et al., 2008;Kumar et al., 2006;Rodrigues et al., 2004).
ESBLs are derivative of common betalactamases that have undergone one or more amino acid substitution near the active site of enzyme, thereby increasing their affinity and hydrolytic activity against third generation cephalosporins and monobactams. However these plasmid mediated enzyme have no detectable activity against carbapenems (Kader et al., 2005;Shobha et al., 2009;Bishara et al., 2005;Briggs et al., 2005).
Being plasmid mediated they are easily transmitted among members of Enterobacteriace thus facilitating the dissemination of resistance not only to βlactam but to other commonly used antibiotics such as quinolones and aminoglycosides.
ESBLs are specifically inhibited by βlactamase inhibitors like clavulanic acid, and this property is commonly utilized for the detection and confirmation of ESBLs in the laboratory (Agrawal et al., 2008;Tsering et al., 2009). As no enough study has been undertaken on prevalence and antibiotic susceptibility pattern of ESBL producing Escherichia coli and Klebsiella pneumoniae in the state of Rajasthan, with the prevalence and antimicrobial susceptibility of ESBLproducing pathogens varying from one region to another, therefore we conducted this study to determine the prevalence and antibiotic susceptibility pattern of ESBL producers among Escherichia coli and Klebsiella pneumoniae isolates in the north west region of the state.

Materials and Methods
This study was carried out in the Department of Microbiology, Sardar Patel Medical college, Bikaner from April 2009 to May 2010 to detect Extended spectrum betalactamase producing strains of Escherichia coli and Klebsiella pneumoniae in a total of 700 clinical isolates. 520 Escherichia coli and 180 Klebsiella pneumoniae were isolated from various clinical specimens such as urine, blood, pus, wound, sputum and other respiratory tract specimen, body fluids, high vaginal swab, stool, semen, prostatic secretion and CSF etc. received from patients attending various outpatient departments, admitted in wards at P.B.M. hospital and associated group of hospitals.

Inclusion Criteria
All consecutive, non-duplicate isolates of Escherichia coli and Klebsiella pneumoniae were collected from various clinical specimens.
Medical and demographic data of the patients were collected using a questionnaire. Data recorded were: demographic characteristics (age, gender); underlying lung diseases, acute suppurative otitis media, diabetes mellitus, chronic renal failure, nephrotic syndrome, connective tissue disease, malignancy, immuno-compromisation, septicemia, burn, eclampsia, puerperal sepsis. pyometra, peritonitis, fractures etc. Presence of intravascular or urinary catheters; prolong hospitalization (> one week); history of intensive care unit (ICU) stay; nursing home residency; being on mechanical ventilation; prior antibiotic use ; and recent surgery (within one month); poor nutritional status; haemodialysis etc. were also noted.

Exclusion Criteria
Isolation of three organism types with no predominating organism and repeated isolate from same patient were excluded from this study.
The samples were processed for the identification of organisms on the basis of conventional microbiological procedures and were screened for ESBLs. All isolates were cultured on Mac Conkey Agar and Blood Agar and urinary isolates on Hichrome UTI media (obtained from Hi-Media, Mumbai, India) also and incubated at 37°C for 24 hrs. They were identified to species level by their characteristic appearances on the media, Gram's stain, Oxidase test, Motility and the pattern of the biochemical reactions. Flow chart was used for preliminary identification of organisms as shown in chart 1 Screening and confirmation of ESBL production by the phenotypic confirmatory (combination disc method) test was done as per the guidelines recommended by CLSI. Control strains, Escherichia coli ATCC 25922 (Beta-Lactamase negative) and Klebsiella pneumoniae ATCC 700603 (ESBL positive) were used for quality control.
Initial Screening Tests (Agrawal et al., 2008;Winn et al., 2006): Inoculum was prepared by suspending few colonies of test strain in 0.9 % sterile saline and turbidity was adjusted to 0.5 Mc Farland turbidity tube. A lawn culture was made from the inoculum using a sterile cotton swab on the surface of Mueller-Hinton agar medium and Ceftazidime (30µg) and Cefotaxime (30µg) discs were applied with all sterile precautions. The plates were incubated for 18-24 hours at 37°C.
According to the CLSI guidelines, isolates showing inhibition zone size of ≤ 22 mm with Ceftazidime (30 µg) and ≤ 27 mm with Cefotaxime (30 µg) were identified as potential ESBL producers and shortlisted for confirmation of ESBL production.
Phenotypic confirmatory test with combination disc: In this test, a thirdgeneration cephalosporin, Ceftazidime (30µg) alone and in combination with clavulanic acid (10 µg) was used. Both the discs were placed at least 25 mm apart, center to center, on a lawn culture of the test isolate on Mueller Hinton Agar (MHA) plate and incubated overnight at 37°C. Difference in zone diameters with and without clavulanic acid was measured.
When there was an increase of ≥ 5 mm in inhibition zone diameter around combination disk of Ceftazidime + Clavulanic acid versus the inhibition zone diameter around Ceftazidime disc alone, was considered as confirmed ESBL producer.

Results and Discussion
Out of 700 isolates of Escherichia coli and Klebsiella pneumoniae, 459 (65.57%) were found ESBLs producers. Out of 520 During past 60 years, bacteria have demonstrated a remarkable ability to resist almost every antibiotic that has been developed.(11,12,13) Extended spectrum beta-lactamases (ESBLs) represent a major group of beta-lactamases, currently being identified worldwide in large numbers, most commonly produced by Klebsiella pneumoniae and Escherichia coli.
The present study was conducted on 700 clinical isolates (520 Escherichia coli and 180 Klebsiella pneumoniae ) recovered from various clinical specimens, from all ages and both sexes attending various outpatient departments and admitted in wards at P.B.M. hospital and associated group of hospitals.
Among ESBL producer high rate of resistance to various antibiotics was seen (cefotaxime, ceftazidime, ceftriaxone, norfloxacin, cotrimoxazole). These high rates of resistance to various antibiotics may be due to various factors like-plasmid mediated transmission of ESBLs, poorly directed therapy and over-the-counter sales of antibiotics.
Foley catheter, intravenous catheter, central venous catheter, intubation, surgery and mechanical ventilation were found as the risk factors for the acquisition of E. coli and K. pneumoniae with ESBLs Ozqunes et al.,       Lautenbach (2001) et al., observed that the prior antibiotic use and longer duration of hospital stay were the risk factor for ESBLproducing E. coli or K. pneumoniae infection.
As ESBL-positive isolates occur in large number of patients and show false susceptibility to expanded-spectrum cephalosporins in standard disk diffusion test, therefore care should be taken by not giving cephalosporins and aztreonam, regardless of the routine susceptibility test results.
In conclusion, Escherichia coli (66.92%) were found to be more extended spectrum βlactamase producer than Klebsiella pneumoniae (61.67%), this may be peculiar to Indian subcontinent. The antimicrobial resistance was significantely higher in ESBL producer than non-ESBL producer.Among Extended spectrum βlactamase producing isolates imipenem and meropenem was most sensitive followed by nitrofurantoin, amikacin and gatifloxacin. Least sensitive antibiotic were cephalosporins (cefotaxime, ceftazidime, ceftriaxone). The risk factors involved in acquisition of ESBL production in the present study were:-severe illness, prior exposure to antibiotics, recent surgery, invasive medical device (urinary catheterization), poor nutritional status, ICU stay, intubation and mechanical ventilation respectively. Therefore clinician should take adequate measures in treating the patients with risk factors.