Dr. K L Shobha (Professor) and Dr Gowrish Rao (Lecturer)
are from the Department of Microbiology,
Melaka Manipal Medical College, Manipal 576104.
Dr. Sugandhi Rao (Professor) and Dr C. K. Sreeja (PG Resident) are from the Department of Microbiology, Kasturba Medical College, Manipal
Corresponding Author: Dr Shobha K.L, Department of Microbiology
Melaka Manipal Medical College(Manipal Campus), Manipal 576104
Gram negative pathogens harbouring Extended Spectrum Beta Lactamases (ESBLs) have caused numerous outbreaks of infection and are becoming an increasing therapeutic problem in many countries. The incidence of ESBL producing strains among the clinical isolates has been steadily increasing over the past years resulting in limitations of therapeutic option. 300 urine samples showing significant bacteriuria by semiquantitative culture technique using a standard calibrated loop techniques were included in the present study. These isolates were tested for antimicrobial susceptibility by Kirby-Bauer disc diffusion technique using Muller Hinton agar. Screening test for ESBL was done according to criteria recommended by CLSI. Phenotypic confirmatory test for ESBL was preformed using modified double disc method Screening test showed 32% Escherichia coli (55/171), 37% Klebsiella (44/119) and 20% Citrobacter species (2/10) to be ESBL positive. Phenotypic confirmatory test showed 35% Escherichia coli (60/171), 41% Klebsiella (49/119) and 30% Citrobacter species (3/10) were found to be ESBL producers. ESBL producers were sensitive to Imipenem followed by Amikacin. Present screening tests are not suitable to pick up all the strains of ESBL producers, clinical laboratories must adopt suitable modifications to detect ESBLs. Imipenem remains the empiric drug of choice for treatment of urinary tract infection.
Key words: Uropathogens , ESBLs
Gram negative pathogens harbouring Extended Spectrum Beta – Lactamases (ESBLs) have caused numerous outbreaks of infection and are becoming an increasing therapeutic problem in many countries. The incidence of ESBL-producing strains among clinical isolates has been steadily increasing over the past years resulting in limitations of therapeutic option1. Over the last fifteen years numerous outbreaks of infection with organisms producing extended spectrum β-Lactamases have been observed world wide2. Microorganisms responsible for urinary tract infection (UTI) such as E.coli, Kebsiella and Citrobacter species have the ability to produce ESBLs in large quantities. These enzymes are plasmid-borne and confer multiple drug resistance, making UTI difficult to treat3. There are not enough data on the prevalence of ESBL producers in UTI in south India. Hence the present study was undertaken to find out prevalence of ESBL producers in urinary isolates of E.coli, K.pneumoniae and Citrobacter species and their antibiotic susceptibility to non-beta lactam antibiotics.
|ESBL producers (60)||53 (88.5%)||18 (30%)||25 (42%)||4 (6.6%)||6 (10%)||4 (6.6%)||60 (100%)|
|Non ESBL producers (111)||104 (94%)||102 (92%)||65 (58%)||74 (67%)||67 (60%)||42 (38%)||111 (100%)|
|Klebsiella species (119)|
|ESBL producers (49)||42 (86%)||15 (31%)||14 (29%)||5 (10%)||3 (6%)||3 (6%)||49 (100%)|
|Citrobacter species (10)|
|ESBL producers (3)||2 (66.66%)||1 (33.33%)||1 (33.33%)||1 (33.33%)||2 (66.66%)||1 (33.33%)||3 (100%)|
|Non ESBL producers (7)||6 (80.57%)||4 (57.14%)||4 (57.14%)||5 (57.44%)||6 (80.57%)||4 (57.44%)||7 (100%)|
300 urine samples showing significant bacteriuria by semiquantitative culture technique using a standard calibrated loop technique4were included in the study. Urine samples were collected from patients attending the Outpatient Department and admitted in the wards at Kasturba Medical College Hospital, Manipal from March 2003 to December 2003. The studied samples included midstream urine and catherized urine. These isolates were tested for antimicrobial susceptibility by Kirby-Bauer5 disc diffusion technique using Muller Hinton agar. The antibiotic discs used were Amikacin (30 μg), Trimethoprim-Sulfamethoxazole (TMP/SMX) (1.25/23.75 μg), Gentamicin (120 μg), Ciprofloxacin (5 μg), Norfloxacin (10 μg), Nalidixic acid (30 μg), Cefotaxime (30 μg), Ceftazidime (30 μg), Imipenem (110 μg). Screening test for ESBL6 was done according to the criteria recommended by CLSI. An inhibition zone of ≤ 27 mm for cefotaxime and ≤ 22 mm for ceftazidime indicated that the strain probably produced ESBL. Phenotypic confirmatory test for ESBL was performed using modified double disc method.7 Muller Hinton agar plates were swabbed with the standard inoculum (corresponding to 0.5 McFarlands standard) to form a lawn culture. A susceptibility disc containing Amoxicillin- Clavulanic acid disc (20/10 μg) was placed in the centre of the plate and a disc of Cefotaxime (30 μg) was placed 20 mm apart, centre to centre, from Amoxicillin-Clavulanic acid disc. The Aztreonam (30 μg) disc was placed at 25 mm and the Ceftazidime (30 μg) disc was placed at 30 mm and Piperacillin (110 μg) Tazobactam (10 μg), Piperacillin/Tazobactam (110 μg) disc was placed 25 mm from Cefepime. Plates were examined for enhancement of zone inhibition of Cefotaxime and Cefepime at the side facing Augmentin and Piperacillin/Tazobactam disc respectively. Organisms that showed a clear extension of inhibition zone towards the disc Augmentin and Piperacillin/Tazobactam were considered ESBL positive.
Of the 300 isolates tested for ESBL production, 171 were Escherichia coli, 119 Klebsiella and 10 Citrobacter species. The screening test showed 55 of the 171 Escherichia coli isolates (32%), 44 of the 119 Klebsiella isolates (37%) and 2 of the 10 Citrobacter species (20%) to be ESBL positive. Phenotypic confirmatory test showed 60 of the 171 Escherichia coli species (35%), 49 of the 119 Klebsiella isolates (41%) and 3 of the 10 Citrobacter species (30%) to be ESBL producers. ESBL producers were sensitive to Imipenem followed by Amikacin. (Table 1).
ESBL are now a significant problem in hospitalized patients throughout the world. The prevalence of ESBLs among clinical isolates varies greatly worldwide and patterns are rapidly changing over time8. By screening test for ESBL, 32% of E.coli, 37% of Klebsiella, and 20% of Citrobacter species were found to be positive. Phenotypic confirmatory test showed 35% E.coli, 41% Klebsiella species and 30% Citrobacter species to be ESBL positive. Our study showed a higher percentage of ESBL producers when compared to reports of Jones et al9, but was in concordance with tSumeetha et al10. Studies conducted by Christopher et al11 and Philip et al12 showed a lower percentage, than our study, of Citrobacter species producing ESBL. As we tested only ten Citrobacter isolates, it is difficult to comment on ESBL producers among the Citrobacter species. Some of the isolates sensitive to cephalosporins, ceftazidime and cefotaxime were ESBL producers by the phenotypic confirmatory method. All the ESBL producers detected by screening test were confirmed to be ESBL producers by the confirmatory test. Antibiotic sensitivity test performed for E.coli, Klebsiella species and Citrobacter species showed lowest sensitivity to ciprofloxacin and nalidixic acid and highest sensitivity to imipenem 100%. These findings were in conformity with the studies conducted by Hansotia et al13 and Abigal et al14
Since a large percentage of urinary isolates was found to be ESBL producers, present screening tests are not suitable to pick up all the strains of ESBL producers, clinical laboratories must adopt modifications to detect ESBLs. Imipenem remains the drug of choice for empiric treatment of urinary tract infection.