Identifying ESBL Enterobacter cloacae

This cross sectional observational study was conducted at the Microbiology Department of The Children’s Hospital and Institute of Child Health Lahore, Pakistan, from April 2011 to March 2012. A total number of 20,257 pathological samples of blood, cerebro-spinal fluid, urine, sputum, peritoneal dialysis catheter, tracheal secretions and pus collected from various wards were analysed. The samples were cultured on solid media as Blood, Chocolate and MacConkey agar. Cystine Lysine Electrolyte Deficient Medium (CLED) was used only for urine culture samples. Enterobacter cloacae were identified by colonial morphology, Gram’s stain, catalase test, oxidase test and API 20E system (bioMerieux). A seven digit number generated on the basis of various biochemical reactions of API 20E system was checked by API 20E software to confirm Enterobacter cloacae.¹⁰ A bacterial suspension of Enterobacter cloacae was made according to the 0.5 McFarland turbidity standard and an even lawn of bacteria was made on the Mueller Hinton agar petri plate (90mm). The screening for ESBL E. cloacae was performed using ceftazidime (30 μg) disk and ceftazidime resistant strains were considered as screen positives. DDST was performed by using disks containing amoxicillin/ clavulanate on Mueller-Hinton agar plate at a 20 mm distance from the indicator drugs; ceftazidime (30 μg) and cefotaxime (30 μg). ESBL production was seen by the clavulanate mediated enhancement of the activity of the indicator drug as a keyhole effect.¹¹ The CLSI confirmatory tests were performed using disks of ceftazidime (30 μg) and cefotaxime (30 μg) alone and in combination with ceftazidime-clavulanate (30/10 μg) and cefotaxime-clavulanate (30/10 μg). The CLSI confirmatory test was considered positive when the inhibition zone produced by the disks in combination clavulanate increased ≥5 mm than the disks without the clavulanate. The results of double disk diffusion test and CLSI test were compared.