Clavulanic Acid

The study was conducted at the Microbiology Department of The Children’s Hospital and Institute of Child Health Lahore, Pakistan, from May 2010 to February 2012. Eleven pathological samples including blood, cerebro-spinal fluid (CSF), urine, sputum, peritoneal dialysis catheters, tracheal secretions and pus were collected from paediatric patients were analysed during the study period. The patient’s history regarding the use of various interventions like intravenous line, urinary catheters, endotracheal tube, lumber puncture, peritoneal dialysis catheters, exchange transfusion, nasogastric tube, surgery, central venous pressure line and tracheostomy was also noted. The Brain Heart Infusion Broth (BHI) was used to inoculate the blood samples and after a period of incubation they were sub-cultured on solid media. All of the clinical samples were cultured on different solid media of Blood, Chocolate and MacConkey agar plates (90mm) while the urine samples were cultured only on Cystine Lysine Electrolyte Deficient Medium (CLED). An overnight incubation of these culture plates was done at 37±1ºC in an incubator. The bacterial strains were identified using API 20E system (bioMerieux). Only the K. pneumoniae strains were included and processed further for ESBL detection. The culture media and antibiotic discs were purchased from Oxoid.
The K. pneumoniae strains were screened as ESBL screening-positive on the basis of resistance to ceftazidime. The screen positive K. pneumoniae strains were further processed for double disc synergy test (DDST) and Clinical Laboratory Standard Institute (CLSI) confirmatory test. In DDST, a disc of amoxicillin-clavulanic acid (AMC) was placed in the center of Mueller-Hinton agar plate (90mm) at 20mm distance to ceftazidime (CAZ 30μg) and cefotaxime (CTX 30μg). ESBL production was detected by the appearance of key hole effect due to the enhanced activity of ceftazidime and cefotaxime with clavulanic acid.
Both DDST positive and negative K. pneumoniae strains were analysed by CLSI confirmatory test. The CLSI confirmatory test was performed with of ceftazidime (CAZ 30μg) and cefotaxime (CTX 30μg) alone and using a combined disc of ceftazidime-clavulanic acid and cefotaxime-clavulanic acid (CAZ/CLA and CTX/CLA 30/10 μg). The CLSI confirmatory test was considered positive when the inhibition zone produced by the combined effect of ceftazidime or cefotaxime and clavulanic acid increased ≥5 mm than ceftazidime or cefotaxime without the clavulanic acid.¹⁰

ESBL-producing isolates were selected from an existing collection of isolates obtained from wastewater and surface water sampled between 2010 and 20125 (link)13 (link). Surface water samples (n = 20) were taken at ten different sites situated in four different regions in the Netherlands (including rivers, canals, lakes, North Sea), wastewater samples (n = 20) included influents and effluents from wastewater treatment plants (WWTP), an international airport WWTP, and from wastewater of health care institutions. Isolates were obtained by filtration of multiple volumes of water samples through 0.45 μm filters, followed by incubation of these filters on selective culture media for the isolation of ESB-producing E. coli: Tryptone Bile X-glucuronide medium supplemented with 1 μg/ml cefotaxime (TBX/CTX) or on ChromID^TM ESBL agar (Biomerieux). Incubation conditions were 18–24 hours at 36 ± 2 °C or 4 to 5 hours at 36 ± 2 °C followed by 18 to 19 hours at 44 ± 0.5 °C. Isolation procedures were based on standard isolation procedures for the selective isolation of E. coli from water and food using chromogenic media (NEN-EN-ISO 9308-1 and ISO 16649-2), adapted to enable selective growth of ESBL-producing variants. Variations in isolation procedures was related to isolates being obtained as part of different projects. Suspected ESBL-E. coli isolates (i.e. the ß-glucuronidase-positive colonies on TBX-CTX as well as on ChromID^TM ESBL agar) were further confirmed as E. coli by testing for indole-production using BBL Dry Slide^TM (BD), and subsequently tested for ESBL-production by disk diffusion following CLSI guidelines14 , using Sensi-Discs^TM (BD, Breda, the Netherlands). Zone diameters were determined for cefotaxime (30 μg) ± clavulanic acid (10 μg), ceftazidime (30 μg) ± clavulanic acid (10 μg). ESBL-producing isolates were defined as strains resistant to cefotaxime (zone diameter ≤22 mm) and/or ceftazidime (zone diameter ≤17 mm), and an increase in zone diameter of ≥5 mm with the disks containing clavulanic acid14 .
From this pre-existing collection of confirmed ESBL-producing E. coli isolates, 93 wastewater and 93 surface water isolates, from 20 samples each, were selected more or less randomly, but taking into account established ABR profiles (when available), to minimize the chance of including duplicate isolates from the same sample. After characterization of phylogenetic groups, ESBL-genes, virulence genes and antibiotic resistance profiles was completed, some isolates were retrospectively identified as duplicates and omitted, leaving 88 wastewater (63 from WWTP, 15 from the international airport and 10 from health care institutions) and 82 surface water isolates for analyses.

Species identification and antimicrobial susceptibility testing were performed using NMIC/ID4 card with BD Phoenix^TM100 Automated Microbiology System (Becton, Dickinson and Company, Sparks, Maryland, USA). The following antimicrobial agents were tested: amikacin (8 ~ 32 μg/mL), amoxicillin/clavulanate (4/2 ~ 16/8 μg/mL), ampicillin (4 ~ 16 μg/mL), ampicillin/sulbactam (4/2 ~ 16/8 μg/mL), aztreonam (2 ~ 16 μg/mL), cefepime (2 ~ 16 μg/mL), cefotaxime (1 ~ 32 μg/mL), ceftazidime (1 ~ 16 μg/mL), ciprofloxacin (0.5 ~ 2 μg/mL), gentamicin (2 ~ 8 μg/mL), imipenem (1 ~ 8 μg/mL), levofloxacin (1 ~ 8 μg/mL), meropenem (1 ~ 8 μg/mL), piperacillin/tazobactam (4/4 ~ 64/4 μg/mL), tetracycline (2 ~ 8 μg/mL), trimethoprim/sulfamethoxazole (0.5/9.5 ~ 2/38 μg/mL), cefotaxime/clavulanate (for ESBL, < 9 μg/mL), ceftazidime/ clavulanate (for ESBL, < 9 μg/mL), cefpodoxime-proxetil (for ESBL, < 9 μg/mL), ceftazidime (for ESBL, < 9 μg/mL) and ceftriaxone/clavulanate (for ESBL, < 9 μg/mL) [15 (link)]. If the MIC of ciprofloxacin was less than or equal to 0.5 μg/mL, or the MIC of levofloxacin was less than or equal to 1 μg/mL, sensitivity or intermediation was confirmed by disk diffusion test (ciprofloxacin (5 μg) and levofloxacin (5 μg), respectively). Phenotypic ESBL confirmation was performed with a double-disk synergy test (cefotaxime (30 μg), cefotaxime/clavulanic acid (30 μg/10 μg), ceftazidime (30 μg) and ceftazidime/clavulanic acid (30 μg/10 μg) disk) following clinical and laboratory standards institute (CLSI) criteria [16 ]. The recently revised CLSI species-specific clinical breakpoints (CBPs) were used to interpret the MIC results. The quality control strains were Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853 and Klebsiella pneumoniae ATCC700603.