Clavulanic acid

Screened-positive bacterial isolates were confirmed for ESBL production using the combined-disk method according to the CLSI guidelines [20 ]. Zones of inhibition were determined for each isolate to antibiotic disks containing 30 μg of cefotaxime, 30 μg of ceftazidime and 10 μg of cefpodozime either alone or in combination with 10μg of clavulanic acid (MAST Group Ltd.). All zones of inhibition which differed by ≥ 5 mm between at least one of the standard antibiotic disks and its corresponding clavulanic combination disks were classified as having an ESBL-producer phenotype. Escherichia coli control strain ATCC 25922 was used to monitor the performance of ESBL detection agents.

To detect ESBLs, all the isolates were tested employing the disk diffusion test (CDDT) containing Ceftazidime (CAZ) 30 µg and Cefotaxime (CTX) 30 µg with a combination of CAZ 30 µg+clavulanic acid (CA) 10 µg and CTX 30 µg+CA 10 µg per disc (Mast Group, Merseyside, UK). Zones of inhibition were compared with the CTX and CAZ discs alone and compared with the combined CAZ 30 µg+CA 10 µg and CTX 30 µg+CA 10 µg discs. An increase in zone diameter of ≥5 mm in the presence of clavulanic acid indicated the existence of ESBL in the test microorganism. Escherichia coli ATCC25922 and Klebsiella pneumonia ATCC700603 were used as negative and positive controls for ESBL production, respectively [25 ].

The Enterobacterales isolates with resistance to one or more of third-generation cephalosporins were investigated for confirmation of ESBL production by double-disc synergy test (DDST), according to CLSI procedure [16 ]. The test was conducted employing ceftazidime (30 µg) and cefotaxime (30 µg) disks separately and each of them in combination with clavulanic acid (10 µg) disk (Mast group, Merseyside, UK). Enhancement of inhibition zone size of ≥ 5 mm in the existence of clavulanic acid was a sign of ESBL positive isolates [16 ]. For the positive ESBL control, Klebsiella pneumoniae ATCC 700,603 and for negative ESBL control, Escherichia coli ATCC 25,922 were utilized. Furthermore, to test the Enterobacterales isolates for AmpC beta-lactamase production, cefoxitin disc (30 µg) was used [18 (link)]. Isolates having zone diameters of less than 18 mm were regarded potential AmpC beta-lactamase producers [18 (link)].

Antibiotic sensitivity profiles of E. coli isolates were examined against 23 antibiotics using the standard disc diffusion method [64 (link)]. The following antibiotic panel (Oxoid™) was used: amoxicillin/clavulanic acid (AMC 20/10 μg), amoxicillin (AMX 10 μg), amikacin (AMK 30 μg), ampicillin (AMP 10 μg), cefepime (FEP 30 μg), cephalothin (CEF 30 μg), cefotaxime (CTX 30 μg), cefoxitin (FOX 30 μg), cefixime (CFM 5 μg), ceftazidime (CAZ 30 μg), cephalexin (LEX 30 μg), cefuroxime (CXM 30 μg), chloramphenicol (CHL 30 μg), ciprofloxacin (CIP 5 μg), gentamicin (GEN 10 μg), imipenem (IPM 10 μg), meropenem (MEM 10 μg), nitrofurantoin (NIT 100 μg), piperacillin (PIP 100 μg), tetracycline (TET 30 μg), tigecycline (TGC 15 μg), nalidixic (NAL 30 μg) and trimethoprim/sulfamethoxazole (SXT 1.25/23.75 μg). Susceptibility profiles were determined according to the Clinical Laboratory Standard Institution (CLSI) guidelines [65 ], and E. coli ATCC 25922 was used for quality control. The double-disc synergy test [66 (link)] using 30 µg cefotaxime in the presence and absence of 10 µg clavulanic acid (MAST Group Ltd., Bootle, UK) was undertaken to identify ESBL-producing isolates. E. coli isolates displaying resistance to one or more antibiotics from three different antibiotic classes were assigned as multidrug-resistant (MDR) isolates.

All ESC-R isolates that generated characteristic ESBL-like colonies on the Brilliance ESBL Agar (n = 104) were subcultured onto SBA and further tested for demonstration of phenotypic ESBL-production with the combination disk test (CDT), including cefpodoxime, ceftazidime, and cefotaxime alone and in combination with clavulanic-acid (MAST Group, United Kingdom). In addition, all Enterobacterales selected on the Brilliance ESBL Agar were subject to further antimicrobial susceptibility testing by disk diffusion on Mueller-Hinton agar (MHA), according to the Clinical and Laboratory Standards Institute methodology [57 ]. The antimicrobial panel included: amoxicillin/clavulanic acid (30 μg), ampicillin (10 μg), aztreonam (30 μg), imipenem (10 μg), trimethoprim/sulfamethoxazole (25 µg), enrofloxacin (5 μg), tetracycline (30 μg), chloramphenicol (30 μg), and gentamicin (10 μg) (all disks and media were from Oxoid, Basingstoke, UK). E. coli ATCC 25,922 was used as the control for the disk diffusion susceptibility testing. Interpretation of the antimicrobial susceptibility results was done according to the CLSI [57 ]. An isolate was considered non-susceptible if it had intermediate or resistant results against the tested antimicrobial agent.