Clavulanate

To provide continuity of monitoring data and allow epidemiological tracing of isolates with particular patterns of resistance (particularly in relation to certain Salmonella serovars), it is recommended that those antimicrobials listed in previous recommendations should remain in future testing requirements. The rationale for inclusion of the antimicrobials recommended for use in current monitoring programmes has been previously described elsewhere (EFSA, 2007, 2008, 2012a), in particular regarding the phenotypic monitoring of the presumptive ESBL‐producing and AmpC β‐lactamase‐producing bacteria in animals and food, and the inclusion of last‐resort antimicrobials in the treatment of certain infections with highly resistant Gram‐negative bacteria in humans, such as the carbapenems and colistin. It is reinforced that isolates are tested for susceptibility and MICs interpreted using the epidemiological cut‐off values and concentration ranges shown in Table 9 to determine the susceptibility of Salmonella spp., and indicator commensal E. coli.
All E. coli isolates deriving from the specific monitoring of ESBL‐/AmpC‐/carbapenemase producing E. coli, as well as those randomly selected isolates of Salmonella spp. and E. coli deriving from the routine monitoring that, after testing with the first panel of antimicrobials are found to be resistant to cefotaxime, ceftazidime or meropenem, should be further tested with a second panel of antimicrobial substances as shown in Table 10. This panel notably includes cefoxitin, cefepime and clavulanate in combination with cefotaxime and ceftazidime for the detection of presumptive ESBL and AmpC producers, as well as imipenem, meropenem and ertapenem to phenotypically identify presumptive carbapenemase producers.

The minimum inhibitory concentration (MIC) as determined by the MHB microdilution method was used to evaluate the antimicrobial susceptibility of 500 UPEC clinical strains according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2016 ). MDR strains were defined as having acquired no susceptibility to at least one antibiotic in three or more classes. XDR strains were defined as having non-susceptibility to at least one agent in all but two or fewer antibiotic classes (Magiorakos et al., 2012 (link)). The MIC for each antibiotic was compared to the standard values of the CLSI. The antibiotic panel that was used included ampicillin (AM; Sigma-Aldrich, St. Louis, MO, USA), amoxicillin-clavulanate (AMC; Great West Road, Brentford Middlesex, UK), ticarcillin-clavulanate (TIM; Gold Biotechnology, Inc., Ashby Road, St. Louis, MO), piperacillin-tazobactam (TZP; Siemens Medical Solutions USA, Inc., Valley Stream Parkway, Malvern, PA, USA), cephalothin (CF; Eli Lilly and Company, S Harding St, Indianapolis, IN, USA), cefaclor (CEC; Phadia Laboratory Systems, Thermo Scientific, Wyman Street, Waltham, MA, USA), ceftazidime (CAZ; Roselle Rd, Schaumburg, IL, USA), aztreonam (ATM; Bristol-Myers Squibb Corporate, Park Avenue, NY, USA), norfloxacin (NOR), ofloxacin (OFX; MP Biomedicals, Solon, OH, USA), meropenem (MEM), imipenem (IPM; AstraZeneca Pharmaceuticals LP, Wilmington, DE, USA), gentamycin (GM; Schering-Plough Pharmaceuticals, Kenilworth, NJ, USA), ceftriaxone (CRO), trimethoprim-sulfamethoxazole (SXT; Roche, Basel, Switzerland), tetracycline (TE; Heritage Pharmaceuticals Inc., Fieldcrest Avenue, Edison, NJ, USA), and nitrofurantoin (F/M; McKesson Pharmaceutical, One Post Street, San Francisco, CA, USA). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as controls.
The extended-spectrum beta-lactamases (ESBLs) were phenotypically detected as previously recommended by CLSI using the double-disc synergy test based on the synergistic effect between clavulanic acid (inhibitor of ESBLs) and β-lactam antibiotics (cefotaxime, CRO, CAZ, cefepime, cefpirome, and ATM). Additionally, ESBLs were detected using an individual disk that was tested with/without clavulanic acid (10 μg/mL) and by the Hodge test using Klebsiella pneumoniae ATCC 700603 (ESBL+) and E. coli ATCC 25922 (ESBL-) as control strains (CLSI, 2016 ).

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.

ESBL confirmatory test involved testing cefotaxime (30 μg) and ceftazidime (30 μg) alone and in combination with clavulanate (10 μg) on Mueller–Hinton agar. If the zone diameter increased ≥ 5 mm in the presence of clavulanate, the isolate was considered ESBL-producing. Escherichia coli ATCC 25,922 and Klebsiella pneumonia ATCC700603 (700,603.18) were used as quality controls.