2.2.1. Characterisation of ESBL Harbouring Gram Negative Bacilli

Identified Enterobacteriaceae were characterized for their resistance pattern by susceptibility testing according to EUCAST (EUCAST V2.0, 2012) [22], with ampicillin (AM), amoxicillin/clavulanic acid (AMC), piperacillin/tazobactam (TZP), cefalexin (CN), cefuroxime (CXM), cefoxitin (FOX), cefotaxime (CTX), ceftazidime (CAZ), cefepime (FEP), imipenem (IPM), meropenem (MEM), gentamicin (GM), trimethoprim/sulfamethoxazole (SXT), nalidixic acid (NA), ciprofloxacin (CIP), moxifloxacin (MOX), tetracycline (TE) and chloramphenicol (C) BD BBLTM Sensi-DiscTM paper discs (BD, Sparks, MD, USA). The inhibition zone diameters were interpreted according to EUCAST guidelines, except Enterobacteriaceae tested for tetracycline, chloramphenicol and nalidixic acid, which were evaluated by the Clinical Laboratory Standards Institute (CLSI, 2011) guidelines [23]. There are no interpretation guidelines for zone diameters of these three antibiotics according to EUCAST.

*E. coli* 25299 was used as reference. The inhibition zone diameters were interpreted according to EUCAST guidelines. The antimicrobials tested and resistance breakpoints applied can be found in the Supplementary Materials (Table S1).

All isolates were screened for ESBL gene families, *bla*CTX-M-1group, *bla*CTX-M-2group, *bla*CTX-M-9group*, bla*GES*, bla*SHV, *bla*TEM, and *bla*VEB by PCR and sequencing as described previously [24,25]. False-positive (not estimated Enterobacteriaceae) strains growing on ESBL-media with a green or brownish colour were identified as Pseudomonadales and Aeromonadales; we decided to include them in the study and therefore these strains were also screened for ESBL genes. Identified Pseudomonadales were characterized for their resistance pattern by susceptibility testing according to EUCAST (EUCAST V2.0, 2012); piperacilin/tazobactam (TZP), ceftazidime (CAZ), cefepime (FEP), meropenem (MEM), imipenem (IPM), amikacin (AN), gentamicin (GM), tobramycin (NN), ciprofloxacin (CIP) and levofloxacin (LEV).
