36 Chapter 3 VC) and incubated for 18–24 hours (35–37°C). In 2013, broths were subcultured on a MacConkey agar containing cefotaxime 1 mg/L (Mediaproducts, Groningen, The Netherlands). In 2014, a switch to a more selective double MacConkey agar plate (containing on one side cefotaxime 1 mg/L, cefoxitin 8 mg/L and on the other side ceftazidime 1 mg/L, cefoxitin 8 mg/L, Mediaproducts, Groningen, the Netherlands) was made to improve sensitivity and specificity of the screening. Broths were simultaneously subcultured on both sides of an EbSA agar plate (AlphaOmega, ‘s-Gravenhage, Netherlands). The Extended Beta-Lactamase Screening Agar (EbSA) plate consists of a split MacConkey agar plate containing ceftazidime (1.0 mg/L) on one side and cefotaxime (1.0 mg/L) on the other side. Both sides contain cloxacillin (400 mg/L) and vancomycin (64 mg/L) for inhibition of AmpC beta-lactamase-producing bacteria and Gram-positive bacteria, respectively. Subsequently, the plates were incubated for 18–24 hours (35–37°C). AmpC producing isolates found in 2013, were retrospectively cultured on the new selective AmpC agar to confirm if they would have been detected using the new agar plate. For all oxidase-negative isolates that grew on either side of the selective agar plates, species identification was performed by MALDI-TOF (bioMérieux, Marcy l’Etoile, France). The presence of AmpC in all E. coli and Klebsiella spp. isolates was phenotypically confirmed using the D68C AmpC & ESBL Detection Set (Mastdiscs, Mastgroup Ltd, Bootle, United Kingdom) and interpreted according to manufacturer’s instructions (Ingram et al. 2011; Nourrisson et al. 2015). The presence of ESBL in isolates with a MIC of > 1 mg/L for ceftazidime and/or cefotaxime was phenotypically confirmed with the combination disk diffusion method for cefotaxime, ceftazidime, and cefepime with and without clavulanic acid (Rosco, Taastrup, Denmark)) and interpreted according to manufacturer’s instructions. Minimal inhibitory concentration (MIC) values for cefotaxime (CTX), ceftazidime (CAZ) and cefoxitin (FOX) were measured using the gradient on a strip method (E-test, bioMérieux, Marcy l’Etoile, France). Genetic confirmation of phenotypically confirmed isolates All phenotypically confirmed E. coli and Klebsiella spp. isolates were screened for the presence of pampC genes using the microarray check MDR CT103 according to the manufacturer’s instructions (Check-Points, Wageningen, the Netherlands) (Cuzon et al. 2012). In addition, all phenotypically confirmed E. coli isolates were subjected to Sanger sequencing of the promoter/attenuator region of the cAmpC gene using M-13 tailed primers as described by Corvec et al. (Stephane Corvec et al. 2002). The obtained sequences of each isolate were assembled and aligned against the promoter/attenuator
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