52 Chapter 4 infection control than pure clonal transfer. Consequently, pAmpC production in E. coli requires active detection and contact precautions for colonized or infected patients, as recommended by different guidelines (Australian Guidelines on Prevention and Control of Infection in Healthcare 2010; RCPI 2012); however, this is often ignored due to the more cumbersome identification in the microbiological laboratory. Current commercial phenotypic AmpC confirmation tests fail to reliably discriminate between pAmpC and constitutive hyperproduction of the chromosomalmediated AmpC (cAmpC) (Ingram et al. 2011). In E. coli, an approach solely based on phenotypic testing has a high sensitivity to detect pAmpC production, but lacks specificity as it detects a high number of isolates that overproduce cAmpC, resulting in unnecessary patient isolation precautions with increased unnecessary healthcare costs. PCR is capable of detecting various pampC genes (Javier Pérez-Pérez and Hanson 2002). The recommended method for detection of pAmpC production in Enterobacterales according to the EUCAST guidelines is to screen isolates for cefoxitin MICs >8 mg/L combined with phenotypic resistance to cefotaxime and/or ceftazidime (Martinez and Simonsen 2017). Confirmation is advised in a two-step algorithm using cloxacillin synergy detection and PCR to discriminate pampC from hyperexpressed campC in E. coli. Several studies suggest the screening of isolates in a similar fashion (Polsfuss et al. 2011; Edquist et al. 2013). However, molecular tests are not always available in laboratories and are relatively expensive and often time-consuming. The aim of this present study was to evaluate various diagnostic approaches through determining the MICs of specific cephalosporins, two commercial AmpC disc-diffusion confirmation tests and WGS to develop an algorithm to detect pAmpC production in ESBL-negative and cefoxitin-resistant E. coli. Materials and methods Overall study design Three datasets consisting of E. coli cefoxitin-resistant and ESBL-negative strains were identified. Most strains were suspected of having either a pAmpC or a cAmpC resistance mechanism. All strains were subjected to WGS to obtain the genotypes [pampC, campC, promoter mutations (hyperproducer) and absence of both (negative)] and subjected to Etests and two AmpC disc-diffusion confirmation tests. The training set contained a wide variety of phenotypes and was used as input for constructing an algorithm to classify the three genotypes (pampC, hyperproducer and negative). The most accurate
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