55 Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC Creating an algorithm based on the training set For the decision tree model, Recursive Partitioning And Regression Trees (RPART), an R package (version 4.1-13), was used; this is an implementation of Classification and Regression Tree (CART), a statistical technique to solve classification problems, developed by Breiman et al (Breiman et al. 1984). RPART was used to create a decision tree model to classify strains based on Etest MICs, AmpC Confirm Kit or D68C test results into a pampC, hyperproducer or negative class. Model optimization and cross-validation were performed within the caret R package (version 6.0-80) in R (version 3.5.1)(R Core Team 2018). The RPART model was trained to optimize for accuracy and by using seed 825 to be able to reproduce model creation. The crossvalidation was performed using a 10-fold three-times-repeated cross-validation using the repeatedcv parameter. Student’s t-test was used to compare model performances (P=0.05). A two-class model was derived from the three-class model by combining the negatives with the hyperproducer class and recalculating the statistics. Results Training set Between January 2014 and March 2018, 267 E. coli strains that had cefoxitin MICs >8 mg/L and were ESBL negative were found in the laboratory information management system at Radboudumc. Out of these strains, 98 were selected for further testing. Eleven of these strains could not be retrieved from the freezer and three strains were identified as not being E. coli by MALDI-TOF MS. This resulted in a training set of 84 E. coli strains. MICs determined using the BD Phoenix System indicated that the training set likely consisted of a wide variety of different resistance phenotypes. A substantial proportion of strains were resistant to both ceftazidime and ceftriaxone (42.9%, n=36) (Table 1), 20 strains (23.8%) were susceptible to 3GCs and 28 strains (33.3%) were intermediate or resistant to at least one of the 3GCs. WGS results revealed that 32 of 84 E. coli strains (38.1%) contained blaCMY-2 and 29.8% (n=25) showed known mutations in the ampC promoter region and were therefore labelled as hyperproducers, 20.2% (n=17) were negative for both pampC genes and mutations in the promoter region of campC and were classified as negative (Figure S1A, available as Supplementary data at JAC Online). 4
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