Use of boronic acid disk methods to detect the combined expression of plasmid-mediated AmpC β-lactamases and extended-spectrum β-lactamases in clinical isolates of Klebsiella spp., Salmonella spp., and Proteus mirabilis

A study using boronic acid (BA), an AmpC enzyme inhibitor, was designed to detect the combined expression of plasmid-mediated AmpC β-lactamases (pAmpCs) and extended-spectrum β-lactamases (ESBLs) in bacterial isolates naturally lacking chromosomal ampC genes. A total of 122 Klebsiella spp., Salmonella spp., and Proteus mirabilis isolates producing or nonproducing pAmpCs and/or ESBLs were analyzed. Detection of genes encoding ESBLs and AmpCs was confirmed by polymerase chain reaction (PCR) followed by sequencing of PCR products. A ≥5-mm increase in zone diameter for i) cefoxitin (FOX) and/or cefotetan (CTT) containing BA versus FOX and/or CTT alone was considered positive for AmpC; ii) ceftazidime (CAZ)-clavulanate (CA) and/or cefotaxime (CTX)–CA tested in combination with BA versus CAZ and/or CTX containing BA was considered positive for ESBL. The disk tests of FOX and/or CTT alone and with BA detected 98.4% of organisms producing pAmpCs. All of the 21 pAmpC and ESBL coproducers were accurately detected ESBL by the disk tests of CTX–CA and/or CAZ–CA containing BA and CTX and/or CAZ containing BA. In conclusion, The BA disk test using Clinical and Laboratory Standards Institute methodology is simple and very efficient method to detect pAmpC and ESBL in organisms naturally lacking chromosomal AmpC enzymes. In particular, the method accurately detects the isolates that harbor both AmpCs and ESBLs.