A Novel Single-Tube Eicosaplex/Octaplex PCR System for the Detection of Extended-Spectrum -Lactamases, Plasmid-Mediated AmpC -Lactamases, and Integrons in Gram-Negative Bacteria

We developed two multiplex polymerase chain reactions (PCRs) for the detection of extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases, aac(6 & PRIME;)-Ib gene, and integrase genes (intI1, intI2, and intI3) in class 1, 2, and 3 integrons in Gram-negative bacteria. We evaluated the PCRs using 109 Gram-negative isolates from non-organic (ANO) and organic (AO) vegetables and fruits. Screening of ANO substances identified five SHV, one TEM-1, one CTX-M, 20 AmpC-CS, and two intI1 positives. DNA sequencing revealed CTX-M in Pantoea spp. was bla(RANH-2), a plasmid-mediated CTX-M related ESBL gene only found in Rahnella spp. Of the 20 AmpC-CS positives, 10 were CMY/MIR/ACT/EC (3 new variants), eight were ACT, one was AZECL, and one was new Pseudomonas-related AmpC family. Screening of AO substances identified 11 SHV, two TEM-1, three CTX-M (one OXY-2, two CTX-M-14/-15), two OXA-9, 13 AmpC-CS and one intI1 positives. The 13 AmpC-CS positives were five CMY/MIR/ACT/EC, three ACT, one MOX-12 variant, and four ADC (one ADC-25 and three new variants). We developed a rapid, easy-to-perform, low-cost, and reliable multiplex PCR system for screening clinically relevant beta-lactamases and integrons in Gram-negative bacteria. We showed the prevalence of ESBLs and AmpC beta-lactamases among our panel of ampicillin-resistant Gram-negative strains and detection of NDM and OXA carbapenemases.