Novel multiplex PCRs for detection of the most prevalent carbapenemase genes in Gram-negative bacteria within Germany

Abstract

Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants. Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available. Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany. Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM, bla OXA-40, bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51, bla IMI, bla FIM and bla DIM. We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM and bla OXA-40, while multiplex-2 included bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51 and bla IMI. Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected. Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.

Plasmid content of carbapenem resistant Acinetobacter baumannii isolates belonging to five International Clones collected from hospitals of Alexandria, Egypt

“…Transconjugants were selected onto MH agar plates supplemented with corresponding antibiotics (ceftazidime-8 µg/mL, meropenem-4 µg/mL) and sodium azide (200 µg/mL). Transconjugants were confirmed by carbapenemase multiplex PCR ( Cerezales et al., 2021 ).…”

Extensive Drug Resistance of Strong Biofilm-Producing Acinetobacter baumannii Strains Isolated from Infections and Colonization Hospitalized Patients in Southern Poland

“…The most common carbapenemases genes in AB in Poland were detected: bla OXA-23, bla OXA-40 , bla OXA-58 , and bla NDM [ 7 ]. Detection of carbapenemasse genes was carried out according out following the protocol described by Cerezales et al [ 29 ]. In the multiplex polymerase chain reaction (PCR), bla OXA-23 (718 bp), bla OXA-40 (413 bp), bla NDM (517 bp), and bla OXA-58 (303 bp) genes were identified ( Table 1 ).…”

Prevalence and characterization of ESBL-producing Escherichia coli in healthy pregnant women and hospital environments in Benin: an approach based on Tricycle

“…The DNA of isolated strains showing phenotypic resistance to beta-lactams and carbapenems was extracted using “Zymo DNA Mini Kit” extraction kits according to the manufacturer’s instructions. PCR was used to detect beta-lactam resistance genes ( bla TEM , bla SHV , bla OXA-1, bla CTXM-1, bla CTXM-9, and bla CTXM-15 ) and carbapenem resistance genes ( bla KPC , bla NDM , bla IMP , bla VIM , and bla OXA-48 ) following the PCR conditions described by Dallenne et al ( 33 ), Memariani et al ( 34 ), and Cerezales et al ( 35 ). All primer sequences are listed in Table 1 .…”