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Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations
Published on: July 24, 2021
Systematic research to overcome newly emerged multidrug-resistant bacteria
1Department of Bacteriology, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Abstract:
In the 1980s, I found that the chromosomal β-lactamase of Klebsiella pneumoniae LEN-1 showed a very high similarity to the R-plasmid-mediated penicillinase TEM-1 on the amino acid sequence level, and this strongly suggested the origination of TEM-1 from the chromosomal penicillinases of K. pneumoniae or related bacteria. Moreover, the chromosomal K1 β-lactamase (KOXY) of Klebsiella oxytoca was found to belong to the class A β-lactamases that include LEN-1 and TEM-1, although KOXY can hydrolyze cefoperazone (CPZ) like the chromosomal AmpC-type cephalosporinases of various Enterobacteriaceae that can hydrolyze several cephalosporins including CPZ. Furthermore, my collaborators and I found plural novel serine-type β-lactamases, such as MOX-1, SHV-24, TEM-91, CTX-M-64, CMY-9, CMY-19, GES-3, GES-4, and TLA-3, mediated by plasmids. Besides these serine-type β-lactamases, we also first identified exogenously acquired metallo-β-lactamases (MBLs), IMP-1 and SMB-1, in imipenem-resistant Serratia marcescens, and the IMP-1-producing S. marcescens TN9106 became the index case for carbapenemase-producing Enterobacteriaceae. I developed the sodium mercaptoacetic acid (SMA)-disk test for the simple identification of MBL-producing bacteria. We were also the first to identify a variety of plasmid-mediated 16S ribosomal RNA methyltransferases, RmtA, RmtB, RmtC, and NpmA, from various Gram-negative bacteria that showed very high levels of resistance to a wide range of aminoglycosides. Furthermore, we first found plasmid-mediated quinolone efflux pump (QepA) and fosfomycin-inactivating enzymes (FosA3 and FosK). We also first characterized penicillin reduced susceptible Streptococcus agalactiae, macrolide-resistant Mycoplasma pneumoniae, as well as Campylobacter jejuni, and Helicobacter pylori, together with carbapenem-resistant Haemophilus influenzae. We constructed a PCR-based open reading frame typing method for rapid identification of Acinetobacter baumannii international clones.
Insights
This research details the discovery of novel antibiotic resistance genes, including beta-lactamases and 16S ribosomal RNA methyltransferases, in various bacteria. It also introduces a simple disk test for identifying metallo-beta-lactamase producing bacteria.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The emergence of antibiotic resistance poses a significant threat to public health.
- Understanding the genetic basis and origins of resistance mechanisms is crucial for developing effective countermeasures.
- Klebsiella pneumoniae and Klebsiella oxytoca are key Gram-negative bacteria often implicated in the spread of resistance.
Purpose of the Study:
- To identify and characterize novel antibiotic resistance genes and enzymes in various bacterial species.
- To investigate the evolutionary origins of antibiotic resistance genes, such as TEM-1 beta-lactamase.
- To develop rapid diagnostic methods for detecting specific resistance mechanisms, like metallo-beta-lactamases.
Main Methods:
- Amino acid sequence analysis to compare beta-lactamases.
- Identification and characterization of plasmid-mediated resistance genes.
- Development of the sodium mercaptoacetic acid (SMA)-disk test for metallo-beta-lactamase detection.
- PCR-based typing for bacterial strain identification.
Main Results:
- Discovery of chromosomal beta-lactamases (LEN-1, KOXY) related to plasmid-mediated TEM-1.
- Identification of numerous novel plasmid-mediated serine-type beta-lactamases (e.g., MOX-1, SHV-24, CTX-M-64).
- First identification of exogenously acquired metallo-beta-lactamases (IMP-1, SMB-1) in Serratia marcescens.
- First identification of plasmid-mediated 16S ribosomal RNA methyltransferases (RmtA-C, NpmA) conferring high-level aminoglycoside resistance.
- Discovery of plasmid-mediated quinolone efflux pump (QepA) and fosfomycin-inactivating enzymes (FosA3, FosK).
- Characterization of resistance in Streptococcus agalactiae, Mycoplasma pneumoniae, Campylobacter jejuni, Helicobacter pylori, and Acinetobacter baumannii.
- Development of the SMA-disk test for simple identification of MBL-producing bacteria.
Conclusions:
- The study highlights the diverse and evolving nature of antibiotic resistance mechanisms in bacteria.
- Plasmid-mediated genes play a significant role in the dissemination of resistance.
- Novel diagnostic tools are essential for timely detection and control of resistant pathogens.
- Further research is needed to understand the clinical and epidemiological impact of these newly identified resistance determinants.
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