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Detection of AmpC β-lactamases in gram-negative bacteria
Qian Zhou1, Mengjun Tang1, Xiaoyan Zhang1
1Jiangsu Institute of Poultry Science, Yangzhou, China.
Abstract:
AmpC β-lactamase genes are clinically important because they often confer resistance to most β-lactams other than 4th-generation cephalosporins and carbapenems. However, traditional and existing detection methods are expensive, labor-intensive and range-limited. We established an efficient multiplex PCR method to simultaneously identify six families of ampC β-lactamase genes, ACC, EBC, CIT, DHA, MOX and FOX, and evaluated the sensitivity and specificity of this assay. The multiplex method could accurately identify ACC, EBC, CIT, DHA, MOX and FOX variants among a total of 175 ampC β-lactamase genes. The minimum concentration of genomic DNA that could be detected was 1.0×103 copies/μL. We subsequently used this method to analyze 2 Salmonella spp. with carrying CMY-2 and DHA-1, and 167 Enterobacteriaceae isolates in blinded PCR testing. Positive isolates produced bright bands that corresponded with their genotype. Results were in concordance with those of the traditional method but showed increased sensitivity and accuracy. This indicates that the newly developed multiplex PCR system could be used as a diagnostic tool to accurately distinguish the six families of ampC β-lactamase genes with high efficiency, wide range, easy operation and good discrimination.
Insights
A new multiplex PCR method efficiently detects six families of AmpC beta-lactamase genes, offering a sensitive and accurate alternative to traditional methods for identifying antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- AmpC beta-lactamase genes are critical in conferring resistance to numerous beta-lactam antibiotics, complicating treatment options.
- Existing methods for detecting AmpC beta-lactamase genes are often costly, time-consuming, and lack comprehensive detection capabilities.
Purpose of the Study:
- To develop and validate an efficient multiplex PCR assay for simultaneous identification of six major AmpC beta-lactamase gene families.
- To evaluate the sensitivity, specificity, and diagnostic utility of the novel multiplex PCR method.
Main Methods:
- Development of a multiplex PCR assay targeting six AmpC beta-lactamase gene families: ACC, EBC, CIT, DHA, MOX, and FOX.
- Validation using 175 characterized AmpC beta-lactamase genes and subsequent testing on clinical isolates, including Salmonella spp. and Enterobacteriaceae.
- Determination of the assay's limit of detection for genomic DNA.
Main Results:
- The multiplex PCR method accurately identified all six targeted AmpC beta-lactamase gene families.
- The assay demonstrated a high sensitivity, detecting down to 1.0x10^3 copies/μL of genomic DNA.
- Results from blinded testing of clinical isolates showed concordance with traditional methods but with enhanced sensitivity and accuracy.
Conclusions:
- The developed multiplex PCR system is a highly efficient, sensitive, and accurate tool for distinguishing the six major AmpC beta-lactamase gene families.
- This assay offers a practical and discriminative diagnostic approach for identifying key antibiotic resistance mechanisms in clinical settings.
