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Published on: May 8, 2013
[Detection methods and changes in metallo-beta-lactamase-producing Gram-negative rod isolates at Nagasaki University
Yoichi Hirakata1, Katsunori Yanagihara, Junichi Matsuda
1Department of Laboratory Medicine, Nagasaki University School of Medicine and Dentistry.
Abstract:
Since plasmid-mediated metallo-beta-lactamase (MBL)-producing Pseudimonas aeruginosa was reported in Japan, MBL-producing Gram-negative rods (GNRs) have emerged worldwide. We developed a way to detect MBL-producing GNRs in routine examination using broth microdilution with the MBL inhibitor sodium mercaptoacetate (SMA) in frozen plates. Between 1996 and 2005, we evaluated this and other methods, including broth microdilution with another MBL inhibitor dipicolinic acid (DPA) in dry plates, conventional PCR, and a combined simple DNA preparation and enzymatic PCR product detection. The combined method is suitable for detecting IMP-type MBL-producing GNRs from numerous isolates. Broth microdilution with SMA at a concentration of 400 microg/mL had high performance and detected most PCR-positive MBL-producing GNRs in routine antimicrobial susceptibility testing. DPA in dry plates at 400 microg/mL yielded false positive results in 11.4% of isolates but worked satisfactorily at 175 microg/mL and 400 microg/mL of SMA in frozen plates. Until 1996, MBL had been detected from only 6 bacterial species, i.e., Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Achromobacter xylosoxidans, Serratia marcescens, and Citrobacter freundii, MBL-producing GNRs were later found in other glucose nonfermenting GNRs such as Acinetobacter baumanii, Acinetobacter lwoffii, and Burkholderia cepacia complex and Enterobacteriaceae. Most MBL-producing bacteria were multidrug resistant and no antimicrobial agents exist that are active against such isolates in monotherapy, making their rapid detection very important in controlling infection control.
Insights
Rapid detection of metallo-beta-lactamase (MBL)-producing Gram-negative rods is crucial. Broth microdilution with sodium mercaptoacetate (SMA) in frozen plates effectively identifies these multidrug-resistant bacteria in routine testing.
Area of Science:
- Microbiology
- Clinical Diagnostics
- Antimicrobial Resistance
Context:
- Metallo-beta-lactamase (MBL)-producing Gram-negative rods (GNRs) have emerged globally since the initial report of plasmid-mediated MBL-producing Pseudomonas aeruginosa in Japan.
- These MBL-producing GNRs are increasingly found in diverse bacterial species, including glucose non-fermenting GNRs and Enterobacteriaceae.
- Most MBL-producing bacteria exhibit multidrug resistance, posing significant challenges for treatment and infection control.
Purpose:
- To develop and evaluate a method for detecting MBL-producing GNRs in routine laboratory examinations.
- To compare the efficacy of broth microdilution with sodium mercaptoacetate (SMA) in frozen plates against other methods, including dipicolinic acid (DPA) in dry plates and PCR.
Summary:
- Broth microdilution using SMA at 400 µg/mL in frozen plates demonstrated high performance in detecting MBL-producing GNRs.
- A combined simple DNA preparation and enzymatic PCR product detection method is suitable for identifying IMP-type MBL-producing GNRs.
- Dipicolinic acid (DPA) in dry plates showed a high false-positive rate (11.4%), whereas SMA in frozen plates yielded satisfactory results.
Impact:
- This study provides a reliable method for the routine detection of MBL-producing GNRs, aiding in infection control.
- The findings highlight the importance of rapid diagnostics for managing infections caused by multidrug-resistant MBL-producing bacteria.
- Effective detection strategies are essential due to the lack of effective monotherapy antimicrobial agents against these resistant isolates.
