Convenient test for screening metallo-beta-lactamase-producing gram-negative bacteria by using thiol compounds

Area of Science:

• Clinical Microbiology
• Antimicrobial Resistance
• Bacterial Pathogenesis

Background:

• Metallo-beta-lactamases (MBLs), particularly IMP-1, confer resistance to beta-lactam antibiotics in Gram-negative bacteria.
• Accurate and rapid detection of MBL-producing bacteria is crucial for effective antimicrobial stewardship and infection control.
• Existing detection methods may be time-consuming or require specialized equipment, limiting their utility in routine clinical settings.

Purpose of the Study:

• To develop and validate a simple, cost-effective disk diffusion test for the detection of IMP-1-type MBL-producing Gram-negative bacteria.
• To evaluate the efficacy of various MBL inhibitors, including thiol compounds like 2-mercaptopropionic acid, in conjunction with ceftazidime.
• To assess the test's ability to differentiate IMP-1 producers from Gram-negative bacteria resistant due to other beta-lactamases (e.g., AmpC, SHV-12).

Main Methods:

• A modified Kirby-Bauer disk diffusion method was employed using two ceftazidime (CAZ) disks and a filter disk impregnated with a metallo-beta-lactamase inhibitor.
• Various inhibitors, including 2-mercaptopropionic acid and mercaptoacetic acid, were tested for their ability to block IMP-1 activity.
• The test was performed on Mueller-Hinton agar, with specific disk placement and incubation conditions to observe zone of inhibition expansion indicative of MBL activity.

Main Results:

• The disk diffusion test successfully detected IMP-1-producing Gram-negative bacteria, characterized by an expanded growth-inhibitory zone between the CAZ and inhibitor disks.
• 2-mercaptopropionic acid and mercaptoacetic acid demonstrated reproducible and clear results in blocking IMP-1 activity.
• The test effectively distinguished IMP-1 producers from CAZ-resistant strains producing AmpC or extended-spectrum beta-lactamases (ESBLs), showing comparable specificity and sensitivity to PCR.

Conclusions:

• A simple disk diffusion test using ceftazidime and a metallo-beta-lactamase inhibitor (2-mercaptopropionic acid or mercaptoacetic acid) is a reliable method for detecting IMP-1-producing Gram-negative bacteria.
• This convenient and cost-effective test is suitable for routine use in clinical laboratories for rapid identification of MBL-producing pathogens.
• The method aids in differentiating MBL-mediated resistance from other beta-lactamase-based resistance mechanisms, supporting appropriate treatment decisions.