Rapid Determination of Benzylpenicillin Resistance in Staphylococcus aureus Bacteraemia Model

Abstract

Rapid determination of antimicrobial susceptibility/resistance is an important factor in selecting an appropriate antimicrobial treatment and eradicating infections promptly. Conventional antimicrobial susceptibility tests (ASTs) are very time consuming. Thus, we developed a liquid chromatography-mass spectrometry (LC-MS/MS) method for rapidly determining the resistance of Staphylococcus aureus to penicillin-G in an animal-infection model. This technique will be able to detect those resistant strains whose resistance mechanism specifically controlled by penicillinase. The resistance status of S. aureus against penicillin-G was determined by conventional AST. Cultured S. aureus cells were inoculated to chicken for developing bacteraemia. The solution of penicillin-G was intravenously administered (10 mg/kg b.w.) to chickens just after infection detection. Blood samples were collected at different intervals after drug administration. The concentration of active penicillin-G and its metabolites were determined from the bacteria-free blood supernatant by utilizing the LC-MS/MS method. Evidence of infection in chicken was observed within 5 h of bacterial inoculation. The penicillinase enzyme generated by S. aureus transforms the active penicillin-G to an inactive metabolite by hydrolysis, which is evident by the mass shift from 335.10600 to 353.11579 Da as quantified using liquid chromatography quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS). The signal intensity of inactive/hydrolysed penicillin-G is several-fold greater than that of the active penicillin-G in the blood sample of chicken infected with resistant strain and treated with penicillin-G. The antimicrobial resistance index (ARI) value of resistant S. aureus strain was more than 1, demonstrating the penicillin-G-resistance pattern of that strain. This method is able to determine the extent of β-lactam antimicrobial resistance within 1.5 h from the patient’s blood and is complementary with those existing AST methods which are usually practicing in the evaluation of β-lactam antibiotic resistance.

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Keywords:

• β-lactamase
• spectrometry
• antibacterial susceptibility test
• chicken infection model
• antibacterial resistance

Acknowledgments

We are grateful to Dr. Suk-Kyung Lim of the Bacterial Disease Division, Animal and Plant Quarantine Agency, Republic of Korea for kindly providing required bacterial isolates.

Ethics Approval

All procedures performed in this study were approved by the ethics committee of Animal and Plant Quarantine Agency, Republic of Korea.

Disclosure

The authors declare that they have no conflicts of interest.