In vitro Synergistic Activities of Fosfomycin in Combination with Other Antimicrobial Agents Against Carbapenem-Resistant Escherichia coli Harboring bla_NDM-1 on the IncN2 Plasmid and a Study of the Genomic Characteristics of These Pathogens

Abstract

Purpose

The spread of New Delhi metallo-β-lactamase (NDM) encoded by the bla_NDM gene has been a global health crisis for many years. Most of bla_NDM-harboring bacteria commonly carry various antimicrobial resistance (AMR) genes on their chromosomes or plasmids, leading to limited treatment options. Thus, we aimed to evaluate the synergistic effects of fosfomycin in combination with other antimicrobial agents against bla_NDM-harboring carbapenem-resistant Escherichia coli (CREC) and to characterize the whole-genome and plasmid sequences of these pathogens.

Methods

Thirty-eight CREC isolates were collected from patients in the Medicine Ward, Songklanagarind Hospital, Thailand. The activity of fosfomycin in combination with other antimicrobial agents against CREC isolates harboring bla_NDM on the plasmid was evaluated using the checkerboard method. In this method, the serial dilutions of two antibiotics were mixed with the cultured CREC, the mixtures were incubated, and FICI was calculated to interpret the synergistic activity of the combination. The whole-genome and particular plasmids of these pathogens were sequenced using next-generation sequencing. Sequence analysis, especially on antimicrobial resistance (AMR) genes, mobile-genetic elements (MGEs), and virulence genes was performed using many bioinformatics tools.

Results

Of the E. coli 38 isolates, only 3 isolates contained the bla_NDM-1 gene, which is located on the IncN2 plasmid. The combinations of fosfomycin with aminoglycosides, colistin, tigecycline, sitafloxacin, and ciprofloxacin were synergies against bla_NDM-1-harboring CREC isolates. Genomic analysis revealed that these isolates harbored many β-lactam resistance genes and other AMR genes that may confer resistance to aminoglycoside, fluoroquinolone, rifampicin, trimethoprim, sulfonamide, tetracycline, and macrolide. Also, various MGEs, especially the bla_NDM-1-bearing IncN2 plasmid, were present in these isolates.

Conclusion

Our study demonstrated some synergistic effects of antimicrobial combination against CREC isolates harboring bla_NDM-1 on the IncN2 plasmid. Also, our data on the whole-genome and plasmid sequences might be beneficial in the control of the spread of bla_NDM-1-harboring CREC isolates. The linkages between bla_NDM-1-carrying plasmid, patient information, and time of collection will be elucidated to track the horizontal gene transfer in the future.

Keywords:

• antimicrobial resistance gene
• checkerboard method
• next-generation sequencing
• bioinformatics tool
• mobile genetic element

Ethical Approval

This study was approved by the Human Research Ethics Committee (HREC), Faculty of Medicine, Prince of Songkla University (Reference Number: 59-352-14-1). Also, the researchers were granted permission to extract the data from the database with waiver of consent because of the observational nature of the study. All data were fully anonymized before the researcher accessed and analyzed.

Acknowledgments

The authors would like to acknowledge the Division of Infectious Diseases, Department of Internal Medicine, Faculty of Medicine, Prince of Songkla University for providing the bacterial isolates. We would also like to thank the Division of Biological Science, the Division of Computational Science, and the Molecular Evolution and Computational Biology (MECoB) Research Unit, Faculty of Science as well as the Department of Biomedical Science and Biomedical Engineering, Faculty of Medicine, Prince of Songkla University for use of their research facilities. Finally, we thank Mr. David Patterson from Department of International Affairs, Faculty of Medicine, Prince of Songkla University, for English language review in this study.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest.

Additional information

Funding

This research was supported by the Human Research Ethics Committee (HREC), Faculty of Medicine, Prince of Songkla University (reference number: 59-352-14-1), and the National Research Council of Thailand (grant number: 1232545). In addition, the study was also supported by a Graduate Scholarship, Faculty of Medicine, Prince of Songkla University, Thailand and the Faculty of Science, Prince of Songkla University, Thailand (grant number: SCI64040135).