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Published on: December 31, 2019
Dissemination of the blaCTX-M-15 gene among Enterobacteriaceae via outer membrane vesicles
Martina Bielaszewska1,2, Ondřej Daniel3, Helge Karch2
1National Reference Laboratory for E. coli and Shigellae, National Institute of Public Health, Šrobárova 48, 100 42 Prague, Czech Republic.
Background:
Bacterial outer membrane vesicles (OMVs) are an emerging source of antibiotic resistance transfer but their role in the spread of the blaCTX-M-15 gene encoding the most frequent CTX-M ESBL in Enterobacteriaceae is unknown.
Objectives:
To determine the presence of blaCTX-M-15 and other antibiotic resistance genes in OMVs of the CTX-M-15-producing MDR Escherichia coli O104:H4 outbreak strain and the ability of these OMVs to spread these genes among Enterobacteriaceae under different conditions.
Methods:
OMV-borne antibiotic resistance genes were detected by PCR; OMV-mediated transfer of blaCTX-M-15 and the associated blaTEM-1 was quantified under laboratory conditions, simulated intraintestinal conditions and under ciprofloxacin stress; resistance to antibiotics and the ESBL phenotype were determined by the CLSI disc diffusion methods and the presence of pESBL by plasmid profiling and Southern blot hybridization.
Results:
E. coli O104:H4 OMVs carried blaCTX-M-15 and blaTEM-1 located on the pESBL plasmid, but not chromosomal antibiotic resistance genes. The OMVs transferred blaCTX-M-15, blaTEM-1 and the associated pESBL into Enterobacteriaceae of different species. The frequencies of the OMV-mediated transfer were significantly increased under simulated intraintestinal conditions and under ciprofloxacin stress when compared with laboratory conditions. The 'vesiculants' (i.e. recipients that received the blaCTX-M-15- and blaTEM-1-harbouring pESBL via OMVs) acquired resistance to cefotaxime, ceftazidime and cefpodoxime and expressed the ESBL phenotype. They were able to further spread pESBL and the blaCTX-M-15 and blaTEM-1 genes via OMVs.
Conclusions:
OMVs are efficient vehicles for dissemination of the blaCTX-M-15 gene among Enterobacteriaceae and may contribute to blaCTX-M-15 transfer in the human intestine.
Insights
Bacterial outer membrane vesicles efficiently spread the blaCTX-M-15 gene, a key driver of antibiotic resistance, among Enterobacteriaceae. These vesicles may play a significant role in the transfer of resistance genes within the human gut.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Bacterial outer membrane vesicles (OMVs) are increasingly recognized for their role in antibiotic resistance gene transfer.
- The specific role of OMVs in the dissemination of the blaCTX-M-15 gene, a prevalent Extended-Spectrum Beta-Lactamase (ESBL) determinant in Enterobacteriaceae, remains largely unknown.
Purpose of the Study:
- To investigate the presence of blaCTX-M-15 and other antibiotic resistance genes within OMVs produced by a multidrug-resistant (MDR) Escherichia coli O104:H4 strain.
- To evaluate the capacity of these OMVs to mediate the transfer of antibiotic resistance genes to other Enterobacteriaceae under various conditions.
Main Methods:
- Polymerase Chain Reaction (PCR) was employed to detect antibiotic resistance genes in OMVs.
- Quantitative analysis of OMV-mediated blaCTX-M-15 and blaTEM-1 gene transfer was performed under laboratory, simulated intraintestinal, and ciprofloxacin-stressed conditions.
- Antibiotic resistance and ESBL phenotype were assessed using CLSI disc diffusion methods, complemented by plasmid profiling and Southern blot hybridization for pESBL presence.
Main Results:
- OMVs from E. coli O104:H4 contained blaCTX-M-15 and blaTEM-1 genes located on the pESBL plasmid, not chromosomal genes.
- OMVs successfully transferred the pESBL plasmid, along with blaCTX-M-15 and blaTEM-1, to diverse Enterobacteriaceae species.
- Gene transfer frequency significantly increased under simulated intestinal conditions and ciprofloxacin stress compared to standard laboratory settings.
- Recipient bacteria acquiring the pESBL plasmid exhibited resistance to cefotaxime, ceftazidime, and cefpodoxime, displaying an ESBL phenotype and further disseminating the resistance genes via OMVs.
Conclusions:
- OMVs serve as highly effective vehicles for the dissemination of the blaCTX-M-15 gene within Enterobacteriaceae populations.
- OMV-mediated blaCTX-M-15 transfer is likely a significant mechanism contributing to antibiotic resistance in the human intestinal environment.
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