Adaptive responses to cefotaxime treatment in ESBL-producing Escherichia coli and the possible use of significantly regulated pathways as novel secondary targets.
The aim of the study was to determine how ESBL-producing Escherichia coli change the expression of metabolic and biosynthesis genes when adapting to inhibitory concentrations of cefotaxime. Secondly, it was investigated whether significantly regulated pathways constitute putative secondary targets that can be used to combat the resistant bacteria.



Strains of E. coli MG1655 encoding blaCTX-M-1 from an IncI1 plasmid and from the chromosome were challenged with cefotaxime corresponding to inhibitory concentrations, and transcriptional patterns were compared with growth without or with very low concentrations of cefotaxime by RNA sequencing. Significantly regulated pathways were inhibited with suitable inhibitors, or genes encoding the enzymes of the regulated pathways were knocked out. The ability of the bacteria to grow in the presence of cefotaxime was determined. Chequerboard assays were utilized to confirm synergies between treatments.



Genes belonging to 16 different functional gene classes were significantly regulated. Protein and peptidoglycan syntheses were up-regulated and low concentrations of chloramphenicol or d-cycloserine, targeting these systems, strongly reduced the MIC of cefotaxime (>32-fold). Inhibition and/or mutations in other genes that were significantly regulated, belonging to energy synthesis, purine synthesis, proline uptake or potassium uptake, also rendered the resistant bacteria more susceptible to cefotaxime.



The results show that ESBL-producing E. coli adapt to treatment with cefotaxime by changing their gene expression patterns and furthermore that targeting regulated adaptive pathways may be a suitable way to identify targets for drugs that will specifically inhibit the resistant bacteria.
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark.
Thea S B
TS
Møller
Department of Systems Biology, Technical University of Denmark, 2800 Lyngby, Denmark.
Martin H
MH
Rau
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark.
Charlotte S
CS
Bonde
Department of Systems Biology, Technical University of Denmark, 2800 Lyngby, Denmark Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2970 Hørsholm, Denmark.
Morten O A
MO
Sommer
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark.
Luca
L
Guardabassi
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg C, Denmark jeo@sund.ku.dk.
John E
JE
Olsen
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002439:Cefotaxime / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008826:Microbial Sensitivity Tests; D013312:Stress, Physiological; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; C055184:beta-lactamase TEM-3; D001618:beta-Lactamases; D002439:Cefotaxime