Genome rearrangements in Escherichia coli during de novo acquisition of resistance to a single antibiotic or two antibiotics successively

Abstract

Background: The ability of bacteria to acquire resistance to antibiotics relies to a large extent on their capacity for genome modification. Prokaryotic genomes are highly plastic and can utilize horizontal gene transfer, point mutations, and gene deletions or amplifications to realize genome expansion and rearrangements. The contribution of point mutations to de novo acquisition of antibiotic resistance is well-established. In this study, the internal genome rearrangement of Escherichia coli during to de novo acquisition of antibiotic resistance was investigated using whole-genome sequencing.

Results: Cells were made resistant to one of the four antibiotics and subsequently to one of the three remaining. This way the initial genetic rearrangements could be documented together with the effects of an altered genetic background on subsequent development of resistance. A DNA fragment including ampC was amplified by a factor sometimes exceeding 100 as a result of exposure to amoxicillin. Excision of prophage e14 was observed in many samples with a double exposure history, but not in cells exposed to a single antibiotic, indicating that the activation of the SOS stress response alone, normally the trigger for excision, was not sufficient to cause excision of prophage e14. Partial deletion of clpS and clpA occurred in strains exposed to enrofloxacin and tetracycline. Other deletions were observed in some strains, but not in replicates with the exact same exposure history. Various insertion sequence transpositions correlated with exposure to specific antibiotics.

Conclusions: Many of the genome rearrangements have not been reported before to occur during resistance development. The observed correlation between genome rearrangements and specific antibiotic pressure, as well as their presence in independent replicates indicates that these events do not occur randomly. Taken together, the observed genome rearrangements illustrate the plasticity of the E. coli genome when exposed to antibiotic stress.

Keywords: Gene amplification; Genome rearrangement; Prophage; de novo resistance.

Fig. 1

Experimental scheme. Wild-type E. coli MG1655 was exposed to stepwise increasing concentrations of amoxicillin (AMX), enrofloxacin (ENRO), kanamycin (KAN), or tetracycline (TET), resulting in strains with a de novo acquired resistance to a single antibiotic. The resistant strains were subsequently made resistant to the three other antibiotics. Each induction of resistance was performed in duplicate, resulting in four replicates with an identical exposure history of the double resistant strains

Fig. 2

Amplification of three different fragments, all including ampC, upon acquisition of resistance to amoxicillin. Fragment B and C were detected in tetracycline resistant strains exposed to amoxicillin, fragment A was detected in all strains with an ampC amplification. See Table 1 for detailed information on prevalence of shown amplifications. The figure depicts genomic organization at point of deletion. The genes involved and the resulting gene products are displayed under the figure. Genes in bold are amplified in all three fragments

Fig. 3

ampC copy number for different strains carrying an ampC amplification. The ampC copy number was determined with qPCR, using untreated wild-type E. coli as a reference. With exception of wild-type (which only acquired resistance to amoxicillin), all strains carried a previous resistance to enrofloxacin (ENRO_R), kanamycin (KAN_R), or tetracycline (TET_R), resulting in a secondary resistance to amoxicillin The number displayed under the strain indicates the concentration amoxicillin used for resistance development. Bars indicate the average copy number from 25 colonies

Fig. 4

Deletions detected in strains with de novo acquired antibiotic resistance a: Deletion of prophage e14 associated genes in strains exposed to any of the four antibiotics. b: Partial deletion of clpS and clpA in strains exposed to enrofloxacin and tetracycline. Figures depict genomic organization at point of deletion. The genes involved and the resulting gene products are displayed under the figure. Prophage associated genes are not shown because the resulting gene products are mostly not characterized. See Table 1 for detailed information on prevalence of the deletions

Fig. 5

Deletions detected in resistant strains made resistant to kanamycin and amoxicillin. a: Deletion of sbmA and surrounding genes in enrofloxacin resistant E. coli exposed to kanamycin. b: Partial or full deletion of 8 genes upon induction of amoxicillin resistance in tetracycline resistant E. coli. Figures depicts genomic organization at point of deletion. The genes involved and the resulting gene products are displayed under the figure. See Table 1 for detailed information on prevalence of the deletions

Fig. 6

Intragenic IS transpositions identified in strains with acquired antibiotic resistance. IS186 insertion was detected in fimA in cells with acquired amoxicillin resistance (a), in yeaR in cells with acquired amoxicillin resistance (b), and in oppB in cells with secondary kanamycin resistance (c). IS1 insertion was found in cyoA in a single kanamycin resistant strain exposed to amoxicillin (d). See Table 1 for detailed information on prevalence of shown IS transpositions

Fig. 7

Intergenic IS transpositions identified in strains with acquired antibiotic resistance. IS5 was found in the 5’ UTR of dcuC and pagP when cells were exposed to kanamycin (a), and mgrB and yobH upon acquisition of resistance to tetracycline (b). IS186 transposition was detected in the 5’ UTR of lon in enrofloxacin resistant cells exposed to tetracycline (c)