Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics

Abstract

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is widely disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6')-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from three hospitals serving a population of 1.2 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, increased transposon copy number is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

Fig 1. Enzymatic assay illustrating ability of AAC(6’)-Ib-cr to modify both kanamycin B and amikacin.

Increasing concentrations of aminoglycoside substrates were incubated with 100 nM of purified enzyme and 0.1 mM acetyl-CoA for 1 h to allow enzymatic reaction to take place. Enzymatic activity was then measured by adding 2 mM DTNB and monitoring the absorbance at 412 nm of the by-product from an acetylation reaction. Enzymatic activity against kanamycin B as a known substrate, and amikacin as a suspected substrate, increased proportional to substrate concentration and above negative control. This suggests AAC(6’)-Ib-cr has the ability to modify amikacin. Negative controls contain no acetyl-coA and thus no measurable acetylation.

Fig 2. Diagrammatic representation of In37 and the derivative transposon and definition of the IS26/In37 hybrid promoter.

The genes making up integron In37 (A) and the derived transposon (B) are presented to scale. In37 is a class 1 integron, with 5’ and 3’ conserved sequences (CS; orange), where the 5’CS encodes the integrase enzyme and an outward facing promoter that drives expression of the gene cassettes as a single transcript (orange arrow to P). In (B) the insertion sites of two copies of IS26 are noted (red), where this generates a composite transposon including a hybrid IS26/In37 promoter sequence (purple arrow to P), and mobilises aac(6’)-Ib-cr and bla_OXA-1 (blue) but excludes the two distal gene cassettes from In37 (green) with the catB3 gene being truncated in the process (noted as catB3Δ). In (C) we confirm the location of the aac(6’)-Ib-cr/bla_OXA-1 operon transcriptional start site using 5’RACE, and present the sequence trace resulting from this experiment, showing poly-C extension and pinpointing the transcriptional start site in complementary sequence relative to the the -10 and -35 promoter elements (bold and underlined) in the IS26/In37 hybrid promoter.

Fig 3. Relative AAC(6’)-lb-cr and OXA-1 protein abundance in IS26/In37 hybrid transposon promoter mutants versus wild-type.

Bar charts show a relative protein abundance per gene copy number of (A) AAC(6’)-lb-cr and (B) OXA-1 with wild-type (WT) promoter and mutated (mutant) promoter, which has a closer match to the -10 E. coli promoter consensus, (TATAAT versus CATAAT as show in Fig 2C). **** indicates p-value ≤ 0.0001 by unpaired t-test.

Fig 4. Association between AAC(6’)-lb-cr abundance, acc(6’)-lb-cr copy number and amikacin susceptibility.

(A) A linear correlation of AAC(6’)-lb-cr abundance and acc(6’)-lb-cr copy number, an outlier was shown in red (B). Association of amikacin susceptibility phenotype with (C) relative AAC(6’)-lb-cr abundance and (D) relative aac(6’)-lb-cr copy number. S, susceptible; R, resistant; **** indicates p-value ≤ 0.0001 and *** indicates p-value ≤ 0.001 by Mann-Whitney test.

Fig 5. Associations between bla_OXA-1 and aac(6’)-lb-cr copy number, OXA-1 abundance and Piperacillin/Tazobactam susceptibility.

A linear correlation of relative OXA-1 and (A) acc(6’)-lb-cr or (B) relative OXA-1 abundance where the outliner shows in red. Association of Piperacillin/Tazobactam susceptibility phenotype with (C) relative OXA-1 abundance and (D) relative OXA-1 gene copy number. S, susceptible; R, resistant; *** indicates p-value ≤ 0.001 and ** indicates p-value ≤ 0.01 by Mann-Whitney test.