Distribution and expression of the aac(6')-Im (aacA16) aminoglycoside resistance gene

Abstract

Background: The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2.

Objectives: To identify sources for the aac(6')-Im fragment found in A. baumannii.

Methods: MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression.

Results: Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different.

Conclusions: The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.

Figure 1.

Context of the the aac(6′)-Im gene in pMRSN791417-8. (a) The ISKpn26 and IS26-bounded region in the pMRSN791417-8 HI2 plasmid containing aac(6′)-Im. Top line, pMRSN791417-8 backbone context, with corresponding regions from R478 (GenBank accession number BX664015) labelled above the line; middle line, detailed resistance region in pMRSN791417-8; bottom line, configuration in the hypothetical F46 (GenBank accession number CP096575) progenitor before an IS26-mediated inversion. The aac(6′)-Im gene is shown as a red arrow, and other resistance genes are shown as blue arrows. IS26 are shown as green boxes with an arrow indicating the orientation of the tnp26 gene. Other IS are shown as white boxes. Gene cassette attC sites are denoted by solid black ovals. Vertical black lines indicate inverted repeats. Known structures are labelled above each line. (b) Alignment of the Pc strong promoter in the aac(6′)-Im containing integron in F46 (GenBank accession number CP096575) with the Pc weak promoter in MRSN365855. (c) Alignment of the integron in the IncR plasmid pAMR200031 (GenBank accession number CP134003) with the hypothetical F46 (GenBank accession number CP096575) progenitor and In2 from Tn21 (GenBank accession number AF071413). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

Transcription analysis of aac(6′)-Im in A. baumannii MRSN825514 (strong promoter), P. putida MRSN365855 (weak P1 and strong P2 promoters) and E. hormaechei MRSN791417 (no promoter). Normalized, log-transformed messenger RNA (mRNA) levels are indicated. Data represent the average from three independent experiments and error bars represent the standard deviation. * and ** indicate P values <0.01 or <0.001, respectively.

Figure 3.

Resistance islands in MRSN365855 (a) The integron in the MRSN365855 chromosome containing the aac(6′)-Im gene cassette. The second structure was generated by reversing the IS6100-mediated inversion and removing one copy of IS6100. Drawn to scale from GenBank accession number CP132007 (bases 4,995,046-5,010,598). (b) A hybrid Tn5393 transposon in the MRSN365855 chromosome. (c) The integron in the pMRSN365855-2 plasmid. The aac(6′)-Im gene is shown as a red arrow, and other resistance genes are shown as blue arrows. intI1 is shown as a purple arrow, with the relevant Pc promoter marked above with a bent arrow. IS26 are shown as green boxes with an arrow indicating the orientation of the tnp26 gene. Other IS are shown as white boxes. Gene cassette attC sites are denoted by solid black ovals. Vertical black lines indicate inverted repeats. Known structures are labelled above or below each line. Where present, the sequences of target site duplications are shown. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 4.

aac(6′)-Im in non-A. baumannii contexts in the GenBank non-redundant nucleotide database. (a) Plasmid-borne examples of aac(6′)-Im in the ARI-B island of IncC plasmid p2 (GenBank accession number CP012989) and in plasmid pCUVET18-1371.1 (GenBank accession number CP115010). (b) Chromosomal examples of aac(6′)-Im in the A. veronii LPH5_2 genomic island AveGI2 (GenBank accession number OR466688), P. aeruginosa AR_0357 (GenBank accession number CP027166) and Aeromonas sp. FDAARGOS_1404 (GenBank accession number CP077237). The aac(6′)-Im gene is shown as a red arrow, and other resistance genes are shown as blue arrows. intI1 is shown as a purple arrow, with the relevant Pc promoter marked above with a bent arrow. IS26 are shown as green boxes with an arrow indicating the orientation of the tnp26 gene. Other IS are shown as white boxes. Gene cassette attC sites are denoted by solid black ovals. Vertical black lines indicate inverted repeats. Known structures are labelled above or below each line. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.