Deletion of TnAbaR23 results in both expected and unexpected antibiogram changes in a multidrug-resistant Acinetobacter baumannii strain

Abstract

Since the 2006 discovery of the Acinetobacter baumannii strain AYE AbaR1 resistance island, similar elements have been reported in numerous members of this species. As AbaR1 is distantly related to Tn7, we have renamed it TnAbaR1. TnAbaR transposons are known to carry multiple antibiotic resistance- and efflux-associated genes, although none have been experimentally studied en bloc. We deleted the TnAbaR transposon in A. baumannii A424, which we have designated TnAbaR23, and characterized independent deletion mutants DCO163 and DCO174. The NotI pulsed-field gel electrophoresis (PFGE) profile of strain DCO174 was consistent with targeted deletion of TnAbaR23 alone, but strain DCO163 apparently harbored a second large genomic deletion. Nevertheless, "subtractive amplification" targeting 52 TnAbaR and/or resistance-associated loci yielded identical results for both mutants and highlighted genes lost relative to strain A424. PCR mapping and genome sequencing revealed the entire 48.3-kb sequence of TnAbaR23. Consistent with TnAbaR23 carrying two copies of sul1, both mutants exhibited markedly increased susceptibility to sulfamethoxazole. In contrast, loss of tetAR(A) resulted in only a minor and variable increase in tetracycline susceptibility. Despite not exhibiting a growth handicap, strain DCO163 was more susceptible than strain DCO174 to 9 of 10 antibiotics associated with mutant-to-mutant variation in susceptibility, suggesting impairment of an undefined resistance-associated function. Remarkably, despite all three strains sharing identical gyrA and parC sequences, the ciprofloxacin MIC of DCO174 was >8-fold that of DCO163 and A424, suggesting a possible paradoxical role for TnAbaR23 in promoting sensitivity to ciprofloxacin. This study highlights the importance of experimental scrutiny and challenges the assumption that resistance phenotypes can reliably be predicted from genotypes alone.

Fig 1

Analysis of the putative TnAbaR23-minus mutants of Acinetobacter baumannii A424. (A) PCR amplification across the targeted deleted locus in DCO163 and DCO174 mutant strains using the primers AbaRUF2 and AbaRDR2 yielded the expected ∼3.7-kb product, while failing to amplify across the A424 TnAbaR23 element. The sequenced strains ATCC 19606 and AYE that lacked and harbored an island within the comM gene were used as positive (2.5-kb band expected) and negative PCR controls, respectively. (B) Pulsed-field gel electrophoresis analysis of NotI- and SfiI-digested A424, DCO163, DCO174, and AYE high-molecular-weight genomic DNA. The three bands with arrows and labels indicate the only observed NotI profile differences between A424 and DCO174. Subsequently available sequence data showed that TnAbaR23 harbors two very closely spaced NotI sites, while the replacement cassette lacks a NotI site. Hence, the observed loss of two A424 bands (825 kb plus 124 kb) and the appearance of a new DCO174-specific band (900 kb), with the difference between the two being the size of TnAbaR23 (48.3 kb). Lanes MM contain molecular size markers.

Fig 2

Schematic map showing the genetic organization of TnAbaR23. Genes are colored by module (see Fig. 3 for details) with key genes labeled. The locations of identified repeat sequences (inverted repeat left [IRL] and inverted repeat right [IRR]) are indicated (positions are shown in thousands[k] from 0k to 50k), and full sequence details are shown in the graph at the bottom of the figure. The red circles shown at both TnAbaR23 ends indicate the 5-bp direct repeat sequences (5′-ACCGC-3′) which flank this element. Study-defined module boundaries are shown as dotted lines (see text and the legend to Fig. 3 for details). This figure is drawn to scale. Full annotation details for TnAbaR23 are available in GenBank (accession number JN676148). Minor annotation changes represented in this figure are listed in Table S4 in the supplemental material. 3′-CS, 3′ conserved sequence.

Fig 3

Comparison of the modular compositions of 15 previously characterized A. baumannii TnAbaR transposons and that of TnAbaR23. A schematic representation of TnAbaR1 drawn to scale with genes colored by module and module boundaries as indicated is shown in the map at the top of the figure; selected genes are labeled. Details of six further modules not represented within TnAbaR1 are shown below the map to the right. Full annotation details of TnAbaR1 and the remaining modules are available via GenBank; accession numbers are listed below or in Table S5 in the supplemental material. Minor annotation changes represented in this figure are listed in Table S4. Modules A to K are shown as equal-sized rectangles when intact or as terminally or internally truncated boxes, as appropriate, to highlight incomplete modules. Rectangles outlined by large dashes represent deleted and/or absent modules. Modules N and L are inserted at sites as shown, while module M in TnAbaR2 occurs in place of a part of module I. Module O is restricted to TnAbaR0 (previously designated Tn6021) and TnAbaR4 and possess an ∼500-bp segment that exhibits high-level identity to a matching segment of the highly conserved module C; corresponding homologous sequences are shown crosshatched. The size to the nearest kilobase and host strain information are as shown. TnAbaR4 is the only characterized member of this family which is not inserted into the comM gene. Additional genetic features are shown where particularly pertinent. The TnAbaR transposons shown above and the GenBank accession numbers of the transposons or associated genomes (shown in parentheses) are as follows: TnAbaR0 (CP000521), TnAbaR1 (CT025832), TnAbaR2 (CP000863), TnAbaR3 (CP001182), TnAbaR4 (CP001182), TnAbaR5 (FJ172370), TnAbaR6 (GQ406245), TnAbaR7 (GQ406246), TnAbaR8 (HM590877), TnAbaR9 (project accession ADGZ01000000), TnAbaR10 (project accession ADHA01000000), TnAbaR12 (JF262168), TnAbaR13 (JF262169), TnAbaR17 (JF262173), TnAbaR20 (HM357806), and TnAbaR23 (JN676148).

Fig 4

Growth curve and Etest analyses of Acinetobacter baumannii A424 and its TnAbaR23-minus mutants. (A) The wild-type strain A424 and DCO163 and DCO174 mutants were grown in 200 μl antibiotic-free LB medium at 37°C with shaking in 96-well plates from standardized overnight cultures and monitored in a Multiskan GO instrument to determine the optical density at 600 nm (OD₆₀₀) measurements at 10-min intervals. The data shown represent the mean values ± standard deviations (error bars) for four independent wells for each strain. No significant difference in growth dynamics of the strains was observed. (B) Images of Etest assays for sulfamethoxazole and ciprofloxacin MICs on A424, DCO163, and DCO174 to highlight both an expected and an unexpected resistance phenotype of DCO174 associated with the loss of TnAbaR23. The edges of the zones of inhibition have been outlined, and the measured MIC values (in micrograms per milliliter) are shown on the white labels at the bottom of the plate image. The switch to sulfamethoxazole susceptibility observed in both DCO163 and DCO174 was entirely predictable from the defined gene content of TnAbaR23 and the PCR-informed absence of a sul1 gene elsewhere on the A424 chromosome. In contrast, the paradoxical changes in ciprofloxacin MIC values observed in DCO163 and DCO174 relative to that of A424 were entirely unexpected.