Abstract
We studied polymyxin B resistance in 10 pairs of clinical Acinetobacter baumannii isolates, two of which had developed polymyxin B resistance in vivo. All polymyxin B-resistant isolates had lower growth rates than and substitution mutations in the lpx or pmrB gene compared to their parent isolates. There were significant differences in terms of antibiotic susceptibility and genetic determinants of resistance in A. baumannii isolates that had developed polymyxin B resistance in vivo compared to isolates that had developed polymyxin B resistance in vitro.
TEXT
Acinetobacter baumannii is a Gram-negative bacillus (GNB) whose strains are increasingly extensively drug resistant (XDR) due to their wide repertoire of antimicrobial resistance mechanisms and ability to acquire new resistance determinants (1–3). Polymyxins are currently the last-line therapeutic option for the treatment of XDR A. baumannii infections. Although surveillance data suggest that <1.0% of current A. baumannii strains are resistant to polymyxins (4, 5), clinical treatment failures have been reported. A. baumannii primarily acquires resistance to polymyxins by alterations to or complete loss of lipopolysaccharide (LPS) (8, 9) mediated via activation of two-component regulatory systems that cause the constitutive activation of LPS-modifying genes (10, 11). Polymyxin-resistant mutants developed in vitro appear to exhibit increased susceptibility to various other classes of antimicrobial compounds but do not necessarily exhibit a corresponding decrease in virulence (12).
We undertook this study to investigate the genetic causes of polymyxin resistance and cell wall physical characteristics in in vivo- and in vitro-derived polymyxin-resistant XDR A. baumannii strains via whole-genome sequencing and electron microscopy and also to assess the biological costs of acquisition of polymyxin resistance.
(This study was presented in part at the 21st European Congress of Clinical Microbiology and Infectious Diseases [ECCMID], Milan, Italy, 7 to 10 May 2011.)
Detailed methodology descriptions are provided in the supplemental material. In brief, 10 epidemiologically unrelated clinical XDR A. baumannii isolates obtained between 2006 and 2009 were selected. Two XDR A. baumannii isolates (isolates 1 and 2) had acquired polymyxin resistance in vivo. The other eight clinical isolates (isolates 3 to 10) were polymyxin B sensitive, with polymyxin B-resistant mutants generated in vitro via passaging on polymyxin B-impregnated Mueller-Hinton agar (MHA) plates at 3 times the MIC for 20 cycles.
MICs of a broad panel of antibiotics (Table 1) were obtained by broth microdilution for all pairs of isolates, with rifampin MICs obtained by a modified broth macrodilution method according to CLSI guidelines (13). The polymyxin B-resistant mutants were subjected to a further 20 days of passaging on drug-free and polymyxin B-impregnated MHA, with susceptibility testing repeated at 10- and 20-day time points to determine the stability of resistant phenotypes.
TABLE 1.
Antibiotic susceptibility testing results for pre- and post-polymyxin B-exposure Acinetobacter baumannii calcoaceticus complex clinical isolates
| Antibiotic(s) | MIC(s) (mg/liter)a |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Isolate 1 | Isolate 2 | Isolate 3 | Isolate 4 | Isolate 5 | Isolate 6 | Isolate 7 | Isolate 8 | Isolate 9 | Isolate 10 | |
| Ampicillin-sulbactam | ≥128/64 (64/32) | ≥128/64 (32/16)b | 64/32 (1/0.5)b | 64/32 (8/4)b | 64/32 (8/4)b | 16/8 (4/2)b | 16/8 (4/2)b | 32/16 (8/4)b | ≥128/64 (8/4)b | 64/32 (16/8) |
| Piperacillin-tazobactam | ≥256/4 | ≥256/4 | ≥256/4 (≤1/4)b | ≥256/4 (≤1/4)b | ≥256/4 (≤1/4)b | ≥256/4 (≤1/4)b | ≥256/4 (≤1/4)b | ≥256/4 (≤1/4)b | ≥256/4 (≤1/4)b | ≥256/4 (≤1/4)b |
| Ceftazidime | ≥128 | ≥128 | ≥128 (≤0.5)b | ≥128 (8)b | ≥128 (4)b | ≥128 (0.5)b | ≥128 (2)b | ≥128 (2)b | ≥128 (≤0.5)b | ≥128 (16)b |
| Cefepime | ≥64 | ≥64 | ≥64 (1)b | ≥128 (1)b | ≥128 (2)b | ≥128 (≤0.5)b | ≥128 (≤0.5)b | ≥128 (1)b | ≥128 (≤0.5)b | ≥128 (4)b |
| Doripenem | ≥16 | ≥16 | ≥16 | ≥16 (≤0.06)b | ≥16 (0.25)b | ≥16 (0.12)b | ≥16 (0.25)b | ≥16 (0.12)b | ≥16 (0.12)b | ≥16 (0.25)b |
| Imipenem | ≥64 (32) | ≥64 | 32 (1)b | 16 (0.25)b | ≥64 (1)b | 32 (0.25)b | 32 (0.5)b | 32 (0.5)b | 32 (0.5)b | 32 (0.5)b |
| Meropenem | ≥64 (32) | ≥64 | ≥64 (0.5)b | 16 (≤0.12)b | ≥64 (0.5)b | 32 (≤0.12)b | 32 (≤0.12)b | 32 (≤0.12)b | ≥64 (≤0.12)b | ≥64 (≤0.12)b |
| Aztreonam | 32 | ≥128 | ≥128 (1)b | ≥128 (1)b | ≥128 (4)b | ≥128 (≤0.5)b | ≥128 (≤0.5)b | 64 (4)b | ≥128 (≤0.5)b | ≥128 (4)b |
| Gentamicin | ≥64 | ≥64 | ≥64 | ≥64 (8)b | ≥64 (16)b | ≥64 (≤0.25)b | ≥64 (0.5)b | ≥64 | ≥64 (8)b | ≥64 (8)b |
| Amikacin | ≥128 | ≥128 | ≥128 (64) | ≥128 (64) | ≥128 | 64 (≤0.1)b | 64 (≤0.1)b | ≥128 (≤0.1)b | ≥128 (32)b | ≥128 (64) |
| Ciprofloxacin | ≥16 | ≥16 | ≥16 | ≥16 (8) | ≥16 | ≥16 | ≥16 | ≥16 | ≥16 (8) | ≥16 |
| Tigecycline | 8 (16) | 4 (4) | 1 (0.25)b | 8 (1)b | 4 (0.5)b | ≥32 (0.5)b | ≥32 (0.5)b | 8 (0.5)b | 8 (0.128)b | 4 (0.064)b |
| Polymyxin B | 2 (≥32)b | 1 (≥32)b | 4 (≥32)b | 0.5 (≥32)b | 0.5 (≥32)b | 1 (≥32)b | 1 (≥32)b | 0.5 (≥32)b | 0.5 (16)b | 0.5 (≥32)b |
Numbers in parentheses indicate the change in MIC in the post-polymyxin B-exposure isolate.
A 3-fold or greater change in the drug MIC was seen with the post-polymyxin B-exposure isolate.
In vitro growth rates were determined for all isolates, and the exponential growth of the bacterial population over 24 h was analyzed using an adapted mathematical model (14).
The paired polymyxin-susceptible and -resistant isolates were sequenced on an Illumina HiSeq2000 platform, assembled de novo using VelvetOptimiser software (https://github.com/tseemann/VelvetOptimiser), and oriented with respect to a finished reference A. baumannii genome (NC_017162.1) with Mauve software (15). Gene annotation was performed using PROKKA (16). In silico multilocus sequence typing (MLST) was used for predictions for each isolate with the MLST 1.7 online software tool (https://cge.cbs.dtu.dk/services/MLST/), selecting the Institut Pasteur MLST scheme (17).
For each XDR A. baumannii lineage, the sequenced reads for both polymyxin-susceptible and polymyxin-resistant isolates were mapped to the draft genome assembly of the susceptible isolate by the use of BWA-MEM (18). Single-nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) were subsequently identified with SAMtools (19) and annotated using SnpEff software (20).
To identify insertion sequences (IS) that differed between the isolates in each pair, we screened all draft genome assemblies to identify every IS present using the ISFinder database (21), determining the presence or absence of these sequences in each isolate read set using ISMapper (22).
Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques were employed to visualize and analyze the polymyxin B resistance mechanisms that developed during XDR A. baumannii post-polymyxin B exposure.
All preexposure isolates were resistant to all antibiotics tested except polymyxin B (Table 1). Increased susceptibility to other antibiotics was generally observed for the in vitro post-polymyxin B-exposure isolates. These phenotypes were stable even after 20 days of passaging. However, the in vivo-derived polymyxin B-resistant isolates (isolates 1B and 2B) showed no significant change in drug MIC results.
We identified between 1 and 35 nonsynonymous mutations and addition of insertion sequence (IS) elements within various genes in each post-polymyxin B-exposure isolate (Table 2). None of the mutations were common between the isolates in any pair, but nonsynonymous genetic mutations were identified in the lipid biosynthesis lpx genes of four postexposure isolates (6B, 7B, 9B, and 10B), including a premature stop codon in lpxA of isolate 9B and a 7-bp sequence insertion resulting in a codon frameshift in lpxC of isolate 10B. Four postexposure isolates (3B, 4B, 5B, and 8B) had an IS (ISAba1) within the lpxA or lpxC gene. Both isolates that developed polymyxin B resistance in vivo (isolates 1B and 2B) harbored mutations at the pmrB locus. Isolate 2B harbored the greatest number of SNPs, most of which were not in genes associated with cell membrane synthesis or regulation, compared to its sensitive parent isolate.
TABLE 2.
Genome-wide mutations found in post-polymyxin B-exposure Acinetobacter baumannii calcoaceticus complex clinical isolates with reference to their pre-polymyxin B-exposure counterparts
| Isolate (inferred ST) | Gene or gene locationa | Annotation or product | Mutation | Reference sequence inference/genomic coordinates of IS “hits” |
|---|---|---|---|---|
| 1 (ST-1) | pmrB | Two-component sensor kinase signal peptide | P233S | YP_003730963.1 |
| NA | Signal peptide | L105F | YP_001712355.1 | |
| NA | Family finger-like domain protein | C8W | YP_003731514.1 | |
| pntA-1 | Pyridine nucleotide transhydrogenase alpha subunit | T157I | YP_047601.1 | |
| NA | TEM-type beta-lactamase | Codon deletion IPFFAAFCL11I | YP_002317692.1 | |
| ABA1_00178 | Laminin-binding surface protein | IS15 insertion | 192134–192126 | |
| 2 (ST-2) | NA | RND transporter/multidrug resistance efflux pump | R172H | YP_001707896.1 |
| NA | Glycosyl transferase | S33F | YP_045879.1 | |
| NA | Putative porin protein associated with imipenem resistance | Stop codon Q36* | YP_003731166.1 | |
| pmrB | Two-component sensor kinase | R263H | YP_003730963.1 | |
| NA | Glycosyltransferase | Frame shift E147E | YP_001083155.1 | |
| mutS | DNA mismatch repair protein | IS15 insertion | 1792809–1792801, 1793624–1793633 | |
| Between papD and fimA | P pilus assembly protein, pilin FimA | ISAba1 insertion | 2395586–2395591 | |
| 3 (ST-2) | NA | Two-component sensor kinase homologous to pmrB | R64C | YP_003730836.1 |
| NA | Phosphatidylserine synthase | P234L | YP_005527355.1 | |
| filE | NA | Codon deletion TAPTAP129− | YP_001845298.1 | |
| lpxA | UDP-N-acetylglucosamine acyltransferase | ISAba1 insertion | 2690056–2690049 | |
| 4 (ST-1) | NA | Outer membrane phospholipase A | Frame shift/base deletion at codon 31 (GT/G) | YP_005526168.1 |
| NA | RND transporter/multidrug resistance efflux pump | Codon insertion T21TPAPA | YP_001707896.1 | |
| lpxC | N-Acetylglucosamine deacetylase | ISAba1 insertion | 3758548–3758539 | |
| 5 (ST-1) | lpxC | N-Acetylglucosamine deacetylase | ISAba1 insertion | 3758550–3758559 |
| 6 (ST-1) | lpxD | UDP-3-O-(3-hydroxymyristoyl) glucosamine N-acyltransferase | S167F | YP_005515126.1 |
| baeR | OmpR family transcriptional regulator | G60A | YP_045370.1 | |
| NA | DNA polymerase III subunit tau | EP435 | YP_002319726.1 | |
| 7 (ST-1) | rpsE | 30S ribosomal protein S6 | Y80D | YP_047025.1 |
| lpxA | UDP-N-acetylglucosamine acyltransferase | G56V | YP_003731736.1 | |
| 8 (ST-1) | NA | Hypothetical protein | Codon insertion D282DDK | WP_000179573 |
| lpxC | N-Acetylglucosamine deacetylase | ISAba1 insertion | Amino acid positions 155–159 | |
| 9 (ST-1) | lpxA | UDP-N-acetylglucosamine acyltransferase | Stop codon E84* | YP_003731736.1 |
| NA | Pilus assembly protein FilE | Codon deletion TAPTAPTAP126 | YP_001714906.1 | |
| Upstream of rpoC | RpoC DNA-directed RNA polymerase, beta′ subunit | ISC1041 insertion | 375323–375326 | |
| 10 (ST-1) | NA | Putative metal transporter | Codon deletion HH171 | YP_005523968.1 |
| lpxC | N-Acetylglucosamine deacetylase | Frame shift with a 7-bp insertion resulting in incomplete codon insertion (Q252QSS) | YP_047978.1 | |
| NA | Peptidase M16C-associated family protein | ISAba125/ISC1041 insertion | 2202796–2202801 | |
| NA | Hypothetical protein | ISAba125 insertion | 3016296–3016300 | |
| NA | Penicillin-binding protein 1A | ISAba1 insertion | 3618404–3618395 |
NA, not available; RND, resistance-nodulation-division.
Representative TEM and SEM images are shown in the figures in the supplemental material. Generally, there were no objective differences between the preexposure and postexposure isolates in TEM. SEM showed that the preexposure isolates had classical smooth and intact surfaces in a diploid structure whereas the polymyxin-B-resistant mutants were more compact in appearance and had multiple dents or craters on their surfaces.
The best-fit growth rate constants for the post-polymyxin B-exposure isolates were lower than those measured for the respective parent isolates (Fig. S3 in the supplemental material), suggesting lower growth rates and lower fitness.
We found significant differences between the isolates based on how the polymyxin resistance was derived, although the number tested was too small for definite conclusions to be reached. The laboratory-induced polymyxin B-resistant isolates became more susceptible to other classes of antibiotics than the preexposure isolates (9, 12), but the isolates that developed resistance in vivo did not exhibit this phenomenon, consistent with other case reports (23–25).
We identified two insertions of IS15 in the mutS gene in clinical polymyxin-resistant isolate 2B, which harbored an excess of SNPs. Truncation of the mutS gene in this isolate may thus explain the large number of SNPs identified in isolate 2B, in similarity to previous reports in A. baumannii (26).
Our EM results had demonstrated morphological differentiation suggestive of LPS loss and altered expression of outer membrane proteins between polymyxin B-susceptible and polymyxin B-resistant A. baumannii isolates (9, 27). This observation was consistent with published reports of Escherichia coli studies where the bacterial cell envelope had been damaged by novel antimicrobial peptides (28).
In conclusion, the more complex selection pressures that occur in vivo likely result in polymyxin-resistant mutants that are different from and perhaps more fit than the in vitro-derived polymyxin-resistant mutants (12, 23–25), suggesting that current in vitro-derived polymyxin-resistant A. baumannii mutants are relatively poor surrogates for the study of polymyxin resistance and its impact on virulence or biofitness.
Supplementary Material
ACKNOWLEDGMENTS
We thank the staff at Singapore General Hospital and Changi General Hospital who had assisted in collecting the organisms for this study as well as Andrea Kwa and Sasikala D/O Suranthran for help with part of the in vitro experimentation.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01884-15.
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