Diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with tigecycline resistance in Acinetobacter baumannii

Diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in... Abstract Objectives To investigate the mechanisms of tigecycline resistance in isogenic Acinetobacter baumannii isolate pairs as well as 65 unique clinical A. baumannii isolates obtained during the MagicBullet clinical trial from Greece, Italy and Spain. Methods A. baumannii isolates were subjected to WGS and the regulatory genes of resistance-nodulation-cell division (RND)-type efflux pumps were analysed. MICs were determined by agar dilution and the expression of RND-type efflux pumps was measured by semi-quantitative RT–PCR. Results In isolate pairs, disruption of adeS or adeN by ISs increased adeB or adeJ expression and conferred increased resistance to at least three antimicrobial classes, respectively. The insertion of ISAba1 in adeN was observed in more than 30% of tested isolates and was the most prevalent IS. Furthermore, the insertion of ISAba125 and ISAba27 into adeN was observed for the first time in A. baumannii isolates. Besides ISs, several different mutations were observed in adeN (e.g. deletions and premature stop codons), all of which led to increased tigecycline MICs. Moreover, several amino acid substitutions were detected in AdeRS, AdeN and AdeL. Of note, the substitutions D21V, G25S and D26N in AdeR were found in multiple sequences and suggest a mutational hotspot. Conclusions This study provides an insight into the different mechanisms associated with tigecycline resistance using a genomic approach and points out the importance of considering adeRS and adeN as markers for tigecycline-resistant A. baumannii isolates. Introduction Acinetobacter baumannii is a serious hospital-acquired pathogen causing wound and urinary tract infections, bloodstream infections, pneumonia and meningitis, especially in patients in ICUs.1 It is of major concern for the healthcare system because of its ability to up-regulate or acquire antimicrobial resistance determinants against multiple antimicrobial classes (e.g. β-lactams, tetracyclines, aminoglycosides, cephalosporins and fluoroquinolones),2 its prolonged survival on dry surfaces and its propensity for epidemic spread.2,3 In recent years, A. baumannii has developed resistance to carbapenems, which is mainly caused by class D β-lactamases such as blaOXA-23-like.4,5 Since the frequency of MDR and in particular carbapenem-resistant strains is increasing, tigecycline and colistin are among the few remaining antibiotics for the treatment of carbapenem-resistant A. baumannii infections. Besides carbapenem resistance, tigecycline and colistin resistance are increasingly being reported.6,7 Tigecycline resistance is often mediated by overexpression of resistance-nodulation-cell division (RND) efflux pumps.8 These tripartite pumps mainly consist of a periplasmic membrane fusion protein (MFP) that interacts with a cytoplasmic membrane-spanning transport protein and an outer membrane pore (OMP).9,10 There are three characterized RND-type efflux pumps in A. baumannii: AdeABC, AdeFGH and AdeIJK.11–13 They are intrinsically encoded on the chromosome and strictly regulated. AdeABC is regulated by the two-component regulatory system AdeRS,14 the LysR-type transcription regulator AdeL is responsible for the regulation of adeFGH expression13 and the TetR-like transcription regulator AdeN represses expression of adeIJK.15 Additionally, a further five as-yet-uncharacterized RND efflux pumps have been reported in A. baumannii.16 Besides RND-type efflux pumps, several other tigecycline resistance mechanisms have been proposed for A. baumannii. Chen et al.17 reported a deletion mutation in the tigecycline-related-methyltransferase (trm) gene, which encodes a SAM-dependent methyltransferase associated with resistance to tigecycline. Moreover, a frameshift mutation in a 1-acyl-sn-glycerol-3-phosphate acyltransferase gene (plsC) was described to increase the MIC of tigecycline.18 The objective of this study was to determine the mechanism of tigecycline resistance in A. baumannii isolates recovered from respiratory and stool samples of patients with ventilator-associated pneumonia (VAP) at hospitals in Greece, Italy and Spain between 2012 and 2015 included in the MagicBullet clinical trial.19,20 Materials and methods Bacterial isolates A. baumannii isolates were recovered from respiratory or stool samples from 65 patients with VAP that were obtained between 2012 and 2015 during the MagicBullet clinical trial.19,20 Initially, we investigated A. baumannii isolates that were consecutively obtained on multiple occasions during a patient’s hospital course from respiratory tract and stool samples. Isolates demonstrating a phenotype shift in tigecycline MIC, i.e. where the initial isolate was tigecycline susceptible or intermediate at an early sampling point and was tigecycline resistant at a later timepoint, were included in this study and termed isolate pairs. In addition, isolates representing isolate pairs were required to demonstrate less than 10 allele differences in both the core and accessory genomes by WGS (see below).21 Furthermore, we analysed 65 unique A. baumannii respiratory tract isolates that were recovered from 65 patients in Greece (n = 37), Italy (n = 14) and Spain (n = 14), respectively. Antimicrobial susceptibility testing Susceptibility to tetracycline, gentamicin (Sigma–Aldrich, Steinheim, Germany), meropenem, tigecycline, amikacin, colistin, minocycline, rifampicin (Molekula, Newcastle upon Tyne, UK), levofloxacin (Sanofi Aventis, Frankfurt, Germany), ciprofloxacin (Bayer Pharma AG, Berlin, Germany), azithromycin (Pfizer Pharma GmbH, Münster, Germany) and erythromycin (AppliChem, Darmstadt, Germany) was determined by agar dilution following the current CLSI guidelines.22 For isolates representing isolate pairs, agar dilution was repeated at least three times. Tigecycline resistance was defined as an MIC of > 2 mg/L using the EUCAST resistance breakpoints for Enterobacteriaceae, as no such breakpoint is available for A. baumannii. WGS and sequence data analysis All respiratory A. baumannii isolates as well as the four isolate pairs were subjected to WGS and analysed as previously described.21 Further analysis was carried out by bioinformatic tools ResFinder for resistome analysis and ISMapper to search for ISs.23,24 Moreover, MLST alleles were extracted using PubMLST (https://pubmlst.org/abaumannii/).25 PCR-based methods to confirm tigecycline resistance determinants All primers used in this study are listed in Table S1 (available as Supplementary data at JAC Online). Crude cell lysates or genomic DNA were used as templates for PCR. Differences in ORFs between isolates representing an isolate pair or the presence of ISs, as determined by WGS, were confirmed by PCR and Sanger sequencing (LGC Genomics GmbH, Berlin, Germany). Putative ISs were further analysed using the BLAST tool of ISfinder (http://www-is.biotoul.fr).29 Semi-quantitative RT–PCR (qRT–PCR) RNA extraction, cDNA synthesis and qRT–PCR to measure the expression of adeB or adeJ were performed, as previously described, using rpoB as the reference gene.26 Primer sequences are shown in Table S1. All qRT–PCR experiments were done in triplicate using freshly prepared RNA and cDNA and were repeated independently at least three times. The relative expression of adeB and adeJ and the number of transcripts were compared between isolates representing an isolate pair. Statistical analysis was done by performing an unpaired t-test using the recorded absolute values. Nucleotide sequence accession number All raw reads generated were submitted to the Sequencing Read Archive (https://www.ncbi.nlm.nih.gov/sra/) of the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA431710. Results Description of isolates To investigate the mechanisms of tigecycline resistance, A. baumannii isolates were initially chosen for comparison in isolate pairs. In total, four isolate pairs were identified that were considered to have the same isogenic background, as demonstrated by WGS, and exhibited an increase in tigecycline MICs (Table 1). Isolate pairs 1 and 2, obtained in Seville, Spain, displayed a shift in tigecycline susceptibility from susceptible or intermediate (MIC 1 or 2 mg/L) to resistant (MIC 16 mg/L), respectively. Isolate pair 3, recovered in Valencia, Spain, exhibited a tigecycline MIC increase from 2 to 4 mg/L. Isolate pair 4, obtained from a hospital in Athens, Greece, also had tigecycline MICs that differed by one dilution step (2 to 4 mg/L). The differences in tigecycline MICs were seen consistently in three independent experiments in all isolate pairs. Table 1. Overview of isolate pairs chosen to investigate tigecycline resistance; clonal lineages were determined by cgMLST and tigecycline MICs were determined by agar dilution Patient Isolate Isolate pair Day of isolationa Sample type Origin Clonal lineage Oxford MLST type Tigecycline MIC (mg/L) 01-001 MB_2 1 3 respiratory Seville, Spain IC2 ST-208 1 MB_5 1 F stool 16 01-002 MB_7 2 8 respiratory Seville, Spain IC2 ST-208 2 MB_43 2 F stool 16 02-077 MB_271 3 3 stool Valencia, Spain IC2 ST-1117 2 MB_273 3 F stool 4 22-168 MB_131 4 0 respiratory Athens, Greece IC1 ST-1567 2 MB_1044 4 F stool 4 Patient Isolate Isolate pair Day of isolationa Sample type Origin Clonal lineage Oxford MLST type Tigecycline MIC (mg/L) 01-001 MB_2 1 3 respiratory Seville, Spain IC2 ST-208 1 MB_5 1 F stool 16 01-002 MB_7 2 8 respiratory Seville, Spain IC2 ST-208 2 MB_43 2 F stool 16 02-077 MB_271 3 3 stool Valencia, Spain IC2 ST-1117 2 MB_273 3 F stool 4 22-168 MB_131 4 0 respiratory Athens, Greece IC1 ST-1567 2 MB_1044 4 F stool 4 F, sample recovered at the end of the patient’s antibiotic treatment. a Hospital day after enrolment into the clinical trial. Table 1. Overview of isolate pairs chosen to investigate tigecycline resistance; clonal lineages were determined by cgMLST and tigecycline MICs were determined by agar dilution Patient Isolate Isolate pair Day of isolationa Sample type Origin Clonal lineage Oxford MLST type Tigecycline MIC (mg/L) 01-001 MB_2 1 3 respiratory Seville, Spain IC2 ST-208 1 MB_5 1 F stool 16 01-002 MB_7 2 8 respiratory Seville, Spain IC2 ST-208 2 MB_43 2 F stool 16 02-077 MB_271 3 3 stool Valencia, Spain IC2 ST-1117 2 MB_273 3 F stool 4 22-168 MB_131 4 0 respiratory Athens, Greece IC1 ST-1567 2 MB_1044 4 F stool 4 Patient Isolate Isolate pair Day of isolationa Sample type Origin Clonal lineage Oxford MLST type Tigecycline MIC (mg/L) 01-001 MB_2 1 3 respiratory Seville, Spain IC2 ST-208 1 MB_5 1 F stool 16 01-002 MB_7 2 8 respiratory Seville, Spain IC2 ST-208 2 MB_43 2 F stool 16 02-077 MB_271 3 3 stool Valencia, Spain IC2 ST-1117 2 MB_273 3 F stool 4 22-168 MB_131 4 0 respiratory Athens, Greece IC1 ST-1567 2 MB_1044 4 F stool 4 F, sample recovered at the end of the patient’s antibiotic treatment. a Hospital day after enrolment into the clinical trial. In an attempt to include as many different tigecycline resistance mechanisms as possible and to determine their prevalence in a large geographical region, 65 unique A. baumannii isolates were analysed. Based on a core-genome MLST (cgMLST) analysis, the majority of isolates belonged to the international clonal lineage IC2 (n = 54, 83.1%), while five isolates (7.7%) belonged to IC1 and six isolates (9.2%) could not be assigned to any known international clonal lineage (Table 2).20 Only two isolates (3.1%) were considered susceptible to tigecycline (MIC ≤ 1 mg/L). Seven isolates (10.8%) showed intermediate MICs of 2 mg/L and the majority of isolates (n = 56, 86.2%) displayed an MIC ≥ 4 mg/L and were therefore considered resistant to tigecycline (Table 2). Table 2. Overview of 65 A. baumannii isolates obtained from patients from Greece, Italy and Spain included in the MagicBullet clinical trial; clonal lineages were determined by cgMLST and tigecycline MICs were determined by agar dilution Country Number of cities (hospitals) Number of patients Clonal lineages (number of isolates) Tigecycline MIC ≤1 mg/L 2 mg/L ≥4 mg/L Greece 4 (7) 37 IC1 (5) 1 (2.7%) 4 (10.8%) 32 (86.5%) IC2 (32) Italy 2 (3) 14 IC2 (10) 1 (7.1%) 0 13 (92.9%) UNa (4) Spain 4 (5) 14 IC2 (12) 0 3 (21.4%) 11 (78.6%) UNa (2) Total 10 (15) 65 IC1 (5) 2 7 56 IC2 (54) UNa (6) Country Number of cities (hospitals) Number of patients Clonal lineages (number of isolates) Tigecycline MIC ≤1 mg/L 2 mg/L ≥4 mg/L Greece 4 (7) 37 IC1 (5) 1 (2.7%) 4 (10.8%) 32 (86.5%) IC2 (32) Italy 2 (3) 14 IC2 (10) 1 (7.1%) 0 13 (92.9%) UNa (4) Spain 4 (5) 14 IC2 (12) 0 3 (21.4%) 11 (78.6%) UNa (2) Total 10 (15) 65 IC1 (5) 2 7 56 IC2 (54) UNa (6) a UN, not related to any of the known international clonal lineages. Table 2. Overview of 65 A. baumannii isolates obtained from patients from Greece, Italy and Spain included in the MagicBullet clinical trial; clonal lineages were determined by cgMLST and tigecycline MICs were determined by agar dilution Country Number of cities (hospitals) Number of patients Clonal lineages (number of isolates) Tigecycline MIC ≤1 mg/L 2 mg/L ≥4 mg/L Greece 4 (7) 37 IC1 (5) 1 (2.7%) 4 (10.8%) 32 (86.5%) IC2 (32) Italy 2 (3) 14 IC2 (10) 1 (7.1%) 0 13 (92.9%) UNa (4) Spain 4 (5) 14 IC2 (12) 0 3 (21.4%) 11 (78.6%) UNa (2) Total 10 (15) 65 IC1 (5) 2 7 56 IC2 (54) UNa (6) Country Number of cities (hospitals) Number of patients Clonal lineages (number of isolates) Tigecycline MIC ≤1 mg/L 2 mg/L ≥4 mg/L Greece 4 (7) 37 IC1 (5) 1 (2.7%) 4 (10.8%) 32 (86.5%) IC2 (32) Italy 2 (3) 14 IC2 (10) 1 (7.1%) 0 13 (92.9%) UNa (4) Spain 4 (5) 14 IC2 (12) 0 3 (21.4%) 11 (78.6%) UNa (2) Total 10 (15) 65 IC1 (5) 2 7 56 IC2 (54) UNa (6) a UN, not related to any of the known international clonal lineages. ISs in regulatory genes of RND-type efflux pumps First, the four isolate pairs were investigated. Analysis using ISMapper revealed the insertion of ISAba1 in adeS in tigecycline-resistant isolates MB_5 (isolate pair 1) and MB_43 (isolate pair 2). The insertion occurred in reverse orientation and the insertion site differed by two nucleotides indicating independent insertion events (Figure S1A). adeN was disrupted by ISAba1 in the tigecycline-resistant isolate MB_273 (isolate pair 3), while ISAba125 was inserted in adeN in the tigecycline-resistant isolate MB_1044 (isolate pair 4) (Figure S1B). These data were confirmed by Sanger sequencing. Moreover, ISAba1 was inserted in the intergenic region between adeS and adeR in isolate pair 3. Since this insertion occurred in both the tigecycline-intermediate isolate (MB_271) and the tigecycline-resistant isolate (MB_273), an association with reduced tigecycline susceptibility was excluded. All other genes of RND-type efflux systems including the five uncharacterized pumps were identical within an isolate pair. Moreover, analysis of genes previously described to be associated with tigecycline resistance17,18 revealed identical plsC in all isolate pairs, while nucleotide 1077 was deleted in trm in both isolates (MB_131 and MB_1044) of isolate pair 4. Since this frameshift occurred in both isolates of the isolate pair, an association with reduced tigecycline susceptibility was excluded in this study. Increased expression of RND-type efflux pumps Since ISs were found in adeS and adeN in tigecycline-resistant isolates of isolate pairs, we investigated the effect of disruption of the regulatory genes on RND-type efflux pump expression. In all four isolate pairs, the expression of the RND-type efflux pump was significantly increased in tigecycline-resistant isolates (P < 0.05). The insertion of ISAba1 in adeS was associated with a 45-fold and 35-fold increase in expression of adeB in isolate pairs 1 and 2, respectively (Figure 1a). Disruption of adeN by ISAba1 or ISAba125 was associated with a 6-fold and 2-fold increase in expression of the efflux pump adeJ in isolate pairs 3 and 4, respectively (Figure 1b). Figure 1. View largeDownload slide Relative adeB and adeJ expression of isolate pairs determined by qRT–PCR. (a) Expression of adeB of isolate pair 1 (MB_2 and MB_5) and isolate pair 2 (MB_7 and MB_43). (b) Expression of adeJ of isolate pair 3 (MB_271 and MB_273) and isolate pair 4 (MB_131 and MB_1044). The number of transcripts in tigecycline-susceptible strains was related to the strains with higher tigecycline MICs after being normalized to the expression of the reference gene rpoB. Results are represented as mean ± SEM. Statistical analysis was done by performing an unpaired t-test using the recorded absolute values. ***P< 0.001, **P< 0.01 *P< 0.05. Figure 1. View largeDownload slide Relative adeB and adeJ expression of isolate pairs determined by qRT–PCR. (a) Expression of adeB of isolate pair 1 (MB_2 and MB_5) and isolate pair 2 (MB_7 and MB_43). (b) Expression of adeJ of isolate pair 3 (MB_271 and MB_273) and isolate pair 4 (MB_131 and MB_1044). The number of transcripts in tigecycline-susceptible strains was related to the strains with higher tigecycline MICs after being normalized to the expression of the reference gene rpoB. Results are represented as mean ± SEM. Statistical analysis was done by performing an unpaired t-test using the recorded absolute values. ***P< 0.001, **P< 0.01 *P< 0.05. Resistome analysis and increased MICs associated with efflux To exclude the effect of other resistance determinants, the presence of acquired resistance genes was analysed using ResFinder and revealed identical resistomes among isolates representing an isolate pair (Figure S2). Moreover, the flavin-dependent monooxygenase TetX, which has been described to be associated with resistance to tigecycline,30 was not detected in any of the A. baumannii isolates by ResFinder or through our own sequence analysis. Therefore, any difference in MICs between isolates of an isolate pair is most likely due to increased adeB or adeJ expression. While the MICs of minocycline, ciprofloxacin, levofloxacin and meropenem in isolates with elevated adeJ expression were increased, no significant changes in MICs of other antimicrobial agents were observed (Table 3). In contrast, we observed an increase in the MICs of amikacin, gentamicin, minocycline, ciprofloxacin, levofloxacin, azithromycin, erythromycin, chloramphenicol and rifampicin in isolates with elevated adeB expression. The increase in MICs was in some cases 4-fold (e.g. gentamicin and minocycline) or higher and changed the phenotype in isolate pair 2 from susceptible to resistant to amikacin (Table 3). Differences in fluoroquinolone MICs between isolates of an isolate pair were not due to gyrA/B or parC/E mutations, since these genes were identical in isolate pairs. These data show that adeS disruption was associated with increased MICs in at least six different antimicrobial classes and adeN disruption in three antimicrobial classes. Table 3. Mean MICs of 12 different antimicrobials for four A. baumannii isolate pairs; all MICs (mg/L) were determined by agar dilution in three independent experiments Isolate pair 1 Isolate pair 2 Isolate pair 3 Isolate pair 4 Antimicrobial MB_2 MB_5 MB_7 MB_43 MB_271 MB_273 MB_131 MB_1044 Tigecycline 1 16 2 16 2 4 2 4 Tetracycline ≥256 ≥256 ≥256 ≥256 ≥256 ≥256 8 8 Minocycline 8 16 4 16 4 32 2 2 Ciprofloxacin 64 ≥256 64 ≥256 128 ≥256 64 128 Levofloxacin 8 64 16 64 8 16 8 32 Amikacin 64 ≥256 8 ≥256 16 16 ≥256 ≥256 Gentamicin 16 128 8 128 ≥256 ≥256 ≥256 ≥256 Chloramphenicol 128 ≥256 128 ≥256 ≥256 ≥256 ≥256 ≥256 Azithromycin 64 128 32 128 32 32 128 128 Erythromycin 32 128 32 128 64 64 64 64 Meropenem 8 8 8 8 128 ≥256 64 64 Rifampicin 128 128 64 128 8 8 4 4 Colistin 2 4 2 2 2 2 ≥256 ≥256 Isolate pair 1 Isolate pair 2 Isolate pair 3 Isolate pair 4 Antimicrobial MB_2 MB_5 MB_7 MB_43 MB_271 MB_273 MB_131 MB_1044 Tigecycline 1 16 2 16 2 4 2 4 Tetracycline ≥256 ≥256 ≥256 ≥256 ≥256 ≥256 8 8 Minocycline 8 16 4 16 4 32 2 2 Ciprofloxacin 64 ≥256 64 ≥256 128 ≥256 64 128 Levofloxacin 8 64 16 64 8 16 8 32 Amikacin 64 ≥256 8 ≥256 16 16 ≥256 ≥256 Gentamicin 16 128 8 128 ≥256 ≥256 ≥256 ≥256 Chloramphenicol 128 ≥256 128 ≥256 ≥256 ≥256 ≥256 ≥256 Azithromycin 64 128 32 128 32 32 128 128 Erythromycin 32 128 32 128 64 64 64 64 Meropenem 8 8 8 8 128 ≥256 64 64 Rifampicin 128 128 64 128 8 8 4 4 Colistin 2 4 2 2 2 2 ≥256 ≥256 Table 3. Mean MICs of 12 different antimicrobials for four A. baumannii isolate pairs; all MICs (mg/L) were determined by agar dilution in three independent experiments Isolate pair 1 Isolate pair 2 Isolate pair 3 Isolate pair 4 Antimicrobial MB_2 MB_5 MB_7 MB_43 MB_271 MB_273 MB_131 MB_1044 Tigecycline 1 16 2 16 2 4 2 4 Tetracycline ≥256 ≥256 ≥256 ≥256 ≥256 ≥256 8 8 Minocycline 8 16 4 16 4 32 2 2 Ciprofloxacin 64 ≥256 64 ≥256 128 ≥256 64 128 Levofloxacin 8 64 16 64 8 16 8 32 Amikacin 64 ≥256 8 ≥256 16 16 ≥256 ≥256 Gentamicin 16 128 8 128 ≥256 ≥256 ≥256 ≥256 Chloramphenicol 128 ≥256 128 ≥256 ≥256 ≥256 ≥256 ≥256 Azithromycin 64 128 32 128 32 32 128 128 Erythromycin 32 128 32 128 64 64 64 64 Meropenem 8 8 8 8 128 ≥256 64 64 Rifampicin 128 128 64 128 8 8 4 4 Colistin 2 4 2 2 2 2 ≥256 ≥256 Isolate pair 1 Isolate pair 2 Isolate pair 3 Isolate pair 4 Antimicrobial MB_2 MB_5 MB_7 MB_43 MB_271 MB_273 MB_131 MB_1044 Tigecycline 1 16 2 16 2 4 2 4 Tetracycline ≥256 ≥256 ≥256 ≥256 ≥256 ≥256 8 8 Minocycline 8 16 4 16 4 32 2 2 Ciprofloxacin 64 ≥256 64 ≥256 128 ≥256 64 128 Levofloxacin 8 64 16 64 8 16 8 32 Amikacin 64 ≥256 8 ≥256 16 16 ≥256 ≥256 Gentamicin 16 128 8 128 ≥256 ≥256 ≥256 ≥256 Chloramphenicol 128 ≥256 128 ≥256 ≥256 ≥256 ≥256 ≥256 Azithromycin 64 128 32 128 32 32 128 128 Erythromycin 32 128 32 128 64 64 64 64 Meropenem 8 8 8 8 128 ≥256 64 64 Rifampicin 128 128 64 128 8 8 4 4 Colistin 2 4 2 2 2 2 ≥256 ≥256 Prevalence of ISs in regulatory genes of RND-type efflux pumps The disruption of adeS or adeN was found in isolate pairs that originated from different patients in different hospitals and cities. In order to investigate the prevalence of ISs in RND-pump regulators in other tigecycline-resistant isolates, a further 65 unique A. baumannii isolates were analysed by WGS. All tigecycline-susceptible and -intermediate isolates displayed undisrupted adeRS, adeN and adeL. In all tigecycline-resistant isolates (n = 56) the regulatory genes adeR and adeL were undisrupted. A disruption of adeS or adeN was detected in a total of 23 tigecycline-resistant isolates (41.1%) (Table 4). ISAba1 was found inserted in adeS in three isolates (5.4%) and was associated with tigecycline MIC values of 16 mg/L. In contrast, adeN was disrupted in 20 isolates (35.7%) and the MICs were 2- to 4-fold lower compared with isolates with disrupted adeS (Figure S3). In the majority of isolates adeN was disrupted by ISAba1 (n = 18, 32.1%), but also ISAba27 (n = 1, 1.8%) and ISAba125 (n = 1, 1.8%) were found. In the isolate with ISAba125 disrupting adeN, ISAba1 was additionally inserted in the intergenic region of adeRS (MIC 8 mg/L). Table 4. Overview of ISs and other differences in the efflux pump regulators adeS and adeN in tigecycline-resistant (MIC > 2 mg/L) A. baumannii isolates Genetic modification Number of isolates adeS adeN ISAba1 3 18 ISAba27 0 1 ISAba125 0 1 ISAba1 insertion in intergenic region of adeRS 1 0 adeRSABC missing or truncated 2 0 1-nucleotide deletion 0 6 6-nucleotide insertion 0 2 Premature stop codon 0 3 Genetic modification Number of isolates adeS adeN ISAba1 3 18 ISAba27 0 1 ISAba125 0 1 ISAba1 insertion in intergenic region of adeRS 1 0 adeRSABC missing or truncated 2 0 1-nucleotide deletion 0 6 6-nucleotide insertion 0 2 Premature stop codon 0 3 Table 4. Overview of ISs and other differences in the efflux pump regulators adeS and adeN in tigecycline-resistant (MIC > 2 mg/L) A. baumannii isolates Genetic modification Number of isolates adeS adeN ISAba1 3 18 ISAba27 0 1 ISAba125 0 1 ISAba1 insertion in intergenic region of adeRS 1 0 adeRSABC missing or truncated 2 0 1-nucleotide deletion 0 6 6-nucleotide insertion 0 2 Premature stop codon 0 3 Genetic modification Number of isolates adeS adeN ISAba1 3 18 ISAba27 0 1 ISAba125 0 1 ISAba1 insertion in intergenic region of adeRS 1 0 adeRSABC missing or truncated 2 0 1-nucleotide deletion 0 6 6-nucleotide insertion 0 2 Premature stop codon 0 3 Other differences in regulatory genes of RND-type efflux pumps In a total of 7 tigecycline-intermediate and 33 tigecycline-resistant isolates, no insertion elements were detected in the regulatory genes of RND-type efflux pumps. Therefore, the regulators were analysed for mutations, deletions or insertions. In two intermediate and in two resistant isolates adeS and adeR could not be detected in draft genomes or by PCR. Further analysis revealed that in three isolates adeC and adeB were at least partially present, whereas adeA was not detectable. In one isolate the whole adeRSABC operon could not be found in the draft genome or by PCR. Instead, these four isolates showed differences in adeN, which included a large 87-nucleotide deletion (position 236–323) and a 6-nucleotide insertion in the other three isolates (Table 4). This 6-nucleotide insertion was found in a total of five isolates, but only in two tigecycline-resistant isolates with MICs of 4 mg/L (Table 4 and Figure S3). Other differences found in adeN included a point mutation creating a premature stop codon at position 592 (n = 3) and the deletion of nucleotide 62 leading to a frameshift mutation (n = 6) (Table 4). In both cases the tigecycline MICs were between 4 and 8 mg/L (Figure S3). Amino acid substitutions in the RND-pump regulators The amino acid sequences of AdeRS, AdeN and AdeL of all 65 isolates were compared with that of the reference strain A. baumannii ACICU. It is important to keep in mind that amino acid substitutions can also be polymorphic, for example in isolates representing different clonal lineages. In order to exclude polymorphisms, the reference strains A. baumannii AYE, ATCC 17978 and ATCC 19606 were included in the analysis. All substitutions found in the four reference strains or in the two susceptible isolates were identified as polymorphisms and excluded. For example, I120V, V136A and L241P were observed in AdeR in the reference strains and the substitutions V14I and V243I were detected in AdeR in one of the susceptible isolates when compared with A. baumannii ACICU. Therefore, these substitutions were considered to be silent polymorphisms. The same procedure was followed for the other regulators. Although a high number of polymorphisms were detected, various amino acid substitutions in AdeRS, AdeN and AdeL were associated with tigecycline resistance (Table S2 and Figure S4). In AdeN the amino acid substitutions H170Y (n = 1), D181N (n = 1) and G215V (n = 3) were detected, while in AdeL only the substitution I37L (n = 6) seemed to be associated with tigecycline resistance. In AdeR, six different substitutions were found (D21V, G25S, D26N, N115K, V119I, E147K), either as a single substitution per isolate or in combinations. The amino acid substitutions D167N and S357P were found in AdeS in two isolates, while the substitutions V137F and A325T were detected in a total of three isolates (Table S2 and Figure S4). The substitution I62M was detected in one tigecycline-resistant isolate as well as in two intermediate isolates. In only 16 isolates were amino acid substitutions associated with tigecycline resistance and MICs of 4–16 mg/L exclusively, i.e. no insertion elements or other mutations were detectable in the RND-type efflux pump regulators (Figure S3). Alternative tigecycline resistance mechanisms An association of trm with increased tigecycline MICs has been excluded in our analysis of isolate pairs. Furthermore, amino acid substitutions were detected in PlsC in seven isolates, including also a tigecycline-susceptible isolate. Therefore, the substitutions are most likely polymorphisms and were excluded. In total, six tigecycline-resistant isolates remained in this study, which carried none of the above-mentioned mutations or other differences in the RND-type efflux pump regulators, trm or plsC, so that a different and hitherto unknown resistance mechanism is suggested. Discussion Previous studies into tigecycline resistance in A. baumannii have mostly concentrated on relatively few isolates and single resistance mechanisms. The use of isolate pairs to investigate tigecycline resistance was first approached by Hornsey et al.31 who found multiple SNPs between their susceptible and resistant isolates, including an alanine to valine substitution in AdeS. Other work also found mutations in AdeS in association with tigecycline resistance,32,33 whereas others have shown the involvement of AdeR.26,34 In the present study we have investigated tigecycline resistance as part of a large multicentre study involving patients from 15 hospitals in Greece, Italy and Spain. We investigated the tigecycline resistance mechanisms using isolate pairs as well as single isolates from 65 patients. Isolate pairs are of great value for analysing the emergence of antimicrobial resistance. These isolates display the same isogenic background and the susceptible isolates precede the resistant isolates so that variations typically seen in epidemiologically unrelated strains are excluded and genetic differences are most likely a result of antimicrobial selection pressure. Our approach revealed that disruption of adeS or adeN by ISs was associated with tigecycline resistance and resulted in increased adeB and adeJ expression, respectively. Furthermore, disruption of adeS also was associated with increased MICs of at least six different antimicrobial classes, which indicates a higher impact of adeB overexpression on antimicrobial resistance in clinical settings. The disruption of adeS by ISAba1 has been reported previously and was associated with elevated MICs of tigecycline and other antimicrobial agents.35,36 The TetR-like transcription regulator adeN is a transcriptional repressor and insertional inactivation has been shown to increase adeJ expression in vitro,15 but overexpression of adeJ has been reported to be toxic for A. baumannii.12 These findings might explain the lower expression of adeJ and lower tigecycline MICs for isolates with disrupted adeN compared with isolates with ISAba1 insertion in adeS. In the 65 unique A. baumannii isolates, ISAba1 was the most common IS in adeN. This is not surprising, since this IS element is one of the most common ISs in A. baumannii and it is found in multiple sites within the genome and the plasmidome.37,38 Furthermore, it has been described in association with an MDR phenotype in A. baumannii,39 since ISAba1 often provides a strong promotor for overexpression of carbapenemases or the −35 region, which can generate a hybrid promotor with a −10 region located downstream of the IS.40 Besides ISAba1, the ISs ISAba125 and ISAba27 were also detected in adeN. To our knowledge this is the first report of ISAba125 and ISAba27 insertions in adeN in clinical A. baumannii isolates. Furthermore, our results show that ISs are more prevalent in adeN than in adeS. Recent studies have shown that disruption of adeN by ISAba1 increased the virulence and pathogenicity of A. baumannii.41,42 This is most concerning, as more than 30% of A. baumannii isolates in this study were found to carry ISAba1 or other ISs in adeN. Since adeS disruption has been associated with high-level drug resistance and adeN disruption with increased virulence and moderate drug resistance, both adeS and adeN disruption might be useful as potential markers of MDR and virulent A. baumannii strains.41,42 In four isolates adeS could not be detected. The loss of adeRS or adeB has recently been reported to significantly alter the transcriptional landscape of two A. baumannii strains including a decreased expression of adeABC and a subsequent decrease in MICs of several antimicrobial agents.43 Since in all isolates with a missing adeRSABC operon, mutations in adeN were detected, an association of adeJ overexpression with elevated tigecycline MICs is likely. For instance, a single nucleotide deletion was detected in adeN in six isolates creating a frameshift and premature stop codon. A similar frameshift mutation was reported by Rosenfeld et al.15 and revealed overexpression of adeJ and suggests similar elevated adeJ expression in our isolates. The effect of the observed adeN mutations on adeJ expression and subsequently on tigecycline resistance remains to be elucidated. Besides insertions or deletions in regulatory genes of efflux pumps, amino acid substitutions in AdeS or AdeR have been described to be associated with increased adeB expression and consequently with an MDR phenotype in A. baumannii.14,26,32–34 Since 16 isolates in this study displayed tigecycline MICs of > 2 mg/L independent of insertions, deletions or frameshifts in adeRS or adeN, amino acid substitutions could also play a role in tigecycline resistance. When analysing such mutations it is necessary to decide if the respective mutation is the cause of resistance or a simple genetic polymorphism. Yoon et al.32 reported that the amino acid substitution A94V in AdeS is such a polymorphism of strains belonging to IC1. These findings correlate well with our analysis of four reference strains and two tigecycline-susceptible isolates. Nevertheless, various substitutions were detected in the RND-pump regulatory proteins. Since none of the substitutions found in our isolates has been described previously, future studies should provide an insight into these mutations and their association with an MDR phenotype of A. baumannii. So far, only the amino acid substitution D20N has been investigated and demonstrated to be the cause of adeB overexpression and an MDR phenotype.34 Recently the crystal structure of AdeR was resolved and important amino acid residues that are associated with activation as well as DNA-binding and dimerization of AdeR were identified.44 The residue D63 was identified as a phosphorylation site and was shown to form a pocket with the residues G19, D20 and L112. In this study we identified several amino acid substitutions in AdeR, which were located near or in the pocket described by Wen et al.44 This includes the substitutions D21V, G25S, D26N, N115K and V119I, which were found as single substitutions or in combination in 25 isolates and might indicate a hotspot for mutations in AdeR in clinical A. baumannii isolates. In conclusion, using a genomic approach our study revealed multiple and diverse tigecycline resistance mechanisms involving the RND-type efflux pump regulators. We found a high prevalence of ISs in adeS and adeN, though disruption of adeS probably has a greater impact on resistance to other antimicrobial agents and therefore in clinical settings. Besides ISs, nucleotide deletions and insertions, premature stop codons and amino acid substitutions were found in AdeRS, AdeL and AdeN. Additionally, a potential hotspot for mutations was identified in AdeR. Our results gave insight into the large diversity of mechanisms associated with tigecycline resistance in A. baumannii isolates, highlighting the importance and potential of genomic approaches in the analysis of antimicrobial resistance in A. baumannii. Acknowledgements We thank the team of curators of the Institut Pasteur Acinetobacter MLST system for curating the data and making them publicly available at http://pubmlst.org/abaumannii/. Funding This work was supported by the German Research Council (DFG) – FOR2251 (www.acinetobacter.de). MagicBullet is a project funded by the European Union – Directorate General for Research and Innovation through the Seventh Framework Program for Research and Development (grant agreement 278232) and has been running since 1 January 2012 (duration, 48 months). Transparency declarations None to declare. Supplementary data Tables S1 and S2 and Figures S1 to S4 appear as Supplementary data at JAC Online. References 1 Peleg AY , Seifert H , Paterson DL. 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Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454 . Antimicrob Agents Chemother 2001 ; 45 : 3375 – 80 . Google Scholar CrossRef Search ADS PubMed 12 Damier-Piolle L , Magnet S , Brémont S et al. AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii . Antimicrob Agents Chemother 2008 ; 52 : 557 – 62 . Google Scholar CrossRef Search ADS PubMed 13 Coyne S , Rosenfeld N , Lambert T et al. Overexpression of resistance-nodulation-cell division pump AdeFGH confers multidrug resistance in Acinetobacter baumannii . Antimicrob Agents Chemother 2010 ; 54 : 4389 – 93 . Google Scholar CrossRef Search ADS PubMed 14 Marchand I , Damier-Piolle L , Courvalin P et al. Expression of the RND-type efflux pump AdeABC in Acinetobacter baumannii is regulated by the AdeRS two-component system . Antimicrob Agents Chemother 2004 ; 48 : 3298 – 304 . 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Tigecycline resistance in Acinetobacter baumannii mediated by frameshift mutation in plsC, encoding 1-acyl-sn-glycerol-3-phosphate acyltransferase . Eur J Clin Microbiol Infect Dis 2014 ; 34 : 625 – 31 . Google Scholar CrossRef Search ADS PubMed 19 Rosso-Fernández C , Garnacho-Montero J , Antonelli M et al. Safety and efficacy of colistin versus meropenem in the empirical treatment of ventilator-associated pneumonia as part of a macro-project funded by the Seventh Framework Program of the European Commission studying off-patent antibiotics: study protocol for a randomized controlled trial . Trials 2015 ; 16 : 102. Google Scholar CrossRef Search ADS PubMed 20 Nowak J , Zander E , Stefanik D et al. High incidence of pandrug-resistant Acinetobacter baumannii isolates collected from patients with ventilator-associated pneumonia in Greece, Italy and Spain as part of the MagicBullet clinical trial . J Antimicrob Chemother 2017 ; 72 : 3277 – 82 . Google Scholar CrossRef Search ADS PubMed 21 Higgins PG , Prior K , Harmsen D et al. Development and evaluation of a core genome multilocus typing scheme for whole-genome sequence-based typing of Acinetobacter baumannii . PLoS One 2017 ; 12 : e0179228 . Google Scholar CrossRef Search ADS PubMed 22 Clinical and Laboratory Standards Institute . Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Sixth Informational Supplement M100S-S26 . CLSI , Wayne, PA, USA , 2016 . 23 Zankari E , Hasman H , Cosentino S et al. Identification of acquired antimicrobial resistance genes . J Antimicrob Chemother 2012 ; 67 : 2640 – 4 . Google Scholar CrossRef Search ADS PubMed 24 Hawkey J , Hamidian M , Wick RR et al. ISMapper: identifying transposase insertion sites in bacterial genomes from short read sequence data . BMC Genomics 2015 ; 16 : 667. Google Scholar CrossRef Search ADS PubMed 25 Jolley KA , Maiden MCJ. BIGSdb: scalable analysis of bacterial genome variation at the population level . BMC Bioinformatics 2010 ; 11 : 595. Google Scholar CrossRef Search ADS PubMed 26 Higgins PG , Schneiders T , Hamprecht A et al. In vivo selection of a missense mutation in adeR and conversion of the novel blaOXA-164 gene into blaOXA-58 in carbapenem-resistant Acinetobacter baumannii isolates from a hospitalized patient . Antimicrob Agents Chemother 2010 ; 54 : 5021 – 7 . Google Scholar CrossRef Search ADS PubMed 27 Hornsey M , Ellington MJ , Doumith M et al. AdeABC-mediated efflux and tigecycline MICs for epidemic clones of Acinetobacter baumannii . J Antimicrob Chemother 2010 ; 65 : 1589 – 93 . Google Scholar CrossRef Search ADS PubMed 28 Bratu S , Landman D , Martin DA et al. Correlation of antimicrobial resistance with β-lactamases, the OmpA-like porin, and efflux pumps in clinical isolates of Acinetobacter baumannii endemic to New York city . Antimicrob Agents Chemother 2008 ; 52 : 2999 – 3005 . Google Scholar CrossRef Search ADS PubMed 29 Siguier P , Perochon J , Lestrade L et al. ISfinder: the reference centre for bacterial insertion sequences . Nucleic Acids Res 2006 ; 34 : D32 – 6 . Google Scholar CrossRef Search ADS PubMed 30 Moore IF , Hughes DW , Wright GD. Tigecycline is modified by the flavin-dependent monooxygenase TetX . Biochemistry 2005 ; 44 : 11829 – 35 . Google Scholar CrossRef Search ADS PubMed 31 Hornsey M , Loman N , Wareham DW et al. Whole-genome comparison of two Acinetobacter baumannii isolates from a single patient, where resistance developed during tigecycline therapy . J Antimicrob Chemother 2011 ; 66 : 1499 – 503 . Google Scholar CrossRef Search ADS PubMed 32 Yoon E-J , Courvalin P , Grillot-Courvalin C. RND-type efflux pumps in multidrug-resistant clinical isolates of Acinetobacter baumannii: major role of AdeABC overexpression and AdeRS mutations . Antimicrob Agents Chemother 2013 ; 57 : 2989 – 95 . Google Scholar CrossRef Search ADS PubMed 33 Montana S , Vilacoba E , Traglia GM et al. Genetic variability of AdeRS two-component system associated with tigecycline resistance in Acinetobacter baumannii isolates . Curr Microbiol 2015 ; 71 : 76 – 82 . Google Scholar CrossRef Search ADS PubMed 34 Nowak J , Schneiders T , Seifert H et al. The Asp20-to-Asn substitution in the response regulator AdeR leads to enhanced efflux activity of AdeB in Acinetobacter baumannii . Antimicrob Agents Chemother 2016 ; 60 : 1085 – 90 . Google Scholar CrossRef Search ADS PubMed 35 Ruzin A , Keeney D , Bradford PA. AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus-Acinetobacter baumannii complex . J Antimicrob Chemother 2007 ; 59 : 1001 – 4 . Google Scholar CrossRef Search ADS PubMed 36 Sun J-R , Perng C-L , Chan M-C et al. A truncated AdeS kinase protein generated by ISAba1 insertion correlates with tigecycline resistance in Acinetobacter baumannii . PLoS One 2012 ; 7 : e49534 . Google Scholar CrossRef Search ADS PubMed 37 Mugnier PD , Poirel L , Nordmann P. Functional analysis if insertion sequence ISAba1, responsible for genomic plasticity of Acinetobacter baumannii . J Bacteriol 2009 ; 191 : 2414 – 8 . Google Scholar CrossRef Search ADS PubMed 38 Ruiz M , Marti S , Fernandez-Cuenca F et al. Prevalence of ISAba1 in epidemiologically unrelated Acinetobacter baumannii clinical isolates . FEMS Microbiol Lett 2007 ; 274 : 63 – 6 . Google Scholar CrossRef Search ADS PubMed 39 Adams MD , Chan ER , Molyneaux ND et al. Genomewide analysis of divergence of antibiotic resistance determinants in closely related isolates of Acinetobacter baumannii . Antimicrob Agents Chemother 2010 ; 54 : 3569 – 77 . Google Scholar CrossRef Search ADS PubMed 40 Turton JF , Warf ME , Woodford N et al. The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii . FEMS Microbiol Lett 2006 ; 258 : 72 – 7 . Google Scholar CrossRef Search ADS PubMed 41 Saranathan R , Pagal S , Sawant AR et al. Disruption of tetR type regulator adeN by mobile genetic element confers elevated virulence in Acinetobacter baumannii . Virulence 2017 ; 8 : 119 . Google Scholar CrossRef Search ADS 42 Oh MH , Choi CH , Lee JC. The effect of ISAba1-mediated adeN gene disruption on Acinetobacter baumannii pathogenesis . Virulence 2017 ; 8 : 1088 – 90 . Google Scholar CrossRef Search ADS PubMed 43 Richmond GE , Evans LP , Anderson MJ et al. The Acinetobacter baumannii two-component system AdeRS regulates genes required for multidrug efflux, biofilm formation, and virulence in a strain-specific manner . mBio 2016 ; 7 : e00430 - 16 . Google Scholar PubMed 44 Wen Y , Ouyang Z , Yu Y et al. Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region . Nucleic Acids Res 2017 ; 45 : 9773 – 87 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with tigecycline resistance in Acinetobacter baumannii

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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0305-7453
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1460-2091
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10.1093/jac/dky083
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Abstract

Abstract Objectives To investigate the mechanisms of tigecycline resistance in isogenic Acinetobacter baumannii isolate pairs as well as 65 unique clinical A. baumannii isolates obtained during the MagicBullet clinical trial from Greece, Italy and Spain. Methods A. baumannii isolates were subjected to WGS and the regulatory genes of resistance-nodulation-cell division (RND)-type efflux pumps were analysed. MICs were determined by agar dilution and the expression of RND-type efflux pumps was measured by semi-quantitative RT–PCR. Results In isolate pairs, disruption of adeS or adeN by ISs increased adeB or adeJ expression and conferred increased resistance to at least three antimicrobial classes, respectively. The insertion of ISAba1 in adeN was observed in more than 30% of tested isolates and was the most prevalent IS. Furthermore, the insertion of ISAba125 and ISAba27 into adeN was observed for the first time in A. baumannii isolates. Besides ISs, several different mutations were observed in adeN (e.g. deletions and premature stop codons), all of which led to increased tigecycline MICs. Moreover, several amino acid substitutions were detected in AdeRS, AdeN and AdeL. Of note, the substitutions D21V, G25S and D26N in AdeR were found in multiple sequences and suggest a mutational hotspot. Conclusions This study provides an insight into the different mechanisms associated with tigecycline resistance using a genomic approach and points out the importance of considering adeRS and adeN as markers for tigecycline-resistant A. baumannii isolates. Introduction Acinetobacter baumannii is a serious hospital-acquired pathogen causing wound and urinary tract infections, bloodstream infections, pneumonia and meningitis, especially in patients in ICUs.1 It is of major concern for the healthcare system because of its ability to up-regulate or acquire antimicrobial resistance determinants against multiple antimicrobial classes (e.g. β-lactams, tetracyclines, aminoglycosides, cephalosporins and fluoroquinolones),2 its prolonged survival on dry surfaces and its propensity for epidemic spread.2,3 In recent years, A. baumannii has developed resistance to carbapenems, which is mainly caused by class D β-lactamases such as blaOXA-23-like.4,5 Since the frequency of MDR and in particular carbapenem-resistant strains is increasing, tigecycline and colistin are among the few remaining antibiotics for the treatment of carbapenem-resistant A. baumannii infections. Besides carbapenem resistance, tigecycline and colistin resistance are increasingly being reported.6,7 Tigecycline resistance is often mediated by overexpression of resistance-nodulation-cell division (RND) efflux pumps.8 These tripartite pumps mainly consist of a periplasmic membrane fusion protein (MFP) that interacts with a cytoplasmic membrane-spanning transport protein and an outer membrane pore (OMP).9,10 There are three characterized RND-type efflux pumps in A. baumannii: AdeABC, AdeFGH and AdeIJK.11–13 They are intrinsically encoded on the chromosome and strictly regulated. AdeABC is regulated by the two-component regulatory system AdeRS,14 the LysR-type transcription regulator AdeL is responsible for the regulation of adeFGH expression13 and the TetR-like transcription regulator AdeN represses expression of adeIJK.15 Additionally, a further five as-yet-uncharacterized RND efflux pumps have been reported in A. baumannii.16 Besides RND-type efflux pumps, several other tigecycline resistance mechanisms have been proposed for A. baumannii. Chen et al.17 reported a deletion mutation in the tigecycline-related-methyltransferase (trm) gene, which encodes a SAM-dependent methyltransferase associated with resistance to tigecycline. Moreover, a frameshift mutation in a 1-acyl-sn-glycerol-3-phosphate acyltransferase gene (plsC) was described to increase the MIC of tigecycline.18 The objective of this study was to determine the mechanism of tigecycline resistance in A. baumannii isolates recovered from respiratory and stool samples of patients with ventilator-associated pneumonia (VAP) at hospitals in Greece, Italy and Spain between 2012 and 2015 included in the MagicBullet clinical trial.19,20 Materials and methods Bacterial isolates A. baumannii isolates were recovered from respiratory or stool samples from 65 patients with VAP that were obtained between 2012 and 2015 during the MagicBullet clinical trial.19,20 Initially, we investigated A. baumannii isolates that were consecutively obtained on multiple occasions during a patient’s hospital course from respiratory tract and stool samples. Isolates demonstrating a phenotype shift in tigecycline MIC, i.e. where the initial isolate was tigecycline susceptible or intermediate at an early sampling point and was tigecycline resistant at a later timepoint, were included in this study and termed isolate pairs. In addition, isolates representing isolate pairs were required to demonstrate less than 10 allele differences in both the core and accessory genomes by WGS (see below).21 Furthermore, we analysed 65 unique A. baumannii respiratory tract isolates that were recovered from 65 patients in Greece (n = 37), Italy (n = 14) and Spain (n = 14), respectively. Antimicrobial susceptibility testing Susceptibility to tetracycline, gentamicin (Sigma–Aldrich, Steinheim, Germany), meropenem, tigecycline, amikacin, colistin, minocycline, rifampicin (Molekula, Newcastle upon Tyne, UK), levofloxacin (Sanofi Aventis, Frankfurt, Germany), ciprofloxacin (Bayer Pharma AG, Berlin, Germany), azithromycin (Pfizer Pharma GmbH, Münster, Germany) and erythromycin (AppliChem, Darmstadt, Germany) was determined by agar dilution following the current CLSI guidelines.22 For isolates representing isolate pairs, agar dilution was repeated at least three times. Tigecycline resistance was defined as an MIC of > 2 mg/L using the EUCAST resistance breakpoints for Enterobacteriaceae, as no such breakpoint is available for A. baumannii. WGS and sequence data analysis All respiratory A. baumannii isolates as well as the four isolate pairs were subjected to WGS and analysed as previously described.21 Further analysis was carried out by bioinformatic tools ResFinder for resistome analysis and ISMapper to search for ISs.23,24 Moreover, MLST alleles were extracted using PubMLST (https://pubmlst.org/abaumannii/).25 PCR-based methods to confirm tigecycline resistance determinants All primers used in this study are listed in Table S1 (available as Supplementary data at JAC Online). Crude cell lysates or genomic DNA were used as templates for PCR. Differences in ORFs between isolates representing an isolate pair or the presence of ISs, as determined by WGS, were confirmed by PCR and Sanger sequencing (LGC Genomics GmbH, Berlin, Germany). Putative ISs were further analysed using the BLAST tool of ISfinder (http://www-is.biotoul.fr).29 Semi-quantitative RT–PCR (qRT–PCR) RNA extraction, cDNA synthesis and qRT–PCR to measure the expression of adeB or adeJ were performed, as previously described, using rpoB as the reference gene.26 Primer sequences are shown in Table S1. All qRT–PCR experiments were done in triplicate using freshly prepared RNA and cDNA and were repeated independently at least three times. The relative expression of adeB and adeJ and the number of transcripts were compared between isolates representing an isolate pair. Statistical analysis was done by performing an unpaired t-test using the recorded absolute values. Nucleotide sequence accession number All raw reads generated were submitted to the Sequencing Read Archive (https://www.ncbi.nlm.nih.gov/sra/) of the National Center for Biotechnology Information (NCBI) under the BioProject accession number PRJNA431710. Results Description of isolates To investigate the mechanisms of tigecycline resistance, A. baumannii isolates were initially chosen for comparison in isolate pairs. In total, four isolate pairs were identified that were considered to have the same isogenic background, as demonstrated by WGS, and exhibited an increase in tigecycline MICs (Table 1). Isolate pairs 1 and 2, obtained in Seville, Spain, displayed a shift in tigecycline susceptibility from susceptible or intermediate (MIC 1 or 2 mg/L) to resistant (MIC 16 mg/L), respectively. Isolate pair 3, recovered in Valencia, Spain, exhibited a tigecycline MIC increase from 2 to 4 mg/L. Isolate pair 4, obtained from a hospital in Athens, Greece, also had tigecycline MICs that differed by one dilution step (2 to 4 mg/L). The differences in tigecycline MICs were seen consistently in three independent experiments in all isolate pairs. Table 1. Overview of isolate pairs chosen to investigate tigecycline resistance; clonal lineages were determined by cgMLST and tigecycline MICs were determined by agar dilution Patient Isolate Isolate pair Day of isolationa Sample type Origin Clonal lineage Oxford MLST type Tigecycline MIC (mg/L) 01-001 MB_2 1 3 respiratory Seville, Spain IC2 ST-208 1 MB_5 1 F stool 16 01-002 MB_7 2 8 respiratory Seville, Spain IC2 ST-208 2 MB_43 2 F stool 16 02-077 MB_271 3 3 stool Valencia, Spain IC2 ST-1117 2 MB_273 3 F stool 4 22-168 MB_131 4 0 respiratory Athens, Greece IC1 ST-1567 2 MB_1044 4 F stool 4 Patient Isolate Isolate pair Day of isolationa Sample type Origin Clonal lineage Oxford MLST type Tigecycline MIC (mg/L) 01-001 MB_2 1 3 respiratory Seville, Spain IC2 ST-208 1 MB_5 1 F stool 16 01-002 MB_7 2 8 respiratory Seville, Spain IC2 ST-208 2 MB_43 2 F stool 16 02-077 MB_271 3 3 stool Valencia, Spain IC2 ST-1117 2 MB_273 3 F stool 4 22-168 MB_131 4 0 respiratory Athens, Greece IC1 ST-1567 2 MB_1044 4 F stool 4 F, sample recovered at the end of the patient’s antibiotic treatment. a Hospital day after enrolment into the clinical trial. Table 1. Overview of isolate pairs chosen to investigate tigecycline resistance; clonal lineages were determined by cgMLST and tigecycline MICs were determined by agar dilution Patient Isolate Isolate pair Day of isolationa Sample type Origin Clonal lineage Oxford MLST type Tigecycline MIC (mg/L) 01-001 MB_2 1 3 respiratory Seville, Spain IC2 ST-208 1 MB_5 1 F stool 16 01-002 MB_7 2 8 respiratory Seville, Spain IC2 ST-208 2 MB_43 2 F stool 16 02-077 MB_271 3 3 stool Valencia, Spain IC2 ST-1117 2 MB_273 3 F stool 4 22-168 MB_131 4 0 respiratory Athens, Greece IC1 ST-1567 2 MB_1044 4 F stool 4 Patient Isolate Isolate pair Day of isolationa Sample type Origin Clonal lineage Oxford MLST type Tigecycline MIC (mg/L) 01-001 MB_2 1 3 respiratory Seville, Spain IC2 ST-208 1 MB_5 1 F stool 16 01-002 MB_7 2 8 respiratory Seville, Spain IC2 ST-208 2 MB_43 2 F stool 16 02-077 MB_271 3 3 stool Valencia, Spain IC2 ST-1117 2 MB_273 3 F stool 4 22-168 MB_131 4 0 respiratory Athens, Greece IC1 ST-1567 2 MB_1044 4 F stool 4 F, sample recovered at the end of the patient’s antibiotic treatment. a Hospital day after enrolment into the clinical trial. In an attempt to include as many different tigecycline resistance mechanisms as possible and to determine their prevalence in a large geographical region, 65 unique A. baumannii isolates were analysed. Based on a core-genome MLST (cgMLST) analysis, the majority of isolates belonged to the international clonal lineage IC2 (n = 54, 83.1%), while five isolates (7.7%) belonged to IC1 and six isolates (9.2%) could not be assigned to any known international clonal lineage (Table 2).20 Only two isolates (3.1%) were considered susceptible to tigecycline (MIC ≤ 1 mg/L). Seven isolates (10.8%) showed intermediate MICs of 2 mg/L and the majority of isolates (n = 56, 86.2%) displayed an MIC ≥ 4 mg/L and were therefore considered resistant to tigecycline (Table 2). Table 2. Overview of 65 A. baumannii isolates obtained from patients from Greece, Italy and Spain included in the MagicBullet clinical trial; clonal lineages were determined by cgMLST and tigecycline MICs were determined by agar dilution Country Number of cities (hospitals) Number of patients Clonal lineages (number of isolates) Tigecycline MIC ≤1 mg/L 2 mg/L ≥4 mg/L Greece 4 (7) 37 IC1 (5) 1 (2.7%) 4 (10.8%) 32 (86.5%) IC2 (32) Italy 2 (3) 14 IC2 (10) 1 (7.1%) 0 13 (92.9%) UNa (4) Spain 4 (5) 14 IC2 (12) 0 3 (21.4%) 11 (78.6%) UNa (2) Total 10 (15) 65 IC1 (5) 2 7 56 IC2 (54) UNa (6) Country Number of cities (hospitals) Number of patients Clonal lineages (number of isolates) Tigecycline MIC ≤1 mg/L 2 mg/L ≥4 mg/L Greece 4 (7) 37 IC1 (5) 1 (2.7%) 4 (10.8%) 32 (86.5%) IC2 (32) Italy 2 (3) 14 IC2 (10) 1 (7.1%) 0 13 (92.9%) UNa (4) Spain 4 (5) 14 IC2 (12) 0 3 (21.4%) 11 (78.6%) UNa (2) Total 10 (15) 65 IC1 (5) 2 7 56 IC2 (54) UNa (6) a UN, not related to any of the known international clonal lineages. Table 2. Overview of 65 A. baumannii isolates obtained from patients from Greece, Italy and Spain included in the MagicBullet clinical trial; clonal lineages were determined by cgMLST and tigecycline MICs were determined by agar dilution Country Number of cities (hospitals) Number of patients Clonal lineages (number of isolates) Tigecycline MIC ≤1 mg/L 2 mg/L ≥4 mg/L Greece 4 (7) 37 IC1 (5) 1 (2.7%) 4 (10.8%) 32 (86.5%) IC2 (32) Italy 2 (3) 14 IC2 (10) 1 (7.1%) 0 13 (92.9%) UNa (4) Spain 4 (5) 14 IC2 (12) 0 3 (21.4%) 11 (78.6%) UNa (2) Total 10 (15) 65 IC1 (5) 2 7 56 IC2 (54) UNa (6) Country Number of cities (hospitals) Number of patients Clonal lineages (number of isolates) Tigecycline MIC ≤1 mg/L 2 mg/L ≥4 mg/L Greece 4 (7) 37 IC1 (5) 1 (2.7%) 4 (10.8%) 32 (86.5%) IC2 (32) Italy 2 (3) 14 IC2 (10) 1 (7.1%) 0 13 (92.9%) UNa (4) Spain 4 (5) 14 IC2 (12) 0 3 (21.4%) 11 (78.6%) UNa (2) Total 10 (15) 65 IC1 (5) 2 7 56 IC2 (54) UNa (6) a UN, not related to any of the known international clonal lineages. ISs in regulatory genes of RND-type efflux pumps First, the four isolate pairs were investigated. Analysis using ISMapper revealed the insertion of ISAba1 in adeS in tigecycline-resistant isolates MB_5 (isolate pair 1) and MB_43 (isolate pair 2). The insertion occurred in reverse orientation and the insertion site differed by two nucleotides indicating independent insertion events (Figure S1A). adeN was disrupted by ISAba1 in the tigecycline-resistant isolate MB_273 (isolate pair 3), while ISAba125 was inserted in adeN in the tigecycline-resistant isolate MB_1044 (isolate pair 4) (Figure S1B). These data were confirmed by Sanger sequencing. Moreover, ISAba1 was inserted in the intergenic region between adeS and adeR in isolate pair 3. Since this insertion occurred in both the tigecycline-intermediate isolate (MB_271) and the tigecycline-resistant isolate (MB_273), an association with reduced tigecycline susceptibility was excluded. All other genes of RND-type efflux systems including the five uncharacterized pumps were identical within an isolate pair. Moreover, analysis of genes previously described to be associated with tigecycline resistance17,18 revealed identical plsC in all isolate pairs, while nucleotide 1077 was deleted in trm in both isolates (MB_131 and MB_1044) of isolate pair 4. Since this frameshift occurred in both isolates of the isolate pair, an association with reduced tigecycline susceptibility was excluded in this study. Increased expression of RND-type efflux pumps Since ISs were found in adeS and adeN in tigecycline-resistant isolates of isolate pairs, we investigated the effect of disruption of the regulatory genes on RND-type efflux pump expression. In all four isolate pairs, the expression of the RND-type efflux pump was significantly increased in tigecycline-resistant isolates (P < 0.05). The insertion of ISAba1 in adeS was associated with a 45-fold and 35-fold increase in expression of adeB in isolate pairs 1 and 2, respectively (Figure 1a). Disruption of adeN by ISAba1 or ISAba125 was associated with a 6-fold and 2-fold increase in expression of the efflux pump adeJ in isolate pairs 3 and 4, respectively (Figure 1b). Figure 1. View largeDownload slide Relative adeB and adeJ expression of isolate pairs determined by qRT–PCR. (a) Expression of adeB of isolate pair 1 (MB_2 and MB_5) and isolate pair 2 (MB_7 and MB_43). (b) Expression of adeJ of isolate pair 3 (MB_271 and MB_273) and isolate pair 4 (MB_131 and MB_1044). The number of transcripts in tigecycline-susceptible strains was related to the strains with higher tigecycline MICs after being normalized to the expression of the reference gene rpoB. Results are represented as mean ± SEM. Statistical analysis was done by performing an unpaired t-test using the recorded absolute values. ***P< 0.001, **P< 0.01 *P< 0.05. Figure 1. View largeDownload slide Relative adeB and adeJ expression of isolate pairs determined by qRT–PCR. (a) Expression of adeB of isolate pair 1 (MB_2 and MB_5) and isolate pair 2 (MB_7 and MB_43). (b) Expression of adeJ of isolate pair 3 (MB_271 and MB_273) and isolate pair 4 (MB_131 and MB_1044). The number of transcripts in tigecycline-susceptible strains was related to the strains with higher tigecycline MICs after being normalized to the expression of the reference gene rpoB. Results are represented as mean ± SEM. Statistical analysis was done by performing an unpaired t-test using the recorded absolute values. ***P< 0.001, **P< 0.01 *P< 0.05. Resistome analysis and increased MICs associated with efflux To exclude the effect of other resistance determinants, the presence of acquired resistance genes was analysed using ResFinder and revealed identical resistomes among isolates representing an isolate pair (Figure S2). Moreover, the flavin-dependent monooxygenase TetX, which has been described to be associated with resistance to tigecycline,30 was not detected in any of the A. baumannii isolates by ResFinder or through our own sequence analysis. Therefore, any difference in MICs between isolates of an isolate pair is most likely due to increased adeB or adeJ expression. While the MICs of minocycline, ciprofloxacin, levofloxacin and meropenem in isolates with elevated adeJ expression were increased, no significant changes in MICs of other antimicrobial agents were observed (Table 3). In contrast, we observed an increase in the MICs of amikacin, gentamicin, minocycline, ciprofloxacin, levofloxacin, azithromycin, erythromycin, chloramphenicol and rifampicin in isolates with elevated adeB expression. The increase in MICs was in some cases 4-fold (e.g. gentamicin and minocycline) or higher and changed the phenotype in isolate pair 2 from susceptible to resistant to amikacin (Table 3). Differences in fluoroquinolone MICs between isolates of an isolate pair were not due to gyrA/B or parC/E mutations, since these genes were identical in isolate pairs. These data show that adeS disruption was associated with increased MICs in at least six different antimicrobial classes and adeN disruption in three antimicrobial classes. Table 3. Mean MICs of 12 different antimicrobials for four A. baumannii isolate pairs; all MICs (mg/L) were determined by agar dilution in three independent experiments Isolate pair 1 Isolate pair 2 Isolate pair 3 Isolate pair 4 Antimicrobial MB_2 MB_5 MB_7 MB_43 MB_271 MB_273 MB_131 MB_1044 Tigecycline 1 16 2 16 2 4 2 4 Tetracycline ≥256 ≥256 ≥256 ≥256 ≥256 ≥256 8 8 Minocycline 8 16 4 16 4 32 2 2 Ciprofloxacin 64 ≥256 64 ≥256 128 ≥256 64 128 Levofloxacin 8 64 16 64 8 16 8 32 Amikacin 64 ≥256 8 ≥256 16 16 ≥256 ≥256 Gentamicin 16 128 8 128 ≥256 ≥256 ≥256 ≥256 Chloramphenicol 128 ≥256 128 ≥256 ≥256 ≥256 ≥256 ≥256 Azithromycin 64 128 32 128 32 32 128 128 Erythromycin 32 128 32 128 64 64 64 64 Meropenem 8 8 8 8 128 ≥256 64 64 Rifampicin 128 128 64 128 8 8 4 4 Colistin 2 4 2 2 2 2 ≥256 ≥256 Isolate pair 1 Isolate pair 2 Isolate pair 3 Isolate pair 4 Antimicrobial MB_2 MB_5 MB_7 MB_43 MB_271 MB_273 MB_131 MB_1044 Tigecycline 1 16 2 16 2 4 2 4 Tetracycline ≥256 ≥256 ≥256 ≥256 ≥256 ≥256 8 8 Minocycline 8 16 4 16 4 32 2 2 Ciprofloxacin 64 ≥256 64 ≥256 128 ≥256 64 128 Levofloxacin 8 64 16 64 8 16 8 32 Amikacin 64 ≥256 8 ≥256 16 16 ≥256 ≥256 Gentamicin 16 128 8 128 ≥256 ≥256 ≥256 ≥256 Chloramphenicol 128 ≥256 128 ≥256 ≥256 ≥256 ≥256 ≥256 Azithromycin 64 128 32 128 32 32 128 128 Erythromycin 32 128 32 128 64 64 64 64 Meropenem 8 8 8 8 128 ≥256 64 64 Rifampicin 128 128 64 128 8 8 4 4 Colistin 2 4 2 2 2 2 ≥256 ≥256 Table 3. Mean MICs of 12 different antimicrobials for four A. baumannii isolate pairs; all MICs (mg/L) were determined by agar dilution in three independent experiments Isolate pair 1 Isolate pair 2 Isolate pair 3 Isolate pair 4 Antimicrobial MB_2 MB_5 MB_7 MB_43 MB_271 MB_273 MB_131 MB_1044 Tigecycline 1 16 2 16 2 4 2 4 Tetracycline ≥256 ≥256 ≥256 ≥256 ≥256 ≥256 8 8 Minocycline 8 16 4 16 4 32 2 2 Ciprofloxacin 64 ≥256 64 ≥256 128 ≥256 64 128 Levofloxacin 8 64 16 64 8 16 8 32 Amikacin 64 ≥256 8 ≥256 16 16 ≥256 ≥256 Gentamicin 16 128 8 128 ≥256 ≥256 ≥256 ≥256 Chloramphenicol 128 ≥256 128 ≥256 ≥256 ≥256 ≥256 ≥256 Azithromycin 64 128 32 128 32 32 128 128 Erythromycin 32 128 32 128 64 64 64 64 Meropenem 8 8 8 8 128 ≥256 64 64 Rifampicin 128 128 64 128 8 8 4 4 Colistin 2 4 2 2 2 2 ≥256 ≥256 Isolate pair 1 Isolate pair 2 Isolate pair 3 Isolate pair 4 Antimicrobial MB_2 MB_5 MB_7 MB_43 MB_271 MB_273 MB_131 MB_1044 Tigecycline 1 16 2 16 2 4 2 4 Tetracycline ≥256 ≥256 ≥256 ≥256 ≥256 ≥256 8 8 Minocycline 8 16 4 16 4 32 2 2 Ciprofloxacin 64 ≥256 64 ≥256 128 ≥256 64 128 Levofloxacin 8 64 16 64 8 16 8 32 Amikacin 64 ≥256 8 ≥256 16 16 ≥256 ≥256 Gentamicin 16 128 8 128 ≥256 ≥256 ≥256 ≥256 Chloramphenicol 128 ≥256 128 ≥256 ≥256 ≥256 ≥256 ≥256 Azithromycin 64 128 32 128 32 32 128 128 Erythromycin 32 128 32 128 64 64 64 64 Meropenem 8 8 8 8 128 ≥256 64 64 Rifampicin 128 128 64 128 8 8 4 4 Colistin 2 4 2 2 2 2 ≥256 ≥256 Prevalence of ISs in regulatory genes of RND-type efflux pumps The disruption of adeS or adeN was found in isolate pairs that originated from different patients in different hospitals and cities. In order to investigate the prevalence of ISs in RND-pump regulators in other tigecycline-resistant isolates, a further 65 unique A. baumannii isolates were analysed by WGS. All tigecycline-susceptible and -intermediate isolates displayed undisrupted adeRS, adeN and adeL. In all tigecycline-resistant isolates (n = 56) the regulatory genes adeR and adeL were undisrupted. A disruption of adeS or adeN was detected in a total of 23 tigecycline-resistant isolates (41.1%) (Table 4). ISAba1 was found inserted in adeS in three isolates (5.4%) and was associated with tigecycline MIC values of 16 mg/L. In contrast, adeN was disrupted in 20 isolates (35.7%) and the MICs were 2- to 4-fold lower compared with isolates with disrupted adeS (Figure S3). In the majority of isolates adeN was disrupted by ISAba1 (n = 18, 32.1%), but also ISAba27 (n = 1, 1.8%) and ISAba125 (n = 1, 1.8%) were found. In the isolate with ISAba125 disrupting adeN, ISAba1 was additionally inserted in the intergenic region of adeRS (MIC 8 mg/L). Table 4. Overview of ISs and other differences in the efflux pump regulators adeS and adeN in tigecycline-resistant (MIC > 2 mg/L) A. baumannii isolates Genetic modification Number of isolates adeS adeN ISAba1 3 18 ISAba27 0 1 ISAba125 0 1 ISAba1 insertion in intergenic region of adeRS 1 0 adeRSABC missing or truncated 2 0 1-nucleotide deletion 0 6 6-nucleotide insertion 0 2 Premature stop codon 0 3 Genetic modification Number of isolates adeS adeN ISAba1 3 18 ISAba27 0 1 ISAba125 0 1 ISAba1 insertion in intergenic region of adeRS 1 0 adeRSABC missing or truncated 2 0 1-nucleotide deletion 0 6 6-nucleotide insertion 0 2 Premature stop codon 0 3 Table 4. Overview of ISs and other differences in the efflux pump regulators adeS and adeN in tigecycline-resistant (MIC > 2 mg/L) A. baumannii isolates Genetic modification Number of isolates adeS adeN ISAba1 3 18 ISAba27 0 1 ISAba125 0 1 ISAba1 insertion in intergenic region of adeRS 1 0 adeRSABC missing or truncated 2 0 1-nucleotide deletion 0 6 6-nucleotide insertion 0 2 Premature stop codon 0 3 Genetic modification Number of isolates adeS adeN ISAba1 3 18 ISAba27 0 1 ISAba125 0 1 ISAba1 insertion in intergenic region of adeRS 1 0 adeRSABC missing or truncated 2 0 1-nucleotide deletion 0 6 6-nucleotide insertion 0 2 Premature stop codon 0 3 Other differences in regulatory genes of RND-type efflux pumps In a total of 7 tigecycline-intermediate and 33 tigecycline-resistant isolates, no insertion elements were detected in the regulatory genes of RND-type efflux pumps. Therefore, the regulators were analysed for mutations, deletions or insertions. In two intermediate and in two resistant isolates adeS and adeR could not be detected in draft genomes or by PCR. Further analysis revealed that in three isolates adeC and adeB were at least partially present, whereas adeA was not detectable. In one isolate the whole adeRSABC operon could not be found in the draft genome or by PCR. Instead, these four isolates showed differences in adeN, which included a large 87-nucleotide deletion (position 236–323) and a 6-nucleotide insertion in the other three isolates (Table 4). This 6-nucleotide insertion was found in a total of five isolates, but only in two tigecycline-resistant isolates with MICs of 4 mg/L (Table 4 and Figure S3). Other differences found in adeN included a point mutation creating a premature stop codon at position 592 (n = 3) and the deletion of nucleotide 62 leading to a frameshift mutation (n = 6) (Table 4). In both cases the tigecycline MICs were between 4 and 8 mg/L (Figure S3). Amino acid substitutions in the RND-pump regulators The amino acid sequences of AdeRS, AdeN and AdeL of all 65 isolates were compared with that of the reference strain A. baumannii ACICU. It is important to keep in mind that amino acid substitutions can also be polymorphic, for example in isolates representing different clonal lineages. In order to exclude polymorphisms, the reference strains A. baumannii AYE, ATCC 17978 and ATCC 19606 were included in the analysis. All substitutions found in the four reference strains or in the two susceptible isolates were identified as polymorphisms and excluded. For example, I120V, V136A and L241P were observed in AdeR in the reference strains and the substitutions V14I and V243I were detected in AdeR in one of the susceptible isolates when compared with A. baumannii ACICU. Therefore, these substitutions were considered to be silent polymorphisms. The same procedure was followed for the other regulators. Although a high number of polymorphisms were detected, various amino acid substitutions in AdeRS, AdeN and AdeL were associated with tigecycline resistance (Table S2 and Figure S4). In AdeN the amino acid substitutions H170Y (n = 1), D181N (n = 1) and G215V (n = 3) were detected, while in AdeL only the substitution I37L (n = 6) seemed to be associated with tigecycline resistance. In AdeR, six different substitutions were found (D21V, G25S, D26N, N115K, V119I, E147K), either as a single substitution per isolate or in combinations. The amino acid substitutions D167N and S357P were found in AdeS in two isolates, while the substitutions V137F and A325T were detected in a total of three isolates (Table S2 and Figure S4). The substitution I62M was detected in one tigecycline-resistant isolate as well as in two intermediate isolates. In only 16 isolates were amino acid substitutions associated with tigecycline resistance and MICs of 4–16 mg/L exclusively, i.e. no insertion elements or other mutations were detectable in the RND-type efflux pump regulators (Figure S3). Alternative tigecycline resistance mechanisms An association of trm with increased tigecycline MICs has been excluded in our analysis of isolate pairs. Furthermore, amino acid substitutions were detected in PlsC in seven isolates, including also a tigecycline-susceptible isolate. Therefore, the substitutions are most likely polymorphisms and were excluded. In total, six tigecycline-resistant isolates remained in this study, which carried none of the above-mentioned mutations or other differences in the RND-type efflux pump regulators, trm or plsC, so that a different and hitherto unknown resistance mechanism is suggested. Discussion Previous studies into tigecycline resistance in A. baumannii have mostly concentrated on relatively few isolates and single resistance mechanisms. The use of isolate pairs to investigate tigecycline resistance was first approached by Hornsey et al.31 who found multiple SNPs between their susceptible and resistant isolates, including an alanine to valine substitution in AdeS. Other work also found mutations in AdeS in association with tigecycline resistance,32,33 whereas others have shown the involvement of AdeR.26,34 In the present study we have investigated tigecycline resistance as part of a large multicentre study involving patients from 15 hospitals in Greece, Italy and Spain. We investigated the tigecycline resistance mechanisms using isolate pairs as well as single isolates from 65 patients. Isolate pairs are of great value for analysing the emergence of antimicrobial resistance. These isolates display the same isogenic background and the susceptible isolates precede the resistant isolates so that variations typically seen in epidemiologically unrelated strains are excluded and genetic differences are most likely a result of antimicrobial selection pressure. Our approach revealed that disruption of adeS or adeN by ISs was associated with tigecycline resistance and resulted in increased adeB and adeJ expression, respectively. Furthermore, disruption of adeS also was associated with increased MICs of at least six different antimicrobial classes, which indicates a higher impact of adeB overexpression on antimicrobial resistance in clinical settings. The disruption of adeS by ISAba1 has been reported previously and was associated with elevated MICs of tigecycline and other antimicrobial agents.35,36 The TetR-like transcription regulator adeN is a transcriptional repressor and insertional inactivation has been shown to increase adeJ expression in vitro,15 but overexpression of adeJ has been reported to be toxic for A. baumannii.12 These findings might explain the lower expression of adeJ and lower tigecycline MICs for isolates with disrupted adeN compared with isolates with ISAba1 insertion in adeS. In the 65 unique A. baumannii isolates, ISAba1 was the most common IS in adeN. This is not surprising, since this IS element is one of the most common ISs in A. baumannii and it is found in multiple sites within the genome and the plasmidome.37,38 Furthermore, it has been described in association with an MDR phenotype in A. baumannii,39 since ISAba1 often provides a strong promotor for overexpression of carbapenemases or the −35 region, which can generate a hybrid promotor with a −10 region located downstream of the IS.40 Besides ISAba1, the ISs ISAba125 and ISAba27 were also detected in adeN. To our knowledge this is the first report of ISAba125 and ISAba27 insertions in adeN in clinical A. baumannii isolates. Furthermore, our results show that ISs are more prevalent in adeN than in adeS. Recent studies have shown that disruption of adeN by ISAba1 increased the virulence and pathogenicity of A. baumannii.41,42 This is most concerning, as more than 30% of A. baumannii isolates in this study were found to carry ISAba1 or other ISs in adeN. Since adeS disruption has been associated with high-level drug resistance and adeN disruption with increased virulence and moderate drug resistance, both adeS and adeN disruption might be useful as potential markers of MDR and virulent A. baumannii strains.41,42 In four isolates adeS could not be detected. The loss of adeRS or adeB has recently been reported to significantly alter the transcriptional landscape of two A. baumannii strains including a decreased expression of adeABC and a subsequent decrease in MICs of several antimicrobial agents.43 Since in all isolates with a missing adeRSABC operon, mutations in adeN were detected, an association of adeJ overexpression with elevated tigecycline MICs is likely. For instance, a single nucleotide deletion was detected in adeN in six isolates creating a frameshift and premature stop codon. A similar frameshift mutation was reported by Rosenfeld et al.15 and revealed overexpression of adeJ and suggests similar elevated adeJ expression in our isolates. The effect of the observed adeN mutations on adeJ expression and subsequently on tigecycline resistance remains to be elucidated. Besides insertions or deletions in regulatory genes of efflux pumps, amino acid substitutions in AdeS or AdeR have been described to be associated with increased adeB expression and consequently with an MDR phenotype in A. baumannii.14,26,32–34 Since 16 isolates in this study displayed tigecycline MICs of > 2 mg/L independent of insertions, deletions or frameshifts in adeRS or adeN, amino acid substitutions could also play a role in tigecycline resistance. When analysing such mutations it is necessary to decide if the respective mutation is the cause of resistance or a simple genetic polymorphism. Yoon et al.32 reported that the amino acid substitution A94V in AdeS is such a polymorphism of strains belonging to IC1. These findings correlate well with our analysis of four reference strains and two tigecycline-susceptible isolates. Nevertheless, various substitutions were detected in the RND-pump regulatory proteins. Since none of the substitutions found in our isolates has been described previously, future studies should provide an insight into these mutations and their association with an MDR phenotype of A. baumannii. So far, only the amino acid substitution D20N has been investigated and demonstrated to be the cause of adeB overexpression and an MDR phenotype.34 Recently the crystal structure of AdeR was resolved and important amino acid residues that are associated with activation as well as DNA-binding and dimerization of AdeR were identified.44 The residue D63 was identified as a phosphorylation site and was shown to form a pocket with the residues G19, D20 and L112. In this study we identified several amino acid substitutions in AdeR, which were located near or in the pocket described by Wen et al.44 This includes the substitutions D21V, G25S, D26N, N115K and V119I, which were found as single substitutions or in combination in 25 isolates and might indicate a hotspot for mutations in AdeR in clinical A. baumannii isolates. In conclusion, using a genomic approach our study revealed multiple and diverse tigecycline resistance mechanisms involving the RND-type efflux pump regulators. We found a high prevalence of ISs in adeS and adeN, though disruption of adeS probably has a greater impact on resistance to other antimicrobial agents and therefore in clinical settings. Besides ISs, nucleotide deletions and insertions, premature stop codons and amino acid substitutions were found in AdeRS, AdeL and AdeN. Additionally, a potential hotspot for mutations was identified in AdeR. Our results gave insight into the large diversity of mechanisms associated with tigecycline resistance in A. baumannii isolates, highlighting the importance and potential of genomic approaches in the analysis of antimicrobial resistance in A. baumannii. Acknowledgements We thank the team of curators of the Institut Pasteur Acinetobacter MLST system for curating the data and making them publicly available at http://pubmlst.org/abaumannii/. Funding This work was supported by the German Research Council (DFG) – FOR2251 (www.acinetobacter.de). MagicBullet is a project funded by the European Union – Directorate General for Research and Innovation through the Seventh Framework Program for Research and Development (grant agreement 278232) and has been running since 1 January 2012 (duration, 48 months). Transparency declarations None to declare. Supplementary data Tables S1 and S2 and Figures S1 to S4 appear as Supplementary data at JAC Online. References 1 Peleg AY , Seifert H , Paterson DL. 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Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region . Nucleic Acids Res 2017 ; 45 : 9773 – 87 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: Mar 14, 2018

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