Role of AxyABM overexpression in acquired resistance in Achromobacter xylosoxidans.

BACKGROUND: Acquired antimicrobial resistance among Achromobacter isolates from cystic fibrosis (CF) patients is frequent. Data concerning the mechanisms involved are scarce. The role of the AxyXY-OprZ and AxyEF-OprN Resistance Nodulation Division (RND) efflux systems has been demonstrated, but not that of AxyABM. OBJECTIVES: To explore the role of efflux systems in the acquired multiresistance observed in a one-step mutant selected after ofloxacin exposure. METHODS: The in vitro resistant mutant NCF-39-Bo2 and its parental strain NCF-39 (MICs of meropenem of 8 and 0.19 mg/L, of ceftazidime of 12 and 3 mg/L, of cefiderocol of 0.094 and 0.032 mg/L and of ciprofloxacin of 8 and 1.5 mg/L, respectively) were investigated by RNA-seq and WGS. Gene inactivation and reverse transcription quantitative PCR (RT-qPCR) were used to explore the role of the efflux systems of interest. RESULTS: RNA-seq showed that the AxyABM efflux system was overproduced (about 40-fold) in the in vitro mutant NCF-39-Bo2 versus its parental strain NCF-39. A substitution in AxyR, the putative regulator of AxyABM, was detected in NCF-39-Bo2. Gene inactivation of axyB (encoding the transporter component) in NCF-39-Bo2 led to a decrease in MICs of ciprofloxacin (5-fold), meropenem (64-fold), ceftazidime (12-fold) and cefiderocol (24-fold). Inactivation of axyB in the clinical isolate AXX-H2 harbouring a phenotype of resistance close to that of NCF-39-Bo2 enhanced the activity of the same molecules, especially meropenem. CONCLUSIONS: AxyABM overproduction is involved in acquired resistance of Achromobacter to ciprofloxacin, meropenem and ceftazidime, antibiotics widely used in CF patients, and increases the MIC of the new promising antibiotic cefiderocol.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Detection of Isolates Belonging to the Epidemic Clones Achromobacter xylosoxidans ST137 and Achromobacter ruhlandii DES from Cystic Fibrosis Patients.

Achromobacter spp. are increasingly reported among cystic fibrosis patients. Genotyping requires time-consuming methods such as multilocus sequence typing or pulsed-field gel electrophoresis. Therefore, data on the prevalence of multiresistant epidemic clones, especially A. xylosoxidans ST137 (AxST137) and the Danish epidemic strain A. ruhlandii (DES), are lacking. We recently developed and published a database for Achromobacter species identification by matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics). The aim of this study was to evaluate the ability of the MALDI-TOF MS to distinguish these multiresistant epidemic clones within Achromobacter species. All the spectra of A. xylosoxidans (n = 1,571) and A. ruhlandii (n = 174) used to build the local database were analyzed by ClinProTools, MALDI Biotyper PCA, MALDI Biotyper dendrogram, and flexAnalysis software for biomarker peak detection. Two hundred two isolates (including 48 isolates of AxST137 and 7 of DES) were tested. Specific biomarker peaks were identified: absent peak at m/z 6,651 for AxST137 isolates and present peak at m/z 9,438 for DES isolates. All tested isolates were well typed by our local database and clustered within distinct groups (ST137 or non-ST137 and DES or non-DES) no matter the MALDI-TOF software or only by simple visual inspection of the spectra by any user. The use of MALDI-TOF MS allowed us to identify isolates of A. xylosoxidans belonging to the AxST137 clone that spread in France and Belgium (the Belgian epidemic clone) and of A. ruhlandii belonging to the DES clone. This tool will help the implementation of segregation measures to avoid interpatient transmission of these resistant clones.

Mobilisation of plasmid-mediated blaVEB-1 gene cassette into distinct genomic islands of Proteus mirabilis after ceftazidime exposure.

OBJECTIVES: We sought to integrate a VEB-1-encoding gene cassette into the integron of the MDR region of genomic islands (GIs) harboured by Proteus mirabilis strains after antibiotic exposure. METHODS: An IncP1 plasmid from Achromobacter xylosoxidans carrying the cassette array dfrA14-blaVEB-1-aadB was introduced by conjugation into five strains of P. mirabilis: PmBRI, PmABB, PmSCO and Pm2CHAMA harbouring Salmonella GI 1 and PmESC harbouring Proteus GI 1. Circular intermediates of the cassettes were amplified by PCR. blaVEB-harbouring P. mirabilis were exposed to increasing concentrations of ceftazidime each day. Presence of blaVEB-1 in the GI was assessed by PCR. The complete MDR regions were mapped and sequenced in positive clones. RESULTS: Circular intermediates were detected for dfrA14 and blaVEB-1-aadB and dfrA14-blaVEB-1-aadB cassettes arrays in A. xylosoxidans, and for aadA2 in P. mirabilis. Insertion of blaVEB-1 into the GIs occurred under ceftazidime pressure. In all cases, the three cassettes from IncP1 were integrated. They replaced the cassette array of PmBRI, PmABB and PmSCO in which floRc, tet(A)G and blaPSE-1 were conserved, whereas they replaced an integron and the IS26-flanked region in Pm2CHAMA. In PmESC, they only replaced aadB, with aadA2 being conserved. blaVEB-1 integration occurred just after conjugation for Pm2CHAMA but required ceftazidime exposure for the other strains. CONCLUSION: Homologous recombination of gene cassettes conferring resistance to clinically important antibiotics may occur under antibiotic pressure between an integron located on a plasmid and a co-resident GI. This feature participates in the acquisition, maintenance and spread of antibiotic resistance genes.

Fluoroquinolone resistance in Achromobacter spp.: substitutions in QRDRs of GyrA, GyrB, ParC and ParE and implication of the RND efflux system AxyEF-OprN.

BACKGROUND: Achromobacter are emerging pathogens in cystic fibrosis patients. Mechanisms of resistance to fluoroquinolones are unknown in clinical isolates. Among non-fermenting Gram-negative bacilli, fluoroquinolone resistance is mostly due to amino acid substitutions in localized regions of the targets (GyrA, GyrB, ParC and ParE) named QRDRs, but also to efflux. OBJECTIVES: To explore quinolone resistance mechanisms in Achromobacter. METHODS: The putative QRDRs of GyrA, GyrB, ParC and ParE were sequenced in 62 clinical isolates, and in vitro one-step mutants obtained after exposure to fluoroquinolones. An in vitro mutant and its parental isolate were investigated by RNASeq and WGS. RT-qPCR and gene inactivation were used to explore the role of efflux systems overexpression. RESULTS: We detected seven substitutions in QRDRs (Q83L/S84P/D87N/D87G for GyrA, Q480P for GyrB, T395A/K525Q for ParE), all in nine of the 27 clinical isolates with ciprofloxacin MIC ≥16 mg/L, whereas none among the in vitro mutants. The RND efflux system AxyEF-OprN was overproduced (about 150-fold) in the in vitro mutant NCF-39-Bl6 versus its parental strain NCF-39 (ciprofloxacin MICs 64 and 1.5 mg/L, respectively). A substitution in AxyT (putative regulator of AxyEF-OprN) was detected in NCF-39-Bl6. Ciprofloxacin MIC in NCF-39-Bl6 dropped from 64 to 1.5 mg/L following gene inactivation of either axyT or axyF. Substitutions in AxyT associated with overexpression of AxyEF-OprN were also detected in seven clinical strains with ciprofloxacin MIC ≥16 mg/L. CONCLUSIONS: Target alteration is not the primary mechanism involved in fluoroquinolone resistance in Achromobacter. The role of AxyEF-OprN overproduction was demonstrated in one in vitro mutant.

Two New SGI1-LK Variants Found in Proteus mirabilis and Evolution of the SGI1-HKL Group of Salmonella Genomic Islands.

Integrative mobilizable elements belonging to the SGI1-H, -K, and -L Salmonella genomic island 1 (SGI1) variant groups are distinguished by the presence of an alteration in the backbone (IS1359 replaces 2.8 kb of the backbone extending from within traN [S005] to within S009). Members of this SGI1-HKL group have been found in Salmonella enterica serovars and in Proteus mirabilis Two novel variants from this group, designated SGI1-LK1 and SGI1-LK2, were found in the draft genomes of antibiotic-resistant P. mirabilis isolates from two French hospitals. Both variants can be derived from SGI1-PmGUE, a configuration found previously in another P. mirabilis isolate from France. SGI1-LK1 could arise via an IS26-mediated inversion in the complex class 1 integron that duplicated the IS26 element and the target site in IS6100 SGI1-LK1 also has a larger 8.59-kb backbone deletion extending from traN to within S013 and removing traG and traH. However, SGI1-LK1 was mobilized by an IncC plasmid. SGI1-LK2 can be derived from a hypothetical progenitor, SGI1-LK0, that is related to SGI1-PmGUE but lacks the aphA1 gene and one copy of IS26. The integron of SGI1-LK2 could arise via deletion of DNA adjacent to an IS26 and a deletion occurring via homologous recombination between duplicated copies of part of the integron 3'-conserved segment. SGI1-K can also be derived from SGI1-LK0. This would involve an IS26-mediated deletion and an inversion via homologous recombination of a segment between inversely oriented IS26s. Similar events can explain the configuration of the integrons in other SGI1-LK variants.IMPORTANCE Members of the SGI1-HKL subgroup of SGI1-type integrative mobilizable elements have a characteristic alteration in their backbone. They are widely distributed among multiply antibiotic-resistant Salmonella enterica serovars and Proteus mirabilis isolates. The SGI1-K type, found in the globally disseminated multiply antibiotic-resistant Salmonella enterica serovar Kentucky clone ST198 (sequence type 198), and various configurations in the original SGI1-LK group, found in other multiresistant S. enterica serovars and Proteus mirabilis isolates, have complex and highly plastic resistance regions due to the presence of IS26 However, how these complex forms arose and the relationships between them had not been analyzed. Here, a hypothetical progenitor, SGI1-LK0, that can be formed from the simpler SGI1-H is proposed, and the pathways to the formation of new variants, SGI1-LK1 and SGI1-LK2, found in P. mirabilis and other reported configurations via homologous recombination and IS26-mediated events are proposed. This led to a better understanding of the evolution of the SGI1-HKL group.

Detection of Achromobacter xylosoxidans in hospital, domestic, and outdoor environmental samples and comparison with human clinical isolates.

Achromobacter xylosoxidans is an aerobic nonfermentative Gram-negative rod considered an important emerging pathogen among cystic fibrosis (CF) patients worldwide and among immunocompromised patients. This increased prevalence remains unexplained, and to date no environmental reservoir has been identified. The aim of this study was to identify potential reservoirs of A. xylosoxidans in hospital, domestic, and outdoor environments and to compare the isolates with clinical ones. From 2011 to 2012, 339 samples were collected in Dijon's university hospital, in healthy volunteers' homes in the Dijon area, and in the outdoor environment in Burgundy (soil, water, mud, and plants). We designed a protocol to detect A. xylosoxidans in environmental samples based on a selective medium: MCXVAA (MacConkey agar supplemented with xylose, vancomycin, aztreonam, and amphotericin B). Susceptibility testing, genotypic analysis by pulsed-field gel electrophoresis, and blaOXA-114 sequencing were performed on the isolates. A total of 50 strains of A. xylosoxidans were detected in hospital (33 isolates), domestic (9 isolates), and outdoor (8 isolates) samples, mainly in hand washing sinks, showers, and water. Most of them were resistant to ciprofloxacin (49 strains). Genotypic analysis and blaOXA-114 sequencing revealed a wide diversity among the isolates, with 35 pulsotypes and 18 variants of oxacillinases. Interestingly, 10 isolates from hospital environment were clonally related to clinical isolates previously recovered from hospitalized patients, and one domestic isolate was identical to one recovered from a CF patient. These results indicate that A. xylosoxidans is commonly distributed in various environments and therefore that CF patients or immunocompromised patients are surrounded by these reservoirs.