Distribution of Achromobacter Species in 12 French Cystic Fibrosis Centers in 2020 by a Retrospective MALDI-TOF MS Spectrum Analysis.

Achromobacter spp. are nonfermenting Gram-negative bacilli mainly studied among cystic fibrosis (CF) patients. The identification of the 19 species within the genus is time-consuming (nrdA-sequencing), thus data concerning the distribution of the species are limited to specific studies. Recently, we built a database using MALDI-TOF mass spectrometry (MS) (Bruker) that allows rapid and accurate species identification and detection of the multiresistant epidemic clones: A. xylosoxidans ST137 spreading among CF patients in various French and Belgium centers, and A. ruhlandii DES in Denmark. Here, we first assessed whether species identification could be achieved with our database solely by analysis of MS spectra without availability of isolates. Then, we conducted a multicentric study describing the distribution of Achromobacter species and of the clone ST137 among French CF centers. We collected and analyzed with our local database the spectra of Achromobacter isolates from 193 patients (528 samples) from 12 centers during 2020. In total, our approach enabled to conclude for 502/528 samples (95.1%), corresponding to 181 patients. Eleven species were detected, only five being involved in chronic colonization, A. xylosoxidans (86.4%), A. insuavis (9.1%), A. mucicolens (2.3%), A. marplatensis (1.1%) and A. genogroup 3 (1.1%). This study confirmed the high prevalence of A. xylosoxidans in chronic colonizations and the circulation of the clone A. xylosoxidans ST137 in France: four patients in two centers. The present study is the first to report the distribution of Achromobacter species from CF patients samples using retrospective MALDI-TOF/MS data. This easy approach could enable future large-scale epidemiological studies.

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.

What is the best technic to dislodge Staphylococcus epidermidis biofilm on medical implants?

BACKGROUND: Bacterial biofilm can occur on all medical implanted devices and lead to infection and/or dysfunction of the device. In this study, artificial biofilm was formed on four different medical implants (silicone, piccline, peripheral venous catheter and endotracheal tube) of interest for our daily clinical and/or research practice. We investigated the best conventional technic to dislodge the biofilm on the implants and quantified the number of bacteria. Staphylococcus epidermidis previously isolated from a breast implant capsular contracture on a patient in the university hospital of Dijon was selected for its ability to produce biofilm on the implants. Different technics (sonication, Digest-EUR®, mechanized bead mill, combination of sonication plus Digest-EUR®) were tested and compared to detach the biofilm before quantifying viable bacteria by colony counting. RESULTS: For all treatments, the optical and scanning electron microscope images showed substantial less biofilm biomass remaining on the silicone implant compared to non-treated implant. This study demonstrated that the US procedure was statistically superior to the other physical treatment: beads, Digest-EUR® alone and Digest-EUR® + US (p < 0.001) for the flexible materials (picc-line, PIV, and silicone). The number of bacteria released by the US is significantly higher with a difference of 1 log on each material. The result for a rigid endotracheal tube were different with superiority for the chemical treatment dithiothreitol: Digest-EUR®. Surprisingly the combination of the US plus Digest-EUR® treatment was consistently inferior for the four materials. CONCLUSIONS: Depending on the materials used, the biofilm dislodging technique must be adapted. The US procedure was the best technic to dislodge S. epidermidis biofilm on silicone, piccline, peripheral venous catheter but not endotracheal tube. This suggested that scientists should compare themselves different methods before designing a protocol of biofilm study on a given material.

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.

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.

Evaluation of the relevance of use of the BD-BACTEC®MycosisIC/F, BD-BACTEC®PlusAerobic/F, BD-BACTEC®Lytic/10 anaerobic/F and BD-BACTEC®PedsPlus/F culture bottle system for fungemia detection: A 4-year retrospective study at the Dijon university hospital, France.

INTRODUCTION: Fungemia is a severe invasive fungal infection that combines rapid progression and a high mortality rate. This type of infection is a vital emergency, and early diagnosis is crucial. Currently, only the BD-BACTEC® Automated Blood Culture System (Becton Dickinson, New Jersey, USA) has a medium specifically dedicated to the detection of fungal agents: the BD-BACTEC®MycosisIC/F bottle. GAP STATEMENT: Thus, it is important to assess the performance of the different bottles offered by the BD-BACTEC® Automated Blood Culture System for the diagnosis of fungemia. AIM: The aim of this study was to evaluate the performance of the BD-BACTEC® MycosisIC/F culture medium in comparison to bacteriologic culture bottle media for the detection of fungemia in different clinical situations. METHODOLOGY: This retrospective study was conducted over a period of 4 years at the Dijon University Hospital. Three hundred and thirty-one pairs of blood cultures (i.e. a BD-BACTEC® MycosisIC/F culture bottle associated with at least one bacteriologic culture media bottle) were included in this study. RESULTS: We showed that the BD-BACTEC® MycosisIC/F culture bottles performed significantly better (i.e. diagnostic advantage either because it was the only positive bottle of the pair or time to positive result was shorter) than the bacteriological culture bottles in 57.7% (191/331) of cases (p <0.01). Multivariate analysis revealed that BD-BACTEC®MycosisIC/F bottles had better diagnostic performance than BD-BACTEC®Bacteriologic bottles in the context of: (i) the initial versus follow-up diagnostic subgroup, (ii) venipuncture or arterial sampling versus other sampling methods, and (iii) detection of filamentous versus yeast fungi. CONCLUSION: We concluded that the use of BD-BACTEC® MycosisIC/F culture bottles is a relevant addition to media optimized for routine bacterial detection.

Epidemiology and antibiotic resistance of prosthetic joint infections according to time of occurrence, a 10-year study.

OBJECTIVES: To describe the microorganisms responsible for prosthetic joint infections (PJIs) and their antimicrobial susceptibilities, and to propose appropriate empirical antimicrobial treatments (EATs) according to time of occurrence METHODS: This 10-year retrospective study presents the bacterial etiology of 282 consecutive PJIs in a French hospital according to time of occurrence (adapted from Zimmerli's classification: early, <3 months; delayed, 3-12 months; late acute, >12 months with hematogenous seeding or contiguous spread; late chronic, >12 months without hematogenous seeding). The expected efficacy of various EATs was analyzed for each PJI. RESULTS: Staphylococci were the most commonly found bacteria (S. aureus (44.3%), coagulase-negative staphylococci (25.2%) with 15.2% and 49.3% methicillin resistance, respectively), followed by Gram-negative bacilli (GNB) (17.7%) and streptococci (14.9%). The distribution of species varied between categories, but antibiotics targeting GNBs were required in all categories. Imipenem-vancomycin was the most effective combination (99.3%) but should be reserved for patients with suspected resistant GNB. Cefotaxime-vancomycin was less effective in early/delayed and late PJIs (91.1% and 86.1%, respectively), due to resistant GNB and polymicrobial infections. Piperacillin/tazobactam-vancomycin appeared to be appropriate in all situations (>96% efficacy). CONCLUSION: Proposing universal recommendations remains challenging, but a good understanding of the local epidemiology is important for optimizing EATs.

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.