Identification of a MarR Subfamily That Regulates Arsenic Resistance Genes.

In this study, comprehensive analyses were performed to determine the function of an atypical MarR homolog in Achromobacter sp. strain As-55. Genomic analyses of Achromobacter sp. As-55 showed that this marR is located adjacent to an arsV gene. ArsV is a flavin-dependent monooxygenase that confers resistance to the antibiotic methylarsenite [MAs(III)], the organoarsenic compound roxarsone(III) [Rox(III)], and the inorganic antimonite [Sb(III)]. Similar marR genes are widely distributed in arsenic-resistant bacteria. Phylogenetic analyses showed that these MarRs are found in operons predicted to be involved in resistance to inorganic and organic arsenic species, so the subfamily was named MarRars. MarRars orthologs have three conserved cysteine residues, which are Cys36, Cys37, and Cys157 in Achromobacter sp. As-55, mutation of which compromises the response to MAs(III)/Sb(III). GFP-fluorescent biosensor assays show that AdMarRars (MarR protein of Achromobacter deleyi As-55) responds to trivalent As(III) and Sb(III) but not to pentavalent As(V) or Sb(V). The results of RT-qPCR assays show that arsV is expressed constitutively in a marR deletion mutant, indicating that marR represses transcription of arsV. Moreover, electrophoretic mobility shift assays (EMSAs) demonstrate that AdMarRars binds to the promoters of both marR and arsV in the absence of ligands and that DNA binding is relieved upon binding of As(III) and Sb(III). Our results demonstrate that AdMarRars is a novel As(III)/Sb(III)-responsive transcriptional repressor that controls expression of arsV, which confers resistance to MAs(III), Rox(III), and Sb(III). AdMarRars and its orthologs form a subfamily of MarR proteins that regulate genes conferring resistance to arsenic-containing antibiotics. IMPORTANCE In this study, a MarR family member, AdMarRars was shown to regulate the arsV gene, which confers resistance to arsenic-containing antibiotics. It is a founding member of a distinct subfamily that we refer to as MarRars, regulating genes conferring resistance to arsenic and antimony antibiotic compounds. AdMarRars was shown to be a repressor containing conserved cysteine residues that are required to bind As(III) and Sb(III), leading to a conformational change and subsequent derepression. Here we show that members of the MarR family are involved in regulating arsenic-containing compounds.

Achromobacter spp. genetic adaptation in cystic fibrosis.

Achromobacter spp. are emerging pathogens in patients with cystic fibrosis (CF) and Achromobacter spp. caused infections are associated with more severe disease outcomes and high intrinsic antibiotic resistance. While conventional CF pathogens are studied extensively, little is known about the genetic determinants leading to antibiotic resistance and the genetic adaptation in Achromobacter spp. infections. Here, we analysed 101 Achromobacter spp. genomes from 51 patients with CF isolated during the course of up to 20 years of infection to identify within-host adaptation, mutational signatures and genetic variation associated with increased antibiotic resistance. We found that the same regulatory and inorganic ion transport genes were frequently mutated in persisting clone types within and between Achromobacter species, indicating convergent genetic adaptation. Genome-wide association study of six antibiotic resistance phenotypes revealed the enrichment of associated genes involved in inorganic ion transport, transcription gene enrichment in ß-lactams, and energy production and translation gene enrichment in the trimethoprim/sulfonamide group. Overall, we provide insights into the pathogenomics of Achromobacter spp. infections in patients with CF airways. Since emerging pathogens are increasingly recognized as an important healthcare issue, our findings on evolution of antibiotic resistance and genetic adaptation can facilitate better understanding of disease progression and how mutational changes have implications for patients with CF.

Antibacterial and antibiofilm activities of ceragenins against Achromobacter species isolated from cystic fibrosis patients.

Achromobacter species, which are recognized as emerging pathogens isolated from patients with cystic fibrosis, are capable of forming biofilm in the respiratory tract in patients and innate multidrug resistance to antimicrobials. CSAs are cationic salt derivatives that mimic the activity of antimicrobial peptides and exhibit antimicrobial activity against bacteria. In this study, the in vitro activities of various ceragenins against Achromobacter-species biofilms were investigated comparatively with a conventional antibiotic (meropenem). Biofilm-formation inhibition and biofilm-adhesion inhibition were investigated on five strong biofilm-producing strains. The lowest MIC50 result was obtained with CSA-13. All of the tested CSAs showed significant biofilm inhibitory activity in the manner of a time- and concentration-dependent effect. To the best of our knowledge, this is the first article to evaluate the antibacterial and antibiofilm activities of tested CSAs against Achromobacter species.

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution.

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild-type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss-of-function mutations, or in a dominant (gain-of-function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.

Expansion and improvement of MALDI-TOF MS databases for accurate identification of Achromobacter species.

Different MALDI-TOF MS databases were evaluated for the identification of Achromobacter species. The in-house and extended database generated in this study rendered more accurate identification (58/64 and 57/64 isolates, respectively) in comparison with the Bruker commercial database (42/64 isolates), especially in those infrequent species that are not available or poorly represented.

Diversity of Achromobacter species recovered from patients with cystic fibrosis, in Argentina / Diversidad de las especies de Achromobacter recuperadas de pacientes con fibrosis quística en Argentina

Abstract Different phenotype-based techniques and molecular tools were used to describe the distribution of different Achromobacter species in patients with cystic fibrosis (CF) in Argentina, and to evaluate their antibiotic resistance profile. Phenotypic identification was performed by conventional biochemical tests, commercial galleries and MALDI-TOF MS. Genetic approaches included the detection of A. xylosoxidans specific marker blaoxa-114, the amplificaron and sequencing of the 16S rRNA gene, nrdA and blaOXA complete sequence, and MLST analysis. Phenotypic approaches, even MALDI-TOF, rendered inconclusive or misleading results. On the contrary, concordant results were achieved with the nrdA sequencing or sequence type (ST) analysis, and the complete blaOXA sequencing, allowing a reliable discrimination of different Achromobacter species. A. xylosoxidans accounted for 63% of Achromobacter infections and A. ruhlandii accounted for 17%. The remaining species corresponded to A. insuavis, A. dolens, A. marplatensis and A. pulmonis. Antimicrobial susceptibilities were determined by the agar dilution method according to CLSI guidelines. Piperacillin, piperacillin/tazobactam and car-bapenems were the most active antibiotics. However, the emergence of carbapenem-resistant isolates was detected. In conclusion, prompt and accurate identification tools were necessary to determine that different Achromobacter species may colonize/infect the airways of patients with CF. Moreover, antimicrobial therapy should be administered based on the susceptibility profile of individual Achromobacter sp. isolates. © 2019 Asociación Argentina de Microbiología. Published by Elsevier España, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Resumen Se emplearon diversas técnicas fenotípicas y moleculares para describir la distribución de diferentes especies del género Achromobacter en pacientes con fibrosis quística (FQ) en Argentina, y se evaluó el perfil de resistencia a los antibióticos. Se realizó la identificación fenotípica por pruebas bioquímicas convencionales, galerías comerciales y MALDI-TOF MS. El enfoque genético incluyó la detección del marcador especie-específico de A. xylosoxidans bla[PRESERVECIRC]tu, la amplificación y la secuenciación de los genes ARNr 16S, nrdA y secuencia completa de blaOXA, y el análisis por MLST. Los enfoques fenotípicos, incluso la técnica de MALDI-TOF, proporcionaron resultados no concluyentes o erróneos. Por el contrario, se obtuvieron resultados concordantes entre la secuenciación del gen nrdA o el análisis de secuenciotipos (ST) y la secuenciación completa de blaOXA, lo que permitió una discriminación confiable de las diferentes especies de Achromobacter. A. xylosoxidans representó el 63% de las infecciones por Achromobacter y A. ruhlandii representó el 17%. Las especies restantes correspondieron a A. insuavis, A. dolens, A. marplatensis y A. pulmonis. Se determinó la sensibilidad a antimicrobianos por el método de dilución en agar de acuerdo al CLSI. Los antibióticos más activos fueron piperacilina, piperacilina/tazobactam y carbapenemes. Sin embargo, se detectó la emergencia de aislamientos resistentes a carbapenemes. En conclusión, resultaron necesarias herramientas de identificación rápida y precisas para determinar las diferentes especies del género Achro-mobacter capaces de colonizar/infectar las vías respiratorias de los pacientes con FQ. Asimismo, la terapia antimicrobiana debería llevarse a cabo en función del perfil de sensibilidad de los aislamientos individuales de Achromobacter spp. © 2019 Asociacion Argentina de Microbiología. Publicado por Elsevier Espana, S.L.U. Este es un artículo Open Access bajo la licencia CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Prevalence of hypermutator isolates of Achromobacter spp. from cystic fibrosis patients.

Bacteria colonising the lungs of cystic fibrosis (CF) patients encounter high selective pressures. Hypermutation facilitates adaptation to fluctuating environments, and hypermutator strains are frequently isolated from CF patients. We investigated the prevalence of hypermutator isolates of Achromobacter spp. among patients affiliated with the CF Centre in Aarhus, Denmark. By exposure to rifampicin, the mutation frequency was determined for 90 isolates of Achromobacter spp. cultured from 42 CF patients; 20 infections were categorised as chronic, 22 as intermittent. The genetic mechanisms of hypermutation were examined by comparing DNA repair gene sequences from hypermutator and normomutator isolates. Achromobacter spp. cultured from 11 patients were categorised as hypermutators, and this phenotype was exclusively associated with chronic infections. Isolates of the Danish epidemic strain (DES) of Achromobacter ruhlandii cultured from patients from both Danish CF centres showed elevated mutation frequencies. The hypermutator state of Achromobacter spp. was most commonly associated with nonsynonymous mutations in the DNA mismatch repair gene mutS; a single clone had developed a substitution in the S-adenosyl-L-methionine-dependent methyltransferase putatively involved in DNA repair mechanisms, but not previously linked to the hypermutator phenotype. Hypermutation is prevalent among clinical isolates of Achromobacter spp. and could be a key determinant for the extraordinary adaptation and persistence of DES.

Diversity of Achromobacter species recovered from patients with cystic fibrosis, in Argentina.

Different phenotype-based techniques and molecular tools were used to describe the distribution of different Achromobacter species in patients with cystic fibrosis (CF) in Argentina, and to evaluate their antibiotic resistance profile. Phenotypic identification was performed by conventional biochemical tests, commercial galleries and MALDI-TOF MS. Genetic approaches included the detection of A. xylosoxidans specific marker blaoxa-114, the amplification and sequencing of the 16S rRNA gene, nrdA and blaOXA complete sequence, and MLST analysis. Phenotypic approaches, even MALDI-TOF, rendered inconclusive or misleading results. On the contrary, concordant results were achieved with the nrdA sequencing or sequence type (ST) analysis, and the complete blaOXA sequencing, allowing a reliable discrimination of different Achromobacter species. A. xylosoxidans accounted for 63% of Achromobacter infections and A. ruhlandii accounted for 17%. The remaining species corresponded to A. insuavis, A. dolens, A. marplatensis and A. pulmonis. Antimicrobial susceptibilities were determined by the agar dilution method according to CLSI guidelines. Piperacillin, piperacillin/tazobactam and carbapenems were the most active antibiotics. However, the emergence of carbapenem-resistant isolates was detected. In conclusion, prompt and accurate identification tools were necessary to determine that different Achromobacter species may colonize/infect the airways of patients with CF. Moreover, antimicrobial therapy should be administered based on the susceptibility profile of individual Achromobacter sp. isolates.

In Vitro Activities of ß-Lactam-ß-Lactamase Inhibitor Antimicrobial Agents against Cystic Fibrosis Respiratory Pathogens.

We tested the in vitro activities of ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam, piperacillin-tazobactam, and 11 other antimicrobial agents against 420 Burkholderia, Achromobacter, Stenotrophomonas, and Pandoraea strains, 89% of which were cultured from respiratory specimens from persons with cystic fibrosis. Among the ß-lactam-ß-lactamase inhibitor agents, meropenem-vaborbactam had the greatest activity against Burkholderia and Achromobacter, including multidrug-resistant and extensively-drug-resistant strains. None of the newer ß-lactam-ß-lactamase combination drugs showed increased activity compared to that of the older agents against Stenotrophomonas maltophilia or Pandoraea spp.

Achromobacter spp. healthcare associated infections in the French West Indies: a longitudinal study from 2006 to 2016.

BACKGROUND: Bacteria of the Achromobacter genus, more particularly xylosoxidans species, are responsible for various healthcare associated infections (HAI) which are increasingly described since the last decade. Cystic fibrosis (CF) patients are considered as potential reservoirs in hospitals. We performed a retrospective study to estimate the frequencies of Achromobacter spp. HAI among patients from French West Indies, to determine characteristics of infected patients and establish a possible link between CF and infections. METHODS: All adults with at least one Achromobacter spp. positive sample and infection criteria in accordance with European official definitions of HAI, hospitalized in University Hospital of Martinique from 2006 to 2016 for more than 48 h, were included. Patient clinical features, immune status and underlying diseases were obtained from medical files. A list of CF patients was given by clinicians. Antibiotic-susceptibility profiles of the strains were determined using an automated method. RESULTS: Mean incidence density was 0.038/1000 days of hospitalization. Achromobacter spp. HAI evolved as an endemic situation with a low but pretty much stable incidence rate over the 11-year observation period. An epidemic peak was noticed in 2013. Among the 66 included patients, 56.1% were immunocompetent and no one had CF. Pneumonia and bacteraemia were the two main HAI. Among the 79 isolated strains, 92.4% were resistant to at least 1 major antibiotic and 16.4% met the definition of multidrug-resistant bacteria. CONCLUSIONS: This microorganism, little known in our country because of the scarcity of CF patients, represents a threat for both immunosuppressed and immunocompetent patients and a therapeutic challenge because of its high resistance.

Replicon typing of plasmids in environmental Achromobacter sp. producing quinolone-resistant determinants.

This study aimed to investigate the antimicrobial resistance profile to quinolones, the presence of quinolone-resistant determinants and the plasmid replicon typing in environmental Achromobacter sp. isolated from Brazil. Soil and water samples were used for bacterial isolation. The antimicrobial susceptibility testing was performed by minimum inhibitory concentration method. The detection of mutations in the quinolone resistance-determining regions (QRDR) genes, the presence of plasmid-mediated quinolone resistance (PMQR) genes, and plasmid replicons were performed by PCR. A total of 16 isolates was obtained from different cultures, cities, and states of Brazil. All isolates were non-susceptible to ciprofloxacin, norfloxacin, and levofloxacin. Some mutations in QRDR genes were found, including Gln-83-Leu and Asp-87-Asn in the gyrA and Gln-80-Ile and Asp-84-Ala in the parC. Different PMQR genes were detected, such as qnrA, qnrB, qnrS, oqxA, and oqxB. Three different plasmid families were detected, being most presented the ColE-like, followed by IncFIB and IncA/C. The presence of different PMQR genes and plasmids in the isolates of the present study shows that environmental bacteria can act as reservoir of important genes of resistance to fluoroquinolones, which is of great concern, due to the potential of horizontal dissemination of these genes. Besides that, there are no studies reporting these results in Achromobacter sp. isolates.

Role of AxyZ Transcriptional Regulator in Overproduction of AxyXY-OprZ Multidrug Efflux System in Achromobacter Species Mutants Selected by Tobramycin.

AxyXY-OprZ is an RND-type efflux system that confers innate aminoglycoside resistance to Achromobacter spp. We investigated here a putative TetR family transcriptional regulator encoded by the axyZ gene located upstream of axyXY-oprZ An in-frame axyZ gene deletion assay led to increased MICs of antibiotic substrates of the efflux system, including aminoglycosides, cefepime, fluoroquinolones, tetracyclines, and erythromycin, indicating that the product of axyZ negatively regulates expression of axyXY-oprZ Moreover, we identified an amino acid substitution at position 29 of AxyZ (V29G) in a clinical Achromobacter strain that occurred during the course of chronic respiratory tract colonization in a cystic fibrosis (CF) patient. This substitution, also detected in three other strains exposed in vitro to tobramycin, led to an increase in the axyY transcription level (5- to 17-fold) together with an increase in antibiotic resistance level. This overproduction of AxyXY-OprZ is the first description of antibiotic resistance acquisition due to modification of a chromosomally encoded mechanism in Achromobacter and might have an impact on the management of infected CF patients. Indeed, tobramycin is widely used for aerosol therapy within this population, and we have demonstrated that it easily selects mutants with increased MICs of not only aminoglycosides but also fluoroquinolones, cefepime, and tetracyclines.

Effect of NaCl on aerobic denitrification by strain Achromobacter sp. GAD-3.

This paper presents the effect of NaCl on aerobic denitrification by a novel aerobic denitrifier strain Achromobacter sp. GAD-3. Results indicated that the aerobic denitrification process was inhibited by NaCl concentrations ≥20 g L-1, leading to lower nitrate removal rates (1.67∼4.0 mg L-1 h-1), higher nitrite accumulation (50.2∼87.4 mg L-1), and increasing N2O emission ratios (13∼72 mg L-1/mg L-1). Poor performance of aerobic denitrification at high salinity was attributed to the suppression of active microbial biomass and electron donating capacity of strain GAD-3. Further studies on the corresponding inhibition of the denitrifying gene expression by higher salinities revealed the significant sensitivity order of nosZ (for N2O reductase) > cnorB (for NO reductase) ≈ nirS (for cytochrome cd(1) nitrite reductase) > napA (for periplasmic nitrate reductase), accompanied with a time-lapse expression between nosZ and cnorB based on reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) analysis. The insights into the effect of NaCl on aerobic denitrification are of great significance to upgrade wastewater treatment plants (WWTPs) containing varying levels of salinity.

Multidrug-resistant Achromobacter animicus causing wound infection in a street child in Mwanza, Tanzania.

Achromobacter animicus (A. animicus) is an aerobic, motile, gram-negative, non-fermenting small bacillus that can also grow anaerobically with potassium nitrate. It has been isolated from sputum of humans suffering from respiratory infections. Literature regarding the role of A. animicus in wound infections is limited. We report a first case of a chronic post-traumatic wound infection caused by a multidrug-resistant A. animicus in a street child from Africa and accompanied diagnostic challenges.

Impact of High Diversity of Achromobacter Populations within Cystic Fibrosis Sputum Samples on Antimicrobial Susceptibility Testing.

Chronic colonization by opportunistic environmental bacteria is frequent in the airways of cystic fibrosis (CF) patients. Studies of Pseudomonas aeruginosa evolution during persistence have highlighted the emergence of pathoadaptive genotypes and phenotypes, leading to complex and diversified inpatient colonizing populations also observed at the intraspecimen level. Such diversity, including heterogeneity in resistance profiles, has been considered an adaptive strategy devoted to host persistence. Longitudinal genomic diversity has been shown for the emergent opportunistic pathogen Achromobacter, but phenotypic and genomic diversity has not yet been studied within a simple CF sputum sample. Here, we studied the genomic diversity and antimicrobial resistance heterogeneity of 132 Achromobacter species strains (8 to 27 strains of identical or distinct colonial morphotypes per specimen) recovered from the sputum samples of 9 chronically colonized CF patients. We highlighted the high within-sample and within-morphotype diversity of antimicrobial resistance (disk diffusion) and genomic (pulsed-field gel electrophoresis) profiles. No sputum sample included strains with identical pulsotypes or antibiotic susceptibility patterns. Differences in clinical categorization were observed for the 9 patients and concerned 3 to 11 antibiotics, including antibiotics recommended for use against Achromobacter Within-sample antimicrobial resistance heterogeneity, not predictable from colonial morphology, suggested that it may represent a selective advantage against antibiotics in an Achromobacter persisting population and potentially compromise the antibiotic management of CF airway infections.

Patterns of virulence factor expression and antimicrobial resistance in Achromobacter xylosoxidans and Achromobacter ruhlandii isolates from patients with cystic fibrosis.

Achromobacter spp. are opportunistic pathogens increasingly recovered from adult patients with cystic fibrosis (CF). We report the characterization of 122 Achromobacter spp. isolates recovered from 39 CF patients by multilocus sequence typing, virulence traits, and susceptibility to antimicrobials. Two species, A. xylosoxidans (77%) and A. ruhlandii (23%) were identified. All isolates showed a similar biofilm formation ability, and a positive swimming phenotype. By contrast, 4·3% and 44·4% of A. xylosoxidans and A. ruhlandii, respectively, exhibited a negative swarming phenotype, making the swimming and swarming abilities of A. xylosoxidans significantly higher than those of A. ruhlandii. A. xylosoxidans isolates from an outbreak clone also exhibited significantly higher motility. Both species were generally susceptible to ceftazidime, ciprofloxacin, imipenem and trimethoprim/sulphamethoxazole and there was no significant difference in susceptibility between isolates from chronic or sporadic infection. However, A. xylosoxidans isolates from chronic and sporadic cases were significantly more resistant to imipenem and ceftazidime than isolates of the outbreak clone.

Purification and characterization of exopolysaccharide bioflocculant produced by heavy metal resistant Achromobacter xylosoxidans.

Optimization of process parameters enhanced bioflocculating activity of 'Achromobacter xylosoxidans strain TERI L1' from 75% to 83.3% in absence of heavy metals, which decreased to 73% in presence of multi-metals. 'TERI L1' could adsorb 90% of multi-metals when grown in presence of 1250 mg L(-1) Zn, 2 mg L(-1) Cd, 30 mg L(-1) Pb, 200 mg L(-1) Ni and 90 mg L(-1) Cu and could adsorb 1100 mg L(-1) of Pb when grown in presence of 1500 ppm lead nitrate. The bioflocculant was purified and characterized. Bioflocculant yield was 5 g L(-1). Fourier transform infrared spectrum indicated presence of carboxyl, hydroxyl, amino groups, typical of glycoprotein. Spectroscopic analysis of bioflocculant by nuclear magnetic resonance revealed that it is a glycoprotein. LC-MS analysis confirmed the bioflocculant as a carbohydrate hetero polymer. Bioflocculant was composed of 75% total sugar with 72.9% neutral sugar and 11.5% protein. Scanning Electron Micrography revealed effective flocculation of kaolin clay by purified exopolysaccharide bioflocculant.

Effect of High-Dose Antimicrobials on Biofilm Growth of Achromobacter Species Isolated from Cystic Fibrosis Patients.

MICs and biofilm inhibitory concentrations (BICs) were measured for 68 cystic fibrosis (CF) Achromobacter isolates for amikacin, aztreonam, colistin, levofloxacin, and tobramycin. With the exception of colistin and levofloxacin, the remaining antibiotics had MIC90s, BICs at which 50% of the isolates were susceptible (BIC50s), and BICs at which 90% of the isolates were susceptible (BIC90s) equal to or above the highest concentrations tested. In a biofilm model, tobramycin was able to significantly increase killing of bacterial cells compared to controls, for intermediate-resistant strains only, at concentrations of 1,000 and 2,000 µg/ml.