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Achromobacter xylosoxidans is one of the nitrogen-fixing bacteria associated with cowpea rhizosphere across Africa. Although its role in improving soil fertility and inducing systemic resistance in plants against pathogens has been documented, there is limited information on its complete genomic characteristics from cowpea roots. Here, we report the complete genome sequence of A. xylosoxidans strain DDA01 isolated from the topsoil of a field where cowpea plants tolerant to cucumber mosaic virus (CMV) were grown in Ibadan, Nigeria. The genome of DDA01 was sequenced via Illumina MiSeq and contained 6,930,067 nucleotides with 67.55% G + C content, 73 RNAs, 59 tRNAs, and 6421 protein-coding genes, including those associated with nitrogen fixation, phosphate solubilization, Indole3-acetic acid production, and siderophore activity. Eleven genetic clusters for secondary metabolites, including alcaligin, were identified. The potential of DDA01 as a plant growth-promoting bacteria with genetic capabilities to enhance soil fertility for resilience against CMV infection in cowpea is discussed. To our knowledge, this is the first complete genome of diazotrophic bacteria obtained from cowpea rhizosphere in sub-Saharan Africa, with potential implications for improved soil fertility, plant disease resistance, and food security., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Achromobacter xylosoxidans is an emerging pathogen that causes airway infections in patients with cystic fibrosis. Knowledge of virulence factors and protein secretion systems in this bacterium is limited. Twin arginine translocation (Tat) is a protein secretion system that transports folded proteins across the inner cell membranes of gram-negative bacteria. Tat has been shown to be important for virulence and cellular processes in many different bacterial species. This study aimed to investigate the role of Tat in iron metabolism and host cell adhesion in A. xylosoxidans . Putative Tat substrates in A. xylosoxidans were identified using the TatFind, TatP, and PRED-Tat prediction tools. An isogenic tatC deletion mutant (ΔtatC) was generated and phenotypically characterized. The wild-type and ΔtatC A. xylosoxidans were fractionated into cytosolic, membrane, and periplasmic fractions, and the expressed proteome of the different fractions was analysed using liquid chromatography-mass spectrometry (LC-MS/MS). A total of 128 putative Tat substrates were identified in the A. xylosoxidans proteome. The ΔtatC mutant showed attenuated host cell adhesion, growth rate, and iron acquisition. Twenty predicted Tat substrates were identified as expressed proteins in the periplasmic compartment, nine of which were associated with the wild type. The data indicate that Tat secretion is important for iron acquisition and host cell adhesion in A. xylosoxidans .

Achromobacter xylosoxidans is an aerobic, catalase-positive, non-pigment-forming, Gram-negative, and motile bacterium. It potentially causes a wide range of human infections in cystic fibrosis and non-cystic fibrosis patients. However, developing a safe preventive or therapeutic solution against A. xylosoxidans remains challenging. This study aimed to construct an epitope-based vaccine candidate using immunoinformatic techniques. A. xylosoxidans was isolated from an auto workshop in Lahore, and its identification was confirmed through 16S rRNA amplification and bioinformatic analysis. Two protein targets with GenBank accession numbers AKP90890.1 and AKP90355.1 were selected for the vaccine construct. Both proteins exhibited antigenicity, with scores of 0.757 and 0.580, respectively and the epitopes were selected based on the IC50 value using the ANN 4.0 and NN-align 2.3 epitope prediction method for MHC I and MHC II epitopes respectively and predicted epitopes were analyzed for antigenicity, allergenicity and pathogenicity. The vaccine construct demonstrated structural stability, thermostability, solubility, and hydrophilicity. The vaccine produced 250 B-memory cells per mm 3 and approximately 16,000 IgM + IgG counts, indicating an effective immune response against A. xylosoxidans. Moreover, the vaccine candidate interacted stably with toll-like receptor 5, a pattern recognition receptor, with a confidence score of 0.98. These results highlight the potency of the designed vaccine candidate, suggesting its potential to withstand rigorous in vitro and in vivo clinical trials. This epitope-based vaccine could serve as the first preventive immunotherapy against A. xylosoxidans infections, addressing this bacterium's health and financial burdens. The findings demonstrate the value of employing immunoinformatic tools in vaccine development, paving the way for more precise and tailored approaches to combating microbial threats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Achromobacter spp. are opportunistic pathogens of environmental origin increasingly isolated in patients with underlying conditions like cystic fibrosis (CF). Despite recent advances, their virulence factors remain incompletely studied, and siderophore production has not yet been investigated in this genus. The aim of this study was to evaluate the production of siderophores in a large collection of Achromobacter spp. and evaluate the variability according to the origin of the strain and species. A total of 163 strains were studied, including 128 clinical strains (CF and non-CF patients) and 35 strains of environmental origin. Siderophores were quantified by the liquid chrome azurol-sulphonate assay. Species were identified by nrdA gene-based phylogeny. Strains were assigned to 20 species, with Achromobacter xylosoxidans being the most represented (51.5% of strains). Siderophore production was observed in 72.4% of the strains, with amounts ranging from 10.1% to 90% siderophore units. A significantly higher prevalence of siderophore-producing strains and greater production of siderophores were observed for clinical strains compared with strains of environmental origin. Highly variable observations were made according to species: A. xylosoxidans presented unique characteristics (one of the highest prevalence of producing strains and highest amounts produced, particularly by CF strains). Siderophores are important factors for bacterial growth commonly produced by members of the Achromobacter genus. The significance of the observations made during this study must be further investigated. Indeed, the differences observed according to species and the origin of strains suggest that siderophores may represent important determinants of the pathophysiology of Achromobacter spp. infections and also contribute to the particular epidemiological success of A. xylosoxidans in human infections., Importance: Achromobacter spp. are recognized as emerging opportunistic pathogens in humans with various underlying diseases, including cystic fibrosis (CF). Although their pathophysiological traits are increasingly studied, their virulence factors remain incompletely described. Particularly, siderophores that represent important factors of bacterial growth have not yet been studied in this genus. A population-based study was performed to explore the ability of members of the Achromobacter genus to produce siderophores, both overall and in relevant subgroups ( Achromobacter species; strain origin, either clinical-from CF or non-CF patients-or environmental). This study provides original data showing that siderophore production is a common trait of Achromobacter strains, particularly observed among clinical strains. The major species, Achromobacter xylosoxidans , encompassed both one of the highest prevalence of siderophore-producing strains and strains producing the largest amounts of siderophores, particularly observed for CF strains. These observations may represent additional advantages accounting for the epidemiological success of this species., Competing Interests: The authors declare no conflict of interest.

Cystic fibrosis (CF) is a genetic disease affecting epithelial ion transport, resulting in thickened mucus and impaired mucociliary clearance. Persons with CF (pwCF) experience life-long infections of the respiratory mucosa caused by a diverse array of opportunists, which are leading causes of morbidity and mortality. In recent years, there has been increased appreciation for the range and diversity of microbes causing CF-related respiratory infections. The introduction of new therapeutics and improved detection methodology has revealed CF-related opportunists such as Achromobacter xylosoxidans ( Ax ). Ax is a Gram-negative bacterial species which is widely distributed in environmental sources and has been increasingly observed in sputa and other samples from pwCF, typically in patients in later stages of CF disease. In this study, we characterized CF clinical isolates of Ax and tested colonization and persistence of Ax in respiratory infection using immortalized human CF respiratory epithelial cells and BALB/c mice. Genomic analyses of clinical Ax isolates showed homologs for factors including flagellar synthesis, antibiotic resistance, and toxin secretion systems. Ax isolates adhered to polarized cultures of CFBE41o- human immortalized CF bronchial epithelial cells and caused significant cytotoxicity and depolarization of cell layers. Ax colonized and persisted in mouse lungs for up to 72 h post infection, with inflammatory consequences that include increased neutrophil influx in the lung, lung damage, cytokine production, and mortality. We also identified genes that are differentially expressed in synthetic CF sputum media. Based on these results, we conclude that Ax is an opportunistic pathogen of significance in CF., Competing Interests: The authors declare no conflict of interest.

Achromobacter is a genus of Gram-negative rods, which can cause persistent airway infections in people with cystic fibrosis (CF). The knowledge about virulence and clinical implications of Achromobacter is still limited, and it is not fully established whether Achromobacter infections contribute to disease progression or if it is a marker of poor lung function. The most commonly reported Achromobacter species in CF is A. xylosoxidans. While other Achromobacter spp. are also identified in CF airways, the currently used Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) method in routine diagnostics cannot distinguish between species. Differences in virulence between Achromobacter species have consequently not been well studied. In this study, we compare phenotypes and proinflammatory properties of A. xylosoxidans, A. dolens , A. insuavis , and A. ruhlandii using in vitro models. Bacterial supernatants were used to stimulate CF bronchial epithelial cells and whole blood from healthy individuals. Supernatants from the well-characterized CF-pathogen Pseudomonas aeruginosa were included for comparison. Inflammatory mediators were analyzed with ELISA and leukocyte activation was assessed using flow cytometry. The four Achromobacter species differed in morphology seen in scanning electron microscopy (SEM), but there were no observed differences in swimming motility or biofilm formation. Exoproducts from all Achromobacter species except A. insuavis caused significant IL-6 and IL-8 secretion from CF lung epithelium. The cytokine release was equivalent or stronger than the response induced by P. aeruginosa. All Achromobacter species activated neutrophils and monocytes ex vivo in a lipopolysaccharide (LPS)-independent manner. Our results indicate that exoproducts of the four included Achromobacter species do not differ consistently in causing inflammatory responses, but they are equally or even more capable of inducing inflammation compared with the classical CF pathogen P. aeruginosa. IMPORTANCE Achromobacter xylosoxidans is an emerging pathogen among people with cystic fibrosis (CF). Current routine diagnostic methods are often unable to distinguish A. xylosoxidans from other Achromobacter species, and the clinical relevance of different species is still unknown. In this work, we show that four different Achromobacter species relevant to CF evoke similar inflammatory responses from airway epithelium and leukocytes in vitro , but they are all equally or even more proinflammatory compared to the classic CF-pathogen Pseudomonas aeruginosa. The results suggest that Achromobacter species are important airway pathogens in CF, and that all Achromobacter species are relevant to treat., Competing Interests: The authors declare no conflict of interest.

Achromobacter species colonization of Cystic Fibrosis respiratory airways is an increasing concern. Two adult patients with Cystic Fibrosis colonized by Achromobacter xylosoxidans CF418 or Achromobacter ruhlandii CF116 experienced fatal exacerbations. Achromobacter spp. are naturally resistant to several antibiotics. Therefore, phages could be valuable as therapeutics for the control of Achromobacter . In this study, thirteen lytic phages were isolated and characterized at the morphological and genomic levels for potential future use in phage therapy. They are presented here as the Achromobacter Kumeyaay phage collection. Six distinct Achromobacter phage genome clusters were identified based on a comprehensive phylogenetic analysis of the Kumeyaay collection as well as the publicly available Achromobacter phages. The infectivity of all phages in the Kumeyaay collection was tested in 23 Achromobacter clinical isolates; 78% of these isolates were lysed by at least one phage. A cryptic prophage was induced in Achromobacter xylosoxidans CF418 when infected with some of the lytic phages. This prophage genome was characterized and is presented as Achromobacter phage CF418-P1. Prophage induction during lytic phage preparation for therapy interventions require further exploration. Large-scale production of phages and removal of endotoxins using an octanol-based procedure resulted in a phage concentrate of 1 × 10 9 plaque-forming units per milliliter with an endotoxin concentration of 65 endotoxin units per milliliter, which is below the Food and Drugs Administration recommended maximum threshold for human administration. This study provides a comprehensive framework for the isolation, bioinformatic characterization, and safe production of phages to kill Achromobacter spp. in order to potentially manage Cystic Fibrosis (CF) pulmonary infections.

Achromobacter xylosoxidans (Ax) is an opportunistic pathogen and causative agent of numerous infections particularly in immunocompromised individuals with increasing prevalence in cystic fibrosis (CF). To date, investigations have focused on the clinical epidemiology and genomic comparisons of Ax isolates, yet little is known about disease pathology or the role that specific virulence factors play in tissue invasion or damage. Here, we model an acute Ax lung infection in immunocompetent C57BL/6 mice and immunocompromised CF mice, revealing a link between in vitro cytotoxicity and disease in an intact host. Mice were intratracheally challenged with sublethal doses of a cytotoxic (GN050) or invasive (GN008) strain of Ax. Bacterial burden, immune cell populations, and inflammatory markers in bronchoalveolar lavage fluid and lung homogenates were measured at different time points to assess disease severity. CF mice had a similar but delayed immune response toward both Ax strains compared to C57BL/6J mice. GN050 caused more severe disease and higher mortality which correlated with greater bacterial burden and increased proinflammatory responses in both mouse models. In agreement with the cytotoxicity of GN050 toward macrophages in vitro , mice challenged with GN050 had fewer macrophages. Mutants with transposon insertions in predicted virulence factors of GN050 showed that disease severity depended on the type III secretion system, Vi capsule, antisigma-E factor, and partially on the ArtA adhesin. The development of an acute infection model provides an essential tool to better understand the infectivity of diverse Ax isolates and enable improved identification of virulence factors important to bacterial persistence and disease., Competing Interests: The authors declare no conflict of interest.

Trimethoprim-sulfamethoxazole (SXT) is the preferable treatment option of the infections caused by Achromobacter spp. Our study aimed to analyze the SXT resistance of 98 Achromobacter spp. isolates from pediatric patients, among which 33 isolates were SXT-resistant. The presence of intI1 was screened by PCR and genome sequence analyses. The intI1 gene was detected in 10 of SXT-resistant isolates that had shorter intI1 PCR fragments named intI1S. Structural changes in intI1S were confirmed by genome sequencing and analyses which revealed 86 amino acids deletion in IntI1S protein compared to canonical IntI1 protein. All IntI1S isolates were of non-CF origin. Pan-genome analysis of intI1S bearing A. xylosoxidans isolates comprised 9052 genes, with the core genome consisting of 5455 protein-coding genes. Results in this study indicate that IntI1S isolates were derived from clinical settings and that cystic fibrosis (CF) patients were potential reservoirs for healthcare-associated infections that occurred in non-CF patients., (© 2022. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

Microbial remediation has become one of the promising ways to eliminate polycyclic aromatic hydrocarbons (PAHs) pollution due to its efficient enzyme metabolism system. Catechol 1,2-dioxygenase (C12O) is a crucial rate-limiting enzyme in the degradation pathway of PAHs in Achromobacter xylosoxidans DN002 that opens the benzene ring through the ortho-cleavage pathway. However, little attention has been given to explore the interaction mechanism of relevant enzyme-substrate. This study aims to investigate the binding interaction between C12O of strain DN002 and catechol by means of a molecular biological approach combined with homology modeling, molecular docking, and multiple spectroscopies. The removal rate of catechol in the mutant strain of cat A deletion was only 12.03%, compared to the wild-type strain (54.21%). A Ramachandran plot of active site regions of the primary amino acid sequences in the native enzyme showed that 93.5% sequences were in the most favored regions on account of the results of homology modeling, while an additional 6.2% amino acid sequences were found in conditionally allowed regions, and 0.4% in generously allowed regions. The binding pocket of C12O with catechol was analyzed to obtain that the catalytic trimeric group of Tyr164-His224-His226 was proven to be great vital for the ring-opening reaction of catechol by molecular docking. In the native enzyme, binding complexes were spontaneously formed by hydrophobic interactions. Binding constants and thermodynamic potentials from fluorescence spectra indicated that catechol effectively quenched the intrinsic fluorescence of C12O in the C12O/catechol complex via conventional static and dynamic quenching mechanisms of C12O. The results of ultraviolet and visible (UV) spectra, synchronous fluorescence, and circular dichroism (CD) spectra revealed conspicuous changes in the local conformation, and site-directed mutagenesis confirmed the role of predicted key residues during catalysis, wherein His 226 had a significant effect on catechol utilization by C12O. This is the first report to reveal interactions of C12O with substrate from the molecular docking results, providing the mechanistic understanding of representative dioxygenases involved in aromatic compound degradation, and a solid foundation for further site modifications as well as strategies for the directed evolution of this enzyme., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: In this study, we sought to determine the source of an outbreak of Achromobacter denitrificans infections in patients at a tertiary-care academic hospital., Design: Outbreak report study with intervention. The study period extended from February 2018 to December 2018., Setting: The study was conducted at a tertiary-care academic hospital in Pretoria, South Africa., Patients and Participants: All patients who cultured A. denitrificans from any site were included in this study. During the study period, 43 patients met this criterion., Interventions: Once an outbreak was confirmed, the microbiology laboratory compiled a list of affected patients. A common agent, chlorhexidine-and-water solution, was used as a disinfectant-antiseptic for all affected patients. The laboratory proceeded to culture this solution. Environmental and surface swabs were also cultured from the hospital pharmacy area where this solution was prepared. Repetitive-element, sequence-based, polymerase chain reaction (rep-PCR) was performed on the initial clinical isolates to confirm the relatedness of the isolates., Results: In total, 43 isolates of A. denitrificans were cultured from patient specimens during the outbreak. The laboratory cultured A. denitrificans from all bottles of chlorhexidine-and-water solutions sampled from the wards and the pharmacy. The culture of the dispenser device used to prepare this solution also grew A. denitrificans. The rep-PCR confirmed the clonality of the clinical isolates with 2 genotypes dominating., Conclusions: Contaminated chlorhexidine-and-water solutions prepared at the hospital pharmacy was determined to be the source of the outbreak. Once this item was removed from the hospital, the laboratory did not culture any further A. denitrificans isolates from patient specimens.

Bacteria belonging to the genus Achromobacter are widely distributed in natural environments and have been recognized as emerging pathogens for their contribution to a wide range of human infections. In particular, patients with cystic fibrosis (CF) are the subjects most frequently colonized by Achromobacter spp., which can cause persistent infections in their respiratory tract. Although many clinical aspects and pathogenic mechanisms still remain to be elucidated, Achromobacter spp. have been a source of expanding interest in recent years. This review examines the current literature regarding Achromobacter spp. role in CF, focusing on taxonomy, prevalence in CF lung infections, genomic characteristics, and adaptation strategies including modifications of metabolism and virulence, acquisition of antibiotic resistance, exchange of mobile genetic elements and development of hypermutation., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Achromobacter xylosoxidans is an opportunistic pathogen implicated in a wide variety of human infections including the ability to colonize the lungs of cystic fibrosis (CF) patients. The role of A. xylosoxidans in human pathology remains controversial due to the lack of optimized in vitro and in vivo model systems to identify and test bacterial gene products that promote a pathological response. We have previously identified macrophages as a target host cell for A. xylosoxidans-induced cytotoxicity. By optimizing our macrophage infection model, we determined that A. xylosoxidans enters macrophages and can reside within a membrane bound vacuole for extended periods of time. Intracellular replication appears limited with cellular lysis preceding an enhanced, mainly extracellular replication cycle. Using our optimized in vitro model system along with transposon mutagenesis, we identified 163 genes that contribute to macrophage cytotoxicity. From this list, we characterized a giant RTX adhesin encoded downstream of a type one secretion system (T1SS) that mediates bacterial binding and entry into host macrophages, an important first step toward cellular toxicity and inflammation. The RTX adhesin is encoded by other human isolates and is recognized by antibodies present in serum isolated from CF patients colonized by A. xylosoxidans, indicating this virulence factor is produced and deployed in vivo . This study represents the first characterization of A. xylosoxidans replication during infection and identifies a variety of genes that may be linked to virulence and human pathology. IMPORTANCE Patients affected by CF develop chronic bacterial infections characterized by inflammatory exacerbations and tissue damage. Advancements in sequencing technologies have broadened the list of opportunistic pathogens colonizing the CF lung. A. xylosoxidans is increasingly recognized as an opportunistic pathogen in CF, yet our understanding of the bacterium as a contributor to human disease is limited. Genomic studies have identified potential virulence determinants in A. xylosoxidans isolates, but few have been mechanistically studied. Using our optimized in vitro cell model, we identified and characterized a bacterial adhesin that mediates binding and uptake by host macrophages leading to cytotoxicity. A subset of serum samples from CF patients contains antibodies that recognize the RTX adhesion, suggesting, for the first time, that this virulence determinant is produced in vivo . This work furthers our understanding of A. xylosoxidans virulence factors at a mechanistic level.

The modulators of severe COVID-19 have emerged as the most intriguing features of SARS-CoV-2 pathogenesis. This is especially true as we are encountering variants of concern (VOC) with increased transmissibility and vaccination breakthroughs. Microbial co-infections are being investigated as one of the crucial factors for exacerbation of disease severity and complications of COVID-19. A key question remains whether early transcriptionally active microbial signature/s in COVID-19 patients can provide a window for future disease severity susceptibility and outcome? Using complementary metagenomics sequencing approaches, respiratory virus oligo panel (RVOP) and Holo-seq, our study highlights the possible functional role of nasopharyngeal early resident transcriptionally active microbes in modulating disease severity, within recovered patients with sub-phenotypes (mild, moderate, severe) and mortality. The integrative analysis combines patients' clinical parameters, SARS-CoV-2 phylogenetic analysis, microbial differential composition, and their functional role. The clinical sub-phenotypes analysis led to the identification of transcriptionally active bacterial species associated with disease severity. We found significant transcript abundance of Achromobacter xylosoxidans and Bacillus cereus in the mortality, Leptotrichia buccalis in the severe, Veillonella parvula in the moderate, and Actinomyces meyeri and Halomonas sp. in the mild COVID-19 patients. Additionally, the metabolic pathways, distinguishing the microbial functional signatures between the clinical sub-phenotypes, were also identified. We report a plausible mechanism wherein the increased transcriptionally active bacterial isolates might contribute to enhanced inflammatory response and co-infections that could modulate the disease severity in these groups. Current study provides an opportunity for potentially using these bacterial species for screening and identifying COVID-19 patient sub-groups with severe disease outcome and priority medical care. IMPORTANCE COVID-19 is invariably a disease of diverse clinical manifestation, with multiple facets involved in modulating the progression and outcome. In this regard, we investigated the role of transcriptionally active microbial co-infections as possible modulators of disease pathology in hospital admitted SARS-CoV-2 infected patients. Specifically, can there be early nasopharyngeal microbial signatures indicative of prospective disease severity? Based on disease severity symptoms, the patients were segregated into clinical sub-phenotypes: mild, moderate, severe (recovered), and mortality. We identified significant presence of transcriptionally active isolates, Achromobacter xylosoxidans and Bacillus cereus in the mortality patients. Importantly, the bacterial species might contribute toward enhancing the inflammatory responses as well as reported to be resistant to common antibiotic therapy, which together hold potential to alter the disease severity and outcome.

Bacteria of the genus Achromobacter are environmental germs, with an unknown reservoir. It can become opportunistic pathogens in immunocompromised patients, causing bacteremia, meningitis, pneumonia, or peritonitis. In recent years, Achromobacter xylosoxidans has emerged with increasing incidence in patients with cystic fibrosis (CF). Recent studies showed that A. xylosoxidans is involved in the degradation of the respiratory function of patients with CF. The respiratory ecosystem of patients with CF is colonized by bacterial species that constantly fight for space and access to nutrients. The type VI secretion system (T6SS) empowers this constant bacterial antagonism, and it is used as a virulence factor in several pathogenic bacteria. This study aimed to investigate the prevalence of the T6SS genes in A. xylosoxidans isolated in patients with CF. We also evaluated clinical and molecular characteristics of T6SS-positive A. xylosoxidans strains. We showed that A. xylosoxidans possesses a T6SS gene cluster and that some environmental and clinical isolates assemble a functional T6SS nanomachine . A. xylosoxidans T6SS is used to target competing bacteria, including other CF-specific pathogens. Finally, we demonstrated the importance of the T6SS in the internalization of A. xylosoxidans in lung epithelial cells and that the T6SS protein Hcp is detected in the sputum of patients with CF. Altogether, these results suggest for the first time a role of T6SS in CF-lung colonization by A. xylosoxidans and opens promising perspective to target this virulence determinant as innovative theranostic options for CF management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Le Goff, Vastel, Lebrun, Mansuelle, Diarra, Grandjean, Triponney, Imbert, Gosset, Dessein, Garnier and Durand.)

Achromobacter xylosoxidans is associated with resilient nosocomial infections, with bacteraemia, pneumonia and chronic cystic fibrosis lung infection being the most common clinical presentations. Innate multi-drug resistance and a suite of virulence factors select for A. xylosoxidans infection during long-term antibiotic therapy, contributing to its persistence, treatment recalcitrance, association with poor clinical outcomes and emergence as a problematic pathogen. Horizontal gene transfer and maintenance of large genomes underpin the resilience and cosmopolitan lifestyle of A. xylosoxidans , and complicate its phylogenetic characterization.

Achromobacter denitrificans is an environmental opportunistic pathogen that is infecting a large number of immunocompromised patients. A more recently identified strain from the historical collection of strains of Achromobacter denitrificans is Achromobacter mucicolens. In hosts with a variety of underlying diseases, Achromobacter spp. can induce a wide spectrum of disorders. Because of the bacterium's intrinsic genetic constitution and resistance gained over time, antibiotics are challenged to handle A. mucicolens. Due to the fact that A. mucicolens is rare and its taxonomy is not completely understood, it is difficult to define clinical symptoms, acquisition risk factors, and thus the best therapeutic course of action. To help comprehend this intrinsic and acquired resistance, we analyzed the entire genome of the A. mucicolens IA strain and utilized bioinformatics methods to estimate the strain's probable drug resistance profile. In our study, we have isolated and cultured a clinically important A. mucicolens strain and subjected it to antimicrobial susceptibility tests against antibiotics in the Vitek 2 testing system. The strain's genome sequence as well as an investigation of 27 of its phenotypic traits provides important information regarding this pathogen. The genome of this A. mucicolens IA strain possesses a number of antibiotic resistance genes that code for efflux pump systems and other antibiotic-regulating as well as -modifying enzymes. Our research analysis predicted genes involved in drug resistance, including genes for efflux pump systems, antibiotic efflux, antibiotic inactivation, and antibiotic target alteration. In vitro studies validated the genomic evidence for its ability to exhibit resistance against a wide range of antibiotics. Our investigation paves the way for more research on understanding the functioning of the key discovered genes that contribute toward the pathogenicity of A. mucicolens and hence gives new information and treatment options for this emerging pathogen. IMPORTANCE Achromobacter species are well-known opportunistic human pathogens that can be found in water and soil and most commonly in hospital settings. They thrive in immunocompromised individuals, producing sporadic cases of pneumonia, septicemia, peritonitis, urinary tract infections, and other illnesses. Achromobacter strains are inherently resistant to a wide spectrum of antibiotics, making them difficult to treat promptly. The strain under study, A. mucicolens , was notably resistant to various antibiotics, and the infection could be controlled only after several rounds of prescription medications at different doses. This consumed a lot of time and put the already immunosuppressed leukemic patient through a great ordeal. The study aimed to raise awareness about the importance of the Achromobacter bacterium's lethality, and doctors should evaluate the bacterium's potential for resistance before prescribing antibiotics. Sanitation and other precautions should also be implemented in hospitals and other public places.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

D-2-Hydroxyglutarate (D-2-HG) is a metabolite involved in many physiological metabolic processes. When D-2-HG is aberrantly accumulated due to mutations in isocitrate dehydrogenase or D-2-HG dehydrogenase, it functions in a pro-oncogenic manner and is thus considered a therapeutic target and biomarker in many cancers. In this study, DhdR from Achromobacter denitrificans NBRC 15125 is identified as an allosteric transcriptional factor that negatively regulates D-2-HG dehydrogenase expression and responds to the presence of D-2-HG. Based on the allosteric effect of DhdR, a D-2-HG biosensor is developed by combining DhdR with amplified luminescent proximity homogeneous assay (AlphaScreen) technology. The biosensor is able to detect D-2-HG in serum, urine, and cell culture medium with high specificity and sensitivity. Additionally, this biosensor is used to identify the role of D-2-HG metabolism in lipopolysaccharide biosynthesis of Pseudomonas aeruginosa, demonstrating its broad usages., (© 2021. The Author(s).)

Arsenotrophic bacteria play an essential role in lowering arsenic contamination by converting toxic arsenite [As (III)] to less toxic and less bio-accumulative arsenate [As (V)]. The current study focused on the qualitative and electrocatalytic detection of the arsenite oxidation potential of an arsenite-oxidizing bacteria A. xylosoxidans BHW-15 (retrieved from As-contaminated tube well water), which could significantly contribute to arsenic detoxification, accumulation, and immobilization while also providing a scientific foundation for future electrochemical sensor development. The minimum inhibitory concentration (MIC) value for the bacteria was 15 mM As (III). Scanning Electron Microscopy (SEM) investigation validated its intracellular As uptake capacity and demonstrated a substantial association with the MIC value. During the stationary phase, the strain's As (III) transformation efficiency was 0.0224 mM/h. Molecular analysis by real-time qPCR showed arsenite oxidase (aioA) gene expression increased 1.6-fold in the presence of As (III) compared to the untreated cells. The immobilized whole-cell also showed As (III) conversion up to 18 days. To analyze the electrochemical oxidation in water, we developed a modified GCE/P-Arg/ErGO-AuNPs electrode, which successfully sensed and quantified conversion of As (III) into As (V) by accepting electrons; implying a functional As oxidase enzyme activity in the cells. To the best of our knowledge, this is the first report on the electrochemical observation of the As-transformation mechanism with Achromobacter sp. Furthermore, the current work highlighted that our isolate might be employed as a promising candidate for arsenic bioremediation, and information acquired from this study may be helpful to open a new window for the development of a cost-effective, eco-friendly biosensor for arsenic species detection in the future., (© 2021. The Author(s).)

A cluster of Achromobacter xylosoxidans, an emerging multidrug-resistant aquaphilic bacterium, was identified in 3 long-term-care facility residents. As Pseudomonas aeruginosa and Serratia marcescens were also present in clinical specimens, we conducted an investigation of all 3 water-associated species and identified P. aerguniosa and S. marcescens contamination at the facility. Sequencing analysis linked P. aeruginosa to a clinical isolate. Findings highlight the need for precautionary measures to prevent transmission of water-associated multidrug-resistant bacteria in long-term-care facilities., (Published by Elsevier Inc.)

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.

Objectives: Achromobacter xylosoxidans is widely distributed in the environment and is increasingly recognised as a significant nosocomial pathogen in healthcare settings. However, the genomic and phylogenetic characteristics of this pathogen are not fully understood. Here we report the first genome sequence of a multidrug-resistant A. xylosoxidans ST273 strain (MTYH1) isolated from a wound infection in China., Methods: Whole-genome sequencing of the strain was performed using an Illumina NovaSeq 6000 platform. Generated short reads were de novo assembled into contigs using SPAdes v.3.13.0. Antimicrobial resistance genes (ARGs) were identified using ResFinder 4.0. Phylogenetic analysis was conducted using NJ/UPGMA phylogeny (MAFFT v.7) based on core genome single nucleotide polymorphism data., Results: The draft genome sequence of A. xylosoxidans ST273 strain MTYH1 consists of 45 contigs comprising 6 643 045 bp with a GC content of 67.5%. According to the Achromobacter spp. multilocus sequence typing (MLST) scheme, MTYH1 belongs to ST273. MTYH1 is resistant to aminoglycosides, ciprofloxacin, aztreonam and cephalosporins. Five ARGs belonging to three different classes were identified in the genome, namely aac(6')-IIa, ant(2'')-Ia, bla CARB-2 , bla OXA-114g and sul1. Phylogenetic analysis showed that MTYH1 was not epidemiologically related to any of the strains retrieved from the NCBI GenBank database., Conclusion: We unravelled the genome sequence of multidrug-resistant A. xylosoxidans ST273 clinical strain MTYH1 recovered from a wound infection in China. Our data may help to understand the antimicrobial resistance mechanisms, genomic features and phylogenetic characteristic of this bacterial pathogen., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In patients with cystic fibrosis (CF), the lung is a remarkable ecological niche in which the microbiome is subjected to important selective pressures. An inexorable colonization by bacteria of both endogenous and environmental origin is observed in most patients, leading to a vicious cycle of infection-inflammation. In this context, long-term colonization together with competitive interactions among bacteria can lead to over-inflammation. While Pseudomonas aeruginosa and Staphylococcus aureus , the two pathogens most frequently identified in CF, have been largely studied for adaptation to the CF lung, in the last few years, there has been a growing interest in emerging pathogens of environmental origin, namely Achromobacter xylosoxidans and Stenotrophomonas maltophilia . The aim of this review is to gather all the current knowledge on the major pathophysiological traits, their supporting mechanisms, regulation and evolutionary modifications involved in colonization, virulence, and competitive interactions with other members of the lung microbiota for these emerging pathogens, with all these mechanisms being major drivers of persistence in the CF lung. Currently available research on A. xylosoxidans complex and S. maltophilia shows that these emerging pathogens share important pathophysiological features with well-known CF pathogens, making them important members of the complex bacterial community living in the CF lung.

Penicillin G acylase (PGA) was an important biocatalyst for enzymatic production of second-generation cephalosporin. PGA from Achromobacter xylosoxidans PX02 (AxPGA) showed relatively lower identity to EcPGA (54.9% in α subunit and 51.7% in β subunit), which could synthesize cefamandole in the kinetically controlled N-acylation (kcNa). Semi-rational design of AxPGA and "small and smart" mutant libraries were developed with minimal screening to improve cefamandole production. A triple mutant αR141A/αF142I/βF24G by combining the mutational sites (βF24, αR141, and αF142) from different subunits of AxPGA showed better performance in cefamandole production, with 4.2-fold of improvement in the (k cat /K m ) AD value for activated acyl donor (R)-Methyl mandelate. Meanwhile, the (k cat /K m ) Ps value for cefamandole by mutant αR141A/αF142I/βF24G was sharply dropped by 25.5 times, indicating its highly synthetic activity and extremely low hydrolysis of cefamandole. Strikingly, the triple mutant αR141A/αF142I/βF24G could form cefamandole with a yield of 85% at an economical substrate ratio (acyl donor/nucleophile) of 1.3:1 (82% at 1.1:1), which advanced the greener and more sustainable process of cefamandole production than the wild type. Furtherly, the improved synthetic ability and lower hydrolysis of cefamandole by mutant were rationalized using molecular docking., (Copyright © 2021. Published by Elsevier B.V.)

Polycyclic aromatic hydrocarbons (PAHs) are widespread and harmful contaminants and are more persistent under anaerobic conditions. The bioremediation of PAHs in anaerobic zones has been enhanced by treating the contamination with nitrate, which is thermodynamically favourable, cost-effective, and highly soluble. However, anaerobic PAHs biotransformation processes that employ nitrate as an electron acceptor have not been fully explored. In this study, we investigated the anaerobic biotransformation of PAHs by strain PheN1, a newly isolated phenanthrene-degrading denitrifier, using phenanthrene as a model compound. PheN1 is phylogenetically closely related to Achromobacter denitrificans and reduces nitrate to nitrite (not N 2 ) during the anaerobic phenanthrene degradation process. Phenanthrene biotransformation processes were detected using gas chromatography-mass spectrometry and were further examined by reverse transcription-quantitative PCR and genome analyses. Carboxylation and methylation were both found to be the initial steps in the phenanthrene degradation process. Downstream biotransformation processed benzene compounds and cyclohexane derivatives. This study describes the isolation of an anaerobic phenanthrene-degrading bacterium along with the pure-culture evidence of phenanthrene biotransformation processes with nitrate as an electron acceptor. The findings in this study can improve our understanding of anaerobic PAHs biodegradation processes and guide PAHs bioremediation by adding nitrate to anaerobic environments., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Achromobacter xylosoxidans DN002 is capable of utilizing numerous aromatic hydrocarbons as sole carbon and energy resource. In this study, the whole genome of strain DN002 was sequenced and analyzed, which consisted of one circular chromosome of 5,943,204 bp and a 278,917 bp plasmid with an average GC content of 65.46 mol%, 5694 protein-coding genes, 13 rRNA genes and 57 tRNA genes. Analysis of cluster of orthologous group (COG) demonstrated that strain DN002 had remarkable gene abundance foramino acid transport and metabolism, transcription, inorganic ion transport and metabolism, energy production and conversion, and carbohydrate transport and metabolism. Genes related to biodegradation of aromatic hydrocarbons, chemotaxis and flagella were identified from the genome, which will advance our fundamental understanding the molecular mechanism for degradation and metabolizing of aromatic hydrocarbons.

Achromobacter spp. are recognized as emerging pathogens in patients with cystic fibrosis (CF). Though recent works have established species-level identification using nrdA sequencing, there is a dearth in knowledge relating to species-level antimicrobial susceptibility patterns and antimicrobial combinations, which hampers the use of optimal antimicrobial combinations for the treatment of chronic infections. The aims of this study were to (i) identify at species-level referred Achromobacter isolates, (ii) describe species-level antimicrobial susceptibility profiles, and (iii) determine the most promising antimicrobial combination for chronic Achromobacter infections. A total of 112 multidrug-resistant (MDR) Achromobacter species isolates from 39 patients were identified using nrdA sequencing. Antimicrobial susceptibility and combination testing were carried out using the Etest method. We detected six species of Achromobacter and found that Achromobacter xylosoxidans was the most prevalent species. Interestingly, sequence analysis showed it was responsible for persistent infection (18/28 patients), followed by Achromobacter ruhlandii (2/3 patients). Piperacillin-tazobactam (70.27%) and co-trimoxazole (69.72%) were the most active antimicrobials. Differences were observed in species-level susceptibility to ceftazidime, carbapenems, ticarcillin-clavulanate, and tetracycline. Antimicrobial combinations with co-trimoxazole or tobramycin demonstrate the best synergy, while co-trimoxazole gave the best susceptibility breakpoint index values. This study enriches the understanding of MDR Achromobacter spp. epidemiology and confirms prevalence and chronic colonization of A. xylosoxidans in CF lungs. It presents in vitro data to support the efficacy of new combinations for use in the treatment of chronic Achromobacter infections., (Copyright © 2020 American Society for Microbiology.)

This study investigated the genetic basis of multidrug resistance in two strains of Achromobacter xylosoxidans isolated from patients attending a hospital in Thailand in 2012. These isolates were highly resistant to cephalosporins, aminoglycosides, fluoroquinolones, co-trimoxazole and carbapenems. Whole genome sequencing revealed that the two isolates were not clonally related and identified a carbapenem resistance gene-habouring integron (In687), residing in a novel genomic island, AcGI1. This In687 shares 100% identical nucleotide sequence with ones found in Acinetobacter baumannii Aci 16, isolated from the same hospital in 2007. We report the first analysis of multidrug-resistant A. xylosoxidans isolated in Thailand, and the first example of this island in A. xylosoxidans. Our data support the idea that resistance has spread in Thailand via horizontal gene transfer between species and suggest the possibility of A. xylosoxidans may serve as a reservoir of antibiotic resistance, especially in hospital setting., (© FEMS 2020.)

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

A structured survey of the cystic fibrosis pathogens Achromobacter, Pandoraea and Ralstonia species from thirteen sentinel hospitals throughout England was undertaken by Public Health England. One isolate per patient of these genera collected from CF patients during the seven-month survey period in 2015 was requested from participating hospitals. Species-level identification was performed using nrdA/gyrB sequence cluster analysis, and genotyping by pulsed-field gel electrophoresis. In total, 176 isolates were included in the survey; 138 Achromobacter spp. (78.4%), 29 Pandoraea spp. (16.5%) and 9 Ralstonia spp. (5.1%). Novel Achromobacter and Pandoraea clusters were identified. High levels of antimicrobial resistance were found, particularly among Pandoraea isolates. Genotyping analysis revealed considerable diversity, however one geographically-widespread cluster of A. xylosoxidans isolates from six hospitals was found, in addition to two other clusters, both comprising isolates from two hospitals, either derived from the same region (A. xylosoxidans), or from hospitals within the same city (P. apista)., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Growth and productivity of rice are negatively affected by soil salinity. However, some salt-tolerant rhizosphere-inhabiting bacteria can improve salt resistance of plants, thereby augmenting plant growth and production. Here, we isolated a total of 53 plant-growth-promoting rhizobacteria (PGPR) from saline and non-saline areas in Bangladesh where electrical conductivity was measured as >7.45 and <1.80 dS/m, respectively. Bacteria isolated from saline areas were able to grow in a salt concentration of up to 2.60 mol/L, contrary to the isolates collected from non-saline areas that did not survive beyond 854 mmol/L. Among the salt-tolerant isolates, Bacillus aryabhattai , Achromobacter denitrificans , and Ochrobactrum intermedium , identified by comparing respective sequences of 16S rRNA using the NCBI GenBank, exhibited a higher amount of atmospheric nitrogen fixation, phosphate solubilization, and indoleacetic acid production at 200 mmol/L salt stress. Salt-tolerant isolates exhibited greater resistance to heavy metals and antibiotics, which could be due to the production of an exopolysaccharide layer outside the cell surface. Oryza sativa L. fertilized with B. aryabhattai MS3 and grown under 200 mmol/L salt stress was found to be favoured by enhanced expression of a set of at least four salt-responsive plant genes: BZ8 , SOS1 , GIG , and NHX1 . Fertilization of rice with osmoprotectant-producing PGPR, therefore, could be a climate-change-preparedness strategy for coastal agriculture.

The immunochromatographic assay NG-Test Carba 5 (NG-Biotech) was evaluated with a collection of 107 carbapenemase-producing nonfermenters (CP-NF) (55 Pseudomonas spp., 51 Acinetobacter spp., and 1 Achromobacter xylosoxidans isolate) and 61 carbapenemase-negative isolates. All KPC, VIM, and NDM carbapenemase producers tested were accurately detected. Of the 16 IMP variants tested, 6 (37.5%) variants were not detected. Considering the epidemiology of CP-NFs in France, the NG-Test Carba 5 would detect 89.4% of CP Pseudomonas spp. but only 12.9% of CP Acinetobacter spp., (Copyright © 2019 American Society for Microbiology.)

Achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. Intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent A. xylosoxidans infection. Still, much of A. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. Here we demonstrate a promising genetic system for use in A. xylosoxidans ; generating a transposon mutant library which was then used to identify genes involved in biofilm development in vitro. We further described the effects of one of the genes found in the mutagenesis screen, encoding a putative enoyl-CoA hydratase, on biofilm structure and tolerance to antimicrobials. Through additional analysis, we find that a fatty acid signaling compound is essential to A. xylosoxidans biofilm ultrastructure and maintenance. This work describes methods for the genetic manipulation of A. xylosoxidans and demonstrated their use to improve our understanding of A. xylosoxidans pathophysiology., Competing Interests: Competing interestsThe authors declare no competing interests.

The aim of this study was to evaluate the contribution of plasmid-mediated genes and efflux to fluoroquinolone resistance in collection of Achromobacter spp. gathered during a 3-year period. Susceptibility to ciprofloxacin and levofloxacin was tested by disk diffusion and microdilution tests for a collection of 98 Achromobacter spp. clinical isolates. Identification of fluoroquinolone-resistant isolates was performed by sequencing and phylogenetic analyses of the nrdA gene. Genetic relatedness among resistant isolates was determined by pulsed-field gel electrophoresis (PFGE) analysis. The influence of an H + conductor cyanide m-chlorophenyl hydrazone (CCCP) and a resistance-nodulation-division-type efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PAβN) on minimal inhibitory concentration (MIC) value was evaluated by broth microdilution. The presence of the plasmid-mediated qnrA, qnrB, qnrC, qnrS, and aac-(6')-Ib-cr genes was investigated by PCR and sequencing. Achromobacter spp. isolates that were resistant or intermediately resistant to fluoroquinolones in disk diffusion tests (44/98) were subjected to microdilution. As a result, 20/98 isolates were confirmed to be resistant to ciprofloxacin while 10/98 was resistant to levofloxacin. CCCP decreased twofold MIC value for ciprofloxacin in six isolates and more than 16 times in one isolate, while MIC value for levofloxacin was decreased in all isolates (twofold to more than eightfold). Fluoroquinolone-resistant isolates were identified as A. xylosoxidans with the nrdA gene sequencing. PFGE revealed that resistant isolates belonged to seven different genotypes. Ten isolates belonging to four genotypes were positive for the aac-(6')-Ib-cr gene. Although resistance to fluoroquinolones was not widespread among analyzed isolates, detected contribution of efflux pumps and the presence of the aac-(6')-Ib-cr gene present a platform for emergence of more resistant strains.

We describe an extremely rare case of mediastinitis superinfected by emerging Achromobacter xylosoxidans. After mitral and aortic valves replacement, the patient first developed a Staphylococcus aureus mediastinitis, and five days after starting adapted antibiotic therapy, superficial pus analysis revealed the presence of Achromobacter xylosoxidans. This superinfection was considered superficial and focus was made on Staphylococcus aureus mediastinitis. Three weeks later, no more Staphylococcus aureus was found in pus samples and the sepsis seemed under control. Unfortunately, blood cultures were again positive for Achromobacter xylosoxidans three weeks later and the patient died from septic shock., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Achromobacter spp. are nonfermentative Gram-negative bacilli considered emergent pathogens in cystic fibrosis (CF). Although some cross-transmission events between CF patients have been described, Achromobacter strains were mostly patient specific, suggesting sporadic acquisitions from nonhuman reservoirs. However, sources of these emergent CF pathogens remain unknown. A large collection of specimens ( n = 273) was sampled in the homes of 3 CF patients chronically colonized by Achromobacter xylosoxidans with the aim of evaluating the potential role of domestic reservoirs in sustaining airway colonization of the patients. Samples were screened for the presence of Achromobacter by using genus-specific molecular detection. Species identification, multilocus genotypes, and antimicrobial susceptibility patterns observed for environmental isolates were compared with those of clinical strains. Patient homes hosted a high diversity of Achromobacter species ( n = 7), including Achromobacter mucicolens and A. animicus , two species previously isolated from human samples only, and genotypes ( n = 15), all showing an overall susceptibility to antimicrobial agents. Achromobacter strains were mostly isolated from indoor moist environments and siphons, which are potential reservoirs for several CF emerging pathogens. A. xylosoxidans , the worldwide prevalent species colonizing CF patients, was not the major Achromobacter species inhabiting domestic environments. A. xylosoxidans genotypes chronically colonizing the patients were not detected in their household environments. These results support the notions that the domestic environment could not be incriminated in sustained patient colonization and that after initial colonization, the environmental survival of A. xylosoxidans clones adapted to the CF airways is probably impaired. IMPORTANCE Achromobacter spp. are worldwide emerging opportunistic pathogens in CF patients, able to chronically colonize the respiratory tract. Apart from regular consultations at the hospital CF center, patients spend most of their time at home. Colonization from nonhuman sources has been suggested, but the presence of Achromobacter spp. in CF patients' homes has not been explored. The domestic environments of CF patients chronically colonized by Achromobacter , especially wet environments, host several opportunistic pathogens, including a large diversity of Achromobacter species and genotypes. However, Achromobacter genotypes colonizing the patients were not detected in their domestic environments, making it unlikely that a shuttle between environment and CF airways is involved in persisting colonization. This also suggests that once the bacteria have adapted to the respiratory tract, their survival in the domestic environment is presumably impaired. Nevertheless, measures for reducing domestic patient exposure should be targeted on evacuation drains, which are frequently contaminated by CF opportunistic pathogens., (Copyright © 2018 American Society for Microbiology.)

High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting steps. In this study, three PGAs isolated from Providencia rettgeri (PrPGA), Alcaligenes faecalis (AfPGA), and Achromobacter xylosoxidans (AxPGA) were efficiently expressed in E. coli by replacing with applicable signal peptide. Different bottlenecks of the expression process were analyzed for PrPGA, AfPGA, and AxPGA. Subsequently, five efficient signal peptides, including OmpA, pelB, Lpp, PhoA, and MalE, were used to replace the original signal peptides of the PGAs. With respect to AfPGA and AxPGA, translocation was the primary limitation, and the use of pelB signal peptide effectively overcame this barrier. For PrPGA, which was almost not expressed in wild type, the translation initiation efficiency was optimized by replacing with MalE signal peptide. In addition, low temperature (20 °C) slowed down the transcription and translation, thereby facilitating the posttranslational process and preventing the formation of inclusion bodies. Furthermore, combined induction with IPTG and arabinose not only enhanced the cell density but also remarkably improved the expression of PGAs. Final specific activities of the three PGAs reached 2100 (PrPGA), 9200 (AfPGA), and 1400 (AxPGA) U/L/OD 600 , respectively. This simple and robust strategy by fitting replacement of signal peptide might dramatically improve the expression of PGAs from various bacteria, which was significant in the production of many valuable β-lactam antibiotics.

The level of catalase and superoxide dismutase induction, as well as generation of superoxide anion radical in cells and accumulation of hydrogen peroxide in the culture medium were researched in three strains of oil-degrading bacteria Achromobacter xylosoxidans at cultivation in rich nutrient medium and in the media with hydrocarbons as the only source of carbon. The effects of pentane, decane, hexadecane, cyclohexane, benzene, naphthalene and diesel fuel were evaluated. It was determined that in the microbial cell on media with hydrocarbons, the generation of superoxide anion radical increases, accumulation of hydrogen peroxide and induction of superoxide dismutase synthesis occur, and catalase activity is reduced. Oxidative stress in the cells of A. xylosoxidans was caused by biotransformation of all the studied hydrocarbons. The most pronounced effect was observed at incubation of bacteria with cyclohexane, pentane, diesel fuel, benzene and naphthalene.

The in vitro activity of anti-pseudomonal β-lactams in combination with avibactam was evaluated against 54 multidrug-resistant non-fermenting Gram-negative bacilli isolated from cystic fibrosis patients. Avibactam increased and/or restored the antibacterial activities of ceftazidime and aztreonam against Pseudomonas aeruginosa and Stenotrophomonas maltophilia, respectively. No β-lactam-avibactam combination was active against Achromobacter xylosoxidans.

We describe the genome sequence of Pseudomonas reinekei MT1 and Achromobacter xylosoxidans MT3, the most abundant members of a bacterial community capable of degrading chloroaromatic compounds. The MT1 genome contains open reading frames encoding enzymes responsible for the catabolism of chlorosalicylate, methylsalicylate, chlorophenols, phenol, benzoate, p-coumarate, phenylalanine, and phenylacetate. On the other hand, the MT3 strain genome possesses no ORFs to metabolize chlorosalicylates; instead the bacterium is capable of metabolizing nitro-phenolic and phenolic compounds, which can be used as the only carbon and energy source by MT3. We also confirmed that MT3 displays the genetic machinery for the metabolism of chlorocathecols and chloromuconates, where the latter are toxic compounds secreted by MT1 when degrading chlorosalicylates. Altogether, this work will advance our fundamental understanding of bacterial interactions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans. Comparative proteomic analysis of consecutively collected susceptible and resistant isolates from the same patient revealed that high Axc protein levels were only observed in the resistant isolate. Heterologous expression of Axc in Escherichia coli significantly increased the resistance towards carbapenems. Importantly, direct Axc mediated hydrolysis of imipenem was demonstrated using pH shift assays and 1 H-NMR, confirming Axc as a legitimate carbapenemase. Whole genome sequencing revealed that the susceptible and resistant isolates were remarkably similar. Together these findings provide a molecular context for the fast development of meropenem resistance in A. xylosoxidans during treatment and demonstrate the use of mass spectrometric techniques in identifying novel resistance determinants.

Objective Achromobacter xylosoxidans (A. xylosoxidans) has been recently reported to have an association with the development of pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma in patients from European countries. However, the prevalence rates for A. xylosoxidans may vary significantly from country to country. To assess this association, the prevalence of A. xylosoxidans was analyzed in Japanese patients with pulmonary B-cell lymphoma. Methods DNA samples were obtained from formalin-fixed, paraffin-embedded sections of pulmonary MALT lymphomas (n=52), diffuse large B-cell lymphomas (DLBCLs, n=18), and benign pulmonary lesions (n=19). All samples were histopathologically reviewed by experienced hematopathologists, and the clonality of all MALT lymphoma cases was confirmed by a polymerase chain reaction (PCR)-based IGH rearrangement clonality assay. They were also tested for the API2-MALT1 fusion transcript. The presence of bacterial DNA was assessed with a nested PCR, and DNA sequencing was performed to confirm the PCR specificity. Results A. xylosoxidans DNA was detected in 1/52 cases of pulmonary MALT lymphoma, 2/18 cases of DLBCL, and 0/19 cases of benign pulmonary lesions. The prevalence of A. xylosoxidans in pulmonary lymphoma was not significantly higher than in benign lesions. Conclusion The present study shows that A. xylosoxidans infection may not be associated with pulmonary B-cell lymphoma in a Japanese case series. Large-scale international studies are needed to clarify the role of A. xylosoxidans in pulmonary lymphoma.

Coprinus comatus is an edible mushroom widely cultivated in China as a delicious food. Various diseases have occurred on C. comatus with the cultivated area increasing. In this study, the pathogenic bacterium JTG-B1, identified as Achromobacter xylosoxidans by 16S rDNA and nrdA gene sequencing, was isolated from edible mushroom Coprinus comatus with serious rot disease on its stipe. A. xylosoxidans has been confirmed as an important opportunistic human pathogenic bacterium and has been isolated from respiratory samples from cystic fibrosis. It is widely distributed in the environment. Here, we first report that fungi can also serve as a host for A. xylosoxidans. We confirmed that it can cross-kingdom infect between animals (mice) and fungi (C. comatus). The results of pathogenicity tests, physiological, biochemical and genotyping analysis of A. xylosoxidans from different hosts suggested that different strain of A. xylosoxidans may have pathogenicity differentiation. A. xylosoxidans not only is pathogenic to C. comatus but also may threaten human health., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

We report the isolation and identification of seven bacterial strains and one fungal strain from dead and diseased Scapteriscus borellii mole crickets collected from a golf course in southern California. Using 16S and 18S rRNA gene sequence analysis we identified the microbes as Serratia marcescens (red), S. marcescens (white), S. marcescens (purple), Achromobacter xylosoxidans, Chryseobacterium sp., Ochrobactrum anthropi, Tsukamurella tryosinosolvens, and Beauveria bassiana. We performed a dose response curve for each of these cricket-associated microbial strains (except T. tryosinosolvens) and two other strains of S. marcescens (DB1140 and ATCC 13880). We found that all of these microbes except O. anthropi were highly pathogenic to D. melanogaster compared to the other strains of S. marcescens. Injecting the mole cricket associated strains of Serratia into flies killed all infected flies in ≤24h. For all other strains, the median time to death of injected flies varied in a dose-dependent manner. In vivo growth assessments of these microbes suggested that the host immune system was quickly overcome. We used disease tolerance curves to better understand the host-microbe interactions. Further studies are necessary to understand in mechanistic detail the virulence mechanisms of these mole cricket associated microbes and how this association may have influenced the evolution of mole cricket immunity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Achromobacter xylosoxidans is a versatile bacterium known for its ability to degrade aromatic compounds. However, its ability to oxidize sulfur compounds for electron and energy source is not reported much. In the present work, the Gram-negative bacterium Achromobacter xylosoxidans strain wsp05 isolated from a waste stabilization ponds (WSPs) system was studied for its ability to oxidize reduced sulfur compounds. The strain was able to oxidize thiosulfate and sodium sulfite. To observe the effect of physicochemical parameters on the rate of sulfur oxidation, strain wsp05 was grown in thiosulfate (20 mM) containing minimal salt medium at varied pH, temperature and ammonium and phosphate ions concentration. Maximum thiosulfate oxidation was observed at 30 °C with initial pH of 7-7.2. The strain was characterized using universal 16S rRNA gene primers revealing high similarity (> 99%) with Achromobacter xylosoxidans NBRC 15126 T belonging to β-proteobacteria. In the present study, we investigated the sulfur oxidation properties of the Achromobacter xylosoxidans strain wsp05, which revealed an ecological and phylogenetic widening over which the thiotrophs are distributed.

Metallo-β-lactamases (MBLs) threaten the effectiveness of β-lactam antibiotics, including carbapenems, and are a concern for global public health. β-Lactam/β-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-β-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC 50 ) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC 50 levels of the new thiol-based inhibitors were 0.66 μM (inhibitor 2a) and 0.62 μM (inhibitor 2b), and the equilibrium dissociation constant ( K D ) of inhibitor 2a was 1.6 μM; thus, both were more potent inhibitors than l-captopril (IC 50 = 47 μM; K D = 25 μM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril., (Copyright © 2017 American Society for Microbiology.)