Complete Genome of Achromobacter xylosoxidans, a Nitrogen-Fixing Bacteria from the Rhizosphere of Cowpea (Vigna unguiculata [L.] Walp) Tolerant to Cucumber Mosaic Virus Infection.

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.

Identification of Virulence Factors Involved in a Murine Model of Severe Achromobacter xylosoxidans Infection.

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.

Pathogenesis of Achromobacter xylosoxidans respiratory infections: colonization, persistence, and transcriptome profiling in synthetic cystic fibrosis sputum medium.

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.

Role of AxyABM overexpression in acquired resistance in Achromobacter xylosoxidans.

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

Anaerobic biodegradation of phenanthrene by a newly isolated nitrate-dependent Achromobacter denitrificans strain PheN1 and exploration of the biotransformation processes by metabolite and genome analyses.

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 N2 ) 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.

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

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

Longitudinal Surveillance and Combination Antimicrobial Susceptibility Testing of Multidrug-Resistant Achromobacter Species from Cystic Fibrosis Patients.

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.

Complete genome sequence of the aromatic-hydrocarbon-degrading bacterium Achromobacter xylosoxidans DN002.

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.

Isolation and identification of salt-tolerant plant-growth-promoting rhizobacteria and their application for rice cultivation under salt stress.

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.

Hematogenous pleural infection caused by Achromobacter xylosoxidans in a patient undergoing maintenance hemodialysis.

A 78-year-old Japanese man, undergoing maintenance hemodialysis for 20 years and having received coronary artery bypass grafting two months before, was hospitalized because of fever with subclinical left-sided pleurisy. Achromobacter xylosoxidans strains exhibiting identical genomic patterns on a macrorestriction analysis were isolated from the blood and the pleural effusion obtained on admission. Physical and radiological examinations did not reveal any lesions in either chest wall or lung adjacent to the effusion, indicating that the organism in the effusion had entered the pleural space via the bloodstream. Immunocompromising conditions due to undergoing maintenance hemodialysis and the presence of the antecedently accumulated pleural effusion may have been associated with the development of hematogenous dissemination. The patient fully recovered only with antibiotic therapy. To our knowledge, the present report is the first describing a case of hematogenous pleural infection caused by A. xylosoxidans.

Evaluation of the Immunochromatographic NG-Test Carba 5 for Rapid Identification of Carbapenemase in Nonfermenters.

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.

Chronic Airway Colonization by Achromobacter xylosoxidans in Cystic Fibrosis Patients Is Not Sustained by Their Domestic Environment.

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.IMPORTANCEAchromobacter 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.

Antioxidant enzymes and reactive oxygen species level of the Achromobacter xylosoxidans bacteria during hydrocarbons biotransformation.

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.

Fitting replacement of signal peptide for highly efficient expression of three penicillin G acylases in E. coli.

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/OD600, 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 ß-lactam antibiotics.

Mediastinitis superinfected by Achromobacter xylosoxidans. A case report.

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.

Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.

Metallo-ß-lactamases (MBLs) threaten the effectiveness of ß-lactam antibiotics, including carbapenems, and are a concern for global public health. ß-Lactam/ß-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-ß-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 (IC50) 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 IC50 levels of the new thiol-based inhibitors were 0.66 µM (inhibitor 2a) and 0.62 µM (inhibitor 2b), and the equilibrium dissociation constant (KD ) of inhibitor 2a was 1.6 µM; thus, both were more potent inhibitors than l-captopril (IC50 = 47 µM; KD = 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.

Microbial associates of the southern mole cricket (Scapteriscus borellii) are highly pathogenic.

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.

Sulfur oxidation by Achromobacter xylosoxidans strain wsp05 reveals ecological widening over which thiotrophs are distributed.

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 15126T belonging to ß-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.

Interspecies Dissemination of a Mobilizable Plasmid Harboring blaIMP-19 and the Possibility of Horizontal Gene Transfer in a Single Patient.

Carbapenemase-producing Gram-negative bacilli have been a global concern over the past 2 decades because these organisms can cause severe infections with high mortality rates. Carbapenemase genes are often carried by mobile genetic elements, and resistance plasmids can be transferred through conjugation. We conducted whole-genome sequencing (WGS) to demonstrate that the same plasmid harboring a metallo-ß-lactamase gene was detected in two different species isolated from a single patient. Metallo-ß-lactamase-producing Achromobacter xylosoxidans (KUN4507), non-metallo-ß-lactamase-producing Klebsiella pneumoniae (KUN4843), and metallo-ß-lactamase-producing K. pneumoniae (KUN5033) were sequentially isolated from a single patient and then analyzed in this study. Antimicrobial susceptibility testing, molecular typing (pulsed-field gel electrophoresis and multilocus sequence typing), and conjugation analyses were performed by conventional methods. Phylogenetic and molecular clock analysis of K. pneumoniae isolates were performed with WGS, and the nucleotide sequences of plasmids detected from these isolates were determined using WGS. Conventional molecular typing revealed that KUN4843 and KUN5033 were identical, whereas the phylogenetic tree analysis revealed a slight difference. These two isolates were separated from the most recent common ancestor 0.74 years before they were isolated. The same resistance plasmid harboring blaIMP-19 was detected in metallo-ß-lactamase-producing A. xylosoxidans and K. pneumoniae Although this plasmid was not self-transferable, the conjugation of this plasmid from A. xylosoxidans to non-metallo-ß-lactamase-producing K. pneumoniae was successfully performed. The susceptibility patterns for metallo-ß-lactamase-producing K. pneumoniae and the transconjugant were similar. These findings supported the possibility of the horizontal transfer of plasmid-borne blaIMP-19 from A. xylosoxidans to K. pneumoniae in a single patient.

Epidemic Achromobacter xylosoxidans strain among Belgian cystic fibrosis patients and review of literature.

BACKGROUND: Achromobacter xylosoxidans is increasingly being recognized as an emerging pathogen in cystic fibrosis. Recent severe infections with A. xylosoxidans in some of our cystic fibrosis (CF) patients led to a re-evaluation of the epidemiology of CF-associated A. xylosoxidans infections in two Belgian reference centres (Antwerp and Ghent). Several of these patients also stayed at the Rehabilitation Centre De Haan (RHC). In total, 59 A. xylosoxidans isolates from 31 patients (including 26 CF patients), collected between 2001 and 2014, were studied. We evaluated Matrix Assisted Laser Desorption Ionisation -Time of Flight mass spectrometry (MALDI-TOF) as an alternative for McRAPD typing. RESULTS: Both typing approaches established the presence of a major cluster, comprising isolates, all from 21 CF patients, including from two patients sampled when staying at the RHC a decade ago. This major cluster was the same as the cluster established already a decade ago at the RHC. A minor cluster consisted of 13 isolates from miscellaneous origin. A further seven isolates, including one from a non-CF patient who had stayed recently at the RHC, were singletons. CONCLUSIONS: Typing results of both methods were similar, indicating transmission of a single clone of A. xylosoxidans among several CF patients from at least two reference centres. Isolates of the same clone were already observed at the RHC, a decade ago. It is difficult to establish to what extent the RHC is the source of transmission, because the epidemic strain was already present when the first epidemiological study in the RHC was carried out. This study also documents the applicability of MALDI-TOF for typing of strains within the species A. xylosoxidans and the need to use the dynamic cutoff algorithm of the BioNumerics® software for correct clustering of the fingerprints.