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.
- MeSH
- Achromobacter denitrificans * genetics MeSH
- Achromobacter * MeSH
- Anti-Bacterial Agents therapeutic use MeSH
- Cystic Fibrosis * MeSH
- Child MeSH
- Genomics MeSH
- Gram-Negative Bacterial Infections * MeSH
- Integrases therapeutic use MeSH
- Integrons genetics MeSH
- Trimethoprim, Sulfamethoxazole Drug Combination MeSH
- Humans MeSH
- Microbial Sensitivity Tests MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Serbia MeSH
BACKGROUND: Achromobacter species are emerging pathogens isolated from respiratory samples of Patients with cystic fibrosis (pwCF) causing growing concerns in the CF community. The epidemiology and the clinical impact of Achromobacter in CF is unclear since data are restricted to small case control studies or selected populations. AIM: To characterize the effect of Achromobacter respiratory infection on CF lung disease. METHODS: European CF Society Patient Registry data was analysed for association between Achromobacter infection and demographic/clinical characteristics and outcomes of pwCF. RESULTS: Of eligible 38,795 patients, Achromobacter infection was reported in 2,093 (prevalence (95% CI) of 5.40% (5.17 - 5.62). The prevalence varied significantly between the countries and increased with age peaking at the age 20-30. Achromobacter infection was more prevalent in pwCF carrying class minimal function mutations, having worse nutrition or lower pulmonary function, and more patients inhaled antibiotics against P. aeruginosa. Patient infected with Achromobacter had similar pulmonary function and BMI to patients infected with P. aeruginosa at all age groups. Being infected with both bacteria was associated with significantly lower pulmonary function and BMI at all age groups. CONCLUSIONS: Achromobacter infection was associated with disease severity similar to infection with P. aeruginosa. Being infected with both bacteria is associated with even more severe disease. This suggests to study if eradication will improve the outcome of pwCF.
- MeSH
- Achromobacter * genetics MeSH
- Cystic Fibrosis * complications epidemiology microbiology MeSH
- Adult MeSH
- Gram-Negative Bacterial Infections * diagnosis drug therapy epidemiology MeSH
- Respiratory Tract Infections * microbiology MeSH
- Humans MeSH
- Young Adult MeSH
- Lung MeSH
- Pseudomonas aeruginosa MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Young Adult MeSH
- Publication type
- Journal Article MeSH
In the present study, bacterial isolates were screened for arsenic resistance efficiency. Environmental isolates were isolated from arsenic-rich soil samples (i.e., from Rajnandgaon district of Chhattisgarh state, India). Amplification and sequencing of 16S rRNA gene revealed that the isolates were of Bacillus firmus RSN1, Brevibacterium senegalense RSN2, Enterobacter cloacae RSN3, Stenotrophomonas pavanii RSN6, Achromobacter mucicolens RSN7, and Ochrobactrum intermedium RSN10. Arsenite efflux gene (arsB) was successfully amplified in E. cloacae RSN3. Atomic absorption spectroscopy (AAS) analysis showed an absorption of 32.22% arsenic by the RSN3 strain. Furthermore, results of scanning electron microscopy (SEM) for morphological variations revealed an initial increase in the cell size at 1 mM sodium arsenate; however, it was decreased at 10 mM concentration in comparison to control. This change of the cell size in different metal concentrations was due to the uptake and expulsion of the metal from the cell, which also confirmed the arsenite efflux system.
Endosulfan is an organochlorine pesticide included in the Stockholm Convention for Persistent Organic Compounds. The utilization of endosulfan as the sole source of carbon and its mineralization was evaluated using pure strains of Bacillus subtilis, Bacillus pseudomycoides, Peribacillus simplex, Enterobacter cloacae, Achromobacter spanius, and Pseudomonas putida, isolated from soil with historical pesticide use. The consumption of the α isomer of endosulfan by five of the six strains studied was higher than 95%, while B. subtilis degraded only 76% of the initial concentration (14 mg/L). On the other hand, the degradation of the β isomer was approximately 86% of the initial concentration (6 mg/L) by B. subtilis, P. simplex, and B. pseudomycoides and 95% by P. putida, E. cloacae, and A. spanius. The ability of A. spanius, P. simplex, and B. pseudomycoides to degrade endosulfan has not been previously reported. The production of endosulfan lactone by the Bacillus strains, as well as A. spanius and P. putida, indicated that endosulfan was degraded by the hydrolytic pathway.
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.
- MeSH
- Achromobacter denitrificans classification drug effects genetics isolation & purification MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Genes, Bacterial * genetics MeSH
- Drug Resistance, Bacterial genetics MeSH
- Fluoroquinolones pharmacology MeSH
- Phylogeny MeSH
- Genotype MeSH
- Gram-Negative Bacterial Infections microbiology MeSH
- Humans MeSH
- Microbial Sensitivity Tests MeSH
- Plasmids genetics MeSH
- Polymerase Chain Reaction MeSH
- Electrophoresis, Gel, Pulsed-Field MeSH
- Sequence Analysis, DNA MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Serbia MeSH
This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZα under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAA expressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGAA production of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGAA is similar but there is a limitation in secretion efficiency.
- MeSH
- Achromobacter genetics metabolism MeSH
- Bioreactors microbiology MeSH
- Gene Expression MeSH
- Genetic Vectors MeSH
- Gene Dosage MeSH
- Cloning, Molecular MeSH
- Codon genetics MeSH
- Penicillin Amidase genetics metabolism MeSH
- Pichia genetics metabolism MeSH
- Promoter Regions, Genetic MeSH
- Industrial Microbiology methods MeSH
- Recombinant Proteins genetics metabolism MeSH
- Transformation, Genetic MeSH
- Publication type
- Journal Article MeSH
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- Keywords
- prodloužená kultivace,
- MeSH
- Achromobacter isolation & purification MeSH
- Time Factors MeSH
- Cystic Fibrosis * microbiology MeSH
- Child MeSH
- Adult MeSH
- Respiratory Tract Infections * microbiology MeSH
- Culture Techniques * methods MeSH
- Humans MeSH
- Specimen Handling MeSH
- Pseudomonas aeruginosa isolation & purification MeSH
- Sputum microbiology MeSH
- Stenotrophomonas maltophilia isolation & purification MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Humans MeSH
- Publication type
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
PbtA, a putative P(1B)-type ATPase from the Gram-negative soil bacterium Achromobacter xylosoxidans A8 responsible for Pb(2+)/Zn(2+)/Cd(2+)-resistance in Escherichia coli, was heterologously expressed in Saccharomyces cerevisiae. When present in Zn(2+)- and Pb(2+)/Cd(2+)-hypersensitive S. cerevisiae strains CM137 and DTY168, respectively, PbtA was able to restore Zn(2+)- and Pb(2+)-resistant phenotype. At the same time, the increase of Pb, Zn, and Cd accumulation in yeast was observed. However, Cd(2+)-tolerance of the pbtA-bearing yeasts dramatically decreased. The PbtA-eGFP fusion protein was localized primarily in the tonoplast and also in the plasma membrane and the perinuclear region corresponding to the endoplasmic reticulum at later growth stages. This indicates that PbtA protein is successfully incorporated into membranes in yeasts. Since PbtA caused a substantial increase of Pb(2+)/Zn(2+)-resistance and accumulation in baker's yeast, we propose its further use for the genetic modification of suitable plant species in order to obtain an effective tool for the phytoremediation of sites polluted by toxic transition metals.
- MeSH
- Achromobacter denitrificans enzymology metabolism MeSH
- Adenosine Triphosphatases metabolism MeSH
- Bacterial Proteins metabolism MeSH
- Cell Membrane enzymology metabolism MeSH
- Endoplasmic Reticulum enzymology metabolism MeSH
- Cadmium metabolism MeSH
- Lead metabolism MeSH
- Saccharomyces cerevisiae enzymology metabolism MeSH
- Zinc metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of β-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160 mM 6-APA, 350 mM acyl donor methylester[Symbol: see text]HCl, pH 6.3, 25 °C, reaction time of 200 min) with immobilized NPGA equaled 96.9 % and 91.1 %, respectively. The enzyme was highly thermostable with maximum activity at 60 °C (pH 8.0) and 65 °C (pH 6.0). Activity half-life at 60 °C (pH 8.0) and at 60 °C (pH 6.0) was 24 min and 6.9 h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230 mM 6-APA, 340 mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5 °C, pH 6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.
- MeSH
- Achromobacter enzymology MeSH
- Amoxicillin metabolism MeSH
- Ampicillin metabolism MeSH
- Anti-Bacterial Agents metabolism MeSH
- beta-Lactams metabolism MeSH
- Biotransformation MeSH
- Enzymes, Immobilized chemistry metabolism MeSH
- Hydrogen-Ion Concentration MeSH
- Penicillanic Acid analogs & derivatives metabolism MeSH
- Penicillin Amidase chemistry isolation & purification metabolism MeSH
- Enzyme Stability MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The cluster of pbtTFYRABC genes is carried by plasmid pA81. Its elimination from Achromobacter xylosoxidans A8 resulted in increased sensitivity towards Pb(2+) and Cd(2+). Predicted pbtTRABC products share strong similarities with Pb(2+) uptake transporter PbrT, transcriptional regulator PbrR, metal efflux P1-ATPases PbrA and CadA, undecaprenyl pyrophosphatase PbrB and its signal peptidase PbrC from Cupriavidus metallidurans CH34. Expression of pbtABC or pbtA in a metal-sensitive Escherichia coli GG48 rendered the strain Pb(2+)-, Cd(2+)- and Zn(2+)-tolerant and caused decreased accumulation of the metal ions. Accumulation of Pb(2+), but not of Cd(2+) or Zn(2+), was promoted in E. coli expressing pbtT. Additional genes of the pbt cluster are pbtF and pbtY, which encode the cation diffusion facilitator (CDF)-like transporter and a putative fatty acid hydroxylase of unknown function, respectively. Expression of pbtF did not confer increased metal tolerance upon E. coli GG48, although the protein showed measurable Pb(2+)-efflux activity. Unlike the pbtT promoter, promoters of pbtABC, pbtF and pbtY contain features characteristic of promoters controlled by metal-responsive transcriptional regulators of the MerR family. Upregulation of pbtABC, pbtF and pbtY upon Pb(2+), Cd(2+) and Zn(2+) exposure was confirmed in wild-type Achromobacter xylosoxidans A8. Gel shift assays proved binding of purified PbtR to the respective promoters.
- MeSH
- Achromobacter denitrificans drug effects genetics MeSH
- Genes, Bacterial MeSH
- Escherichia coli drug effects genetics MeSH
- Gene Expression MeSH
- Cadmium toxicity MeSH
- Cloning, Molecular MeSH
- Multigene Family MeSH
- Lead toxicity MeSH
- Plasmids MeSH
- Gene Expression Regulation, Bacterial drug effects MeSH
- Gene Expression Profiling MeSH
- Drug Tolerance * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
