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 * genetika   MeSH
• Achromobacter * MeSH
• antibakteriální látky terapeutické užití   MeSH
• cystická fibróza * MeSH
• dítě MeSH
• genomika MeSH
• gramnegativní bakteriální infekce * MeSH
• integrasy terapeutické užití   MeSH
• integrony genetika   MeSH
• kombinace léků trimethoprim a sulfamethoxazol MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Srbsko MeSH

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 klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální geny * genetika   MeSH
• bakteriální léková rezistence genetika   MeSH
• fluorochinolony farmakologie   MeSH
• fylogeneze MeSH
• genotyp MeSH
• gramnegativní bakteriální infekce mikrobiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• plazmidy genetika   MeSH
• polymerázová řetězová reakce MeSH
• pulzní gelová elektroforéza MeSH
• sekvenční analýza DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Srbsko 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 enzymologie   metabolismus   MeSH
• adenosintrifosfatasy metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčná membrána enzymologie   metabolismus   MeSH
• endoplazmatické retikulum enzymologie   metabolismus   MeSH
• kadmium metabolismus   MeSH
• olovo metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   metabolismus   MeSH
• zinek metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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 účinky léků   genetika   MeSH
• bakteriální geny MeSH
• Escherichia coli účinky léků   genetika   MeSH
• exprese genu MeSH
• kadmium toxicita   MeSH
• klonování DNA MeSH
• multigenová rodina MeSH
• olovo toxicita   MeSH
• plazmidy MeSH
• regulace genové exprese u bakterií účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• tolerance léku * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Achromobacter xylosoxidans strain A8 was isolated from soil contaminated with polychlorinated biphenyls. It can use 2-chlorobenzoate and 2,5-dichlorobenzoate as sole sources of carbon and energy. This property makes it a good starting microorganism for further development toward a bioremediation tool. The genome of A. xylosoxidans consists of a 7-Mb chromosome and two large plasmids (98 kb and 248 kb). Besides genes for the utilization of xenobiotic organic substrates, it contains genes associated with pathogenesis, toxin production, and resistance. Here, we report the complete genome sequence.

• MeSH
• Achromobacter denitrificans genetika   izolace a purifikace   metabolismus   MeSH
• bakteriální chromozomy MeSH
• chlorbenzoáty metabolismus   MeSH
• DNA bakterií chemie   genetika   MeSH
• energetický metabolismus MeSH
• genom bakteriální MeSH
• molekulární sekvence - údaje MeSH
• plazmidy MeSH
• půdní mikrobiologie MeSH
• sekvenční analýza DNA MeSH
• uhlík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The complete 98,192bp nucleotide sequence was determined for plasmid pA81, which is harbored by the haloaromatic acid-degrading bacterium Achromobacter xylosoxidans A8. The majority of the 103 open reading frames identified on pA81 could be categorized as either "backbone" genes, genes encoding (halo)aromatic compound degradation, or heavy metal resistance determinants. The backbone genes controlled conjugative transfer, replication and plasmid stability, and were well conserved with other IncP1-beta plasmids. Genes encoding (halo)aromatic degradation were clustered within a type I transposon, TnAxI, and included two ring-hydroxylating oxygenases (ortho-halobenzoate oxygenase, salicylate 5-hydroxylase) and a modified ortho-cleavage pathway for chlorocatechol degradation. The cluster of heavy metal resistance determinants was contained within a Type II transposon TnAxII, and included a predicted P-type ATPase and cation diffusion facilitator system. Genes identical to those carried by TnAxI and TnAxII were identified on other biodegradative/resistance plasmids and genomic islands, indicating an evolutionary relationship between these elements. Collectively, these insights further our understanding of how mobile elements, and interactions between mobile elements affect the fate of organic and inorganic toxicants in the environment.

• MeSH
• Achromobacter denitrificans genetika   MeSH
• aromatické uhlovodíky metabolismus   MeSH
• bakteriální geny MeSH
• DNA bakterií genetika   chemie   MeSH
• financování organizované MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• multigenová rodina MeSH
• otevřené čtecí rámce MeSH
• oxygenasy se smíšenou funkcí genetika   MeSH
• oxygenasy genetika   MeSH
• plazmidy genetika   chemie   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• těžké kovy metabolismus   MeSH
• transpozibilní elementy DNA MeSH