Assuntos
Antibacterianos/farmacologia , Bovinos/microbiologia , Farmacorresistência Bacteriana , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Microbiologia da Água , Animais , Proteínas de Bactérias/genética , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Humanos , Camundongos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , VirulênciaRESUMO
From the Central-East European region the first VIM metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa strains were published from Croatia, Poland and Hungary. The aim of this study was to assess the contribution of MBL-production to carbapenem-resistance among P. aeruginosa clinical isolates in the Military Medical Academy (MMA) in Belgrade, Serbia between August 2004 and September 2007. Only one P. aeruginosa isolate with strain number 722 proved MBL-positive that harboured a novel class 1 integron with a bla(VIM-2)-like cassette in the first position, followed by orfD, a putative gene with unknown function. Our data indicate that MBL-producing strains occur at a prevalence of less than 1% among imipenem-nonsusceptible P. aeruginosa clinical isolates in this Belgrade hospital. The newly identified VIM MBL-producing P. aeruginosa strain 722 could be assigned to serotype O11, and it was panresistant to all antimicrobials tested. The isolate displayed sequence type ST235 by multilocus sequence typing which is the founder sequence type of the previously identified international clonal complex CC11 that already contains bla(VIM)-positive isolates from Italy, Greece, Sweden, Hungary and Poland. In conclusion, this is the first report of VIM MBL-producing P. aeruginosa from Serbia and also of the occurrence of such isolates belonging to the international clonal complex CC11 in this country.
Assuntos
Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana/genética , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , beta-Lactamases/genética , Hospitais Militares , Humanos , Imipenem/farmacologia , Integrons/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Sérvia/epidemiologia , SorotipagemRESUMO
Our aim was to characterise by molecular techniques group A streptococci isolated from invasive infections in Hungary in 2004-2005. Twenty-six nonduplicate invasive GAS isolates were selected and examined. The mortality rate proved high (52.3%) for those cases (n = 21) where data were available. Predominant emm types were emm1 (n = 13, 50%) and emm80 (n = 5, 19.2%), but other M types (emm4, emm28, emm66, emm81.1, emm82, emm84) were also identified. Eight different PFGE types were distinguished, and each emm type showed an individual PFGE pattern. Our results show that--similarly to results obtained in several other countries--emm type 1 strains predominate among invasive GAS isolates, and that emm 1 type strains recovered from severe streptococcal infections were associated with the presence of the speA gene. The rate for macrolide resistance proved low: only two isolates showed elevated MICs for erythromycin.
Assuntos
Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Proteínas de Transporte/genética , Criança , Pré-Escolar , Análise por Conglomerados , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Exotoxinas/genética , Feminino , Genótipo , Humanos , Hungria , Masculino , Proteínas de Membrana/genética , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Infecções Estreptocócicas/mortalidade , Streptococcus pyogenes/isolamento & purificaçãoRESUMO
Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' desaturases. These enzymes are also characterized by the presence of a cytochrome b5 domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a 'front end' desaturase, the borage Delta6-fatty acid desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue.
Assuntos
Ácidos Graxos Dessaturases/genética , Plantas/enzimologia , Aminoácidos , Clonagem Molecular , Citocromos b5/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Expressão Gênica , Histidina/genética , Linoleoil-CoA Desaturase , Microssomos , Mutagênese , Plantas/genética , Saccharomyces cerevisiae/genéticaRESUMO
The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.
Assuntos
Magnoliopsida/enzimologia , Oxirredutases/isolamento & purificação , Esfingosina/análogos & derivados , Sequência de Aminoácidos , Clonagem Molecular , Magnoliopsida/genética , Dados de Sequência Molecular , Especificidade de Órgãos , Oxirredutases/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Esfingosina/biossínteseRESUMO
The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids. However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6. These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site. This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Magnoliopsida/enzimologia , Oxirredutases/metabolismo , Ácidos Graxos Dessaturases/genética , Linoleoil-CoA Desaturase , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oxirredutases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae , Especificidade por SubstratoRESUMO
The consensus sequence of the third histidine box of a range of Delta(5), Delta(6), Delta(8) and sphingolipid desaturases differs from that of the membrane-bound non-fusion Delta(12) and Delta(15) desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b(5) fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Delta(6) desaturase.
