RESUMO
Molecular and morphological methods were evaluated to distinguish between Haemonchus contortus and Haemonchus placei species. A total of 141 H. contortus and 89 H. placei male adult specimens collected from artificially infected lambs were identified individually by PCR analysis, using a species-specific primer pair. These PCR results were used as gold standard for Haemonchus spp. identification. Haemonchus placei presented higher mean spicule and barb lengths than H. contortus (P<0.05). However, some measurements overlapped. For this reason, a discriminate function did not allow the correct identification of 13 H. contortus and one H. placei specimen. The sheath tail length of the third stage larvae (L3), which comprises the distance between the tip of the larval tail and the end of the sheath tail, were measured. Only three of the 485 H. placei larvae (0.619%) had a sheath tail shorter than 85 µm, while only four of the 500 H. contortus larvae (0.8%) presented a sheath tail longer than 85 µm. The results indicated that 6.09% of the male adult specimens would be misclassified based on the discriminate function, while only 0.71% of infective larvae would be misclassified. Therefore, identification of L3 can be used as the first method to indicate the presence of H. placei and/or H. contortus in a population of domestic ruminants.
Métodos moleculares e morfológicos foram avaliados para a identificação de Haemonchus contortus e Haemonchus placei. No total, 141 H. contortus e 89 H. placei machos adultos, obtidos de cordeiros artificialmente infectados, foram identificados individualmente por PCR com o emprego de um par de “primers” espécie-específico. Esses resultados da análise por PCR foram considerados como padrão para a identificação das espécies de Haemonchus. Haemonchus placei apresentou valores médios de espículos e ganchos superiores aos de H. contortus (P<0,05). Entretanto, houve sobreposição de alguns valores. Por essa razão, a função discriminante não permitiu a identificação correta de 13 exemplares de H. contortus e de um, de H. placei. Foi medida a cauda da bainha de larvas infectantes (L3), que compreende a distância entre a ponta da cauda da larva e a ponta da cauda da bainha. Apenas três das 485 L3 de H. placei (0,619%) apresentaram a cauda da bainha com medida inferior a 85 µm e somente em quatro das 500 L3 de H. contortus (0,8%) essa medida foi superior a 85 µm. Os resultados demonstraram que 6,09% dos machos adultos seriam identificados erroneamente com base na função discriminante, enquanto a identificação incorreta de L3 seria de apenas 0,71%. Portanto, a identificação de L3 pode ser utilizada como método inicial para indicar a presença de H. placei e/ou H. contortus em uma população de ruminantes domésticos.
Assuntos
Adolescente , Adulto , Criança , Humanos , Pessoa de Meia-Idade , Aminoaciltransferases , Proteínas de Bactérias , Hexosiltransferases , Peptidil Transferases , Resistência às Penicilinas/genética , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Alelos , Proteínas de Transporte/genética , Cefotaxima/farmacologia , Cefalosporinas/farmacologia , Doenças Transmissíveis Emergentes/epidemiologia , DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado , Testes de Sensibilidade Microbiana , Muramilpentapeptídeo Carboxipeptidase/genética , Proteínas de Ligação às Penicilinas , Infecções Pneumocócicas/epidemiologia , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/efeitos dos fármacos , Estados Unidos/epidemiologiaRESUMO
To make progress in understanding peptidoglycan metabolism, we will reconstitute in vitro the assembly process and the molecular machineries that carry out this formidable task. We review here the reports of isolation of complexes comprising penicillin-binding proteins (PBPs), the enzymes that synthesize the peptidoglycan from its lipid-linked precursor.
Assuntos
Parede Celular/metabolismo , Escherichia coli/metabolismo , Peptidoglicano/biossíntese , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Parede Celular/química , Parede Celular/genética , Cromatografia de Afinidade , Escherichia coli/genética , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Imunoprecipitação , Muramilpentapeptídeo Carboxipeptidase/genética , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Coloração e RotulagemRESUMO
The binding of the essential cell division protein FtsN of Escherichia coli to the murein (peptidoglycan) sacculus was studied. Soluble truncated variants of FtsN, including the complete periplasmic part of the protein as well as a variant containing only the C-terminal 77 amino acids, did bind to purified murein sacculi isolated from wild-type cells. FtsN variants lacking this C-terminal region showed reduced or no binding to murein. Binding of FtsN was severely reduced when tested against sacculi isolated either from filamentous cells with blocked cell division or from chain-forming cells of a triple amidase mutant. Binding experiments with radioactively labeled murein digestion products revealed that the longer murein glycan strands (>25 disaccharide units) showed a specific affinity to FtsN, but neither muropeptides, peptides, nor short glycan fragments bound to FtsN. In vivo FtsN could be cross-linked to murein with the soluble disulfide bridge containing cross-linker DTSSP. Less FtsN, but similar amounts of OmpA, was cross-linked to murein of filamentous or of chain-forming cells compared to levels in wild-type cells. Expression of truncated FtsN variants in cells depleted in full-length FtsN revealed that the presence of the C-terminal murein-binding domain was not required for cell division under laboratory conditions. FtsN was present in 3,000 to 6,000 copies per cell in exponentially growing wild-type E. coli MC1061. We discuss the possibilities that the binding of FtsN to murein during cell division might either stabilize the septal region or might have a function unrelated to cell division.
Assuntos
Divisão Celular , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Reagentes de Ligações Cruzadas , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hexosiltransferases/química , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Muramilpentapeptídeo Carboxipeptidase/química , Muramilpentapeptídeo Carboxipeptidase/genética , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Proteínas de Ligação às Penicilinas , Peptidoglicano/química , Peptidil Transferases/química , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , FenótipoRESUMO
The enzymes involved in the biosynthesis of peptidoglycan are targets for the development of new antibiotics. The bifunctional high molecular weight (HMW) penicillin-binding proteins (PBPs), which contain both glycosyltransferase (GTase) and transpeptidase (TPase) activities, are particularly attractive targets because of their extracellular location. However, there is limited mechanistic or structural information about the GTase modules of these enzymes. In this paper, we describe the overexpression and characterization of the GTase module of Escherichia coli PBP1b, a paradigm of the HMW PBPs. We define the C-terminal boundary of the GTase module and show that the isolated module can be overexpressed at significantly higher levels than the full-length protein. The catalytic efficiency and other characteristics of the isolated module are comparable in most respects to the full-length enzyme. This work lays the groundwork for mechanistic and structural analysis of GTase modules.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Escherichia coli/genética , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Hexosiltransferases/química , Hexosiltransferases/metabolismo , Muramilpentapeptídeo Carboxipeptidase/química , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Peptidoglicano Glicosiltransferase , Peptidil Transferases/química , Peptidil Transferases/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Catálise/efeitos dos fármacos , Detergentes/farmacologia , Glicosiltransferases/isolamento & purificação , Hexosiltransferases/genética , Cinética , Metais/farmacologia , Estrutura Molecular , Muramilpentapeptídeo Carboxipeptidase/genética , Proteínas de Ligação às Penicilinas , Peptidil Transferases/genética , Estrutura Secundária de Proteína , Deleção de SequênciaRESUMO
In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Hexosiltransferases/metabolismo , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Peptidoglicano Glicosiltransferase , Peptidil Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Divisão Celular/fisiologia , Parede Celular/enzimologia , Parede Celular/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Genes Reporter , Proteínas de Fluorescência Verde , Hexosiltransferases/química , Hexosiltransferases/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Muramilpentapeptídeo Carboxipeptidase/química , Muramilpentapeptídeo Carboxipeptidase/genética , Mutação , Proteínas de Ligação às Penicilinas , Peptidoglicano/biossíntese , Peptidil Transferases/química , Peptidil Transferases/genética , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Low-level penicillin resistance in an international Streptococcus pneumoniae serotype 19F clone emerging in Switzerland was characterized by mutations in the penicillin-binding protein PBP2x. Some isolates of this clone had evolved to higher resistance levels (penicillin MICs of 0.094 and 1 microg/ml), probably by acquisition of pbp2x fragments from local nonencapsulated pneumococci.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Genes Bacterianos/genética , Hexosiltransferases/genética , Muramilpentapeptídeo Carboxipeptidase/genética , Resistência às Penicilinas/genética , Peptidil Transferases/genética , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Alelos , Testes de Sensibilidade Microbiana , Mutação , Nasofaringe/microbiologia , Proteínas de Ligação às Penicilinas , Infecções Pneumocócicas/transmissão , Polimorfismo de Fragmento de Restrição , SuíçaRESUMO
Penicillin-resistant Streptococcus pneumoniae isolates (n = 76) from clinical samples of patients admitted to Hacettepe University Hospital between January 1997 and December 2001 were included in the study. MICs of penicillin G, erythromycin A, clindamycin, cefaclor, cefotaxime, vancomycin, chloramphenicol, tetracycline, ciprofloxacin and rifampicin were determined by agar dilution. The isolates were serogrouped on the basis of the Neufeld Quellung reaction and were typed by BOX-PCR. Genetic polymorphism of the penicillin resistance genes pbp2b and pbp2x was investigated by restriction fragment length polymorphism (RFLP) analysis. Of the 76 isolates tested, 64 (84.2%) showed intermediate resistance to penicillin, while 12 (15.8%) were resistant to higher levels of penicillin (MIC > or = 2 mg/L). The resistance patterns of the isolates revealed six different resistance profiles. There were 22 different serotypes, with c. 55% of the isolates belonging to serotypes 23B, 19A, 19F, 14, 6 A and 9V. Five distinct patterns for pbp2b and 12 distinct patterns for pbp2x were obtained by RFLP analysis of penicillin-binding protein genes. The combination of these patterns allowed isolates to be classified into 22 fingerprint subgroups. BOX-PCR analysis showed that the isolates fell into 14 distinct BOX genotypes, with 33 subtypes. Serotype 9V isolates with pbp genotype 2-6 and BOX-PCR type 4, 4.1 or 4.2 were related to the pandemic clone Spain(9V)-3. No relatedness to other international clones was detected among the other study strains, but genetic relatedness was observed among some of the serotype 19A and 23B isolates. Overall, the results demonstrated that most of the penicillin-resistant pneumococcal isolates in Turkey, other than those belonging to serotypes 9V, 19A and 23B, were derived from several independent clones, possibly resulting from multiple importation of strains originating from outside the country. Differences in pbp patterns, serotypes and resistance profiles among isolates that showed similar BOX-PCR patterns supported the hypothesis that horizontal transfer of capsular genes, pbp genes and other genetic determinants between S. pneumoniae and viridans group streptococci may have occurred.
Assuntos
Aminoaciltransferases , Hospitais Universitários , Epidemiologia Molecular , Resistência às Penicilinas/genética , Infecções Pneumocócicas/epidemiologia , Streptococcus pneumoniae/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Farmacorresistência Bacteriana Múltipla , Hexosiltransferases/genética , Humanos , Testes de Sensibilidade Microbiana , Muramilpentapeptídeo Carboxipeptidase/genética , Proteínas de Ligação às Penicilinas , Peptidil Transferases/genética , Infecções Pneumocócicas/microbiologia , Polimorfismo de Fragmento de Restrição , Sorotipagem , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Turquia/epidemiologiaRESUMO
The SOS response aids bacterial propagation by inhibiting cell division during repair of DNA damage. We report that inactivation of the ftsI gene product, penicillin binding protein 3, by either beta-lactam antibiotics or genetic mutation induces SOS in Escherichia coli through the DpiBA two-component signal transduction system. This event, which requires the SOS-promoting recA and lexA genes as well as dpiA, transiently halts bacterial cell division, enabling survival to otherwise lethal antibiotic exposure. Our findings reveal defective cell wall synthesis as an unexpected initiator of the bacterial SOS response, indicate that beta-lactam antibiotics are extracellular stimuli of this response, and demonstrate a novel mechanism for mitigation of antimicrobial lethality.
Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Peptidoglicano Glicosiltransferase , Resposta SOS em Genética , beta-Lactamas/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Divisão Celular , Parede Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Óperon Lac , Muramilpentapeptídeo Carboxipeptidase/genética , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Mutação , Óperon , Proteínas de Ligação às Penicilinas , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transdução de Sinais , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , beta-Galactosidase/biossíntese , beta-Lactamas/metabolismoRESUMO
Resistance genes were determinded for 81 strains of Streptococcus pneumoniae isolated from Ehime University hospital, during 2002 and 2003 by various clinical material. In penicillin-binding proteins of mutation, there were 74 strains; pbp2x mutation 23 strains (28.4%), pbp2b mutation one strain (1.2%), pbp1a + pbp2x mutations 5 strains (6.2%), pbp2x + pbp2b mutations 18 strains (22.2%) and all mutations 27 strains (33.3%). As for the result of macrolide resistance genes, there were 67 strains; mefA gene 20 strains (24.7%), ermB gene 46 strains (56.8%) and both gene one strain (1.2%). In the analysis of gyrA gene and parC gene, 3 strains (3.7%) had both gene mutations, and 26 strains (32.1%) had only parC gene mutation. There was more of an increase than before in isolates, two or more mutation strains with PBPs gene, ermB gene holding strains and the levofloxacin resistance strain. These results suggest that the gyrA gene or parC gene mutation strains hold PBPs gene mutation and macrolide resistance genes in a high rate, and there will be more drug resistance in the future.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Hexosiltransferases/genética , Macrolídeos/farmacologia , Muramilpentapeptídeo Carboxipeptidase/genética , Mutação/genética , Peptidil Transferases/genética , Streptococcus pneumoniae/genética , Resistência beta-Lactâmica/genética , DNA Girase/genética , DNA Topoisomerase IV/genética , Humanos , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all beta-lactams. Affinity for penicillin generally correlated with beta-lactam MICs for the mutants, but these associations were not strictly proportional.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Hexosiltransferases/genética , Hexosiltransferases/fisiologia , Muramilpentapeptídeo Carboxipeptidase/genética , Muramilpentapeptídeo Carboxipeptidase/fisiologia , Mutação/genética , Mutação/fisiologia , Peptidil Transferases/genética , Peptidil Transferases/fisiologia , Resistência beta-Lactâmica/genética , Resistência a Ampicilina/genética , Cristalografia por Raios X , Vetores Genéticos/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Proteínas de Ligação às Penicilinas , Penicilinas/metabolismo , Plasmídeos/genética , Ligação ProteicaRESUMO
OBJECTIVE: The emergence of multidrug resistance within Streptococcus pneumoniae population was analysed, correlating penicillin resistance Pen(R) with secondary antibiotic resistance, capsular serotype, and genetic diversity among isolates. METHODS: DNA fingerprinting, following macro-restriction enzyme digestion and pulse field gel electrophoresis (PFGE), and restriction fragment analysis of the PBP 2b gene, following PCR amplification, were performed on the Pen(R) S. pneumoniae, among 377 clinical isolates obtained from the clinical microbiology laboratory (University of Michigan Medical Center). RESULTS: Overall 35% of the isolates were Pen(R) of which 45% demonstrated high-level penicillin (Pen(R)-R, MIC>1). Respiratory isolates were more likely to be Pen(R) (p <0.001) than non-respiratory isolates and the rate of Pen(R)-R was significantly increased in children <10 years of age (59.6%, p <0.02). Secondary antibiotic resistance was more frequently associated with Pen(R)-R. Genomic DNA fingerprinting analysis and restriction fragment analysis of the PBP 2b gene demonstrated genomic divergence with discrete conserved pattern in the PBP 2b gene among the resistant isolates. CONCLUSION: The emergence of multidrug resistance in the S. pneumoniae population in SE Michigan is not due to expansion of a single or limited number of resistant clones, is occurring most frequently in the paediatric population and is associated with a decreased susceptibility to penicillin.
Assuntos
Streptococcus pneumoniae/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Distribuição de Qui-Quadrado , Impressões Digitais de DNA , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Variação Genética , Hexosiltransferases/genética , Humanos , Michigan , Muramilpentapeptídeo Carboxipeptidase/genética , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas , Peptidil Transferases/genética , Polimorfismo de Fragmento de Restrição , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
The importance of amino acid sequence differences in the C-terminal part and levels of mRNA expression of penicillin-binding protein 5 (PBP5) for ampicillin resistance in Enterococcus faecium was investigated. Seventeen isolates from Norwegian hospitalized patients (ampicillin MIC 0.064->256 mg/L) with different C-terminal pbp5 DNA sequences encoding 11 different amino acid sequences were analyzed with a 14C-radiolabeled penicillin- binding assay to PBP5 and with real-time PCR quantification of pbp5 mRNA expression. Using multiple logistic regression analysis the amino acid substitution Met 485 was linked to ampicillin MIC and levels of 14C-radiolabeled penicillin bound to PBP5; however, there were isolates with identical PBP5 alleles and different ampicillin MICs. There was no relation between the quantity of pbp5 mRNA transcripts and ampicillin resistance. The results cannot explain ampicillin resistance in Norwegian clinical strains of E. faecium and indicate that other factors besides the properties of the C-terminal part of PBP5 are most likely involved.
Assuntos
Substituição de Aminoácidos , Resistência a Ampicilina/genética , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Enterococcus faecium/genética , Hexosiltransferases/genética , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidil Transferases/genética , Sequência de Bases , Primers do DNA , Enterococcus faecium/enzimologia , Enterococcus faecium/isolamento & purificação , Humanos , Análise Multivariada , Noruega , Proteínas de Ligação às Penicilinas , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Reto/microbiologia , beta-Lactamases/genéticaRESUMO
OBJECTIVES: To determine the epidemiological relationship between non-invasive penicillin non-susceptible Streptococcus pneumoniae isolates collected in the Christchurch community between 1997 and 2001. METHODS: One hundred and ninety-seven pneumococcal isolates were examined by macrorestriction profile analysis of SmaI-digested genomic DNA separated by PFGE and restriction fragment length polymorphism analysis of penicillin binding protein genes. RESULTS: Four major clonal lineages were identified, the largest and most homogeneous containing 95 (48.2%) of the isolates, the bulk of which (93.7%), had identical macrorestriction patterns. Members of this clonal group were multidrug-resistant and exhibited high resistance to third-generation cephalosporins, with MICs > or =8.0 mg/L not uncommon (23.1%). Two of the clonal groups, each containing 24 (12.2%) isolates, appeared indistinguishable from the globally widespread Spain23F-1 and France9V-3 strains, respectively. The fourth (12.7% of isolates) multidrug-resistant clone possessed intermediate penicillin susceptibility (MIC 0.12 mg/L). CONCLUSIONS: This study shows that several distinct penicillin-resistant pneumococcal clones are present in the Christchurch community, most of which appear to have been imported into New Zealand.
Assuntos
Resistência às Penicilinas/genética , Penicilinas/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Criança , Pré-Escolar , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Farmacorresistência Bacteriana Múltipla , Eletroforese em Gel de Campo Pulsado , Feminino , Hexosiltransferases/genética , Humanos , Lactente , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Família Multigênica/genética , Muramilpentapeptídeo Carboxipeptidase/genética , Nova Zelândia/epidemiologia , Proteínas de Ligação às Penicilinas , Peptidil Transferases/genética , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologiaRESUMO
Detection of oxacillin resistance in coagulase-negative staphylococci (C-NS) by phenotypic methods is often difficult. The present study compared the National Committee for Clinical Laboratory Standards (NCCLS) revised guidelines of phenotypic methods with a mecA-based polymerase chain reaction (PCR) for C-NS. Ninety clinical C-NS isolates were tested for oxacillin resistance by disk diffusion (1-microg disk), minimum inhibitory concentration (MIC) breakpoint (0.5 microg/ml) after 24 h, and mecA-based PCR. The sensitivity and specificity of disk diffusion was 80% and 93%, and the sensitivity and specificity of the MIC breakpoint after 24 h was 84% and 91%, respectively, against PCR as gold standard. Eleven strains (7 mecA-positive and 4 mecA-negative) showed discordant results between MIC breakpoint after 24 h and PCR. Six of the 7 mecA-positive and all 4 mecA-negative discordant strains had inducible oxacillin resistance and beta-lactamase hyperproduction, respectively. The present study concludes that inducible oxacillin resistance and beta-lactamase hyperproduction are the major causes of discordant results between phenotypic methods and mecA-based PCR, and need special attention.
Assuntos
Antibacterianos/farmacologia , Coagulase/metabolismo , Resistência a Meticilina/genética , Oxacilina/farmacologia , Reação em Cadeia da Polimerase/métodos , Staphylococcus/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Muramilpentapeptídeo Carboxipeptidase/genética , Fenótipo , Sensibilidade e Especificidade , Infecções Estafilocócicas/microbiologia , Staphylococcus/enzimologiaRESUMO
All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S. pneumoniae isolates collected in Japan, were completely sequenced. The MICs of penicillin for these strains varied between 0.015 and 8 microg/ml. In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains. These six strains were highly resistant (MICs >/= 2 microg/ml) to cefotaxime. The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains. In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 microg/ml) was the highest imipenem MIC detected. In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were >/=1 microg/ml. In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 microg/ml) was the highest cefotaxime MIC detected. On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed. The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S. pneumoniae resistance to beta-lactam antibiotics. Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with beta-lactam resistance.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , DNA Bacteriano/genética , Genes Bacterianos/genética , Hexosiltransferases/genética , Muramilpentapeptídeo Carboxipeptidase/genética , Peptídeo Sintases , Peptidil Transferases/genética , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Substituição de Aminoácidos , Antibacterianos/farmacologia , Sequência de Bases , Primers do DNA , DNA Bacteriano/química , Japão , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação/genética , Proteínas de Ligação às Penicilinas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Certain mutants in Escherichia coli lacking multiple penicillin-binding proteins (PBPs) produce misshapen cells containing kinks, bends and branches. These deformed regions exhibit two structural characteristics of normal cell poles: the peptidoglycan is inert to dilution by new synthesis or turnover, and a similarly stable patch of outer membrane caps the sites. To test the premise that these aberrant sites represent biochemically functional but misplaced cell poles, we assessed the intracellular distribution of proteins that localize specifically to bacterial poles. Green fluorescent protein (GFP) hybrids containing polar localization sequences from the Shigella flexneri IcsA protein or from the Vibrio cholerae EpsM protein formed foci at the poles of wild-type E. coli and at the poles and morphological abnormalities in PBP mutants. In addition, secreted wild-type IcsA localized to the outer membrane overlying these aberrant domains. We conclude that the morphologically deformed sites in these mutants represent fully functional poles or pole fragments. The results suggest that prokaryotic morphology is driven, at least in part, by the controlled placement of polar material, and that one or more of the low-molecular-weight PBPs participate in this process. Such mutants may help to unravel how particular proteins are targeted to bacterial poles, thereby creating important biochemical and functional asymmetries.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Hexosiltransferases/genética , Muramilpentapeptídeo Carboxipeptidase/genética , Mutação , Peptidil Transferases/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas , Peptidoglicano/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Shigella flexneri/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoAssuntos
Aminoaciltransferases , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Hexosiltransferases/genética , Muramilpentapeptídeo Carboxipeptidase/genética , Resistência às Penicilinas/genética , Peptidil Transferases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Streptococcus pneumoniae/efeitos dos fármacos , Mutação/genética , Mutação/fisiologia , Proteínas de Ligação às Penicilinas , Valor Preditivo dos TestesRESUMO
pbp2b gene alterations were analyzed in 102 clinical isolates of Streptococcus pneumoniae (30 penicillin susceptible, 23 intermediate, and 49 resistant) from Korea. On the basis of PBP2B amino acid sequences, penicillin-nonsusceptible isolates of S. pneumoniae belonged to six groups, and 76% of the isolates in groups I to IV showed the same divergent block of amino acid alterations. Thirteen isolates (group II) also possessed a divergent block that was identical to that of Streptococcus oralis. The pbp2b genes of most Korean isolates showed novel mosaic mutations due to horizontal gene transfer. The Thr252 --> Ala substitution, previously thought to be associated only with penicillin-nonsusceptible strains, was also found in three penicillin-susceptible strains. On the basis of their pbp2b nucleotide sequences, all penicillin-nonsusceptible isolates can be detected by multiplex PCR, which can be used as a novel method for detection of antibiotic-resistant pneumococcal strains in clinical specimens.
Assuntos
Aminoaciltransferases , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Variação Genética , Hexosiltransferases/genética , Muramilpentapeptídeo Carboxipeptidase/genética , Resistência às Penicilinas/genética , Peptidil Transferases/genética , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Sequência de Aminoácidos , Humanos , Coreia (Geográfico) , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Proteínas de Ligação às Penicilinas , Penicilinas/farmacologia , Reação em Cadeia da Polimerase/métodos , Alinhamento de Sequência , Análise de Sequência de DNA , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genéticaRESUMO
The antibiotic susceptibilities, genotypes of penicillin (PEN)-binding protein genes (pbp), and serotype distributions of Streptococcus pneumoniae isolates from meningitis patients were investigated by a nationwide surveillance group in Japan between 1999 and 2002. We analyzed 146 isolates from children (/=18 years old). Isolates with or without abnormal pbp1a, pbp2x, or pbp2b genes identified by PCR were classified into six genotype patterns and 90% MIC (MIC(90)) values for PEN: (i) strains with three normal genes (17.2% of isolates; MIC(90), 0.031 micro g/ml); (ii) strains with abnormal pbp2x (22.1%, 0.063 micro g/ml); (iii) strains with abnormal pbp2b (1.0%, 0.125 micro g/ml); (iv) strains with abnormal pbp2x and pbp2b (7.4%, 0.25 micro g/ml); (v) strains with abnormal pbp1a and pbp2x (12.7%, 0.25 micro g/ml); and (vi) strains with three abnormal PBP genes (39.7%, 4 micro g/ml), which are termed genotypic PEN-resistant S. pneumoniae (gPRSP). Panipenem, a carbapenem, showed an excellent MIC(90) (0.125 micro g/ml) against gPRSP, followed by meropenem and vancomycin (0.5 micro g/ml), cefotaxime and ceftriaxone (1 micro g/ml), and ampicillin (4 micro g/ml). Strains of gPRSP were significantly more prevalent in children (45.2%) than in adults (27.4%). The most frequent serotypes were 6B, 19F, 23F, 6A, and 14 in children and 23F, 22, 3, 10, 6B, and 19F in adults. Serotypes 6B, 6A, 19F, 23F, and 14 predominated among gPRSP. In children, 7- and 11-valent pneumococcal conjugate vaccines would cover 76.2 and 81.3% of isolates, respectively, although coverage would be lower in adults (43.9 and 56.0%, respectively). These findings suggest the need for early introduction of pneumococcal conjugate vaccines and continuous bacteriological surveillance for meningitis.
Assuntos
Aminoaciltransferases , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Hexosiltransferases/genética , Meningite Pneumocócica/genética , Muramilpentapeptídeo Carboxipeptidase/genética , Peptidil Transferases/genética , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Farmacorresistência Bacteriana , Feminino , Genótipo , Humanos , Lactente , Japão/epidemiologia , Lactamas/farmacologia , Masculino , Meningite Pneumocócica/epidemiologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , N-Acetil-Muramil-L-Alanina Amidase/genética , Proteínas de Ligação às Penicilinas , Vigilância da População , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorotipagem , Fatores de Tempo , Vancomicina/farmacologiaRESUMO
A total of 395 Haemophilus influenzae strains from 226 Japanese institutions participating in the Nationwide Surveillance Study Group for Bacterial Meningitis were received from 1999 to 2002. All strains were analyzed by PCR to identify the resistance genes, and their susceptibilities to beta-lactam agents were determined. Of these strains, 29.1% were beta-lactamase nonproducing and ampicillin (AMP) susceptible (BLNAS) and lacked all resistance genes; 15.4% were beta-lactamase producing and AMP resistant and had the bla(TEM-1) gene; 30.6% were beta-lactamase nonproducing and AMP resistant (low-BLNAR) and had a Lys-526 or His-517 amino acid substitution in ftsI encoding PBP 3; 13.9% were beta-lactamase nonproducing and AMP resistant (BLNAR) and had an additional substitution of Thr-385 in ftsI; 9.1% were amoxicillin-clavulanic acid resistant (BLPACR I) and had the bla(TEM-1) gene and a Lys-526 or His-517 amino acid substitution in ftsI; and 1.8% showed resistance similar to that of the BLPACR I group (BLPACR II) but had bla(TEM-1) gene and ftsI substitutions, as was the case for the BLNAR strains. All but three strains were serotype b. The prevalence of BLNAR strains has increased rapidly: 0% in 1999, 5.8% in 2000, 14.1% in 2001, and 21.3% in 2002. The MICs at which 90% of BLNAR isolates were inhibited were as follows: AMP, 16 micro g/ml; cefotaxime, 1 micro g/ml; ceftriaxone, 0.25 micro g/ml; and meropenem, 0.5 micro g/ml. All of these values were higher than those for the BLNAS counterpart strains. The relatively wide distributions of the beta-lactam MICs for BLNAR strains presumably reflect variations in ftsI gene mutations. Pulsed-field gel electrophoresis suggested the rapid spread of specific H. influenzae type b strains throughout Japan. Expedited vaccination, rapid identification, and judicious antibiotic use could slow their spread.
