RESUMEN
The objective of this study was to develop a canonical, parsimoniously-informative SNP panel for subtyping Shiga-toxin producing Escherichia coli (STEC) O157:H7 that would be consistent with epidemiological, PFGE, and MLVA clustering of human specimens. Our group had previously identified 906 putative discriminatory SNPs, which were pared down to 391 SNPs based on their prevalence in a test set. The 391 SNPs were screened using a high-throughput form of TaqMan PCR against a set of clinical isolates that represent the most diverse collection of O157:H7 isolates from outbreaks and sporadic cases examined to date. Another 30 SNPs identified by others were also screened using the same method. Two additional targets were tested using standard TaqMan PCR endpoint analysis. These 423 SNPs were reduced to a 32 SNP panel with the almost the same discriminatory value. While the panel partitioned our diverse set of isolates in a manner that was consistent with epidemiological data and PFGE and MLVA phylogenies, it resulted in fewer subtypes than either existing method and insufficient epidemiological resolution in 10 of 47 clusters. Therefore, another round of SNP discovery was undertaken using comparative genomic resequencing of pooled DNA from the 10 clusters with insufficient resolution. This process identified 4,040 potential SNPs and suggested one of the ten clusters was incorrectly grouped. After its removal, there were 2,878 SNPs, of which only 63 were previously identified and 438 occurred across multiple clusters. Among highly clonal bacteria like STEC O157:H7, linkage disequilibrium greatly limits the number of parsimoniously informative SNPs. Therefore, it is perhaps unsurprising that our panel accounted for the potential discriminatory value of numerous other SNPs reported in the literature. We concluded published O157:H7 SNPs are insufficient for effective epidemiological subtyping. However, the 438 multi-cluster SNPs we identified may provide the additional information required.
Asunto(s)
Escherichia coli O157/genética , Polimorfismo de Nucleótido Simple , Escherichia coli Shiga-Toxigénica/genética , Infecciones por Escherichia coli/microbiología , HumanosRESUMEN
Since the 1980s, pertussis notifications in the United States have been increasing. To determine the types of Bordetella pertussis responsible for these increases, we divided 661 B. pertussis isolates collected in the United States during 1935-2009 into 8 periods related to the introduction of novel vaccines or changes in vaccination schedule. B. pertussis diversity was highest from 1970-1990 (94%) but declined to ≈ 70% after 1991 and has remained constant. During 2006-2009, 81.6% of the strains encoded multilocus sequence type prn2-ptxP3-ptxS1A-fim3B, and 64% were multilocus variable number tandem repeat analysis type 27. US trends were consistent with those seen internationally; emergence and predominance of the fim3B allele was the only molecular characteristic associated with the increase in pertussis notifications. Changes in the vaccine composition and schedule were not the direct selection pressures that resulted in the allele changes present in the current B. pertussis population.
Asunto(s)
Bordetella pertussis/genética , Bordetella pertussis/aislamiento & purificación , Variación Genética , Tos Ferina/microbiología , Alelos , Antígenos Bacterianos/genética , Bordetella pertussis/clasificación , Bordetella pertussis/inmunología , Notificación de Enfermedades/estadística & datos numéricos , Proteínas Fimbrias/genética , Humanos , Repeticiones de Minisatélite/genética , Tipificación de Secuencias Multilocus , Toxina del Pertussis/genética , Vacuna contra la Tos Ferina/administración & dosificación , Vacuna contra la Tos Ferina/inmunología , Estados Unidos/epidemiología , Vacunación/estadística & datos numéricos , Factores de Virulencia de Bordetella/genética , Tos Ferina/epidemiología , Tos Ferina/prevención & controlRESUMEN
AmpD indirectly regulates the production of AmpC beta-lactamase via the cell wall recycling pathway. Recent publications have demonstrated the presence of multiple ampD genes in Pseudomonas aeruginosa and Escherichia coli. In the prototype P. aeruginosa strain, PAO1, the three ampD genes (ampD, ampDh2, and ampDh3) contribute to a stepwise regulation of ampC beta-lactamase and help explain the partial versus full derepression of ampC. In the present study, the roles of the three ampD homologs in nine clinical P. aeruginosa isolates with either partial or full derepression of ampC were evaluated. In eight of nine isolates, decreased RNA expression of the ampD genes was not associated with an increase in ampC expression. Sequence analyses revealed that every derepressed isolate carried mutations in ampD, and in two fully derepressed strains, only ampD was mutated. Furthermore, every ampDh2 gene was of the wild type, and in some fully derepressed isolates, ampDh3 was also of the wild type. Mutations in ampD and ampDh3 were tested for their effect on function by using a plasmid model system, and the observed mutations resulted in nonfunctional AmpD proteins. Therefore, although the sequential deletion of the ampD homologs of P. aeruginosa can explain partial and full derepression in PAO1, the same model does not explain the overproduction of AmpC observed in these clinical isolates. Overall, the findings of the present study indicate that there is still an unknown factor(s) that contributes to ampC regulation in P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , beta-Lactamasas/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Bacterianos , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , N-Acetil Muramoil-L-Alanina Amidasa/genética , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Homología de Secuencia de Aminoácido , Resistencia betalactámica/genética , beta-Lactamasas/genéticaRESUMEN
Two Pseudomonas aeruginosa mutants exhibiting increased expression of ampC were selected during exposure to ciprofloxacin. These mutants also exhibited significant increases in mexCD-oprJ expression, but further studies failed to show a link between the increased expression of mexCD-oprJ and ampC. Increased ampC expression was not related to mutations within ampR, the ampC-ampR intergenic region, ampD, ampDh2, or ampDh3 or to changes in the levels of expression of these amidase genes. However, ampD complementation restored wild-type levels of ampC expression and ceftazidime susceptibility, suggesting alternative mechanisms of ampC regulation.
Asunto(s)
Antiinfecciosos/farmacología , Proteínas Bacterianas/metabolismo , Ciprofloxacina/farmacología , Mutación , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Regulación hacia Arriba , beta-Lactamasas/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , beta-Lactamasas/genéticaRESUMEN
Mutations within the structural gene of ampD can lead to AmpC overproduction and increases in beta-lactam MICs in organisms with an inducible ampC. However, identification of mutations alone cannot predict the impact that those mutations have on AmpD function. Therefore, a model system was designed to determine the effect of ampD mutations on ceftazidime MICs using an AmpD(-) mutant Escherichia coli strain which produced an inducible plasmid-encoded AmpC. ampD genes were amplified by PCR from strains of E. coli, Citrobacter freundii, and Pseudomonas aeruginosa. Also, carboxy-terminal truncations of C. freundii ampD genes were constructed representing deletions of 10, 21, or 25 codons. Amplified ampD products were cloned into pACYC184 containing inducible bla(ACT-1)-ampR. Plasmids were transformed into E. coli strains JRG582 (AmpD(-)) and K-12 259 (AmpD(+)). The strains were evaluated for a derepressed phenotype using ceftazidime MICs. Some mutated ampD genes, including the ampD gene of a derepressed C. freundii isolate, resulted in substantial decreases in ceftazidime MICs (from >256 microg/ml to 12 to 24 microg/ml) for the AmpD(-) strain, indicating no role for these mutations in derepressed phenotypes. However, ampD truncation products and ampD from a partially derepressed P. aeruginosa strain resulted in ceftazidime MICs of >256 microg/ml, indicating a role for these gene modifications in derepressed phenotypes. The use of this model system indicated that alternative mechanisms were involved in the derepressed phenotype observed in strains of C. freundii and P. aeruginosa. The alternative mechanism involved in the derepressed phenotype of the C. freundii isolate was downregulation of ampD transcription.