RESUMEN
Trimethoprim and sulfamethoxazole are used commonly together as cotrimoxazole for the treatment of urinary tract and other infections. The evolution of resistance to these and other antibacterials threatens therapeutic options for clinicians. We generated and analysed a chemical-biology-whole-genome data set to predict new targets for antibacterial combinations with trimethoprim and sulfamethoxazole. For this we used a large transposon mutant library in Escherichia coli BW25113 where an outward-transcribing inducible promoter was engineered into one end of the transposon. This approach allows regulated expression of adjacent genes in addition to gene inactivation at transposon insertion sites, a methodology that has been called TraDIS-Xpress. These chemical genomic data sets identified mechanisms for both reduced and increased susceptibility to trimethoprim and sulfamethoxazole. The data identified that over-expression of FolA reduced trimethoprim susceptibility, a known mechanism for reduced susceptibility. In addition, transposon insertions into the genes tdk, deoR, ybbC, hha, ldcA, wbbK and waaS increased susceptibility to trimethoprim and likewise for rsmH, fadR, ddlB, nlpI and prc with sulfamethoxazole, while insertions in ispD, uspC, minC, minD, yebK, truD and umpG increased susceptibility to both these antibiotics. Two of these genes' products, Tdk and IspD, are inhibited by AZT and fosmidomycin respectively, antibiotics that are known to synergise with trimethoprim. Thus, the data identified two known targets and several new target candidates for the development of co-drugs that synergise with trimethoprim, sulfamethoxazole or cotrimoxazole. We demonstrate that the TraDIS-Xpress technology can be used to generate information-rich chemical-genomic data sets that can be used for antibacterial development.
Asunto(s)
Antibacterianos/farmacología , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Sulfametoxazol/farmacología , Trimetoprim/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Biblioteca de Genes , Silenciador del Gen , Humanos , Pruebas de Sensibilidad Microbiana , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Secuenciación Completa del GenomaRESUMEN
Live attenuated oral enterotoxigenic Escherichia coli (ETEC) vaccines have been demonstrated to be safe and immunogenic in human volunteers and to provide a viable approach to provide protection against this important pathogen. This report describes the construction of new ETEC vaccine candidate strains from recent clinical isolates and their characterization. All known genes for ETEC toxins were removed, and attenuating deletion mutations were made in the aroC, ompC, and ompF chromosomal genes. An isolate expressing coli surface antigen 2 (CS2), CS3, heat-labile toxin (LT), heat-stable toxin (ST), and enteroaggregative Escherichia coli heat-stable toxin 1 (EAST1) was attenuated to generate ACAM2007. The subsequent insertion of the operon encoding CS1 created ACAM2017, and this was further modified by the addition of an expression cassette containing the eltB gene, encoding a pentamer of B subunits of LT (LTB), to generate ACAM2027. Another isolate expressing CS5, CS6, LT, ST, and EAST1 was attenuated to generate ACAM2006, from which a lysogenic prophage was deleted to create ACAM2012 and an LTB gene was introduced to form ACAM2022. Finally, a previously described vaccine strain, ACAM2010, had the eltB gene incorporated to generate ACAM2025. All recombinant genes were incorporated into the chromosomal sites of the attenuating mutations to ensure maximal genetic stability. The expression of the recombinant antigens and the changes in plasmids accompanying the deletion of toxin genes are described. Strains ACAM2025, ACAM2022, and ACAM2027 have been combined to create the ETEC vaccine formulation ACE527, which has recently successfully completed a randomized, double-blind, placebo-controlled phase I trial and is currently undergoing a randomized, double-blind placebo-controlled phase II challenge trial, both in healthy adult volunteers.
Asunto(s)
Toxinas Bacterianas/genética , Escherichia coli Enterotoxigénica/genética , Vacunas contra Escherichia coli/genética , Factores de Virulencia/genética , Toxinas Bacterianas/inmunología , Escherichia coli Enterotoxigénica/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/inmunología , Humanos , Plásmidos , Ensayos Clínicos Controlados Aleatorios como Asunto , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Combinadas/administración & dosificación , Vacunas Combinadas/genética , Vacunas Combinadas/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Factores de Virulencia/inmunologíaRESUMEN
A global collection of plasmids of the IncHI1 incompatibility group from Salmonella enterica serovar Typhi were analyzed by using a combination of DNA sequencing, DNA sequence analysis, PCR, and microarrays. The IncHI1 resistance plasmids of serovar Typhi display a backbone of conserved gene content and arrangement, within which are embedded preferred acquisition sites for horizontal DNA transfer events. The variable regions appear to be preferred acquisition sites for DNA, most likely through composite transposition, which is presumably driven by the acquisition of resistance genes. Plasmid multilocus sequence typing, a molecular typing method for IncHI1 plasmids, was developed using variation in six conserved loci to trace the spread of these plasmids and to elucidate their evolutionary relationships. The application of this method to a collection of 36 IncHI1 plasmids revealed a chronological clustering of plasmids despite their difference in geographical origins. Our findings suggest that the predominant plasmid types present after 1993 have not evolved directly from the earlier predominant plasmid type but have displaced them. We propose that antibiotic selection acts to maintain resistance genes on the plasmid, but there is also competition between plasmids encoding the same resistance phenotype.
Asunto(s)
Farmacorresistencia Bacteriana/genética , Plásmidos/genética , Salmonella typhi/genética , Fiebre Tifoidea/epidemiología , Fiebre Tifoidea/microbiología , ADN Bacteriano/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vietnam/epidemiologíaRESUMEN
OBJECTIVES: To determine the contribution to fluoroquinolone resistance of point mutations in the gyrA and parC genes of Salmonella Typhi. METHODS: Point mutations that result in Ser-83-->Phe, Ser-83-->Tyr and Asp-87-->Asn amino acid substitutions in GyrA and Glu-84-->Lys in ParC were introduced into a quinolone-susceptible, attenuated strain of Salmonella Typhi using suicide vector technology. This is the first time that this approach has been used in Salmonella and abrogates the need for selection with quinolone antibacterials in the investigation of resistance mutations. RESULTS: A panel of mutants was created using this methodology and tested for quinolone resistance. The ParC substitution alone made no difference to quinolone susceptibility. Any single GyrA substitution resulted in resistance to nalidixic acid (MIC >or= 512 mg/L) and increased by up to 23-fold the MIC of the fluoroquinolones ofloxacin (MIC
Asunto(s)
Antibacterianos/farmacología , Topoisomerasa de ADN IV/genética , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Mutación Puntual , Salmonella typhi/efectos de los fármacos , Sustitución de Aminoácidos , Ciprofloxacina/farmacología , Girasa de ADN/genética , Humanos , Pruebas de Sensibilidad Microbiana , Ácido Nalidíxico/farmacología , Salmonella typhi/enzimología , Salmonella typhi/genética , Salmonella typhi/crecimiento & desarrolloRESUMEN
A gene cassette incorporating the crs-rsd site-specific recombination system from the Salmonella enterica subsp. enterica serovar Dublin virulence plasmid improved the inheritance in S. enterica serotype Typhi strain CVD908-htrA of a multicopy plasmid expression vector. Use of this recombination cassette may improve expression of heterologous antigens from multicopy plasmid expression vectors in attenuated bacterial vaccine strains.
Asunto(s)
Antígenos Bacterianos/genética , Antígenos Heterófilos/genética , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Vacunas contra la Salmonella/genética , Salmonella typhi/genética , Antígenos Bacterianos/inmunología , Antígenos Heterófilos/inmunología , Vectores Genéticos/metabolismo , Helicobacter pylori/genética , Recombinación Genética , Vacunas contra la Salmonella/inmunología , Salmonella typhi/inmunología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
A vaccine against enterotoxigenic Escherichia coli (ETEC) is needed to prevent diarrheal illness among children in developing countries and at-risk travelers. Two live attenuated ETEC strains, PTL002 and PTL003, which express the ETEC colonization factor CFA/II, were evaluated for safety and immunogenicity. In a randomized, double-blind, placebo-controlled trial, 19 subjects ingested one dose, and 21 subjects ingested two doses (days 0 and 10) of PTL-002 or PTL-003 at 2 x 10(9) CFU/dose. Anti-CFA/II mucosal immune responses were determined from the number of antibody-secreting cells (ASC) in blood measured by enzyme-linked immunospot assay, the antibody in lymphocyte supernatants (ALS) measured by enzyme-linked immunosorbent assay (ELISA), and fecal immunoglobulin A (IgA) levels determined by ELISA. Time-resolved fluorescence (TRF) ELISA was more sensitive than standard colorimetric ELISA for measuring serum antibody responses to CFA/II and its components, CS1 and CS3. Both constructs were well tolerated. Mild diarrhea occurred after 2 of 31 doses (6%) of PTL-003. PTL-003 produced more sustained intestinal colonization than PTL-002 and better IgA response rates: 90% versus 55% (P = 0.01) for anti-CFA/II IgA-ASCs, 55% versus 30% (P = 0.11) for serum anti-CS1 IgA by TRF, and 65% versus 25% (P = 0.03) for serum anti-CS3 IgA by TRF. Serum IgG response rates to CS1 or CS3 were 55% in PTL-003 recipients and 15% in PTL-002 recipients (P = 0.02). Two doses of either strain were not significantly more immunogenic than one. Based on its superior immunogenicity, which was comparable to that of a virulent ETEC strain and other ETEC vaccine candidates, PTL-003 will be developed further as a component of a live, oral attenuated ETEC vaccine.
Asunto(s)
Vacunas contra Escherichia coli/efectos adversos , Vacunas contra Escherichia coli/inmunología , Escherichia coli/inmunología , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/inmunología , Adolescente , Adulto , Anticuerpos Antibacterianos/análisis , Anticuerpos Antibacterianos/sangre , Células Productoras de Anticuerpos , Método Doble Ciego , Enterotoxinas/metabolismo , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/administración & dosificación , Heces/microbiología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Persona de Mediana Edad , Vacunas Atenuadas/administración & dosificaciónRESUMEN
Oral delivery of toxin-negative derivatives of enterotoxigenic Escherichia coli (ETEC) that express colonization factor antigens (CFA) with deletions of the aroC, ompC, ompF, and toxin genes may be an effective approach to vaccination against ETEC-associated diarrhea. We describe the creation and characterization of an attenuated CFA/I-expressing ETEC vaccine candidate, ACAM2010, from a virulent isolate in which the heat-stable enterotoxin (ST) and CFA/I genes were closely linked and on the same virulence plasmid as the enteroaggregative E. coli heat-stable toxin (EAST1) gene. A new suicide vector (pJCB12) was constructed and used to delete the ST and EAST1 genes and to introduce defined deletion mutations into the aroC, ompC, and ompF chromosomal genes. A phase I trial, consisting of an open-label dose escalation phase in 18 adult outpatient volunteers followed by a placebo-controlled double-blind phase in an additional 31 volunteers, was conducted. The vaccine was administered in two formulations, fresh culture and frozen suspension. These were both well tolerated, with no evidence of significant adverse events related to vaccination. Immunoglobulin A (IgA) and IgG antibody-secreting cells specific for CFA/I were assayed by ELISPOT. Positive responses (greater than twofold increase) were seen in 27 of 37 (73%) subjects who received the highest dose level of vaccine (nominally 5 x 10(9) CFU). Twenty-nine of these volunteers were secreting culturable vaccine organisms at day 3 following vaccination; five were still positive on day 7, with a single isolation on day 13. This live attenuated bacterial vaccine is safe and immunogenic in healthy adult volunteers.
Asunto(s)
Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/efectos adversos , Vacunas contra Escherichia coli/inmunología , Proteínas Fimbrias/efectos adversos , Proteínas Fimbrias/inmunología , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Niño , Método Doble Ciego , Escherichia coli/genética , Escherichia coli/inmunología , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/efectos adversos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Vacunas contra Escherichia coli/administración & dosificación , Vacunas contra Escherichia coli/genética , Femenino , Proteínas Fimbrias/administración & dosificación , Proteínas Fimbrias/genética , Eliminación de Gen , Humanos , Inmunización , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Plásmidos , Resultado del Tratamiento , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/efectos adversos , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
A recombinant strain of attenuated Salmonella enterica serovar Typhi surface-expressing Yersinia pestis F1 antigen was generated by transforming strain BRD1116 (aroA aroC htrA) with plasmid pAH34L encoding the Y. pestis caf operon. BRD1116/pAH34L was stable in vitro and in vivo. An immunisation regimen of two intranasal doses of 1 x 10(8) cfu of BRD1116/pAH34L given intranasally to mice 7 days apart induced the strongest immune response compared to other regimens and protected 13 out of 20 mice from lethal challenge with Y. pestis. Intranasal immunisation of mice constitutes a model for oral immunisation with Salmonella vaccines in humans. Thus, the results demonstrate that attenuated strains of S. enterica serovar Typhi which express Y. pestis F1 antigen may be developed to provide an oral vaccine against plague suitable for use in humans.
Asunto(s)
Antígenos Bacterianos/inmunología , Peste/prevención & control , Vacunas contra la Salmonella/inmunología , Yersinia pestis/química , Administración Oral , Animales , Antígenos Bacterianos/genética , Cápsulas Bacterianas/inmunología , Ratones , Vacunas contra la Salmonella/administración & dosificación , Vacunación , Yersinia pestis/inmunologíaRESUMEN
Recombinant vaccine strains of Salmonella enterica serovar Typhi capable of expressing Helicobacter pylori urease were generated by transforming strains CVD908 and CVD908-htrA with a plasmid harboring the ureAB genes under the control of an in vivo-inducible promoter. The plasmid did not interfere with the ability of either strain to replicate and persist in human monocytic cells or with their transient colonization of mouse lungs. When administered to mice intranasally, both recombinant strains elicited antiurease immune responses skewed towards a Th1 phenotype. Vaccinated mice exhibited strong immunoglobulin G2a (IgG2a)-biased antiurease antibody responses as well as splenocyte populations capable of proliferation and gamma interferon (IFNgamma) secretion in response to urease stimulation. Boosting of mice with subcutaneous injection of urease plus alum enhanced immune responses and led them to a more balanced Th1/Th2 phenotype. Following parenteral boost, IgG1 and IgG2a antiurease antibody titers were raised significantly, and strong urease-specific splenocyte proliferative responses, accompanied by IFNgamma as well as interleukin-4 (IL-4), IL-5, and IL-10 secretion, were detected. Neither immunization with urease-expressing S. enterica serovar Typhi alone nor immunization with urease plus alum alone conferred protection against challenge with a mouse-adapted strain of H. pylori; however, a vaccination protocol combining both immunization regimens was protective. This is the first report of effective vaccination against H. pylori with a combined mucosal prime-parenteral boost regimen in which serovar Typhi vaccine strains are used as antigen carriers. The significance of these findings with regard to development of a human vaccine against H. pylori and modulation of immune responses by heterologous prime-boost immunization regimens is discussed.