RESUMEN
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
Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified approximately 500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs.
Asunto(s)
Variación Genética , Genoma de Protozoos , Plasmodium falciparum/genética , Transportadoras de Casetes de Unión a ATP/genética , Alelos , Animales , Resistencia a Medicamentos/genética , Evolución Molecular , GTP Ciclohidrolasa/genética , Amplificación de Genes , Eliminación de Gen , Tolerancia Inmunológica/genética , Inmunidad/genética , Familia de Multigenes , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Plasmodium falciparum/fisiología , Proteínas Protozoarias/genéticaRESUMEN
The need to understand the genetic basis of drug resistance in human pathogens has never been greater. The global incidence of drug-resistant organisms, such as those that cause malaria, continues to rise, while the repertoire of effective, inexpensive drugs is declining. Genomic technologies, such as DNA microarrays and full-genome sequencing offer new hope in advancing our understanding of the underlying genetic processes that facilitate a resistance phenotype. Importantly, evidence that drug resistance in many organisms can be a multigene, complex phenomenon implies that unbiased, genome-wide scans of diversity will be required to fully understand the molecular mechanisms of both established and novel resistance traits. While the potential application of full-genome approaches for deciphering mechanisms of drug resistance has yet to be fully realized, this review evaluates drug resistance in human malaria parasites and discusses the exciting role genome-based systems could play in monitoring drug resistance, as well as guiding the implementation of efficient therapeutic strategies for malaria. The approaches reviewed within this article will be applicable to all known or emerging microbial pathogens.
Asunto(s)
Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Genoma , Malaria/parasitología , Plasmodium/efectos de los fármacos , Plasmodium/genética , Animales , Variación Genética , HumanosRESUMEN
DNA microarrays, initially designed to measure gene expression levels, also provide an ideal platform for determining genetic diversity. Oligonucleotide microarrays, predominantly high-density oligonucleotide arrays, have emerged as the principal platforms for performing genome-wide diversity analysis. They have wide-ranging potential applications including comparative genomics, polymorphism discovery and genotyping. The identification of inheritable genetic markers also permits the analysis of quantitative traits, population studies and linkage analysis. In this review, we will discuss the application of oligonucleotide arrays, in particular high-density oligonucleotide arrays for elucidating genetic diversity and highlight some of the directions that the field may take.
Asunto(s)
Ligamiento Genético , Variación Genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Marcadores Genéticos , Genoma , Humanos , Patrón de Herencia , Sitios de Carácter CuantitativoRESUMEN
The accurate identification of Salmonella enterica subsp. enterica serovar Typhi variants that fail to express the capsular polysaccharide, Vi, is an important and much discussed issue for medical microbiology. We have tested a multiplex PCR method which shows the presence or absence of the genetic locus required for Vi expression. Of 2,222 Salmonella serovar Typhi clinical isolates collected from patients' blood over a 4-year period in a region of Pakistan where typhoid is endemic, 12 tested negative for Vi expression by serological agglutination. However, only 1 of these 12 was Vi negative by the multiplex PCR method. This result was confirmed by immunofluorescence, the most sensitive method for Vi characterization in Salmonella serovar Typhi. The multiplex PCR described therefore represents a simple and accurate method for surveillance for Vi-negative variants of Salmonella serovar Typhi in Pakistan. Testing of clinical isolates of Salmonella serovar Typhi, before subculture, from other regions where Vi-negative Salmonella serovar Typhi has been described should be carried out so that the impact of vaccination with purified Vi antigen on the levels of Vi-negative Salmonella serovar Typhi in bacterial populations can be assessed.
Asunto(s)
Antígenos Bacterianos/análisis , Polisacáridos Bacterianos/análisis , Salmonella typhi/inmunología , Aglutinación , Antígenos Bacterianos/genética , Secuencia de Bases , Medios de Cultivo , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polisacáridos Bacterianos/genética , Salmonella typhi/crecimiento & desarrolloRESUMEN
Resistance to chloramphenicol was reported in Salmonella Typhi in 1950 but it was not until 22 years later that the first outbreaks of chloramphenicol-resistant typhoid fever occurred. Multidrug-resistant (MDR) Salmonella Typhi emerged in the 1980s and today has an almost worldwide distribution. Genome analysis of Salmonella Typhi strain CT18, an MDR isolate from a patient admitted to The Centre for Tropical Diseases, Ho Chi Minh City, Viet Nam, in December 1993 revealed that the resistance plasmid pHCM1 is very closely related to plasmid R27 which was first isolated in 1961. There is a core region shared by the two plasmids with five regions of variation. Two of these regions contain the genes encoding resistance. The largest region is 34.955 kbp in length, is bordered by two almost identical IS10 elements and contains several integron-like structures including a truncated Tn10 element. The second region is 14.75I kbp and encodes a trimethoprim-resistance gene, dfrA14, associated with a class one integrase. Restriction enzyme analysis has shown that the variation in Salmonella Typhi plasmids, collected during the emergence of resistant Salmonella Typhi in Viet Nam, maps to five variable regions. These regions appear to be hot spots for DNA acquisition in IncHI1 plasmids.
Asunto(s)
Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/genética , Salmonella typhi/efectos de los fármacos , Fiebre Tifoidea/tratamiento farmacológico , Antibacterianos/economía , Costo de Enfermedad , Genoma Bacteriano , Humanos , Plásmidos/genética , Salmonella typhi/genética , Fiebre Tifoidea/economía , Fiebre Tifoidea/genéticaRESUMEN
The first outbreak of multidrug-resistant (MDR) typhoid fever in Vietnam was in 1993, and by 1995 nearly 90% of cases were MDR. Plasmid HCM1, sequenced in full, is an incHI1 plasmid from Salmonella enterica serovar Typhi strain CT18, isolated in Vietnam in 1993. Restriction analysis shows that pHCM1 shares a restriction fragment length polymorphism (RFLP) pattern with plasmids isolated from the first outbreak and 10 of 17 MDR plasmids isolated from sporadic cases occurring at the same time in Vietnam. A core region of pHCM1 has significant DNA sequence similarity to plasmid R27, isolated in 1961 from S. enterica in the United Kingdom. There are five regions of DNA in pHCM1 which are not present in R27. Two of these are putative acquisition regions; the largest is 34.955 kbp in length and includes sequences of several antibiotic resistance genes and several insertion sequences. The borders of this region are defined by two identical IS10 left elements, associated with an inversion of DNA or with a truncated Tn10 element. The second, smaller region is 14.751 kbp and carries a trimethoprim resistance gene dfr14A cassette associated with a class 1 integrase. In 1993 to 1994, restriction analysis revealed some variations in the structures of Salmonella serovar Typhi MDR plasmids which were mapped to the two putative acquisition regions and three smaller variable regions. In 1996 a single RFLP type, RFLP7, was found to carry the dfrA7 and sul-1 genes, which were not present on R27 or pHCM1. This plasmid type appears to have a selective advantage over other plasmids with the same resistance phenotype.
Asunto(s)
Genes MDR/genética , Plásmidos/genética , Salmonella typhi/efectos de los fármacos , Fiebre Tifoidea/microbiología , Conjugación Genética , ADN Bacteriano/análisis , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad Microbiana , Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
pHCM2 is a 106 kbp cryptic plasmid harboured by Salmonella typhi CT18, originally isolated from a typhoid patient in Vietnam. The genome of S. typhi CT18, including pHCM2, has recently been completely sequenced and annotated. Bioinformatic analysis revealed that 57% of the coding sequences (CDSs) encoded on pHCM2 display over 97% DNA sequence identity to the virulence-associated plasmid of Yersinia pestis, pFra. pHCM2 encodes no obvious virulence-associated determinants or antibiotic resistance genes but does encode a wide array of putative genes directly related to DNA metabolism and replication. PCR analysis of a series of S. typhi isolates from Vietnam detected pHCM2-related DNA sequences in some S. typhi isolated before, but not after, 1994. Similar pHCM2-related sequences were also detected in S. typhi isolated from other regions of South East Asia and Pakistan but not elsewhere in the world.
Asunto(s)
Plásmidos/genética , Salmonella typhi/genética , Secuencia de Bases , Mapeo Cromosómico , Biología Computacional , Replicación del ADN , Evolución Molecular , Genes Bacterianos/genética , Humanos , Datos de Secuencia Molecular , Fiebre Tifoidea/microbiología , VietnamRESUMEN
A global collection of 26 isolates of Salmonella typhi was investigated by sequencing a total of 3336 bp in seven housekeeping genes. Only three polymorphic sites were found and the isolates fell into four sequence types. These results show that S. typhi is a recent clone whose last common ancestor existed so recently that multiple mutations have not yet accumulated. Based on molecular clock rates for the accumulation of synonymous polymorphisms, we estimate that the last common ancestor of S. typhi existed 15,000-150,000 years ago, during the human hunter-gatherer phase and prior to the development of agriculture and the domestication of animals.