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INTRODUCTION: Polymyxins, the cationic lipopeptide antibiotics, are the last line of therapeutics against the MDR Gram-negative bacterial (GNB) pathogens. Unfortunately, the rising cases of polymyxin-resistant strains from across the globe have adversely impacted their utility. While the molecular mechanisms responsible for developing polymyxin resistance (PolR) are largely understood, the prevalence of PolR strains in India has not been investigated systematically. The current study was undertaken to primarily determine the prevalence of PolR strains in India. Moreover, the extent of the spread of mobile colistin resistance (mcr) genes among the GNB strains in India was also determined. METHOD: A systematic search for articles using the relevant inclusion and exclusion criteria was performed in the applicable databases for the period January 2015 to December 2023. The included 41 studies were subjected to a meta-analysis using the Comprehensive Meta-Analysis software (V4.0). Publication biases were assessed using funnel plots and Egger's regression analysis. RESULT: Considering a total of 41 studies including 24â589 bacterial isolates the present meta-analysis found the rate of PolR bacteria in India to be at 15.0% (95% CI: 11.2 to 19.8). Among the Indian States, Tamil Nadu topped with the highest prevalence of PolR at 28.3%. Investigating the contribution of the mcr genes, it was observed that among the PolR strains, 8.4% (95% CI: 4.8 to 14.3) were mcr positive. CONCLUSION: The study determined the prevalence of PolR strains in India at 15.0%, which is higher than that of the global average at 10%. The study also determined that 8.4% of the PolR strains carried the mcr genes. The mcr-positive strains reported from India could be an underestimation of the actual numbers due to the non-inclusion of mcr screening in many previous studies. This study provides insight into the state of the PolR situation in India, which may be useful to develop a monitoring strategy to contain the spread of such strains and preserve the efficacy of the polymyxins.
Assuntos
Antibacterianos , Infecções por Bactérias Gram-Negativas , Polimixinas , Índia/epidemiologia , Humanos , Prevalência , Antibacterianos/farmacologia , Polimixinas/farmacologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/epidemiologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/classificação , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana Múltipla/genética , Colistina/farmacologiaRESUMO
With the rising incidences of antimicrobial resistance (AMR) and the diminishing options of novel antimicrobial agents, it is paramount to decipher the molecular mechanisms of action and the emergence of resistance to the existing drugs. Polymyxin, a cationic antimicrobial lipopeptide, is used to treat infections by Gram-negative bacterial pathogens as a last option. Though polymyxins were identified almost seventy years back, their use has been restricted owing to toxicity issues in humans. However, their clinical use has been increasing in recent times resulting in the rise of polymyxin resistance. Moreover, the detection of "mobile colistin resistance (mcr)" genes in the environment and their spread across the globe have complicated the scenario. The mechanism of polymyxin action and the development of resistance is not thoroughly understood. Specifically, the polymyxin-bacterial lipopolysaccharide (LPS) interaction is a challenging area of investigation. The use of advanced biophysical techniques and improvement in molecular dynamics simulation approaches have furthered our understanding of this interaction, which will help develop polymyxin analogs with better bactericidal effects and lesser toxicity in the future. In this review, we have delved deeper into the mechanisms of polymyxin-LPS interactions, highlighting several models proposed, and the mechanisms of polymyxin resistance development in some of the most critical Gram-negative pathogens.
Assuntos
Lipopolissacarídeos , Polimixinas , Humanos , Polimixinas/farmacologia , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Colistina/farmacologiaRESUMO
Infections caused by multi-drug resistant (MDR) bacterial pathogens are a leading cause of mortality and morbidity across the world. Indiscriminate use of broad-spectrum antibiotics has seriously affected this situation. With the diminishing discovery of novel antibiotics, new treatment methods are urgently required to combat MDR pathogens. Polymyxins, the cationic lipopeptide antibiotics, discovered more than half a century ago, are considered to be the last-line of antibiotics available at the moment. This antibiotic shows a great bactericidal effect against Gram-negative bacteria. Polymyxins primarily target the bacterial membrane and disrupt them, causing lethality. Because of their membrane interacting mode of action, polymyxins cause nephrotoxicity and neurotoxicity in humans, limiting their usability. However, recent modifications in their chemical structure have been able to reduce the toxic effects. The development of better dosing regimens has also helped in getting better clinical outcomes in the infections caused by MDR pathogens. Since the mid1990s the use of polymyxins has increased manifold in clinical settings, resulting in the emergence of polymyxin-resistant strains. The risk posed by the polymyxin-resistant nosocomial pathogens such as the Enterobacteriaceae group, Pseudomonas aeruginosa, and Acinetobacter baumannii, etc. is very serious considering these pathogens are resistant to almost all available antibacterial drugs. In this review article, the mode of action of the polymyxins and the genetic regulatory mechanism responsible for the emergence of resistance are discussed. Specifically, this review aims to update our current understanding in the field and suggest possible solutions that can be pursued for future antibiotic development. As polymyxins primarily target the bacterial membranes, resistance to polymyxins arises primarily by the modification of the lipopolysaccharides (LPS) in the outer membrane (OM). The LPS modification pathways are largely regulated by the bacterial two-component signal transduction (TCS) systems. Therefore, targeting or modulating the TCS signalling mechanisms can be pursued as an alternative to treat the infections caused by polymyxin-resistant MDR pathogens. In this review article, this aspect is also highlighted.
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Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Polimixinas/farmacologia , HumanosRESUMO
In Caulobacter crescentus the combined action of chromosome replication and the expression of DNA methyl-transferase CcrM at the end of S-phase maintains a cyclic alternation between a full- to hemi-methylated chromosome. This transition of the chromosomal methylation pattern affects the DNA-binding properties of the transcription factor GcrA that controls the several key cell cycle functions. However, the molecular mechanism by which GcrA and methylation are linked to transcription is not fully elucidated yet. Using a combination of cell biology, genetics, and in vitro analysis, we deciphered how GcrA integrates the methylation pattern of several S-phase expressed genes to their transcriptional output. We demonstrated in vitro that transcription of ctrA from the P1 promoter in its hemi-methylated state is activated by GcrA, while in its fully methylated state GcrA had no effect. Further, GcrA and methylation together influence a peculiar distribution of creS transcripts, encoding for crescentin, the protein responsible for the characteristic shape of Caulobacter cells. This gene is duplicated at the onset of chromosome replication and the two hemi-methylated copies are spatially segregated. Our results indicated that GcrA transcribed only the copy where coding strand is methylated. In vitro transcription assay further substantiated this finding. As several of the cell cycle-regulated genes are also under the influence of methylation and GcrA-dependent transcriptional regulation, this could be a mechanism responsible for maintaining the gene transcription dosage during the S-phase.
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Caulobacter crescentus/genética , Metilação de DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Transcrição Gênica/genética , DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/genética , Regiões Promotoras Genéticas/genética , Fator sigma/genéticaRESUMO
The LiaSR two-component signal transduction system regulates cellular responses to several environmental stresses, including those that induce cell envelope damages. Downstream regulons of the LiaSR system have been implicated in tolerance to acid, antibiotics and detergents. In the dental pathogen Streptococcus mutans, the LiaSR system is necessary for tolerance against acid, antibiotics, and cell wall damaging stresses during growth in the oral cavity. To understand the molecular mechanisms by which LiaSR regulates gene expression, we created a mutant LiaR in which the conserved aspartic acid residue (the phosphorylation site), was changed to alanine residue (D58A). As expected, the LiaR-D58A variant was unable to acquire the phosphate group and bind to target promoters. We also noted that the predicted LiaR-binding motif upstream of the lia operon does not appear to be well conserved. Consistent with this observation, we found that LiaR was unable to bind to the promoter region of lia; however, we showed that LiaR was able to bind to the promoters of SMU.753, SMU.2084 and SMU.1727. Based on sequence analysis and DNA binding studies we proposed a new 25-bp conserved motif essential for LiaR binding. Introducing alterations at fully conserved positions in the 25-bp motif affected LiaR binding, and the binding was dependent on the combination of positions that were altered. By scanning the S. mutans genome for the occurrence of the newly defined LiaR binding motif, we identified the promoter of hrcA (encoding a key regulator of the heat shock response) that contains a LiaR binding motif, and we showed that hrcA is negatively regulated by the LiaSR system. Taken together our results suggest a putative role of the LiaSR system in heat shock responses of S. mutans.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Resposta ao Choque Térmico/fisiologia , Streptococcus mutans/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Genoma Bacteriano/fisiologia , Motivos de Nucleotídeos/fisiologia , Streptococcus mutans/genéticaRESUMO
In Caulobacter crescentus, methylation of DNA by CcrM plays an important part in the regulation of cell cycle progression. Thanks to this methyltransferase, the activity of which is cell cycle regulated, the chromosome transitions between a hemimethylated state in the S-phase to a fully methylated condition in the G1 and G2 phases. Any perturbation in CcrM expression, such as depletion or constitutive expression, causes severe developmental defects. Several studies suggest that the role of CcrM is conserved across the Alphaproteobacteria. In the past few years, the importance of methylation on the expression of cell cycle regulated genes has emerged, suggesting that CcrM-dependent methylation can direct the binding of transcription factors to specific methylated sequences and affect the expression of genes depending on the methylation state of their promoters. CcrM activity has recently been linked to GcrA, a cell cycle master regulator that controls the expression of several genes during S-phase. Here, we review recent findings that establish the global role of methylation in cell cycle progression, and also explore the significance of a CcrM-GcrA epigenetic module that has co-evolved in Alphaproteobacteria, including Caulobacter, in controlling several genes involved in cell division, polarity, and motility.
Assuntos
Alphaproteobacteria/genética , Caulobacter/genética , Ciclo Celular/genética , Metilação de DNA , Modelos Genéticos , Alphaproteobacteria/citologia , Caulobacter/citologia , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Processamento de Proteína Pós-Traducional , Sinorhizobium meliloti/fisiologia , Simbiose , Medicago sativa/microbiologia , Fosforilação , Sinorhizobium meliloti/genéticaRESUMO
Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA-binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.
Assuntos
Caulobacter crescentus/genética , Metilação de DNA/genética , Metiltransferases/genética , Transcrição Gênica , Adenosina/genética , Alphaproteobacteria/crescimento & desenvolvimento , Sequência de Aminoácidos , Caulobacter crescentus/crescimento & desenvolvimento , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Metiltransferases/metabolismo , Regiões Promotoras GenéticasRESUMO
CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (â¼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Repressoras/metabolismo , Streptococcus mutans/metabolismo , Proteínas de Bactérias/genética , Eletroforese em Gel Bidimensional , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Regulon/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Streptococcus mutans/genéticaRESUMO
In this study, we report the presence of the El Tor CTXΦ and classical CTXΦ in Vibrio cholerae O1 strains isolated from Varanasi, India. Polymerase chain reaction, DNA sequencing and restriction fragment length polymorphism revealed that, although ctx-positive strains isolated after 1990 contain CTXΦ harbouring El Tor type of rstR and classical ctxB, strains isolated before 1990 contain El Tor type of rstR and El Tor ctxB. Two V. cholerae O1 strains (VC104 and VC106) represent an altered/hybrid strain containing the RS1 element followed by CTXΦ prophage harbouring El Tor type rstR and classical ctxB on the chromosome-I and RS2 element followed by second copy of CTXΦ prophage harbouring classical type rstR and classical ctxB on the chromosome-II. This is the first report of occurrence of El Tor CTXΦ harbouring classical ctxB and classical CTXΦ harbouring classical ctxB in chromosome-I and -II, respectively in diarrhoeal isolates of V. cholerae O1 El Tor strains from Varanasi, India, and that had been isolated in 1992.
RESUMO
We examined the effect of storage and sodium chloride on excision of CTXPhi or pre-CTXPhi and CTXPhi from Vibrio cholerae O139 strains. We found that one strain of V. cholerae O139 VO146P showed loss of the complete phage array, and other strain VO170P showed partial loss of the phage array giving rise to altered strains designated as VO146N and VO170N. Results of PCR and RFLP analysis revealed that both strains (VO146P and VO170P) possessed a single copy of pre-CTX(ET)Phi and two copies of CTXPhi comprising CTX(Class)Phi and CTX(Calc)Phi arranged in tandem, and integrated in the large chromosome. The presence of classical ctxB was detected in CTX(Calc)Phi of both V. cholerae O139 strains. Nucleotide sequencing of three housekeeping genes showed no difference between parent and altered strains of V. cholerae O139.
Assuntos
Toxina da Cólera/genética , Prófagos/genética , Cloreto de Sódio/farmacologia , Vibrio cholerae O139/virologia , Ativação Viral/fisiologia , DNA Viral/química , DNA Viral/isolamento & purificação , Variação Genética , Genoma Bacteriano , Genoma Viral , Humanos , Dados de Sequência Molecular , Família Multigênica , Preservação Biológica/métodos , Prófagos/efeitos dos fármacos , Prófagos/fisiologia , Fatores de Tempo , Vibrio cholerae O139/classificação , Vibrio cholerae O139/efeitos dos fármacos , Ativação Viral/efeitos dos fármacosRESUMO
In this study, we report the presence of SXT in environmental Vibrio cholerae O1 El Tor strains isolated before 1992 from Varanasi, India. All isolates, except one, were resistant to Tm, and/or Sul, Sm, Fr, Na and Am. None contained plasmids. PCR and DNA sequencing revealed the presence of SXT containing dfrA1 and/or sulII, strAB in six isolates and dfr18, sulII and strAB in five isolates. Three clinical V. cholerae O1 isolated during 1992 contained the antibiotic resistance gene cassette aadA1 in the class 1 integron. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence of the transferable nature of SXT and associated antibiotic resistance genes, and its integration into the prfC site. Results of phylogenetic analysis of the intSXT gene of clonally similar V. cholerae showed a clear difference between dfr18(+) and dfrA1(+) V. cholerae O1 isolates. This is the first report of the occurrence of SXT harbouring sulII, strAB, dfr18 and/or dfrA1 genes in environmental V. cholerae O1 isolated prior to 1992 from Varanasi, India, and suggests emergence of SXT(+) antibiotic-resistant V. cholerae O139 and O1 from an environmental V. cholerae progenitor by acquisition of SXT and antibiotic-resistant gene clusters.
RESUMO
A multiplex PCR was developed to detect pre-CTXΦ and CTXΦ in Vibrio cholerae. A total of 115 V. cholerae were tested, of which 42 V. cholerae O1 and 18 V. cholerae O139 contained CTXΦ. Six V. cholerae O139 contained only pre-CTXΦ and three V. cholerae O1 and 23 V. cholerae O139 contained both pre-CTXΦ and CTXΦ. None of the V. cholerae non-O1 and non-O139 that were tested had pre-CTXΦ or CTXΦ. Results of Restriction Fragment Length Polymorphism (RFLP) analysis revealed the V. cholerae isolates possessed single or multiple copies of pre-CTXΦ and CTXΦ, always proceeded by a tandemly arranged RS1 element. Comparative nucleotide sequence analyses of the core region genes, orfU and zot, of 15 V. cholerae showed pre-CTX(ET) Φ and CTX(ET) Φ lineage with V. cholerae El Tor and pre-CTX(Class) Φ, pre-CTX(Calc) Φ, and CTX(Calc) Φ with classical V. cholerae O1 and O139. Two distinct types of ctxB were detected in V. cholerae O139. Multi-locus Sequence Typing (MLST) of seven V. cholerae housekeeping genes indicated clonal origin, irrespective of the presence of pre-CTXΦ and/or CTXΦ.
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Sequencing of three housekeeping genes, mdh, dnaE and recA, and ribotyping for seven non-toxigenic Vibrio cholerae O1 strains isolated from different geographic sources indicate a phylogenetic relationship among the strains. Results of MLST and ribotyping indicate a clear difference between three toxigenic strains (N16961, O395, and 569B) and three non-toxigenic strains from India (GS1, GS2, and GW87) and one Guam strain (X392), the latter of which were similar in both MLST and ribotyping, while two other non-toxigenic strains from the USA and India (2740-80 and OR69) appeared to be more closely related to toxigenic strains than to non-toxigenic strains, although this was not supported by ribotyping. These results provide clues to the emergence of toxigenic strains from a non-toxigenic progenitor by acquisition of virulence gene clusters. Results of split decomposition analysis suggest that widespread recombination occurs among the three housekeeping genes and that recombination plays an important role in the emergence of toxigenic strains of V. cholerae O1.
Assuntos
Filogenia , Ribotipagem , Vibrio cholerae O1/genética , Sequência de Bases , DNA Polimerase III/genética , DNA Bacteriano/genética , Genes Bacterianos , Variação Genética , Malato Desidrogenase/genética , Dados de Sequência Molecular , Família Multigênica , Recombinases Rec A/genética , Recombinação Genética , Análise de Sequência de DNA , Vibrio cholerae O1/classificação , Vibrio cholerae O1/patogenicidade , VirulênciaRESUMO
In this study, we report the presence of the SXT element and Class I integron in Vibrio cholerae non-O1, non-O139 strains isolated from Varanasi, India. Isolates were resistant to cotrimoxazole, trimethoprim and/or streptomycin, furazolidone and ampicillin. None contained plasmids. Polymerase chain reaction (PCR) and DNA sequencing revealed the presence of antibiotic resistance gene cassettes, aadA1, aadA2, aadA5 and dfrA15, in the Class I integron and SXT, an integrative conjugative element containing dfr18, sulII and strAB, in three and six of the isolates respectively. Conjugation experiments, followed by PCR analysis of transconjugants, provided evidence for the transferable nature of intSXT and associated antibiotic resistance gene cassettes. This is the first report of the occurrence of SXT ICE, dfr18, sulII, strAB and aadA5 genes in environmental V. cholerae non-O1, non-O139 strains from Varanasi, India, that had been isolated before 1992.
Assuntos
Antibacterianos/farmacologia , Vibrioses/microbiologia , Vibrio cholerae não O1/efeitos dos fármacos , Vibrio cholerae não O1/genética , Ampicilina/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Antagonistas do Ácido Fólico/farmacologia , Furazolidona/farmacologia , Humanos , Índia , Integrases/genética , Integrons/genética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Família Multigênica , Estreptomicina/farmacologia , Trimetoprima/farmacologia , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Vibrio cholerae não O1/isolamento & purificação , Microbiologia da ÁguaRESUMO
Isolates of Vibrio cholerae O1 biotype El Tor serotype Inaba associated with an outbreak of cholera in Trivandrum, southern India, were characterized. PCR testing revealed that all five isolates examined carried the TCP pathogenicity island, the CTX genetic element and the RTX toxin, and produced cholera toxin (CT). RFLP analysis revealed that these Inaba isolates possessed a single copy of the CTX element flanked by two tandemly arranged copies of the RS element upstream of the core region. The isolates were resistant to ampicillin, nalidixic acid, trimethoprim, sulfamethoxazole, streptomycin and the vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129). Ribotyping of these Inaba isolates revealed a hybridization profile similar to a strain of serotype Ogawa prevalent in southern India.
Assuntos
Antibacterianos/farmacologia , Cólera/microbiologia , Farmacorresistência Bacteriana , Vibrio cholerae O1/classificação , Vibrio cholerae O1/genética , Cólera/epidemiologia , Toxina da Cólera/genética , DNA Bacteriano/genética , Surtos de Doenças , Dosagem de Genes , Genes Bacterianos , Ilhas Genômicas/genética , Humanos , Índia/epidemiologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Ribotipagem , Sorotipagem , Vibrio cholerae O1/efeitos dos fármacos , Vibrio cholerae O1/isolamento & purificaçãoRESUMO
In this study, we describe a septaplex PCR assay for rapid identification of Vibrio cholerae including detection of the virulence and intsxt genes. Conditions were optimized to amplify fragments of ISRrRNA (encoding for 16S-23S rRNA gene, Intergenic spacer regions), O1rfb (O1 serogroup specific rfb), O139rfb (O139 serogroup specific rfb), ctxA (cholera toxin subunit A), tcpA (toxin coregulated pilus), and intsxt (sxt integron) simultaneously in a single PCR. The septaplex PCR was evaluated using 211 strains of V. cholerae and six water samples for in situ testing. PCR results were correlated with genotype data obtained by individual PCR and slot-blot assays. The one-step PCR described here can be used to identify V. cholerae accurately and rapidly. Also, the virulence and intsxt genes can be simultaneously detected, providing a useful method for monitoring pathogenic, intsxt-positive and nonpathogenic, intsxt-negative V. cholerae serogroups both in the environment and clinical settings.