Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellín, Colombia

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylococcus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA isolates (83% vs. 73%). MRSA strains harboured SCCmec types IVc (60%), I (30%), IVa (7%) and V (3%). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.

Molecular divergence in the timeless and cpr genes among three sympatric cryptic species of the Anopheles triannulatus complex

Anopheles triannulatus s.l. is a malaria vector with a wide geographic distribution, ranging from Argentina-Nicaragua and Trinidad. Here we analysed sequences of two genes, timeless and cpr, to assess the genetic variability and divergence among three sympatric cryptic species of this complex from Salobra, central-western Brazil. The timeless gene sequences did not conclusively differentiate Anopheles halophylus and An. triannulatus species "C". However, a partial separation has been observed between these species and An. triannulatus s.s. Importantly, the analysis of the cpr gene sequences revealed fixed differences, no shared polymorphisms and considerable genetic differentiation among the three species of the An. triannulatus complex. The results confirm that An. triannulatus s.s., An. halophylus and An. triannulatus species C are distinct taxa, with the latter two likely representing a more recent speciation event.

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.

Shigella in Brazilian children with acute diarrhoea: prevalence, antimicrobial resistance and virulence genes

Diarrhoeal disease is still considered a major cause of morbidity and mortality among children. Among diarrhoeagenic agents, Shigella should be highlighted due to its prevalence and the severity of the associated disease. Here, we assessed Shigella prevalence, drug susceptibility and virulence factors. Faeces from 157 children with diarrhoea who sought treatment at the Children's Hospital João Paulo II, a reference children´s hospital in Belo Horizonte, state of Minas Gerais, Brazil, were cultured and drug susceptibility of the Shigella isolates was determined by the disk diffusion technique. Shigella virulence markers were identified by polymerase chain reaction. The bacterium was recovered from 10.8% of the children (88.2% Shigella sonnei). The ipaH, iuc, sen and ial genes were detected in strains isolated from all shigellosis patients; set1A was only detected in Shigella flexneri. Additionally, patients were infected by Shigella strains of different ial, sat, sen and set1A genotypes. Compared to previous studies, we observed a marked shift in the distribution of species from S. flexneri to S. sonnei and high rates of trimethoprim/sulfamethoxazole resistance.

Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes

In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae). The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes). This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes) and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.

In house reverse membrane hybridisation assay versus GenoType MTBDRplus and their performance to detect mutations in the genes rpoB, katG and inhA

Drug-resistant tuberculosis (TB) threatens global TB control and is a major public health concern in several countries. We therefore developed a multiplex assay (LINE-TB/MDR) that is able to identify the most frequent mutations related to rifampicin (RMP) and isoniazid (INH) resistance. The assay is based on multiplex polymerase chain reaction, membrane hybridisation and colorimetric detection targeting of rpoB and katG genes, as well as the inhA promoter, which are all known to carry specific mutations associated with multidrug-resistant TB (MDR-TB). The assay was validated on a reference panel of 108 M. tuberculosis isolates that were characterised by the proportion method and by DNA sequencing of the targets. When comparing the performance of LINE-TB/MDR with DNA sequencing, the sensitivity, specificity and agreement were 100%, 100% and 100%, respectively, for RMP and 77.6%, 90.6% and 88.9%, respectively, for INH. Using drug sensibility testing as a reference standard, the performance of LINE-TB/MDR regarding sensitivity, specificity and agreement was 100%, 100% and 100% (95%), respectively, for RMP and 77%, 100% and 88.7% (82.2-95.1), respectively, for INH. LINE-TB/MDR was compared with GenoType MTBDRplus for 65 isolates, resulting in an agreement of 93.6% (86.7-97.5) for RIF and 87.4% (84.3-96.2) for INH. LINE-TB/MDR warrants further clinical validation and may be an affordable alternative for MDR-TB diagnosis.

Oxidative stress enhances the expression of sulfur assimilation genes: preliminary insights on the Enterococcus faecalis iron-sulfur cluster machinery regulation

The Firmicutes bacteria participate extensively in virulence and pathological processes. Enterococcus faecalis is a commensal microorganism; however, it is also a pathogenic bacterium mainly associated with nosocomial infections in immunocompromised patients. Iron-sulfur [Fe-S] clusters are inorganic prosthetic groups involved in diverse biological processes, whose in vivo formation requires several specific protein machineries. Escherichia coli is one of the most frequently studied microorganisms regarding [Fe-S] cluster biogenesis and encodes the iron-sulfur cluster and sulfur assimilation systems. In Firmicutes species, a unique operon composed of the sufCDSUB genes is responsible for [Fe-S] cluster biogenesis. The aim of this study was to investigate the potential of the E. faecalis sufCDSUB system in the [Fe-S] cluster assembly using oxidative stress and iron depletion as adverse growth conditions. Quantitative real-time polymerase chain reaction demonstrated, for the first time, that Gram-positive bacteria possess an OxyR component responsive to oxidative stress conditions, as fully described for E. coli models. Likewise, strong expression of the sufCDSUB genes was observed in low concentrations of hydrogen peroxide, indicating that the lowest concentration of oxygen free radicals inside cells, known to be highly damaging to [Fe-S] clusters, is sufficient to trigger the transcriptional machinery for prompt replacement of [Fe-S] clusters.

CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco, Northeast Region of Brazil: a case-control study

Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection.

Micro-organismos da subclasse Coccidia: resistência e implicações para o processamento de materiais de assistência à saúde

Este estudo teórico propõe uma reflexão sobre a resistência intrínseca da subclasse Coccidia, particularmente o gênero Cryptosporidium, considerado como um agente potencialmente patogênico para pacientes imunocomprometidos, e suas repercussões na prática assistencial. Atualmente, as diretrizes internacionais e nacionais aprovam como procedimento seguro a desinfecção química de alto nível de endoscópios digestivos, após sua limpeza. No entanto, estudos evidenciaram que micro-organismos da subclasse Coccidia, especificamente o Cryptosporidium, responsável por infecção entérica, são mais resistentes que as micobactérias e não são inativados pelos desinfetantes químicos de alto nível, exceto pelo Peróxido de Hidrogênio a 6% e 7,5%, formulação ainda não disponível no Brasil. Conclui-se que a legislação deve incluir este agente entre os micro-organismos teste para aprovação de desinfetantes químicos de alto nível e que as autoridades sanitárias devem se esforçar para garantir que os estabelecimentos de assistência à saúde tenham acesso a produtos eficazes contra o Cryptosporidium.

Utilização de bioensaios e marcadores moleculares para detecção da resistência de coleópteros de produtos armazenados a inseticidas

The qualitative and quantitative losses caused by stored product insects are of great concern, and since there is only a few active ingredients available for their control it is very important to have a frequent insect resistance monitoring. The objective of this research is to evaluate combination of bioassays and molecular marker techniques to detect insecticide resistance in stored product beetles. The Coleoptera species used for the tests were Sitophilus oryzae (L.) (Curculionidae), Rhyzopertha dominica (F.) (Bostrichidae) and Oryzaephilus surinamensis (L.) (Silvanidae). For the bioassays it was used the impregnated filter paper technique, applying 1 mL of deltamethrin (K-Obiol 25 CE TM) using four concentrations and five replicates, including a control with solvent only. Ten adults of each species were liberated separately on each dish. The mortality was evaluated after 24 h and resistance determined by probit analysis. The samples used for the PCR-RAPD were either in vivo or preserved in 70% ethanol, kept in -18°C freezer. After extraction, quantification and DNA quality analysis, the 25 µL samples had the DNA amplified and tested with six primers. The bioassays showed a crescent mortality proportional to insecticide concentration. The resistance factor for R. dominica, S. zeamais and S. oryzae were: 2,2; 3,2 and 9,2, respectively, compared to the susceptible populations of each species. The PCR-RAPD analysis revealed bands which indicate inter and intraspecific variability in the populations, but it was not possible to correlate them to resistance. The association of bioassay and PCR-RAPD represents a precise and valuable tool for resistance management of stored product insects, but more populations and primers should be tested.

Efeito do sinergista butóxido de piperonila na resistência de Oryzaephilus surinamensis (L.) (Coleoptera, Silvanidae) a deltametrina e fenitrotiom

A utilização de sinergistas é uma importante ferramenta para determinar os mecanismos envolvidos na resistência de insetos. Nesta pesquisa, o sinergista butóxido de piperonila (PBO) foi usado, em diferentes proporções, para avaliar a contribuição relativa de enzimas oxidases no metabolismo do inseticida organofosforado fenitrotiom e do piretróide deltametrina, em quatro populações de Oryzaephilus surinamensis: OS1 (suscetível) e OS2, OS3 e OS4 (resistentes). O sinergista aumentou, significativamente, a toxicidade da deltametrina nas populações resistentes, indicando que as oxidases exercem uma importante função na resistência a este inseticida. Para o fenitrotiom, o PBO apresentou um efeito antagonista, diminuindo significativamente a toxicidade do inseticida em todas as populações, indicando que este sinergista não é o mais apropriado para a mistura com compostos organofosforados.

Crescimento de raízes de trigo afetado pela resistência mecânica do solo

A compactação do solo é um problema comum que afeta diversas propriedades físicas do solo e o crescimento das plantas. Este trabalho foi desenvolvido em câmara de crescimento na Universidade Federal do Rio Grande do Sul, em Porto Alegre, em 1994, durante 35 dias (530 GD, 0ºC de temperatura base) e objetivou determinar os efeitos da resistência mecânica do solo no crescimento das raízes de trigo. Para tanto, foram utilizados vasos dispostos em delineamento completamente casualizado preenchidos com solo Podzólico Vermelho-Escuro álico. Os tratamentos constaram de compactação do solo que resultou em resistências de 1,0; 2,0; 3,5 e 5,5 MPa. A combinação de umidade gravimétrica e densidade do solo foi estabelecida para minimizar os possíveis efeitos de falta de água e oxigênio. Com o aumento da resistência do solo diminuíram o comprimento, a superfície e a matéria seca das raízes, aumentando, porém, seu raio. A menor exploração do solo pelas raízes causou o menor acúmulo de matéria seca na parte aérea e, especialmente, nas raízes. Os efeitos foram percebidos logo no início do desenvolvimento da planta. A resistência do solo, isoladamente, caracterizou-se como um fator de diminuição do crescimento radicular.

Expressão dos genes nod de Rhizobium tropici, R. etli e R. leguminosarum bv. phaseoli e estabelecimento da nodulação do feijoeiro na presença de exsudatos de sementes de Mimosa flocculosa e Leucaena leucocephala

Na etapa inicial da troca de sinais moleculares entre macro e microssimbiontes, a interação do feijoeiro e estirpes de Rhizobium tropici, R. etli e R. leguminosarum bv. phaseoli foi avaliada pela expressão dos genes nod de estirpes bacterianas, contendo a fusão nodA::gusA. Esta avaliação foi efetuada por meio da atividade da enzima ß-glucuronidase, utilizando, como indutores, exsudatos liberados pelas sementes de Mimosa flocculosa e Leucaena leucocephala. Além disso, avaliou-se o efeito da adição desses exsudatos no estabelecimento da nodulação do feijoeiro, cv. Carioca. Nos testes "in vitro", a mistura de exsudatos de sementes de feijoeiro e M. flocculosa promoveu aumentos sinergísticos significativos na expressão dos genes nod, tanto das estirpes de R. tropici (CIAT 899/pGUS 32 e F 98.5/pGUS 32) quanto de R. etli (CFN 42/pGUS 32). Em condições controladas, a adição dos exsudatos, tanto de M. flocculosa quanto de L. leucocephala, proporcionou aumento significativo na nodulação inicial do feijoeiro, quando foi inoculada a estirpe CFN 42 (R. etli). A nodulação do feijoeiro cultivado em vasos com solo não foi inibida pelo suprimento de N-mineral, quando se inoculou a estirpe CIAT 899 (R. tropici) e foram fornecidos exsudatos de sementes de M. flocculosa.

Resistência à penetração e permeabilidade de latossolo vermelho distrófico típico sob sistemas de manejo na região dos cerrados

As conseqüências diretas do manejo inadequado do solo são a erosão, a redução da produtividade e a perda da sustentabilidade. Objetivou-se com este estudo avaliar a resistência à penetração (RP) e a permeabilidade do solo à água (PER) sob sistemas de manejo em uso na região dos cerrados. O estudo foi realizado na Embrapa Milho e Sorgo, em Sete Lagoas (MG), em Latossolo Vermelho distrófico típico textura muito argilosa fase cerrado. O cerrado nativo propiciou menor densidade do solo, maior macroporosidade, maior volume total de poros, conseqüentemente, menor resistência à penetração (0,84 a 2,09 MPa) e maior permeabilidade (95 mm h-1). Foram verificados maiores valores de resistência à penetração vertical para o sistema com preparo convencional com arado de discos e cultivo em rotação com milho e feijão, na profundidade de 15-30 cm no solo, sendo o valor 3,04 MPa classificado como alto, podendo ser um indicativo de restrição ao desenvolvimento radicular e compactação do solo. De modo geral, ao longo do perfil do solo, os maiores valores de RP foram observados para o plantio direto. Não houve diferenças significativas na PER entre os sistemas de manejo, estando os valores na faixa de 6 a 14 mm h-1, sendo a mesma classificada como lenta; estes valores foram bem inferiores aos do sistema em equilíbrio. Os atributos físicos utilizados neste estudo, como indicadores da qualidade do solo, apresentaram boa performance na distinção dos efeitos proporcionados pelos sistemas de manejo em relação ao sistema em equilíbrio, contribuindo para o monitoramento do manejo sustentável de solos da região dos cerrados.