The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains

This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5% of the samples were positive for tetA, and also 37.5% were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.

Importance of TNF-alpha in the course of acute infection with Trypanosoma cruzi: influence of its inhibition by pentoxifylline treatment

Infection of C3H/He mice with the Peruvian strain of Trypanosoma cruzi (Biodeme type I, Z2b), a macrophagotropic strain, determined severe parasitism of macrophages, necrosis of the spleen, and high host mortality. In the present study, pentoxifylline (PTX), an inhibitor of TNF-alpha was investigated on its action upon splenic necrosis, parasitemia and host survival. Immunohistochemical data suggested the importance of this cytokine in parasite destruction and decreasing of parasitemia, although paradoxically contributing to the high mortality of infected mice. Necrotic lesions involving several organs, specially the heart, in acute Chagas disease, are important aggravating factors, increasing cardiac morbidity. Advantage of inhibiting TNF-alpha action was herein investigated. Infected mice were divided into two groups: untreated (n = 24), and PTX treated mice (n = 25). PTX was administered in two daily doses of 30 mg/kg/bw, by intraperitoneal route. Normal controls either treated with PTX or saline were also included. Histopathology of the spleen and in situ immunolabeling of TNF-alpha, using anti-TNF-alpha monoclonal antibody, were performed. Necrotic areas were evaluated by morphometry. Mice treated with PTX showed a significant decrease of necrotic areas and diminution of TNF-alpha expression in spleen tissue, suggesting that PTX treatment could control TNF-alpha effects, and thus be used as an adjuvant in the treatment of acute Chagas' disease.

Polymorphisms of toll-like receptor 2 and 4 genes in Chagas disease

The aim of this study was to test the possible implication of toll-like receptor 2 (TLR2) and TLR4 gene polymorphisms in determining the susceptibility to Chagas' disease. Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 475 individuals from Colombia, 143 seropositive with chagasic cardiomyopathy, 132 seropositive asymptomatic and 200 seronegative. The TLR2 arginine to glutamine substitution at residue 753(Arg753Gln) polymorphism was absent in the groups analyzed. The TLR4 Asp299Gly and Thr399Ile polymorphisms are in linkage disequilibrium and we observed a very low frequency of these polymorphisms in our study population (2.6% and 1.8% respectively). The overall TLR2 and TLR4 alleles and genotype distribution in seronegative and seropositive were not significantly different. We compared the frequencies between asymptomatic patients and those with chagasic cardiomyopathy and we did not observe any significant differences in the distribution of alleles or genotypes. In summary, this study corroborates the low frequency of TLR2 and TLR4 polymorphisms observed in other populations and suggest that these do not play an important role in Chagas' disease. The validation of these findings in independent cohorts is needed to firmly establish a role for TLR2 and TLR4 variants in Chagas' disease.

Transcription of meiotic-like-pathway genes in Giardia intestinalis

The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasite's differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process.

Characterization of Shigella spp. by antimicrobial resistance and PCR detection of ipa genes in an infantile population from Porto Velho (Western Amazon region), Brazil

The incidence of Shigella spp. was assessed in 877 infants from the public hospital in Rondônia (Western Amazon region, Brazil) where Shigella represents the fourth cause of diarrhea. Twenty-five isolates were identified: 18 were Shigella flexneri, three Shigella sonnei, three Shigella boydii and one Shigella dysenteriae. With the exception of S. dysenteriae, all Shigella spp. isolated from children with diarrhea acquired multiple antibiotic resistances. PCR detection of ipa virulence genes and invasion assays of bloody diarrhea and fever (colitis) were compared among 25 patients testing positive for Shigella. The ipaH and ipaBCD genes were detected in almost all isolates and, unsurprisingly, all Shigella isolates associated with colitis were able to invade HeLa cells. This work alerts for multiple antibiotic resistant Shigella in the region and characterizes presence of ipa virulence genes and invasion phenotypesin dysenteric shigellosis.

Mutations in rpoB and katG genes in Mycobacterium isolates from the Southeast of Mexico

The most frequent mutations associated with rifampin and isoniazid resistance in Mycobacterium are the substitutions at codons 531 and 315 in the rpoB and katG genes, respectively. Hence, the aim of this study was to characterize these mutations in Mycobacterium isolates from patients suspected to be infected with drug-resistant (DR) pulmonary tuberculosis (TB) in Veracruz, Mexico. Drug susceptibility testing of 25 clinical isolates revealed that five were susceptible while 20 (80%) were DR (15% of the annual prevalence for Veracruz). Of the DR isolates, 15 (75%) were resistant to rifampin, 17 (85%) to isoniazid and 15 (75%) were resistant to both drugs (MDR). Sequencing analysis performed in the isolates showed that 14 (93%) had mutations in the rpoB gene; seven of these (47%) exhibited a mutation at 531 (S[L). Ten (58%) of the 20 resistant isolates showed mutations in katG; nine (52%) of these 10 exhibited a mutation at 315 (S[T). In conclusion, the DR profile of the isolates suggests a significant number of different DR-TB strains with a low frequency of mutation at codons 531 and 315 in rpoB and katG, respectively. This result leads us to consider different regions of the same genes, as well as other genes for further analysis, which is important if a genetic-based diagnosis of DR-TB is to be developed for this region.

Preferential transcription of Paracoccidioides brasiliensis genes: host niche and time-dependent expression

Paracoccidioides brasiliensis causes infection through inhalation by the host of airborne propagules from the mycelium phase of the fungus. This fungus reaches the lungs, differentiates into the yeast form and is then disseminated to virtually all parts of the body. Here we review the identification of differentially-expressed genes in host-interaction conditions. These genes were identified by analyzing expressed sequence tags (ESTs) from P. brasiliensis cDNA libraries. The P. brasiliensis was recovered from infected mouse liver as well as from fungal yeast cells incubated in human blood and plasma, mimicking fungal dissemination to organs and tissues and sites of infection with inflammation, respectively. In addition, ESTs from a cDNA library of P. brasiliensis mycelium undergoing the transition to yeast were previously analyzed. Together, these studies reveal significant changes in the expression of a number of genes of potential importance in the host-fungus interaction. In addition, the unique and divergent representation of transcripts when the cDNA libraries are compared suggests differential gene expression in response to specific niches in the host. This analysis of gene expression patterns provides details about host-pathogen interactions and peculiarities of sites within the host.

Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates

Plasmodium vivax control is now being hampered by drug resistance. Orthologous Plasmodium falciparum genes linked to chloroquine or sulfadoxine-pyrimethamine chemoresistance have been identified in P. vivax parasites, but few studies have been performed. The goal of the present work is to characterise pvmdr1 and pvdhfr genes in parasite isolates from a Brazilian endemic area where no molecular investigation had been previously conducted. The pvmdr1 analysis revealed the existence of single (85.7%) and double (14.3%) mutant haplotypes, while the pvdhfr examination showed the presence of double (57.2%) and triple (42.8%) mutant haplotypes. The implications of these findings are discussed.

blaCTX-M-2 and blaCTX-M-28 extended-spectrum β-lactamase genes and class 1 integrons in clinical isolates of Klebsiella pneumoniae from Brazil

Twenty-eight Klebsiella pneumoniae clinical isolates that exhibited an extended-spectrum cephalosporin-resistance profile from a city in the Northeast of Brazil were analysed by PCR and DNA sequencing in order to determine the occurrence of blaCTX-M genes and class 1 integrons. We determined the occurrence of the blaCTX-M-2 gene in six K. pneumoniae isolates and describe the first detection of the blaCTX-M-28 gene in South America. Seven isolates carried class 1 integrons. Partial sequencing analysis of the 5'-3'CS variable region in the class 1 integrons of three isolates revealed the presence of aadA1, blaOXA-2 and dfr22 gene cassettes.

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is mediated by CD4+ Th1 cells that mainly secrete IFN-γ and TNF-α, important cytokines in the pathophysiology of the disease. Spontaneous remission is, in part, attributed to the down regulation of IFN-γ and TNF-α by TGF-β. In the current paper, we compared weight, histopathology and immunological parameters during the acute and recovery phases of EAE to establish the best biomarker for clinical remission. Female Lewis rats were immunised with myelin basic protein (MBP) emulsified with complete Freund's adjuvant. Animals were evaluated daily for clinical score and weight prior to euthanisation. All immunised animals developed the expected characteristics of EAE during the acute phase, including significant weight loss and high clinical scores. Disease remission was associated with a significant reduction in clinical scores, although immunised rats did not regain their initial weight values. Brain inflammatory infiltrates were higher during the acute phase. During the remission phase, anti-myelin antibody levels increased, whereas TNF-α and IFN-γ production by lymph node cells cultured with MBP or concanavalin A, respectively, decreased. The most significant difference observed between the acute and recovery phases was in the induction of TNF-α levels in MBP-stimulated cultures. Therefore, the in vitro production of this cytokine could be used as a biomarker for EAE remission.

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale

The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.

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.

An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates

An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95% concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).