Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

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.

Molecular epidemiology of extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae over a 10 year period in Calgary, Canada

A study was designed to investigate the molecular epidemiology of extended-spectrum -lactamase (ESBL)-producing Klebsiella pneumoniae isolated in a centralized region over a 10 year period (200009). Molecular characterization was done using isoelectric focusing, PCR and sequencing for bla(CTX-M), bla(TEM) and bla(SHV) genes and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with PFGE using XbaI and multilocus sequencing typing. A total of 89 patients with incident infections were identified; the majority presented with hospital-onset urinary tract infections. The absolute number of ESBL-producing isolates remained very low until 2003, increased slightly in 2004, remained stable until 2008 and then in 2009 there was an abrupt increase in the numbers of ESBL producers identified. The majority of K. pneumoniae produced CTX-M-14 and -15, and have replaced SHV-12-producing isolates since 2005. We identified four different major sequence types (STs) among 32 of isolates (i.e. ST17, ST20, and the new ST573 and ST575) and provided insight into their clinical and molecular characteristics. The ST isolates were more likely to produce community-onset infections, were associated with bla(CTX-M) and emerged during the latter part of the study period. ST17 produced CTX-M-15 and SHV-12, and was more likely to be positive for qnrB; ST20 produced CTX-M-14 and was positive for qnrS. The multiresistant ST575 that produced CTX-M-15 appeared in 2009. Our study highlights the importance of molecular epidemiology in providing insight into the emergence, characteristics and distribution of STs among ESBL-producing K. pneumoniae.

The characteristics of VIM-1-producing Klebsiella pneumoniae from South Africa

A study was designed to characterize a carbapenem-resistant Klebsiella pneumoniae (KPSA01) isolated from a patient in Gauteng, South Africa without recent travel outside South Africa. Molecular characterization was done using isoelectric focusing, polymerase chain reaction and sequencing for bla(VIM), bla(IMP), bla(NDM), bla(CTX-Ms), bla(OXAs), bla(TEMs), and bla(SHV), plasmid-mediated quinolone resistance determinants, multilocus sequencing typing, plasmid replicon typing, and addiction factors. KPSA01 produced VIM-1 and belonged to the newly described sequence type ST569. The plasmid that harboured bla(VIM) typed within the narrow host range IncF replicon group, contained the aadA1 gene cassette, and tested positive for the vagCD and ccdAB addiction systems. This is the first report of VIM-1-producing K. pneumoniae outside Europe. It is important that surveillance studies be undertaken in Africa to determine if VIM-1-producing K. pneumoniae are present in significant numbers.

The clinical impact of chromosomal rearrangements with breakpoints upstream of the SOX9 gene: two novel de novo balanced translocations associated with acampomelic campomelic dysplasia

Abstract Background The association of balanced rearrangements with breakpoints near SOX9 [SRY (sex determining region Y)-box 9] with skeletal abnormalities has been ascribed to the presumptive altering of SOX9 expression by the direct disruption of regulatory elements, their separation from SOX9 or the effect of juxtaposed sequences. Case presentation We report on two sporadic apparently balanced translocations, t(7;17)(p13;q24) and t(17;20)(q24.3;q11.2), whose carriers have skeletal abnormalities that led to the diagnosis of acampomelic campomelic dysplasia (ACD; MIM 114290). No pathogenic chromosomal imbalances were detected by a-CGH. The chromosome 17 breakpoints were mapped, respectively, 917–855 kb and 601–585 kb upstream of the SOX9 gene. A distal cluster of balanced rearrangements breakpoints on chromosome 17 associated with SOX9-related skeletal disorders has been mapped to a segment 932–789 kb upstream of SOX9. In this cluster, the breakpoint of the herein described t(17;20) is the most telomeric to SOX9, thus allowing the redefining of the telomeric boundary of the distal breakpoint cluster region related to skeletal disorders to 601–585 kb upstream of SOX9. Although both patients have skeletal abnormalities, the t(7;17) carrier presents with relatively mild clinical features, whereas the t(17;20) was detected in a boy with severe broncheomalacia, depending on mechanical ventilation. Balanced and unbalanced rearrangements associated with disorders of sex determination led to the mapping of a regulatory region of SOX9 function on testicular differentiation to a 517–595 kb interval upstream of SOX9, in addition to TESCO (Testis-specific enhancer of SOX9 core). As the carrier of t(17;20) has an XY sex-chromosome constitution and normal male development for his age, the segment of chromosome 17 distal to the translocation breakpoint should contain the regulatory elements for normal testis development. Conclusions These two novel translocations illustrate the clinical variability in carriers of balanced translocations with breakpoints near SOX9. The translocation t(17;20) breakpoint provides further evidence for an additional testis-specific SOX9 enhancer 517 to 595 kb upstream of the SOX9 gene.

BF Integrase Genes of HIV-1 Circulating in Sao Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naive patients from the Sao Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naive and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population.

Molecular evolution of a malaria resistance gene (DARC) in primates

Genes involved in host-pathogen interactions are often strongly affected by positive natural selection. The Duffy antigen, coded by the Duffy antigen receptor for chemokines (DARC) gene, serves as a receptor for Plasmodium vivax in humans and for Plasmodium knowlesi in some nonhuman primates. In the majority of sub-Saharan Africans, a nucleic acid variant in GATA-1 of the gene promoter is responsible for the nonexpression of the Duffy antigen on red blood cells and consequently resistance to invasion by P. vivax. The Duffy antigen also acts as a receptor for chemokines and is expressed in red blood cells and many other tissues of the body. Because of this dual role, we sequenced a 3,000-bp region encompassing the entire DARC gene as well as part of its 5' and 3' flanking regions in a phylogenetic sample of primates and used statistical methods to evaluate the nature of selection pressures acting on the gene during its evolution. We analyzed both coding and regulatory regions of the DARC gene. The regulatory analysis showed accelerated rates of substitution at several sites near known motifs. Our tests of positive selection in the coding region using maximum likelihood by branch sites and maximum likelihood by codon sites did not yield statistically significant evidence for the action of positive selection. However, the maximum likelihood test in which the gene was subdivided into different structural regions showed that the known binding region for P. vivax/P. knowlesi is under very different selective pressures than the remainder of the gene. In fact, most of the gene appears to be under strong purifying selection, but this is not evident in the binding region. We suggest that the binding region is under the influence of two opposing selective pressures, positive selection possibly exerted by the parasite and purifying selection exerted by chemokines.

Haplotype frequencies based on eight polymorphic sites at the 3' untranslated region of the HLA-G gene in individuals from two different geographical regions of Brazil

The Brazilian population represents an admixture of native Amerindians, Portuguese settlers and Africans who were brought as slaves during the colonization period that began in the 16th century and was followed by waves of immigrations of Europeans and Asians in the 20th century. The contribution of these different ethnic groups to the constitution of Brazilian populations from different geographic regions is variable and, in addition to environmental factors, might act by determining different allele profiles among Brazilian populations from different regions. We studied polymorphic sites at the 3' untranslated region of the HLA-G gene in individuals from a Northeastern Brazilian region and compared them to our previously published data about a Southeastern Brazilian region, located at a distance of 2589 km. Our results showed that most polymorphic sites present a similar distribution in both populations, except for the lower frequency of the +3003C allele in the Northeastern population compared to the Southeastern population. Although differences in genotypic distribution were only significant for the +3003 locus (P = 0.0201), the diversity of haplotypes was distinct for each population. These results are important for casecontrol studies on the association of human leucocyte antigen-G polymorphism with disease and also in terms of the genetic structure of two distinct Brazilian populations.

Analysis of Hepatitis C Virus Intrahost Diversity across the Coding Region by Ultradeep Pyrosequencing

Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naive patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.

Alterations in vasoconstrictor responses to the endothelium-derived contracting factor uridine adenosine tetraphosphate are region specific in DOCA-salt hypertensive rats

Uridine adenosine tetraphosphate (Up(4)A) has been recently identified as a novel and potent endothelium-derived contracting factor and contains both purine and pyrimidine moieties, which activate purinergic P2X and P2Y receptors. The present study was designed to compare contractile responses to Up(4)A and other nucleotides such as ATP (P2X/P2Y agonist), UTP (P2Y(2)/P2Y(4) agonist), UDP (P2Y(6) agonist), and alpha,beta-methylene ATP (P2X(1) agonist) in different vascular regions [thoracic aorta, basilar, small mesenteric, and femoral arteries] from deoxycorticosterone acetate-salt (DOCA-salt) and control rats. In DOCA-salt rats [vs. control uninephrectomized (Uni) rats]: (1) in thoracic aorta, Up(4)A-, ATP-, and UP-induced contractions were unchanged; (2) in basilar artery, Up(4)A-, ATP-, UTP- and UDP-induced contractions were increased, and expression for P2X(1), but not P2Y(2) or P2Y(6) was decreased; (3) in small mesenteric artery, Up(4)A-induced contraction was decreased and UDP-induced contraction was increased; expression of P2Y(2) and P2X(1) was decreased whereas P2Y(6) expression was increased; (4) in femoral artery, Up(4)A-. UTP-, and UDP-induced contractions were increased, but expression of P2Y(2), P2Y(6) and P2X(1) was unchanged. The alpha,beta-methylene ATP-induced contraction was bell-shaped and the maximal contraction was reached at a lower concentration in basilar and mesenteric arteries from Uni rats, compared to arteries from DOCA-salt rats. These results suggest that Up(4)A-induced contraction is heterogenously affected among various vascular beds in arterial hypertension. P2Y receptor activation may contribute to enhancement of Up(4)A-induced contraction in basilar and femoral arteries. These changes in vascular reactivity to Up(4)A may be adaptive to the vascular alterations produced by hypertension. (C) 2011 Elsevier Ltd. All rights reserved.

Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus × Bos indicus)

Abstract Background In tropical countries, losses caused by bovine tick Rhipicephalus (Boophilus) microplus infestation have a tremendous economic impact on cattle production systems. Genetic variation between Bos taurus and Bos indicus to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (Bos indicus) × Holstein (Bos taurus) cross. Results Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23. Conclusions The experimental F2 population derived from Gyr × Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.

Clinical, epidemiological and molecular features of the HIV-1 subtype C and recombinant forms that are circulating in the city of São Paulo, Brazil

Abstract Background The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite, several studies involving molecular epidemiology, lack of data regarding a large cohort study has not been published from this city. Objectives This study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistance mutations, according to subtype, with emphasis on subtype C and BC recombinants in the city of São Paulo, Brazil. Study design RNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which 211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences were used in phylogenetic analyses for subtyping and identification of drug resistance mutations. The envelope gene of subtype C and BC samples was also sequenced. Results From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%) were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). The subtype C and BC recombinants were mainly identified in drug-naïve patients (72.7%) and the heterosexual risk exposure category (86.3%), whereas for subtype B, these values were 69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend of subtype C and BC recombinants was observed (p < 0.01). Conclusion The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city of São Paulo, principally among the heterosexual population. These findings may have an impact on preventive measures and vaccine development in Brazil.