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-resistancedetermining 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.

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-resistancedetermining 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.

Background Molecular studies of Bacillus diversity in various environments have been reported. However, there have been few investigations concerning Bacillus in steel plant environments. In this study, genotypic and phenotypic diversity and phylogenetic relationships among 40 bacterial isolates recovered from steel plant waste were investigated using classical and molecular methods. Results 16S rDNA partial sequencing assigned all the isolates to the Bacillus genus, with close genetic relatedness to the Bacillus subtilis and Bacillus cereus groups, and to the species Bacillus sphaericus. tDNA-intergenic spacer length polymorphisms and the 16S–23S intergenic transcribed spacer region failed to identify the isolates at the species level. Genomic diversity was investigated by molecular typing with rep (repetitive sequence) based PCR using the primer sets ERIC2 (enterobacterial repetitive intergenic consensus), (GTG)5, and BOXAIR. Genotypic fingerprinting of the isolates reflected high...

Aim: To study genetic diversity of Chromobacterium haemolyticum isolates recovered from a natural tropical lake. Methods and Results: A set of 31 isolates were recovered from a bacterial freshwater community by conventional plating methods and subjected to genetic and phenotypic characterization. The 16S ribosomal RNA (rRNA) gene phylogeny revealed that the isolates were related most closely with C. haemolyticum. In addition to the molecular data, our isolates exhibited strong b-haemolytic activity, were nonviolacein producers and utilized i-inositol, d-mannitol and d-sorbitol in contrast with the other known chromobacteria. Evaluation of the genetic diversity in the 16S rRNA gene, tRNA intergenic spacers (tDNA) and 16S-23S internal transcribed spacers (ITS) unveiled different levels of genetic heterogeneity in the population, which were also observed with repetitive extragenic palindromic (rep)-PCR genomic fingerprinting using the BOX-AR1 primer. tDNA-and ITS-PCR analyses were partially congruent with the 16S rRNA gene phylogeny. The isolates exhibited high resistance to b-lactamic antibiotics. Conclusion: The population genetic heterogeneity was revealed by 16S rRNA gene sequence, ITS and BOX-PCR analysis. Significance and Impact of the Study: This study provides for the first time an insight into the genetic diversity of phylogenetically close isolates to C. haemolyticum species.

The physical and chemical variables of the water, and the composition and structure of the zooplankton communities, in Três Marias and Furnas Reservoirs in Minas Gerais, Brazil, were compared to characterize these environments in relation to their trophic state. Higher values of electrical conductivity and chlorophyll-a, total solids, suspended organic matter and total nitrogen concentrations were recorded in Três Marias Reservoir. Higher water transparency and nitrite and nitrate concentrations were observed in Furnas (P < 0.000). Higher zooplankton densities were always obtained in Três Marias Reservoir and, during the rainy period (P < 0.000), with mean values in the dry and rainy periods of 23 721 and 90 872 org m )3 , respectively, in Três Marias Reservoir and 9022 and 40 434 org m )3 , respectively, in Furnas Reservoir. Copepoda was the dominant group in both reservoirs, mainly the younger stages (nauplii and copepodids). Based on the absolute and relative values, the contribution of rotifers was higher in Três Marias Reservoir than in Furnas Reservoir. Although the Trophic State Index, based on water transparency and chlorophyll-a and total phosphorus concentrations, indicated an oligotrophic state for both reservoirs, the higher densities of the zooplankton community in Três Marias Reservoir, as well as the predominance of cyclopoids and smaller-sized species such as bosminids, characterized this environment as mesotrophic. Larger-sized species such as calanoids, daphniids and sidids in Furnas Reservoir characterized the better water quality of this reservoir.

Lactic acid bacteria were isolated and identified in the faeces of Chinese Crested and Yorkshire terrier pups and their probiotic features were investigated in vitro. Thirty seven isolates were identified as Lactobacillus or Enterococcus. Out of these isolates, 31 were lactic acid bacteria (LAB) and belonged to the species Lactobacillus reuteri (16/37; 43.3%), Lactobacillus animalis (7/37; 18.9%), Lactobacillus acidophilus (3/37; 8.1%), Lactobacillus sanfranciscensis (2/37; 5.4%), Lactobacillus murinus (2/37; 5.4%), and Lactobacillus paraplantarum (1/37; 2.7%), while six other LAB isolates were Enterococcus spp. (6/37; 16.2%). Strains were tested for resistance to gastric acidity (pH 2.5 for 3 h) and bile salts (0.3% ox gall), cell surface hydrophobicity by microbial adhesion to solvents, antagonism against pathogenic bacteria (Staphylococcus aureus, Enterococcus faecalis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria...

Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.

Lactic acid bacteria strains are commonly used for animal and human consumption due to their probiotic properties. One of the major genera used is Lactobacillus, a highly diverse genus comprised of several closely related species. The selection of new strains for probiotic use, especially strains of Lactobacillus, is the focus of several research groups. Accurate identification to species level is fundamental for research on new strains, as well as for safety assessment and quality assurance. The 16S-23S internal transcribed spacer (ITS-1) is a deeply homologous region among prokaryotes that is commonly used for identification to the species level because it is able to acquire and accumulate mutations without compromising general bacterial metabolism. In the present study, 16S-23S ITS regions of 45 Lactobacillus species (48 strains) were amplified and subjected to independent enzymatic digestions, using 12 restriction enzymes that recognise six-base sequences. Twenty-nine species showed unique restriction patterns, and could therefore be precisely identified solely by this assay (64%). This approach proved to be reproducible, allowing us to establish simplified restriction patterns for each evaluated species. The restriction patterns of each species were similar among homologous strains, and to a large extent reflected phylogenetic relationships based on 16S rRNA sequences, demonstrating the promising nature of this region for evolutionary studies.

Agradeço ao professor Dr.Álvaro Cantini Nunes, o acolhimento, a orientação e por ter propiciado a oportunidade de renovação da minha experiência. Também pelo aprendizado em Biologia Molecular, contribuindo na minha formação profissional. À Dra. Maria Rosa Quaresma Bomfim, eu agradeço a co-orientação e disponibilidade em todas as etapas desta pós-graduação, ensinando, auxiliando e incentivando o desenvolvimento dos trabalhos, além da ótima convivência. Meu agradecimento a toda equipe do Laboratório de Genética Molecular de Protozoários Parasitas, que direta ou indiretamente auxiliaram o desenvolvimento desse trabalho ou que contribuíram para um convívio harmônico e produtivo durante as atividades. Em especial quero agradecer aos colegas Sávio Cicco Sandes,

Cyclopes didactylus, commonly called silky anteater, is the smallest and least studied of the anteaters. It is an arboreal species occurring in rainforests, ranging from southern Mexico to Central and South America, with an apparently disjoint distribution between Amazon and Atlantic rainforests in Brazil. Although seven subspecies are recognized, little is known about its geographical variation. Thus, to evaluate the population dynamics and evolutionary history of the South American silky anteater, we analyzed 1542 bp sequences of the mitochondrial control region (CR), COI and Cyt-b genes of 32 individuals. Haplotype network, AMOVA and molecular dating analyses were performed and identified seven geographic clusters. The split of lineages separating Cyclopedidae (Cyclopes) and Myrmecophagidae (Myrmecophaga and Tamandua genera) was estimated around 41 million years ago (mya), and the intraspecific lineage diversification of C. didactylus began in the Miocene around 13.5 mya, likely ...

Objective To evaluate if body surface temperature close to the central venous catheter insertion area is different when patients develop catheter-related bloodstream infections.Methods Observational cross-sectional study. Using a non-contact infrared thermometer, 3 consecutive measurements of body surface temperature were collected from 39 patients with central venous catheter on the following sites: nearby the catheter insertion area or totally implantable catheter reservoir, the equivalent contralateral region (without catheter), and forehead of the same subject.Results A total of 323 observations were collected. Respectively, both in male and female patients, disregarding the occurrence of infection, the mean temperature on the catheter area minus that on the contralateral region (mean ± standard deviation: -0.3±0.6°C versus-0.2±0.5ºC; p=0.36), and the mean temperature on the catheter area minus that on the forehead (mean ± standard deviation: -0.2±0.5°C versus-0.1±0.5ºC; p=0.3) ...

The armadillo genus Dasypus is the most species-rich and widely distributed genus of the order Cingulata and it has a dynamic taxonomic history. Recent morphology-based studies have proposed new taxonomic arrangements, but these were not yet assessed with molecular data. The two comprehensive phylogenetic hypotheses available for the genus are conflicting and were each based on a subset of taxa, hampering a proper evaluation of species boundaries. Using a multilocus molecular dataset, based on the broadest geographic sampling of Dasypus to date, we inferred the phylogenetic relationships of all species of the genus, including the recently reinstated D. beniensis and D. pastasae. We tested recent taxonomic hypotheses using several species-delimitation approaches. Our phylogeny recovered three main lineages of long-nosed armadillos that we treat as subgenera (Hyperoambon, Muletia and Dasypus) and identified the majority of its diversification as having occurred during the Pliocene. Molecular species delimitation supported morphological evidence in assigning D. hybridus as a subspecies of D. septemcinctus and confirming the split of the D. kappleri complex into three species. Our results strongly support the recognition of Guiana Shield populations formerly assigned to D. novemcinctus as a distinct species. The phylogenetic positions of D. mazzai and D. sabanicola remain uncertain. Further investigation using faster-evolving genes and additional samples may help to clarify the relationships of these young species.

Brazilian kefir is a homemade fermented beverage that is obtained by incubating milk or a brown sugar
solution with kefir grains that contribute their different microbiological compositions. It is highly
important to isolate and characterize microorganisms from Brazilian kefir grains to obtain starter cultures
for the industrial production of a standardized commercial kefir. Thus, the present study aimed to
isolate lactic acid bacteria from eight kefir grains that were propagated in milk or sugar solutions from
five different locations in Brazil and to select Lactobacillus isolates based on desirable in vitro probiotic
properties. One hundred eight isolates from both substrates were identified by amplified ribosomal DNA
restriction analysis and/or 16S rRNA gene sequencing and were determined to belong to the following 11
species from the genera: Lactococcus, Leuconostoc, Lactobacillus (L.), and Oenococcus. Leuconostoc mesenteroides,
Lactobacillus kefiri, and Lactobacillus kefiranofaciens were isolated only from milk grains,
whereas Lactobacillus perolens, Lactobacillus parafarraginis, Lactobacillus diolivorans, and Oenococcus oeni
were isolated exclusively from sugar water grains. When the microbial compositions of four kefir grains
were evaluated with culture-independent analyses, L. kefiranofaciens was observed to predominant in
milk grains, whereas Lactobacillus hilgardii was most abundant in sugar water kefir. Unfortunately,
L. hilgardii was not isolated from any grain, although this bacteria was detected with a cultureindependent
methodology. Fifty-two isolated Lactobacilli were tested for gastric juice and bile salt
tolerance, antagonism against pathogens, antimicrobial resistance, and surface hydrophobicity. Three
Lactobacillus strains (L. kefiranofaciens 8U, L. diolivorans 1Z, and Lactobacillus casei 17U) could be classified
as potential probiotics. In conclusion, several lactic acid bacteria that could be used in combination with
yeasts as starter cultures for both milk kefir and sugar water kefir were characterized, and the functional
properties of several of the lactobacilli isolated from the kefir grains were suggestive of their possible use
as probiotics in both kefir and other dairy products.

Two strains of Lactobacillus, previously isolated from bovine faeces and tested in vitro for properties desired in probiotics, were evaluated for their in vivo effectiveness in protecting against experimental salmonellosis. L. salivarius L38 and L. acidophilus L36 previously demonstrated the ability to successfully colonize the gastrointestinal tract of germ-free mice and stimulate the immune system associated with the intestinal mucosa. L38- or L36-feeding showed no detrimental effect on the general health indicators and did not induce changes in normal architecture of liver and small intestine, indicating that the use of these strains is apparently safe. In control animals fed L38 strain, several cytokines had augmented mRNA levels that can be associated with a homeostatic state of intestinal mucosa, while L36 had less diverse regulation. IgA production and secretion in the intestinal lumen induced by infection was abrogated by pretreating with both lactobacilli. In addition, liver and small intestine histological scores and, translocation of Salmonella cells to liver and spleen, indicated that these strains did not confer protection against the infection. So, the IL-12:IL-18àIFN-g axis, essential for an effective immune response against Salmonella, was not favored with L38 or L36 strains. However, increased expression of IL-10 in different portions of the gastrointestinal tract of L38-fed animals is indicative of anti-inflammatory effect to be explored furthermore.