Antimicrobial resistance and genetic relationships of enterococci from siblings and non-siblings Heliconius erato phyllis caterpillars.

BACKGROUND: Studies evaluating bacteria in insects can provide information about host-microorganism-environment interactions. The gut microbial community has a profound effect on different physiological functions of insects. Enterococcus spp. are part of the gut community in humans and other animals, as well as in insects. The presence and antimicrobial resistance profile of enterococci are well studied in different animals; however, data for Heliconius erato phyllis (Lepidoptera: Nymphalidae) do not yet exist. Therefore, the aims of this study were to evaluate the distribution of enterococcal species, their antimicrobial resistance profile and virulence genes, and the genetic relationships between enterococci isolated from fecal samples from sibling and non-sibling H. erato phyllis caterpillars collected from different sites in South Brazil. METHODS: Three H. erato phyllis females were captured (two from a forest fragment and one from an urban area), and kept individually in open-air insectaries. Eggs were collected and caterpillars (siblings and non-siblings) were fed daily with Passiflora suberosa leaves. Fecal samples (n = 12) were collected from fifth-instar caterpillars, inoculated in selective medium, and 15 bacterial colonies were randomly selected from each sample. Enterococci were identified by PCR and MALDI-TOF, analyzed by disk diffusion antimicrobial susceptibility tests, and screened for resistance and virulence genes by PCR. The genetic relationships between the strains were determined using pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 178 enterococci strains were identified: E. casseliflavus (74.15%; n = 132), E. mundtii (21.34%; n = 38), E. faecalis (1.12%; n = 2) and Enterococcus sp. (3.37%; n = 6). High rates of resistance to rifampicin (56%) and erythromycin (31%) were observed; 120 (67.41%) of the isolates showed resistance to at least one antibiotic and six (3.37%) were multidrug-resistant.None of the erythromycin-resistant strains was positive for the erm(B) and msrC genes. The virulence genes esp, ace, and gelE were observed in 35%, 7%, and 1% of the strains, respectively. PFGE separated the enterococci into 22 patterns, four being composed of strains from sibling caterpillars. CONCLUSION: Enterococcus casseliflavus was the dominant species in fecal samples of fifth-instar caterpillars. Resistant enterococci strains may be related to environmental pollution or the resistome. The PFGE analysis showed genetic relationships between some strains, suggesting that the enterococci isolated from fecal samples of the sibling caterpillars might have come from common sources, e.g., via diet (herbivory) and/or vertical transmission (through the egg surface). Further studies will be conducted to better understand the role of Enterococcus in the microbial community of the gastrointestinal tract of these insects, and the mechanisms involved in acquisition and maintenance of enterococci.

Perfil de suscetibilidade aos antimicrobianos e diversidade das espécies de enterococos isolados de leite cru de búfalas no Sul do Brasil

O leite de búfala é utilizado principalmente para a produção de queijo mussarela. A procura por produtos derivados de leite de búfala tem aumentado no mercado, consideravelmente, nas últimas décadas. Este trabalho teve como objetivo avaliar a diversidade e o perfil de suscetibilidade a antimicrobianos de enterococos isolados de amostras de leite cru de búfalas no Sul do Brasil. Amostras de leite cru refrigerado contendo leite de diversas búfalas cedidas pela Cooperativa Sulriograndense de Bubalinocultores Ind. Com. Ltda, durante junho a agosto de 2012, foram empregadas para isolar as bactérias do gênero Enterococcus. Os microrganismos foram identificados em nível de espécie através de suas características fenotípicas, e seus perfis de suscetibilidade a antimicrobianos foram analisados através do método de disco-difusão em ágar. Oitenta colônias foram selecionadas aleatoriamente das amostras de leite cru e as espécies mais frequentes nestas foram Enterococcus faecalis (63,75%), seguida de Enterococcus faecium (28,75%), Enterococcus durans (2,5%), Enterococcus spp. (3,75%) e Lactococcus sp. (1,25%). Os resultados dos testes de sensibilidade revelaram que a maioria dos enterococos era sensível aos antimicrobianos testados, entretanto 13,9% apresentaram perfil de resistência a nitrofurantoína, 12,7% a tetraciclina, 1,3% a eritromicina, 1,3% a norfloxacina, 1,3% a cloranfenicol e 1,

Viral load and epidemiological profile of patients infected by pandemic influenza a (H1N1) 2009 and seasonal influenza a virus in Southern Brazil.

Correlation between virologic profile and clinical features of patients infected by influenza virus provides important information for epidemiological control and clinical management of future disease outbreaks. Samples from patients in Southern Brazil, from June to December 2009, were examined and the viral load was correlated with epidemiological data. All samples were analyzed by qRT-PCR for detection of the 2009-pandemic Influenza A (H1N1). Relative viral loads were assessed based on the 2(-ΔCT) method and epidemiological data were obtained for each patient, following ethical policies. A total of 933 samples were positive for pH1N1 (2009) influenza; 172 were positive for seasonal influenza A; 13 were undetermined; 1992 samples were negative for influenza A. Combined molecular and epidemiological data were available for 38 seasonal and 198 pandemic samples. The median viral load was higher in pandemic than in seasonal influenza samples; in patients infected with pH1N1 (2009), viral load associated positively with chills, myalgia and rhinorrhea, and negatively with dyspnea, but no association was observed with other symptoms, nor with clinical conditions such as pregnancy, smoking, immunodepression and co-morbidities. Regarding patients infected with seasonal influenza, viral loads did not show statistically significant association with any of the symptoms. This is the first study in Brazil that examines epidemiological and molecular data from the 2009 influenza pandemic. The results may serve as a basis for developing strategies to control human-to-human infection and viral dissemination, and for implementing effective measures and public health policies against future novel disease outbreaks.

Typing of Staphylococcus epidermidis clinical strains by a selected panel of Brazilian killer yeasts.

Discrimination of multi-resistant microorganisms in small clinical microbiology laboratories is frequently based on the biologic profile (biotype) of phenotypic markers, such as antimicrobial susceptibility patterns (antibiograms) and serologic or enzymatic typing, but few use indicative microorganisms. The purpose of this study was to evaluate the power of a panel of selected killer yeasts for differentiating and discriminating clinical isolates of Staphylococcus epidermidis from two care hospitals and clinical microbiology laboratories from the South of Brazil. The short panel of eleven killer yeasts was capable of discriminating 100% of the sensitive strains of S. epidermidis using quantitative data matrix (QDM) and differentiating them from strains of coagulase-positive Staphylococcus. Therefore, this phenotypic methodology proved to be valid as a discriminatory tool when applied to these clinical bacteria strains, besides being simple and feasible for routine use even in microbiological laboratories with minimal resources.

Phenotypic and genotypic heterogeneity of Enterococcus species isolated from food in Southern Brazil.

Enterococcus is an important group of lactic acid bacteria (LAB), which inhabits the gastrointestinal tract of humans and animals. These microorganisms can also be found in large groups of foods where they can play a beneficial role during food maturation processes or, conversely, can be used as a food contamination indicator. These microorganisms have an additional importance in various aspects of clinical microbiology. The aim of this study was to investigate and determine the phenotypic and genetic diversity in 55 enterococci isolated from different food sources. Phenotypic characteristics based upon substrate hydrolysis differences were used to identify different Enterococcus species. Analysis of the resultant data divided these species into eight Enterococcus phenotype groups. E. faecalis was the food isolate species with the greatest phenotypic variability. Fifty-five previously isolated Enterococcus strains were re-confirmed as belonging to this genus by PCR techniques. Randomly amplified polymorphic DNA (RAPD-PCR) was used to study the genetic variability using M13 primers and the resultant DNA fragments produced a database of different fingerprints. After statistical analyses of the RAPD-PCR profiles, 42 patterns were obtained and 6 different Enterococcus RAPD clusters (ERC) were identified. Genetic diversity was highest in ERC I, which grouped together approximately 40% of the E. faecium and E. faecalis isolates obtained from dairy products. Samples isolated from meat and vegetables offered the greatest genotype variability. Results of the present study suggest the presence of both phenotypic and genotypic variability within enterococci strains isolated from diverse sources of food common to Southern Brazil.

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci.

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 microg) and cefoxitin (30 microg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.