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

Multidrug resistance in Enterococci isolated from wild pampas foxes (Lycalopex gymnocercus) and Geoffroy's Cats (Leopardus geoffroyi) in the Brazilian pampa biome

Enterococci are ubiquitous microorganisms present in various environments and within the gastrointestinal tracts of humans and other animals. Notably, fecal enterococci are suitable indicators for monitoring antimicrobial resistance dissemination. Resistant bacterial strains recovered from the fecal samples of wild animals can highlight important aspects of environmental disturbances. In this report, we investigated antimicrobial susceptibility as well as resistance and virulence genes in fecal enterococci isolated from wild Pampas foxes (Lycalopex gymnocercus) (n = 5) and Geoffroy's cats (Leopardus geoffroyi) (n = 4) in the Brazilian Pampa biome. Enterococci were isolated from eight out of nine fecal samples and Enterococcus faecalis was identified in both animals. However, E. faecium and E. durans were only detected in Pampas foxes, while E. hirae was only detected in Geoffroy's cats. Antimicrobial susceptibility analysis showed resistance to rifampicin (94%), erythromycin (72.6%), ciprofloxacin/norfloxacin (40%), streptomycin (38%), and tetracycline (26%). The high frequency of multidrug-resistant enterococci (66%) isolated in this study is a matter of concern since these are wild animals with no history of therapeutic antibiotic exposure. The tetM/tetL and msrC/ermB genes were detected in most tetracycline- and erythromycin-resistant enterococci, respectively. The gelE, ace, agg, esp, and clyA virulence genes were also detected in enterococci. In conclusion, our data suggest that habitat fragmentation and anthropogenic activities in the Pampa biome may contribute to high frequencies of multidrug-resistant enterococci in the gut communities of wild Pampas foxes and Geoffroy's cats. To the best of the authors' knowledge, this is the first report of antimicrobial-resistant enterococci in the Pampa biome

Determination of clonal relationship and virulence factors in enterococci isolated from immatures of Heliconius erato phyllis (Lepidoptera – Nymphalidae)

Enterococcus estão presentes no trato gastrointestinal (TGI) de animais vertebrados e invertebrados, os quais podem adquirir estas bactérias através de mecanismos de transferência horizontal e vertical de micro-organismos. Pesquisas recentes descreveram a presença de Enterococcus em lepidópteros, porém estudos com borboletas do gênero Heliconius são escassos. Este trabalho teve como objetivo, a partir de uma bacterioteca de 178 Enterococcus sp. previamente isolados de fezes de imaturos de Heliconius erato phyllis detectar a presença de genes de resistência e virulência por PCR; avaliar fenotipicamente os fatores de virulência e a produção de compostos com atividade antimicrobiana; e avaliar o perfil clonal das cepas pela técnica de PFGE. Dos 178 enterococos, 55 apresentavam resistência a eritromicina e 11 a norfloxacina e/ou ciprofloxacina, porém não foi detectado nenhum gene relacionado a estes fenótipos de resistência. Quanto aos fatores de virulência testados nos 178 enterococos, 63 isolados amplificaram para o gene esp, 12 para ace e 2 para gelE. Os genes agg e cylA não foram detectados. Na análise fenotípica dos fatores de virulência, os dois E. faecalis positivos para gelE também foram positivos para a degradação da gelatinase e 130 enterococos apresentaram capacidade de hidrolisar hemácias. Para investigar a produção de substância antagonista, foram selecionados 26 enterococos; destes, 15 apresentaram atividade contra a bactéria indicadora Listeria monocytogenes. Oitenta e seis isolados foram selecionados para avaliar a relação clonal por PFGE, e foi possível observar a partir do dendrograma que os enterococos no TGI dos imaturos têm origem alimentar e materna. Conclui-se que os enterococos provenientes de amostras fecais de imaturos de H. erato phyllis possuem poucos genes relacionados a mecanismos de virulência comuns na área clínica. É possível que os Enterococcus produtores de substância antagonista desempenhem um papel importante no equilíbrio das comunidades microbianas do TGI deste inseto, e que ocorra a transferência vertical de micro-organismos das fêmeas para a prole para a espécie H. erato phyllis.Enterococcus is present in the gastrointestinal (GI) tract of vertebrates and invertebrates, which can acquire these bacteria through mechanisms of horizontal and vertical transfer of microrganisms. Recent research has described the presence of Enterococcus in Lepidoptera, but studies with butterflies of the genus Heliconius are scarce. This study had as objective, from a bacterioteca of 178 Enterococcus sp. previously isolated from fecal samples of immature Heliconius erato phyllis detect the presence of resistance and virulence genes by PCR; phenotypically assess the virulence factors and the production of compounds with antimicrobial activity; and to evaluate the clonal profile of the strains by PFGE. Of the 178 enterococci, 55 were resistant to erythromycin and 11 resistant to norfloxacin and/or ciprofloxacin, however was not detected any gene related to these resistance phenotypes. The virulence factors were tested in 178 enterococci, and 63 isolated amplified for the esp gene, 12 for ace and 2 for gelE. The agg and cylA genes were not detected. In the phenotypic analyzes, both E. faecalis positive to gelE gene were also positive for the degradation of gelatinase, and 130 enterococci showed ability to hydrolyze red blood cells. To investigate the production of antagonistic substance, 26 enterococci were selected and of these, 15 showed activity against the indicator bacteria Listeria monocytogenes. Eighty-six isolates were selected to evaluate the clonal profile by PFGE, and it was possible to observe in the dendrogram that the origin of the enterococci in GIT of immatures was the herbivory and maternal origin. It is concluded that the enterococci from fecal samples of immature H. erato phyllis possess few mechanisms of virulence-related genes, common in the clinical area. It is possible that the antagonistic substance-producing enterococci could play an important role in the equilibrium of the microbial communities of the GIT of this insect, and there is vertical transmission of enterococci from females to their offspring to the specie H. erato phyllis

Determination of clonal relationship and virulence factors in enterococci isolated from immatures of Heliconius erato phyllis (Lepidoptera – Nymphalidae)

Enterococcus estão presentes no trato gastrointestinal (TGI) de animais vertebrados e invertebrados, os quais podem adquirir estas bactérias através de mecanismos de transferência horizontal e vertical de micro-organismos. Pesquisas recentes descreveram a presença de Enterococcus em lepidópteros, porém estudos com borboletas do gênero Heliconius são escassos. Este trabalho teve como objetivo, a partir de uma bacterioteca de 178 Enterococcus sp. previamente isolados de fezes de imaturos de Heliconius erato phyllis detectar a presença de genes de resistência e virulência por PCR; avaliar fenotipicamente os fatores de virulência e a produção de compostos com atividade antimicrobiana; e avaliar o perfil clonal das cepas pela técnica de PFGE. Dos 178 enterococos, 55 apresentavam resistência a eritromicina e 11 a norfloxacina e/ou ciprofloxacina, porém não foi detectado nenhum gene relacionado a estes fenótipos de resistência. Quanto aos fatores de virulência testados nos 178 enterococos, 63 isolados amplificaram para o gene esp, 12 para ace e 2 para gelE. Os genes agg e cylA não foram detectados. Na análise fenotípica dos fatores de virulência, os dois E. faecalis positivos para gelE também foram positivos para a degradação da gelatinase e 130 enterococos apresentaram capacidade de hidrolisar hemácias. Para investigar a produção de substância antagonista, foram selecionados 26 enterococos; destes, 15 apresentaram atividade contra a bactéria indicadora Listeria monocytogenes. Oitenta e seis isolados foram selecionados para avaliar a relação clonal por PFGE, e foi possível observar a partir do dendrograma que os enterococos no TGI dos imaturos têm origem alimentar e materna. Conclui-se que os enterococos provenientes de amostras fecais de imaturos de H. erato phyllis possuem poucos genes relacionados a mecanismos de virulência comuns na área clínica. É possível que os Enterococcus produtores de substância antagonista desempenhem um papel importante no equilíbrio das comunidades microbianas do TGI deste inseto, e que ocorra a transferência vertical de micro-organismos das fêmeas para a prole para a espécie H. erato phyllis.Enterococcus is present in the gastrointestinal (GI) tract of vertebrates and invertebrates, which can acquire these bacteria through mechanisms of horizontal and vertical transfer of microrganisms. Recent research has described the presence of Enterococcus in Lepidoptera, but studies with butterflies of the genus Heliconius are scarce. This study had as objective, from a bacterioteca of 178 Enterococcus sp. previously isolated from fecal samples of immature Heliconius erato phyllis detect the presence of resistance and virulence genes by PCR; phenotypically assess the virulence factors and the production of compounds with antimicrobial activity; and to evaluate the clonal profile of the strains by PFGE. Of the 178 enterococci, 55 were resistant to erythromycin and 11 resistant to norfloxacin and/or ciprofloxacin, however was not detected any gene related to these resistance phenotypes. The virulence factors were tested in 178 enterococci, and 63 isolated amplified for the esp gene, 12 for ace and 2 for gelE. The agg and cylA genes were not detected. In the phenotypic analyzes, both E. faecalis positive to gelE gene were also positive for the degradation of gelatinase, and 130 enterococci showed ability to hydrolyze red blood cells. To investigate the production of antagonistic substance, 26 enterococci were selected and of these, 15 showed activity against the indicator bacteria Listeria monocytogenes. Eighty-six isolates were selected to evaluate the clonal profile by PFGE, and it was possible to observe in the dendrogram that the origin of the enterococci in GIT of immatures was the herbivory and maternal origin. It is concluded that the enterococci from fecal samples of immature H. erato phyllis possess few mechanisms of virulence-related genes, common in the clinical area. It is possible that the antagonistic substance-producing enterococci could play an important role in the equilibrium of the microbial communities of the GIT of this insect, and there is vertical transmission of enterococci from females to their offspring to the specie H. erato phyllis

Diversidade e perfil antimicrobiano de Enterococcus spp. isolados de fezes de imaturos de Heliconius erato phyllis (LEPIDOPTERA-NYMPHALIDAE)

Borboletas do gênero Heliconius (Lepidoptera-Nymphalidae) estão presentes desde florestas até ambientes urbanos e são organismos modelo para estudos ecológicos e evolutivos, porém pouco se sabe a respeito da microbiota destes insetos. Um trabalho recente identificou Enterococcus como o gênero de bactérias mais frequente em amostras de lagartas e adultos de Heliconius. Micro-organismos do gênero Enterococcus são gram-positivos, catalase negativos, com morfologia celular de diplococos ou cocos de cadeias curtas que toleram concentrações de 6,5% de NaCl e variações de temperatura de 10˗45ºC, além de possuírem resistência intrínseca e adquirida a diversos antimicrobianos. Estas bactérias são muitas vezes relacionadas a infecções humanas ou ao trato gastrointestinal de animais de sangue quente, porém podem ser encontradas em diversas fontes não entéricas como solo, vegetação e ambientes aquáticos. No presente trabalho, lagartas de quinto estádio provenientes de três locais do Rio Grande do Sul, Brasil, foram analisadas quanto à presença de diferentes espécies de Enterococcus. Pelo menos duas espécies desta bactéria foram encontradas em fezes de lagartas provenientes de fêmeas das três localidades. Além disso, foi avaliado o perfil de resistência destes isolados frente a 11 antimicrobianos de importância clínica e veterinária; os isolados apresentaram resistência ao antibiótico rifampicina e resistência intermediária a eritromicina, norfloxacina e ciprofloxacina

Diversidade e perfil antimicrobiano de Enterococcus spp. isolados de fezes de imaturos de Heliconius erato phyllis (LEPIDOPTERA-NYMPHALIDAE)

Borboletas do gênero Heliconius (Lepidoptera-Nymphalidae) estão presentes desde florestas até ambientes urbanos e são organismos modelo para estudos ecológicos e evolutivos, porém pouco se sabe a respeito da microbiota destes insetos. Um trabalho recente identificou Enterococcus como o gênero de bactérias mais frequente em amostras de lagartas e adultos de Heliconius. Micro-organismos do gênero Enterococcus são gram-positivos, catalase negativos, com morfologia celular de diplococos ou cocos de cadeias curtas que toleram concentrações de 6,5% de NaCl e variações de temperatura de 10˗45ºC, além de possuírem resistência intrínseca e adquirida a diversos antimicrobianos. Estas bactérias são muitas vezes relacionadas a infecções humanas ou ao trato gastrointestinal de animais de sangue quente, porém podem ser encontradas em diversas fontes não entéricas como solo, vegetação e ambientes aquáticos. No presente trabalho, lagartas de quinto estádio provenientes de três locais do Rio Grande do Sul, Brasil, foram analisadas quanto à presença de diferentes espécies de Enterococcus. Pelo menos duas espécies desta bactéria foram encontradas em fezes de lagartas provenientes de fêmeas das três localidades. Além disso, foi avaliado o perfil de resistência destes isolados frente a 11 antimicrobianos de importância clínica e veterinária; os isolados apresentaram resistência ao antibiótico rifampicina e resistência intermediária a eritromicina, norfloxacina e ciprofloxacina

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42·4% vs 44·2%; absolute difference -1·69 [-9·58 to 6·11] p=0·67; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5-8] vs 6 [5-8] cm H2O; p=0·0011). ICU mortality was higher in MICs than in HICs (30·5% vs 19·9%; p=0·0004; adjusted effect 16·41% [95% CI 9·52-23·52]; p<0·0001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0·80 [95% CI 0·75-0·86]; p<0·0001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status