Resistance determinants of emerging pathogens isolated from an intensive care unit as a parameter of population health conditions of the Legal Amazon microregion / Determinantes de resistência a patógenos emergentes isolados de uma unidade de terapia intensiva como parâmetro das condições de saúde da população da microrregião da Amazônia Legal

Bacteria responsible for causing infections are common in hospital environments, water, soil, and food products. The infection risk is intensified by the absence of public sanitation, poor quality of life, and food scarcity. These external factors promote the dissemination of pathogens by direct contamination or biofilm formation. In this work, we identified bacterial isolates obtained from intensive care units in the southern region of Tocantins, Brazil. We compared matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques and 16S ribosomal ribonucleic acid (rRNA) molecular analysis; we also performed phenotypic characterization. Fifty-six isolates characterized using morphotinctorial tests were classified as gram-positive (80.4%; n = 45) and gram-negative (19.6%; n = 11) and were resistant to several antibiotic classes; notably, we identified the blaOXA-23 resistance gene in the ILH10 isolate. Microbial identification using MALDI-TOF MS resulted in the identification of Sphingomonas paucimobilis and Bacillus circulans. 16S rRNA sequencing revealed four isolates belonging to the genera Bacillus and Acinetobacter. The similarity was superior to 99% for Acinetobacter schindleri in the Basic Local Alignment Search Tool (BLAST), grouped in the clade superior to 90%. Several strains isolated from intensive care units (ICU) were resistant to various antibiotic classes. These techniques allowed for the identification of several microorganisms of importance in public health, enabling improvements in human infection control and proving the quality of inputs, food, and water.

As bactérias responsáveis por causar infecções são comuns em ambientes hospitalares, água, solo e produtos alimentícios. O risco de infecção é intensificado pela ausência de saneamento público, má qualidade de vida e escassez de alimentos. Esses fatores externos promovem a disseminação de patógenos por contaminação direta ou formação de biofilme. Neste trabalho, identificamos isolados bacterianos obtidos de unidades de terapia intensiva na região sul do Tocantins, Brasil. Comparamos técnicas de espectrometria de massa de tempo de voo com ionização por dessorção a laser assistida por matriz (MALDI-TOF MS) e análise molecular de ácido ribonucleico ribossômico 16S (rRNA); também realizamos caracterização fenotípica. Cinquenta e seis isolados caracterizados por testes morfotintoriais foram classificados como gram-positivos (80,3%; n = 45) e gram-negativos (19,6%; n = 11) e foram resistentes a várias classes de antibióticos; notavelmente, identificamos o gene de resistência blaOXA-23 no isolado de ILH10. A identificação microbiana usando MALDI-TOF MS resultou na identificação de Sphingomonas paucimobilis e Bacillus circulans. O sequenciamento do 16S rRNA revelou quatro isolados pertencentes aos gêneros Bacillus e Acinetobacter. A similaridade foi superior a 99% para Acinetobacter schindleri no BLAST, agrupado no clado superior a 90%. Várias cepas isoladas de ICU foram resistentes a várias classes de antibióticos. Essas técnicas permitiram a identificação de diversos microrganismos de importância em saúde pública, possibilitando melhorias no controle de infecções humanas e comprovando a qualidade dos insumos, alimentos e água.

Microbial diversity in artisanal cheese produced and commercialized in Vale do Taquari in southern Brazil / Diversidade microbiana encontrada em queijos artesanais produzidos e comercializados no Vale do Taquari no sul do Brasil

Researchers have been utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify bacteria and fungi directly from isolates obtained on culture plates; the resulting mass spectrum is then compared with spectra stored in the instrument software. Hence, a fast analytical response is obtained, and the more spectra are known to compare, the safer and more reliable the interpretation of the method will be. Thus, this study sought to identify the diversity of strains found in 10 samples of artisan cheese produced and commercialized in Vale do Taquari (Rio Grande do Sul State, Brazil) using MALDI-TOF MS. From the analyzed cheese, 22 strains were identified at the species level; one sample presented the pathogen Staphylococcus aureus, two showed the presence of lactic acid bacteria (Lactococcus lactis), and the vast majority (68.18%) of strains were composed of species of the Enterobacteriaceae family, with the prevalence of the genera Escherichia, Enterobacter, and Klebsiella. Escherichia coli was present in 50% of the samples analyzed. This demonstrates the need for greater control during all stages of artisanal cheese production and evaluation of the raw material, including safe practices during milking, so that the product meets the identity and quality parameters suitable for human consumption.

MALDI-TOF MS vendo sendo utilizado em laboratórios para identificar bactérias e fungos diretamente de isolados obtidos em placas de cultura. O espectro de massa resultante é então comparado com espectros armazenados no software do equipamento. Assim, obtém-se uma resposta analítica rápida, sendo que, quanto mais espectros forem conhecidos para comparar, mais seguro e confiável será a interpretação do método. Desta maneira, o presente trabalho teve por objetivo, identificar por MALDI-TOF MS, a diversidade de cepas encontrados em 10 amostras de queijos artesanais produzidos e comercializados no Vale do Taquari, Rio Grande do Sul, Brasil. Dos queijos analisados, 22 cepas foram identificadas a nível de espécie, sendo uma (1) amostra apresentou o patógeno Staphylococcus aureus, duas a presença da bactéria ácido láctica (Lactococcus lactis) e a grande maioria (68,18%) das cepas era composta por espécies da família Enterobacteriaceae, com prevalência dos gêneros Escherichia, Enterobacter e Klebsiella. E. coli estava presente em 50% das amostras analisadas. Isso demonstra a necessidade de um maior controle durante todas as etapas de produção do queijo artesanal, bem como a avaliação da matéria-prima, incluindo práticas seguras durante a ordenha para que o produto atenda aos parâmetros de identidade e qualidade, sendo apto ao consumo humano.

Urethral obstruction secondary to Staphylococcus felis chronic urethritis and hepatoid gland adenoma in a captive ocelot (Leopardus pardalis)

A male adult ocelot (Leopardus pardalis) kept under human care developed anuria, which progressed to death. Grossly, the urinary bladder was markedly dilated and filled with red discolored urine containing blood clots. In addition, the animal had a hepatoid cell adenoma adjacent to the urethra, which likely caused partial urethral occlusion. Microscopically, there was a predominantly neutrophilic, fibrinous and hemorrhagic urethritis, cystitis, and pyelonephritis with intralesional gram-positive cocci. Microbiologic culture followed by MALDI-TOF MS analysis resulted in the identification of isolates from the urine and urethra as Staphylococcus felis.(AU)

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines

Abstract Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Resumo Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Use of MALDI-TOF for identification and surveillance of gramnegative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram-negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Abstract Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.

Species identification and antimicrobial susceptibility profile of bacteria associated with cow mastitis in southern Brazil / Identificação de espécies e perfil de suscetibilidade aos antimicrobianos de bactérias associadas à mastite bovina no sul do Brasil

Bovine mastitis is the most common disease in dairy cattle and responsible for economic losses in the milk industry. The present study aimed to identify the main species and to evaluate the antimicrobial susceptibility of bacterial isolates from cow herds with mastitis in dairy farms from southern Brazil. A total of 107 milk samples were collected from different cow herds in one important dairy producing region in southern Brazil, including farms located in ten cities from the Northeast region in the Rio Grande do Sul state. Bacterial strains were isolated and submitted to presumptive identification by classical bacteriological methods. Bacterial species were also identified by MALDI-TOF MS and antimicrobial susceptibility testing was performed with 12 antimicrobials commonly used in dairy farms. Fifty-one bacterial strains were isolated and the presumptive identification demonstrated the occurrence of Staphylococcus spp. (82.3%), Bacillus spp. (3.9%), Klebsiella spp. (3.9%), Streptococcus spp. (3.9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) and Serratia sp. (2%). Forty-one isolates were successfully identified in the MALDI-TOF analysis, including 35 isolates from eleven different bacterial species. Importantly, there were eight different Staphylococcus species, with a high frequency of Staphylococcus chromogenes (48.6%) and Staphylococcus aureus (20%). Overall, bacterial isolates demonstrated resistance to penicillin (46.3%), tetracycline (39%), amoxicillin (36.6%), ampicillin (34.1%) and sulfamethoxazole/trimethoprim (31.7%). Enrofloxacin was the unique antimicrobial that all isolates were susceptible. In addition, there were six multidrug resistant isolates (five S. chromogenes and one S. aureus). This study highlights that bacterial pathogens with resistance to several antimicrobials were identified in cows from dairy farms in a very important milk producing region located in southern Brazil. Microbial identification of the bovine mastitis pathogens and determination of the antimicrobial profile is necessary for the rational use of the medicines.

A mastite bovina é a doença mais comum em gado leiteiro e responsável por perdas econômicas na indústria de laticínios. O presente estudo teve como objetivo identificar as principais espécies e avaliar a suscetibilidade antimicrobiana de isolados bacterianos de rebanhos bovinos com mastite em fazendas leiteiras no sul do Brasil. Um total de 107 amostras de leite foram coletadas em diferentes rebanhos bovinos em uma importante região produtora de leite do sul do Brasil, incluindo fazendas localizadas em 10 cidades da região Nordeste do estado do Rio Grande do Sul. As cepas bacterianas foram isoladas e submetidas à identificação presuntiva por métodos bacteriológicos clássicos. A identificação bacteriana foi confirmada por MALDI-TOF MS e o teste de sensibilidade antimicrobiana foi realizado com antimicrobianos comumente usados em fazendas leiteiras. Cinquenta e uma cepas bacterianas foram isoladas e a identificação presuntiva demonstrou a ocorrência de Staphylococcus spp. (82,3%), Bacillus spp. (3,9%), Klebsiella spp. (3,9%), Streptococcus spp. (3,9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) e Serratia sp. (2%). Os 41 isolados foram identificados com sucesso na análise MALDI-TOF, incluindo 35 isolados de onze espécies bacterianas diferentes. É importante ressaltar que houve a ocorrência de oito espécies diferentes de Staphylococcus, com alta frequência de Staphylococcus chromogenes (48,6%) e Staphylococcus aureus (20%). No geral, os isolados bacterianos tiveram alta resistência à penicilina (46,3%), tetraciclina (39%), amoxicilina (36,6%), ampicilina (34,1%) e sulfametoxazol/trimetoprima (31,7%). A enrofloxacina foi o único antimicrobiano que todos os isolados foram suscetíveis. Além disso, havia seis isolados multirresistentes (cinco S. chromogenes e um S. aureus). Este estudo destaca que os patógenos bacterianos com resistência aos antimicrobianos estão presentes em fazendas leiteiras de subsistência em uma importante região produtora no sul do Brasil. É necessário o monitoramento constante dos patógenos da mastite bovina e a determinação de seu perfil antimicrobiano para o uso racional dos medicamentos.

Species identification and antimicrobial susceptibility profile of bacteria associated with cow mastitis in southern Brazil / Identificação de espécies e perfil de suscetibilidade aos antimicrobianos de bactérias associadas à mastite bovina no sul do Brasil

Bovine mastitis is the most common disease in dairy cattle and responsible for economic losses in the milk industry. The present study aimed to identify the main species and to evaluate the antimicrobial susceptibility of bacterial isolates from cow herds with mastitis in dairy farms from southern Brazil. A total of 107 milk samples were collected from different cow herds in one important dairy producing region in southern Brazil, including farms located in ten cities from the Northeast region in the Rio Grande do Sul state. Bacterial strains were isolated and submitted to presumptive identification by classical bacteriological methods. Bacterial species were also identified by MALDI-TOF MS and antimicrobial susceptibility testing was performed with 12 antimicrobials commonly used in dairy farms. Fifty-one bacterial strains were isolated and the presumptive identification demonstrated the occurrence of Staphylococcus spp. (82.3%), Bacillus spp. (3.9%), Klebsiella spp. (3.9%), Streptococcus spp. (3.9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) and Serratia sp. (2%). Forty-one isolates were successfully identified in the MALDI-TOF analysis, including 35 isolates from eleven different bacterial species. Importantly, there were eight different Staphylococcus species, with a high frequency of Staphylococcus chromogenes (48.6%) and Staphylococcus aureus (20%). Overall, bacterial isolates demonstrated resistance to penicillin (46.3%), tetracycline (39%), amoxicillin (36.6%), ampicillin (34.1%) and sulfamethoxazole/trimethoprim (31.7%). Enrofloxacin was the unique antimicrobial that all isolates were susceptible. In addition, there were six multidrug resistant isolates (five S. chromogenes and one S. aureus). This study highlights that bacterial pathogens with resistance to several antimicrobials were identified in cows from dairy farms in a very important milk producing region located in southern Brazil. Microbial identification of the bovine mastitis pathogens and determination of the antimicrobial profile is necessary for the rational use of the medicines.(AU)

A mastite bovina é a doença mais comum em gado leiteiro e responsável por perdas econômicas na indústria de laticínios. O presente estudo teve como objetivo identificar as principais espécies e avaliar a suscetibilidade antimicrobiana de isolados bacterianos de rebanhos bovinos com mastite em fazendas leiteiras no sul do Brasil. Um total de 107 amostras de leite foram coletadas em diferentes rebanhos bovinos em uma importante região produtora de leite do sul do Brasil, incluindo fazendas localizadas em 10 cidades da região Nordeste do estado do Rio Grande do Sul. As cepas bacterianas foram isoladas e submetidas à identificação presuntiva por métodos bacteriológicos clássicos. A identificação bacteriana foi confirmada por MALDI-TOF MS e o teste de sensibilidade antimicrobiana foi realizado com antimicrobianos comumente usados em fazendas leiteiras. Cinquenta e uma cepas bacterianas foram isoladas e a identificação presuntiva demonstrou a ocorrência de Staphylococcus spp. (82,3%), Bacillus spp. (3,9%), Klebsiella spp. (3,9%), Streptococcus spp. (3,9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) e Serratia sp. (2%). Os 41 isolados foram identificados com sucesso na análise MALDI-TOF, incluindo 35 isolados de onze espécies bacterianas diferentes. É importante ressaltar que houve a ocorrência de oito espécies diferentes de Staphylococcus, com alta frequência de Staphylococcus chromogenes (48,6%) e Staphylococcus aureus (20%). No geral, os isolados bacterianos tiveram alta resistência à penicilina (46,3%), tetraciclina (39%), amoxicilina (36,6%), ampicilina (34,1%) e sulfametoxazol/trimetoprima (31,7%). A enrofloxacina foi o único antimicrobiano que todos os isolados foram suscetíveis. Além disso, havia seis isolados multirresistentes (cinco S. chromogenes e um S. aureus). Este estudo destaca que os patógenos bacterianos com resistência aos antimicrobianos estão presentes em fazendas leiteiras de subsistência em uma importante região produtora no sul do Brasil. É necessário o monitoramento constante dos patógenos da mastite bovina e a determinação de seu perfil antimicrobiano para o uso racional dos medicamentos.(AU)

In vitro selection of autochthonous bacterium with probiotic potential for the neotropical fish piauçu Megaleporinus microcephalus / [Seleção in vitro de bactéria autóctone com potencial probiótico para o peixe neotropical piauçu Megaleporinus macrocephalus]

The study aimed to isolate, identify, and apply in vitro tests on bacteria with autochthonous probiotic potential isolated from fifteen healthy specimens of Megaleporinus macrocephalus. The strains were selected from the intestinal tract of fish and inoculated in the Petri plate containing Sharp Man Rogosa Agar (MRS) for (48 hours at 35ºC). They were isolated based on a test of catalase, Gram stain, tolerance to different gradients NaCl (1, 2 and 3%), pH (4, 5, 6, 8 and 9) values and bile salts (2.5 and 5%), in addition to the inhibition zone against pathogens. Of the 42 strains isolated, ST1 and ST9 had higher values (p<0.05) for total viable cells (31.80±0.07 and 32.51±0.05 CFU/mL × 108) respectively. In the resistance tests, strains ST1 and ST9 presented the best results, with emphasis on ST9 in the gradients of pH, high values of bile salts and larger inhibition zones against Aeromonas hydrophila and Aeromonas jandaei. The strains with the best results in the tests, ST1 and ST9, were identified by the MALDI-TOF-MS method as Enterococcus faecium. Thus, the isolated E. faecium bacteria, may be recommended as for probiotic use in farming the M. macrocephalus.

O presente estudo visou isolar, identificar e aplicar testes in vitro em bactérias com potencial probiótico, autóctones, isoladas de 15 espécimes saudáveis de Megaleporinus macrocephalus. As cepas foram selecionadas do trato intestinal dos peixes e inoculadas em placas de Petri contendo Man Rogosa Sharped Agar (MRS), por 48 horas, a 35ºC. Foram isoladas com base em teste de catalase, coloração de Gram, tolerância a diferentes gradientes de NaCl (1, 2 e 3%), valores de pH (4, 5, 6, 8 e 9) e sais biliares (2,5 e 5%), além do halo de inibição contra patógenos. Das 42 cepas isoladas, ST1 e ST9 apresentaram maiores valores (P<0,05) para células viáveis totais (31,80±0,07 e 32,51±0,05 UFC/mL × 108), respectivamente. Nos testes de resistência, as cepas ST1 e ST9 apresentaram os melhores resultados, com destaque para ST9 nos gradientes de pH, altos valores de sais biliares e maiores halos de inibição contra Aeromonas hydrophila e Aeromonas jandaei. As cepas com melhores resultados nos testes, ST1 e ST9, foram identificadas pelo método de MALDI-TOF-MS como Enterococcus faecium. Assim, a bactéria isolada Enterococcus faecium, pode ser recomendada para uso probiótico na criação do M. macrocephalus.

Use of MALDI-TOF for identification and surveillance of gramnegative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.(AU)

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.(AU)

Atypical mandibular osteomyelitis in an ewe caused by coinfection of Pseudomonas aeruginosa and Lactococcus raffinolactis

Background: Osteomyelitis is defined as a bone inflammation involving the cortical and medullary regions, usually caused by the local invasion of opportunistic microorganisms. The inflammatory reaction of bone may extend to the periosteum and soft tissues, compromising adjacent structures far from the initially infected foci. Different classifications of transmission routes, gravity levels, and tissues involved in animal and human osteomyelitis are available. In humans, the infection can reach bone tissue by exogenous or hematogenous pathways. This paper reports an atypical case of mandibular pyogranulomatous osteomyelitis in an ewe caused by concomitant Pseudomonas aeruginosa and Lactococcus raffinolactis infection. Case: The animal presented a 1-month history of progressive mandibular enlargement refractory to conventional therapy. In a physical examination, an increased volume located in the ventrolateral region of the right ramus of the mandible was observed. Fine-needle aspiration of the lesion enabled isolation in bacteriological culture of Pseudomonas aeruginosa and Lactococcus raffinolactis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS). Besides support care procedures and antimicrobial treatment approaches for the sheep based on in vitro tests, the animal died due to the severity of the clinical signs and the progressive worsening of the general health status. The radiographic image examination of the mandibular region revealed a severe and infiltrative periodontal reaction, with a predominance of a great number of neutrophils and macrophages, necrotic areas, and bone destruction, characterized histologically as a pyogranulomatous rection. At post mortem examination, a large pyogranuloma was observed in the entire horizontal branch of the mandible as well, showing a dark yellowish content of coarse consistency, caseous appearance, and bone fragmentation. Discussion: Ovine mandibular osteomyelitis is a well-established bone inflammation involving the cortical and medullary regions, characterized clinically by local enlargement, asymmetry, pain sensitivity, edema, hyperthermia, infiltrate caseous or suppurative material, and bone rarefaction. In the current report, 1-month history of progressive enlargement of the mandibular region, prostration, and weight loss in an ewe were referred. Where clinical and epidemiological features, bacteriological, cytological, histological, and mass spectrometry diagnostic approaches were assessed to diagnostic. Most reports involving the etiology of ovine mandibular osteomyelitis have been diagnosed based on classical phenotypic tests. Here, the concomitant identification of P. aeruginosa and L. raffinolactis infection was possible using mass spectrometry (MALDI-TOF), highlighting the importance of molecular methods in the diagnosis of animal diseases. In addition, the differentiation between Lactococcus and Enterococcus species is difficult, which could underestimate the diagnosis of Lactococcus species as a primary pathogen from animal diseases. We report, for the first time, a fatal case of mandibular pyogranulomatous osteomyelitis in a sheep caused by Pseudomonas aeruginosa and Lactococcus raffinolactis coinfection.

Isolation and antimicrobial resistance of coagulase-negative staphylococci recovered from healthy tortoises in Minas Gerais, Brazil / Isolamento e resistência aos antimicrobianos de estafilococos coagulase negativos recuperados de jabutis saudáveis em Minas Gerais, Brasil

In the last few decades, there has been a global increase in the adoption of reptiles as companion animals, mainly turtles and tortoises. Considering the popularity of reptiles as pets in Brazil, and a notable lack of data about potentially pathogenic staphylococci in these animals, this study isolated and evaluate the antimicrobial susceptibility of staphylococcal species from healthy tortoises (Chelonoidis carbonaria) in Brazil. During a 12-month period (February 2019 to February 2020), cloacal swabs from 66 healthy tortoises were collected at the Wild Animals Screening Center in Belo Horizonte, Minas Gerais, Brazil. The swabs were plated onto mannitol salt agar for staphylococci isolation, and species identification was performed using MALDI-TOF MS. Antimicrobial susceptibility was investigated using the disk diffusion method, and the presence of the mecA gene was investigated by PCR to detect methicillin resistance. Of the tested animals, 72.7% were positive for staphylococcal isolation. All isolates were coagulase-negative staphylococci (CoNS), and Staphylococcus sciuri (81.3%), and S. xylosus (12.5%) were the most frequently isolated species. The majority of the isolates (56%) were resistant to at least one antimicrobial agent. A high frequency of resistance was observed for penicillin (35.5%) and tetracycline (29.1 %). All strains were susceptible to cefoxitin, chloramphenicol, ciprofloxacin, erythromycin, and gentamicin. All isolates were negative for the mecA gene. The present work suggests that healthy tortoises are mainly colonized by CoNS, especially S. sciuri. Half of the isolates were resistant to at least one antimicrobial, raising questions regarding the possible role of these animals as reservoirs of antimicrobial resistance genes.

Nas últimas décadas, houve um aumento global na adoção de répteis como animais de companhia, principalmente tartarugas e jabutis. Considerando a popularidade dos répteis como animais de estimação no Brasil e a notável falta de dados sobre estafilococos potencialmente patogênicos nesses animais, o objetivo deste estudo foi isolar e avaliar a susceptibilidade antimicrobiana de espécies estafilocócicas de jabutis (Chelonoidis carbonaria) saudáveis no Brasil. Durante um período de 12 meses (fevereiro de 2019 a fevereiro de 2020), suabes cloacais de 66 jabutis saudáveis foram coletados no Centro de Triagem de Animais Silvestres em Belo Horizonte, Minas Gerais, Brasil. Os suabes foram plaqueados em ágar manitol salgado para isolamento de estafilococos e a identificação das espécies foi realizada usando MALDI-TOF MS. A susceptibilidade aos antimicrobianos foi investigada pelo método de difusão em disco, e a presença do gene mecA foi investigada por PCR para detectar resistência à meticilina. Dos animais testados, 72,7% foram positivos para o isolamento estafilocócico. Todos os isolados eram estafilococos coagulase-negativos (CoNS), sendo Staphylococcus sciuri (81,3%) e S. xylosus (12,5%) as espécies mais frequentemente isoladas. A maioria dos isolados (56%) foi resistente a pelo menos um antimicrobiano. Alta frequência de resistência foi observada para penicilina (35,5%) e tetraciclina (29,1%). Todas as estirpes foram sensíveis à cefoxitina, cloranfenicol, ciprofloxacina, eritromicina e gentamicina. Todos os isolados foram negativos para o gene mecA. O presente trabalho sugere que jabutis saudáveis são colonizados principalmente por CoNS, especialmente S. sciuri. Metade dos isolados foram resistentes a pelo menos um antimicrobiano, levantando questões sobre o possível papel desses animais como reservatórios de genes de resistência aos antimicrobianos.

Mannheimia varigena as the etiologic agent of lameness and coronary band lesion in cattle / Mannheimia varigena como agente etiológico de claudicação e lesão na banda coronária em bovinos

Mannheimia varigena was identified as the etiologic agent of lameness and coronary band lesion in 30% of cattle in a farm located in Rio Grande do Sul, Brazil. Swab samples from the lesions were cultured in McConkey Agar and Blood Agar for microbiological identification. Culture growth was submitted to Gram staining and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) identification. Antimicrobial susceptibility test based on disc diffusion was performed for three antibiotics: ceftiofur, gentamicin and florfenicol. Furthermore, molecular characterization of 16S rDNA gene sequencing was performed and the data was used in a phylogenetic analysis. For that purpose, total DNA was extracted by thermo extraction directly from the bacterial colonies and the polymerase chain reaction (PCR) was performed. Gram-negative Mannheimia varigena strain LBV010/22 was identified as the causative of the lesions. The strain was susceptible to all antibiotics tested. The phylogenetic analysis demonstrated that the analyzed strain is closely related to M. varigena strains from pyelonephritis and respiratory tract. Overall, this is the first report of M. varigena as the causative agent of coronary band injury in bovine. Therefore, our findings show the importance of an accurate microbiological identification of infectious agent in lameness cases in order to prevent the occurrence and perform an appropriate treatment in the future.

Mannheimia varigena foi identificada como agente etiológico de claudicação e lesão de banda coronária em 30% dos bovinos de uma fazenda localizada no Rio Grande do Sul, Brasil. Amostras de swab das lesões foram cultivadas em Ágar McConkey e Ágar Sangue para identificação microbiológica. O crescimento da cultura foi submetido à coloração de Gram e identificação por Espectrometria de Massa de Ionização por Dessorção a Laser Assistida por Matriz (MALDI-TOF MS). O teste de suscetibilidade antimicrobiana baseado na difusão em disco foi realizado para três antibióticos: ceftiofur, gentamicina e florfenicol. Além disso, foi realizada a caracterização molecular do sequenciamento do gene 16S rDNA e o resultado utilizado para análise filogenética. Para tanto, o DNA total foi extraído por termoextração diretamente das colônias bacterianas e uma reação em cadeia da polimerase (PCR) foi realizada. Foi identificada como causadora das lesões a cepa gram-negativa de Mannheimia varigenaLBV010/22. Ela foi suscetível a todos os antibióticos testados. A análise filogenética demonstrou que a cepa analisada está intimamente relacionada às M. varigena presentes em pielonefrite e no trato respiratório. No geral, este é o primeiro relato de M. varigenacomo agente causador de lesão de banda coronária em bovinos. Portanto, nossos achados mostram a importância de uma identificação microbiológica precisa do agente infeccioso nos casos de claudicação, a fim de prevenir a ocorrência e realizar um tratamento adequado no futuro.

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.(AU)

Biofilm production by Staphylococcus spp. isolated from bovine mastitis in dairy herds in state of Acre, Brazil and its implications / Produção de biofilme por Staphylococcus spp. isolados de mastite bovina, em rebanhos leiteiros do estado do Acre, Brasil, e suas implicações

This study aimed to identify the Staphylococcus species responsible for bovine mastitis in dairy herds in northern Brazil, to investigate the capacity of biofilm production, and to analyze the association of biofilm production with multiresistance and intensity of California Mastitis Tests (CMT) reactions that can make treatment more difficult and cause misdiagnoses, respectively. Milk samples were collected from 23 dairy farms located in five municipalities in the state of Acre. A total of 339 crossbred cows were tested by CMT, with 109 animals (229 udder ceilings) reacting to the test. After bacterial isolation in blood agar, the catalase-positive and gram-positive cocci were submitted for identification by MALDI-TOF MS. Of 103 strains identified as staphylococci, Staphylococcus chromogenes (58.3%) and Staphylococcus aureus (19.4%) were the most prevalent species. Biofilm production was quantitatively evaluated using a microplate adherence test. Among the Staphylococcus strains, 71.8% were biofilm producers. Most strains of S. chromogenes (68.3%) had the capacity to produce biofilms, ranging from weak (43.3%), moderate (13.3%), and strong (11.7%) producers. Among S. aureus strains, 50% were non-biofilm producers, and none were strong producers. Our data showed an association between biofilm production capacity and multidrug resistance. In addition, there was a reduction in the response to the CMT test, which can mask the diagnosis.

O presente estudo teve como objetivos identificar as espécies de estafilococos envolvidas com mastite em rebanhos leiteiros no norte do Brasil, investigar a capacidade de produção de biofilme e analisar a associação da produção de biofilme com multirresistência e intensidade de reações de CMT que podem dificultar o tratamento e causar erros de diagnósticos, respectivamente. Amostras de leite foram coletadas em 23 propriedades leiteiras, situadas em cinco municípios do estado do Acre. Um total de 339 vacas mestiças foram testadas pelo CMT, sendo detectados 109 animais (229 tetos) reagentes ao teste. Após isolamento em ágar sangue, os cocos Gram positivos produtores de catalase foram submetidos à identificação por MALDI TOF MS. Um total de 103 estirpes foram identificadas como estafilococos, sendo mais prevalentes as espécies Staphylococcus chromogenes (58,3%) e S. aureus (19,4%). A produção de biofilme foi avaliada quantitativamente pelo teste de aderência em microplaca. Dentre todas as amostras de estafilococos, 71,8% foram produtoras de biofilme. A maioria das amostras de S. chromogenes (68,3%) apresentou capacidade de produzir biofilme, variando de fraco (43,3%), moderado (13,3%) a forte (11,7%) produtor. Entre as amostras de S. aureus, 50% não foram produtoras e nenhuma foi produtora forte. Essa capacidade de produção de biofilme mostrou-se associada à multirresistência a antimicrobianos, além de diminuir a intensidade no teste CMT, podendo mascarar o diagnóstico.

Staphylococcus aureus-induced pyogranulomatous dermatitis, osteomyelitis, and meningitis with Splendore-Hoeppli reaction in a cat coinfected with the feline leukemia virus and Leishmania sp

This report describes a case of a pyogranulomatous dermatitis, osteomyelitis, and meningitis with Splendore-Hoeppli reaction caused by Staphylococcus aureus in a sheltered female adult cat coinfected with the feline leukemia virus (FeLV) and Leishmania sp. The cat had a mild anemia and marked increased total leukocytes, particularly band and segmented neutrophils. The cat had laboratorial diagnosis of FeLV and Leishmania sp. infections. Clinically, the cat had extensive and multifocal areas of ulceration in cranial region. Due to the progression of cutaneous lesions, progressive weight loss, and the risk for other sheltered animals, the cat was euthanized. Microscopically, there was marked pyogranulomatous ulcerative dermatitis, osteomyelitis and meningitis, with multiple large intralesional colonies of Gram-positive cocci associated with Splendore-Hoeppli reaction. Aerobic bacterial isolates were identified as S. aureus by MALDI-ToF MS. Leishmania sp. DNA sequences were detected in liver and spleen, and amastigotes were demonstrated in skin sections by immunohistochemistry. In conclusion, here we describe a case of S. aureus-induced pyogranulomatous meningitis with SH reaction in a cat naturally coinfected with FeLV and Leishmania sp(AU)

Characterization of the first two toxins isolated from the venom of the ancient scorpion Tityus (Archaeotityus) mattogrossensis (Borelli, 1901)

Abstract Background: Almost all Tityus characterized toxins are from subgenera Atreus and Tityus, there are only a few data about toxins produced by Archaeotityus, an ancient group in Tityus genus. Methods: Tityus (Archaeotityus) mattogrossensis crude venom was fractionated by high performance liquid chromatography, the major fractions were tested in a frog sciatic nerve single sucrose-gap technique. Two fractions (Tm1 and Tm2) were isolated, partially sequenced by MALDI-TOF/MS and electrophysiological assayed on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells. Results: The sucrose-gap technique showed neurotoxicity in four fractions. One fraction caused a delay of action potential repolarization and other three caused a reduction in amplitude. An electrophysiological assay showed that Tm1 is active on HEK293 Nav 1.3, HEK293 Nav 1.6, DUM and DRG cells, and Tm2 on HEK293 Nav 1.3 and DRG cells, but not in HEK293 Nav 1.6. In addition, Tm1 and Tm2 did promote a shift to more negative potentials strongly suggesting that both are -NaScTx. Conclusion: Although Tityus (Archaeotityus) mattogrossensis is considered an ancient group in Tityus genus, the primary structure of Tm1 and Tm2 is more related to Tityus subgenus. The patch clamp electrophysiological tests suggest that Tm1 and Tm2 are NaScTx, and also promoted no shift to more negative potentials, strongly suggesting that both are -NaScTx. This paper aimed to explore and characterize for the first time toxins from the ancient scorpion Tityus (Archaeotityus) mattogrossensis.

Proteomics characterization of Staphylococcus spp. from goat mastitis and phenogeno-typical assessment of resistance to beta-lactamics

ABSTRACT: Mastitis occupies a prominent place among the diseases that affect dairy herds due to economic problems and public health. Staphylococcus spp. are infectious agents more involved in the etiology of caprine mastites, especially coagulase-negative Staphylococcus. Nineteen isolates of Staphylococcus spp. were obtained from subclinical caprine mastitis. All isolates were characterized by MALDI-TOF MS, being 47.36% (9/19) identified for S. epidermidis, 15.78% (3/19) for S. warneri, 10.52% (2/19) for S. aureus and S. caprae and 5.26% (1/19) for S. lugdunensis, S. simulans, and S. cohnii. All isolates characterized by MALDI-TOF were subjected a to polymerase chain reaction (PCR) for the 16S rRNA gene of Staphylococcus spp. to confirm the gender. After determining the species, tests for phenotypic detection of resistance to beta-lactams were carried out simple disk diffusion oxacillin, cefoxitin, penicillin G and amoxicillin + clavulanic acid, agar screen oxacillin and microdilution (MIC) cefoxitin. The disk diffusion test showed a strength of 58% (11/19) for penicillin G, 26.31% (5/19) for cefoxitin and 26.31% (5/19) for oxacillin. All strains were susceptible to amoxicillin + clavulanic acid and agar screen oxacillin. In the MIC, 63.15% (12/19) of the samples were cefoxitin resistant (MIC >4.0g/ml). Then isolates were subjected to detection of the mecA resistance genes and regulators (mecl and mecRI), mecC and blaZ. Two samples of Staphylococcus epidermidis had the mecA gene. All isolates were negative for the mecA gene variant, mecl, mecRI, mecC and blaZ. These findings reinforce the importance of this group of microorganisms in the etiology of subclinical mastitis in goats and open perspectives for future research to investigate the epidemiology of the disease.

RESUMO: A mastite ocupa lugar de destaque entre as doenças que acometem o rebanho leiteiro, em virtude de problemas econômicos e de saúde pública. Staphylococcus spp. são os agentes infecciosos mais envolvidos na etiologia das mastites caprinas, principalmente Staphylococcus coagulase negativo. Dezenove isolados de Staphylococcus spp. foram obtidos a partir de mastite caprina subclínica. Todos os isolados foram caracterizados por MALDI-TOF MS, sendo 47,36% (9/19) identificadas como S. epidermidis, 15,78%(3/19) como S. warneri, 10,52% (2/19) como S. caprae e S. aureus e 5,26% (1/19) tanto para S. lugdunensis, como para S. simulans e S. cohnii. Todos os isolados caracterizados pelo MALDI-TOF foram submetidos a reação em cadeia da polimerase (PCR) para o gene 16rRNA de Staphylococcus spp. para a confirmação do gênero. Após a determinação da espécie, foram realizadas as provas para a detecção fenotípica de resistência aos beta-lactâmicos: difusão em disco simples de oxacilina, cefoxitina, penicilina G e amoxacilina +ácido clavulânico, ágar screen de oxacilina e microdiluição em caldo (MIC) de cefoxitina. O teste de difusão em disco demonstrou resistência de 58% (11/19) para penicilina G, 26,31% (5/19) para cefoxitina e 26,31% (5/19) para oxacilina. Todas as amostras foram sensíveis a amoxicilina + ácido clavulânico e ao ágar screen de oxacilina. Pelo MIC, 63,15% (12/19) das amostras foram resistentes a cefoxitina (MIC >4,0g/ml). Em seguida os isolados foram submetidos a detecção dos genes de resistência mecA e seus reguladores (mecl e mecRI), mecC e blaZ. Duas amostras de S. epidermidis apresentaram o gene mecA. Todos os isolados foram negativos para a variante do gene mecA, mecl, mecRI, mecC e blaZ. Tais achados reforçam a importância deste grupo de microrganismos na etiologia da mastite subclínica em caprinos e abre perspectivas para futuras pesquisas para a investigação da epidemiologia da doença.

Neuroactive venom compounds obtained from Phlogiellus bundokalbo as potential leads for neurodegenerative diseases: insights on their acetylcholinesterase and beta-secretase inhibitory activities in vitro

Spider venom is a rich cocktail of neuroactive compounds designed to prey capture and defense against predators that act on neuronal membrane proteins, in particular, acetylcholinesterases (AChE) that regulate synaptic transmission through acetylcholine (ACh) hydrolysis - an excitatory neurotransmitter - and beta-secretases (BACE) that primarily cleave amyloid precursor proteins (APP), which are, in turn, relevant in the structural integrity of neurons. The present study provides preliminary evidence on the therapeutic potential of Phlogiellus bundokalbo venom against neurodegenerative diseases. Methods Spider venom was extracted by electrostimulation and fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by matrix-assisted laser desorption ionization-time flight mass spectrometry (MALDI-TOF-MS). Neuroactivity of the whole venom was observed by a neurobehavioral response from Terebrio molitor larvae in vivo and fractions were screened for their inhibitory activities against AChE and BACE in vitro. Results The whole venom from P. bundokalbo demonstrated neuroactivity by inducing excitatory movements from T. molitor for 15 min. Sixteen fractions collected produced diverse mass fragments from MALDI-TOF-MS ranging from 900-4500 Da. Eleven of sixteen fractions demonstrated AChE inhibitory activities with 14.34% (± 2.60e-4) to 62.05% (± 6.40e-5) compared with donepezil which has 86.34% (± 3.90e-5) inhibition (p > 0.05), while none of the fractions were observed to exhibit BACE inhibition. Furthermore, three potent fractions against AChE, F1, F3, and F16 displayed competitive and uncompetitive inhibitions compared to donepezil as the positive control. Conclusion The venom of P. bundokalbo contains compounds that demonstrate neuroactivity and anti-AChE activities in vitro, which could comprise possible therapeutic leads for the development of cholinergic compounds against neurological diseases.(AU)