Pan-genomic analysis of Corynebacterium amycolatum gives insights into molecular mechanisms underpinning the transition to a pathogenic phenotype.

Corynebacterium amycolatum is a nonlipophilic coryneform which is increasingly being recognized as a relevant human and animal pathogen showing multidrug resistance to commonly used antibiotics. However, little is known about the molecular mechanisms involved in transition from colonization to the MDR invasive phenotype in clinical isolates. In this study, we performed a comprehensive pan-genomic analysis of C. amycolatum, including 26 isolates from different countries. We obtained the novel genome sequences of 8 of them, which are multidrug resistant clinical isolates from Spain and Tunisia. They were analyzed together with other 18 complete or draft C. amycolatum genomes retrieved from GenBank. The species C. amycolatum presented an open pan-genome (α = 0.854905), with 3,280 gene families, being 1,690 (51.52%) in the core genome, 1,121 related to accessory genes (34.17%), and 469 related to unique genes (14.29%). Although some classic corynebacterial virulence factors are absent in the species C. amycolatum, we did identify genes associated with immune evasion, toxin, and antiphagocytosis among the predicted putative virulence factors. Additionally, we found genomic evidence for extensive acquisition of antimicrobial resistance genes through genomic islands.

Genome Report of Emergent Fish Pathogen Edwardsiella piscicida Recovered from Pseudoplatystoma corruscans in Brazil.

Edwardsiella piscicida is a Gram-negative bacteria belonging to the Hafniaceae family which affects several species of marine and freshwater fish. We present the complete genome of E. piscicida strain BEP80 recovered from the Brazilian catfish named Surubim (Pseudoplatystoma corruscans), consisting a chromosome of 3,883,256 bp and no plasmids.

Characterization of Multidrug-Resistant Staphylococcus aureus Isolates and Comparison of Methods of Susceptibility to Vancomycin.

S. aureus are among the main bacteria causing problems related to multidrug resistance in nosocomial infections. Therefore, it is necessary to carry out a reliable and rapid diagnosis for the identification of the bacteria and characterization of its susceptibility profile, especially vancomycin, which is an alternative treatment against multidrug-resistant (MDR) S. aureus. Thus, the goal of this study was to characterize isolates of S. aureus regarding the resistance and virulence and to check the susceptibility to vancomycin, through different methods, for comparative purposes. Seventeen antimicrobials were tested to assess the susceptibility profile. It was evaluated the presence of identification (nuc), resistance (mecA and blaZ), biofilm (icaA and icaD) and siderophore (sfaD and sbnD) genes. The susceptibility to vancomycin was evaluated by Minimum Inhibitory Concentration (MIC) by broth microdilution (BMD), E-test, commercial panel (Kit), and Phoenix equipment. Most S. aureus (93,33%) was classified as MDR. These isolates were 100% positive for nuc, mecA, icaA, icaD, and sfaD genes; 96.67% for sbnD and 33.33% for blaZ. In relation to BMD, all methods correctly classified the susceptibility of the isolates; however, regarding the exact MIC value for vancomycin, Phoenix showed agreement of 63.33%, E-test (33.33%) and Kit (26.66%). In conclusion, most of S. aureus was considered MDR. Also, they presented resistance, biofilm production, and siderophores genes, showing the pathogenic potential of these bacteria. Besides, the Phoenix test was considered the most effective, as it presents advantages, such as identification of the microorganism and a greater number of antimicrobials tested at a time.

Antibacterial and antibiofilm activities and synergism with florfenicol from the essential oils of Lippia sidoides and Cymbopogon citratus against Aeromonas hydrophila.

AIMS: Aeromonas hydrophila is an opportunistic bacterium, with a high capacity for biofilm production, which can cause severe damage in aquaculture. The objective of this study was to identify the chemical compounds of the essential oils of Lippia sidoides (EOLS) and Cymbopogon citratus (EOCC), and to evaluate the biocidal, antibiofilm and synergistic action with the antimicrobial florfenicol of these essential oils (EOs) against A. hydrophila. METHODS AND RESULTS: The antibacterial activity of EOLS and EOCC was verified by the minimum bactericidal concentration and by the action of these EOs against both forming and consolidated biofilms. The synergistic activity of EOs with florfenicol was performed using the checkerboard technique. The main component of EOLS and EOCC was carvacrol (44.50%) and α-citral (73.56%), respectively. Both EOs showed weak inhibitory activity (≥3125.00 µg ml-1 ). Two bacterial isolates were able to produce biofilm, and EOLS and EOCC acted upon the bacterial isolates to prevent biofilm formation. A bactericidal effect was verified for EOLS in the previously consolidated biofilm for both isolates and for EOCC in only one of the isolates. In general, EOLS had a synergistic effect with florfenicol, while EOCF had an additive effect. CONCLUSIONS: Both EOs were able to interfere with biofilm formation and did not have an antagonistic effect in combination with florfenicol. The best results were found for EOLS, which showed a synergistic effect with florfenicol and the ability to interfere in the formation of consolidated biofilm. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the potential of EOLS and EOCC to interfere in biofilm and act in synergy with florfenicol to reduce the occurrence of A. hydrophila. Development of these compounds may contribute to the development of herbal medicines in aquaculture.

Phytochemical identification and quantification of quercetin in Triplaris gardneriana wedd. leaves by HPLC-DAD with evaluation of antibacterial activity.

Triplaris gardneriana is used by traditional medicine. The objective of this work was the leaves chemical study with isolate, identify and quantify the chemical constituent, validate the analytical method and evaluate the antibacterial activity. The ethyl acetate and chloroform fractions were subjected to column chromatography for isolation of the compounds quercetin and lupeol, respectively. For the identification, quantification of quercetin in the samples and validation of method were performed using HPLC-DAD. The antibacterial activity was evaluated by the microdilution method. The isolated phytochemicals are being reported for the first time in the species. The ethyl acetate fraction showed a higher content of quercetin with 9.967 ± 1.01 mg.g-1. The method was validated. The samples showed good antibacterial activity. In this study, quercetin was isolated and quantified in the species being a great alternative as a producer of this secondary metabolite, which can be safely applied in the quality control analysis.

Cymbopogon flexuosus essential oil as an additive improves growth, biochemical and physiological responses and survival against Aeromonas hydrophila infection in Nile tilapia.

The objective of the present study was to evaluate growth, biochemical, hematological and intestinal enzymes responses and survival of Nile tilapia juveniles fed a diet containing the essential oil of lemongrass Cymbopogum flexuosus (EOCF) and infected by Aeromonas hydrophila. Five diets were evaluated (in quadruplicate) with increasing levels of EOCF (0.0 - control; 0.25; 0.50; 1.0 and 2.0 mL kg diet-1). On day 45, eight fish per treatment were sampled and blood, liver and intestine samples were taken. Others eight fish per treatment were infected with A. hydrophila followed by a 15-day period of observation. Citral is the main constituent of EOCF. The inclusion of 2.0 mL EOCF kg diet-1 increased specific growth rate and survival after A. hydrophila infection and decreased feed conversion ratio of Nile tilapia. In general, higher concentrations of EOCF in the diet reduced plasma glucose and triglycerides levels, and increased plasma amino acids, alanine aminotransferase (ALT) and hepatic ALT levels, hematological parameters, and the activity of intestinal enzymes. It was concluded that the inclusion of 2.0 mL EOCF kg diet-1 improved growth performance, biochemical and physiological responses and decreased mortality of Nile tilapia after A. hydrophila infection.

Low frequency of antibodies anti-Neospora caninum in rodents in Fernando de Noronha Island, Brazil.

This study aimed to evaluate the frequency of Neospora caninum antibodies in rodents from the Island of Fernando de Noronha, Brazil, and to contribute to epidemiological data on neosporosis in this region. Blood samples were collected from 154 black-rats (Rattus rattus) and 12 rock-cavies (Kerodon rupestris). The search for anti-N. caninum antibodies was made using the NAT technique, with a cut-off of 1:20. The frequency of positive mice was 5.19% (8/154 - 1:20 titer) and all rock-cavies were negative. The low frequency of antibodies against N. caninum in rodents found in the Island shows the low contamination of the environment where these animals live.

Antimicrobial activity of propolis extract fractions against Staphylococcus spp. isolated from goat mastitis / Atividade antimicrobiana de frações do extrato de própolis frente à Staphylococcus spp. isolados de mastite caprina

The indiscriminate use of antibiotics in the treatment of caprine mastitis causes the appearance of resistant microorganisms, besides leaving residues in milk, putting at risk to human health. In this way, propolis is an alternative in the treatment of diseases because it has antimicrobial activity, mainly because of the presence of flavonoids in its composition. The aim of this study was to evaluate the antimicrobial potential of propolis to Staphylococcus spp. Isolated from cases of goat mastitis and qualify the crude ethanoic extract by high performance liquid chromatography (HPLC). In this study, the minimum bactericidal concentration values of propolis extracts in ethanol, ethyl acetate and hexane showed that the best concentrations capable of promoting the highest mortality of the isolates of Staphylococcus spp. from mastitis in goats, were 6250, 3125 and 1562.5µg/mL, respectively. By the microplate adherence test, it was found that 20.78% isolates were not able to form biofilm, 14.70% were classified as moderate and 64.70% were weak and none as a strong biofilm producer. Propolis in its different diluents was able to affect the formation of biofilm and showed a pronounced marked antimicrobial activity against Staphylococcus spp. strains and may be indicated for use in in vivo studies.(AU)

O uso indiscriminado de antibióticos no tratamento de mastite caprina leva ao desenvolvimento de micro-organismos resistentes que poderão estar presentes em alimentos, colocando em risco a saúde humana. Dessa forma, a própolis surge como uma alternativa para o tratamento de doenças por possuir uma ação antimicrobiana, principalmente pela presença de flavonoides em sua composição. O objetivo desse estudo foi avaliar o potencial antimicrobiano da própolis frente à Staphylococcus spp. isolados de casos de mastite caprina e qualificar o extrato etanoico bruto por cromatografia líquida de alta eficiência (CLAE-DAD). Neste estudo, os valores de concentração bactericida mínima (CBM) dos extratos de própolis em álcool etílico, acetato de etila e hexano nos isolados foram de 6250, 3125 e 1562,5µg/mL, respectivamente. Pelo teste de aderência à microplacas, observou-se que 20,78% dos microorganismos, não foram capazes de formar biofilme, 14,70% foram classificados como moderados, 64,70% em fracos e nenhum como forte produtor de biofilme. A própolis em seus diferentes diluentes foi capaz de afetar a formação de biofilme e apresentou significativa atividade antimicrobiana frente a cepas de Staphylococcus spp., podendo ser indicada para utilização em estudos "in vivo".(AU)

Improvement of Baker's yeast-based fuel cell power output by electrodes and proton exchange membrane modification.

The production of more efficient yeast-based fuel cells (YFCs) depends on a combination of effective proton exchange membranes, electron mediators and current collectors. The adhesion of organisms on electrode surface plays a key role in the electron transfer process optimizing the generated power density. In this work, it is reported the preparation of a new YFC prototype using membranes of polyvinyl alcohol/ phosphoric acid and anodes of carbon nanotubes/polyurethane. The high surface area for yeast adhesion and the strong interaction established between cells/carbon nanotubes favor the energy generation in fuel cell. To evaluate the influence of external mediators and the consumption of feed solution (glucose) on performance of YFC, the kinetics of current generation of resulting fuel cells was analyzed. Results reveal that increases in the impedance of electrodes on generated power can be minimized by periodical infusion of feed fuel, preserving 70% of maximum power, representing an important condition for prolonged activity of fuel cell.

Chemical composition and antimicrobial activity of two extract of propolis against isolates of Staphylococcus spp. and multiresistant bacterials / Composição química e atividade antimicrobiana de dois extratos de própolis contra isolados de Staphylococcus spp. e bactérias multirresistentes

There is a growing need to discover and develop alternative therapies for the treatment of mastitis caused by Staphylococcus spp. and multidrug-resistant bacterial infections. This study examined the chemical composition and antimicrobial potential of two propolis extracts (EPA and EPB) against seventy-seven isolates of Staphylococcus spp. obtained from subclinical bovine mastitis; three clinical strains of MRSA and two from clinical strains of S. aureus ATCC, identified as S. aureus ATCC 6538 and S. aureus ATCC 25923. The total phenolic content was determined by the Folin-Ciocalteau method, the total flavonoid content by the Dowd method and the phenolic profile was quantified by HPLC-DAD. The MBC values of the extracts were evaluated by broth microdilution method. The amount of total phenolic and flavonoid compounds was higher in EPA than EPB. Both extracts revealed the presence of caffeic, coumaric, cinnamic, ferulic and 3,4-dihydroxybenzoic acids, with higher concentrations of coumaric and cinnamic acids. Staphylococcus spp. isolates were susceptible to EPA (90.9%), EPB (83.1%) and oxacillin (80.5%). The oxacillin susceptible isolates were also susceptible to EPA (70.1%) and EPB (80.6%), whereas those oxacillin-resistant strains were also susceptible to EPA (40.0%) and to EPB (26.7%). MBC ranged from 34.3 to 68.7µm/mL for EPA and from 68.7 to 137.5µg/mL for EPB. Both extracts inhibited significantly (100%) the clinical strains of MRSA, S. aureus ATCC 6538 and S. aureus ATCC 25923 at the concentration of 68.7µg/mL. It is concluded that both extracts of propolis, whose main constituents are coumaric and cinnamic acids, have high antimicrobial activity against the microorganisms studied, and EPA also against oxacillin-resistant strains. These findings reinforce its potential use for the treatment of bovine mastitis.(AU)

É cada vez mais oportuna a necessidade de descobrir e desenvolver terapias alternativas para tratamento da mastite causada por Staphylococcus spp. e de infecções bacterianas multirresistentes. Este estudo examinou a composição química e o potencial antimicrobiano de dois extratos etanólicos de própolis (EPA e EPB) contra setenta e sete isolados de Staphylococcus spp. obtidos a partir de mastite bovina subclínica; três estirpes clínicas de MRSA e duas de linhagens clínicas de S. aureus ATCC, identificadas como, S. aureus ATCC 6538 e S. aureus ATCC 25923, ambas metacilina resistentes. O teor total de fenólicos foi determinado pelo método de Folin-Ciocalteau, o teor de flavonoides totais pelo método Dowd e o perfil fenólico foi quantificado por HPLC-DAD. CBM dos extratos foi avaliada pelo método de microdiluição em caldo. A quantidade total de compostos fenólicos e flavonoides foi maior no EPA do que no EPB. Ambos os extratos revelaram a presença dos ácidos cafeico, cumárico, cinâmico, ferúlico e 3,4-di-hidroxibenzóico, com maiores concentrações de ácidos cumárico e cinâmico. Os isolados de Staphylococcus spp. foram sensíveis a EPA (90,9%), EPB (83,1%) e oxacilina (80,5%). Os isolados suscetíveis à oxacilina também foram suscetíveis ao EPA (70,1%) e ao EPB (80,6%), enquanto os do resistente à oxacilina foram suscetíveis ao EPA (40,0%) e ao EPB (26,7%). MBC variou de 34,3 a 68,7µm/mL para EPA e de 68,7 a 137,5µg/mL para EPB. Ambos os extratos inibiram significativamente (100%) as linhagens clínicas de MRSA, S. aureus ATCC 6538 e S. aureus ATCC 25923 na concentração de 68,7µg/mL. Conclui-se que os extratos etanólicos da própolis, cujos principais constituintes são os ácidos cumário e cinâmico, possuem atividade antimicrobiana contra os micro-organismos estudados, e o EPA também contra as cepas resistentes à oxacilina. Estes achados reforçam seu potencial uso para o tratamento da mastite bovina.(AU)

Morphology and biometry of the reproductive organs of adult males of Trachemys scripta elegans reared in São Paulo state, Brazil / Morfologia e biometria dos órgãos reprodutivos de machos adultos de Trachemys scripta elegans criados em São Paulo, Brasil

Trachemys scripta elegans is an American underwater chelonian illegally marketed in Brazilian pet shops. When abandoned in nature, it compromises native species, threatening local biodiversity. However, little is known about the body development and structure of its reproductive tract. The objective of the present study was to investigate the morphology and biometry of testis, epididymis and penis, as well as the biometry of the body and secondary sexual characters in this species. Twenty-seven adult males were used aiming to contribute to preservation actions in captivity, population control, and scientific research, as well as to interspecific comparisons. Sex identification by the third claw length was effective, and the specimens presented harmonious and positive body development between mass, carapace, plastron, and height, with unimodal tendency and higher frequency of maximum carapace length at 15cm. The testes and epididymides presented biometric similarity between the antimeres and anatomical and histological structure similar to that of other species of chelonians and mammals, except for the type of epithelium. The findings suggest that there is conserved morphology between slider turtles and homology in relation to mammals. Histological similarity to the reproductive organs of other amniotes, including humans, may give rise to scientific and comparative studies, essential for the establishment of conservation strategies in reptiles.(AU)

Trachemys scripta elegans é um quelônio subaquático americano ilegalmente comercializado em pet shops brasileiros. Ao ser abandonado na natureza, compromete as espécies nativas, ameaçando à biodiversidade local. No entanto, pouco se conhece sobre o desenvolvimento corporal e a estrutura do seu aparelho reprodutor. O objetivo do presente trabalho foi investigar a morfologia e a biometria dos testículos, epidídimos e pênis, a biometria corporal e dos caracteres sexuais secundários. Foram utilizados 27 machos adultos desta espécie, visando contribuir com ações de preservação em cativeiro, controle populacional e pesquisas científicas, além de comparações interespecíficas. A identificação sexual pelo comprimento da terceira garra foi efetiva e os espécimes apresentaram desenvolvimento corporal harmônico e positivo entre massa, carapaça, plastrão e altura, com tendência unimodal e maior frequência de comprimento máximo de carapaça em 15,0cm. Testículos e epidídimos apresentaram semelhança biométrica entre os antímeros e estrutura anatômica e histológica semelhantes à de outras espécies de quelônios e mamíferos, excetuando-se pelo tipo de epitélio. Os achados sugerem haver morfologia conservada entre os cágados e homologia em relação aos mamíferos. A semelhança histológica com os órgãos reprodutivos de outros amniotas, incluindo os humanos, pode dar ensejo a estudos científicos e comparativos, essenciais para estabelecimento de estratégias de conservação em répteis.(AU)

Low intensity electric field inactivation of Gram-positive and Gram-negative bacteria via metal-free polymeric composite.

The adhesion of pathogenic bacteria in medical implants and surfaces is a health-related problem that requires strong inhibition against bacterial growth and attachment. In this work, we have explored the enhancement in the antibacterial activity of metal free-based composites under external electric field. It affects the oxidation degree of polypyrrole-based electrodes and consequently the antibacterial activity of the material. A conductive layer of carbon nanotubes (graphite) was deposited on porous substrate of polyurethane (sandpaper) and covered by polypyrrole, providing highly conductive electrodes characterized by intrinsic antibacterial activity and reinforced by electro-enhanced effect due to the external electric field. The bacterial inhibition of composites was monitored from counting of viable cells at different voltage/time of treatment and determination of biofilm inhibition on electrodes and reactors. The external voltage on electrodes reduces the threshold time for complete bacterial inactivation of PPy-based composites to values in order of 30 min for Staphylococcus aureus and 60 min for Escherichia coli.

Inclusion of discarded banana in sugarcane silage decreases dry matter losses and improves its nutritional value / La inclusión de banano de descarte en el ensilaje de caña de azúcar reduce las pérdidas de materia seca y mejora su valor nutricional / A inclusão de banana de descarte em silagem de cana de açúcar decresce as perdas de matéria seca e melhora o seu valor nutricional

Abstract Background: Sugarcane silage (SCS) has often been characterized by large dry matter (DM) losses associated with reduction in soluble nutrients and increase in indigestible fiber. Objective: To evaluate the effect of including discarded fruits on the losses and nutritional value in SCS. Methods: A completely randomized design was used with five treatments and five replications. Silages were added with 15% (as-fed basis) papaya (Carica papaya) (SP), acerola (Malpighia glabra) (SA), banana (Musa sp.) (SB) or tomato (Solanum lycopersicum) (ST). The control treatment was composed only of SCS. Twenty-five (25-L) plastic buckets were used to make the experimental silos, which were opened on the 90th day after their closure. Data were analyzed by analysis of variance. Results: Gas losses and total DM losses were lower and DM recovery was higher in SB and SA than in the control (p<0.05). The SB had lower content of neutral detergent fiber and acid detergent fiber compared to SP, SA, ST and the control group (p<0.05). There was no effect of treatment for ethanol content (p>0.05). However, there was treatment effect for lactic and acetic acids, ammonia nitrogen contents, lactic acid bacteria, and yeasts and mold counts (p<0.05). Conclusion: The inclusion of 15% banana or acerola in SCS decreases losses and increases DM recovery. Furthermore, the inclusion of 15% banana improves the nutritional value of SCS.

Resumen Antecedentes: El ensilaje de caña de azúcar (SCS) es frecuentemente caracterizado por grandes pérdidas de materia seca (DM) asociadas con reducciones en los nutrientes solubles y aumentos en la fibra indigestible. Objetivo: Evaluar el efecto de la inclusión de frutas de descarte sobre las perdidas y el valor nutricional del SCS. Métodos: Se utilizó un diseño completamente aleatorizado con cinco tratamientos y cinco repeticiones. Fueron adicionados a los ensilajes, 15 % (materia natural) de papaya (Carica papaya) (SP), acerola (Malpighia glabra) (SA), banano (Musa sp.) (SB) o tomate (Solanum lycopersicum) (ST). El control estuvo compuesto apenas por SCS. Veinticinco baldes de 25 L fueron usados para hacer los silos, los cuales se abrieron al día 90 posterior a su cierre. Los datos fueron analizados por análisis de varianza. Resultados: Las pérdidas de gases y de DM total fueron menores y la recuperación de DM fue mayor en SB y SA que en el grupo control (p<0,05). Los contenidos de fibra detergente neutra y ácida fueron menores en SB comparados con SP, SA, ST y el grupo control (p<0,05). No hubo efecto para el contenido de etanol (p>0,05). Sin embargo, hubo efecto de tratamiento para los contenidos de ácidos láctico y acético, y nitrógeno amoniacal y recuento de bacterias ácido lácticas, levaduras y hongos (p<0,05). Conclusiones: La inclusión de 15% de banano y acerola en SCS decreció las perdidas, y aumentó la recuperación de DM. Además, la inclusión de un 15% de banano mejoró el valor nutricional de SCS.

Resumo Antecedentes: A silagem de cana de açúcar (SCS) é frequentemente caracterizada por grandes perdas de matéria seca (DM) associadas com reduções nos nutrientes solúveis e aumento na fibra indigestível. Objetivo: Avaliar o efeito da inclusão de frutas de descarte sobre as perdas e o valor nutricional de SCS. Métodos: Um delineamento completamente casualizado foi utilizado, com cindo tratamentos e cinco repetições. As silagens foram adicionadas com 15% (matéria natural) de mamão (Carica papaya) (SP), acerola (Malpighia glabra) (SA), banana (Musa sp.) (SB) ou tomate (Solanum lycopersicum) (ST). O controle foi composto apenas por SCS. Vinte e cinco baldes de 25 L foram usados para fazer os silos, que foram abertos no 90º dia após o fechamento deles. Os dados foram analisados por meio de análise de variância. Resultados: As perdas por gases e de DM total foram menores e a recuperação de DM foi maior em SB e SA que no controle (p<0,05). Os conteúdos de fibra em detergente neutro e ácido foram menores em SB comparado a SP, SA, ST e o grupo de controle (p<0,05). Não houve efeito para conteúdo de etanol (p>0,05). Contudo, houve efeito de tratamento para os conteúdos de ácidos lático e acético, e nitrogênio amoniacal e contagens de bactérias láticas, e leveduras e fungos (p<0,05). Conclusões: A inclusão de 15% de banana e acerola em SCS decresceu as perdas, e aumentou a recuperação de DM. Adicionalmente, a inclusão de 15% de banana melhorou o valor nutricional da SCS.

Frequency of Staphylococcus aureus virulence genes in milk of cows and goats with mastitis / Frequência de genes fatores de virulência de Staphylococcus aureus em leite de vacas e cabras com mastite

The present study determined the frequency of Staphylococcus aureus virulence genes in 2,253 milk samples of cows (n=1000) and goats (n=1253) raised in three different geographical regions of the state Pernambuco, Brazil. The presence of genes of virulence factors associated to adhesion to host cells (fnbA, fnbB, clfA and clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla and hlb), and capsular polysaccharide (cap5 and cap8) was evaluated by PCR. A total of 123 and 27 S. aureus strains were isolated from cows' and goats' milk, respectively. The sec and tsst genes were detected exclusively in goats' isolates, while the seh gene was only identified in cows' isolates. The number of toxin genes per strain showed that goats' isolates are likely more toxic than bovines' isolates. The cap5 genotype predominated in both host species, especially in strains collected from cows raised in the Agreste region. The cap8 genotype is likely more virulent due to the number of virulence genes per strain. The results of the present study demonstrate that S. aureus may pose a potential threat to human health in Brazil, and, therefore, these results should support actions related to mastitis control programs.(AU)

O presente estudo determinou a frequência de genes de virulência de Staphylococcus aureus em 2253 amostras de leite, sendo de vacas n=1000 e de cabras n=1253, procedentes das três regiões geográficas do estado de Pernambuco, Brasil. A presença de genes de fatores de virulência associados à adesão às células hospedeiras (fnbA, fnbB, clfA e clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla e hlb) e polissacarídeo capsular (cap5 e cap8) foram avaliadas por PCR. Um total de 123 e 27 cepas de S. aureus foram isoladas do leite de vacas e cabras, respectivamente. Os genes sec e tsst foram detectados exclusivamente em isolados de cabras, enquanto o gene seh foi identificado apenas em isolados de vaca. O número de genes de toxina por cepa mostrou que os isolados de cabras são potencialmente mais tóxicos do que os isolados obtidos de bovinos. O genótipo cap5 predominou em ambas as espécies hospedeiras, especialmente em cepas coletadas de vacas criadas na região Agreste. O genótipo cap8 é potencialmente mais virulento devido ao número de genes de virulência por isolado. Os resultados do presente estudo demonstram que S. aureus pode representar uma ameaça potencial para a saúde humana no Brasil e, portanto, estes resultados devem subsidiar ações relacionadas aos programas de controle de mastite.(AU)

Influência do polipirrol e dos níveis de salinidade na formação de biofilme em Aeromonas spp / Influence of polypyrrole and salinity levels on biofilm formation in Aeromonas spp

Bactérias do gênero Aeromonas são patógenos altamente disseminados no ambiente aquático, responsáveis por grandes perdas econômicas na piscicultura de diversos países. São micro-organismos oportunistas e sua patogenicidade está ligada a alguns fatores de virulência, como a formação de biofilme. O estresse salino é um dos fatores que favorecem a formação dessas colônias e, consequentemente, o aumento de infecções. Essas infecções quando estão associadas ao biofilme são ainda mais resistentes aos antimicrobianos. Nesse contexto, o polipirrol destaca-se como uma alternativa antimicrobiana por possuir vários atributos terapêuticos e não apresentar toxicidade aos organismos. Dessa forma, o objetivo desse estudo foi avaliar o perfil de susceptibilidade e a capacidade de formação de biofilme dos isolados de Aeromonas spp. associados aos diferentes níveis de salinidade e polipirrol. Determinou-se a atividade antibacteriana dos isolados e ensaios de motilidade foram realizados com bactérias que carreavam o gene fla. Também verificou-se a capacidade do cloreto de sódio e polipirrol em interferir na formação do biofilme. Os resultados foram evidenciados com a microscopia eletrônica de varredura. As concentrações de 2 e 3% de NaCl inibiram a motilidade bacteriana. Na formação do biofilme, 83% dos isolados bacterianos induziram a produção na concentração de 0,25%. O polipirrol causou a morte de todos os isolados testados na concentração de 125μg/mL. Além disso, esse composto diminuiu a motilidade bacteriana nas concentrações de 0,25 a 3%, sendo que em relação à produção de biofilme, não houve interferência. Esses resultados evidenciam que os diferentes níveis de NaCl influenciam na formação do biofilme favorecendo a persistência da infecção. Este estudo também realçou a potencialidade do polipirrol como agente bactericida, sendo uma alternativa eficaz às drogas antimicrobianas para o tratamento das infecções causadas por Aeromonas spp.(AU)

Bacteria of the genus Aeromonas are highly disseminated pathogens in the aquatic environment, responsible for great economic losses in the pisciculture of several countries. They are opportunistic microorganisms and their pathogenicity is linked to some virulence factors, such as biofilm formation. Saline stress is one of the factors that favor the formation of these colonies and, consequently, the increase of infections. These infections, when associated with biofilm, are even more resistant to antimicrobials. In this context, polypyrrole stands out as an antimicrobial alternative because it has several therapeutic attributes and does not present toxicity to organisms. Thus, the objective of this study was to evaluate the susceptibility profile and the biofilm formation capacity of Aeromonas spp. associated with different levels of salinity and polypyrrole. The antibacterial activity of the isolates was determined and motility assays were performed with bacteria bearing the fla gene. The ability of sodium chloride and polypyrrole to interfere with biofilm formation has also been demonstrated. The results were evidenced with scanning electron microscopy. Concentrations of 2 and 3% of NaCl inhibited bacterial motility. In the biofilm formation, 83% of the bacterial isolates induced production at the concentration of 0.25%. Polypyrrole caused the death of all the isolates tested at the concentration of 125μg/mL. In addition, this compound decreased bacterial motility at concentrations of 0.25 to 3%, and no biofilm was produced. These results show that the different levels of NaCl influence in the formation of the biofilm favoring the persistence of the infection. This study also highlighted the potential of polypyrrole as a bactericidal agent, being an effective alternative to antimicrobial drugs for the treatment of infections caused by Aeromonas spp.(AU)

Resistance of Corynebacterium pseudotuberculosis in the Brazilian semiarid environment / Resistência de Corynebacterium pesudotuberculosis no ambiente do semiárido brasileiro

The semiarid northeast of Brazil contains a unique biome known as caatinga, with a maximum temperature of 40 ºC and a relativity humidity of 56%. The caatinga is characterized by a variety of plants, including Cereus jamacaru Dc (mandacaru), Poincianella microphylla Mart. ex G. Don (catingueira), Pilosocereus gounellei FAC Weber (xique-xique) and Mimosa tenuiflora (Willd.) Poir (jurema preta). Sheep and goat industries are economically strong in that region, despite the fact that caseous lymphadenitis is highly prevalent. The aim of the present study was to assess the survival and biofilm production of Corynebacterium pseudotuberculosis isolates in the environment and under controlled temperatures (28°C, 37°C and 42°C) under different surfaces (plants, soil, wood, wire and thorns). In addition, we investigated the effects of applying the disinfectants chlorhexidine, hypochlorite and quaternary ammonia in soil, tiles, wood and vegetation cover. Four strains of C. pseudotuberculosis were selected (two from goats and two from sheep) for inoculation according to their in vitro biofilm production. Adherence to microplates was used to assess the biofilm-forming ability of the bacteria. Lower survival rates were observed when isolates of C. pseudotuberculosis were subjected to a temperature of 42°C. In terms of caatinga biome plants, contamination of jurema-preta plants resulted in the lowest survival rates. The disinfectant quaternary ammonia promoted a lower inoculum survival in all surfaces. The disinfectants and the higher temperature contributed to the reduction of biofilm production in isolates of C. pseudotuberculosis. knowledge of these patterns is important for the establishment of disease control measures, given the questionable efficacy of the treatment and the immuno-prophylaxis of caseous lymphadenitis.(AU)

O semiárido nordestino do Brasil possui um bioma exclusivo, a caatinga, que apresenta temperatura máxima de 40oC e uma umidade relativa do ar de 56%. A caatinga é caracterizada por uma diversidade de plantas, entre elas Cereus jamacaru DC. (mandacaru), Poincianella microphylla Mart ex G. Don (catingueira), Pilosocereus gounellei F.A.C. Weber (xique-xique) e Mimosa tenuiflora (Willd.) Poir (jurema preta). A produção de ovinos e caprinos está em franca expansão, porém a linfadenite caseosa é uma enfermidade de alta prevalência na região. Objetiva-se com o presente estudo, verificar a sobrevivência e produção de biofilme em isolados de Corynebacterium pseudotuberculosis em temperaturas de 28oC, 37oC e 42oC, quando inoculado em superfícies de solo, madeira, arame e espinho e em plantas nas condições ambientais da caatinga. Além disto, foi verificado o efeito da aplicação dos desinfetantes clorexidine, hipoclorito e amônia quaternária sobre o solo, piso (lajota), madeira e vegetação de cobertura do solo. Foram selecionadas quatro amostras de C. pseudotuberculosis (dois caprinos e dois ovinos) para inoculação de acordo com a sua produção in vitro de biofilme. A adesão a microplacas foi utilizado para avaliar a capacidade de formação de biofilme das bactérias. As menores taxas de sobrevivência foram observadas quando isolados de C. pseudotuberculosis foram submetidos a uma temperatura de 42°C. Com relação as plantas do bioma caatinga, a contaminação na planta jurema-preta apresentou menores índices de sobrevivência. O desinfetante amônia quartenária promoveu uma menor sobrevivência do inóculo em todas as superfícies. Os desinfetantes e temperatura contribuíram para a redução na produção de biofilme nos isolados de Corynebacterium pseudotuberculosis. O conhecimento destes padrões é importante para o estabelecimento de medidas de controle da enfermidade, dada a eficiência questionável do tratamento e imunoprofilaxia da linfadenite caseosa.(AU)

Usnic acid-loaded polyaniline/polyurethane foam wound dressing: preparation and bactericidal activity.

The improved bactericidal activity of new composites for wound dressing prototypes represents an important strategy for development of more efficient devices that make use of synergistic interaction between components. The doping level of polyaniline represents a critical parameter for its corresponding biologic activity. In this work, it is explored the doping effect of usnic acid on undoped polyaniline, that introduces important advantages namely, improved bactericidal activity of polyaniline and the anti-biofilm properties of lichen derivative. The deposition of the resulting material on polyurethane foam potentializes its applicability as wound dressing, characterizing a new platform for application against Escherichia coli and Staphylococcus aureus.

Atividade antimicrobiana e antibiofilme de nanopartículas de prata sobre isolados de Aeromonas spp. obtidos de organismos aquáticos / Antimicrobial and antibiofilm activity of silver nanoparticles against Aeromonas spp. isolated from aquatic organisms

O uso indiscriminado de antimicrobianos tem proporcionado a algumas bactérias patogênicas a seleção de cepas multirresistentes, situação que pode ser agravada pela formação do biofilme. Desta forma, as nanopartículas de prata (AgNPs) vêm se destacando como uma alternativa inovadora, de baixo custo e eficiente contra doenças causadas por bactérias. O objetivo deste estudo foi determinar a atividade antimicrobiana das AgNPs e a interferência na formação do biofilme de Aeromonas spp. obtidas de organismos aquáticos. As AgNPs foram sintetizadas quimicamente utilizando como agente redutor o citrato trissódico e caracterizadas por espectrofotometria ultravioleta-visível (UV-Vis). A atividade antimicrobiana foi realizada contra três isolados pelo método de microdiluição em caldo para determinar a concentração bactericida mínima (CBM) e um cultivo com CCCP, um inibidor da bomba de efluxo, foi realizado para complementar o efeito das AgNPs. A interferência no biofilme foi realizada segundo o protocolo de formação e consolidado, além da caracterização desta estrutura de resistência por microscopia eletrônica de varredura. No teste da CBM, as AgNPs não foram capazes de inativar o crescimento dos isolados, ao passo que o nitrato de prata obteve eficiência em diferentes concentrações. Na presença do inibidor de bomba de efluxo, dos isolados analisados, um passou de resistente a sensível na presença das nanopartículas. As AgNPs foram eficazes em diminuir a formação de biofilme, como também atuaram sobre o biofilme consolidado em todos os isolados testados. Estes resultados indicam o potencial das nanopartículas de prata em interferir com o biofilme de Aeromonas spp. de organismos aquáticos e seres humanos.(AU)

The indiscriminate use of antibiotics has selected some pathogenic bacteria being multidrug-resistant, a situation that can be exacerbated by biofilms formation. Thus, silver nanoparticles (AgNPs) have been highlighted as an innovative alternative, low-cost and effective against bacterial diseases. The aim of this study was to determine the antimicrobial activity of AgNPs and the interference in Aeromonas spp. biofilm formation. The strains were obtained from aquatic organisms. The AgNPs were chemically synthesized using as reducing agent trisodium citrate and characterized by ultraviolet-visible spectroscopy (UV-Vis). The antimicrobial activity was carried out against three isolates by the microdilution broth method for determining minimum bactericidal concentration (CBM) and cultivation of CCCP, an inhibitor of the efflux pump, was carried out to complement the effect of AgNPs. Interference in the biofilm formation was performed according to the protocol and consolidated, within the resistance structure characterization by scanning electron microscopy. In the test of the CBM, the AgNPs were unable to inactivate the growth of the isolates, while the silver nitrate obtained efficiency in different concentrations. In the efflux pump inhibitor presence the isolates were analyzed, one went from resistant to nanoparticles to sensitive. The AgNPs were effective in reducing of biofilm formation and acted on the consolidated biofilm in all tested isolates. These results indicate the silver nanoparticles to interfere with Aeromonas spp. biofilm from aquatic organisms and human bodies.(AU)

Gene floR e a resistência ao florfenicol em isolados de Aeromonas spp. autóctones de organismos aquáticos / Gene floR and resistance to florfenicol in isolated Aeromonas spp. indigenous aquatic organisms

O gene floR descrito é descrito pela literatura como o responsável pela resistência ao florfenicol, que é um antimicrobiano amplamente utilizado na aquicultura. Esse gene já foi relatado em muitas espécies de bactérias, inclusive no gênero Aeromonas. Essas bactérias causam alta mortalidade na piscicultura trazendo prejuízos econômicos. É importante que haja estudos sobre esse gene e possíveis mutações que possam levar a alterações na estrutura e função da proteína. Os objetivos desse estudo foram caracterizar o gene floR em isolados de Aeromonas spp. obtidas do Vale do São Francisco e verificar se a presença desse gene está associada com a resistência ao florfenicol. Foram realizadas reações em cadeia da polimerase (PCR) para a presença do gene floR em 27 isolados de Aeromonas spp.. Amostras positivas para a presença do gene foram sequenciadas e analisadas quanto à presença de polimorfismos por meio de alinhamentos. Os diferentes haplótipos detectados foram utilizados para análises com os programas SIFT e PolyPhen para predição de alteração de função proteica. A modelagem estrutural da proteina codificada pelo gene floR foi realizada com o programa Modeller e, os modelos foram avaliados pelo Procheck, Verify3D e Whatif. A similaridade da estrutura tridimensional da proteína referência com as estruturas tridimensionais das proteínas codificadas pelos diferentes haplótipos foi comparada através do TM-align. A resistência das bactérias ao florfenicol foi avaliada através do teste de microdiluição em caldo, o qual também foi realizado na presença do carbonil cianeto m-clorofenil hidrazona para verificar o efeito da inibição da bomba de efluxo sobre tal resistência. Dos vinte e sete isolados avaliados quanto a presença do gene floR, 14 isolados foram positivos e 10 foram sequenciados, o que permitiu a identificação de três polimorfismos no gene floR, que levaram a construção de três haplótipos diferentes (TAA, TTA e CTG). As análises realizadas com os programas SIFT e PolyPhen apontaram que os haplótipos TTA e TAA provavelmente poderiam alterar a estrutura e função da proteína. As proteínas modeladas para os três haplótipos demonstraram possuir praticamente a mesma conformação estrutural entre si. Todos os isolados que apresentaram o gene foram resistentes ao florfenicol e aqueles que não apresentavam foram sensíveis. O teste na presença do Carbonil Cianeto m-Clorofenil Hidrazona foi realizado para três isolados, cada isolado representando um haplótipo, sendo possível observar a inibição do crescimento bacteriano em todas as concentrações independente do haplótipo. Os resultados obtidos nesse estudo mostram que a resistência ao flofenicol em Aeromonas spp. pode ser explicada pela presença do gene floR, e que esse gene está relacionado com uma bomba de efluxo. As mutações verificadas no gene floR, parecem não estar envolvidas com alteração de estrutura e função da proteína codificada por esse gene.(AU)

The floR gene is described in related literature as responsible for resistance to florfenicol, which is a widely used antimicrobial agent in aquaculture. This gene has been reported in many species of bacteria, including the genus Aeromonas. These bacteria cause high mortality in fish farming bringing economic losses. It is important that studies of this gene and possible mutations that can lead to changes in the structure and function of the protein. The aim of this study was to characterize the floR gene in isolates of Aeromonas spp. and check if the presence of this gene is associated with resistance to florfenicol in Aeromonas spp. obtained from the San Francisco Valley. PCR (Polymerase Chain Reaction) were also performed to verify the presence of the floR gene in 27 isolates of Aeromonas spp. Positive samples for the presence of the gene were sequenced and analyzed for the presence of polymorphisms using alignments. Different haplotypes detected were used for analysis with the SIFT and PolyPhen programs for prediction of changes in protein function. The structural modeling of protein encoded by the floR gene was performed using the Modeller software, and the models were evaluated by Procheck, Verify3D and Whatif. The similarity of the dimensional structure of reference protein with the dimensional structures of the proteins encoded by the different haplotypes was compared by TM-align. Bacterial resistance to florfenicol was assessed by the microdilution test, which was also performed in the presence of carbonyl cyanide m-chlorophenyl hydrazone to verify the effect of inhibiting the efflux pump. 14 isolates were positive for the presence of floR gene and 10 were sequenced and allowed the identification of three polymorphisms in the floR gene, which led to construction of three different haplotypes (TAA TTA and CTG). The analyzes carried out with the SIFT and PolyPhen programs showed that the TTA and TAA haplotypes could probably change the protein structure-function. Proteins modeled for the three haplotypes were found to have substantially the same structural conformation with each other. All isolates presenting the gene were resistant to florfenicol and those who did not have were sensitive. The test in the presence of carbonyl cyanide m-chlorophenylhydrazone was conducted for three isolates, representing each single haplotype and was observed inhibition of bacterial growth at all concentrations independent of the haplotype. The results of this study show that resistance to flofenicol in Aeromonas spp. may be explained by the presence of floR gene and that this gene is associated with an efflux pump. Mutations observed in floR gene do not appear to be involved with chenges in structure and function of the protein encoded by gene.(AU)

Atividade antimicrobiana in vitro do extrato etanólico bruto da folha da Hymenaea martiana Hayne frente às Staphylococcus spp. e avaliação de seu potencial como desinfetante em cabras / In vitro antimicrobial activity of ethanolic extract of Hymenaea martiana Hayne leaf on strains of Staphylococcus spp. and evaluation of its potential as a disinfectant in goats

Este estudo objetivou avaliar a ação antimicrobiana e antisséptica do extrato etanólico bruto da folha da Hymenaea martiana (Jatobá). O estudo foi realizado no Laboratório de Microbiologia e Imunologia da UNIVASF, na cidade de Petrolina-PE. Os extratos foram preparados utilizando diferentes diluentes, sendo estes: álcool etílico absoluto e a água destilada. Em seguida, foi empregada a técnica da Concentração Inibitória Mínima (MIC) e da Concentração Bactericida Mínima (CBM). Todos os ensaios foram realizados em triplicata. A CBM média do extrato diluído em etanol foi de 358µg/µL e do extrato diluído em água destilada foi igual a 520,82µg/mL. Não houve diferença (P<0,05) quanto à inibição bacteriana para o extrato diluído em álcool etílico absoluto ou água destilada autoclavada. Ao comparar a atividade do extrato diluído em álcool etílico absoluto e a relação com a presença do gene blaZ, observou-se que os isolados negativos para o gene pesquisado apresentaram uma CBM igual a 412,3µg/mL, e, quando comparadas aos que foram positivos para o gene blaZ, que foi de 308,80µg/mL, contudo sem diferença estatística. Quanto à inibição das bactérias utilizando extrato aquoso, a atividade foi igual para as bactérias com ou sem o gene (520,82µg/mL). Desse modo, tanto o extrato diluído em álcool etílico absoluto quanto em agua destilada autoclavada demonstrou atividade antimicrobiana, sugerindo que ocorreu extração de substâncias bioativas. Em relação ao potencial antisséptico, H. martiana teve ação pareada com o cloro, contudo aquele agiu mais rapidamente, enquanto o cloro agiu de modo ideal uma hora após a aplicação; ambos os resultados destacam que o extrato etanólico bruto das folhas de H. martiana possui potencial de combate à proliferação de bactérias ambientais e infecciosas, surgindo como uma forma de prevenir a mastite.(AU)

This study aimed to evaluate the antimicrobial and antiseptic action of the crude ethanolic extract of Hymenaea martiana leaves. The study was conducted at the Laboratory of Microbiology and Immunology of UNIVASF, city of Petrolina, state o Pernambuco. The extracts were prepared using different solvents, such as absolute ethyl alcohol and distilled water. Then, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) techniques were used. All assays were performed in triplicate. The average of MBC extract diluted in ethanol was 358µg/mL, and the extract diluted in distilled water was equal to 520.82µg/mL. There was no difference (P<0.05) and bacterial inhibition to extract diluted in absolute ethanol or autoclaved distilled water. Comparing the activity of the extract diluted in ethanol and the relation with the presence of blaZ gene, it was observed that the negative strains for there searched gene showed a MBC equal to 412.3µg/mL in relation to those that were positive for blaZ gene, that was 308.80µg/µL, and, however, there was no statistical difference. The bacterial inhibition activity using an aqueous extract was equal for the bacteria that had or not the blaZ gene (520.82µg/mL). Thus, the extract diluted in absolute ethanol in autoclaved distilled water as demonstrated antimicrobial activity, suggesting that occurred extraction of bioactive substances. Regarding the antiseptic potential, H. martiana had the same action of chlorine, although, this acted immediately, while the chlorine action happened properly an hour after the application. Both results pointed out that the crude ethanolic extract of H. martiana leaves has potential to combat the proliferation of environmental and infectious bacteria, emerging as a way to prevent mastitis.(AU)