Desenvolvimento e caracterização de nanocápsulas poliméricas contendo carvacrol para inibição de Salmonella enteritidis / Development and characterization of polymeric nanocapsules containing carvacrol for inhibition from Salmonella enteritidis

Avaliou-se caracterização físico-química, ação bactericida e estabilidade de nanocápsulas poliméricas de Eudragit contendo carvacrol preparadas utilizando técnica de deposição interfacial do polímero pré-formado. As nanocápsulas apresentaram diâmetro médio de 146 nm, PDI de 0,181 e potencial zeta de + 23,44 mV e a concentração bactericida mínima necessária para inativar Salmonella Enteritidis foi de 0,331 mg/mL. A solução contendo nanocápsulas manteve suas características físico-químicas e atividade bactericida inalteradas durante os 45 dias do teste de estabilidade, demonstrando características promissoras para o desenvolvimento de um sanitizantes para uso em indústria produtora de ovos e frigorífico de aves.

Encapsulação de antimicrobianos naturais em nanolipossomas para inibição de bactérias causadoras de surtos alimentares / Encapsulation of natural antimicrobials in nanoliposomas for the inhibition of bacterials causing foodborne disease

Avaliou-se ação bactericida e caracterização físico-química de duas diferentes formulações de nanolipossomas preparados utilizando técnica de hidratação do filme lipídico. Nanolipossomas contendo carvacrol apresentaram diâmetro médio de 270,83 nm, PDI de 0,20 e potencial zeta de + 8,64 mV. Nanolipossomas contendo carvacrol e nisina apresentaram diâmetro médio de 205,25 nm, PDI de 0,31 e potencial zeta de - 22,48 mV. Para ambas formulações a concentração bactericida mínima foi de 3,53 mg/mL para Salmonella e 5,3 mg/mL para Escherichia coli e Staphylococcus aureus. Não ocorreu efeito sinérgico entre carvacrol e nisina, porém os nanolipossomas contendo carvacrol apresentaram ação bactericida contra os microrganismos testados, demonstrando potencial para sua utilização no desenvolvimento de um sanitizante para uso em indústrias de alimentos.

Evaluation antibacterial and antibiofilm activity of the antimicrobial peptide P34 against Staphylococcus aureus and Enterococcus faecalis

ABSTRACT The adhesion ability of bacteria to abiotic surfaces has important implications in food industries, because these organisms can survive for long periods through the biofilm formation. They can be transferred from one place to another in the industry causing contamination of the food processing environment. In this study, the antibacterial and antibiofilm activities of the antimicrobial peptide P34, characterized as a bacteriocin-like substance (BLS P34) were tested against planktonic and sessile cells of Staphylococcus aureus and Enterococcus faecalis isolated from foods. The BLS P34 showed inhibitory effect against all planktonic cells of E. faecalis. The inhibition of biofilm formation and the eradication of pre-formed biofilm were evaluated with the crystal violet assay and with the reduction of 3-bromide [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. The BLS P34 promoted a reduction of percentage of adhered microbial cells on the surface, not being able to perform the complete elimination of biofilm formation. The metabolic activity of S. aureus biofilms decreased considerably between 41-95%. However, E. faecalis cells showed up metabolically stimulated. The BLS P34 has the potential antibiofilm for the species S. aureus. Studies suggest more detailed approaches to a better understanding of the interactions between the antimicrobial and bacterial cells within the biofilm structure.

Commonly screened antibiotics in raw milk from dairy plants under State Inspection in Rio Grande do Sul, Brazil / Antibióticos comumente investigados no leite cru em laticínios sujeitos a Inspeção Estadual no Rio Grande do Sul, Brasil

This study aimed at identifying the commonly screened antibiotics during the dairy processing routine in raw-milk receiving points in plants inspected by the official services in the state of Rio Grande do Sul (RS), from January 2014 to February 2015. Among the 36 participating industries, the most commonly screened antibiotics were beta lactams (100%) and tetracyclines (69%). The antibiotics screened at the milk receiving point were chosen because of the practicality and speed in performing the screening (67%), rather than specific knowledge on which antibiotics the milk suppliers used. (22%).(AU)

Este estudo teve por objetivo identificar os antibióticos comumente investigados durante a rotina de processamento do leite cru recebido em laticínios sob Inspeção Estadual no Rio Grande do Sul (RS), entre janeiro de 2014 e fevereiro de 2015. Entre as 36 indústrias participantes, os antibióticos mais comumente investigados foram beta-lactâmicos (100%) e tetraciclinas (69%). A seleção por quais antibióticos investigar no recebimento do leite foi influenciada pela praticidade e rapidez na execução da análise (67%), em detrimento do conhecimento específico sobre quais antibióticos eram utilizados pelos produtores de leite (22%).(AU)

Activity of the antimicrobial peptide P34 against bovine alphaherpesvirus type 1 / Atividade do peptídeo antimicrobiano P34 contra o alfaherpesvírus bovino tipo 1

ABSTRACT: Previous studies have demonstrated the antimicrobial activity of the peptide P34. In this study, the antiviral potential of P34 and the in vitro mechanism of action were investigated against bovine alphaherpesvirus type 1 (BoHV1). P34 exhibited low toxicity, a high selectivity index (22.9) and a percentage of inhibition of up to 100% in MDBK cells. Results from antiviral assays indicated that P34 did not interact with cell receptors, but it was able to inhibit the viral penetration immediately after pre-adsorption. In addition, BoHV1 growth curve in MDBK cells in the presence of P34 revealed a significant reduction in virus titer only 8h post-infection, also suggesting an important role at late stages of the replicative cycle. Virucidal effect was observed only in cytotoxic concentrations of the peptide. These findings showed that the antimicrobial peptide P34 may be considered as a potential novel inhibitor of in vitro herpesviruses and must encourage further investigation of its antiherpetic activity in animal models as well as against a wide spectrum of viruses.

RESUMO: A atividade antimicrobiana do peptídeo P34 já foi previamente demonstrada. Neste estudo, o potencial antiviral do P34 e o mecanismo de ação in vitro contra o alfaherpesvírus bovino tipo 1 (BoHV1) foram investigados. O P34 exibiu baixa toxicidade, alto índice de seletividade (22.9) e percentagem de inibição viral de até 100% em células MDBK. Os resultados dos ensaios antivirais indicaram que não interage com receptores celulares, mas é capaz de inibir a penetração viral, imediatamente após a pré-adsorção. Além disso, a curva de crescimento do BoHV1 em células MDBK na presença do P34 revelou uma significativa redução no título somente após 8h de infecção, sugerindo também uma importante atividade do peptídeo nas fases finais do ciclo replicativo. Efeito virucida frente / BoHV1 foi observado apenas em concentrações citotóxicas do peptídeo. Os dados obtidos indicam que o peptídeo antimicrobiano P34 pode ser considerado um potencial composto inibidor de herpesvírus, in vitro, e estimulam posteriores investigações sobre sua atividade anti-herpética em modelos animais, bem como contra outros vírus.

Antiviral activity of a Bacillus sp: P34 peptide against pathogenic viruses of domestic animals

P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity against EAV and FHV-1. The peptide P34 inhibited the replication of EAV by 99.9% and FHV-1 by 94.4%. Virucidal activity was detected only against EAV. When P34 and EAV were incubated for 6 h at 37 °C the viral titer reduced from 10(4.5) TCID50 to 10(2.75) TCID50, showing a percent of inhibition of 98.6%. In conclusion, our results demonstrated that P34 inhibited EAV and FHV-1 replication in infected cell cultures and it showed virucidal activity against EAV. Since there is documented resistance to the current drugs used against herpesviruses and there is no treatment for equine viral arteritis, it is advisable to search for new antiviral compounds to overcome these infections.

Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage

The antimicrobial activity of the bacteriocin-like substance (BLS) P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g-1) previously inoculated with a suspension of 10² cfu g-1 of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe) against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products.

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

Inhibition of listeria monocytogenes in minas frescal cheese by free and nanovesicle-encapsulated nisin

The effectiveness of free and nanovesicle-encapsulated nisin to control Listeria monocytogenes in Minas Frescal cheese was investigated. Commercial nisin was encapsulated into liposomes of partially purified soy lecithin. Free (0.1 mg/mL and 0.25 mg/mL) and nanovesicle-encapsulated nisin (0.25 mg/mL) were applied onto the surface of cheese samples, and L. monocytogenes was inoculated before incubation at 6-8°C for 28 days. A bactericidal effect was observed with 0.25 mg/mL free nisin; a bacteriostatic effect was observed for liposome-encapsulated nisin and 0.1 mg/mL free nisin. Free nisin was more efficient than nisin-loaded liposomes in controlling L. monocytogenes. Possible reasons for this behavior, and also the significance of nisin to soft cheeses are discussed. Nisin acted as a suitable barrier within hurdle technology, potentially extending the shelf-life and safety of fresh cheeses.

Hydrolytic potential of a psychrotrophic Pseudomonas isolated from refrigerated raw milk

The production of extracellular hydrolases by a psychrotrophic bacterium isolated from refrigerated raw milk, and identified as a Pseudomonas sp. belonging to the Pseudomonas jenssenii group, was studied. This bacterium produced proteolytic and lipolytic enzymes in all media investigated (skim milk, cheese whey, casein broth, and tryptone soy broth). High levels of á-glucosidase were produced in skim milk broth. Hydrolytic enzymes detected in skim milk broth are of particular concern, indicating that these enzymes could be produced by Pseudomonas sp. during the cold storage of raw milk, contributing to the spoilage problem in milk and dairy products.

Perfil de resistência a antimicrobianos de cepas Pseudomonas aeruginosa isoladas de ralos e pias de enfermarias hospitalares em Santa Catarina, Brasil / Antimicrobial resistance profile of Pseudomonas aeruginosa strains isolated from sinks and drains in hospital wards in Santa Catarina, Brazil

Pseudomonas aeruginosa é um micro-organismo oportunista que contribui para ocorrência de infecções hospitalares. A ocorrência desta bactéria foi investigada em 104 amostras isoladas de pias e ralos de enfermarias de dois hospitais de Santa Catarina. P. aeruginosa foi identificada em 47% das amostras. A suscetibilidade a antimicrobianos foi avaliada através do teste de difusão em agar, segundo recomendações do Commitee for Clinical Laboratory Standards (CLSI). Observou-se elevada resistência para cefalotina(100%), seguida pelo imipenem (34,7%), gentamicina (26,5%) e ceftriaxona (18,3%). As linhagens isoladas apresentaram elevadasensibilidade à ciprofloxacina e norfloxacina, 100% e 95,9%, respectivamente. Cerca de 40% das linhagens apresentaram multirresistência. Os resultados de resistência foram correlacionados com os principais antimicrobianos utilizados nos hospitais. Esses resultados indicam que pias e ralos em enfermarias hospitalares representam um risco potencial para a saúde pública, por apresentarem bactérias com elevada resistência a antimicrobianos, demonstrando a necessidade do uso racional de antibióticos nos hospitais edesinfecção adequada desses locais

Pseudomonas aeruginosa is an opportunistic microorganism that contribute to the occurrence of hospital infections. The occurrence of this bacterium was investigated in 104 samples isolated from sinks and drains of two hospitals in Santa Catarina. P. aeruginosa was identified in 47% of the samples. The antimicrobial susceptibility was evaluated by the agar diffusiontest, according to recommendations of the Commitee for Clinical Laboratory Standard (CLSI). It was observed a high resistance to cefalotin (100%), followed by imipenem (34.7%), gentamycin (26.5%) and ceftriaxone (18.3%). The strains showed elevatedsensitivity to ciprofloxacin and norfloxacin, 100% and 95.9%, respectively. Multidrug resistance was observed in about 40% of strains. The results for antimicrobial resistance correlate with the major antimicrobial agents used in the hospitals. Theseresults indicate that sinks and drains in hospital wards represent a potential hazard to public health, to present potentially dangerous bacteria with resistance to antibiotics, demonstrating the need for rational use of antibiotics in hospitals and disinfection of those locations

Efficacy of modified atmosphere packaging to control Sitophilus spp. in organic maize grain

The effect of modified atmosphere packaging on the mortality of Sitophilus spp. in organic maize grain was studied. Maize grains were packed with five different atmospheres consisting of 20 percent O2, or 20, 40, 60, 80 percent CO2 with remaining N2. The packages were stored for 30 days at 26°C. CO2 and O2 concentrations were monitored inside the packages during the storage. The moisture content, titratable acidity and pH were analysed on the first and thirtieth day. At 5, 15 and 30 days of storage no significant differences were observed in the percentages of dead insects when CO2 was used. The results of progeny indicated that from the fifth day the number of emerging insects was low at 20, 60 and 80 percent CO2. Complete inhibition of the insects was achieved with 30 days of exposure in CO2 atmospheres.

O efeito da embalagem em atmosferas modificadas sobre os insetos Sitophilus spp. nos grãos de milho orgânico foi estudado. Os grãos de milho foram empacotados em cinco atmosferas iniciais diferentes de 20 por cento de O2, ou 20, 40, 60, 80 por cento de CO2 com balanço de N2. As embalagens foram armazenadas por 30 dias a 26ºC. As concentrações de CO2 e O2 foram monitoradas dentro das embalagens durante o armazenamento. As análises de teor de umidade, acidez e pH foram também realizados no primeiro e trigésimo dia. Os resultados mostraram que no quinto, décimo quinto e trigésimo dia de estocagem não foram encontrados diferenças significativas nas porcentagens de insetos mortos quando CO2 foi usado. Os resultados relativos à progênie indicaram que no quinto dia o número de insetos emergentes foi menor a 20, 60 e 80 por cento CO2. A completa inibição dos insetos foi atingida com 30 dias de exposição em atmosferas de CO2.

Production of bacteriocin-like substances by lactic acid bacteria isolated from regional ovine cheese

Lactic acid bacteria (LAB) were isolated from ovine milk and cheeses manufactured in the South Region of Brazil. Among 112 bacterial isolates investigated, 59 were chosen through a screening for LAB. Among these 59 strains of LAB, 21 percent showed antimicrobial, proteolytic and lipolytic activities. Based on this screening, Lactobacillus plantarum LCN 17 and Lactobacillus rhamnosus LCN 43 were selected and tested for the production of bacteriocin-like substances (BLS). The BLS produced by both isolates showed antimicrobial activity against Listeria monocytogenes, whereas that produced by L. plantarum LCN 17 presented higher stability to different temperature, pH and enzyme treatments. These strains present potential for production of BLS, and for use as starter cultures.

Adhesion and biocides inactivation of Salmonella on stainless steel and polyethylene

The adhesion of Salmonella (S.) strains to stainless steel and polyethylene and their inactivation by biocides used in food industry was investigated. Coupons of stainless steel and polyethylene were immersed in bacterial suspensions of S. Enteritidis, S. Typhimurium, and S. Bredeney during 15, 30, and 60 minutes, and submitted to different concentrations of peracetic acid (PAA), sodium hypochlorite (NaOCl), and quaternary ammonium (Quat) sanitizers. Hydrophobicity of the surfaces was evaluated by contact angle measurements using the sessile drop method and bacterial adhesion was accompanied through bacterial counts and scanning electron microscopy (SEM). Results indicated that the three serovars of Salmonella presented similar adhesion to both materials (5.0 to 6.5 log cfu cm-2). The time of exposure did not influence the counts of adhered cells on both surfaces, however SEM revealed larger clusters of S. Enteritidis on both materials, not found for the other serovars. S. Enteritidis presented lower sessile drop angle on polyethylene, indicating hydrophilic properties of this material. The biocides were not able to inactivate all the microorganisms adhered on both surfaces. At least 1 log cfu cm-2 of all serovars tested remained viable after the exposure to different biocide concentrations. In general, higher counts of survivors were observed on polyethylene disinfected with different concentrations of biocides. S. Bredeney e S. Typhimurium were more resistant than S. Enteritidis to PAA, whilst S. Enteritidis presented smaller reduction rates to NaOCl. This last biocide was able to reduce Salmonella counts in approximately 3.0 to 4.0 log cm-2. When adhered to polyethylene, the serovars S. Typhimurium and S. Enteritidis were more resistant to Quat than S. Bredeney in all concentrations tested, and the numbers of S. Enteritidis remained almost unaltered. On stainless steel disinfected by Quat, S. Bredeney presented higher numbers of survivors.

Avaliação da resistência de Salmonella à ação de desinfetantes ácido peracético, quaternário de amônio e hipoclorito de sódio / The resistance of Salmonella strains to the biocides peracetic acid, quaternary ammonium and sodium hypochlorite

Este estudo avaliou a resistência de três cepas de Salmonella frente aos desinfetantes ácido peracético, quaternário de amônio e hipoclorito de sódio. S. Enteritidis (SE86), responsável por mais de 90% dos surtos alimentares ocorridos no Rio Grande do Sul (RS), foi comparada com S. Typhimurium e S. Bredeney, ambas não envolvidas na ocorrência de salmonelose. A resistência aos desinfetantes foi avaliada por meio de teste de suspensão, conforme preconizado pelo Ministério da Agricultura, Pecuária e Abastecimento. Todos os desinfetantes avaliados, nas concentrações indicadas pelos fabricantes, foram capazes de inativar as três cepas de Salmonella, entretanto, SE86 apresentou maior resistência ao hipoclorito de sódio a 400 ppm, além de ser a única cepa bacteriana capaz de sobreviver por até 15 minutos de exposição a 200 ppm desse desinfetante. Uma vez que o hipoclorito de sódio, nestas condições, é empregado com frequência como agente de desinfecção em indústrias de alimentos e serviços de alimentação, as características acima mencionadas podem contribuir para que esse microrganismo seja responsável pelos surtos de salmoneloses ocorridos no RS. Em função da importância desses micro-organismos como agentes patogênicos alimentares, cuidados especiais devem ser tomados nos processos de desinfecção e contaminação cruzada por Salmonella.

The resistance of Salmonella strains to the biocides peracetic acid, quaternary ammonium and sodiumhypochlorite was assessed. S. Enteritidis (SE86), which is responsible for more than 90% of food-borneoutbreaks occurred in Rio Grande do Sul (RS) was compared to S. Typhimurium and S. Bredeney, bothnot related to salmonellosis. The bacterial resistance to biocides was evaluated using the suspension test asrecommended by The Brazilian Agriculture Ministry. The three biocides were able to inactivate all of themicroorganisms tested, when the concentrations indicated by the manufacturers were used. However, SE86was resistant to 400 ppm sodium hypochlorite, and survived for up to 15 minutes of exposure to 200 ppmof this biocide, which was not demonstrated by the other bacteria strains. Since the sodium hypochlorite,at such concentration and time of exposure, is frequently used at food industries and food services, andowing to the above cited resistance characteristic, this condition could be contributing for the SE86 withfood-borne salmonellosis outbreak occurred in RS. In view of the importance of these microorganismsas alimentary pathogens, special cares in the food disinfection processes and cross-contamination withSalmonella must be taken.

Poultry feather hydrolysate as a protein source for growing rats / Hidrolisado de penas como fonte de proteína para ratos

Feather protein has been considered as a protein complement for animal diets, since it is largely available as a by-product of poultry processing. In this work, a feather protein hydrolysate produced by the keratinolytic microorganism Vibrio sp. kr2 was evaluated as a feed additive. Wistar rats were fed seven experimental diets (n = 6 rats perdiet) containing different protein sources: casein (CAS), soybean protein, feather hydrolysate, feather meal, and soybean protein supplemented with 10 or 20% (w/w) feather hydrolysate, and 20% feather hydrolysate supplemented methionine. Values for weight gain, feed ingest, true digestibility, feed: gain ratio, Protein Efficiency Ratioand Net Protein Ratio were similar for diets containing soybean proteinand 20% feather hydrolysate supplemented methionine. Lowest values for all nutritional parameters were observed for diets containing soybean protein supplemented with 10 or 20% (w/w) feather hydrolysate, feather hydrolysate and feather meal. Protein source had a considerable influence in the final weight of liver, kidney and hearth, although no significant differences were observed for brains. These results indicate that feather hydrolysate may be useful as supplementary protein in feed formulations.

A proteína da pena é uma boa fonte proteica para dietas de animais, sendo um material de grande disponibilidade como subproduto da produção avícola. Neste trabalho, um hidrolisado protéico de penas produzido pelo microrganismo queratinolítico Vibrio sp. kr2 foi avaliado como suplemento em rações. Ratos da linhagem Wistar foram alimentados com sete dietas experimentais (n = 6 ratos pordieta) contendo diferentes fontes de proteína: caseina (CAS), proteína de soja, hidrolisado de pena, farinha de pena, e proteína de soja suplementada com 10 ou 20% (w/w) hidrolisado de pena, ou 20% hidrolisado de pena suplementado com metionina. Os valores de ganho de peso, consumo, digestibilidade verdadeira, coeficiente deeficiência alimentar, coeficiente de eficiência proteica (PER) e eficiência líquida proteica (NPR) foram similares para as dietas contendo proteína de soja e proteína de soja suplementada com 20% hidrolisado de pena e metionina. Valores inferiores para todos parâmetros nutricionais foram observados para as dietas contendo 10 ou 20% hidrolisado de pena, hidrolisado de pena e fariha de pena. A fonte protéica influenciou no peso final do fígado, rins e coração, porém as diferenças não foram significativas para cérebros. Estes resultados indicam que o hidrolisado de penas pode ser usado como fonte de proteína suplementar na formulação de rações desde que suplementados com metionina.

Phenotypic and genotypic characterization of Salmonella Enteritidis isolates

In order to study the epidemiology of Salmonella Enteritidis outbreaks and determine the source of contamination so that a recurrence can be avoided, detailed characterization is necessary. Thus, the purpose of this study was to verify whether rep-PCR was able to discriminate among Salmonella Enteritidis isolates. Phage typing, detection of virulence genes and antimicrobial resistance testing were also associated to rep-PCR results. One hundred and two S. Enteritidis isolates from broiler carcasses, food, human, pigs, poultry-related samples, and nine isolates from other countries were genotypically typed by REP-PCR, ERIC-PCR and BOX-PCR, collectively called rep-PCR. Phage typing, detection of virulence genes and antimicrobial resistance testing were also performed. Only three fingerprinting profiles were obtained with each rep-PCR method, with the majority of isolates belonging to the same profile. No relationship was observed between genotypic profile and year, place of isolation or source of infection. However, the less frequent rep-PCR profiles showed single antimicrobial resistance patterns. Although few strains isolated from swine were analyzed, different antimicrobial resistance patterns were observed. Furthermore, phage type 4 was not found in swine isolates. rep-PCR showed a lower discriminatory power as compared with antimicrobial resistance and phage typing, but the combination of genotypic and phenotypic methods was more discriminatory than any method alone, resulting in 48 different types.

Uma caracterização detalhada de Salmonella Enteritidis é necessária para que possa ser desenvolvido o estudo da epidemiologia dos surtos causados por este organismo, bem como a determinação da fonte de contaminação, evitando que ocorram novos surtos. Assim, o objetivo deste estudo foi verificar se a rep-PCR era capaz de diferenciar isolados de S. Enteritidis. A fagotipagem, a detecção de genes de virulência e a determinação de resistência antimicrobiana foram associadas aos resultados da rep-PCR. Cento e duas S. Enteritidis isoladas de carcaças de frango, alimentos prontos para consumo, humanos suínos, amostras relacionadas a aves, e nove isolados de outros países foram genotipicamente tipados por REP-PCR, ERIC-PCR e BOX-PCR, juntamente chamados de rep-PCR. A fagotipagem, a detecção de genes de virulência e a determinação de resistência antimicrobiana também foram realizadas. Somente três padrões de fingerprinting foram obtidos com cada método de rep-PCR, sendo que a maioria dos isolados pertenceu ao mesmo perfil. Nenhuma relação foi observada entre o perfil genotípico e o ano, o local de isolamento e a fonte de infecção. Entretanto, os perfis menos freqüentes de rep-PCR apresentaram padrões de resistência antimicrobiana únicos. Embora poucas amostras de suínos tenham sido analisadas, diferentes padrões de resistência antimicrobiana foram observados. Além disso, o fagotipo 4 não foi encontrado em isolados de suínos. A rep-PCR apresentou um menor poder discriminatório quando comparada com a resistência antimicrobiana e com a fagotipagem, mas a combinação dos métodos genotípicos e fenotípicos foi mais discriminatória do que qualquer método isolado, resultando em 48 tipos diferentes.

Keratinolytic bacteria isolated from feather waste

The aim of this study was to characterize keratinolytic bacteria isolated from feather waste. Four isolates were selected after growth on solid medium with feather meal as sole carbon and nitrogen source and screened for proteolytic activity on milk agar plates. Three isolates were Gram-negative (belonged to the genera Burkholderia, Chryseobacterium and Pseudomonas) and one was Gram-positive (Microbacterium sp.). These bacteria grew on diverse keratin wastes such as feather meal, raw feathers, chicken nails, hair and wool. Keratinase activity was detected during growth, but the complete degradation of these substrates was not always achieved. The proteolytic character of crude enzymes was assessed using azokeratin and azocasein as substrates. The keratinases were active on both substrates and were similar in keratin hydrolysis when compared with commercially available microbial peptidases. These novel keratinolytic isolates have potential biotechnological use in processes involving keratin hydrolysis.

O objetivo deste estudo foi caracterizar bactérias queratinolíticas isoladas resíduos de penas. Quatro isolados foram selecionados após crescimento em meio sólido contendo farinha de pena como única fonte de carbono e nitrogênio e avaliados quanto a atividade proteolítica em placas de ágar leite. Foram identificadas três linhagens Gram-negativas (pertencentes aos gêneros Burkholderia, Chryseobacterium e Pseudomonas) e uma Gram-positiva (Microbacterium sp.). Estas bactérias cresceram em vários resíduos queratinosos como farinha de pena, penas de frango, unhas de frango, pelos e lã. A atividade queratinolítica foi observada durante crescimento, mas a degradação completa dos substratos não foi observada em todos os casos. O caráter proteolítico das enzimas foi determinado usando azoqueratina e azocaseína como substratos. As queratinases foram ativas em ambos substratos e apresentaram hidrólise de queratina comparável a peptidases microbianas disponíveis comercialmente. Estes novos isolados queratinolíticos apresentam potencial uso biotecnológico em processos relacionados com hidrólise de queratina.

Production of an extracellular keratinase from Chryseobacterium sp. growing on raw feathers

The strain Chryseobacterium sp. kr6 shown to be useful for biotechnological purposes such as hydrolysis of poultry feathers and de-hairing of bovine pelts. The effect of temperature, initial pH and media composition on protease production by this keratinolytic strain was studied. The enzyme was produced between 25 and 37ºC, with maximum activity and yield at 30ºC. When protease production was tested in media with different initial pH, maximum activity was observed when cultivation was carried out at 30ºC and initial pH ranging from 6.0 to 8.0. Higher activity was observed when feathers or feather meal were used as growth substrates, followed by soybean meal. The addition of carbohydrates or surfactants to feather broth resulted in decrease in keratinolytic activity.

Screening for antimicrobial activity among bacteria isolated from the Amazon Basin

Bactérias produtoras de atividade antimicrobiana foram identificadas entre 86 isolados de ambientes aquáticos da Bacia Amazônica. Destes, 59 isolados (68.6 per center) apresentaram atividade antimicrobiana contra pelo menos uma bactéria indicadora. A atividade inibitória foi principalmente observada contra bactérias Gram-positivas, como Listeria monocytogenes e Bacillus cereus. As substâncias antimicrobianas produzidas por 19 linhagens que demonstraram maior atividade inibitória foram parcialmente caracterizadas, apresentando resistência térmica até 100ºC e resistência parcial ao tratamento proteolítico. Algumas substâncias foram parcialmente inativadas somente quando tratadas com ácido tricloroacético ou com pronase E na concentração de 2 mg ml-1. A detecção da atividade antimicrobiana em géis de poliacrilamida mostrou que os compostos apresentaram peso molecular inferior à 14 kDa. Várias linhagens apresentaram atividade antibacteriana, que em alguns casos estaria relacionada com peptídios antimicrobianos. O potencial destes microrganismos para produzir substâncias antimicrobianas é grande e merece ser mais explorado.