Phenotypic and genotypic characterization of clinically relevant bacteria isolated from dental waste and waste workers' hands, mucosas and coats.

Infectious wastes are potential sources of pathogenic micro-organisms, which may represent a risk to the professionals who manage them. In this study, we aimed to characterize the infectious bacteria present in dental waste and waste workers. The dental waste produced over 24 h was collected and waste workers were sampled by swabbing. Isolate resistance profiles were characterized by Vitek® and PCR and biofilm formation by Congo Red agar, string test and microtitre assay. To assess similarity between the waste and the workers' samples, a random amplified polymorphic DNA test was used. Twenty-eight bacteria were identified as clinically relevant. The most frequent gene was blaTEM present in five Gram-negative micro-organisms, and one blaSHV in Klebsiella pneumoniae. All Pseudomonas aeruginosa were positive to extracellular polymeric substances formation, except one isolated from a worker. Klebsiella pneumoniae had negative results for the string test. Pseudomonas aeruginosa showed better adherence at 25°C after 48 h of incubation and K. pneumonia had the best biofilm formation at the same temperature, after 24 h. The similarity between P. aeruginosa recovered from dental waste and from workers was low, however, it is important to note that a pathogen was found on a worker's hands and that improvements in biosafety are required. SIGNIFICANCE AND IMPACT OF THE STUDY: Infectious dental waste can contain clinically relevant bacteria with important resistance and biofilm profiles. These micro-organisms could be transmitted to waste workers, other professionals and patients if the principles of biosafety measures are neglected. To our knowledge, no study has ever evaluated the microbial characterization and the potential contamination risk of dental infectious waste and waste handlers. The presence of clinically relevant bacteria in the hands and nasal mucosa of waste workers highlights the need for studies in this field to clarify the risk of these pathogens in dental healthcare services, and to stress the need for an efficient waste management.

Biofilm and toxin profile: A phenotypic and genotypic characterization of coagulase-negative staphylococci isolated from human bloodstream infections.

Coagulase-negative staphylococci (CNS) represent one of the most prevalent microorganisms in nosocomial infections worldwide, nevertheless little is known about their pathogenicity features. Thus, our aim was to characterize virulence aspects of CNS isolated from patients with bloodstream infections assisted in hospitals of Belo Horizonte, MG, Brazil. Strains were identified using bioMérieuxVitek® and for biofilm production evaluation, Congo Red Agar (CRA) and polystyrene plates were used. PCR was applied to detect icaA, icaB, icaC, atlE, sea, sec, sed, tsst-1 and agr. For statistical analyses were used hierarchical cluster, chi-square test and correspondence. 59 strains were analyzed, being S. haemolyticus the most prevalent. On CRA, 96.5% were biofilm producer, whereas on polystyrene plate, 100% showed adhesion at different times evaluated. Regarding genotypic analyses, 15.2%, 38.9%, 8.4%, 49.1%, 76.2%, 23.7%, 1.6%, 30.5% and 38.9% were positive for icaA, icaB, icaC, atlE, sea, sec, sed, tsst-1 and agr, respectively. Six clusters were formed and frequency distributions of agr, atlE, icaA, icaB, sea, sec, tsst-1 differed (P < 0.001). In conclusion, all strains were biofilm producer, with high prevalence of atlE, and had potential of toxin production, with high prevalence of sea. According to the group-analyses, icaB showed relationship with the strong adherence in samples.

TSST-1, enterotoxin and bacteriocin-like substance production by Staphylococcus aureus isolated from foods / TSST-1, enterotoxinas e substâncias tipo bacteriocina produzidas por Staphylococcus aureus isolados de alimentos

The production of Toxic Shock Syndrome Toxin-1 (TSST-1), enterotoxins and bacteriocin-like substances was evaluated in 95 strains of Staphylococcus aureus recovered from raw bovine milk (n=31) and from food samples involved in staphylococcal food poisoning (n=64). Enterotoxigenicity tests with the membrane over agar associated to optimal sensibility plate assays were performed and showed that 96.77% of strains recovered from milk and 95.31% from food samples produced enterotoxins A, B, C, D or TSST-1. Reference strains S. epidermidis, Bacillus cereus, Listeria monocytogenes, Lactobacillus casei, Pseudomonas aeruginosa, S. aureus, Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis and Bacteroides fragilis were used as indicator bacteria in the antagonistic assays, the first five being sensitive to antagonistic substances. Brain heart infusion agar, in pH values ranging from 5.0 to 7.0 in aerobic atmosphere showed to be the optimum condition for antagonistic activity as evaluated with the best producer strains against the most sensitive indicator bacterium, L. monocytogenes. Sensitivity to enzymes confirmed the proteinaceous nature of these substances. Neither bacteriophage activity nor fatty acids were detected and the antagonistic activity was not due to residual chloroform. Results did not establish a positive correlation between the bacteriocinogenic profile and toxigenicity in the tested S. aureus strains.(AU)

Avaliou-se a produção de toxina-1 da síndrome do choque tóxico (TSST-1), enterotoxinas e substâncias antagonistas tipo bacteriocina em 95 amostras de Staphylococcus aureus recuperadas de leite bovino in natura (n=31) e de alimentos envolvidos em surto de intoxicação (n=64). Testes de enterotoxigenicidade pelo método da membrana sobre ágar, associado à técnica da sensibilidade ótima em placa, revelaram que 96,77% das amostras do leite e 95,31% daquelas dos alimentos produziram enterotoxinas estafilocócicas tipos A, B, C, D ou TSST-1. Nos ensaios de antagonismo, foram utilizadas como reveladoras amostras de referência de S. epidermidis, Bacillus cereus, Listeria monocytogenes, Lactobacillus casei, Pseudomonas aeruginosa, S. aureus, Salmonella typhimurium, Escherichia coli, Enterococcus faecalis e Bacteroides fragilis, sendo as cinco primeiras sensíveis às substâncias produzidas. As condições ótimas para a atividade antagonista, avaliadas com as melhores produtoras contra a indicadora mais sensível, L. monocytogenes, foram observadas em aerobiose, em ágar infuso de cérebro-coração, nos valores de pH entre 5,0 e 7,0. A sensibilidade a enzimas confirmou a natureza proteica destas substâncias. Não foram detectadas atividades de bacteriófagos nem de ácidos graxos, e a atividade antagonista não foi devido ao clorofórmio residual. Os resultados não mostraram correlação entre o perfil bacteriocinogênico e a toxigenicidade nas amostras de Staphylococcus testadas.(AU)

TSST-1, enterotoxin and bacteriocin-like substance production by Staphylococcus aureus isolated from foods / TSST-1, enterotoxinas e substâncias tipo bacteriocina produzidas por Staphylococcus aureus isolados de alimentos

The production of Toxic Shock Syndrome Toxin-1 (TSST-1), enterotoxins and bacteriocin-like substances was evaluated in 95 strains of Staphylococcus aureus recovered from raw bovine milk (n=31) and from food samples involved in staphylococcal food poisoning (n=64). Enterotoxigenicity tests with the membrane over agar associated to optimal sensibility plate assays were performed and showed that 96.77% of strains recovered from milk and 95.31% from food samples produced enterotoxins A, B, C, D or TSST-1. Reference strains S. epidermidis, Bacillus cereus, Listeria monocytogenes, Lactobacillus casei, Pseudomonas aeruginosa, S. aureus, Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis and Bacteroides fragilis were used as indicator bacteria in the antagonistic assays, the first five being sensitive to antagonistic substances. Brain heart infusion agar, in pH values ranging from 5.0 to 7.0 in aerobic atmosphere showed to be the optimum condition for antagonistic activity as evaluated with the best producer strains against the most sensitive indicator bacterium, L. monocytogenes. Sensitivity to enzymes confirmed the proteinaceous nature of these substances. Neither bacteriophage activity nor fatty acids were detected and the antagonistic activity was not due to residual chloroform. Results did not establish a positive correlation between the bacteriocinogenic profile and toxigenicity in the tested S. aureus strains.

Avaliou-se a produção de toxina-1 da síndrome do choque tóxico (TSST-1), enterotoxinas e substâncias antagonistas tipo bacteriocina em 95 amostras de Staphylococcus aureus recuperadas de leite bovino in natura (n=31) e de alimentos envolvidos em surto de intoxicação (n=64). Testes de enterotoxigenicidade pelo método da membrana sobre ágar, associado à técnica da sensibilidade ótima em placa, revelaram que 96,77% das amostras do leite e 95,31% daquelas dos alimentos produziram enterotoxinas estafilocócicas tipos A, B, C, D ou TSST-1. Nos ensaios de antagonismo, foram utilizadas como reveladoras amostras de referência de S. epidermidis, Bacillus cereus, Listeria monocytogenes, Lactobacillus casei, Pseudomonas aeruginosa, S. aureus, Salmonella typhimurium, Escherichia coli, Enterococcus faecalis e Bacteroides fragilis, sendo as cinco primeiras sensíveis às substâncias produzidas. As condições ótimas para a atividade antagonista, avaliadas com as melhores produtoras contra a indicadora mais sensível, L. monocytogenes, foram observadas em aerobiose, em ágar infuso de cérebro-coração, nos valores de pH entre 5,0 e 7,0. A sensibilidade a enzimas confirmou a natureza proteica destas substâncias. Não foram detectadas atividades de bacteriófagos nem de ácidos graxos, e a atividade antagonista não foi devido ao clorofórmio residual. Os resultados não mostraram correlação entre o perfil bacteriocinogênico e a toxigenicidade nas amostras de Staphylococcus testadas.

Characteristics of Lactobacillus and Gardnerella vaginalis from women with or without bacterial vaginosis and their relationships in gnotobiotic mice.

The objectives of the present study were to evaluate in vitro the production of antagonistic compounds against Gardnerella vaginalis by Lactobacillus strains isolated from women with or without bacterial vaginosis (BV), and to select one of the better Lactobacillus producers of such a substance to be tested in vivo using a gnotobiotic animal model challenged with one of the more sensitive G. vaginalis isolates. A total of 24 isolates from women with and without BV were identified as G. vaginalis. A higher frequency (P<0.05) of this bacterium was observed in women with BV (56.7%) when compared to healthy women (17.6%). A total of 86 strains of Lactobacillus were obtained from healthy women and women with BV. Lactobacillus strains were more frequently present (P<0.05) in healthy women (97.5%) than in women with BV (76.7%). Lactobacillus crispatus was the predominating strain in both healthy women and women with BV. Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus gasseri and Lactobacillus vaginalis were isolated with an intermediate frequency in the two groups. In vitro antagonism assays were performed using as indicators 17 reference strains and the G. vaginalis strains isolated from women with BV and from healthy women. Lactobacillus isolated from healthy women showed the higher antagonistic activity against all the indicator strains when compared with isolates from women with BV. Concerning the indicator strains, G. vaginalis found in women with BV was more resistant to the antagonism, particularly when Lactobacillus isolates from women with BV were used as producer strains. A high vaginal population level of G. vaginalis was obtained by intravaginal inoculation of germ-free mice, and this colonization was accompanied by vaginal histopathological lesions. A tenfold decrease in vaginal population level of G. vaginalis and a reduction of histological lesions were observed when the pathogenic challenge was performed in mice previously monoassociated with an L. johnsonii strain. Concluding, results of the present study suggest that progression of G. vaginalis-associated BV depends in part on a simultaneous presence of Lactobacillus populations with a low antagonistic capacity and of a G. vaginalis strain with a high resistance to this antagonism. The results could also explain why G. vaginalis is frequently found in the vaginal ecosystem of healthy women.

Isolation, identification and antimicrobial susceptibility of Bacteroides fragilis group strains recovered from broiler faeces.

1. The objective was to evaluate the occurrence of cultivable components of the Bacteroides fragilis group in faeces of broiler chickens and their antimicrobial susceptibility patterns. 2. Faecal samples of 36 × 45-d-old Cobb broilers of both sexes from 15 different flocks on one farm were diluted 10-fold and plated on to Bacteroides-bile-esculin agar for colony count and isolation. Identification was by molecular methods and antimicrobial susceptibility in the agar dilution assay. 3. A total of 236 isolates was recovered from a mean population of 3·32 × 10(7 )colony-forming units/g of faeces. B. fragilis was shown to be the predominant Bacteroides species (45·3%), followed by B. distasonis (35·6%), B. vulgatus (8·9%), B. ovatus (2·5%) and B. stercoris (1·3%). 4. Among 204 bacterial isolates tested, high resistance to ampicillin (98·5%), norfloxacin (95·1%) and tetracycline (88·2%) were observed. High (89·7%) multi-drug resistance was observed to 3-7 of the tested drugs. 5. Components of the B. fragilis group were sub-dominant in broiler faecal microbiota, with a different species pattern compared with human and high antimicrobial multi-drug resistance.

In-house validation of PremiTest, a microbiological screening test with solvent extraction, for the detection of antimicrobial residues in poultry muscles.

PremiTest, a microbial inhibition test for the screening of antimicrobial residues, was validated according to the criteria established by Decision 2002/657/EC. Sensitivity, detection capability (CCß), specificity, selectivity, robustness and applicability were evaluated. The methodology involves the technique of solvent extraction, which increases the detection capability of the test for a wider range of antibiotics. The following CCß values in poultry muscle were found: penicillin G ≤ 12.5 µg kg(-1), total sulfonamides ≤ 75 µg kg(-1), erythromycin 75 µg kg(-1) and lincomycin 50 µg kg(-1). The detection capability of chlortetracycline was equal to its maximum residue limit (100 µg kg(-1)) and the method did not detect gentamicin (1000 µg kg(-1)), for which no MRL is established in poultry muscle. Specificity evaluated in relation to different analytes and matrices did not detect any interferences in the tests results; whilst the robustness showed that the pH neutralisation point of the extract affects the analytical results and the kits' performance. Only the screening of tetracyclines requires the analysis of extracts without pH neutralisation. The results of the validation process showed that this method is acceptable for screening ß-lactam, sulfonamide and macrolide antimicrobial groups in the National Residues and Contaminants Control Programme (PNCRC), and that for this it is fit for purpose.

Phenotypic changes in a laboratory-derived ertapenem-resistant Escherichia coli strain.

The aim of this study was to identify phenotypic changes in a laboratory-derived strain of ertapenem-resistant Escherichia coli (Ec-ERT) when compared to its susceptible parent strain (Ec-WT). In both strains, we assessed both the effects of ertapenem via time-kill curves and the occurrence of cross resistance with other beta-lactams. The strains were compared based on growth pattern, biochemical-physiological profile and changes in the subproteome using 2D-DIGE followed by MALDI-TOF/TOF MS. To assess virulence, we employed a murine model of intraperitoneal infection in which we investigated the invasiveness of both strains. Growth persistence of the laboratory-derived resistant strain was observed via the time-kill curve assay, but cross resistance was not observed for other beta-lactams. We also observed a slower growth rate and changes in the biochemical and physiological characteristics of the drug-resistant bacteria. In the resistant strain, a total of 51 protein spots were increased in abundance relative to the wild-type strain, including an outer membrane protein A, which is related to bacterial virulence. The mouse infection assay showed a higher invasiveness of the Ec-ERT strain in relation to the Ec-WT strain. In conclusion, the alterations driven by ertapenem in E. coli reinforce the idea that antimicrobial agents may interfere in several aspects of bacterial cell biology, with possible implications for host-bacteria interactions.

Isolation of clinically relevant fungal species from solid waste and environment of dental health services.

AIMS: This study was undertaken to detect, identify and determine antifungal susceptibility of yeast strains isolated from dental solid waste and to evaluate airborne fungi in the Brazilian dental health care environment and in the waste storage room. METHODS AND RESULTS: A group of 17 yeast strains were identified by macroscopic and microscopic characteristics, API 20C Aux system and Multiplex PCR. All 104 airborne fungal colonies were identified by macroscopic and microscopic morphology. The CLSI broth microdilution method was utilized as the susceptibility test. Candida parapsilosis was the prevailing yeast species recovered from waste, followed by Rhodotorula glutinis. Three strains of Candida guilliermondii presented minimal inhibitory concentration values considered to be susceptible dose dependent (2 µg ml(-1)) to voriconazole. Of all airborne fungal species, 69% were recovered from the waste storage room and 31% were recovered from the clinical/surgical environment. Most of them were identified as Cladosporium spp. CONCLUSIONS: These findings reinforce the potential risk of waste handling and point out the need for safe management to minimize the spread of these agents to the environment. Filamentous fungi isolation in almost all sampled environments indicates that a periodic monitoring of airborne microbiota in the dental health care service environment is required. SIGNIFICANCE AND IMPACT OF THE STUDY: The survival of yeast strains for 48 h suggests that dental waste should be carefully controlled and monitored.

Antagonism and synergism in Gardnerella vaginalis strains isolated from women with bacterial vaginosis.

Antagonistic and synergistic substances are important for interactions between micro-organisms associated with human body surfaces, either in healthy or in diseased conditions. In the present study, such compounds produced by Gardnerella vaginalis strains isolated from women with bacterial vaginosis (BV) were detected in vitro and the antagonistic ones were partially characterized. Among 11 G. vaginalis strains tested, all showed antagonistic activity against at least one of the 22 indicator bacteria assayed. Interestingly, for some of these strains, antagonism reverted to synergism, favouring one of the indicator strains (Peptostreptococcus anaerobius) when the growth medium was changed. Partial characterization of antagonistic substances suggested a bacteriocin-like chemical nature. Depending on growth conditions, G. vaginalis isolated from women with BV produced antagonistic or synergistic compounds for other bacterial components of the vaginal ecosystem. This is the first report to our knowledge of the production of antagonistic and/or synergistic substances by G. vaginalis. This ability may be a pivotal factor in understanding BV and the ecological role of this bacterium in the vaginal environment.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

Identification and in vitro production of Lactobacillus antagonists from women with or without bacterial vaginosis.

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

LyeTx I, a potent antimicrobial peptide from the venom of the spider Lycosa erythrognatha.

LyeTx I, an antimicrobial peptide isolated from the venom of Lycosa erythrognatha, known as wolf spider, has been synthesised and its structural profile studied by using the CD and NMR techniques. LyeTx I has shown to be active against bacteria (Escherichia coli and Staphylococcus aureus) and fungi (Candida krusei and Cryptococcus neoformans) and able to alter the permeabilisation of L: -alpha-phosphatidylcholine-liposomes (POPC) in a dose-dependent manner. In POPC containing cholesterol or ergosterol, permeabilisation has either decreased about five times or remained unchanged, respectively. These results, along with the observed low haemolytic activity, indicated that antimicrobial membranes, rather than vertebrate membranes seem to be the preferential targets. However, the complexity of biological membranes compared to liposomes must be taken in account. Besides, other membrane components, such as proteins and even specific lipids, cannot be discarded to be important to the preferential action of the LyeTx I to the tested microorganisms. The secondary structure of LyeTx I shows a small random-coil region at the N-terminus followed by an alpha-helix that reached the amidated C-terminus, which might favour the peptide-membrane interaction. The high activity against bacteria together with the moderate activity against fungi and the low haemolytic activity have indicated LyeTx I as a good prototype for developing new antibiotic peptides.

Purification and partial characterization of a bacteriocin produced by an oral Fusobacterium nucleatum isolate.

AIMS: To purify and partially characterize a bacteriocin produced by a Fusobacterium nucleatum strain. METHODS AND RESULTS: Following protein precipitation the effect of different treatments on a bacteriocin produced by a F. nucleatum strain named P12.2 isolated from a patient with periodontitis was evaluated. The antagonistic activity of the intracellular fraction obtained at 80% ammonium sulphate was preserved at pH values from 6.0 to 9.0 and showed to be sensitive to high temperatures and to treatment with proteases. The fraction was submitted to sequential steps of gel filtration, ion exchange, and reverse phase chromatography, and SDS-PAGE. Data obtained by mass spectrometry revealed that the molecular mass of the protein was 27,296 Da. CONCLUSIONS: For the first time a bacteriocin produced by a F. nucleatum strain was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description on characterization of a bacteriocin produced by F. nucleatum. It is possible that the bacteriocin plays a role in the regulation of population levels of periodontopathic organisms.

Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.

Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined.

Determinação do perfil protéico da membrana externa da Leptospira interrogans sorovariedade Hardjoprajitno / Protein profile of the outer membrane of Leptospira interrogans serovar Hardjoprajitno

Estudou-se o perfil das proteínas da membrana externa (PME) da Leptospira interrogans sorovariedade Hardjoprajitno por meio da eletroforese bidimensional. Foram utilizadas técnicas de extração das PME com Triton x114 e precipitação com acetona. Os géis foram corados com nitrato de prata e as imagens analisadas para determinação da massa molecular das proteínas detectadas. Foram visualizadas 35 bandas protéicas, sendo que cinco delas se destacaram por estarem em maior quantidade: 22,54KDa (LipL22), 30/26KDa (LipL32), 34,41KDa (PME34), 42,75KDa (LipL41) e 58,59KDa (LipL63).(AU)

The protein profile of the outer membrane of Leptospira interrogans serovar Hardjoprajitno was determined by two-dimensional gel electrophoresis. The outer membrane was extracted with Triton x 114 and the proteins were precipitated with acetone. The images were analyzed for the determination of the molecular weight of the detected proteins. Thirty-five spots for the proteins that are predominant in the outer membrane of this Leptospira were observed and five proteins were found in higher quantities: 22.54KDa (LipL22), 30/26KDa (LipL32), 34.41KDa (PME34) (2), 42.75KDa (LipL41), and 58.59KDa (LipL63).(AU)

Determinação do perfil protéico da membrana externa da Leptospira interrogans sorovariedade Hardjoprajitno / Protein profile of the outer membrane of Leptospira interrogans serovar Hardjoprajitno

Estudou-se o perfil das proteínas da membrana externa (PME) da Leptospira interrogans sorovariedade Hardjoprajitno por meio da eletroforese bidimensional. Foram utilizadas técnicas de extração das PME com Triton x114 e precipitação com acetona. Os géis foram corados com nitrato de prata e as imagens analisadas para determinação da massa molecular das proteínas detectadas. Foram visualizadas 35 bandas protéicas, sendo que cinco delas se destacaram por estarem em maior quantidade: 22,54KDa (LipL22), 30/26KDa (LipL32), 34,41KDa (PME34), 42,75KDa (LipL41) e 58,59KDa (LipL63).

The protein profile of the outer membrane of Leptospira interrogans serovar Hardjoprajitno was determined by two-dimensional gel electrophoresis. The outer membrane was extracted with Triton x 114 and the proteins were precipitated with acetone. The images were analyzed for the determination of the molecular weight of the detected proteins. Thirty-five spots for the proteins that are predominant in the outer membrane of this Leptospira were observed and five proteins were found in higher quantities: 22.54KDa (LipL22), 30/26KDa (LipL32), 34.41KDa (PME34) (2), 42.75KDa (LipL41), and 58.59KDa (LipL63).

Purification and partial characterization of a bacteriocin produced by Eikenella corrodens.

AIMS: The purpose of this study was to purify and characterize a bacteriocin produced by Eikenella corrodens A32E2. METHODS AND RESULTS: Peptostreptococcus anaerobius ATCC27337 was used as indicator strain in antagonistic assays for bacteriocin-producing E. corrodens A32E2. Protein extraction was influenced by pH and buffer composition. The protein was active in the pH range 6-8. Inhibitory activity was lost by both heating and treatment with proteolytic enzymes and decreased with organic solvents. The substance is rather unstable but maintains 100% of its activity after being exposed to acetone and when stored at -70 degrees C. The antagonistic substance was first precipitated by ammonium sulfate and further partially purified by Mono-Q FPLC and C-18 HPLC. Mass spectrometry analysis showed that the molecular mass was 23 625 Da, and the sequence obtained for the N-terminus was: Met-Asn-Phe-Asp-Glu-Lys-Val-Gly-Lys-Val-X-Phe-Lys-Val-Gly-Asp. CONCLUSIONS: The evidence presented in this study supports the idea that an antagonistic substance produced by E. corrodens A32E2 isolated from a periodontal diseased site is a novel bacteriocin, which we designate corrodecin. SIGNIFICANCE AND IMPACT OF THE STUDY: We anticipated that corrodecin might play an important role at the periodontal site. This compound could also be attractive in biotechnological applications as an interesting tool for oral ecosystem control.

The influence of molecular oxygen exposure on the biology of Prevotella intermedia, with emphasis on its antibiotic susceptibility.

AIM: This study focuses on investigating the molecular and physiological characteristics of Prevotella intermedia after molecular oxygen exposure (MOE) and the effect on drug susceptibility patterns. METHODS AND RESULTS: Samples of P. intermedia were used as parent strains: ATCC25611 and four clinical isolates. Strains adapted to oxidative stress by MOS were obtained by the enrichment technique. Drug susceptibility was evaluated by minimal inhibitory concentrations (MIC) using agar dilution. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to evaluate the genetic diversity of all strains and physiological analyses were made by sodiumdodecylsulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis of crude, cell-free extracts. The genetic profile showed that lineages with altered MIC values were selected after MOE. Overall, we found significant decrease in drug susceptibility for the aero-strains against all tested antimicrobials (amoxicillin, amoxicillin+clavulanic acid, clindamycin, chloramphenicol, ertapenen and metronidazole). We also observed markedly different protein expression patterns between the parent and selected aero-strains. CONCLUSIONS: MOE induces changes in the genetic profile and protein expression patterns of P. intermedia that may also be linked to its drug resistance mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of MOE on anaerobic bacterial physiology and behaviour may influence antimicrobial susceptibility patterns with potential consequences to antimicrobial chemotherapy.

Production of antagonistic substance by Eikenella corrodens isolated from the oral cavity of human beings with and without periodontal disease.

AIMS: Antagonistic abilities may confer ecological advantages for micro-organisms in competitive ecosystems. However, reports regarding this phenomenon in Eikenella corrodens are not available. METHODS AND RESULTS: Nineteen E. corrodens strains, isolated from the oral cavity of human beings without periodontal disease (n = 5) and with aggressive (n = 9) and chronic (n = 5) periodontitis, as well as a reference strain (E. corrodens ATCC23834), were evaluated for antagonistic activity. The following indicators were used: Porphyromonas gingivalis FDC381, Prevotella intermedia ATCC25611, Actinomyces israelii ATCC12102, Eubacterium lentum ATCC25559, Peptostreptococcus anaerobius ATCC27337, Actinobacillus actinomycetemcomitans FDCY4, Fusobacterium nucleatum ATCC10953, Streptococcus sanguinis ATCC10557, Streptococcus uberis ATCC9927, Streptococcus mutans IM/UFRJ, Staphylococcus aureus ATCC33591 and Candida albicans ATCC18804. All the strains showed antagonism against at least one of the indicator strains. This phenomenon was more frequently observed for strains isolated from patients with chronic periodontitis (36.4%), than those from healthy subjects (20.6%) and those with aggressive periodontitis (10.8%). CONCLUSIONS: The heterogeneous antagonistic spectrum exhibited by E. corrodens isolates suggests their ability to produce more than one antagonistic substance, whose ecological relevance is yet to be demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description of antagonistic compound production by E. corrodens and its relationships with the clinical status of the patients.