In vitro evaluation of physical and chemical parameters involved in aPDT of Aggregatibacter actinomycetemcomitans.

Periodontitis is an infectious disease characterized by the destruction of supporting tissues. Antimicrobial photodynamic therapy (aPDT) has been proposed as an improved method for eliminating microorganisms. Its efficiency depends on the correct use of physical and chemical parameters. Thus, these parameters and their relations were evaluated in this study with the purpose of establishing lethal conditions for combating bacterial agents. Diode lasers and light-emitting diodes (LEDs) were characterized to evaluate the absorption profile and resonance of methylene blue (MB) and toluidine blue O (TBO). The relations between light energy density and photosensitizer absorption were determined. Two methodologies were used to evaluate the effects of aPDT against Aggregatibacter actinomycetemcomitans. LED light exhibited a broad emission spectrum with a peak light wavelength of 637 nm and 99% purity. The resonance intensity of MB was higher with diode laser irradiation, and TBO showed higher resonance intensity with LED irradiation. There was no difference in the absorption profile of photosensitizers using diode lasers or LEDs, and variations in power density did not result in an increasing or decrease in light absorption. A. actinomycetemcomitans was susceptible to photodynamic processes. Emission spectra and peak light wavelengths of light sources combined with the absorption profiles of photosensitizers were the main parameters involved in determining the efficiency of photodynamic effects. Power density did not alter the light absorption of photosensitizers. The association between adequate irradiation characteristics and photosensitizer absorption results in complete inactivation of A. actinomycetemcomitans. In addition, the bactericidal effect was not altered by an increase in energy densities.

Molecular association of pathogenicity and resistance to multiple antimicrobials in Acinetobacter baumannii strains recovered from patients with diverse infectious diseases / Associação molecular de fatores de patogenicidade e resistência a múltiplos antimicrobianos em linhagens de Acinetobacter baumannii recuperados de pacientes com doenças infecciosas diversas

ABSTRACT INTRODUCTION: The success of Acinetobacter baumannii infections can be attributed to its various virulence factors and antimicrobial resistance mechanisms. OBJECTIVE: To evaluate the presence and correlation between different resistance and virulence factors in clinical A. baumannii strains. METHODS: Study conducted at a University Hospital in Belo Horizonte, Minas Gerais, Brazil. The confirmation of Acinetobacter baumannii-calcoaceticus complex was performed by detecting the blaOXA-51 gene through the polymerase chain reaction (PCR), as well as the search for genes: blaOXA-23, 24, 58, 143, blaVIM-1, csuE, ompA and ISAba1. Antimicrobials and metallo-betalactamase (MβL) expression were evaluated by E-test®; and genetic diversity, by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Biofilm formation was classified into four categories according to the mean optical density obtained. RESULTS: 98.4% (61/62) of the strains were resistant to meropenem; 71%, to ceftazidime; and 61.3%, to ampicillin-sulbactam; while 98.4% were sensitive to polymyxin B; and 48.4%, to tigecycline. The production of MβL was detected in 95.2% of the strains. The blaOXA-51 gene was detected in all strains tested; blaVIM-1, in 83.9%; and ISAba1, in 90.3%. On the other hand, the csuE and ompA genes were present in 43.5% and 53.2% of the strains, respectively. CONCLUSION: There was a possible correlation between gentamicin resistant samples and those that were positive for the ompA gene. The csuE gene correlated positively with ISAba1.

RESUMO INTRODUÇÃO: O sucesso das infecções por Acinetobacter baumannii pode ser atribuído a seus vários fatores de virulência e a mecanismos de resistência a antimicrobianos. OBJETIVO: Avaliar a presença e a correlação entre diferentes fatores de resistência e virulência em amostras clínicas de A. baumannii. MÉTODOS: Estudo conduzido em um hospital universitário em Belo Horizonte, Minas Gerais, Brasil. A confirmação do complexo Acinetobacter baumannii-calcoaceticus foi realizada pela detecção do gene blaOXA-51, por meio da reação em cadeia da polimerase (PCR), assim como a pesquisa dos genes: blaOXA-23, 24, 58, 143, blaVIM-1, csuE, ompA e ISAba1. Os antimicrobianos e a expressão das metalobetalactamases (MβL) foram avaliados pelo E-test®; e a diversidade genética, por enterobacterial repetitive intergenic consensus (ERIC)-PCR. A formação de biofilme foi classificada em quatro categorias de acordo com a média da densidade ótica obtida. RESULTADOS: Do total de amostras, 98, 4% (61/62) foram resistentes ao meropenem; 71%, a ceftazidime; e 61, 3%, a ampicilina-sulbactam; enquanto 98, 4% foram sensíveis a polimixina B; e 48, 4%, a tigeciclina. A produção de MβL foi detectada em 95, 2% das amostras. O gene blaOXA-51 foi detectado em todas as amostras testadas; blaVIM-1, em 83, 9%; e ISAba1, em 90, 3%. Por outro lado, os genes csuE e ompA estiveram presentes em 43, 5% e 53, 2% das amostras, respectivamente. CONCLUSÃO: Houve uma possível correlação entre as amostras resistentes a gentamicina e aquelas positivas para o gene ompA. O gene csuE correlacionou-se positivamente com ISAba1.

5-Nitroimidazole-derived Schiff bases and their copper(II) complexes exhibit potent antimicrobial activity against pathogenic anaerobic bacteria.

In the present work a family of novel secnidazole-derived Schiff base compounds and their copper(II) complexes were synthesized. The antimicrobial activities of the compounds were evaluated against clinically important anaerobic bacterial strains. The compounds exhibited in vitro antibacterial activity against Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Parabacteroides distasonis and Fusubacterium nucleatum pathogenic anaerobic bacteria. Upon coordination to copper(II) the antibacterial activity significantly increased in several cases. Some derivatives were even more active than the antimicrobial drugs secnidazole and metronidazole. Therefore, the compounds under study are suitable for in vivo evaluation and the microorganisms should be classified as susceptible to them. Electrochemical studies on the reduction of the nitro group revealed that the compounds show comparable reduction potentials, which are in the same range of the bio-reducible drugs secnidazole and benznidazole. The nitro group reduction potential is more favorable for the copper(II) complexes than for the starting ligands. Hence, the antimicrobial activities of the compounds under study might in part be related to intracellular bio-reduction activation. Considering the increasing resistance rates of anaerobic bacteria against a wide range of antimicrobial drugs, the present work constitutes an important contribution to the development of new antibacterial drug candidates.

Coagulase-Negative Staphylococci Isolated from Human Bloodstream Infections Showed Multidrug Resistance Profile.

Coagulase-negative staphylococci (CNS) are important pathogens causing nosocomial infections worldwide with increasing resistance to antimicrobials. The aim of this study was to characterize resistance aspects of CNS isolated from patients with bloodstream infections acquired in hospitals in Belo Horizonte, MG, Brazil. Staphylococcus strains were characterized using repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting with (GTG)5 primer. Phenotypic resistance was analyzed using AST-P5085 card (bioMérieuxVitek®). PCR was used to detect mecA, vanA, blaZ, ermA/B/C, aac-aphD, and SCC-mec. For statistical analyses, we used hierarchical cluster, chi-square test (χ2), and correspondence. Several clusters were formed within the same species using (GTG)5 primer, and strains showed resistance to the following antimicrobials: benzylpenicillin (100%); oxacillin (93.1%); gentamicin (36.3%); ciprofloxacin (63.7%); moxifloxacin (32.7%); norfloxacin (81.0%); erythromycin (86.2%); clindamycin (75.8%); linezolid, teicoplanin and vancomycin (1.7%); tigecycline (0%); fusidic acid (10.35%); rifampicin (13.7%); and trimethoprim/sulfamethoxazole (46.5%). Regarding genotypic analyses, 40%, 0%, 78%, 42%, 100%, 24%, and 30% were positive for mecA, vanA, blaZ, ermA, ermB, ermC, and aac-aphD, respectively. Regarding staphylococcal cassette mec (SCCmec) type, 3.4% presented type I; 5.0% type II; 27.1% type III; 20.3% type IIIA; and 32.2% type IIIB. Six clusters were formed and frequency distributions of resistant strains to oxacillin, gentamicin, ciprofloxacin, moxifloxacin, norfloxacin, erythromycin, clindamycin, linezolid, teicoplanin, vancomycin, fusidic acid, rifampicin, and trimethoprim/sulfamethoxazole, and mecA, blaZ, ermC, aac-aphD, and SCCmec type differed (p < 0.001). In conclusion, the strains investigated in this study were multidrug resistant and carried multiple antibiotic resistance genes.

Antidiarrheal activity of extracts from Maytenus gonoclada and inhibition of Dengue virus by lupeol.

Diarrhea is an infectious disease caused by bacterial, virus, or protozoan, and dengue is caused by virus, included among the neglected diseases in several underdeveloped and developing countries, with an urgent demand for new drugs. Considering the antidiarrheal potential of species of Maytenus genus, a phytochemical investigation followed by antibacterial activity test with extracts of branches and heartwood and bark of roots from Maytenus gonoclada were conducted. Moreover, due the frequency of isolation of lupeol from Maytenus genus the antiviral activity against Dengue virus and cytotoxicity of lupeol and its complex with ß-cyclodextrins were also tested. The results indicated the bioactivity of ethyl acetate extract from branches and ethanol extract from heartwood of roots of M. gonoclada against diarrheagenic bacteria. The lupeol showed potent activity against Dengue virus and low cytotoxicity in LLC-MK2 cells, but its complex with ß-cyclodextrin was inactive. Considering the importance of novel and selective antiviral drug candidates the results seem to be promising.

Antidiarrheal activity of extracts from Maytenus gonoclada and inhibition of Dengue virus by lupeol

ABSTRACT Diarrhea is an infectious disease caused by bacterial, virus, or protozoan, and dengue is caused by virus, included among the neglected diseases in several underdeveloped and developing countries, with an urgent demand for new drugs. Considering the antidiarrheal potential of species of Maytenus genus, a phytochemical investigation followed by antibacterial activity test with extracts of branches and heartwood and bark of roots from Maytenus gonoclada were conducted. Moreover, due the frequency of isolation of lupeol from Maytenus genus the antiviral activity against Dengue virus and cytotoxicity of lupeol and its complex with β-cyclodextrins were also tested. The results indicated the bioactivity of ethyl acetate extract from branches and ethanol extract from heartwood of roots of M. gonoclada against diarrheagenic bacteria. The lupeol showed potent activity against Dengue virus and low cytotoxicity in LLC-MK2 cells, but its complex with β-cyclodextrin was inactive. Considering the importance of novel and selective antiviral drug candidates the results seem to be promising.

Antimicrobial susceptibility profile of enterotoxigenic and enteropathogenic Escherichia coli isolates obtained from fecal specimens of children with acute diarrhea / Perfil de suscetibilidade a drogas antimicrobianas de amostras de Escherichia coli enterotoxigênica e enteropatogênica isoladas de espécimes fecais de crianças com doença diarreica aguda

ABSTRACT Among the diseases which etiopathogenesis is associated with Escherichia coli, acute diarrhea stands out. Studies on the characterization of the antimicrobial susceptibility profile contribute to the selection of appropriate empirical antimicrobial therapy. In this study, the antimicrobial susceptibility profile of 98 enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) strains isolated from fecal specimens of children with acute diarrhea was evaluated. The resistance rates to ampicillin, sulfamethoxazole/trimethoprim, amoxicillin/clavulanate, and nalidixic acid were high, ranging from 34.7% to 10.2%. The result of this research recommends the use of cefotaxime and ceftriaxone for the empirical treatment of children with acute diarrhea which the etiology suggested is ETEC or EPEC.

RESUMO Entre as doenças cuja etiopatogenia está associada à Escherichia coli, destaca-se a doença diarreica aguda. Estudos que visam à caracterização do perfil de suscetibilidade antimicrobiana contribuem para o delineamento de antibioticoterapia empírica eficaz. Neste estudo, foi avaliado o perfil de suscetibilidade a antimicrobianos de 98 amostras de E. coli enterotoxigênica (ETEC) e E. coli enteropatogênica (EPEC) isoladas de crianças com doença diarreica. As frequências de resistência a ampicilina, sulfametoxazol-trimetoprima, amoxicilina-clavulanato e ácido nalidíxico foram elevadas, variando entre 34,7% e 10,2%. Esta pesquisa recomenda o emprego de cefotaxima e ceftriaxona para o tratamento empírico de crianças com quadro de diarreia cuja etiologia sugerida seja ETEC ou EPEC.

Cytokine Expression in Patients Hospitalized for Severe Odontogenic Infection in Brazil.

INTRODUCTION: Severe odontogenic infections remain an important public health concern and a significant economic burden to public health care facilities. Despite this, several aspects of the disease, such as its immune response profile, remain poorly understood. The aim of this study was to search for an association between mRNA levels of the cytokines interferon-γ, interleukin (IL)-1ß, tumor necrosis factor-α, IL-17A, IL-10, and transforming growth factor-ß and the chemokines IL-8, CCL2/MCP-1, and CCL5 and odontogenic infection. METHODS: The case group was composed of 12 patients hospitalized in consequence of severe odontogenic infection, and our control group included 12 individuals with healthy periapical tissues. Clinical samples were taken from the case (drainage site) and control (periapical interstitial fluid) groups with the aid of paper points. Total RNA was extracted, complementary DNA was synthesized, and mRNA levels were determined by quantitative polymerase chain reaction. Data analysis was performed by using SPSS, and the Wilcoxon signed rank test was used to determine statistical significance (P < .05). RESULTS: Data generated showed a significantly increased expression of proinflammatory cytokines (interferon-γ, IL-1ß, tumor necrosis factor-α, and IL-17A), IL-8, and CCL2/MCP-1 in odontogenic infection patients. The mRNA levels of IL-10, transforming growth factor-ß, and CCL5 were similar in both study groups. CONCLUSIONS: In general, individuals presenting with odontogenic infections exhibited extraordinary proinflammatory cytokine profiles paralleled with unaltered expression of regulatory mediators.

Phenotypic and genotypic diversity of multidrug-resistant Pseudomonas aeruginosa Isolates from bloodstream infections recovered in the Hospitals of Belo Horizonte, Brazil.

BACKGROUND: Pseudomonas aeruginosa commonly causes nosocomial bloodstream infections and the emergence of a variety of ß-lactamases (BLs) is worrying. In 5 hospitals in Belo Horizonte, Brazil, the presence of phenotypes encoding BL genes was established and the genetic diversity of the P. aeruginosa strains recovered from bloodstream infections was analyzed. MATERIALS AND METHODS: The isolates were investigated using a disk diffusion (DD) method and the Etest, for encoding metallo-ß-lactamases (MBLs), oxacillinases and cephalosporinases. Genes and genetic diversity were evaluated by random amplified polymorphic DNA (RAPD) genotyping and enterobacterial repetitive intergenic consensus (ERIC)-PCR. RESULTS: Twelve strains (30%) were positive for MBLs by Etest and DD, 15 were cephalosporinase-positive and 87.5% were positive for blaSPM-1 and blaVIM-1. Twenty-three strains (57.5%) were grouped into profile A, 32.5% into profile B and 10% into profile C by RAPD genotyping. ERIC-PCR revealed a varying degree of similarity between strains, ranging from 45 to 100%. CONCLUSIONS: The results suggest distinct clonal populations in the 5 hospitals studied, indicating a potentially problematic epidemiological situation in Belo Horizonte, Brazil.

Resistance markers and genetic diversity in Acinetobacter baumannii strains recovered from nosocomial bloodstream infections.

In this study, phenotypic and genotypic methods were used to detect metallo-ß-lactamases, cephalosporinases and oxacillinases and to assess genetic diversity among 64 multiresistant Acinetobacter baumannii strains recovered from blood cultures in five different hospitals in Brazil from December 2008 to June 2009. High rates of resistance to imipenem (93.75%) and polymyxin B (39.06%) were observed using the disk diffusion (DD) method and by determining the minimum inhibitory concentration (MIC). Using the disk approximation method, thirty-nine strains (60.9%) were phenotypically positive for class D enzymes, and 51 strains (79.6%) were positive for cephalosporinase (AmpC). Using the E-test, 60 strains (93.75%) were positive for metallo-ß-lactamases (MßLs). All strains were positive for at least one of the 10 studied genes; 59 (92.1%) contained blaVIM-1, 79.6% contained blaAmpC, 93.7% contained blaOXA23 and 84.3% contained blaOXA51. Enterobacteria Repetitive Intergenic Consensus (ERIC)-PCR analysis revealed a predominance of certain clones that differed from each other. However, the same band pattern was observed in samples from the different hospitals studied, demonstrating correlation between the genotypic and phenotypic results. Thus, ERIC-PCR is an appropriate method for rapidly clustering genetically related isolates. These results suggest that defined clonal clusters are circulating within the studied hospitals. These results also show that the prevalence of MDR A. baumannii may vary among clones disseminated in specific hospitals, and they emphasize the importance of adhering to appropriate infection control measures.

Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry.

Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells.

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis.

BACKGROUND: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. RESULTS: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. CONCLUSIONS: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.

Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction.

Chronic periodontitis is a highly prevalent endogenous polymicrobial disease. To better understand the etiology of the disease a quantitative approach is mandatory and real-time PCR is the molecular technique currently preferred to achieve this purpose. Taking into account that such a kind of study is still scarce, we aimed to evaluate the association between periodontal microbiota and chronic periodontitis. A total of 60 low-income age-matched female adults, 30 with chronic periodontitis and 30 without periodontal disease, were enrolled. DNA obtained from subgingival specimens was used for quantification of Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia by real-time PCR. A. actinomycetemcomitans, E. corrodens, and F. nucleatum were detected in all subjects, P. gingivalis was observed in 70.0% and 46.6% and P. intermedia in 90.0% and 80.0% of chronic periodontitis patients and periodontally healthy subjects, respectively. P. gingivalis mean count was significantly higher in patients with chronic periodontitis than in periodontally healthy individuals. Accurate detection and quantification of five putative periodontal pathogens was feasible using a simple and fast real-time PCR protocol. Although P. gingivalis and P. intermedia have been found more commonly in chronic periodontitis patients, no statistical difference was observed between periodontally diseased and healthy groups. Quantitative data indicated association between P. gingivalis and chronic periodontitis. However, because of its uneven distribution, it should not be solely taken as a marker of periodontal status.

Use of 2-D electrophoresis and ESI mass spectrometry techniques to characterize Fusobacterium nucleatum proteins up-regulated after oxidative stress.

The genus Fusobacterium belongs to the Fusobacteriaceae family and is a Gram-negative obligate anaerobic bacterium found in the human oral microbiota. Even that Fusobacterium nucleatum cannot grow under aerobic conditions, they may exhibit aerotolerance as an adaptive response which could figure as an important virulence factor, during the stages of infection, when these anaerobes are shifted to aerobic conditions. In this regard, little is known about bacterial oxidative stress adaptive response and the influence of this adaptation on the host-bacteria relationship. We aimed to use both techniques 2-DE and Electrospray Ionization Mass Spectrometry (ESI-MS) to characterize proteins in F. nucleatum, after oxidative stress. We related three different proteins which were up-regulated by oxidative stress. As its genome is already sequenced, these proteins were found in data base search, by homology. Thus, by using techniques as ESI-Q/TOF-MS, in addition to 2-DE, the opportunity exists to gain a more holistic view of the bacterial proteome of human pathogens, to achieve a better understanding of species diversity and to elucidate the role of specific proteins in disease. This work represents one of the first studies using genetic and physiological approaches to understand the phenomenon of oxidative stress in F. nucleatum.

In vitro antioxidant potential of medicinal plant extracts and their activities against oral bacteria based on Brazilian folk medicine.

BACKGROUND AND OBJECTIVES: This study aims to determine antibacterial activities of Cocos nucifera (husk fiber), Ziziphus joazeiro (inner bark), Caesalpinia pyramidalis (leaves), aqueous extracts and Aristolochia cymbifera (rhizomes) alcoholic extract against Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Lactobacillus casei. The antioxidant activity and acute toxicity of these extracts were also evaluated. MATERIAL AND METHODS: The plant extracts antibacterial activity was evaluated in vitro and the minimal inhibitory concentration (MIC) was determined by the broth micro-dilution assay. The bacterial killing kinetic was also evaluated for all extracts. In addition, the antibacterial effect of the extracts was tested in vitro on artificial oral biofilms. The acute toxicity of each extract was determined in according to Lorke [Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol 1983;54:275-87] and the antioxidant activity was evaluated by DPPH photometric assay [Mensor LL, Menezes FS, Leitão GG, Reis AS, Santos TC, Coube CS, et al. Screening of Brazilian plants extract for antioxidant activity by the use of DPPH free radical method. Phytother Res 2001;15:127-30]. RESULTS: MIC and the bactericidal concentrations were identical, for each evaluated extract. However, microbes of artificial biofilms were less sensitive to the extracts than the planktonic strains. A. cymbifera extract induced the highest bactericidal effect against all tested bacteria, followed by C. nucifera, Z. joazeiro and C. pyramidalis extracts, respectively. All extracts showed good antioxidant potential, being C. nucifera and C. pyramidalis aqueous extracts the most active ones. CONCLUSION: In conclusion, all oral bacteria tested (planktonic or in artificial biofilms) were more susceptible to, and rapidly killed in presence of A. cymbifera, C. pyramidalis and C. nucifera than Z. joazeiro extracts, respectively. Thus, these extracts may be of great interest for future studies about treatment of oral diseases, considering their potent antioxidant activity and low toxicity.

Effects of oxidative stress on the virulence profile of Prevotella intermedia during experimental infection in gnotobiotic mice.

Prevotella intermedia is a component of the indigenous microbiota but is also responsible for anaerobic infections of the gastrointestinal tract and oral cavity. The aim of the present study was to investigate the influence of oxidative stress on the in vivo pathogenicity of P. intermedia. Germ-free mice were challenged intraperitoneally with parental (wt) or oxidative stress adapted (aero) strains. Bacterial virulence was evaluated by histopathology, hyperaemia and blood analysis [C-reactive protein (CRP), serum albumin and white blood cells (WBCs)], 3 and 10 days after challenge. CRP levels and WBC count were higher in animals challenged with the aero strain, and the albumin level was lower in this group, only 10 days after infection (P<0.05). Body weight gain was significantly reduced whereas hyperaemia and ratios of spleen/organ weight were increased in animals challenged with the aero strain (P<0.05). The liver of animals challenged with the aero strain showed hyperaemia, vasodilatation as well as an increase in the number of inflammatory cells and liver/organ weight ratio (P<0.05). Similar, but more discrete, alterations were observed in the small intestine of animals challenged with the aero strain. Studies on stress responses of this putative pathogen may help to better understand the aggressive potential and virulence markers of anaerobic bacteria.

Identification and antimicrobial susceptibility of micro-organisms recovered from cutaneous lesions of human American tegumentary leishmaniasis in Minas Gerais, Brazil.

An evaluation of the microbiota present in cutaneous ulcers from 31 patients with a clinical and parasitological diagnosis of American tegumentary leishmaniasis (ATL) was carried out by the standard filter paper disc technique, including antimicrobial susceptibility of the bacterial isolates. Microbial examination indicated that 21 patients (67.7%) were contaminated with one to four bacteria and some of them also with yeast. A total of 142 micro-organisms were isolated. Staphylococcus aureus was the most frequently recovered bacterium (95.2% of positive patients) and was found to produce type B (70% of the staphylococcal isolates) and type C (50%) enterotoxins as well as toxic shock syndrome toxin (60%). Proteus mirabilis (33.3% of the positive patients), Streptococcus pyogenes (19.0 %), H(2)S-negative Proteus species (19.0%), Klebsiella oxytoca (14.3%), Enterobacter species (9.5%), Peptostreptococcus species (9.5%), Pseudomonas species (4.8%), Prevotella bivia (4.8%), Escherichia coli (4.8%), Streptococcus agalactiae (4.8%), Bacteroides fragilis (4.8%), Candida albicans (9.5%) and Candida tropicalis (4.8%) were also isolated. Surprisingly, Staph. aureus isolates were susceptible to almost all tested drugs, although some of them were resistant to penicillin (69%) and ampicillin + sulbactam (68%). Concerning obligate anaerobes, all the Gram-negative isolates (25% of the total) were resistant to metronidazole. The results of the present study show that microbial secondary contaminants, particularly Staph. aureus, should be considered in the diagnosis and treatment of ATL lesions.

Enhanced pathogenicity of Fusobacterium nucleatum adapted to oxidative stress.

Fusobacterium nucleatum is an obligate anaerobic bacterium found in the indigenous human microbiota but also recovered from several anaerobic infections. Considering the biological and medical relevance of F. nucleatum, the characterization of its response to oxidative stress is needed in order to understand how this anaerobic bacterium survives during an invasive process of oxygenated tissues. Influence of oxidative stress by atmospheric oxygen exposure on cellular morphology and pathogenicity of F. nucleatum were investigated. The wild-type F. nucleatum ATCC 25586 (wt-strain) was exposed to oxidative stress to select an adapted strain (aero-strain). Conventional NIH Swiss mice were split in two experimental groups which were challenged intraperitoneally with wt-strain and aero-strain, respectively, and a control group, unchallenged. Histopathological and hyperemia analysis were performed by day 30 after infection. Gram stain of aero-strain showed drastic changes in cellular morphology when compared to wt-strain. A significant increase of liver weight/body weight ratio (P < 0.05) as well as a tendency (P = 0.16) to higher spleen weight/body weight ratio were observed for the mice challenged with aero-strain when compared to the two other animal groups. Additionally, these animals also showed hyperemia in the spleen and liver as well as an increased number of inflammatory cells and steatosis in the liver. The results showed that, in addition to extensive changes in cell morphology, the adaptation to oxidative stress might also influence the pathogenicity of F. nucleatum. These findings have clinical implications since in the host tissues this indigenous putative pathogen is exposed to more or less oxygenated environments found on the different anatomic sites invaded by the bacterium.

Differential gene expression in a Bacteroides fragilis metronidazole-resistant mutant.

OBJECTIVES: The current work focused on molecular changes in a spontaneous Bacteroides fragilismutant selected by low concentrations of metronidazole as an adaptive response to the drug. METHODS: A metronidazole-resistant strain derived from B. fragilis ATCC 25285 was selected by passage in the presence of drug using 0-4 mg/L gradient plates. Using a combination of proteomics for identification of differentially expressed proteins by two-dimensional electrophoresis and selected mutational analyses by single cross-over insertion and an allelic exchange, we have identified genes involved in the adaptive response to metronidazole. RESULTS: There are significant changes in the protein profiles of resistant strains. These changes appeared to affect a wide range of metabolic proteins including lactate dehydrogenase (up-regulated) and flavodoxin (down-regulated), which may be involved in electron transfer reactions. Also, the enzymic activity of the pyruvate-ferredoxin oxidoreductase (PorA) complex was impaired. Mutant strains lacking the genes for flavodoxin and PorA were less susceptible to metronidazole than the sensitive parent, and a double flavodoxin/PorA mutant had even less susceptibility but none of the mutants were as resistant as the spontaneous metronidazole-resistant strain. CONCLUSIONS: Overall, the data indicated that there were global changes in the regulation of the physiology of the metronidazole-resistant strain. In addition, flavodoxin was identified as an important contributor to metronidazole sensitivity in B. fragilis.

Enhanced pathogenicity of susceptible strains of the Bacteroides fragilis group subjected to low doses of metronidazole.

Different concentrations of metronidazole are used widely to treat protozoan and fungal infections. As an antibacterial drug, metronidazole is mainly used against anaerobes, of which the Bacteroides fragilis group is the most important in terms of the frequency of recovery and antimicrobial resistance patterns. The objective of this study was to investigate (1) in vivo metronidazole-induced modifications in the B. fragilis group reflected by altered virulence, and (2) the interference of metronidazole in cellular viability of these samples when subjected in vitro to human polymorphonuclear leukocytes (PMNs). Strains adapted to low metronidazole concentrations were observed to be more virulent, as demonstrated experimentally in mice by weight loss, quantitative evidence of tissue damage, hemorrhage and anatomopathology of spleen, liver and small intestine samples. A significant increase (P < 0.05) in mean bacterial viability rate of about 2.62-fold was observed for all the drug-adapted strains after contact with human PMNs. However, the level of this phenomenon was quite different among the tested species. These results draw attention to the risk that prolonged therapy, even with low concentrations of metronidazole, may affect the pathogenicity of Bacteroides strains, producing changes in host-bacteria relationships.