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Introduction: Staphylococcus aureus is a major cause of health care associated infections worldwide. Objective: The aim of this work was to evaluate epidemiological characteristics and antimicrobial susceptibility of Methicillin-resistant S . aureus (MRSA) strains isolates from infections in a Brazilian tertiary hospital. Methods: Clinical and epidemiological data of the patients were collected. Bacterial strains were isolated and identified using the classical identification tests. Antimicrobial susceptibility assays were performed using the disc-diffusion method. Findings: A total of 590 samples of S. aureus were isolated from patients and 42.5% were characterized as MRSA. Considering the clinical specimens, most of samples were isolated from blood and tracheal secretion, catheter tip, surgical site swabs, wound secretion, exudates and urine. Overall, a high frequency of resistance was observed against ciprofloxacin, clindamycin, erythromycin, amikacin, gentamicin, trimethoprim-sulfamethoxazole, tetracycline and chloramphenicol. Association between death and multidrug-resistance in elderly patients, and death and occurrence of bacteremia by multidrug-resistant MRSA was observed. Conclusions: Our data are highly relevant for surveillance systems and to map on a wider scale the dynamics of circulation of MRSA and raise discussions on containment strategies and rational use of empiric chemotherapy.

As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host–bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.

Metronidazole is used in clinical practice for the treatment of protozoan and anaerobic infections. Under anaerobic conditions, 5-nitroimidazoles are reduced to cytotoxic nitroradicals which have been shown to act by non-specific binding to, and inactivation of the organism's DNA and enzymes. Among anaerobes, the Bacteroides fragilis group is the most relevant both in terms of frequency of isolation and antimicrobial resistance. In the present study we investigated the mechanism of action of metronidazole in the B. fragilis group. We evaluated chromosomal DNA integrity in susceptible and resistant strains during a period of 5 h after metronidazole exposure and we quantified the drug remaining in the medium after microbial growth. Metronidazole was not reduced in resistant cells which remained metabolically active and with their entire genetic material throughout the experiment. On the other hand, susceptible cells presented chromosomal breakage, a rapid consumption of dissolved metronidazole and a mortality of 90% of the bacterial population during the first 30 minutes of exposure. This interaction between metronidazole and DNA molecules that suggests strands breakage has been previously demonstrated in cell-free extract and in Escherichia coli cells. Our results show this phenomenon also occurring inBacteroides group what was not previously observed in the literature.

We report in this work the antibacterial evaluation of 12 lipophilic fluoroquinolone derivatives containing diaminoalkyl side chains at C-7 position. The compounds were investigated against 15 bacterial strains including gram-negative and gram-positive species of clinical and microbiological importance. Three compounds (5, 10 and 11) were as active as or more efficient than gatifloxacin against gram-positive bacteria M. lentus. When compared with gatifloxacin compound 10 was 16 times more active. Two compounds (11 and 12) were twice more active than the reference compound against S. aureus.

Aims This study was focused on evaluating the persistency of antimicrobial resistance (AR) genes and putative pathogenic bacteria in an anaerobic digesters operating at mesophilic ambient temperature, in two different year seasons: summer and winter. Methods and Results Abundance and dynamic of AR genes encoding resistance to macrolides (ermB), aminoglycosides (aphA2) and beta-lactams (blaTEM-1) and persistency of potentially pathogenic bacteria in pilot-scale anaerobic digesters were investigated. AR genes were determined in the influent and effluent in both conditions. Overall, after 60 days, reduction was observed for all evaluated genes. However, during the summer, anaerobic digestion was more related to the gene reduction as compared to winter. Persistency of potentially pathogenic bacteria was also evaluated by metagenomic analyses compared to an in-house created database. Clostridium, Acinetobacter and Stenotrophomonas were the most identified. Conclusions Overall, considering the mesophilic ambient temperature during anaerobic digestion (summer and winter), a decrease in pathogenic bacteria detection through metagenomic analysis and AR genes is reported. Although the mesophilic anaerobic digestion has been efficient, the results may suggest medically important bacteria and AR genes persistency during the process. Significance and Impact of the Study This is the first report to show AR gene dynamics and persistency of potentially pathogenic bacteria through metagenomic approach in cattle manure ambient temperature anaerobic digestion.

Introduction: Healthcare waste (HCW) might potentially harbor infective viable microorganisms in sanitary landfills. We investigated the antimicrobial susceptibility patterns and the occurrence of the mecA gene in coagulase-negative Staphylococcus strains (CoNS) recovered from the leachate of the HCW in an untreated sanitary landfill. Methodology: Bacterial identification was performed by physiological and molecular approaches, and minimum inhibitory concentrations (MICs) of antimicrobial drugs were determined by the agar dilution method according to CLSI guidelines. All oxacillin-resistant bacteria were screened for the mecA gene. Results: Out of 73 CoNS, seven different species were identified by 16S rDNA sequencing: Staphylococcus felis (64.4%; n = 47), Staphylococcus sciuri (26.0%; n = 19), Staphylococcus epidermidis (2.7%; n = 2), Staphylococcus warneri (2.7%; n = 2), Staphylococcus lentus (1.4%; n = 1), Staphylococcus saprophyticus (1.4%; n = 1), and Staphylococcus haemolyticus (1.4%; n = 1). Penicillin was the least effective antimicrobial (60.3% of resistance; n = 44) followed by erythromycin (39.8%; n = 29), azithromycin (28.8%; n = 21), and oxacillin (16.5%; n = 12). The most effective drug was vancomycin, for which no resistance was observed, followed by gentamicin and levofloxacin, for which only intermediate resistance was observed (22%, n = 16 and 1.4%, n = 1, respectively). Among the oxacillin-resistant strains, the mecA gene was detected in two isolates. Conclusions: Considering the high antimicrobial resistance observed, our results raise concerns about the survival of putative bacterial pathogens carrying important resistance markers in HCW and their environmental spread through untreated residues discharged in sanitary landfills.

To investigate the etiology and the epidemiological profile of patients with vulvovaginal candidiasis (VVC) and predisposing factors.Vaginal secretions were streaked in Sabouraud agar and yeast samples were isolated and identified by Polymerase Chain Reaction (PCR). Demographic and clinical data were obtained with a questionnaire. For statistical analysis, the Student's t-test, the χ² and Fischer tests were applied as needed using the Statistical Package for Social Sciences (SPSS) software, with the level of significance set at 5%.Sixty-nine patients aged from 15 to 52 years were evaluated. They were predominantly white (79.7%), with higher education (58%), married (56.5%) and sexually active (97.1%). Among them, 34.8% were pregnant, 7.2% diabetic, 1.4% seropositive for AIDS, and 36.2% were using oral contraceptives. Recent antibiotic therapy was mentioned by 13% of the patients, and antifungal or anti-trichomonas therapy was mentioned by 5.8 and 1.4% of the patients, respectively. Corticosteroid use was reported by 2.9% and antineoplastic by 1.4%. Vaginal discharge and itching were the main complaints (97.1 and 73.9%), followed by burning (63.8%) and erythema (63.8%). When present, the vaginal flow was predominantly white (88.1%) or lumpy (86.6%). The diagnosis was confirmed by culture in 55 (79.7%) patients, with mixed infections in 4 patients. The most prevalent species was C. albicans, followed by C. glabrata (one monoinfection and two mixed infections with C. albicans). C. lusitaniae and C. albicans were also identified in mixed infections (two patients).Despite the high culture positivity and clinical data characteristic of VVC, the symptoms were not pathognomonic. C. albicans is the most prevalent species, but other species are also involved in VVC etiology, such as the emergence of C. lusitaniae.

Anaerobic digestion figures as a sustainable alternative to avoid discharge of cattle manure in the environment, which results in biogas and biofertilizer. Persistence of potentially pathogenic and drug-resistant bacteria during anaerobic digestion of cattle manure was evaluated. Selective cultures were performed for enterobacteria (ENT), non-fermenting Gram-negative rods (NFR) and Gram-positive cocci (GPC). Antimicrobial susceptibility patterns were determined and a decay of all bacterial groups was observed after 60days. Multidrug-resistant bacteria were detected both the influent and effluent. GPC, the most prevalent group was highly resistant against penicillin and levofloxacin, whereas resistance to ampicillin, ampicillin-sulbactam and chloramphenicol was frequently observed in the ENT and NFR groups. The data point out the need of discussions to better address management of biodigesters and the implementation of sanitary and microbiological safe treatments of animal manures to avoid consequences to human, animal and environmental health.

Title compounds (III) display a higher antibacterial activity than chloramphenicol but they are less active than the reference drug vancomycin.

International audience; This paper describes the synthesis and in vitro biological activities of imidazolidine and hexahydropyrimidine derivatives against bacteria (Escherichia coli, Staphylococcus aureus and Mycobacterium tuberculosis) and Leishmania protozoa. Out of sixteen heterocyclic derivatives tested, none were cytotoxic against mammalian cells. The compounds showed significant bacterial effects and leishmanicidal activity. Compounds 4a and 4c were active against S. aureus and E. coli, respectively. Compounds 3a-3f, 4h and 4i presented promising results against M. tuberculosis, with MIC values ranging from 12.5 to 25.0 μg/mL, comparable to the "first and second line" drugs used to treat tuberculosis. Compounds 4a, 4c and 4e were active against L major. Three of them were structurally characterized by single-crystal X-ray diffraction.

Coagulase-negative staphylococci (CoNS), which are generally neglected as foodborne bacteria, are emerging as significant opportunistic pathogens that may be highly resistant to available antimicrobial drugs. In this study, antimicrobial susceptibility patterns, mecA gene occurrence, and virulence-associated characteristics were evaluated in CoNS isolated from soft cheese in Brazil. A total of 227 bacterial isolates were recovered from 35 cheese samples belonging to 5 batches with 7 different trademarks. The CoNS counts ranged from 10(6) to 10(7) CFU/g. High antimicrobial resistance percentages were observed for oxacillin (76.2%), penicillin (78.5%), erythromycin (67.8%), gentamicin (47.2%), clindamycin (35.7%), rifampicin (26.8%), azithromycin (14.7%), tetracycline (14.7%), levofloxacin (14.2%), and sulfamethoxazole-trimethoprim (11.9%). A low antimicrobial resistance percentage was observed for chloramphenicol (2.3%), and all of the tested bacteria were susceptible to vancomycin and linezolid. In total, a multiple antibiotic resistance (MAR) index of0.2 was observed for 80.6% of the isolated CoNS. However, the MAR index ranged from 50% to 92.6% when only bacterial cheese isolates belonging to the same trademark were considered. Regarding to the prevalence of CoNS carrying mecA gene, 81.5% of the isolated strains were mecA(+) , and 76.2% of these were phenotypically resistant to oxacillin. Three isolates carried the enterotoxin A gene (sea), 29.5% produced biofilm in a laboratory test, and α- or ß-hemolysis were observed for 3% and 5.2%, respectively. This study highlights the extent of the antimicrobial resistance phenomenon in neglected foodborne microorganisms and the potential public health risks that are related to the consumption of CoNS-contaminated soft cheese.

Urban pigeons (Columba livia) are birds that coexist with humans, especially in urban environments. It is accepted that their adaption to these environments is mainly related to three basic reasons: (i) the architecture of urban buildings, monuments and works of engineering has gaps, cracks and spaces that can be used for landing, nesting and shelter, protecting the birds from the weather; (ii) the absence of natural predators; and (iii) availability of food, as these birds are omnivorous, somewhat selective in their feeding (23). In this way pigeons may have an important role in spreading zoonotic agents to humans as they are a natural reservoir of many microorganisms, including pathogenic enterobacteria and other microbial groups such as Enterococcus(6, 19, 41). The transmission of infectious diseases happens usually by either ingestion of food, water, the handling of products contaminated with their feces. The dispersion of wind-dried pigeon droppings should also be considered as a route of pathogen spread (29, 36). Enterococci are Gram-positive, facultative anaerobes, arranged in pairs and short chains. These bacteria were considered as harmless inhabitants of the gastrointestinal tracts of humans and animals, being commonly isolated from feces, water, plants, soil and food products (14, 33). Despite their low virulence, studies have documented that enterococci have been emerging as significant human pathogens (21, 25). Due to their frequent association with infections and their increasing antimicrobial resistance, enterococci have become a serious problem in human medicine as a pathogenic microorganism, as well as nosocomial infection-associated bacteria. Besides natural antimicrobial resistance among Enterococcus species to several antibiotics currently used, they may also acquire resistance determinants through horizontal gene transfer (5). Furthermore, especially regarding bacteria spread environmentally, the release of toxic and heavy metals in various forms into the environment may contribute to the rise of antimicrobial resistance. In the last decade it was stated that antibiotic-resistant bacteria could arise in the environment through co- or cross-resistance to metals (30). Considering global issues, such as public health, urban planning and environmental problems, as pigeon populations increase, especially in Brazilian cities, prospective studies are needed to better assess the risks of acquired infections related to multidrug-resistant bacteria that might be spread or released by urban birds (29, 41, 44). This study aimed to evaluate Enterococcus spp. occurrence in fresh fecal samples isolated from urban pigeons in a southeastern Brazilian city, their antimicrobial susceptibility and their toxic metal tolerance patterns.

Distribution of ESBL-producing enterobacteria associated to community-acquired monomicrobial urinary tract infections and antimicrobial susceptibility trends over a 9-year period Vanessa Cordeiro Dias, Vânia Lucia da Silva, Eduarda de Oliveira Firmo, Lucas Quinet de Andrade Bastos, Andre Netto Bastos, Ricardo Villela Bastos, Claudio Galuppo Diniz Department of Parasitology, Microbiology and Immunology, Institute of Biological Sciences, Federal University of Juiz de Fora, Minas Gerais, Brazil, Cortes Villela Clinical Laboratory, Juiz de Fora, Minas Gerais, Brazil

Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of β-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host-bacteria relationship.

Despite the importance of gastrointestinal diseases and their global distribution, affecting millions of individuals around the world, the role and antimicrobial susceptibility patterns of anaerobic bacteria such as those in the Bacteroides fragilis group (BFG) are still unclear in young children. This study investigated the occurrence and distribution of species in the BFG and enterotoxigenic strains in the fecal microbiota of children and their antimicrobial susceptibility patterns. Diarrheic (n=110) and non-diarrheic (n=65) fecal samples from children aged 0-5 years old were evaluated. BFG strains were isolated and identified by conventional biochemical, physiological and molecular approaches. Alternatively, bacteria and enterotoxigenic strains were detected directly from feces by molecular biology. Antimicrobial drug susceptibility patterns were determined by the agar dilution method according to the guidelines for isolated bacteria. BFG was detected in 64.3% of the fecal samples (55% diarrheic and 80.4% non-diarrheic), and 4.6% were enterotoxigenic. Antimicrobial resistance was observed against ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, ceftriaxone, clindamycin and chloramphenicol. The data show that these bacteria are prevalent in fecal microbiota at higher levels in healthy children. The molecular methodology was more effective in identifying the B. fragilis group when compared to the biochemical and physiological techniques. The observation of high resistance levels stimulates thoughts about the indiscriminate use of antimicrobial drugs in early infancy. Further quantitative studies are needed to gain a better understanding of the role of these bacteria in acute diarrhea in children.

The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. In addition to the difficulties in controlling infectious diseases, the phenotype of resistance can generate metabolic changes which, in turn, can interfere with host-pathogen interactions. The aim of the present study was to identify changes in the subproteome of a laboratory-derived piperacillin/tazobactam-resistant strain of Escherichia coli (minimal inhibitory concentration [MIC] = 128 mg/L) as compared with its susceptible wild-type strain E. coli ATCC 25922 (MIC = 2 mg/L) using 2-D fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF MS). In the resistant strain, a total of 12 protein species were increased in abundance relative to the wild-type strain, including those related to bacterial virulence, antibiotic resistance and DNA protection during stress. Fourteen proteins were increased in abundance in the wild-type strain compared to the resistant strain, including those involved in glycolysis, protein biosynthesis, pentose-phosphate shunt, amino acid transport, cell division and oxidative stress response. In conclusion, our data show overall changes in the subproteome of the piperacillin/tazobactam-resistant strain, reporting for the first time the potential role of a multidrug efflux pump system in E. coli resistance to piperacillin/tazobactam.

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.

Os resíduos de serviços de saúde suscitam polêmica quanto a importância para a saúde humana, animal e ambiental. Avaliou-se a ocorrência de bactérias clinicamente relevantes na pilha de resíduos de serviços de saúde em um aterro sanitário e seu perfil de susceptibilidade aos antimicrobianos. Alíquotas de chorume foram processadas para isolamento seletivo de Staphylococcus sp, bastonetes Gram negativos da família Enterobacteriaceae e não fermentadores. Resistência bacteriana a todos os antimicrobianos testados foi observada em todos os grupos microbianos, além de resistência a mais de uma droga. Os resultados permitem sugerir que bactérias viáveis nos resíduos de serviços de saúde representam riscos à saúde humana e animal. Além disso, a ocorrência de linhagens multirresistentes sustenta a hipótese dos resíduos de serviços de saúde atuarem como reservatórios de marcadores de resistência, com impacto ambiental. A falta de legislação regional de segregação, tratamento e destino de resíduos podem expor diferentes populações a riscos de transmissão de doenças infecciosas associadas a microrganismos multirresistentes. Health service waste gives rise to controversy regarding its importance for human, animal and environmental health. Occurrences of clinically relevant bacteria in piles of health service waste in a sanitary landfill and their antimicrobial susceptibility profile were evaluated. Aliquots of leachate were processed for selective isolation of Staphylococcus sp, Gram-negative rods of the Enterobacteriaceae family and non-fermenters. Bacterial resistance to all the antimicrobials tested was observed in all microbial groups, including resistance to more than one drug. The results make it possible to suggest that viable bacteria in health service waste represent risks to human and animal health. Furthermore, occurrences of multiresistant strains support the hypothesis that health service waste acts as a reservoir for resistance markers, with an environmental impact. The lack of regional legislation concerning segregation, treatment and final disposal of waste may expose different populations to risks of transmission of infectious diseases associated with multiresistant microorganisms.

Health service waste gives rise to controversy regarding its importance for human, animal and environmental health. Occurrences of clinically relevant bacteria in piles of health service waste in a sanitary landfill and their antimicrobial susceptibility profile were evaluated. Aliquots of leachate were processed for selective isolation of Staphylococcus sp, Gram-negative rods of the Enterobacteriaceae family and non-fermenters. Bacterial resistance to all the antimicrobials tested was observed in all microbial groups, including resistance to more than one drug. The results make it possible to suggest that viable bacteria in health service waste represent risks to human and animal health. Furthermore, occurrences of multiresistant strains support the hypothesis that health service waste acts as a reservoir for resistance markers, with an environmental impact. The lack of regional legislation concerning segregation, treatment and final disposal of waste may expose different populations to risks of transmission of infectious diseases associated with multiresistant microorganisms.

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

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.

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

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 (P0.05) 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.

Background A considerable number of tests are recommended in the literature to evaluate in vitro commercial chemical solutions. The variety of tests reflects their limitations and the need to enhance disinfection process. Methods In this study the efficacy of 4 chemical disinfectants selected by their practical use in Health Care Services and by literature recommendation in aerobic and in strict anaerobic bacteria were evaluated by their practical use in Health Care Services and by literature recommendation in aerobic and in strict anaerobic bacteria. Viability was tested in biofilms grown on glass and rubber tip carriers. Results The results showed microbial growth in chemical solutions at concentrations recommended by the literature or at very close concentrations to them. Viable cells were recovered from biofilms after 30 minutes ( Bacteroides fragilis ) and 60 minutes ( Streptococcus mutans and Salmonella thyphimurium ) contact with 2.4% glutaraldehyde and after 60 minutes ( S typhimurium ) in 2.0% glutaraldehyde. In 70% ethyl alcohol, S tiphymurium was viable up to 10 minutes, Eschericia coli up to 30 minutes, and S mutans up to 60 minutes. In 1% sodium hypochlorite, S mutans was viable up to 30 minutes and S tiphymurium up to 45 minutes. Detection of cell viability could be related to methodologic differences, including biofilm formation, as demonstrated by scanning electron microscopy. It should be emphasized that B fragilis , the most clinically relevant obligate anaerobe, remained viable in one routinely used solution. Conclusion These findings pointed out the need of periodic surveillance of disinfectants' activity used in Health Care Services and the need of reviewing routines of disinfection protocols.

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.

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.

Metronidazole is widely used to treat protozoan and fungal infections. As an antibacterial drug, it is used mainly against anaerobes. Among anaerobes, the Bacteroides fragilis group is the most relevant in terms of frequency of recovery and antimicrobial resistance patterns. The use of metronidazole and other antimicrobial drugs induces morphological changes in this bacterial group. The present study investigated in vivo if these morphological modifications were accompanied by changes in virulence patterns by using germfree mice experimentally challenged with metronidazole-resistant Bacteroides strains before and after exposure to metronidazole. It was observed that metronidazole-resistant strains were more virulent after contact with the drug, as demonstrated by anatomicopathologic data for spleen, liver, and small intestine samples. These results suggest that long-term therapy and high metronidazole concentrations could interfere with microbial pathogenicity, resulting in changes to host-bacterium relationships.

The aim of this study was to use the fluorescence in situ hybridization (FISH) technique to test the hypothesis of qualitative and quantitative differences of 8 periodontopathogens between pregnant and non-pregnant women. This cross-sectional study included 20 pregnant women in their second trimester of pregnancy and 20 non-pregnant women. Probing depth, bleeding on probing, clinical attachment level, and presence of calculus were recorded. Subgingival plaque samples were collected and the FISH technique identified the presence and numbers of Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus, Porphyromonas gingivalis, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia and Prevotella nigrescens. The Mann-Whitney U-test was applied to compare the data between the two groups. The mean age, ethnicity, marital status, education, and economic level in both groups were similar. The clinical parameters showed no significant differences between pregnant and non-pregnant women. The numbers of subgingival periodontopathogens were not found to be significantly different between groups, despite the higher mean counts of P. intermedia in pregnant women. Colonization patterns of the different bacteria most commonly associated with periodontal disease were not different in the subgingival plaque of pregnant and non-pregnant women.

Group B Streptococcus (GBS) is still not routinely screened during pregnancy in Brazil, being prophylaxis and empirical treatment based on identification of risk groups. This study aimed to investigate GBS prevalence in Brazilian pregnant women by culture or polymerase chain reaction (PCR) associated to the enrichment culture, and to determine the antimicrobial susceptibility patterns of isolated bacteria, so as to support public health policies and empirical prophylaxis. After an epidemiological survey, vaginal and anorectal specimens were collected from 221 consenting laboring women. Each sample was submitted to enrichment culture and sheep blood agar was used to isolate suggestive GBS. Alternatively, specific PCR was performed from enrichment cultures. Antimicrobial susceptibility patterns were determined for isolated bacteria by agar diffusion method. No risk groups were identified. Considering the culture-based methodology, GBS was detected in 9.5% of the donors. Twenty five bacterial strains were isolated and identified. Through the culture-PCR methodology, GBS was detected in 32.6% specimens. Bacterial resistance was not detected against ampicillin, cephazolin, vancomycin and ciprofloxacin, whereas 22.7% were resistant to erythromycin and 50% were resistant to clindamycin. GBS detection may be improved by the association of PCR and enrichment culture. Considering that colony selection in agar plates may be laboring and technician-dependent, it may not reflect the real prevalence of streptococci. As in Brazil prevention strategies to reduce the GBS associated diseases have not been adopted, prospective studies are needed to anchor public health policies especially considering the regional GBS antimicrobial susceptibility patterns.