Simone Gonçalves dos Santos

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis

BackgroundBacterial 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.ResultsAn 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.ConclusionsComparative 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.

Composition analysis of dental solid waste in Brazil

When developing proper waste management strategies, it is essential to characterize the volume and composition of solid waste. The aim of this work was to evaluate the composition of dental waste produced by three dental health services in Belo Horizonte, Minas Gerais State, Brazil. Two universities, one public and one private, and one public dental health service were selected. Waste collection took place from March to November 2007. During this period, three samples were collected from each dental health service. The total amount of dental waste produced in one day of dental work was manually separated into three categories: infectious and potentially infectious waste, accounting for 24.3% of the total waste; non-infectious waste, accounting for 48.1%; and domestic-type waste, accounting for 27.6% (percentages are for mean weights of solid waste). Our results showed that most of the waste considered as biomedical may be misclassified, consequently making the infectious waste amount appear much larger. In addition, our results suggest that the best waste minimization method is recycling, and they help to define an appropriate waste management system in all three of the dental health services involved in this study.

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-β-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-β-lactamases (MβLs). 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.

Count, identification and antimicrobial susceptibility of bacteria recovered from dental solid waste in Brazil.

In Brazil, few studies on microbial content of dental solid waste and its antibiotic susceptibility are available. An effort has been made through this study to evaluate the hazardous status of dental solid waste, keeping in mind its possible role in cross-infection chain. Six samples of solid waste were collected at different times and seasons from three dental health services. The microbial content was evaluated in different culture media and atmospheric conditions, and the isolates were submitted to antibiotic susceptibility testing. A total of 766 bacterial strains were isolated and identified during the study period. Gram-positive cocci were the most frequent morphotype isolated (48.0%), followed by Gram-negative rods (46.2%), Gram-positive rods (5.0%), Gram-negative-cocci (0.4%), and Gram-positive coccobacillus (0.1%). Only two anaerobic bacteria were isolated (0.3%). The most frequently isolated species was Staphylococcus epidermidis (29.9%), followed by Stenotrophomonas maltophilia (8.2%), and Enterococcus faecalis (6.7%). High resistance rate to ampicillin was observed among Gram-negative rods (59.4%) and Gram-positive cocci (44.4%). For Gram-negative rods, high resistance was also noted to aztreonam (47.7%), cefotaxime (47.4%), ceftriaxone and cefazolin (43.7%), and ticarcillin-clavulanic acid (38.2%). Against Gram-positive cocci penicillin exhibit a higher resistance rate (45.0%), followed by ampicillin, erythromycin (27.2%), and tetracycline (22.0%). The present study demonstrated that several pathogenic bacteria are present in dental solid waste and can survive after 48 h from the waste generation time and harbor resistance profiles against several clinical recommended antibiotics.

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 β-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-β-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.

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.

Analysis of microbial load on surgical instruments after clinical use and following manual and automated cleaning

BACKGROUND We aimed to monitor the microbial load and identify the microorganisms recovered from surgical instruments after clinical use and following manual and automated cleaning. METHODS This experimental study was carried out in the Laboratory of Oral Microbiology and Anaerobes at the Federal University of Minas Gerais in Brazil. Microbial samples were taken from 125 surgical instruments used in 25 types of gastrointestinal surgeries. RESULTS The average microbial load was 93.1 CFU/100 mL after clinical use and 41 CFU/100 mL and 8.24 CFU/100 mL on instruments following 2 sequential steps of manual cleaning, respectively, and 75 CFU/100 mL and 16.1 CFU/100 mL on instruments after automated cleaning. Surgical wound classification significantly affected the microbial load recovered on instruments. Coagulase-negative Staphylococcus, Escherichia coli, Pseudomonas spp, Stenotrophomonas maltophilia, and Acinetobacter baumannii complex were recovered. CONCLUSIONS The average microbial load observed after the cleaning steps decreased, and the decrease in microbial load was more pronounced using the manual method compared with that observed using the automated method.

Alteration in the endogenous intestinal flora of swiss webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.

Phenotypic and molecular detection of the bla KPC gene in clinical isolates from inpatients at hospitals in São Luis, MA, Brazil

BackgroundBacteria that produce Klebsiella pneumoniae carbapenemases (KPCs) are resistant to broad-spectrum β-lactam antibiotics. The objective of this study was to phenotypically and genotypically characterize the antibiotic susceptibility to carbapenems of 297 isolates recovered from clinical samples obtained from inpatients at 16 hospitals in São Luis (Maranhão, Brazil).MethodsThe study was conducted using phenotypic tests and molecular methods, including polymerase chain reaction (PCR), sequencing and enterobacterial repetitive intergenic consensus (ERIC)-PCR. The nonparametric chi-square test of independence was used to evaluate the associations between the bacterial blaKPC gene and the modified Hodge test, and the chi-square adherence test was used to assess the frequency of carbapenemases and their association with the blaKPC gene.ResultsThe most frequently isolated species were Acinetobacter baumannii (n = 128; 43.0%), K. pneumoniae (n = 75; 25.2%), and Pseudomonas aeruginosa (n = 42; 14.1%). Susceptibility assays showed that polymixin B was active against 89.3% of the bacterial isolates. The Acinetobacter spp. and K. pneumoniae strains were susceptible to amikacin and tigecycline, and Pseudomonas spp. were sensitive to gentamicin and amikacin. Among the 297 isolates, 100 (33.7%) were positive for the blaKPC gene, including non-fermentative bacteria (A. baumannii) and Enterobacteriaceae species. Among the isolates positive for the blaKPC gene, K. pneumoniae isolates had the highest positivity rate of 60.0%. The blaKPC gene variants detected included KPC-2, which was found in all isolates belonging to species of the Enterobacteriaceae family. KPC-2 and KPC-3 were observed in A. baumannii isolates. Importantly, the blaKPC gene was also detected in three Raoultella isolates and one isolate of the Pantoea genus. ERIC-PCR patterns showed a high level of genetic diversity among the bacterial isolates; it was capable of distinguishing 34 clones among 100 strains that were positive for blaKPC and were circulating in 11 of the surveyed hospitals.ConclusionsThe high frequency of the blaKPC gene and the high degree of clonal diversity among microorganisms isolated from patients from different hospitals in São Luis suggest the need to improve the quality of health care to reduce the incidence of infections and the emergence of carbapenem resistance in these bacteria as well as other Gram-negative pathogens.

Chemical Composition and Antimicrobial Activity of the Essential Oil from Microlicia crenulata

Abstract The chemical compositions of essential oils extracted by hydrodistillation and of volatile substances extracted by HS-SPME from aerial parts of Microlicia crenulata were studied. The antimicrobial activity of the essential oil was investigated. The essential oils of M. crenulata consist of a complex mixture, composed mainly of terpenoid compounds. There is a slight variation in the yield and chemical composition of the essential oils according to the phenological stages of the year. The oil presented moderate inhibitory activity against Pseudomonas aeruginosa, but did not inhibit the growth of Bacillus cereus var. mycoides, Escherichia coli and Staphylococcus aureus. The oil showed moderate antimicrobial activity against Sacharomyces cerevisiae and Aspergillus flavus fungi, weak activity against Aspergillus niger and was not active against species of Candida and Cryptococcus at the highest concentration tested (250 µg/mL). This study is the first to report the composition and antimicrobial properties of the essential oil of M. crenulata.