Flávia L. Carmo

Mangrove bacterial diversity and the impact of oil contamination revealed by pyrosequencing: Bacterial proxies for oil pollution

Background Mangroves are transitional coastal ecosystems in tropical and sub-tropical regions and represent biologically important and productive ecosystems. Despite their great ecological and economic importance, mangroves are often situated in areas of high anthropogenic influence, being exposed to pollutants, such as those released by oil spills. Methodology/Principal Findings A microcosm experiment was conducted, which simulated an oil spill in previously pristine mangrove sediment. The effect of the oil spill on the extant microbial community was studied using direct pyrosequencing. Extensive bacterial diversity was observed in the pristine mangrove sediment, even after oil contamination. The number of different OTUs only detected in contaminated samples was significantly higher than the number of OTUs only detected in non-contaminated samples. The phylum Proteobacteria, in particular the classes Gammaproteobacteria and Deltaproteobacteria, were prevalent before and after the simulated oil spill. On the other hand, the order Chromatiales and the genus Haliea decreased upon exposure to 2 and 5% oil, these are proposed as sensitive indicators of oil contamination. Three other genera, Marinobacterium, Marinobacter and Cycloclasticus increased their prevalence when confronted with oil. These groups are possible targets for the biomonitoring of the impact of oil in mangrove settings. Conclusions/Significance We suggest the use of sequences of the selected genera as proxies for oil pollution, using qPCR assessments. The quantification of these genera in distinct mangrove systems in relation to the local oil levels would permit the evaluation of the level of perturbance of mangroves, being useful in field monitoring. Considering the importance of mangroves to many other environments and the susceptibility of such areas to oil spills this manuscript will be of broad interest.

Supplementing the Antimicrobial Effects of Chemomechanical Debridement with Either Passive Ultrasonic Irrigation or a Final Rinse with Chlorhexidine: A Clinical Study

INTRODUCTION The ability of 2 different approaches to supplement the antimicrobial effects of chemomechanical debridement in infected root canals was compared in vivo. METHODS Samples from necrotic root canals of teeth with apical periodontitis were taken at the baseline (S1), after preparation with rotary nickel-titanium BioRaCe instruments and 2.5% NaOCl irrigation (S2), and then after either passive ultrasonic irrigation (PUI) for activation of NaOCl (n = 13) or a final rinse with 2% chlorhexidine (CHX) (n = 14) (S3). The incidence of positive culture for bacteria and fungi as well as positive broad-range polymerase chain reaction (PCR) results for bacteria, fungi, and archaea was determined. RESULTS All S1 samples were positive for bacteria in all methods. Fungi were not detected, and archaea occurred in only one S1 sample. Treatment procedures were significantly effective in reducing the incidence of positive culture and PCR results. Although both supplementary approaches reduced the incidence of positive bacteriologic results when compared with postinstrumentation samples, reduction was not statistically significant (P > .05). There was no significant difference for intergroup comparisons either (P > .05). CONCLUSIONS Although supplementary disinfection with either PUI or a final rinse with CHX can reduce the number of cases with positive culture and PCR results for bacteria, many cases still remain with detectable bacteria in the main root canal. Research on alternative or supplementary antimicrobial methods or substances should be encouraged.

Climate change affects key nitrogen-fixing bacterial populations on coral reefs

Coral reefs are at serious risk due to events associated with global climate change. Elevated ocean temperatures have unpredictable consequences for the ocean’s biogeochemical cycles. The nitrogen cycle is driven by complex microbial transformations, including nitrogen fixation. This study investigated the effects of increased seawater temperature on bacteria able to fix nitrogen (diazotrophs) that live in association with the mussid coral Mussismilia harttii. Consistent increases in diazotroph abundances and diversities were found at increased temperatures. Moreover, gradual shifts in the dominance of particular diazotroph populations occurred as temperature increased, indicating a potential future scenario of climate change. The temperature-sensitive diazotrophs may provide useful bioindicators of the effects of thermal stress on coral reef health, allowing the impact of thermal anomalies to be monitored. In addition, our findings support the development of research on different strategies to improve the fitness of corals during events of thermal stress, such as augmentation with specific diazotrophs.

Identification of herpesviruses types 1 to 8 and human papillomavirus in acute apical abscesses.

INTRODUCTION It has been suggested that viruses, especially herpesviruses, can play a role in the pathogenesis of marginal and apical periodontitis. This study aimed to detect herpesviruses types 1 to 8, namely herpes simplex virus (HSV-1/2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), human herpesvirus-7 (HHV-7), and human herpesvirus-8 (HHV-8) as well as human papillomavirus (HPV) in acute apical abscesses. METHODS Twenty-four samples were taken by aspiration of the purulent exudate from acute apical abscesses. DNA extracted from clinical samples served as a template in single or nested polymerase chain reaction (PCR) assays for the detection of the target viruses. RESULTS Control PCR reactions with β-globin gene primers revealed that all samples but one had detectable human DNA. Of the 23 abscess samples positive for the β-globin gene, 14 (61%) were positive for at least one of the target human viruses. Thirteen (56.5%) cases had herpesvirus: HHV-8 occurred in 11 (48%), VZV and HHV-6B in two (9%), and HHV-7 and HSV-1/2 in one (4%). EBV and HCMV were not present in any of the examined samples. HPV was detected in three (13%) abscess samples. Viral coinfection was found in five cases, with one case harboring three of the targeted viruses. CONCLUSION A large number of abscess samples were positive for at least one target virus. Unexpectedly, HHV-8 was for the first time detected and in a high prevalence. Papillomavirus and other herpesviruses were also found for the first time in endodontic abscesses. Although these findings suggest an association, the specific role of viruses in the pathogenesis of acute apical abscesses awaits further clarification.

Bacterial community profiling of cryogenically ground samples from the apical and coronal root segments of teeth with apical periodontitis.

Bacteria located at the apical part of infected root canals are arguably directly involved in the pathogenesis of apical periodontitis. This study was conducted to profile and further compare the bacterial communities established at the apical and middle/coronal segments of infected root canals. Extracted teeth with attached apical periodontitis lesions were sectioned so as to obtain two root fragments representing the apical third and the coronal two thirds. Root fragments were subjected to a cryogenic grinding approach. DNA was extracted from root powder samples and used as a template for bacterial community profiling using a 16S ribosomal RNA gene-based seminested polymerase chain reaction/denaturing gradient gel electrophoresis approach. The mean number of bands in apical samples from teeth with primary infections was 28, ranging from 18 to 48, whereas in the middle/coronal samples, it was also 28, ranging from 19 to 36. Findings showed that the profile of bacterial community colonizing the apical third of infected root canals is as diverse as that occurring at the middle/coronal thirds. A high variability was observed for both interindividual (samples from the same region but from different patients) and intraindividual (samples from different regions of the same tooth) comparisons. The methodology used to prepare and analyze samples was highly effective in disclosing a previously unanticipated broad diversity of endodontic bacterial communities, especially at the apical part of infected root canals.

Profiling of root canal bacterial communities associated with chronic apical periodontitis from Brazilian and Norwegian subjects.

The aim of this study was to compare the bacterial community profiles of the root canal microbiota associated with chronic apical periodontitis from Brazilian and Norwegian patients using the denaturing gradient gel electrophoresis (DGGE) and the ribosomal intergenic spacer analysis (RISA) approaches. DNA extracted from root canal samples was subjected to polymerase chain reaction using primers appropriate for further DGGE or RISA analysis. The resulting banding patterns representative of the bacterial community structures in samples from the two locations were compared. DGGE and RISA fingerprints showed a great interindividual variability in the bacterial community profiles, irrespective of the geographic location of the patient. However, similarities among the bacterial community DGGE profiles revealed the existence of a geography-related pattern.

Molecular microbiological evaluation of passive ultrasonic activation as a supplementary disinfecting step: a clinical study.

INTRODUCTION This in vivo study used molecular microbiology methods to evaluate the effects of passive ultrasonic irrigation (PUI) as a supplementary disinfecting step after root canal preparation. METHODS Samples were taken from 10 necrotic root canals of teeth with apical periodontitis before (S1) and after rotary nickel-titanium instrumentation using 2.5% NaOCl as the irrigant (S2) and then after PUI for NaOCl activation (S3). The parameters examined included the incidence of positive broad-range polymerase chain reaction (PCR) results for bacterial presence, the impact on bacterial diversity evaluated by PCR-denaturing gradient gel electrophoresis (DGGE), the quantitative bacterial reduction determined by real-time PCR, and the identification of persistent species by clone library analysis. RESULTS All S1 samples were positive for bacteria in all tests. Treatment procedures were significantly effective in reducing the incidence of positive results for bacteria, the number of bacterial cells (infectious bioburden), and the bacterial diversity (number of species and abundance). However, the supplementary PUI approach did not succeed in significantly enhancing disinfection beyond that achieved by chemomechanical preparation. Several bacterial species/phylotypes were identified in post-treatment samples that were positive for bacteria. CONCLUSIONS Findings from this clinical study including a small sample size suggest that PUI can be ineffective in significantly improving disinfection of the main root canal after chemomechanical procedures.

Clinical antimicrobial efficacy of NiTi rotary instrumentation with NaOCl irrigation, final rinse with chlorhexidine and interappointment medication: a molecular study

AIM To evaluate clinically the antibacterial effects of root canal treatment procedures using molecular microbiology analyses. METHODOLOGY Samples were taken from 14 necrotic root canals of teeth with apical periodontitis before (S1) and after instrumentation with NaOCl irrigation (S2), a final rinse with chlorhexidine (CHX) (S3) and then one-week interappointment medication with calcium hydroxide/CHX paste (S4). The parameters examined included the following: incidence of positive broad-range PCR results for bacterial presence; impact on bacterial community structures evaluated by PCR-Denaturing Gradient Gel Electrophoresis (DGGE); quantitative bacterial reduction determined by real-time PCR; and identification of bacterial persisters by cloning and sequencing. Data from the different tests were subjected to statistical analyses and diversity indicator calculations. RESULTS All S1 samples were positive for bacteria in all tests. Treatment procedures promoted a decrease in microbial diversity and significantly reduced the incidence of positive results and the bacterial counts (P < 0.05). In general, each subsequent treatment step improved disinfection. No specific taxon or community pattern was associated with post-treatment samples. CONCLUSION Supplementary steps consisting of a final rinse with CHX followed by calcium hydroxide interappointment medication promoted further decrease in the bacterial bioburden to levels significantly below those achieved by the chemomechanical procedures alone. Because the long-term outcome of root canal treatment is dependent upon maximal bacterial reduction, the present results are of clinical relevance.

Viral-bacterial associations in acute apical abscesses.

OBJECTIVE Viral-bacterial and bacterial synergism have been suggested to contribute to the pathogenesis of several human diseases. This study sought to investigate the possible associations between 9 candidate endodontic bacterial pathogens and 9 human viruses in samples from acute apical abscesses. STUDY DESIGN DNA extracts from purulent exudate aspirates of 33 cases of acute apical abscess were surveyed for the presence of 9 selected bacterial species using a 16S ribosomal RNA gene-based nested polymerase chain reaction (PCR) approach. Single or nested PCR assays were used for detection of the human papillomavirus (HPV) and herpesviruses types 1 to 8. RESULTS Two-thirds of the abscess samples were positive for at least one of the target viruses. Specifically, the most frequently detected viruses were HHV-8 (54.5%); HPV (9%); and varicella zoster virus (VZV), Epstein-Barr virus (EBV), and HHV-6 (6%). Bacterial DNA was present in all cases and the most prevalent bacterial species were Treponema denticola (70%), Tannerella forsythia (67%), Porphyromonas endodontalis (67%), Dialister invisus (61%), and Dialister pneumosintes (57.5%). HHV-8 was positively associated with 7 of the target bacterial species and HPV with 4, but all these associations were weak. Several bacterial pairs showed a moderate positive association. Viral coinfection was found in 6 abscess cases, but no significant viral association could be determined. CONCLUSIONS Findings demonstrated that bacterial and viral DNA occurred concomitantly in two-thirds of the samples from endodontic abscesses. Although this may suggest a role for viruses in the etiology of apical abscesses, the possibility also exists that the presence of viruses in abscess samples is merely a consequence of the bacterially induced disease process. Further studies are necessary to clarify the role of these viral-bacterial interactions, if any, in the pathogenesis of acute apical abscesses.

Bacterial communities reflect the spatial variation in pollutant levels in Brazilian mangrove sediment.

The majority of oil from oceanic oil spills converges on coastal ecosystems such as mangrove forests. A major challenge to mangrove bioremediation is defining the mangrove’s pollution levels and measuring its recuperation from pollution. Bioindicators can provide a welcome tool for defining such recovery. To determine if the microbial profiles reflected variation in the pollutants, samples from different locations within a single mangrove with a history of exposure to oil were chemically characterised, and the microbial populations were evaluated by a comprehensive range of conventional and molecular methods. Multivariate ordination of denaturing gradient gel electrophoresis (DGGE) microbial community fingerprints revealed a pronounced separation between the sediment and rhizosphere samples for all analysed bacterial communities (Bacteria, Betaproteobacteria, Alphaproteobacteria, Actinobacteria and Pseudomonas). A Mantel test revealed significant relationships between the sediment chemical fertility and oil-derived pollutants, most of the bacterial community fingerprints from sediment samples, and the counts by different cultivation strategies. The level of total petroleum hydrocarbons was significantly associated with the Bacteria and Betaproteobacteria fingerprints, whereas anthracene and the total level of polycyclic aromatic hydrocarbons were associated with the Actinobacteria. These results show that microbial communities from the studied mangrove reflect the spatial variation of the chemicals in the sediment, demonstrating the specific influences of oil-derived pollutants.