Assessment of virulence potential of uncharacterized Enterococcus faecalis strains using pan genomic approach - Identification of pathogen-specific and habitat-specific genes.

Enterococcus faecalis, a leading nosocomial pathogen and yet a prominent member of gut microbiome, lacks clear demarcation between pathogenic and non-pathogenic strains at genome level. Here we present the comparative genome analysis of 36 E. faecalis strains with different pathogenic features and from different body-habitats. This study begins by addressing the genome dynamics, which shows that the pan-genome of E. faecalis is still open, though the core genome is nearly saturated. We identified eight uncharacterized strains as potential pathogens on the basis of their co-segregation with reported pathogens in gene presence-absence matrix and Pathogenicity Island (PAI) distribution. A ~7.4 kb genomic-cassette, which is itself a part of PAI, is found to exist in all reported and potential pathogens, but not in commensals and other uncharacterized strains. This region encodes four genes and among them, products of two hypothetical genes are predicted to be intrinsically disordered that may serve as novel targets for therapeutic measures. Exclusive existence of 215, 129, 4 and 1 genes in the blood, gastrointestinal tract, urogenital tract, oral cavity and lymph node derived E. faecalis genomes respectively suggests possible employment of distinct habitat-specific genetic strategies in the adaptation of E. faecalis in human host.

In vitro assessment of the recurrent doses of topical gaseous ozone in the removal of Enterococcus faecalis biofilms in root canals.

AIM: To evaluate the potential antibacterial effect of recurrent doses of topical gaseous ozone on the Enterococcus faecalis biofilms0 growth in human root canals in vitro. MATERIALS AND METHODS: One hundred and thirty four human single-rooted mandibular premolars were enlarged to a size 35 K-File. Each root canal were inoculated with an overnight culture of Enterococcus faecalis 0ATCC 29212 in tryptic soy broth for 24 hours and incubated for 7 days at 37°C. At 7-day interval, 4 specimens were prepared for Scanning Electron Microscope (SEM) analysis to confirm the presence and purity of biofilms whilst the other contaminated root canals were irrigated and disinfected. One hundred root canals of total 134 specimens were selected to create the experimental groups and divided into 5 subgroups. In each experimental group ( n = 20) root canals), recurrent ozone doses were applied with different irrigation and disinfection protocols in 5 different time intervals. Bacterial growth was analyzed by counting viable E. faecalis on tryptic soy agar plates. RESULTS: According to intergroup comparison results observed in the final sample collection analysis, the amount of remaining bacteria in the positive control group were found to be significantly higher compared to Groups 1, 2, 3, 4, 5 and the material control group ( P < 0.01). The remaining amount of bacteria in the last count of Group 1 were found to be significantly higher compared to Group 2 ( P < 0.05), Group 4 ( P < 0.01), Group 5 (P < 0.05) and the material control group (P < 0.01). CONCLUSION: The application of topical gaseous ozone in recurrent doses provides a positive effect in the removal of E. faecalis biofilm from root canals. However, during disinfection procedure, the combined use of recurrent doses of topical gaseous ozone with 2% NaOCl enhanced its antibacterial effect against E. faecalis biofilm.

Characterization of lead-resistant river isolate Enterococcus faecalis and assessment of its multiple metal and antibiotic resistance.

Contamination of surface waters has a direct impact on the public health of entire communities. Microorganisms inhabiting contaminated surface waters have developed mechanisms of coping with a variety of toxic metals and drugs. Investigations were carried out to isolate and identify lead-resistant bacteria from the river Kizilirmak along the city of Kirikkale, Turkey. Of the 33 lead-resistant isolates, one isolate with a minimal inhibitory concentration of 1,200 mg L(-1) was isolated and identified as Enterococcus faecalis by using biochemical tests and 16S rRNA sequencing. Lead-resistant E. faecalis isolate was found out to be resistant to other heavy metals like aluminum, lithium, barium, chromium, iron, silver, tin, nickel, zinc, and strontium and to drugs like amikacin, aztreonam, and gentamicin. E. faecalis harbored four plasmids with the molecular sizes of 1.58, 3.06, 22.76, and 28.95 kb. Plasmid profile analyses of cured derivatives revealed that the lead resistance ability of E. faecalis was still existing despite the elimination of all the plasmids. Moreover, the antibiotic resistance pattern of the cured derivatives did not demonstrate any change from the parental strain. Our findings indicated that the lead resistance genes of E. faecalis were located on the chromosomal DNA rather than the plasmid.

The influence of cone-beam computed tomography and periapical radiographic evaluation on the assessment of periapical bone destruction in dog's teeth.

OBJECTIVE: The aim of this study was to determine the influence of periapical radiographs, cone beam computed tomography (CBCT) sections, and cone beam volumetric data on the determination of periapical bone destruction in endodontically treated distal root canals of premolar canine teeth. Nontreated mesial roots were used as controls. STUDY DESIGN: Enterococcus faecalis strain (ATCC 29212) was inoculated into 30 root canals of 2 mongrel dogs to induce apical periodontitis. After 60 days, the root canals of the distal roots of the 11 mandibular and 4 maxillary premolars were endodontically treated (n = 15). The mesial root canals were used as controls (no treatment). The bone destruction was evaluated after 6 months by 5 evaluators using periapical radiographs and by CBCT (coronal and sagittal sections). After the experimental period, the area of the lesions in periapical radiographs and CBCT sections were measured in mm(2) using the ImageTool software. A single evaluator measured the volumetric data using the OsiriX software. The comparison between the diagnosis methods in treated root canals and controls was performed using parametric and nonparametric criteria. The Pearson correlation coefficient was computed between radiographic values and CBCT volumetric data in treated root canals and controls. RESULTS: The results showed the presence of chronic apical periodontitis in every inoculated tooth. After 6 months, periapical radiographs, coronal CBCT sections, and volumetric data showed lower bone destruction in endodontically treated teeth in comparison with the control group (P < .05). The 5 evaluators found no differences between the apical periodontitis area of treated teeth and controls when CBCT sagittal sections were used (P > .05). No correlation was found between x-ray and CBCT volumetric values in treated root canals. CONCLUSIONS: Although selected CBCT sagittal sections showed similar values of bone destruction in endodontically and nontreated root canals, volumetric CBCT data showed that periapical lesions of endodontically treated root canals had half of the volume of periapical lesions in nontreated root canals. No relationship could be found between the periapical values of bone destruction and volumetric data found in CBCT of treated rood canals.

Assessment of survival of Listeria monocytogenes, Salmonella Infantis and Enterococcus faecalis artificially inoculated into experimental waste or compost.

AIMS: To evaluate survival of pathogenic strains, Listeria monocytogenes and Salmonella Infantis and a sanitation indicator Enterococcus faecalis in composts at different stages of the composting process and during storage. METHODS AND RESULTS: The studied pathogenic and indicator strains, originally isolated from compost, were inoculated into compost samples from the various stages of the composting process. During incubation, indigenous microflora diversity was monitored with DGGE analysis. After 90 days of incubation, strain survival was observed in compost sampled before the beginning of the cooling phase, and DGGE analysis demonstrated an increase of microbial diversity up to the cooling phase. However, inoculated strains were not detected in composts after 30, 60 or 90 days of incubation in compost sampled after the start of the cooling phase. Microbial diversity also became stable, and DGGE profiles reached a maximum number of bands at this stage. CONCLUSIONS: Strain survival was not observed in stabilized composts. The cooling phase seems to be the turning point for pathogen survival and at this stage the indigenous microflora appeared to play a significant role in suppression. SIGNIFICANCE AND IMPACT OF THE STUDY: The importance of indigenous microflora in the survival of pathogens in four different composts was demonstrated. Stabilized composts were recommended for spreading on land.

DIAGNOdent laser fluorescence assessment of endodontic infection.

INTRODUCTION: Real-time assessment of the microbial status of the root canal system would be useful in clinical endodontic practice for determining endpoints of biomechanical treatment. This laboratory study used an existing laser fluorescence device, the DIAGNOdent (KaVo, Biberach, Germany), in a proof-of-concept study. METHODS: Visible laser red light (wavelength 655 nm) was used to elicit fluorescence emissions in the near-infrared range from infected and uninfected root canals. A prototype sapphire tip designed for periodontal assessment was used to analyze the pulp chamber and coronal third of the root canal system in extracted teeth. The fluorescence properties of bacterial cultures, monospecies biofilms in root canals, pulpal soft tissues, and sound dentin were also evaluated, together with 50 extracted teeth with known endodontic pathology. RESULTS: Sound dentin and healthy pulpal soft tissue gave an average fluorescence reading of 5 (on a scale of 100), whereas biofilms of Enterococcus faecalis and Streptococcus mutans established in root canals showed a progressive increase in fluorescence over time. Fluorescence readings reduced to the "healthy" threshold reading of 5 when root canals were endodontically treated, and the experimentally created bacterial biofilms were removed completely. High fluorescence readings were recorded in the root canals and pulp chambers of extracted teeth with radiographic evidence of periapical pathology and scanning electron microscopy evidence of bacterial infection. CONCLUSIONS: The use of the DIAGNOdent fluorescence approach for the assessment of the status of the pulp chamber and root canal system holds promise for clinical application; once more, flexible tips can be developed for gaining greater penetration into middle and apical thirds of the root canal.

Assessment of pheromone response in biofilm forming clinical isolates of high level gentamicin resistant Enterococcus faecalis.

Twenty five clinical isolates of high level gentamicin resistant Enterococcus faecalis were tested for their biofilm formation and pheromone responsiveness. The biofilm assay was carried out using microtiter plate method. Two isolates out of the 25 (8%) were high biofilm formers and 19 (76%) and four (16%) isolates were moderate and weak biofilm formers respectively. All the isolates responded to pheromones of E. faecalis FA2-2 strain. On addition of pheromone producing E. faecalis FA2-2 strain to these isolates, seven of 19 (37%) moderate biofilm formers developed into high biofilm formers. Similarly one of the 4 (25%) weak biofilm formers developed into high level biofilm former. Twelve (48%) of the 25 isolates were transconjugated by cross streak method using gentamicin as selective marker. This proves that the genetic factor for gentamicin resistance is present in the pheromone responsive plasmid. Among these twelve transaconjugants, seven isolates and one isolate were high biofilm formers on addition of E. faecalis FA2-2 and prior to its addition respectively. Out of the total 25 isolates, eight transconjugants for gentamicin resistance could turn to high biofilm formers on addition of the pheromone producing strain. All the isolates were resistant to more than two antibiotics tested. All the isolates were sensitive to vancomycin. The results indicate the significance of this nosocomial pathogen in biofilm formation and the role of pheromone responding clinical isolates of E. faecalis in spread of multidrug resistance genes.

Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry.

The commercially available LIVE/DEAD BacLight kit is enjoying increased popularity among researchers in various fields of microbiology. Its use in combination with flow cytometry brought up new questions about how to interpret LIVE/DEAD staining results. Intermediate states, normally difficult to detect with epifluorescence microscopy, are a common phenomenon when the assay is used in flow cytometry and still lack rationale. It is shown here that the application of propidium iodide in combination with a green fluorescent total nucleic acid stain on UVA-irradiated cells of Escherichia coli, Salmonella enterica serovar Typhimurium, Shigella flexneri, and a community of freshwater bacteria resulted in a clear and distinctive flow cytometric staining pattern. In the gram-negative bacterium E. coli as well as in the two enteric pathogens, the pattern can be related to the presence of intermediate cellular states characterized by the degree of damage afflicted specifically on the bacterial outer membrane. This hypothesis is supported by the fact that EDTA-treated nonirradiated cells exhibit the same staining properties. On the contrary, this pattern was not observed in gram-positive Enterococcus faecalis, which lacks an outer membrane. Our observations add a new aspect to the LIVE/DEAD stain, which so far was believed to be dependent only on cytoplasmic membrane permeability.

Assessment of the effects of Nurmi-type cultures and a defined probiotic preparation on a Salmonella typhimurium 29E challenge in vivo.

The effects of treatment with an undefined commercial Nurmi-type culture (NTC), cultured cecal contents, and a dual-strain probiotic, containing Enterococcus faecalis and Pediococcus pentosaceus, on Salmonella Typhimurium colonization were evaluated in a specific-pathogen-free bird model. Two sets of trials were performed, and each study was arranged as a randomized complete block design with three treatments. Treatments consisted of (i) control, (ii) commercial NTC, and (iii) cultured cecal contents in the first set of trials and (i) control, (ii) defined probiotic, and (iii) cultured cecal contents in the second set. On day 1, birds were administered 1.2 x 10(7) CFU of the appropriate treatment by oral gavage. On day 3, all birds were challenged with 1 x 10(6) CFU of Salmonella Typhimurium 29E (nalidixic acid resistant). Chicks were asphyxiated with argon gas on day 10, and ceca were aseptically removed. Salmonella Typhimurium counts (CFU per milliliter of cecal contents) were determined on brilliant green agar containing 30 mg of nalidixic acid per liter, and CFU counts were log transformed prior to analysis. Cecal pH and volatile fatty acid concentrations were also determined. Data were analyzed by one-way analysis of variance, and means were compared by Tukey's pairwise analysis. Commercial NTC and cultured cecal contents treatments resulted in a significant decrease (P < or = 0.05) in Salmonella Typhimurium 29E colonization, with the NTC offering a higher level of protection. In the second set of trials, the defined probiotic tended to reduce colonization by Salmonella Typhimurium (P = 0.07), while chicks treated with cultured cecal contents displayed a significant decrease (P = 0.03) when compared to the negative control. No significant change was observed in cecal pH or in acetate and propionate concentrations; however, a significant increase in butyrate concentrations in both the cultured cecal contents and defined probiotic treatment groups was observed when compared to the control birds. These observations suggest that defined cultures are less effective Salmonella control agents than are preparations generated from the complete cecal microflora.