Successful use of a phage endolysin for treatment of chronic pelvic pain syndrome/chronic bacterial prostatitis.

Chronic prostatitis (CP) is a common inflammatory condition of the prostate that is estimated to effect 2%-10% of the world's male population. It can manifest as perineal, suprapubic, or lower back pain and urinary symptoms occurring with either recurrent bacterial infection [chronic bacterial prostatitis (CBP)] or in the absence of evidence of bacterial infection [chronic pelvic pain syndrome (CPPS)]. Here, in the case of a 39 years-old CBP patient, we report the first successful use of a bacteriophage-derived muralytic enzyme (endolysin) to treat and resolve the disease. Bacteriological analysis of the patient's prostatic secretion and semen samples revealed a chronic Enterococcus faecalis prostate infection, supporting a diagnosis of CBP. The patient's E. faecalis strain was resistant to several antibiotics and developed resistance to others during the course of treatment. Previous treatment with multiple courses of antibiotics, bacteriophages, probiotics, and immunologic stimulation had failed to achieve long term eradication of the infection or lasting mitigation of the symptoms. A cloned endolysin gene, encoded by E. faecalis bacteriophage ϕEf11, was expressed, and the resulting gene product was purified to electrophoretic homogeneity. A seven-day course of treatment with the endolysin resulted in the elimination of the E. faecalis infection to below culturally detectable levels, and the abatement of symptoms to near normal levels. Furthermore, during the endolysin treatment, the patient experienced no untoward reactions. The present report demonstrates the effectiveness of an endolysin as a novel modality in managing a recalcitrant infection that could not be controlled by conventional antibiotic therapy.

Morphological alterations in tongue epithelial cells infected by SARS-CoV-2: A case-control study.

OBJECTIVE: The aim of the present case-control study was to evaluate the morphological aspects of the epithelial cells from the dorsum of the tongue and the expression of the SARS-CoV-2 Spike protein in these cells, in patients with and without COVID-19 infection. METHODS: 24 individuals with at least one symptom of COVID-19 were recruited among inpatients from Hospital Universitário Pedro Ernesto (Rio de Janeiro, Brazil). 14 patients who tested positive for COVID-19 by RT-PCR were included in the case group, and 10 patients who tested negative were included in the control group. Cytological smears from the dorsum of the tongue were obtained from all patients and analyzed using immunohistochemistry directed against SARS-CoV-2-Spike protein. Morphological changes in epithelial cells were analyzed using light microscopy. RESULTS: Immunohistochemistry showed that 71% of the COVID-19 patients presented epithelial cells positive for the presence of the SARS-CoV-2 Spike protein, and all cells coming from patients in the control group were negative. Cytological analysis showed significant differences when comparing epithelial cells from COVID-19-positive and COVID-19-negative patients. CONCLUSION: COVID-19 may generate dimensional changes in tongue epithelial cells; however, further studies are necessary to understand how this happens.

Intrinsic resistance of Enterococcus faecalis strains to ΦEf11 phage endolysin is associated with the presence of ΦEf11 prophage.

The use of bacteriophage-encoded murein hydrolases (endolysins) is being actively explored as a means of controlling multidrug-resistant pathogens. Previously, we isolated and characterized one such enzyme, the phage ΦEf11 ORF28 lysin, which demonstrated profound antimicrobial activity against many strains of Enterococcus faecalis. Although the lysin is eminently active against many vancomycin-resistant enterococal (VRE) strains, and displays lower minimum inhibitory concentrations than vancomycin against vancomycin-sensitive strains, there is a subset of E. faecalis strains that is not affected by the lysin. Currently, there is no explanation for the disparate sensitivity to ORF28 lysin among E. faecalis strains. In the present investigation, we show that the intrinsic insensitivity of the insusceptible strains to the lysin is associated with the presence of a ΦEf11 prophage. Of the strains harboring phage ΦEf11 genes (N = 28), 68% were insensitive to the lysin, whereas 91% of the strains (N = 75) lacking detectable ΦEf11 genes demonstrated lysin sensitivity. Furthermore, curing a lysin-resistant, lysogenic E. faecalis strain resulted in a lysin-sensitive derivative, whereas lysogenizing a wild-type non-lysogenic strain converted it from lysin sensitivity to lysin resistance. Our results suggest that lysin resistance comes about through lysogenic conversion of non-lysogenic, lysin-sensitive strains.

The prevalence and impact of lysogeny among oral isolates of Enterococcus faecalis.

Bacterial phenotypic properties are frequently influenced by the uptake of extrachromosomal genetic elements, such as plasmids and bacteriophage genomes. Such modifications can result in enhanced pathogenicity due to toxin production, increased toxin release, altered antigenicity, and resistance to antibiotics. In the case of bacteriophages, the phage genome can stably integrate into the bacterial chromosome as a prophage, to produce a lysogenic cell. Oral enterococcal strains have been isolated from subgingival plaque and the root canals of endodontically-treated teeth that have failed to heal. Previously, we isolated a bacteriophage, phage ɸEf11, induced from a lysogenic Enterococcus faecalis strain recovered from the root canal of a failed endodontic case. PCR analysis using phage ɸEf11-specific oligonucleotide primers, disclosed that lysogens containing ɸEf11 prophages were commonly found among oral E. faecalis strains, being detected in 19 of 61 (31%) strains examined. Furthermore, in comparison to an isogenic cured strain, cultures of a lysogen harboring an ɸEf11 prophage exhibited altered phenotypic characteristics, such as increased persistence at high density, enhanced biofilm formation, and resistance to a bacteriophage lytic enzyme. From these results we conclude that lysogeny is common among oral E. faecalis strains, and that it alters properties of the lysogenic cell.

Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins.

ϕEf11 is a temperate Siphoviridae bacteriophage that infects strains of Enterococcus faecalis The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for an N-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathione S-transferase tag. It produced rapid, profound lysis in E. faecalis populations and was active against 73 of 103 (71%) E. faecalis strains tested. In addition, it caused substantial destruction of E. faecalis biofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+ Liquid chromatography-mass spectrometry analysis of E. faecalis cell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as an N-acetylmuramidase, an endo-ß-N-acetylglucosaminidase, and an endopeptidase, rather than an N-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterized E. faecalis bacteriophages.IMPORTANCE The emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains of Enterococcus faecalis The lysin is broadly active against most of the tested E. faecalis strains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced by E. faecalis bacteriophages.

Structural proteins of Enterococcus faecalis bacteriophage ϕEf11.

ϕEf11, a temperate Siphoviridae bacteriophage, was isolated by induction from a root canal isolate of Enterococcus faecalis. Sequence analysis suggested that the ϕEf11 genome included a contiguous 8 gene module whose function was related to head structure assembly and another module of 10 contiguous genes whose products were responsible for tail structure assembly. SDS-PAGE analysis of virions of a ϕEf11 derivative revealed 11 well-resolved protein bands. To unify the deduced functional gene assignments emanating from the DNA sequence data, with the structural protein analysis of the purified virus, 6 of the SDS-PAGE bands were subjected to mass spectrometry analysis. 5 of the 6 protein bands analyzed by mass spectrometry displayed identical amino acid sequences to those predicted to be specified by 4 of the ORFs identified in the ϕEf11 genome. These included: ORF8 (predicted scaffold protein), ORF10 (predicted major head protein), ORF15 (predicted major tail protein), and ORF23 (presumptive antireceptor).

Prophage induction in lysogenic Aggregatibacter actinomycetemcomitans cells co-cultured with human gingival fibroblasts, and its effect on leukotoxin release.

Lysogeny is common among strains of the periodontal pathogen Aggregatibacter actinomycetemcomitans. Since lysogenic induction is known to result in the increased synthesis and release of bacterial toxins from lysogens, it would be important to elucidate the conditions under which induction of these bacteria may occur. Co-cultures of A. actinomycetemcomitans strains (either lysogenic or non-lysogenic) and human cells (either gingival fibroblasts or pharyngeal epithelial cells) were prepared. Following incubation, bacteriophage titers of up to 6.2 × 10(7) pfu/ml were detected in the cell-free, spent culture media from the co-cultures of the lysogenic A. actinomycetemcomitans strains and the fibroblasts. Little (maximum of 2 × 10(0) pfu/ml) or no titers of phage could be detected in the mono-cultures of the lysogenic A. actinomycetemcomitans strains alone. In contrast, no phage were detectable in the cell-free spent culture media of the lysogens cocultured with the epithelial cells. Futhermore, co-culture of the A. actinomycetemcomitans lysogens with the fibroblasts resulted in enhanced release of the A. actinomycetemcomitans leukotoxin into the culture medium, in comparison with the spent culture media from mono-cultures of the lysogens alone. These results are consistent with the concept that interaction with fibroblasts may mediate prophage induction in lysogenic strains of A. actinomycetemcomitans, and that leukotoxin release is greatly augmented following induction of the lysogens.

The annotated complete DNA sequence of Enterococcus faecalis bacteriophage φEf11 and its comparison with all available phage and predicted prophage genomes.

φEf11 is a temperate Siphoviridae bacteriophage isolated by induction from a lysogenic Enterococcus faecalis strain. The φEf11 DNA was completely sequenced and found to be 42,822 bp in length, with a G+C mol% of 34.4%. Genome analysis revealed 65 ORFs, accounting for 92.8% of the DNA content. All except for seven of the ORFs displayed sequence similarities to previously characterized proteins. The genes were arranged in functional modules, organized similar to that of several other phages of low GC Gram-positive bacteria; however, the number and arrangement of lysis-related genes were atypical of these bacteriophages. A 159 bp noncoding region between predicted cI and cro genes is highly similar to the functionally characterized early promoter region of lactococcal temperate phage TP901-1, and possessed a predicted stem-loop structure in between predicted P(L) and P(R) promoters, suggesting a novel mechanism of repression of these two bacteriophages from the λ paradigm. Comparison with all available phage and predicted prophage genomes revealed that the φEf11 genome displays unique features, suggesting that φEf11 may be a novel member of a larger family of temperate prophages that also includes lactococcal phages. Trees based on the blast score ratio grouped this family by tail fiber similarity, suggesting that these trees are useful for identifying phages with similar tail fibers.

Presence of Helicobacter pylori in supragingival dental plaque of individuals with periodontal disease and upper gastric diseases.

BACKGROUND: Helicobacter pylori is a Gram-negative microorganism which is able to colonize the gastric mucosa and is associated with peptic ulcer, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Several studies have detected this bacterium in the oral cavity, suggesting it as a potential reservoir. The aim of this study was to investigate the presence of H. pylori in the oral cavity of individuals with periodontal disease and gastric diseases. METHODS: 115 individuals, with mean age 49.6 (±5.8) years, were divided in 4 groups: (A) with gastric diseases and periodontal disease; (B) with gastric diseases and no periodontal disease; (C) without gastric diseases and without periodontal disease, (D) without gastric diseases and with periodontal disease. Supra and subgingival plaque samples were collected from posterior teeth of the individuals with sterile paper points, and prepared for Polymerase Chain Reaction analysis. Fisher's exact test was used for detecting statistical differences between groups (p<0.05). RESULTS: H. pylori was detected in supragingival plaque of 9/36 (25%) of group A, 1/31 (0.3%) of group B, 0 (0%) of group C and 3/36 (8.3%) of group D. No subgingival samples were positive for H. pylori. There was a statistically higher prevalence of H. pylori in groups A and D when compared to B and C (p<0.05). CONCLUSION: H. pylori was detected in the supragingival plaque, but not in the subgingival plaque, of individuals with periodontal disease and upper gastric diseases. There was an association between the supragingival colonization of H. pylori and oral hygiene parameters such as the presence of plaque and gingival bleeding.

Detection of cytotoxin genotypes of Helicobacter pylori in stomach, saliva and dental plaque.

BACKGROUND: The aim of this study was to detect the presence of Helicobacter pylori and its virulent cagA genes in the oral cavity of individuals with upper gastric diseases. Sixty-two individuals (42+/-2.3 years) with dispepsy symptoms, referred for gastroscopy and who were H. pylori positive in the gastric biopsy, were recruited and separated in two groups: case group-individuals with gastric disease (n = 30); control group-individuals with no gastric disease (n = 32); saliva, dental plaque and biopsy samples were collected from all individuals. Oral and biopsy samples were analyzed by PCR using specific primers for H. pylori 16S ribosomal and cagA genes. PCR products were sequenced for DNA homology confirmation. H. pylori was detected neither in dental plaque nor in saliva in the control group. In the case group H. pylori DNA was detected in 16/30 (53.3%) saliva samples and in 11/30 (36.6%) dental plaque samples. The cagA gene was detected in 13/30 (43.3%) gastric biopsies, in 7/16 (43.8%) saliva samples, and in 3/11 (27.3%) dental plaque samples. Eighteen (60.0%) individuals in the case group were H. pylori positive both in oral and biopsy samples, and 8 (26.6%) of those were positive for cagA-H. pylori DNA. H. pylori and its virulent clone showed a higher prevalence in the oral cavity of individuals in the case group than in the control group (p < 0.05). Our results suggest that dental plaque and saliva may serve as temporary reservoir for H. pylori and its virulent cagA variant in individuals with gastric disease.

Higher levels of salivary MUC5B and MUC7 in individuals with gastric diseases who harbor Helicobacter pylori.

OBJECTIVE: The aim of the present study was to assess the salivary levels of MUC5B and MUC7 in individuals with dyspeptic disease and Helicobacter pylori (H. pylori) in the stomach, compared to individuals without dyspeptic disease. METHODS: 30 individuals with dyspeptic disease, who underwent endoscopy for upper gastrointestinal complaints at Hospital Pedro Ernesto-RJ, Brasil and tested positive for H. pylori, and 23 controls with no dyspeptic disease, with mean age 53.5+/-4.4 years, were included in the study. Saliva samples and 3 antral biopsy were taken for PCR analysis and histologic examination. In addition, saliva samples were tested by ELISA with F2 monoclonal antibody and EU7A antibody against MUC7, to determine MUC5B and MUC7 levels, prior to endoscopic examination. The expression pattern of the proteins was quantified by comparison to a pooled saliva sample of 19 healthy volunteers. RESULTS: MUC5B and MUC7 salivary levels were higher in the individuals with dyspeptic disease than in controls (p<0.0001). 33.3% (9/30) of the dyspeptic individuals and 0% of the controls had H. pylori in the oral cavity. CONCLUSIONS: Individuals with gastric diseases, with H. pylori in the stomach, showed higher levels of salivary H. pylori receptors-MUC5B and MUC7-than individuals without gastric diseases. These results suggest that higher levels of specific salivary mucins could be useful as risk indicators for infection by H. pylori.

The antimicrobial efficacy of the erbium, chromium:yttrium-scandium-gallium-garnet laser with radial emitting tips on root canal dentin walls infected with Enterococcus faecalis.

BACKGROUND: The authors used an in vitro model to investigate the ability of an erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser with radial emitting tips to disinfect Enterococcus faecalis-infected dentin. MATERIALS AND METHODS: The in vitro infected-dentin model system consisted of a dentin cylinder, prepared from a human anterior tooth root, cemented into a sealable two-chamber device fabricated from a syringe needle cap. The model's lower chamber contained a buffer solution, and the dentin cylinder was placed between the upper and lower chambers. After sterilization, the authors inoculated the root canal of each dentin cylinder with E. faecalis. They used an Er,Cr:YSGG laser with radial emitting tips to irradiate the root canal of each infected dentin cylinder (varying laser power and exposure time). After laser treatment, the authors machined the root canal dentin walls and collected the resulting dentin fillings in the buffer-reservoir. They quantified the E. faecalis titer of each buffer-reservoir by using selective agar plates. RESULTS: The authors found that bacterial recovery decreased when laser irradiation duration or power increased. A greater degree of disinfection was achieved with a 120-second application of laser than with sodium hypochlorite treatment. Finally, they found that a 99.7 percent reduction in bacterial counts could be obtained using the laser. CONCLUSION: The results of this study suggest that the Er,Cr:YSGG laser with a radial emitting tip has a significant antimicrobial effect on dentinal tubules infected with E. faecalis. CLINICAL IMPLICATIONS: Er,Cr:YSGG laser treatment could be a valuable tool for root canal disinfection during endodontic treatment.

A comparison of the machining efficiency of two brands of stainless steel endodontic hand files.

The purpose of this study was to compare the machining efficiency of a flexible stainless steel K-type hand file recently introduced by Brasseler USA ("F-Style files"), with that of a well-studied Endodontic instrument ("Flex-o-files," Maillefer Dentsply), that has been on the market for more than 20 years. The comparison of machining efficiencies of these two brands of files was conducted on both dentin and plexiglas substrates, using an apparatus that allowed a constant force to be applied from each file being tested. The results indicated that the Brasseler instruments had poorer machining efficiency than those of the Maillefer files.

An objective evaluation comparing the physical properties of two brands of stainless steel endodontic hand files.

The purpose of this study was to evaluate the physical properties of a flexible stainless steel K-type hand file recently introduced by Brasseler USA (F-Style files; Brasseler USA, Savannah, GA), in comparison to those of a well-studied instrument (Flex-o-files; Maillefer-Dentsply, Ballaigues, Switzerland), that has been on the market for more than 20 yr. The physical properties measured included torque at failure, angular deflection at failure, flexibility, and consistency of diameter at 3 mm from the cutting tip; and the evaluations were carried out on size #10 through #50 files of each of the two brands being tested. The results indicated that the Brasseler instruments were inherently more flexible, but had smaller diameters, lower torque, and angular deflection values at failure, than those of the Maillefer files.

Efficacy of bacterial removal from instrumented root canals in vitro related to instrumentation technique and size.

OBJECTIVE: This in vitro investigation assessed the efficacy of removing radioactively labeled bacteria from infected canals with 2 engine-driven rotary nickel titanium instrumentation techniques differing in sequence and apical enlargement size. STUDY DESIGN: A standard quantity of (3)H-thymidine-labeled Enterococcus faecalis (3.70 x 10(4) cpm, 2.0 x 10(7) colony-forming units) was used to inoculate the mesiobuccal canals of 50 extracted mandibular molars. The teeth were incubated for 5 days to allow infection of the surrounding dentin from the canals. Five of the teeth were used as controls to determine the number of cycles of irrigation and drying necessary to reduce the (3)H counts recovered from the canals to baseline levels. After this process, the unbound bacteria in the root canals of the remaining 45 teeth then were washed out with buffer until baseline levels of radioactivity were obtained. The mesiobuccal root of 1 of these 45 teeth was removed, decalcified, and digested, and the total radioactivity released from the root dentin was measured. Of the remaining 44 teeth, 22 then were instrumented with GT and Profile (Dentsply/Tulsa Dental Co, Tulsa, Okla) instruments to apical size #35 (group 1) and 22 teeth with Pow-R instruments (Moyco/Union Broach, York, Pa) to apical size #50 (group 2), in the presence of a standard quantity of phosphate-buffered saline solution placed in the canal. After instrumentation, the medium from each canal was collected with paper points and its radioactivity was counted with liquid scintillation spectrometry. RESULTS: The mean (3)H level recovered with instrumentation of canals in group 1 was 75 cpm (+/- 29, standard deviation) and in group 2 was 123 cpm (+/- 50, standard deviation). A 2-tailed Mann-Whitney test indicated that the radioactivity of samples from group 2 was significantly higher than that of samples from group 1. CONCLUSION: The results suggested that instrumentation to an apical size of #50, as performed with the Pow-R instruments, was more effective in debriding infected root canals than instrumentation to an apical size of #35, as performed with the GT and Profile instruments.