Antimicrobial efficacy of sodium hypochlorite and hyper-pure chlorine dioxide in the depth of dentin tubules in vitro.

OBJECTIVES: The study aimed to compare the antibacterial effect of a novel disinfectant, hyper-pure chlorine dioxide (hClO2) to sodium hypochlorite (NaOCl) in various depths of dentin tubules. MATERIALS AND METHODS: The distal root of the extracted lower molars was infected artificially with Enterococcus faecalis. The control group was rinsed with saline, and the test groups were irrigated with either 5% NaOCl or 0.12% hClO2. The longitudinally split teeth were stained by viability stain. The coronal third of the root was scanned with a confocal laser scanning microscope. The fluorescent intensities were measured, and the percentage of dead bacteria was calculated at depths up to 950 µm along the dentin tubules. The effect of penetration depth, irrigants, and their interaction on antimicrobial efficacy was determined by the linear mixed model. RESULTS: The percentage of dead bacteria was higher both in the NaOCl (45.1 ± 2.3%, p < 0.01) and in the hClO2 (44.6 ± 3.8%, p < 0.01) irrigant groups compared to saline (23 ± 4.5%); however, there was no difference between them. The percentage of killed bacteria was not correlated with the depths in any group (p = 0.633). CONCLUSIONS: Our results suggest that the functional penetration depth of NaOCl is at least 2-3 times more than published to date. There is no difference in disinfection effectiveness along the dentin tubules between NaOCl and hClO2 until at least the measured 950 µm. However, both were only able to eradicate the intratubular bacteria partially. CLINICAL RELEVANCE: Hyper-pure ClO2 could be used as an alternative or final adjuvant irrigant in endodontic treatment.

Short- and long term antibacterial effects of a single rinse with different mouthwashes: A randomized clinical trial.

Objectives: Reducing the microbial level in the aerosol created during dental procedures is essential to avoiding infections. The aim of this study was to examine the change in Streptococcus mutans (S. mutans) and the total bacterial load in human saliva in vivo after a single rinse with different mouthwashes. Material and methods: One mL of unstimulated saliva was collected from volunteers with poor oral hygiene at baseline and 5 min after a 1-min rinsing with diluted Solumium Oral® (hyper-pure 0.0015% chlorine dioxide; ClO2), Listerine Total Care®, Corsodyl® (0.2% chlorhexidine-digluconate; CHX), or BioGate Si*CLEAN for bacterial investigation. In a second study, volunteers rinsed with 0.003% ClO2 or CHX for 1 min, and saliva was collected at baseline, after 5 min, and after 90 min. After plating, the total plate and S. mutans colony numbers were determined. Results: In the first study, ClO2 and CHX similarly reduced both total germ and S. mutans numbers, while Listerine Total Care® decreased only the S. mutans counts. BioGate Si*Clean had no effect on either the total germ or S. mutans numbers. In the second study, an increasing tendency toward bacterial regrowth was observed with CHX after 90 min compared to the 5-min value, while no change was measured after ClO2 rinsing. Conclusion: Hyper-pure ClO2 rinsing may be a new promising preventive and therapeutic adjuvant in dental practice, as it is similar in effectiveness to the gold standard CHX-containing mouthwashes, especially in patients concerned with taste or tooth discoloration during oral health therapy.

Formulation of Chlorine-Dioxide-Releasing Nanofibers for Disinfection in Humid and CO2-Rich Environment.

Background: Preventing infectious diseases has become particularly relevant in the past few years. Therefore, antiseptics that are harmless and insusceptible to microbial resistance mechanisms are desired in medicine and public health. In our recent work, a poly(ethylene oxide)-based nanofibrous mat loaded with sodium chlorite was formulated. Methods: We tested the chlorine dioxide production and bacterial inactivation of the fibers in a medium, modeling the parameters of human exhaled air (ca. 5% (v/v) CO2, T = 37 °C, RH > 95%). The morphology and microstructure of the fibers were investigated via scanning electron microscopy and infrared spectroscopy. Results: Smooth-surfaced, nanoscale fibers were produced. The ClO2-producing ability of the fibers decreased from 65.8 ppm/mg to 4.8 ppm/mg with the increase of the sample weight from 1 to 30 mg. The effect of CO2 concentration and exposure time was also evaluated. The antibacterial activity of the fibers was tested in a 24 h experiment. The sodium-chlorite-loaded fibers showed substantial antibacterial activity. Conclusions: Chlorine dioxide was liberated into the gas phase in the presence of CO2 and water vapor, eliminating the bacteria. Sodium-chlorite-loaded nanofibers can be sources of prolonged chlorine dioxide production and subsequent pathogen inactivation in a CO2-rich and humid environment. Based on the results, further evaluation of the possible application of the formulation in face-mask filters as medical devices is encouraged.

Prevalence of Staphylococcus aureus in wild hedgehogs (Erinaceus europaeus) and first report of mecC-MRSA in Hungary.

In 2011 mecC, a new mecA gene homologue, was described in a bovine isolate in the UK. Since then, mecC-positive methicillin-resistant Staphylococcus aureus (mecC-MRSA) has also been found in wild animals. An especially high prevalence of mecC-MRSA has been reported among hedgehogs in Sweden (64%) and Denmark (61%). Based on these findings we aimed to survey the hedgehog population for mecC-MRSA in Hungary. Altogether 200 hedgehogs were screened for Staphylococcus aureus using a culture-based method. The antibiotic susceptibility of the isolates to nine drugs was determined, their genetic relatedness was established by PFGE and spa-typing, and virulence genes were identified by PCR. Whole genome sequencing was performed for the single mecC-MRSA isolate found. Of the 200 animals, 13 were carriers of S. aureus (6.5%). Among these, one isolate was mecA positive and one was mecC positive. The isolates were susceptible to non-beta-lactam antibiotics. Toxin genes were not found, but the majority carried genes responsible for adhesion and biofilm production. The mecC-MRSA isolate was a single-locus variant of ST130, had a new spa type (t19701) and belonged to SCCmec type XI. It carried a recently described, novel exfoliative toxin (etE). This is the first report of mecC-MRSA in Hungary and the first survey of staphylococcus carriage among wild animals in the country. The mecC prevalence was much lower than in Northern European countries and rather similar to other countries in our region. MecC-MRSA could potentially emerge as a novel human pathogen, especially where close contact occurs between humans and animals.

Whole genome sequencing of coagulase positive staphylococci from a dog-and-owner screening survey.

BACKGROUND: Staphylococcus aureus and S. pseudintermedius are the two most common coagulase positive staphylococci (CPS). S. aureus is more prevalent among humans, whereas S. pseudintermedius is more commonly isolated from dogs, however, both can cause various community and hospital acquired diseases in humans. METHODS: In the current study we screened 102 dogs and 84 owners in Hungary. We tested the antibiotic susceptibility of the strains and in order to get a better picture of the clonal relationship of the strains, we used pulsed-field gel electrophoresis. In addition, three pairs of isolates with identical PFGE patterns were whole genome sequenced, MLST and spa types were established. RESULTS: Carriage rate of S. aureus was 23.8% in humans and 4.9% in dogs and two cases of co-carriage were found among dogs and owners. S. pseudintermedius carriage rate was 2.4% and 34.3%, respectively, with only one co-carriage. The isolates were generally rather susceptible to the tested antibiotics, but high tetracycline resistance of S. pseudintermedius strains was noted. The co-carried isolates shared almost the same resistance genes (including tet(K), bla(Z), norA, mepR, lmrS, fosB) and virulence gene pattern. Apart from the common staphylococcal enzymes and cytotoxins, we found enterotoxins and exfoliative toxins as well. The two S. aureus pairs belonged to ST45-t630, ST45-t671 and ST15-t084, ST15-t084, respectively. The co-carried S. pseudintermedius isolates shared the same housekeeping gene alleles determining a novel sequence type ST1685. CONCLUSIONS: Based on the genomic data, dog-owner co-carried strains displayed only insignificant differences therefore provided evidence for potential human-to-dog and dog-to-human transmission.

Dissemination of VanA-Type Enterococcus faecium Isolates in Hungary.

Although vanA carrying Enterococcus faecium human clinical isolates have been rarely found in Hungary before 2012, they have been detected in continuously increasing numbers since then. To identify factors associated with their dissemination, we investigated the clonal relatedness and plasmids of 30 vanA carrying E. faecium isolates originating from different Hungarian healthcare institutions from 2012 to 2014. Molecular typing of the isolates (n = 30) was performed with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, Tn1546 polymerase chain reaction mapping, plasmid restriction fragment length polymorphism analysis, and sequencing. A single Tn1546 variant was detected in all of the isolates. It harbored IS1251 in the vanS-vanH intergenic region, had an entire deletion of the transposase gene and a partial deletion of the resolvase gene, and was located on a pRUM-like plasmid. Based on PFGE, the isolates could be grouped into 13 pulsotypes. Representative strains of these pulsotypes belonged to ST17, ST18, ST80, ST117, and ST203, which are known to be part of the hospital-adapted clades. The increase in the number of vanA carrying E. faecium clinical isolates in Hungary could be explained by the dissemination of pRUM-like vancomycin resistance plasmids in hospital-adapted clonal lineages.

Effect of dentin powder on the antimicrobial properties of hyperpure chlorine-dioxide and its comparison to conventional endodontic disinfecting agents.

INTRODUCTION: Previously we found that the high purity chlorine-dioxide(ClO2) has a very potent disinfectant efficacy on oral pathogenic microorganisms and as a root canal irrigant it is able to eliminate the experimental Enterococcus faecalis(E. faecalis) infection from the root canal system. This study examines whether the presence of dentin powder influences the antibacterial efficacy of ClO 2. METHODS: In an in vitro dentin powder model the following irrigants were tested against planktonic E. faecalis: 2% chlorhexidine (CHX), 2.5% sodium hypochlorite (NaOCl), 0.12%ClO2 (Solumium) and one local root canal medicament: saturated Ca(OH)2. Survival of bacteria exposed to agents without and with human dentin powder or preincubated with dentin powder was investigated. The effect of the dentin powder on ClO2 concentration was investigated by titrations. RESULTS: Without dentin powder ClO 2 killed all E. faecalis and delivered the best result already after 1 minute; however, after longer contact time with dentin the difference between the disinfectants disappeared. The presence of dentin powder decreased the concentration of ClO 2 and attenuated the antibacterial efficiency of ClO2 and Ca(OH)2, but did not decrease of CHX and NaOCl.Preincubation with dentin powder caused significant loss of antibacterial activity of all investigated agents, ClO2 and Ca(OH)2 having the highest reduction. CONCLUSION: As the presence of dentin powder had a negative effect on the efficacy of disinfectants, the importance of elimination of dentin scrapings and smear layer from the root canal system during endodontic treatments is highly recommended. ClO 2 can be effective for a final rinse.

The effect of indomethacin, myeloperoxidase, and certain steroid hormones on bactericidal activity: an ex vivo and in vivo experimental study.

BACKGROUND: The role of myeloperoxidase (MPO) is essential in the killing of phagocytosed bacteria. Certain steroid hormones increase MPO plasma concentration. Our aim was to test the effect of MPO, its inhibitor indomethacin, and certain steroid hormones on bactericidal activity. METHODS: Human polymorphonuclear leukocytes (PMN) were incubated with opsonised Escherichia coli and either MPO, indomethacin, estradiol, or hydrocortisone. Intracellular killing capacity was evaluated with UV microscopy after treatment with fluorescent dye. Next, an in vivo experiment was performed with nine groups of rats: in the first phase of the study indomethacin treatment and Pasteurella multocida infection (Ii), indomethacin treatment without infection (I0), untreated control with infection (Mi) and untreated control without infection (M0); in the second phase of the study rats with infection and testosterone treatment (NT), castration, infection and testosterone treatment (CT), castration, infection and estradiol treatment (CE), non-castrated infected control (N0), and castrated infected control (C0). After treatment bacteria were reisolated from the liver and heart blood on agar plates, and laboratory parameters were analyzed. For the comparison of laboratory results ANOVA or Kruskal-Wallis test and LSD post hoc test was used. RESULTS: Indomethacin did not have a remarkable effect on the bacterial killing of PMNs, while the other compounds increased bacterial killing to various degrees. In the animal model indomethacin and infection caused a poor clinical state, a great number of reisolated bacteria, elevated white blood cell (WBC) count, decreased C-reactive protein (CRP) and serum albumin levels. Testosterone treatment resulted in less bacterial colony numbers in group NT, but not in group CT compared to respective controls (N0, C0). Estradiol treatment (CE) decreased colony numbers compared to control (C0). Hormone administration resulted in lower WBC counts, and in group CE, a decreased CRP. CONCLUSIONS: MPO, estradiol, and hydrocortisone improve bacterial killing activity of PMNs. Indomethacin treatment and castration weaken immune responses and clinical state of infected rats, while testosterone and estradiol have a beneficial effect.

Comparing the efficacy of hyper-pure chlorine-dioxide with other oral antiseptics on oral pathogen microorganisms and biofilm in vitro.

This study examines the antibacterial properties of sodium hypochlorite (NaOCl), chlorhexidine gluconate (CHX), Listerine®, and high purity chlorine dioxide (Solumium, ClO2) on selected common oral pathogen microorganisms and on dental biofilm in vitro. Antimicrobial activity of oral antiseptics was compared to the gold standard phenol. We investigated Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, Veillonella alcalescens, Eikenella corrodens, Actinobacillus actinomycetemcomitans and Candida albicans as some important representatives of the oral pathogens. Furthermore, we collected dental plaque from the upper first molars of healthy young students. Massive biofilm was formed in vitro and its reduction was measured after treating it with mouthrinses: CHX, Listerine® or hyper pure ClO2. Their biofilm disrupting effect was measured after dissolving the crystal violet stain from biofilm by photometer. The results have showed that hyper pure ClO2 solution is more effective than other currently used disinfectants in case of aerobic bacteria and Candida yeast. In case of anaerobes its efficiency is similar to CHX solution. The biofilm dissolving effect of hyper pure ClO2 is significantly stronger compared to CHX and Listerine® after 5 min treatment. In conclusion, hyper pure ClO2 has a potent disinfectant efficacy on oral pathogenic microorganisms and a powerful biofilm dissolving effect compared to the current antiseptics, therefore high purity ClO2 may be a new promising preventive and therapeutic adjuvant in home oral care and in dental or oral surgery practice.

Effectiveness of a high purity chlorine dioxide solution in eliminating intracanal Enterococcus faecalis biofilm.

We investigated the effectiveness of chlorine dioxide (ClO2) solution in comparison to sodium hypochlorite (NaOCl) and chlorhexidine gluconate (CHX) in the elimination of intracanal Enterococcus faecalis biofilm. Extracted human teeth were inoculated with E. faecalis. After preparation the canals were irrigated with ClO2, NaOCl, CHX or physiologic saline for control. Two and five days later bacterial samples were collected and streaked onto Columbia agar. CFU/mL were counted. The canal walls were investigated by scanning electron microscopy (SEM). The gas phase was investigated in an upside down Petri dish where E. faecalis was inoculated onto blood agar. The irrigants were placed on absorbent paper into the cover. Bacteria were detectable in the control group, but not in any of the irrigants groups. There was a massive reinfection 2 or 5 days after irrigation in the control group. The lowest reinfection was found after the ClO2 treatment. These findings were confirmed by SEM images. We observed an antibacterial effect of ClO2 and NaOCl gas phases on E. faecalis growth, but not of CHX. ClO2 eliminates intracanal biofilm and keeps canal nearly free from bacteria. We suggest the use of high purity ClO2 as a root canal irrigant in clinical practice.

Antiretroviral effect of 4-thio-uridylate against human immunodeficiency virus type 1.

Antiretroviral effect of thiolated nucleotide 4-thio-uridylate (S4UMP, designated as UD29) against human immunodeficiency virus type 1 (HIV-1) have been quantitatively determined in cell-based viral infectivity assays. In syntitium inhibition assay on MT-2 human T-cell line UD29 prevented cell fusion and formation of syntitia induced by HIV-1IIIB with IC50 values of 11.7 µg/ml. In a single-cycle viral infection assay (MAGI assay) UD29 proved to have a potent inhibitory effect against HIV-1IIIB on HeLaCD4-LTR/ß-gal cells, which was dose dependent with IC50 values of 4.75 µg/ml and IC90 of 39.7 µg/ml. UD29 showed a most prominent antiviral effect when administered 30 min prior HIV-1 infection. As HIV entry requires thiol/disulfide exchange process, results suggest that reactive -SH group of enol-form of the thiolated nucleotide may interfere with the function of cell surface proteins. UD29 cannot penetrate into cells and may have an interactive role in redox processes active in viral entry.

Distribution and genetic relatedness of vancomycin-resistant enterococci (VRE) isolated from healthy slaughtered chickens in Hungary from 2001 to 2004.

The presence of the vanA gene was determined in enterococci from healthy poultry, originating from the Hungarian resistance monitoring system between 2001 and 2004. Enterococci (n = 562) were collected from intestinal samples of slaughtered broiler chickens. The presence of van genes was detected by polymerase chain reaction (PCR). The vancomycin-resistant enterococcus (VRE) strains carried only the vanA gene. Genus- and species-level identification of the vanA gene carrier strains was carried out by PCR using specific primers. In 2001, 25 out of the 289 isolated strains (8.6%) were vanA carriers (1 Enterococcus mundtii, 13 E. durans and 11 E.faecium). In 2002 (n = 87), 20 (23%) strains were vanA positive (11 E. durans and 9 E. faecium). In 2003 and 2004, none of the strains (n = 95 and 91, respectively) were positive for the most common van genes. In 2003, there was only one strain for which higher minimum inhibitory concentrations (MIC) of vancomycin (4 mg/L) and teicoplanin (8 mg/L) were found. In 2004 there were three strains for which the MIC of vancomycin was 8 mg/L, and 2 strains and 1 strain with teicoplanin MICs of 4 mg/L and 8 mg/L, respectively. The potential similarity of these strains was studied by pulsed-field gel electrophoresis (PFGE). The VRE strains were not closely related to one another. The annual data of vancomycin resistance indicate an association between the recovery of vancomycin-resistant enterococci and the use of avoparcin in animal feeds. This study indicates that with the reduced use of antibiotics in food animals, it is possible to decrease the rate of resistant bacteria. Although the use of avoparcin had been banned in 1998, the VRE strains disappeared only five years later.

Rapid detection of methicillin resistance in teicoplanin-resistant coagulase-negative staphylococci by a penicillin-binding protein 2' latex agglutination method.

Thirty-five clinical isolates of coagulase-negative staphylococci with decreased glycopeptide sensitivity were examined by a penicillin-binding protein (PBP2') latex agglutination (LA) test and were compared to the detection of the mecA gene by PCR, and oxacillin susceptibility determined minimum inhibitory concentrations. The latex test demonstrated high sensitivity and specificity for detecting methicillin resistance in coagulase-negative staphylococci after PBP2' induction with oxacillin.