Antimicrobial susceptibility of commensal Neisseria in a general population and men who have sex with men in Belgium.

Non-pathogenic Neisseria are a reservoir of antimicrobial resistance genes for pathogenic Neisseria meningitidis and Neisseria gonorrhoeae. Men who have sex with men (MSM) are at risk of co-colonization with resistant non-pathogenic and pathogenic Neisseria. We assessed if the antimicrobial susceptibility of non-pathogenic Neisseria among MSM differs from a general population and if antimicrobial exposure impacts susceptibility. We recruited 96 participants at our center in Belgium: 32 employees, 32 MSM who did not use antibiotics in the previous 6 months, and 32 MSM who did. Oropharyngeal Neisseria were cultured and identified with MALDI-TOF-MS. Minimum inhibitory concentrations for azithromycin, ceftriaxone and ciprofloxacin were determined using E-tests® and compared between groups with non-parametric tests. Non-pathogenic Neisseria from employees as well as MSM were remarkably resistant. Those from MSM were significantly less susceptible than employees to azithromycin and ciprofloxacin (p < 0.0001, p < 0.001), but not ceftriaxone (p = 0.3). Susceptibility did not differ significantly according to recent antimicrobial exposure in MSM. Surveilling antimicrobial susceptibility of non-pathogenic Neisseria may be a sensitive way to assess impact of antimicrobial exposure in a population. The high levels of antimicrobial resistance in this survey indicate that novel resistance determinants may be readily available for future transfer from non-pathogenic to pathogenic Neisseria.

Prevention of the proliferation of oral pathogens due to prolonged mask use based on α-cyclodextrin and hydroxytyrosol mouthwash.

OBJECTIVE: Face masks help contain the aerosol-mediated transmission of infectious viral particles released from individuals via cough and sneezes. However, the prolonged use of face masks has raised concerns regarding oral hygiene. Here, we present a mouthwash formulation based on α-cyclodextrin and hydroxytyrosol that can maintain healthy oral microbiota. MATERIALS AND METHODS: We isolated and cultured Candida albicans, Staphylococcus aureus, and a mix of Streptococcus sp., Staphylococcus sp. and Neisseria sp. from oral and throat swabs. The microorganisms were cultured in a standard medium with or without the mouthwash. To evaluate the effect of the mouthwash on the oral microbiota, the DNA from the saliva of 3 volunteers that used the mouthwash was extracted. Then, the DNA was amplified using primer pairs specific for bacterial and fungal DNA. Twelve further volunteers were offered to use the mouthwash and a questionnaire was submitted to them to assess the possible beneficial effects of mouthwash on halitosis and other oral disturbances. RESULTS: The bacteria and fungi cultured in media containing the mouthwash showed a growth reduction ranging from 20 to 80%. The PCR amplification of fungal and bacterial DNA extracted from volunteers that used the mouthwash showed a reduction of both bacteria and fungi. Volunteers that used the mouthwash reported a tendency towards a reduction of halitosis, gingival and mouth inflammation, and dry mouth. CONCLUSIONS: The use of a mouthwash containing α-cyclodextrin and hydroxytyrosol is not aggressive against oral mucosa; it is safe and effective to reduce the bacterial and fungal load due to the continuous use of face masks.

A Comprehensive Review on the Interplay between Neisseria spp. and Host Sphingolipid Metabolites.

Sphingolipids represent a class of structural related lipids involved in membrane biology and various cellular processes including cell growth, apoptosis, inflammation and migration. Over the past decade, sphingolipids have become the focus of intensive studies regarding their involvement in infectious diseases. Pathogens can manipulate the sphingolipid metabolism resulting in cell membrane reorganization and receptor recruitment to facilitate their entry. They may recruit specific host sphingolipid metabolites to establish a favorable niche for intracellular survival and proliferation. In contrast, some sphingolipid metabolites can also act as a first line defense against bacteria based on their antimicrobial activity. In this review, we will focus on the strategies employed by pathogenic Neisseria spp. to modulate the sphingolipid metabolism and hijack the sphingolipid balance in the host to promote cellular colonization, invasion and intracellular survival. Novel techniques and innovative approaches will be highlighted that allow imaging of sphingolipid derivatives in the host cell as well as in the pathogen.

Meningococcal Quinolone Resistance Originated from Several Commensal Neisseria Species.

Quinolone resistance is increasing in Neisseria meningitidis, with its prevalence in China being high (>70%), but its origin remains unknown. The aim of this study was to investigate the donors of mutation-harboring gyrA alleles in N. meningitidis A total of 198 N. meningitidis isolates and 293 commensal Neisseria isolates were collected between 2005 and 2018 in Shanghai, China. The MICs of ciprofloxacin were determined using the agar dilution method. The resistance-associated genes gyrA and parC were sequenced for all isolates, while a few isolates were sequenced on the Illumina platform. The prevalences of quinolone resistance in the N. meningitidis and commensal Neisseria isolates were 67.7% (134/198) and 99.3% (291/293), respectively. All 134 quinolone-resistant N. meningitidis isolates possessed mutations in T91 (n = 123) and/or D95 (n = 12) of GyrA, with 7 isolates also harboring ParC mutations and exhibiting higher MICs. Phylogenetic analysis of the gyrA sequence identified six clusters. Among the 71 mutation-harboring gyrA alleles found in 221 N. meningitidis isolates and genomes (n = 221), 12 alleles (n = 103, 46.6%) were included in the N. meningitidis cluster, while 20 alleles (n = 56) were included in the N. lactamica cluster, 27 alleles (n = 49) were included in the N. cinerea cluster, and 9 alleles (n = 10) were included in the N. subflava cluster. Genomic analyses identified the exact N. lactamica donors of seven mutation-harboring gyrA alleles (gyrA92, gyrA97, gyrA98, gyrA114, gyrA116, gyrA151, and gyrA230) and the N. subflava donor isolate of gyrA171, with the sizes of the recombinant fragments ranging from 634 to 7,499 bp. Transformation of gyrA fragments from these donor strains into a meningococcal isolate increased its ciprofloxacin MIC from 0.004 µg/ml to 0.125 or 0.19 µg/ml and to 0.5 µg/ml with further transformation of an additional ParC mutation. Over half of the quinolone-resistant N. meningitidis isolates acquired resistance by horizontal gene transfer from three commensal Neisseria species. Quinolone resistance in N. meningitidis increases in a stepwise manner.

Evaluation of different testing tools for the identification of non-gonococcal Neisseria spp. isolated from Lebanese male semen: a strong and significant association with infertility.

PURPOSE: The aim was to evaluate several microbiological tools for the identification of non-gonococcal Neisseria spp. isolated from semen samples from Lebanese men and to determine the putative link between the presence of Neisseria commensal species and infertility. METHODOLOGY: Within a cross-sectional retrospective study design, the whole population included in this investigation was divided in 2 categories: 173 patients with symptoms of infertility and 139 patients with normal seminograms. Epidemiological and microbiological investigations were performed for 59 strains of Neisseria through several phenotypic and genotypic tools, including seminograms, an analytical profile index of Neisseria and Haemophilus (API-NH), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), porA PCR, 16S rRNA and rplF gene sequencing, and antimicrobial susceptibility testing. RESULTS: The risk of Neisseria infection was twice as high in infertile patients compared to the control group [odds ratio (OR): 1.95, confidence interval (CI): 1.05-3.65, P =0.03]. Unreliable diagnosis of Neisseria urogenital infection has serious health and social consequences. Our findings showed that API-NH and 16S rRNA sequencing are poor tools to identify Neisseria at the species level. Therefore, reliable diagnosis of cases using MALDI-TOF MS and/or rplF sequencing is needed to provide critical treatment decisions and prevent antimicrobial resistance spreading in the community. CONCLUSION: This work predicted a strong and significant association between the presence of Neisseria spp. in semen and male infertility among the Lebanese population. For a better understanding of this association, it is recommended that more genomic and large-scale epidemiological investigations are undertaken to reach definitive conclusions.

Celiac disease-associated Neisseria flavescens decreases mitochondrial respiration in CaCo-2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial-induced cellular imbalance.

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis.

Unusual Neisseria species as a cause of infection in patients taking eculizumab.

BACKGROUND: Non-meningococcal, non-gonococcal Neisseria spp. are typically commensal and rarely cause invasive disease. Eculizumab is a terminal complement inhibitor that increases susceptibility to meningococcal disease, but data on disease caused by typically-commensal Neisseria spp. are lacking. This series describes postmarketing reports of typically-commensal Neisseria spp. disease in patients receiving eculizumab. METHODS: We searched the FDA Adverse Event Reporting System (FAERS) and medical literature for reports of commensal Neisseria spp. disease in patients receiving eculizumab, from eculizumab U.S. approval (2007) through January 31, 2018. RESULTS: We identified seven FAERS reports (including one case also reported in the literature) of non-meningococcal, non-gonococcal Neisseria disease, including N. sicca (mucosa)/subflava (n = 2), N. cinerea (n = 2), N. sicca (mucosa) (n = 1), N. mucosa (n = 1, with concurrent alpha-hemolytic Streptococcus bacteremia), and N. flavescens (subflava) (n = 1). Four cases had sources of patient immunosuppression in addition to eculizumab. Three patients had sepsis (n = 2) or septic shock (n = 1). Five patients were bacteremic. All patients were hospitalized; the infections resolved with antibiotics. CONCLUSIONS: Our search identified seven cases of disease from typically commensal Neisseria spp. in eculizumab recipients. These findings suggest that any Neisseria spp. identified from a normally sterile site in an eculizumab recipient could represent true infection warranting prompt treatment.

Variability in nitrate-reducing oral bacteria and nitric oxide metabolites in biological fluids following dietary nitrate administration: An assessment of the critical difference.

There is conflicting evidence on whether dietary nitrate supplementation can improve exercise performance. This may arise from the complex nature of nitric oxide (NO) metabolism which causes substantial inter-individual variability, within-person biological variation (CVB), and analytical imprecision (CVA) in experimental endpoints. However, no study has quantified the CVA and CVB of NO metabolites or the factors that influence their production. These data are important to calculate the critical difference (CD), defined as the smallest difference between sequential measurements required to signify a true change. The main aim of the study was to evaluate the CVB, CVA, and CD for markers of NO availability (nitrate and nitrite) in plasma and saliva before and after the ingestion of nitrate-rich beetroot juice (BR). We also assessed the CVB of nitrate-reducing bacteria from the dorsal surface of the tongue. It was hypothesised that there would be substantial CVB in markers of NO availability and the abundance of nitrate-reducing bacteria. Ten healthy male participants (age 25 ±â€¯5 years) completed three identical trials at least 6 days apart. Blood and saliva were collected before and after (2, 2.5 and 3 h) ingestion of 140 ml of BR (∼12.4 mmol nitrate) and analysed for [nitrate] and [nitrite]. The tongue was scraped and the abundance of nitrate-reducing bacterial species were analysed using 16S rRNA next generation sequencing. There was substantial CVB for baseline concentrations of plasma (nitrate 11.9%, nitrite 9.0%) and salivary (nitrate 15.3%, nitrite 32.5%) NO markers. Following BR ingestion, the CVB for nitrate (plasma 3.8%, saliva 12.0%) and salivary nitrite (24.5%) were lower than baseline, but higher for plasma nitrite (18.6%). The CD thresholds that need to be exceeded to ensure a meaningful change from baseline are 25, 19, 37, and 87% for plasma nitrate, plasma nitrite, salivary nitrate, and salivary nitrite, respectively. The CVB for selected nitrate-reducing bacteria detected were: Prevotella melaninogenica (37%), Veillonella dispar (35%), Haemophilus parainfluenzae (79%), Neisseria subflava (70%), Veillonella parvula (43%), Rothia mucilaginosa (60%), and Rothia dentocariosa (132%). There is profound CVB in the abundance of nitrate-reducing bacteria on the tongue and the concentration of NO markers in human saliva and plasma. Where these parameters are of interest following experimental intervention, the CD values presented in this study will allow researchers to interpret the meaningfulness of the magnitude of the change from baseline.

An Evolutionarily Conserved Mechanism for Intrinsic and Transferable Polymyxin Resistance.

Polymyxins, a family of cationic antimicrobial cyclic peptides, act as a last line of defense against severe infections by Gram-negative pathogens with carbapenem resistance. In addition to the intrinsic resistance to polymyxin E (colistin) conferred by Neisseria eptA, the plasmid-borne mobilized colistin resistance gene mcr-1 has been disseminated globally since the first discovery in Southern China, in late 2015. However, the molecular mechanisms for both intrinsic and transferable resistance to colistin remain largely unknown. Here, we aim to address this gap in the knowledge of these proteins. Structural and functional analyses of EptA and MCR-1 and -2 have defined a conserved 12-residue cavity that is required for the entry of the lipid substrate, phosphatidylethanolamine (PE). The in vitro and in vivo data together have allowed us to visualize the similarities in catalytic activity shared by EptA and MCR-1 and -2. The expression of either EptA or MCR-1 or -2 is shown to remodel the surface of enteric bacteria (e.g., Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, etc.), rendering them resistant to colistin. The parallels in the PE substrate-binding cavities among EptA, MCR-1, and MCR-2 provide a comprehensive understanding of both intrinsic and transferable colistin resistance. Domain swapping between EptA and MCR-1 and -2 reveals that the two domains (transmembrane [TM] region and phosphoethanolamine [PEA] transferase) are not functionally exchangeable. Taken together, the results represent a common mechanism for intrinsic and transferable PEA resistance to polymyxin, a last-resort antibiotic against multidrug-resistant pathogens.IMPORTANCE EptA and MCR-1 and -2 remodel the outer membrane, rendering bacteria resistant to colistin, a final resort against carbapenem-resistant pathogens. Structural and functional analyses of EptA and MCR-1 and -2 reveal parallel PE lipid substrate-recognizing cavities, which explains intrinsic and transferable colistin resistance in gut bacteria. A similar mechanism is proposed for the catalytic activities of EptA and MCR-1 and -2. Together, they constitute a common mechanism for intrinsic and transferable polymyxin resistance.

Neisseria flavescens: A Urease-Expressing Potential Pathogen Isolated from Gastritis Patients.

Parasitic pathogens, such as H. pylori (Helicobacter pylori), are considered as primary elements for causing stomach infection and leading to chronic gastritis or ulcers. Here, an unreported urease- and oxidase-producing Neisseria flavescens-like bacteria was isolated from the gastroscopic biopsies of 14C-UBT-positive gastritis patients. The isolate expressed the activity of urease, which is a pathogenic factor and considered as a reliable marker for diagnosis of H. pylori infection. However, the isolate didn't express the key functional genes of H. pylori including vacA and hpaA, and also the morphological feature of isolate was significantly different with H. pylori. Eventually, the 16S rDNA of isolate was sequenced and its sequence shared about 99.8% similarity with the N. flavescens standard strains, but about 20.8% similarity with the H. pylori. Further study of antibiotics-resistance revealed the N. flavescens isolate is high resistant to metronidazole, but highly sensitive to ampicillin sodium. To summarize, a urease-expressing N. flavescens strain was isolated and identified from Chinese gastritis patients; the encouraging results provides an important reference for the further study of its pathogenicity and the reasonable diagnosis and use of antibiotics clinically.

Neisseria models of infection and persistence in the upper respiratory tract.

The Gram-negative bacteria genus Neisseria includes both pathogenic and commensal species that are found primarily in the upper respiratory tract of humans and animals. The development of animal models to study neisserial pathogenesis has focused almost exclusively on two species that cause disease in humans. These include Neisseria meningitidis, an obligate commensal that can cause invasive disease, and N. gonorrhoeae, the causative agent of gonorrhea. Both pathogens can persist in the upper respiratory tract. This article will give a brief overview of the genus Neisseria. The anatomy of the upper respiratory tract and its use as a niche for bacteria will be discussed. Next, studies that provide insight about the first stage of upper respiratory tract infection, namely colonization, will be reviewed. Most studies of upper respiratory tract infection have focused on N. meningitidis infections of laboratory mice. This review will also discuss models of respiratory tract persistence by Neisseria species, including commensals, in mice, non-human primates and human volunteers. The article includes a section that discusses the future utility of upper respiratory tract models in informing the development of effective antimicrobial therapies. Such knowledge is needed to minimize the dissemination of antimicrobial resistance from respiratory reservoirs.

Acute Cystitis Caused by Commensal Neisseria oralis: A Case Report and Review of the Literature.

BACKGROUND: Neisseria are usually harmless inhabitants of otherwise asymptomatic persons' upper respiratory mucosal surfaces. METHOD: It is, therefore, expected that a disturbance in the physiology leads to nongonococcal, non-meningococcal Neisseria becoming pathogenic. RESULT: We report the case of a diabetic man who initially presented with nonspecific symptoms and was later found to have cystitis caused by N. oralis. CONCLUSION: We also review the pertinent literature and discuss available evidence on pathophysiological mechanisms of infection with such commensal bacteria.

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria.

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.

Microbial Flora and Antibiotic Resistance in Peritonsillar Abscesses in Upstate New York.

OBJECTIVES: To identify the common bacteria in recent peritonsillar abscesses and the prevalence of antibiotic resistance and compare both between adults and children. METHODS: This is a retrospective chart review at a single academic institution of patients who underwent either incision and drainage or tonsillectomy for a peritonsillar abscess between 2002 and 2012 (n=69). Medical records were reviewed for cultures, comorbidities, and drainage procedures. RESULTS: Cultures obtained from 62.32% of peritonsillar abscesses were polymicrobial, and 34.78% were monomicrobial. The most common pathogens were ß-hemolytic Streptococcus (31.88%), α-hemolytic Streptococcus (21.74%), Neisseria (14.49%), and Streptococcus milleri (13.04%). Group A ß-hemolytic streptococcus was more common in children and Streptococcus milleri was more common in adults. Alpha-hemolytic streptococcus was resistant to clindamycin (6.67%) and erythromycin (6.67%). Streptococcus milleri was resistant to clindamycin (11.11%) and erythromycin (11.11%). Staphylococcus was resistant to penicillin (37.5%), oxacillin (25%), erythromycin (25%), and clindamycin (12.5%). CONCLUSIONS: ß- and α-hemolytic Streptococci, Neisseria, and Streptococcus milleri are the most common pathogens. Streptococcus milleri is more common in adults, and ß-hemolytic streptococcus is more common in children. Resistance to clindamycin and erythromycin is common in Streptococci and Staphylococci, and penicillin resistance is common in Staphylococci.

Prevalence and genotyping of commensal Neisseria with reduced susceptibility to penicillin.

We analyzed 85 Neisseria spp. strains collected by swabbing from neutropenic patients to determine the prevalence of reduced susceptibility to penicillin and to ascertain the clonal relationship between these strains. High genetic diversity and an elevated level of penicillin resistance were found among commensal Neisseria clinical isolates.

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.

Effect of stoned olive pomace on rumen microbial communities and polyunsaturated fatty acid biohydrogenation: an in vitro study.

BACKGROUND: Stoned olive pomace (SOP), which represents approximately 50% of the conversion process of olives to olive oil, is largely not utilised and creates costs for its disposal and has negative environmental impacts. In vitro trial experiments were employed to study the effect of feeds integrated with this bio-waste, which is rich in polyphenols, on rumen biohydrogenation, using sheep rumen liquor as inoculum. RESULTS: Fatty acid (FA) analysis and a polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) approach aimed at characterising the microbial community indicated that including SOP in feeds at the level of 50 g/kg and 90 g/kg induced changes in the FA profile and microbial populations. The simultaneous decrease of Butyrivibrio proteoclasticus and accumulation of vaccenic acid was observed. A depression in the populations of Neisseria weaveri, Ruminobacter amylophilus and other unclassified bacteria related to members of the Lachnospiraceae and Pasteurellaceae families was detected, suggesting that these microbial groups may be involved in rumen biohydrogenation. CONCLUSIONS: Supplementation of feeds with SOP alters the rumen bacterial community, including bacteria responsible for the hydrogenation of vaccenic acid to stearic acid, thereby modifying the FA profile of the rumen liquor. Hence, a use of SOP aimed to produce meat or dairy products enriched in functional lipids can be hypothesised.

Survival of microorganisms on complete dentures following ultrasonic cleaning combined with immersion in peroxide-based cleanser solution.

OBJECTIVES: To compare ultrasonic cleaning combined with immersion in a commercially available peroxide-based cleanser solution (Polident(®) ) with other denture cleaning methods, we examined the quantity of micro-organisms that survived on dentures before and after various cleaning methods. SUBJECTS AND METHODS: One hundred complete dentures belonging to 50 nursing home residents (mean age, 84.6 years) were randomly assigned to five groups according to the cleaning method employed: (A) immersion in Polident(®) solution alone, (B) brushing with water, (C) ultrasonic cleaning with water, (D) method (A) followed by method (B) and (E) ultrasonic cleaning combined with immersion in Polident(®) solution. Before and after the dentures had been cleaned, denture biofilm was collected from the mucosal surface of each lateral half of the examined dentures. The collected micro-organisms were cultured, presumptively identified by standard methods and quantified. Comparisons between the five cleaning methods were carried out using the Kruskal-Wallis test and Dunn's multiple comparisons test. RESULTS: The denture cleaning methods involving the use of Polident(®) solution (methods A, D and E) were significantly more effective at denture disinfection than the other methods (p < 0.05); in particular, the quantity of Candida spp. was lowest after method E (median, 0.00; significantly lower than those observed after methods A, B and C; p < 0.05). CONCLUSION: It was concluded that ultrasonic cleaning combined with immersion in a peroxide-based cleanser solution effectively reduces the quantity of micro-organisms surviving on dentures and is a suitable method for elderly individuals who find brushing their dentures difficult.

Chitosan-thioglycolic acid as a versatile antimicrobial agent.

As functionalized chitosans hold great potential for the development of effective and broad-spectrum antibiotics, representative chitosan derivatives were tested for antimicrobial activity in neutral media: trimethyl chitosan (TMC), carboxy-methyl chitosan (CMC), and chitosan-thioglycolic acid (TGA; medium molecular weight: MMW-TGA; low molecular weight: LMW-TGA). Colony forming assays indicated that LMW-TGA displayed superior antimicrobial activity over the other derivatives tested: a 30 min incubation killed 100% Streptococcus sobrinus (Gram-positive bacteria) and reduced colony counts by 99.99% in Neisseria subflava (Gram-negative bacteria) and 99.97% in Candida albicans (fungi). To elucidate LMW-TGA effects at the cellular level, microscopic studies were performed. Use of fluorescein isothiocyanate (FITC)-labeled chitosan derivates in confocal microscopy showed that LMW-TGA attaches to microbial cell walls, while transmission electron microscopy indicated that this derivative severely affects cell wall integrity and intracellular ultrastructure in all species tested. We therefore propose LMW-TGA as a promising and effective broad-band antimicrobial compound.