Scalp microbiome composition changes and pathway evaluations due to effective treatment with Piroctone Olamine shampoo.

OBJECTIVE: To characterize the scalp microbial composition, function, and connection to dandruff severity using a metagenomics approach and to understand the impact of a Piroctone Olamine containing anti-dandruff shampoo on the scalp microbiome. METHODS: Shotgun metagenomics was used to characterize the composition of the scalp microbiomes from 94 subjects with and without clinically defined dandruff. Furthermore, the microbiome of the scalps of 100 dandruff sufferers before and after 3 weeks of treatment with either control or anti-dandruff shampoo containing 0.5% Piroctone Olamine (PO) was characterized and compared to identify microorganisms associated with the dandruff condition and the associated pathways and processes that may contribute to PO's effect on scalp microbiome. RESULTS: A higher relative abundance of Malassezia restricta and Staphylococcus capitis and a lower abundance of Cutibacterium acnes were associated with the dandruff scalps relative to the no-dandruff scalps. A 3-week PO shampoo treatment reduced the relative abundance of Malassezia species and Staphylococcus capitis and increased the relative abundance of Cutibacterium acnes. This change to the scalp microbiome composition is consistent with a return to a healthy no-dandruff microbiome and improved clinical signs and symptoms as measured by adherent scalp flaking score (ASFS) compared with the control shampoo. Functional genomics analysis showed that the PO shampoo treatment reduced oxidative stress-associated genes and decreased the abundance of protease, urease, and lipase genes. These changes correlated positively to improvements in dandruff severity. PO treatment favourably shifted scalp microbiomes in dandruff subjects toward the no-dandruff state. CONCLUSION: Our results suggest that part of the aetiology of dandruff can be attributed to dysbiosis of the scalp microbiome. PO treatment can restore a healthier microbiome, reducing oxidative stress and promoting better scalp health.

OBJECTIF: Caractériser la composition microbienne du cuir chevelu, sa fonction et son lien avec la sévérité des pellicules à l'aide d'une approche métagénomique. Comprendre l'impact d'un shampooing antipelliculaire à base de piroctone olamine sur le microbiome du cuir chevelu. MÉTHODES: La métagénomique shotgun a été utilisée pour caractériser la composition des microbiomes du cuir chevelu de 94 sujets avec et sans pellicules définies cliniquement. Par ailleurs, le microbiome des cuirs chevelus de 100 personnes ayant des pellicules avant et après trois semaines de traitement par un shampooing témoin ou un shampooing antipelliculaire contenant 0,5 % de piroctone olamine (PO) a été caractérisé et comparé pour identifier les micro­organismes associés à l'état pelliculaire, et les voies et processus associés pouvant contribuer à l'effet de la PO sur le microbiome du cuir chevelu. RÉSULTATS: Une abondance relative plus élevée de Malassezia restricta et de Staphylococcus capitis, et une abondance plus faible de Cutibacterium acnes étaient associées aux cuirs chevelus avec des pellicules par rapport aux cuirs chevelus sans pellicules. Un traitement avec un shampooing contenant de la PO de 3 semaines a réduit l'abondance relative des espèces Malassezia et Staphylococcus capitis, et a augmenté l'abondance relative de Cutibacterium acnes. Cette modification de la composition du microbiome du cuir chevelu est cohérente avec un retour à un microbiome sain sans pellicules, et une amélioration des signes et symptômes cliniques mesurés par le score de desquamation du cuir chevelu adhérent (Adherent Scalp Flaking Score, ASFS) par rapport au shampooing témoin. L'analyse génomique fonctionnelle a montré que le traitement avec un shampooing contenant de la PO réduisait les gènes associés au stress oxydatif et diminuait l'abondance des gènes de la protéase, de l'uréase et de la lipase. Ces modifications étaient corrélées positivement à des améliorations de la sévérité des pellicules. Le traitement avec la PO a favorisé l'évolution des microbiomes du cuir chevelu des sujets ayant des pellicules vers un état sans pellicules. CONCLUSION: Nos résultats suggèrent qu'une partie de l'étiologie des pellicules peut être attribuée à la dysbiose du microbiome du cuir chevelu. Le traitement avec la PO peut rétablir un microbiome plus sain, en réduisant le stress oxydatif et en favorisant une meilleure santé du cuir chevelu.

Quantitation of Risk Reduction of E. coli Transmission After Using Antimicrobial Hand Soap.

Handwashing with soap is an effective and economical means to reduce the likelihood of Escherichia coli infection from indirect contact with contaminated surfaces during food preparation. The purpose of this study was to conduct a quantitative microbial risk assessment (QMRA) to evaluate the risk of infection from indirect contact with fomites contaminated with E. coli after hand washing with antimicrobial hand soaps. A Monte Carlo simulation was done with a total of 10,000 simulations to compare the effectiveness of two antimicrobial and one control (non-antimicrobial) bar soaps in reducing the exposure and infection risk compared to no hand washing. The numbers of E. coli on several fomites commonly found in household kitchens, as well as the transfer rates between fomites and onto fingertips, were collected from the literature and experimental data. The sponsor company provided the E. coli survival on hands after washing with antimicrobial and control soaps. A number of scenarios were evaluated at two different exposure doses (high and low). Exposure scenarios included transfer of E. coli between meat-to-cutting board surface-to-hands, meat-to-knife surface-to-hands, and from a countertop surface-to-hands, kitchen sponge-to-hands, hand towel-to-hands, and dishcloth-to-hands. Results showed that the risks of illness after washing with the control soap was reduced approximately 5-fold compared to no handwashing. Washing with antimicrobial soap reduced the risk of E. coli infection by an average of about 40-fold compared with no handwashing. The antimicrobial soaps ranged from 3 to 32 times more effective than the non-antimicrobial soap, depending on the specific exposure scenario. Importance: The Centers for Disease Control and Prevention indicate the yearly incidence rate of Shiga Toxin producing E. coli infections is about 1.7/100,000, with about 10% of cases leading to life-threatening hemolytic uremic syndrome and 3-5% leading to death. Our findings confirm handwashing with soap reduces the risks associated with indirect transmission of E. coli infection from contact with fomites during food preparation. Further, in these exposure scenarios, antimicrobial soaps were more effective overall than the non-antimicrobial soap in reducing exposure to E. coli and risk of infection.

Evaluation of the Influenza Risk Reduction from Antimicrobial Spray Application on Porous Surfaces.

Antimicrobial spray products are used by millions of people around the world for cleaning and disinfection of commonly touched surfaces. Influenza A is a pathogen of major concern, leading to up to 49,000 deaths and 114,000 hospitalizations per year in the United States alone. One of the recognized routes of transmission for influenza A is by transfer of viruses from surfaces to hands and subsequently to mucous membranes. Therefore, routine cleaning and disinfection of surfaces is an important part of the environmental management of influenza A. While the emphasis is generally on spraying hard surfaces and laundering cloth and linens with high temperature machine drying, not all surfaces can be treated in this manner. The quantitative microbial risk assessment (QMRA) approach was used to develop a stochastic risk model for estimating the risk of infection from indirect contact with porous fomite with and without surface treatment with an antimicrobial spray. The data collected from laboratory analysis combined with the risk model show that influenza A infection risk can be lowered by four logs after using an antimicrobial spray on a porous surface. Median risk associated with a single touch to a contaminated fabric was estimated to be 1.25 × 10-4 for the untreated surface, and 3.6 × 10-8 for the treated surface as a base case assumption. This single touch scenario was used to develop a generalizable model for estimating risks and comparing scenarios with and without treatment to more realistic multiple touch scenarios over time periods and with contact rates previously reported in the literature. The results of this study and understanding of product efficacy on risk reduction inform and broaden the range of risk management strategies for influenza A by demonstrating effective risk reduction associated with treating nonporous fomites that cannot be laundered at high temperatures.

Bacteriophages safely reduce Salmonella contamination in pet food and raw pet food ingredients.

Contamination of pet food with Salmonella is a serious public health concern, and several disease outbreaks have recently occurred due to human exposure to Salmonella tainted pet food. The problem is especially challenging for raw pet foods (which include raw meats, seafood, fruits, and vegetables). These foods are becoming increasingly popular because of their nutritional qualities, but they are also more difficult to maintain Salmonella-free because they lack heat-treatment. Among various methods examined to improve the safety of pet foods (including raw pet food), one intriguing approach is to use bacteriophages to specifically kill Salmonella serotypes. At least 2 phage preparations (SalmoFresh® and Salmonelex™) targeting Salmonella are already FDA cleared for commercial applications to improve the safety of human foods. However, similar preparations are not yet available for pet food applications. Here, we report the results of evaluating one such preparation (SalmoLyse®) in reducing Salmonella levels in various raw pet food ingredients (chicken, tuna, turkey, cantaloupe, and lettuce). Application of SalmoLyse® in low (ca. 2-4×106 PFU/g) and standard (ca. 9×106 PFU/g) concentrations significantly (P < 0.01) reduced (by 60-92%) Salmonella contamination in all raw foods examined compared to control treatments. When SalmoLyse®-treated (ca. 2×107 PFU/g) dry pet food was fed to cats and dogs, it did not trigger any deleterious side effects in the pets. Our data suggest that the bacteriophage cocktail lytic for Salmonella can significantly and safely reduce Salmonella contamination in various raw pet food ingredients.

Cetylpyridinium chloride mouth rinses alleviate experimental gingivitis by inhibiting dental plaque maturation.

Oral rinses containing chemotherapeutic agents, such as cetylpyridinium chloride (CPC), can alleviate plaque-induced gingival infections, but how oral microbiota respond to these treatments in human population remains poorly understood. Via a double-blinded, randomised controlled trial of 91 subjects, the impact of CPC-containing oral rinses on supragingival plaque was investigated in experimental gingivitis, where the subjects, after a 21-day period of dental prophylaxis to achieve healthy gingivae, received either CPC rinses or water for 21 days. Within-subject temporal dynamics of plaque microbiota and symptoms of gingivitis were profiled via 16S ribosomal DNA gene pyrosequencing and assessment with the Mazza gingival index. Cetylpyridinium chloride conferred gingival benefits, as progression of gingival inflammation resulting from a lack of dental hygiene was significantly slower in the mouth rinse group than in the water group due to inhibition of 17 gingivitis-enriched bacterial genera. Tracking of plaque α and ß diversity revealed that CPC treatment prevents acquisition of new taxa that would otherwise accumulate but maintains the original biodiversity of healthy plaques. Furthermore, CPC rinses reduced the size, local connectivity and microbiota-wide connectivity of the bacterial correlation network, particularly for nodes representing gingivitis-enriched taxa. The findings of this study provide mechanistic insights into the impact of oral rinses on the progression and maturation of dental plaque in the natural human population.

Microbiota-based Signature of Gingivitis Treatments: A Randomized Study.

Plaque-induced gingivitis can be alleviated by various treatment regimens. To probe the impacts of various anti-gingivitis treatments on plaque microflora, here a double blinded, randomized controlled trial of 91 adults with moderate gingivitis was designed with two anti-gingivitis regimens: the brush-alone treatment and the brush-plus-rinse treatment. In the later group, more reduction in both Plaque Index (TMQHI) and Gingival Index (mean MGI) at Day 3, Day 11 and Day 27 was evident, and more dramatic changes were found between baseline and other time points for both supragingival plaque microbiota structure and salivary metabonomic profiles. A comparison of plaque microbiota changes was also performed between these two treatments and a third dataset where 50 subjects received regimen of dental scaling. Only Actinobaculum, TM7 and Leptotrichia were consistently reduced by all the three treatments, whereas the different microbial signatures of the three treatments during gingivitis relieve indicate distinct mechanisms of action. Our study suggests that microbiota based signatures can serve as a valuable approach for understanding and potentially comparing the modes of action for clinical treatments and oral-care products in the future.

Bacteriophage cocktail for biocontrol of Salmonella in dried pet food.

Human salmonellosis has been associated with contaminated pet foods and treats. Therefore, there is interest in identifying novel approaches for reducing the risk of Salmonella contamination within pet food manufacturing environments. The use of lytic bacteriophages shows promise as a safe and effective way to mitigate Salmonella contamination in various food products. Bacteriophages are safe, natural, highly targeted antibacterial agents that specifically kill bacteria and can be targeted to kill food pathogens without affecting other microbiota. In this study, we show that a cocktail containing six bacteriophages had a broadspectrum activity in vitro against a library of 930 Salmonella enterica strains representing 44 known serovars. The cocktail was effective against 95% of the strains in this tested library. In liquid culture dose-ranging experiments, bacteriophage cocktail concentrations of ≥10(8) PFU/ml inactivated more than 90% of the Salmonella population (10(1) to 10(3) CFU/ml). Dried pet food inoculated with a mixture containing equal proportions of Salmonella serovars Enteritidis (ATCC 4931), Montevideo (ATCC 8387), Senftenberg (ATCC 8400), and Typhimurium (ATCC 13311) and then surface treated with the six-bacteriophage cocktail (≥2.5 ± 1.5 × 10(6) PFU/g) achieved a greater than 1-log (P < 0.001) reduction compared with the phosphate-buffered saline-treated control in measured viable Salmonella within 60 min. Moreover, this bacteriophage cocktail reduced natural contamination in samples taken from an undistributed lot of commercial dried dog food that tested positive for Salmonella. Our results indicate that bacteriophage biocontrol of S. enterica in dried pet food is technically feasible.

Characterization of bacterial isolates from the microbiota of mothers' breast milk and their infants.

This investigation assessed the potential of isolating novel probiotics from mothers and their infants. A subset of 21 isolates among 126 unique bacteria from breast milk and infant stools from 15 mother-infant pairs were examined for simulated GI transit survival, adherence to Caco-2 cells, bacteriocin production, and lack of antibiotic resistance. Of the 21 selected isolates a Lactobacillus crispatus isolate and 3 Lactobacillus gasseri isolates demonstrated good profiles of in vitro GI transit tolerance and Caco-2 cell adherence. Bacteriocin production was observed only by L. gasseri and Enterococcus faecalis isolates. Antibiotic resistance was widespread, although not universal, among isolates from infants. Highly similar isolates (≥ 97% similarity by barcode match) of Bifidobacterium longum subsp. infantis (1 match), Lactobacillus fermentum (2 matches), Lactobacillus gasseri (6 matches), and Enterococcus faecalis (1 match) were isolated from 5 infant-mother pairs. Antibiotic resistance profiles between these isolate matches were similar, except in one case where the L. gasseri isolate from the infant exhibited resistance to erythromycin and tetracycline, not observed in matching mother isolate. In a second case, L. gasseri isolates differed in resistance to ampicillin, chloramphenicol and vancomycin between the mother and infant. In this study, gram positive bacteria isolated from mothers' breast milk as well as their infants exhibited diversity in GI transit survival and acid inhibition of pathogens, but demonstrated limited ability to produce bacteriocins. Mothers and their infants offer the potential for identification of probiotics; however, even in the early stages of development, healthy infants contain isolates with antibiotic resistance.

Effects of genetic, processing, or product formulation changes on efficacy and safety of probiotics.

Commercial probiotic strains for food or supplement use can be altered in different ways for a variety of purposes. Production conditions for the strain or final product may be changed to address probiotic yield, functionality, or stability. Final food products may be modified to improve flavor and other sensory properties, provide new product formats, or respond to market opportunities. Such changes can alter the expression of physiological traits owing to the live nature of probiotics. In addition, genetic approaches may be used to improve strain attributes. This review explores whether genetic or phenotypic changes, by accident or design, might affect the efficacy or safety of commercial probiotics. We highlight key issues important to determining the need to re-confirm efficacy or safety after strain improvement, process optimization, or product formulation changes. Research pinpointing the mechanisms of action for probiotic function and the development of assays to measure them are greatly needed to better understand if such changes have a substantive impact on probiotic efficacy.

Bacteriophages lytic for Salmonella rapidly reduce Salmonella contamination on glass and stainless steel surfaces.

A cocktail of six lytic bacteriophages, SalmoFresh™, significantly (p < 0.05) reduced the number of surface-applied Salmonella Kentucky and Brandenburg from stainless steel and glass surfaces by > 99% (2.1-4.3 log). Both strains were susceptible to SalmoFresh™ in the spot-test assay. Conversely, SalmoFresh™ was unable to reduce surface contamination with a Salmonella Paratyphi B strain that was not susceptible to the phage cocktail in the spot-test assay. However, by replacing two SalmoFresh™ component phages with two new phages capable of lysing the Paratyphi B strain in the spot-test assay, the target range of the cocktail was shifted to include the Salmonella Paratyphi B strain. The modified cocktail, SalmoLyse™, was able to significantly (p < 0.05) reduce surface contamination of the Paratyphi B strain by > 99% (2.1-4.1 log). The data show that both phage cocktails were effective in significantly reducing the levels of Salmonella on hard surfaces, provided the contaminating strains were susceptible in the spot-test (i.e., spot-test susceptibility was indicative of efficacy in subsequent surface decontamination studies). The data also support the concept that phage preparations can be customized to meet the desired antibacterial application.

Fecal excretion of Bifidobacterium infantis 35624 and changes in fecal microbiota after eight weeks of oral supplementation with encapsulated probiotic.

Certain randomized, placebo-controlled trials of oral supplementation with B. infantis 35624 have demonstrated the amelioration of symptoms of irritable bowel syndrome. Potential GI colonization by B. infantis 35624 or effects of supplementation on resident GI microbiota may pertain to these clinical observations. In this study, fecal excretion of B. infantis 35624 before, during and after 8 weeks of daily treatment was compared in subjects with IBS who received either the encapsulated oral supplement (n = 39) or placebo (n = 37) and in healthy subjects who received the supplement (n = 41). Secondarily, changes in assessed fecal microbiota and IBS symptoms were determined. Supplementation significantly increased fecal B. infantis 35624 excretion vs. placebo in IBS subjects; excretion in healthy subjects receiving supplement was quantitatively similar. Fecal levels of the probiotic declined and approached baseline once dosing ceased, documenting that colonization is transient. Although supplementation increased numbers of B infantis 35624 within the GI tract, limited changes in 10 other fecal taxa were observed either in healthy subjects or those with IBS. No impact on IBS symptoms was observed. Detection of bacterial DNA in fecal samples suggests that the probiotic is able to survive transit through the GI tract, although strain selective culture techniques were not performed to confirm viability of B. infantis 35624 in the feces. Continuous probiotic administration was necessary to maintain steady-state transit. Given the complex spectrum of GI microbiota, however, monitoring perturbations in selected taxa may not be not a useful indicator of probiotic function.

Rapid detection of Salmonella in pet food: design and evaluation of integrated methods based on real-time PCR detection.

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead-based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix-associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.

Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus.

Contaminated hands or inanimate surfaces can act as a source of infection during outbreaks of human norovirus infection. We evaluated the virucidal efficacy of seven hand sanitizers containing various active ingredients, such as ethanol, triclosan, and chlorhexidine, and compared their effectiveness against feline calicivirus (FCV), murine norovirus (MNV), and a GII.4 norovirus fecal extract. We also tested the efficacy of 50, 70, and 90% of ethanol and isopropanol. Reduction of viral infectivity was measured by plaque assay, and the number of genomic copies was determined with a TaqMan real-time reverse transcription PCR assay. Based on the results of a quantitative suspension test, only one ethanol-based product (72% ethanol, pH 2.9) and one triclosan-based product (0.1% triclosan, pH 3.0) reduced the infectivity of both MNV and FCV (by >2.6 and ≥3.4 log units, respectively). Four of the seven products were effective against either MNV or FCV, whereas chlorhexidine was ineffective against both viruses. For these hand sanitizers, no correlation was found between reduced infectivity and decline of viral RNA. Ethanol and isopropanol concentrations ≥70% reduced the infectivity of MNV by ≥2.6 log units, whereas 50 and 70% ethanol reduced the infectivity of FCV by ≥2.2 log units after exposure for 5 min. The susceptibility of FCV to low pH and the relative high susceptibility of MNV to alcohols suggest that both surrogate viruses should be considered for in vitro testing of hand sanitizers.

Low pH gel intranasal sprays inactivate influenza viruses in vitro and protect ferrets against influenza infection.

BACKGROUND: Developing strategies for controlling the severity of pandemic influenza is a global public health priority. In the event of a pandemic there may be a place for inexpensive, readily available, effective adjunctive therapies to support containment strategies such as prescription antivirals, vaccines, quarantine and restrictions on travel. Inactivation of virus in the intranasal environment is one possible approach. The work described here investigated the sensitivity of influenza viruses to low pH, and the activity of low pH nasal sprays on the course of an influenza infection in the ferret model. METHODS: Inactivation of influenza A and avian reassortment influenza was determined using in vitro solutions tests. Low pH nasal sprays were tested using the ferret model with an influenza A Sydney/5/97 challenge. Clinical measures were shed virus, weight loss and body temperature. RESULTS: The virus inactivation studies showed that influenza viruses are rapidly inactivated by contact with acid buffered solutions at pH 3.5. The titre of influenza A Sydney/5/97 [H3N2] was reduced by at least 3 log cycles with one minute contact with buffers based on simple acid mixtures such as L-pyroglutamic acid, succinic acid, citric acid and ascorbic acid. A pH 3.5 nasal gel composition containing pyroglutamic acid, succinic acid and zinc acetate reduced titres of influenza A Hong Kong/8/68 [H3N2] by 6 log cycles, and avian reassortment influenza A/Washington/897/80 X A Mallard/New York/6750/78 [H3N2] by 5 log cycles, with 1 min contact.Two ferret challenge studies, with influenza A Sydney/5/97, demonstrated a reduction in the severity of the disease with early application of low pH nasal sprays versus a saline control. In the first study there was decreased weight loss in the treatment groups. In the second study there were reductions in virus shedding and weight loss, most notably when a gelling agent was added to the low pH formulation. CONCLUSION: These findings indicate the potential of a low pH nasal spray as an adjunct to current influenza therapies, and warrant further investigation in humans.

Efficacy of an encapsulated probiotic Bifidobacterium infantis 35624 in women with irritable bowel syndrome.

BACKGROUND: Probiotic bacteria exhibit a variety of properties, including immunomodulatory activity, which are unique to a particular strain. Thus, not all species will necessarily have the same therapeutic potential in a particular condition. We have preliminary evidence that Bifidobacterium infantis 35624 may have utility in irritable bowel syndrome (IBS). OBJECTIVES: This study was designed to confirm the efficacy of the probiotic bacteria B. infantis 35624 in a large-scale, multicenter, clinical trial of women with IBS. A second objective of the study was to determine the optimal dosage of probiotic for administration in an encapsulated formulation. METHODS: After a 2-wk baseline, 362 primary care IBS patients, with any bowel habit subtype, were randomized to either placebo or freeze-dried, encapsulated B. infantis at a dose of 1 x 10(6), 1 x 10(8), or 1 x 10(10), cfu/mL for 4 wk. IBS symptoms were monitored daily and scored on to a 6-point Likert scale with the primary outcome variable being abdominal pain or discomfort. A composite symptom score, the subject's global assessment of IBS symptom relief, and measures of quality of life (using the IBS-QOL instrument) were also recorded. RESULTS: B. infantis 35624 at a dose of 1 x 10(8) cfu was significantly superior to placebo and all other bifidobacterium doses for the primary efficacy variable of abdominal pain as well as the composite score and scores for bloating, bowel dysfunction, incomplete evacuation, straining, and the passage of gas at the end of the 4-wk study. The improvement in global symptom assessment exceeded placebo by more than 20% (p < 0.02). Two other doses of probiotic (1 x 10(6) and 1 x 10(10)) were not significantly different from placebo; of these, the 1 x 10(10) dose was associated with significant formulation problems. No significant adverse events were recorded. CONCLUSIONS: B. infantis 35624 is a probiotic that specifically relieves many of the symptoms of IBS. At a dosage level of 1 x 10(8) cfu, it can be delivered by a capsule making it stable, convenient to administer, and amenable to widespread use. The lack of benefits observed with the other dosage levels of the probiotic highlight the need for clinical data in the final dosage form and dose of probiotic before these products should be used in practice.

Effects of triclosan-containing rinse on the dynamics and antimicrobial susceptibility of in vitro plaque ecosystems.

Dental plaque microcosms were established under a feast-famine regimen within constant-depth film fermentors and exposed four times daily postfeeding to a triclosan (TR)-containing rinse (dentifrice) (TRD). This was diluted so that the antimicrobial content was 0.6 mg/ml. Microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the V2-V3 region of the eubacterial 16S rRNA gene (rDNA). Dominant isolates and PCR amplicons were identified by partial sequencing of 16S rDNA. TRD caused considerable decreases in the counts of both gram-negative organisms and total anaerobic cells, transiently lowered the numbers of streptococci and actinomycetes, and markedly increased the proportion of lactobacilli. DGGE indicated the presence of putatively unculturable bacteria and showed that a Porphyromonas sp. and Selenomonas infelix had been inhibited by TRD. Pure culture studies of 10 oral bacteria (eight genera) showed that Neisseria subflava, Prevotella nigrescens, and Porphyromonas gingivalis were highly susceptible to TR, while the lactobacilli and streptococci were the least susceptible. Clonal expansion of the lactobacilli in the pulsed microcosm could be explained on the basis of TR activity. The mean MICs of TR, chlorhexidine, erythromycin, penicillin V, and vancomycin for the population before and after 5 days of exposure to TRD showed few significant changes. In conclusion, changes in plaque microcosm populations following repeated exposure to TRD showed inhibition of the most susceptible flora and clonal expansion of less susceptible species.

Exposure of sink drain microcosms to triclosan: population dynamics and antimicrobial susceptibility.

Recent concern that the increased use of triclosan (TCS) in consumer products may contribute to the emergence of antibiotic resistance has led us to examine the effects of TCS dosing on domestic-drain biofilm microcosms. TCS-containing domestic detergent (TCSD) markedly lowered biofouling at 50% (wt/vol) but was poorly effective at use levels. Long-term microcosms were established and stabilized for 6 months before one was subjected to successive 3-month exposures to TCSD at sublethal concentrations (0.2 and 0.4% [wt/vol]). Culturable bacteria were identified by 16S rDNA sequence analysis, and their susceptibilities to four biocides and six antibiotics were determined. Microcosms harbored ca. 10 log(10) CFU/g of biofilm, representing at least 27 species, mainly gamma proteobacteria, and maintained dynamic stability. Viable cell counts were largely unaffected by TCSD exposure, but species diversity was decreased, as corroborated by denaturing gradient gel electrophoresis analysis. TCS susceptibilities ranged widely within bacterial groups, and TCS-tolerant strains (including aeromonads, pseudomonads, stenotrophomonads, and Alcaligenes spp.) were isolated before and after TCSD exposure. Several TCS-tolerant bacteria related to Achromobacter xylosoxidans became clonally expanded during dosing. TCSD addition did not significantly affect the community profiles of susceptibility to the test biocides or antibiotics. Several microcosm isolates, as well as reference bacteria, caused clearing of particulate TCS in solid media. Incubations of consortia and isolates with particulate TCS in liquid led to putative TCS degradation by the consortia and TCS solubilization by the reference strains. Our results support the view that low-level exposure of environmental microcosms to TCS does not affect antimicrobial susceptibility and that TCS is degradable by common domestic biofilms.

Effects of a chlorhexidine gluconate-containing mouthwash on the vitality and antimicrobial susceptibility of in vitro oral bacterial ecosystems.

Oral bacterial microcosms, established using saliva inocula from three individuals, were maintained under a feast-famine regime within constant-depth film fermenters. Steady-state communities were exposed four times daily, postfeeding, to a chlorhexidine (CHX) gluconate-containing mouthwash (CHXM) diluted to 0.06% (wt/vol) antimicrobial content. The microcosms were characterized by heterotrophic plate counts and PCR-denaturing gradient gel electrophoresis (DGGE). CHXM caused significant decreases in both total anaerobe and total aerobe/facultative anaerobe counts (P < 0.05), together with lesser decreases in gram-negative anaerobes. The degree of streptococcal and actinomycete inhibition varied considerably among individuals. DGGE showed that CHXM exposure caused considerable decreases in microbial diversity, including marked reductions in Prevotella sp. and Selenomonas infelix. Pure-culture studies of 10 oral bacteria (eight genera) showed that Actinomyces naeslundii, Veillonella dispar, Prevotella nigrescens, and the streptococci were highly susceptible to CHX, while Lactobacillus rhamnosus, Fusobacterium nucleatum, and Neisseria subflava were the least susceptible. Determination of the MICs of triclosan, CHX, erythromycin, penicillin V, vancomycin, and metronidazole for microcosm isolates, before and after 5 days of CHXM exposure, showed that CHXM exposure altered the distribution of isolates toward those that were less susceptible to CHX (P < 0.05). Changes in susceptibility distributions for the other test agents were not statistically significant. In conclusion, population changes in plaque microcosms following repeated exposure to CHXM represented an inhibition of the most susceptible flora with a clonal expansion of less susceptible species.

Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women.

Urogenital infections afflict an estimated one billion people each year. The size of this problem and the increased prevalence of multi-drug resistant pathogens make it imperative that alternative remedies be found. A randomized, placebo-controlled trial of 64 healthy women given daily oral capsules of Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14 for 60 days showed no adverse effects. Microscopy analysis showed restoration from asymptomatic bacterial vaginosis microflora to a normal lactobacilli colonized microflora in 37% women during lactobacilli treatment compared to 13% on placebo (P=0.02). Lactobacilli were detected in more women in the lactobacilli-treated group than in the placebo group at 28 day (P=0.08) and 60 day (P=0.05) test points. Culture findings confirmed a significant increase in vaginal lactobacilli at day 28 and 60, a significant depletion in yeast at day 28 and a significant reduction in coliforms at day 28, 60 and 90 for lactobacilli-treated subjects versus controls. The combination of probiotic L. rhamnosus GR-1 and L. fermentum RC-14 is not only safe for daily use in healthy women, but it can reduce colonization of the vagina by potential pathogenic bacteria and yeast.

Microbial characterization of biofilms in domestic drains and the establishment of stable biofilm microcosms.

We have used heterotrophic plate counts, together with live-dead direct staining and denaturing gradient gel electrophoresis (DGGE), to characterize the eubacterial communities that had formed as biofilms within domestic sink drain outlets. Laboratory microcosms of these environments were established using excised biofilms from two separate drain biofilm samples to inoculate constant-depth film fermentors (CDFFs). Drain biofilms harbored 9.8 to 11.3 log(10) cells of viable enteric species and pseudomonads/g, while CDFF-grown biofilms harbored 10.6 to 11.4 log(10) cells/g. Since live-dead direct staining revealed various efficiencies of recovery by culture, samples were analyzed by DGGE, utilizing primers specific for the V2-V3 region of eubacterial 16S rDNA. These analyses showed that the major PCR amplicons from in situ material were represented in the microcosms and maintained there over extended periods. Sequencing of amplicons resolved by DGGE revealed that the biofilms were dominated by a small number of genera, which were also isolated by culture. One drain sample harbored the protozoan Colpoda maupasi, together with rhabtidid nematodes and bdelloid rotifers. The microcosm enables the maintenance of stable drain-type bacterial communities and represents a useful tool for the modeling of this ecosystem.