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In the lungs of patients with cystic fibrosis (CF), the main pathogen Pseudomonas aeruginosa is often co-isolated with other microbes, likely engaging in inter-species interactions. In the case of chronic co-infections, this cohabitation can last for a long time and evolve over time, potentially contributing to the clinical outcome. Interactions involving the emerging pathogens Achromobacter spp. have only rarely been studied, reporting inhibition of P. aeruginosa biofilm formation. To evaluate the possible evolution of such interplay, we assessed the ability of Achromobacter spp. isolates to affect the biofilm formation of co-isolated P. aeruginosa strains during long-term chronic co-infections. We observed both competition and cohabitation. An Achromobacter sp. isolate secreted exoproducts interfering with the adhesion ability of a co-isolated P. aeruginosa strain and affected its biofilm formation. Conversely, a clonal Achromobacter sp. strain later isolated from the same patient, as well as two longitudinal strains from another patient, did not show similar competitive behavior against its P. aeruginosa co-isolates. Genetic variants supporting the higher virulence of the competitive Achromobacter sp. isolate were found in its genome. Our results confirm that both inter-species competition and cohabitation are represented during chronic co-infections in CF airways, and evolution of these interplays can happen even at the late stages of chronic infection.

Background Paranasal sinuses act as bacterial reservoirs and contribute to transmitting bacteria to the lower airway of patients with cystic fibrosis (CF). Also, passage of bacteria from the oral cavity to the lungs may occur. Methods We evaluated the presence of Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and Serratia marcescens in sputum and nasal lavage of 59 patients with CF, and also collected saliva and used toothbrushes from 38 of them. We assessed the clonal identity of the strains isolated from the different samples by pulsed-field gel electrophoresis. Results About 80% of the patients were positive for at least one of the bacterial species examined in nasal lavage and sputum. Among the subjects with positive sputum, 74% presented the same species in the nasal lavage and saliva, and 26% on their toothbrush. S. aureus was the most abundant species in all samples. Clonal identity (≥80% similarity) of the strains isolated among the different samples from each patient was confirmed in almost all cases. Longitudinal observation helped to identify five patients who were colonised in the lower airways after an initial period of nasal or oral colonisation. Conclusion Nasal and oral sites act as bacterial reservoirs, favouring the transmission of potentially pathogenic microorganisms to the lower airway. The lack of eradication from these sites might undermine the antibiotic therapy applied to treat the lung infection, allowing the persistence of the bacteria within the patient if colonisation of these sites is not assessed, and no specific therapy is performed., Nasal and oral sites act as bacterial reservoirs favouring the transmission of potentially pathogenic microorganisms to the lower airways of patients with cystic fibrosis https://bit.ly/2TMomjk

Recent findings indicate that the oral cavity acts as a bacterial reservoir and might contribute to the transmission of bacteria to the lower airways. Control of a potentially pathogenic microbiota might contribute to prevent the establishment of chronic infection in cystic fibrosis. We evaluated the presence of CF microorganisms in saliva and toothbrushes of CF patients and verify their possible transmission to lower airways. Methods: We assessed the presence of P. aeruginosa, S. aureus, S. maltophilia, A. xylosoxidans, S. marcescens, and yeasts in saliva, toothbrushes and sputum of 38 CF patients and assessed the clonal identity of the strains occurring contemporary in multiple sites by PFGE. Results: At least one of the investigated species was isolated from 60 saliva samples and 23 toothbrushes. S. aureus was the most abundant species, followed by Candida spp. 31 patients contemporary had the same species in sputum and saliva/toothbrush: in most cases, clonal identity of the strains among the different sites was confirmed. Conclusion: Toothbrushes may be sources of oral contamination and might act as reservoirs favoring transmission of potentially pathogenic microorganisms from the environment to the oral cavity and eventually to the LAW. Oral hygiene and toothbrush care are important strategies to prevent CF lung infections.

The present study investigated the compounds present in the low molecular mass fraction of Lentinus edodes mushroom (shiitake) extract and their anti-virulence activity against oral pathogens (reference and clinical Streptococcus mutans, Actinomyces naeslundii, and Prevotella intermedia strains). Oxalic, succinic, and quinic acids, and adenine, inosine, and uridine were identified by HPLC-DAD-ESI-MS/MS. Their anti-biofilm production and preformed biofilm disaggregation activities were studied using commercial standard compounds at different concentrations. As regards S. mutans, the highest activity was shown by adenine at 5 mg mL-1 both in the biofilm inhibition (BI 50%) and biofilm disaggregation tests (BD 20%). Considering A. naeslundii, BI values close to 80% were registered for oxalic acid at 1 mg mL-1 and 2 mg mL-1 and BD 50% for quinic acid at 3 mg mL-1. A weaker activity was found against P. intermedia. Furthermore, different mixtures of the commercial standards were tested showing that the activity of a compound can be strongly and sometimes negatively affected by the presence of the other compounds.

The low molecular mass (LMM) extract of Cichorium intybus var. silvestre (red chicory) has been shown to inhibit virulence-linked properties of oral pathogens including Streptococcus mutans, Actinomyces naeslundii and Prevotella intermedia. In the present study HPLC-DAD-ESI/MS(2) was used to investigate the compounds contained in this extract for their anti-virulence activity. The extract contained a number of components, including oxalic, succinic, shikimic and quinic acids, which interfere with the growth and virulence traits (i.e., biofilm formation, adherence to epithelial cells and hydroxyapatite) of oral pathogens involved in gingivitis and tooth decay. Succinic and quinic acid seem to be the most potent, mainly by interfering with the ability of oral pathogens to form biofilms (either through inhibition of their development or promotion of their disruption). Our findings suggest that one or more of these compounds may modulate plaque formation in vivo, which is a prerequisite for the development of both caries and gingivitis.

Contrary to the common assumption that food has a negative impact on oral health, research has shown that several foods contain a number of components with antibacterial and antiplaque activity. These natural compounds may be useful for improving daily oral hygiene. In this study we evaluate the mode of antimicrobial action of fractions of mushroom and red chicory extracts on Prevotella intermedia, a periodontopathogenic bacterium. The minimal inhibitory concentration corresponded to 0.5x compared to the natural food concentration for both extracts. This concentration resulted in a bacteriostatic effect in mushroom extract and in a slightly bactericidal effect in chicory extract. Cell mass continued to increase even after division stopped. As regards macromolecular synthesis, DNA was almost totally inhibited upon addition of either mushroom or chicory extract, and RNA to a lesser extent, while protein synthesis continued. Cell elongation occurred after septum inhibition as documented by scanning electron microscopy and cell measurement. The morphogenetic effects are reminiscent of the mode of action of antibiotics such as quinolones orβ-lactams. The discovery of an antibiotic-like mode of action suggests that these extracts can be advantageously employed for daily oral hygiene in formulations of cosmetic products such as mouthwashes and toothpastes.

Caries and gingivitis are the most prevalent oral infectious diseases of humans and are due to the accumulation of dental plaque (a microbial biofilm) on the tooth surface and at the gingival margin, respectively. Several in vitro and in vivo studies have shown that many natural components of foods and beverages inhibit the adhesion of and/or exert activity against oral bacteria. These biological activities have mainly been attributed to the polyphenol fraction. In order to explore the possibility that diet can alter the dental plaque community, in this study we evaluated the composition of the microbiota of supra- and subgingival plaque samples collected from 75 adult subjects with different drinking habits (drinkers of coffee, red wine, or water for at least 2 years) by analyzing the microbial population through the separation of PCR-amplified fragments using the denaturing gradient gel electrophoresis (DGGE) technique. The mean numbers of bands of the DGGE profiles from all three categories were evaluated. There were no significant differences between the two kinds of plaque collected from the control group (water drinkers), and this group showed the highest number of bands (supragingival plaque, 18.98 ± 3.16 bands; subgingival plaque, 18.7 ± 3.23 bands). The coffee and wine drinker groups generated the lowest numbers of bands for both supragingival plaque (coffee drinkers, 8.25 ± 3.53 bands; wine drinkers, 7.93 ± 2.55 bands) and subgingival plaque (coffee drinkers, 8.3 ± 3.03 bands; wine drinkers, 7.65 ± 1.68 bands). The differences between coffee drinkers or wine drinkers and the control group (water drinkers) were statistically significant. A total of 34 microorganisms were identified, and the frequency of their distribution in the three subject categories was analyzed. A greater percentage of subjects were positive for facultative aerobes when supragingival plaque was analyzed, while anaerobes were more frequent in subgingival plaque samples. It is noteworthy that the frequency of identification of anaerobes was significantly reduced when the frequencies for coffee and wine drinkers were compared with the frequencies for subjects in the control group. The DGGE profiles of the organisms in both plaque samples from all groups were generated and were used to construct dendrograms. A number of distinct clusters of organisms from water, coffee, and wine drinkers were formed. The clustering of some of the DGGE results into cohort-specific clusters implies similarities in the microbiotas within these groups and relevant differences in the microbiotas between cohorts. This supports the notion that the drinking habits of the subjects may influence the microbiota at both the supragingival and the subgingival levels.

Medically important bacteria can persist in surface waters longer than was previously thought, by activating specific survival strategies and, thus, may represent a further threat to human health, in that they are non-detectable by the traditional culture methods currently used for the evaluation of microbiological quality. Combining microbial physiology, microbial biochemistry, microbial genetics, microbial ecology and molecular biology techniques allow us to achieve more accurate detection of human pathogens located in natural environments external to the human body.

The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stresses capable of inducing cell growth inhibition and cell death. This state can be summarized as a quiescent form of life waiting for suitable conditions. This strategy has been shown to be activated by medically important bacteria either when present in natural environments or in the human body during the infection process. In this study we have evaluated the effects of antibiotics acting on peptidoglycan or protein synthesis of Enterococcus faecalis in the VBNC state. The activity of the antibiotics was determined by their ability both to inhibit resuscitation (i.e. recovery of cell division) and to bind the molecular target of action. Benzylpenicillin, piperacillin and gentamicin block cell resuscitation at the minimal inhibitory concentrations (MICs) of growing cells, while vancomycin acts only at doses 500 times higher than the MIC. This different behaviour is discussed taking into consideration the mode of action of the antibiotics.

This paper reports the content in macronutrients, free sugars, polyphenols, and inorganic ions, known to exert any positive or negative action on microbial oral disease such as caries and gingivitis, of seven food/beverages (red chicory, mushroom, raspberry, green and black tea, cranberry juice, dark beer). Tea leaves resulted the richest material in all the detected ions, anyway tea beverages resulted the richest just in fluoride. The highest content in zinc was in chicory, raspberry and mushroom. Raspberry is the richest food in strontium and boron, beer in selenium, raspberry and mushroom in copper. Beer, cranberry juice and, especially green and black tea are very rich in polyphenols, confirming these beverages as important sources of such healthy substances. The fractionation, carried out on the basis of the molecular mass (MM), of the water soluble components occurring in raspberry, chicory, and mushroom extracts (which in microbiological assays revealed the highest potential action against oral pathogens), showed that both the high and low MM fractions are active, with the low MM fractions displaying the highest potential action for all the fractionated extracts. Our findings show that more compounds that can play a different active role occur in these foods.

When exposed to stress-provoking environmental conditions such as those of ground waters, many medically important bacteria have been shown to be capable of activating a survival strategy known as the viable but non-culturable (VBNC) state. In this state bacteria are no longer culturable on conventional growth media, but the cells maintain their viability and pathogenicity genes/factors and can start dividing again, in a part of the cell population, upon restoration of favourable environmental conditions. Little is known about the genetic mechanisms underlying the VBNC state. In this study we show evidence of involvement of the rpoS gene in persistence of Escherichia coli in the VBNC state. The kinetics of entry into the non-culturable state and duration of cell viability were measured in two E. coli mutants carrying an inactivated rpoS gene and compared with those of the parents. For these experiments, laboratory microcosms consisting of an artificial oligotrophic medium incubated at 4 degrees C were used. The E. coli parental strains reached the non-culturable state in 33 days when the plate counts were evaluated on Luria-Bertani agar containing sodium pyruvate, whereas cells of the rpoS mutants lost their culturability in only 21 days. Upon reaching unculturability the parents yielded respiring cells and cells with intact membranes for at least the next three weeks and resuscitation was allowed during this time. In contrast, the RpoS- mutant cells demonstrated intact membranes for only two weeks and a very restricted (

The aim of this study was to analyse the chemical composition of peptidoglycan and the state of some of the enzymes involved in its metabolism in Escherichia coli KN126 in the viable but nonculturable (VBNC) state which is a survival strategy adopted by bacteria (including those of medical interest) when exposed to environmental stresses. When entering the VBNC state, E. coli cells miniaturised and became coccus-shaped. Analysis of peptidoglycan chemical composition, by separation in HPLC of muropeptides released by muramidase digestion of purified peptidoglycan, indicated a high degree of cross-linking, a threefold increase in unusual DAP-DAP cross-linking, an increase in muropeptides bearing covalently bound lipoprotein, and a shortening of the average length of glycan strands in comparison with dividing cells. Analysis of penicillin-binding proteins (PBPs), enzymes involved in the terminal stage of peptidoglycan assembly showed the disappearance of high-molecular-weight PBPs 1A, 1B, 2, and 3 in VBNC cells. Finally, VBNC cells displayed an autolytic capability which was far higher than that of exponentially growing cells. It is suggested that part of these alterations of peptidoglycan may be connected with the VBNC state.

BackgroundIn previous works we have shown that a low-molecular-mass (LMM) fraction from mushroom (Lentinus edodes) homogenate interferes with binding of Streptococcus mutans to hydroxyapatite and Prevotella intermedia to gingival cells. Additionally, inhibition of biofilm formation of both odonto- and periodonto-pathogenic bacteria and detachment from preformed biofilms have been described for this compound. Further purification of mushroom extract has been recently achieved and a sub-fraction (i.e. # 5) has been identified as containing the majority of the mentioned biological activities. The aim of this study was to characterise the bacterial receptors for the purified mushroom sub-fraction #5 in order to better elucidate the mode of action of this compound when interfering with bacterial adhesion to host surfaces or with bacteria-bacteria interactions in the biofilm state.MethodsCandidate bacterial molecules to act as target of this compound were bacterial surface molecules involved in cell adhesion and biofilm formation, and, thus, we have considered cell wall associated proteins (CWPs), teichoic acid (TA) and lipoteichoic acid (LTA) of S. mutans, and outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of P. intermedia.ResultsFifteen S. mutans CWPs and TA were capable of binding sub-fraction #5, while LTA did not. As far as P. intermedia is concerned, we show that five OMPs interact with sub-fraction # 5. Capacity of binding to P. intermedia LPS was also studied but in this case negative results were obtained.ConclusionsBinding sub-fraction # 5 to surface molecules of S. mutans or P. intermedia may result in inactivation of their physiological functions. As a whole, these results indicate, at molecular level, the bacterial surface alterations affecting adhesion and biofim formation. For these antimicrobial properties, the compound may find use in daily oral hygiene.

Penicillin-binding proteins 5 (PBP5s) of enterococci are structurally and immunologically related proteins that are characterized by their low affinity for penicillin. For this reason, they are mainly involved in penicillin resistance, due essentially to their ability to take over the function of all other PBPs already bound and inhibited by the beta-lactam. It has been demonstrated that penicillin resistance in enterococci is acquired either by overproduction of PBP5 or by the presence of specific amino acid sequences in the protein that further decrease the affinity for penicillin. In particular, a specific amino acid box (ANNGA) previously identified in Enterococcus faecium is responsible for the high penicillin resistance displayed by this species. Here, we describe the insertion of the PBP5 amino acid box ANNGA in Enterococcus faecalis, an enterococcal species usually more sensitive to penicillin, by site-directed mutagenesis. Mutagenized PBP5 was re-introduced into a pbp5 mutant of E. faecalis obtained by insertion of transposon Tn916. Data indicate that this amino acid box brings about no reduction in penicillin sensitivity in the recipient E. faecalis strain, but, paradoxically, dramatically lowers the penicillin minimal inhibitory concentration caused by the native PBP5. We deduce that, although enterococcal PBP5s are a family of closely related proteins as far as biological function is concerned, differences exist in their three-dimensional structure that affect penicillin affinity.

Among the survival strategies developed by bacteria when faced with adverse environmental conditions, the viable but nonculturable (VNC) state has been described. In this state, bacteria are unable to form colonies but are still alive and capable of metabolic activity. The VNC state has been described in numerous Gram-negative species, but recently also in Enterococcus faecalis, a Gram-positive species which can be found in the environment. In this study we describe a competitive PCR (cPCR) protocol to detect and quantify a specific sequence of DNA from culturable and nonculturable E. faecalis cells present in water samples. The protocol was found to be specific and capable of detecting amounts of DNA up to 0.1 pg corresponding to approximately 2 cells ml(-1). Moreover, it allows an internal standard to be used to quantify the amount of specific DNA present in samples from different environments. The application of this cPCR method to water samples from Lake Garda enabled us to demonstrate the presence of nonculturable forms of E. faecalis in lake water and to quantify their DNA and the corresponding concentration of nonculturable cells.

Bacterial rod morphogenesis was studied in synchronously growing cells of Escherichia coli C600 during the reshaping process that follows the removal of mecillinam, a beta-lactam antibiotic that specifically inhibits lateral wall formation of gram-negative rods and causes transition to coccal shape. Removal of mecillinam after 30 min of action did not affect the timing of subsequent cell division, but removal after 50 min delayed resumption of cell division for approximately one generation time. In order to study the interplay between lateral wall elongation and septum formation in determining and maintaining the bacterial rod shape, we evaluated the effect of re-adding mecillinam or of adding aztreonam (a specific inhibitor of septum formation) at various stages of the reshaping process. We conclude that mecillinam was active only during the reshaping process, while aztreonam was active only later when the cells were close to dividing again. These results provide further evidence for our previous proposal according to which elongation and septation are two alternating and competing events of the cell cycle and are linked to each other to force bacterial rods to grow to a given length.

Dental caries is an infectious disease which results from the acidic demineralisation of the tooth enamel and dentine as a consequence of the dental plaque (a microbial biofilm) accumulation. Research showed that several foods contain some components with antibacterial and antiplaque activity. Previous studies indicated antimicrobial and antiplaque activities in a low-molecular-mass (LMM) fraction of extracts from either an edible mushroom (Lentinus edodes) or from Italian red chicory (Cichorium intybus). MethodsWe have evaluated the antimicrobial mode of action of these fractions on Streptococcus mutans, the etiological agent of human dental caries. The effects on shape, macromolecular syntheses and cell proteome were analysed. ResultsThe best antimicrobial activity has been displayed by the LMM mushroom extract with a bacteriostatic effect. At the MIC of both extracts DNA synthesis was the main macromolecular synthesis inhibited, RNA synthesis was less inhibited than that of DNA and protein synthesis was inhibited only by roughly 50%. The partial inhibition of protein synthesis is compatible with the observed significant increase in cell mass. The increase in these parameters is linked to the morphological alteration with transition from cocci of the untreated control to elongated cells. Interestingly, these modifications were also observed at sub-MIC concentrations. Finally, membrane and cytosol proteome analysis was conducted under LMM mushroom extract treatment in comparison with untreated S. mutans cells. Significant changes were observed for 31 membrane proteins and 20 of the cytosol fractions. The possible role of the changed proteins is discussed. ConclusionsThis report has shown an antibiotic-like mode of action of mushroom and chicory extracts as demonstrated by induced morphogenetic effects and inhibition of specific macromolecular synthesis. This feature as well as the safe use of this extract as result of its natural origin render the LMM both mushroom and chicory extracts suitable for the formulation into products for daily oral hygiene such as mouthwashes or toothpastes.

Peptidoglycan synthesis and its fine chemical composition were studied in dividing cocci of Escherichia coli carrying the lov-1 mutation and in which the coccal shape was obtained either by mecillinam treatment or by transferring a pbpA mutation (penicillin-binding protein 2- phenotype), as compared to normal rods and non-dividing cocci. Synchronously dividing cocci showed peptidoglycan synthesis only in the cell cycle phase corresponding to cell septation. During the phase corresponding to lateral wall elongation, peptidoglycan synthesis was strongly reduced. This type of synthesis suggests that the dividing cocci consisted only of the two poles. Analysis of the muropeptide composition revealed a specific fourfold increase in the tetra-tetra-tetra trimer in dividing cocci as compared to non-dividing cocci or parental rods. We postulate that, in E. coli, the chemical composition of septal peptidoglycan differs from that of lateral wall peptidoglycan.

The main objective was to investigate whether low-molecular-weight fraction of edible mushroom shiitake extract (Lentinus edodes) possesses caries-preventive properties. The study was designed as a double-blind, three-leg, cross-over, randomized, controlled clinical trial carried out on two series of volunteers at the University of Gothenburg, and the Academic Centre for Dentistry Amsterdam. Volunteers rinsed twice daily with a solution containing low-molecular-weight fraction of edible mushroom, placebo (negative control without active ingredients), or Meridol (positive control, AmF-SnF2) for two weeks, with a two-week washout period between each rinsing period. Changes in the acidogenicity of dental plaque before and after a sucrose challenge, shifts in microbial composition, and plaque scores were determined. Frequent rinses with shiitake reduced the metabolic activity of dental plaque. No reduction of plaque scores and no inhibition of the production of organic acids in plaque was found. Minor differences in microbial composition between test sessions were found. To conclude, the results indicate that shiitake extract has anticariogenic potential, but not to the same extent as the positive control.

Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in beta-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in beta-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not strictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.

Fecal indicator bacteria, including total and fecal coliforms, Escherichia coli, and enterococci have been used to indicate the presence of fecal contamination in water used for drinking, shellfishing, and recreation. The assumptions behind the concept of using fecal indicator bacteria (total coliforms, fecal coliforms, E. coli, and enterococci) are that these organisms are normal inhabitants of the gastrointestinal tracts of humans and animals and are incapable of long-term survival or replication outside of their hosts. These assumptions must be valid if FIB levels in water are used to ensure the microbial safety in water and to identify “fecally contaminated” areas, which is essential to microbial source tracking (MST) studies. However, after utilization of total coliforms in the 1920s and fecal coliforms in the 1980s, studies showed that these groups of indicator bacteria failed to meet these assumptions because they include strains that are naturally occurring in nonfecal sources (i.e., soil, vegetation, and water) and are capable of persisting and regrowing in the environment. In the 1980s, E. coli and enterococci were recommended for use as FIB because they were believed to be more specific to fecal waste. Recent studies have shown that these bacteria also failed to meet the criteria for FIB.

Low molecular mass (LMM) fractions obtained from extracts of raspberry, red chicory, and Shiitake mushrooms have been shown to be an useful source of specific antibacterial, antiadhesion/coaggregation, and antibiofilm agent(s) that might be used for protection towards caries and gingivitis. In this paper, the effects of such LMM fractions on human gingival KB cells exposed to the periodontal pathogensPrevotella intermediaandActinomyces naeslundiiwere evaluated. Expression of cytokeratin 18 (CK18) andβ4 integrin (β4INT) genes, that are involved in cell proliferation/differentiation and adhesion, and of the antimicrobial peptideβ2 defensin (HβD2) in KB cells was increased upon exposure to either live or heat-killed bacteria. All LMM fractions tested prevented or reduced the induction of gene expression byP. intermediaandA. naeslundiidepending on the experimental conditions. Overall, the results suggested that LMM fractions could modulate the effects of bacteria associated with periodontal disease in gingival cells.

Gingivitis is a preventable disease characterised by inflammation of the gums due to the buildup of a microbial biofilm at the gingival margin. It is implicated as a precursor to periodontitis, a much more serious problem which includes associated bone loss. Unfortunately, due to poor oral hygiene among the general population, gingivitis is prevalent and results in high treatment costs. Consequently, the option of treating gingivitis using functional foods, which promote oral health, is an attractive one. Medicinal mushrooms, including shiitake, have long been known for their immune system boosting as well as antimicrobial effects; however, they have not been employed in the treatment of oral disease. In the current study, the effectiveness of shiitake mushroom extract was compared to that of the active component in the leading gingivitis mouthwash, containing chlorhexidine, in an artificial mouth model (constant depth film fermenter). The total bacterial numbers as well as numbers of eight key taxa in the oral community were investigated over time using multiplex qPCR. The results indicated that shiitake mushroom extract lowered the numbers of some pathogenic taxa without affecting the taxa associated with health, unlike chlorhexidine which has a limited effect on all taxa.

The aim of the current study was to investigate the anticariogenic potential of the (sub)fractions obtained from the edible mushroom shiitake (Lentinula edodes) inin vitrocaries model. We used a modified constant depth film fermentor (CDFF) with pooled saliva as the inoculum and bovine dentin as a substratum. The test compounds were low molecular weight fraction (MLMW) of the shiitake extract and subfractions 4 and 5 (SF4 and SF5) of this fraction. Chlorhexidine (CHX) and water served as a positive and a negative control, respectively. Dentin mineral loss was quantified (TMR), microbial shifts within the microcosms were determined (qPCR), and the acidogenicity of the microcosms was assessed (CIA). From the compounds tested, the SF4 of shiitake showed strong inhibiting effect on dentin demineralization and induced microbial shifts that could be associated with oral health. The acid producing potential was increased, suggesting uncoupling of the glycolysis of the microbiota by the exposure to SF4. In conclusion, the results suggest that SF4 of shiitake has an anticariogenic potential.

Red wine is a widely consumed beverage with multiple beneficial effects on human health. In the present paper, the anticaries properties of red wine were studied in vitro and ex vivo. Our in vitro findings shows that dealcoholised red wine, besides exerting antibacterial activity, strongly interferes with Streptococcus mutans adhesion to saliva-coated hydroxyapatite (sHA) beads, promotes its detachment from sHA, and powerfully inhibits in vitro biofilm formation. The main components responsible for such activities were found to be proanthocyanidins. The ability of red wine to inhibit ex vivo S. mutans biofilm formation on the occlusal surface of natural human teeth also was demonstrated. Our data indicates that protection of the oral cavity from the cariogenic action of S. mutans may be another beneficial effect of the moderate consumption of red wine.

The classicGram-positive bacteria used as fecal contamination indicators are enterococci (fecal streptococci) and clostridia. However, many other Gram-positive bacteria of human origin or potentially human pathogens, such as staphylococci, listeriae, mycobacteria, bifidobacteria, may be present in seawater. The route for human infection by these microorganisms may be either by contactwithwater (Mycobacteriummarinum,Mycobacteriumbalnei, Staphylococcus aureus) or by ingestion of contaminatedwater or seafood (classicwater-borne diseases). Thus, a correct evaluation of the microbiological quality of marine water is fundamental for preventing human infection. The methods currently used to assess the microbiological quality of seawater are based only on culture, thus implying that nongrowing bacteria (unable to form colonies) are non-viable, i.e. dead. As is the case with many Gram-negative bacteria, certain survival strategies (e.g. the viable but nonculturable (VBNC) state) activated also by Gram-positive bacteria are at present under investigation. Such strategies allow the bacteria to survive and persist in a viable state even in oligotrophic stress-inducing conditions such as those present in the marine environment. In these conditions, many bacteria are shown to maintain their pathogenic potential (i.e. infectivity and transmissibility), and resuscitation (i.e. recovery of cell division) has been described upon restoration of suitable environmental conditions that coincides, for example, with ingestion by humans. As these bacterial forms may no longer be recoverable by culture, one could argue that culture methods alone would not be considered enough to protect human health. Thus, it could be postulated that culture-independent methods would be preferable. In this chapter, we describe the current knowledge regarding the presence and methods of detection of medically important Gram-positive bacteria in the marine environment, but, above all, we would emphasize the modern concepts of the survival strategies activated by those microorganisms (especially enterococci) for which such research is already in progress. A knowledge of the strategies activated by these bacteria will allow us to better understand bacterial viability and survival strategies with a view to find suitable molecular target(s) for the identification of bacteria that do not divide but are still viable, i.e. still potentially pathogenic.

Several human pathogens and fecal-pollution indicators may persist as viable organisms in natural environments, owing to their ability to activate different types of survival strategies. These strategies include adhesion on both abiotic and biotic surfaces and the entrance to the so-called viable but nonculturable (VBNC) state. In an 18-month survey for the detection of enterococci in both lake water and seawater, C. Signoretto et al. (Appl. Environ. Microbiol. 70:6892-6896, 2004) have shown that Enterococcus faecalis was detected mostly bound to plankton and in the VBNC state. In the present study, we show that in vitro adhesion of E. faecalis to copepods accelerated the entry of cells into the VBNC state relative to that of planktonic bacteria. VBNC E. faecalis cells maintained their adhesive properties to copepods and chitin (the main component of the copepod carapace), though to a reduced extent in comparison with growing cells. Sugar competition experiments showed interference with adhesion to both copepods and chitin by GlcNAc and only to copepods by d -mannose. Four enterococcal cell wall proteins present in both growing and VBNC cells and lipoteichoic acid were shown to be capable of binding chitin. The results indicate that copepods may represent an additional environmental reservoir of enterococci, thus suggesting the advisability of redesigning the protocols currently used for microbial detection during the evaluation of the microbiological quality of environmental samples.

Aims: The current standard culture methods are unable to detect nongrowing bacteria and, thus, might not be sufficient for precise monitoring of the microbiological quality of waters. The use of a molecular method such as PCR could be a valid alternative to detect bacterial faecal contamination indicators such as Escherichia coli and Enterococcus faecalis and reveal the presence of culturable and nonculturable bacterial forms. Methods and Results: The presence of E. coli and Ent. faecalis cells in 30 groundwater samples was evaluated with the standard culture method and compared with a specific PCR protocol. A substantial percentage (50%) of the samples not containing culturable cells proved positive in the search for Ent. faecalis DNA by PCR. Quantification by competitive PCR (cPCR) of the DNA detected allowed us to calculate the number of nonculturable cells present in water samples: the number varied from 2 to 120 cells ml−1. Only four samples were positive for E. coli DNA and the corresponding nonculturable cells varied from 24 to 70 ml−1. Conclusions: This study demonstrates that the standard culture methods in use are unable to detect a substantial proportion of the bacterial population which is nonculturable but, as previously demonstrated, potentially still viable and able to express those pathogenic factors needed for causing infections in humans. Significance and Impact of the Study: To protect human health it is necessary to develop and use methods which detect the nonculturable as well as culturable bacteria present in water.

The presence of enterococci in lake and seawater in an 18-month survey comparing molecular (PCR and quantitative PCR) and culture methods was evaluated, as well as the possibility that zooplankton could act as reservoirs for enterococci. Samples of both water and zooplankton were collected monthly from a Lake Garda site and an Adriatic Sea site. In lake water, the positive samples numbered 13 of 54 (24%) by culture and 32 of 54 (59%) when PCR was applied. In seawater, they numbered 0 of 51 by culture and 18 of 51 (35%) by PCR. Enterococci were found either totally bound to plankton or totally in water, depending on the presence or absence of plankton, respectively. These results clearly indicate that the PCR assay is a powerful tool for detecting fecal indicators and pathogens in the environment, thus providing a much more sensitive method than culture.

The effect of exposure to artificial sea water (ASW) on the ability of classical Vibrio cholerae O1 cells to interact with chitin-containing substrates and human intestinal cells was studied. Incubation of vibrios in ASW at 5 degrees C and 18 degrees C resulted in two kinds of cell responses: the viable but non-culturable (VBNC) state (i.e.

Stressed vancomycin-resistant enterococci (VRE) can activate a survival strategy known as the viable but nonculturable (VBNC) state and are able to maintain vancomycin resistance. During restoration of division they continue to express the vancomycin resistance trait. We suggest that VBNC enterococci may constitute further reservoirs of VRE and therefore represent an additional risk for human health.

Aims: The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. Methods and Results: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1 : 10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). Conclusions: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. Significance and Impact of the Study: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.

The ability of viable but nonculturable (VBNC) Enterococcus faecalis to adhere to Caco-2 and Girardi heart cultured cells and to urinary tract epithelial cells (ECs) was studied. Enterococci were harvested during the vegetative growth phase (early exponential and stationary), in the VBNC state, and after recovery of the ability to divide. VBNC bacteria maintained their adherence capability but the efficiency of attachment was reduced by about 50 to 70%, depending on the target cell employed. The decrease was transient, since enterococci that regained their culturability showed adherence values similar to those observed for actively growing cells. Analysis of the invasive properties of E. faecalis revealed that the VBNC state caused a decrease in the number of bacteria that entered the cultured HEK cells as a result of the reduction in the number of adhering bacteria. These results highlight the importance of studies of the VBNC phenomenon, with respect to both microbial survival in the environment and the impact on human health.

The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.

The viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria when they are exposed to hostile environmental conditions. It has been shown that VBNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve pathogenic factors and/or genes. It is thus necessary to develop methods capable of detecting nonculturable bacteria and of establishing their viability when the microbiological quality of environments is monitored. In this study we demonstrated that a gene was expressed during the VBNC state in a low-nutrient-concentration microcosm through detection of Enterococcus faecalis pbp5 mRNA by reverse transcription-PCR over a 3-month period. The presence of mRNA correlated with metabolic activity and resuscitation capability, indicating the viability of the VBNC cells.

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in beta-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of beta-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function.

Recently, in Escherichia coli was cloned a Sau3AI 3.4-kb fragment containing the gene encoding for penicillin-binding protein 5 (PBP5) of Enterococcus faecalis. The structural gene for the PBP of E. faecalis and the flanking regions were entirely sequenced (C. Signoretto, M. Boaretti, and P. Canepari, FEMS Microbiol. Lett. 123:99-106, 1994). When the entire cloned E. faecalis DNA insert, labeled with digoxigenin, was used as a probe to detect a homology gene in enterococci, it was observed that only DNAs of all the E. faecalis strains reacted to the probe. The same results were obtained when a HindIII fragment of 0.35 kb from the entire insert of 3.4 kb was used. In this study we tested a total of 62 clinically isolated enterococcal strains, belonging to the species E. faecalis (36 strains), E. faecium (13), E. gallinarum (6), E. bovis (2) E. avium (3), E. hirae (1), and E. casseliflavus (1). The results indicate that both the entire segment and the HindIII fragment may be useful for preparing a species-specific probe for rapid identification of E. faecalis species.