Pietro Canepari

Publications of Pietro Canepari

• Functional foods and strategies contrasting bacterial adhesion.

  Authors: Caterina Signoretto, Pietro Canepari, Monica Stauder, Luigi Vezzulli, Carla Pruzzo

  Current opinion in biotechnology. 09/2011;

  Antibacterial strategies targeting bacterial adhesion to substrates are considered a valuable alternative to traditional antibiotic therapy, in view of the great advantage they bring in combating theAntibacterial strategies targeting bacterial adhesion to substrates are considered a valuable alternative to traditional antibiotic therapy, in view of the great advantage they bring in combating the infectious process at the very early stage without selecting for drug resistant cells. Amongst bioactive compounds with activity against bacterial adhesion, several are found in natural food and beverages, such as cranberry, tea, coffee, wine and milk. For the analysis of their anti-infective potential, successful experimental models can be conducted using different substrates from the oral cavity. Studies conducted so far in this field allowed the discovery of a variety of anti-adhesive fractions and compounds proven to be effective against bacterial traits involved in the development of oral pathologies such as caries and gingivitis/periodontitis. Discovering new anti-adhesive compounds from natural products, unravelling and testing their prophylactic and therapeutic values, and improving their use in the general population are promising new frontiers in the global fight against human infectious diseases.

• Testing a low molecular mass fraction of a mushroom (Lentinus edodes) extract formulated as an oral rinse in a cohort of volunteers.

  Authors: Caterina Signoretto, Gloria Burlacchini, Anna Marchi, Marcello Grillenzoni, Giacomo Cavalleri, Lena Ciric, Peter Lingström, Elisabetta Pezzati, Maria Daglia, Egija Zaura, Jonathan Pratten, David A Spratt, Michael Wilson, Pietro Canepari

  Journal of biomedicine & biotechnology. 01/2011; 2011:857987.

  Although foods are considered enhancing factors for dental caries and periodontitis, laboratory researches indicate that several foods and beverages contain components endowed with antimicrobial andAlthough foods are considered enhancing factors for dental caries and periodontitis, laboratory researches indicate that several foods and beverages contain components endowed with antimicrobial and antiplaque activities. A low molecular mass (LMM) fraction of an aqueous mushroom extract has been found to exert these activities in in vitro experiments against potential oral pathogens. We therefore conducted a clinical trial in which we tested an LMM fraction of shiitake mushroom extract formulated in a mouthrinse in 30 young volunteers, comparing the results with those obtained in two identical cohorts, one of which received water (placebo) and the other Listerine. Plaque index, gingival index and bacterial counts in plaque samples were determined in all volunteers over the 11 days of the clinical trial. Statistically significant differences (P < 0.05) were obtained for the plaque index on day 12 in subjects treated with mushroom versus placebo, while for the gingival index significant differences were found for both mushroom versus placebo and mushroom versus Listerine. Decreases in total bacterial counts and in counts of specific oral pathogens were observed for both mushroom extract and Listerine in comparison with placebo. The data suggest that a mushroom extract may prove beneficial in controlling dental caries and/or gingivitis/periodontitis.

• Effects of mushroom and chicory extracts on the physiology and shape of Prevotella intermedia, a periodontopathogenic bacterium.

  Authors: Caterina Signoretto, Anna Marchi, Anna Bertoncelli, Gloria Burlacchini, Francesco Tessarolo, Iole Caola, Elisabetta Pezzati, Egija Zaura, Adele Papetti, Peter Lingström, Jonathan Pratten, David A Spratt, Michael Wilson, Pietro Canepari

  Journal of biomedicine & biotechnology. 01/2011; 2011:635348.

  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.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 or β-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.

• Drinking habits are associated with changes in the dental plaque microbial community.

  Authors: Caterina Signoretto, Franco Bianchi, Gloria Burlacchini, Francesca Sivieri, Dave Spratt, Pietro Canepari

  Journal of clinical microbiology. 12/2009;

  Caries and gingivitis are the most prevalent infectious diseases of humans and are due to the accumulation of dental plaque (a microbial biofilm) on tooth surface and at the gingival margin,Caries and gingivitis are the most prevalent infectious diseases of humans and are due to the accumulation of dental plaque (a microbial biofilm) on 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 an antimicrobial activity against oral bacteria. These biological activities have been attributed mainly 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 collected from 75 adult subjects with different drinking habits (drinkers of coffee, or red wine or water for two years at least) by analysing the microbial population through separation of PCR-amplified fragments using the denaturing gradient gel electrophoresis (DGGE) technique. The mean numbers of the bands of the DGGE profiles from all the three categories was evaluated. There were no significant differences between the two kinds of plaque collected from the control (water) group and this group showed the highest number of bands (supragingival = 18.98+/-3.16 and subgingival = 18.7+/-3.23). The coffee and wine drinker groups generated the lowest numbers of bands for both supra- (coffee = 8.25+/-3.53 and wine = 7.93+/-2.55) and sub-gingival (coffee = 8.3+/-3.03 and wine = 7.65+/-1.68) plaque. The differences between coffee drinkers or wine drinkers vs. the control (water) group, respectively, were statistically significant. A total of 34 microorganisms were identified and their frequency of distribution into the three subject categories was analysed. A greater percentage of subjects were positive for facultative aerobes when supragingival plaque was analysed while anaerobes were more frequent in subgingival samples. Relevantly, the frequency of anaerobe identification was significantly reduced when coffee and wine drinkers were compared with the subjects of the control group. DGGE profiles of both plaques samples from all groups were generated and used to construct dendrograms. A number of distinct clusters of water or coffee or wine drinkers were formed. The clustering of the some of DGGE results into cohort specific clusters implies similarities in the microbiota within these groups and relevant differences in the microbiota between cohorts. This supports the notion that the drinking habits of the subjects may influence the microbiota at both a supraginigival and subgingival level.

• Support for the role of Candida spp. in extensive caries lesions of children.

  Authors: Caterina Signoretto, Gloria Burlacchini, Fiorenzo Faccioni, Massimiliano Zanderigo, Nicolò Bozzola, Pietro Canepari

  The new microbiologica : official journal of the Italian Society for Medical, Odontoiatric, and Clinical Microbiology (SIMMOC). 02/2009; 32(1):101-7.

  Candida spp. are frequently detected in the mouths of children with extensive caries lesions compared with caries-free subjects. In this study we evaluated the presence of Candida spp. in associationCandida spp. are frequently detected in the mouths of children with extensive caries lesions compared with caries-free subjects. In this study we evaluated the presence of Candida spp. in association with mutans streptococci and lactobacilli in the saliva of children with dental decay, before and after anti-caries treatment. Samples of saliva from 14 children with caries lesions and from 13 caries-free subjects were evaluated for the presence of mutans streptococci, lactobacilli and Candida spp. by culture. Eleven of 14 carious subjects hosted Candida spp. in their saliva as against only 2 out of 13 subjects without caries lesions. Carious subjects were treated by adopting a conventional protocol for caries disease (rinses with a mouthwash containing 0.2% chlorhexidine and fluorine). After treatment, the salivary bacterial counts decreased for mutans streptococci and in some cases for lactobacilli, but large numbers of Candida spp. remained in the saliva of several children. The latter were treated with the antifungal drug nystatin (oral rinses) and evaluation of the level of yeasts in the saliva showed disappearance of the microorganism in several cases. The results indicate that antiseptic treatment alone for dental decay is not sufficient for the eradication of microorganisms potentially responsible for caries lesions, in particular when yeasts are present. We hypothesize that the oral cavity of children could act as a reservoir of fungi, and eradication could be needed to prevent both exacerbation of caries lesions, and colonization by Candida spp. of other host sites.

• Towards more accurate detection of pathogenic Gram-positive bacteria in waters.

  Authors: Caterina Signoretto, Pietro Canepari

  Current opinion in biotechnology. 07/2008; 19(3):248-53.

  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, inMedically 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.

• Human pathogens in water: insights into their biology and detection.

  Authors: Pietro Canepari, Carla Pruzzo

  Current opinion in biotechnology. 07/2008; 19(3):241-3.

• Microbiological evaluation of the effects of hyperbaric oxygen on periodontal disease.

  Authors: Caterina Signoretto, Franco Bianchi, Gloria Burlacchini, Pietro Canepari

  The new microbiologica : official journal of the Italian Society for Medical, Odontoiatric, and Clinical Microbiology (SIMMOC). 11/2007; 30(4):431-7.

  The term periodontitis indicates a variety of clinical manifestations of infectious disorders in which the supporting tissues of the teeth are attacked. The initiation and progression of periodontalThe term periodontitis indicates a variety of clinical manifestations of infectious disorders in which the supporting tissues of the teeth are attacked. The initiation and progression of periodontal disease are attributed to the presence of elevated levels of pathogenic bacteria within the gingival crevice. Approaches to periodontal treatment range from surgical to regenerative therapy and anti-infective chemotherapy. Anti-infective drug therapy should be rationally based on the composition of the pathogenic microbiota. It is also important to recognize that the periodontopathic plaque constitutes a bacterial biofilm infection that may render the resident microorganisms more resistant than the same organisms grown planktonically. Hyperbaric oxygen (HBO) has been successfully used in several medical applications. The therapeutic effect is related to elevated partial oxygen pressure in the tissues. The aim of this study was to evaluate the effects of HBO on a selected number of patients suffering from adult chronic periodontitis in comparison with surgical intervention (scaling and root planning, SRP), as well as the effects of a combination of both therapies on the evolution over time of the microflora of the periodontal pockets. Bacteria were detected either by culture or by a molecular method (PCR). Microbiological data indicate that the combination of HBO and SRP substantially reduced (by up to 99.9%) the gram-negative anaerobe loads of the subgingival microflora. The low values of pathogens persisted for at least two months after the therapy. HBO or SRP alone produced a temporarily more limited effect on periodontal anaerobes. Additional experimental confirmation of these results was provided by molecular detection of the main periodontopathogenic bacteria with a significant reduction in the number of dental sites which harboured them. It is also shown that HBO both alone and in combination with SRP reduced the Gingival Index value to zero and gingival health persisted for 3 months at least. Thus, in parallel with the loss of periodontopathogenic bacteria, a substantial improvement in oral health was observed. In conclusion, this study has shown that HBO may represent a useful aid, especially in combination with SRP, as far as non-surgical periodontal therapy is concerned.

• Adhesion to medical device materials and biofilm formation capability of some species of enterococci in different physiological states.

  Authors: Mar Lleo, Barbara Bonato, Maria Carla Tafi, Greta Caburlotto, Dennis Benedetti, Pietro Canepari

  FEMS microbiology letters. 10/2007; 274(2):232-7.

  Enterococci may survive in adverse environments including the human body where bacteriocins, antibiotics, iron-limitation and immune response represent stressing conditions for bacteria that causeEnterococci may survive in adverse environments including the human body where bacteriocins, antibiotics, iron-limitation and immune response represent stressing conditions for bacteria that cause division block. In those conditions, bacteria present in the human body would hardly be in an exponentially growing phase but would mostly be in physiological states such as starvation or the viable but nonculturable (VBNC) state. The possibility that the starved and VBNC bacteria can maintain their ability to adhere to living and inanimate substrates is the first mandatory step for them potentially to cause an infection process. In this study it is shown that starved and stationary enterococcal cells are able to form biofilms on plastic material albeit with reduced efficiency as compared to growing cells. Moreover, although VBNC enterococcal forms are not capable of forming biofilms, Enterococcus faecalis and other enterococcal species of medical interest maintain their ability to synthesize the polymeric matrix for a limited period of time under adverse environmental conditions. The data presented, together with those regarding the maintenance of the division recovery potential already proved in nonculturable bacteria, further support the possibility for the VBNC and other nondividing bacterial forms to have a role as infectious agents and to constitute a risk to human health.

• Inhibition of the resuscitation from the viable but non-culturable state in Enterococcus faecalis.

  Authors: Maria del Mar Lleò, Dennis Benedetti, Maria Carla Tafi, Caterina Signoretto, Pietro Canepari

  Environmental microbiology. 10/2007; 9(9):2313-20.

  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 canThe 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.

• Differences in microbiological composition of saliva and dental plaque in subjects with different drinking habits.

  Authors: Caterina Signoretto, Gloria Burlacchini, Franco Bianchi, Giacomo Cavalleri, Pietro Canepari

  The new microbiologica : official journal of the Italian Society for Medical, Odontoiatric, and Clinical Microbiology (SIMMOC). 11/2006; 29(4):293-302.

  Several foods have been shown to contain natural components (especially polyphenols) which display anti-adhesive properties against Streptococcus mutans, the aetiological agent responsible for dentalSeveral foods have been shown to contain natural components (especially polyphenols) which display anti-adhesive properties against Streptococcus mutans, the aetiological agent responsible for dental crown caries, as well as inhibition of glucosyltransferases, which are the S. mutans enzymes involved in the synthesis of an adherent, water-insoluble glucan from sucrose. Other studies have demonstrated an in vitro action on oral plaque biofilm formation and desorption. This study evaluated whether the activity displayed in vitro by food compounds could affect the microbiological composition of saliva and dental plaque of subjects with a diet rich in these foods, comparing the results with those obtained from subjects with a different diet. The foods considered were: coffee, barley coffee, tea and wine. A total of 93 subjects were recruited into the study. Six samples of both plaque and saliva were collected from each subject at roughly one-monthly intervals. Total bacteria, total streptococci, S. mutans and lactobacilli counts were determined by culture in both saliva and dental plaque. The highest bacterial titres were recorded for the control population, while each drinking habit subgroup showed counts roughly one log lower than the controls. These differences in bacterial counts proved statistically significant (P<0.05). As far as dental plaque was concerned, while total counts did not significantly vary per mg of plaque in the subjects belonging to the different drinking habit subgroups, a significant decrease (P<0.05) was observed in those subjects drinking coffee, tea, barley coffee and wine when mutans streptococci and lactobacilli were evaluated. In several cases a more than one log decrease was observed. Plaque indices were also determined, and a significant (P<0.05) reduction in values was recorded in the subjects belonging the specific drinking habit subgroups compared to the control group. This study indicates that there is a correlation between consumption of specific foods and oral health in terms of reduced plaque deposition and lower counts of odontopathogens.

• Survival of enterococcal species in aquatic environments.

  Authors: Maria del Mar Lleò, Barbara Bonato, Dennis Benedetti, Pietro Canepari

  FEMS microbiology ecology. 11/2005; 54(2):189-96.

  Analysis of the survival ability of faecal streptococci/enterococci in the environment has almost invariably been conducted using the standard culture method (CFU counts) despite the demonstrationAnalysis of the survival ability of faecal streptococci/enterococci in the environment has almost invariably been conducted using the standard culture method (CFU counts) despite the demonstration that these microorganisms are capable of entering a viable but nonculturable (VBNC) state. In this study we evaluated the fate, in terms of culturability and viability, of different enterococcal species under laboratory stress conditions mimicking those of the aquatic environment. The results indicate that enterococcal species may activate two different survival strategies, namely starvation and the VBNC state, depending on the specific environmental condition. Moreover, the different enterococcal species can be divided into three groups on the basis of the time needed to activate the VBNC state and the resuscitation capability. The differences in activation of the two survival strategies and the different kinetics observed among the enterococcal species reaching the VBNC state should be taken into consideration when the microbiological quality of waters has to be evaluated and because of their role as faecal contamination indicators.

• Persistence of Enterococcus faecalis in aquatic environments via surface interactions with copepods.

  Authors: Caterina Signoretto, Gloria Burlacchini, Carla Pruzzo, Pietro Canepari

  Applied and environmental microbiology. 06/2005; 71(5):2756-61.

  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. TheseSeveral 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.

• Adhesion of Enterococcus faecalis in the nonculturable state to plankton is the main mechanism responsible for persistence of this bacterium in both lake and seawater.

  Authors: Caterina Signoretto, Gloria Burlacchini, Maria del Mar Lleò, Carla Pruzzo, Massimiliano Zampini, Luigi Pane, Giorgio Franzini, Pietro Canepari

  Applied and environmental microbiology. 12/2004; 70(11):6892-6.

  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 zooplanktonThe 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.

• Persistence of adhesive properties in Vibrio cholerae after long-term exposure to sea water.

  Authors: Carla Pruzzo, Renato Tarsi, Maria Mar Lleò, Caterina Signoretto, Massimiliano Zampini, Luigi Pane, Rita R Colwell, Pietro Canepari

  Environmental microbiology. 10/2003; 5(10):850-8.

  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.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. <0.1 colony forming unit ml-1) at 5 degrees C, and starvation (i.e. maintenance of culturability of the population) at 18 degrees C. The latter remained rod shaped and, after 40 days' incubation, presented a 47-58% reduction in the number of cells attached to chitin, a 48-53% reduction in the number of bacteria adhering to copepods, and a 48-54% reduction in the number of bacteria adhering to human cultured intestinal cells, compared to control cells not suspended in ASW. Bacteria suspended in ASW at 5 degrees C became coccoid and, after 40 days, showed 34-42% fewer cells attached to chitin, 52-55% fewer adhering to copep-ods, and 45-48% fewer cells adhering to intestinal cell monolayers, compared to controls. Sarkosyl-insoluble membrane proteins that bind chitin particles were isolated and analysed by SDS-PAGE. After 40 days incubation in ASW at both 5 degrees C and 18 degrees C vibrios expressed chitin-binding ligands similar to bacteria harvested in the stationary growth phase. It is concluded that as vibrios do not lose adhesive properties after long-term exposure to ASW, it is important to include methods for VBNC bacteria when testing environmental and clinical samples for purposes of public health safety.

• Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state.

  Authors: Marzia Boaretti, Maria Mar Lleò, Barbara Bonato, Caterina Signoretto, Pietro Canepari

  Environmental microbiology. 10/2003; 5(10):986-96.

  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 asWhen 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 (<7 days) resuscitation capability. Guanosine 3',5'-bispyrophosphate (ppGpp) acts as a positive regulator during the production and functioning of RpoS. A mutant deficient in ppGpp production behaved like the rpoS mutants, while overproducers of ppGpp displayed a vitality at least comparable to that of RpoS+ strains. These results suggest that the E. coli parental strains enter the VBNC state which lasts for, at least, three weeks, after which apparently all the cells die. The rpoS mutants do not activate this survival strategy and early die. This implies involvement of the rpoS gene in E. coli persistence in the VBNC state.

• Vancomycin resistance is maintained in enterococci in the viable but nonculturable state and after division is resumed.

  Authors: Maria Mar Lleò, Barbara Bonato, Caterina Signoretto, Pietro Canepari

  Antimicrobial agents and chemotherapy. 04/2003; 47(3):1154-6.

  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 restorationStressed 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.

• The viable but nonculturable state and starvation are different stress responses of Enterococcus faecalis, as determined by proteome analysis.

  Authors: Sabina Heim, Maria Mar Lleo, Barbara Bonato, Carlos A. Guzman, Pietro Canepari

  Journal of bacteriology. 01/2003; 184(23):6739-45.

  The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between theThe protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between the VBNC state and other stress responses. The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria. This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of E. faecalis, which is activated in response to multiple environmental stresses.

• In vitro adhesion to human cells by viable but nonculturable Enterococcus faecalis.

  Authors: Carla Pruzzo, Renato Tarsi, Maria Mar Lleò, Caterina Signoretto, Massimiliano Zampini, Rita R Colwell, Pietro Canepari

  Current microbiology. 09/2002; 45(2):105-10.

  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 wereThe 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.

• Modification of the peptidoglycan of Escherichia coli in the viable but nonculturable state.

  Authors: Caterina Signoretto, Maria Mar Lleò, Pietro Canepari

  Current microbiology. 03/2002; 44(2):125-31.

  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 butThe 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.