N-Carboxybutyl chitosan, a modified chitin of crustacean origin, displayed inhibitory, bactericidal, and candidacidal activities when tested against 298 cultures of various pathogens. Examination by electron microscopy showed that microbial cells exposed to N-carboxybutyl chitosan underwent marked morphological alterations. The data are of importance in defining the suitability of N-carboxybutyl chitosan as a wound dressing.A number of polysaccharides, mainly cross-linked dextran, calcium alginate, carboxymethyl starch, modified agar, and carboxymethyl cellulose, are used in wound treatment (13,17). These polysaccharides, however, do not seem to combine favorable physical forms with functional properties; rather, besides the hemostatic action of oxycellulose, no functional property has been reported (17). The same is true for gelatin and collagen (15). On the other hand, chitin derivatives are not in widespread use as wound dressings, and relevant studies on wound healing have been confined to a few recent articles (1, 11). Interestingly, some antibacterial and antifungal activities have been described with chitosan and modified chitosans (5,8,9,18).N-Carboxybutyl chitosan is more versatile than any other polysaccharide currently used in wound management and lends itself to the manufacture of wound dressings with peculiar characteristics. Such dressings, in particular, can stimulate ordered regeneration (3) and vascularization (10) of tissue and allow gaseous exchange and turn into a gel form when they are in contact with body fluids (14), thus permitting dressing removal without disturbing the newly formed tissues. We undertook the present study to define the antimicrobial properties of N-carboxybutyl chitosan and to test it in different physical forms in view of its use in wound management. N-Carboxybutyl chitosan, which was prepared from crustacean chitosan (degree of deacetylation, 0.73) according to a proprietary procedure (R. Muzzarelli, U.S. patent 4,835,265, June 1986) and as described previously (12, 14), was tested against a variety of gram-positive and gramnegative pathogens and Candida spp. A total of 298 strains were used, all of which were freshly isolated from clinical material and identified according to conventional laboratory criteria.Two different methods were used to assess the antimicrobial activity of N-carboxybutyl chitosan. The first method was a quantitative assay based on conventional agar dilution tests (20), with final concentrations being 2, 4, 6, 8, and 9 mg/ml (concentrations above 9 mg/ml did not permit full solubilization of N-carboxybutyl chitosan into the test medium). The inoculum suspensions were prepared from fresh broth cultures and adjusted to obtain a concentration of approximately 107 CFU/ml. Test plates were inoculated by using an automatic replicating device (Titertek; Flow Labo-* Corresponding author. ratories, Inc., McLean, Va.) that delivered 1 1.l of bacterial suspension per spot. A control plate with no N-carboxybutyl chitosan was inoculated first, and a second co...

Green and roasted coffees of the two most used species, Coffea arabica and Coffea robusta, several commercial coffee samples, and known coffee components were analyzed for their ability to interfere with Streptococcus mutans' sucrose-independent adsorption to saliva-coated hydroxyapatite (HA) beads. All coffee solutions showed high antiadhesive properties. The inhibition of S. mutans' adsorption to HA beads was observed both when coffee was present in the adsorption mixture and when it was used to pretreat the beads, suggesting that coffee active molecules may adsorb to a host surface, preventing the tooth receptor from interacting with any bacterial adhesions. Among the known tested coffee components, trigonelline and nicotinic and chlorogenic acids have been shown to be very active. Dialysis separation of roasted coffee components also showed that a coffee component fraction with 1000 Da < MW < 3500 Da, commonly considered as low MW coffee melanoidins, may sensibly contribute to the roasted coffee's antiadhesive properties. The obtained results showed that all coffee solutions have antiadhesive properties, which are due to both naturally occurring and roasting-induced molecules.

Culturable vibrios were isolated from water and plankton fractions collected during an 18-month sampling study performed along the north-central coast of the Adriatic Sea (Italy). Unculturable Vibrio vulnificus and V. parahaemolyticus were detected in plankton fractions by polymerase chain reaction amplification of DNA sequences for cytotoxin-haemolysin and thermolabile haemolysin respectively. The presence of V. parahaemolyticus, V. vulnificus and V. cholerae virulence genes and the expression of pathogenicity-associated traits were analysed in all isolates. The results showed the spreading of these properties among the environmental isolates and confirm the need of both monitoring the presence of vibrios in coastal areas and studying their pathogenicity potential in order to properly protect human health.

The role of Streptococcus mutans in the initiation of dental caries has been recognized and attributed, at least in part, to its ability to colonize the tooth surface. Therefore, factors which prevent S. mutans attachment to hydroxyapatite (HA) are of considerable interest for the prophylaxis of this infectious disease. Chitosan, a chitin derivative by N-deacetylation, is an interesting candidate in this respect, since it stimulates the ordered regeneration of oral soft tissues, prevents the deleterious action of organic acid, and exhibits bactericidal action against several pathogens. In the present work, the efficacy of a low-molecular-weight chitosan (LMWC) and its derivatives N-carboxymethyl chitosan (NCMC) and imidazolyl chitosan (IMIC) in preventing S. mutans attachment to HA beads was assessed. The effects of chitosan on both sucrose-dependent and -independent adherence were evaluated. In both cases, when saliva-coated or uncoated HA beads were treated with any of the chitosans, a reduction in S. mutans adsorption ranging from 47 to 66% was observed. When HA beads were coated with saliva after the treatment with chitosan, neither carbohydrate caused a statistically significant reduction in S. mutans adsorption, suggesting that saliva deposition restores HA binding properties. Bacteria grown in the presence of chitosan subminimal inhibitory concentrations (sub-MICs) ranging from 12 to 500 micrograms mL-1 adsorbed poorly to HA and exhibited a lower affinity toward xylene than untreated controls. In the presence of chitosan sub-MICs up to 60 micrograms mL-1, an increase in the percentage of detached bacteria from two- to nine-fold was observed. The desorptive effect of chitosan was weaker when S. mutans had adhered to saliva-coated HA in the presence of sucrose. These results demonstrate that the presence of minor amounts of modified chitosans prevents S. mutans adsorption to HA and suggest that colonization of the tooth surface might be impaired by the use of toothpastes, mouthrinses, or chewing gums containing any of the tested polysaccharides.

Five chemically modified chitosans were tested for their antifungal activities against Saprolegnia parasitica by the radial growth assay in chitosan-bearing agar, and the fungal growth assay in chitosan-bearing broth. Results indicated that methylpyrrolidinone chitosan, N-carboxymethyl chitosan and N-phosphonomethyl chitosan exerted effective fungistatic action against S. parasitica: in fact the radial growth was nil for 50 h at 20 degrees C, and the fungus was precipitated when the Bacto YM broth contained one of these chitosans. Electron microscopy observations (TEM and SEM) provided evidence of ultrastructural alterations, damaged fungal structures, uptake of modified chitosans, and hyphal distortion and retraction.

The role of type 1 fimbriae in the interactions between Escherichia coli and Mytilus galloprovincialis Lam. hemocytes was evaluated. The association of fimbriated strain MG155 with hemocyte monolayers at 18°C was 1.5-and 3-to 4-fold greater than the association of unfimbriated mutant AAEC072 in artificial seawater and in hemolymph serum, respectively. Such differences were apparently due to different adhesive properties since MG155 adhered more efficiently than AAEC072 when hemocytes were incubated at 4°C to inhibit the internalization process. Hemolymph serum increased both association and adherence of MG155 two-to threefold but did not affect association and adherence of AAEC072. MG155 was also 1.5-to 1.7-fold more sensitive to killing by hemocytes than AAEC072, as evaluated by the number of culturable bacteria after 60 and 120 min of incubation. The role of type 1 fimbriae in MG155 interactions with hemocytes was confirmed by the inhibitory effect of D-mannose. In in vivo experiments MG155 cells were cleared from circulating hemolymph more rapidly than AAEC072 cells were cleared. These results confirm that surface properties are crucial in influencing bacterial persistence and survival within mussel hemolymph.

The role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied. Bacterial adherence to and association with hemocytes were evaluated at 4 and 18°C, respectively. In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA. In artificial seawater (ASW), no significant differences between the two strains were observed. N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively. The addition of D-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant. A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria. In contrast, serum adsorbed with either wild-type V. cholerae El Tor or wild-type Escherichia coli carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes. The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins.

Forty-one Tnpho A mutants of Vibrio cholerae O1 classical strain CD81 were analyzed for their ability to interact with chitin particles, Tigriopus fulvus copepods and the Intestine 407 cell line compared to the parent strain. Thirteen mutants were less adhesive than CD81; in particular, T21, T33 and T87 were less adhesive towards all substrates and insensitive to inhibition by N-acetyl glucosamine (GlcNAc). By SDS-PAGE analysis of sarkosyl-insoluble membrane proteins (siMPs) isolated from mutants and parent, it was found that a 53 kDa siMP is missing in T21, T33 and T87 mutants. It is hypothesized that this protein might have the function to mediate adherence to GlcNAc-containing substrates both in the aquatic environment and in human intestine.