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Chronic wounds represent an important burden on the healthcare system, requiring frequent hospitalizations and expensive treatments. It is now recognized that a primary factor contributing to a non-healing trajectory and a low therapeutic response is the biofilm infection. The purpose of this study was to identify the bacterial isolates collected from chronic skin wounds of hospitalized patients and to evaluate their antimicrobial susceptibility profiles, virulence factors, as well as the ability to develop biofilms in vitro. A number of 44 wound samples were collected from 39 patients. The isolated strains belonged to seven different microbial species, i.e.: Staphylococcus aureus (32 strains), followed by Pseudomonas aeruginosa (4), Escherichia coli (3), Klebsiella pneumoniae (2), Proteus mirabilis (1), Citrobacter freundii (1), group G β-hemolytic streptococci (1). In comparison to the other isolates, P. aeruginosa strains exhibited the highest capacity to develop complex biofilm structures in vitro, followed by S. aureus, with insignificant differences between MRSA and non-methicillin resistant isolates. The Enterobacteriaceae strains expressed less virulent phenotypes, lower adherence to epithelial cells and biofilm forming capacity, but also significant resistance phenotypes with a potential of unfavorable epidemiological outcome. The isolation of MRSA, ESBL-producing microorganisms and multiple antibiotic resistant P. aeruginosa suggests the potential risk of nosocomial spread and the potential severe outcome in case of bacteremia and sepsis. This study represents an important step in elucidating the host-wound microbiome interaction, by describing various resistance and virulence threats of microorganisms colonizing and/or infecting the chronic wounds. However, in order to establish a statistical relevant correlation, larger studies are needed.

The aim of present study was to investigate the microbial colonization of worn contact lenses (CLs) and to evaluate the inhibitory effect of pomelo (Citrus maxima) peels essential oil on the biofilm development on unworn CLs. The essential oil was isolated by steam distillation and analyzed by gas chromatography coupled with mass spectrometry, twenty compounds being isolated. The antimicrobial activity of pomelo oil was tested against S. epidermidis and P. aeruginosa strains, known for their ability to develop biofilms on prosthetic devices, by qualitative screening methods and quantitative assay of the minimal inhibitory concentrations (MIC) in order to evaluate the antibiofilm activity. Our study revealed that all worn CLs where 100% colonized by staphylococci and Enterobacteriaceae strains. The pomelo essential oil inhibited the development of bacterial biofilms formed by Gram-positive and Gram-negative microorganisms on soft CLs, its antibiofilm activity being specific and dependent on different physical parameters (contact time and temperature). The architecture of bacterial biofilms developed on soft contact lenses was analyzed using confocal scanning laser microscopy (CSLM).

Recent public announcements stated that 60% to 85% of all microbial infections involve biofilms developed on natural tissues (skin, mucosa, endothelial epithelia, teeth, bones) or artificial devices (central venous, peritoneal and urinary catheters, dental materials, cardiac valves, intrauterine contraceptive devices, contact lenses, different types of implants). Prosthetic medical devices are risk factors of chronic infections in developed countries and these infections are characterized by slow onset, middle intensity symptoms, chronic evolution and resistance to antibiotic treatment. In case of biofilm development, a series of genes (40-60% of the prokaryotic genome) are modulated (activated/inhibited) by complex cell to cell signalling mechanisms and the biofilm cells become phenotypically distinct from their counterpart--free cells, being more resistant to stress conditions (including all types of antimicrobial substances); this resistance is phenotypical, behavioural and, more recently, called TOLERANCE. Four major mechanisms can account for biofilm antibiotic tolerance: (1) the failure of antibiotic penetration into the depth of a mature biofilm due to the biofilm matrix; (2) the accumulation of high levels of antibiotic degrading enzymes; (3) in the depth of biofilm, cells are experiencing nutrient limitation entering in a slow-growing or starved state; slow-growing or non-growing cells being not highly susceptible to antimicrobial agents, this phenomenon could be amplified by the presence of phenotypic variants or "persisters" and (4) biofilm's bacteria can turn on stress-response genes and switch to more tolerant phenotypes on exposure to environmental stresses; (5) genetic changes, probably selected by different stress conditions, such as mutations and gene transfer could occur inside the biofilm. In these conditions, biofilm associated infections require a different approach, both clinically and paraclinically.

The microbial biofilm, composed by a single or multiple species, is defined as a sessile community of microbial cells irreversibly attached to a substratum or an interface and also among them, embedded in a matrix of extracellular polymeric substances as their own products, exhibiting a modified phenotype concerning the rate of growth and gene transcription. The biofilm is considered a primitive form of cellular differentiation, with primitive circulatory system, homeostasis and "integrality", similar to eukaryotic tissues in their intercellular cooperation. A microbial biofilm is considered to be the most successful and competitive expression of the prokaryotic genome--biofilm cells being metabolically more efficient and well protected, exhibiting resistance to different stress factors, including host defence mechanisms and antibiotics. The ability of the bacterial cells to behave as a community is the result of a complex intra- and inter-cellular communication based on a signaling system regulated by quorum-sensing and response (QS), mechanism ubiquitous in bacteria, and implicated in the regulation of very different and complex physiological processes, depending on cellular density. The language used for this intercellular communication is based on small, self-generated signal molecules known as bacterial pheromones with different chemical structures (N-homoserine lactones and derivatives in Gram-negative, and octapeptides, amino acids respectively in Gram-positive bacteria). The signal molecules level required for a specific response is very low in comparison with those of intracellular hormones. The aim of this review is to present the development, architecture and physiology of microbial biofilms.

Regulatory constraints and environmental and human health concerns have promoted the search for alternative bio-control strategies of fire blight, a destructive disease of rosaceous plants which produces serious losses in apple and pear orchards all over the world. The aim of this study was to establish the antimicrobial activity of Citrus maxima essential oil against Erwinia amylovora. An agar diffusion method was used for the screening of the inhibitory effect of Citrus maxima essential oil on bacterial strains growth. The quantitative inhibitory effect of pomelo oil on in vitro biofilm development was established by a microtiter colorimetric assay. In order to investigate the ability of pomelo oil to interfere with bacterial adherence and subsequent biofilm development on leaves obtained from different pomaceous fruit trees species and cultivars: Pyrus (Napoca, Williams), Malus (Golden Delicious) and Cydonia (Aromate), leaves were immersed in pomelo oil for 1, 2, 3, 5 and 10 minutes before exposing them to bacterial colonization. The architecture of bacterial biofilms developed on leaf surface was analyzed using Confocal Scanning Laser Microscopy (CSLM). Our results showed that Citrus maxima essential oil inhibited the development of bacterial biofilms on leaves, pomelo oil being more active on Cydonia (Aromate) leaves when the leaves were treated for 5 minutes. The results obtained from this study may contribute to the development of new bio-control agents as alternative strategies to protect fruit trees from fire blight disease.

The vegetal extracts are used as an ecological alternative to classical anti-infectious treatments based on antibiotics, exhibiting the advantage of reduced secondary effects. Most of these compounds are secondary metabolites, especially aromatic substances synthesized by plants in a reduced concentration. The aim of this study was to investigate the influence of usnic acid against quorum sensing and response mechanisms involved in the initiation and development of the dental plaque biofilm and its tolerance to antimicrobials. Three samples of super-gingival dental plaque were treated for different time intervals with usnic acid at 200 ig/ml in dimethyl-sulfoxide, representing the MIC value. Each dental plaque sample was inoculated in Brain Heart Infusion medium to establish the microbial growing curve by viable cells counts using the tenfold microdilutions method. For strains identification there were used the microtest galleries: API 20Strep, API Staph, API 20NE, API 20E. MIC value for usnic acid was determined by twofold microdilution technique. Usnic acid selectively inhibited the biofilm development by Gram positive bacteria and the expression of haemolytic properties of strains isolated from the dental plaque. The growth curve of the isolated strains was affected by usnic acid, the changes consisting of the lag phase extension to 6-10 h (this time interval being considered as the persistence time of antimicrobial activity) and the significant decrease of the viable cell number and consecutively, the prolongation of the generation time. These effects are demonstrating the interference of the usnic acid with the intra- and interspecies signalling mechanisms based on quorum sensing and response and dependent on cell density, giving the possibility to use them as an active principle in some new pharmaceutical formula intended for the prevention and treatment of gingival and periodontal pathologies.

The aim of the present study was to investigate the dental plaque formed on natural teeth surfaces by optic and confocal laser scanning microscopy (CLSM), to quantify the microbial density by viable cell counts, to identify the recovered microbial strains, the antibiotic susceptibility testing and their pathogenicity features (i.e. adherence and invasion capacity on HeLa cells, adherence on prostetic substrata used in oral medicine by original experimental models, production of extracellular enzymes and exotoxins). results: The CLSM revealed a very complex and highly organized architecture of dental plaque. The direct optic examination of Gram-stained smears revealed a great diversity of morphological types (82.5% of cases), with comparative levels of microbial densities. 50% of recovered microbial strains exhibited ability to adhere to three different polimeric inert substrata used in oral medicine, but reduced adherence and invasion capacity of HeLa cells and scared expression of soluble enzymatic factors.

The emergence of the bacterial antibiotic multi-resistance made more and more stringent the developing of new anti-microbial strategies. The purpose of the present study was to investigate the antimicrobial potential of six (6) newly synthesized chemical compounds (derivating from phenantroline and dimethylguanin-copper complex combinations) versus 97 enterobacterial strains isolated from the hospital environment. The qualitative screening of the antimicrobial activity of the chemical compounds was performed by an adapted diffusion method. The minimal inhibitory concentrations (MIC) of the active chemical compounds were established by Mueller Hinton broth microdillution method. The tested chemical compounds were also tested for their ability to inhibit microbial adherence and biofilm development on inert substrata by a simple microtiter method. All six chemical compounds proved to have antimicrobial activity versus the most of the tested strains, the phenantroline derivatives exhibiting higher antimicrobial activity than the dimethylguanidine-copper complex combinations. The subinhibitory concentrations of the tested chemical products slightly inhibited the adherence ability of the bacterial strains to the inert substratum. Our results demonstrated that phenantroline derivatives may represent a new strategy of antimicrobial treatment, simultaneously with the bactericidal effect, the subinhibitory concentrations of these newly synthesized chemical compounds decreasing the adherence ability of bacteria to the inert substratum.

Bacterial adherence to the cellular substrate (skin and mucosa) represents a precondition of infectious pathology. It was demonstrated that bacteria which adhere and form biofilms on catheters and other inert materials used in medicine are resistant to the therapeutic antibiotic concentrations being protected by the biofilm mathrix and generating severe and hard to treat infections. There are only few studies on the influence of antibiotics on the bacterial adhesins synthesis and bacterial adherence to the cellular substrate. The purpose of this study was to investigate the influence of subinhibitory concentrations of antibiotics on adherence capacity of Listeria monocytogenes, Vibrio cholerae and Aeromonas hydrophyla to the cellular substrate represented by HEp-2 cells. Suspensions (approximately 10(10) cells/ml) of bacterial cultures developed on solid media were incubated for 30 minutes in the presence of subinhibitory concentrations of penicillin, ampicillin, amoxicilin with clavulanic acid, ceftazidim, norfloxacin, kanamycin, chloramphenicole and vancomycin. Study of bacterial adherence to the cellular substrate was done by Cravioto's modified method. The quantitative evaluation of adherence/invasion capacity of bacterial suspensions pretreated with antibiotics was done by comparing the adherence/invasion index with controls without antibiotics. Penicillin, amoxicillin with clavulanic acid and vancomycin have significantly stimulated the adherence of Listeria monocytogenes strain and inhibited the adherence of Vibrio cholerae and Aeromonas hydrophyla strains. Ampicillin and chloramphenicole exhibited no significant effect on bacterial adherence capacity. The influence of kanamycin, ceftazidim and norfloxacin could not be interpreted due to the occurrence of a severe cytotoxic effect manifested by cell monolayer detaching, probably due to the action of antibiotic suspensions or to the increase of bacterial virulence under the selective pressure of the antibiotic.