Efficacy of acoustic waves in preventing Streptococcus mutans adhesion on dental unit water line.

BACKGROUND: The quality and health safety of water used for refrigeration and flushing of the handpieces, water-syringes and other components of dental units is of considerable importance. Water crosses these devices by a system of intersected small plastic tubes (about 2 mm of diameter), named dental unit water lines (DUWLs). DUWLs may be heavily colonized by many bacterial species in a planktonic phase, adherent or in biofilm lifestyle, resulting in a potential risk of infection, not only for all professionals who routinely use these devices, but also for occasional-patients, especially immunocompromised patients. Contamination of DUWLs can be prevented or reduced with the use of disinfectants, but the eradication of microorganisms, especially which those are adherent or living in biofilm lifestyle on the inner surfaces of DUWLs is challenging and often, the normal methods of water disinfection are not effective. Moreover, disinfectants routinely used to disinfect DUWLs may alter the bond strength of the dentine bonding agent used for restorative practice in dentistry. STUDY DESIGN: To identify an innovative and alternative strategy, able to prevent bacterial adhesion to DUWL surfaces through a physical approach, which is more effective in overcoming the problem of DUWL contamination and the risk of infection compared to the standard methods already in use. In this pilot study we tested a member of the oral streptococci family, that is not a component of the biofilm detected on the walls of DUWL, but is frequently detected in water samples from DUWL, due to human fluid retraction during dental therapy. Namely, the pathogenic bacterial species Streptococcus mutans. METHODS: We employ elastic acoustic waves at high-energy in preventing S. mutans adhesion to the inner walls of an experimental water circuit reproducing a DUWLs. To stress the capability of acoustic waves to interfere with bacterial adhesion also in extreme conditions, a high S. mutans contamination load was adopted. RESULTS: We observe a significant decrease of adherent bacteria exposed to acoustic waves treatment respect to control. CONCLUSION: This study demonstrates the effectiveness of acoustic waves in counteracting the adhesion of S. mutans to the inner walls of an experimental water circuit reproducing a DUWL, opening up new prospects for future practical applications. The interesting results, so far obtained, require an in-depth analysis of the methods regarding both the various bacterial species involved and the infective charges to be used.

The potential human exposure to antibiotic resistant-Escherichia coli through recreational water.

It is important that bathing water sites are free as possible from antibiotic resistant bacteria (ARB) to prevent the spread of difficult to treat infections throughout the population. This study examines the possible human exposure to antibiotic resistant Escherichia coli (AR-E. coli) through recreational activities at two different bathing water sites located near wastewater treatment plants (WWTPs). A quantitative risk assessment model was created to model the pathway of the AR-E. coli from the WWTPs effluent water through to the bathing water sites. Both sampling data and data from scientific literature were used. The main steps considered for the model were: the dilution and decay of the AR-E. coli from the WWTPs effluent water into the river; the dilution of the river into the bathing water sites and the human exposure to AR-E. coli through recreational activities at the bathing water sites (as a result of water ingestion). The results show the mean predicted human exposure levels ranged between 0.45 and 345.09 cfu/100 ml. A back calculation method determined that in accordance with the European Bathing Water Directive (2006/7/EC) (BWD) to be considered "poor" water quality, the concentration of AR-E. coli in WWTP effluent water would need to exceed 2.45 log cfu/ml at site 1 and exceed 2.71 log cfu/ml at site 2. This study provides valuable information for regulatory bodies and policy makers on the possible human exposure levels to AR-E. coli and the maximum permissible concentrations in WWTP effluent water to ensure compliance with relevant bathing water legislation.

Human exposure to antibiotic resistant-Escherichia coli through irrigated lettuce.

Antibiotic resistant bacteria (ARB) have been found on fresh fruit and vegetables globally. These types of ARB infections are spreading rapidly and are a major human health threat. A quantitative human exposure assessment model was created using scenario analysis to investigate the potential human exposure to antibiotic resistant Escherichia coli (AR-E. coli) through the consumption of lettuce irrigated with surface water. Scientific literature and site specific data were collected to model each process from farm to fork to calculate the concentration of AR-E. coli on the lettuce at the point of human consumption. The processes examined were the adhesion, colonisation and viability of bacteria on the lettuce; the effect of different post-harvest cleaning processes; the effect of consuming the lettuce before, on or after the expiry date; and the effect of the consumer washing the lettuce. The results show the mean human exposure levels ranged between 1.00 × 10-2 and 1.35 × 106 colony forming units (CFU) of AR-E. coli per 100 g of surface water irrigated lettuce for the different scenarios investigated. The mean probability of illness from consuming 100 g of lettuce contaminated with potential pathogenic antibiotic-sensitive E. coli was between 1.46 × 10-9 to 1.88 × 10-2. A back calculation revealed that in order for the EC No 1441/2007 regulation to be exceeded (≥1000 CFU/g of E. coli on lettuce at the manufacturing stage), the mean contamination levels required in the irrigation water would need to be 2.7, 3.1 or 4.8 log CFU/ml using the post-harvest treatments of washing with water, rapid cooling with water and washing with a chlorine solution respectively. The information generated from this model could help to set guidelines for producers on maximum permissible AR-E. coli contamination levels in irrigation water and provides recommendations on the best post-harvest treatment to use.

Thickness measurement of soft thin films on periodically patterned magnetic substrates by phase difference magnetic force microscopy.

The need for accurate measurement of the thickness of soft thin films is continuously encouraging the development of techniques suitable for this purpose. We propose a method through which the thickness of the film is deduced from the quantitative measurement of the contrast in the phase images of the sample surface acquired by magnetic force microscopy, provided that the film is deposited on a periodically patterned magnetic substrate. The technique is demonstrated by means of magnetic substrates obtained from standard floppy disks. Colonies of Staphylococcus aureus adherent to such substrates were used to obtain soft layers with limited lateral (a few microns) and vertical (hundreds of nanometers) size. The technique is described and its specific merits, limitations and potentialities in terms of accuracy and measurable thickness range are discussed. These parameters depend on the characteristics of the sensing tip/cantilever as well as of the substrates, the latter in terms of spatial period and homogeneity of the magnetic domains. In particular, with the substrates used in this work we evaluated an uncertainty of about 10%, a limit of detection of 50-100 nm and an upper detection limit (maximum measurable thickness) of 1 µm, all obtained with standard lift height values (50-100 nm). Nonetheless, these parameters can be easily optimized by selecting/realizing substrates with suitable spacing and homogeneity of the magnetic domains. For example, the upper detection limit can be increased up to 25-50 µm while the limit of detection can be reduced to a few tens of nanometers or a few nanometers.

Analytical techniques to study microbial biofilm on abiotic surfaces: pros and cons of the main techniques currently in use.

Biofilm is a bacterial lifestyle widespread in microbial world and represents a concern in health care. Despite the great life expectancy related to advanced health care, the increasing numbers of biofilm-mediated infections remain a significant public health challenge. Moreover, the problem of biofilm-mediated infections becomes much more severe when biofilm colonizes medical devices and biomaterials. The public health risk due to microbial biofilm-related infections is a concern that requires full attention. However, the complexity of biofilm makes difficult its exhaustive analysis. Although biofilm represents a major challenge in both microbiological and hygiene areas, at now methods aimed to analyse biofilm formation and development are not standardized yet. Different methods have been employed to qualitatively and quantitatively evaluate biofilm each of which is useful to estimate a peculiar aspect of biofilm lifestyle. In the present review, fifteen assays for the qualitative and quantitative evaluation of bacterial biofilm colonizing abiotic substrates, such as medical devices, prosthesis or surfaces for food production together with advantages and limitations of each method were described and compared. Some methods are suited to quantify biofilm matrix while others are capable to evaluate both living and dead cells or quantify exclusively viable cells in biofilm. In particular, colorimetric methods to evaluate biofilm matrix (crystal violet; 1,9-dimethyl methylen blue and fluorescein-di-acetate methods) or viable cells (LIVE/DEAD BacLight, BioTimer Assay, resazurin, tetrazolium hydroxide salt methods) and genetic methods to estimate the bacterial population (PCR and FISH) are reported. Moreover, a section is dedicated to examine the performances of advanced microscopic techniques employed to study microbial biofilms (mass spectrometry; confocal laser scanning microscopy; Raman spectroscopy and electron microscopy). Because of its complexity, an exhaustive study of biofilm requires a combination of different experimental approaches as biochemical, genetic or physical ones.

Lactoferrin decreases inflammatory response by cystic fibrosis bronchial cells invaded with Burkholderia cenocepacia iron-modulated biofilm.

In cystic fibrosis (CF) high iron concentration in airway secretion plays a pivotal role in bacterial multiplication and biofilm formation as well as in inflammatory response. Burkholderia cenocepacia, an opportunistic facultative pathogen responsible for chronic lung infections and cepacia syndrome, recurrently infects CF patients. Lactoferrin (Lf), an iron binding multifunctional glycoprotein synthesized by exocrine glands and neutrophils, has been found at higher concentration in the airway secretions of infected CF patients than in healthy subjects. Here the influence of milk derivative bovine lactoferrin (bLf), an emerging important regulator of iron and inflammatory homeostasis, on invasiveness of B. cenocepacia iron-modulated biofilm, as well as on inflammatory response by infected CF bronchial (IB3-1) cells, is reported. bLf did not significantly affect invasion efficacy by biofilmforming B. cenocepacia clinical strains. Conversely, the addition of bLf to cell monolayers during infection significantly decreased the pro-inflammatory Interleukin (IL)-1beta and increased the anti-inflammatory IL-11 expression compared to that observed in cells infected in the absence of bLf. The bLf ability to modulate genes expressed following B. cenocepacia infection seems related to its localization to the nucleus of infected IB3-1 cells. These results provide evidence for a role of bLf in the protection of infected CF cells from inflammation-related damage, thus extending the therapeutic potential of this multifunctional natural protein.

Detection of different Borrelia burgdorferi genospecies in serum of people with different occupational risks: short report.

This study is aimed at applying a previously described PCR-based method to detect B. burgdorferi sensu lato and different Borrelia genospecies in total DNA preparations of serum samples collected from people with different occupational risks for tick bite and with serological evidence of borreliosis. Among the seropositive samples, the PCR for B. burgdorferi confirmed the positivity in 65 percent of the forestry workers and in 60 percent of the subjects living in the same area. None of the seronegative subjects belonging to the control group showed the presence of B. burgdorferi sensu lato DNA. Results on genospecies distribution show that B. afzelii was the predominant species, followed by B. garinii and finally by B. valaisiana.

Violacein and biofilm production in Janthinobacterium lividum.

AIMS: To analyse the environmental stimuli modulating violacein and biofilm production in Janthinobacterium lividum. METHODS AND RESULTS: Violacein and biofilm production by J. lividum DSM1522(T) was assayed in different growth conditions. Our data suggest that violacein and biofilm production is controlled by the carbon source, being inhibited by glucose and enhanced by glycerol. J. lividum produced violacein also in the presence of different sub-inhibitory concentrations of ampicillin. As opposite, the production of N-acylhomoserine lactone(s), quorum sensing regulators was shown to be positively regulated by glucose. Moreover, violacein-producing cultures of J. lividum showed higher CFU counts than violacein-nonproducing ones. CONCLUSIONS: Taken together, our results suggest that violacein and biofilm production could be regulated by a common metabolic pathway and that violacein as well as biofilm could represent a response to environmental stresses and a key factor in the survival mechanisms of J. lividum. SIGNIFICANCE AND IMPACT OF THE STUDY: Although several recent studies disclosed a number of interesting biological properties of violacein, few data are reported on the physiologic function of violacein in J. lividum. This paper adds new information on the complex mechanisms allowing and regulating bacterial life in hostile environments.

Multifactorial aspects of antimicrobial activity of propolis.

We investigated the antibacterial activity of sub-inhibitory concentrations of ethanolic extract of propolis (EEP), and its effect on the antibacterial activity of some antibiotics. Some clinically isolated Gram-positive strains were used. Moreover, sub-inhibitory concentrations of EEP were used to value its action on some important virulence factors like lipase and coagulase enzymes, and on biofilm formation in Staphylococcus aureus. Our results indicated that EEP had a significant antimicrobial activity towards all tested clinical strains. Adding EEP to antibacterial tested drugs, it drastically increased the antimicrobial effect of ampicillin, gentamycin and streptomycin, moderately the one of chloramphenicol, ceftriaxon and vancomycin, while there was no effect with erithromycin. Moreover, our results pointed out an inhibitory action of EEP on lipase activity of 18 Staphylococcus spp. strains and an inhibitory effect on coagulase of 11 S. aureus tested strains. The same EEP concentrations showed a negative interaction with adhesion and consequent biofilm formation in S. aureus ATCC 6538P.

Metallo-beta-lactamase producers in environmental microbiota: new molecular class B enzyme in Janthinobacterium lividum.

Eleven environmental samples from different sources were screened for the presence of metallo-beta-lactamase-producing bacteria by using a selective enrichment medium containing a carbapenem antibiotic and subsequently testing each isolate for production of EDTA-inhibitable carbapenemase activity. A total of 15 metallo-beta-lactamase-producing isolates, including 10 Stenotrophomonas maltophilia isolates, 3 Chryseobacterium spp., one Aeromonas hydrophila isolate, and one Janthinobacterium lividum isolate (a species in which production of metallo-beta-lactamase activity was not previously reported), were obtained from 8 samples. In the J. lividum isolate, named JAC1, production of metallo-beta-lactamase activity was elicited upon exposure to beta-lactams. Screening of a JAC1 genomic library for clones showing a reduced imipenem susceptibility led to the isolation of a metallo-beta-lactamase determinant encoding a new member (named THIN-B) of the highly divergent subclass B3 lineage of metallo-beta-lactamases. THIN-B is most closely related (35.6% identical residues) to the L1 enzyme of S. maltophilia and more distantly related to the FEZ-1 enzyme of Legionella gormanii (27.8% identity) and to the GOB-1 enzyme of Chryseobacterium meningosepticum (24.2% identity). Sequences related to bla(THIN-B), and inducible production of metallo-beta-lactamase activity, were also detected in the J. lividum type strain DSM1522. Expression of the bla(THIN-B) gene in Escherichia coli resulted in decreased susceptibility to several beta-lactams, including penicillins, cephalosporins (including cephamycins and oxyimino cephalosporins), and carbapenems, revealing a broad substrate specificity of the enzyme. The results of this study indicated that metallo-beta-lactamase-producing bacteria are widespread in the environment and identified a new molecular class B enzyme in the environmental species J. lividum.

Invasion of human cultured cells by Prevotella nigrescens.

Invasion of gingival and junctional epithelial cells has been recently proposed as a potentially relevant mechanism in the pathogenesis and recurrence of periodontal disease. The gram negative anaerobe Prevotella nigrescens was shown to be involved in the development of periodontal lesions in man, suggesting a possible involvement of invasivity as a mean to circumvent the host immune surveillance and other hostile factors. Appropriately designed invasion assays demonstrated that P. nigrescens efficiently invades human epithelial cells, through a mechanism whose efficiency is influenced by the phase of growth, by the multiplicity of infection, and by the cell line used, and that requires microfilament integrity, but is not affected by an impairment of microtubule organization. Intracellular replication assays suggested that P. nigrescens probably multiplies within Kb epithelial cells, causing extensive cell alterations. Invasion of gingival epithelial cells could consequently be a basic step in the virulence mechanism of the species.

Production of bacteriolytic enzymes as a tool for characterizing enterococci.

Bacteriolytic enzymes secreted by log-phase cultures of enterococci (Enterococcus faecalis, Ent. faecium, Ent. durans, Ent. hirae, Ent. casseliflavus, Ent. avium, Ent. mundtii) were analysed by means of a zymogram technique to resolve activities according to the size of their polypeptide component and their specificities towards different substrates. Heterogeneous patterns of lytic activity were observed with different species. For each test substrate, homogeneous patterns of lytic activities were observed with strains of the same species, except for Ent. faecalis strains, which showed heterogeneous lytic patterns even towards the same substrate, and could be divided into at least four different groups according to their lytic pattern. No lytic activity was common to all strains tested. Results of zymogram analysis of Enterococcus bacteriolytic enzymes were consistent with current knowledge on enterococcal taxonomy, indicating that this analytical approach may be a useful tool for fine-tuned characterization of different enterococcal strains.

A new approach to use of bacteriolytic enzymes as a tool for species identification: selection of species-specific indicator strains with bacteriolytic activity towards Enterococcus strains.

We describe the bacteriolytic activity of 377 group D Enterococcus isolates expressed towards 25 Enterococcus strains belonging to different species and Micrococcus luteus ATCC 4698. Of the 26 indicator strains used to reveal bacteriolytic activity, 5 were lysed by all of the strains of some species and were not lysed by all of the strains of other species. The use of these indicator strains allowed us to devise a new method to differentiate group D Enterococcus strains, based on qualitative analysis (lysis or no lysis of the indicator strains) of bacteriolytic activity. The bacteriolytic patterns obtained fell into six bacteriolytic groups corresponding (98% agreement) to species or groups of enterococci as determined by a comparison with data from a phenetic similarity study.

Modified MacConkey medium which allows simple and reliable identification of Providencia stuartii.

This work describes a modified MacConkey medium (MCP medium) enabling the simple identification of Providencia stuartii, an emerging nosocomial pathogen. A total of 813 strains, belonging to the families Enterobacteriaceae and Pseudomonadaceae, were tested on MCP medium; all P. stuartii strains were phosphatase positive, as were 97.5% of Morganella morganii strains, in contrast with all other tested organisms. A simple discriminating test, such as the ornithine or citrate test, allowed identification of strains of these species. We have also compared the reliabilities of P. stuartii identification by commercial kits (API 20E system) by using a standard MacConkey or MCP medium. Sixteen and three-tenths percent of P. stuartii strains were misidentified by using the former procedure, while with the latter all strains were correctly identified. Finally, the MCP medium was used over a 6-month period in our routine clinical laboratory. Of a total of 1,278 seeded urine samples from elderly patients, we isolated 103 P. stuartii strains which were all correctly identified by coupling MCP medium and the API 20E system. Seventeen and one-half percent of these strains were misidentified when the API 20E system was used in combination with standard MacConkey medium.