Controlled electrophoretic deposition of bacteria to surfaces for the design of biofilms

In this report, the formation of ordered clusters of both spherical and rod-shaped bacteria on an electrode during electrophoretic deposition is described. Inside clusters, adhering bacteria are regularly spaced with an interbacterial distance that can be controlled by adjusting the ionic strength of the suspending solution and the DC density used. Formed clusters can be immobilized on the surface by applying a sufficiently high current density. This method enables the design of bacterial biofilms for biotechnological and biomedical applications. When AC fields were used, rod-shaped bacteria adhering on the electrode were seen to align parallel to the applied field.     

Dot assay for determining adhesive interactions between yeasts and bacteria under controlled hydrodynamic conditions

Different methods for cell disintegration were tested for their efficacy on filamentous fungi, including percussion grinding, homogenization using an Ultra-Turrax, chemical treatment and lyophylization. The release of protein from Ganoderma applanatum and Pycnoporus cinnabarinus and the activity of cytoplasmatic glucose-6-phosphate dehydrogenase in the crude extracts were monitored to determine the efficiency of each disintegration technique used. Fungal cells proved to be particularly resistant towards some disintegration methods commonly used for yeasts and bacteria. Best results were obtained using a percussion grinder, if necessary, in combination with an Ultra-Turrax pretreatment.     

Organ specificity and transcriptional control of metabolic routes revealed by expression QTL profiling of source-sink tissues in a segregating potato population

Background  

With the completion of genome sequences belonging to some of the major crop plants, new challenges arise to utilize this data for crop improvement and increased food security. The field of genetical genomics has the potential to identify genes displaying heritable differential expression associated to important phenotypic traits. Here we describe the identification of expression QTLs (eQTLs) in two different potato tissues of a segregating potato population and query the potato genome sequence to differentiate between cis- and trans-acting eQTLs in relation to gene subfunctionalization.     

Synthesis and Biological Evaluation of Gramicidin S‐Inspired Cyclic Mixed α/β‐Peptides

Via a Mannich reaction involving a dibenzyliminium species and the titanium enolates of Evans' chiral acylated oxazolidinones the β²‐amino acids (R)‐ and (S)‐Fmoc‐β²homovaline and (R)‐Fmoc‐β²homoleucine are synthesized. These building blocks were used, in combination with commercially available α‐ and β³‐amino acids, for the synthesis of the cyclo‐(αβ³αβ²α)₂ peptide 2 and the cyclo‐(αβ²αβ³α)₂ peptides 3 – 5 . The peptides 2 – 5 were screened for their ability to inhibit a small panel of Gram‐negative and Gram‐positive bacterial strains.     

Role of structure and glycosylation of adsorbed protein films in biolubrication

Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy), we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, meanwhile decreasing their structural softness. High-molecular-weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra-orally, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation of adsorbed protein films, providing an important clue to design effective therapeutics to restore biolubrication in patients with insufficient biolubrication.     

Vaginal epithelial cells regulate membrane adhesiveness to co‐ordinate bacterial adhesion

Vaginal epithelium is colonized by different bacterial strains and species. The bacterial composition of vaginal biofilms controls the balance between health and disease. Little is known about the relative contribution of the epithelial and bacterial cell surfaces to bacterial adhesion and whether and how adhesion is regulated over cell membrane regions. Here, we show that bacterial adhesion forces with cell membrane regions not located above the nucleus are stronger than with regions above the nucleus both for vaginal pathogens and different commensal and probiotic lactobacillus strains involved in health. Importantly, adhesion force ratios over membrane regions away from and above the nucleus coincided with the ratios between numbers of adhering bacteria over both regions. Bacterial adhesion forces were dramatically decreased by depleting the epithelial cell membrane of cholesterol or sub‐membrane cortical actin. Thus, epithelial cells can regulate membrane regions to which bacterial adhesion is discouraged, possibly to protect the nucleus.     

Effect of Cinnamon Oil on icaA Expression and Biofilm Formation by Staphylococcus epidermidis

Staphylococcus epidermidis is notorious for its biofilm formation on medical devices, and novel approaches to prevent and kill S. epidermidis biofilms are desired. In this study, the effect of cinnamon oil on planktonic and biofilm cultures of clinical S. epidermidis isolates was evaluated. Initially, susceptibility to cinnamon oil in planktonic cultures was compared to the commonly used antimicrobial agents chlorhexidine, triclosan, and gentamicin. The MIC of cinnamon oil, defined as the lowest concentration able to inhibit visible microbial growth, and the minimal bactericidal concentration, the lowest concentration required to kill 99.9% of the bacteria, were determined using the broth microdilution method and plating on agar. A checkerboard assay was used to evaluate the possible synergy between cinnamon oil and the other antimicrobial agents. The effect of cinnamon oil on biofilm growth was studied in 96-well plates and with confocal laser-scanning microscopy (CLSM). Biofilm susceptibility was determined using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Real-time PCR analysis was performed to determine the effect of sub-MIC concentrations of cinnamon oil on expression of the biofilm-related gene, icaA. Cinnamon oil showed antimicrobial activity against both planktonic and biofilm cultures of clinical S. epidermidis strains. There was only a small difference between planktonic and biofilm MICs, ranging from 0.5 to 1% and 1 to 2%, respectively. CLSM images indicated that cinnamon oil is able to detach and kill existing biofilms. Thus, cinnamon oil is an effective antimicrobial agent to combat S. epidermidis biofilms.Staphylococcus epidermidis is a gram-positive bacterium and an important agent of nosocomial infections worldwide. Treatment of these infections is increasingly problematic because of the resistance of clinical isolates to an increasing number of antimicrobial agents and, more importantly, due to its ability to grow as a biofilm. Biofilm formation by S. epidermidis (35) can be governed in part by the production of polysaccharide intercellular adhesin. Polysaccharide intercellular adhesin is produced by enzymes encoded by the ica operon which comprises four intercellular adhesion genes: icaA, icaB, icaC, and icaD. The expression of the ica operon and biofilm formation are tightly regulated by icaR under in vitro conditions (15). Biofilm formation can be influenced by changing environmental conditions, such as the presence of subinhibitory concentrations of antimicrobials like tetracycline and quinopristin-dalfopristin, as well as high temperatures, anaerobiosis, ethanol stress, and osmolarity (8, 9, 26, 37).Previous studies have demonstrated that microorganisms within biofilms are less susceptible to antimicrobial treatment than their planktonic counterparts (4), probably due to a combination of poor antimicrobial penetration, nutrient limitation, adaptive stress responses, induction of phenotypic variability, and persister cell formation (28). For this reason, current research has been focused on identifying new compounds that have antimicrobial activity against microorganisms, both in planktonic and biofilm modes of growth. Plant essential oils have been used in food preservation, pharmaceutical therapies, alternative medicine, and natural therapies for many thousands of years (23, 36).Cinnamon oil is one of the essential oils commonly used in the food industry because of its special aroma (6). Cinnamomum is a genus in the family Lauraceae, many species of which are used for spices. One of the species is Cinnamomum burmannii from Indonesia, also called Indonesian cassia (the commercial name is “cinnamon stick”). Several publications have demonstrated the antibacterial activity of cinnamon oil isolated from the bark of this species (12, 18, 22, 39). Cinnamon oil was also shown to be effective against biofilm cultures of Streptococcus mutans and Lactobacillus plantarum (14). In addition, essential oil derived from the leaves of another closely related species within this plant family, Cinnamomum osmophloeum (endemic to Taiwan), had an excellent inhibitory effect on planktonic cultures of nine gram-positive and gram-negative bacteria, including methicillin-resistant Staphylococcus aureus and S. epidermidis (6). Previous studies reported that the predominant active compound found in cinnamon oil was cinnamaldehyde (36, 39). Cinnamaldehyde causes inhibition of the proton motive force, respiratory chain, electron transfer, and substrate oxidation, resulting in uncoupling of oxidative phosphorylation, inhibition of active transport, loss of pool metabolites, and disruption of synthesis of DNA, RNA, proteins, lipids, and polysaccharides (11, 13, 33). In addition, an important characteristic of volatile oils and their components is their hydrophobicity, which enables them to partition into and disturb the lipid bilayer of the cell membrane, rendering them more permeable to protons. Extensive leakage from bacterial cells or the exit of critical molecules and ions ultimately leads to bacterial cell death (36).The susceptibility of S. epidermidis to cinnamon oil derived from the bark of Cinnamomum burmannii, however, has never been published, neither for planktonic organisms nor for staphylococci in a biofilm mode of growth. Hence, the current study was undertaken to establish the efficacy of this oil as an antimicrobial agent against clinical S. epidermidis isolates in planktonic and biofilm cultures. Chlorhexidine, triclosan, and gentamicin were used as positive controls in addition to examination of possible synergistic effects by combining cinnamon oil with any of these clinically used antimicrobials.     

Malaria vectors and transmission dynamics in coastal south-western Cameroon

Background

Malaria is a major public health problem in Cameroon. Unlike in the southern forested areas where the epidemiology of malaria has been better studied prior to the implementation of control activities, little is known about the distribution and role of anophelines in malaria transmission in the coastal areas.

Methods

A 12-month longitudinal entomological survey was conducted in Tiko, Limbe and Idenau from August 2001 to July 2002. Mosquitoes captured indoors on human volunteers were identified morphologically. Species of the Anopheles gambiae complex were identified using the polymerase chain reaction (PCR). Mosquito infectivity was detected by the enzyme-linked immunosorbent assay and PCR. Malariometric indices (plasmodic index, gametocytic index, parasite species prevalence) were determined in three age groups (<5 yrs, 5–15 yrs, >15 yrs) and followed-up once every three months.

Results

In all, 2,773 malaria vectors comprising Anopheles gambiae (78.2%), Anopheles funestus (17.4%) and Anopheles nili (7.4%) were captured. Anopheles melas was not anthropophagic. Anopheles gambiae had the highest infection rates. There were 287, 160 and 149 infective bites/person/year in Tiko, Limbe and Idenau, respectively. Anopheles gambiae accounted for 72.7%, An. funestus for 23% and An. nili for 4.3% of the transmission. The prevalence of malaria parasitaemia was 41.5% in children <5 years of age, 31.5% in those 5–15 years and 10.5% in those >15 years, and Plasmodium falciparum was the predominant parasite species.

Conclusion

Malaria transmission is perennial, rainfall dependent and An. melas does not contribute to transmission. These findings are important in the planning and implementation of malaria control activities in coastal Cameroon and West Africa.

     

Physico-chemical surface characteristics and adhesive properties of Streptococcus salivarius strains with defined cell surface structures

Physico-chemical surface characteristics and adhesive properties of a series of mutants of Streptococcus salivarius HB with defined cell surface structures were determined. Zeta potentials showed no relation either with the presence or absence of specific antigens on the bacterial cell surface, or with the adhesive properties of the cells. Hydrophobicity was assessed by surface free energy determination from measured contact angles, by adsorption to hexadecane and by hydrophobic interaction chromatography. Generally, the progressive removal of fibril subclasses from the cell surface resulted in a reduced hydrophobicity. However, specific fibrillar subclasses appeared to contribute to surface hydrophobicity to widely different extents. Bacterial adhesion to polymethylmethacrylate increased with increasing hydrophobicity of the mutants. However, adhesion to a more complex biological substratum, such as saliva-coated hydroxyapatite, correlated only partly with hydrophobicity. The organism, deprived of most of its fibrillar surface structures, clearly showed the least adhesion to hydrophobic ligands, to both polymethylmethacrylate and saliva-coated hydroxyapatite, and had a significantly higher surface free energy than the other mutants and the parent strain.