Moshe Shemesh

The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM; OMIM 600737) is a unique group of neuromuscular disorders characterized by adult onset, slowly progressive distal and proximal weakness and a typical muscle pathology including rimmed vacuoles and filamentous inclusions. The autosomal recessive form described in Jews of Persian descent is the HIBM prototype. This myopathy affects mainly leg muscles, but with an unusual distribution that spares the quadriceps. This particular pattern of weakness distribution, termed quadriceps-sparing myopathy (QSM), was later found in Jews originating from other Middle Eastern countries as well as in non-Jews. We previously localized the gene causing HIBM in Middle Eastern Jews on chromosome 9p12–13 (ref. 5) within a genomic interval of about 700 kb (ref. 6). Haplotype analysis around the HIBM gene region of 104 affected people from 47 Middle Eastern families indicates one unique ancestral founder chromosome in this community. By contrast, single non-Jewish families from India, Georgia (USA) and the Bahamas, with QSM and linkage to the same 9p12–13 region, show three distinct haplotypes. After excluding other potential candidate genes, we eventually identified mutations in the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene in the HIBM families: all patients from Middle Eastern descent shared a single homozygous missense mutation, whereas distinct compound heterozygotes were identified in affected individuals of families of other ethnic origins. Our findings indicate that GNE is the gene responsible for recessive HIBM.

Genetic and Physiological Effects of Noncoherent Visible Light Combined with Hydrogen Peroxide on Streptococcus mutans in Biofilm

ABSTRACT Oral biofilms are associated with the most common infections of the oral cavity. Bacteria embedded in the biofilms are less sensitive to antibacterial agents than planktonic bacteria are. Recently, an antibacterial synergic effect of noncoherent blue light and hydrogen peroxide (H2O2) on planktonic Streptococcus mutans was demonstrated. In this study, we tested the effect of a combination of light and H2O2 on the vitality and gene expression of S. mutans embedded in biofilm. Biofilms of S. mutans were exposed to visible light (wavelengths, 400 to 500 nm) for 30 or 60 s (equivalent to 34 or 68 J/cm2) in the presence of 3 to 300 mM H2O2. The antibacterial effect was assessed by microbial counts of each treated sample compared with that of the control. The effect of light combined with H2O2 on the different layers of the biofilm was evaluated by confocal laser scanning microscopy. Gene expression was determined by real-time reverse transcription-PCR. Our results show that noncoherent light, in combination with H2O2, has a synergistic antibacterial effect through all of the layers of the biofilm. Furthermore, this treatment was more effective against bacteria in biofilm than against planktonic bacteria. The combined light and H2O2 treatment up-regulated the expression of several genes such as gtfB, brp, smu630, and comDE but did not affect relA and ftf. The ability of noncoherent visible light in combination with H2O2 to affect bacteria in deep layers of the biofilm suggests that this treatment may be applied in biofilm-related diseases as a minimally invasive antibacterial procedure.

Durable contact active antimicrobial materials formed by a one-step covalent modification of polyvinyl alcohol, cellulose and glass surfaces.

In this work we have applied a direct covalent linkage of quaternary ammonium salts (QAS) to prepare a series of contact active antimicrobial surfaces based on widely utilized materials. Formation of antimicrobial polyvinyl alcohol (PVA-QAS), cellulose (cellulose-QAS) and glass (glass-QAS) surfaces was achieved by one step synthesis with no auxiliary linkers. The X-ray photoelectron spectroscopy (XPS) revealed tridentate binding mode of the antimicrobial agent. The antimicrobial activity of the prepared materials was tested on Bacillus cereus, Alicyclobacillus acidoterrestris, Escherichia coli and Pseudomonas aeruginosa. Active site density of the modified materials was examined and found to correlate with their antimicrobial activity. Stability studies at different pH values and temperatures confirmed that the linkage of the bioactive moiety to the surface is robust and resistant to a range of pH and temperatures. Prolonged long-term effectiveness of the contact active materials was demonstrated by their repeated usage, without loss of the antimicrobial efficacy.

Physical and transcriptional map of the hereditary inclusion body myopathy locus on chromosome 9p12-p13.

Hereditary inclusion body myopathy (HIBM) is a group of neuromuscular disorders characterised by adult-onset, slowly progressive distal and proximal muscle weakness and typical muscle pathology. Previously, we have mapped the gene responsible for a recessive form of HIBM to chromosome 9p1 and narrowed the interval to one single YAC clone of 1 Mb in size. As a further step towards the identification of the HIBM gene, we have constructed a detailed physical and transcriptional map of this region. A high resolution BAC contig that includes the HIBM critical region, flanked by marker 327GT4 and D9S1859, was constructed. This contig allowed the precise localisation of 25 genes and ESTs to the proximal region of chromosome 9. The expression pattern of those mapped genes and ESTs was established by Northern blot analysis. In the process of refining the HIBM interval, 13 new polymorphic markers were identified, of which 11 are CA-repeats, and two are single nucleotide polymorphisms. Certainly, this map provides an important integration of physical and transcriptional information corresponding to chromosome 9p12-p13, which is expected to facilitate the cloning and identification not only of the HIBM gene, but also other disease genes which map to this region.

Bioinspired passive anti-biofouling surfaces preventing biofilm formation

Biofilm formation enables bacteria to grow under unfavorable conditions, provides them with protection, and increases their resistance to antimicrobial agents. Once a biofilm has formed, it is difficult, and in some systems, impossible to treat. Strategies based on the release of biocidal agents have shown only transient efficiency. Herein, we present a novel bioinspired passive approach to the prevention of surface biofilm attachment by exploiting superhydrophobic surfaces formed via the self-assembly of paraffin or fluorinated wax crystals. Our surfaces show exceptional ability to inhibit biofilm formation of both Gram-positive Bacillus cereus and Gram-negative Pseudomonas aeruginosa over a 7 day period (up to 99.9% inhibition).

The LuxS Based Quorum Sensing Governs Lactose Induced Biofilm Formation by Bacillus subtilis

Bacillus species present a major concern in the dairy industry as they can form biofilms in pipelines and on surfaces of equipment and machinery used in the entire line of production. These biofilms represent a continuous hygienic problem and can lead to serious economic losses due to food spoilage and equipment impairment. Biofilm formation by Bacillus subtilis is apparently dependent on LuxS quorum sensing (QS) by Autoinducer-2 (AI-2). However, the link between sensing environmental cues and AI-2 induced biofilm formation remains largely unknown. The aim of this study is to investigate the role of lactose, the primary sugar in milk, on biofilm formation by B. subtilis and its possible link to QS processes. Our phenotypic analysis shows that lactose induces formation of biofilm bundles as well as formation of colony type biofilm. Furthermore, using reporter strain assays, we observed an increase in AI-2 production by B. subtilis in response to lactose in a dose dependent manner. Moreover, we found that expression of eps and tapA operons, responsible for extracellular matrix synthesis in B. subtilis, were notably up-regulated in response to lactose. Importantly, we also observed that LuxS is essential for B. subtilis biofilm formation in the presence of lactose. Overall, our results suggest that lactose may induce biofilm formation by B. subtilis through the LuxS pathway.

DNA-microarrays identification of Streptococcus mutans genes associated with biofilm thickness

BackgroundA biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans biofilms.ResultsComparative transcriptome analyses indicated that expression of 29 genes was differentially altered in 400- vs. 100-microns depth and 39 genes in 200- vs. 100-microns biofilms. Only 10 S. mutans genes showed differential expression in both 400- vs. 100-microns and 200- vs. 100-microns biofilms. All of these genes were upregulated.As sucrose is a predominant factor in oral biofilm development, its influence was evaluated on selected genes expression in the various depths of biofilms. The presence of sucrose did not noticeably change the regulation of these genes in 400- vs. 100-microns and/or 200- vs. 100-microns biofilms tested by real-time RT-PCR.Furthermore, we analyzed the expression profile of selected biofilm thickness associated genes in the luxS- mutant strain. The expression of those genes was not radically changed in the mutant strain compared to wild-type bacteria in planktonic condition. Only slight downregulation was recorded in SMU.2146c, SMU.574, SMU.609, and SMU.987 genes expression in luxS- bacteria in biofilm vs. planktonic environments.ConclusionThese findings reveal genes associated with the thickness of biofilms of S. mutans. Expression of these genes is apparently not regulated directly by luxS and is not necessarily influenced by the presence of sucrose in the growth media.

External pH Is a Cue for the Behavioral Switch That Determines Surface Motility and Biofilm Formation of Alicyclobacillus acidoterrestris

Bacteria use different strategies to survive unfavorable environmental conditions. Alicyclobacillus acidoterrestris is a bacterium capable of surviving extremely harsh conditions, for instance, during industrial food processing. A. acidoterrestris is a spore-forming, thermoacidophilic, nonpathogenic bacterium that commonly contaminates commercial pasteurized fruit juices and is, therefore, considered a major microbiological contaminant in the juice industry. The purpose of this study was to elucidate whether A. acidoterrestris is capable of multicellular behavior by testing its ability of biofilm formation and surface motility. A. acidoterrestris was found to be proficient in migration over a surface that is apparently powered by flagella. It was further shown that lowering the external pH leads to inhibition in surface motility of these bacteria. Concomitantly, the reduction in the external pH triggered biofilm formation of A. acidoterrestris cells. Thus, although no significant biofilm was formed at pH 4.5, robust cell adhesion and confluent biofilm formation was seen below the pH 3.6. These findings indicate that the reduction of external pH is an environmental cue for the behavioral switch that inhibits surface motility and triggers biofilm formation of A. acidoterrestris. Gaining insight into the multicellular behavior that facilitates A. acidoterrestris survival in food contact surfaces may contribute to the development of novel antimicrobial means to prevent cross-contamination caused by this bacterium.

Development of a Method to Determine the Effectiveness of Cleaning Agents in Removal of Biofilm Derived Spores in Milking System

Microbial damages caused by biofilm forming bacteria in the dairy industry are a fundamental threat to safety and quality of dairy products. In order to ensure the optimal level of equipment hygiene in the dairy industry, it is necessary to determine the biofilm removal efficiency of cleaning agents used for cleaning-in-place (CIP) procedures. However, currently there is no standard method available for evaluating and comparing cleaning agents for use in CIP procedures in the dairy industry under realistic conditions. The present study aims to establish a CIP model system to evaluate the effectiveness of cleaning agents in removal of biofilm derived spores from the surfaces of stainless steel which is the predominant substrate in milking equipment on dairy farms. The system is based on Bacillus subtilis spores surrounded with exopolymeric substances produced by bacteria during biofilm formation. The spores applied on sampling plates were mounted on T-junctions protruding 1.5–11-times the milk pipe diameter from the main loop to resemble different levels of cleaning difficulty. The cleaning tests were conducted using commercial alkaline detergents and caustic soda at conditions which are relevant to actual farm environment. The spores removal effect was evaluated by comparing the number of viable spores (attached to sampling plates) before and after cleaning. Evaluation of the cleaning and disinfecting effect of cleaning agents toward biofilm derived spores was further performed, which indicates whether spores elimination effect of an agent is due to killing the spores or removing them from the surfaces of dairy equipment. Moreover, it was established that the presence of extracellular matrix is an important factor responsible for high level of cleaning difficulty characteristic for surface attached spores. In overall, the results of this study suggest that the developed model system simulates actual farm conditions for quantitative evaluation of the effectiveness of cleaning and disinfecting agents and their cleaning and disinfecting effect on removal of biofilm derived spores.

A contact active bactericidal stainless steel via a sustainable process utilizing electrodeposition and covalent attachment in water

A contact active bactericidal stainless steel was synthesized in water utilizing phenol electrodeposition, followed by covalent attachment of quaternary ammonium salts. The approach minimizes the amount of the antimicrobial agent and avoids its release into the environment. Gram negative and Gram positive bacteria were inactivated upon contact with the modified surface.