Effect of daptomycin and vancomycin on Staphylococcus epidermidis biofilms: An in vitro assessment using fluorescence in situ hybridization

Colonization of in-dwelling catheters by microbial biofilms is a major concern in patient health eventually leading to catheter-related blood stream infections. Biofilms are less susceptible to standard antibiotic therapies that are effective against planktonic bacteria. Standard procedure for the detection of microorganisms on the catheter tip is culture. However, viable but non-culturable cells (VBNCs) may be missed. The aim of this study was to evaluate the use of fluorescence in situ hybridization (FISH) as an indicator to visualize and quantify the effect of the antibiotics daptomycin and vancomycin on biofilms in situ. We established an in vitro catheter biofilm model of Staphylococcus epidermidis biofilms on polyurethane catheters. Biofilm activity was measured by FISH and correlated to colony forming units (CFU) data. Digital image analysis was used for quantification of total biofilm mass and the area of the FISH positive biofilm cells. FISH showed a pronounced effect of both antibiotics on the biofilms, with daptomycin having a significantly stronger effect in terms of both reduction of biofilm mass and number of FISH-positive cells. This supports the anti-biofilm capacity of daptomycin. Interestingly, neither antibiotic was able to eradicate all of the FISH-positive cells. In summary, FISH succeeded in visualization, quantification, and localization of antibiotic activity on biofilms. This technique adds a new tool to the arsenal of test systems for anti-biofilm compounds. FISH is a valuable complementary technique to CFU since it can be highly standardized and provides information on biofilm architecture and quantity and localization of survivor cells

Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was observed. Finally, all formulations proved to be biocompatible with both ISO compliant L929 fibroblasts and human MG63 osteoblast-like cells

The immune reconstitution inflammatory syndrome in whipple disease: a cohort study.

Whipple disease, which is caused by infection with Tropheryma whipplei, can be treated effectively with antimicrobials. Occasionally, inflammation reappears after initial improvement; this is often interpreted as refractory or recurrent disease. However, polymerase chain reaction for T. whipplei in tissue is sometimes negative during reinflammation, indicating absence of vital bacteria, and this reinflammation does not respond to antimicrobials but does respond to steroids.To demonstrate that the immune reconstitution inflammatory syndrome (IRIS) occurs in patients treated for Whipple disease.Cohort study. (International Standard Randomised Controlled Trial Number Register registration number: ISRCTN45658456)2 academic medical centers in Germany.142 patients treated for Whipple disease out of a cohort of 187 were observed for reappearance of inflammatory signs after effective antibiotic therapy. Definitions of IRIS in HIV infection, tuberculosis, and leprosy were adapted for application to Whipple disease.On the basis of study definitions, IRIS was diagnosed in 15 of 142 patients. Symptoms included fever, arthritis, pleurisy, erythema nodosum, inflammatory orbitopathy, small-bowel perforation, and a hypothalamic syndrome. Two patients died. There was a positive correlation with previous immunosuppressive treatment and a negative correlation with previous diarrhea and weight loss.The study was observational and thus has inherent weaknesses, such as incomplete and potentially selective data recording.The immune reconstitution inflammatory syndrome was diagnosed in about 10\% of patients with Whipple disease in the study cohort; the outcome varied from mild to fatal. Patients who had had previous immunosuppressive therapy were at particular risk. An immune reconstitution syndrome should be considered in patients with Whipple disease in whom inflammatory symptoms recur after effective treatment. Early diagnosis and treatment with steroids may be beneficial; prospective studies are needed.European Commission and Deutsche Forschungsgemeinschaft

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

Oral Biofilm Architecture on Natural Teeth

Periodontitis and caries are infectious diseases of the oral cavity in which oral biofilms play a causative role. Moreover, oral biofilms are widely studied as model systems for bacterial adhesion, biofilm development, and biofilm resistance to antibiotics, due to their widespread presence and accessibility. Despite descriptions of initial plaque formation on the tooth surface, studies on mature plaque and plaque structure below the gum are limited to landmark studies from the 1970s, without appreciating the breadth of microbial diversity in the plaque. We used fluorescent in situ hybridization to localize in vivo the most abundant species from different phyla and species associated with periodontitis on seven embedded teeth obtained from four different subjects. The data showed convincingly the dominance of Actinomyces sp., Tannerella forsythia, Fusobacterium nucleatum, Spirochaetes, and Synergistetes in subgingival plaque. The latter proved to be new with a possibly important role in host-pathogen interaction due to its localization in close proximity to immune cells. The present study identified for the first time in vivo that Lactobacillus sp. are the central cells of bacterial aggregates in subgingival plaque, and that Streptococcus sp. and the yeast Candida albicans form corncob structures in supragingival plaque. Finally, periodontal pathogens colonize already formed biofilms and form microcolonies therein. These in vivo observations on oral biofilms provide a clear vision on biofilm architecture and the spatial distribution of predominant species

Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle

© 2008 Pringle et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Uncultivated Microbial Eukaryotic Diversity: A Method to Link ssu rRNA Gene Sequences with Morphology

Protists have traditionally been identified by cultivation and classified taxonomically based on their cellular morphologies and behavior. In the past decade, however, many novel protist taxa have been identified using cultivation independent ssu rRNA sequence surveys. New rRNA “phylotypes” from uncultivated eukaryotes have no connection to the wealth of prior morphological descriptions of protists. To link phylogenetically informative sequences with taxonomically informative morphological descriptions, we demonstrate several methods for combining whole cell rRNA-targeted fluorescent in situ hybridization (FISH) with cytoskeletal or organellar immunostaining. Either eukaryote or ciliate-specific ssu rRNA probes were combined with an anti-α-tubulin antibody or phalloidin, a common actin stain, to define cytoskeletal features of uncultivated protists in several environmental samples. The eukaryote ssu rRNA probe was also combined with Mitotracker® or a hydrogenosomal-specific anti-Hsp70 antibody to localize mitochondria and hydrogenosomes, respectively, in uncultivated protists from different environments. Using rRNA probes in combination with immunostaining, we linked ssu rRNA phylotypes with microtubule structure to describe flagellate and ciliate morphology in three diverse environments, and linked Naegleria spp. to their amoeboid morphology using actin staining in hay infusion samples. We also linked uncultivated ciliates to morphologically similar Colpoda-like ciliates using tubulin immunostaining with a ciliate-specific rRNA probe. Combining rRNA-targeted FISH with cytoskeletal immunostaining or stains targeting specific organelles provides a fast, efficient, high throughput method for linking genetic sequences with morphological features in uncultivated protists. When linked to phylotype, morphological descriptions of protists can both complement and vet the increasing number of sequences from uncultivated protists, including those of novel lineages, identified in diverse environments