The improvement of a phenotype microarray protocol for the chemical sensitivity analysis of Streptococcus thermophilus

Phenotype MicroArray (PM) permits the characterisation of bacteria under nearly 2000 culture conditions. The PM standard procedure for the chemical sensitivity analysis of Gram-positive bacteria failed in the analysis of Streptococcus thermophilus. Therefore, we developed an efficient and reproducible protocol to obtain a chemically sensitive profile of S. thermophilus using PM.     

Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications

Cell labeling is a preliminary step in multiple biophysical approaches, including the solid-state nuclear magnetic resonance (NMR) study of bacteria in vivo. Deuterium solid-state NMR has been used in the past years to probe bacterial membranes and their interactions with antimicrobial peptides, following a standard labeling protocol. Recent results from our laboratory on a slow-growing bacterium has shown the need to optimize this protocol, especially the bacterial growth time before harvest and the concentration of exogenous labeled fatty acids to be used for both Escherichia coli and Bacillus subtilis. It is also essential for the protocol to remain harmless to cells while providing optimal labeling. We have therefore developed a fast and facile approach to monitor the lipid composition of bacterial membranes under various growth conditions, combining solution ³¹P NMR and GCMS. Using this approach, the optimized labeling conditions of Escherichia coli and Bacillus subtilis with deuterated palmitic acid were determined. Our results show a modification of B. subtilis phospholipid profile as a function of the growth stage, as opposed to E. coli. Our protocol recommends low concentrations of exogenous palmitic acid in the growth medium, and bacteria harvest after the exponential phase.     

Localization of Ruminal Cellulolytic Bacteria on Plant Fibrous Materials as Determined by Fluorescence In Situ Hybridization and Real-Time PCR

To visualize and localize specific bacteria associated with plant materials, a new fluorescence in situ hybridization (FISH) protocol was established. By using this protocol, we successfully minimized the autofluorescence of orchard grass hay and detected rumen bacteria attached to the hay under a fluorescence microscope. Real-time PCR assays were also employed to quantitatively monitor the representative fibrolytic species Fibrobacter succinogenes and Ruminococcus flavefaciens and also total bacteria attached to the hay. F. succinogenes was found firmly attached to not only the cut edges but also undamaged inner surfaces of the hay. Cells of phylogenetic group 1 of F. succinogenes were detected on many stem and leaf sheath fragments of the hay, even on fragments on which few other bacteria were seen. Cells of phylogenetic group 2 of F. succinogenes were often detected on hay fragments coexisting with many other bacteria. On the basis of 16S rRNA gene copy number analysis, the numbers of bacteria attached to the leaf sheaths were higher than those attached to the stems (P < 0.05). In addition, R. flavefaciens had a greater tendency than F. succinogenes to be found on the leaf sheath (P < 0.01) with formation of many pits. F. succinogenes, particularly phylogenetic group 1, is suggested to possibly play an important role in fiber digestion, because it is clearly detectable by FISH and is the bacterium with the largest population size in the less easily degradable hay stem.     

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (> 98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences.     

Development of a modified DNA extraction method for pulsed-field gel electrophoresis analysis of Staphylococcus aureus and enterococci without using lysostaphin

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to clinical isolates of Staphylococcus aureus and enterococci to reduce the cost of using lysostaphin. This protocol reduces the expenses of PFGE typing of S. aureus and enterococci as it removes the use of lysostaphin during the spheroplast formation from these bacteria.     

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2'',7''-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.     

A generally applicable cryopreservation method for nitrite-oxidizing bacteria

Nitrite-oxidizing bacteria are key members of the global nitrogen cycle but their study is hampered by their limited availability in culture, mostly due to laborious cultivation procedures and the lack of stable preservation methods. In this study, it was demonstrated that long-term cryopreservation of nitrite-oxidizing bacteria assigned to the genera Nitrobacter, Nitrospina, Nitrococcus, Nitrotoga and Nitrospira was possible using a simple and rapid protocol. Their survival was tested with different cryoprotecting agents, DMSO and Hatefi, and in various carbon-rich preservation media, ten-fold diluted TSB, and ten-fold diluted TSB supplemented with 1% trehalose, and 1% sucrose. Optimal preservation conditions were strain-dependent and marine strains appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide with or without ten-fold diluted trypticase soy broth as a preservation medium allowed successful preservation of all tested strains.     

Quantitative and Qualitative Analysis of Bacteria in Er(III) Solution by Thin-Film Magnetopheresis

Magnetic deposition, quantitation, and identification of bacteria reacting with the paramagnetic trivalent lanthanide ion, Er³⁺, was evaluated. The magnetic deposition method was dubbed thin-film magnetopheresis. The optimization of the magnetic deposition protocol was accomplished with Escherichia coli as a model organism in 150 mM NaCl and 5 mM ErCl₃ solution. Three gram-positive bacteria, Staphylococcus epidermidis, Staphylococcus saprophyticus, and Enterococcus faecalis, and four gram-negative bacteria, E. coli, Pseudomonas aeruginosa, Proteus mirabilis, and Klebsiella pneumoniae, were subsequently investigated. Quantitative analysis consisted of the microscopic cell count and a scattered-light scanning of the magnetically deposited material aided by the computer data acquisition system. Qualitative analysis consisted of Gram stain differentiation and fluorescein isothiocyanate staining in combination with selected antisera against specific types of bacteria on the solid substrate. The magnetic deposition protocol allowed quantitative detection of E. coli down to the concentration of 10⁵ CFU ml^-1, significant in clinical diagnosis applications such as urinary tract infections. Er³⁺ did not interfere with the typical appearance of the Gram-stained bacteria nor with the antigen recognition by the antibody in the immunohistological evaluations. Indirect antiserum-fluorescein isothiocyanate labelling correctly revealed the presence of E. faecalis and P. aeruginosa in the magnetically deposited material obtained from the mixture of these two bacterial species. On average, the reaction of gram-positive organisms was significantly stronger to the magnetic field in the presence of Er³⁺ than the reaction of gram-negative organisms. The thin-film magnetophoresis offers promise as a rapid method for quantitative and qualitative analysis of bacteria in solutions such as urine or environmental water.     

Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR

A PMA (propidium monoazide) pretreatment protocol, in which PMA is applied directly to membrane filters, was developed for the PCR-based quantification (PMA-qPCR) of viable Legionella pneumophila. Using this method, the amplification of DNA from membrane-damaged L. pneumophila was strongly inhibited for samples containing a small number of dead bacteria.     

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.     

Labelling strategy and membrane characterization of marine bacteria Vibrio splendidus by in vivo 2H NMR

Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo ²H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. ²H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. In this study, we showed that V. splendidus can be labelled using deuterated palmitic acid, and demonstrated the importance of surfactant choice in the labelling protocol. Moreover, we assessed the impact of lipid deuteration on the general fitness of the bacteria, as well as the saturated-to-unsaturated fatty acid chains ratio and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show larger membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions - an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies.     

An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells

To reduce the interference in MTT reduction assay, we developed an improved protocol for evaluating the viability of Escherichia coli cells by conducting MTT reduction in 1.5-mL centrifuge tubes and formazan dissolution in test tubes. Our study is helpful in developing protocols for measuring the viability of other gram-negative bacteria.     

A standardized method for the sampling of rhizosphere and rhizoplan soil bacteria associated to a herbaceous root system

Plants-microorganisms interactions play a fundamental role in terrestrial ecosystems and various methods have been reported for plant-associated bacteria extraction. However, these methods exhibit notable variations and lack of some procedural details that may impact the interpretations of results. We propose here a standardized and detailed protocol for the independent extraction of bulk, rhizosphere and rhizoplan soil fractions. This protocol was applied to the sampling of different polluted soil fractions collected in the vicinity of Arabidopsis halleri dense root system. It allowed us to determine the cultivable bacterial densities in each fraction and to confirm the existence of a bacterial gradient linked to roots distance, with a higher amount of bacteria in the rhizospheric area. We suggest to use this unified procedure as a common basis for soil sampling and bacterial communities analysis from other roots systems.     

Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification

Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7% ± 18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.     

An exhaustive yet simple virtual screening campaign against Sortase A from multiple drug resistant Staphylococcus aureus

Methicillin resistant Staphylococcus aureus (MRSA) is one of the challenging bacterial pathogen due to its acquired resistance to the β lactam antibiotics. The Sortase A is an enzyme of Gram-positive bacteria including S. aureus to anchor surface proteins to the cell wall. Sortase A is well studied enzyme and considered as the drug target against MRSA. Sortase A plays active role in anchoring the virulence proteins on the cell wall of the Gram-positive bacteria. The inhibition of Sortase A activity results in the separation of S. aureus from the host cells and ultimately alleviation of the infection. Here, we adapted a structure-based virtual screening protocol which helped in identification of novel potential inhibitors of Sortase A. The protocol involved the docking of a chemical library of druglike compounds with the Sortase A binding site represented by multiple crystal structures. The compounds were ranked by multiple scoring functions and shortlisted for future experimental screening. The method resulted in shortlisting of three compounds as potential novel inhibitors of Sortase A out of a large chemical library. The high rankings of shortlisted compounds estimated by multiple scoring functions showed their binding potential with Sortase A. The results are proved to be a simple yet efficient choice of structure-based virtual screening. The identified compounds are druglike and show high rankings among all set protocols of the virtual screening. We hope that the study would eventually help to expedite the discovery of novel drug candidates against MRSA.     

A simple protocol to obtain highly pure Wolbachia endosymbiont DNA for genome sequencing

Most genome sequencing projects using intracellular bacteria face difficulties in obtaining sufficient bacterial DNA free of host contamination. We have developed a simple and rapid protocol to isolate endosymbiont DNA virtually free from fly and mosquito host DNA. We purified DNA from six Wolbachia strains in preparation for genome sequencing using this method, and achieved up to 97% pure Wolbachia sequence, even after using frozen insects. This is a significant improvement for future Wolbachia and other endosymbiont genome projects.     

Genetic Diversity of the Biofilm Covering Montacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.     

Do wheelchairs spread pathogenic bacteria within hospital walls?

Transmission of nosocomial pathogens has been linked to transient colonization of health care workers, medical devices and other constituents of patients’ environment. In this paper we present our findings concerning the presence of pathogenic bacteria on wheelchairs, and the possibility that wheelchairs constitute a reservoir of these bacteria and a means of spreading them. In this work we examined four wheelchairs, each from a different location: the internal medicine ward, the emergency department, the general surgery ward and wheelchair stockpile of the transportation unit of the hospital. The samples were collected and cultured on different media. Bacterial identification and antimicrobial sensitivity testing were carried out using accepted practices in the microbiology laboratory. We found that wheelchairs are contaminated with several pathogenic bacteria, among them antibiotic-resistant strains such as MRSA, Pseudomonas aeruginosa, Acinetobacter baumanni etc. Since there is no specific guideline protocol that deals with disinfection and cleaning frequency of wheelchairs in hospitals, we suggest each hospital to write one.     

Magnetic Nanoparticles from Magnetospirillum gryphiswaldense Increase the Efficacy of Thermotherapy in a Model of Colon Carcinoma

Magnetic nanoparticles (MNPs) are capable of generate heating power under the influence of alternating magnetic fields (AMF); this behaviour recently opened new scenarios for advanced biomedical applications, mainly as new promising tumor therapies. In this paper we have tested magnetic nanoparticles called magnetosomes (MNs): a class of MNPs naturally produced by magnetotactic bacteria. We extracted MNs from Magnetospirillum gryphiswaldense strain MSR-1 and tested the interaction with cellular elements and anti-neoplastic activity both in vitro and in vivo, with the aim of developing new therapeutic approaches for neoplastic diseases. In vitro experiments performed on Human Colon Carcinoma HT-29 cell cultures demonstrated a strong uptake of MNs with no evident signs of cytotoxicity and revealed three phases in the interaction: adherence, transport and accumulation in Golgi vesicles. In vivo studies were performed on subcutaneous tumors in mice; in this model MNs are administered by direct injection in the tumor volume, then a protocol consisting of three exposures to an AMF rated at 187 kHz and 23kA/m is carried out on alternate days, over a week. Tumors were monitored by Magnetic Resonance Imaging (MRI) to obtain information about MNs distribution and possible tissue modifications induced by hyperthermia. Histological analysis showed fibrous and necrotic areas close to MNs injection sites in mice subjected to a complete thermotherapy protocol. These results, although concerning a specific tumor model, could be useful to further investigate the feasibility and efficacy of protocols based on MFH. Magnetic nanoparticles naturally produced and extracted from bacteria seem to be promising candidates for theranostic applications in cancer therapy.