Finding gene-expression patterns in bacterial biofilms

The production of biofilms by bacteria is a lifestyle that is thought to require or involve a differential gene expression compared with that of planktonic bacteria. Recently, we have witnessed a change of focus from the simple hunt for hypothetical essential biofilm genes to the identification of late and more complex biofilm functions. However, finding common bacterial biofilm gene-expression patterns through global expression analysis remains difficult. Owing to the apparently minimal overlap between functions involved in biofilm formation by different bacteria, exploring the biofilm lifestyle could prove to be a case-by-case task for which global approaches show their limits.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of mycobacteria in routine clinical practice

Background

Non-tuberculous mycobacteria recovered from respiratory tract specimens are emerging confounder organisms for the laboratory diagnosis of tuberculosis worldwide. There is an urgent need for new techniques to rapidly identify mycobacteria isolated in clinical practice. Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) has previously been proven to effectively identify mycobacteria grown in high-concentration inocula from collections. However, a thorough evaluation of its use in routine laboratory practice has not been performed.

Methodology

We set up an original protocol for the MALDI-TOF MS identification of heat-inactivated mycobacteria after dissociation in Tween-20, mechanical breaking of the cell wall and protein extraction with formic acid and acetonitrile. By applying this protocol to as few as 10⁵ colony-forming units of reference isolates of Mycobacterium tuberculosis, Mycobacterium avium, and 20 other Mycobacterium species, we obtained species-specific mass spectra for the creation of a local database. Using this database, our protocol enabled the identification by MALDI-TOF MS of 87 M. tuberculosis, 25 M. avium and 12 non-tuberculosis clinical isolates with identification scores ≥2 within 2.5 hours.

Conclusions

Our data indicate that MALDI-TOF MS can be used as a first-line method for the routine identification of heat-inactivated mycobacteria. MALDI-TOF MS is an attractive method for implementation in clinical microbiology laboratories in both developed and developing countries.     

Esp-independent biofilm formation by Enterococcus faecalis

Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.     

Investigation of the mesoscale structure and volumetric features of biofilms using optical coherence tomography

Optical coherence tomography (OCT) was successfully applied to visualize the mesoscale structure of three different heterotrophic biofilms. For this purpose, biofilm volumes of 4 × 4 × 1.6 mm³ were scanned with spatial resolutions lower than 20 µm within an acquisition time of 2 min. A heterogeneous structure was detected for biofilms cultivated in laminar as well as transient flow conditions. The structure was found to be more homogeneous for the biofilm grown in turbulent flow. This biofilm structure was characterized by a volumetric porosity of 0.36, whereas the porosity calculated for biofilms grown in laminar and transient conditions was 0.65. These results were directly generated from the distribution of porosity calculated from the OCT images acquired and can be linked to structural properties. Up to now, the mesoscale biofilm structure was only observable with time‐consuming and expensive studies, for example, magnetic resonance microscopy. OCT will most certainly be helpful for improved understanding and prediction of biofilm physics with respect to macroscale processes, for example, mass transfer and detachment as the information about mesoscale is easily accessible using this method. In the context of this study, we show that CLSM images do not necessarily provide an accurate representation of the biofilm structure at the mesoscale. Additionally, the typical characteristic parameters obtained from CLSM image stacks differ largely from those calculated from OCT images. Nevertheless, to determine the local distribution of biofilm constituents, microscopic methods such as confocal laser scanning microscopy are required. Biotechnol. Bioeng. 2010;107: 844–853. © 2010 Wiley Periodicals, Inc.     

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.     

An in vitro dynamic microcosm biofilm model for caries lesion development and antimicrobial dose-response studies

Some dynamic biofilm models for dental caries development are limited as they require multiple experiments and do not allow independent biofilm growth units, making them expensive and time-consuming. This study aimed to develop and test an in vitro dynamic microcosm biofilm model for caries lesion development and for dose-response to chlorhexidine. Microcosm biofilms were grown under two different protocols from saliva on bovine enamel discs for up to 21 days. The study outcomes were as follows: the percentage of enamel surface hardness change, integrated hardness loss, and the CFU counts from the biofilms formed. The measured outcomes, mineral loss and CFU counts showed dose-response effects as a result of the treatment with chlorhexidine. Overall, the findings suggest that biofilm growth for seven days with 0.06 ml min^?1 salivary flow under exposure to 5% sucrose (3 × daily, 0.25 ml min^?1, 6 min) was suitable as a pre-clinical model for enamel demineralization and antimicrobial studies.     

生物被膜态益生菌控制病原菌感染及其开发策略

生物被膜是细菌的一种特殊生存方式。生物被膜感染在临床中的高耐药性问题、反复性问题、迁延性问题等是临床中亟待解决的问题。益生菌作为机体共生微生物的一部分,能够通过多种方式对抗病原菌。本文就临床生物被膜治疗中亟待解决的问题做出了部分总结,归纳总结了部分益生菌生物被膜对抗病原菌生物被膜的作用机制,而且还从改进益生菌生物被膜研究方法、增强益生菌生物被膜稳定性和开发新型生物被膜态益生菌的角度出发,就如何开发生物被膜态益生菌的策略方法进行了综述。     

Assessing the Contamination Potential of Freshly Extracted Escherichia coli Biofilm Cells by Impedancemetry

Planktonic bacteria passing to a sessile state during the formation of a biofilm undergo many gene expression and phenotypic changes. These transformations require a significant time to establish. Inversely, cells extracted from a biofilm should also require a significant time before acquiring the same physiological characteristics as planktonic cells. Relatively few studies have addressed the kinetics of this inverse transformation process. We tested one aspect, namely, the contamination potential of freshly extracted Escherichia coli biofilm cells, precultured in a synthetic medium, in a rich liquid growth medium. We compared the time between inoculation and the beginning of the growth phase of freshly extracted biofilm cells, and suspended exponential and suspended stationary phase cells precultured in the same synthetic medium. Unexpectedly, the lag time for the extracted biofilm cells was the same as the lag time of the suspended exponential phase cells and significantly less than the lag time of the suspended stationary phase cells. The lag times were determined by an impedance technique. Cells extracted from biofilms, i.e., biofilms formed in canalizations and broken up by hydrodynamic forces, are an important source of contamination. Our work shows, in the case of E. coli, the high potential of freshly extracted biofilm cells to reinfect a new medium.     

Development and structure of drinking water biofilms and techniques for their study

Drinking water systems are known to harbour biofilms, even though these environments are oligotrophic and often contain a disinfectant. Control of these biofilms is important for aesthetic and regulatory reasons. Study of full-scale systems has pointed to several factors controlling biofilm growth, but cause-and-effect relationships can only be established in controlled reactors. Using laboratory and pilot distribution systems, along with a variety of bacterial detection techniques, insights have been gained on the structure and behaviour of biofilms in these environments. Chlorinated biofilms differ in structure from non-chlorinated biofilms, but often the number of cells is similar. The number and level of cellular activity is dependent on the predominant carbon source. There is an interaction between carbon sources, the biofilm and the type of pipe material, which complicates the ability to predict biofilm growth. Humic substances, which are known to sorb to surfaces, appear to be a usable carbon source for biofilms. The finding offers an explanation for many of the puzzling observations in full scale and laboratory studies on oligotrophic biofilm growth. Pathogens can persist in these environments as well. Detection requires methods that do not require culturing.     

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms

Aims: It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. Methods and Results: It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3′‐[1‐[(phenylamino)carbonyl]‐3,4‐tetrazolium]Bis(4‐methoxy)‐6‐nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. Conclusions: We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. Significance and Impact of the Study: This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.     

Candida biofilms on implanted biomaterials: a clinically significant problem

In recent years there has been an increasing appreciation that microbial biofilms are ubiquitous, which has resulted in a number of studies on infectious diseases from a biofilm perspective. Biofilms are defined as structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. A wide range of biomaterials used in clinical practice have been shown to support colonization and biofilm formation by Candida spp., and the increase in Candida infections in the last decades has almost paralleled the increase and widespread use of a broad range of medical implant devices, mainly in populations with impaired host defenses. Formation of Candida biofilms has important clinical repercussions because of their increased resistance to antifungal therapy and the ability of cells within biofilms to withstand host immune defenses. Further recognition and understanding of the role of Candida biofilms in human infection should help in the clinical management of these recalcitrant infections.     

Proteomic and functional analyses reveal a unique lifestyle for Acinetobacter baumannii biofilms and a key role for histidine metabolism

Biofilm formation is one of the main causes for the persistence of Acinetobacter baumannii, a pathogen associated with severe infections and outbreaks in hospitals. Here, we performed comparative proteomic analyses (2D-DIGE and MALDI-TOF/TOF and iTRAQ/SCX-LC-MS/MS) of cells at three different conditions: exponential, late stationary phase, and biofilms. These results were compared with alterations in the proteome resulting from exposure to a biofilm inhibitory compound (salicylate). Using this multiple-approach strategy, proteomic patterns showed a unique lifestyle for A. baumannii biofilms and novel associated proteins. Several cell surface proteins (such as CarO, OmpA, OprD-like, DcaP-like, PstS, LysM, and Omp33), as well as those involved in histidine metabolism (like Urocanase), were found to be implicated in biofilm formation, this being confirmed by gene disruption. Although l-His uptake triggered biofilms efficiently in wild-type A. baumannii, no effect was observed in Urocanase and OmpA mutants, while a slight increase was observed in a CarO deficient strain. We conclude that Urocanase plays a crucial role in histidine metabolism leading to biofilm formation and that OmpA and CarO can act as channels for L-His uptake. Finally, we propose a model in which novel proteins are suggested for the first time as targets for preventing the formation of A. baumannii biofilms.     

Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology

Until recently, microbial identification in clinical diagnostic laboratories has mainly relied on conventional phenotypic and gene sequencing identification techniques. The development of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) devices has revolutionized the routine identification of microorganisms in clinical microbiology laboratories by introducing an easy, rapid, high throughput, low-cost, and efficient identification technique. This technology has been adapted to the constraint of clinical diagnostic laboratories and has the potential to replace and/or complement conventional identification techniques for both bacterial and fungal strains. Using standardized procedures, the resolution of MALDI-TOF MS allows accurate identification at the species level of most Gram-positive and Gram-negative bacterial strains with the exception of a few difficult strains that require more attention and further development of the method. Similarly, the routine identification by MALDI-TOF MS of yeast isolates is reliable and much quicker than conventional techniques. Recent studies have shown that MALDI-TOF MS has also the potential to accurately identify filamentous fungi and dermatophytes, providing that specific standardized procedures are established for these microorganisms. Moreover, MALDI-TOF MS has been used successfully for microbial typing and identification at the subspecies level, demonstrating that this technology is a potential efficient tool for epidemiological studies and for taxonomical classification.     

Enhanced visualization of microbial biofilms by staining and environmental scanning electron microscopy

Bacterial biofilms, i.e. surface-associated cells covered in hydrated extracellular polymeric substances (EPS), are often studied with high-resolution electron microscopy (EM). However, conventional desiccation and high vacuum EM protocols collapse EPS matrices which, in turn, deform biofilm appearances. Alternatively, wet-mode environmental scanning electron microscopy (ESEM) is performed under a moderate vacuum and without biofilm drying. If completely untreated, however, EPS is not electron dense and thus is not resolved well in ESEM. Therefore, this study was towards adapting several conventional SEM staining protocols for improved resolution of biofilms and EPS using ESEM. Three different biofilm types were used: 1) Pseudomonas aeruginosa unsaturated biofilms cultured on membranes, 2) P. aeruginosa cultured in moist sand, and 3) mixed community biofilms cultured on substrates in an estuary. Working with the first specimen type, a staining protocol using ruthenium red, glutaraldehyde, osmium tetroxide and lysine was optimized for best topographic resolution. A quantitative image analysis tool that maps relief, newly adopted here for studying biofilms, was used to compare micrographs. When the optimized staining and ESEM protocols were applied to moist sand cultures and aquatic biofilms, the smoothening effect that bacterial biofilms have on rough sand, and the roughening that aquatic biofilms impart on initially smooth coupons, were each quantifiable. This study thus provides transferable staining and ESEM imaging protocols suitable for a wide range of biofilms, plus a novel tool for quantifying biofilm image data.     

A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms

Aims: Research on biofilms requires validated quantitative models that focus both on matrix and viable bacterial mass. In this study, a new microplate model for the detection of Staphylococcus aureus biofilms was developed. Methods and Results: Dimethyl methylene blue (DMMB) dye was used to quantify biofilm matrix colorimetrically. Initially developed for the detection of glycosaminoglycans, the DMMB protocol was optimized for S. aureus biofilm research. In addition, the redox indicator resazurin was used to determine the viable bacterial biofilm burden. Conclusion: A new, simple and reproducible microplate test system based on DMMB and resazurin, offering a reliable differentiation between biofilm matrix and cellular activity, was developed and validated for the detection of S. aureus biofilms. Significance and Impact of the Study: The DMMB–resazurin microtitre plate model is a valuable tool for high capacity screening of biocides and for the development of synergistic mixtures of biocides, destroying both biofilm matrix and bacteria.     

MALDI-TOF MS and Filamentous Fungal Identification: A Success Story?

Purpose of Review

The diagnosis of invasive fungal disease remains challenging in the clinical laboratory. In this paper, the use of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of filamentous fungi as well as its application for antifungal resistance testing and strain typing is evaluated.

Recent Findings

Most studies report very high accuracy for the identification of filamentous fungi by MALDI-TOF MS. Its cost effectiveness, short analysis time, and low error rate and the fact that it can also discriminate between closely related and cryptic species make it appropriate for implementation in the clinical routine. Two drawbacks remain in the availability of extended reference spectra databases and the fact that this technique can only be applied on isolates.

Summary

More work on (simultaneous) antifungal susceptibility testing and strain typing is needed. The application of MALDI-TOF MS directly on clinical specimens would further improve the diagnosis of invasive fungal disease and improve its successful management.

     

Carbon Catabolite Control is Important for Listeria monocytogenes Biofilm Formation in Response to Nutrient Availability

The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. The biofilm formation is strongly influenced by the availability of nutrients and environmental conditions, and particularly enhanced in poor minimal essential medium (MEM) containing glucose rather than in rich brain heart infusion (BHI) broth. To gain better insight into the conserved protein expression profile in these biofilms, the proteomes from biofilm- and planktonic-grown cells from MEM with 50?mM glucose or BHI were compared using two-dimensional polyacrylamide gel electrophoresis followed by MALDI-TOF/TOF analysis. 47 proteins were successfully identified to be either up (19 proteins) or down (28 proteins) regulated in the biofilm states. Most (30 proteins) of them were assigned to the metabolism functional category in cluster of orthologous groups of proteins. Among them, up-regulated proteins were mainly associated with the pentose phosphate pathway and glycolysis, whereas a key enzyme CitC involved in tricarboxylic acid cycle was down-regulated in biofilms compared to the planktonic states. These data implicate the importance of carbon catabolite control for L. monocytogenes biofilm formation in response to nutrient availability.