Studying bacterial infections through culture-independent approaches

Molecular Microbiology Research Laboratory, Pharmaceutical Science Division, Franklin-Wilkins Building, King's College London, 150 Stamford Street, London SE1 9NH, UK.
Journal of Medical Microbiology (Impact Factor: 2.25). 06/2009; 58(Pt 11):1401-18. DOI: 10.1099/jmm.0.013334-0
Source: PubMed


The ability to characterize accurately the cause of infection is fundamental to effective treatment. The impact of any antimicrobial agents used to treat infection will, however, always be constrained by both the appropriateness of their use and our ability to determine their effectiveness. Traditional culture-based diagnostic microbiology is, in many cases, unable to provide this information. Molecular microbiological approaches that assess the content of clinical samples in a culture-independent manner promise to change dramatically the types of data that are obtained routinely from clinical samples. We argue that, in addition to the technical advance that these methodologies offer, a conceptual advance in the way that we reflect on the information generated is also required. Through the development of both of these advances, our understanding of infection, as well as the ways in which infections can be treated, may be improved. In the analysis of the microbiological content of certain clinical samples, such as blood, cerebrospinal fluid, brain and bone biopsy, culture-independent approaches have been well documented. Herein, we discuss how extensions to such studies can shape our understanding of infection at the many sites of the human body where a mixed flora, or in more ecological terms, a community of microbes, is present. To do this, we consider the underlying principles that underpin diagnostic systems, describe the ways in which these systems can be applied to community characterization, and discuss the significance of the data generated. We propose that at all locations within the human body where infection is routinely initiated within the context of a community of microbes, the same principles will apply. To consider this further, we take insights from areas such as the gut, oral cavity and skin. The main focus here is understanding respiratory tract infection, and specifically the infections of the cystic fibrosis lung. The impact that the use of culture-independent, molecular analyses will have on the way we approach the treatment of infections is also considered.

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    • "D. nodosus and F. necrophorum have been detected using a range of culture-dependent and -independent techniques from both swabs and biopsies collected from the interdigital skin of sheep with ID and SFR (Bennett et al., 2009; Calvo-Bado et al., 2011; Witcomb et al., 2014). The advantages and disadvantages of culture-dependent and independent methods have been discussed elsewhere (Rogers et al., 2009), but due to the fastidious nature of both anaerobes, PCR is more sensitive than culturing methods (Moore et al., 2005). Additionally problematic is the marked pleomorphism of F. necrophorum and other bacteria and the limited morphologies presented within a genus; making reliable identification of F. necrophorum and D. nodosus using morphology alone prone to error (Hofstad, 2006; Young, 2007). "
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    ABSTRACT: Analysis of bacterial populations in situ provides insights into pathogen population dynamics and potential reservoirs for disease. Here we report a culture-independent study of ovine footrot (FR); a debilitating bacterial disease that has significant economic impact on sheep farming worldwide. Disease begins as an interdigital dermatitis (ID), which may then progress to separation of the hoof horn from the underlying epidermis causing severe footrot (SFR). Dichelobacter nodosus is the causative agent of ovine FR, however, the role of Fusobacterium necrophorum and other bacteria present in the environment and on the feet of sheep is less clear. The objective of this study was to use fluorescence in situ hybridisation (FISH) to detect, localise and quantify D. nodosus, F. necrophorum and the domain Bacteria from interdigital skin biopsies of healthy, ID- and SFR-affected feet. D. nodosus and F. necrophorum populations were restricted primarily to the epidermis, but both were detected more frequently in feet with ID or SFR than in healthy feet. D. nodosus cell counts were significantly higher in feet with ID and SFR (p<0.05) than healthy feet, whereas F. necrophorum cell counts were significantly higher only in feet with SFR (p<0.05) than healthy feet. These results, together with other published data, indicate that D. nodosus likely drives pathogenesis of footrot from initiation of ID to SFR; with D. nodosus cell counts increasing prior to onset of ID and SFR. In contrast, F. necrophorum cell counts increase after SFR onset, which may suggest an accessory role in disease pathogenesis, possibly contributing to the severity and duration of SFR. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
    Veterinary Microbiology 02/2015; 176(3-4). DOI:10.1016/j.vetmic.2015.01.022 · 2.51 Impact Factor
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    • "This is because the largest portion of microorganisms (generally more that 99%) cannot be cultivated under standard laboratory conditions (Amann et al. 1995). Well studied environments that display viable but not culturable microorganisms include soil (Torsvik et al. 1990); the activated-sludge process for waste-water treatment (Wagner et al. 1993); clinical samples exhibiting mixed communities of biofilm-forming bacteria resistant to antibiotics (Rogers et al. 2009); and foods and beverages (Mamlouk et al. 2009). For these reasons, a combination of culture-dependent and independent methods can maximize the estimation of microbial richness in complex ecosystems, as well as the detection of non-cultivable but functional microbes. "
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    ABSTRACT: The paper production process is significantly affected by direct and indirect effects of microorganism proliferation. Microorganisms can be introduced in different steps. Some microorganisms find optimum growth conditions and proliferate along the production process, affecting both the end product quality and the production efficiency. The increasing need to reduce water consumption for economic and environmental reasons has led most paper mills to reuse water through increasingly closed cycles, thus exacerbating the bacterial proliferation problem. In this work, microbial communities in a paper mill located in Italy were characterized using both culture-dependent and independent methods. Fingerprinting molecular analysis and 16S rRNA library construction coupled with bacterial isolation were performed. Results highlighted that the bacterial community composition was spatially homogeneous along the whole process, while it was slightly variable over time. The culture-independent approach confirmed the presence of the main bacterial phyla detected with plate counting, coherently with earlier cultivation studies ( Proteobacteria, Bacteroidetes, and Firmicutes), but with a higher genus diversification than previously observed. Some minor bacterial groups, not detectable by cultivation, were also detected in the aqueous phase. Overall, the population dynamics observed with the double approach led us to hypothesize a possible role of suspended bacteria in the re-formation mechanisms of resistant biofilms.
    Bioresources 02/2014; 9(2):2541-2559. DOI:10.15376/biores.9.2.2541-2559 · 1.43 Impact Factor
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    • "Although a limited number of bacterial species including Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus and Haemophilus influenzae have been established as important CF pathogens [1], it is now appreciated that CF airway infections are more broadly polymicrobial in nature. Furthermore, culture-independent methodologies have revealed that the bacterial communities present are even more diverse than previously realised [2], [3]. A range of these techniques has been deployed: generation of 16S rRNA clone libraries [4], [5], terminal restriction fragment length polymorphism analysis [2], [6], microarray hybridisation [7], phylochip analysis [8] and pyrosequencing [9]. "
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    PLoS ONE 12/2013; 8(12):e82432. DOI:10.1371/journal.pone.0082432 · 3.23 Impact Factor
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