Phase-variable bacteria simultaneously express multiple capsules

Capsular polysaccharides (CPSs) protect bacteria from host and environmental factors. Many bacteria can express different CPSs and these CPSs are phase variable. For example, Bacteroides thetaiotaomicron (B. theta) is a prominent member of the human gut microbiome and expresses eight different capsular polysaccharides. Bacteria, including B. theta, have been shown to change their CPSs to adapt to various niches such as immune, bacteriophage, and antibiotic perturbations. However, there are limited tools to study CPSs and fundamental questions regarding phase variance, including if gut bacteria can express more than one capsule at the same time, remain unanswered. To better understand the roles of different CPSs, we generated a B. theta CPS1-specific antibody and a flow cytometry assay to detect CPS expression in individual bacteria in the gut microbiota. Using these novel tools, we report for the first time that bacteria can simultaneously express multiple CPSs. We also observed that nutrients such as glucose and salts had no effect on CPS expression. The ability to express multiple CPSs at the same time may provide bacteria with an adaptive advantage to thrive amid changing host and environmental conditions, especially in the intestine.

High-Resolution Imaging of Bacterial Spatial Organization with Vertical Cell Imaging by Nanostructured Immobilisation (VerCINI)

Light microscopy is indispensable for analysis of bacterial spatial organization. However, imaging in bacteria is difficult due to their small sizes and the fact that most species are non-spherical, meaning they typically lie horizontally on a microscope coverslip. This is especially problematic when considering that many essential bacterial processes—such as cell division—occur along the short axes of these cells, and so are viewed side-on by standard microscopy. We recently developed a pair of methods to overcome this problem by forcing cells to stand vertically during imaging, named VerCINI (Vertical Cell Imaging by Nanostructured Immobilisation) and µVerCINI (Microfluidic VerCINI). The concept behind both methods is that cells are imaged while confined vertically inside cell traps made from a nanofabricated mould. By doing so, the short axes of the cells are rotated parallel to the microscope imaging plane and are imaged with high resolution. μVerCINI combines the vertical cell confinement with microfluidics so that vertical imaging can be done during fluid exchange, such as during antibiotic perturbations. Here, we provide a practical guide to implementing both VerCINI and µVerCINI, with detailed protocols and experience-based tips so that interested researchers can easily use one or both imaging methods to complement their current approaches.

Manipulation of the Gut Microbiota Reveals Role in Colon Tumorigenesis

ABSTRACT Mounting evidence indicates that alterations to the gut microbiota, the complex community of bacteria that inhabits the gastrointestinal tract, are strongly associated with the development of colorectal cancer. We used antibiotic perturbations to a murine model of inflammation-driven colon cancer to generate eight starting communities that resulted in various severities of tumorigenesis. Furthermore, we were able to quantitatively predict the final number of tumors on the basis of the initial composition of the gut microbiota. These results further bolster the evidence that the gut microbiota is involved in mediating the development of colorectal cancer. As a final proof of principle, we showed that perturbing the gut microbiota in the midst of tumorigenesis could halt the formation of additional tumors. Together, alteration of the gut microbiota may be a useful therapeutic approach to preventing and altering the trajectory of colorectal cancer. There is growing evidence that individuals with colonic adenomas and carcinomas harbor a distinct microbiota. Alterations to the gut microbiota may allow the outgrowth of bacterial populations that induce genomic mutations or exacerbate tumor-promoting inflammation. In addition, it is likely that the loss of key bacterial populations may result in the loss of protective functions that are normally provided by the microbiota. We explored the role of the gut microbiota in colon tumorigenesis by using an inflammation-based murine model. We observed that perturbing the microbiota with different combinations of antibiotics reduced the number of tumors at the end of the model. Using the random forest machine learning algorithm, we successfully modeled the number of tumors that developed over the course of the model on the basis of the initial composition of the microbiota. The timing of antibiotic treatment was an important determinant of tumor outcome, as colon tumorigenesis was arrested by the use of antibiotics during the early inflammation period of the murine model. Together, these results indicate that it is possible to predict colon tumorigenesis on the basis of the composition of the microbiota and that altering the gut microbiota can alter the course of tumorigenesis. IMPORTANCE Mounting evidence indicates that alterations to the gut microbiota, the complex community of bacteria that inhabits the gastrointestinal tract, are strongly associated with the development of colorectal cancer. We used antibiotic perturbations to a murine model of inflammation-driven colon cancer to generate eight starting communities that resulted in various severities of tumorigenesis. Furthermore, we were able to quantitatively predict the final number of tumors on the basis of the initial composition of the gut microbiota. These results further bolster the evidence that the gut microbiota is involved in mediating the development of colorectal cancer. As a final proof of principle, we showed that perturbing the gut microbiota in the midst of tumorigenesis could halt the formation of additional tumors. Together, alteration of the gut microbiota may be a useful therapeutic approach to preventing and altering the trajectory of colorectal cancer.

Antibiotic-Induced Alterations of the Murine Gut Microbiota and Subsequent Effects on Colonization Resistance against Clostridium difficile

ABSTRACTPerturbations to the gut microbiota can result in a loss of colonization resistance against gastrointestinal pathogens such asClostridium difficile. AlthoughC. difficileinfection is commonly associated with antibiotic use, the precise alterations to the microbiota associated with this loss in function are unknown. We used a variety of antibiotic perturbations to generate a diverse array of gut microbiota structures, which were then challenged withC. difficilespores. Across these treatments we observed thatC. difficileresistance was never attributable to a single organism, but rather it was the result of multiple microbiota members interacting in a context-dependent manner. Using relative abundance data, we built a machine learning regression model to predict the levels ofC. difficilethat were found 24 h after challenging the perturbed communities. This model was able to explain 77.2% of the variation in the observed number ofC. difficileper gram of feces. This model revealed important bacterial populations within the microbiota, which correlation analysis alone did not detect. Specifically, we observed that populations associated with thePorphyromonadaceae,Lachnospiraceae,Lactobacillus, andAlistipeswere protective and populations associated withEscherichiaandStreptococcuswere associated with high levels of colonization. In addition, a population affiliated with theAkkermansiaindicated a strong context dependency on other members of the microbiota. Together, these results indicate that individual bacterial populations do not drive colonization resistance toC. difficile. Rather, multiple diverse assemblages act in concert to mediate colonization resistance.IMPORTANCEThe gastrointestinal tract harbors a complex community of bacteria, known as the microbiota, which plays an integral role preventing its colonization by gut pathogens. This resistance has been shown to be crucial for protection againstClostridium difficileinfections (CDI), which are the leading source of hospital-acquired infections in the United States. Antibiotics are a major risk factor for acquiring CDI due to their effect on the normal structure of the indigenous gut microbiota. We found that diverse antibiotic perturbations gave rise to altered communities that varied in their susceptibility toC. difficilecolonization. We found that multiple coexisting populations, not one specific population of bacteria, conferred resistance. By understanding the relationships betweenC. difficileand members of the microbiota, it will be possible to better manage this important infection.

Temporal profiling of the bacterial and fungal communities in ΔF508 adult cysticfibrosis sputum

Aims: The purpose of this study was to analyse the bacterial and fungal turnover in the lungs of cystic fibrosis patients who were ΔF508 homo- and hetero-zygotes. Further to this we wanted to identify the effects that Intravenous (IV) antibiotic perturbations had on the community and most importantly, whether exacerbations in these patients could be attributed to microbial species or communities. Methods: A total of 149 samples were collected from 18 adult CF patients attending a clinic at the RVI hospital, Newcastle upon Tyne. The samples were subject to DNA extraction followed by bacterial and fungal community DGGE analysis as well as qPCR analysis of the bacterial load. Results: We have found that bacterial and fungal communities present in the CF lung are not different when patients are suffering an exacerbation. Further to this, we have found that bacterial communities in the CF lung are disturbed by IV antibiotic administration and cause increased species turnover. We have shown that fungal taxa are capable of chronically colonising the CF lung. Conclusions: Our study adds further evidence to the assertion that changes in bacterial communities are not the cause of CF exacerbations. However, we were able to demonstrate that acquisition of new bacterial taxa was strongly associated with exacerbations in one patient. This study is the first to illustrate that fungi can persist in the CF lung but are not associated with clinical status.

Temporal profiling of the bacterial and fungal communities in ΔF508 adult cysticfibrosis sputum

Aims: The purpose of this study was to analyse the bacterial and fungal turnover in the lungs of cystic fibrosis patients who were ΔF508 homo- and hetero-zygotes. Further to this we wanted to identify the effects that Intravenous (IV) antibiotic perturbations had on the community and most importantly, whether exacerbations in these patients could be attributed to microbial species or communities. Methods: A total of 149 samples were collected from 18 adult CF patients attending a clinic at the RVI hospital, Newcastle upon Tyne. The samples were subject to DNA extraction followed by bacterial and fungal community DGGE analysis as well as qPCR analysis of the bacterial load. Results: We have found that bacterial and fungal communities present in the CF lung are not different when patients are suffering an exacerbation. Further to this, we have found that bacterial communities in the CF lung are disturbed by IV antibiotic administration and cause increased species turnover. We have shown that fungal taxa are capable of chronically colonising the CF lung. Conclusions: Our study adds further evidence to the assertion that changes in bacterial communities are not the cause of CF exacerbations. However, we were able to demonstrate that acquisition of new bacterial taxa was strongly associated with exacerbations in one patient. This study is the first to illustrate that fungi can persist in the CF lung but are not associated with clinical status.