Tag Archives: bacteria

Microbial forensics: the science behind the Amerithrax investigation

Nearly a decade after the postal anthrax attacks in the USA that killed 5 individuals and infected more than 20 people, scientists have revealed the measures used to trace the Bacillus anthracis strain used in the bioterror attack in a new paper available online for free from Proceedings of the National Academy of Sciences. A groundbreaking mix of genomics and microbiology were used as part of the criminal investigation  into the 2001 anthrax attacks (called Amerithrax); microbial forensics proved key to identifying the exact flask from which the anthrax spores were taken.

Rasko and colleagues used highly accurate whole-genome sequencing and comparative genomics (against the B. anthracis Ames ancestor, believed to be the progenitor of all Ames lab samples and used as a gold standard reference strain in the USA) to determine the source strain of B. anthracis used in the letter attacks. First, the scientists took spore samples from some of the letters and grew them in the lab. A number of morphological variants were observed in these letter-isolated bacterial samples (yellow or yellow–grey coloured rather than the usual grey–white of wild-type anthrax colonies) and all had diminished abilities to sporulate. These variants were then sequenced and compared with genomes sequences of the gold standard Ames ancestor to identify four distinct loci with genetic mutations (three of which were in B. anthracis sporulation pathways, specifically regulation of a key protein, Spo0F) in the morpholigical variants—features unique to the isolated anthrax variants. None of these variants were found to be prevalent in the environment (even in the areas associated with the Amerithrax investigation).

Ultimately, using comparisons with genomes of repository anthrax sources, the anthrax spores used to lace the letters were found to have a unique genetic fingerprint; anthrax batches were eventually traced back to a source flask (RMR-1029) in the lab of Dr Bruce Ivins (a key suspect in the subsequent criminal investigation who later committed suicide before a criminal case could be brought to trial).

The study authors conclude that the B. anthracis bioterror attack investigations “taught us important lessons about the integration of whole-genome sequencing for forensic applications”, although they do concede that their methods might not applicable to other bioterror agents.

ResearchBlogging.orgRasko, D., Worsham, P., Abshire, T., Stanley, S., Bannan, J., Wilson, M., Langham, R., Decker, R., Jiang, L., Read, T., Phillippy, A., Salzberg, S., Pop, M., Van Ert, M., Kenefic, L., Keim, P., Fraser-Liggett, C., & Ravel, J. (2011). Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1016657108

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CFTR aids Listeria escape into cell cytosol

Courtesy of CDC/ Dr. Balasubr Swaminathan; Peggy Hayes

The intracellular pathogen Listeria monocytogenes must escape the vacuole formed during entry into the host cell to replicate in its preferred environment—the cell cytosol—and continues its life cycle. Although the pore-forming bacterial toxin listeriolysin O is vital for Listeria escape and virulence, new research by Radtke and colleagues published online in PNAS shows that a host cell protein, CFTR (cystic fibrosis transmembrane conductance regulator, which forms a chloride ion channel that, incidentally, when dysfunctional results in cystic fibrosis), promotes escape of L. monocyotgenes from intracellular vacuoles.

Radtke et al. reasoned that, as the intravacuolar environment is dynamic and likely modulated by a variety of proteins, regulation of ion flux whilst Listeria is inside a vacuole could affect its subsequent escape from this membrane-bound organelle. The researchers confirmed that CFTR was endogenously expressed by mouse macrophages and addition of a CFTR inhibitor did not affect uptake of Listeria into host cells but did reduce the number of intracellular bacteria, indicating that the bacteria might be trapped within the vacuole. Using macrophages isolated from either wild-type mice or mice carrying the CFTR mutation associated with human cystic fibrosis, they found that defects in CFTR led to delayed intracellular replication (indicative of a defect in vacuole escape). Finally, the researchers conclude that CFTR potentially promotes escape of Listeria by controlling the flux of chlorides into the vacuole—a high chloride concentration seems to increase both the oligomerisation and haemolytic activity of listeriolysin O, the key bacterial toxin needed for escape.


Little is known about the role of ion transport in the context of bacterial infection and it would be interesting to see whether other ion channels and transporters also contribute to the virulence of Listeria and other intracellular bacteria.


 ResearchBlogging.orgRadtke, A., Anderson, K., Davis, M., DiMagno, M., Swanson, J., & O’Riordan, M. (2011). Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1013262108

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Spreading Salmonella—hyper-replicating bacteria act as a reservoir for dissemination

New research reveals how Salmonella enterica spread in the gut and gallbladder—a subpopulation of Salmonella primed for invasion rapidly replicate in the host cell cytosol such that bacteria-laden cells are extruded out of the epithelial-cell layer releasing invasive Salmonella into the gastrointestinal and biliary lumen. Leigh Knodler and colleagues write that other mucosal-dwelling pathogens could use this “host cell process as an exit strategy”.

Salmonella species can cause a range of infections from typhoid fever to food poisoning. Ordinarily, the intracellular bacteria Salmonella enterica resides and replicates within a membrane-bound vacuole in epithelial cells. During its life cycle, the bacteria are adapted to survive within a wide range of environmental niches within the human host (including cells such as enterocytes and macrophages and organs such as the spleen and gastrointestinal tract).

Knodler et al. observed a subpopulation of Salmonella that were ‘hyper-replicating’; these bacteria were doubling in number at almost five times the rate of the overall population of bacteria in the epithelial cell. Not only that, these bacteria were rapidly proliferating not in the Salmonella-containing vacuole, but in the host cell cytosol (which is believed to be nutrient rich) and were ready to invade other cells (they expressed type III secretion system 1 components and flagella, virulence factors that are required for invasion). Moreover, epithelial cells overloaded with these hyper-replicating cytosolic Salmonella were forced out of the apical side of the epithelial-cell layer—just as when dying cells are extruded out of the epithelium during the normal rapid turnover of epithelial cells that occurs to maintain the gut epithelium. Subsequently, invasive bacteria are released into the lumen and are primed and ready to infect new cells. The extruded host cells then die in a caspase-1-dependent manner and trigger the production of the proinflammatory cytokine interleukin 18—a process which could, in part, explain the high levels of mucosal inflammation observed in Salmonella infections of the gut and gallbladder.

ResearchBlogging.orgKnodler, L., Vallance, B., Celli, J., Winfree, S., Hansen, B., Montero, M., & Steele-Mortimer, O. (2010). Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia Proceedings of the National Academy of Sciences, 107 (41), 17733-17738 DOI: 10.1073/pnas.1006098107

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Filed under Infectious Disease, Microbiology, Uncategorized

Walking with bacteria

They swim, they swarm, they twitch and glide…they even ride on comet tails, and now it seems that bacteria can ‘walk’ as Maxsim Gibiansky and colleagues demonstrate in their short but sweet research published in Science.

Gibiansky et al. studied the behaviour of Pseudomonas aeruginosa, a bacteria that is ordinarily found in soil and water, but has increasingly been associated with opportunistic infections in humans (and is a particular problem in those with cystic fibrosis). A key feature of P. aeruginosa is that these bacteria form multicellular, surface-bound communities called biofilms and are able to move within these communities by twitching motility owing to their type IV pili (hair-like structures on bacteria that can extend, tether to a surface and then retract to move bacteria along). The researchers studied microscopy movies of the P. aeruginosa biofilms and used computer software to track how the bacteria transitioned from planktonic state (that is, freely suspended in liquid) to the surface-bound biofilm.

Two different surface motility mechanisms were observed just after P. aeruginosa bacteria attached to a surface, but before a microcolony of bacteria were formed. The scientists studied mutant bacteria lacking flagella (a tail-like bacterial appendage that can also enable bacteria to move) that can only move using their type IV pili. These bacteria tended to ‘crawl’ in one direction when positioned horizontal to the surface and ‘walked’ in all directions when attached vertically to the surface by one end of the bacteria. Each movement mechanism was useful for surface exploration; crawling enabled directional movement across larger areas (6 μm distance) than walking, which enabled rapid exploration in local areas (up to 2 μm distance). Furthermore, these same movements were observed in wild-type bacteria. Moreover, the orientation of bacteria influenced biofilm morphology. Surface detachment was facilitated by type IV pili by tilting bacteria from horizontal to vertical positions and after bacterial division newborn bacteria detach and then ‘walk’ away. Finally, bacteria lacking type IV pili could neither ‘crawl’ or ‘walk’.

Scientific observations like this brevia report add to the understanding of bacterial behaviour in biofilms and could eventually lead to useful, new treatments against biofilm-forming pathogens.


ResearchBlogging.orgGibiansky, M., Conrad, J., Jin, F., Gordon, V., Motto, D., Mathewson, M., Stopka, W., Zelasko, D., Shrout, J., & Wong, G. (2010). Bacteria Use Type IV Pili to Walk Upright and Detach from Surfaces Science, 330 (6001), 197-197 DOI: 10.1126/science.1194238

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Altruistic bacterial charity workers help protect their vulnerable stressed out kin

US scientists have found that a small minority of highly antibiotic-resistant bacteria will produce and share a molecule, indole, that can activate survival mechanisms in less-resistant cells to enable the whole bacterial population to survive stressful environments despite the fact that production of this signalling molecule weakens the fitness of bacteria.

The increasing incidence of antibiotic resistance and the emergence of so-called ‘superbugs’ are of huge importance to medicine and society as a whole with the ever-increasing likelihood of a return to a world without antibiotics. This potentially disastrous public health crisis led the Infectious Diseases Society of America to launch the “10x’20” initiative in which they call for a global commitment to research and develop 10 new, effective antibiotic drugs by 2020. As a complement to this drug development, research into how bacteria develop this resistance could provide crucial clues for the rational design of new antimicrobial agents.

Henry Lee and colleagues investigated the population dynamics of antibiotic resistance. They grew a vat of E. coli with increasing amounts of the antibiotic norfloxacin and then took samples of the bacteria and monitored the percentage of bacteria that became resistant to the antibiotic. The scientists found an individual isolate that was highly resistant to norfloxacin (even higher than the greatest norfloxacin levels tested in their bioreactor). These bacteria produced indole, which is known to aid tolerance to stress in E. coliindole induces anti-stress mechanisms such as drug efflux pumps that help drive out toxic substances from the bacterial cell—although its production can reduce the overall fitness of the bacteria. Moreover, indole boosts the antibiotic resistance of the whole bacterial population and not just the select few that produce it. This population-based resistance was not drug specific and was even observed when the scientists challenged E. coli with gentamicin, which is a different type of antibiotic (with a different mode of action) to the quinolone norfloxacin.

The researchers conclude that under antibiotic stress, a few drug-resistant mutants will endure a fitness cost to produce and share the benefits of the metabolite indole to “shield the less-resistant bacteria from antibiotic insult” and enable these ‘weak’ bacteria to survive.

ResearchBlogging.orgLee HH, Molla MN, Cantor CR, & Collins JJ (2010). Bacterial charity work leads to population-wide resistance. Nature, 467 (7311), 82-5 PMID: 20811456

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The MetaHIT catalogue 2010— your gut microbiome directory

An international team of scientists have produced a catalogue of genes from the micro-organisms that live in our gut (the gut microbiome), and it is the first published work from the MetaHIT (Metagenomics of the Human Intestinal Tract) project. “This gene catalogue contains virtually all of the prevalent gut microbial genes in our cohort, provides a broad view of the functions important for bacterial life in the gut and indicates that many bacterial species are shared by different individuals,” write Junjie Qin and colleagues.

The research, published last week in the journal Nature, pieces together a staggering 576.6 gigabases of gene sequence to assemble and characterise 3.3 million non-redundant microbial genes from faecal samples from124 European individuals. The results provide a vital clue to the microbial species which are prevalent, and surprisingly common between different individuals, in the human gut.

The human body hosts trillions of micro-organism, most of which live in our gut. These gut bacteria are hugely important for human life, not only do they help us to get vital energy from the food we eat but changes in the types of micro-organisms in the gut are thought to contribute to bowel disease and obesity.

The researchers used an Illumina Genome Analyser to deep sequence DNA from faecal samples from Danish and Spanish adults who were healthy, overweight and obese, or had inflammatory bowel disease. This approach is called metagenomics and directly analyses genetic material from environmental samples, which means that organisms can be studied in their natural habitat and allows otherwise difficult-to-culture micro-organisms to be studied.

Qin et al. generated almost 200 times more metagenomic sequence data from the gut than had been produced in previous studies. The scientists found that their gene set was 150 times bigger than the human gene complement and included most of the known human intestinal microbial genes. Furthermore, their analysis revealed that 99% of the genes they identified were bacterial and that a common core of bacterial species existed in each person— including members of the Bacteriodetes and the Firmicutes, which have already been shown to be abundant in the gut environment. Finally, they used their gene catalogue to uncover the bacterial functions which are important for life in this habitat, such as synthesis of short-chain fatty acids, vital amino acids and vitamins, and the breakdown of complex polysaccharides.

“We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively,” conclude the investigators who hope that their extensive catalogue of the human gut microbiome will enable future studies of the association between microbial genes and human phenotypes, disease and living habits from birth to old age.

ResearchBlogging.orgQin, J., Li, R., Raes, J., Arumugam, M., Burgdorf, K., Manichanh, C., Nielsen, T., Pons, N., Levenez, F., Yamada, T., Mende, D., Li, J., Xu, J., Li, S., Li, D., Cao, J., Wang, B., Liang, H., Zheng, H., Xie, Y., Tap, J., Lepage, P., Bertalan, M., Batto, J., Hansen, T., Le Paslier, D., Linneberg, A., Nielsen, H., Pelletier, E., Renault, P., Sicheritz-Ponten, T., Turner, K., Zhu, H., Yu, C., Li, S., Jian, M., Zhou, Y., Li, Y., Zhang, X., Li, S., Qin, N., Yang, H., Wang, J., Brunak, S., Doré, J., Guarner, F., Kristiansen, K., Pedersen, O., Parkhill, J., Weissenbach, J., Antolin, M., Artiguenave, F., Blottiere, H., Borruel, N., Bruls, T., Casellas, F., Chervaux, C., Cultrone, A., Delorme, C., Denariaz, G., Dervyn, R., Forte, M., Friss, C., van de Guchte, M., Guedon, E., Haimet, F., Jamet, A., Juste, C., Kaci, G., Kleerebezem, M., Knol, J., Kristensen, M., Layec, S., Le Roux, K., Leclerc, M., Maguin, E., Melo Minardi, R., Oozeer, R., Rescigno, M., Sanchez, N., Tims, S., Torrejon, T., Varela, E., de Vos, W., Winogradsky, Y., Zoetendal, E., Bork, P., Ehrlich, S., & Wang, J. (2010). A human gut microbial gene catalogue established by metagenomic sequencing Nature, 464 (7285), 59-65 DOI: 10.1038/nature08821


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M cells, gatekeepers or gateway to the gut

Glycoprotein 2 is the M cell receptor for type I pili on bacteria and is important for the immune response to these bacteria, according to research by Hase and colleagues published last week in the journal Nature.

The mucosal immune system is one of the largest components of our immune system and is hugely important for protecting mucosal surfaces (like our gastrointestinal tract) from harmful pathogens. Our guts are home to trillions of commensal bacteria which live quite happily there causing us no harm whatsoever. The gastrointestinal tract is protected from these bacteria (or other damaging substances) by a layer of tightly packed epithelial cells which form a barrier against any bacteria or molecules penetrating the gut. However, Microfold (M) cells are specialised intestinal cells located over mucosal lymphoid tissue called Peyer’s patches which are potential entry points into the host. M cells sample microorganisms or molecules in our gut and help transport them across the epithelial cell barrier (a process called antigen transcytosis) to deliver to professional immune cells (like macrophages, T cells or dendritic cells) to stimulate a protective immune response. In essence, M cells act like CCTV cameras to survey the gut area for anything that is out of the ordinary, or potentially harmful, and then present them to our immune system (essentially the police and the law courts) to sort those bad ‘uns out.

Previous work had shown that antigen transcytosis by M cells is important for mucosal immune responses but little was known about the mechanism involved. The researchers used microarray to scan the entire genome for specific molecules associated with M cells and found that glycoprotein 2 (GP2) was expressed in M cells in both human and mouse Peyer’s patches. Using immunoelectron microscopy they showed that GP2 was localised to the apical surface of M cells (the surface exposed to the commensal bacteria in the gut). Furthermore, they found that GP2 bound a variety of commensal and pathogenic enterobacteria (Escherichia coli, Salmonella enterica serovar Enteritidis and Salmonella Typhimurium), and more specifically bound to FimH expressed in the bacterial type I pilus (filamentous projections on the bacterial surface which are important for adhesion). Three-dimensional imaging revealed that GP2 accumulates around E. coli and S. Typhimurium as they are internalised in M cells and deletion of GP2 in mice reduced the uptake of type-I-piliated bacteria. After bacteria are translocated through M cells in a GP2-dependent manner they were captured by dendritic cells. Furthermore, GP2 was important for induction of mucosal immune responses against specific bacterial antigens (proteins that stimulate an immune response). Bacteria deficient in FimH lost the ability to bind GP2, had reduced entry into Peyer’s patches and induced a weak helper T cell and antibody immune response. Similarly, mice lacking GP2 had reduced helper T cell and antibody immune responses after challenge with bacteria expressing FimH.

This paper highlights the biological importance of GP2-dependent M cell antigen transcytosis as part of immunosurveillance in the intestine for bacteria expressing FimH. More work is needed to fully understand exactly what happens to the bacteria after they are delivered to immune cells and tissues by the M cells. Finally, M cells are thought to be a promising target for oral vaccinations to induce a protective immune response and this work shows that GP2 may be a possible vaccine target.

Although M cells act as a great surveillance system for the gut there are always a few bacteria that abuse the system. Shigella is one particular deviant bacterium that cannot normally invade the apical surface of intestinal epithelial cells and so uses the M cells to breach the epithelial cell barrier and gain access to their basolateral surface. Here, they can successfully invade, replicate in the intestinal epithelium, and wreak havoc on the gut by causing shigellosis or bacillary dysentery.

Hase, K., Kawano, K., Nochi, T., Pontes, G., Fukuda, S., Ebisawa, M., Kadokura, K., Tobe, T., Fujimura, Y., Kawano, S., Yabashi, A., Waguri, S., Nakato, G., Kimura, S., Murakami, T., Iimura, M., Hamura, K., Fukuoka, S., Lowe, A., Itoh, K., Kiyono, H., & Ohno, H. (2009). Uptake through glycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response Nature, 462 (7270), 226-230 DOI: 10.1038/nature08529

Further reading

Jang, M.H. et al., (2004) Intestinal villous M cells: An antigen entry site in the mucosal epithelium. Proceedings of the National Academy of Sciences, 101, p.6110-6115.

Schroeder, G.N. and Hilbi, H. (2008) Molecular pathogenesis of Shigella spp.: Controlling host cell signalling, invasion and death by Type III secretion. Clinical Reviews Microbiology, 21, p.134-156.

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