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Hi All

For the next #microtwjc session on Tuesday 3rd Feb 8pm GMT we will be discussing the following paper

Granulocytes Impose a Tight Bottleneck upon the Gut Luminal Pathogen Population during Salmonella Typhimurium Colitis

Published in December in PLoS Pathogens

The paper is available from the link below:

http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1004557

Sorry Im posting it so close to the session I forgot it was my turn!

Abstract

Topological, chemical and immunological barriers are thought to limit infection by enteropathogenic bacteria. However, in many cases these barriers and their consequences for the infection process remain incompletely understood. Here, we employed a mouse model forSalmonella colitis and a mixed inoculum approach to identify barriers limiting the gut luminal pathogen population. Mice were infected via the oral route with wild type S. Typhimurium (S.Tm) and/or mixtures of phenotypically identical but differentially tagged S. Tm strains (“WITS”, wild-type isogenic tagged strains), which can be individually tracked by quantitative real-time PCR. WITS dilution experiments identified a substantial loss in tag/genetic diversity within the gut luminal S. Tm population by days 2–4 post infection. The diversity-loss was not attributable to overgrowth by S. Tm mutants, but required inflammation, Gr-1+ cells (mainly neutrophilic granulocytes) and most likely NADPH-oxidase-mediated defense, but not iNOS. Mathematical modelling indicated that inflammation inflicts a bottleneck transiently restricting the gut luminalS. Tm population to approximately 6000 cells and plating experiments verified a transient, inflammation- and Gr-1+ cell-dependent dip in the gut luminal S. Tm population at day 2 post infection. We conclude that granulocytes, an important clinical hallmark of S. Tm-induced inflammation, impose a drastic bottleneck upon the pathogen population. This extends the current view of inflammation-fuelled gut-luminal Salmonella growth by establishing the host response in the intestinal lumen as a double-edged sword, fostering and diminishing colonization in a dynamic equilibrium. Our work identifies a potent immune defense against gut infection and reveals a potential Achilles’ heel of the infection process which might be targeted for therapy.

Author Summary:

Salmonella Typhimurium can colonize the human intestine and cause severe diarrhea. In recent years, it has become clear that this pathogen profits from inflammatory changes in the intestinal lumen, as the inflamed gut helps Salmonella to out-compete the resident microbiota. Granulocytes transmigrating into the gut lumen were found to “foster” luminal Salmonellagrowth by providing nutrients (used by Salmonella, not the microbiota) and by releasing growth inhibitors affecting the microbiota, but not the pathogen. In this study, we extend this “fostering” concept by showing that gut luminal Salmonella Typhimurium population is itself surprisingly vulnerable to the host’s inflammatory response. Indeed, inflammation reduces the size of the gut luminal Salmonella population by as much as 105-fold at day 2 post infection. Thus, triggering of mucosal inflammation is in fact a double-edged sword by providing S. Typhimurium with a relative growth advantage against the microbiota in the gut lumen and by killing 99.999% of the gut luminal pathogen population at day 2. However, the pathogen population can recover and grow up again during the subsequent days. This changes the current view: Inflammation is not simply “beneficial” for the pathogen in the gut lumen. Instead, pathogen growth in the inflamed gut must be considered as an equilibrium between inflammation-inflicted killing and fostering growth of the surviving bacteria.

Discussion points:

1. Is the paper well written and concise?

2. Are the experiments well designed?

3. Do the results further our knowledge?

4. Anything you would have done differently?

If there is anything else you would like to discuss please use the comments box below.

See you all on Tues

Our next #microtwjc session will be on Tuesday 8th July 8pm (BST).

We will be discussing the following paper Fructose-Asparagine Is a Primary Nutrient during Growth of Salmonella in the Inflamed Intestine by Ali et al published in PLoS pathogens in June 2014. The link to the paper is here http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1004209

Abstract

Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10−/− mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1− SPI2− or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.

Author Summary

It has long been thought that the nutrient utilization systems of Salmonella would not make effective drug targets because there are simply too many nutrients available to Salmonella in the intestine. Surprisingly, we have discovered that Salmonella relies heavily on a single nutrient during growth in the inflamed intestine, fructose-asparagine (F-Asn). A mutant of Salmonella that cannot obtain F-Asn is severely attenuated, suggesting that F-Asn is the primary nutrient utilized by Salmonella during inflammation. No other organism has been reported to synthesize or utilize this novel biological compound. The novelty of this nutrient and the apparent lack of utilization systems in mammals and most other bacteria suggest that the F-Asn utilization system represents a specific and potent therapeutic target for Salmonella.

Discussion points
1. Is the paper well written and easy to follow and understand?
2. Are the methods adequate?
3. Do the results further our knowledge?
4. Any other experiments you would do?

For the next session (Tuesday 4th March, 8pm GMT) we will be discussing the following paper: Effect of spaceflight on Pseudomonas aeruginosa final cell density is modulated by nutrient and oxygen availability available from the following link http://www.biomedcentral.com/1471-2180/13/241

Abstract

Background: Abundant populations of bacteria have been observed on Mir and the International Space Station.
While some experiments have shown that bacteria cultured during spaceflight exhibit a range of potentially
troublesome characteristics, including increases in growth, antibiotic resistance and virulence, other studies have
shown minimal differences when cells were cultured during spaceflight or on Earth. Although the final cell density
of bacteria grown during spaceflight has been reported for several species, we are not yet able to predict how
different microorganisms will respond to the microgravity environment. In order to build our understanding of how
spaceflight affects bacterial final cell densities, additional studies are needed to determine whether the observed
differences are due to varied methods, experimental conditions, or organism specific responses.

Results: Here, we have explored how phosphate concentration, carbon source, oxygen availability, and motility
affect the growth of Pseudomonas aeruginosa in modified artificial urine media during spaceflight. We observed
that P. aeruginosa grown during spaceflight exhibited increased final cell density relative to normal gravity controls
when low concentrations of phosphate in the media were combined with decreased oxygen availability. In
contrast, when the availability of either phosphate or oxygen was increased, no difference in final cell density was
observed between spaceflight and normal gravity. Because motility has been suggested to affect how microbes
respond to microgravity, we compared the growth of wild-type P. aeruginosa to a ΔmotABCD mutant deficient in
swimming motility. However, the final cell densities observed with the motility mutant were consistent with those
observed with wild type for all conditions tested.

Conclusions: These results indicate that differences in bacterial final cell densities observed between spaceflight and
normal gravity are due to an interplay between microgravity conditions and the availability of substrates essential for
growth. Further, our results suggest that microbes grown under nutrient-limiting conditions are likely to reach higher cell
densities under microgravity conditions than they would on Earth. Considering that the majority of bacteria inhabiting
spacecrafts and space stations are likely to live under nutrient limitations, our findings highlight the need to explore the
impact microgravity and other aspects of the spaceflight environment have on microbial growth and physiology

Discussion points?

Was the paper well written?
Were the methods used appropiate?
Were the experiments carried out adequately?
What else can be done?

The paper up for discussion next time is Staphylococcus aureus small colony variants are susceptible to light activated antimicrobial agents and is available from the following link

http://www.biomedcentral.com/1471-2180/13/201

Abstract

Background

Antibiotic therapy can select for small colony variants of Staphylococcus aureus that are more resistant to antibiotics and can result in persistent infections, necessitating the development of more effective antimicrobial strategies to combat small colony variant infections. Photodynamic therapy is an alternative treatment approach which utilises light in combination with a light-activated antimicrobial agent to kill bacteria via a non-specific mechanism of action. In this study, we investigated whether the combination of 665 nm laser light and the light-activated antimicrobial agent methylene blue was able to successfully kill S. aureus small colony variants. S. aureus and isogenic stable small colony variant were exposed to varying doses (1.93 to 9.65 J/cm2) of 665 nm laser light in the presence of varying concentrations (1 to 20 μM) of methylene blue. dynamic therapy

Results

The combination of 665 nm laser light and methylene blue was found to be an effective strategy for the killing of small colony variants. At the highest light dose (9.65 J/cm2) and methylene blue concentration (20 μM) tested, the number of viable bacteria decreased by approximately 6.9 log10 for the wild type and approximately 5 log10 for the small colony variant.

Conclusions

These results suggest that photodynamic therapy has potential for use in the treatment of superficial infections caused by small colony variants of S. aureus and supports further research in this field.

Discussion points
1. Views on the paper and experimental design?
2. Use of photodynamic therapy as treatment
3. Can this therapy lead to a reduction in resistant bacteria?
4. Are there other alternatives to antibiotic therapy?
5. What should be done next?

For next weeks #microtwjc session (8pm BST 23rd July) I thought we would cool things down a little and discuss sequencing bacteria and eukarya in ice taken from a lake in Antarctica. The paper was recently published in PLOS one and is available from the following link http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0067221

Abstract:
Lake Vostok, the 7th largest (by volume) and 4th deepest lake on Earth, is covered by more than 3,700 m of ice, making it the
largest subglacial lake known. The combination of cold, heat (from possible hydrothermal activity), pressure (from the
overriding glacier), limited nutrients and complete darkness presents extreme challenges to life. Here, we report
metagenomic/metatranscriptomic sequence analyses from four accretion ice sections from the Vostok 5G ice core. Two
sections accreted in the vicinity of an embayment on the southwestern end of the lake, and the other two represented part
of the southern main basin. We obtained 3,507 unique gene sequences from concentrates of 500 ml of 0.22 mm-filtered
accretion ice meltwater. Taxonomic classifications (to genus and/or species) were possible for 1,623 of the sequences.
Species determinations in combination with mRNA gene sequence results allowed deduction of the metabolic pathways
represented in the accretion ice and, by extension, in the lake. Approximately 94% of the sequences were from Bacteria and
6% were from Eukarya. Only two sequences were from Archaea. In general, the taxa were similar to organisms previously
described from lakes, brackish water, marine environments, soil, glaciers, ice, lake sediments, deep-sea sediments, deep-sea
thermal vents, animals and plants. Sequences from aerobic, anaerobic, psychrophilic, thermophilic, halophilic, alkaliphilic,
acidophilic, desiccation-resistant, autotrophic and heterotrophic organisms were present, including a number from
multicellular eukaryotes.

Discussion points:

1. Was the paper well written, easy to understand/follow and the data presented well?

2. Were the methods appropriate? Anything else you would have liked the authors to do?

3. Are the results useful? Do they help us to understand microbial communities?

4. What next?