scholarly journals The Streptomycin-Treated Mouse Intestine SelectsEscherichia coli envZMissense Mutants That Interact with Dense and Diverse Intestinal Microbiota

2012 ◽  
Vol 80 (5) ◽  
pp. 1716-1727 ◽  
Author(s):  
Mary P. Leatham-Jensen ◽  
Jakob Frimodt-Møller ◽  
Jimmy Adediran ◽  
Matthew E. Mokszycki ◽  
Megan E. Banner ◽  
...  
Keyword(s):  
Treated Mouse ◽  
Bacterial Biofilms ◽  
Deletion Mutants ◽  
Content Type ◽  
E Coli ◽  
Minority Member ◽  
Mouse Intestine ◽  
Colicin V ◽  
Colonizing Ability

ABSTRACTPreviously, we reported that the streptomycin-treated mouse intestine selected nonmotileEscherichia coliMG1655flhDCdeletion mutants ofE. coliMG1655 with improved colonizing ability that grow 15% fasterin vitroin mouse cecal mucus and 15 to 30% faster on sugars present in mucus (M. P. Leatham et al., Infect. Immun. 73:8039–8049, 2005). Here, we report that the 10 to 20% remaining motileE. coliMG1655 areenvZmissense mutants that are also better colonizers of the mouse intestine thanE. coliMG1655. One of theflhDCmutants,E. coliMG1655 ΔflhD, and one of theenvZmissense mutants,E. coliMG1655 mot-1, were studied further.E. coliMG1655 mot-1 is more resistant to bile salts and colicin V thanE. coliMG1655 ΔflhDand grows ca. 15% slowerin vitroin mouse cecal mucus and on several sugars present in mucus compared toE. coliMG1655 ΔflhDbut grows 30% faster on galactose. Moreover,E. coliMG1655 mot-1 andE. coliMG1655 ΔflhDappear to colonize equally well in one intestinal niche, butE. coliMG1655 mot-1 appears to use galactose to colonize a second, smaller intestinal niche either not colonized or colonized poorly byE. coliMG1655 ΔflhD. Evidence is also presented thatE. coliMG1655 is a minority member of mixed bacterial biofilms in the mucus layer of the streptomycin-treated mouse intestine. We offer a hypothesis, which we call the “Restaurant” hypothesis, that explains how nutrient acquisition in different biofilms comprised of different anaerobes can account for our results.

scholarly journals An Escherichia coli Nissle 1917 Missense Mutant Colonizes the Streptomycin-Treated Mouse Intestine Better than the Wild Type but Is Not a Better Probiotic

2013 ◽  
Vol 82 (2) ◽  
pp. 670-682 ◽  
Author(s):  
Jimmy Adediran ◽  
Mary P. Leatham-Jensen ◽  
Matthew E. Mokszycki ◽  
Jakob Frimodt-Møller ◽  
Karen A. Krogfelt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Probiotic Strain ◽  
Treated Mouse ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
E Coli ◽  
Two Component Signal Transduction ◽  
Mouse Intestine ◽  
Colicin V

ABSTRACTPreviously we reported that the streptomycin-treated mouse intestine selected for two differentEscherichia coliMG1655 mutants with improved colonizing ability: nonmotileE. coliMG1655flhDCdeletion mutants that grew 15% fasterin vitroin mouse cecal mucus and motileE. coliMG1655envZmissense mutants that grew slowerin vitroin mouse cecal mucus yet were able to cocolonize with the faster-growingflhDCmutants. TheE. coliMG1655envZgene encodes a histidine kinase that is a member of theenvZ-ompRtwo-component signal transduction system, which regulates outer membrane protein profiles. In the present investigation, theenvZP41Lgene was transferred from the intestinally selectedE. coliMG1655 mutant toE. coliNissle 1917, a human probiotic strain used to treat gastrointestinal infections. Both theE. coliMG1655 andE. coliNissle 1917 strains containingenvZP41Lproduced more phosphorylated OmpR than their parents. TheE. coliNissle 1917 strain containingenvZP41Lalso became more resistant to bile salts and colicin V and grew 50% slowerin vitroin mucus and 15% to 30% slower on several sugars present in mucus, yet it was a 10-fold better colonizer thanE. coliNissle 1917. However,E. coliNissle 1917envZP41Lwas not better at preventing colonization by enterohemorrhagicE. coliEDL933. The data can be explained according to our “restaurant” hypothesis for commensalE. colistrains, i.e., that they colonize the intestine as sessile members of mixed biofilms, obtaining the sugars they need for growth locally, but compete for sugars with invadingE. colipathogens planktonically.

scholarly journals Screening of Escherichia coli Species Biodiversity Reveals New Biofilm-Associated Antiadhesion Polysaccharides

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Olaya Rendueles ◽  
Laetitia Travier ◽  
Patricia Latour-Lambert ◽  
Thierry Fontaine ◽  
Julie Magnus ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Bacterial Adhesion ◽  
Bioactive Molecules ◽  
Bacterial Biofilms ◽  
Gram Positive ◽  
Content Type ◽  
E Coli ◽  
Species Biodiversity

ABSTRACTBacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted fromin vitromature biofilms formed by 122 naturalEscherichia coliisolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animalE. colistrain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion ofStaphylococcus aureusfrom mixedE. coliandS. aureusbiofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices.IMPORTANCEWe sought to demonstrate that bacterial biofilms are reservoirs for unknown molecules that antagonize bacterial adhesion. The use of natural strains representative ofEscherichia colispecies biodiversity showed that nonbiocidal antiadhesion polysaccharides are frequently found in mature biofilm extracts (bacterium-free suspensions which contain soluble molecules produced within the biofilm). Release of an antiadhesion polysaccharide confers a competitive advantage upon the producing strain against clinically relevant pathogens such asStaphylococcus aureus. Hence, exploring the biofilm environment provides a better understanding of bacterial interactions within complex communities and could lead to improved control of pathogen colonization.

scholarly journals Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

2007 ◽  
Vol 75 (7) ◽  
pp. 3315-3324 ◽  
Author(s):  
Eric J. Gauger ◽  
Mary P. Leatham ◽  
Regino Mercado-Lubo ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Flagellar Motor ◽  
Wild Type ◽  
E Coli ◽  
Mouse Intestine ◽  
Flagellum Biosynthesis ◽  
Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

scholarly journals Anaerobic Respiration of Escherichia coli in the Mouse Intestine

2011 ◽  
Vol 79 (10) ◽  
pp. 4218-4226 ◽  
Author(s):  
Shari A. Jones ◽  
Terri Gibson ◽  
Rosalie C. Maltby ◽  
Fatema Z. Chowdhury ◽  
Valley Stewart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Electron Flow ◽  
Anaerobic Respiration ◽  
Fumarate Reductase ◽  
Treated Mouse ◽  
Content Type ◽  
E Coli ◽  
Facultative Anaerobes ◽  
Mouse Intestine

ABSTRACTThe intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such asEscherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochromebdoxidase and the anaerobic terminal reductases. We found thatE. coliuses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamineN-oxide. Competitive colonizations revealed that cytochromebdoxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence ofE. coli, we conclude thatE. coliis the only bacterium in the streptomycin-treated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases thatE. coliuses to maximize its competitiveness and achieve the highest possible population in the intestine.

scholarly journals Escherichia coli Pathotypes Occupy Distinct Niches in the Mouse Intestine

2014 ◽  
Vol 82 (5) ◽  
pp. 1931-1938 ◽  
Author(s):  
Jessica P. Meador ◽  
Matthew E. Caldwell ◽  
Paul S. Cohen ◽  
Tyrrell Conway
Keyword(s):  
Escherichia Coli ◽  
Infection Process ◽  
Treated Mouse ◽  
Colonization Resistance ◽  
Content Type ◽  
E Coli ◽  
Similar Strategy ◽  
Mouse Intestine

ABSTRACTSince the first step of the infection process is colonization of the host, it is important to understand howEscherichia colipathogens successfully colonize the intestine. We previously showed that enterohemorrhagic O157:H7 strainE. coliEDL933 colonizes a niche in the streptomycin-treated mouse intestine that is distinct from that of human commensal strains, which explains howE. coliEDL933 overcomes colonization resistance imparted by some, but not all, commensalE. colistrains. Here we sought to determine if otherE. colipathogens use a similar strategy. We found that uropathogenicE. coliCFT073 and enteropathogenicE. coliE2348/69 occupy intestinal niches that are distinct from that ofE. coliEDL933. In contrast, two enterohemorrhagic strains,E. coliEDL933 andE. coliSakai, occupy the same niche, suggesting that strategies to prevent colonization by a given pathotype should be effective against other strains of the same pathotype. However, we found that a combination of commensalE. colistrains that can prevent colonization byE. coliEDL933 did not prevent colonization byE. coliCFT073 orE. coliE2348/69. Our results indicate that development of probiotics to target multipleE. colipathotypes will be problematic, as the factors that govern niche occupation and hence stable colonization vary significantly among strains.

scholarly journals Precolonized Human Commensal Escherichia coli Strains Serve as a Barrier to E. coli O157:H7 Growth in the Streptomycin-Treated Mouse Intestine

2009 ◽  
Vol 77 (7) ◽  
pp. 2876-2886 ◽  
Author(s):  
Mary P. Leatham ◽  
Swati Banerjee ◽  
Steven M. Autieri ◽  
Regino Mercado-Lubo ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Treated Mouse ◽  
Colonization Resistance ◽  
Limited Growth ◽  
Metabolic Capacity ◽  
Isogenic Strain ◽  
E Coli ◽  
Probiotic Strains ◽  
Mouse Intestine

ABSTRACT Different Escherichia coli strains generally have the same metabolic capacity for growth on sugars in vitro, but they appear to use different sugars in the streptomycin-treated mouse intestine (Fabich et al., Infect. Immun. 76:1143-1152, 2008). Here, mice were precolonized with any of three human commensal strains (E. coli MG1655, E. coli HS, or E. coli Nissle 1917) and 10 days later were fed 105 CFU of the same strains. While each precolonized strain nearly eliminated its isogenic strain, confirming that colonization resistance can be modeled in mice, each allowed growth of the other commensal strains to higher numbers, consistent with different commensal E. coli strains using different nutrients in the intestine. Mice were also precolonized with any of five commensal E. coli strains for 10 days and then were fed 105 CFU of E. coli EDL933, an O157:H7 pathogen. E. coli Nissle 1917 and E. coli EFC1 limited growth of E. coli EDL933 in the intestine (103 to 104 CFU/gram of feces), whereas E. coli MG1655, E. coli HS, and E. coli EFC2 allowed growth to higher numbers (106 to 107 CFU/gram of feces). Importantly, when E. coli EDL933 was fed to mice previously co-colonized with three E. coli strains (MG1655, HS, and Nissle 1917), it was eliminated from the intestine (<10 CFU/gram of feces). These results confirm that commensal E. coli strains can provide a barrier to infection and suggest that it may be possible to construct E. coli probiotic strains that prevent growth of pathogenic E. coli strains in the intestine.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Activity of Ceftazidime-Avibactam against Fluoroquinolone-Resistant Enterobacteriaceae and Pseudomonas aeruginosa

2015 ◽  
Vol 59 (6) ◽  
pp. 3059-3065 ◽  
Author(s):  
C. Pitart ◽  
F. Marco ◽  
T. A. Keating ◽  
W. W. Nichols ◽  
J. Vila
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistant Mutant ◽  
Fluoroquinolone Resistance ◽  
Cross Resistance ◽  
Content Type ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
The Impact

ABSTRACTCeftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200Enterobacteriaceaeand 25Pseudomonas aeruginosastrains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistantEnterobacteriaceaestrains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBLEscherichia coli(MIC90of 0.25 mg/liter), ESBLKlebsiella pneumoniae(MIC90of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC90of 1 mg/liter), non-ESBLE. coli(MIC90of ≤0.125 mg/liter), non-ESBLK. pneumoniae(MIC90of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC90of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistantP. aeruginosastrains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC90of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtainedin vitrofrom two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC90values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains ofEnterobacteriaceaeandP. aeruginosawere ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affectEnterobacteriaceaeandP. aeruginosasusceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.

scholarly journals Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Derek Fleming ◽  
Laura Chahin ◽  
Kendra Rumbaugh
Keyword(s):  
Glycoside Hydrolase ◽  
Bacterial Population ◽  
Chronic Wounds ◽  
Glycoside Hydrolases ◽  
Bacterial Biofilms ◽  
Planktonic Cells ◽  
Content Type ◽  
Biomass Dissolution

ABSTRACT The persistent nature of chronic wounds leaves them highly susceptible to invasion by a variety of pathogens that have the ability to construct an extracellular polymeric substance (EPS). This EPS makes the bacterial population, or biofilm, up to 1,000-fold more antibiotic tolerant than planktonic cells and makes wound healing extremely difficult. Thus, compounds which have the ability to degrade biofilms, but not host tissue components, are highly sought after for clinical applications. In this study, we examined the efficacy of two glycoside hydrolases, α-amylase and cellulase, which break down complex polysaccharides, to effectively disrupt Staphylococcus aureus and Pseudomonas aeruginosa monoculture and coculture biofilms. We hypothesized that glycoside hydrolase therapy would significantly reduce EPS biomass and convert bacteria to their planktonic state, leaving them more susceptible to conventional antimicrobials. Treatment of S. aureus and P. aeruginosa biofilms, grown in vitro and in vivo, with solutions of α-amylase and cellulase resulted in significant reductions in biomass, dissolution of the biofilm, and an increase in the effectiveness of subsequent antibiotic treatments. These data suggest that glycoside hydrolase therapy represents a potential safe, effective, and new avenue of treatment for biofilm-related infections.

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