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1.  Oral-Derived Bacterial Flora Defends Its Domain by Recognizing and Killing Intruders—A Molecular Analysis Using Escherichia coli as a Model Intestinal Bacterium 
Microbial Ecology  2010;60(3):655-664.
Within the same human gastrointestinal tract, substantial differences in the bacterial species that inhabit oral cavity and intestinal tract have been noted. Previous research primarily attributed the differences to the influences of host environments and nutritional availabilities (“host habitat” effect). Our recent study indicated that, other than the host habitat effect, an existing microbial community could impose a selective pressure on incoming foreign bacterial species independent of host-mediated selection (“community selection” effect). In this study, we employed in vitro microbial floras representing microorganisms that inhabit the oral cavities and intestinal tract of mice in combination with Escherichia coli as a model intestinal bacterium and demonstrated that E. coli displays a striking community preference. It thrived when introduced into the intestinal microbial community and survived poorly in the microbial flora of foreign origin (oral community). A more detailed examination of this phenomenon showed that the oral community produced oxygen-free radicals in the presence of wild-type E. coli while mutants deficient in lipopolysaccharides (LPS) did not trigger significant production of these cell-damaging agents. Furthermore, mutants of E. coli defective in the oxidative stress response experienced a more drastic reduction in viability when cocultivated with the oral flora, while the exogenous addition of the antioxidant vitamin C was able to rescue it. We concluded that the oral-derived microbial community senses the E. coli LPS and kills the bacterium with oxygen-free radicals. This study reveals a new mechanism of community invasion resistance employed by established microflora to defend their domains.
Electronic supplementary material
The online version of this article (doi:10.1007/s00248-010-9708-4) contains supplementary material, which is available to authorized users.
PMCID: PMC2954290  PMID: 20625713
2.  In vitro communities derived from oral and gut microbial floras inhibit the growth of bacteria of foreign origins 
Microbial ecology  2010;60(3):665-676.
The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-DGGE analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions towards each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: they persisted when introduced into the bacterial community of the same origin, while their viable count declined more than 3 orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.
PMCID: PMC2954289  PMID: 20625712
3.  In Vitro Communities Derived from Oral and Gut Microbial Floras Inhibit the Growth of Bacteria of Foreign Origins 
Microbial Ecology  2010;60(3):665-676.
The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract, substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-based denaturing gradient gel electrophoresis analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions toward each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: They persisted when introduced into the bacterial community of the same origin, while their viable count declined more than three orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.
Electronic supplementary material
The online version of this article (doi:10.1007/s00248-010-9711-9) contains supplementary material, which is available to authorized users.
PMCID: PMC2954289  PMID: 20625712
4.  A Product of Heme Catabolism Modulates Bacterial Function and Survival 
PLoS Pathogens  2013;9(7):e1003507.
Bilirubin is the terminal metabolite in heme catabolism in mammals. After deposition into bile, bilirubin is released in large quantities into the mammalian gastrointestinal (GI) tract. We hypothesized that intestinal bilirubin may modulate the function of enteric bacteria. To test this hypothesis, we investigated the effect of bilirubin on two enteric pathogens; enterohemorrhagic E. coli (EHEC), a Gram-negative that causes life-threatening intestinal infections, and E. faecalis, a Gram-positive human commensal bacterium known to be an opportunistic pathogen with broad-spectrum antibiotic resistance. We demonstrate that bilirubin can protect EHEC from exogenous and host-generated reactive oxygen species (ROS) through the absorption of free radicals. In contrast, E. faecalis was highly susceptible to bilirubin, which causes significant membrane disruption and uncoupling of respiratory metabolism in this bacterium. Interestingly, similar results were observed for other Gram-positive bacteria, including B. cereus and S. aureus. A model is proposed whereby bilirubin places distinct selective pressure on enteric bacteria, with Gram-negative bacteria being protected from ROS (positive outcome) and Gram-positive bacteria being susceptible to membrane disruption (negative outcome). This work suggests bilirubin has differential but biologically relevant effects on bacteria and justifies additional efforts to determine the role of this neglected waste catabolite in disease processes, including animal models.
Author Summary
Bilirubin is the terminal breakdown product of heme, which is deposited at high concentrations in the human intestine, where it can come into contact with host cells, the gastrointestinal (GI) microflora, and invading pathogens. Here, we report that bilirubin can act as a protectant for the Gram-negative bacterial pathogen E. coli O157:H7, which causes severe hemorrhagic diarrhea and life-threatening kidney damage. Paradoxically, bilirubin is highly toxic towards another enteric opportunistic pathogen, the Gram-positive bacterium E. faecalis. Whereas the protection of E. coli stems from the neutralization of host reactive oxygen species, bilirubin's toxicity toward E. faecalis is rooted in its lipophilic properties, which drives the rapid association of bilirubin with bacteria, leading to disrupted cell membranes and concomitant death. These results suggest small molecule metabolites can modulate bacterial communities in the intestine, a finding that may have important implications for diseases caused by enteric bacteria and disrupted flora.
PMCID: PMC3723568  PMID: 23935485
5.  Ethanolamine Controls Expression of Genes Encoding Components Involved in Interkingdom Signaling and Virulence in Enterohemorrhagic Escherichia coli O157:H7 
mBio  2012;3(3):e00050-12.
Bacterial pathogens must be able to both recognize suitable niches within the host for colonization and successfully compete with commensal flora for nutrients in order to establish infection. Ethanolamine (EA) is a major component of mammalian and bacterial membranes and is used by pathogens as a carbon and/or nitrogen source in the gastrointestinal tract. The deadly human pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) uses EA in the intestine as a nitrogen source as a competitive advantage for colonization over the microbial flora. Here we show that EA is not only important for nitrogen metabolism but that it is also used as a signaling molecule in cell-to-cell signaling to activate virulence gene expression in EHEC. EA in concentrations that cannot promote growth as a nitrogen source can activate expression of EHEC’s repertoire of virulence genes. The EutR transcription factor, known to be the receptor of EA, is only partially responsible for this regulation, suggesting that yet another EA receptor exists. This important link of EA with metabolism, cell-to-cell signaling, and pathogenesis, highlights the fact that a fundamental means of communication within microbial communities relies on energy production and processing of metabolites. Here we show for the first time that bacterial pathogens not only exploit EA as a metabolite but also coopt EA as a signaling molecule to recognize the gastrointestinal environment and promote virulence expression.
In order to successfully cause disease, a pathogen must be able to sense a host environment and modulate expression of its virulence genes as well as compete with the indigenous microbiota for nutrients. Ethanolamine (EA) is present in the large intestine due to the turnover of intestinal cells. Here, we show that the human pathogen Escherichia coli O157:H7, which causes bloody diarrhea and hemolytic-uremic syndrome, regulates virulence gene expression through EA metabolism and by responding to EA as a signal. These findings provide the first information directly linking EA with bacterial pathogenesis.
PMCID: PMC3372972  PMID: 22589288
6.  Community-based interference against integration of Pseudomonas aeruginosa into human salivary microbial biofilm 
Molecular Oral Microbiology  2011;26(6):337-352.
As part of the human gastrointestinal tract, the oral cavity represents a complex biological system and harbors diverse bacterial species. Unlike the gut microbiota which is often considered a health asset, studies of the oral commensal microbial flora have been largely limited to their implication in oral diseases such as dental caries and periodontal diseases; Little emphasis has been given to their potential beneficial roles, especially the protective effects against oral colonization by foreign/pathogenic bacteria. In this study, we used the salivary microbiota derived from healthy human subjects to investigate protective effects against the colonization and integration of Pseudomonas aeruginosa, an opportunistic bacterial pathogen, into developing and pre-formed salivary biofilms. When co-cultivated in saliva medium, P. aeruginosa persisted in the planktonic phase, but failed to integrate into salivary microbial community during biofilm formation. Furthermore, in the saliva medium supplemented with 0.05% (w/v) sucrose, the oral flora inhibited the growth of P. aeruginosa by producing lactic acid. More interestingly, while pre-formed salivary biofilms were able to prevent P. aeruginosa colonization, the same biofilms recovered from mild chlorhexidine gluconate treatment displayed a shift in microbial composition and showed a drastic reduction in protection. Our study indicates that normal oral communities with balanced microbial compositions could be important in effectively preventing the integration of foreign/pathogenic bacterial species, such as P. aeruginosa.
PMCID: PMC3327514  PMID: 22053962
bacterial interference; microbial flora; oral cavity; Pseudomonas aeruginosa; salivary biofilm
7.  The Role of Innate Immune Responses in the Outcome of Interspecies Competition for Colonization of Mucosal Surfaces 
PLoS Pathogens  2005;1(1):e1.
Since mucosal surfaces may be simultaneously colonized by multiple species, the success of an organism may be determined by its ability to compete with co-inhabitants of its niche. To explore the contribution of host factors to polymicrobial competition, a murine model was used to study the initiation of colonization by Haemophilus influenzae and Streptococcus pneumoniae. Both bacterial species, which occupy a similar microenvironment within the nasopharynx, persisted during colonization when given individually. Co-colonization, however, resulted in rapid clearance of S. pneumoniae from the upper respiratory tract, associated with increased recruitment of neutrophils into paranasal spaces. Systemic depletion of either neutrophil-like cells or complement was sufficient to eliminate this competitive effect, indicating that clearance was likely due to enhanced opsonophagocytic killing. The hypothesis that modulation of opsonophagocytic activity was responsible for host-mediated competition was tested using in vitro killing assays with elicited neutrophil-like cells. Components of H. influenzae (but not S. pneumoniae) stimulated complement-dependent phagocytic killing of S. pneumoniae. Thus, the recruitment and activation of neutrophils through selective microbial pattern recognition may underlie the H. influenzae–induced clearance of S. pneumoniae. This study demonstrates how innate immune responses may mediate competitive interactions between species and dictate the composition of the colonizing flora.
Bacterial infection commonly begins with organisms that colonize and proliferate on mucosal surfaces. These microenvironments may be occupied by multiple microbial species, suggesting that successful colonizers are distinguished by their capacity to prevail over their competitors. This study examines interactions between two bacterial species that both colonize and infect the human upper respiratory tract. In a mouse model, strains of both Haemophilus influenzae and Streptococcus pneumoniae efficiently colonize the nasal mucosa when tested individually. In contrast, following co-inoculation, H. influenzae rapidly and completely outcompetes S. pneumoniae. This competitive effect is dependent on the local responses from the host in the form of a specific type of white blood cell (neutrophil) that acts to engulf and kill microorganisms that have been labeled by proteins that bind to microbial surfaces (complement). The results of this study show that recognition of microbial products from one species may activate inflammatory responses that promote the clearance of another competing species. This study also demonstrates how manipulations such as antibiotics or vaccines, which are meant to diminish the presence of a single pathogen, may inadvertently alter the competitive interactions of complex microbial communities.
PMCID: PMC1238736  PMID: 16201010
8.  Population Dynamics of Streptococcus mitis in Its Natural Habitat 
Infection and Immunity  2001;69(10):6055-6063.
The purpose of this study was to examine the genetic structure of the typical commensal Streptococcus mitis biovar 1 in its natural habitat in the human oral cavity and pharynx and to investigate the role that selected microbial properties and host, spatial, and temporal factors play in determining the structure of the bacterial population. Consecutive samples were collected from buccal and pharyngeal mucosal surfaces of two infants, their four parents, and two elderly individuals over a period of approximately 1 year. A total of 751 isolates identified as S. mitis biovar 1 were typed by restriction endonuclease analysis (REA) and representative clones were typed by multilocus enzyme electrophoresis (MLEE). The genetic diversity of the S. mitis biovar 1 isolates collected from single infant hosts over a period of 9 to 10 months was found to be between 0.69 and 0.76, which is considerably higher than that previously observed for intestinal populations of Escherichia coli. The study provides evidence of the existence of both transient and persistent clones in adult individuals. In the two infants, however, none of 42 demonstrated clones were detected on more than a single occasion. Statistical calculations showed that the ability to persist was not distributed at random in the S. mitis biovar 1 population. However, neither immunoglobulin A1 protease activity nor the ability to bind α-amylase from saliva was a preferential characteristic of persistent genotypes. In contrast to current concepts of climax ecosystems, the species niche in the habitat appears to be maintained predominantly by a succession of clones rather than by stable strains. Several lines of evidence suggest that the major origin of “new” clones is the many other habitats in the respiratory tract that are occupied by this species.
PMCID: PMC98734  PMID: 11553543
9.  Colonization resistance of the digestive tract in conventional and antibiotic-treated mice 
The Journal of Hygiene  1971;69(3):405-411.
The effect of oral administration of antibiotics on the intestinal flora of conventional mice and their resistance to colonization by orally introduced Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa was studied. Colonization resistance (CR) was expressed as the log of the oral bacterial dose followed by a persistent take in 50% of the contaminated animals. The intestinal flora was virtually eliminated by the antibiotics and this elimination was accompanied by a precipitous fall of CR. CR gradually returned to normal values during the period of repopulation of the intestinal tract by the organisms surviving the treatment. Antibiotic treatment resulted in the disappearance of Enterobacteriaceae, enterococci, staphylococci and yeasts and, under appropriate housing conditions, the animals remained free of these organisms indefinitely. Germ-free mice contaminated with the intestinal flora of an antibiotic-treated animal and their offspring housed in a germ-free isolator showed high values of CR. Their intestinal flora consisted of anaerobic bacteria only. Apparently, these anaerobes are responsible for CR in these and in conventional mice.
PMCID: PMC2130899  PMID: 4999450
10.  Preferential Use of Central Metabolism In Vivo Reveals a Nutritional Basis for Polymicrobial Infection 
PLoS Pathogens  2015;11(1):e1004601.
The human genitourinary tract is a common anatomical niche for polymicrobial infection and a leading site for the development of bacteremia and sepsis. Most uncomplicated, community-acquired urinary tract infections (UTI) are caused by Escherichia coli, while another bacterium, Proteus mirabilis, is more often associated with complicated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent requirements for specific central pathways in vivo despite colonizing and occupying the same host environment. Using mutants of specific central metabolism enzymes, we determined glycolysis mutants lacking pgi, tpiA, pfkA, or pykA all have fitness defects in vivo for P. mirabilis but do not affect colonization of E. coli during UTI. Similarly, the oxidative pentose phosphate pathway is required only for P. mirabilis in vivo. In contrast, gluconeogenesis is required only for E. coli fitness in vivo. The remarkable difference in central pathway utilization between E. coli and P. mirabilis during experimental UTI was also observed for TCA cycle mutants in sdhB, fumC, and frdA. The distinct in vivo requirements between these pathogens suggest E. coli and P. mirabilis are not direct competitors within host urinary tract nutritional niche. In support of this, we found that co-infection with E. coli and P. mirabilis wild-type strains enhanced bacterial colonization and persistence of both pathogens during UTI. Our results reveal that complementary utilization of central carbon metabolism facilitates polymicrobial disease and suggests microbial activity in vivo alters the host urinary tract nutritional niche.
Author Summary
The human urinary tract is a leading source for polymicrobial infections and for the development of bacteremia and sepsis. Treating these potentially dangerous infections have recently become more challenging due to the appearance of uropathogenic strains that are resistant to the many of the most commonly prescribed antibiotics. The majority of urinary tract infections (UTI) are caused by Escherichia coli, while another bacterium, Proteus mirabilis, is more likely to cause catheter-associated UTI. Here, we report that uropathogenic E. coli and P. mirabilis have divergent nutritional requirements despite growing in the same host environment. This result indicates that E. coli and P. mirabilis do not directly compete for nutrients during UTI. Indeed, we found that persistence of both pathogens is enhanced when they co-colonize the host. This work represents an important step toward understanding the basic nutritional requirements for two major pathogens that cause UTI and shows how mixed infections can change these requirements. Understanding how bacteria grow during infections is fundamental to ultimately uncover new ways to combat increasingly drug-resistant bacterial infections.
PMCID: PMC4287612  PMID: 25568946
11.  Salmonella enterica Serovar Typhimurium Exploits Inflammation to Compete with the Intestinal Microbiota 
PLoS Biology  2007;5(10):e244.
Most mucosal surfaces of the mammalian body are colonized by microbial communities (“microbiota”). A high density of commensal microbiota inhabits the intestine and shields from infection (“colonization resistance”). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10−/−, VILLIN-HACL4-CD8) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen.
Author Summary
A dense microbial community colonizes the intestinal tract of mammals, contributing to health and nutrition and conferring efficient protection against most pathogenic intruders. Intestinal pathogens can overcome this colonization resistance and cause disease; however, the mechanisms used to do this are still elusive. In this study we analyzed intestinal infection by the model pathogen Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm). We show that the virulent wild-type pathogen overcomes colonization resistance by inducing the host's inflammatory immune response and exploiting it for its purpose. In contrast, an avirulent Salmonella mutant defective in triggering inflammation was unable to overcome colonization resistance by itself. Colonization by this mutant was restored if inflammation was provided concomitantly, in mice with inflammatory bowel disease (genetic and inducible) or by co-infection with wild-type S. Tm. These findings reveal a previously unrecognized strategy by which pathogenic bacteria overcome colonization resistance: abusing the host's inflammatory immune response to gain an edge against the normal microbial community of the gut. This represents a first step towards unravelling the molecular mechanisms underlying this three-way interaction of host, microbiota, and pathogens.
Inducing inflammation is key to the ability of the virulent pathogen Salmonella enterica serovar Typhimurium to outcompete the protective resident microbiota in a race to colonize the gut.
PMCID: PMC1951780  PMID: 17760501
12.  Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide 
Antimicrobial Agents and Chemotherapy  2006;50(11):3651-3657.
Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into “probiotic” antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora.
PMCID: PMC1635210  PMID: 17060534
13.  Anaerobic Respiration of Escherichia coli in the Mouse Intestine ▿ 
Infection and Immunity  2011;79(10):4218-4226.
The intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia 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 cytochrome bd oxidase and the anaerobic terminal reductases. We found that E. coli uses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bd oxidase 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 of E. coli, we conclude that E. coli is 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 that E. coli uses to maximize its competitiveness and achieve the highest possible population in the intestine.
PMCID: PMC3187261  PMID: 21825069
14.  A Novel Two-Component Signaling System That Activates Transcription of an Enterohemorrhagic Escherichia coli Effector Involved in Remodeling of Host Actin▿  
Journal of Bacteriology  2007;189(6):2468-2476.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for worldwide outbreaks of bloody diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome. After colonizing the large intestine, EHEC forms attaching and effacing (AE) lesions on intestinal epithelial cells. These lesions cause destruction of the microvilli and elicit actin rearrangement to form pedestals that cup each bacterium individually. EHEC responds to a signal produced by the intestinal microbial flora, autoinducer-3 (AI-3), and the host hormones epinephrine and norepinephrine to activate transcription of the genes involved in AE lesion formation. These three signals, involved in interkingdom communication, are sensed by bacterial sensor kinases. Here we describe a novel two-component system, QseEF (quorum-sensing E. coli regulators E and F), which is part of the AI-3/epinephrine/norepinephrine signaling system. QseE is the sensor kinase and QseF the response regulator. The qseEF genes are cotranscribed, and transcription of qseEF is activated by epinephrine through the QseC sensor. A qseF mutant does not form AE lesions. QseF activates transcription of the gene encoding EspFu, an effector protein translocated to the host cell by the EHEC, which mimics a eukaryotic SH2/SH3 adapter protein to engender actin polymerization during pedestal formation. Expression of the espFu gene from a plasmid restored AE lesion formation to the qseF mutant, suggesting that lack of espFu expression in this mutant was responsible for the loss of pedestal formation. These findings suggest the QseEF is a two-component system involved in the regulation of AE lesion formation by EHEC.
PMCID: PMC1899401  PMID: 17220220
15.  Topographical Continuity of Bacterial Populations in the Healthy Human Respiratory Tract 
Rationale: Defining the biogeography of bacterial populations in human body habitats is a high priority for understanding microbial–host relationships in health and disease. The healthy lung was traditionally considered sterile, but this notion has been challenged by emerging molecular approaches that enable comprehensive examination of microbial communities. However, studies of the lung are challenging due to difficulties in working with low biomass samples.
Objectives: Our goal was to use molecular methods to define the bacterial microbiota present in the lungs of healthy individuals and assess its relationship to upper airway populations.
Methods: We sampled respiratory flora intensively at multiple sites in six healthy individuals. The upper tract was sampled by oral wash and oro-/nasopharyngeal swabs. Two bronchoscopes were used to collect samples up to the glottis, followed by serial bronchoalveolar lavage and lower airway protected brush. Bacterial abundance and composition were analyzed by 16S rDNA Q-PCR and deep sequencing.
Measurements and Main Results: Bacterial communities from the lung displayed composition indistinguishable from the upper airways, but were 2 to 4 logs lower in biomass. Lung-specific sequences were rare and not shared among individuals. There was no unique lung microbiome.
Conclusions: In contrast to other organ systems, the respiratory tract harbors a homogenous microbiota that decreases in biomass from upper to lower tract. The healthy lung does not contain a consistent distinct microbiome, but instead contains low levels of bacterial sequences largely indistinguishable from upper respiratory flora. These findings establish baseline data for healthy subjects and sampling approaches for sequence-based analysis of diseases.
PMCID: PMC3208663  PMID: 21680950
healthy lung colonization; microbiome; 16S rDNA; pyrosequencing
16.  Targeted Antimicrobial Treatment to Re-establish a Healthy Microbial Flora for Long-term Protection 
Advances in Dental Research  2012;24(2):94-97.
Streptococcus mutans has been implicated as the major acid-producing (cariogenic) bacterium. Dietary sugars and other factors may cause an imbalance of oral microflora that enables S. mutans to become dominant in the multi-species biofilms on the tooth surface, which could lead to dental caries. The application of broad-spectrum antimicrobials often results in re-colonization and re-dominance of S. mutans within oral flora, while in contrast, therapies capable of selective elimination of S. mutans from oral microbial communities may help to re-establish the normal flora and provide long-term protection. C16G2, a novel synthetic antimicrobial peptide with specificity for S. mutans, was found to have robust killing efficacy and selectivity for S. mutans in vitro. A subsequent pilot human study found that a single application of C16G2 in the oral cavity (formulated in a mouthrinse vehicle) was associated with a reduction in plaque and salivary S. mutans, lactic acid production, and enamel demineralization during the entire 4-day testing period. C16G2 is now being developed as a new anticaries drug.
PMCID: PMC3420366  PMID: 22899688
microbial ecology; microbiology; microbial genetics; caries; dental biofilm; microbiota
17.  Escherichia coli O157:H7 Super-Shedder and Non-Shedder Feedlot Steers Harbour Distinct Fecal Bacterial Communities 
PLoS ONE  2014;9(5):e98115.
Escherichia coli O157:H7 is a major foodborne human pathogen causing disease worldwide. Cattle are a major reservoir for this pathogen and those that shed E. coli O157:H7 at >104 CFU/g feces have been termed “super-shedders”. A rich microbial community inhabits the mammalian intestinal tract, but it is not known if the structure of this community differs between super-shedder cattle and their non-shedding pen mates. We hypothesized that the super-shedder state is a result of an intestinal dysbiosis of the microbial community and that a “normal” microbiota prevents E. coli O157:H7 from reaching super-shedding levels. To address this question, we applied 454 pyrosequencing of bacterial 16S rRNA genes to characterize fecal bacterial communities from 11 super-shedders and 11 contemporary pen mates negative for E. coli O157:H7. The dataset was analyzed by using five independent clustering methods to minimize potential biases and to increase confidence in the results. Our analyses collectively indicated significant variations in microbiome composition between super-shedding and non-shedding cattle. Super-shedders exhibited higher bacterial richness and diversity than non-shedders. Furthermore, seventy-two operational taxonomic units, mostly belonging to Firmicutes and Bacteroidetes phyla, were identified showing differential abundance between these two groups of cattle. The operational taxonomic unit affiliation provides new insight into bacterial populations that are present in feces arising from super-shedders of E. coli O157:H7.
PMCID: PMC4032279  PMID: 24858731
18.  Development of the Human Infant Intestinal Microbiota 
PLoS Biology  2007;5(7):e177.
Almost immediately after a human being is born, so too is a new microbial ecosystem, one that resides in that person's gastrointestinal tract. Although it is a universal and integral part of human biology, the temporal progression of this process, the sources of the microbes that make up the ecosystem, how and why it varies from one infant to another, and how the composition of this ecosystem influences human physiology, development, and disease are still poorly understood. As a step toward systematically investigating these questions, we designed a microarray to detect and quantitate the small subunit ribosomal RNA (SSU rRNA) gene sequences of most currently recognized species and taxonomic groups of bacteria. We used this microarray, along with sequencing of cloned libraries of PCR-amplified SSU rDNA, to profile the microbial communities in an average of 26 stool samples each from 14 healthy, full-term human infants, including a pair of dizygotic twins, beginning with the first stool after birth and continuing at defined intervals throughout the first year of life. To investigate possible origins of the infant microbiota, we also profiled vaginal and milk samples from most of the mothers, and stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns of the twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract.
Author Summary
It has been recognized for nearly a century that human beings are inhabited by a remarkably dense and diverse microbial ecosystem, yet we are only just beginning to understand and appreciate the many roles that these microbes play in human health and development. Knowing the composition of this ecosystem is a crucial step toward understanding its roles. In this study, we designed and applied a ribosomal DNA microarray-based approach to trace the development of the intestinal flora in 14 healthy, full-term infants over the first year of life. We found that the composition and temporal patterns of the microbial communities varied widely from baby to baby, supporting a broader definition of healthy colonization than previously recognized. By one year of age, the babies retained their uniqueness but had converged toward a profile characteristic of the adult gastrointestinal tract. The composition and temporal patterns of development of the intestinal microbiota in a pair of fraternal twins were strikingly similar, suggesting that genetic and environmental factors shape our gut microbiota in a reproducible way.
Microarray profiling of the microbial communities of infant guts throughout the first year shows initial variation then convergence on the adult flora, providing new insight into this human ecosystem.
PMCID: PMC1896187  PMID: 17594176
19.  Role of anaerobic flora in the translocation of aerobic and facultatively anaerobic intestinal bacteria. 
Infection and Immunity  1987;55(11):2689-2694.
It is thought that the normal enteric microflora acts not only to prevent intestinal colonization but also to prevent subsequent systemic dissemination of ingested, potentially pathogenic bacteria. To determine the relative roles of specific components of the intestinal bacterial flora in bacterial translocation out of the gut, mice were given various antimicrobial agents to selectively eliminate specific groups of intestinal bacteria. The cecal flora and the translocating bacteria in mesenteric lymph nodes were monitored both before and after oral inoculation with antibiotic-resistant Escherichia coli C25. Orally administered streptomycin selectively eliminated cecal facultative gram-negative bacilli, orally administered bacitracin-streptomycin eliminated all cecal bacterial species except low numbers of aerobic sporeformers, and parenterally administered metronidazole selectively eliminated cecal anaerobic bacteria. Compared with control mice, only metronidazole-treated mice had significantly increased rates of dissemination of intestinal bacteria into mesenteric lymph nodes, indicating that the exclusive absence of anaerobic bacteria facilitated the translocation of the intestinal facultative bacteria. In a parallel experiment with streptomycin-resistant E. coli C25 as a marker, parallel results were obtained. Metronidazole increased the translocation of the marker strain and the indigenous strains of intestinal bacteria. Thus, anaerobes appeared to play a key role in confining indigenous bacteria to the gut. However, intestinal colonization and translocation of E. coli C25 occurred most readily after bacitracin-streptomycin treatment, suggesting that in addition to anaerobic bacteria, other bacterial groups may play a role in limiting the intestinal colonization and extraintestinal dissemination of E. coli C25.
PMCID: PMC259962  PMID: 3666959
20.  Inflammation-Induced Acid Tolerance Genes gadAB in Luminal Commensal Escherichia coli Attenuate Experimental Colitis 
Infection and Immunity  2013;81(10):3662-3671.
Dysregulated immune responses to commensal intestinal bacteria, including Escherichia coli, contribute to the development of inflammatory bowel diseases (IBDs) and experimental colitis. Reciprocally, E. coli responds to chronic intestinal inflammation by upregulating expression of stress response genes, including gadA and gadB. GadAB encode glutamate decarboxylase and protect E. coli from the toxic effects of low pH and fermentation acids, factors present in the intestinal lumen in patients with active IBDs. We hypothesized that E. coli upregulates gadAB during inflammation to enhance its survival and virulence. Using real-time PCR, we determined gadAB expression in luminal E. coli from ex-germfree wild-type (WT) and interleukin-10 (IL-10) knockout (KO) (IL-10−/−) mice selectively colonized with a commensal E. coli isolate (NC101) that causes colitis in KO mice in isolation or in combination with 7 other commensal intestinal bacterial strains. E. coli survival and host inflammatory responses were measured in WT and KO mice colonized with NC101 or a mutant lacking the gadAB genes (NC101ΔgadAB). The susceptibility of NC101 and NC101ΔgadAB to killing by host antimicrobial peptides and their translocation across intestinal epithelial cells were evaluated using bacterial killing assays and transwell experiments, respectively. We show that expression of gadAB in luminal E. coli increases proportionately with intestinal inflammation in KO mice and enhances the susceptibility of NC101 to killing by the host antimicrobial peptide cryptdin-4 but decreases bacterial transmigration across intestinal epithelial cells, colonic inflammation, and mucosal immune responses. Chronic intestinal inflammation upregulates acid tolerance pathways in commensal E. coli isolates, which, contrary to our original hypothesis, limits their survival and colitogenic potential. Further investigation of microbial adaptation to immune-mediated inflammation may provide novel insights into the pathogenesis and treatment of IBDs.
PMCID: PMC3811779  PMID: 23876805
21.  Ecology and evolution of viruses infecting uncultivated SUP05 bacteria as revealed by single-cell- and meta-genomics 
eLife  2014;3:e03125.
Viruses modulate microbial communities and alter ecosystem functions. However, due to cultivation bottlenecks, specific virus–host interaction dynamics remain cryptic. In this study, we examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria isolated from a model marine oxygen minimum zone (OMZ) to identify 69 viral contigs representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. Infection frequencies suggest that ∼1/3 of SUP05 bacteria is viral-infected, with higher infection frequency where oxygen-deficiency was most severe. Observed Microviridae clonality suggests recovery of bloom-terminating viruses, while systematic co-infection between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. Analyses of 186 microbial and viral metagenomes revealed that SUP05 viruses persisted for years, but remained endemic to the OMZ. Finally, identification of virus-encoded dissimilatory sulfite reductase suggests SUP05 viruses reprogram their host's energy metabolism. Together, these results demonstrate closely coupled SUP05 virus–host co-evolutionary dynamics with the potential to modulate biogeochemical cycling in climate-critical and expanding OMZs.
eLife digest
Microorganisms help to drive a number of processes that recycle energy and nutrients, including elements such as carbon, nitrogen, and sulfur, around the Earth's ecosystems. Viruses that infect microbes can also affect these cycles by killing and breaking open microbial cells, or by reprogramming the cell's metabolism. However, as there are many different species of microbes and viruses —the vast majority of which cannot easily be grown in the laboratory— little is known about most virus–host interactions in natural ecosystems, especially in the oceans.
In the world's oceans, the concentration of oxygen dissolved in the water changes in different regions and at different depths. ‘Oxygen minimum zones’ occur globally throughout the oceans at depths of 200–1000 meters, and climate change is causing these zones to expand and intensify. Although a lack of oxygen is sometimes considered detrimental to living organisms, oxygen minimum zones appear to be rich with microbial life that is adapted to thrive under oxygen-starved conditions.
Sulfur-oxidizing bacteria are one of the most abundant groups of microbes in these oxygen minimum zones, and several of these bacteria are known to influence the recycling of chemical substances. Now, Roux et al. introduce a new method to identify viruses that infect the microbes in this environment, including those microbes that cannot be grown in the laboratory and which have previously remained largely unexplored.
The genomes of 127 individual bacterial cells —collected from an oxygen minimum zone in western Canada— were examined. Roux et al. estimate that about a third of the sulfur-oxidizing bacterial cells are infected by at least one virus, but often multiple viruses infected the same bacterium. Five new genera (groups of one or more species) of viruses were also discovered and found to infect these bacteria. Looking for these new viral sequences in the DNA of this oxygen minimum zone's microbial community revealed that these newly discovered viruses persist in this region over several years. It also revealed that these viruses appear to only be found within the oxygen minimum zone. Roux et al. uncovered that these viruses carry genes that could manipulate how an infected bacterium processes sulfur-containing compounds; this is similar to previous observations showing that other viruses also influence cellular process (such as photosynthesis) in infected bacteria. As such, these newly discovered viruses might also influence the recycling of chemical elements within oxygen minimum zones.
Together, Roux et al.'s findings provide an unprecedented look into a wild virus community using a method that can be generalized to uncover viruses in a data type that is quickly becoming more widespread: single cell genomes. This effort to understand virus–host interactions by looking in the genomes of individual cells now sets the stage for future efforts aimed to uncover the impact of viruses on bacteria in other environments across the globe.
PMCID: PMC4164917  PMID: 25171894
SUP05; bacteriophages; viruses; single cell genomics; oxygen minimum zone; viral dark matter; other
22.  Bacterial Community Assembly and Turnover within the Intestines of Developing Zebrafish 
PLoS ONE  2012;7(1):e30603.
The majority of animal associated microorganisms are present in digestive tract communities. These intestinal communities arise from selective pressures of the gut habitats as well as host's genotype are regarded as an extra ‘organ’ regulate functions that have not evolved wholly on the host. They are functionally essential in providing nourishment, regulating epithelial development, and influencing immunity in the vertebrate host. As vertebrates are born free of microorganisms, what is poorly understood is how intestinal bacterial communities assemble and develop in conjunction with the development of the host.
Methodology/Principal Findings
Set within an ecological framework, we investigated the bacterial community assembly and turnover within the intestinal habitats of developing zebrafish (from larvae to adult animals). Spatial and temporal species-richness relationships and Mantel and partial Mantel tests revealed that turnover was low and that richness and composition was best predicted by time and not intestinal volume (habitat size) or changes in food diet. We also observed that bacterial communities within the zebrafish intestines were deterministically assembled (reflected by the observed low turnover) switching to stochastic assembly in the later stages of zebrafish development.
This study is of importance as it provides a novel insight into how intestinal bacterial communities assemble in tandem with the host's development (from early to adult stages). It is our hope that by studying intestinal microbiota of this vertebrate model with such or some more refined approaches in the future could well provide ecological insights for clinical benefit. In addition, this study also adds to our still fledgling knowledge of how spatial and temporal species-richness relationships are shaped and provides further mounting evidence that bacterial community assembly and dynamics are shaped by both deterministic and stochastic considerations.
PMCID: PMC3261916  PMID: 22276219
23.  Recolonization and colonization resistance of the large bowel after three methods of preoperative preparation of the gastrointestinal tract for elective colorectal surgery. 
The Journal of Hygiene  1986;97(1):49-59.
The impact of three current types of preoperative large bowel preparation on the microbial flora and the colonization resistance (CR) was investigated in 15 volunteers. In the first group a whole gut irrigation was performed without administration of antibiotics (group WGI). In the second group 0.5 g/l metronidazole and 1 g/l neomycin was added to the irrigation fluid (group WGI + AB). A whole gut irrigation with prior oral administration of 1 l mannitol 10% was performed in the third group. The antibiotic prophylaxis in this group consisted of two doses of 80 mg gentamicin i.v. and 500 mg metronidazole orally 24 h after lavage (group Mann + AB). One hour after the mechanical cleansing procedure was finished all volunteers were orally contaminated with one dose of an Escherichia coli test strain. The aerobic faecal reduction due to the cleansing procedure was 2-3 logs, while for the anaerobes it was 4-5 logs. The anaerobic flora in group WGI recovered within 24 h, while the aerobes showed a transient 'overgrowth' for the period of 2 days. The overgrowth of aerobes in group WGI + AB was observed for more than a week and the total numbers of aerobes started gradually to decline after the anaerobic flora had reached pretreatment levels at day three or four. Despite the normal numbers of anaerobes present 24 h after treatment, overgrowth of E. coli was seen in the group Mann + AB, probably due to residual mannitol left in the intestinal tract. The test strain of E. coli was excreted for a period of 1 week by the volunteers in the groups WGI and Mann + AB, but it was isolated for more than 10 weeks in the group WGI + AB. It is thought that all three methods of preoperative large bowel preparation decreased the CR of the gastrointestinal tract because of a disturbance of the interaction between aerobic and anaerobic microorganisms and alterations of the colonic wall. The anaerobic microflora, however, appeared to be primarily responsible for the maintenance of the CR. Antimicrobial prophylaxis should consist of a high dose, short term, systemic antibiotic regimen, not only because an adequate serum level of an appropriate drug at the time of operation substantially decreases the incidence of postoperative septic complications but also because a systemic regimen scarcely influences the CR of the gastrointestinal tract. beta-Aspartylglycine appeared to be a specific but not very sensitive marker for decreased CR.
PMCID: PMC2082873  PMID: 3734441
24.  Evolutionary Relationships of Wild Hominids Recapitulated by Gut Microbial Communities 
PLoS Biology  2010;8(11):e1000546.
Although bacteria are continually acquired over the lifetime of an individual, the phylogenetic relationships of great ape species is mirrored in the compositions of their gut microbial communities.
Multiple factors over the lifetime of an individual, including diet, geography, and physiologic state, will influence the microbial communities within the primate gut. To determine the source of variation in the composition of the microbiota within and among species, we investigated the distal gut microbial communities harbored by great apes, as present in fecal samples recovered within their native ranges. We found that the branching order of host-species phylogenies based on the composition of these microbial communities is completely congruent with the known relationships of the hosts. Although the gut is initially and continuously seeded by bacteria that are acquired from external sources, we establish that over evolutionary timescales, the composition of the gut microbiota among great ape species is phylogenetically conserved and has diverged in a manner consistent with vertical inheritance.
Author Summary
The microbial communities that inhabit the gastrointestinal tract of humans and other mammals are complex, dynamic, and critical to both health and disease. The composition and constituents of these communities are influenced by multiple factors such as host diet, geography, physiology, and disease state. Given the central role of the gut microbiota in the physiology of the host, it is important to determine whether it is predictable and substantially determined by the host, or variable and largely determined by the external environment (including diet) experienced by the host. A valuable way of determining the relative contributions of such factors is by comparing gut microbial communities in closely related host species. Applying a high-throughput sequencing approach, we profiled the distal gut microbiotae of great ape species sampled in their native ranges and then employed a parsimony-based analysis of phylogenetically informative phylotypes (i.e., bacterial taxa residing in multiple individuals) to determine the relationships among the diverse microbial communities. Our analyses revealed a clear species-specific signature of microbial community structure. Moreover, the pattern of relationships among the five great ape species (Homo sapiens, Pan troglodytes, P. paniscus, Gorilla gorilla, and G. beringei) inferred from their fecal microbial communities was identical to that inferred from host mitochondrial DNA, indicating that host phylogeny shapes the gut microbiota over evolutionary timescales. It seems after all that you are not what you eat.
PMCID: PMC2982803  PMID: 21103409
25.  Comparative Genomic and Functional Analysis of 100 Lactobacillus rhamnosus Strains and Their Comparison with Strain GG 
PLoS Genetics  2013;9(8):e1003683.
Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.
Author Summary
Some bacterial species are specialists and adapted to a single niche, while others are generalists and able to grow in various environmental conditions. Lactobacillus rhamnosus is a generalist and its members can often be found in different human cavities but also in various artisanal and industrial dairy products. To gain insights into the genetic complexity and ecological versatility of this species, we collected 100 L. rhamnosus strains from different niches. Genomic and functional analysis of these revealed a dichotomy within the species that reflected its adaptation to particular niches. The variable regions identified in the L. rhamnosus genome encode lifestyle traits that allowed us to demonstrate that some L. rhamnosus isolates possibly resided in multiple habitats. Our work brings valuable data on the ecological dynamics and adaptability of the species and provides a basis for a model explaining the ecology of L. rhamnosus in an anthropocentric perspective. Finally, we observed that a set of pheno-genomic markers, i.e. CRISPR oligotyping or carbohydrate metabolism, would be sufficient and among the best ways to differentiate the L. rhamnosus strains, providing a general approach to select the highest diversity in these and other bacterial species.
PMCID: PMC3744422  PMID: 23966868

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