Mouse Model Of Colonization Research Articles

The Rgg1518 transcriptional regulator is a necessary facet of sugar metabolism and virulence in Streptococcus pneumoniae.

Rggs are a group of transcriptional regulators with diverse roles in metabolism and virulence. Here, we present work on the Rgg1518/SHP1518 quorum sensing system of Streptococcus pneumoniae. The activity of Rgg1518 is induced by its cognate peptide, SHP1518. In vitro analysis showed that the Rgg1518 system is active in conditions rich in galactose and mannose, key nutrients during nasopharyngeal colonization. Rgg1518 expression is highly induced in the presence of these sugars and its isogenic mutant is attenuated in growth on galactose and mannose. When compared with other Rgg systems, Rgg1518 has the largest regulon on galactose. On galactose it controls up- or downregulation of a functionally diverse set of genes involved in galactose metabolism, capsule biosynthesis, iron metabolism, protein translation, as well as other metabolic functions, acting mainly as a repressor of gene expression. Rgg1518 is a repressor of capsule biosynthesis, and binds directly to the capsule regulatory region. Comparison with other Rggs revealed inter-regulatory interactions among Rggs. Finally, the rgg1518 mutant is attenuated in colonization and virulence in a mouse model of colonization and pneumonia. We conclude that Rgg1518 is a virulence determinant that contributes to a regulatory network composed of multiple Rgg systems.

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor.

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.

Lipidation of Pneumococcal Antigens Leads to Improved Immunogenicity and Protection.

Streptococcus pneumoniae infections lead to high morbidity and mortality rates worldwide. Pneumococcal polysaccharide conjugate vaccines significantly reduce the burden of disease but have a limited range of protection, which encourages the development of a broadly protective protein-based alternative. We and others have shown that immunization with pneumococcal lipoproteins that lack the lipid anchor protects against colonization. Since immunity against S. pneumoniae is mediated through Toll-like receptor 2 signaling induced by lipidated proteins, we investigated the effects of a lipid modification on the induced immune responses in either intranasally or subcutaneously vaccinated mice. Here, we demonstrate that lipidation of recombinant lipoproteins DacB and PnrA strongly improves their immunogenicity. Mice immunized with lipidated proteins showed enhanced antibody concentrations and different induction kinetics. The induced humoral immune response was modulated by lipidation, indicated by increased IgG2/IgG1 subclass ratios related to Th1-type immunity. In a mouse model of colonization, immunization with lipidated antigens led to a moderate but consistent reduction of pneumococcal colonization as compared to the non-lipidated proteins, indicating that protein lipidation can improve the protective capacity of the coupled antigen. Thus, protein lipidation represents a promising approach for the development of a serotype-independent pneumococcal vaccine.

Immune exclusion by naturally acquired secretory IgA against pneumococcal pilus-1.

Successful infection by mucosal pathogens requires overcoming the mucus barrier. To better understand this key step, we performed a survey of the interactions between human respiratory mucus and the human pathogen Streptococcus pneumoniae. Pneumococcal adherence to adult human nasal fluid was seen only by isolates expressing pilus-1. Robust binding was independent of pilus-1 adhesive properties but required Fab-dependent recognition of RrgB, the pilus shaft protein, by naturally acquired secretory IgA (sIgA). Pilus-1 binding by specific sIgA led to bacterial agglutination, but adherence required interaction of agglutinated pneumococci and entrapment in mucus particles. To test the effect of these interactions in vivo, pneumococci were preincubated with human sIgA before intranasal challenge in a mouse model of colonization. sIgA treatment resulted in rapid immune exclusion of pilus-expressing pneumococci. Our findings predict that immune exclusion would select for nonpiliated isolates in individuals who acquired RrgB-specific sIgA from prior episodes of colonization with piliated strains. Accordingly, genomic data comparing isolates carried by mothers and their children showed that mothers are less likely to be colonized with pilus-expressing strains. Our study provides a specific example of immune exclusion involving naturally acquired antibody in the human host, a major factor driving pneumococcal adaptation.

Candida albicans Morphogenesis Programs Control the Balance between Gut Commensalism and Invasive Infection

Candida albicans is a gut commensal and opportunistic pathogen. The transition between yeast and invasive hyphae is central to virulence but has unknown functions during commensal growth. In a mouse model of colonization, yeast and hyphae co-occur throughout the gastrointestinal tract. However, competitive infections of C.albicans homozygous gene disruption mutants revealed an unanticipated, inhibitory role for the yeast-to-hypha morphogenesis program on commensalism. We show that the transcription factor Ume6, a master regulator of filamentation, inhibits gut colonization, not by effects on cell shape, but by activating the expression of a hypha-specific pro-inflammatory secreted protease, Sap6, and a hyphal cell surface adhesin, Hyr1. Like a ume6 mutant, strains lacking SAP6 exhibit enhanced colonization fitness, whereas SAP6-overexpression strains are attenuated in the gut. These results reveal a tradeoff between fungal programs supporting commensalism and virulence in which selection against hypha-specific markers limits the disease-causing potential of this ubiquitous commensal-pathogen.

Immunogenicity and protective efficacy of monovalent PCVs containing 22F and 33F polysaccharides in mouse models of colonization and co-infection

BackgroundIn the current transmission, we studied the immunogenicity and protective efficacy of serotypes 22F and 33F in the prevention of colonization and of invasive Streptococcus pneumoniae (Spn) pathogenesis during an influenza co-infection. Serotypes 22F and 33F are emerging Spn serotypes, which are not part of currently administered pneumococcal conjugate vaccine formulations (PCVs). Spn serotype 6A is an ingredient in the currently administered PCV13 vaccine and was therefore included in the study as a control. MethodsAdult (six weeks) and infant (two weeks) C57BL/6 mice were intranasally infected in the nasopharynx (NP) with Spn serotypes 22F, 33F, or 6A. Influenza A H1N1 A/Puerto Rico/8/193 virus (PR8) was introduced one day after the NP Spn colonization. In an immunization challenge study, mice were vaccinated with monovalent 22F, 33F, or 6A polysaccharide conjugated to the CRM197 antigen. The immunized mice were colonized or co-infected to study the vaccines efficacy. ResultsAll three Spn serotypes established colonization in adult and infant mice. The co-infected mice showed an increase in Spn NP density. Invasive Spn infection (bacteremia) was observed following the co-infection with serotypes 22F and 6A but not 33F in adult mice, whereas infant mice developed bacteremia following co-infection with all three Spn serotypes. The vaccinations led to robust serum antibody responses to capsular polysaccharides 22F, 6A, and less for 33F. The vaccinations resulted in reductions of Spn NP colonization density for all three serotypes, prevention of bacteremia, and increased survival with Spn serotypes 22F and 6A. Passive transfer of antisera was associated with a reduction of Spn colonization densities in infant mice. ConclusionVaccinations with monovalent 22F, 33F, or 6A formulations protect against Spn colonization, and the efficacy of the 22F vaccination was comparable to the 6A vaccination in preventing an invasive Spn bacterial infection during an influenza co-infection.

Targeting Clostridium difficile Surface Components to Develop Immunotherapeutic Strategies Against Clostridium difficile Infection.

New therapies are needed to prevent and treat Clostridium difficile infection and to limit the rise in antibiotic resistance. Besides toxins, several surface components have been characterized as colonization factors and have been shown as immunogenic. This review will focus on passive and active immunization strategies targeting C. difficile surface components to combat C. difficile. Concerning passive immunization, the first strategies used antisera raised against the entire bacterium to prevent infection in the hamster model. Then, surface components such as the flagellin and the S-layer proteins were used for immunization and the passive transfer of antibodies was protective in animal models. Passive immunotherapy with polyvalent immunoglobulins was used in humans and bovine immunoglobulin concentrates were evaluated in clinical trials. Concerning active immunization, vaccine assays targeting surface components were tested mainly in animal models, mouse models of colonization and hamster models of infection. Bacterial extracts, spore proteins and surface components of vegetative cells such as cell wall proteins, flagellar proteins, and polysaccharides were used as vaccine targets. Vaccine assays were performed by parenteral and mucosal routes of immunization. Both gave promising results and pave the way to development of new vaccines.

Type IV Pili Promote Clostridium difficile Adherence and Persistence in a Mouse Model of Infection

Cyclic diguanylate (c-di-GMP) is a second messenger that regulates the transition from motile to sessile lifestyles in numerous bacteria and controls virulence factor production in a variety of pathogens. In Clostridium difficile, c-di-GMP negatively regulates flagellum biosynthesis and swimming motility and promotes the production of type IV pili (TFP), biofilm formation, and surface motility in vitro Flagella have been identified as colonization factors in C. difficile, but the role of TFP in adherence to host cells and in colonization of the mammalian gut is unknown. Here we show that c-di-GMP promotes adherence to epithelial cells in vitro, which can be partly attributed to the loss of flagella. Using TFP-null mutants, we demonstrate that adherence to epithelial cells is partially mediated by TFP and that this TFP-mediated adherence requires c-di-GMP regulation. In a mouse model of colonization, the TFP-null mutants initially colonized the intestine as well as the parental strain but were cleared more quickly. Moreover, compared to the parent strain, C. difficile strains lacking TFP were particularly deficient in association with the cecal mucosa. Together these data indicate that TFP and their positive regulation by c-di-GMP promote attachment of C. difficile to the intestinal epithelium and contribute to persistence of C. difficile in the host intestine.

Rgg-Shp regulators are important for pneumococcal colonization and invasion through their effect on mannose utilization and capsule synthesis

Microbes communicate with each other by using quorum sensing (QS) systems and modulate their collective ‘behavior’ for in-host colonization and virulence, biofilm formation, and environmental adaptation. The recent increase in genome data availability reveals the presence of several putative QS sensing circuits in microbial pathogens, but many of these have not been functionally characterized yet, despite their possible utility as drug targets. To increase the repertoire of functionally characterized QS systems in bacteria, we studied Rgg144/Shp144 and Rgg939/Shp939, two putative QS systems in the important human pathogen Streptococcus pneumoniae. We find that both of these QS circuits are induced by short hydrophobic peptides (Shp) upon sensing sugars found in the respiratory tract, such as galactose and mannose. Microarray analyses using cultures grown on mannose and galactose revealed that the expression of a large number of genes is controlled by these QS systems, especially those encoding for essential physiological functions and virulence-related genes such as the capsular locus. Moreover, the array data revealed evidence for cross-talk between these systems. Finally, these Rgg systems play a key role in colonization and virulence, as deletion mutants of these QS systems are attenuated in the mouse models of colonization and pneumonia.

Surface Proteins and Pneumolysin of Encapsulated and Nonencapsulated Streptococcus pneumoniae Mediate Virulence in a Chinchilla Model of Otitis Media.

Streptococcus pneumoniae infections result in a range of human diseases and are responsible for almost one million deaths annually. Pneumococcal disease is mediated in part through surface structures and an anti-phagocytic capsule. Recent studies have shown that nonencapsulated S. pneumoniae (NESp) make up a significant portion of the pneumococcal population and are able to cause disease. NESp lack some common surface proteins expressed by encapsulated pneumococci, but express surface proteins unique to NESp. A chinchilla model of otitis media (OM) was used to determine the effect various pneumococcal mutations have on pathogenesis in both NESp and encapsulated pneumococci. Epithelial cell adhesion and invasion assays were used to examine the effects in relation to deletion of intrinsic genes or expression of novel genes. A mouse model of colonization was also utilized for comparison of various pneumococcal mutants. It was determined that pneumococcal surface protein K (PspK) and pneumolysin (Ply) affect NESp middle ear pathogenesis, but only PspK affected epithelial cell adhesion. Experiments in an OM model were done with encapsulated strains testing the importance of native virulence factors and treatment of OM. First, a triple deletion of the common virulence factors PspA, PspC, and Ply, (ΔPAC), from an encapsulated background abolished virulence in an OM model while a PspC mutant had detectable, but reduced amounts of recoverable bacteria compared to wildtype. Next, treatment of OM was effective when starting antibiotic treatment within 24 h with resolution by 48 h post-treatment. Expression of NESp-specific virulence factor PspK in an encapsulated strain has not been previously studied, and we showed significantly increased adhesion and invasion of human epithelial cells by pneumococci. Murine colonization was not significantly increased when an encapsulated strain expressed PspK, but colonization was increased when a capsule mutant expressed PspK. The ability of PspK expression to increase colonization in a capsule mutant despite no increase in adhesion can be attributed to other functions of PspK, such as sIgA binding or immune modulation. OM is a substantial economic burden, thus a better understanding of both encapsulated pneumococcal pathogenesis and the emerging pathogen NESp is necessary for effective prevention and treatment.

Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance.

The fungal pathogen Candida glabrata has emerged as a major health threat since it readily acquires resistance to multiple drug classes, including triazoles and/or echinocandins. Thus far, cellular mechanisms promoting the emergence of resistance to multiple drug classes have not been described in this organism. Here we demonstrate that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata clinical isolates. Strains carrying alterations in mismatch repair gene MSH2 exhibit a higher propensity to breakthrough antifungal treatment in vitro and in mouse models of colonization, and are recovered at a high rate (55% of all C. glabrata recovered) from patients. This genetic mechanism promotes the acquisition of resistance to multiple antifungals, at least partially explaining the elevated rates of triazole and multi-drug resistance associated with C. glabrata. We anticipate that identifying MSH2 defects in infecting strains may influence the management of patients on antifungal drug therapy.

Immunization with Pneumococcal Surface Protein K of Nonencapsulated Streptococcus pneumoniae Provides Protection in a Mouse Model of Colonization.

Current vaccinations are effective against encapsulated strains of Streptococcus pneumoniae, but they do not protect against nonencapsulated Streptococcus pneumoniae (NESp), which is increasing in colonization and incidence of pneumococcal disease. Vaccination with pneumococcal proteins has been assessed for its ability to protect against pneumococcal disease, but several of these proteins are not expressed by NESp. Pneumococcal surface protein K (PspK), an NESp virulence factor, has not been assessed for immunogenic potential or host modulatory effects. Mammalian cytokine expression was determined in an in vivo mouse model and in an in vitro cell culture system. Systemic and mucosal mouse immunization studies were performed to determine the immunogenic potential of PspK. Murine serum and saliva were collected to quantitate specific antibody isotype responses and the ability of antibody and various proteins to inhibit epithelial cell adhesion. Host cytokine response was not reduced by PspK. NESp was able to colonize the mouse nasopharynx as effectively as encapsulated pneumococci. Systemic and mucosal immunization provided protection from colonization by PspK-positive (PspK(+)) NESp. Anti-PspK antibodies were recovered from immunized mice and significantly reduced the ability of NESp to adhere to human epithelial cells. A protein-based pneumococcal vaccine is needed to provide broad protection against encapsulated and nonencapsulated pneumococci in an era of increasing antibiotic resistance and vaccine escape mutants. We demonstrate that PspK may serve as an NESp target for next-generation pneumococcal vaccines. Immunization with PspK protected against pneumococcal colonization, which is requisite for pneumococcal disease.

Effect of nonheme iron-containing ferritin Dpr in the stress response and virulence of pneumococci.

Streptococcus pneumoniae (pneumococcus) produces hydrogen peroxide as a by-product of metabolism and provides a competitive advantage against cocolonizing bacteria. As pneumococci do not produce catalase or an inducible regulator of hydrogen peroxide, the mechanism of resistance to hydrogen peroxide is unclear. A gene responsible for resistance to hydrogen peroxide and iron in other streptococci is that encoding nonheme iron-containing ferritin, dpr, but previous attempts to study this gene in pneumococcus by generating a dpr mutant were unsuccessful. In the current study, we found that dpr is in an operon with the downstream genes dhfr and clpX. We generated a dpr deletion mutant which displayed normal early-log-phase and mid-log-phase growth in bacteriologic medium but survived less well at stationary phase; the addition of catalase partially rescued the growth defect. We showed that the dpr mutant is significantly more sensitive to pH, heat, iron concentration, and oxidative stress due to hydrogen peroxide. Using a mouse model of colonization, we also showed that the dpr mutant displays a reduced ability to colonize and is more rapidly cleared from the nasopharynx. Our results thus suggest that Dpr is important for pneumococcal resistance to stress and for nasopharyngeal colonization.

Piliation of invasive Streptococcus pneumoniae isolates in the era before pneumococcal conjugate vaccine introduction in Malawi.

The pneumococcal pilus has been shown to be an important determinant of adhesion and virulence in mouse models of colonization, pneumonia, and bacteremia. A pilus is capable of inducing protective immunity, supporting its inclusion in next-generation pneumococcal protein vaccine formulations. Whether this vaccine target is common among pneumococci in sub-Saharan Africa is uncertain. To define the prevalence and genetic diversity of type I and II pili among invasive pneumococci in Malawi prior to the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into routine childhood immunization, we examined 188 Streptococcus pneumoniae isolates collected between 2002 and 2008 (17% serotype 1). In this region of high disease burden, we found a low frequency of invasive piliated pneumococci (14%) and pilus gene sequence diversity similar to that seen previously in multiple global pneumococcal lineages. All common serotypes with pilus were covered by PCV13 and so we predict that pilus prevalence will be reduced in the Malawian pneumococcal population after PCV13 introduction.

Export Requirements of Pneumolysin in Streptococcus pneumoniae

Streptococcus pneumoniae is a major causative agent of otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolysins (CDCs), is produced by virtually all clinical isolates of S. pneumoniae, and ply mutant strains are severely attenuated in mouse models of colonization and infection. In contrast to all other known members of the CDC family, Ply lacks a signal peptide for export outside the cell. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains undefined. We show that an exogenously added signal sequence is not sufficient for Sec-dependent Ply secretion in S. pneumoniae but is sufficient in the surrogate host Bacillus subtilis. Previously, we showed that Ply is localized primarily to the cell wall compartment in the absence of detectable cell lysis. Here we show that Ply released by autolysis cannot reassociate with intact cells, suggesting that there is a Ply export mechanism that is coupled to cell wall localization of the protein. This putative export mechanism is capable of secreting a related CDC without its signal sequence. We show that B. subtilis can export Ply, suggesting that the export pathway is conserved. Finally, through truncation and domain swapping analyses, we show that export is dependent on domain 2 of Ply.

Nasopharyngeal Colonization and Invasive Disease Are Enhanced by the Cell Wall Hydrolases LytB and LytC of Streptococcus pneumoniae

Background Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen.Methodology/Principal FindingsWe have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs) LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection.Conclusions/SignificanceThis study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factors involved in the colonization of the nasopharynx and in the progress of invasive disease by avoiding host immunity.

Functional characteristics of a protective monoclonal antibody against serotype A and C lipooligosaccharides from Moraxella catarrhalis.

A monoclonal antibody (MAb), designated MAb 8E7 (immunoglobulin G3), specific for Moraxella catarrhalis lipooligosaccharide (LOS) was evaluated for its functional activity in vitro and in a mouse model of colonization. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the MAb 8E7 could be prepared to a high titer against LOS of the homologous strain 035E, and that it had bactericidal activity. MAb 8E7 reacted with M. catarrhalis serotype A and C LOSs but not serotype B LOS, as measured by ELISA and Western blotting. On the basis of published structures of LOSs, this suggests that the epitope recognized by MAb 8E7 is directed to a common sequence of either alpha-GlcNAc-(1-->2)-beta-Glc-(1--> at the branch substituting position 4 of the trisubstituted Glc residue or a terminal tetrasaccharide alpha-Gal-(1-->4)-beta-Gal-(1-->4)-alpha-Glc-(1-->2)-beta-Glc-(1--> at the branch substituting position 6 of the trisubstituted Glc residue. In a whole-cell ELISA, MAb 8E7 reacted with 70% of the 30 wild-type strains and clinical isolates tested. Immuno-electron microscopy demonstrated that MAb 8E7 reacted with a cell surface-exposed epitope of LOS on strain O35E. MAb 8E7 inhibited the adherence of strain O35E to Chang conjunctival epithelial cells by 90%. Passive immunization with MAb 8E7 could significantly enhance the clearance of strain O35E from mouse lungs in an aerosol challenge mouse model. This enhanced bacterial clearance was inhibited when MAb 8E7 was absorbed by M. catarrhalis serotype A LOS, indicating that the M. catarrhalis LOS-directed antibody may play a major role in the enhancement of M. catarrhalis clearance from lungs. These data suggest that MAb 8E7, which recognizes surface-exposed LOS of M. catarrhalis, is a protective antibody against M. catarrhalis.

Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 isolates produce Shiga-like toxins and carry a 60-megadalton plasmid which encodes an adhesin for Henle 407 intestinal cells. A streptomycin-treated mouse model was used to compare the intestinal colonizing capacity of E. coli O157:H7 strain 933 with that of its 60-megadalton plasmid-cured derivative, strain 933cu. When fed individually to mice, both 933 and 933cu maintained a stable number of organisms per gram of feces, and the greatest numbers of 933 or 933cu were isolated from cecal and proximal colonic epithelial cells. When 933 and 933cu were simultaneously fed to mice, 933cu was unable to maintain a stable level of colonization in about two-thirds of the mice tested. However, in one-third of the mice, the number of 933cu in feces began to increase rapidly until a stable level of co-colonization with 933 was attained. The isolate from these mice, 933cu-rev, was excreted in high numbers when fed alone to mice and was found on epithelial cells throughout the entire large bowel and distal small intestine. Moreover, 933cu-rev grew in mucus from all segments of the intestine and at higher levels than strain 933 or 933cu. Only mice fed strain 933cu-rev died. Histopathological studies confirmed that mice fed 933cu-rev died from bilateral renal cortical tubular necrosis consistent with toxic insult, perhaps due to Shiga-like toxins. The virulence of 933cu-rev may reflect its ability to grow well in mucus and colonize the small as well as large bowel.