Enteric salmonellosis disrupts the microbial ecology of the murine gastrointestinal tract

The commensal microbiota protects the murine host from enteric pathogens. Nevertheless, specific pathogens are able to colonize the intestinal tract and invade, despite the presence of an intact biota. Possibly, effective pathogens disrupt the indigenous microbiota, either directly through pathogen-commensal interaction, indirectly via the host mucosal immune response to the pathogen, or by a combination of these factors. This study investigates the effect of peroral Salmonella enterica serovar Typhimurium infection on the intestinal microbiota. Since the majority of the intestinal microbiota cannot be cultured by conventional techniques, molecular approaches using 16S rRNA sequences were applied. Several major bacterial groups were assayed using quantitative PCR. Administration of either the 50% lethal dose (LD₅₀) or 10× LD₅₀ of Salmonella enterica serovar Typhimurium caused changes in the microbiota throughout the intestinal tract over the time course of infection. A 95% decrease in total bacterial number was noted in the cecum and large intestine with 10× LD₅₀ S. enterica serovar Typhimurium challenge at 7 days postinfection, concurrent with gross evidence of diarrhea. In addition, alterations in microbiota composition preceded the onset of diarrhea, suggesting the involvement of pathogen-commensal interactions and/or host responses unrelated to diarrhea. Microbiota alterations were not permanent and reverted to the microbiota of uninfected mice by 1 month postinfection. Infection with a Salmonella pathogenicity island 1 (SPI1) mutant did not result in microbiota alterations, while SPI2 mutant infections triggered partial changes. Neither mutant was capable of prolonged colonization or induction of mucosal inflammation. These data suggest that several Salmonella virulence factors, particularly those involved in the local mucosal host response, are required for disruption of the intestinal ecosystem.     

Citrobacter rodentium: a model enteropathogen for understanding the interplay of innate and adaptive components of type 3 immunity

Citrobacter rodentium is a natural murine intestinal pathogen that shares a core set of virulence factors with the related human pathogens enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). C. rodentium is now the most widely used small animal model for studying the molecular underpinnings of EPEC and EHEC infections in vivo, including: enterocyte attachment; virulence; colonization resistance; and mucosal immunity. In this review, we discuss type 3 immunity in the context of C. rodentium infection and discuss recent publications that use this model to understand how the innate and adaptive components of immunity intersect to mediate host protection against enteric pathogens and maintain homeostasis with the microbiota.     

Epithelial barrier: an interface for the cross-communication between gut flora and immune system

Large numbers of environmental antigens, including commensal bacteria and food-derived antigens, constitutively interact with the epithelial layer of the gastrointestinal (GI) tract. Commensal bacteria peacefully cohabit with the host GI tract and exert multiple beneficial or destructive effects on their host. Intestinal epithelial cells (IECs) constitute the first physical and immunological protective wall against invasive pathogens and a cohabitation niche for commensal bacteria. As the physiological homeostasis of IECs is maintained by multiple biological processes such as apoptosis, autophagy, and the handling of endoplasmic reticulum stress, the aberrant kinetics of these biological events, which have genetic and environmental causes, leads to the development of host intestinal pathogenesis such as inflammatory bowel disease. In addition, IECs recognize and interact with commensal bacteria and give instructions to mucosal immune cells to initiate an immunological balance between active and quiescent conditions, eventually establishing intestinal homeostasis. The mucosal immune system regulates the homeostasis of gut microbiota by producing immunological molecules such as secretory immunoglobulin A, the production of which is mediated by IECs. IECs therefore play a central role in the creation and maintenance of a physiologically and immunologically stable intestinal environment.     

The gut mucus network: A dynamic liaison between microbes and the immune system

A complex mucus network made up of large polymers of the mucin-family glycoprotein MUC2 exists between the large intestinal microbial mass and epithelial and immune cells. This has long been understood as an innate immune defense barrier against the microbiota and other luminal threats that reinforces the barrier function of the epithelium and limits microbiota contact with the tissues. However, past and recent studies have provided new evidence of how critical the mucus network is to act as a ‘liaison’ between host and microbe to mediate anti-inflammatory, mutualistic interactions with the microbiota and protection from pathogens. This review summarizes historical and recent insights into the formation of the gut mucus network, how the microbes and immune system influence mucus, and in turn, how the mucus influences immune responses to the microbiota.     

Antibiotic treatment alters the colonic mucus layer and predisposes the host to exacerbated Citrobacter rodentium-induced colitis

Antibiotics are often used in the clinic to treat bacterial infections, but the effects of these drugs on microbiota composition and on intestinal immunity are poorly understood. Citrobacter rodentium was used as a model enteric pathogen to investigate the effect of microbial perturbation on intestinal barriers and susceptibility to colitis. Streptomycin and metronidazole were used to induce alterations in the composition of the microbiota prior to infection with C. rodentium. Metronidazole pretreatment increased susceptibility to C. rodentium-induced colitis over that of untreated and streptomycin-pretreated mice, 6 days postinfection. Both antibiotic treatments altered microbial composition, without affecting total numbers, but metronidazole treatment resulted in a more dramatic change, including a reduced population of Porphyromonadaceae and increased numbers of lactobacilli. Disruption of the microbiota with metronidazole, but not streptomycin treatment, resulted in an increased inflammatory tone of the intestine characterized by increased bacterial stimulation of the epithelium, altered goblet cell function, and thinning of the inner mucus layer, suggesting a weakened mucosal barrier. This reduction in mucus thickness correlates with increased attachment of C. rodentium to the intestinal epithelium, contributing to the exacerbated severity of C. rodentium-induced colitis in metronidazole-pretreated mice. These results suggest that antibiotic perturbation of the microbiota can disrupt intestinal homeostasis and the integrity of intestinal defenses, which protect against invading pathogens and intestinal inflammation.     

Pathobionts of the gastrointestinal microbiota and inflammatory disease

Our immune system is charged with the vital mission of identifying invading pathogens and mounting proper inflammatory responses. During the process of clearing infections, the immune system often causes considerable tissue damage. Conversely, if the target of immunity is a member of the resident microbiota, uncontrolled inflammation may lead to host pathology in the absence of infectious agents. Recent evidence suggests that several inflammatory disorders may be caused by specific bacterial species found in most healthy hosts. Although the mechanisms that mediate pathology remain largely unclear, it appears that genetic defects and/or environmental factors may predispose mammals to immune-mediated diseases triggered by potentially pathogenic symbionts of the microbiota. We have termed this class of microbes 'pathobionts', to distinguish them from acquired infectious agents. Herein, we explore burgeoning hypotheses that the combination of an immunocompromised state with colonization by pathobionts together comprise a risk factor for certain inflammatory disorders and gastrointestinal (GI) cancer.     

Intestinal colonization resistance

Dense, complex microbial communities, collectively termed the microbiota, occupy a diverse array of niches along the length of the mammalian intestinal tract. During health and in the absence of antibiotic exposure the microbiota can effectively inhibit colonization and overgrowth by invading microbes such as pathogens. This phenomenon is called ‘colonization resistance’ and is associated with a stable and diverse microbiota in tandem with a controlled lack of inflammation, and involves specific interactions between the mucosal immune system and the microbiota. Here we overview the microbial ecology of the healthy mammalian intestinal tract and highlight the microbe–microbe and microbe–host interactions that promote colonization resistance. Emerging themes highlight immunological (T helper type 17/regulatory T‐cell balance), microbiota (diverse and abundant) and metabolic (short‐chain fatty acid) signatures of intestinal health and colonization resistance. Intestinal pathogens use specific virulence factors or exploit antibiotic use to subvert colonization resistance for their own benefit by triggering inflammation to disrupt the harmony of the intestinal ecosystem. A holistic view that incorporates immunological and microbiological facets of the intestinal ecosystem should facilitate the development of immunomodulatory and microbe‐modulatory therapies that promote intestinal homeostasis and colonization resistance.     

Regulation of the polymeric immunoglobulin receptor and IgA transport: new advances in environmental factors that stimulate pIgR expression and its role in mucosal immunity

Secretory IgA (SIgA) antibodies represent the first line of antigen-specific immune defense protecting the mucosal surfaces against environmental pathogens and antigens, and maintaining homeostasis with the commensal microbiota. The polymeric immunoglobulin receptor (pIgR) has the dual role of transporting locally produced dimeric IgA across mucosal epithelia, and serving as the precursor of secretory component, a glycoprotein that enhances the immune functions of SIgA. The complex regulation of pIgR expression and transcytosis by host and microbial factors is finely tuned to optimize the role of SIgA in mucosal immunity. Disruption of this regulatory network in disease states similar to inflammatory bowel disease can result in profound consequences for mucosal homeostasis and systemic sequelae. Future research into the function and regulation of pIgR and SIgA may offer new insights into the prevention and treatment of infectious and inflammatory diseases that originate at mucosal surfaces.     

Interactions among strategies associated with bacterial infection: pathogenicity,epidemicity, and antibiotic resistance

Infections have been the major cause of disease throughout the history of human populations. With the introduction of antibiotics, it was thought that this problem should disappear. However, bacteria have been able to evolve to become antibiotic resistant. Nowadays, a proficient pathogen must be virulent, epidemic, and resistant to antibiotics. Analysis of the interplay among these features of bacterial populations is needed to predict the future of infectious diseases. In this regard, we have reviewed the genetic linkage of antibiotic resistance and bacterial virulence in the same genetic determinants as well as the cross talk between antibiotic resistance and virulence regulatory circuits with the aim of understanding the effect of acquisition of resistance on bacterial virulence. We also discuss the possibility that antibiotic resistance and bacterial virulence might prevail as linked phenotypes in the future. The novel situation brought about by the worldwide use of antibiotics is undoubtedly changing bacterial populations. These changes might alter the properties of not only bacterial pathogens, but also the normal host microbiota. The evolutionary consequences of the release of antibiotics into the environment are largely unknown, but most probably restoration of the microbiota from the preantibiotic era is beyond our current abilities.     

Complement evasion of pathogens: Common strategies are shared by diverse organisms

Infectious diseases represent a major health problem. Based on the limited efficacy of existing drugs and vaccines and the increasing antibiotic resistance new strategies are needed to fight infectious diseases. A better understanding of pathogen–host interaction is one important aspect to identify new virulence factors and antimicrobial and anti-inflammatory compounds utilized by pathogens represent an additional source for effective anti-inflammatory compounds. Complement forms a major defense line against invading microbes, and pathogens have learned during evolution to breach this defense line. The characterization of how pathogens evade complement attack is a rapidly developing field of current research. Pathogens mimic host surfaces and bind host complement regulators. Similarly pathogens utilize a number of complement inhibitory molecules which help to evade complement attack and which display anti-inflammatory activity. The molecular identification of these molecules, as well as the functional characterization of their roles at the pathogen–host interface is an important and emerging field of infection biology. In addition, pathogens utilize multiple sets of such regulators as redundancy and multiplicity is important for immune and complement evasion. Here we summarize the current scenarios of this emerging field which identifies multiple virulence factors and complement evasion strategies, but which at the same time reveals common mechanisms for immune and complement defense.     

Infectious disorders of the upper gastrointestinal tract (excluding Helicobacter pylori)

The upper gastrointestinal (GI) tract, including the oesophagus, stomach and small intestine, is subject to a vast array of pathogens. While some may be a reflection of disseminated infection, others produce disease specific to the upper GI tract. This review focuses on the most common infectious disorders of the upper GI tract that may be encountered by the general surgical pathologist, including viral, bacterial, fungal and parasitic organisms. Clinical and diagnostic histological features are discussed, as well as useful ancillary diagnostic techniques.     

Complement evasion of pathogens: common strategies are shared by diverse organisms

Infectious diseases represent a major health problem. Based on the limited efficacy of existing drugs and vaccines and the increasing antibiotic resistance new strategies are needed to fight infectious diseases. A better understanding of pathogen–host interaction is one important aspect to identify new virulence factors and antimicrobial and anti-inflammatory compounds utilized by pathogens represent an additional source for effective anti-inflammatory compounds. Complement forms a major defense line against invading microbes, and pathogens have learned during evolution to breach this defense line. The characterization of how pathogens evade complement attack is a rapidly developing field of current research. Pathogens mimic host surfaces and bind host complement regulators. Similarly pathogens utilize a number of complement inhibitory molecules which help to evade complement attack and which display anti-inflammatory activity. The molecular identification of these molecules, as well as the functional characterization of their roles at the pathogen–host interface is an important and emerging field of infection biology. In addition, pathogens utilize multiple sets of such regulators as redundancy and multiplicity is important for immune and complement evasion. Here we summarize the current scenarios of this emerging field which identifies multiple virulence factors and complement evasion strategies, but which at the same time reveals common mechanisms for immune and complement defense.     

Infectious disorders of the upper gastrointestinal tract (excluding Helicobacter pylori)

The upper gastrointestinal (GI) tract, including the oesophagus, stomach and small intestine, is host to numerous microorganisms. Some reflect gastrointestinal involvement by systemic disease, but others are primary digestive disorders that first present at GI sites. This review focuses on the most common infectious disorders of the upper GI tract encountered in general surgical pathology practice, including viral, bacterial, fungal and parasitic organisms. Clinical and histological features are discussed, as well as useful ancillary diagnostic techniques.     

The streptomycin mouse model for Salmonella diarrhea: functional analysis of the microbiota, the pathogen's virulence factors, and the host's mucosal immune response

The mammalian intestine is colonized by a dense microbial community, the microbiota. Homeostatic and symbiotic interactions facilitate the peaceful co-existence between the microbiota and the host, and inhibit colonization by most incoming pathogens ('colonization resistance'). However, if pathogenic intruders overcome colonization resistance, a fierce, innate inflammatory defense can be mounted within hours, the adaptive arm of the immune system is initiated, and the pathogen is fought back. The molecular nature of the homeostatic interactions, the pathogen's ability to overcome colonization resistance, and the triggering of native and adaptive mucosal immune responses are still poorly understood. To study these mechanisms, the streptomycin mouse model for Salmonella diarrhea is of great value. Here, we review how S. Typhimurium triggers mucosal immune responses by active (virulence factor elicited) and passive (MyD88-dependent) mechanisms and introduce the S. Typhimurium mutants available for focusing on either response. Interestingly, mucosal defense turns out to be a double-edged sword, limiting pathogen burdens in the gut tissue but enhancing pathogen growth in the gut lumen. This model allows not only studying the molecular pathogenesis of Salmonella diarrhea but also is ideally suited for analyzing innate defenses, microbe handling by mucosal phagocytes, adaptive secretory immunoglobulin A responses, probing microbiota function, and homeostatic microbiota-host interactions. Finally, we discuss the general need for defined assay conditions when using animal models for enteric infections and the central importance of littermate controls.     

A murine model of chronic mucosal colonization by Pseudomonas aeruginosa.

Chronic mucosal colonization by Pseudomonas aeruginosa is an integral part of the pathologic process associated with disease due to infection with this organism. We have adapted the streptomycin-treated murine model of chronic mucosal colonization by enteric pathogens to study colonization by P. aeruginosa. Mice first received 1 mg of streptomycin per ml of drinking water for 2 to 5 days and then ingested 10(7) CFU of P. aeruginosa per ml of drinking water for a minimum of 5 days. The result of this regimen was chronic mucosal colonization with P. aeruginosa for up to 10 weeks, which was determined by fecal cultures and confirmed by culture of the intestines after killing of the experimental animals. Bacterial counts were highest in the cecum and colon, with some evidence for extraintestinal bacterial translocation as well. Use of P. aeruginosa mutants deficient in the production of colonization factors such as pili and those dependent on the rpoN gene product resulted in a lower level of chronic colonization. Immune responses to type-specific lipopolysaccharide, pili, and flagellar antigens were measured, and increases in both serum and intestinal antibodies were usually elicited when a strain elaborated a given antigen. This model represents an easy method of routinely achieving chronic mucosal colonization by P. aeruginosa and should prove useful for the study of both bacterial virulence factors and host responses associated with this infectious process.     

Pathogen-induced actin filament rearrangement in infectious diseases

Host defence mechanisms involve the establishment and maintenance of numerous barriers to infectious microbes, including skin and mucosal surfaces, connective tissues, and a sophisticated immune system to detect and destroy invaders. Defeating these defence mechanisms and breaching the cell membrane barrier is the ultimate challenge for most pathogens. By invading the host and, moreover, by penetrating into individual host cells, pathogens gain access to a protective niche, not only to avoid immune clearance, but also to replicate and to disseminate from cell to cell within the infected host. Many pathogens are accomplishing these challenges by exploiting the actin cytoskeleton in a highly sophisticated manner as a result of having evolved common as well as unique strategies.     

Oral immunization using live attenuated Salmonella spp. as carriers of foreign antigens.

A variety of techniques, including the use of live oral vaccines, have been used to deliver antigens to the gut-associated lymphoid tissues in an attempt to initiate production of specific secretory immunoglobulin A for protection against pathogens that colonize or cross mucosal surfaces to initiate infection. A number of attenuated Salmonella mutants are able to interact with the lymphoid tissues in the Peyer's patches but are not able to cause systemic disease. Some of these mutants are effective as live vaccines (i.e., able to protect against infection with the virulent Salmonella parent) and are candidates for use as carriers for virulence determinants of other mucosal pathogens. This has been shown to be an effective means of stimulating significant levels of specific mucosal secretory immunoglobulin A directed against the carrier strains and against a variety of heterologous antigens and has been shown to stimulate production of serum antibodies and cell-mediated responses as well. This review examines the history of this mechanism of vaccine delivery and summarizes the most recent applications of this evolving technology. This is a technique for vaccine delivery with significant potential for influencing the management of infectious diseases on a large scale. It can be used not only for vaccines against enteric bacterial pathogens but also for vaccines against a variety of other bacteria, viruses, and parasites. The results obtained to date are encouraging, and there is great potential for development of safe, effective, affordable vaccines.     

Preventing staphylococcal disease by disarming the immune responses to infection

Use of experimental models of staphylococcal infections clarified several bacterial virulence factors as well as many hematopoetic cell types and their products that are involved in the pathogenesis of infection. For many decades it has been believed that antibody mediated response to staphylococci and their products was the major, if not the only one, hallmark of immune reactivity during infection. Recent studies have documented that T cell mediated responses to superantigens produced by staphylococci are not only prominent but also decisive with respect to sequels. Also the nonantigen specific immune responsiveness to staphylococcal infection is reviewed including roles of neutrophils, complement system and nitric oxide. The knowledge gained regarding staphylococcal virulence factors and the host immune responses has prompted researchers to develop new strategies how to interact in vivo witl the infectious process. Some of these approaches are commented in this review regarding e.g. vaccination procedures in order to prevent severe infections as well as therapeutic procedures to minimize organ damage during an ongoing infectious process.     

The front line of enteric host defense against unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and microbiota

The intestinal tract is a complex ecosystem that combines resident microbiota and the cells of various phenotypes with complex metabolic activities that line the epithelial wall. The intestinal cells that make up the epithelium provide physical and chemical barriers that protect the host against the unwanted intrusion of microorganisms that hijack the cellular molecules and signaling pathways of the host and become pathogenic. Some of the organisms making up the intestinal microbiota also have microbicidal effects that contribute to the barrier against enteric pathogens. This review describes the two cell lineages present in the intestinal epithelium: the goblet cells and the Paneth cells, both of which play a pivotal role in the first line of enteric defense by producing mucus and antimicrobial peptides, respectively. We also analyze recent insights into the intestinal microbiota and the mechanisms by which some resident species act as a barrier to enteric pathogens. Moreover, this review examines whether the cells producing mucins or antimicrobial peptides and the resident microbiota act in partnership and whether they function individually and/or synergistically to provide the host with an effective front line of defense against harmful enteric pathogens.     

A 66-kilodalton heat shock protein of Salmonella typhimurium is responsible for binding of the bacterium to intestinal mucus.

Salmonella typhimurium infections have increased during the last few years. However, the interplay of virulence factors in S. typhimurium pathogenesis is still poorly understood, particularly with regard to the mechanisms and components of the bacterium which are involved in its interaction with the intestinal mucus. We have observed that S. typhimurium is aggregated by incubation with colonic mucus (guinea pig model). To quantify this phenomenon, an aggregation assay was established. By using this assay, it was found that the aggregation profile of S. typhimurium strains freshly isolated from patients (age 9 and older) with salmonellosis correlated with the severity of the disease. An isolate with high aggregation behavior was chosen for characterization of the bacterial component involved in binding to colonic mucus material. The component of S. typhimurium responsible for aggregation was purified and characterized as a 66-kDa protein which was able to completely inhibit mucus-mediated bacterial aggregation. This protein was recognized by monoclonal antibodies against the 65-kDa heat shock protein (HSP) of Mycobacterium leprae. The 66-kDa protein of S. typhimurium was inducible by incubating the bacteria at 50 degrees C and was secreted into the supernatant, from which it could be isolated in both dimeric and polymeric forms. The monoclonal anti-HSP 65, as well as a polyclonal antibody against the 66-kDa protein of S. typhimurium, caused dose-dependent inhibition of the aggregation of S. typhimurium by crude mucus preparations. This is the first report showing that a bacterial HSP is involved in mucus-mediated interaction of pathogens with the host.