MicrobiologyBytes

Salmonella enterica serotype Typhimurium (S. Typhimurium) is an important food-borne pathogen that in humans causes a self-limited gastroenteritis, characterized by fever, acute intestinal inflammation, diarrhea, and the presence of neutrophils in stool samples. In addition, S. Typhimurium is a model organism for studying bacterial genetics and microbial pathogenesis. As the frontier in bacterial pathogenesis research is moving towards understanding the complexity of host-pathogen interaction at the tissue level, studies on the pathogenesis of S. Typhimurium gastroenteritis using animal models have helped establish important new concepts that exert a strong influence on the research field. Recent studies on S. Typhimurium pathogenesis reveal how tissue-specific host factors and the presence of other bacterial species shape the outcome of host-pathogen interaction in the intestinal lumen. This review discusses these new paradigms for the interplay between the pathogen, the host and its resident microbial community.
Salmonella, the host and its microbiota. Curr Opin Microbiol. Oct 24 2011
The intestine is host to a diverse bacterial community whose structure, at the phylum level, is maintained through unknown mechanisms. Acute inflammation triggered by enteric pathogens, such as Salmonella enterica serotype Typhimurium (S. Typhimurium), is accompanied by changes in the bacterial community structure marked by an outgrowth of the pathogen. Recent studies show that S. Typhimurium can harness benefit from the host response to edge out the beneficial bacterial species that dominate in the healthy gut. The elucidation of how S. Typhimurium alters the bacterial community structure during gastroenteritis is beginning to provide insights into mechanisms that dictate the balance between the host and its microbiota.

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Interfering RNAs (RNAi) are meant to match the sequence of the messenger RNAs made from genes, and then to block or inactivate the mRNA, keeping it from being translated into a harmful protein. One of the main hurdles has been delivering the agents specifically to the cells in which they are needed. An article in this Tuesday’s New York Times drew attention to this problem when reporting that many pharmaceutical companies have suspended their research into RNA interference. But what if you could use a common bacteria to deliver the payload? In work reported in this week’s Proceedings of the National Academies of Sciences, researchers led by Fenyong Liu at UC Berkeley made a modified strain of Salmonella to deliver interfering RNA exactly where it was needed. The result: they inhibited cytomegaloviral infection in mice.

ArsTechnica: Meet the newest virus fighter: Salmonella

See: Oral delivery of RNase P ribozymes by Salmonella inhibits viral infection in mice. PNAS USA February 7, 2011 doi: 10.1073/pnas.1014975108

Several important pathogens cause disease by surviving and replicating within host cells. Bacterial proliferation is the product of both replication and killing undergone by the population. However, these processes are difficult to distinguish, and are usually assessed together by determination of net bacterial load. In addition, measurement of net load does not reveal heterogeneity within pathogen populations. This is particularly important in persistent infections in which slow or nongrowing bacteria are thought to have a major impact. This paper reports the development of a reporter system based on fluorescence dilution that enables direct quantification of the replication dynamics of Salmonella enterica serovar Typhimurium (S. Typhimurium) in murine macrophages at both the population and single-cell level. The authors used this technique to demonstrate that a major S. Typhimurium virulence determinant, the Salmonella pathogenicity island 2 type III secretion system, is required for bacterial replication but does not have a major influence on resistance to killing. On entry into macrophages, many of the bacteria do not replicate, but appear to enter a dormant-like state. These could represent an important reservoir of persistent bacteria. The approach could be extended to other pathogens to study the contribution of virulence and host resistance factors to replication and killing, and to identify and characterize nonreplicating bacteria associated with chronic or latent infections.

Dynamics of intracellular bacterial replication at the single cell level. PNAS USA February 2 2010, doi: 10.1073/pnas.1000041107

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• Salmonella
• Life in the inflamed intestine, Salmonella style
• Sept. 23, 1869: Here Comes Typhoid Mary

Swarming is flagella-driven bacterial group motility over a surface, which is observed in the laboratory on media solidified with agar. The percentage of agar is critical for enabling swarming. Some bacteria like Vibrio parahaemolyticus and Proteus mirabilis can swarm readily on higher percentage agar, whereas others like Salmonella, Escherichia coli, Serratia, Pseudomonas, and Bacillus swarm only on lower percentage agar. Hard-agar swarmers differentiate into specialized swarm cells that are elongated and have increased flagella. Medium-agar swarmers generally do not display a similar differentiated morphology. In many of the latter class of swarmers (e.g. Serratia, Pseudomonas, Bacillus), movement is enabled by powerful extracellular surfactants whose synthesis is under quorum-sensing control. Surfactants lower surface tension and allow rapid colony expansion. Salmonella and E. coli do not appear to make such surfactants.

Swarming bacteria exhibit adaptive resistance to multiple antibiotics. Analysis of this phenomenon has revealed the protective power of high cell densities to withstand exposure to otherwise lethal antibiotic concentrations. This paper shows that that high cell densities promote bacterial survival, even in a nonswarming state, but that the ability to move, as well as the speed of movement, confers an added advantage, making swarming an effective strategy for prevailing against antimicrobials. There is no evidence of induced resistance pathways or quorum-sensing mechanisms controlling this group resistance, which occurs at a cost to cells directly exposed to the antibiotic. This work is relevant to the adaptive antibiotic resistance of bacterial biofilms.

Cell density and mobility protect swarming bacteria against antibiotics. PNAS USA February 2 2010 doi: 10.1073/pnas.0910934107

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Non-typhoidal Salmonella serotypes (NTS) are a leading cause of acute food borne disease worldwide. The most common human clinical isolates are Salmonella enterica serotypes Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis). In immunocompetent individuals, NTS are associated with gastroenteritis, a localized infection of the terminal ileum and colon that manifests as fever, diarrhea and intestinal cramping. However, a breach of mucosal barrier functions in immunocompromised individuals can result in the development of a life-threatening bacteremia. Current research on S. Typhimurium pathogenesis is beginning to paint a novel picture of the unique challenges and opportunities encountered during life in the inflamed intestine. Recent studies have identified host factors that are crucial for activating mucosal barrier functions in the inflamed intestine of immunocompetent hosts. In turn, these findings provide clues about the identity of mucosal barrier defects that put immunocompromised hosts at risk of developing bacteremia. New insights into the consequences consequences of inflammation on the growth conditions encountered by microbes residing in the intestinal lumen reveal how the pathogen might benefit from inducing antimicrobial host responses.

The lower gastrointestinal tract is densely populated with resident microbial communities (microbiota), which do not elicit overt host responses but rather provide benefit to the host, including niche protection from pathogens. However, introduction of bacteria into the underlying tissue evokes acute inflammation. Non-typhoidal Salmonella serotypes (NTS) elicit this stereotypic host response by actively penetrating the intestinal epithelium and surviving in tissue macrophages. Initial responses generated by bacterial host cell interaction are amplified in tissue through the interleukin (IL)-18/interferon-gamma and IL-23/IL-17 axes, resulting in the activation of mucosal barrier functions against NTS dissemination. However, the pathogen is adapted to survive antimicrobial defenses encountered in the lumen of the inflamed intestine. This strategy enables NTS to exploit inflammation to outcompete the intestinal microbiota, and promotes the Salmonella transmission by the fecal/oral route.

Life in the inflamed intestine, Salmonella style. Trends Microbiol. Oct 9 2009

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• Novel genetic tools for studying food-borne Salmonella
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Nontyphoidal Salmonellae are responsible for an estimated 1.4 million cases of gastrointestinal disease with 500 associated deaths in the United States, at a cost of $2 billion. The number of cases worldwide probably exceeds 100 million each year. Infection generally occurs after the ingestion of contaminated food or water, and usually leads to a self-limiting enterocolitis. The disease is characterized by diarrhea, abdominal cramps, nausea, fever, vomiting, and headache lasting 7–10 days, followed by a longer period of subclinical fecal shedding. Infants, the elderly, and immunocompromised individuals are at risk for serious systemic complications and death as a result of infection.

Contaminated foods, including beef, pork, poultry, and egg products are frequent vectors responsible for the transmission of these organisms to humans. Livestock can harbor Salmonellae subclinically resulting in carcass contamination at slaughter and in the laying of contaminated eggs. In recent years, as the traditional routes of infection are better controlled, large outbreaks of nontyphoidal Salmonella infection in the United States have been attributed to fruits, vegetables, and processed foods including jalapeño peppers, cantaloupe, cereal, and peanut butter (CDC).

Serology based on surface antigens is the standard method of classification of Salmonella. The host-range and disease can differ considerably between serovars, making such classification important. Throughout the world, the most prevalent nontyphoidal serovars isolated from human sources are serovars Typhimurium and Enteritidis and these two serovars comprise nearly 40% of isolations from human sources in the United States. These serovars can be harbored subclinically in livestock for prolonged periods of time and are thus very difficult to eradicate in the absence of a detailed knowledge of the biology of the organism in this niche.

The bacterial factors necessary for Salmonellae to persist subclinically in the gastrointestinal tract of livestock and to survive and grow in other reservoirs such as crops and processed foods are only beginning to be elucidated. This knowledge will allow the development of new strategies and the identification of points in the production chain where producers can intervene to improve the safety of foods. We review the current status as well as the uses of complete genome sequence information for Salmonellae, and enhancements of genetic techniques that may rapidly increase our knowledge of the biology of this organism in these important food safety niches.

Complete genome sequencing of Salmonellae is allowing us to better understand their genetic diversity, to develop novel tools, and to improve existing genetic techniques to understand the complex biology of these important food-borne pathogens. Approximately half of the genes in Salmonella still have no known phenotype in the environment. Frontiers for further study of Salmonella for improved food safety using modern genetic tools are likely to include determination of the genes necessary for environments where Salmonella must survive outside the host, such as in feces, soil, water, and plants. Understanding how Salmonella completes its entire host-to-host life cycle in agriculture may reveal previously unknown vulnerabilities that will be susceptible to novel intervention and allow us to break the chain of transmission.

Novel genetic tools for studying food-borne Salmonella. Curr Opin Biotechnol. 2009 Apr;20(2):149-57.
Nontyphoidal Salmonellae are highly prevalent food-borne pathogens. High-throughput sequencing of Salmonella genomes is expanding our knowledge of the evolution of serovars and epidemic isolates. Genome sequences have also allowed the creation of complete microarrays. Microarrays have improved the throughput of in vivo expression technology (IVET) used to uncover promoters active during infection. In another method, signature tagged mutagenesis (STM), pools of mutants are subjected to selection. Changes in the population are monitored on a microarray, revealing genes under selection. Complete genome sequences permit the construction of pools of targeted in-frame deletions that have improved STM by minimizing the number of clones and the polarity of each mutant. Together, genome sequences and the continuing development of new tools for functional genomics will drive a revolution in the understanding of Salmonellae in many different niches that are critical for food safety.

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• MicrobiologyBytes: Salmonella
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• Pathogens in raw foods

Despite improvements in agricultural practices, leafy greens, tomatoes, salad crops and nuts were among the foods linked to recent outbreaks of gastrointestinal illnesses caused by Escherichia coli O157:H7 and non-typhoidal Salmonella (Human enteric pathogens in produce: un-answered ecological questions with direct implications for food safety. Curr Opin Biotechnol. April 4 2009). Because plants are not traditionally considered as hosts for human enteric pathogens, recent produce-associated outbreaks highlight important deficiencies in our understanding of the ecology of enteric pathogens outside of their human and animal hosts. The ongoing food safety debate focuses on answering the question whether plants are true alternate hosts for Salmonella or E. coli, or whether they are simply matrices where these organisms persist.

In a survey of several farms, up to 43% of produce sampled in the field was positive for Salmonella enterica, and the pathogen was found in the soil, irrigation water and on the hands of agricultural laborers. Following the 2006 E. coli O157:H7 spinach outbreak in the United States, the pathogen was isolated from cattle and feral swine faeces, river sediment, pasture soil, and surface water near the implicated fields. Human enteric pathogens are often recovered from surface water and untreated waste water used for irrigation. These reports establish that enteric pathogens in various environmental reservoirs may lead to food-associated outbreaks. Once deposited in soils, enteric bacteria persist for periods of time that range from a few weeks to several years. In field studies, both E. coli and Salmonella from raw manure were capable of colonizing the root zone and above ground parts of plants, supporting the hypothesis that pre-harvest contamination in the field could be a plausible route of produce contamination.

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For an enteric bacterium, it may make evolutionary sense to colonize vegetative and reproductive parts of plants that are then eaten by animals. If so, then enteric bacteria should have sophisticated, co-evolved mechanisms for getting into plants, spreading and multiplying in edible plant tissues to levels capable of populating guts of their herbivorous hosts. Salmonella enterica and enterovirulent E. coli are able to colonize tissues of plants quite effectively. This suggests that under favorable conditions enterics can exist as epi- or endophytes. If endophytic growth is truly a part of the life cycle of enterics, then this explains why current surface salad crop decontamination procedures may not be very effective. Conversely, if endo- or epiphytic growth is an important part of the life cycle of enterics, then we should be able to uncover evidence of specificity in the bacterial attachment, colonization and avoidance of plant defenses.

Recent laboratory studies identified a few of the genes and mechanisms that enterics use to colonize external surfaces of host plants. It appears that bacterial polymers and aggregative fimbriae were involved in the attachment of E. coli and/or Salmonella to plant seedlings. In their reliance on cellulose for attachment to plant surfaces, enteric pathogens are similar to plant symbiotic and pathogenic bacteria that also use cellulose fibrils to anchor themselves to plant surfaces.

Most plant pathogens and endophytes also produce hemicellulases and pectinases, enzymes that degrade polymers in plant cell walls. Unlike closely related members of the Enterobacteriaceae, Salmonella and E. coli do not seem to produce such enzymes and their genomes do not encode homologs of these enzymes. It is not yet clear whether Salmonella has unknown classes of cell wall degrading enzymes, whether it manages to gain entry and spread in plant tissues without such enzymes by moving intercellularly, or whether it relies on enzymes from the host or from other endophytes or plant pathogens to degrade plant cell walls. Regardless of their route of entry, enteric bacteria that were present inside plant seedlings were found in the intercellular spaces between host cell walls.

Although recent research has established that Salmonella and enterovirulent E. coli are capable of spending at least a part of their life cycle as plant-associated endo- or epiphytes, several important questions about the genetics and physiology of these interactions still need to be answered before plants are designated as true alternate hosts for these bacteria. Because there is evidence of specificity in the interactions of plant genotypes with enterics, defining the genetic basis and molecular markers associated with resistance to enterics may help identify crop cultivars that are less conducive to supporting growth of human pathogens. Further characterization of the attachment to plant surfaces and interactions with the resident microbiota will likely help improve pre- and post-harvest treatments to ensure safety of produce for human consumption.

Related:

• Pathogens in raw foods
• E. coli O157:H7 Spinach Outbreak

Approximately 50 years ago the Pillsbury Company was asked to develop protocols to ensure that astronauts would be free from food-borne illness during space travel. The entire process of production, harvesting, processing, and preparation of food was critically analyzed in order to identify control points that might be susceptible to the introduction of microbial contamination. Thus, Hazard Analysis of Critical Control Point (HACCP) was born. Risk assessment within HACCP was used to bypass end-product testing, which was deemed to be too expensive and essentially impractical for both NASA and the food industry. Global recognition of standardized protocols to eliminate risk at every step from “farm to fork” has translated into our national food safety policy. Unfortunately, raw foods have thrown a major linchpin into this vastly effective policy because of the lack of a verifiable kill step to ensure the elimination of food-borne disease transmission. Salmonella outbreaks in leafy greens, tomatoes, and other produce exemplify food safety issues related to the consumption of raw foods. Produce at harvest will contain indigenous bacteria and viruses, but their numbers are presumed to be relatively low and devoid of human pathogens. These assumptions have kept raw foods under the HACCP radar, and problems are exacerbated by low infectious dose for some strains and the establishment of Salmonella in environmental reservoirs. Issues with trace-back, such as co-mingling of produce lots from multiple farms before retail sale, globalization of food markets, and the short shelf-life of fresh-cut products further exacerbate the problem. These issues are complex, and easy solutions are not in sight.

Pathogens in raw foods: what the salad bar can learn from the raw bar. Curr Opin Biotechnol. Apr 14 2009
Recent Salmonella outbreaks associated with consumption of fresh produce have increased public concern for the safety of raw food products, perhaps signaling a paradigm shift in approaches to food safety. Limitations to our capacity to ensure that raw foods are safe for the consumer include the availability of sufficiently rapid and reliable technology for prevention, intervention, and risk assessment. Other food products, such as shellfish, with greater historical precedent for real or perceived public health risk may offer perspective and insight into strategies for meeting these challenges. This review documents current practices for pathogen prevention and detection in raw oysters and presents technological advances and impediments that determine the application of these methods.

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• E. coli O157:H7 Spinach Outbreak
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