MicrobiologyBytes

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

Campylobacter jejuni, a Gram-negative bacterium, is one of the leading bacterial causes of food-borne human gastroenteritis. C. jejuni is currently estimated to cause 5–14% of diarrhoea worldwide, which translates into 400–500 million cases per year. Most cases of C. jejuni mediated gastroenteritis (campylobacteriosis) are characterized by nausea, abdominal cramps, diarrhea, and fatigue. While outbreaks of campylobacteriosis occur predominantly through consumption of contaminated milk and untreated water, most Campylobacter infections are sporadic in nature and linked to the improper handling and consumption of poultry. The linkage between human infection and the handling of raw poultry is not unexpected, as C. jejuni is a common commensal organism of chickens. In fact, C. jejuni colonize the intestinal tract of a variety of animals, including common livestock (cattle, sheep, pigs), domestic animals (dogs, cats), poultry, and wildlife (rabbits, pheasant, quail).

A number of methods (e.g. serotyping, short variable region [SVR] sequencing, pulsed-field gel electrophoresis [PFGE] and multilocus sequence typing [MLST]) are useful for the discrimination of C. jejuni isolates in epidemiological investigations. These methods have enabled investigators to identify the strain responsible for an outbreak. The use of MLST in particular has provided researchers with the benefit of a defined molecular fingerprint to compare strains. The recent explosion of genome sequences and comparative genomic data has increased our understanding of the epidemiology and metabolic capacity of this organism.

The identification of genetic markers predictive of ecological source and virulence potential are important to detecting and preventing the dissemination of C. jejuni via food sources. Comparative genomic studies have demonstrated that the C. jejuni population structure relates to ecological source (livestock versus non-livestock sources). Additionally, DNA sequence analysis implicates phase and allelic variation as possible mechanisms for altered gene expression and protein synthesis.

In spite of recent advances, significant gaps still exist in our knowledge of C. jejuni biology. First, researchers have yet to uncover a correlation between genomic diversity and disease severity. Second, C. jejuni virulence and disease pathology are not yet predictable on the basis of genotype. Third, the core genes necessary for disease and the variable (i.e. dispensable) genes whose products contribute to C. jejuni disease are not known. Fourth, on the basis of the observation that nucleotide changes in certain genes alter a strain’s pathogenicity, studies are needed to identify additional genes/proteins whose expression/function is influenced by nucleotide/residue variations. To address these questions, a small infectious disease animal model is needed to test the pathogenic potential of C. jejuni isolates. Continued work focusing on the relationship of genotype to phenotype is important in understanding this enigmatic organism.

Comparative studies of Campylobacter jejuni genomic diversity reveal the importance of core and dispensable genes in the biology of this enigmatic food-borne pathogen. Curr Opin Biotechnol. Apr 3 2009
MLST, DNA microarrays, and genome sequencing has allowed for a greater understanding of the metabolic capacity and epidemiology of Campylobacter jejuni. While strain-specific genes may provide an isolate a selective advantage in environments and contribute to the organism’s pathogenicity, recent work indicates that C. jejuni pathogenicity is dictated by variations in the nucleotide sequence of core genes. Challenges facing C. jejuni researchers include determining (a) the degree to which genomic diversity enables this bacterium to persist in particular environments; (b) if C. jejuni virulence and disease severity can be predicted on the basis of genotype; (c) the set of core and variable genes whose products contribute to virulence; and (d) the genes in which nucleotide changes can affect a strain’s pathogenicity.

Related:

• Campylobacter jejuni
• Animals farmed for meat are the main source of Campylobacter infections
• How Campylobacter jejuni survives within cells
• Protein promotes antibiotic resistance in Campylobacter

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.

Related:

• E. coli O157:H7 Spinach Outbreak
• Food Biosafety & Disgruntled Teenagers

BBC News

Obesity is an enormous public health problem, arising as a consequence of alterations in eating behavior and how the body regulates energy intake, expenditure, and storage. Although an increased intake of energy-dense foods, especially when combined with reduced physical activity, contributes to the high prevalence of obesity, the existence of complex systems that regulate energy balance requires that this paradigm be considered in a larger context. In particular, recent evidence suggests that the gut microbiota may play a role in obesity by increasing the host’s energy-harvesting efficiency.

The treatment of obesity is challenging. Various surgical procedures designed to interfere with the ingestion and/or absorption of foods have been developed over the last 60 years. The Roux-en-Y gastric bypass (RYGB), currently the most commonly performed operation, involves creating a small gastric pouch from the stomach. This surgery leads to changes in acid exposure to the gastric remnant and proximal small bowel, restricts the amount and types of food that can be comfortably ingested, promotes a modest degree of nutrient malabsorption by shortening the length of the small bowel, and may result in intestinal dysmotility, all of which might be expected to alter the gut microbiota. Presently, very little is known about the changes in the gut microbiota that occur after RYGB, and no information has been published on changes in microbial diversity after RYGB in humans.

A recent study used the traditional Sanger and high-throughput 454 pyrosequencing methods to analyze the human gut microbiota in 9 individuals, 3 in each of the categories of normal weight, morbidly obese, and post-gastric bypass surgery. The goals were to identify specific microbial lineages that may play important roles in the development of obesity and also to determine whether the presence or abundance of these microorganisms changes after RYGB.

Human gut microbiota in obesity and after gastric bypass. PNAS USA January 21, 2009
Recent evidence suggests that the microbial community in the human intestine may play an important role in the pathogenesis of obesity. We examined 184,094 sequences of microbial 16S rRNA genes from PCR amplicons by using the 454 pyrosequencing technology to compare the microbial community structures of 9 individuals, 3 in each of the categories of normal weight, morbidly obese, and post-gastric-bypass surgery. Phylogenetic analysis demonstrated that although the Bacteria in the human intestinal community were highly diverse, they fell mainly into 6 bacterial divisions that had distinct differences in the 3 study groups. Specifically, Firmicutes were dominant in normal-weight and obese individuals but significantly decreased in post-gastric-bypass individuals, who had a proportional increase of Gammaproteobacteria. Numbers of the H2-producing Prevotellaceae were highly enriched in the obese individuals. Unlike the highly diverse Bacteria, the Archaea comprised mainly members of the order Methanobacteriales, which are H2-oxidizing methanogens. Using real-time PCR, we detected significantly higher numbers of H2-utilizing methanogenic Archaea in obese individuals than in normal-weight or post-gastric-bypass individuals. The coexistence of H2-producing bacteria with relatively high numbers of H2-utilizing methanogenic Archaea in the gastrointestinal tract of obese individuals leads to the hypothesis that interspecies H2 transfer between bacterial and archaeal species is an important mechanism for increasing energy uptake by the human large intestine in obese persons. The large bacterial population shift seen in the post-gastric-bypass individuals may reflect the double impact of the gut alteration caused by the surgical procedure and the consequent changes in food ingestion and digestion.

Related:

• Infectobesity: the bugs that make you fat
• Gut Feeling
• Antibiotics, your gut, and you
• Probiotics – weapons in the war against gut pathogens

What is genomics? How will it affect our lives? In this primer on the genomics revolution, entrepreneur Barry Schuler says we can at least expect healthier, tastier food. He suggests we start with the pinot noir grape, to build better wines.

Bees are important contributors to the economies of many countries, but as Travis Glare and Maureen O’Callaghan discuss in this article in Microbiology Today, they are many threats to the survival on the humble bee, including the risk of disease from micro-organisms:

There are many threats to bee survival, including the risk of disease caused by micro-organisms. The vast majority of our knowledge of bee diseases focuses on the honey bee, Apis mellifera, although there are actually over 20,000 species, both stingless and stinging, from those with solitary lifestyles to complex societies such as honey bee hives. Viruses, fungi, protozoa and bacteria are all known to cause infections in bees, sometimes leading to collapse of colonies, and causing serious threats to the bee-keeping industry. Bees have two distinct life forms, brood (egg, larva and pupal stages which develop within the hive) and adult. Most diseases are specific to just one of these life stages. While the list of diseases is quite long, only a few are of serious concern to apiculturists.

Read more

Related:

• Colony Collapse Disorder
• The great bee swindle – colony collapse disorder

Nice microbiology video from the Royal Sussex County Hospital NHS Trust.

A decade ago, a new variant form of Creutzfeldt-Jakob disease was identified. The emergence of this prion disease in humans was the consequence of the zoonotic transmission of bovine spongiform encephalopathy through dietary exposure. Since then, the control of human exposure to prions has become a priority, and a policy based on the exclusion of known infectious materials from the food chain has been implemented. Because all investigations carried out failed to reveal evidence of infectivity in milk from affected ruminants, this product has continuously been considered as safe. In this study, researchers demonstrate the presence of prions in colostrum and milk from sheep incubating natural scrapie and displaying apparently healthy mammary glands. This finding indicates that milk from small ruminants could contribute to the transmission of prion disease between animals. It also raises some concern with regard to the risk to humans associated with milk products from ovine and other dairy species.

Prions in Milk from Ewes Incubating Natural Scrapie. 2008 PLoS Pathog 4(12): e1000238
Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.

Related:

• The Origin of BSE
• A novel approach in the molecular differentiation of prion strains
• Oral transmission of prions is enhanced by binding to soil
• What the heck are prions for?
• Alzheimer’s Disease and Prions