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

We all know that China is changing – fast. With the largest population in the world and the speed of urbanization, the potential for the spread of human immunodeficiency virus (HIV) is worrying. By some estimates, over a million people in China could be infected with HIV, but reliable figure on the prevalence of HIV/AIDS in China have always been hard to come by. The most recent data from CDC estimates that in 2007, an estimated 700,000 people in China were living with HIV. An estimated 50,000 new HIV infections and 20,000 deaths related to acquired immunodeficiency syndrome (AIDS) occurred in 2007, and an estimated 71% of persons with HIV infection were unaware of their HIV status. In 2007, 40% of those living with HIV had been infected through heterosexual transmission and 38% through injection-drug use (HIV Infection – Guangdong Province, China, 1997-2007. MMWR 58(15) 396-400 April 24, 2009).

Guangdong Province in southeastern China is the country’s most heavily populated region, with an estimated 76 million permanent residents and 17 million migrants. Guangdong has undergone rapid economic development in recent years. The number of HIV infections in Guangdong increased from 102 recorded in 1997 to 4,593 in 2007, although this increase is partly due to increased testing and surveillance. Among males classified by HIV transmission category, 82% of newly diagnosed infections were attributed to injection-drug use, and among females, 53% engaged in high-risk heterosexual conduct.

During the ten years from 1997-2007, an aggregated total of 22,571 newly diagnosed HIV cases were reported in Guangdong, 82% in males and 18% in females. Every year, injection-drug use was the most commonly reported HIV transmission category.

The recent increase in reported HIV cases attributed to high-risk heterosexual contact and the decline in cases attributed to injection-drug use might suggest a shift in Guangdong’s HIV epidemic similar to the national trend, in which heterosexual transmission is the main transmission category in China. In the central region of Guangdong Province, where approximately 80% of the province’s HIV cases were reported in 2007, rapid economic growth has led to an influx of migrant workers. Migrant women who lack appropriate job skills might seek to supplement their income by becoming sex workers, and migrant men living apart from their spouses might become clients of sex workers.

The findings in this report are subject to at least three limitations. First, large percentages of the data were missing key elements. For example, in approximately 22% of cases, the patient’s age group was unknown, and approximately 38% of patients were not classified by transmission category. Second, because definitions for sex worker and injection-drug user were institution based, verification was not possible. Finally, because HIV-positive people in China are required by law to report their names and national identification numbers, those consenting to HIV testing likely represent a sample that is biased in unpredictable ways.

More community-based sampling of populations at high risk are being planned to provide a more complete picture of the HIV epidemic in Guangdong. Surveillance methods should be redefined so that they rely less extensively on institutions and more accurately represent those populations at greatest risk. Finally, because an estimated 71% of people with HIV infection in China are unaware of their status, more HIV counseling and testing should be urgently provided.

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Related:

• The changing face of HIV in China. 2008 Nature 455: 609-11
• HIV treatment reduces death rates in Africa
• Why is HIV a pathogen?
• How does HIV cause AIDS?

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Related:

• 10 things you should know about H1N1 (swineflu)
• 10 more things you should know about H1N1 (swineflu)

Ebola and Marburg virus are filoviruses that cause outbreaks of highly lethal haemorrhagic fever. Mortality rates in these diseases average more than 50%, with the highest recorded rates seen for Ebola Zaire virus (88%) and Marburg Angola virus (90%). Infection with these filoviruses produces a very high fever followed by interference with blood coagulation and vascular permeability, causing internal bleeding, bruising and skin rashes. After an asymptomatic incubation period, which can last days to weeks, symptoms of a typical filovirus infection emerge; headache, nausea, fever and malaise followed by more serious haemorrhagic symptoms and, in fatal cases, death results from multi-organ failure owing to shock.

Present treatments for filovirus infection are palliative, and consist primarily of supportive care, including hydration and pain management. There is no effective treatment or cure for these diseases. Therefore, vaccine development is crucially important as a strategy for fighting filovirus outbreaks. However, vaccine efficacy testing for Ebola virus is very difficult. There is no readily identifiable high-risk human population that can be targeted for a placebo-controlled clinical trials because disease outbreaks are unpredictable and sporadic, both geographically and temporally. Normally, clinical trials of medicines and vaccines intended for human use follow a lengthy but predictable sequence of safety and efficacy testing.

Because of its sporadic nature, the incidence of Ebola virus infection in human populations is not predictable and does not allow for adequate testing. Moreover, the immune correlates of protection from filovirus disease in humans remain unknown and therefore cannot be used to assess candidate vaccine efficacy. To facilitate the licensing of medicines when efficacy cannot be evaluated in the setting of natural infection, the U.S. Food and Drug Administration (FDA) introduced a new regulation in 2002 as an alternative licensing pathway for pharmaceutical products that target highly lethal pathogens. The FDA’s “animal rule” allows approval based on animal efficacy data. The animal rule is intended to be used as a pathway for regulatory approval only when there is no other way to licence a vaccine (Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule. 2009 Nature Reviews Microbiology 7: 393-400).

In the case of Ebola virus, the relevant animal models are non-human primates and mice. The immune correlates of Ebola virus infection consist of immunoglobulin G responses, although other factors, such as T cells, are also likely to be important in a successful immune response. Current vaccine candidates against Ebola virus include the virus glycoprotein and nucleocapsid proteins. Initial animal testing of Ebola vaccines has shown a protective effect in non-human primates and positive antibody titres in humans.

To date, no vaccines have received regulatory approval and been licensed using the FDA animal rule. This pathway does not diminish the level of regulatory contol required for vaccine approval; extensive human testing is still required to demonstrate safety and immunogenicity. The predictive relationship between animals and humans for protective efficacy is unknown, and therefore an immune correlate is used to bridge the gap between animal efficacy studies and human immunogenicity trials. It has not yet been determined what level of efficacy in animals will be required for vaccine approval, but other vaccines currently administered to the U.S. population have shown efficacies in human trials that are as low as 18%. Even this level of efficacy will provide a benefit against pathogens such as filoviruses with high mortality rates, and therefore may be acceptable against emerging natural infections or bioterrorism threats.

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Related:

• Congo confirms Ebola outbreak
• New species of Ebola virus discovered
• Marburg haemorrhagic fever in Uganda
• Deadly Marburg virus linked to fruit bat

Bluetongue is a disease of ruminants caused by Bluetongue virus (BTV) and it is transmitted by biting midges. In sheep, clinical signs of BTV infection can include fever, vasodilation, swelling and, in severe cases, death, although the severity of symptoms varies with the breed of sheep, the individual animal and the strain of virus involved. Cattle are a major reservoir host for BTV infection, primarily because of the less obvious clinical signs in these animals. After an exhaustive search for a natural agent of transmission, Culicoides midges were shown to be the vectors for Bluetongue virus (Culicoides and the emergence of bluetongue virus in northern Europe. Trends Microbiol 2009 17(4): 172-178).

Although BTV infection was initially centred in Africa, the virus was first detected in Greece in 1989, from where it has spread steadily north. Since the disease is spread exclusively by insects, and because the virus is quite specific about which midge species can be used as vectors, predictions about the spread of the disease were based on known ranges of different midge species, which in turn depends on climate. However, midges can sometimes be carried over very long distances by weather systems, or by ships or aircraft.

Despite widespread speculation regarding the exact origin of BTV-8 as the strain of the virus found in northern Europe, no single convincing hypothesis has been proposed. Although future full-genome sequencing might assist this task (as was the case in the incursion of West Nile virus into North America), the small number of reference strains of BTV-8 from areas of potential origin collected before the incursion into northern Europe makes it unlikely that this approach will provide unambiguous evidence. As long as our understanding of the potential routes of virus introduction remains poor, we will be unable to accurately estimate the potential for future introductions of BTV, as has been illustrated by the more recent detection of BTV-6 in Europe, or of other midge-borne arboviruses, such as African horse sickness virus (AHSV).

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Although the technology to produce safe, effective, inactivated vaccines existed, no coordinated action was taken by any Member State of the European Union (EU) to begin production of a BTV-8 vaccine until late 2007, when the full damage began to become evident. This was in part due to the assumption that the virus would not overwinter under northern European conditions (despite the fact that BTV had been documented overwintering successfully in other areas with far cooler winter temperatures). In the absence of an available vaccine, knowledge concerning the entomology of the insects involved in BTV transmission became paramount.

The spread of BTV has provided a severe test of the way in which the movement of vector-borne pathogens is predicted, identified and controlled in Europe. There are many arobovirus diseases (spread by arthropod vectors such as midges, mosquitos and ticks), affecting human as well as animal health. Whether BTV represents a herald for future incursions by other arboviruses into Europe remains difficult to know. It is clear that there exists a similar potential for emergence of other insect-borne pathogens on grounds of climate alone, but where different vectors are used – for example, in the case of AHSV – the dynamics of the current BTV outbreak cannot easily be used to estimate risk. What has been shown by this outbreak is that arbovirus–vector relationships are highly dynamic and extremely difficult to combat. Unless regions that are potentially at risk of transmission are prepared to invest the resources required to provide adequate information regarding vectors and suitable control methods, this will remain the case.

Related:

• Bluetongue virus
• How does bluetongue virus survive the winter?
• Bluetongue virus polymerase

Stanley Prusiner was awarded the first ever SGM Prize Medal (to a microbiologist of international standing whose work has had a far-reaching impact beyond microbiology) at the SGM Spring meeting at Harrogate on 1st April 2009. MicrobiologyBytes was there and this is a summary of his Prize lecture.

Prions are infectious proteins which multiply by binding to a host cell protein and converting it into insolubile fibrils (“amyloid“). Prions are associated with infectious, inherited and sporadic diseases – a feature unique to these entities. Tikvah Alper was the first person to identify prions in the 1960s, but when Prusiner started working on them in 1974, at first he didn’t believe the protein-only hypothesis. After eight years of failing to be able to identify any nucleic acid associated with them, in 1982 he changed his mind and invented the name prion (“pree-on”).

In prion diseases, the cellular form of the protein, PrP^c, is converted into a disease-associated form, PrP^Sc. If prions really are infectious proteins, PrP^Sc produced in bacteria should be able to cause disease – and it does. It is also possible to produce synthetic amyloids with different biological properties – essentially strains of the protein.

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Quinacrine cures cultured cells of prions. In mice, the drug increases survival time of infected animals by up to 20%, but recently concluded clinical trials in humans have shown little effect. Prusiner’s group have found that quinacrine does work in stationary phase cells – such as those in the brain. Future trials of anti-prion (or amyloid) drugs need to be carried out in stationary cells. The latest assay uses genetically-modified mice which express luciferase when glial cells are disturbed. The resulting luminescence can be detected in the brains of live mice, and signs of disease can be recorded even before any neurological symptoms appear. This is up to eight times faster than waiting for the mice to die and examining their brains, and only requires one tenth of the animals. Prusiner hopes to use this approach to study Alzheimer’s and Parkinson’s disease, which also involve brain injury and amyloid deposits.

Stanley Prusiner’s take home message to all the students present was: it’s important to be lucky! But as Robin Weiss, SGM President, pointed out, Pasteur said: Fortune favours the prepared mind!

Related:

• Alzheimer’s Disease and Prions
• The Origin of BSE
• What the heck are prions for?
• Drugs for treatment of prion infections
• What drove the cow mad? Lessons from a fish

Spring 2009 Meeting, Harrogate International Centre, 30 March – 2 April 2009

We all live in an information rich, highly interconnected world, and the success of evolution can be measured in terms of how living organisms make sense of and respond to information. Past posts on quorum sensing are some of the most popular out of all the subjects I have covered on MicrobiologyBytes. Quorum sensing is the use of small molecules by bacteria to coordinate behavior by groups of individual cells and carry out decision-making processes.

Bacteria have evolved a number of communication systems which can be broadly described as contact-independent and contact-dependent signaling mechanisms. Quorum sensing is a contact-independent process since it involves transfer of secreted molecules called autoinducers. As autoinducer levels increase throughout a growing bacterial population, changes in gene transcription are triggered resulting in altered growth rates and group dynamics. There is an energy cost in producing these compounds and throwing them out of the cell, and in some conditions, the secretion of autoinducers may attract unwanted attention from competitors (Bacterial landlines: contact-dependent signaling in bacterial populations. Curr Opin Microbiol. Feb 24 2009). Contact-dependent signaling methods allow bacteria to carry out more direct, and possibly less costly, communication between cells – it’s the landline alternative to expensive cellphone bills.

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Known methods of contact-dependent signaling include C-signaling in Myxococcus xanthus which allows groups of cells to coordinate motile behavior. The process is mediated by a non-diffusable 17 kDa surface protein encoded by the csgA gene. Contact between neighboring myxobacteria initiates a p17-dependent signaling cascade resulting in expression of genes required for control of motility or for sporulation.

The Gram-positive soil bacterium Bacillus subtilis also undergoes a contact-dependent differentiation process as a means to produce dormant spores when faced with starvation. Under nutrient poor conditions, vegetative B. subtilis cells divide asymmetrically, forming a large mother cell and a smaller daughter cell called a forespore. Despite their intimate association, the mother cell and the forespore remain separated by two membranes and maintain distinct gene expression profiles. Endospore formation is an energy intensive process that is coordinated by multiple signaling pathways. Contact-dependent signaling plays an important role in allowing the cells to coordinate this process.

Contact-dependent inhibition also occurs in E. coli, where a single E. coli cell in the logarithmic phase of growth can use a CDI system to inhibit the growth of hundreds of susceptible target cells in mixed cultures, forcing them to enter a viable but non-replicating state. However, one of the first recognized instances of contact-dependent communication between bacteria was, arguably, conjugation mediated by sex (F) pili. Bacteria encode a large variety of other pilus types and adhesive molecules, many of which have been studied primarily with respect to their abilities to modulate bacteria–host cell interactions. However, it is feasible that some of these organelles also function in inter-bacterial communication. For example, recent studies indicate that several types of soil bacteria can express complex networks of electrically conductive pili known as nanowires.

Although quorum sensing has been getting all the attention recently, we have known about contact-dependent communication mechanisms in bacteria for far longer. Perhaps only now are we realizing how these complimentary systems might fit together and how they could shed light on the development of true multicellularity during evolution.

Related:

• Quorum Sensing in Bacteria: We Two Are One
• Pneumococcus: the quorum-sensing killer
• Quorum sensing in Serratia
• Cracking the cholera code

In the last week there has been some fairly wild speculation in the media about viruses which “cause” diabetes. The fuss came from the publication of a paper which claimed to have detected virus proteins in the pancreases of diabetes patients (The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes. Diabetologia 6 March 2009). At the same time, a separate study found four rare mutations in a gene which is thought to reduce the risk of developing type 1 diabetes and may be involved in the immune response to infection with enteroviruses (Rare Variants of IFIH1, a Gene Implicated in Antiviral Responses, Protect Against Type 1 Diabetes. Science Mar 5 2009).

The press was buzzing with speculation about the chances of a vaccine to prevent diabates. Very good news for diabetics? Well not so fast. Before we look at the science, let me tell you two things about myself. First, I have two close relatives who are affected by diabetes, so this is a disease I care a lot about. Second, I’ve been in the virology business a long time – and we’ve been here before.

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The new paper claimed to have detected “enterovirus capsid protein vp1″ in 44 out of 72 pancreases from children who had died of type-1 diabetes shortly after becoming ill, but in only three out of 50 neonatal and paediatric normal control specimens. Statistically there is a strong correlation in this study between diabetes and the presence of the virus protein, but a correlation does not indicate a cause. Are diabetics more susceptible to enterovirus infection? We don’t know. While it’s not ethically possible to satisfy Koch’s postulates in humans, we need to be very careful in inferring from small scale studies such as this one:

1. The microorganism must be found in abundance in all organisms suffering from the disease.
2. The microorganism must be isolated from a diseased organism and grown in pure culture.
3. The cultured microorganism should cause disease when introduced into a healthy organism.
4. The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

There are over a hundred different enteroviruses and the antibody used for detection of virus protein in this study (yes, that’s right, just one non-specific antibody) does not identify the virus involved. Vaccine against diabetes? I don’t think so.

But as I said, we’ve been here before. There are reports of viruses associated with diabetes dating from the 1960s, and a very well known model of Coxsackie virus B4 causing diabetes in mice dating from the 1970s (Coxsackie Viruses and Diabetes Mellitus. BMJ 1973 November 3; 4(5887): 260–262). So does the latest work add anything new, and is a vaccine against diabetes just around the corner? No. I wish it was.

Related:

• Can you have confidence in the news?
• Media coverage affects how people perceive the threat of disease