Awards

Andrew Greenhill of Federation University Australia in Australia, along with partners at the Papua New Guinea Institute of Medical Research, will use advanced environmental microbiology methods to study microbial community dynamics associated with survival of the typhoid fever-causing bacterium Salmonella Typhi in aquatic environments in Papua New Guinea. "Typhoid Mary" Mallon was an Irish-American cook, written into infectious disease folklore as the first asymptomatic carrier of S. Typhi. More than eighty years after her death, little is still known about how the bacteria persists in environmental niches such as contaminated water, which is a major route of disease transmission. They will collect and filter water samples from streams in areas where the disease is common over an eight-month period covering both wet and dry seasons and analyze the microbial communities within by qPCR. This will be combined with physiochemical parameters of the water (temperature, stream height, photosynthetic activity) collected using DIMPP - a low-cost, late-stage prototype suitable for use in low-resource environments - to build network models. These models will be used to identify mathematical connections between environmental factors, aquatic populations, and the presence of antibiotic resistance genes. These connections could be used to develop an early warning system for impending outbreaks.

Jason Andrews of Stanford University in the U.S. will study the association between the typhoid fever-causing bacterium Salmonella Typhi and its bacteriophage in both aquatic environments and the human gastrointestinal tract to see if they influence geographic and seasonal disease outbreaks in Bangladesh. The ecology and evolution of many know bacterial pathogens including V. cholerae are affected by the viruses (bacteriophage) that infect them. Indeed, seasonal cholera epidemics are inversely correlated with phage prevalence in water. They will study this relationship in S. Typhi, which contaminates half of city water supplies in Bangladesh. They will generate a library of local phage strains infecting S. Typhi and use a computational approach to identify indicator S. Typhi strains that may be susceptible to these phage, which they will then test experimentally. They will also characterize the abundance and strains of phage in municipal water supplies, and in stool samples from typhoid cases, and compare these with clinical cases of typhoid to determine if they shape temporal and spatial patterns of typhoid.

Andrew Jackson of the University of Liverpool in the United Kingdom will determine whether the amoeba, Acanthamoeba, which is commonly found in water and soil, acts as a host for Salmonella Typhi bacteria, which cause typhoid fever, to support growth and disease spread in Malawi. Typhoid fever is a systemic, potentially fatal illness, usually contracted by consuming contaminated drinking water. An estimated 11-21 million cases occur worldwide each year. Acanthamoeba is known as the 'Trojan Horse' of the microbial world for its ability to host a number of human pathogens, including S. Typhi. It is speculated that Acanthamoeba acts as an environmental reservoir to facilitate the survival of S. Typhi, and perhaps other human pathogens. They will prove the widespread presence of Acanthamoeba-Salmonella associations directly by using single-cell DNA sequencing. Individual amoeba will be isolated from water and soil samples from typhoid hotspots in Malawi using fluorescence-activated cell sorting. Total DNA in each amoeba will be sequenced in order to identify carriage of S. Typhi strains. Single nucleotide polymorphism analysis will be used to compare these with bacteria in local clinical isolates to determine the role of Acanthamoeba in disease transmission.

Gemma Langridge of Quadram Institute Bioscience in the United Kingdom, along with co-investigators Aaron Jenkins of the University of Sydney in Australia and France Daigle of the University of Montreal in Canada, will collect different isolates of S. Typhi, which causes typhoid fever, to analyze genomic structure, growth, and gene expression to better understand how it can survive at low levels in water, and determine how it can be reactivated for monitoring. Typhoid fever is a potentially fatal disease associated with exposure to contaminated water. S. Typhi, the bacteria causing the disease, exist in water in a so-called viable-but-non-culturable (VBNC) state, which makes it difficult to monitor for control efforts. Their previous analyses have shown that the VBNC bacteria undergo structural rearrangements of their genomes and changes in gene expression, which may explain the reduced growth. They will analyze the correlation between genome structure and growth of different S. Typhi isolates using samples isolated from around 25 typhoid fever cases, and a further 75 stored isolates across the endemic region. Of these, a selection will be tested for their ability to enter a VBNC state and survive in water from areas of high and low typhoid incidence. One isolate that has entered VBNC with a defined structural genotype will be tested to establish the conditions most suitable for resuscitation.

Dilip Abraham of Christian Medical College in India will analyze water samples from peri-urban and rural areas in India to study whether and how the typhoid fever-causing bacterium S. Typhi survives by living inside the common amoebae, Acanthamoeba. Acanthamoeba spp. are known to internalize S. Typhi, and may provide an intracellular environmental niche and extend survival of the bacteria. They will collect samples from drinking water sources and sewage lines in peri-urban and rural sites in Southern India. From these samples, they will recover Acanthamoeba and detect any internalized S. Typhi by culture and PCR. Whole genome sequencing will be used to conduct comparative genomic analysis of intracellular S. Typhi to identify any major virulence markers that promote its ability to be internalized by and remain viable within Acanthamoeba. This would help explain why persistence of S. Typhi in the environment can lead to sporadic outbreaks, and allow officials to plan preventative strategies beyond vaccines for the control and elimination of typhoid.

Denise Monack of Stanford University in the U.S. will use a genetic approach to identify the molecular mechanisms that enable the typhoid fever-causing bacterium S. Typhi to survive in aquatic environments and to rapidly adapt to transmission to humans. Annually, S. Typhi causes over 20 million infections and 200,000 deaths, mostly among populations that lack access to clean drinking water. Understanding how S. Typhi persists in water and then quickly adapts to its human host is critical for controlling transmission. Bacteria use various mechanisms to adapt to environmental changes, including so-called RNA thermometers (RNATs), which form secondary structures in mRNAs that can rapidly activate gene expression when temperatures change. They will use their established genetic screening approach to identify new RNATs in S. Typhi and validate their ability to promote bacterial persistence within aquatic microbial communities by generating mutants. They will also follow up on past work in which a bioinformatics approach identified new RNATs that may regulate the expression of the chitinase enzyme, which is used by the cholera-causing bacterium to bind to plankton and create a protective environmental niche. They will evaluate whether chitin is also important for S. Typhi persistence and transmission.

Windy Tanner, formerly at the University of Utah and now at Yale University in the U.S., together with Jim VanDerslice of the University of Utah and colleagues from Mehran University of Engineering and Technology in Pakistan, will analyze water samples to determine the conditions that promote the survival of the typhoid fever causing bacterium Salmonella Typhi, and they will use metagenomic deconvolution to identify any gene exchange from other microbial species that may produce drug-resistant strains. S. Typhi is responsible for over 100,000 deaths each year, mostly in the developing world where fecal contamination of food and drinking water is common. The emergence of drug-resistant strains has limited the available treatment options. Biofilms are environmental niches with complex microbial communities and are ubiquitous in the environments where S. Typhi is commonly found. They will sample water and biofilms from a variety of these environments along the fecal-drinking water transmission route in the Sindh province of Pakistan and test for the presence of S. Typhi using qPCR and culture methods. They will also evaluate whether specific organisms stabilize and protect S. Typhi in these biofilms and could cause resistance gene exchange.

Robert Kingsley of the Quadram Institute Bioscience in the United Kingdom will locate the typhoid fever-causing bacteria S. Typhi in water reservoirs in Harare, Zimbabwe, and identify any associated protozoa species present in the water that may be supporting disease spread. Typhoid fever is endemic in Zimbabwe, with several major outbreaks reported in the last decade. The bacteria persist in unclean aquatic environments, possibly supported by protozoa, and are transmitted to humans through ingestion of contaminated drinking water. They will detect S. Typhi in sewage effluent and low-quality drinking water in hotspots of typhoid transmission by enrichment culture and PCR, and use whole genome sequencing to establish the phylogenetic relationship between these bacteria and clinical typhoid isolates in the same city. They will also amplify 18S rDNA from the sewage and drinking water samples to characterize the microbial community in water and define the protozoa population. These data will help identify potential synergistic interactions between S. Typhi and other microbes to inform prevention strategies.

France Daigle of the University of Montreal in Canada will identify the microorganisms that enable the survival of the typhoid fever-causing bacterium, Salmonella enterica serovar Typhi, at low levels in water, and thereby enhances disease spread. Typhoid fever spreads through contaminated food and water, and results in over 125,000 deaths annually worldwide. S. Typhi are so-called auxotrophic bacteria because they rely on an external source of the essential amino acids that they need to grow. Microbial interactions may provide nutrients and also increase bacterial fitness and support persistence by protecting them from the environment, thereby increasing the rate of disease transmission. They will assemble a microbial community in water consisting of three components: one protozoan (from a group known to promote bacterial survival); a defined consortium of bacteria representative of the human fecal microbiota; and fluorescently-tagged S. Typhi. They will evaluate the ability of S. Typhi to grow in these microcosms, and how they grow, such as in biofilms or inside the protozoa. They will also determine whether these persistent S. Typhi are better able to infect and survive in human cells. Finally, water samples from an endemic region in East Africa will be analyzed for the presence of S. Typhi and identified beneficial microbial partners using quantitative PCR.

Aaron Jenkins of the University of Sydney in Australia will combine genomics approaches with physical chemistry to identify the organisms and environmental factors in riverbeds that support the survival and spread of the bacterium, Salmonella enterica serovar Typhi, which causes typhoid. Aquatic environments are a major reservoir of typhoid, but how the bacteria survive in these conditions is unclear, making it difficult to prevent the disease spreading to humans. They hypothesize that S. Typhi survive in biofilms associated with sediments in riverbeds, and that the composition of this niche promotes its ability to infect humans. To test this, they will sample aquatic biofilms from areas of high, low, and zero typhoid incidence in Fiji, and identify the microbial communities supporting S. Typhi survival using antibody capture and metagenomics. They will also use fluorescence in situ hybridization to determine the spatial organization of S. Typhi in multispecies biofilms. In addition, they will analyze the composition of the sediments and soil of the riverbeds, and the nutrients being taken up by resident fish and crustaceans. By combining these results with their epidemiological data, they can identify the ecological niches that support high typhoid incidence, which will help develop and guide intervention strategies to block transmission to humans.