Environ. Sci. Technol. 2006, 40, 7150-7156
Environment Arrays: A Possible Approach for Predicting Changes in Waterborne Bacterial Disease Potential† J A C K A . H E I N E M A N N * A N D H A° K A N R O S EÄ N School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand MARION SAVILL Environmental Science Research, Ltd., Christchurch, New Zealand SOFIA BURGOS-CARABALLO AND GARY A. TORANZOS Department of Biology, University of Puerto Rico, San Juan, Puerto Rico
Current molecular techniques for identifying bacteria in water have proven useful, but they are not reliably predictive of impending disease outbreaks. Genomics-based approaches will help to detect the presence of pathogens quickly and well before they grow into a population that poses a risk to public health. We suggest that genomics is only one component of the toolbox that will be needed to identify emerging waterborne threats. We propose a methodology beyond genomics, based on activity in the mobile genome. This approach makes use of a new device called an environment array. The array will depend upon the same research necessary for genomics-based detection, but will not require an a priori knowledge of virulence genes. Environment arrays are assembled from molecular profiles of the infectious elements that transfer between bacteria. The advantage of the array is that it monitors the activity of the mobile genome, rather than the presence of particular DNA sequences. Environmental arrays should thus be many times more sensitive than traditional hybridization or PCR-based techniques that target alreadyknown DNA sequences. Mobile elements are known to respond to new environmental conditions that may correlate with a chemical contamination or the bloom of bacterial pathogens, potentially allowing for a much broader application in detecting unknown or unanticipated biological and chemical contaminants.
Introduction The ability to prevent outbreaks, or even individual cases, of disease is the goal of all public health workers. The best public health management practices allow us to treat diseases or sometimes even prevent outbreak dispersion. Prevention is often based on early warning: that is, detecting diseasecausing microorganisms while populations of these organisms are small and harmless minorities of an ecosystem (1). Current microbiological techniques can provide early warnings when the infectious agents are well-known in advance. Genomics-based approaches are expected to push the limits of detection further, but they, too, are still only able to detect what we know to look for. In this regard, we believe it is already timely to suggest post-genomic ap†
This review is part of the Emerging Contaminants Special Issue. * Corresponding author phone: +64 3 364 2590; fax: +64 3 364 2590; e-mail:
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proaches to monitoring the emergence of both biological and chemical agents that are deemed harmful to human health or the environment. The approach we suggest (Figure 1), detailed below, is based on monitoring indicators of change at the genome level, rather than searching for DNA sequences per se, as genomics based approaches must do. The challenge of environmental monitoring using current techniques is in detecting small populations, or rare genes, in a large volume of material. For example, as few as 60-100 cells may be sufficient for some gastrointestinal pathogens to cause disease (2). Yet this number of disease-causing bacteria within even the amount of water consumed by the average cow or human per day would be extremely challenging to detect, especially if they were not easily cultured, were not easy to separate from the many other microorganisms in the sample, were not evenly distributed, or if we had little existing knowledge about the genes they might be carrying. That is why having an indicator that was spread across both threatening and benign microorganisms would “ramp up” the signal. For this purpose we propose to monitor changes in the genes that compose the mobile genome. That genome is the vectors, such as plasmids, transposable elements, integrons, phages, and the like, and the genes they carry. These genes move and change rapidly and rarely accumulate to great numbers within species. Nevertheless, they can cause the rapid adaptation of bacteria to new environments. This rapid adaptation is demonstrated by the antibiotic resistance genes, icons of the mobile genome, which began flowing to bacteria that are pathogens of humans around 60 years ago (3, 4). Recent research suggests that even small and local environmental changes provoke changes in the mobile genome (5-7). Since all species in a changing environment must adapt or die, a flux of adaptive genes will change the genomic profile of more than just the potential pathogens. Seeing changes in many microorganisms at once can be a rather large signal, because the genes can transfer hundreds to thousands of times even among bacteria that are not dividing. In contrast, one would have to wait for many generations of cell division to significantly increase the copy number of a particular (e.g., virulence) gene in an environment. Genes that ultimately adapt bacteria to new environments often become citizens of the genome only after they achieve a certain population density on mobile vectors. Later, like many antibiotic resistance genes and restriction-modification systems (8), these genes settle onto chromosomes. A method for quickly characterizing the population of genes on mobile 10.1021/es060331x CCC: $33.50
2006 American Chemical Society Published on Web 10/12/2006
FIGURE 1. Top: Single cassette diversity map of a terrestrial plot (64). Like a microarray, black indicates nothing detected. Bottom: Environmental array data shown using analogy of a microarray. The array indicates changes in the cassette cluster diversity profile over time. These changes are detected by a subtractive hybridization of labeled PCR products from two different samples, or some other technology that reveals changes in the community of genes in integrons. To the extent that the number of cassette clusters of a particular size class at one location has remained the same in time, the array is yellow (or black if no cassettes were ever detected at that site). If more clusters of a particular size are found at subsequent times, the additional cap the spot as green. If fewer clusters of a particular size remain at a site at later times, the site is capped red. vectors would provide a type of “snapshot” of the mobile genome in that environment. We consider this analogous to a microarray tuned to changes in gene movements, so we call it an environment array. This snapshot would serve as a baseline description of an environment that could be sampled later in time, compared over distances, and even to develop comparisons between ecosystems. Changes in the structure of the mobile population would announce new selective forces, ushered in by environmental change. The environment array is an approach for exploring risk. The environment array would best be used to indicate what environments were undergoing rapid change and thus might deserve a more focused investigation for the causes of those changes, including the potential for that environment to give rise to new or expanded populations of pathogens. Environment arrays are complements to virulence-factor activity relationships (VFAR, see below).
Scientific Gaps in the VFAR Concept A report by the U.S. National Research Council advocates using the power of genome sequencing and functional genomics focused on environmental monitoring to provide early indicators of suspected virulence genes accumulating in water with the aim of alerting us to the possibility that there exists a threat to human health or the environment. The report suggests an approach which they call VFAR, an idea based on the chemical concept called QSAR, for quantitative structure-activity relationships. VFAR is a threatprediction approach specifically applied to waterborne
pathogens. The argument is made that potential pathogens can be identified based on the finding of genes or gene products that share structural or functional similarities with genes or gene products known to be virulence factors (1). Detection of Important Genes. Different isolates of the same bacterial species can have genomes that vary by 20% (9). Even smaller changes can be relevant. For example, the difference between a deterministic pathogen like shiga toxin producing E. coli and our normal colonic E. coli can be just a few, even a single, gene (10). VFAR cannot proactively search for currently undescribed combinations of genes and bacteria that could produce an emergent pathogen. VFAR will be highly dependent on the construction of its databases and the power of its defining gene-identification algorithms. Often, these different genes are present in plasmids or phage, mobile gene vectors that cross strain, species, and even biological kingdom genetic boundaries, and are not evenly represented in all genomes of a single species (1113). Mobile genes may convert saprophytic microorganisms into deterministic pathogens (14). The difference between non-pathogenic aquatic or estuarine water Vibrio cholerae and the pathogenic form is a phage genome. Pathogenic forms are able to secrete cholera toxin, resulting in large numbers of V. cholerae being excreted during diarrheal disease, increasing the rate of their spread to new hosts (15). Like the Council, we are optimistic that the ability to detect such low numbers will improve as the power of molecular approaches becomes more useful. But these examples illustrate how tremendously difficult it is to pre-assign the role of pathogen or saprophyte to any given representative of a bacterial species or genus. It also begs the question of whether the large genome sequencing projectssthose necessary to fill the gene databases upon which the functional genomics behind VFAR will be basedswill catalog these potentially unidentified mobile genes, even if they can be detected at relevant concentrations in environmental samples.
The Mobile Genomic Vault The mobile genome is composed of the genes that do not call a particular genome home and are therefore not necessarily evenly represented in isolates of the same species (14). The mobile genome reproduces by horizontal gene transfer (HGT), the movement of genes (or subgene sequences) between organisms asynchronous from cell or organism reproduction (20, 21). HGT often provides new combinations of genes for virulence. But what makes an organism a likely threat to human health or the environment is not just the last genes transferred in by phage or plasmids, but the combination of historical acquisitions of genes from gene vectors along with other chromosomal genes (22). The history of each bacterium in an environment cannot be known in advance, so sampling the mobile genome for activity would significantly extend warning times and detection limits. HGT is revealed in the short term (50 fluoresence units), cassette amplifications were more than 95% reproducible. Doing replicated DNA extractions from the samples and searching VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Logarithmic increase in the number of yearly publications containing the keywords “integron” or “integrons” (as searched at scholar.google.com). for less abundant cassettes reduced the reproducibility of the results, but the cassette “fingerprints” acquired were, as the authors state, “readily compared with similarly acquired fingerprints from other samples” (64). This, or a similar technique, could be adapted and used to build environmental arrays. Not only could cassettes be compared at certain points in space, but also in time. There is, at this point, no proof-of-concept that arrays built on integrons and gene cassettes will necessarily be sensitive enough to detect the changes in the mobile genome required. Research into the phenomenon of integrons is still in its infancy. Searching Google Scholar (scholar.google.com) for publications concerning integrons and another genetic elements, namely transposons, clearly illustrates this point. Using the terms “integron” or “integrons” for a year-by-year search reveals a logarithmic growth in the number of papers published in this area (Figure 3), with roughly 2300 papers published since 1989. There has been more than a 100-fold increase in annual publications comparing 1989 to 2005. The number of published papers on transposons over the same timespan, while growing at a similar absolute rate, increased by roughly 2500 publications between 1989 and 2005. But this has only been a 4-fold increase in yearly publications. The number of publications on integrons is in 2005 the number of papers on transposons in 1982; research on integrons is roughly 25 years behind the research into transposons. This illustrates how new the field of integrons is and how much there is still to learn. The environment array has several advantages. First, there is no need to culture the organisms from which gene cassettes are to be amplified because the primers can be applied directly to DNA purified from the environment (65). This is important since there can be up to 500 000 species of microorganisms per gram of soil (66); we have so far only been able to study 0.1-1% of microorganisms using culturing techniques (37, 67). The drawback of using cultureindependent techniques and having one set of primers amplifying DNA from several species of bacteria is that it is extremely difficult to identify the organism(s) from which the gene cassettes were extracted (if one would wish to do so). Also, PCR is biased toward isolating shorter DNA fragments so there is a risk that larger cassettes might go unnoticed or have started to degrade in their environmental conditions. Second, using only a small set of primers, the integrongene cassette system allows for the amplification of intact horizontally mobile genes from a wide range of bacteria (63). There is no need to have any previous knowledge about the 7154
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TABLE 1. Genera of Bacteria Where Integrons Have Been Found (58, 71, 72, 74) gram-negative
gram-negative (cont)
gram-positive
Acinetobacter Aeromonas Alcaligenes Burkholderia Campylobacter Citrobacter Corynebacterium Enterobacter Escherichia Geobacter Klebsiella
Listonella Mycobacterium Nitrosomonas Pseudomonas Salmonella Serratia Shewanella Shigella Treponema Vibrio Xanthomonas
Aerococcus Brevibacterium Corynebacterium Enterococcus Mycobacterium Staphylococcus
sequences of the actual genes being isolated or what they do. Thus, this approach greatly increases our power to isolate a wide, and potentially more representative, sample from the environment in question, and a range of genes. The integron-gene cassette system therefore makes an excellent target for the characterization of horizontally mobile genes targeted by environment arrays. Integrons appear to be the primary system for the capture of antibiotic resistance and virulence genes in gram-negative enterobacteria (68). Integrons were first discovered because of their involvement in the dissemination of antibiotic resistance genes (69). Integrons were later found to be widespread in environmental samples (70) and it was quickly realized that they offered an easy way to isolate entire genes from environmental samples (65). Although any kind of gene can potentially be found in a cassette, specific examples so far isolated include genes for antibiotic resistance, DNA metabolism, and potential virulence genes (53). Integrons are a common element in bacteria and are virtually ubiquitous in antibiotic-resistant gram-negative clinical isolates (Table 1). They have so far been found in more than 20 bacterial genera such as Acinetobacter, Campylobacter, Salmonella, and Escherichia (71). It is often reported that integrons are typically only found in gramnegative bacteria and until recently, there had only been spurious reports regarding the existence of integrons in grampositive bacteria. The observation that integrons appear to be common in gram-positive bacteria infecting poultry has, however, greatly expanded the host range for gene cassette genes (72). Nonetheless, waterborne bacterial pathogens are often gram negative members of the Enterobacteriaceae.
Where Will This Take Us? In the future, the power of genomics will be coupled with a vast capacity to monitor the physical environment in real time. Yet to make value of that capacity, prediction tools will have to significantly advance in sophistication (73). Our environment array could yield relevant data by monitoring trends in the activity of elements moving in and between genomes, using isolates of the same environment at different times or isolates taken in different places at the same time. This advances over the limitations of VFAR, which makes extrapolations from DNA sequence to protein structure to function. Since the environment array will be monitoring a biological activity (gene mobility), it will not be accepting or rejecting genes based on a priori conceptions about their functions. The same information used by VFAR will be needed even if environment arrays are adopted, so that the specific threat can be identified. Without complementary genomics-based methods, the environment array is likely to yield too many false positives to be effective. Environment arrays will never replace VFAR and other genomics-based approaches for monitoring environments for threats we already know about. Therefore, a significant attribute of VFAR is that it can maintain an incentive for basic research on structurefunction predictions. VFAR will come online one day, and we are preparing for that day by having already in place a complementary “technology” that will help VFAR achieve its goals. The primary innovation of this article is to shift the focus from particular DNA sequences being used to monitor environments and predict the potential for certain environments to become a risk to human health, in the way that microbiological culturing data has always been used, to one on horizontal gene transfer as an active ecological process that can be monitored in real time to detect ecosystem-level changes. A key insight has been to link the power of using DNA (and DNA-based technologies such as PCR and hybridization) to monitor gene ecology rather than to monitor changes in concentrations of certain genes or organisms per se. Monitoring dedicated only to DNA sequences is sure to miss important developments. Environment arrays are envisioned to be one means of capturing ecological changes using DNA sequences, particularly those found in integrons, as tags. This may not be the best or only technology that could be developed for this purpose. It is proposed here that environment array approaches be used to go beyond traditional genetic approaches for the exploration of bacterial pathogens in the environmentsand water in particularsto provide a greater understanding of changes and insight into the emergence of bacteria disease.
Acknowledgments We thank B. Kurenbach for helpful comments on the manuscript. J.A.H. acknowledges the support of the Erskine Fund of the University of Canterbury which supported his visit to The University of Puerto Rico and U6570 from the University of Canterbury.
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Received for review February 14, 2006. Revised manuscript received August 21, 2006. Accepted September 4, 2006. ES060331X
