lederberg experiment on bacteria conjugation

An official website of the United States government

The .gov means it's official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you're on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings
• Browse Titles

NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Institute of Medicine (US) Forum on Microbial Threats. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington (DC): National Academies Press (US); 2009.

Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary.

• Hardcopy Version at National Academies Press

4 Antibiotic Resistance: Origins and Countermeasures

From Joshua Lederberg’s appreciation of the microbial world’s immense and fluid genetic resources came his recognition that humans, despite their dominion over “higher” forms of life, remain prey to microscopic predators. After a few decades in which it appeared that human ingenuity, in the form of antibiotics, had outwitted the pathogens—but during which Lederberg and others warned of our distinct disadvantage in an escalating “arms race” with infectious microbes—antibiotic-resistant bacterial strains, or “superbugs,” have now become ubiquitous. As Stanley Cohen of Stanford University observes in his contribution to this chapter, “It seems quite remarkable that despite the enormous progress made in the treatment of infectious disease during Joshua Lederberg’s lifetime, the ominous microbial threat discussed by Lederberg on multiple occasions continues.” The papers collected in this chapter explore the evolutionary origins of the antibiotic resistance phenomenon, take its measure as a present and future threat to public health, and propose scientific approaches to addressing it, including investigating environmental reservoirs of antibiotic resistance, identifying sources of novel antibiotics, and developing alternatives to conventional antibiotic therapies.

In the chapter’s first paper, workshop speaker Julian Davies, of the University of British Columbia, reviews the history of the development of antibiotic resistance, beginning in the early twentieth century. “Although we have gained considerable understanding of the biochemical and genetic bases of antibiotic resistance,” he writes, “we have failed dismally to control the development of antibiotic resistance, or to stop its transfer among bacterial strains.” This failure is perhaps understandable given the ubiquity of resistance genes in the environment—a vast collection of genes referred to by Davies and others as the “resistome”—and the many opportunities for genetic exchange that both nature and man have availed microbes. Thus, as Davies notes, “any drug usage, no matter how well controlled, inevitably leads to the selection of multidrug-resistant pathogens.”

Davies describes the spectrum of resistance mechanisms and places them in an evolutionary context, from their function in “virgin” (antibiotic-free) environments to their potential for transmission and reassortment with other resistance elements in human-created environments, such as wastewater treatment systems. He also considers the function of the natural bioactive compounds from which many clinical antibiotics are derived and, in particular, the low-dose effects of natural antibiotics, which appear to differ significantly from the therapeutic effects of clinical antibiotics administered at high doses.

In the next contribution to this chapter, Cohen contends that antibiotic resistance has become a global public health threat “largely as a consequence of the conventional approach used to treat infections, which is to attack the pathogen in the hope that the host will not be harmed by the drug.” He proposes a different approach to dealing with microbial pathogens: by interfering with the cooperative relationship that many of them have with their hosts. Host cells, he notes, furnish many invading pathogens with genes and gene products necessary for pathogen propagation and transmission.

Cohen describes the strategy by which he and coworkers have identified several such pathogen-exploited host genes and discusses their prospects as therapeutic targets. “There is hope that devising antimicrobial therapies that target host cell genes exploited by pathogens, rather than—or in addition to—targeting the pathogens themselves may help in the effort to thwart the microbial threat,” he concludes. However, he also acknowledges that it remains to be determined whether such host-oriented therapies will, as expected, be less vulnerable to circumvention by pathogen mutations.

The final paper in this chapter, by Jo Handelsman of the University of Wisconsin, describes research in her laboratory on two topics relevant to antibiotic resistance: the use of metagenomics to discover (potentially transferrable) resistance functions in soil bacteria; and the potential for manipulating endogenous microbial communities so that they will defend their hosts against pathogens, thus eliminating the need for antibiotic therapy.

In order to understand the process of host invasion from the perspective of the commensal microbes involved in such events, Handelsman and coworkers are characterizing the composition, dynamics, and functions of model endogenous communities. Their studies of the microbial community of the gypsy moth gut have yielded intriguing evidence that commensal bacteria interact in ways that can influence host health, sometimes in surprising ways—including collaborating with invaders to kill their hosts. Handelsman’s group has also found that treatment with antibiotics alters the response of their model microbial community to pathogens. These findings and observations have led to further inquiries into the nature of community robustness and the genetic attributes of invading microbes that permit them to overcome a robust community.

• ANTIBIOTIC RESISTANCE AND THE FUTURE OF ANTIBIOTICS

Julian Davies, Ph.D. 1

University of British Columbia

Naturally occurring small molecules, and their synthetic and semisynthetic derivatives, have been used as the foundation of infectious disease therapy since the late 1930s. Following the introduction of the “wonder drugs” penicillin and streptomycin, dozens of novel and derived bioactive compounds have been developed and used for the treatment of microbial maladies in humans, animals, and plants. Figure 4-1 shows a brief history of antibiotic development from the pre-antibiotic era to the present. The use of these important therapeutic agents for nonhuman applications, such as animal feed additives, started in the early 1950s and has expanded enormously. More than half of the total annual production volumes of all antimicrobials today are employed for nontherapeutic use as growth promotants and prophylactics in the food animal and aquaculture industries and for many other agricultural purposes.

Major classes of antimicrobials and the year of their discovery.

In 2000, more than 25 million pounds of antibiotics were manufactured in the United States alone. Over a half century, this equates to about 1 million metric tons! Considering the fact that Russia, China, and India each currently produces more antibiotics than the United States, the amounts of these compounds made worldwide is very significant. 2 From an ecological point of view, the dispersion of these bioactive compounds into the environment has created increasing pressure for the selection of antibiotic-resistant microbes throughout the entire biosphere. This selection pressure is clearly most crucial in specific therapeutic situations, such as hospitals and their associated intensive care units, but it is evident that antibiotic-resistant bacteria are now ubiquitous! The increasing use of antibiotics, since the 1950s, for both appropriate and inappropriate applications, has led almost simultaneously to increases in antibiotic-resistant bacteria, and the spectrum of resistance determinants has grown correspondingly. Little wonder that antibiotic resistance is a continuing and major threat concomitant with efforts to cure human disease.

A series of epidemics of resistant organisms have marked the antibiotic era: penicillin-resistant Staphylococcus aureus , methicillin-resistant Staphylococcus aureus (MRSA), 3 vancomycin-intermediate Staphylococcus aureus (VISA), 4 drug-resistant Vibrio cholerae , multidrug-resistant (MDR) 5 and extensively drug-resistant (XDR) 6 Mycobacterium tuberculosis (hereinafter, MDR- and XDR-TB), CTX-M resistant Escherichia coli and Klebsiella pneumoniae , Clostridium difficile, and many others. Reports of new outbreaks of these so-called “superbugs” in the popular press are regular events.

The CTX-M family of extended-spectrum β -lactamases is of particular interest and concern. These enzymes inactivate the extended-spectrum (third-generation) cephalosporins of the cefotaxime class that were first introduced for the treatment of infections caused by gram-negative organisms in the 1990s. The increasing use of these antibiotics led to the appearance of resistant strains in several countries. As indicated by the frequent reports of the increasing prevalence of extended-spectrum β -lactamases, this resistance mechanism is now endemic in hospitals and the community throughout the world ( Livermore et al., 2007 ; Queenan and Bush, 2007 ). Moreover, families of CTX-M enzymes that differ in amino acid sequence and catalytic activity have been reported in almost every country. Isolates of pathogenic Enterobacteriaceae carrying these resistance determinants, in particular K. pneumoniae , are essentially untreatable. In a number of hospitals and among certain populations, such as the military, the appearance of multidrug-resistant Acinetobacter baumannii isolates carrying a CTX-M enzyme has been of particular concern: this is an emerging pathogen capable of heightened mortality and morbidity ( Peleg et al., 2008 ).

Antibiotic-resistant infections are commonplace. Human and nonhuman use of antimicrobial agents has guaranteed this status quo; bacteria and other microbes evolve rapidly to adapt to diverse environments and novel stresses and remain alive. Nonetheless, in industrialized nations “old” antibiotics, penicillin and the sulfonamides, are effective most of the time treating in routine outpatient. Until recently the pharmaceutical industry has survived by developing novel agents (usually by synthetic chemical modification of existing compounds) in order to counter new types of infections; this continuing arms race has permitted modern medicine to keep up, in some measure, with bacterial genetics. Occasionally, structurally novel compounds have been isolated that offer a narrow window of therapeutic success, but this has become less common and there is no panacea yet.

Given the seriousness of the current situation, a number of well-considered proposals have been mooted with the objective of controlling resistance development and restoring and maintaining the efficacy of treatments against infectious diseases ( Norrby et al., 2005 ; Spellberg et al., 2008 ). But any drug usage, no matter how well controlled, inevitably leads to the selection of drug-resistant pathogens.

Antibiotic Resistance in the Environment

Since the first report of a penicillin-destroying enzyme (penicillinase) in a bacterial strain ( Abraham and Chain, 1940 ), antibiotic resistance traits have been found in many environmental bacteria isolates ( Table 4-1 ).

Reports on Antibiotic Resistance Genes Isolated from the Environment.

It is generally believed that pristine environments represent the major reservoir of resistance genes to be acquired by bacterial pathogens. The identification of the “resistome” expanded the range of antibiotic resistance determinants and soil-derived actinomycete strains involved ( D’Costa et al., 2006 ). More recently, the demonstration that a proportion of the microbes in the environment are capable of metabolizing antibiotics provided additional evidence for the existence of enzymatic mechanisms for the biodegradation of antibiotics that enable bacteria to subsist on antibiotic substrates (the “subsistome”); this work has revealed the presence of a great diversity of antibiotic-modifying enzymes in nature, and these enzymes provide the basis for a wide range of antibiotic resistance mechanisms ( Dantas et al., 2008 ). In addition, it has been shown that bacterial genomes possess a significant number of genes that, if over-expressed in other hosts, would be candidate antibiotic resistance genes (intrinsic resistance; Tamae et al., 2008 ). In these latter cases, it has not yet been demonstrated that these genes are bona fide resistance genes; however, they have the potential to be. The existence of a universe of latent resistance genes in pathogens, commensals, and environmental organisms provides further evidence that resistance genes are common in nature; resistance is everywhere genotypically, if not phenotypically.

In an ideal world the identification of a new antibiotic resistance phenotype might provide the basis for “early-warning” measures in clinical situations and could guide the development of preventive measures that should be taken to avoid dissemination of the determinant. However, this is difficult to achieve in practice, given that resistance genes may spread rapidly and become well established in the population before they are detected definitively. In addition, it is debatable how much of the environmental and intrinsic resistance is significant in clinical terms. The strategy certainly could have worked in the case of CTX-M determinants, since their putative source was first identified in environmental Kluyvera spp. and later in enteric pathogens.

As has been known since the first use of antibiotics in research and the clinic, random mutation (spontaneous or induced) gives rise to antibiotic resistance. The mutations can occur in the genes encoding drug targets, drug export, or other mechanisms ( Table 4-2 ). These mutations (usually point mutations) are pleiotropic; 7 it could be argued, conversely, that resistant mutants are frequently the result of point mutations selected by nutritional or other pressures. Indeed, the pleiotropic effects may influence a wide variety of other functions. Even resistant strains that are detected in the environment (i.e., the resistome or subsistome) may, in reality, be the result of multifunctional activities, such as nutritional balances. Experiments performed in our laboratory demonstrate that antibiotic-resistant mutants may have multiple phenotypes that could act as means of resistance selection depending on the nutrients available in the environment. Which came first?

Mechanisms of Resistance, 2008.

Cryptic resistance genes that are present in bacteria or bacterial populations may be revealed by metagenomic cloning and expression ( Riesenfeld et al., 2004 ), although it will be necessary to employ a wide range of expression hosts, in addition to E. coli , to identify functional resistance cloned from diverse bacterial genera. We do not yet know the mechanisms for gene “pick-up” from environmental sources, nor in what way such genes are “tailored” for efficient expression in heterologous hosts, such as bacterial pathogens. Environmental bacteria vary greatly in their G+C 8 content and codon usage; converting the transferred genes into functional genes in new hosts must require extensive “tailoring” by mutation and recombination. Native promoters need to be altered to permit transcription and translation in the new hosts. A good example of the way in which “new” genes may be acquired and expressed is provided by the omnipresent integron-mediated acquisition and expression system. In principle, any open reading frame can be inserted as a cassette into an attachment site associated with an integron recombinase and become a functional gene ( Figure 4-2 ).

Integron mechanism of gene capture. Integron-mediated gene capture and the model for cassette exchange. Outline of the process by which circular antibiotic resistance gene cassettes ( antR ) are repeatedly inserted at the specific attI site in a class 1 (more...)

The integron cassette promoter is strong and very effective in a variety of microbial hosts. However, the acquisition process is not well understood, and essentially all aspects of the origins and evolution of antibiotic resistance genes remain unsolved. Integron-encoded gene cassettes are widespread in all natural environments (marine and terrestrial), with the vast majority of such genes being of unknown function ( Koenig et al., 2008 ). Although primarily of gram-negative bacterial origin, there have been reports of resistance integrons in gram-positive bacteria ( Nandi et al., 2004 ). Gene exchange between bacterial genera is an evolutionary axiom, but it is still necessary to solve the dilemma of access to the resistance gene sources.

Antibiotic Resistance in Urban Environments

Urban areas, constantly exposed to the large variety of antibiotics that are commonly used in the hospital and the community, have considerable reservoirs of resistance. In large cities, thousands of people receive antibiotic treatment every day (e.g., in hospitals, in nursing homes, and at home), and accordingly, many antibiotics are released as part of the waste stream of the sanitary sewer system into wastewater. The “hotspots” are generally considered to be hospital-associated, but given that the main disposal route is through sewers, it is very likely that wastewater treatment plants (WWTPs) are also hotspots, with significant concentrations of antibiotic-resistant microbes containing multiple resistance genes from a wide variety of antibiotics and myriad potential vectors.

That WWTPs provide ideal environments for gene exchange and gene acquisition has been confirmed by studies of antibiotic resistance plasmids isolated from WWTP bacterial cultures ( Tennstedt et al., 2005 ). Sequencing of purified plasmid DNAs demonstrated that very complex genomes are formed, as evidenced by the combinations of resistance genes, transfer factors, transposases, integrons and their associated integrases, bacteriophage remnants, and other potential mobile elements. Thus, a considerable level of genetic mixing and matching occurs, with the consequence that novel combinations of antibiotic resistance determinants may be discharged into WTTP effluents ( Schlüter et al., 2007 ). It is also possible that virulence genes in the form of pathogenicity islands are acquired by plasmids.

While the roles of such newly formed resistance elements may be a matter of speculation there is absolutely no doubt that they are produced, and could contribute to, the gene pool responsible for increasing antibiotic resistance in the human community.

Antibiotic Hormesis

Antibiotics have long been known to have multiple effects on target cells. For many years, clinicians and a growing number of microbiologists and biochemists have reported that sub-inhibitory concentrations of commonly used antibiotics may affect microbial cell growth, morphology, structure, adhesion, virulence, and a variety of other phenotypes ( Davies et al., 2006 ). In the majority of studies the changes induced were advantageous to the microbes. For example, a number of so-called antibiotics induce the formation of biofilms that permit microbial communities to survive under adverse conditions; others may promote swarming and motility, perhaps enhancing nutrient accessibility; even protein synthesis inhibitors may cause changes in cell-wall structure and function. Recently, detailed studies of transcriptional and proteomic changes (rather than phenotypic ones) have confirmed the wide range of responses of microbes to bioactive small molecules.

Almost all antibiotics have side effects that in many cases require careful dosage monitoring; these effects often occur at sub-inhibitory concentrations. The recent demonstration that these compounds exert the phenomenon of hormesis 9 provides an explanation for the dual activities of microbially-derived natural products ( Davies, 2006 ). The differences are due to transcriptional effects that are concentration-dependent. At low concentrations, antibiotics cause strong stimulation of transcription from specific groups of promoters. At higher concentrations (near-inhibitory), the transcription patterns change, as illustrated in Figure 4-3 . Such dose-response dependence has been demonstrated by the use of promoter-reporter constructs or microarray analyses in the laboratory. We have proposed that these hormetic effects of antibiotics account for the differences in activity of low-molecular-weight compounds in their natural environment (soils, etc.) compared to their role in the treatment of infectious diseases.

Concentration dependence of transcription modulation by antibiotics. MIC = minimal inhibitory concentrations; SMs = small molecules. SOURCE: Adapted from Yim et al. (2006).

In pristine environments, complex microbial populations can be assumed to exist in some form of homeostatic equilibrium that change as a result of fluctuations in nutritional resources. Stability in the population is probably maintained by cell-to-cell signaling that modulates their metabolic activity. When soil isolates of bacteria producing useful bioactive compounds (such as antibiotics) are transplanted to a completely different environment (a laboratory), and exposed to potent mutagens and fermentation on unusual substrates to very high densities under artificial conditions, the yields of small molecules are often amplified considerably, generating sufficiently large amounts of the desired compounds to permit their testing as antibiotics at elevated (inhibitory) concentrations.

Antibiotics and Other Anthropomorphisms

The microbiological literature is spiced with comments such as “the bacteria have to make a decision” or “the microbe has made a choice.” These statements are patently ridiculous. Microbes are genetically programmed to respond to different external environments; they do not make choices. Perhaps the most pervasive of such comments refers to the activity of microbially-produced small molecules. Selman Waksman’s seminal work on the discovery of potent compounds such as neomycin and streptomycin led him to define them as antibiotics. He later realized that this definition was based on laboratory studies of soil microbes, grown in media containing exotic substrates and tested for their activities against human pathogens, not environmental bacteria. Waksman (1961) subsequently made a different judgment:

The existence of microbes that have the capacity to produce antibiotics in artificial culture cannot be interpreted as signifying that such phenomena are important in controlling microbial populations in nature…. Unless one accepts the argument that laboratory environments are natural, one is forced to conclude that antibiotics play no part in modifying or influencing living processes that normally occur in nature.

We may disagree with his conclusion, in the light of current knowledge, but must concur with the distinction between the laboratory and complex natural environments.

Another “misinterpretation” is that all of these compounds kill bacteria. On the contrary, it is well known that most of the antimicrobial agents used therapeutically do not kill other bacteria—they only inhibit the growth of the target organism. This is an important aspect of antimicrobial therapy: the drug retards or inhibits bacterial growth and virulence and allows the human immune system to eliminate the weakened pathogens. 10

The Global Microbiome

Bacteria are the most abundant living organisms on this planet and, given that they are essential to the maintenance of all other living organisms, they are the most important. This is becoming increasingly evident as results of studies of the human microbiome demonstrate that the vast microbial population of the human gastrointestinal tract varies drastically with changes in age, diet, and disease ( Bäckhed et al., 2005 ).

The microbial world is immense in number and diversity; specific details are subject to debate, but estimates indicate that there are between 10,000 to 50,000 taxa per gram in different soil types ( Quince et al., 2008 ), while the bacterial density may be as large as 10 9 total bacteria per gram of soil. These are communities, not isolated organisms! In many environments, in the human intestinal tract for example, a small number of dominant phyla may constitute as much as 90 percent of the total population. Are these populations in constant conflict (the “war metaphor”), or do they undergo flux and coexist as controlled communities? It seems most logical that the latter is true, in which case some form of signaling process would be required to modulate the mixed populations under differing conditions. We believe that the signaling is primarily chemical, involving a variety of naturally occurring, low-molecular-weight compounds; after all, this is the predominant form of signaling in higher organisms. Of course, other forms of interactions, such as electrical connections between bacteria, are not excluded; the latter notion is supported by the demonstration of nanowires linking bacterial cells in biofilms ( Gorby et al., 2006 ).

There is increasing evidence that the bioactive molecules generally classified as antibiotics play roles as chemical signals. Based on estimates of the putative small molecule biosynthetic clusters predicted from the nucleotide sequences of bacterial genomes, most soil and other environmental microbes have the genetic capacity to produce a number of bioactive compounds that are active at very low concentrations. Since many soil bacteria have the capacity to produce up to 20 different bioactive compounds, the notion of community structures controlled by small molecules becomes obvious. Welcome to the world of chemical biology!

Although these compounds may have antibiotic activity at high and often nonphysiological concentrations, their functions as chemical signals or modulators, as with the well-known quorum-sensing autoinducers, may be paramount in the environment. Even in the case of microcins 11 or bacteriocins, 12 which are natural peptides having potent in vitro activity as antibiotics, one can question whether specific bacterial killing in mixed bacterial populations is their natural function. As mentioned earlier, we and others have shown that at low concentrations (several log units below the determined minimum inhibitory concentration), these highly potent molecules modulate transcription in target bacteria without any deleterious effects (inhibition or killing); each antibiotic induces specific groups of genes, depending on the type of compound and its concentration. Until the concentrations of microcins and antibiotics in the gut (and other environments) are determined accurately, we will not be able to make any predictions about the potential roles of these molecules. It is probable that they also modulate functions in the epithelial cells lining the gastrointestinal (GI) tract as well as in the bacterial population. The signaling is inter-generic!

It all comes down to the question of concentration; the activities of all bioactive small molecules are dependent on the dose used. It is possible to study the range of responses in the laboratory, but at present this is not possible in complex natural environments. Current studies with antibiotics and other bioactive molecules suggest that synergy may be favored over antagonism in bacterial populations in nature. As observed in an earlier Institute of Medicine Forum workshop summary report Ending the War Metaphor ( IOM, 2006 ), bacteria are not necessarily involved in extensive warfare in the GI tract; rather they use the bioactive small molecules to sense and tune into each other in a state of coexistence in microbial communities. It could be that the considerable distress suffered by many people with the ingestion of oral antibiotics is due to the fact that the presence of additional signaling compounds in the medicines creates mixed “signals” in the gut. Increasing knowledge of the natural biological activities of microbes may provide novel methods for their detection and isolation; in all likelihood, they will continue to be the source of agents with indispensable therapeutic value—until inevitably they develop resistance, of course.

The Pre-Antibiotic Era and Antibiotic Virgin Lands

Finally, coming back to the environmental origins of antibiotic resistance, there have been a number of studies in which archived bacteria, isolated before the extensive use of antibiotics, were examined for the presence of plasmids and resistance genes. In a seminal study by V. Hughes and N. Datta (1983) , a collection of bacteria isolated from the 1920s (the Murray Collection) were found to carry plasmids, but no resistance genes; a number of the plasmids were shown to be transferable by conjugation. In another study by D. H. Smith (1967) using a collection of E. coli strains lyophilized in 1946 (some from Joshua Lederberg), R-factors were found in one strain; subsequent conjugation studies showed inter-strain transfer of resistance to tetracycline and streptomycin. Thus, one can conclude that plasmids were present, but resistance determinants were rare. These were all human isolates. It might be worthwhile to have another look at these collections using modern molecular biology techniques. The uropathogenic strains of E. coli in the Murray Collection have been found to contain pathogenicity islands typical of modern isolates (Dobrindt and Davies, unpublished).

Studies have also been done with bacteria isolated from native communities that have had no apparent contact with other humans and no exposure to antibiotics. The first such study, conducted in 1969, demonstrated that the indigenous population in the Solomon Islands had intestinal bacteria carrying R-factors that mediated resistance to streptomycin and tetracycline; the streptomycin resistance was due to a streptomycin phosphotransferase found in resistant strains in the United States ( Gardner et al., 1969 ). More recent investigations examining the inhabitants of remote villages in South America found that as many as 90 percent of the population harbored multidrug-resistant strains (as detected in stool samples); Class I integrons were also identified ( Bartoloni et al., 2009 ; Pallecchi et al., 2007 ). Resistance to tetracycline, ampicillin, sulfatrimethoprim, chloramphenicol, and streptomycin was frequent. These latter studies had the benefit of using sensitive detection technologies that were not available previously. None of the work examined the presence of resistance genes in associated environmental samples such as soils or in animals. These results imply that antibiotic resistance is at least maintained in the absence of antibiotic exposure. However, there is the possibility that occasional use of antibiotics, or that rare contact with humans who have used antibiotics, triggers the selection of populations of resistant bacteria in naïve populations. It may also be that therapeutic use of plant products by native populations selected for integron-encoded resistance elements, since the Class I integrons carry efflux genes with a wide substrate range.

Other studies have examined the bacterial populations of wild animals (birds, mammals, rodents, fish, reptiles, etc.) with similar results: resistance genes are present in all species tested. The extent to which these animals encountered human detritus is not known, but they clearly play a role in the global dissemination of antibiotic resistance. Seagulls and geese have close contact with humans and migrate over long distances! Suffice it to say, antibiotic resistance plasmids, integrons, transposons, and so forth, are widespread in bacteria. The natural resistome is ubiquitous.

Conclusions

Antibiotics are indispensable in the treatment of infectious and other diseases. Their discovery was essentially the result of human intervention, the consequence of a laboratory phenomenon that developed into an industry. Unfortunately, although antibiotic production and use have been of enormous medical and commercial value in transforming the therapy of infectious diseases in the last 60 years, the extensive use of antimicrobial agents during this period has not led to the eradication of any one bacterial pathogen. Moreover, pharmaceutical companies are discontinuing their infectious disease programs, largely for economic reasons. Discoveries of new, broad-spectrum antibiotics are very rare. The widespread development of antibiotic resistance in hospitals and the community makes it imperative that the search for new, novel bioactive compounds be maintained.

The pharmaceutical industry has screened hundreds of thousands of compounds for antibiotic activity over the last 50 years. Many antimicrobial agents were discovered that did not appear to be useful or were not competitive for one reason or another. They might have had undesirable side effects and/or an unacceptable toxicity profile. Could some of these agents be used effectively for a short time in cases of life-threatening infections with multidrug-resistant pathogens? In circumstances where the currently available antibiotics are not effective, might some of the rejected compounds be resurrected for use at sub-inhibitory concentrations, alone or in synergistic combination with known compounds so that toxic side effects would be minimized?

National and international agencies have been seeking ways to maintain appropriate levels of work on antibiotic discovery (and recovery) while coping with the problem of antibiotic resistance ( Norrby et al., 2005 ; Spellberg et al., 2008 ). The Infectious Disease Society of America and the Federation of European Microbiological Societies have made a number of rational recommendations, but their implementation appears to have been less successful than had been hoped. Questions still abound. What definitive actions can be taken? Containment procedures to date have successfully curtailed the spread of some viral infections. Any appearance of bird flu, for instance, has been met with rapid and drastic action and the disease has not yet reached a significant number of the human population. Should the same tactics be employed with the appearance of all new antibiotic resistance determinants? Could the CTX-M β -lactamase pandemic have been arrested had the first identified outbreak been contained by strict isolation measures? Is there an active and effective early-warning system for new resistant pathogens worldwide? What agencies would be the most effective in exercising this oversight?

Surely governments, the pharmaceutical industry, and academia can cooperate in actions that will curtail resistance development in pathogens and support efforts to provide a continuing supply of potent antimicrobial agents. Such a concerted effort is urgent. As Joshua Lederberg said, “[I]n that natural evolutionary competition, there is no guarantee that we will find ourselves the survivor” ( Lederberg, 1988 ).

• MICROBIAL DRUG RESISTANCE: AN OLD PROBLEM IN NEED OF NEW SOLUTIONS

Stanley N. Cohen, M.D. 13

Stanford University

Barring geno-suicide, the human dominion is challenged by only pathogenic microbes, for whom we remain the prey; they the predator. Joshua Lederberg, May 1993

As anyone who knew Joshua Lederberg is aware, he was not prone to hyperbole or overstatement, so this pronouncement is sobering. The confrontation between microbes and humanity that Lederberg referred to has had a long history—from the biblical Plague of the Philistines described in the Book of Samuel, to the first documented instance of plague, Justinian’s Plague, in the sixth century A.D., through a series of major pandemics that decimated Western European populations in the centuries that followed. In modern times, perhaps the greatest plague was that of pandemic influenza, which occurred during the winter of 1918–1919.

The challenge from microbes is multifaceted. An early instance of biological warfare occurred during the bubonic plague epidemic known as the Black Death during the mid-fourteenth century. During the Siege of Caffa (now the Ukranian city of Feodosija) in 1346, the invading Mongols, who were suffering from Plague, catapulted their comrades’ corpses over the walls of Caffa in the hope that the rotting corpses would poison the city’s water supply and destroy its defenders ( Wheelis, 2002 ). 14

Human intervention against microbial disease commenced with the practice of variolation—the subcutaneous inoculation of smallpox-susceptible persons with material taken from a pustule of an individual afflicted with smallpox—an approach that can be traced to ancient folk practices in China, Turkey, and Africa ( Riedel, 2005 ). A modern understanding of infectious disease began with the visualization of microbes by van Leeuwenhoek in 1683. In the 1870s, Louis Pasteur and Robert Koch first advanced the germ theory, which focused on the causal role of microbes in infectious disease. Half a century later, Griffith (1928) showed that disease-producing properties could be transferred between bacteria by a substance they produced. Fifteen years after that observation came the dramatic discovery by Avery, MacLeod, and McCarty that the genetic information responsible for such disease-producing properties resides in DNA, rather than in proteins, as had previously been suspected ( Avery et al., 1944 ). This discovery set the stage for the seminal experiments of Lederberg and Edward Tatum on conjugation and recombination in bacteria, which depended significantly on extrachromosomal elements carried by these microbes. In an article published in the journal Physiological Reviews in October 1952, Lederberg invented the name “plasmids” for these extrachromosomal elements, and subsequent studies from multiple laboratories showed that plasmids were circles of DNA that could be passed among bacteria conjugally by hair-like structures called pili (for a review, see Cohen, 1993 ).

As Lederberg was conducting his investigations of bacterial conjugation and recombination, antibiotics were introduced for the clinical treatment of infectious diseases. The use of penicillin saved many lives near the close of World War II, and it was widely anticipated at the time that antibiotic use would soon lead to the worldwide eradication of bacterial infections. This hope quickly dissolved with the appearance of antibiotic resistance. Multidrug-resistant bacteria were observed first in Japan in the late 1950s and soon were detected also in other countries throughout the world. Multidrug-resistant bacteria quickly emerged as a medical problem because the drug resistance genes they carry could pass horizontally via conjugation to other bacteria—some of which were more pathogenic than the original plasmid host—as well as linearly to the progeny of the resistant cells. The spread of bacterial resistance to antimicrobial drugs has spawned years of research on mechanisms underlying such resistance. These investigations have shown that resistance to antimicrobials has become common worldwide among bacterial populations largely as a consequence of the conventional approach used to treat infections, which is to attack the pathogen in the hope that the host will not be harmed by the drug. However, notwithstanding this hope, antibacterial and antiviral drugs clearly do have toxic effects on the host; moreover, those bacteria that acquire mutations that result in insensitivity to the antimicrobial can continue to reproduce while most individuals in the bacterial population are killed or their growth inhibited. This leads to the take-over of bacterial populations by resistant microbes. Another limitation of the conventional approach to antimicrobial therapy, in light of the increasing threat of bioterrorism, is the potential for intentional alteration of pathogens so as to render them drug-resistant.

From the early stages of his career, Joshua Lederberg was interested in learning how drug-resistant bacterial populations evolve. With his first wife, Esther Lederberg, he developed a beautifully simple, effective, and inexpensive procedure called replica plating to investigate this: a sterile piece of velvet attached to a cylinder is touched to a plate on which bacterial colonies are growing; the velvet is then touched to multiple sterile growth plates, which in the Lederbergs’ experiment contained antibiotics not present on the original plate. Using this method, the Lederbergs found that some bacteria present on the original plate contained mutations that resulted in resistance to an antibiotic that the bacteria had not been exposed to ( Lederberg and Lederberg, 1952 ). Based on these findings and on subsequent investigations of mutations that lead to antibiotic resistance, research by the scientific community has focused on heritable resistance. It is worth noting, however, that the Lederbergs left open the possibility that resistance to antibiotic exposure or other temporary environmental challenges that disappear after several generations of bacterial growth under nonselective conditions might also occur ( Lederberg and Lederberg, 1952 ). Such adaptive resistance, which results from antibiotic-induced altered expression of nonmutated genes, is in fact an additional problem that has in recent years been the subject of increasing scientific attention.

Host-Oriented Therapeutics

Host-microbe interactions generally are viewed as adversarial, and research on such interactions has largely focused on enhancing the ability of the host to halt invasion by pathogens ( Beutler, 2004 ; Ishii et al., 2008 ). Pathogens can also exploit the normal functions of host cells for propagation, and the cellular genes exploited by pathogens (CGEPs) are potential targets for antimicrobial therapies that may be less subject to mutational circumvention by pathogens. Moreover, certain host cell genes are required by multiple pathogens, providing the potential for broad-spectrum therapeutic effects from the targeting of a single gene or gene product.

Consider the example of viral propagation and the pathogenesis of viral disease, which require that the infecting virus bind to host cells, that the viral genome be internalized, and that its genes be expressed and its proteins processed. To produce disease, the virus must then replicate, produce a pathological event, undergo morphogenesis, and be released in order to infect other cells of the host. All of these events require the functions of host cells and recruitment of these functions by the virus. At least some of these host cell functions are implicated in the pathogenesis of diseases caused by bacterial pathogens. Similarly, the effects of microbial toxins are also dependent on host functions: anthrax toxicity, for example, requires the recruitment of host proteins to enable uptake and processing of the toxin, as shown in Figure 4-4 .

The road to anthrax toxicity. (1) Anthrax protective antigen (PA) binds to receptors on the surface of the host cell, and (2) is cleaved in two by furin. (3) The remaining 63-kilodalton subunit (PA 63 ) heptamerizes. (4) The heptamer interacts with the (more...)

When I raise the prospect of developing antimicrobial therapies that target host cell functions recruited by pathogens, there typically are questions about whether interference with such host functions will result in unacceptable drug toxicity. Possible toxicity is an issue that must of course be addressed with any medication, and for infectious diseases, this is true whether a host function or the pathogen itself is the primary target of the therapy. However, drugs that treat disease by targeting normal cell functions routinely are used for all other types of illness, from high blood pressure to neuropsychiatric disorders. The goal of treatment has been to find agents that differentially—but not necessarily exclusively—affect the pathogenic process. There is no inherent novelty to host-oriented therapy. Rather, only in the infectious disease arena are therapies historically not host oriented.

Finding host genes recruited by pathogens is practical using gene inactivation approaches. However, as mammalian cells normally contain two copies of each gene, inactivation of both copies ordinarily is necessary to produce biological effects. Whereas such homozygous gene inactivation can be accomplished readily when the gene is known and the DNA sequence is available, the use of random inactivation of genes in a mammalian cell population as a tool for the discovery of gene function is a more formidable task. Some years ago, Limin Li and I reported a retrovirus-based strategy for accomplishing this ( Li and Cohen, 1996 ) using viruses having an RNA genome ( Figure 4-5 ).

Diagrammatic representation of the site of chromosomal insertion of a lentiviral GSV. A regulated promoter located in the GSV is used to generate antisense transcripts that functionally inactivate both copies of the chromosomal gene containing the insertion. (more...)

When such viruses, which are called retroviruses, infect cells, a DNA copy of their genomes is inserted into the chromosome of the infected cell. In large cell populations, inserts can occur in nearly every gene. The inserted DNA copy of the retrovirus genome, which in Figure 4-5 is termed a Gene Search Vector (GSV), inactivates one of the two copies of the chromosomal gene into which it has inserted. The GSV contains a regulated antisense promoter that initiates transcription extending outward from the insert into the adjacent chromosomal DNA segment. This generates an RNA transcript that is complementary to that chromosomal DNA sequence. As the transcript initiated in the GSV is complementary also to mRNA coming from the second copy of the chromosomal gene containing the GSV insertion, the second copy of the chromosomal gene is also inactivated (silenced). The GSV-treated cell population can then be screened, for example, for individual cells that survive the lethal effects of a pathogen or a toxin, and genes containing GSV insertions can be identified and characterized. The overall procedure has been referred to as Random Homozygous Knock Out (RHKO) ( Li and Cohen, 1996 ). The biological effects of gene inactivation observed during the screening can be confirmed by inactivating the same chromosomal gene in naïve cells—using antisense approaches, small interfering RNA, or homologous recombination.

The RHKO strategy enables the identification of genes and genetic pathways required for pathogenicity. Functional homozygous gene inactivation also has proved practical using expressed sequence tag (EST) libraries ( Lu et al., 2003 ) or small interfering RNA libraries ( Cherry et al., 2005 ; Dunn et al., 2004 ; Hao et al., 2008 ). However, for EST-based and RNA interference (RNAi)-based methods, the scope of gene inactivation is limited by the contents of the RNAi or EST sequences used to create the library. To the best of my knowledge, the RHKO strategy currently is the only way to comprehensively inactivate mammalian genes randomly.

CGEPs Identified Using Gene Inactivation Approaches

The first gene isolated by the RHKO strategy—tumor susceptibility gene 101 (Tsg101)—was identified using a tumor susceptibility screen ( Li and Cohen, 1996 ). Further study showed that Tsg101 encodes a variant of ubiquitin conjugase enzymes ( Koonin and Abagyan, 1997 ; Ponting et al., 1997 ) and, importantly, that it has a key role in endocytic trafficking ( Babst et al., 2000 ; Bishop and Woodman, 2001 ). An initial clue that Tsg101 gene might be involved in endocytic functions had come from two-hybrid analysis for identification of other cellular proteins that interact with Tsg101; one of the Tsg101-binding proteins detected in such experiments was Hrs ( Lu et al., 2003 ), which was known to have a role in the trafficking of receptors to the cell surface (for a review, see Raiborg and Stenmark, 2002 ). The trafficking process is mediated by multicomponent protein complexes, which have been termed ESCRT (Endosomal Sorting Complexes Required for Transport) complexes, and Tsg101 is an ESCRT complex protein ( Babst et al., 2000 ; Katzmann et al., 2001 ). Viral budding is topologically similar to receptor trafficking to the cell surface, and virus-encoded proteins can interact with Tsg101 ( VerPlank et al., 2001 ) and other components of ESCRT complexes and usurp ESCRT-complex functions to enable viral egress from cells ( Garrus et al., 2001 ; Goff et al., 2003 ; Martin-Serrano et al., 2001 ). Mutations in Tsg101 can interfere with the interaction between Tsg101 and virus-encoded egress proteins ( Garrus et al., 2001 ; Goff et al., 2003 ; Martin-Serrano et al., 2001 ) and, consequently, can inhibit viral release. Recent evidence indicates that externally-administered agents such as cyclic peptides can inhibit interaction of Tsg101 with its human immunodeficiency virus (HIV) partner, the egress-promoting Gag protein, and thus interfere with Gag-promoted release of virus-like particles at an inhibitor concentration that does not detectably affect the trafficking of cellular receptors ( Tavassoli et al., 2008 ). Antibodies that interact with Tsg101 on the cell surface have also been found to interfere with virus release ( Bonavia et al., 2008 ). Collectively, such findings support the notion that host-targeting therapies may prove useful in combating infectious pathogens.

The Search for CGEPs Continues: Anthrax and Tuberculosis

Assay development is crucial to the search for CGEPs. Assays can be as simple as the selection of survivors to pathogen exposure, or can be more complex, for example, involving the identification of cells expressing a “reporter” protein that has been linked to a CGEP-regulated gene. To identify CGEPs involved in the lethal effects of anthrax toxin, we used an EST-based antisense approach to host gene inactivation and an assay that identified cells surviving the lethal effects of anthrax toxin. These investigations have led to the discovery that ARAP3, a phosphoinoside-binding protein previously implicated in membrane vesicle trafficking and cytoskeletal organization, and LRP6, a lipoprotein-receptor-related protein also present on cell surfaces, affect internalization and the consequent lethality of anthrax toxin ( Lu et al., 2004 ; Wei et al., 2006 ). LRP6 was previously known to be a co-receptor for signaling by the Wnt protein, which has an important role in embryogenesis, differentiation, colorectal cancer, adipogenesis, and osteogenesis (for a review, see He et al., 2004 ). Reconstitution of ARAP3 or LRP6 deficiency in naïve cells by using antisense methods or small interfering RNAs (siRNAs) has confirmed the role of these genes in anthrax toxin lethality, and confocal imaging studies have shown that internalization of the cell surface-binding component of anthrax toxin ( Figure 4-4 ) is impaired in cells deficient in ARAP3 function or LRP6 function ( Lu et al., 2004 ; Wei et al., 2006 ). Antibodies generated against LRP6 were found to offer partial protection of cells from toxin-induced death ( Wei et al., 2006 ).

Similar studies to identify CGEPs for Mycobacterium tuberculosis currently are under way in my laboratory, in collaboration with the laboratory of Carl Nathan at the Weil School of Medicine at Cornell University. Assays have been established to identify macrophages whose ability to survive infection by M. tuberculosis is altered by host gene inactivation. Preliminary experiments have identified host genes that putatively modulate macrophage killing by M. tuberculosis and have revealed that some of these genes have pathways in common.

It seems quite remarkable that despite the enormous progress made in the treatment of infectious diseases during Joshua Lederberg’s lifetime, the ominous microbial threat discussed by Lederberg on multiple occasions continues. A simple fact underlies this threat: for every pathogen-encoded protein or nucleic acid targeted by a therapeutic agent, there is the potential for a target-altering mutation that can render the therapy ineffective. Thus far, the strategy for coping with drug-resistant microbes has been to discover or design new pathogen-targeting drugs, but there is concern that the time of mutational resistance to even “drugs of last resort” is approaching.

There is hope that devising antimicrobial therapies that target host cell genes exploited by pathogens, rather than—or in addition to—targeting the pathogens themselves, may help in the effort to thwart the microbial threat. Here, I have reviewed some of the evidence indicating that such CGEPs can be identified by genetic screening strategies and that the pathways encoded by CGEPs can offer approachable targets for antimicrobial therapies. Whether therapeutic measures aimed at interfering with pathogen exploitation of host function will be less subject to circumvention by pathogen mutations has yet to be determined. However, host-oriented therapies clearly should not select for host mutations that increase susceptibility to a pathogen, and microbial mutations that enable the pathogen to propagate by exploiting alternative host cell functions may prove to be less common than mutations that simply modify the pathogen site targeted by a drug.

Acknowledgments

I thank multiple colleagues in my laboratory at Stanford University who have participated in studies reviewed here, and the U.S. National Institutes of Health (NIH), the Defense Advanced Products Research Agency (DARPA), 15 and the Defense Threat Reduction Agency (DTRA) 16 for research support.

• EXPANDING THE MICROBIAL UNIVERSE: METAGENOMICS AND MICROBIAL COMMUNITY DYNAMICS

Jo Handelsman, Ph.D. 17

University of Wisconsin

In the real world, microbial processes occur within the context of a community. Most of our understanding of microorganisms is derived from bacteria in pure culture. It is critical, if we are to manage microbes to improve human health, that we develop a complementary understanding of microbial behavior in complex communities. In this essay, I will discuss studies in two different areas of microbial community research in my laboratory: antibiotic resistance and host-microbe interactions. I will begin with a brief overview of our work on the discovery of antibiotic resistance genes in communities of soil bacteria, and proceed to a more detailed discussion of a model system that we have developed to study endogenous microbial communities and their role in host health and disease.

The Metagenomics of Resistance

Where does antibiotic resistance come from? We know quite a lot about the clinical sources of antibiotic resistance as it affects humans, and we know a bit about the agricultural reservoirs of resistance, particularly in livestock systems. We really know nothing, however, about the source of antibiotic resistance, except that it must come from the environment, because the genes that confer antibiotic resistance vastly predate the use of antibiotics.

So, where do these genes come from? Soil is a likely source for many of them, because most of the antibiotics that we use today are derived from soil microbes (see the contribution to this chapter by Julian Davies). Soil is an extremely species-rich environment. Our models and statistical analyses suggest that a single gram of soil may contain between 5,000 and 40,000 species (conservatively defined) of microbes ( Schloss and Handelsman, 2006 ). In addition to being the richest source of new microbes we have on Earth, soil is also one of the most difficult environments in which to study microbes, because the vast majority of them—probably more than 99.9 percent—cannot currently be cultured. We know these uncultured organisms only by their molecular signatures, which indicate that they are very different from the species that we can culture (see contributions by David Relman in Chapter 2 and Jonathan Eisen in Chapter 5 ).

Metagenomics was developed in order to study the collective genomes of an assemblage of organisms as a single entity, or a metagenome; the name comes from the Greek, meta meaning “transcendent” or “overarching.” My group takes a particular approach to such studies, which can be described as functional metagenomics (in contrast to the sequence-driven metagenomic studies, discussed by workshop speaker Jill Banfield, as described in the workshop overview and in Chapter 2 ). The method we use is very simple and, on the face of it, quite crude. We extract DNA directly from the soil; most of what we obtain comes from Bacteria and Archaea. We clone the extracted DNA in Escherichia coli, and then screen the resulting library for an expressed activity, such as antibiotic production or antibiotic resistance.

We have searched for genes that confer resistance to various antibiotics. We have found genes for all three types of enzymes known to modify aminoglyco-side antibiotics (e.g., kanamycin) in our libraries. We have also founds genes that confer resistance to β -lactam antibiotics (e.g., penicillin and cephalosporin) in a library derived from soil sampled at a pristine site in Alaska: an island in the middle of the fast-moving, high-volume Tanana River, very far from human activity (except for our visits, and we do not bring antibiotics with us). The β -lactam-resistant clones isolated from this location represent a broad spectrum of resistance mechanisms. Some clones contain gene sequences resembling those of known resistance genes from clinical isolates; these genes express enzymes (such as β -lactamases, pencillinases, and carbapenemases) that degrade β -lactam antibiotics. Other clones contain genes that diverge deeply from known β -lactam resistance genes from cultured organisms; we think these genes may confer resistance through a regulatory function.

One of the most interesting clones that we obtained from the Alaskan soil encodes the first known example of a hybrid β -lactamase: two different kinds of β -lactamases fused into a single protein. One end confers resistance to the penicillin-like compounds, and the other end confers resistance to the cephalosporin-like compounds; together, they confer very broad resistance to β -lactam antibiotics ( Allen et al., 2009 ). This is a gene that we hope to never see in clinically important pathogens, but based on what we know about the transfer of antibiotic resistance determinants, this is entirely possible. We should, therefore, continue to examine and anticipate the broad range of antibiotic resistance genes in our environment. Essential in this exploration is that we are mindful of antibiotic resistance in communities and that we account for both culturable and nonculturable members of those communities since either type of organism could provide an important source of resistance to human pathogens.

Phalanx or Traitors?

The second part of this essay concerns the nature of the microbial communities that comprise the normal gastrointestinal flora of animals. Do these commensal communities protect their hosts from disease, or do they encourage the disease process?

My group is interested in commensal gut communities for a variety of reasons. We know from many studies 18 in mammals, as well as in invertebrates, that the normal gut flora influences host physiology. There is evidence that endogenous microbial communities contribute to obesity, diabetes, and high cholesterol ( Ley et al., 2006 ); there is also evidence that the microbiota is essential to the health of the host. We also know that most pathogens live as commensals and then—under the right conditions—they become invasive, and thereby pathogenic. We are very interested in that switch, because if we want to use microbial communities to protect us from disease (e.g., through the use of probiotics or through the deliberate manipulation of microbial community dynamics), we need to understand how the opposite sometimes happens. We study the role of microorganisms in communities that inhabit the midguts of gypsy moth and cabbage white butterfly larvae. These are good model systems, as the larvae are easy to rear and dissect and their gut microbial communities can be readily and naturally manipulated via host feeding. Moreover, the intestinal epithelium of these insects is surprisingly similar to that of humans and other mammals, in terms of both its anatomy and immunological function. Using the larval model systems, we have explored the role of microbial communities in health and disease of the host.

Who Is There?

We use both culture-based and culture-independent methods to examine the composition of communities. We find uncultured organisms that are readily recognizable as members of phylogenetic groups that have many culturable members, but these particular organisms cannot themselves be cultured, for largely unknown reasons.

Table 4-3 shows the diversity of bacterial phylotypes that we have identified in the gypsy moth system. Most of these are members of two phyla: the Proteobacteria and the Firmicutes. Over time and under different feeding regimes, we have observed changes in community composition at the species level, but rarely at the phylum level.

Phylogeny of Cultured and Uncultured Bacteria from Third Instar Gypsy Moth Midguts Feeding on an Artificial Diet.

Are Signals Exchanged in the Gut Community?

Many disease processes induced by bacteria are dependent on microbial communication through small molecules. Some bacterial species use a mechanism, called quorum sensing, to gauge the density of their populations ( Engebrecht and Silverman, 1984 ; Fuqua et al., 1994 ; Miller and Bassler, 2001 ). Our first question was: Does quorum sensing occur in the larval midgut?

Quorum sensing is mediated by small molecules called homoserine lactones. Some thought it was impossible that these molecules—and therefore quorum sensing—could be viable in the lepidopteran (moth and butterfly) midgut, because it is an extremely alkaline environment, typically reaching pH 10 to 12, a pH expected to destroy lactones. Nevertheless, we found that quorum sensing did indeed occur in the gut.

To determine whether a molecular signal was being exchanged between cells in the bacterial community, we used a simple luminescence-based reporter system, described in Figure 4-6 . This reporter system enabled us to visualize the impact of homoserine lactone signal exchange among cells within the larval gut. We transformed bacterial cells in the midgut with biosensors that emit light only if they receive quorum-sensing signals sent by another cell. We detected luminescence in whole, living, larvae: clear evidence that the members of the gut bacterial community are communicating with each other in the highly alkaline gut.

Detection of quorum-sensing activity and signal exchange in the guts of cabbage white butterfly larvae. Bioluminescence was detected in the individual guts of larvae fed Pantoea pSB401 (top row), Pantoea mixed with Pantoea panI ::Tn 5 pSB401 (middle row) (more...)

What is the biological role of these signals in the insect midgut? We asked whether quorum-sensing signals that are required for Pseudomonas aeruginosa infection in other systems are also required in the cabbage white larval gut system; as Figure 4-7 illustrates, the answer was a resounding “yes.” We then demonstrated that we could chemically inhibit the larval gut quorum-sensing system using a known quorum-sensing inhibitor synthesized in the laboratory of our colleague at the University of Wisconsin, Helen Blackwell. Both approaches indicated that quorum-sensing is required for the virulence of P. aeruginosa in the larval gut. Furthermore, these results suggest that the gut microbes are, in fact, acting as a community and are not merely coexisting.

Mortality of cabbage white butterfly larvae fed P. aeruginosa strains and the quorum-sensing analog indole inhibitor. Treatments include P. aeruginosa PAO1, P. aeruginosa PAO1 and indole inhibitor, P. aeruginosa PAO1-JP2 ( lasI rhlI N -acyl-L-lomoserine (more...)

Does the Community Affect the Health of the Host?

Bacillus thuringiensis (Bt) is a well-characterized pathogen of gypsy moth and, in fact, of all members of the order Lepidoptera. The bacterium produces rhomboid protein crystals that are highly toxic only to lepidopterans. The Bt toxin is known to resemble bacterial pore-forming toxins that affect mammals; thus, we are interested in exploring the interaction of Bt, its toxin, and the microbial community in which they function as a model for mammalian gut disease.

Bt has been used widely for the last 50 years to control insect pests that cannot be deterred or eradicated by other means, and in organic agriculture in lieu of synthetic pesticides. Interestingly, despite this broad usage, resistance to Bt has not developed in the field. Over the last decade, crop plants have been genetically engineered to express the Bt toxin gene and cultivated according to strict guidelines designed to prevent the development of resistance to the toxin. Although Bt toxin-expressing crops are now grown on a massive scale in the United States, resistance has not become a problem. Why this is the case, even though antibiotic resistance is rampant, is not known.

Bt was discovered nearly 100 years ago, and both the bacterium and the interaction between its toxin and the cells of the lepidopteran gut epithelium—where the toxin forms pores—have been very well studied. However, the pathway by which pore formation (which damages the intestinal epithelium) leads to the animal’s death is not known. It has been assumed that either bacteremia caused by Bt or starvation (because pore formation is associated with reduced feeding) is the proximal cause of death.

Neither of those putative mechanisms satisfied one of my graduate students, Nichole Broderick, whose research had shown that some compounds known to enhance insects’ sensitivity to Bt toxin also affect the bacterial community of the gut. As a result, she developed the hypothesis that the gut microbial community acts as a protection—a phalanx—against infection by Bt, and she predicted that eliminating the gut bacteria would enhance Bt activity.

To test this hypothesis, we treated insects with increasing concentrations of an antibiotic cocktail that we knew would kill all of the bacteria that we could detect in the gut ( Broderick et al., 2006 ). However, antibiotic treatment did not enhance Bt killing; instead, the more antibiotic the larvae received in their diet, the greater the larval survival following exposure to Bt ( Figure 4-8 ). That result led us to the traitor hypothesis: if Bt is inactive in the absence of the endogenous microbial community, then perhaps community members actually collaborate with Bt in a multispecies infection. If so, one would predict that if we introduced members of the gut bacterial community into antibiotic-treated larvae that lacked a gut flora and were resistant to Bt, sensitivity to Bt would be restored.

Gypsy moth larvae reared on antibiotics are not susceptible to Bt. The vertical line indicates the concentration of antibiotic at which no bacteria are detected in the midgut. IU = international units. SOURCE: Broderick et al. (2006).

We tested this hypothesis by rearing larvae on high levels of the antibiotic cocktail ( Figure 4-9 ), letting the antibiotic clear, then feeding the larvae with Enterobacter , a gram-negative bacterium that we have found consistently in the gypsy moth gut; we then assessed the susceptibility of these larvae to Bt ( Broderick et al., 2004 ). In control experiments with larvae raised without antibiotics, Bt and Bt plus Enterobacter both kill nearly 100 percent of the larvae ( Broderick et al., 2006 ). Antibiotics reduced killing by Bt, and the addition of Enterobacter prior to Bt exposure restored the killing. This suggests that Enterobacter and Bt work together to kill the insects.

Restoration of B. thuringiensis toxicity by an Enterobacter spp. after elimination of the detectable gut flora and B. thuringiensis activity by antibiotics. Lymantria dispar larvae were reared until the third instar on a sterile artificial diet amended (more...)

Our work with the lepidopteran gut system is at an early stage. We have learned that the microbial assemblage it contains is relatively simple, comprising two phyla that are also found among the human gut microbiota. Chemical signals are exchanged between bacterial cells in the gut, and when this signaling process is inhibited chemically or genetically, the pathogenesis of P. aeruginosa is attenuated.

The microbial community affects host health. When the pathogen Bt is introduced, the normally benign gut microbiota mediate pathogenesis. In the absence of the normal gut microbiota, Bt does not induce killing and reintroduction of normal gut residents restores killing. This system might provide a model for studying the common phenomenon that commensal microbes can act as pathogens under the right conditions.

The insect gut system and the soil metagenomic analysis of antibiotic resistance genes both reveal the importance of accounting for communities in the analysis of microbial behavior and genetic potential in biological systems.

Davies References

• Abraham EP, Chain E. An enzyme from bacteria able to destroy penicillin. Nature. 1940; 146 (3713):837. [ PubMed : 3055168 ]
• Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. 2005; 307 (5717):1915–1920. [ PubMed : 15790844 ]
• Bartoloni A, Pallecchi L, Rodriguez H, Fernandez C, Mantella A, Bartalesi F, Strohmeyer M, Kristiansson C, Gotuzzo E, Paradisi F, Rossolini GM. Antibiotic resistance in a very remote Amazonas community. International Journal of Antimicrobial Agents. 2009; 33 (2):125–129. [ PubMed : 18947984 ]
• Dantas G, Sommer MOA, Oluwasegun RD, Church GM. Bacteria subsisting on antibiotics. Science. 2008; 320 (5872):100–103. [ PubMed : 18388292 ]
• Davies J. Are antibiotics naturally antibiotics? Journal of Industrial Microbiology and Bio-technology. 2006; 33 (7):496–499. [ PubMed : 16552582 ]
• Davies J, Spiegelman GB, Yim G. The world of subinhibitory antibiotic concentrations. Current Opinion in Microbiology. 2006; 9 (5):1–9. [ PubMed : 16942902 ]
• D’Costa VM, McGrann KM, Hughes DW, Wright GD. Sampling the antibiotic resistome. Science. 2006; 311 (5759):374–377. [ PubMed : 16424339 ]
• Gardner P, Smith DH, Beer H, Moellering, Jr RC. Recovery of resistance (R) factors from a drug-free community. Lancet. 1969; 2 (7624):774–776. [ PubMed : 4186024 ]
• Gorby YA, Yanina S, McLean JS, Rosso KM, Moyles D, Dohnalkova A, Beveridge TJ, Chang IS, Kim BH, Kim KS, Culley DE, Reed SB, Romine MF, Saffarini DA, Hill EA, Shi L, Elias DA, Kennedy DW, Pinchuk G, Watanabe K, Ishii S, Logan B, Nealson KH, Fredrickson JK. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proceedings of the National Academy of Sciences. 2006; 103 (30):11358–11363. [ PMC free article : PMC1544091 ] [ PubMed : 16849424 ]
• Hughes VM, Datta N. Conjugative plasmids in bacteria of the “pre-antibiotic” era. Nature. 1983; 302 (5910):725–726. [ PubMed : 6835408 ]
• IOM (Institute of Medicine). Ending the war metaphor: the changing agenda for unraveling the host-microbe relationship. Washington, DC: The National Academies Press; 2006. [ PubMed : 21850787 ]
• Koenig JE, Boucher Y, Charlebois RL, Nesbo C, Zhaxybayeva O, Bapteste E, Spencer M, Joss MJ, Stokes HW, Doolittle WF. Integron-associated gene cassettes in Halifax Harbour: assessment of a mobile gene pool in marine sediments. Environmental Microbiology. 2008; 10 (4):1024–1038. [ PubMed : 18190517 ]
• Lederberg J. Medical science, infectious disease and the unity of humankind. Journal of the American Medical Association. 1988; 260 (5):684–685. [ PubMed : 3392795 ]
• Livermore DM, Canton R, Gniadkowski M, Nordmann P, Rossolini GM, Arlet G, Ayala J, Coque TM, Kern-Zdanowicz I, Luzzaro F, Poirel L, Woodford N. CTX-M: changing the face of ESBLs in Europe. Journal of Antimicrobial Chemotherapy. 2007; 59 (2):165–174. [ PubMed : 17158117 ]
• Nandi S, Maurer JJ, Hofacre C, Summers AO. Gram-positive bacteria are a major reservoir of Class 1 antibiotic resistance integrons in poultry litter. Proceedings of the National Academy of Sciences. 2004; 101 (18):7118–7122. [ PMC free article : PMC406475 ] [ PubMed : 15107498 ]
• Norrby SR, Nord CE, Finch R. European Society of Clinical Microbiology and Infectious Diseases. Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infectious Diseases. 2005; 5 (2):115–119. [ PubMed : 15680781 ]
• Pallecchi L, Lucchetti C, Bartoloni A, Bartalesi F, Mantella A, Gamboa H, Carattoli A, Paradisi F, Rossolini GM. Population structure and resistance genes in antibiotic-resistant bacteria from a remote community with minimal antibiotic exposure. Antimicrobial Agents and Chemotherapy. 2007; 51 (4):1179–1184. [ PMC free article : PMC1855465 ] [ PubMed : 17220407 ]
• Peleg A, Seifert H, Paterson DL. Acinetobacter baumannii : emergence of a successful pathogen. Clinical Microbiology Reviews. 2008; 21 (3):538–582. [ PMC free article : PMC2493088 ] [ PubMed : 18625687 ]
• Queenan AM, Bush K. Carbapenemases: the versatile β -lactamases. Clinical Microbiology Reviews. 2007; 20 (3):440–458. [ PMC free article : PMC1932750 ] [ PubMed : 17630334 ]
• Quince C, Curtis TP, Sloan WT. The rational exploration of microbial diversity. The ISME Journal. 2008; 2 (10):997–1006. [ PubMed : 18650928 ]
• Riesenfeld CS, Goodman RM, Handelsman J. Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Environmental Microbiology. 2004; 6 (9):981–989. [ PubMed : 15305923 ]
• Schlüter A, Szczepanowski R, Pühler A, Top EM. Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plant provides evidence for a widely accessible drug resistance gene pool. FEMS Microbiology Reviews. 2007; 31 (4):449–477. [ PubMed : 17553065 ]
• Smith DH. R factor infection of Escherichia coli lyophilized in 1946. Journal of Bacteriology. 1967; 94 (6):2071–2072. [ PMC free article : PMC276944 ] [ PubMed : 4864414 ]
• Spellberg B, Guidos R, Gilbert D, Bradley J, Boucher HW, Scheld WM, Bartlett JG, Edwards J. the Infectious Diseases Society of America. The epidemic of antibiotic-resistant infections: a call to action for the medical community from the Infectious Diseases Society of America. Clinical Infectious Diseases. 2008; 46 (2):155–164. [ PubMed : 18171244 ]
• Tamae C, Liu A, Kim K, Sitz D, Hong J, Becket E, Bui A, Solaimani P, Tran KP, Yang H, Miller JH. Determination of antibiotic hypersensitivity among 4,000 single-gene-knockout mutants of Escherichia coli . Journal of Bacteriology. 2008; 190 (17):5981–5988. [ PMC free article : PMC2519525 ] [ PubMed : 18621901 ]
• Tennstedt T, Szczepanowski R, Krahn I, Pühler A, Schlüter A. Sequence of the 68,869 bp IncP-1alpha plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, a Tn 402 -like integron and other transposable elements. Plasmid. 2005; 53 (3):218–238. [ PubMed : 15848226 ]
• Yim G, Huimi Wang H, Davies J. The truth about antibiotics. International Journal of Medical Microbiology. 2006; 296 (2–3):163–170. [ PubMed : 16503195 ]
• Waksman SA. The role of antibiotics in nature. Perspectives in Biology and Medicine. 1961; 4 (3):271–286.

Cohen References

• Avery OT, MacLeod CM, McCarty M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Journal of Experimental Medicine. 1944; 79 :137–158. [ PMC free article : PMC2135445 ] [ PubMed : 19871359 ]
• Babst M, Odorizzi G, Estepa EJ, Emr SD. Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic. 2000; 1 (3):248–258. [ PubMed : 11208108 ]
• Beutler B. Toll-like receptors and their place in immunology. Where does the immune response to infection begin? Nature Reviews Immunology. 2004; 4 (7):498. [ PubMed : 18293536 ]
• Bishop N, Woodman P. Tsg101/mammalian vps23 and mammalian vps28 interact directly and are recruited to vps4-induced endosomes. Journal of Biological Chemistry. 2001; 276 (15):11735–11742. [ PubMed : 11134028 ]
• Bishop RA. Graduate School of Biomedical Sciences. Houston: University of Texas Health Science Center; 2003. [accessed July 31, 2008]. The history of bubonic plague. http://dpalm ​.med.uth ​.tmc.edu/courses/BT2003 ​/BTstudents2003_files%5CPlague2003 ​.htm .
• Bonavia A, Santos D, Bamba D, Li L, Gu J, Kinch M, Goldblatt M. Recruitment of the TSG101/ESCRT-1 machinery in host cells by influenza virus: implications for broad spectrum therapy. Abstract W9-2 for the 27th Annual Meeting of the American Society for Virology; July 12–16, 2008; Ithaca, NY. 2008. p. 91.
• Cherry S, Doukas T, Armknecht S, Whelan S, Wang H, Sarnow P, Perrimon N. Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition. Genes and Development. 2005; 19 (4):445–452. [ PMC free article : PMC548945 ] [ PubMed : 15713840 ]
• Cohen SN. Bacterial plasmids: their extraordinary contribution to molecular genetics. Gene. 1993; 135 (1–2):67–76. [ PubMed : 8276280 ]
• Collier RJ, Young JAT. Anthrax toxin. Annual Review of Cell and Developmental Biology. 2003; 19 :45–70. [ PubMed : 14570563 ]
• Dunn SJ, Khan IH, Chan UA, Scearce RL, Melara CL, Paul AM, Sharma V, Bih F.-Y, Holzmayer TA, Luciw PA, Abo A. Identification of cell surface targets for HIV-1 therapeutics using genetic screens. Virology. 2004; 321 (2):260–273. [ PubMed : 15051386 ]
• Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, Wettstein DA, Stray KM, Cote M, Rich RL, Myszka DG, Sundquist WI. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell. 2001; 107 (1):55–65. [ PubMed : 11595185 ]
• Goff A, Ehrlich LS, Cohen SN, Carter CA. Tsg101 control of human immunodeficiency virus type 1 Gag trafficking and release. Journal of Virology. 2003; 77 (17):9173–9182. [ PMC free article : PMC187429 ] [ PubMed : 12915533 ]
• Griffith F. The significance of pneumococcal types. Journal of Hygiene. 1928; 27 :113–159. [ PMC free article : PMC2167760 ] [ PubMed : 20474956 ]
• Hao L, Sakurai A, Watanabe T, Sorensen E, Nidom CA, Newton MA, Ahlquist P, Kawaoka Y. Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature. 2008; 454 (7206):890–893. [ PMC free article : PMC2574945 ] [ PubMed : 18615016 ]
• He X, Semenov M, Tamai K, Zeng X. LDL receptor-related proteins 5 and 6 in wnt/beta-catenin signaling: arrows point the way. Development. 2004; 131 (8):1663–1677. [ PubMed : 15084453 ]
• Ishii KJ, Koyama S, Nakagawa A, Coban C, Akira S. Host innate immune receptors and beyond: making sense of microbial infections. Cell Host and Microbe. 2008; 3 (6):352–363. [ PubMed : 18541212 ]
• Katzmann DJ, Babst M, Emr SD. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, escrt-i. Cell. 2001; 106 (2):145–155. [ PubMed : 11511343 ]
• Koonin EV, Abagyan RA. TSG101 may be the prototype of a class of dominant negative ubiquitin regulators. Nature Genetics. 1997; 16 (4):330–331. [ PubMed : 9241264 ]
• Lederberg J. Cell genetics and hereditary symbiosis. Physiological Reviews. 1952; 32 (4):403–430. [ PubMed : 13003535 ]
• Lederberg J. AIDS pandemic provokes alarming reassessments of infectious disease. The Scientist. 1993; 7 (14):11.
• Lederberg J, Lederberg EM. Replica plating and indirect selection of bacterial mutants. Journal of Bacteriology. 1952; 63 (3):399–406. [ PMC free article : PMC169282 ] [ PubMed : 14927572 ]
• Li L, Cohen SN. Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells. Cell. 1996; 85 (3):319–329. [ PubMed : 8616888 ]
• Lu Q, Hope LW, Brasch M, Reinhard C, Cohen SN. TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation. Proceedings of the National Academy of Sciences. 2003; 100 (13):7626–7631. [ PMC free article : PMC164637 ] [ PubMed : 12802020 ]
• Lu Q, Wei W, Kowalski PE, Chang AC, Cohen SN. EST-based genome-wide gene inactivation identifies ARAP3 as a host protein affecting cellular susceptibility to anthrax toxin. Proceedings of the National Academy of Sciences. 2004; 101 (49):17246–17251. [ PMC free article : PMC534609 ] [ PubMed : 15569923 ]
• Martin-Serrano J, Zang T, Bieniasz PD. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nature Medicine. 2001; 7 (12):1313–1319. [ PubMed : 11726971 ]
• Ponting CP, Cai YD, Bork P. The breast cancer gene product Tsg101: a regulator of ubiquitination? Journal of Molecular Medicine. 1997; 75 (7):467–469. [ PubMed : 9253709 ]
• Raiborg C, Stenmark H. Hrs and endocytic sorting of ubiquitinated membrane proteins. Cell Structure and Function. 2002; 27 (6):403–408. [ PubMed : 12576633 ]
• Riedel S. Edward Jenner and the history of smallpox and vaccination. Proceedings (Baylor University Medical Center). 2005; 18 (1):21–25. [ PMC free article : PMC1200696 ] [ PubMed : 16200144 ]
• Tavassoli A, Lu Q, Gam J, Pan H, Benkovic SJ, Cohen SN. Inhibition of HIV budding by a genetically selected cyclic peptide targeting the Gag-Tsg101 interaction. ACS Chemical Biology. 2008; 3 (12):757–764. [ PubMed : 19053244 ]
• VerPlank L, Bouamr F, LaGrassa TJ, Agresta B, Kikonyogo A, Leis J, Carter CA. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proceedings of the National Academy of Sciences. 2001; 98 (14):7724–7729. [ PMC free article : PMC35409 ] [ PubMed : 11427703 ]
• Wei W, Lu Q, Chaudry GJ, Leppla SH, Cohen SN. The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin. Cell. 2006; 124 (6):1141–1154. [ PubMed : 16564009 ]
• Wheelis M. Biological warfare at the 1346 siege of Caffa. Emerging Infectious Diseases. 2002; 8 (9):971–975. [ PMC free article : PMC2732530 ] [ PubMed : 12194776 ]

Handelsman References

• Allen HK, Moe LA, Rodbumrer J, Gaarder A, Handelsman J. Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil. The ISME Journal. 2009; 3 (2):243–251. [ PubMed : 18843302 ]
• Borlee BR, Geske GD, Robinson CJ, Blackwell HE, Handelsman J. Quorum-sensing signals in the microbial community of the cabbage white butterfly larval midgut. The ISME Journal. 2008; 2 (11):1101–1111. [ PMC free article : PMC3904650 ] [ PubMed : 18650927 ]
• Bregman E, Kirsner JB. Amino acids of colon and rectum. Possible involvement of diaminopimelic acid of intestinal bacteria in antigenicity of ulcerative colitis colon. Proceedings of the Society for Experimental Biology and Medicine. 1965; 118 :727–731. [ PubMed : 14264542 ]
• Broderick NA, Raffa KF, Goodman RM, Handelsman J. Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods. Applied Environmental Microbiology. 2004; 70 (1):293–300. [ PMC free article : PMC321235 ] [ PubMed : 14711655 ]
• Broderick NA, Raffa KF, Handelsman J. Midgut bacteria required for Bacillus thuringiensis insecticidal activity. Proceedings of the National Academy of Sciences. 2006; 103 (41):15196–15199. [ PMC free article : PMC1622799 ] [ PubMed : 17005725 ]
• Engebrecht J, Silverman M. Identification of genes and gene products necessary for bacterial bioluminescence. Proceedings of the National Academy of Sciences. 1984; 81 (13):4154–4158. [ PMC free article : PMC345387 ] [ PubMed : 6377310 ]
• Fleming TJ, Wallsmith DE, Rosenthal RS. Arthropathic properties of gonococcal peptidoglycan fragments: implications for the pathogenesis of disseminated gonococcal disease. Infection and Immunity. 1986; 52 (2):600–608. [ PMC free article : PMC261043 ] [ PubMed : 3084386 ]
• Fuqua WC, Winans SC, Greenberg EP. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. Journal of Bacteriology. 1994; 176 (2):269–275. [ PMC free article : PMC205046 ] [ PubMed : 8288518 ]
• Hirayama K, Rafter J. The role of lactic acid bacteria in colon cancer prevention: mechanistic considerations. Antonie van Leeuwenhoek. 1999; 76 (1–4):391–394. [ PubMed : 10532395 ]
• Hooper LV. Bacterial contributions to mammalian gut development. Trends in Microbiology. 2004; 12 (3):129–134. [ PubMed : 15001189 ]
• Hooper LV, Gordon JI. Commensal host-bacterial relationships in the gut. Science. 2001; 292 (5519):1115–1118. [ PubMed : 11352068 ]
• Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science. 2001; 291 (5505):881–884. [ PubMed : 11157169 ]
• Hooper LV, Midtvedt T, Gordon JI. How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annual Review of Nutrition. 2002; 22 :283–307. [ PubMed : 12055347 ]
• Johannsen L, Toth LA, Rosenthal RS, Opp MR, Obal F Jr, Cady AB, Kreuger JM. Somnogenic, pyrogenic, and hematologic effects of bacterial peptidoglycan. American Journal of Physiology. 1990; 258 (1 Pt. 2):R182–186. [ PubMed : 2105668 ]
• Krueger JM, Pappenheimer JR, Karnovsky ML. Sleep-promoting effects of muramyl peptides. Proceedings of the National Academy of Sciences. 1982; 79 (19):6102–6106. [ PMC free article : PMC347061 ] [ PubMed : 6964403 ]
• Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature. 2006; 444 (7122):1022–1023. [ PubMed : 17183309 ]
• Miller MB, Bassler BL. Quorum sensing in bacteria. Annual Review of Microbiology. 2001; 55 :165–199. [ PubMed : 11544353 ]
• Rath HC, Herfarth HH, Ikeda JS, Grenther WB, Hamm TE Jr, Balish E, Tauroq JD, Hammer RE, Wilson KH, Sartor RB. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human β 2 microglobulin transgenic rats. Journal of Clinical Investigation. 1996; 98 (4):945–953. [ PMC free article : PMC507509 ] [ PubMed : 8770866 ]
• Schloss PD, Handelsman J. Toward a census of bacteria in soil. PLoS Computational Biology. 2006; 2 (7):e92. [ PMC free article : PMC1513271 ] [ PubMed : 16848637 ]

Emeritus professor of microbiology and immunology.

Accurate production figures are difficult to obtain.

MRSA is a type of S. aureus that is resistant to antibiotics called β -lactams. β -lactam antibiotics include methicillin and other more common antibiotics such as oxacillin, penicillin, and amoxicillin (for more information, see http://www ​.cdc.gov/ncidod ​/dhqp/ar_MRSA_ca_public.html#2 ).

VISA and vancomycin-resistant S. aureus (VRSA) are specific types of antimicrobial-resistant staphylococcal bacteria. Although most staphylococci are susceptible to the antimicrobial agent vancomycin, some have developed resistance to vancomycin. Infections caused by VISA and VRSA isolates cannot be successfully treated with vancomycin because these organisms are no longer responsive to vancomycin. However, to date, all VISA and VRSA isolates have been susceptible to other Food and Drug Administration (FDA)-approved drugs (for more information, see http://www ​.cdc.gov/ncidod ​/dhqp/ar_visavrsa_FAQ.html ).

MDR-TB is tuberculosis that is resistant to at least two of the best anti-tuberculosis drugs, isoniazid and rifampin. These drugs are considered first-line drugs and are used to treat all individuals with tuberculosis (for more information, see http://www ​.cdc.gov/tb ​/pubs/tbfactsheets/mdrtb.htm ).

XDR-TB is a relatively rare type of MDR-TB. XDR-TB is defined an M. tuberculosis isolate that is resistant to isoniazid and rifampin plus any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin; for more information see http://www ​.cdc.gov/tb ​/pubs/tbfactsheets/mdrtb.htm ).

Producing more than one effect; having multiple phenotypic expressions (see http://www ​.merriam-webster ​.com/dictionary/pleiotropic ).

guanine-cytosine.

Hormesis defines a dose-response activity of antibiotics (and other agents). It usually refers to a positive (beneficial) effect at low concentrations and a negative (toxic or inhibitory) effect at higher concentrations ( Yim et al., 2006 ).

For this reason, treatment with compounds with a cidal action is favored for patients who are immunosuppressed or in cases of critical infections caused by highly virulent organisms.

Microcins are essentially bacteriocins that contain a smaller number of amino acids.

Bacteriocins are bacterially produced, small, heat-stable peptides that are active against other bacteria and to which the producer has a specific mechanism of immunity. Bacteriocins can have a narrow or a broad target spectrum (see http://www ​.nature.com ​/nrmicro/journal/v3 ​/n10/glossary/nrmicro1273_glossary.html ).

Kwoh-Ting Li Professor in the School of Medicine; Professor of Genetics and Professor of Medicine.

The Black Death killed an estimated 43 million people worldwide, including 25 million people in Europe (one-third of the population); devastated commerce; and led to social upheavals ( Bishop, 2003 ).

An agency of the U.S. Department of Defense.

Howard Hughes Medical Institute Professor, Department of Bacteriology.

See Bregman and Kirsner (1965) , Fleming et al. (1986) , Hirayama and Rafter (1999) , Hooper (2004) , Hooper and Gordon (2001) , Hooper et al. (2001 , 2002) , Johannsen et al. (1990) , Krueger et al. (1982) , Ley et al. (2006) , and Rath et al. (1996) .

• Cite this Page Institute of Medicine (US) Forum on Microbial Threats. Microbial Evolution and Co-Adaptation: A Tribute to the Life and Scientific Legacies of Joshua Lederberg: Workshop Summary. Washington (DC): National Academies Press (US); 2009. 4, Antibiotic Resistance: Origins and Countermeasures.
• PDF version of this title (21M)

In this Page

Other titles in this collection.

• The National Academies Collection: Reports funded by National Institutes of Health

Related information

• PMC PubMed Central citations
• PubMed Links to PubMed

Recent Activity

• Antibiotic Resistance: Origins and Countermeasures - Microbial Evolution and Co-... Antibiotic Resistance: Origins and Countermeasures - Microbial Evolution and Co-Adaptation

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

Connect with NLM

National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894

Web Policies FOIA HHS Vulnerability Disclosure

Help Accessibility Careers

The rockefeller university

1958 nobel prize in physiology or medicine.

By the mid-20th century, science had established several crucial facts about the capabilities of bacteria, a seemingly primitive, unicellular organism first classified in 1676. Some bacteria, scientists understood, can cause life-threatening disease; some are resistant to even the strongest antibiotics; and some that are neither virulent nor resistant to begin with can gain both virulence and resistance. The question of how bacteria accomplish such sleight of hand, which had been subject to decades of logical but inaccurate speculation, was resolved by a 22-year-old graduate student in 1947. Joshua Lederberg, Rockefeller University’s fifth president, won a share of the 1958 Nobel Prize in Physiology or Medicine for his discoveries of genetic transfer in bacteria.

Through the 1940s, scientific wisdom had it that bacteria do not have genetic mechanisms similar to those of higher organisms. The prevailing hypothesis, taught in Dr. Lederberg’s Columbia University medical school classes, classed bacteria with schizomycetes, organisms that reproduce by cloning. The 1944 discovery of Rockefeller scientists Oswald T. Avery, Maclyn McCarty and Colin MacLeod that deoxyribonucleic acid, or DNA, is the genetic material in Pneumococcus proved that bacteria have genes and thus drew an unexpected parallel between bacteria and higher organisms. But their discovery, unconnected to a method of proliferation, was met with widespread skepticism. Inspired by the new evidence, Dr. Lederberg interrupted medical school to pursue experimental genetics with Edward L. Tatum, the Yale University chemist with whom he would later share the Nobel Prize.

Initial experiments with the intestinal bacteria Escherichia coli led Dr. Lederberg to estimate that only one in 20 strains are fertile, and that if bacteria mate, they do so only during a particular phase of their life cycle. After crossing two strains of E. coli, each with different mutations for nutritional deficiencies, he found that some of the offspring of each strain had regained the ability to produce the nutrients its parent could not. When that ability continued to be inherited by successive generations, Dr. Lederberg had effectively proved the textbooks wrong. He named the bacterial mating process conjugation, received his Ph.D. for this research and officially left medical school to continue in bacterial genetics. We now understand that bacterial mating occurs only through cell-to-cell contact, when a bridge is formed between the two cells that transports genetic information from the donor cell to the recipient.

Dr. Lederberg’s experiments also identified E. coli as a haploid that carries only a single chromosome and suggested that conjugation is a form of unequal horizontal gene transfer: Rather than exchanging genes equally, the mating bacteria transfer partial genetic material from one parent to the other. He also developed a technique that allowed for the identification of antibiotic- or bacteriophage-resistant strains without exposing the bacteria to the phage or the drug, and proved that resistance is a genetic mutation rather than an adaptation.

Following his seminal research at Yale, Dr. Lederberg accepted a position to chair the newly founded department of genetics at the University of Wisconsin, Madison. With his graduate student Norton Zinder — later a colleague at Rockefeller University — Dr. Lederberg showed that bacteriophages can transfer genetic information between cells in Salmonella. The process, which they named transduction, was the first demonstration that it is possible to introduce new genes into an organism and in other ways manipulate its genetic material. The discovery explained how different species of bacteria can so quickly gain resistance to the same antibiotic.

The scientific contributions of Dr. Lederberg’s pathbreaking foray into bacterial genetics are legion. His work gave scientists an experimental model whose simplicity and rapid growth made it ideal for genetic studies. His description of bacterial conjugation led directly to the distinction denoted since 1962 by the terms prokaryotic and eukaryotic. His findings led to research that elucidated the mechanisms of bacteriophages and other viruses; explained how cell growth is interrupted; and clarified how cancer progresses. And his description of transduction led to the development of gene therapy and contributed to the boom in biotechnology and genetic engineering in the 1970s. Dr. Lederberg received half of the 1958 Nobel Prize “for his discoveries concerning genetic recombination and the organization of the genetic material of bacteria. Dr. Tatum and his colleague George Wells Beadle received the second half of the 1958 prize “for their discovery that genes act by regulating definite chemical events.”

Born in 1925 and raised in New York City, Dr. Lederberg received his Ph.D. from Yale University in 1947 and then joined the University of Wisconsin, Madison, where he founded the department of medical genetics 10 years later. In 1959, he moved to Stanford University, where he was chair of the newly established department of genetics. There he also expanded his research into the fields of artificial intelligence and exobiology. In 1978, Dr. Lederberg became the fifth president of The Rockefeller University, a position he held until 1990, when he retired from the presidency and became University Professor and head of the Laboratory of Molecular Genetics and Informatics, where his research continued. Throughout his later research career, Dr. Lederberg was highly active in international science and human rights advocacy, serving as a public policy adviser to nine United States presidential administrations and authoring a weekly Washington Post column, “Science and Man,” for six years. He was a member of the National Academy of Sciences and a foreign member of The Royal Society. In addition to the Nobel Prize, he received the National Medal of Science and the Presidential Medal of Freedom. He died in New York in 2008.

Recent News

Is metastasis a hereditary disorder, researchers discover a genetic predisposition increasing the risk of breast cancer metastasis, new center for ambitious neuroscience projects comes to campus.

• View all news
• Nobel Prize 20/26

American Society for Microbiology

Esther lederberg and the rise of microbial genetics.

Oct. 4, 2023

Scientific Beginnings

Lederberg found a little lambda.

The Nobel Laureate's Wife

And Yet, She Persisted

Out of the shadows.

• Undergraduate Student
• Escherichia coli
• Graduate Student
• Molecular Biology
• Molecular Biology & Physiology

Author: Madeline Barron, Ph.D.

Share Your Research at ASM Microbe 2025

Discover asm membership, get published in an asm journal.

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals
• Explore content
• About the journal
• Publish with us
• Sign up for alerts
• Published: 18 December 1986

Forty years of genetic recombination in bacteria

• Joshua Lederberg 1   na1  

Nature volume  324 ,  pages 627–628 ( 1986 ) Cite this article

402 Accesses

19 Citations

Metrics details

Between April and June 1946, Joshua Lederberg and Edward L. Tatum carried out a series of experiments that proved that bacteria can exchange their genes by sexual crossings. The experiments were reported in Nature just 40 years ago 1 . In the following pair of articles, Joshua Lederberg first provides a personal reminiscence of the circumstances of the discovery and then, together with Harriet Zuckerman, considers it as a possible case of ‘postmature’ scientific discovery.

This is a preview of subscription content, access via your institution

Access options

Subscribe to this journal

Receive 51 print issues and online access

185,98 € per year

only 3,65 € per issue

Buy this article

• Purchase on SpringerLink
• Instant access to full article PDF

Prices may be subject to local taxes which are calculated during checkout

Lederberg, J. & Tatum, E.L. Nature 158 , 558 (1946).

Article   ADS   CAS   Google Scholar  

Beadle, G.W. & Tatum, E.L. Proc. natn. Acad. Sci. U.S.A. 27 , 499 – 506 (1941).

Avery, O.T., MacLeod, C.M. & McCarty, M. J. exp. Med. 79 , 137 – 158 (1944).

Article   CAS   Google Scholar  

Ryan, F.J. & Lederberg, J. Proc. natn. Acad. Sci. U.S.A. 32 , 163–173 (1946).

Article   ADS   Google Scholar  

Dubos, R. The Bacterial Cell (Harvard, Cambridge, 1945).

Google Scholar  

Winge & Lausten, O. C.R. Lab. Carlsberg, Ser, physiol. , 22 , 99–119, (1937).

Sherman, J.M. & Wing, H.U. J. Bact. 33 , 315 – 321 (1937).

CAS   PubMed   Google Scholar  

Gowen, J.W. & Lincoln, R.E. J. Bact. 44 , 551 – 554 (1942).

Gray, C.H. & Tatum, E.L. Proc. natn. Acad. Sci. U.S.A. 30 , 404–410 (1944).

Lederberg, J. in University on the Heights , (ed. First, W.) 105 – 109. (Doubleday, Garden City, New York, 1969).

Lederberg, J. A. Rev. Genet. 13 , 1–5 (1979).

Lederberg, J. Science 114 , 68 – 69 (1951).

Tatum, E.L. & Bonner, D.M. Proc. natn. Acad. Sci. U.S.A. 30 , 30 – 37 (1944).

Bachmann, B.J. Microb. Revs. 47 , 180 – 230 (1983).

CAS   Google Scholar  

Download references

Author information

Joshua Lederberg: The research summarized in this article was supported in 1946 by a fellowship of the Jane Coffin Childs Fund for Medical Research.

Authors and Affiliations

Rockefeller University, New York, New York, 10021

Joshua Lederberg

You can also search for this author in PubMed   Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article.

Lederberg, J. Forty years of genetic recombination in bacteria. Nature 324 , 627–628 (1986). https://doi.org/10.1038/324627a0

Download citation

Issue Date : 18 December 1986

DOI : https://doi.org/10.1038/324627a0

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

This article is cited by

How the choice of experimental organism matters: biological practices and discipline boundaries.

• Richard M. Burian

Synthese (1992)

Ethical and political problems in third world biotechnology

• Daniel J. Goldstein

Journal of Agricultural Ethics (1989)

Bacterial sex timely

• MURIEL LEDERMAN

Nature (1987)

Quick links

• Explore articles by subject
• Guide to authors
• Editorial policies

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Advertisement

Lederberg on bacterial recombination, Haldane, and cold war genetics: an interview

• Notes and Comments
• Published: 20 September 2014
• Volume 36 , pages 280–288, ( 2014 )

Cite this article

• Sahotra Sarkar 1  

218 Accesses

2 Citations

Explore all metrics

Joshua Lederberg (1925–2008) was one of the pioneers of molecular genetics perhaps best known for his discovery of genetic recombination in bacteria which earned him a Nobel Prize in 1958 (shared with George Beadle and Edward Tatum). Lederberg’s interests were broad including the origin of life, exobiology (a term that he coined) and emerging diseases and artificial intelligence in his later years. This article contains the transcription of an interview in excerpts, documenting the interactions between Lederberg and fellow biologist J.B.S. Haldane which lasted from 1946 until Haldane’s death in Kolkata (then Calcutta) in 1964.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save.

• Get 10 units per month
• Download Article/Chapter or eBook
• 1 Unit = 1 Article or 1 Chapter
• Cancel anytime

Price includes VAT (Russian Federation)

Instant access to the full article PDF.

Rent this article via DeepDyve

Institutional subscriptions

Similar content being viewed by others

Recombineering and MAGE

Lamarckian realities: the CRISPR-Cas system and beyond

For contemporary analysis of that controversy, see Sarkar ( 1990 , 1991b , 1992c ); for a reappraisal, see Sarkar ( 2004 ), Chap. 13.

Haldane had just married Helen Spurway (Clark 1968 ).

The reference is to Haldane’s unpublished paper on the Luria-Delbrück distribution (see Sect. 2 ).

This is the high variance of the Luria-Delbrück distribution—see Sect. 2 .

See Novick and Szilard ( 1950 , 1951 ).

See Haldane ( 1939 ); for comments, see Sarkar ( 1992d ) and Shapiro ( 1993 ).

Paul ( 1983 ) analyzes this episode in detail.

See Haldane ( 1949 ).

Biographical details on Lindegren is from Mortimer ( 1993 ).

Biographical details on Sonneborn are from Beale (1982).

Cairns, J., Overbaugh, J., & Miller, S. (1988). The origin of mutants. Nature, 335 , 142–145.

Cavalli-Sforza, L. L., & Lederberg, J. (1956). Isolation of preadaptive mutants through sib selection. Genetics, 41 , 367–381.

Google Scholar  

Clark, R. (1968). J.B.S.: The life and work of J.B.S. Haldane . London: Hodder and Stoughton.

Crow, J. F. (2008). Obituary: Joshua Lederberg, 1925–2008. Nature Genetics, 40 , 486.

Article   Google Scholar  

Haldane, J. B. S. (1932). The causes of evolution . London: Harper and Brothers.

Haldane, J. B. S. (1939). The Marxist philosophy and the sciences . New York: Random House.

Haldane, J. B. S. (1949). In defense of genetics. Modern Quarterly, 4 , 194–202.

Lea, D. E., & Coulson, C. A. (1949). The distribution of mutants in bacterial populations. Journal of Genetics, 49 , 264–285.

Lederberg, J., & Lederberg, E. M. (1952). Replica plating and indirect selection of bacterial mutants. Journal of Bacteriology, 63 , 399–406.

Lederberg, J., & Tatum, E. L. (1946). Gene recombination in Escherichia coli . Nature, 158 , 558.

Lindegren, C. C. (1949). The yeast cell, its genetics and cytology . St. Louis: Educational Press.

Lindegren, C. C. (1966). The cold war in biology . Ann Arbor: Planarian Press.

Luria, S. E., & Delbrück, M. (1943). Mutations of bacteria from virus sensitivity to virus resistance. Genetics, 28 , 491–511.

Ma, W. T., Sandri, G. V., & Sarkar, S. (1992). Analysis of the Luria-Delbrück distribution using discrete convolution powers. Journal of Applied Probability, 29 , 255–267.

Mortimer, R. K. (1993). Carl C. Lindegren: Iconoclastic Father of Neurospora and Yeast Genetics. In M. N. Hall & P. Linder (Eds.), The early days of yeast genetics (pp. 22–38). Plainview: Cold Spring Harbor Press.

Novick, A., & Szilard, L. (1950). Experiments with the chemostat on spontaneous mutations of bacteria. Proceedings of the National Academy of Sciences (USA), 36 , 708–719.

Novick, A., & Szilard, L. (1951). Experiments with on spontaneous and chemically induced mutations of bacteria growing in a chemostat. Cold Spring Harbor Symposia on Quantitative Biology, 16 , 337–343.

Paul, D. B. (1983). A war on two fronts: J.B.S. Haldane and the response to Lysenkoism in Britain. Journal of the History of Biology, 16 , 1–37.

Provine, W. B. (1971). The origins of theoretical population genetics . Chicago: University of Chicago Press.

Sarkar, S. (1990). On the possibility of directed mutations in bacteria: Statistical analyses and reductionist strategies. In A. Fine, M. Forbes, & L. Wessels (Eds.), PSA 1990 : Proceedings of the 1990 biennial meeting of the philosophy of science association (Vol. 1, pp. 111–124). East Lansing: Philosophy of Science Association.

Sarkar, S. (1991a). Haldane’s solution of the Luria-Delbrück distribution. Genetics, 127 , 257–261.

Sarkar, S. (1991b). Lamarck contre Darwin, reduction versus statistics: Conceptual issues in the controversy over directed mutagenesis in bacteria. In A. I. Tauber (Ed.), Organism and the origins of self (pp. 235–271). Dordrecht: Kluwer.

Chapter   Google Scholar  

Sarkar, S. (Ed.). (1992a). The founders of evolutionary genetics . Dordrecht: Kluwer.

Sarkar, S. (1992b). Haldane as Biochemist: The Cambridge Decade, 1923–1932. In S. Sarkar (Ed.), The founders of evolutionary genetics (pp. 53–81). Dordrecht: Kluwer.

Sarkar, S. (1992c). Neo-darwinism and the problem of directed mutations. Evolutionary Trends in Plants, 6 , 73–79.

Sarkar, S. (1992d). Science, philosophy, and politics in the work of J.B.S. Haldane, 1922–1937. Biology and Philosophy, 7 , 385–409.

Sarkar, S. (2004). Molecular models of life: Philosophical papers on molecular biology . Cambridge: MIT Press.

Sarkar, S. (2007). Haldane and the emergence of modern evolutionary theory. In M. Matthen & C. Stephens (Eds.) Handbook of the philosophy of science: Philosophy of biology (Vol. 3, pp. 49–86). New York: Elsevier.

Sarkar, S., Ma, W. T., & Sandri, Gv. (1992). On fluctuation analysis: A new, simple and efficient method for computing the expected number of mutants. Genetica, 85 , 173–179.

Shapiro, A. M. (1993). Haldane, Marxism, and the conduct of research. Quarterly Review of Biology, 68 , 69–77.

Download references

Author information

Authors and affiliations.

University of Texas at Austin, Austin, TX, USA

Sahotra Sarkar

You can also search for this author in PubMed   Google Scholar

Corresponding author

Correspondence to Sahotra Sarkar .

Rights and permissions

Reprints and permissions

About this article

Sarkar, S. Lederberg on bacterial recombination, Haldane, and cold war genetics: an interview. HPLS 36 , 280–288 (2014). https://doi.org/10.1007/s40656-014-0029-7

Download citation

Received : 06 December 2013

Accepted : 11 December 2013

Published : 20 September 2014

Issue Date : October 2014

DOI : https://doi.org/10.1007/s40656-014-0029-7

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Joshua Lederberg
• J.B.S. Haldane
• Helen Spurway
• Molecular genetics
• Find a journal
• Publish with us
• Track your research

Lederberg on bacterial recombination, Haldane, and cold war genetics: an interview

• PMID: 25515361
• DOI: 10.1007/s40656-014-0029-7

Joshua Lederberg (1925-2008), was one of the pioneers of molecular genetics perhaps best known for his discovery of genetic recombination in bacteria which earned him a Nobel Prize in 1958 (shared with George Beadle and Edward Tatum). Lederberg's interests were broad including the origin of life, exobiology (a term that he coined) and emerging diseases and artificial intelligence in his, later years. This article contains the transcription of an interview in excerpts, docu- menting the interactions between Lederberg and fellow biologist J.B.S. Haldane wlich lasted from 1946 until Haldane's death in Kolkata (then Calcutta) in 1964.

Publication types

• Historical Article
• Biology / history*
• Genome, Bacterial / genetics*
• History, 19th Century
• History, 20th Century
• Molecular Biology / history*
• Recombination, Genetic / genetics*
• United States

Personal name as subject

• Joshua Lederberg
• J B S Haldane