Esther Lederberg found that this curious F factor, bestowing the capacity to initiate mating, could be acquired by a bacterium that didn’t have it. How? Through a different mechanism, which I’ve just mentioned: transduction. That is, carried in by a virus. This was a dizzying compoundment of two kinds of horizontal gene transfer, functioning as a double-stroke process to move DNA between microbes. Take a deep breath and relax with your puzzlement as I say it again: transfer of the F factor—whatever it was—from one bacterium to another, by a virus, gave the second bacterium an ability to mate with still other bacteria. An F- bacterium became F+. A virgin became a player.
But wait. Let’s pause here and remember that “sex” is only a metaphor for what these bacteria were doing. It applies nicely in some ways but in others does not. The Lederbergs tended to treat the term literally, speaking often in their work about bacterial sex, but other biologists disagree, noting the most important distinctions. Bacterial “sex” doesn’t involve the fusion of two gametes, egg and sperm, each bearing a half share of an entire genome. And it doesn’t result in reproduction. A bacterium generates offspring by splitting, not by mating. The net result of conjugation is genetic recombination—mixing—which often proves helpful in the evolutionary struggle. But conjugation itself doesn’t produce babies.
It all sounds weird because it is weird. Esther Lederberg published her peculiar discovery, in collaboration with her husband and one other coauthor, and near the end of their paper, they noted passingly that the ability to conjugate among E. coli was conferred by some other sort of “infective hereditary factor.” Her husband, as sole author of another paper published just a month earlier, had alluded likewise to “infective heredity.” Transduction was infective, just as conjugation was sexy. And the phrase had an enduring ring.
Norton Zinder got his PhD in Wisconsin, and by 1952, he was back in New York as an assistant professor at the Rockefeller Institute, where Oswald Avery had worked. One year later, he published an overview of this whole perplexing business, genetic transfer among bacteria. He meant to sort things out. There were three modes of such sideways inheritance, Zinder explained. The first was conjugation, as discovered by Tatum in collaboration with Zinder’s mentor, Joshua Lederberg. The second mode was transformation, as discovered by Griffith and illuminated by Avery’s team. The third mode was transduction, as discovered (though he didn’t crow) by him and Lederberg. Conjugation was analogous to sex. But the other two were different, involving processes more like infection, as the Lederberg team had suggested passingly. These other two processes deserved their own descriptive category, their own metaphor. Zinder, picking up on the Lederberg’s phrase, called them infective heredity.
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Bacteria, bacteria. Bacterial sex. Bacterial transformation. Dead bacteria, live bacteria, virulent and mild. Bacterial DNA. A person might get the impression that this whole subject of horizontal gene transfer is merely a matter of bacteria, by bacteria, for bacteria.
But that impression would last only until a person came to the work of Tsutomu Watanabe, a bacteriologist at Keio University in Tokyo during the 1950s and 1960s. In 1963 Watanabe alerted his fellow scientists to an urgently human implication of the new bacterial discoveries: that resistance to multiple antibiotics among bacteria spreads horizontally. It can happen by conjugation. It can happen by transduction. It can happen in a sudden leap. Consequently, it has become a dire problem. And the problem is especially severe in hospitals, where such huge volumes and such variety of antibiotics are used, selecting for resistant bacterial strains that then infect people who are already ill. The World Health Organization now considers antibiotic resistance one of the biggest threats to global health in the twenty-first century. Watanabe saw it coming. He understood why such resistance would spread so fast and so widely. Adopting the terminology of Zinder and the Lederbergs, Watanabe called it “an example of ‘infective heredity.’ ”
Antibiotic resistance was already a serious and growing problem, just after World War II, in Japan and around the world. People were dying again of diseases, such as bacillary dysentery, that the great revolution in antibiotics was supposed to have tamed. The first sulfa drugs had been introduced in the late 1930s, and immediately there were reports of bacterial strains with resistance. Penicillin was discovered in 1928 and developed for human use beginning in 1942; initially, it was a very potent weapon against Staphylococcus of various sorts; but by 1955, penicillin-resistant strains of staph were turning up, especially in hospitals, from Sydney to Seattle. Methicillin became available in 1959, valued highly as an answer to forms of staph—especially Staphylococcus aureus—that had acquired resistance to penicillin. But resistance to methicillin also appeared soon and spread fast, so that by 1972 methicillin-resistant Staphylococcus aureus (infamous now as MRSA) was a concern in England, the United States, Poland, Ethiopia, India, and Vietnam. And by the early twenty-first century, MRSA was killing more Americans per year than AIDS. Despite some success at reducing MRSA transmission in hospitals, by a more recent tally the numbers were still bad: more than 23,000 deaths annually in the United States and seven hundred thousand deaths globally from infection by unstoppable strains of bacteria.
What has driven this grim, costly trend is not just the use of antibiotics but also the reckless overuse of them for foolish or unnecessary purposes—doctors pandering to patients, for instance, by prescribing an antibiotic for those who want to believe it will cure a viral infection. (Antibiotics target bacteria exclusively and have zero effect on viruses. You might just as well try to hose the dirt off your driveway using a flashlight.) Another contributing factor is agricultural use: feeding low doses of antibiotics routinely to domestic livestock, because that somehow increases their rate of growth. In the United States during a recent year, more than 32 million pounds of antibiotics were sold for use in livestock, and most of that went for growth promotion and preventive dosing of food-animal populations, regardless of whether the individuals were sick. Globally, total consumption of antimicrobials (that is, drugs against dangerous microbial fungi as well as bacteria) by livestock was roughly 126 million pounds, with China using even more than the United States, and Brazil in third place. Most of that total goes into cattle, chickens, and pigs. A significant fraction of it involves drugs that are also important in human medicine.
So there’s an extraordinary amount of evolutionary pressure, out there in the world, forcing bacteria to acquire resistance or die. But the most startling aspects of the trend have been how speedily resistance has spread and how many different kinds of bacteria have acquired multiple resistance—that is, resistance not just to one antibiotic but also to whole arsenals of different kinds. The danger of multiple resistance is that whatever antibiotic may be prescribed, whatever pharmaceutical may be thrown at it, the bacteria just keep eating a person’s flesh or blood or guts, sometimes to the death. The death of the patient is no dead end to the strain of bacteria, of course, if it has managed to infect other victims in the meantime. And the appearance of resistance to each drug so quickly, in one strain of bacteria and another, as occurred in the 1940s and 1950s, was a phenomenon that couldn’t be explained by the slow Darwinian process of mutation, natural selection, and ordinary inheritance, occurring independently in each case. Darwinian selection was certainly involved, but selection can act only on variation: genetic differences between one individual and another. What was the source of the variation? Mutation alone couldn’t account for the appearance of so many new genes, so fast, in so many different organisms. It had to be something else—something that moved speedily and sideways, even between members of different bacterial species. Tsutomu Watanabe recognized the alternate explanation and, based on research by him and his Japanese colleagues, laid it out in English for the first time.
The Japanese work began after World War II in response to an increase in cases of dysentery, an intestinal affliction resulting in bloody diarrhea and other symptoms. Postwar deprivation, dislocat
ion, and disruption of sanitary and health services probably exacerbated the problem, but its proximate cause was a bacterium, Shigella dysenteriae. The preferred treatment at first involved various forms of sulfa drugs, but Shigella strains soon showed resistance to those sulfas, so medical people turned to newer antibiotics, such as streptomycin and tetracycline. By 1953, strains of Shigella showed resistance also to both of those. Each bacterial strain, though, was resistant to only one drug. It could still be stopped by the others. Then in 1955 a Japanese woman returned from a stay in Hong Kong, sick with dysentery, and Shigella from her feces tested resistant to multiple antibiotics. From that point, resistance spread fast, shockingly fast, and during the late 1950s, Japan suffered a wave of dysentery outbreaks caused by Shigella superbugs resistant to four kinds of antibiotic: sulfas, streptomycin, tetracycline, and chloramphenicol. Could these strains have acquired such multiple resistance so quickly by incremental mutations alone—one misplaced A, C, G, or T at a time? The odds against that were so high you’d need a string of twenty-eight zeros to print them. But if not, what was happening?
The alarm bell rang louder when researchers discovered that this phenomenon wasn’t confined to Shigella. Some cultures of Escherichia coli, taken from patients with resistant Shigella, showed resistance to the same drugs. E. coli had shared. A whole set of resistance genes had evidently moved sideways, in the depths of the patients’ guts, from one kind of bacterium to another. Two teams of Japanese researchers then reproduced the phenomenon in their laboratories, showing similar transfer between bacterial strains cultured together in flasks or dishes, and concluded that the capacity for multiple resistance was passed by conjugation. Yes, a sizable packet of genes, not just one bit of DNA, was moving across. And the exchange wasn’t limited to Shigella and Escherichia. Further research showed that the packet could cross boundaries between other species, even from genus to genus, among almost every group in the enteric bacteria, a large family of bugs that live within human bellies.
What exactly was this packet of genes that traveled so easily across boundaries? Watanabe and a colleague, Toshio Fukasawa, in earlier work, had offered a hypothesis: it was an episome, a sort of autonomous genetic element that floats free within a bacterial cell, unattached to the cell’s single circular chromosome. An episome is sublimely selfish DNA. It carries extra information beyond what’s absolutely necessary to assemble and operate the cell. It codes for traits that might be useful in emergencies. It can hold multiple genes, exist in many copies within a cell, replicate independently apart from the chromosome, and send a copy of itself into another cell during conjugation. It might be lost entirely from a strain of bacteria when its genes aren’t needed, because of changing environmental conditions, and then, when conditions shift again, reacquired from other strains. Wow: wildly mobile DNA. Esther Lederberg’s F factor was such an episome, though she didn’t realize that at the time of discovery. The concept didn’t exist until 1958. But now Watanabe declared to the scientific world in his 1963 paper what Fukasawa and he had already said in Japanese: multiple resistance, to streptomycin and those three other antibiotics, was coded on an episome. They gave the episome a name: resistance transfer factor. It became known as R factor, for short, in parallel to Esther Lederberg’s F factor.
This R factor could be transferred by conjugation. It could be transferred (at least in lab experiments) by transduction. It explained how harmless bacteria such as ordinary Escherichia coli could convey genes for multiple antibiotic resistance, across species boundaries, into dangerous bacteria such as Shigella dysenteriae, in a blink. Its medical significance was “limited to Japan at present,” Watanabe wrote, but R factor and episomes like it “could become a serious and world-wide problem in the future.” That was prescient and understated.
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Word of the Japanese discoveries spread, thanks to Watanabe’s publications, though word didn’t spread so quickly and broadly as bacterial resistance did. Unless you were a reader of Bacteriological Reviews or ate lunch with bacterial geneticists, you probably would have been unaware, in the early 1960s, of the way horizontal gene transfer was carrying this problem around the globe.
A young American named Stuart B. Levy, on leave from medical school, heard about it while on a research fellowship at the Pasteur Institute in Paris around that time. A Japanese researcher at the Pasteur gave an informal talk on multiple drug resistance, describing what his countrymen had learned, and Levy approached him afterward. Levy was fascinated by the work and especially interested in Tsutomu Watanabe. “Do you know him?” Levy asked. The Japanese colleague, a man named Takano, knew him very well. Watanabe was at Keio University in Minato Ward, part of Tokyo, and had hosted some of Takano’s own work. “If you want, I’ll write a letter for you,” Takano said. That led to an invitation, and Levy finagled another getaway from med school so he could work for a few months in Watanabe’s lab. It was a formative experience.
Stuart Levy MD is nowadays a professor at Tufts University School of Medicine and an internationally renowned authority on antibiotic use, overuse, and resistance. Old photos from fieldwork and conference events show him as a jaunty young man with a big, dark mustache and the good sense to smile and relax when the work was done. His twin brother, Jay, is also a medical researcher—one of the three scientists whose labs first isolated the causal virus of AIDS. While Jay stayed with viruses, Stuart focused on bacteria. He cofounded the Alliance for the Prudent Use of Antibiotics (APUA) in 1981 and still serves as its president. He’s also a past president of the American Society for Microbiology, a huge and august organization with an international membership. He reminisced about Watanabe when I visited him in his office, on the eighth floor of a drab building just outside Boston’s Chinatown. By this time, Dr. Levy was in his midseventies, clean shaven, with thinning hair and brown eyes that looked sad in their deep sockets above his mild, welcoming smile. He had seen a lot since Paris and Tokyo in the early sixties.
“We worked in the lab without air-conditioning,” he said of his time with Watanabe. “It was very, very hot. Hot and humid.” Levy’s lab bench was on an upper level, with a sort of overlook from which, glancing down, he could see Professor Watanabe doing experiments in shirt sleeves, “because it was so hot.” Periodically, someone would bring forth a hose and spray the professor with water to cool him off. He was a small man, an inch or two shorter than Levy, who spoke impeccable English and had a simple, straightforward manner toward students and postdocs. He would bicycle through the streets of Minato with his junior colleagues and sometimes took three or four of them out to a bar, Levy recalled, for an evening of karaoke. “We would be singing English songs, and he was reading, he was directing it. And it was . . .”—for a moment’s pause, Levy left me to picture the professor, arms waving, as he joyously followed the bouncing ball and crooned something from Roy Orbison or the Honeycombs—“ . . . unbelievable moments.” On a visit to Philadelphia for a scientific meeting, a few years later, Watanabe stayed with Levy’s parents, nearby, in Wilmington, Delaware. “I was delighted that Watanabe would come,” Levy said, “because I worshipped him, in a weird way.” A lively mentor, a focused and dignified Japanese scientist. What became of him? I wondered.
“He passed away of stomach cancer,” said Levy. “He probably was in his forties, early fifties.”
Before that occurred, though, Levy himself had coauthored a paper or two with Watanabe on resistance factors. “One is in Japanese,” Levy recalled. “Don’t ask me what it says.” So I didn’t, and it has never been translated, but the title in English suggests they were investigating possible ways to fight resistant infections by preventing the bacteria from replicating their DNA. Levy had returned to the United States by then and resumed his medical studies, pointed toward a career that mixed research on bacterial resistance with some clinical practice and a sense of mission. His mission, pursued through publications, lectures, and APUA, was to protect the world against bacterial superbugs by devising defen
sive therapies and raising awareness about the scope and the consequences of needless antimicrobial excess.
In his research, Levy focused especially on resistance to tetracycline, and in the mid-1970s he led a pioneering study of how such resistance could be transferred from the gut bacteria of poultry to the gut bacteria of humans. That work, published in the New England Journal of Medicine, showed that intestinal bacteria of chickens, if the birds ate tetracycline-laced feed, acquired resistance to the antibiotic within a week. Less expected, more worrying, was that bacteria in the bowels of farm workers on the same site acquired the same resistance over a period of months. Soon after the early farm studies, Levy’s lab also discovered just how those resistant bacteria evade tetracycline: by pumping it back out through the cell wall, with a sort of efflux mechanism. This mechanism is coded by a single gene on an episome (by that time, the word episome had been replaced by a synonym, plasmid) that sometimes travels laterally, carrying other genes for resistance to other antibiotics as well as the tetracycline-evader gene. A plasmid, as known ever since, is a short stretch of DNA, sometimes circular like a bracelet, that exists and replicates in a cell independently of the cell chromosome. That independence facilitates its lateral passage to other cells and helps explain how the efflux mechanism against tetracycline moved sideways so fast, from one bacterium to another, and from Leghorns into the bellies of people.
Levy’s research continued, likewise his leadership as a voice of concern, and in 1992 he published a book titled The Antibiotic Paradox. The paradox is that these drugs, antibiotics, which made human lives so much better and longer during much of the twentieth century, have also been making our bacterial enemies so much more formidable. The book was updated in 2002, and in that edition he said:
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