“The discovery of transferable R factors, forty years ago, opened the eyes of microbiologists and medical scientists to a breadth of gene spread never before imagined. Transfer of resistance genes could occur among bacterial species more genetically and evolutionarily distant than a horse is from a cow.”
The implications of those discoveries weren’t fully realized at the time, Levy added, but they presaged the spread of antibiotic resistance, by horizontal gene transfer, around the planet.
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All this talk about bacteria, which are invisible to the naked eye but nearly ubiquitous—on our skin, in our guts, throughout our environment, and spanning the history of life as we know it—made me want to see some. Gene sequences from bacteria and journal papers on bacterial dynamics are all very well, but I hankered for a glimpse of their corporeal presence. So I flew to London and connected to Porton Down, a high-security science compound located discreetly in the Wiltshire countryside, amid rolling fens and stubble fields, near the city of Salisbury in southwestern England. There, behind a tall chain-link fence, lies the National Collection of Type Cultures (NCTC), one of four major repositories of cell lines and microbes under custodianship of an agency called Public Health England. The other three, encompassing cell lines for medical research, and infectious viruses, and fungi, are tucked elsewhere around the country. NCTC, with its primary site at Porton Down, is devoted to bacteria.
I got through the security gatehouse because a genial press liaison, Isobel Atkin, had arranged for me to be welcomed. Inside the secure perimeter, I found a long, severe redbrick structure, looking like a Dickensian shoe factory, and a number of functional, metal-box modular buildings, some of them stacked two high for efficient use of space. The Communications Office, including Atkin’s desk and others in a single crowded room, was on the second floor of one stack, reachable by a metal stairway. At the far end of the row sat a larger metal box, formally known as Building 17. Less formally: the Warehouse, within which rests the Ultra-Low Temperature Storage Facility (ULTSF). That’s where the bacteria sleep, and that’s where I spent most of my day.
Building 17 is carefully compartmented, a repository of records as well as specimens, and as you move through it, you penetrate deeper into zones of secure containment, cryogenic preservation, and British medical history. Atkin and I were accompanied by Julie Russell, head of Culture Collections for Public Health England. Just inside, we passed among office carrels and then buzzed our way through a locked door into the storage area, the ULTSF, and its first big section, known as the Tank Room. This was a hangar-like space filled with dozens of freezer tanks—great steel barrels with tightly sealed lids, containing liquid nitrogen chilled to about minus 190 degrees Celsius. Within each tank rested about 25,000 ampoules, sealed glass tubes of frozen bacteria, neatly boxed and labeled and racked. These were production samples of various bacterial strains—including many strains of medical importance—grown and packaged at another facility, from historical strains kept here at Porton Down. They were available, at modest cost, for use by researchers around the world. (NCTC formerly gave away its bacterial samples upon request to reputable labs; nowadays the samples are sold for a reasonable recoup of expenses, but their genomic sequence data are given freely.) Producing such viable samples and making them available for all manner of scientific study is one of the chief purposes of the NCTC. Storage of the original strains, literally frozen in time—frozen in evolutionary stasis as they existed decades ago—is the other chief purpose. Those original strains have their own storage rooms, more like bank vaults than airplane hangars, deeper within the building.
Two staff people greeted us to give the tour. Steve Grigsby, supervisor of the ULTSF, with his chiseled features and vice-grip handshake, looked like an aging Daniel Craig in a black turtleneck. Jodie Roberts, senior cryostore technician, was a smart young woman with a multicolored ponytail who appreciated the subtleties of bacterial evolution as well as storage. Roughly a million ampoules of bacteria are stored in this room, Grigsby said. Roberts opened one tank, and we watched the frozen vapors rise. The tanks are on an alarm system, Grigsby explained; if one of them loses power and starts to warm up—to a dangerously balmy minus 153 degrees C. or above—an alert clangs and brings him running at whatever time of day or night. The room itself has a separate alarm for the safety of human personnel. If the ambient nitrogen level rises above a certain threshold, another alarm tells whoever is present that they are about to asphyxiate; windows open automatically, fans bring in outside air. He demonstrated for my benefit, with the windows and fans, a generous use of Public Health England electricity.
Beyond the Tank Room was another door, giving into a sanctum called Room 17/11. A special badge was needed here (Atkin’s wouldn’t open the lock), and Jodie Roberts buzzed us in. The temperature was just below 5 degrees C. (roughly 40 degrees Fahrenheit, like inside your refrigerator), which is much warmer than the tanks but cold enough for long-term preservation if the bacteria are lyophilized (freeze-dried) in their vacuum ampoules. The ceiling was low, and along the left wall stood eight storage cabinets holding many of the British collection’s more notable samples. These included a sample of Oswald Avery’s Streptococcus pneumoniae (known here by its accession number, NCTC13276), descended straight from the strain in which he discovered transformation, and another Streptococcus strain (NCTC8303) worked on by Fred Griffith. Freeze-dried securely, they were stored in the cabinets for future reference and research. There was also a specimen of Haemophilus influenzae, a bug that lives harmlessly in many people but sometimes causes nasty infections; this sample (NCTC4842) was isolated in 1935 by Alexander Fleming, the discoverer of penicillin, reportedly from his own nose. Eventually I got to see them all.
Roberts opened a cabinet, within which were nested a number of cartons. She lifted out one carton and raised its top, revealing dozens of delicate, balsa-wood boxes, each roughly the size to hold a single Montblanc pen. The preferred term for these little boxes is “coffins.” It seemed nicely appropriate; but they were Dracula coffins, holding dangerous creatures quite ready to wake up. Tiny labels on the lids, hand lettered, told which coffin was which. Each coffin contained a glass outer tube protecting the delicate ampoule, which was a smaller tube, round at one end, the other end heat sealed to a needle point. Inside the ampoule, under vacuum conditions, was the bacterial sample. All you could see was a dab of yellow material, barely more than a smudge, at the ampoule’s round end. The yellow stuff was mostly precipitate of a nutrient broth, a special mix used to protect the sample when it was freeze-dried. Within the smudge lurked dormant bacteria.
We test the integrity of the vacuum, Roberts explained, by sparking the ampoule. If it’s good, it will fluoresce. Then she showed me, applying her electric spark-testing wand to one little tube. It did indeed fluoresce, with a gentle blue light, indicating a good vacuum. If an ampoule didn’t fluoresce, that meant the vacuum was lost, she said, and the sample was spoiled. They would dispose of it.
I had asked in advance to see one particular sample, a historical treasure, and Roberts obliged. Wearing orange medical gloves, she gingerly lifted the coffin of a specimen known as NCTC1. Its number 1 testified that it was the very first sample acquired, back in 1920, when NCTC was founded. It was a strain of Shigella flexneri, a bacterium that causes shigellosis, a form of dysentery, similar to what Watanabe saw in Japan. Shigellosis manifests as bowel inflammation and diarrhea, and the infectious dose for a human is low—only a trace needed to take hold and bloom in someone’s gut—meaning that it is highly transmissible through tainted water or food. The diarrhea can be bad, especially in the absence of decent medical care: shigellosis still kills more than a million people a year, most of them children in developing nations. In early 1915 it afflicted a British solider in France, and that’s how the sample later known as NCTC1 came to be gathered.
The soldier was one Private Ernest Cable, twenty-eight years old, serving with a regiment out of East Surrey. No
photo survives, and almost nothing is known about Private Cable or his next of kin; he had lodged with a family in England before joining the army, the family had a toddler, and to that toddler he wrote his will. Cable arrived sick at a military hospital in Wimereux, France, just down the coast from Calais, thirty or forty miles behind the trench lines of the Western Front. The hospital was in a converted hotel, formerly the Grand Hotel of Wimereux, and devoted entirely to infectious diseases, not battlefield injuries. Although his clinical records have disappeared, Cable probably suffered bloody diarrhea and gut cramps. The diagnosis was dysentery. On March 13, 1915, as the Allies pushed an offensive in Artois and Champagne, a hundred miles south, Cable died—but not before a stool sample was taken. The stool sample went to Lieutenant William Broughton-Alcock, a bacteriologist at the Wimereux hospital, who isolated a bacterium. That isolate was later placed to the species Shigella flexneri and identified as serotype 2A. Five years later, it went into the collection as NCTC1. What makes it interesting is not its numerical priority but what was found a century later about its biology and its genome.
Private Cable’s Shigella was resistant to antibiotics that hadn’t yet been invented. More precisely, to antibiotic substances not yet discovered by humans. More precisely still, Cable’s strain was resistant in 1915 to penicillin and erythromycin, which went into use against human infections in 1942 and 1952, respectively. This seeming reversal of cause-and-effect order was deduced by a team based at the Wellcome Trust Sanger Institute, in Hinxton, a little village just south of Cambridge. Kate S. Baker was the first author on their paper, published in 2014.
Baker and her colleagues took an old ampoule of NCTC1 from Porton Down, broke it open, revived the bacterium, and cultured it, waking the old bug from its time capsule. They plated out samples of the growing bacteria onto separate dishes of nutrient agar, then challenged them with a whole list of modern antimicrobials. Baker’s team found “intrinsic antimicrobial resistance” against those two drugs, penicillin and erythromycin. Intrinsic? It seems befuddling—like an intrinsic proclivity to wear bulletproof vests before guns were invented. How and why would anyone do that? In this case, such preparedness reflects the fact that resistance to antibiotics does exist in the wild because antibiotics exist in the wild. Some bacteria produce antibiotic substances as natural weapons, deterrents, for use in their competitive struggles against other bacteria. Resistance likewise arises naturally, slowly, as an evolved trait, for defense against such weapons.
Some evidence of this phenomenon came in 1969 from the Solomon Islands, a remote archipelago in the southwest Pacific. Modern medicine hadn’t arrived, and the people of the Solomons were still “an antibiotic virgin population.” Despite the absence of lab-produced drugs, a group of American researchers found bacteria, in a soil sample, that proved resistant to both tetracycline and streptomycin. How did antibiotic resistance arrive before manufactured antibiotics? Unknown, but again, the answer probably lay in the natural battles among bacteria.
In this Solomon Islands case, the same double resistance—against tetracycline and streptomycin—turned up also among human-dwelling bacteria. In a fecal sample from one native islander, a person “from the innermost bush country,” the researchers found E. coli resistant to both the same drugs. Had it somehow been transferred sideways from soil bacteria to bacteria in the human gut? Although the American team couldn’t answer that question, not in 1969, they did attribute the resistance in both bugs to R factors, transferable genes on little plasmids, of just the sort that Watanabe and Stuart Levy had deduced.
Another antibiotic, vancomycin, has an origin story that also traces to distant soils. Vancomycin was developed from a natural substance generated by bacteria found in dirt from Borneo. In 1952 a missionary sent this smidgen of dirt to one of his friends, an organic chemist in the United States. The friend worked at Eli Lilly, a pharmaceutical company then sorely interested in discovering antibiotics that would work against staphylococcus strains resistant to penicillin. The natural substance produced by the Borneo bug, known first as compound 05865, was purified and modified slightly, then named vancomycin because of what it could vanquish: the gonorrhea bug and other troublesome bacteria, including staph—even staph strains that resisted penicillin. But again defenses arose against the vanquisher, in the hospitals of Japan and the United States, if not naturally in the soils of Borneo. By the late 1980s, vancomycin resistance had shown up among bacteria of one genus, Enterococcus. The first identified gene for resistance in Enterococcus (eventually there were several such genes) was called vanA, and because enterococci contain plasmids and other systems for horizontal gene transfer, it was probably just a matter of time—and not much time—before vanA dodged sideways into other kinds of bacteria. Sure enough, it soon went across genus boundaries from Enterococcus into staph, including Staphylococcus aureus.
This was bad news for human medicine. Back in 1982, vancomycin had been one of the go-to weapons against methicillin-resistant Staphylococcus aureus (the dreaded MRSA), but by 1996, in Japan, there were staph infections with reduced susceptibility to the drug, and soon afterward vancomycin-resistant staph started showing up in the United States. A patient in Michigan, a poor soul with multiple health problems, including diabetes, kidney failure, and chronic infected foot ulcers, yielded the first American sample of vancomycin-resistant Staphylococcus aureus (a new demon to fear: VRSA), some of it isolated from the infected foot. That patient had been treated with vancomycin among other antibiotics for infections following an earlier toe amputation, so the staph may have acquired its resistance gene by horizontal transfer within the patient’s own body. Oy vey. But the resistant staph might just as likely have been ambient in the hospital, having recently snatched up its new gene while abiding within somebody else’s multiply infected body. Soon afterward, another VRSA strain was isolated from a woman in Pennsylvania, this time despite the fact that she herself hadn’t recently been treated with vancomycin. The Pennsylvania patient, like the one in Michigan, was suffering an infected foot ulcer, and from that ulcer, again, came the resistant staph.
These two foot-eating strains of VRSA both carried the vanA gene, suggesting they had each gotten their resistance by transfer from Enterococcus bacteria, probably in separate events. The US Centers for Disease Control and Prevention promptly issued a warning that, because this gene was jumping around so deftly, from gut-dwelling bacteria into skin-rotting bacteria, “additional VRSA infections are likely to occur.” No one’s feet were safe, and especially not those of anyone foolish enough to walk barefoot in a hospital.
Private Ernest Cable’s fatal dysentery, back in 1915, seems like a warning signal of all this travail, but only in retrospect. Its meaning couldn’t be divined at the time because human-made antibiotics didn’t yet exist—and therefore neither did the problems of their use, their overuse, and the resistance genes that spread so quickly from natural beginnings. Cable’s case merely represents an obscure step along the way. Its particulars were unexceptional—a man’s death in wartime, a bacterial sample archived routinely—and their implications weren’t recognized until a century later, when Kate Baker and her colleagues regrew the bug, extracted its DNA, and sequenced its genome. They found genes that would have defended Cable’s original bug from penicillin and erythromycin, if any such drugs had existed in that Wimereux hospital. Private Cable, even if given such treatment, probably would have died. But we’ll never know.
He was buried in the Wimereux cemetery, and his Shigella strain, after lab culturing by Lieutenant Broughton-Alcock, plus a few other steps along the way, was buried in its own tiny coffin within a box, within a cabinet, within a very cold room, within Building 17/11, within the NCTC compound at Porton Down. The orange-gloved hands of Jodie Roberts now held that coffin for me to see. I bent close and read the label aloud: “Shigella flexneri, type 2A, strain Cable.”
“We know that it’s still viable,” said Julie Russell, the head of collections, “because there was
more than one.” More than one such coffin, that is, each containing an ampoule of Cable’s bug, taken from the same early batch, reawakened briefly and freeze-dried for better preservation, by the latest new method in 1951, then reburied in the cold. “And we’ve grown it from the other one,” she said. That was for the Baker team’s work, when they broke open one ampoule, cultured it, and looked at its deep identity. Now, besides this one, Russell added, there is one more ampoule of NCTC1, strain Cable, from the earliest batch, under storage here at NCTC. Two left, total.
Are they precious? I asked.
“That’s right, yeah,” she said casually, and what she meant was: they can be very damn useful.
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During the late 1960s and early 1970s, scientists began to realize that the implications of horizontal gene transfer go far beyond the problem of bacterial resistance to antibiotics. Those implications include the whole matter of how evolution works—by Darwinian mechanisms, or otherwise?—and how it has worked for much of the past four billion years. A British bacteriologist named Ephraim S. Anderson hinted at this in 1968.
Anderson, born in 1911, came from an Estonian Jewish immigrant family in a working-class neighborhood of Newcastle upon Tyne, an unadvantaging start for an aspiring British scientist in the hard years between the world wars. He showed his brilliance in school, won a scholarship to study medicine, then struggled to find work, disfavored for at least some positions because he was a Jew. He joined the Royal Army Medical Corps and spent five years in Cairo, Egypt, tracing typhoid outbreaks among British troops. Returning to England, he took a research job at the Enteric Reference Laboratory, a national facility with a practical mission: identifying and characterizing strains of intestinal bacteria that threaten human health. Within a few years, Anderson became its director. By then, he was an expert on enteric bacteria such as the Salmonella group, which includes the typhoid bug, and during the 1960s he emerged as an influential voice on public health, warning early and loudly about the dangers of antibiotic resistance. He was known for his brusqueness, his feistiness, his “great gift” for rubbing people the wrong way, and for his strong opposition to the routine use of antibiotics for growth promotion in livestock. He was among the earliest bacteriologists in England who recognized what Watanabe and his Japanese colleagues had seen: that resistance genes could spread quickly, from strain to strain, from species to species, on plasmids. For that alone, he would be notable. But in one of his journal papers, Anderson went a step further, speculating that another important effect of such transfer factors “is their possible importance in bacterial evolution in general.”
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