“No change since you were here yesterday morning, I’m told,” Chris said solemnly. Still unresponsive.
When he’d been admitted to the TICU in December, Tom had confessed that he hated to go to sleep at night because he was afraid he’d go to sleep and never wake up. It seemed that most people who died in the ICU died at night—literally during the graveyard shift. He tried not to pay attention to the code blues and the gurneys that rolled in with skeletal people lying on them and rolled out with covered-up cadavers, and the smell of frankincense in the air, from the priest who came to give some their last rites. But it was hard not to watch. “We’re the nearly deads and the newly deads,” he’d declared. The fine line between the two was becoming more frightening by the day.
I plunked down in the chair beside Tom’s bed, exhausted. “No change” left us in limbo, but suddenly the sight of Chris tending calmly to Tom, and Tom resting quietly and not dead, let me step out of panic mode. That afternoon, there was no way to sugarcoat the results from the latest CT. A new fluid collection was expanding in the back of Tom’s pancreas, and another one where the new feeding tube had leaked. Two more drains were inserted, for a total of five. Tom now looked like a pincushion. He had lost 100 pounds, and his skin took on the pale and waxy look of a corpse. The hum of the monitors that had been so annoying before had become the only note of reassurance that he was, at least, still alive. All hope now rested on the phages.
18
PANNING FOR GOLD
March 1–March 11, 2016
Wherever you find bacteria, you find the phages that prey on them. If you want to find phages with an appetite for intestinal bacteria, a pile of poop is a good place to start. For A. baumannii, you go low. Or, as one Navy scientist summed it up, “some of the worst places you would ever want to go.” These include sewage treatment plants, standing cesspools, or dumps filled with dirty diapers and other fecal-tainted debris, rotting garbage, the occasional dead animal, and maybe wastewater runoff from a local hospital or animal farm. The Navy’s environmental phage sources include ships that travel internationally. Since some nasty superbugs have been found in the pipes and sewers connected to the NIH’s hospital in Bethesda, they also collect samples from nearby sewage treatment facilities.
The other place you can find phages is the opposite of these retch-worthy locales, where the dirty work has already been done. These were the spotlessly clean, well-ordered phage “libraries,” a walk-in fridge in some low-traffic corner of a microbiology lab. In this library, the collection consists not of volumes but vials, most no bigger than your pinkie, labeled and stored for easy reference and access—just what we needed now.
Whether sifting through library or lagoon, the search for the particular A. baumannii phages active against Tom’s specific isolate was going to be painstaking. With time such a critical factor, we primarily focused on sources in the continental United States, where transport would be easiest. And we got lucky. Between Texas A&M’s CPT and the Navy’s Biological Defense Research Directorate (BDRD) lab, we’d tapped into some of the most experienced and respected phage scientists on the planet.
Ry had devoted his long career to studying how phages manage to detonate bacterial cells (lysis) when replication is complete and the new progeny are ready for release. Tough bacterial cell walls are able to withstand enormous pressure, but somehow phages are able not only to cause the infected bacterium to explode but to do it on a timer, chosen to optimize the number of virions released.
At the Navy’s lab and NIH before that, Dr. Biswajit Biswas, Carl Merril’s former protégé and now Theron’s phage team leader at the Navy lab, had worked for decades to develop ways to effectively select the best phages for treatment. Among other things, he had helped develop a faster system to grow cultures and phages, using an automated incubator with a sophisticated camera and computer to monitor and analyze the data in real time. All this meant the lab could judge the therapeutic efficacy of different phages and different combinations of phages to kill target bacteria in hours instead of days. In recent years, the Navy’s extensive work on therapeutic uses of phages to treat wounds had involved A. baumannii, with the intention of eventually using phage therapy to treat multi-drug-resistant infections in service members or VA patients. But so far, they’d only tried it on lab animals. No humans. Tom would be the first.
Both teams were packed with skilled, passionate scientists, each with a distinct focus, history, and strengths that complemented the others. Suddenly, it felt like a dream team had materialized out of nowhere.
Their mission was straightforward but not simple. They had to find the phages that would target Tom’s multi-drug-resistant strain of A. baumannii, then grow the phages in quantity, purify them, and get them to the UCSD’s investigational pharmacy to be prepped. Ordinarily, all of this would take weeks, but with daily, sometimes hourly, updates and consults with Chip and me, the team knew Tom was failing fast. They had to find ways to hurry the process without sacrificing quality—and Tom. They couldn’t hurry Mother Nature; phages need time to replicate, about twenty to forty minutes once they infect a bacterial cell. Harvesting them, repeating the propagation process, and the rounds to purify them eat up the clock. So everyone opted to work harder and faster to close the routine gaps, doubling up on the numbers of assays underway at one time and working through nights to eliminate downtime between steps. There’d be time to sleep later, Theron joked. For now, the race was on, from bog to bedside.
One team in Maryland, the other in Texas, the medical team and the rest of us in San Diego, and others online were different time zones and miles apart but up against A. baumannii in a single race against the same clock. If Tom survived the first round of phage therapy, once the battle was underway, win or lose, we were all on the same team.
While Chip and I forged ahead on the regulatory and administrative fronts, the CPT and Navy teams set out to find and prepare the phages as rapidly as possible. Both labs had a combined collection of hundreds of phages that were active against a number of pathogens, but at the time I contacted Ry, they had only a few A. baumannii phages on hand, most left over from various phage hunts in the past few years.
“We’re lucky that we never clean out our fridge,” Jason quipped. Pickings were slim simply because the Texas group hadn’t done a concerted phage hunt for A. baumannii—until now.
The Navy, on the other hand, had been doing precisely that. Many sailors and other service members had returned from Middle East wars with Iraqibacter infections. They usually weren’t as virulent as Tom’s infection, but their existence had spurred the Navy’s search effort, and we were now the beneficiaries. The Navy had extensive libraries of a few thousand phage specimens from environmental sources—those collected from the muck. Of the some three hundred partially characterized phages in its collection, one hundred fifty were active against various strains of A. baumannii. Since A. baumannii phages were more finicky than most, there was no telling if any would match Tom’s strain.
Biswajit was known among peers as the Phage Whisperer, or as Carl Merril put it, a kind of phage matchmaker who had an intuitive sense of how to wrangle phages to get the best results. From a family of veterinarians in India, he had begun a medical career as a vet himself there. But he was also a born tinkerer and in his long career as a scientist had earned a reputation as an inventor. He had a sixth sense when it came to choosing phages that kept the selective pressure on an evolving bacterial infection.
In a way, Biswajit had been preparing for this moment for nearly twenty years. He had worked with one of the pioneering phage companies in the US in 1994, and although his animal studies were promising, like Carl, he hadn’t been able to get any traction for phage therapy in the medical community at the time. Although he had remained convinced that phage therapy could be a life-saving medicine, eventually he shelved the work. With the Navy BDRD, his work with phages had included those that kill anthrax, a bioterrorism concern. When the call came to develop a ph
age cocktail for use in Tom’s case, the long hiatus was over. His time had finally come.
The Texas and Navy labs differed in their setups and processes to get the job done, but once you had phage in hand, the basic steps of a phage selection process began the same. You screen the phages to be sure that the phage you carry forward is a lytic active killer phage and not a temperate phage that embeds its genetic material into the bacteria and hits the snooze button. The thinking was that temperate phages could make matters worse, because they often transmit toxin genes as well as antibiotic resistance genes they’ve picked up from other bacteria.
Once you’ve isolated a phage that’s active against the target bacteria, you have to grow lots more of it to obtain enough for a therapeutic supply. Since phages can only grow by infecting and killing bacteria, you must mix the phage and bacteria together in a culture. Nothing about the basic interaction between phages and bacteria had changed since Félix’s day, when researchers worked with a clutter of Petri dishes to culture organisms. In contemporary labs, though, where work involves sophisticated technology and is on a larger scale, Petri dishes are now microtiter plates the size of an iPhone, each with ninety-six dimpled wells for culturing bacteria and isolating phages, then culturing again to amplify the phages’ effective against the target bacteria. Walk past a few of the clear-covered plating panels laid on a lab counter, and it’s a little like an aerial view of Midwestern farm fields in late spring, a blanket of neat, dotted rows of carefully cultivated crops, row after row after row.
When the phages finish growing, you can see the glossy plaques where they’ve destroyed the bacteria. Researchers then harvest the phages with a glass pipette. After growing and harvesting many rounds from the bacterial “lawn” on the dimpled plating panels, you’ve got a flask full of phages—as many as ten billion per milliliter. But the other product of phage growth is a lot of dead bacteria, cell parts, and environmental flotsam and jetsam. Part of that cell debris is endotoxin, a toxic part of the bacterial cell wall that must be separated from the phages to minimize the risk that it might trigger septic shock in the patient. Purification also removes other potentially harmful residuals from the process. Carl later told me that in several of the early phage therapy experiments in the 1930s, phage concoctions may have killed more people than they cured, because no one knew that they needed to be purified or how to do it. These days, purifying the phages was the hardest part.
Typically, researchers put the crude phage preparations in a large centrifuge tube and then use a powerful spin cycle that forces the phage particles and other debris to sink to the bottom of the tube, leaving the debris behind. Ry fondly described his vintage 1970s centrifuge as something like a souped-up Cadillac engine in an old Maytag washer. When it was cranked up to full speed, you didn’t want to open the lid on that g-force. Once spun, the phage concentrates are dissolved in a sterile liquid, which must be done carefully to avoid damaging the phages themselves. The next step is to mix them with an organic alcohol to remove the endotoxin. The final phage prep would be tested again and treated again, if needed, to bring the endotoxin levels down.
There are a few different options for purifying phages, and the Texas and Navy labs each had their own processes, but ultimately, they’d need to meet the FDA requirement for safe levels of endotoxin in a product for human use. No one really knew how pure the phage preps needed to be for treating humans safely, so the cleaner the better. We wanted squeaky clean, virulent phages, ready to take A. baumannii down fast, and preferably an assortment that had evolved to overcome A. baumannii defenses by targeting different receptors. The more phages found that were active against Tom’s A. baumannii and the more varied their weaponry against it, the better. That would keep the bacteria engaged in a constant battle on different fronts, meaning its own pathogenic arsenal would be spread thin, increasing the chances that the phages could exploit a vulnerable flank.
In the days ahead, as the labs began their work of hunting, harvesting, and processing phages, Chip and I each had a list of administrative checkpoints requiring procedural paperwork, permissions, clinical plans, committee reviews and approvals, and legal papers that ensured that those who supplied the phages would not be held legally culpable if Tom died. We used every connection and every ounce of goodwill that we had at the university to get the approvals we needed. The paperwork for this typically took weeks or months, but we managed obtain it within two days, thanks to countless hidden heroes, those who do the chop-wood-carry-water administrative work from the inside.
In the middle of the night in early March, three weeks into the phage hunt, Jason emailed Chip and me with both good news and bad news from Texas A&M. They had tested a small sample of the Belgian phage, but unfortunately, the phages weren’t active against Tom’s A. baumannii. The good news was the team had found four phages that were active against Tom’s isolate. One was a phage from AmpliPhi Biosciences, a phage therapy startup—in San Diego, no less—specializing in the development of phage-based therapies for clinical use. AmpliPhi hadn’t surfaced in my search because I’d focused on labs that were working on Acinetobacter phages. AmpliPhi’s focus was not on these phages, but they happened to have one that originated from a patient in Australia, which they gave to CPT. When Ry contacted the AmpliPhi CEO for permission to use it in Tom’s cocktail, he instantly gave his okay and, understanding the urgency, required none of the usual paperwork.
Three other phages were found de novo, from environmental samples pooled from sewage, soil, watering holes, and poop collected from swine and cattle barns near their lab at College Station, Texas. In total, these three new phages and the phage from AmpliPhi were the first phages selected for the Texas cocktail. Even through email, Ry’s reply in the morning bristled with excitement at the realization that his lab had identified three new A. baumannii phages in just a matter of days. All they needed now was to purify and brew them. Texas tea.
On March 11, not quite three weeks from the start of the phage hunt, we received an email from Biswajit at the Navy lab. Pending the formal go-ahead, Biswajit and his lab staff had conducted preliminary tests of the A. baumannii phages in their collection against Tom’s bacterial isolate, now formally named TP1. They quickly identified ten highly virulent phages that killed Tom’s bacterial isolate. Within eighteen hours Biswajit had carefully selected the most virulent four phages they would carry forward to make their first cocktail.
Theron had some red tape to wrangle, but he found an ally in the BDRD director, Dr. Alfred Mateczun. Mateczun was a retired Navy captain who was also an MD and had a reputation for integrity and decisive action. He had been heavily involved in responding to the 2001 anthrax attack aimed at several US politicians, shifting from research to sample analysis for almost one year. When Theron contacted him about Tom’s case, Mateczun’s response was characteristically blunt.
“Well,” Mateczun said matter-of-factly, “he’s been in a coma for what, six weeks? He’s going into organ failure, and may die soon.” He boiled the rest down to two questions.
“Do we have the bacteria from the infection?”
“Yes, sir,” said Theron.
“And do we have phage that will kill it?”
“Yes, we do, sir.”
“Okay, we have nothing to lose. Send the phage.”
With that, Mateczun hit the Go button. The Navy had answered our SOS.
Now, as the Biolog automated system incubated, monitored, photographed, and charted the phage-bacteria interaction every fifteen minutes, the Navy lab harvested and cultivated the fittest of the fittest phages. From small-scale harvesting of the phages from plaques, the lab infected flasks full of A. baumannii to produce 3.6 liters—almost a gallon—of the raw mixture. A sixteen-hour spin cycle in the centrifuge produced about 10 milliliters—two concentrated teaspoons of the active phage preparation, called lysate—and they were good to go.
I’d only worked with phages in one undergrad class, and it wasn’t particularly memorable—
except for the intense smell of those bacterial cultures we’d used in phage experiments. That was unforgettable. The Petri dishes were incubated at body temperature in what resembled a microwave. When you opened the door, a waft of warm air rushed out and you were overcome with an odor something like a mix of old socks, armpits, and decay. When I heard one day that Biswajit had joked about how smelly the lab had become, I had to laugh. I remembered the long nights babysitting putrid plates of bacterial broth. But to a diehard phage researcher, that’s the smell of potential. And Biswajit was a guy who lived and breathed phages. A sewage treatment plant in his community wasn’t far away, and he remarked that on some days, when the blistering summer temperatures were just right, he’d catch a whiff and think, “Ah, it’s a perfect day to hunt for phages!”
As I worked through the formal paperwork required of me as Tom’s wife, small things constantly reminded me of my strange double life as scientist and spouse. I was now over any hesitation about pressing in my professional capacity for information or procedural responses to expedite the administrative approval processes. Meanwhile, every form approving treatment or interventions for Tom called for me to sign as his spouse, “the patient’s wife.” Just seeing the words made my stomach clench. Every minute of every day was a walk on a high wire, emotionally. I could put one foot in front of the other just well enough—as long as I didn’t look down. Focusing strictly on what needed to be done next seemed to work: Scientist-Me could maintain a semblance of objectivity and detachment, and Wife-Me was able to stop short of the unthinkable outcome of Tom dying.
The Perfect Predator Page 19