VA RESEARCH: DRUG-RESISTANT INFECTIOUS DISEASES

By Craig Collins

THE DEPARTMENT OF VETERANS AFFAIRS’ (VA) infectious disease research program is all encompassing, involving fundamental investigations of what makes a bacterium, virus, fungus, or parasite into a disease-causing pathogen; of how infectious diseases are passed from person to person; and of the effectiveness of preventive strategies, vaccines, and drugs. In recent years, the infectious disease research community has turned much of its attention to multidrug-resistant organisms (MDROs), a group of pathogens that has led to an increase in microbial infections over the last few decades.

For a number of reasons, our arsenal of antimicrobial drugs, so effective for decades in treating infectious microorganisms, has not evolved – but in the meantime, the organisms they were designed to kill have adapted. The U.S. Centers for Disease Control and Prevention (CDC) estimates that at least 2 million people are infected by MDROs annually, and that at least 23,000 die each year as a result of these infections. An additional 15,000 die every year from Clostridium difficile (C. diff), a pathogen associated with long-term antibiotic use in health care settings.

MDROs are a significant concern for the VA medical community, whose patient population is statistically older and more vulnerable to infection than other Americans due to battle injuries, comorbid diseases, and other factors. Many MDROs are implicated in health care-acquired infections (HAIs), which occur when pathogens colonize and linger in health care settings. One of the most virulent MDROs, methicillin-resistant Staphylococcus aureus, or MRSA, can survive for more than nine months on inadequately disinfected surfaces. About three-fourths of all HAIs are from organisms that resist first-line antibiotics. A 2012 study conducted by the Alliance for Aging Research found that 99,000 Americans died of HAIs annually, and that MRSA infections killed more than emphysema, HIV/AIDS, Parkinson’s disease, and homicide combined.

The VA’s attack on MDROs is two pronged. Its MDRO Prevention Initiative, a collection of procedural guidance for professionals throughout the Veterans Health Administration (VHA), has greatly reduced infection rates for organisms such as MRSA and C. diff throughout its health care facilities. Updates and improvements to the program continue, along with a robust research program into effective infection control techniques and protocols.

In the absence of effective antimicrobial treatments, VA investigators are also probing the weaknesses of these MDROs in basic research that may lead to effective vaccines or drug treatments. Researchers are focusing particular attention on a group of organisms known as the ESKAPE pathogens, highly resistant pathogens responsible for the majority of HAIs:

• Enterococcus faecium (E. faecium)

• Staphylococcus aureus (S. aureus)

These two organisms are unusual for Gram-positive bacteria, which respond to Gram staining due to their lack of a rugged outer cell membrane. Gram-positive bacteria have historically been vulnerable to penicillin-derived antibiotics such as methicillin, but E. faecium and S. aureus have evolved a resistance to these and other drugs.

• Klebsiella pneumoniae

• Acinetobacter baumannii

• Pseudomonas aeruginosa

• Enterobacter species These four Gram-negative bacteria have a built-in defense against many classes of antibiotics. Broad-spectrum “lastline” antibiotics, such as carbapenems or cephalosporins, that used to be effective against these organisms are losing their edge, and in many cases, are contributing to their evolution into “superbugs.”

VA researchers, however, are discovering new vulnerabilities and possible modes of attack against these organisms, often at the molecular level.

The increasing resistance of infectious organisms to known antibiotics has led investigators to explore other ways of penetrating and killing pathogens. In some cases, promising treatments have been suggested by findings in cancer research.

“One of the biggest areas of research over the last 10 years that has transformed cancer treatment, to some degree,” said Robert Striker, M.D., Ph.D., associate professor at the University of Wisconsin School of Medicine and a researcher with the William S. Middleton Memorial Veterans Hospital, “has been a class of drugs called kinase inhibitors.” Kinase, an enzyme, works as a kind of switch to turn certain cell functions, such as protein formation, on or off. In cancer cells, kinases have become “dysregulated,” leading to unchecked cell growth. Kinase inhibitors target this dysregulation, essentially turning the switch to the “off” position.

According to Striker, there are more than 500 kinases at work in human cells, and cancer researchers have so far formulated about 25 FDA-approved kinase inhibitors that will target specific switches on cancer cells, slowing or stopping tumor growth while leaving the rest of the body’s cells alone. In his laboratory, Striker’s team has discovered that some of these drugs can hit a kinase switch on the MRSA organism and re-sensitize it to methicillin, which kills S. aureus by preventing it from building a cell wall.

Methicillin is one of a family of antibiotics known as the beta-lactams, commonly denoted as β-lactams, which also include penicillins, cephalosporins, carbapenems, and monobactams. One of the oldest and most successful classes of antibiotics, the β-lactams have also contributed significantly to the evolution of drug resistance among pathogenic organisms. Striker and his team have used kinase inhibitors to re-sensitize other organisms, such as Streptococcus pneumoniae and Listeria monocytogenes, to β-lactams.

The switch targeted by Striker is known as the PASTA kinase: “A penicillin-associated serine/threonine kinase,” said Striker. “The acronym doesn’t quite work.” It’s unique to Gram-positive organisms, including some, such as tuberculosis, that haven’t been treated with β-lactam antibiotics in the past – but which can be made sensitive to them now, Striker said, with the use of kinase inhibitors. “We’re not just creating new antibiotics,” he said, “but we’re creating antibiotics that will rescue some of the old antibiotics and make them more active against bacteria that contain these PASTA kinases.”

In her work as chief of the Division of Infectious Disease at the Stony Brook University School of Medicine and as a researcher with the Northport VA Medical Center, Bettina Fries, M.D., has studied the effectiveness of fighting Klebsiella pneumoniae, the most common drug-resistant Gram-negative bacterium, with a cancer treatment pioneered by Nobel laureates in the 1970s: hybridoma technology, which boosts the immune system’s ability to attack and kill invasive cells.

When an animal is injected with a substance that provokes an immune response, specialized white bloods cells known as B cells produce antibodies that bind to the injected antigen, and then antibody-producing B cells are then harvested from the spleen of the animal – typically, a mouse. These B cells are then fused with mutated B cells known as myelomas, producing a cell line called a hybridoma, which both produces antibodies and reproduces like a cancer cell. These hybridomas, also known as monoclonal antibodies, are chemically identical and built to target specific cells.

Klebsiella is an organism that’s often harmless, commonly found on the skin or in the mouth or gut, but can turn virulent and, when found in the lungs, is a major cause of HAIs. It has developed a resistance to broad-spectrum carbapenems, one of the last lines of defense in treating MDROs in hospital patients, and is associated with high mortality rates.

Fries and her colleagues are studying ways to get around this drug resistance by boosting the body’s own immune response, creating monoclonal antibodies that target the polysaccharide capsule of Klebsiella, part of the bacterium’s outer envelope. “When the antibody binds to the bacteria, then the bacteria are taken up by inflammatory cells in the patient and killed,” said Fries.

People make antibodies on their own all the time, when they’re vaccinated or infected, but in many Klebsiella infections, Fries said, the antibodies come too late. “If you can make the antibodies in the laboratory and give them to the patient early in the infection, when they haven’t made their own yet, you can help them fight the infection.” So far, her team has produced monoclonal Klebsiella antibodies in mice, and are now investigating ways of “humanizing” the antibody, or changing its protein structure to make it match the structure of a human B cell. “Once you humanize the antibodies,” she said, “then you have a product you can get FDA approved for use in human patients.”

An ESKAPE pathogen of particular concern to VA researchers is Acinetobacter baumannii, a Gram-negative bacterium that can form biofilms and adhere to surfaces for extended periods of time, or become airborne in water vapor. A. baumannii’s natural habitat is still unknown, as it’s rarely found outside hospital environments. Last year, the World Health Organization named multidrug-resistant A. baumannii as one of its three most critical priorities. In health care settings, the organism is notoriously difficult to eradicate, and has evolved a vigorous resistance to antibiotics, including carbapenems.

In the mid-2000s, A. baumannii was the cause of an outbreak among wounded service members returning from the Middle East, particularly from Iraq, earning it the nickname “Iraqibacter.” As patients moved from one level of care to another – from forward facilities to Landstuhl Regional Medical Center in Germany to stateside hospitals such as Walter Reed Army Medical Center and Brooke Army Medical Center – many became infected and brought the organism with them, where it infected others.

A. baumannii’s strong antibiotic resistance has turned an organism with a relatively low level of virulence into a killer, said Philip Rather, Ph.D., professor of microbiology at Emory University School of Medicine and a research career scientist at the Atlanta VA Medical Center. Estimates of the mortality rates of A. baumannii-infected patients vary widely; Rather puts it at between 25 and 60 percent, depending on the patient population. Most who are infected with A. baumannii are already sick or wounded, “so, they’re already weakened to begin with,” Rather said. “And some of these strains now are just very difficult to treat with antibiotics. And in fact, some strains are completely resistant to every available antibiotic.”

As Rather and his investigative team have discovered, one of the features that makes A. baumannii so dangerous is its ability to switch back and forth from an avirulent form – one that does not cause disease – to a virulent pathogen. Either form will be attacked by the body’s immune system, he said, but the virulent form typically survives by throwing up defenses. At lower temperatures in the surrounding environment, Rather’s team believes, the avirulent form has a survival advantage, lying low until it’s taken up by a host.

In spring 2018, Rather and his team at Emory’s Antibiotic Resistance Center, which includes another VA investigator, David Weiss, Ph.D., reported not only that they’d discovered how A. baumannii performs this switch – with a regulatory gene, ABUW_1645 – but also that they’d figured out how to manipulate that switch, turning the virulent form back into an avirulent one. “We’ve now been able to show that when you lock cells into that avirulent form,” Rather said, “they act as an incredibly effective vaccine, at least in animal models.”

After Rather’s team has demonstrated the vaccine’s effectiveness and ability to keep the avirulent cells “locked” in animal models, it hopes to move on to clinical trials. The ability to flip A. baumannii’s virulence switch may also enable treatments for already infected patients. “We could identify chemicals we could treat humans with,” he said, “and if the chemical causes all the virulent cells to switch to avirulent, our immune system would clear them almost immediately, and it could be a next generation of therapies for this bacterium.”

Among the first VA researchers to study A. baumannii isolates from U.S. military hospitals was Robert A. Bonomo, M.D., professor at Case Western Reserve University (CWRU) School of Medicine and chief of medical service at the Louis Stokes Cleveland VA Medical Center. By mapping the genes of these organisms, Bonomo and Dr. Mark Adams, also of CWRU, found that not only were A. baumannii strains evolving to become more resistant, but that several strains had multiple genes conferring resistance to multiple antibiotics. “There were some isolates that had never seen certain antibiotics before,” he said, “that were already resistant to them, even before those antibiotics were used to treat them.”

Bonomo and Adams mapped out the entire genome of one of these early multidrug-resistant organisms from the outbreak at Walter Reed Army Medical Center, and have now published about 70 papers on A. baumannii, attempting to understand its resistance genes and looking at novel combinations to treat multidrug-resistant strains. Two years ago, they were part of a U.S.-Argentinian study that discovered how an A. baumannii bacillus and other MDROs could make themselves immune to antibiotics they had never seen before: They had devised several ways of sharing genes with other bacteria, one of which was a new wrinkle in bacterial genetic exchanges. The team, led by Dr. Alejandro Vila, observed MDROs sloughing off little bits of their protective outer membranes and sharing them with other bacteria: “… kind of like little blebs of lipids, with enzymes or genes in them,” said Bonomo, “that go off the surface of MDROs onto the surface of another cell and fuse with the surface of that cell, and directly transfer the gene, as well as the protein, over to the other recipient.”

Understanding this particular defense mechanism may aid researchers in designing an attack, and Bonomo, Vila, and colleagues have since widened their focus. “We’ve also, with the support of the VA, tried to understand how other carbapenem-resistant bacteria, or bacteria that are resistant to our last-resort antibiotics, evolved.” They’ve studied carbapenem-resistant K. pneumoniae and P. aeruginosa, and recently began working with Brad Spellberg, M.D., an immunologist at the University of Southern California, to generate a monoclonal antibody that might be used as a vaccine against A. baumannii. In collaboration with Rather at Emory, Bonomo also studies the impact of new drugs on bacteria with altered transport systems.

Bonomo is a standout among VA researchers: In 2017, he received the William S. Middleton Award, the highest honor awarded annually by the department’s Biomedical Laboratory R&D Service to senior research scientists for their outstanding contributions in areas of prime importance to the VA research mission. But like many VA investigators, Bonomo is also a clinician, and he and his colleagues never lose sight of what this micro-level research is all about: “The people who do research in the VA are very dedicated to the welfare of their patients, and are hopeful of eventual cures. Our lab is driven not just by science, but by a desire to bring good therapies to patients, to prolong life, and mitigate suffering,” he said. “We’re privileged to take care of veterans.”