The Basics

Clostridium difficile (320x240)
credit: CDC/ James ArcherIllustration of a Clostridium difficile bacterium

Antibiotic resistance is the ability of bacteria to resist the effects of antibiotics, a type of drug - such as penicillin or ciprofloxacin - that kills or stops the growth of bacteria. When bacteria become resistant to antibiotics, it makes it more difficult to treat diseases caused by those bacteria and limits the treatment options. Alternative treatments may be costly and, in some cases, toxic. 

Other classes of microorganisms can also become resistant to the drugs that are used to treat them (antibiotics are effective against bacteria, but not most other types of microorganisms). For example, viruses can develop resistance to antivirals and fungi can become resistant to antifungals. Antimicrobial resistance is the general term used to refer to the resistance of microorganisms to drugs used to treat disease. Microorganisms that develop drug resistance are sometimes called "superbugs".

The resistance of some infections to effective treatment with existing antibiotics is allowing some infectious diseases, that previously could be brought under control, to re-emerge.

History of antibiotics

The first antibiotic, penicillin, was discovered by Sir Alexander Fleming in 1928, but it was not widely distributed until 1945 when it used to treat infections among soldiers during World War II. At the time, it was considered a "miracle drug." Hope for a future free from infectious diseases was short-lived, however, because by the 1950s resistance to penicillin had become a substantial clinical problem.

New antibiotics, such as tetracycline, erythromycin, and methicillin were developed, and there was new hope that infectious diseases could be vanquished. But, once again, problems of bacterial resistance became evident. In 1962, the first case of methicillin-resistant Staphylococcus aureus (MRSA) was identified. Resistance of certain bacteria to other antibiotics was also reported.

For several decades starting in the late 1960s, many new antibiotics, of different classes that target different bacterial components or processes, were introduced, although in more recent decades, development of new antibiotics has slowed. Eventually, however, resistance was observed for virtually all available antibiotics. Therefore, some bacterial infections that had been treatable with antibiotics in the past, but have now become resistant, are an emerging threat.

Causes of antibiotic resistance

Antibiotic resistance is a natural process that occurs over time when antibiotics are used, usually due to genetic changes in the bacteria. However, antibiotic resistance is being accelerated through the overuse and misuse of antibiotics. The overuse of antibiotics can kill off the drug-sensitive bacteria and allow the resistant bacteria to remain and flourish.

Taking antibiotics for a viral infection like a cold or the flu (for which antibiotics are ineffective) is an example of misuse of antibiotics. Another example is giving antibiotics to healthy animals to promote their growth and prevent disease. Antibiotic use in farm animals allows the resistant bacteria to thrive within the animals, and then the drug-resistant bacteria can be transferred to humans when they consume meat products. As a result, infections in humans harboring resistant forms of bacteria become more difficult to treat.  

Other factors that allow the spread of antibiotic resistance are poor infection control, inadequate sanitary conditions, and inappropriate food handling. In the absence of proper hygienic conditions, resistant strains of bacteria can spread from person to person. 

Mechanisms of antibiotic resistance

Bacteria employ a variety of strategies to modify a bacterial component or process so that antibiotics can no longer stop their growth or kill them. One means is to restrict entry of the antibiotic into the cell by limiting or changing the size of the openings in the cell wall. Other bacteria can use pumps in their cell walls to eliminate drugs that have already entered.

Some resistant bacteria use enzymes to break down and destroy the antibiotic or alter the drug in some way so that it is no longer functional.

To counteract antibiotics that are designed to disrupt an essential process that bacteria need to survive, such as the process of making nutrients, resistant bacteria can develop a new, alternative way of performing the same function. Some bacteria can overcome antibiotics that are designed to target a specific component of the bacterium by changing the look of the target so that it is no longer recognized by the drug.

These resistance mechanisms are encoded in their genes. Often, the genes are found on plasmids; these circular pieces of DNA can be passed from one bacterium to another within the same species and sometimes across bacterial families. This allows the resistance mechanism to be transferred to other bacteria.

Infections resistant to antimicrobial drugs

Antibiotic resistance has become a widespread problem in the United States - and worldwide - as a growing number of infections become resistant to the drugs that have been used to treat them. The list includes pneumonia, tuberculosis, gonorrhea, blood poisoning, and food-borne diseases.

Resistance to drugs used to treat infections caused by other types of microorganisms, such as viruses (for example, HIV), parasites (malaria), and fungi (Candida auris) is posing a further challenge to public health.

The Centers for Disease Control and Prevention has identified the top 18 drug-resistant threats to the United States and classified them into three categories based on their level of concern. The highest risk categories, "urgent" and "serious," require more monitoring and prevention activities than those in the "concerning" group. The list is re-evaluated periodically and modified as new threats emerge. The CDC is expected to release a new list in the fall of 2019. 

CDC Categorization of the top drug-resistant threats in the United States

Hazard Level

Examples

Urgent

Clostridium Difficile

Carbapenem-Resistant Enterobacteriaceae

Neisseria gonorrhoeae

Serious

Multidrug-Resistant Acinetobacter

Extended Spectrum Enterobacteriaceae

Vancomycin-Resistant Enterococcus

Methicillin-Resistance Staphylococcus aureus

Drug-Resistant Tuberculosis 

Concerning

Vancomycin-Resistant Staphylococcus Aureus

Erythromycin-Resistant Streptococcus Group A

The Problem

Antibiotic resistance is one of the biggest threats to public health today. The CDC estimates that each year at least 2 million people in the United States become infected with bacteria that are resistant to antibiotics and that, at a minimum, 23,000 people die as a direct result of these infections.

As an increasing number of infections become resistant to available antibiotics (and antivirals and antifungals), the ability to effectively prevent and treat a growing list of diseases is being compromised. These diseases become harder, and sometimes impossible, to treat. The diminishing number of effective antibiotics also increases the risk of many medical procedures including surgery, chemotherapy, and organ transplants. 

When first-line antibiotics can no longer be used to treat infections, alternate drugs must be used which are more expensive and require a longer course of treatment, often resulting in longer hospital stays. In some cases, the alternate antibiotics may be more toxic. Together, these factors place a considerable economic burden on the patients, their families, and the healthcare system. 

Unfortunately, the development of new antibiotics has been scaled back. Research to develop new antibiotics and bring them to market is expensive and generally not as profitable to the pharmaceutical industry as are other drugs.

The Research

E. Coli (320x240)
credit: CDC/ James ArcherIllustration of drug-resistant E. coli

The problem of antibiotic resistance is an active area of research within the department. Some scientists are investigating the details and mechanisms of how bacteria acquire resistance to different groups of antibiotics. Others are looking for new ways to combat infections that resist the available antibiotic drugs. One of these groups is employing bacteriophages, while another group is working towards developing a probiotic cocktail that could be used to treat drug-resistant diseases. 

Resistance to fluoroquinolone antibiotics

Dr. Lynn Zechiedrich has been studying the problem of antibiotic resistance for over 25 years. One research focus in her laboratory has been toward understanding the mechanism of action of and the mechanisms of resistance to the antibiotics known as fluoroquinolones, some of the most potent, widely prescribed, and broad-spectrum antibiotics in use world-wide. The fluoroquinolone class of antibiotics includes ciprofloxacin and levofloxacin, among others.

The fluoroquinolones act by targeting two essential bacterial enzymes known as topoisomerases - gyrase and topoisomerase IV - which help control the winding and unwinding of DNA strands during important cellular processes such as DNA replication, recombinationtranscription, and chromosome segregation. During these cellular processes, DNA strands are temporarily broken by topoisomerases. The broken DNA intermediate form is normally short-lived, but in the presence of the fluoroquinolone antibiotics, the intermediate form is stabilized, resulting in bacterial cell death. The Zechiedrich laboratory uses biochemical, biophysical, and genetic approaches to determine how topoisomerases carry out their cellular roles and how drugs block their function. 

In another line of research, Dr. Zechiedrich and colleagues are investigating whether the drug ciclopirox, an off-patent anti-fungal agent, could be repurposed as a candidate for antibiotic use, particularly against multidrug resistant gram-negative bacteria. The ability to use a drug designed to combat one type of microorganism for use against another could circumvent the time and money associated with developing a new drug.

The Zechiedrich group reported that ciclopirox inhibited the growth of even multidrug-resistant gram-negative bacteria Acinetobacter baumanniiEscherichia coli, and Klebsiella pneumoniae. They further investigated the mechanism of action and found that the drug affected sugar metabolism in the bacteria, altering the composition of molecules in the outer membrane of the bacterial cell. In their current research in this area, they are pursuing the drug target as a potentially new target for new antibiotic development.

Resistance to beta-lactam antibiotics

Beta-lactam antibiotics are the most widely used class of drugs for the treatment of bacterial infections. They include penicillin and its derivatives, such as methicillin and amoxicillin, as well as other groups of antibiotics known as the cephalosporins, carbapenems, and monobactams. The beta-lactam ring portion of the antibiotic targets the penicillin-binding proteins (PBP), found in the bacterial cell membrane, which function in the synthesis of the cell wall. Binding of the antibiotic to the PBPs prevents the PBPs from performing their essential role and results in the death of the bacterial cell.

Dr. Timothy Palzkill, professor of Pharmacology and Chemical Biology and Molecular Virology and Microbiology, and his research team have been studying mechanisms of resistance to the beta-lactam antibiotics. In gram-negative bacteria, the most common mechanism of resistance is the hydrolysis, or breaking apart, of the antibiotics by enzymes referred to as the beta-lactamases. There are two broad classes of beta-lactamases, the serine-β-lactamases and the metallo-β-lactamases. Dr. Palzkill and his group use a variety of advanced genetic, biochemical, and physical techniques to understand details about the structure and function of both groups of the beta-lactamases and their interactions with drug-resistant forms of antibiotics that are subject to drug resistance.

Instead of drug resistance through the action of beta-lactamases, gram-positive bacteria acquire resistance to beta-lactam antibiotics through the production of a protein called PBP2a, which is able to avoid the inhibitory effects of the antibiotics. This is the mechanism by which methicillin-resistant Staphylococcus aureus (MRSA) is able to persist despite treatment with multiple beta-lactam antibiotics. Dr. Palzkill and coworkers conducted a study in which they found that the protein BLIP-II was able to weakly bind and inhibit PBP2a, making it susceptible to beta-lactam antibiotics. They are continuing this line of research by searching for mutations that increase the affinity of BLIP-II to PBP2a.

Resistance to colistin 

The reduction in treatment options due to the increased prevalence beta-lactamases that break down beta-lactam antibiotics has led to the increased use of polymyxin antibiotics such as colistin. Polymyxins are polypeptides that act by binding to and subsequently disrupting the bacterial membrane. The recent emergence and spread of a plasmid-encoded, transferable colistin resistance gene, mcr-1, is a cause for concern. The mcr-1 gene encodes an enzyme, MCR-1, that modifies a component of the membrane and blocks colistin binding. 

Dr. Palzkill and his group have determined the X-ray structure of one important functional region of the MCR-1 protein. In order to more fully understand the mechanism of MCR-1, they are working to determine the structure of the full-length MCR-1 enzyme. In addition, the laboratory is working to discover inhibitors of the MCR-1 enzyme that would circumvent resistance and broaden treatment options for colistin.

Use of bacteriophages to combat antibiotic-resistant bacteria

Bacteriophages are viruses that specifically kill bacteria. Drs. Anthony Maresso,  Frank Ramig, and Barbara Trautner and their colleagues have been investigating the feasibility of using bacteriophages, or phages, to combat drug-resistant bacteria. This idea was originally proposed by Felix d'Herelle in 1926, but following the discovery and initial successes of antibiotics, this approach was largely dropped (although several Eastern European nations have used this approach to successfully treat some bacterial infections). 

Given the current limitations in treating drug-resistant bacterial infections, the researchers have revisited this idea and set about to determine whether phages can be effective at killing a large group of bacteria that are resistant to antibiotics. Their bacterial target was a specific group of Escherichia coli, called ST131, that colonize the gastrointestinal tract, but can infect sites outside the intestines (this category of E. coli is known as extraintestinal pathogenic E. coli), and is considered the predominant cause of all antibiotic-resistant E. coli infections in the United States. The bacteria are multi-drug resistant, in addition to producing deadly virulence factors; failure to control their growth can lead to sepsis, which can be fatal. 

In their study, they sought to identify phages that would kill 12 strains of antibiotic-resistant bacteria that were isolated from patients. They did this by first isolating phages from the feces of birds and dogs, which are known to be reservoirs for the E. coli ST131 bacteria, and then testing to see if the phages lysed, or killed, the bacteria in a laboratory test. Although no single phage could kill all 12 bacterial strains in lab cultures, they found combinations of two or three phages that were effective against all the bacteria they tested.

Next, they tested the phages to find out if they could also kill the antibiotic-resistant bacteria in a mouse model of sepsis. When delivered into the animals, the phages reduced the levels of bacteria and substantially improved the health of the mice. The results demonstrated that phages isolated from the environment, with little experimental manipulation, can be effective in combating even very serious infections by E. coli superbugs.

There are several advantages to using phages instead of antibiotics to combat bacterial infections. Phages do not infect human cells. They are very specific for certain species or strains of bacteria, so that they can be used to target the "bad" bacteria, while not harming the "good" intestinal microbiota. However, they can be made to act broadly via cocktails, if desired. In addition, phages can evolve, so should resistance against a set of phages develop, new phages could be identified in the environment or evolved in the laboratory in a matter of days, unlike antibiotics which can take many years, at great cost, to develop. While the scientists are still somewhat cautious about this approach, as sometimes a host's immune system can neutralize the activities of phages and some phages may not work well in animals, they are continuing to explore this option.

In another study, these researchers found that certain metals enhance the killing of E. coli ST131 bacteria by phages in blood. They observed that when they treated E. coli ST131 with phage, the phage effectively killed the bacteria in culture medium but not in blood. The blood samples contained a chemical called EDTA which was used to prevent clotting and is known to bind to metals. They saw more efficient bacterial killing in blood treated with heparin, a natural anti-clotting factor, suggesting that the differing outcomes may be due to the level of metals in the blood. 

When they added the metals calcium, magnesium, and iron, which are commonly found in blood, they found that the inhibition of ST131 killing by EDTA was overcome by the addition of the metals. Furthermore, metal-enhanced killing was observed for several other strains of extraintestinal pathogenic E. coli, not only ST131. Metals also enhanced ST131 killing in a mouse model system. This work points to the essential role of metals for bacterial killing by phage in blood.

Antibiotic treatment alters the intestinal microbiota resulting in hard-to-treat Clostridium difficile infections

Clostridium difficile is a gram-positive, spore-forming bacterium that is considered to be one of the three highest risk drug-resistant infections in the United States, as classified by the CDC. It is an opportunistic infection that infects the colon of patients following antibiotic treatment. The microbiota that inhabit the gut normally prevent C. difficile colonization and suppress C. difficile-associated disease, but treatment with antibiotics results in changes to the composition of microbiota that allow C. difficile to grow and cause disease.

C. difficile produces toxins that damage intestinal cells and cause inflammation, producing diarrhea, and can be fatal. Of nearly 500,000 infections each year, approximately 29,000 result in death. It is the most common cause of hospital-acquired infections in developed countries. The infection is very difficult to treat, with many patients suffering from recurrent infections. The bacterium is naturally resistant to many common antibiotics, such as the fluoroquinolones, so investigators are searching for alternate ways to treat the disease. 

Dr. Robert Britton and his research group are interested in understanding how the intestinal microbiota provides a barrier to incoming pathogens and how perturbation of the microbiota can result in infections, primarily C. difficile infectionsThey have developed mini-bioreactors and mice colonized with a human intestinal microbiota to determine which members of the microbial community are responsible for inhibiting C. difficile invasion. Their goal is to develop a probiotic cocktail, derived from the human intestinal microbiota, that will suppress C. difficile invasion.  

Researchers have found that two C. difficile lineages, RT027 and RT078, have become more predominant and virulent in the last couple of decades, causing major outbreaks. To identify factors that have enhanced the virulence of these two lineages, Dr. Britton and his colleagues investigated what sources of food RT027 and RT078 preferred. Their study showed that the virulent lineages were highly efficient in their use of the dietary sugar trehalose (which is found in diet soda and is used to stabilize processed foods), giving these microbes a competitive advantage over other, less virulent bacteria.

Using their mouse model, they found that the mice receiving trehalose in their diet had more severe disease, with higher mortality rates, and they produced higher levels of toxins. These results suggest that the introduction of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to virulence. This study further demonstrates that diet can influence the composition of the microbiota and response to infection.