Infection treatment without anti-biotic

Infection treatment without anti-biotic

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When we have some bacterial infection, say throat infection (pharyngitis) we take antibiotics as treatment. I was wondering how throat infection was treated when there was no antibiotics at all, before 1940's I think.

Throat infection is just example, It can be post surgical operation, urinary track infection etc, basically anything where we use antibiotics currently. So in a way replacement of antibiotic tablet when antibiotic wasn't available.

Interestingly, treatment of mild pharyngitis before the advent of antibiotics wasn't too different from what it is today - it was (and still is) mostly symptomatic. First of all, viral pharyngitis, which is quite common (Hidreth et al., 2015), is not affected by antibiotics (no viral infection is), and they are not indicated in such situations. Second, mild and self-limiting bacterial infections, including pharyngitis, most often do not warrant prescription of antibiotics, as this leads to the growing problem of antibiotic resistance, which, according to the World Health Organization is "one of the biggest threats to global health." Many guidelines can be quoted, urging clinicians to prescribe antibiotics sparingly, but pharyngitis is so common (literally everyone has had it, multiple times) that one can simply turn to one's own experience - simply put, when you have a mild case of soar throat, your doctor doesn't give you antibiotics (if you even bother going to the doctor about it), most often you are advised to take only symptomatic medications (many of which are OTC). These most often include compounds with analgesic, antiseptic, and/or anti-inflammatory properties. Interestingly, such compounds were widely available long before the advent of antibiotics and even the pharmaceutical industry itself.

Menthol, for example, is a selective agonist of κ-opioid (Galeotti et al., 2002) and a modulator of GABBAa receptors (Watt et al., 2008), thereby eliciting analgesic and anaesthetic effects, respectively. Moreover, by disrupting bacterial cell membranes, menthol (and similar compounds, such as thymol, for example), elicit antibacterial activity, as well (Trombetta et al., 2005). Thus, wild mint, peppermint, and thyme are much more often indicated in cases of soar throat than antibiotics! Their use in the form of infusions and decoctions likely predates most, if not all, modern countries. Moreover, the analgesic and anti-inflammatory properties of willow bark, which are due to the salicylic acid contained therein, were known to Hippocrates (Norn et al., 2009), quite possibly dating back much earlier. Furthermore, it is known that caffeine potentiates the analgesic effects of many nonsteroidal anti-inflammatory drugs, including salicilic acid (Granados-Soto and Castañeda-Hernández, 1999). Unsurprisingly, this combination is often found in many modern drugs (many OTC), being marketed all over the world. Whether this effect was known before the advent of the pharma industry, however, is hard to say. Willow bark has been used as an analgesic and anti-inflammatory medication since ancient times, but coffee was brought to the West in the XVI century.

This is only a handful of traditional medications for pharyngitis, and I'm sure many more can be found, as well as many other treatments for many other conditions. The point is that treatments of mild pharyngitis were available before the advent of antibiotics, as a matter of fact, many of those treatments are still first-line therapy in most cases, even to this day. Bacteriophages. Institute in Georgia

Bacteriophages were used to treat infectious diseases in some places, most notably the Republic of Georgia, before antibiotics became popular. The advent of antibiotics, which many thought heralded the end of infectious diseases, unfortunately resulted in less interest in therapeutic bacteriophages.

'Anti-antibiotic' allows for use of antibiotics without driving resistance

IMAGE: Researchers have found that an inexpensive, FDA-approved drug for treating cholesterol -- cholestyramine -- taken in conjunction with an antibiotic prevents the antibiotic from driving antimicrobial resistance in the gut. view more

Credit: Andrew Cheshire, Penn State

UNIVERSITY PARK, Pa. -- An inexpensive, FDA-approved drug -- cholestyramine -- taken in conjunction with an antibiotic prevents the antibiotic from driving antimicrobial resistance, according to new research by scientists at Penn State and the University of Michigan. The team's findings appear today (Dec. 1) in the journal eLife.

"Antimicrobial resistance is a serious problem that has led to people dying from common bacterial infections," said Andrew Read, Evan Pugh Professor of Biology and Entomology and director of the Huck Institutes of the Life Sciences, Penn State. "Many of our most important antibiotics are failing, and we are beginning to run out of options. We have created a therapy that may help in the fight against antimicrobial resistance, an 'anti-antibiotic' that allows antibiotic treatment without driving the evolution and onward transmission of resistance."

According to Valerie Morley, postdoctoral scholar in the Huck Institutes of the Life Sciences, Penn State, an important cause of antibiotic-resistant infections in healthcare settings is vancomycin-resistant [VR] Enterococcus faecium.

"E. faecium is an opportunistic pathogen that colonizes the human gastrointestinal tract and spreads via fecal-oral transmission," she said. "The bacterium is asymptomatic in the gut but can cause serious infections, such as sepsis and endocarditis, when introduced to sites like the bloodstream or the spinal cord."

Morley noted that daptomycin is one of the few remaining antibiotics to treat VR E. faecium infection, yet VR E. faecium is quickly becoming resistant to daptomycin as well. Daptomycin is administered intravenously to treat infections caused by VR E. faecium. The antibiotic is mostly eliminated by the kidneys, but 5-10% of the dose enters the intestines, where it can drive the evolution of resistance.

To investigate whether systemic daptomycin treatment does, indeed, drive an increase in daptomycin-resistant VR E. faecium, the team inoculated mice orally with different strains of daptomycin-susceptible VR E. faecium. Beginning one day after inoculation, the researchers gave the mice daily doses of either subcutaneous daptomycin, oral daptomycin or a control mock injection for five days. The team used a range of doses and routes of administration, including those that would be similar to clinical human doses, to maximize the likelihood of observing resistance emergence. Next, they collected fecal samples from the mice to measure the extent of VR E. faecium shedding into the environment and to determine daptomycin susceptibility of the E. faecium bacteria that were present in the feces.

The researchers found that only the highest doses of daptomycin consistently reduced fecal VR E. faecium below the level of detection, whereas lower doses resulted in VR E. faecium shedding. From the bacteria that were shed, the team found that one strain acquired a mutation in a gene that had previously been described in association with daptomycin resistance, while another acquired several mutations that had not previously been associated with daptomycin resistance.

"Our experiments show that daptomycin resistance can emerge in E. faecium that has colonized the GI tract, and that this resistance can arise through a variety of genetic mutations," said Morley.

The team also observed that daptomycin-resistant bacteria were shed even when the daptomycin was administered subcutaneously.

Finally, the team investigated whether the orally administered adjuvant cholestyramine -- an FDA-approved bile-acid sequestrant -- could reduce daptomycin activity in the GI tract and prevent the emergence of daptomycin-resistant E. faecium in the gut. They found that cholestyramine reduced fecal shedding of daptomycin-resistant VR E. faecium in daptomycin-treated mice by up to 80-fold.

"We have shown that cholestyramine binds the antibiotic daptomycin and can function as an 'anti-antibiotic' to prevent systemically administered daptomycin from reaching the gut," said Read.

Amit Pai, professor and chair of the Department of Clinical Pharmacy, University of Michigan, noted that no new strategies have been developed to reduce antimicrobial resistance beyond the use of combination therapy, the development of vaccines for upper and lower respiratory tract infections and simply reducing the unnecessary use of antibiotics.

"These are blunt instruments for antimicrobial resistance reduction at the population level but do not readily translate to an intervention that can be used in individuals," said Pai. "Reducing selective antibiotic pressure on bacteria that reside in the colon is a potential individual-level strategy that deserves greater attention."

Other Penn State authors on the paper include Derek Sim, senior research assistant Samantha Olson, undergraduate student Lindsey Jackson, undergraduate student Elsa Hansen, assistant research professor Grace Usher, graduate student and Scott Showalter, professor of chemistry. Authors from the University of Michigan include Clare Kinnear, postdoctoral research fellow, and Robert Woods, assistant professor of internal medicine.

The Penn State Eberly College of Science and the Eberly Family Trust supported this research.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.

Molecular tweezers that attack antibiotic resistant bacteria

Prof. Raz Jelinek (left) and his student Ravit Malishev. Credit: Dani Machlis/BGU

Researchers from Ben-Gurion University (BGU), together with American and German colleagues, have developed new "molecular tweezers" to combat antibiotic-resistant bacteria. Their recently announced findings were published in Cell Chemical Biology.

For years, medical professionals have struggled with bacterial infections becoming increasingly resistant to antibiotics. These molecular tweezers may be the key to battling one of greatest public health issues of the 21st century.

"Our discovery prevents infection without building up antibiotic resistance, and it might even be preferable to develop treatments based on molecular tweezers rather than antibiotics," said BGU Department of Chemistry Prof. Raz Jelinek.

The research team, led by Prof. Jelinek and his Ph.D. student Ravit Malishev, tested their molecular tweezers on the Staphylococcus aureus (Staph) bacteria. In the U.S. staph infections have an estimated mortality rate of over 25%, and 40% for drug-resistant strains.

The tweezers target biofilm, a thin layer of fibers that protects the bacteria. By gripping the fibers and destroying the protective layer, the tweezers impair the bacteria without directly attacking it, which prevents resistance from occurring.

Prof. Jelinek, who is also BGUs vice president of research and development and a member of the Ilse Katz Institute for Nanoscale Science and Technology, explained, "The tweezers are just like your home tweezers but a million times smaller, and instead of plucking hairs they attack fibers of the bacteria's biofilm." By doing that they break the biofilm, making it more vulnerable to human immune defenses and external substances that are used against bacteria like antibiotics."

"The success of the study indicates an innovative direction of antibiotic treatments against pathogenic bacteria. We found that binding the tweezers to the biofilm disrupts its protective capabilities. Consequently, the bacterial pathogens become, less virulent to the human body, and, more vulnerable to elimination by the immune system. This breakthrough may open up new ways to fight antibiotic-resistant bacteria." Prof. Jelinex hopes that following further testing, a pill containing millions of ingestible tweezers could identify biofilms in the body and break them apart.

Empirical treatment with non-anti-tuberculosis antibiotics decreased microbiological detection in cervical tuberculous lymphadenitis

Diagnosis of cervical tuberculous lymphadenitis (CTL), the most commonly occurring form of extrapulmonary tuberculosis, remains as a challenge in clinic. Detection of the presence of Mycobacterium tuberculosis (Mtb) in fine needle aspiration cytology (FNAC) samples is one golden criterion to confirm the CTL diagnosis. Due to the non-specific clinical presentation, CTL might be confused with other lymph node enlargement diseases therefore empirical treatment with non-anti-TB antibiotics is often initially administered. However, it is still unclear whether this diagnostic antibiotic treatment affects the positivity of Mtb detection in FNAC. The demographics and clinical characteristics of 732 lymph node enlargement patients who had underwent FNAC were retrospectively analyzed and 605 (82.65%) of them were diagnosed as CTL. A total of 279 CTL cases (279/605, 46.11%) with completion of three Mtb tests (AFB, NAAT, and Mtb culture) in FNAC samples were selected for analyzing the effect of empirical antibiotic treatment on the positivity of Mtb tests. Compared to CTL patients without antibiotic treatment prior to FNAC, patients received empirical non anti-TB treatment had significantly lower positivity for acid fast bacilli staining (adjusted OR 0.11, 95% CI 0.06-0.21), nucleic acid amplification test (NAAT) (adjusted OR 0.38, 95% CI 0.21-0.71), and Mtb culture (adjusted OR 0.11, 95% CI 0.06-0.19). In conclusion, this study demonstrated that empirical non anti-TB antibiotic treatment reduced the opportunity to confirm CTL by microbiological analysis. Patients with cervical lymph node enlargement should undergo FNAC for Mtb tests prior to initiation of empirical non anti-TB treatment.

Keywords: Cervical tuberculous lymphadenitis Empirical treatment Lymph node enlargement Microbiological detection Non-anti-tuberculosis.

Health Solutions From Our Sponsors

Jose Rosa-Oliveras, et. al. Otitis Media: To Treat, To Refer, To Do Nothing: A Review for the Practioner. Pediatrics in Review. November 2016, 36 (11), 480-488.

Lieberthal, AS, et al. The Diagnosis and Management of Acute Otitis Media. Pediatrics. March 2013. Vol:131/Issue 3.

Top Ear Infection Home Treatment Related Articles

Earwax Removal

Earwax (ear wax) is a natural substance secreted by special glands in the skin on the outer part of the ear canal. It repels water, and traps dust and sand particles. Usually a small amount of wax accumulates, dries up, and then falls out of the ear canal carrying with it unwanted particles. Under ideal circumstances, you should never have to clean your ear canals. The absence of ear wax may result in dry, itchy ears, and even infection. Ear wax may accumulate in the ear for a variety of reasons including narrowing of the ear canal, production of less ear wax due to aging, or an overproduction of ear wax in response to trauma or blockage within the ear canal.

Eustachian Tube Dysfunction

The Eustachian tube is a membrane lined tube that connects the middle ear space to the back of the nose. Symptoms of Eustachian tube dysfunction or blockage include popping and/or clicking in the ear, and ear fullness and/or pain.

Causes of Eustachian tube dysfunction or blockage include allergies, sinus infections, ear infections, and the common cold.

Treatment includes home remedies to relieve pain and several maneuvers (swallowing, chewing gum, yawning etc.), which can be done to improve Eustachian tube function. In severe cases surgery may be necessary.

Granulomatosis With Polyangiitis

Granulomatosis with polyangiitis may be fatal within months without treatment. Treatment aims to stop inflammation with high doses of prednisone and cyclophosphamide.

Swimmer's Ear (External Otitis)

Swimmer's ear (external otitis) is an infection of the skin that covers the outer ear canal.

Causes of swimmer's ear include excessive water exposure that leads to trapped bacteria in the ear canal. Symptoms of simmer's include a feeling of fullness in the ear, itching, and ear pain. Chronic swimmer's ear may be caused by eczema, seborrhea, fungus, chronic irritation, and other conditions.

Common treatment includes antibiotic ear drops.

Ruptured (Perforated) Eardrum (Symptoms, Treatment, Surgery)

A perforated (ruptured, punctured) eardrum (tympanic membrane) is a hole or tear in the eardrum. The eardrum separates the ear canal and middle ear. Most ruptured eardrums do not cause pain, however, the condition can be uncomfortable. Bacteria, viral, and fungal infections are the most common causes a ruptured eardrum. Earwax removal attempts, changes in air pressure, and trauma are other causes of a ruptured eardrum.

  • Ear pain
  • Partial or full hearing loss
  • A mucousy or pus-like blood-tinged discharge from you ear
  • Bleeding from the ear
  • Ringing in the ear
  • Vertigo
  • Nausea
  • Vomiting
  • Middle ear infection

REFERENCE: Cleveland Clinic. "Ruptured Eardrum (Perforated Tympanic Membrane)." Updated: Aug 208, 2014.

Sore Throat

Sore throat (throat pain) usually is described as pain or discomfort in the throat area. A sore throat may be caused by bacterial infections, viral infections, toxins, irritants, trauma, or injury to the throat area. Common symptoms of a sore throat include a fever, cough, runny nose, hoarseness, earaches, sneezing, and body aches. Home remedies for a sore throat include warm soothing liquids and throat lozenges. OTC remedies for a sore throat include OTC pain relievers such as ibuprofen or acetaminophen. Antibiotics may be necessary for some cases of sore throat.


Clinical diagnosis. Clinical findings are unreliable for establishing a diagnosis of intravascular device-related infection, because of their poor specificity and sensitivity. For example, the most sensitive clinical findings, such as fever with or without chills, have poor specificity, and inflammation or purulence around the intravascular device and bloodstream infection have greater specificity but poor sensitivity [ 2]. Blood culture results that are positive for S. aureus, coagulase-negative staphylococci, or Candida species, in the absence of any other identifiable source of infection, should increase the suspicion for catheter-related bloodstream infection [ 3, 5, 11]. Current evidence-based recommendations for diagnosis are summarized in table 5.

Current evidence-based recommendations for the diagnosis and management of catheter-related infections.

Current evidence-based recommendations for the diagnosis and management of catheter-related infections.

Rapid diagnostic techniques. Gram stain may be helpful for the diagnosis of local infections, but it is significantly less sensitive than are quantitative methods for the diagnosis of catheter-related infections [ 55]. In one study, use of acridine orange stains for rapid diagnosis resulted in a positive predictive value of 91% and a negative predictive value of 97% [ 16].

Cultures of samples of iv catheters. Laboratory criteria for the diagnosis of intravascular catheter-related infections are precise, but differences in the definitions and methodologies used in various studies have made data difficult to compare [ 2, 3]. Semiquantitative (roll plate) or quantitative (vortex or sonication methods) catheter culture techniques are the most reliable diagnostic methodologies, because they have greater specificity in the identification of catheter-related infection, in comparison with qualitative cultures, in which a single contaminating microbe can result in a positive culture result ( table 5) [ 13, 14]. The predictive value of quantitative or semiquantitative culture methods may vary depending on the type and location of the catheter, the culture methodology used, and the source of catheter colonization [ 56]. For example, a recently inserted catheter (duration of placement, >1 week) is most commonly colonized by a skin microorganism along the external surface of the catheter, so the roll plate method will be quite sensitive in the identification of such colonization. For longer-dwelling catheters (duration of placement, >1 week), in which intraluminal spread from the hub may be the dominant mechanism for catheter colonization, the roll plate method is less sensitive, and methods that obtain samples of both the internal and external surfaces for culture are more sensitive [ 56]. As use of antimicrobial-coated catheters becomes more prevalent, the existing definitions of catheter colonization and catheter-related infection may need to be modified, because such coatings may lead to false-negative culture results [ 57, 58].

The most widely used laboratory technique for the clinical diagnosis of catheter-related infection is the semiquantitative method, in which the catheter segment is rolled across the surface of an agar plate and colony-forming units are counted after overnight incubation [ 14]. Quantitative culture of the catheter segment requires either flushing the segment with broth, or vortexing, or sonicating it in broth, followed by serial dilutions and surface plating on blood agar [ 13, 59, 60]. A yield of ⩾15 cfu from a catheter, by means of semiquantitative culture, or a yield of ⩾10 2 cfu from a catheter, by means of quantitative culture, with accompanying signs of local or systemic infection, is indicative of catheter-related infection. In a prospective study that compared the sonication, flush culture, and roll plate methods, the sonication method was 20% more sensitive for the diagnosis of catheter infection than was the roll plate method, and it was >20% more sensitive than was the method of flushing the individual catheter lumens [ 6]. If only catheter-related bloodstream infections are considered, the sensitivities of the 3 methods are as follows: sonication, 80% roll plate method, 60% and flush culture, 40%–50%.

Summary receiver operating characteristic-curve analysis has been recommended as a potentially more rigorous method for comparing the accuracy of different diagnostic tests for the same condition, because a given test may have one sensitivity at a given specificity and a different sensitivity at another specificity [ 61]. A meta-analysis confirmed that quantitative cultures of catheter segments were more accurate than were the roll plate and qualitative methods in a receiver operating characteristic-curve analysis (P = .03) [ 12]. At this time, it is unclear whether any of these differences are clinically significant.

Paired cultures of blood drawn through the iv catheter and percutaneously. Patients with suspected iv catheter-related infection should have 2 sets of blood samples drawn for culture, with at least 1 set drawn percutaneously. The clinical usefulness of cultures of blood samples drawn from an indwelling CVC was assessed in a study of hospitalized patients with cancer [ 17]. In the study, the positive predictive value of catheter and peripheral blood cultures was 63% and 73%, respectively, and the negative predictive value was 99% and 98%, respectively. Therefore, a positive culture result for a blood sample drawn through a catheter requires clinical interpretation, but a negative result is helpful for excluding catheter-related bloodstream infection.

Quantitative cultures of peripheral and CVC blood samples. Quantitative blood culturing techniques have been developed as an alternative for the diagnosis of catheter-related bloodstream infection in patients for whom catheter removal is undesirable because of limited vascular access. This technique relies on quantitative culture of paired blood samples, one of which is obtained through the central catheter hub and the other from a peripheral venipuncture site. In most studies, when blood obtained from the CVC yielded a colony count at least 5–10-fold greater than that for blood obtained from a peripheral vein, this was predictive of catheter-related bloodstream infection [ 19]. Among tunneled catheters, for which the method is most accurate, a quantitative culture of blood from the CVC that yields at least 100 cfu/mL may be diagnostic without a companion culture of a peripheral blood sample [ 62].

Differential time to positivity for CVC versus peripheral blood cultures. This new method, which correlates well with quantitative blood cultures, makes use of continuous blood-culture monitoring for positivity (e.g., radiometric methods) and compares the differential time to positivity for qualitative cultures of blood samples drawn from the catheter and a peripheral vein. When studied with tunneled catheters, this method has offered accuracy comparable to that of quantitative cultures of blood samples and has had greater cost-effectiveness [ 20, 21]. In a study of differential time to positivity, a definite diagnosis of catheter-related bacteremia could be made in 16 of the 17 patients who had a positive result of culture of a blood sample from the CVC at least 2 h earlier than they had a positive result of a peripheral blood culture the overall sensitivity was 91% and specificity was 94% [ 20]. Most hospitals do not have quantitative blood culture methodologies, but many will be able to use differential time to positivity for diagnosis.

Infusate-related bloodstream infection. Infusate-related bloodstream infection is uncommon and is defined as the isolation of the same organism from both infusate and separate percutaneous blood cultures, with no other source of infection. The sudden onset of symptoms of bloodstream infection soon after the initiation of an infusion, resulting from the administration of contaminated iv fluid, is often diagnostic [ 2]. When this diagnosis is suggested, cultures of iv fluid should be part of an investigation of potential sources of infection.

Home Care for a Staph Infection

If you do develop a staph infection on your skin, some basic hygiene measures will encourage healing and help to prevent the spread of infection:

  • Keep it clean. Follow your doctor’s instructions on how to clean your wound or skin condition.
  • Keep it covered. Cover the affected area with gauze or a bandage, as recommended by your doctor, to protect it and avoid spreading the infection to other people.
  • Don’t touch it. Avoid touching the area, so you don’t spread the bacteria to other parts of your body.
  • Use towels only once. After you bathe, dry yourself off, then wash the towel in hot water before using it again. ( 12 )

NIH scientists identify nutrient that helps prevent bacterial infection

Taurine, which helps the body digest fats and oils, could offer treatment benefit.

Colorized scanning electron micrograph showing carbapenem-resistant Klebsiella pneumoniae interacting with a human neutrophil. NIAID

Scientists studying the body’s natural defenses against bacterial infection have identified a nutrient — taurine — that helps the gut recall prior infections and kill invading bacteria, such as Klebsiella pneumoniae (Kpn). The finding, published in the journal Cell by scientists from five institutes of the National Institutes of Health, could aid efforts seeking alternatives to antibiotics.

Scientists know that microbiota — the trillions of beneficial microbes living harmoniously inside our gut — can protect people from bacterial infections, but little is known about how they provide protection. Scientists are studying the microbiota with an eye to finding or enhancing natural treatments to replace antibiotics, which harm microbiota and become less effective as bacteria develop drug resistance.

The scientists observed that microbiota that had experienced prior infection and transferred to germ-free mice helped prevent infection with Kpn. They identified a class of bacteria — Deltaproteobacteria — involved in fighting these infections, and further analysis led them to identify taurine as the trigger for Deltaproteobacteria activity.

Taurine helps the body digest fats and oils and is found naturally in bile acids in the gut. The poisonous gas hydrogen sulfide is a byproduct of taurine. The scientists believe that low levels of taurine allow pathogens to colonize the gut, but high levels produce enough hydrogen sulfide to prevent colonization. During the study, the researchers realized that a single mild infection is sufficient to prepare the microbiota to resist subsequent infection, and that the liver and gallbladder — which synthesize and store bile acids containing taurine — can develop long-term infection protection.

The study found that taurine given to mice as a supplement in drinking water also prepared the microbiota to prevent infection. However, when mice drank water containing bismuth subsalicylate — a common over-the-counter drug used to treat diarrhea and upset stomach — infection protection waned because bismuth inhibits hydrogen sulfide production.

Scientists from NIH’s National Institute of Allergy and Infectious Diseases led the project in collaboration with researchers from the National Institute of General Medical Sciences the National Cancer Institute the National Institute of Diabetes and Digestive and Kidney Diseases and the National Human Genome Research Institute.


A Stacy et al. Infection trains the host for microbiota-enhanced resistance to pathogens. Cell DOI: 10.1016/j.cell.2020.12.011 (2021).

Yasmine Belkaid, Ph.D., chief of NIAID’s Metaorganism Immunity Section in the Laboratory of Immune System Biology, is available to comment.

This news release describes a basic research finding. Basic research increases our understanding of human behavior and biology, which is foundational to advancing new and better ways to prevent, diagnose, and treat disease. Science is an unpredictable and incremental process — each research advance builds on past discoveries, often in unexpected ways. Most clinical advances would not be possible without the knowledge of fundamental basic research. To learn more about basic research at NIH, visit

NIAID conducts and supports research — at NIH, throughout the United States, and worldwide — to study the causes of infectious and immune-mediated diseases, and to develop better means of preventing, diagnosing and treating these illnesses. News releases, fact sheets and other NIAID-related materials are available on the NIAID website.

Molecular Mechanisms of Antibiotic Resistance in Enterococci

As previously noted, enterococci exhibit significant resistance to a wide variety of antimicrobial agents. This resistance is almost certainly relevant in most natural ecological settings in which enterococci dwell. As normal commensals of the human gastrointestinal tract, enterococci are routinely exposed to a myriad of antibiotics in the course of contemporary medical treatment, and enterococcal resistance plays a key role in the ecological dynamics that occur during and after antibiotic therapy. In addition, their resistance has confounded the best efforts of contemporary medicine to cope with infections caused by enterococci.

Intrinsic resistance—that which is encoded within the core genome of all members of the species𠅍iffers from acquired resistance, in that the latter is present in only some members of the species and is obtained via the horizontal exchange of mobile genetic elements (or via selection upon antibiotic exposure). A great deal of effort has been devoted to understanding the molecular mechanisms of resistance in enterococci. This has resulted in identification of determinants that specify resistance for many antibiotics, including those that are (or once were) clinically useful as therapeutics to treat enterococcal infections, as well as those to which enterococci, as commensals of humans, are incidentally exposed in the course of therapy for infections caused by other bacteria. In many cases, this research has led to the development of an understanding of the regulation and biochemical activities of the resistance determinants, and, in selected cases, has provided insight into the consequences of antibiotic resistance on the biological fitness of enterococci. This section will provide an overview of mechanisms of resistance that have been examined in the past 10 years.

Glycopeptide resistance

The glycopeptides vancomycin, teicoplanin, and newer derivatives, are used to treat serious infections due to resistant Gram-positive bacteria. Most Gram-negative bacteria are not susceptible to glycopeptides because their outer membrane prevents access to the peptidoglycan targets located in the periplasmic space. Glycopeptides inhibit bacterial growth by interfering with peptidoglycan biosynthesis. The antibiotics form complexes with the D-Ala-D-Ala peptide termini of peptidoglycan precursors on the outer surface of the cell, which prevents the cell wall biosynthetic enzymes (i.e., the PBPs) from using them as substrates for transglycosylation and transpeptidation and, hence, impairment of cell wall integrity.

Glycopeptide resistance has been extensively reviewed (Arthur & Courvalin, 1993 Arthur & Quintiliani, Jr., 2001 Courvalin, 2005 Courvalin, 2006 Depardieu, Podglajen, Leclercq, Collatz, & Courvalin, 2007 Jaspan, et al., 2010). The biochemical basis for resistance derives from modification of the antibiotic target. Glycopeptide-resistant enterococci produce altered peptidoglycan precursors in which the D-Ala-D-Ala termini have been modified such that they terminate in either D-Ala-D-lactate or D-Ala-D-Ser. These substitutions reduce the binding affinity of the antibiotics for the peptidoglycan precursors (

1000 fold reduction for D-Ala-D-lac

7 fold for D-Ala-D-Ser). The altered precursors can still serve as substrates for the cell wall biosynthetic enzymes to enable the construction of functional peptidoglycan, but the reduced affinity of glycopeptides renders the drugs unable to inhibit cell wall biosynthesis. The capacity to produce the alternative glycopeptide-resistant peptidoglycan precursors is encoded by resistance operons usually encoded on mobile genetic elements (and thus transferable to otherwise susceptible hosts). Specific types of glycopeptide resistance are encoded in the chromosome as part of the core genome of certain enterococcal species.

Overview of genetic mechanisms of glycopeptide resistance

Nine distinct gene clusters conferring glycopeptide resistance have been described in enterococci. These determinants differ from each other both genetically and phenotypically, based on their physical location (encoded on a mobile genetic element or in the core genome) the specific glycopeptides to which they confer resistance (often distinguished operationally as providing resistance to both vancomycin and teicoplanin, or providing resistance to vancomycin but not teicoplanin) the level of resistance they confer whether resistance is inducible or constitutively expressed and the type of peptidoglycan precursor that is produced by their gene products. The Van gene clusters encode several functions: (i) a regulatory module, namely a two-component signal transduction system that is responsible for sensing the presence of glycopeptides and activating expression of the resistance genes in inducible Van types (ii) enzymes that produce the modified peptidoglycan precursors, including enzymatic machinery that is required to produce the appropriate substitute (D-Lac or D-Ser), and a ligase that fuses D-Ala to either D-Lac or D-Ser to make the corresponding dipeptide that can be incorporated into peptidoglycan precursors via the normal biosynthetic machinery of the cell and (iii) D,D-carboxypeptidases that eliminate any of the normal (unmodified) peptidoglycan precursor synthesized by the natural biosynthetic machinery of the cell, thereby ensuring that nearly all precursors reaching the cell surface are of the modified variety. The Van gene clusters are typically referred to by the names given to the ligases they encode (VanA, VanB, VanC, and so on). The VanA and VanB types are the most common among clinical isolates and have been studied in the greatest detail.

The VanA determinant (Figure 1) confers a high level of resistance to vancomycin and teicoplanin. VanA is typically encoded on Tn1546 or related transposons, and includes seven open reading frames transcribed from two separate promoters. The regulatory apparatus is encoded by the VanR (response regulator) and VanS (sensor kinase) two-component system, which are transcribed from a common promoter, while the remaining genes are transcribed from a second promoter. Gene products that specify the production of modified peptidoglycan precursors include VanH (dehydrogenase that converts pyruvate to lactate) and VanA (ligase that forms D-Ala-D-Llac dipeptide). The VanX (dipeptidase that cleaves D-Ala-D-Ala) and VanY (D,D-carboxypeptidase) peptidases serve to eliminate the natural peptidoglycan precursors from the cell. The 7th gene, VanZ, is often referred to as an �ssory” function, but its role in resistance is not fully understood.

Figure 1.

Regulation of vancomycin resistance gene clusters. Comparison of VanA regulation (panel A) and VanB regulation (panel B). The VanS (or VanSB) sensor kinases are anchored in the cytoplasmic membrane by two transmembrane segments (TM) that flank the predicted (more. )

The VanB locus (Figure 1) confers moderate to high-level resistance to vancomycin, but is not induced by teicoplanin. VanB is usually acquired onTn5382/Tn1549 type transposons, which occur on plasmids or in the chromosome of the host. The genetic organization of VanB is similar to that of VanA, in that it contains two distinct promoters transcribing seven open reading frames, but there are some significant differences. For example, although VanB encodes a two-component system (named VanRB and VanSB), this signaling system is considerably different from that encoded in VanA. VanB encodes homologs of VanH and the D-Ala-D-Ala ligase (encoded by VanB), as well as the peptidases (VanX and VanY). However VanB lacks a homolog of VanZ, and instead encodes a protein named VanW, whose role in resistance is not fully understood.

Regulation of glycopeptide resistance

Expression of vancomycin resistance is controlled via the VanR/VanS two-component signal transduction system, shown in Figure 1. The VanS sensor kinase is thought to recognize a (poorly defined) stimulus that signals the presence of vancomycin in the environment. VanS thereby becomes activated and autophosphorylates a conserved histidine residue on the cytoplasmic side of the protein. That phosphoryl group can be transferred to a conserved aspartate residue on the VanR response regulator, which leads to VanR dimerization, enhanced VanR binding to the 2 promoters located in the Van locus, and consequently, an increased transcription of both the Van resistance genes as well as the regulatory genes (Depardieu, Courvalin, & Kolb, 2005). Additionally, there is substantial evidence that VanS serves as a phosphatase for VanR under non-inducing conditions, to prevent activation of Van expression via spurious phosphorylation from other sensor kinases in the cell (cross-talk), or via autophosphorylation of VanR from acetyl-phosphate (Depardieu, Courvalin, & Kolb, 2005). As a result, the phosphatase activity of VanS is critical to maintain the signaling pathway in the off state in the absence of an inducing antibiotic. Mutations that impair the phosphatase activity of VanS (or remove VanS completely) lead to constitutive expression of the resistance genes (Arthur & Quintiliani, Jr., 2001). This is now known to be true for many of the Van clusters (Depardieu, Kolbert, Pruul, Bell, & Courvalin, 2004 Panesso, et al., 2010). In fact, constitutively resistant mutants that carry lesions in VanS can be isolated from patients during glycopeptide therapy. For example, examination of successive isolates of E. faecium obtained from a patient suffering from an infection with a VanB strain revealed that a short deletion in the VanSB kinase led to the loss of phosphatase activity and constitutive glycopeptide resistance (Depardieu, Courvalin, & Msadek, 2003).

Although both VanA and VanB rely on two-component signaling systems to control Van expression, it is clear that there are important differences between these regulatory systems. For example, the VanS and VanSB sensor kinases exhibit relatively little sequence identity in the N-terminal portion that serves as the site of stimulus recognition. In fact, the amino acid sequence of the predicted extracellular ligand-binding domain of VanS is short, and is likely to comprise only a short loop that connects the two transmembrane helices outside the membrane, which suggests that VanS belongs to the intramembrane-sensing family of sensor kinases (Mascio, Alder, & Silverman, 2007), whereas the predicted extracellular domain of VanSB is substantially larger and likely constitutes an independently-folded extracellular domain that serves to recognize cognate signals. Given the distinct architecture of these two sensor kinases, it seems plausible that they recognize and respond to different molecular signals to trigger kinase activation and expression of the resistance genes. In fact, this predicted difference in ligand binding𠅊nd consequently, in the inducibility of the signaling system—underlies the difference in teicoplanin susceptibility of enterococci that contain VanA vs. those that contain VanB. Although the molecular identity of the actual inducing signal(s) remain unclear, the VanA resistance genes are induced by the presence of both vancomycin and teicoplanin (thereby conferring resistance to both), but the VanB resistance genes are only induced by vancomycin—hence, VanB strains remain susceptible to teicoplanin. Of note, VanSB can acquire mutations of various types that lead to constitutive expression of the resistance genes or to inducibility by teicoplanin, thereby altering the phenotype of such mutants carrying the VanB locus.

Regulation by host factors?

Some evidence suggests that one or more sensor kinases encoded in the genome of the enterococcal host can contribute to the regulation of the Van resistance genes. For example, VanRB-dependent gene expression remains inducible even in the absence of VanSB (Baptista, Rodrigues, Depardieu, Courvalin, & Arthur, 1999), suggesting that another sensor kinase can phosphorylate and activate VanRB. Similarly, the VanE cluster in E. faecalis encodes a VanSE kinase that is predicted to be nonfunctional due to a premature stop codon (Abad໚ Patiño, Courvalin, & Perichon, 2002), but such resistance is nevertheless inducible by vancomycin (Foucault, Depardieu, Courvalin, & Grillot-Courvalin, 2010). Such findings suggest that enterococci encode endogenous two-component signaling systems whose natural function is to monitor the integrity of the cell wall for perturbations, and activate appropriate adaptive responses to ensure cell wall maintenance and further, that the glycopeptide resistance gene cassettes have managed to exploit these endogenous systems to assist in the regulation of glycopeptide resistance. Other host factors may also play a role in regulation of Van expression. For example, expression of the VanE vancomycin resistance genes may be influenced by the alteration of DNA supercoiling in E. faecalis (Paulsen, et al., 2003).

The expanding Van alphabet

While the VanA- and VanB-type vancomycin resistance clusters continue to be the predominant forms that account for vancomycin resistance in hospitals, new Van resistance gene clusters have been recently described, which brings the number of known gene clusters capable of conferring Van resistance to nine. Lebreton and colleagues (Lee, Huda, Kuroda, Mizushima, & Tsuchiya, 2003) recently described such a new gene cluster, named VanN, that specifies incorporation of D-Ala-D-Ser at the terminus of the peptidoglycan precursors. VanN joins other recently described Van clusters (Boyd, Willey, Fawcett, Gillani, & Mulvey, 2008 Xu, et al., 2010) known to specify incorporation of either D-Ala-D-Ser (VanC, VanE, VanG, and VanL types) or D-Ala-D-Lac (VanA, VanB, VanD, and VanM types) into peptidoglycan precursors.

Fitness cost of vancomycin resistance

Despite the complex mechanism that underlies glycopeptide resistance, resistant enterococci have disseminated worldwide, suggesting that resistance imposes little or no biological cost to the bacteria. This hypothesis was carefully examined by using pairs of isogenic enterococcal strains with specific mutations, to evaluate the fitness cost of vancomycin resistance during growth in vitro and in vivo (Franke & Clewell, 1981). The investigators found that expression of vancomycin resistance imposed a significant fitness cost, both when expression is induced by the antibiotic and when expression is constitutive due to mutation of the regulatory apparatus. However, uninduced vancomycin resistance did not impose a measurable fitness cost. Thus, these results offer a strong evolutionary rationale for the tight regulation of vancomycin resistance by the VanS/VanR two-component signaling system found in Van gene clusters.

Alternative mechanism of glycopeptide resistance

A novel mechanism of glycopeptide resistance has been described in laboratory-selected vancomycin-resistant mutants of E. faecium (Cremniter, et al., 2006). This mechanism is unrelated to that encoded by the Van gene clusters (namely, those with production of peptidoglycan precursors containing D-Lac or D-Ser substitutions). The investigators selected highly resistant mutants in vitro and performed extensive analysis of peptidoglycan structure in the mutants. Their analysis revealed that the beta-lactam insensitive L,D-transpeptidase pathway (discussed in more detail below, under Ampicillin resistance) was activated. This alternative transpeptidase (named Ldtfm) is capable of crosslinking enterococcal peptidoglycan using the L-Lys found at the 3 rd position of the peptide stem (rather than the D-Ala found at position 4, as is typical of most PBPs). The investigators found that a cryptic D,D-carboxypeptidase was activated in the glycopeptide-resistant mutants, whose activity resulted in production of peptidoglycan peptide stem precursors that are tetrapeptides (lacking the terminal D-Ala), rather than pentapeptides. Such precursors are not substrates for binding by glycopeptide antibiotics, but can be cross-linked by the Ldtfm transpeptidase. However, it remains unknown whether this mechanism of glycopeptide resistance is relevant in clinical isolates.

Daptomycin resistance

Daptomycin is a lipopeptide antibiotic with potent in vitro bactericidal activity against Gram-positive bacteria. The mechanism of antimicrobial action for daptomycin has not been unequivocally established, but is thought to involve calcium-dependent insertion into the cytoplasmic membrane followed by membrane depolarization, release of intracellular potassium ions, and rapid cell death (Alborn, Jr., Allen, & Preston, 1991 Matsumura & Simor, 1998 Silverman, Perlmutter, & Shapiro, 2003). Because its mechanism of action is distinct from those of other antibiotics, daptomycin is useful for treatment of infections that are caused by multidrug-resistant Gram-positive strains. Daptomycin resistance has been observed in clinical isolates following daptomycin therapy, typically as a result of mutations in chromosomal genes. In Staphylococcus aureus, resistance is associated with mutations in genes encoding proteins such as MprF, a lysylphosphatidylglycerol synthetase YycG, a sensor histidine kinase and RpoB and RpoC, the β and β′ subunits of RNA polymerase (Galimand, et al., 2011).

Daptomycin-nonsusceptible clinical E. faecium strains have been described (Muller, Le Breton, Morin, Benachour, Auffray, & Rincé, 2006). The investigators determined that these strains did not carry mutations in homologs of genes known to confer nonsusceptibility to daptomycin in S. aureus (yycG, mprF, rpoB, rpoC were evaluated in this study), which suggests the existence of one or more novel mechanisms of daptomycin resistance in enterococci. However, the genes responsible for resistance were not identified.

Recent studies have begun to explore the genetic basis of daptomycin resistance in enterococci (Arias, et al., 2011). The genomes of a pair of E. faecalis strains isolated from the same patient before and after daptomycin therapy were sequenced to identify polymorphisms contributing to resistance (Arias, et al., 2011). In that study, unique-sequence polymorphisms were found in cls, gdpD (both thought to be involved in phospholipid metabolism) and liaF, but no polymorphisms were found in homologs of the genes identified in daptomycin-resistant S. aureus isolates. Follow up analysis with in vitro selection for daptomycin-resistant variants of the original susceptible strain led to the identification of mutations in liaF and gdpD. Importantly, the daptomycin-resistant phenotype was determined to be a consequence of the identified mutations, as site-directed mutagenesis to recapitulate these mutations in an otherwise daptomycin-susceptible host conferred enhanced daptomycin resistance, demonstrating that mutations in these genes confer resistance. Similarly, in a clinical strain pair of E. faecium recovered from a patient both before and after daptomycin therapy, a polymorphism in cls was identified (but not in liaFSR or gdpD) in the daptomycin-resistant derivative. Changes in cls, liaF, liaS, or liaR were also identified in other daptomycin-resistant clinical isolates of enterococci, which suggests that such mutations play a key role in the development of daptomycin resistance in vivo. Development of daptomycin resistance appeared to be associated with profound ultrastructural changes in the cell envelope and septal apparatus, although it remains unclear if these changes are functionally important for resistance or merely an incidental consequence of the mutations. LiaF is part of the three-component LiaFSR regulatory system, which is known to coordinate the response of the cell envelope to antibiotics and antimicrobial peptides in some Gram-positive bacteria, which suggests that perturbations in the activity of this signaling system may alter envelope properties in a such a way that daptomycin can no longer interact with, or insert into, the membrane efficiently.

In parallel studies, Palmer and coworkers systematically selected daptomycin-resistant variants of E. faecalis in vitro (Palmer, Kos, & Gilmore, 2010) in a stepwise manner, and characterized the order of appearance of mutations that correspond to increased resistance to daptomycin. Whole-genome sequence comparison identified mutations in seven genes, including cls, rpoN, and additional genes whose cellular functions in other contexts have not been established. Transfer of the cls mutant allele to a susceptible E. faecalis strain conferred enhanced resistance to daptomycin, which unambiguously proved that the cls mutation is sufficient for resistance. Time-resolved analysis of the emergence of mutations during daptomycin exposure revealed that cls mutations appeared early in multiple independent selections, which highlights its importance. Additional daptomycin-resistant mutants were obtained that lacked such cls mutation, which shows that alternative paths to daptomycin resistance also exist. Collectively, the results from these two studies suggest that, while the underlying mechanisms are genetically distinct from those identified in S. aureus, a role for altered membrane phospholipid composition and/or surface properties in both staphylococcal and enterococcal daptomycin resistance is likely.

Aminoglycoside resistance

Aminoglycosides act by binding to the 16S rRNA of the 30S ribosomal subunit and interfering with protein synthesis. Enterococci generally exhibit a moderate level of intrinsic aminoglycoside resistance that has been attributed to poor uptake of antibiotics. For example, analysis of selected mutants that exhibited enhanced gentamicin resistance in vitro suggested that impaired uptake of gentamicin can contribute directly to enhanced resistance (Aslangul, et al., 2006), although the mutations or genes responsible for the alteration in uptake were not unambiguously identified. However, some evidence suggests that other mechanisms may contribute to, or even be primarily responsible for, intrinsic resistance of some enterococci to aminoglycosides.

Moderate species-specific intrinsic resistance to aminoglycosides in E. faecium is enhanced by a chromosomally encoded rRNA methyltransferase, EfmM (Galloway-Pe༚, Roh, Latorre, Qin, & Murray, 2012) that uses S-adenosyl methionine as a methyl donor to methylate a specific residue on 16S rRNA, in the context of the 30S ribosomal subunit. The inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and conversely, expression of a recombinant version of efmM in Escherichia coli confers enhanced resistance to these drugs. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. In addition, a chromosomally encoded 6′-N-aminoglycoside acetyltransferase (aac(6′)-Ii) confers low-level intrinsic resistance (Costa, Galimand, Leclercq, Duval, & Courvalin, 1993). The physiological effects of these two factors seems to be additive, in the sense that mutation of both genes led to the largest reduction in resistance. However, it remains unclear if aminoglycoside resistance is the primary function of EfmM. The position of m5C1404 at the junction between the ribosomal A-site and the P-site in 16S rRNA suggests that this modification might also play a more basic role in protein synthesis by influencing codon-anticodon interactions.

High-level resistance to aminoglycosides (HLGR) is conferred by a mechanism distinct from those described above, and importantly, abolishes the synergistic bactericidal activity of aminoglycosides in combination with cell-wall�tive agents that are important in the treatment of severe enterococcal infections, such as endocarditis. HLGR is usually acquired on a mobile element that encodes an aminoglycoside-modifying enzyme. Such enzymes can be phosphotransferases (APHs) that use ATP to phosphorylate a hydroxyl group on the antibiotic, acetyltransferases (AACs) that use acetyl-CoA to acetylate an amino group on the antibiotic, or nucleotidyltransferases (ANTs) that use ATP to adenylylate a hydroxyl group on the antibiotic. HLGR is most often associated with members of the APH(2ʹʹ)-I phosphotransferase family or the bifunctional AAC(6ʹ)-Ie-APH(2ʹʹ)-Ia family that are encoded on various transposons or conjugative plasmids (reviewed in (Kak, Donabedian, Zervos, Kariyama, Kumon, & Chow, 2000)).

Rifampicin resistance

Rifampicin inhibits bacterial growth by binding to the beta subunit of RNA polymerase (RpoB) and preventing initiation of transcription (Wehrli, Knüsel, Schmid, & Staehelin, 1968). Rifampicin has been used for decades as part of an antibiotic cocktail to treat infections caused by Mycobacterium tuberculosis, and has recently found increasing use in the treatment of staphylococcal infections associated with indwelling medical devices, such as artificial joints. Most resistance to rifampicin results from mutations of specific sites in the gene encoding the beta subunit of the RNA polymerase, which reduces the affinity of rifampicin for the polymerase. Mutations in RpoB responsible for rifampicin resistance have been identified in numerous and diverse species of bacteria. Additionally, enzymatic inactivation of rifampicin has been observed in a handful of cases.

Although rifampicin has not been used extensively to treat enterococcal infections, acquired resistance to rifampicin is nonetheless common in enterococci—nearly 79% of 71 clinical isolates were found to exhibit rifampicin resistance (Andrews, Ashby, Jevons, Marshall, Lines, & Wise, 2000), as well as 㹗% of a diverse collection of isolates from six countries in Europe (Lautenbach, Schuster, Bilker, & Brennan, 1998). Presumably this is at least partially a consequence of commensal enterococci being exposed to rifampicin during treatment for non-enterococcal infections, but other as-yet-unknown factors may contribute to the occurrence of resistant enterococcal isolates as well. In E. faecium, substitutions in RpoB are associated with rifampicin resistance, and most of the identified RpoB polymorphisms have been previously implicated in conferring resistance in other species of bacteria. The biological cost of rifampicin resistance is variable, depending on the particular mutation in RpoB, as well as other potential compensatory mutations that may occur elsewhere in the genome (Enne, Delsol, Roe, & Bennett, 2004).

Spontaneous rifampicin-resistant mutants of E. faecalis and E. faecium are readily isolated in vitro (Kristich, Little, Hall, & Hoff, 2011). Mutations were identified in the rpoB gene of all such mutants at sites known to be associated with resistance to rifampicin in other species of bacteria. For two particular mutants, confirmation that the rpoB point mutations are indeed responsible for rifampicin resistance was obtained by expressing the mutant alleles in an otherwise rifampicin-susceptible host. One unexpected observation was that some RpoB mutations led to an alteration in cephalosporin resistance in an allele-specific manner. In particular, the rpoB H486Y mutation conferred a substantially enhanced resistance to cephalosporins in multiple lineages of E. faecalis, as well as in E. faecium, whereas other Rif-resistant substitutions in rpoB did not affect cephalosporin resistance. The mechanistic basis for this observation is not known, but the rpoB H486Y substitution may alter rates of transcription of genes that contribute to intrinsic cephalosporin resistance in enterococci.

Rifampicin resistance in some isolates of E. faecium can be reversed by inclusion of subinhibitory concentrations of daptomycin. This phenomenon could not be explained by 1) alteration of rifampicin transport into, or efflux from, the cell by daptomycin 2) the direct inactivation of rifampicin nor 3) mutations at the rifampicin-binding site in rpoB (Reynolds & Courvalin, 2005). The molecular explanation for this effect remains unknown and authors speculate that there may be another mechanism for rifampicin resistance besides the two known mechanisms (namely, mutation in the rpoB gene and rifampicin inactivation). This third mechanism has been dubbed �ptomycin-reversible resistance,” and further investigation will be needed to define its basis.

Quinolone resistance

Quinolones generally exhibit only moderate activity against enterococci. Quinolones inhibit the growth of bacteria by interfering with DNA replication, specifically by binding to the type II topisomerases that control DNA supercoiling (DNA gyrase and DNA topoisomerase IV) and inhibiting their function, leading to lethal double-strand breaks in the DNA. Quinolone resistance in many species of bacteria occurs via mutations in the “quinolone resistance determining regions” of the genes that encode gyrase and topoisomerase IV. These mutations prevent efficient binding of the antibiotic to the enzyme, which enables DNA replication to continue despite the presence of the antibiotic. Such mutations have been observed in clinical and lab-derived quinolone-resistant isolates of enterococci (Kak, Donabedian, Zervos, Kariyama, Kumon, & Chow, 2000 Oyamada Y. , Ito, Inoue, & Yamagishi, 2006 Palmer, Daniel, Hardy, Silverman, & Gilmore, 2011 Werner, Fleige, Ewert, Laverde-Gomez, Klare, & Witte, 2010), and presumably act to confer enhanced quinolone resistance through a similar mechanism.

A second mechanism known to contribute to quinolone resistance in other species of bacteria is efflux of the antibiotic out of the cell. Such efflux is often a function of pumps with relatively broad or nonspecific substrate specificities, which are sometimes referred to as multidrug-resistance efflux pumps (MDRs). Genes encoding MDRs are usually found on the bacterial chromosome. Although the primary physiological functions for most MDRs remain unclear, these proteins are known to actively transport toxic compounds out of the cell. The genome of E. faecalis V583 is predicted to encode 34 MDRs (Davis, et al., 2001), which suggests that drug efflux may play an important role in antibiotic resistance. Two of these pumps have been experimentally implicated in promoting quinolone resistance. The first is EmeA, a homolog of Staphylococcus aureus MDR NorA. Mutation of EmeA resulted in modest increases in susceptibility to several compounds, including quinolones (Jung, et al., 2010), and treatment of wild-type E. faecalis OG1RF with known MDR inhibitors (reserpine, lansoprazole, and verapamil) inhibits the efflux of the quinolone norfloxacin, as well as the toxic compound ethidium bromide. The second pump is EfrAB, an ABC-type transporter that enhances resistance to a variety of structurally unrelated compounds, including quinolones, when expressed in E. coli (Lefort, Saleh-Mghir, Garry, Carbon, & Fantin, 2000). Its function in enterococci was not investigated.

A new mechanism of quinolone resistance has been identified—protection of DNA gyrase and topoisomerase IV from inhibition by quinolones. The activity is provided by members of the Qnr protein family that were originally identified in enterobacteria as a transmissible type of quinolone resistance (Mascher, Helmann, & Unden, 2006). The natural function of Qnr proteins remains unclear. These proteins are characterized by tandem pentapeptide repeats, and homologs of Qnr appear to be encoded in the genomes of various bacteria. Inactivation of a homolog of Qnr identified in the genome of E. faecalis, comprised of 42 predicted pentapeptide repeats, resulted in a modest decrease in resistance to fluoroquinolones. Overexpression of the corresponding gene yielded an increase in resistance. Furthermore, expression of the E. faecalis gene in heterologous organisms, including S. aureus and Lactococcus lactis, increased the level of quinolone resistance in those hosts (Arsène & Leclercq, 2007). Purified EfsQnr inhibited the ATP-dependent DNA supercoiling activity of E. coli gyrase (Hellinger, Rouse, Rabadan, Henry, Steckelberg, & Wilson, 1992), suggesting that EfsQnr may protect E. faecalis from the effects of quinolones by modulating the action of gyrase in cells.

Macrolide, lincosamide, and streptogramin resistance

Macrolides, lincosamides, and streptogramin antibiotics inhibit protein synthesis by binding to the 50S subunit of the ribosome. Macrolides and lincosamides are not used to treat enterococcal infections, but resistance to them is nonetheless widespread (Jonas, Murray, & Weinstock, 2001). The most common form of acquired resistance to macrolides is production of an enzyme that methylates a specific adenine in the 23S rRNA of the 50S ribosomal subunit, which reduces the binding affinity of the macrolide for the ribosome. This modification also reduces the binding of lincosamide and streptogramin B antibiotics to the ribosome. The responsible enzyme is typically encoded by the ermB gene, and the phenotype is often referred to as MLSB. An efflux pump, encoded by the transferrable mefA gene, is also known to pump macrolides out of the cell, but confers a lower level of resistance than ermB (Clancy, et al., 1996).

E. faecalis and E. faecium are known to exhibit different intrinsic susceptibilities to quinupristin-dalfopristin (Q-D), members of the streptogramin family that act synergistically. E. faecalis is sufficiently intrinsically resistant that these antibiotics cannot be used therapeutically, whereas E. faecium is usually susceptible. The molecular basis for this difference appears to stem from the existence of a chromosomally encoded putative ABC transporter in the E. faecalis genome, named Lsa (198). Lsa is encoded in the genome of all isolates of E. faecalis evaluated (n=180), but in none of the genomes of other enterococci (n=189). Disruption the lsa gene in E. faecalis OG1RF resulted in a 㹀-fold decrease in MIC to Q-D. In addition, expression of the E. faecalis V583 lsa gene in otherwise susceptible E. faecium led to a 6-fold increase in MIC. Expression of lsa in the heterologous host Lactococcus lactis also moderately increased Q-D resistance (Singh & Murray, 2005). Further support for a role of Lsa in Q-D resistance stems from the observation that clinical isolates of E. faecalis that are susceptible to lincosamides and dalfopristin harbor mutations in lsa that result in premature stop codons (Dina, Malbruny, & Leclercq, 2003). However, the influence of Lsa on efflux or transport of the antibiotics has not been directly evaluated. Such studies will be helpful in determining the mechanism by which Lsa provides resistance, as the protein itself contains ATP-binding domains characteristic of ABC transporters, but lacks identifiable transmembrane sequences that would be expected of an authentic efflux pump. As such, the molecular mechanism of Lsa action remains unknown. Acquired resistance to Q-D has emerged in E. faecium, and is mediated by members of the streptogramin acetyltransferase family of enzymes that acetylate streptogramin A (such as VatH), and by the Vga genes, which encode an ABC transporter that presumably function to export the antibiotic from the cell (Kak & Chow, 2002).

Beta-lactam resistance

Antibiotics in the beta-lactam family inhibit bacterial growth by serving as suicide substrates for the D,D-transpeptidases (also known as penicillin-binding proteins, or PBPs) that catalyze cross-linking of peptidoglycan peptide side chains during the synthesis of mature peptidoglycan. Once modified by a beta-lactam antibiotic, PBPs are inactivated, thereby preventing continued cell wall synthesis. Enterococci exhibit intrinsic nonsusceptibility to beta-lactam antibiotics, but the extent of nonsusceptibility varies among the different classes of beta-lactams and between enterococcal species: penicillins have the most activity against enterococci (and E. faecium is inherently a bit more resistant than E. faecalis), carbapenems slightly less, and cephalosporins exhibit the least activity. This spectrum of activity is reflected in the utility of these drugs for treatment, insofar as ampicillin remains an effective therapy for susceptible enterococcal infections, but cephalosporins are completely ineffective against enterococci. In fact, prior use of cephalosporins is a major risk factor for the acquisition of an enterococcal infection (reviewed in (Shepard & Gilmore, 2002)).

The intrinsic nonsuceptibility of enterococci to beta-lactams involves the production of the low-affinity class B penicillin-binding protein 5 (Pbp5), an ortholog of the low-affinity Pbp2a expressed by methicillin-resistant isolates of Staphylococcus aureus (Gonzales, Schrekenberger, Graham, Kelkar, DenBesten, & Quinn, 2001). Due to its relatively low affinity for beta-lactams, the chromosomally encoded Pbp5 is capable of carrying out peptidoglycan synthesis at concentrations of beta-lactam antibiotics that saturate all of the other enterococcal PBPs (Canepari, Lleò, Cornaglia, Fontana, & Satta, 1986), and therefore is required for beta-lactam resistance (Arbeloa, et al., 2004 Rice L. B., Carias, Rudin, Lakticová, Wood, & Hutton-Thomas, 2005). Pbp5 is required for both the intrinsic beta-lactam resistance traits of enterococci, such as their intrinsic cephalosporin resistance, as well as for the acquired (enhanced) resistance to members of the beta-lactam family (such as ampicillin), to which enterococci are otherwise clinically susceptible. Below, we discuss resistance to two different families of beta-lactam antibiotics (cephalosporins and ampicillin) in more detail.

Intrinsic cephalosporin resistance

E. faecalis and E. faecium are naturally (intrinsically) resistant to cephalosporins. This trait has been known for decades and is encoded by chromosomal determinants in the core genome of these organisms, but its molecular basis remains incompletely understood. Thus far, in E. faecalis, a handful of genetic determinants have been shown to be required for intrinsic cephalosporin resistance: the low-affinity penicillin-binding protein Pbp5 a two-component signal transduction system, CroRS a transmembrane Ser/Thr kinase, IreK and one of the early enzymes involved in synthesis of peptidoglycan precursors, MurAA. The role of these determinants has been best studied in E. faecalis, although it appears likely that similar mechanisms are present in in E. faecium.

Genetic studies on isogenic mutants of E. faecalis and E. faecium provide clear evidence of a requirement for Pbp5 in intrinsic cephalosporin resistance (Arbeloa, et al., 2004 Rice L. B., Carias, Rudin, Lakticová, Wood, & Hutton-Thomas, 2005). Deletion mutants that lack pbp5 exhibit large reductions in the level of cephalosporin resistance. The deletion mutants also exhibit a reduction in resistance to the non-cephalosporin beta-lactam, ampicillin, although the magnitude of the reduction is more modest (especially for E. faecalis). High-molecular-weight PBPs are often categorized as Class A (bifunctional, exhibiting both transglycosylase and transpeptidase activities) and Class B (monofunctional, exhibiting transpeptidase activity but lacking transglycosylase activity). As a Class B PBP, Pbp5 contains a transpeptidase domain, but lacks a transglycosylase domain, which is necessary for the initial polymerization of the disaccharide moiety of peptidoglycan precursors. Therefore, even though Pbp5 is capable of synthesizing crosslinks between the peptide side chains of peptidoglycan, Pbp5 must cooperate with one or more transglycosylases for cell wall biosynthesis. Candidate transglycosylase partners include the three bifunctional Class A enterococcal PBPs (or in principle, monofunctional transglycosylases analogous to Mgt of Staphylococcus aureus, although no examples have yet been identified in enterococci). Analysis of isogenic deletion mutants lacking one or more Class A PBPs in both E. faecalis and E. faecium revealed that Pbp5 cooperates with one or both Class A PBPs, either PbpF or PonA, to permit growth in the presence of cephalosporins. The third Class A PBP (PbpZ) encoded by these organisms is unable to provide transglycosylase activity in the presence of cephalosporins. Triple mutants of either E. faecalis or E. faecium that lack all three class A PBPs are viable (although susceptible to cephalosporins), which indicates that as-yet-unidentified additional transglycosylases capable of peptidoglycan polymerization must exist. Given that double mutants lacking pbpF and ponA are susceptible to cephalosporins, it appears that these unidentified transglycosylases do not have the capacity to participate in functional interactions with Pbp5. Of note, while much of the PBP deletion analysis indicates substantial similarities in the underlying mechanisms of Pbp5 function in E. faecalis and E. faecium, deletion of the Class A PBPs in E. faecium revealed an unexpected dissociation between the expression of resistance to ceftriaxone and ampicillin that was not observed in E. faecalis (Rice L. B., Carias, Rudin, Lakticová, Wood, & Hutton-Thomas, 2005), suggesting that Pbp5-mediated crosslinking of peptidoglycan in E. faecium was differentially susceptible to beta-lactams, depending upon partner glycosyltransferase.


The genome of E. faecalis V583 encodes 17 two-component signal transduction systems (TCSs) (Hancock & Perego, 2004). Systematic inactivation of these TCSs and phenotypic characterization of the mutants revealed that the inactivation of CroRS rendered E. faecalis susceptible to extended spectrum cephalosporins, but not to a panel of antibiotics that perturb other cellular processes (Hartmann, et al., 2010). These results are consistent with studies in another lineage of E. faecalis, in which deletion of the genes encoding the CroRS TCS resulted in a loss of resistance to ceftriaxone (Comenge, et al., 2003). Further analysis of the deletion mutant revealed that the loss of CroRS function did not alter the expression of Pbp5, peptidoglycan precursor production, or peptidoglycan crosslinking. The CroRS TCS appeared to function according to conventional models for TCSs, in that the CroS kinase could autophosphorylate itself in an ATP-dependent manner, followed by the transfer of the phosphoryl group to the CroR response regulator. The CroR response regulator contains a functional DNA binding domain, which suggests that transcriptional remodeling is necessary for adaptation to the stress imposed by cephalosporins. However, only a few genes controlled directly by CroR have been identified thus far. These include salB, encoding a secreted protein that does not contribute to cephalosporin resistance (Murray, 1990), croRS itself (Murray, 1990), and genes that encode a putative glutamine transporter (Lebreton, et al., 2011) with no obvious connection to cephalosporin resistance. Treatment of E. faecalis cells with a panel of cell-wall-active antibiotics resulted in induction of a CroR-dependent promoter, but nothing more is known about the nature of the physiological signal(s) that influences CroS kinase regulation of cephalosporin resistance.

In addition to the CroRS TCS, a second signal transduction protein (IreK) is required for cephalosporin resistance in E. faecalis. IreK exhibits a characteristic bipartite domain architecture that includes a 𠇎ukaryotic-type” Ser/Thr kinase coupled, through a putative transmembrane segment, to a series of five repeats of the PASTA domain. Homologs of IreK are nearly universal among the genomes of Gram-positive bacteria. The function of the extracellular PASTA domains is not well understood, but it has been proposed that they bind to peptidoglycan or fragments thereof (Moellering, Jr. & Weinberg, 1971 Squeglia, et al., 2011 Yeats, Finn, & Bateman, 2002), which suggests that IreK could serve as a transmembrane receptor kinase that senses damage or perturbation of the peptidoglycan and initiates a signaling circuit to restore cell wall integrity. Consistent with that view, a homolog of IreK in Bacillus subtilis (PrkC) responds to fragments of peptidoglycan released by growing cells, which signals exit from dormancy by B. subtilis spores (Shah, Laaberki, Popham, & Dworkin, 2008). Analysis of an E. faecalis deletion mutant revealed that IreK is required for intrinsic cephalosporin resistance and for resistance to certain other cell-envelope stresses, such as detergents that are present in bile salts (Kuch, et al., 2012). IreK exhibits protein kinase activity in vitro, and its kinase activity is required to promote cephalosporin resistance in E. faecalis cells (Kristich, Wells, & Dunny, 2007). As with other members of this kinase family, IreK can catalyze autophosphorylation of threonine residues contained with a specific segment of the kinase domain known as the �tivation loop”. Phosphorylation at these sites is usually thought to lead to a conformational change, which results in enhanced activity of the kinase (i.e., �tivation”). Analysis of site-directed mutants bearing phosphomimetic substitutions at these sites in IreK support the hypothesis that phosphorylation of the IreK activation loop enhances kinase activity in vivo and leads to increased cephalosporin resistance (Kristich, Wells, & Dunny, 2007). Other than itself, physiological substrates of IreK in E. faecalis cells that are important for cephalosporin resistance have not yet been described.

E. faecalis IreK and its homologs in other low-GC Gram-positive bacteria are encoded immediately adjacent to a gene that encodes a PP2C-type protein phosphatase (called IreP in E. faecalis). IreP can dephosphorylate both IreK and substrates of IreK in vitro, and analysis of deletion mutants lacking IreP indicate that this activity is important in vivo. IreP mutants exhibit substantial hyperresistance to cephalosporins, a finding which is consistent with hyperactivation of the IreK kinase (Kristich, Wells, & Dunny, 2007). Furthermore, mutants that lack IreP exhibit a large reduction in fitness in the absence of cephalosporins, as compared to wild-type E. faecalis, which indicates that uncontrolled activation of cephalosporin resistance mechanisms imparts a significant fitness cost to the cell. The complex regulatory circuitry controlling intrinsic cephalosporin resistance in E. faecalis may therefore stem from the fitness cost that is associated with expression of this phenotype.


A recent transposon mutagenesis screen in E. faecalis revealed a new determinant of intrinsic cephalosporin resistance in enterococci (Vesić & Kristich, 2012). As with most low-GC Gram-positive bacteria, the genome of E. faecalis encodes two homologs (annotated as MurAA and MurAB) of the enzyme that catalyzes the first committed step in the synthesis of the peptidoglycan precursor UDP-N-acetylglucosamine 1-carboxyvinyl transferase, which performs PEP-dependent conversion of UDP-N-acetylglucosamine to UDP-N-acetylglucosamine-enolpyruvate. Deletion of murAA, but not murAB, led to an increased susceptibility of E. faecalis to cephalosporins. This enhanced cephalosporin susceptibility does not reflect a general growth or cell-wall synthesis defect of the mutant, because the deletion mutant is not sensitized to antibiotics in general—or even to all antibiotics that inhibit cell wall biosynthesis𠅋ut exhibits a loss of resistance specifically for extended spectrum cephalosporins and for fosfomycin (an antibiotic known to target MurA homologs). Chemical genetic analysis revealed synergistic action of ceftriaxone with fosfomycin that was also observed with two strains of E. faecium, suggesting that MurAA of E. faecium functions in a similar manner to promote cephalosporin resistance. In addition, expression of murAA-enhanced cephalosporin resistance in an E. faecalis mutant that lacked IreK, which suggests that MurAA may function downstream of IreK in a pathway that leads to cephalosporin resistance. Further genetic analysis revealed that MurAA catalytic activity is necessary, but not sufficient, for this role.

Ampicillin resistance

Modifications in Pbp5 are associated with increased resistance to beta-lactams, such as ampicillin. For example, the Pbp5-encoding gene found in hospital-associated, ampicillin-resistant strains of E. faecium differs by

5% from the corresponding gene in community-associated, ampicillin-susceptible strains (Garnier, Taourit, Glaser, Courvalin, & Galimand, 2000). Most studies that report an association between mutations in Pbp5 and enhanced ampicillin resistance have been performed on non-isogenic clinical isolates, in which unknown factors other than Pbp5 may influence resistance. To circumvent this limitation, Rice and colleagues (Rice, Calderwood, Eliopoulos, Farber, & Karchmer, 1991) used a plasmid-based pbp5 expression system to explore the impact of specific amino acid substitutions in Pbp5 on ampicillin resistance.

Substitutions that had previously been implicated as contributing to ampicillin resistance in clinical strains conferred modest levels of resistance when expressed from the plasmid-borne pbp5 in an otherwise-susceptible E. faecium host, thereby providing direct evidence of their influence. Combinations of point mutations, especially Pbp5 M485A with a Ser insertion at position 466, yielded substantially enhanced levels of resistance. Furthermore, a correlation was established between the affinity of purified, recombinant Pbp5 mutants for antibiotic binding, with resistance levels provided by these alleles. Further analysis revealed that the chromosomally-encoded pbp5 determinant could be transferred between strains of E. faecium (Rice L. B., 2005) by conjugation, which suggests a mechanism by which high-level ampicillin resistance conferred by mutant pbp5 alleles could be disseminated among clinical isolates. Although the mechanism of conjugative transfer was not established in that study, a plausible mechanism could involve mobilization mediated by co-integrated enterococcal conjugative plasmids, as recently described in E. faecalis (Marshall, Donskey, Hutton-Thomas, Salata, & Rice, 2002). Similar to E. faecium, mutations in Pbp5 of clinical isolates of E. faecalis may also lead to enhanced resistance to beta-lactam antibiotics, such as imipenem and ampicillin (Oyamada Y. , et al., 2006).

L,D transpeptidation

A multi-step in vitro selection process was used to generate a highly ampicillin-resistant mutant of E. faecium (Mainardi, et al., 2002). The mutant was found to contain exclusively L-Lys-3-D-Asx-L-Lys cross-links in its peptidoglycan (a product of L,D-transpeptidation, rather than the typical D,D-transpeptidation carried out by PBPs), and peptidoglycan composition was unaffected by the presence of ampicillin. Analysis of peptidoglycan composition of strains with intermediate levels of resistance obtained during the selection process revealed that the balance between D,D-transpeptidation and L,D-transpeptidation influences ampicillin resistance (Manson, Hancock, & Gilmore, 2010). High-level resistance requires elevated activity of a beta-lactam insensitive D,D-carboxypeptidase, which cleaves the peptide stem termini of normal peptidoglycan precursors to generate the substrate for the L,D transpeptidase. The metallo-D,D-carboxypeptidase (DdcY) responsible for cleavage of the peptidoglycan precursors has been identified (183), and its enhanced expression appears to be mediated by mutations that result in activation of a putative cryptic two-component signal transduction system (DdcSR). The enzyme responsible for the formation of the L,D- crosslinks (called Ldtfm) has been identified (Mainardi, Gutmann, Acar, & Goldstein, 1995) and appears to be constitutively expressed in E. faecium. Ldtfm is evolutionarily unrelated to the D,D-transpeptidases, in that it uses an active-site Cys nucleophile, rather than a Ser nucleophile. In addition, homologs of Ldtfm are encoded in the genomes of a variety of Gram-positive bacteria (Mainardi, et al., 2005 Mainardi, Gutmann, Acar, & Goldstein, 1995). The normal physiological function of the L,D-transpeptidase is unclear, but it has been proposed to have a role in the maintenance of peptidoglycan structure in stationary-phase cells. Surprisingly, although beta-lactams are usually thought to specifically inhibit the D,D-transpeptidase activity of PBPs, a particular sub-class of the beta-lactam antibiotic family was found to inhibit Ldtfm via covalent modification of the active site Cys (Mainardi, Legrand, Arthur, Schoot, van Heijenoort, & Gutmann, 2000). As noted above, activation of the L,D transpeptidase cross-linking pathway can result in emergence of cross-resistance to beta-lactams and glycopeptides.

Linezolid resistance

Linezolid is a member of the oxazolidinone family of antibiotics developed for use against multidrug-resistant Gram-positive bacteria. Linezolid interferes with bacterial growth by inhibiting protein synthesis through interaction with the translational initiation complex. Resistance to linezolid can be selected in vitro, and has also been observed in clinical settings (89, 164). Mutations within the central loop of domain V of 23S rRNA, including a G2576U mutation, are associated with resistance to linezolid, and presumably prevent or reduce binding of the antibiotic to the ribosomal subunit. Analysis of linezolid-resistant E. faecalis and E. faecium isolates selected during therapy, or obtained from patients following linezolid treatment failure, revealed a direct correlation between the percentage of rRNA genes that carry a G2576U mutation (each genome encodes several copies of the rRNA genes𠅏our in E. faecalis and six in E. faecium) and the phenotypic level of linezolid resistance, which suggests that the percentage of ribosomes that carry rRNA with the G2576U substitution is the primary determinant for the level of linezolid resistance (Martínez-Martínez, Pascual, & Jacoby, 1998). A similar correlation was observed for linezolid-resistant mutants of E. faecalis selected in vitro in a recombination-proficient genetic background (Lu, Chang, Perng, & Lee, 2005). Attempts to derive linezolid resistant mutants in a recombination-deficient mutant genetic background did not yield resistant mutants at comparable frequencies. Collectively, these results suggest that recombination between rRNA genes after the emergence of the G2576U mutation may enable the amplification of the level of linezolid resistance in enterococci under the selective pressure imposed by antibiotic treatment.

More Resources

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