Anthony R. M. Coates

Antibiotic resistance—the need for global solutions

The causes of antibiotic resistance are complex and include human behaviour at many levels of society; the consequences affect everybody in the world. Similarities with climate change are evident. Many efforts have been made to describe the many different facets of antibiotic resistance and the interventions needed to meet the challenge. However, coordinated action is largely absent, especially at the political level, both nationally and internationally. Antibiotics paved the way for unprecedented medical and societal developments, and are today indispensible in all health systems. Achievements in modern medicine, such as major surgery, organ transplantation, treatment of preterm babies, and cancer chemotherapy, which we today take for granted, would not be possible without access to effective treatment for bacterial infections. Within just a few years, we might be faced with dire setbacks, medically, socially, and economically, unless real and unprecedented global coordinated actions are immediately taken. Here, we describe the global situation of antibiotic resistance, its major causes and consequences, and identify key areas in which action is urgently needed.

The future challenges facing the development of new antimicrobial drugs

The emergence of resistance to antibacterial agents is a pressing concern for human health. New drugs to combat this problem are therefore in great demand, but as past experience indicates, the time for resistance to new drugs to develop is often short. Conventionally, antibacterial drugs have been developed on the basis of their ability to inhibit bacterial multiplication, and this remains at the core of most approaches to discover new antibacterial drugs. Here, we focus primarily on an alternative novel strategy for antibacterial drug development that could potentially alleviate the current situation of drug resistance — targeting non-multiplying latent bacteria, which prolong the duration of antimicrobial chemotherapy and so might increase the rate of development of resistance.

Novel classes of antibiotics or more of the same

The world is running out of antibiotics. Between 1940 and 1962, more than 20 new classes of antibiotics were marketed. Since then, only two new classes have reached the market. Analogue development kept pace with the emergence of resistant bacteria until 10–20 years ago. Now, not enough analogues are reaching the market to stem the tide of antibiotic resistance, particularly among gram‐negative bacteria. This review examines the existing systemic antibiotic pipeline in the public domain, and reveals that 27 compounds are in clinical development, of which two are new classes, both of which are in Phase I clinical trials. In view of the high attrition rate of drugs in early clinical development, particularly new classes and the current regulatory hurdles, it does not seem likely that new classes will be marketed soon. This paper suggests that, if the world is to return to a situation in which there are enough antibiotics to cope with the inevitable ongoing emergence of bacterial resistance, we need to recreate the prolific antibiotic discovery period between 1940 and 1962, which produced 20 classes that served the world well for 60 years. If another 20 classes and their analogues, particularly targeting gram‐negatives could be produced soon, they might last us for the next 60 years. How can this be achieved? Only a huge effort by governments in the form of finance, legislation and providing industry with real incentives will reverse this. Industry needs to re‐enter the market on a much larger scale, and academia should rebuild its antibiotic discovery infrastructure to support this effort. The alternative is Medicine without effective antibiotics.

Sterilizing Activities of Fluoroquinolones against Rifampin-Tolerant Populations of Mycobacterium tuberculosis

ABSTRACT The bactericidal activities of ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin, and gatifloxacin were tested in three models of rifampin-tolerant Mycobacterium tuberculosis persisters. Model 1 was a 100-day-old, unshaken, anaerobically adapted culture in which serial dilutions of the quinolones were incubated for 5 days and CFU counts were then done In models 2 and 3, 100 mg of rifampin/liter was added to the 100-day culture for 5 or 7 days to produce tolerant organisms that did not grow on plates; the rifampin was then washed off, fresh medium was added to allow recovery of growth on plates, and the culture was incubated for 7 days before CFU counts. In model 2, the quinolones were added after rifampin had been washed off, whereas in model 3 the quinolones were added to the cultures containing rifampin. In models 1 and 2, ciprofloxacin had the least bactericidal activity, ofloxacin and levofloxacin had greater activities, and moxifloxacin and gatifloxacin had the greatest activities. In model 3, ofloxacin had no detectable activity whereas moxifloxacin killed about log10 0.279 CFU of the persisters per ml at concentrations attainable in lesions; isoniazid had virtually no activity. These findings predict that ofloxacin will not be found to have effective sterilizing activity in clinical studies now planned whereas moxifloxacin will be able to shorten treatment.

Circulating Human Heat Shock Protein 60 in the Plasma of British Civil Servants Relationship to Physiological and Psychosocial Stress

Background—The Whitehall cohort studies (I and II) of British civil servants have identified sociodemographic, psychosocial, and biological risk factors for coronary heart disease (CHD). To identify mechanisms responsible for susceptibility to CHD, specific biological markers of stress are increasingly being measured. One marker linked to susceptibility to CHD is heat shock protein (Hsp) 60. Methods and Results—Blood was taken from 229 civil servants (126 men and 103 women) in the Whitehall II cohort drawn equally from the range of employment grades. Plasma was assayed for levels of Hsp60, tumor necrosis factor &agr; (TNF&agr;), C-reactive protein, von Willebrand factor, high density lipoprotein (HDL), total cholesterol, and total/HDL ratio. Psychosocial measures included socioeconomic status, psychological distress, and social isolation. The majority of the participants had Hsp60 in their plasma, and ≈20% had >1000 ng/mL of this protein (a concentration likely to induce biological effects). A positive association between plasma Hsp60 and TNF&agr; and a negative association with von Willebrand factor was found. There was also a significant association between elevated Hsp60 levels, low socioeconomic status, and social isolation, together with an association with psychological distress in women. Conclusions—The majority of participants exhibited Hsp60 in their plasma, and there was evidence of an association between levels of this stress protein and the proinflammatory cytokine, TNF&agr;, and with various psychosocial measures.

Detection of mRNA Transcripts and Active Transcription in Persistent Mycobacterium tuberculosis Induced by Exposure to Rifampin or Pyrazinamide

Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.

Novel approaches to developing new antibiotics for bacterial infections

Antibiotics are an essential part of modern medicine. The emergence of antibiotic‐resistant mutants among bacteria is seemingly inevitable, and results, within a few decades, in decreased efficacy and withdrawal of the antibiotic from widespread usage. The traditional answer to this problem has been to introduce new antibiotics that kill the resistant mutants. Unfortunately, after more than 50 years of success, the pharmaceutical industry is now producing too few antibiotics, particularly against Gram‐negative organisms, to replace antibiotics that are no longer effective for many types of infection. This paper reviews possible new ways to discover novel antibiotics. The genomics route has proven to be target rich, but has not led to the introduction of a marketed antibiotic as yet. Non‐culturable bacteria may be an alternative source of new antibiotics. Bacteriophages have been shown to be antibacterial in animals, and may find use in specific infectious diseases. Developing new antibiotics that target non‐multiplying bacteria is another approach that may lead to drugs that reduce the emergence of antibiotic resistance and increase patient compliance by shortening the duration of antibiotic therapy. These new discovery routes have given rise to compounds that are in preclinical development, but, with one exception, have not yet entered clinical trials. For the time being, the majority of new antibiotics that reach the marketplace are likely to be structural analogues of existing families of antibiotics or new compounds, both natural and non‐natural which are screened in a conventional way against live multiplying bacteria.

Nasal decolonization of Staphylococcus aureus with mupirocin: strengths, weaknesses and future prospects

Staphylococcus aureus in the nose is a risk factor for endogenous staphylococcal infection. UK guidelines recommend the use of mupirocin for nasal decolonization in certain groups of patients colonized with methicillin-resistant S. aureus (MRSA). Mupirocin is effective at removing S. aureus from the nose over a few weeks, but relapses are common within several months. There are only a few prospective randomized clinical trials that have been completed with sufficient patients, but those that have been reported suggest that clearance of S. aureus from the nose is beneficial in some patient groups for the reduction in the incidence of nosocomial infections. There is no convincing evidence that mupirocin treatment reduces the incidence of surgical site infection. New antibiotics are needed to decolonize the nose because bacterial resistance to mupirocin is rising, and so it will become less effective. Furthermore, a more bactericidal antibiotic than mupirocin is needed, on the grounds that it might reduce the relapse rate, and so clear the patient of MRSA for a longer period of time than mupirocin.

Chaperonins are cell-signalling proteins: the unfolding biology of molecular chaperones

The chaperonins are a subgroup of oligomeric molecular chaperones; the best-studied examples are chaperonin 60 (GroEL) and chaperonin 10 (GroES), both from the bacterium Escherichia coli. At the end of the 20th century, the paradigm of chaperonins as protein folders had emerged, but it is likely that during the 21st century these proteins will come to be viewed as intercellular signals. Indeed, it is possible that the chaperonins were among the first intercellular signalling proteins to evolve. During the past few years, it has emerged that chaperonin 10 and chaperonin 60 can be found on the surface of various prokaryotic and eukaryotic cells, and can even be released from cells. Secreted chaperonins can interact with a variety of cell types, including leukocytes, vascular endothelial cells and epithelial cells, and activate key cellular activities such as the synthesis of cytokines and adhesion proteins. Much has been made of the high degree of sequence conservation among the chaperonins, particularly in terms of the immunogenicity of these proteins. However, different chaperonin 60 proteins can bind to different cell-surface receptors, including the Toll-like receptors, suggesting that this family of proteins cannot be treated as one biological entity and that several subfamilies may exist. Chaperonins have been implicated in human diseases on the basis of their immunogenicity. The finding that chaperonins can also induce tissue pathology suggests that they may play roles in infections and in idiopathic diseases such as atherosclerosis and arthritis.

Stress wars: the direct role of host and bacterial molecular chaperones in bacterial infection.

Since 1962, when Ferruccio Ritossa discovered new puffing patterns in the polytene chromosomes of Drosophila incubated at an elevated temperature (153), we have been aware that stress at the cellular level is answered by the production of specific gene products. These products are variously termed heat shock proteins (Hsps) or cell stress proteins and were originally identified as molecules that are produced in response to the presence of unfolded proteins within the cell (2). However, it was not until the pioneering work of the groups of Laskey, Ellis, and Georgopoulos that the relationship between the generation of correctly assembled macromolecules and the proteins that function to ensure correct assembly was established. Laskey and coworkers studied the nuclear protein nucleoplasmin, which ensures correct assembly of histones and DNA into nucleosomes. Laskey termed nucleoplasmin a molecular chaperone as it mimicked the function of a human chaperone who ensures correct interactions between people (98). Ellis and Georgopoulos studied the protein which eventually was known as chaperonin 60 (Cpn60) and which was responsible for initiating the vast flood of papers on molecular chaperones over the past two decades (58). Currently, a molecular chaperone is defined by Ellis as “one of a large and diverse group of proteins that share the property of assisting the non-covalent assembly/disassembly of other macromolecular structures, but which are not permanent components of these structures when they are performing their normal biological functions” (58). A further refinement of terminology is necessary as certain molecular chaperones are produced constitutively and the concentrations within the cell do not increase in response to stress. These proteins are defined as molecular chaperones but are not Hsps or stress proteins. Molecular chaperones, whose concentrations increase in response to stress, are both chaperones and stress proteins/heat shock proteins. The first protein-folding molecular chaperone to be discovered was Cpn60 (58). Since the identification of this protein as a molecular chaperone in 1988, many more proteins with actual or putative molecular chaperone functions have been discovered, and the term currently applies to 25 families of proteins (Table ​(Table1).1). In all three kingdoms of life molecular chaperones are classified as essential proteins, and there is significant conservation of sequences between proteins used by prokaryotes and proteins used by eukaryotes (such as thioredoxin [Trx] family members, cyclophilins, chaperonins, Hsp70, and Hsp90). Eukaryotic cells have multiple compartments (cytosol, endoplasmic reticulum, mitochondria, nucleus), and in these compartments stress-induced protein folding is known as the unfolded protein response (158). The unfolded protein responses are an important element in the integrated biology of the cell, are linked to key intracellular signaling pathways, and are now being associated with human disease states (158). Implicit in the definition of molecular chaperones was that they were intracellular proteins involved in the folding of client proteins within cellular compartments, which, because of the high protein concentration (on the order of 200 to 400 mg/ml), favor inappropriate protein-protein interactions, resulting in significant protein denaturation (58). However, it is becoming clear that many molecular chaperones can exist outside the cell and participate in nonfolding actions. TABLE 1. Eukaryotic and prokaryotic molecular chaperone and stress protein families Analysis of immune responses to bacteria in the 1970s identified what was termed a “common antigen” in many bacterial species (81). Patients infected with Mycobacterium tuberculosis or Mycobacterium leprae exhibit significant antibody responses to a 65-kDa antigen (189). Subsequent work identified this antigen (and the common antigen) as the molecular chaperone Cpn60 (193). It has now been established that a number of molecular chaperones from bacteria and protozoan parasites (Cpn60, Hsp70, and Hsp90) are (i) potent immunogens, (ii) active immunomodulators, and (iii) inducers of cross-reactive immunity and autoimmunity (176). The mammalian immune system recognizes the molecular chaperones of infecting parasites as particularly strong immunological signals, which is surprising in view of the significant homology between host and parasite proteins. This profound immune responsiveness should be useful in developing vaccines against pathogens. The Cpn60 (Hsp65) protein of M. tuberculosis has also proven to be an extremely powerful immunomodulator that is able to protect against a number of experimental autoimmune diseases in rodents, including diabetes. In a recent phase II study, immunization of individuals who had newly developed type I diabetes with a peptide derived from human Cpn60 proved to be effective in limiting the progression of this disease (146). Most researchers studying molecular chaperones work within the paradigm that these proteins are present solely in intracellular compartments. However, in 1989, just 1 year after the identification of Cpn60 as a molecular chaperone, Japanese scientists found that the protein-folding catalyst and stress protein thioredoxin was secreted by T cells from patients with a certain form of leukemia and was able to induce T cells to express one of the subunits of the interleukin-2 (IL-2) receptor (167). Subsequently, the human protein was found to be a potent chemoattractant for neutrophils, monocytes, and T lymphocytes with a unique mechanism of action (124). Since this initial discovery, a growing number of mammalian molecular chaperones have been found to be secreted onto the cell surface or into the extracellular milieu, either tissue culture fluid (with cultured cells) or biological fluids such as blood, synovial fluid, or bronchoalveolar secretions (Table ​(Table2).2). Most of these secreted proteins have agonist activity with a mammalian cell population(s), normally myeloid and lymphoid cells and/or vascular endothelial cells (VECs) (62). Although less attention has been paid to the cell-cell signaling activity of bacterial molecular chaperones, bacterial Cpn10, Cpn60, and Hsp70 have been reported to stimulate or inhibit the proinflammatory actions of myeloid cells and VECs (62). Thus, it is becoming clear that molecular chaperones are examples of moonlighting proteins, that is, proteins which have more than one function. The enzymes of glycolysis are the prototypic moonlighting proteins. For example, secreted phosphoglucoisomerase has been identified as three distinct cytokines: neuroleukin, autocrine motility factor, and differentiation and maturation mediator. This protein also acts as an implantation factor (76). Perhaps the most bizarre example of the moonlighting functions of molecular chaperones is the neurotoxin used by a hunting insect, the antlion or doodlebug, to paralyze its prey. This toxin is produced by a symbiotic bacterium, Enterobacter aerogenes, which lives in the insects saliva. It has been determined that this toxin is a molecular chaperone, Cpn60. The E. aerogenes Cpn60 is almost identical to the Escherichia coli Cpn60 protein GroEL. Surprisingly, single-residue substitutions in GroEL can change it from an inactive protein into a potent insect neurotoxin (187). TABLE 2. Molecular chaperones found on the cell surface and/or secreted by cells and/or found in extracellular fluids In the last decade there have been a number of reports which support the hypothesis that inducible molecular chaperones, produced both by bacteria and by hosts, function as intracellular, cell surface, or extracellular signals which are involved in the control of the infectious process. This suggests that infection, among other things, is a contest of stress mechanisms with a multitude of unexpected evolutionary twists and turns. In this review we focus on bacterial infection but discuss examples from eukaryotic parasites that exemplify particular mechanisms. The molecular chaperones involved in the formation of the type III secretion system, while indirectly contributing to bacterial virulence, are not included here as they have been extensively reviewed elsewhere (48).