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Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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2
Microbe Resistance

OVERVIEW

Since the discovery and subsequent widespread use of antimicrobials, a variety of pathogenic viruses, bacteria, protozoa, and helminths have developed numerous mechanisms that render them resistant to some—and, in certain cases, to nearly all—antimicrobial agents. The focus of this session of the workshop was on exploring some of the latest information emerging about how various important pathogens develop resistance to drugs and how such resistance might be overcome.

The bacterial strains staphylococci, enterococci, and pneumococci pose some of the most serious problems in terms of antimicrobial resistance. Scientists have now acquired detailed information about how these bacteria develop drug resistance. In staphylococci, for example, optimization of resistance depends on the operation of a complex pathway involving a central resistance gene and a number of auxiliary genes. Thus, developing drugs that specifically target any of these genes holds potential for reducing the microbe’s drug resistance. A second novel intervention would target the ecology of these types of bacteria. For example, penicillin-resistant strains of pneumonia bacteria have been found to breed prolifically in the nasopharynx of preschool-age children, particularly those who attend day care centers. Devising interventions to limit antimicrobial exposure might help reduce the genetic propensity of these bacteria to develop drug resistance.

Malaria and schistosomiasis are major health threats in the developing

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

world. Chloroquine was historically the primary drug for treating malaria, but its widespread use has led to increasing microbial resistance. Scientists have now identified a particular type of mutation at a specific location on a single gene as being critical in the development of resistance, and efforts are now under way to develop new drugs that target this resistance mechanism. Praziquantel is the only drug now available to treat schistosomiasis. Since the drug has been in use for more than two decades, concerns are mounting that the parasitic worms that transmit the disease from snails to humans are beginning to become resistant. Among the immediate needs, praziquantel’s effectiveness can be prolonged by more selective use, with treatment targeted only to those people at greatest risk for heavy infection and morbidity, as well as by the use of integrated disease management practices, such as snail control, health education, and improved sanitation. At the same time, new drug development needs to continue in anticipation of the eventual failure of praziquantel efficacy.

Influenza is a global threat to health. Vaccines represent the first line of defense against the flu, with a new vaccine being developed and distributed each year in response to the changing genetic composition of the causative virus. Still, vaccines are not a total answer, and several classes of antiviral drugs have been developed to treat infected individuals. Two antivirals— amantadine and rimantadine—have been around since the 1960s. Although effective in some circumstances, both types of drugs suffer from drug-resistance problems. Another family of newer drugs, called neuraminidase inhibitors, shows even more promise, as these formulations appear to pose a reduced risk of triggering resistance. A major problem, however, is that the pharmaceutical companies that produce these newer drugs are not making enough doses to cover medical needs in the event—certain to happen at some point—that a highly modified and virulent form of the influenza virus emerges from the animal world and spreads among the human population worldwide.

Adding to concerns about antimicrobial resistance is the possibility that terrorists or a rogue nation might use “bioweapons” to expose large numbers of people to genetically engineered drug-resistant pathogens in order to trigger large-scale disease outbreaks. This scenario was brought into sharp perspective in autumn 2001 by the intentional distribution through the U.S. mail of envelopes containing spores of Bacillus anthracis. One issue considered during this session involved the effects of exposure to both anthrax and ionizing radiation at the same time, conditions that military personnel, in particular, might someday face. Based on a recent study in mice, scientists have been able to identify some fundamental factors that contribute to increased susceptibility to bacterial infections in general, and to B. anthracis in particular, after ionizing radiation, as well as to make some general

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

recommendations about effective methods of therapy and prophylaxis following such combined exposures.

NEW STRATEGIES AGAINST MULTI-DRUG-RESISTANT BACTERIAL PATHOGENS

Alexander Tomasz, Ph.D.

The Rockefeller University, New York, NY

A major impact of the “chemical warfare” that humanity has been waging against the microbial world on an escalating scale since the discovery of antibiotics is the emergence of a vast variety of resistance mechanisms that have moved into virtually all pathogenic species—viruses, bacteria, and protozoa alike. This emergence has occurred with a swiftness that, on an evolutionary scale, is truly remarkable.

The rapid progression from a uniformly antibiotic-sensitive bacterium to a uniformly antibiotic-resistant species is well demonstrated by the case of Staphylococcus aureus, which is a primary agent of hospital-acquired infections. In the early 1940s, when penicillin was introduced into therapy, all strains of staphylococci were highly sensitive to this antibiotic. In less than a decade, S. aureus acquired the penicillinase-based resistance mechanism from an unknown “extra species” source. Penicillin resistance spread across the entire species with the “plasmid epidemic,” and by the late 1950s penicillin was useless against S. aureus.

The final stage of this remarkable and sweeping genetic change, propelled by the pressure of antibiotic use, is documented in a recent study conducted in Portugal (Sá-Leão et al., 2001). Screening the S. aureus nasal flora recovered from 1,000 young and healthy volunteers who had never received antibiotics showed that 97 percent of the S. aureus colonizing these individuals produced penicillinase and were resistant to penicillin (Sá-Leão et al., 2001). Clearly, the extra-species drug-resistance gene penicillinase has become a domesticated genetic component of S. aureus without causing any survival deficit to the cells. The penicillin-resistant S. aureus, which was originally associated only with patients in hospitals, has managed to move into the community within 50 years of its appearance on the scene.

Equally fast was the response of S. aureus and other staphylococci to the introduction in 1959 of semisynthetic ß-lactam antibiotics, such as methicillin. The first methicillin-resistant S. aureus (MRSA) was detected in the United Kingdom in 1961 (Jevons, 1961). By the 1990s, MRSA had become a globally spread pathogen, making the management of nosocomial S. aureus infections complicated and expensive.

Essentially the same phenomena were observed in Streptococcus

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

pneumoniae, one of the major community-acquired pathogens of our era. S. pneumoniae are responsible for a series of potentially life-threatening diseases that together cause an estimated 1 million to 3 million deaths worldwide annually. The first penicillin-resistant pneumococci (PRSP) were detected in 1965 (Hansman et al., 1974), followed by increasing numbers of reports on the detection of resistant strains. By the mid-1990s, penicillin-resistant strains had spread globally.

In both MRSA and PRSP, antibiotic resistance has unfolded in stages, on a rapid time and geographic scale. Initial detection of MRSA and PRSP was followed by reports on geographic spread. Next came reports on the increase in resistance level and in the frequency of resistant isolates. Eventually, multi-drug-resistant strains carrying resistance traits to different classes of antimicrobial agents also began to appear.

In 1993, a small group of international experts, including microbiologists, physicians, and public health personnel, gathered at Rockefeller University for a workshop to survey data on the accelerating spread of multi-drug-resistant pathogens (Tomasz, 1994). By this time, the specter of untreatable bacterial infections had appeared on the horizon as a clear possibility. Strains of common community-acquired and nosocomial pathogens equipped with multi-drug-resistant traits had been identified, with some clinical isolates retaining susceptibility to only a single antimicrobial agent.

The workshop participants identified a number of specific genetic events which, if they occurred, could precipitate a genuine public health crisis. Examples of such events include the acquisition of high-level vancomycin resistance among MRSA or pneumococci, and the acquisition of ß-lactamase plasmid by group A streptococci. The alarm sounded at the workshop was recently echoed by the World Health Organization (WHO): “Increasingly drug-resistant infections in rich and developing nations alike are threatening to make once treatable diseases incurable (WHO, 2000). Tables 2-1 and 2-2 illustrate the multi-drug resistance phenomenon: the strikingly successful adaptation of two major human pathogens—S. aureus and S. pneumoniae—to a planetary environment that became saturated with highly toxic substances due to the immense quantities of antimicrobial agents deployed in human and veterinary medicine, in agribusiness, and in virtually the entire biosphere.

What can one do in this situation? Clearly the backlash of multi-drug resistance has caught the pharmaceutical chemists and infectious diseases specialists by surprise. In retrospect, it seems that the antibiotic era had two interrelated “cardinal sins.” One sin was the neglect and sometimes complete abandonment of preventive measures in favor of a single-minded antibiotic strategy against bacterial infections. The second was the failure to seriously consider consequences of the fact that the overwhelming major

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

TABLE 2-1 Development of Multi-Drug Resistance by S. aureus and S. epidermidis (S: susceptible, R: resistant)

 

S. aureus ATCC 6538 (1930)

MRSA Brazilian epidemic clone (1994)

Methicillin-resistant S. epidermidis New York Hospital (1996)

Amikacin

S

R

R

Amp/Sulbactam

-

R

R

Ampicillin

S

R

R

Cephalothin

S

R

R

Cefotaxime

S

-

-

Chloramphenicol

S

R

R

Ciprofloxacin

S

R

R

Clindamycin

S

R

R

Erythromycin

S

R

R

Gentamicin

S

R

R

Imipenem

S

R

R

Oxacillin

S

R

R

Rifampin

S

R

R

Vancomycin

S

S

S

Teicoplanin

S

S

-

Tetracycline

S

R

R

Trimeth/Sulfa

S

R

-

Mupirocine (topical)

S

R

R

ity of both the most effective antibiotics and resistance mechanisms are actually products of the microbial world. Antibiotics are produced in tiny quantities and on a microscopic scale by some microbes—presumably for the control of the “quorum” of their habitat—and the producer microbes also invented self-protective resistance mechanisms against their own products. The reintroduction of these highly toxic agents into the biosphere in enormous quantities was a major violation of this quorum sensing. It has amplified local wars among microbes to a global conflict between human and microbe, a chemical warfare in which both offensive and defensive (resistance) armaments came from the microbial world (Tomasz, 2000).

The current genomic revolution may offer clues for the production of new antimicrobial agents that would not have been invented by the microbial world during evolution. Development and introduction of such novel agents would be a welcome development indeed. However, it would be naïve to think that the microbial world already “awakened” by the antimicrobial armaments race would simply submit to such new onslaughts. Antibiotic-resistance mechanisms have emerged rapidly in the past, even against completely synthetic agents, such as trimethoprim and fluoro

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

TABLE 2-2 Development of Multi-Drug Resistance by S. pneumoniae and Enterococcus faecium (S: susceptible, R: resistant)

 

S. pneumoniae D39 (1949)

S. pneumoniae 6B Dallas, Texas (1992)

Enterococcus faecium (VRE) (Tn5482) Memorial Hospital (1996)

Amikacin

S

-

R

Amp/Sulbactam

S

R

R

Ampicillin

S

R

R

Cephalothin

S

-

R

Cefotaxime

S

R

-

Chloramphenicol

S

R

S

Ciprofloxacin

S

R

R

Clindamycin

S

R

R

Erythromycin

S

R

R

Gentamicin

S

-

R

Imipenem

S

S

R

Oxacillin

S

R

R

Rifampin

S

-

R

Vancomycin

S

S

R

Teicoplanin

S

S

R

Tetracycline

S

R

S

Trimeth/Sulfa

S

R

R

Mupirocine (topical)

S

-

-

quinolones. Thus, a possible deployment of new antimicrobial agents would not solve the basic dilemma of the antimicrobial armaments race that originates from its erroneous core philosophy: namely, the indiscriminate killing of bacteria by wide-spectrum antimicrobial agents. The fallacies of this philosophy have been pointed out repeatedly (Tomasz, 2000).

There are a number of antimicrobial strategies, not yet exploited, that would be more discriminatory and therefore less likely to provoke another wave of drug resistance. The pharmaceutical industry’s traditional approach to drug development has been either to find new wide-spectrum drugs against bacterial targets or to reconfigure old drugs against targets that became inaccessible due to drug resistance. Examples of these two strategies would be the development of new classes of fluoroquinolones and the semisynthetic modification of ß lactams to accommodate the penicillinase-based resistance mechanism.

However, there are at least two completely different strategies that offer promise. The first strategy would target the resistance phenotype; the second would target the ecology of resistant bacteria.

Recent studies have shown that in both MRSA and PRSP, high-level

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

antibiotic resistance requires more than the presence of the central drug-resistance determinant (the mecA gene in the case of MRSA, and the mosaic PBP genes that encode low affinity binding proteins in PRSP). Expression of an optimal high-level antibiotic-resistant phenotype also requires the assistance of a number of additional genetic determinants, the functioning of which is critical for the generation of antibiotic resistance, although the protein products of these genes do not react with the antimicrobial agent. Transposon mutagenesis of a highly methicillin-resistant MRSA strain has identified over 20 such “auxiliary genes” (De Lencastre et al., 1999). Inactivation of these genes had no effect on the transcription of the resistance gene mecA to its gene product (the low affinity penicillin binding protein PBP2A), yet phenotypic resistance of the bacteria was drastically reduced. Recent observations in PRSP identified a similar phenomenon. Inactivation of the small pneumococcal operon murMN, responsible for the production of branched structured components in the bacterial cell wall, caused a complete collapse of the penicillin resistant phenotype in spite of the fact that the primary resistance determinants (the low affinity PBPs) remained unchanged in the mutant bacteria (Filipe and Tomasz, 2000).

These observations indicate that reversal of drug resistance is possible by two completely different ways: either by inactivation of the central genetic determinant and its gene product, or by inactivation of the products of auxiliary genes (see Figure 2-1). It follows that auxiliary genes represent novel types of antibacterial targets. Compounds capable of inactivating the products of these genes should represent synergistic agents that together with ß-lactam antibiotics would render resistant bacteria sensitive again to these classical antimicrobial agents.

FIGURE 2-1 Two ways to reverse drug resistance.

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

A major roadblock to development of such agents, however, is that this approach seems to collide head-on with the central philosophy of the pharmaceutical industry, which is only willing to invest in the development of wide-spectrum antimicrobial agents that can assure a market in the range of $1 billion a year. Clearly, the types of agents described here would be specific for the particular bacterial pathogen and therefore would be outside such marketing interest. While the position of “big pharma” on this issue is based on complex economic realities, I believe that the future points in a different direction: the development of highly specific narrow-spectrum agents, the deployment of which would not challenge the entire microbial world each time they are used in therapy. Such development will be hastened by current progress in devising highly sensitive molecular techniques for rapidly detecting and identifying bacterial pathogens—a capability that may lie in the not too distant future. With rapid and safe diagnostics at hand, the use of wide-spectrum antimicrobial agents should be reserved to special cases only because of their indiscriminate challenge to both harmful and harmless bacteria.

A second novel intervention with bacterial pathogens, particularly drug-resistant strains, would target the ecology of these bacteria. Recent observations indicate that the overwhelming majority of diseases caused by resistant strains of S. aureus are linked to a surprisingly few epidemic clones or genetic lineages that have immense geographic spread (Oliveira et al., 2002) and that appear to combine in their genetic backgrounds not only determinants of antibiotic resistance but also genes that assure ecological success (i.e., spread and colonization) of the bacteria (see Figure 2-2). Similar observations also have been made for penicillin-resistant S. pneumoniae (Sá-Leão et al., 2000). Clearly, identification of determinants of epidemicity may provide completely new targets—vaccines or chemical agents—against specific multi-drug-resistant clones that are responsible for most of the hardships of resistant disease.

Following up on this ecological reasoning raises questions related to the ecological reservoirs of bacterial pathogens, particularly the drug-resistant clones. It has been clearly shown that in the case of PRSP a major sanctuary and breeding ground of drug-resistant strains is the nasopharynx of pre-school-age children, particularly those who attend day care centers. All children have immature immune systems. When this natural condition is combined with the close contact among children that is characteristic of day care centers, the high frequency of viral respiratory diseases in such centers, and the use (and misuse) of immense quantities of antimicrobial agents, the result is the creation of a bona fide “factory” of resistant pneumococci (Sá-Leão et al., 2000). Similar studies could not identify a comparable reservoir of MRSA among healthy carriers (Sá-Leão et al., 2001).

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

FIGURE 2-2 Geographic spread of pandemic MRSA clones.

Rather, it seems that for resistant staphylococci the ecological reservoir is the hospital itself.

A novel and potentially effective intervention to reduce the spread of resistant forms of these two important pathogens would involve intervention at the level of their ecological reservoirs—namely, lowering the carriage rate of resistant bacteria. The European Community has recently initiated such a major project (EURIS, European Resistance Intervention Study), which is aimed at identifying the most effective intervention strategies by which carriage of resistant pneumococci could be reduced among children attending day care centers in member countries (Sá-Leão et al., 2000). An analogous attempt for MRSA would zero in on the hospital itself by introducing rigorous infection-control measures, such as those that have been successfully tested and advocated by several recent studies (Farr and Jarvis, 2002; Pittet, 2002).

MALARIA AND THE PROBLEM OF CHLOROQUINE RESISTANCE

Thomas E. Wellems, M.D., Ph.D.

Laboratory of Malaria and Vector Research National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, MD

The discovery of chloroquine nearly 70 years ago had a considerable impact against the morbidity and mortality of malaria. This impact had

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

such effect that chloroquine became recognized as one of the most successful and important drugs ever deployed against an infectious disease. Massive use of the drug, however, eventually produced resistant malaria strains (Peters, 1989). First reports of chloroquine resistance were with Plasmo dium falciparum, the species responsible for the most acute and deadly form of human malaria (Payne, 1987). By the 1970s, resistant P. falciparum strains were established in South America, India, Southeast Asia, and Papua New Guinea. Africa was spared until the late 1970s, when resistance was detected in Kenya and Tanzania, seeding the spread of resistance across the continent within a decade (Peters, 1987). In the absence of a replacement drug with the low cost and reliability of chloroquine, the morbidity and mortality from malaria resurged in Africa (Greenberg et al., 1989; Trape et al., 1998).

Molecular Basis of Chloroquine Action and Resistance

Chloroquine interrupts hematin detoxification in malaria parasites as they grow within their host red blood cells (Chou et al., 1980) (see Figure 2-3).

FIGURE 2-3 The pathway of hemoglobin digestion and hematin polymerization in a P. falciparum-infected red blood cell. Chloroquine accumulates in the acid digestive food vacuole of sensitive parasites and interferes with polymerization. Chloroquine-resistant parasites reduce this accumulation and thereby reduce drug toxicity.

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

Hematin, a toxic ferriprotoporphyrin product released from digested host hemoglobin, is normally detoxified in the parasite’s acid food vacuole by polymerization into innocuous pigment crystals (Dorn et al., 1998). Chloroquine interferes with polymerization and poisons the parasite by complexing with hematin and adsorbing to the growing faces of the crystals (Sullivan et al., 1996; Pagola et al., 2000).

Chloroquine-resistant P. falciparum survives drug exposure by reducing the accumulation of chloroquine in the digestive food vacuole (Verdier et al., 1985). The mechanism of this reduction, not yet established, may involve changes in digestive vacuole pH or a direct effect on drug flux across the digestive vacuole membrane.

Chloroquine resistance results from multiple mutations in PfCRT, a P. falciparum protein located at the parasite’s digestive vacuole membrane (Fidock et al., 2000). PfCRT contains 10 predicted transmembrane segments and has a structure consistent with a transporter or channel (Nomura et al., 2001) (see Figure 2-4). Although the exact patterns of PfCRT mutations differ according to the geographic origin of chloroquine-resistant parasites, all of these patterns include a key substitution for lysine at position

FIGURE 2-4 Schematic representation of PfCRT and positions of mutations associated with chloroquine resistance. The critical K76T mutation occurs in the first of the ten predicted transmembrane domains. Filled circles show the positions of all other PfCRT mutations that have been identified in different chloroquine-resistant isolates. These mutations may compensate for the K76T change or help maintain critical functional properties of the PfCRT molecule in resistant parasites.

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

76. This substitution is universally K76T in naturally resistant P. falciparum. In drug pressure experiments, chloroquine-resistant parasites have been selected that carry alternative K76I and K76N substitutions in PfCRT, but such changes have not been detected outside the laboratory (Cooper et al., 2002). These results suggest that loss of lysine’s positive charge at PfCRT position 76 is a central feature of the chloroquine resistance mechanism.

The fact that K76T is always found in the context of additional PfCRT mutations suggests that this key substitution requires accommodative or compensatory adjustments elsewhere in the protein. Different patterns of mutations can evidently serve in these adjustments, as characteristic PfCRT types occur in the Eastern and Western hemispheres (Fidock et al., 2000; Carlton et al., 2001; Wellems and Plowe, 2001). At least four independent foci of chloroquine resistance have been deduced from these PfCRT types: two in South America; one in Southeast Asia that eventually spread to Africa; and one in or near Papua New Guinea with the same substitutions as a South American form. Drug pressure has driven population sweeps of chloroquine-resistant P. falciparum from these foci into nearly all malaria regions (Wootton et al., 2002) (see Figure 2-5). An exception in these sweeps is Central America, where P. falciparum populations are still sensitive to chloroquine and may be isolated by a genetic or transmission restriction against the entry of resistant strains.

FIGURE 2-5 Map of the spread of chloroquine-resistant Plasmodium falciparum from four foci identified by PfCRT alleles and polymorphic markers.

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

Role of PfCRT Mutations and Malaria Immunity in Chloroquine Treatment Outcomes

Epidemiological investigations have confirmed the association of chloroquine treatment failures with the PfCRT K76T mutation. In a study in Mali, this mutation and other possible markers of resistance were evaluated against chloroquine treatment outcomes in cases of uncomplicated malaria (Djimdé et al., 2001).The PfCRT K76T mutation was found in 100 percent of the chloroquine treatment failures, compared to a baseline prevalence of 41 percent K76T in patients before treatment. However, not all infections with chloroquine-resistant parasites persisted or recurred after chloroquine treatment: 27 percent of patients with these infections showed clinical resolution of symptoms and parasitological clearance. These clearances after drug treatment correlated with age, consistent with an important influence of immunity that develops in children over time from repeated malaria episodes (Djimdé et al., 2001; Wellems and Plowe, 2001).

The association between mutant PfCRT and chloroquine treatment outcomes has also been evaluated in the Cameroon (Basco and Ringwald, 2001), Sudan (Babiker et al., 2001), Mozambique (Mayor et al., 2001), and Papua Indonesia (Maguire et al., 2001), where chloroquine-sensitive and -resistant P. falciparum strains both remain common; and in Brazil (Vieira et al., 2001), Uganda (Dorsey et al., 2001a), Laos (Pillai et al., 2001), Thailand, and Papua New Guinea (Chen et al., 2001), where chloroquine-resistant parasites now predominate. Results from these studies support a universal association of the PfCRT K76T mutation with chloroquine treatment failures and show that additional factors including immunity can affect the outcome resistant infections after treatment.

The importance of immunity in treatment outcome has recently been demonstrated in a rodent model of drug-resistant malaria. Results with this model showed that rodents with partial immunity from previous infections benefited from drug treatment and cleared resistant Plasmodium yoelii parasites (Cravo et al., 2001).

Other Factors That May Affect Rates of Chloroquine Treatment Failure

The uptake, distribution, and metabolism of chloroquine can have a significant effect on treatment outcomes. Concentrations of chloroquine and its principal monodesethylchloroquine (mono-DEC) metabolite are reported to exhibit inter-individual variations that can influence classifications of resistance in vivo (Hellgren et al., 1989).

Chloroquine resistance lines that have been adapted to in vitro culture conditions frequently show differences in laboratory measures of chloroquine response (as measured by IC50 values). The question is often raised

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

whether these in vitro variations might be associated with additional P. falciparum determinants that can modulate the effect of PfCRT mutations and increase treatment failure rates. The Pgh-1 P-glycoprotein encoded by the pfmdr1 gene is a possible example of such a determinant (Cowman et al., 1991). Certain substitutions in Pgh-1 can affect the chloroquine IC50s of parasites resistant (but not sensitive) to chloroquine and are associated with in vitro mefloquine and quinine responses (Reed et al., 2000). Pgh-1 N86Y is a widespread polymorphism in Asia and Africa that has been associated with chloroquine-resistant strains in some studies but not in others (Dorsey et al., 2001b). In Mali, a 50 percent prevalence of Pgh-1 N86Y in patients before treatment rose to 86 percent (17 percent mixed type) in cases of chloroquine failure (Djimdé et al., 2001). However, multivariate analyses of PfCRT K76T and Pgh-1 N86Y showed no independent effect of Pgh-1 N86Y on treatment failure rates and no strengthening of the association of PfCRT K76T with these failure rates. These results indicate that the Pgh-1 N86Y polymorphism does not have an important effect in chloroquine treatment failure but may relate to fitness adaptations in response to physiological changes from PfCRT mutations.

Whether clinical chloroquine resistance might be affected by other Pgh-1 polymorphisms or modulatory determinants elsewhere in the P. falciparum genome will require additional epidemiological studies of candidate mutations and case treatment outcomes. Such studies will also help clarify uncertainties about the relative importance of the different factors that modulate in vitro drug responses in the laboratory and factors that affect treatment failure rates in vivo.

Points of Observation and Recommendation

  • Tests that rapidly detect the PfCRT K76T molecular marker have several advantages and applications in the surveillance of chloroquine-resistant P. falciparum. By making use of PCR-amplified DNA from small blood spots on filter paper, tests that are now available significantly reduce the time, labor, cost, and operative limitations of in vivo or in vitro assays with live parasites. Molecular marker data from large numbers of blood samples can therefore be efficiently collected and used in treatment policy decisions where the prevalence of resistant strains is unknown or changing. Fresh malaria outbreaks or drug-resistant epidemics are important situations that can benefit from such data. PfCRT K76T surveillance may also be useful in regions where chloroquine use has been stopped because of problems with resistance. Such surveillance may detect declining rates of chloroquine resistance, perhaps allowing the reconsideration of chloroquine in combination with other antimalarial drugs.

  • The importance of the interface between drug action and immunity

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
×

is highlighted by the relief from chloroquine-resistant malaria that immune individuals can often experience after treatment. Chloroquine is therefore still commonly used as a first-choice drug by malaria-experienced individuals in regions of drug resistance, especially where supplies of alternative drugs are expensive and must be targeted to young children and malaria-naive patients who lack protective immunity. An interesting question is whether vaccines will be able to improve therapeutic responses against malaria. Better understanding of the interface between drug action and immunity will help address this possibility.

  • New drugs that act on hematin but are not subject to the chloroquine resistance mechanism should be of great benefit. Indeed, certain 4-aminoquinoline compounds with side chain variations, such as short- and long-chain analogs of chloroquine (De et al., 1996; Ridley et al., 1996) and amodiaquine derivatives (Hawley et al., 1996), are known to be effective against chloroquine-resistant parasites. This may be because the chloroquine resistance mechanism is sensitive to the side chain structure of chloroquine while the inhibition of hematin polymerization depends largely on p-p recognition between the 7-chloro-substituted quinoline ring and hematin µ-oxodimers (Vippagunta et al., 1999). Hematin remains attractive as a target because it is a molecule the parasite cannot mutate. The search for new compounds with desirable pharmacokinetic profiles, low toxicity, and low production costs will be helped by improved understanding of the resistance mechanism at the molecular level.

  • Chemosensitizing agents that can be co-administered with chloroquine may have promise against chloroquine-resistant P. falciparum. Several studies have shown that resistance can be partially reversed by a range of structurally diverse agents that include calcium antagonists such as verapamil, various tricyclic compounds, and some plant alkaloids (Martin et al., 1987; Rasoanaivo et al., 1996). One of these compounds, chlorpheniramine, has been combined with chloroquine and tested in a study of African children (Sowunmi et al., 1997). The combination was suggested to have some benefit against chloroquine-resistant infections but requires further study. Better understanding of the mechanism by which chloroquine-chemosensitizing agents act may provide new leads for drug development.

  • Plasmodium vivax causes a debilitating form of malaria less fatal than P. falciparum but nevertheless still responsible for tremendous impact on the economy and life in afflicted regions. Despite comparable exposure of P. vivax and P. falciparum to chloroquine pressure, no P. vivax strain was reported to be chloroquine-resistant until 1989 (Rieckmann et al., 1989). Resistant P. vivax is present today in several regions of Southeast Asia (Baird et al., 1997), and some evidence suggests that it also occurs in South America (Whitby, 1997). Although chloroquine is thought to have the same action on hematin in P. vivax and P. falciparum, chloroquine

Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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resistance in these two major agents of malaria differs in that the homolog of the PfCRT transporter in P. vivax does not have reading-frame mutations associated with chloroquine treatment failures (Nomura et al., 2001). Investigations of P. vivax determinants responsible for resistance are presently limited by the lack of a practical in vitro culture system and genetic crosses for linkage mapping. These tools of investigation are needed. Advances against chloroquine-resistant malaria, including diagnostics and analogs of chloroquine against the different forms of P. vivax and P. falciparum, will benefit from basic research in this direction.

    DRUG RESISTANCE IN TREATMENT OF SCHISTOSOMIASIS

    Charles H. King, M.D., M.S.

    Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, OH

    The Worm Challenge

    The helminthic parasites, commonly referred to as “worms,” are among the most common of human infections. Worldwide, over 1 billion people are infected with Ascaris roundworms, 1.2 billion carry hookworm, and an estimated 200 million people are infected with blood flukes of Schistosoma spp. (WHO, 1993; Chan, 1997). Although worm infections are rarely lethal, they produce a significant spectrum of disease and disability, ranging from moderate exercise intolerance up to severe anemia and organ damage, with the resulting loss of millions of productive life-years globally (de Silva et al., 1997). Poor sanitation favors the simultaneous transmission of many parasites, and it is common for one person to harbor infection with multiple worms of many different helminth species. What is more, even after successful treatment, symptomatic, high-level worm infection may return in as few as three to six months after cure.

    The 1970s and 1980s saw the development of a number of safe and effective oral anthelmintics including mebendazole, albendazole, ivermectin, and praziquantel. Together, these broad-spectrum agents proved capable of giving effective treatment for almost any human helminth infection. The economics of treating human worms were not favorable, however. The limited resources of the developing countries, where worms are endemic, coupled with the significant overhead required to provide repeated drug therapy, severely limited the fundamental demand for these drugs. Although wide-scale, population-based programs were recommended by the WHO as the best means of controlling helminth-related diseases, in practice, many health ministries chose to focus on other priorities.

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    With the initially limited use of anthelmintics, serious drug resistance was not observed and was not thought to be a problem. An unfortunate consequence of the drugs’ initial success, combined with their limited use, was the disinclination of pharmaceutical researchers to pursue additional, new classes of anthelmintic drugs for human use. Both antiparasitic drug use and the roster of available anthelmintic drugs remained stable until the 1990s, when national and international programs began to provide widespread treatment for onchocerciasis and lymphatic filariasis. Newer programs, now being implemented, aim to provide widespread “deworming” of children at risk (Partnership for Child Development, 1997). These, too, will greatly expand the human use of anthelmintic drugs, raising the concern that resistance to anthelmintics will soon be with us.

    Common Threads of Drug Resistance in Helminths and Bacteria

    In a sense, anti-parasite drug treatment poses many of the same challenges found in treating multiply-resistant bacterial infections:

    1. A single, often expensive agent is used for almost all therapy;

    2. Older, alternative drugs are being dropped from production;

    3. With limited market potential, new drug development is not perceived as a priority;

    4. Pathogen isolates with intermediate-level drug resistance are beginning to be found in heavily treated areas.

    As a result, clinically significant resistance to our best anthelmintics is expected to occur in the near future (Brindley, 1994).

    Unique Features of Drug Resistance in Helminths: The Schistosomiasis Experience

    How does the problem of drug resistance differ for helminthic infections? In practical terms, anthelmintic resistance is difficult to detect, because we have no easy means of performing in vitro culture and sensitivity testing for worms. Early, low-level resistance will be difficult to detect, after which the typically rapid shift from 10 percent to 90 percent resistance will come as a surprise to many practitioners.

    In parasitic infections such as schistosomiasis, treatment does not have to be fully curative to control disease—the antischistosomal drug praziquantel is accepted as “highly effective” even though it is never 100 percent effective in eliminating infection (King and Mahmoud, 1989). Its therapeutic effect is obtained because, in schistosomiasis, unlike bacterial, viral, or even malarial infection, the pathogen does not reproduce within the human

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    body (King, 2001). With substantial suppression of infection, the net result is effective prevention of disease (Warren, 1982).

    Complicating the search for praziquantel resistance, the clinical efficacy of praziquantel therapy varies spontaneously from person to person. Host immune responses are essential to the anti-schistosomal effect of the drug, and their effectiveness appears to vary from strain to strain and from person to person. Further, not all stages of the schistosome parasite are drug-sensitive (Cioli, 1998)—if a person is infected with both the praziquantel-sensitive adult worm stages and with praziquantel-insensitive immature worms (schistosomula), examination three months after treatment may show a “persistently” positive egg count, suggesting drug failure. In fact, the eggs detected at three months post-treatment are likely to be due to the maturation and mating of these initially insensitive immature forms, which are now grown to adulthood and are fully susceptible to praziquantel retreatment (Gryseels et al., 2001).

    In contemplating the phenomenon of bacterial drug resistance, the functional ecosystem for the emerging resistant strain is the individual human body. Although there is potential for spread to other humans, the foremost factor in the survival of a resistant bacterium is its success within the treated human host. By contrast, schistosomes divide their life cycle between free-living forms (miracidia, cercariae), a form parasitic for snails (sporocysts), and the forms parasitic for humans (schistosomula larvae and adult schistosomes) (Sturrock, 2001). The transmission of schistosomiasis is integrally linked both to environmental factors and to human susceptibility factors (King, 2001). For schistosomes, reproductive success depends on many more external environmental factors, and the emergence or persistence of resistant strains requires relative success in all aspects of its life cycle.

    There is an uneven distribution of worm burden across infected human populations (King et al., 2000). Even in a highly endemic area, most people harbor light infections, while a small minority (5–10 percent) harbor very heavy worm burdens. For schistosomes, reproduction requires obligate sexual mating. The clustering of parasite distribution means that worm mating tends to be nonrandom. This phenomenon, combined with a long generation time (6–12 months), undoubtedly results in the slowing of the spread of resistance traits in schistosomiasis.

    Experience with Drug Resistance in Schistosomiasis

    Hycanthone

    In the 1970s and early 1980s, the drug hycanthone was used as population-based treatment for Schistosoma mansoni. Lab studies in animals indicated 10–20 percent of worms survived therapy, and it was found that

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    the progeny of these survivors were resistant to hycanthone (Cioli and Pica Mattoccia, 1984). Further study indicated that the resistance trait was heritable, autosomal recessive, inducible by drug exposure, and not intrinsic (Brindley, 1994). Hycanthone was ultimately withdrawn for safety reasons before resistance became a clinically significant issue, but concern remained over the relative ease with which resistance occurred.

    Concern About Praziquantel

    Over the last several years, reports have begun to emerge of evident praziquantel “failures” in treating schistosomiasis mansoni. Given the baseline variability in praziquantel cure rates, as described above, there was considerable debate about the implications of these reported failures. In a new focus in Senegal, reports of very low cure rates (<40 percent) in large-scale treatment of S. mansoni raised concerns about declining drug efficacy (Cioli, 1998; Gryseels et al., 2001). Eventually, the apparently poor response to praziquantel at this site was found to be due to ongoing heavy reinfection, but the event elicited international concern and prompted the European Community to found a concerted action network on “Patterns of praziquantel usage and monitoring of possible resistance in Africa” to periodically review the issue (Renganathan and Cioli, 1998).

    Laboratory studies have identified tolerant and relatively resistant S. mansoni strains from clinical isolates. Some S. mansoni strains taken from Egyptian subjects who failed multiple courses of praziquantel therapy demonstrated moderate- to high-level resistance to praziquantel therapy (William et al., 2001). Of note, this resistance phenotype is sometimes lost in later generations after serial passaging in mice. It was noted that tolerant strains are less fecund, releasing fewer eggs per adult worm pair (Fallon et al., 1997). Reports also indicate that at the life cycle stage of asexual multiplication, which occurs in the intermediate snail host, tolerant strains produce fewer of the cercarial forms that are infectious for humans (William et al., 2001). Although these studies suggest that low-level praziquantel resistance is already present in the field environment, they also indicate that tolerant strains are less fit to compete with drug-sensitive schistosomes. To date, such resistance is very uncommon, and does not affect the continuing recommendation that praziquantel be used in mass treatment programs for schistosomiasis (Bennett et al., 1997).

    Is this tolerance universal? Review of our own long-term (eight-year) experience in treating urinary schistosomiasis due to S. haematobium in Kenya does not show a similar pattern (King et al., 2000). Although there is year-to-year variation in cure rates, there have been no treated participants who have failed to eliminate their infection after repeated praziquantel therapy. Furthermore, children who became reinfected after the start of the

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    treatment program had the same curative response to praziquantel as children who were treated for the first time.

    In order to address the reasons why resistance was not observed, we turned to mathematical modeling of resistance transmission, using a dynamic model in which the features of parasite clustering and sexual reproduction could be incorporated. Our modeling analysis indicated that there were several features of the parasite/drug treatment ecology that were likely to explain the absence, so far, of any clinically apparent resistance. First, there was incomplete exposure of the parasite population to the drug—our school-based program left adult age groups untreated so that 25–50 percent of worms went unexposed, creating a “refuge” for sensitive worm genes (Van Wyk, 2001); second, emergence of clinically detectable resistance was estimated to require 7 to 10 generations, and, given the 6- to 12-month generation time for S. haematobium, there was probably not sufficient time for this to occur; third, if the resistance trait is recessive or polygenic, sexual reproduction was predicted to slow its emergence by several generations; and fourth, crowding effects on fecundity in heavily infected humans was likely to delay the transmission of resistance genes as well. Most important, reduced reproductive fitness of the resistant strains was seen to substantially delay the arrival of clinically significant levels of drug resistance (King et al., 2000).

    Conclusions Based on Field Experience and Modeling Analysis

    Praziquantel resistance is not yet a clinical reality, but it is expected to emerge within the next 10 years (King et al., 2000). Resistance becomes more likely as drug usage becomes more widespread (Bennett et al., 1997; Van Wyk, 2001). Praziquantel’s pivotal role in schistosomiasis therapy means that we must find means to extend its useful life span. In addition, drugs must be developed to replace praziquantel when praziquantel resistance begins to dominate at the clinical level (Doenhoff et al., 2000).

    For now, praziquantel’s effectiveness can be prolonged by more selective use, with treatment targeted only to those patients at greatest risk for heavy infection and morbidity. Resistance can also be delayed by the use of integrated disease management techniques, including snail control, water development, health education, sanitation, alternative drug use, and, possibly, by the development of effective vaccination strategies.

    Immediate Needs

    Loss of praziquantel effectiveness could become an operational catastrophe for schistosomiasis control programs. For now, there are several immediate needs that must be addressed. The new wave of inexpensive

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    generic formulations needs to be monitored for potency and for quality, so that subtherapeutic praziquantel dosing does not become the norm (Doenhoff et al., 2000). Operational research programs should be implemented, employing effective sampling techniques for field monitoring in order to detect and quickly report treatment failures. Desktop decision tools are needed to optimize the impact of treatment programs, and backup plans are needed for production of the WHO-designated “essential drugs,” oxamniquine and metrifonate, when praziquantel effectiveness declines significantly. Finally, new drug development needs to continue in anticipation of the eventual failure of praziquantel efficacy.

    In summary, it is now essential that, as national and international control programs become a reality, the value of praziquantel is not prematurely lost. Prudent use will extend the usefulness of this very safe and effective drug. However, such measures will only delay and not prevent the eventual emergence of drug resistance. It is imperative that public health planners and health providers anticipate the emergence of praziquantel resistance, and the final need for alternative agents.

    BACTERIAL INFECTION IN IRRADIATED MICE: THERAPY AND PROPHYLAXIS (ANTHRAX, A SPECIAL CONSIDERATION)1

    Thomas B. Elliott, Ph.D.2

    Armed Forces Radiobiology Research Institute, Bethesda, MD

    Military personnel will be exposed to common infectious agents and may be exposed to biological weapons and ionizing radiation. Ionizing radiation depresses hemopoiesis in bone marrow and compromises innate and acquired immune responses. Neutropenia and thrombocytopenia contribute to the reduced innate responses. Consequently, concurrent infection and sublethal irradiation are synergistic (Elliott et al., 1990).

    The number of leukocytes and thrombocytes in blood of mice given 6.5 Gy 60Co gamma photons is shown versus time in days after irradiation in Figure 2-6 (Elliott et al., 1990). The leukocytes decline rapidly to almost

    1  

    For additional data and analysis on the findings of this research, see Brook et al., 2001a.

    2  

    Studies of this nature are complex and require the cooperation of many persons. Contributors to studies of post-irradiation infections during the past several years include: GD Ledney, I Brook, GS Madonna, RA Harding, WE Jackson, III, GB Knudson, MO Shoemaker, SS Bouhaouala, RE Ruiz, J Deen, CE Inal, S Leppla, J Rogers, SJ Peacock, YA Golubeva, BT Gnade, JH Thakar, AFRRI 60Co Radiation Staff, Veterinary Staff, and Graphics Staff.

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    FIGURE 2-6 The number of leukocytes and thrombocytes in blood of B6D2F1/J female mice given 6.5 Gy 60Co gamma photons is shown versus time in days after irradiation. SOURCE: Elliott et al., 1990.

    undetectable concentration by Day 3. They remain low for almost two weeks, when they begin a gradual recovery. The thrombocytes remain stable for approximately 5 days, and then decline to a low concentration between 8–10 days and then begin to recover.

    The gastrointestinal mucous layer is reduced within 3 days in gamma-photon-irradiated mice. The resident enteric microflora change and, thereby, resistance to colonization with exogenous bacteria is reduced. As shown in Figure 2-7, the indigenous microflora decline from 1010–12 CFU/g to 104–6 CFU/g within 4 days (Brook et al., 1988). Whereas the anaerobic bacteria (primarily Bacteroides species) remain low for over two weeks following irradiation, facultative bacteria, primarily the Enterobacteriaceae, in the ileum recover to 109 CFU/g by Day 12. Bacteria translocate from intestines 5–8 days after lethal doses of gamma radiation.

    We developed animal models of infection following irradiation to evaluate efficacy of therapeutic agents against endogenous and exogenous infections following sublethal and lethal doses of ionizing radiation (Brook et al., 1999). In particular, we developed a model to assess susceptibility of irradiated mice to a biological warfare (BW) agent, Bacillus anthracis spores,

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    FIGURE 2-7 Indigenous intestinal microflora decline from 1010-12 CFU/g to 104-6 CFU/g within 4 days in irradiated mice. SOURCE: Brook et al., 1988.

    which were inoculated intratracheally in a measured dose to simulate inhalation of an aerosol (Brook et al., 2001a).

    There are several reasons for performing these studies in an animal model. We need to limit and control the variable factors. Animal models offer a compromise between clinical reality and experimental simplicity. They include the complex pathophysiological and immunological interactions and features of infectious disease, which cannot be mimicked together in vitro. We use whole-body, acute doses to simulate real worst-case military scenarios, whereas whole-body, acute doses are not commonly used in human medicine. In oncology, radiation therapy is focused and partial-body. For transplantations, irradiation is whole-body, but fractionated by giving one-tenth of the total dose per session, and almost always (90–95 percent of cases) chemotherapy is provided during the intervals between radiation sessions in order to ablate the bone marrow.

    Microbiology of B. anthracis -Induced Polymicrobial Sepsis

    The experimental design for collecting scheduled microbiological specimens from sublethally irradiated mice that were challenged with intratracheal B. anthracis Sterne spores, is shown in Figure 2-8. Following irradiation and spore challenge, we collected tissues aseptically from five mice per treatment group, which were euthanized at scheduled times after spore challenge. We cultured homogenized lung and spleen as well as blood.

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    FIGURE 2-8 The experimental design for collecting scheduled microbiological specimens from irradiated and challenged mice.

    When we gave mice only a 0-, 3-, 5-, or 7-Gy dose of gamma photons, no bacteria were isolated from tissues. When we gave mice only a dose of B. anthracis Sterne spores, we only isolated B. anthracis from tissues. But when we gave mice a dose of spores four days after irradiation, we isolated not only B. anthracis, but several other bacterial species as well (e.g., En terococcus faecalis, Erysipelothrix rhusiopathiae, Staphylococcus sp., Acinetobacter lwoffi, Enterobacter cloacae, Klebsiella pneumoniae, and Escherichia coli)—as many as three species in a single animal—which caused a polymicrobial sepsis. This is a unique finding in sublethally irradiated mice, in our experience, which complicates successful antimicrobial therapy for anthrax following irradiation (Brook et al., 2001a).

    To summarize the susceptibility of irradiated mice to infectious agents, ionizing radiation impairs innate immune responses, decreases colonization resistance of endogenous bacteria, and reduces the threshold of sepsis caused by exogenous bacteria.

    Following lethal doses, ionizing radiation induces translocation of endogenous bacteria from intestines to the bloodstream. Furthermore, combined sublethal radiation and Bacillus anthracis spores decrease the threshold for translocation of intestinal bacteria and the consequent bacteremia. Animals develop a polymicrobial infection caused by exogenous B. anthracis together with endogenous enteric gram-positive and gram-negative bacteria. This is a unique finding.

    Antimicrobial Therapy for Sepsis After Irradiation

    The successful management of post-irradiation sepsis is a difficult challenge (Brook et al., 1988; Brook and Elliott, 1991). Antimicrobial agents

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    together with basic clinical support are fundamental. Quinolones have been recommended for preventing sepsis by selective decontamination of the intestinal tract. Although anti-gram-positive antibiotics could be used to supplement other antimicrobial agents, it is imperative that the anaerobic microflora in the intestine not be suppressed because ionizing irradiation reduces them by several logarithms and they are required to provide colonization resistance.

    Selected non-specific biological response modifiers (BRMs), which enhance innate immunity by inducing cytokines naturally, or specific BRMs, such as cytokines, could augment specific antimicrobial agents, but this combined approach to therapy following irradiation remains essentially experimental (Peterson et al., 1994). Probiotics, such as specific strains of Lactobacillus, might also offer an advantage by enhancing colonization resistance and restoring the intestinal microflora.

    Factors that influence therapy for post-irradiation infection include: (i) reduced innate immune responses, particularly the decreased number of phagocytic cells; (ii) the pathogenesis of microorganisms; (iii) coverage by selected bactericidal (not bacteriostatic) antimicrobial agents, which cover facultative gram-positive and gram-negative (but not anaerobic) bacteria; (iv) the half-life of the selected antimicrobial agent (which in turn determines the dose schedule); (v) the route of administration (oral is optimal following irradiation, especially for large numbers of casualties); and (vi) the starting time and duration of antimicrobial therapy.

    The general principles of pharmacokinetics and pharmacodynamics must be considered as well. Figure 2-9 shows the concept that the concentrations of the selected antimicrobial agents must remain above the threshold minimum bactericidal concentration to achieve successful therapy after irradiation because of the absence of an effective innate response and to maintain as high an area under the curve divided by the MBC (AUBC) as practical. That is, in principle, the higher the AUBC, the greater the cidal effect against the bacteria.

    Selection of an effective antimicrobial chemotherapeutic regimen also depends upon (i) the cidal mechanisms of action of the selected agents, whether by inhibiting formation of cell wall, interrupting cell membrane function, interfering with DNA function or replication, inhibiting protein synthesis, or antagonizing metabolism, and (ii) microbial drug resistance of bacteria, whether by selection of a pre-existing genetic ability in a population or by mutation, which changes the genetic ability of the microorganism. For antimicrobial therapy for sepsis after irradiation, second-generation quinolones, either ciprofloxacin or levofloxacin, are recommended as the first choice, third- or fourth-generation cephalosporins, either ceftriaxone (third-generation) or cefepime (fourth-generation) as a second choice, or aminoglycosides, either gentamicin or amikacin, as a third choice,

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    FIGURE 2-9 Concept of the relationship between pharmacokinetic factors, minimum inhibitory concentration (or minimum bactericidal concentration) of an antimicrobial agent and the area under the curve versus time after administration of the drug.

    with or without amoxicillin or vancomycin as an adjunct, for a duration of 21 days (Brook and Ledney, 1992).

    Experimental Antimicrobial Therapy for B. anthracis Sterne-Induced Polymicrobial Sepsis After Irradiation

    The currently recommended treatment for anthrax for up to 60 days is the quinolone, ciprofloxacin i.v., penicillin G i.v., or the tetracycline, doxycycline i.v. (Dixon et al., 1999; Inglesby et al., 1999). Figure 2-10 depicts our experimental design for evaluating antimicrobial therapeutic agents against B. anthracis Sterne infection in sublethally irradiated (7 Gy) B6D2F1/ J female mice. Antimicrobial agents were given for 7, 14, or 21 days after intratracheal spore challenge (Elliott et al., 2002).

    To determine the effect of starting time of therapy on survival, irradiated mice, 12 per group, were given 7.8 × 108 CFU B. anthracis Sterne spores i.t. Penicillin G, 62.5 mg i.m., was started 6, 24, or 48 hours after spore challenge and continued for 7 days through day 11. Survival was prolonged when penicillin was started 6 or 24 hours after challenge, but when penicillin G therapy was delayed for 48 hours, survival was essentially the same as the control (Elliott et al., 2002).

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    FIGURE 2-10 An experimental design for evaluating antimicrobial therapeutic agents against B. anthracis Sterne infection in sublethally irradiated (7 Gy) B6D2F1/ J female mice. SOURCE: Elliott et al., 2002.

    To compare survival following therapy with penicillin and the two quinolones, ofloxacin and trovafloxacin, irradiated mice, 19 or 20 per group, were given 4.1 × 108 CFU B. anthracis Sterne spores i.t. The quinolone ofloxacin, 40 mg/kg p.o., and penicillin G, 125 mg i.m., were given either separately or in combination, and the quinolone, trovafloxacin, 20 mg/kg, was given either s.c. or p.o. Administration of the agents was started 24 hours after i.t. spore challenge. No control mice survived. Survival was 20 and 25 percent in mice given ofloxacin or penicillin G separately. When these two agents were combined, survival was increased to 55 percent. Survival in mice that were given trovafloxacin p.o. or s.c. was 95 and 100 percent, respectively (Elliott et al., 2002).

    To evaluate efficacy of macrolides against B. anthracis infection, irradiated mice, 20 per group, were given 1.8 × 108 CFU B. anthracis Sterne spores i.t. Mice were given doses of macrolides s.c. or p.o., which were based on allometric scaling to 10 times the equivalent doses in humans, starting 24 hours after i.t. spore challenge for 14 days through day 18. Sterile water was given either s.c. or p.o. to control groups of mice. Azithromycin (AZM, 50 mg/kg) was given either s.c. or p.o. Clarithromycin (CLR, 150 mg/kg) and erythromycin (ERY, 500 mg/kg) were given only p.o., and the quinolone, trovafloxacin (TVA, 20 mg/kg) was given p.o. for comparison with previous results and as a sort of “positive” control. Only one of 40 water-treated control mice survived. Few mice that were given the macrolides survived (between 0 and 15 percent). Survival was 80 percent in mice given trovafloxacin for comparison. The macrolides may be ineffective because they tend to accumulate in tissues and, so, concentrations in serum are low (Elliott et al., 2002).

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Antimicrobial Resistance in B. anthracis Sterne in Vitro

    We have not observed a change in antimicrobial susceptibility in B. anthracis Sterne against penicillin G, ciprofloxacin, levofloxacin, and vancomycin during the course of 21 days of antimicrobial therapy. However, we evaluated the potential for B. anthracis Sterne to develop antimicrobial resistance in vitro by passing growth of the bacteria sequentially in minimally inhibiting concentrations of quinolones and doxycycline. Each drug was diluted two-fold as depicted in Figure 2-11. A 0.1-ml amount of a suspension of bacteria, which contained approximately 2.0 × 107 CFU B. anthracis Sterne, was added to each tube in the series and allowed to incubate at 35°C. The MIC was determined by visual observation of microbial growth at 24 and 48 hours.

    The highest subinhibitory concentration of each antimicrobial agent in which microbial growth was observed was then used as the inoculum for the next series of dilutions. This macrodilution method (Davies et al., 1999) was performed in duplicate with each antimicrobial agent for 21 serial passages to simulate 21 days of therapy.

    The graph in Figure 2-12 shows the results from the evaluation with alatrofloxacin, a prodrug of trovafloxacin. The MIC began to increase four times or greater than the initial MIC by the ninth passage. So, it appears that a subpopulation of B. anthracis Sterne possesses the propensity to develop resistance against alatrofloxacin in vitro. Results with ciprofloxacin,

    FIGURE 2-11 Two-fold macrodilution of an antimicrobial agent in vitro to determine minimum inhibitory concentration (MIC) of the drug against a strain of bacteria.

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    FIGURE 2-12 Change in minimum inhibitory concentration of alatrofloxacin against Bacillus anthracis Sterne in brain heart infusion during the course of 21 serial passages of the microorganism performed in duplicate. SOURCE: Elliott, unpublished data; Brook et al., 2001b.

    gatifloxacin, and ofloxacin were similar but we observed only a minimal increase of the MIC for doxycycline. Cross-susceptibility was also determined with the cultures from the twenty-first passage among the quinolones. All substrain isolates that were grown in the presence of one quinolone were also resistant to the other quinolones (Brook et al., 2001b).

    Antimicrobial agents alone are not likely to resolve infection by B. anthracis, particularly following irradiation, because pathogenesis and death from B. anthracis is mediated by lethal and edema toxins together with a polymicrobial sepsis. Therefore, once the toxins are formed, antimicrobial agents alone are not adequate to prevent mortality. By extending the general approach to treating sepsis following irradiation, successful management of anthrax following irradiation will include the following elements discussed below.

    New quinolones are effective against both B. anthracis and endogenous bacteria. Agents with a wide spectrum of activity and high concentration in serum are more effective than agents with limited spectrum (e.g., penicillin and early quinolones) or low serum concentration (e.g., macrolides). Bacil lus anthracis could develop antimicrobial resistance with prolonged therapy. Vaccination early during therapy or injection of anti-serum could be a valuable adjunct to inactivate lethal and edema toxins. The standard, initial vaccination requires three injections two weeks apart. Horse anti-serum was used during the treatment of victims of the release of virulent B.

    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
    ×

    anthracis spores in Sverdlovsk, USSR, in 1979, but the patients died, so anti-serum seems to have limited value (Abramova et al., 1993).

    Summary

    We demonstrated (i) that low-level, acute radiation combined with endemic and BW agents, B. anthracis in particular, increases mortality synergistically; (ii) improved, effective therapy, that is, recent quinolones, to control mixed, polymicrobial infection with intestinal bacteria, which is induced by B. anthracis spore challenge after irradiation; and (iii) development of antimicrobial resistance in vitro in B. anthracis Sterne.

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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    Suggested Citation:"2. Microbe Resistance." Institute of Medicine. 2003. The Resistance Phenomenon in Microbes and Infectious Disease Vectors: Implications for Human Health and Strategies for Containment: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/10651.
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    The resistance topic is timely given current events. The emergence of mysterious new diseases, such as SARS, and the looming threat of bioterrorist attacks remind us of how vulnerable we can be to infectious agents. With advances in medical technologies, we have tamed many former microbial foes, yet with few new antimicrobial agents and vaccines in the pipeline, and rapidly increasing drug resistance among infectious microbes, we teeter on the brink of loosing the upperhand in our ongoing struggle against these foes, old and new. The Resistance Phenomenon in Microbes and Infectious Disease Vectors examines our understanding of the relationships among microbes, disease vectors, and human hosts, and explores possible new strategies for meeting the challenge of resistance.

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