The Microbial “RESISTOME” Although human activity has accelerated the spread of antibiotic resistance, the problem has long existed in nature. JULI A N JOSEPHSON
Staphylococcus aureus bacteria are becoming increasingly resistant to -lactam antibiotics such as methicillin. The bacteria secrete stickylooking substances called biofilms, which are woven between the bacteria to protect them from antibiotics. PHOTO COURTESY OF CDC
W
hen antibiotics first appeared on the pharmaceutical market during the 1940s and 1950s, many health-care professionals cheered, believing that the bacterial enemy had been
flung back and that, in the foreseeable future, infectious disease would be a thing of the unlamented past. This enemy, however, fought back gamely. The increasing development of microbial resistance to drugs is dealing humanity a major setback in combating infectious diseases and seems to be outpacing the treatments and patient management strategies available to the physician. Moreover, resistant bacteria are migrating beyond the hospital, sickroom, and farm setting and into water, soil, and the community at large. © 2006 American Chemical Society
November 1, 2006 / Environmental Science & Technology n 6531
Genes from seemingly unlikely sources Although human activity has markedly enhanced the evolution and distribution of resistant bacteria in hospitals and the environment in general, it is not necessarily the only, or even the proximate, cause. The development of microbial resistance to drugs had been going on in nature long before antibiotics came into medicinal use. Apparently, bacteria, including human pathogens, can acquire resistance genes in natural environments, particularly in soils. Certain organisms that generate antibiotics live in soils; most susceptible bacteria in their vicinity, including human pathogens, die off, but some develop resistance to these natural products. A portion of the resistance genes in human pathogens may have been transferred from what would, at first glance, seem to be unlikely sources, such as one described by Ronald Jones, a principal of the Jones Group/JMI Laboratories. At the National Foundation for Infectious Diseases (NFID) Conference on Antimicrobial Resistance, held in June 2005, he reported the transfer of genes from the plant pathogen Xanthomonas campestris citri, the cause of citrus canker, to the human pathogen Pseudomonas aeruginosa. Citrus canker can devastate whole citrus groves and spread rampantly throughout a citrusgrowing region. P. aeruginosa is mainly an “opportunistic” hospital pathogen that causes nosocomial diseases, such as fever and septicemia (blood poisoning). Nosocomial diseases are typically bacterial infections that are contracted during a hospital stay. Such infections often affect immunocompromised patients, particularly those suffering from burns or undergoing chemotherapy.
Resistant bacteria are migrating beyond the hospital, sickroom, and farm setting and into water, soil, and the community at large. Mark Toleman, a research fellow in microbiology at the University of Bristol (U.K.), also believes that the genes from X. campestris citri almost certainly “jumped off a plasmid into the chromosome of P. aeruginosa.” Moreover, he advocates that this gene transfer occurred in the environment, rather than in a hospital setting. “Precisely how and where this transfer occurred remains unclear,” he explains. The X. campestris citri–P. aeruginosa gene transfer is not unique. At the NFID meeting, Jacques Acar, a professor emeritus at the Université Pierre et Marie Curie in Paris, speculated that genetic material from a microbe in the Mediterranean Sea could be transferred to a human pathogen. Toleman says that these transfers have been proven. The microbe is Shewanella algae, and the human pathogens are E. coli and Klebsiella species. The lat6532 n Environmental Science & Technology / November 1, 2006
ter two bacteria are human commensals. Diseases attributable to E. coli have been well documented. Klebsiella often affects immunocompromised patients and can cause severe pneumonia. The genes from S. algae, particularly one labeled qnrA, may have conferred on E. coli and Klebsiella resistance to “cipro” (ciprofloxacin) and nalidixic acid, both of which are antibiotics of the quinolone class. One can only speculate on the circumstances under which contact was made between these organisms. Could E. coli and Klebsiella have found their way from ill patients to the Mediterranean Sea via storm-water runoff, and, through a malign coincidence, encountered the S. algae, as Acar hinted? Or did the gene transfer occur within the body of a patient, as Toleman suggests, after which the altered E. coli and Klebsiella made their way into the community? (Some species of Shewanella can cause meningitis, intra-abdominal sepsis, ear infections, and bacteremia.) Do analogous phenomena between human pathogens and other organisms in the environment confer drug resistance on the pathogens, and if so, how might such exchanges be traced and prevented in the future? The P. aeruginosa containing the X. campestris citri gene was first observed in a hospital in Newton, Texas. The genetic culprit is known as bla VIM-7, a gene that codes for an enzyme that neutralizes antibiotics, mainly of the -lactam class. Among antibiotics of this class are penicillin, methicillin, and amoxicillin. “Both sides of the bla VIM-7 gene are traceable to X. campestris,” Toleman explains. “Perhaps bla VIM-7 came to X. campestris from another bacterium in the environment, but which one has not been determined,” he added. “The bla VIM-7 gene has never been observed anywhere in the world before, so it is likely that it originated initially in an unknown soil bacterium before being transferred to X. campestris citri and then to the common soildwelling bacterium P. aeruginosa.” P. aeruginosa is a Gram-negative bacterium. Tole man explains that Gram-negative bacteria seem considerably faster at developing resistance to drugs than do Gram-positive bacteria. A salient example of a Gram-positive microbe that developed resistance to antibiotics is MRSA, methicillin-resistant Staphylococcus aureus, now spread out into the community. “But it took S. aureus 40 years to develop resistance to methicillin, which had been in use since the 1950s,” Toleman observes. Moreover, antibiotics are available to treat infections caused by Gram-positive bacteria, and new antibiotics are in the pipeline. This is not true for Gram-negative bacteria, and currently several resistance mechanisms are emerging in such microbes, enabling them to become virtually untreatable (1, 2). What is more, “There are currently no new drugs in the pipeline for Gram-negative bacteria,” he says. Among other Gram-negative that have rapidly developed resistance to -lactam antibiotics are Acinetobacter baumannii and species of Enterobacter. A. baumannii (family Enterobacteraceae) also is a hospital pathogen of opportunity that causes skin and soft-tissue infections and blood poisoning in
immunocompromised patients. Moreover, P. aeruginosa and A. baumannii “can live almost anywhere” in the environment, Toleman notes. They are oligotrophs, able to scavenge from low concentrations of nutrients. This attribute could help these types of resistant bacteria to spread from the hospital setting and get out into the community in much the same manner as MRSA has. Microbial resistance has been building up against more than -lactam- and quinolone-class antibiotics. For instance, bacteria have been observed to develop resistance against aminoglycosides such as streptomycin, and macrolides such as erythromycin.
Resistance: natural origins plus human “help” Although resistance to drugs has long existed in nature, human activity has strongly amplified its development and its spread in and beyond hospitals and farms. The relevant literature and discussions at meetings, such as those sponsored by NFID, abound with explanations and examples of how human activity has brought about, accelerated, and enhanced the development of microbial resistance to drugs. One example entails the overuse and misuse of antibiotics of all classes in hospitals and other health-care settings. Not only have target microbes developed resistance to one or more drugs in these settings, but some diseases that heretofore were easily cured have become increasingly serious or even fatal. These include diseases caused by P. aeruginosa and A. baumannii (as mentioned above), MRSA, and, more recently, those caused by vancomycin-resistant Enterococcus. Some resistant microbes have migrated out into the community. MRSA is frequently found in crowded environments, such as military installations, gyms, and prisons. Moreover, the community-associated MRSA strain appears to be more virulent than the hospital-associated MRSA strain, and genes have been observed to migrate between the two strains. Another contributing human activity is the heavy use of antibiotics in domesticated animals to suppress disease and to fatten the animals and enhance their appearance and that of their meat. There, too, target microbes have developed resistance and have migrated into the overall environment, especially into water and soil. Perhaps for these reasons, some countries—Sweden, for example—have severely limited or even banned most uses of antibiotics in animal farming. Resistance in the human and natural environments developed in several ways. In some cases, a target microbe may have had its own genes that conferred resistance. In other cases, antibiotic-susceptible bacteria had contact with resistant bacteria; the resistant strains transferred resistance genes to the susceptible strains in packets of genetic material, known as plasmids. In some instances, the resistance genes so received became an integral part of the newly resistant microbe’s chromosomes; these genes are known as integrons. In several papers, Toleman and his colleagues describe the various mechanisms by which resistance genes blend into a microbe’s genome (1).
The development of microbial resistance to drugs in nature, however, may long predate that attributable to human activity, according to Gerard Wright of McMaster University (Canada). Rather, human activity may have sharply enhanced and accelerated the pace. Wright, Vanessa D’Costa, and their colleagues found that such resistance exists in bacteria collected in remote parts of northern Ontario; in forests, where one would not normally expect to encounter antibiotics; and in farmyards where livestock are fed—many experts say overfed—antibiotics.
The development of microbial resistance to drugs had been going on in nature long before antibiotics came into medicinal use. “Most clinically relevant antibiotics originate from soil-dwelling actinomycetes,” D’Costa and Wright note (3), also citing research conducted by Tobias Kieser and others (4). They write: “Soildwelling bacteria produce and encounter a myriad of antibiotics, evolving corresponding sensing and evading techniques.” They also suggest that “the soil could serve as an unrecognized reservoir for resistance that has already emerged or has the potential to emerge in clinically important bacteria. Consequently, an understanding of resistance determinants present in the soil—the soil ‘resistome’—will provide information not only about frequencies of antibiotic resistance emergence but also about new mechanisms that may emerge as clinical problems.” Examples of the evolution of highly specific resistance elements include vancomycin resistance in Streptomyces coelicolor, Paenibacillus (which can cause meningitis, pneumonia, and septicemia in humans), and Rhodococcus, a cause of infection in grazing animals (3, 5, 6). Wright and his team harvested microbes from soils in Northern Ontario; in forests, where human habitation is sparse; and on farms where antibiotics are commonly administered to livestock. They tested 21 drugs typical of the most widely prescribed antibiotics. They found that the level of resistance was the same in microbes from the north and from the farms. Indeed, all strains were resistant to at least 2 antibiotics and some to as many as 15, with an average of 7 or 8. In addition, some bacteria were resistant to antibiotics they had never before encountered. Wright noted that soil-based organisms are the source of perhaps 70% of antibiotics currently in use. The kicker is that these organisms secrete antibiotics to protect themselves in their environment. However, they had to develop resistance to their own secretions so that they themselves would not November 1, 2006 / Environmental Science & Technology n 6533
die from their own weapons. Wright observes that most microbes in the environment generate defenses against not only their own secretions but also chemicals from other microbes. Hence, resistance developed in nature: even a human pathogen in the environment can develop resistance against its neighbors’ biochemicals. It should be noted that the Wright study was not set up to compare farms with forest environments. It was, rather, a general survey of numerous environments, including urban, rural—where antibiotic use was suspected—and remote forest areas. No significant difference in antibiotic resistance was found among these environments.
The administration of antibiotics should be restricted to cases of real necessity. As Alexander Tomasz of Rockefeller University observes, the Wright study “illuminates the dark side of the antibiotic paradigm. Microbes that synthesize the sophisticated chemicals that have been key to humankind’s success in controlling bacterial disease also possess equally sophisticated mechanisms to protect themselves against their own products” (7). Tomasz warns, “These mechanisms represent formidable weaponry that could annul the successes of antimicrobial therapy if they were to find their way to human pathogens.”
New drugs or approaches? Currently, prospects for new drugs appear bleak. Andrew Simor, the head of microbiology at Sunnybrook Health Sciences Center (Canada), hopes that research on the mechanism of resistance development will help pharmaceutical companies design badly needed new antibiotics. Like many infectiousdisease experts, he worries that resistance to existing antibiotics is rising and that few new drugs are in the pipeline. Moreover, as Toleman explains, “For a pharmaceutical firm, developing a new drug aimed at replacing what [still] works is not an attractive investment, for legislative and regulatory as well as financial reasons. But if resistance [to drugs] becomes worse, it would be years before a new drug comes along—a minimum of 5 years, and more like 10.” Developing innovative approaches that lead to new drugs or treatments could be a very long-term proposition. Possibly, one path would be to discover how to interdict the means by which resistant bacteria express their resistance to drugs, such as enzymatic reactions, efflux, and impermeable cell walls. Such research has been under way for the past several years, but progress appears to be uneven so far. Even if such paths of interdiction show promise for the development of new, effective drugs, many years of lead time will remain, not to mention questions of safety and high cost. 6534 n Environmental Science & Technology / November 1, 2006
Researchers at the University of Utrecht and other centers in The Netherlands are trying to combine biomedical and statistical techniques to devise and perfect a “search-and-destroy” (S&D) infection-control measure aimed at nosocomial MRSA (8). Utrecht researchers constructed a stochastic three-hospital model and an analytic one-hospital model aimed at quantifying the effectiveness of different infection-control measures and predicting the effects of rapid diagnostic testing (RDT) on patients’ isolation needs. Their model suggests that systematic application of S&D and RDT can reduce the incidence of nosocomial MRSA, perhaps to
