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for reducing the problem of resistance The relatively modest increments in minimum inhibi-tory concentration (MIC) associated with single topoisomerase point mutations have led to the popu-larity of the concept of the “mutant prevention concentration” or MPC (Drlica and Zhao, 2007).

The logic behind identifying the MPC is to admin-ister a dose of fluoroquinolone that will result in local fluoroquinolone concentrations that exceed the highest MIC conferred by a first-order mutant

with a single-point mutation. By doing this, the growth of mutant strains can be inhibited and thereby the emergence of resistance prevented. The MPC is a logical and compelling theory and has been shown to be quite effective in animal models of infection treated by fluoroquinolones. Yet fluoroquinolone resistance is now widespread. Why?

There are several reasons why the MPC concept has not prevented the emergence of resistance to fluoroquinolones. The first and most obvious is that the vast majority of fluoroquinolone prescrip-tions are not designed with the concept of suppressing resistance in mind. Standard doses are administered to a variety of individuals, with a variety of weights, renal functions, and metabolisms, and with infec-tions in a variety of body sites. As a result, the tar-get concentrations for suppressing mutants may not have been achieved in many cases. The second reason is that we now understand that there are more “auxiliary” mechanisms of resistance to fluo-roquinolones than we appreciated when we first started using these agents (Strahilevitz et al., 2009).

These mechanisms include the expression of efflux pumps, some intrinsic and some acquired, the acquisition of genes that confer protection to the topoisomerases, and the acquisition of a modifying gene that inactivates ciprofloxacin and norfloxacin.

The effect of these mechanisms is to increase the

“baseline” MIC of the susceptible organism, a fact that will not be appreciated in the clinical setting because the information available to the clinician is not sufficiently detailed to make that judgment (Rice, 2012). In the presence of these auxiliary mechanisms, the impact of a single-point mutation may be to increase the MIC to beyond what had been identified as the MPC of a susceptible strain.

The final reason is that the problem of resistance in the hospital setting is, in actuality, only uncom-monly associated with the emergence of resistance in the infecting strain at the time of the infection.

Resistance emerges and is acquired elsewhere, in places like the GI tract, where our ability to control the concentrations of antibiotic is limited, and our ability to predict or control the flora is minimal.

Concept 2a: Emergence of resistance frequently occurs at sites distant to and in bacteria unrelated to those causing the

infection being treated

As we discuss the emergence and spread of antimi-crobial resistance, we must first acknowledge that

antibiotics given to patients do not target only the microorganisms at the site of infection. Antibiotics that achieve significant clinical use are selected for their broad volume of distribution, and they inhibit and kill in an indiscriminate manner. As a result, areas of the body colonized by microorganisms unre-lated to any disease process will experience selective pressure similar to that experienced at the site of infection. These areas include the skin, the oral and nasal cavities, the upper respiratory tract, the upper and lower GI tract, the perineum, and the urinary tract, among others. Depending on the specific anti-microbial agent being used, there may be subinhibi-tory or superinhibisubinhibi-tory concentrations found in many of these locations. Subinhibitory or barely inhibitory concentrations may select out resistant mutants of the resident bacteria, while superinhibi-tory concentrations will reduce the normal popula-tion and provide an environment where naturally resistant or newly resistant bacteria can thrive.

One salient example of such a phenomenon is the emergence of resistance to ciprofloxacin in staphy-lococci. When ciprofloxacin was first introduced into clinical use, it was truthfully marketed as the first effective oral therapy for the treatment of methicillin-resistant S. aureus infections (MRSA).

However, within 1 year of its clinical use, medical centers were reporting dramatic increases in the rates of ciprofloxacin resistance in MRSA strains (Blumberg et al., 1991). In most cases, this resist-ance was due to the accumulation of point muta-tions in the cellular topoisomerase genes, resulting in amino acid changes that lowered the affinity for the fluoroquinolone. The rapidity with which skin or nasal colonization with resistant staphylococci occurred in one clinical study was striking, and consistent with the rapid emergence observed in the clinical setting (Kotilainen et al., 1990). Unlike many antibiotics, the fluoroquinolones permeate many regions of the body, including the skin and mucous membranes. Yet the concentrations of anti-biotics achieved in these regions are difficult to predict, and they are very possibly at a level that could promote selection of first-order mutants.

These areas of typical staphylococcal colonization were, therefore, turned into natural selection regions in patients treated by ciprofloxacin or other fluoro-quinolones. Subsequent volunteer studies documented the rapid emergence of fluoroquinolone resistance in viridans streptococcal strains colonizing the human pharynx after the experimental administration of levofloxacin (Fantin et al., 2009).

Another of the major locations for emergence of resistance is the human GI tract, which is rou-tinely inhabited by large numbers of diverse types of bacteria. The bulk of these bacteria are anaer-obes and are not (to our knowledge) pathogenic to humans. They are, in fact, beneficial to us in that they maintain the homeostasis of our digestive system, helping us digest different kinds of foods and to manufacture and absorb important vita-mins. Increasing evidence also suggests that our microflora is critical to the normal development of the immune system (Purchiaroni et al., 2013).

Some antibiotics achieve very high concentrations in the human GI tract after intravenous adminis-tration. Ceftriaxone, for example, achieves con-centrations as high as 5000 μg/ml in the bile after routine dosing (Hayton et al., 1986). Such concen-trations are more than sufficient to suppress the growth of many GI colonizers, but they do not suppress the growth of ampicillin-resistant E.

faecium, whose ceftriaxone MICs can exceed 10,000 μg/ml (Donskey et al., 1999b). Early stud-ies associated ampicillin-resistant E. faecium GI colonization with the administration of extended-spectrum cephalosporins, and in some hospitals these pathogens became prevalent coin-cident with the increased use of these agents (Grayson et al., 1991).

The emergence of vancomycin resistance in E. faecium presents an interesting counterpoint to the ampicillin resistance story. Vancomycin was intro-duced into clinical use in the late 1950s as a treat-ment for penicillin-resistant S. aureus, which was becoming an increasingly prevalent nosocomial problem. After nearly 30 years of use, no resistance to vancomycin had been reported in either staphy-lococci or enterococci, but in the late 1980s, reports emerged from Europe of the isolation of enterococ-cal strains expressing high-level resistance to vanco-mycin (Shlaes et al., 1989a,b). Two major determinants (VanA and VanB) were described, both of which were transferable between enterococcal strains, suggesting that they were acquired determinants. In Europe, the emergence of these strains was attrib-uted to the use of avoparcin, a vancomycin analogue, to promote growth in food animals (Woodford, 1998). In support of this epidemiology was the identification of vancomycin-resistant enterococci in the feces of food animals, in food items sold in grocery stores, and in the feces of community dwellers (Woodford, 1998). There was logic to the association: vancomycin administered intravenously

achieved negligible concentrations in the GI tract, at least for the first several days, and so had not historically exerted significant selective pressure favoring the emergence of resistance in GI coloniz-ers. The poor oral absorption of avoparcin admin-istered to animals by mouth guaranteed very high concentrations in the GI tract, leading to the colo-nization of food animals by naturally resistant strains. Enterococci, which are tolerant to the action of glycopeptides such as avoparcin or vanco-mycin, were inhibited but not killed and, therefore, were available to acquire resistance determinants from naturally resistant bacteria. The fact that these determinants were present on transferable elements promoted the acquisition. Curiously, though, rates of infection by these bacteria in European hospitals were negligible in the early years (Woodford, 1998).

The emergence of vancomycin-resistant entero-cocci in the US followed a very different pattern.

Avoparcin has never been licensed in the US, and when animals in the US were tested for coloniza-tion by vancomycin-resistant enterococci, none were found (Coque et al., 1996). Similarly, vanco-mycin-resistant enterococci were not found colo-nizing community dwellers in the US (Coque et al., 1996). However, in contrast to Europe, vancomy-cin-resistant enterococci quickly became important pathogens in US hospitals, particularly in immuno-compromised patients (Vergis et al., 2001). Also in contrast to Europe, US vancomycin-resistant ente-rococci expressed very high levels of resistance to ampicillin (Centers for Disease Control and Prevention, 1993; Descheemaeker et al., 1999).

Clinical studies began to associate colonization and infection by vancomycin-resistant enterococci with cephalosporin use (Moreno et al., 1995; Bonten et al., 1998b). Eventually, it was recognized that in the US, vancomycin-resistant enterococcal strains emerged from a clonal complex that had not only acquired high-level ampicillin resistance, but had also acquired putative virulence determinants that promoted infection in hospitalized patients (Top et al., 2008). These strains had become prevalent in US hospitals in association with increased use of extended-spectrum cephalosporins, and so were available to acquire mobile vancomycin-resistance determinants when selective pressure was applied by oral vancomycin treatment of Clostridium dif-ficile infections in the 1980s and 1990s (Bartlett et al., 1978). The differences in these two epidemi-ologies reinforces two points: that the mammalian GI tract is an important site for the emergence of

resistance in enterococci; and that the impact of antimicrobial administration on the emergence of resistant strains is often unpredictable.

The impact of cephalosporins on colonization by ampicillin- and vancomycin-resistant E. faecium strains was examined in a series of animal studies using a murine model of enterococcal colonization (Donskey et al., 1999a, 2000b; Rice et al., 2004;

Lakticova et al., 2006). These studies showed that cephalosporins such as ceftriaxone promoted the establishment of high-level colonization of the mouse GI tract by E. faecium after the introduction of minimal numbers of organisms to the stomach, but that ceftriaxone did not promote the persis-tence of high-level colonization over time. The persistence of high-level colonization was pro-moted by the administration of agents with potent activity against anaerobic bacteria. Agents with poor enterococcal activity in the upper GI tract and potent activity against anaerobic bacteria (such as cefotetan and clindamycin) promoted both the establishment and persistence of enterococcal colo-nization. Subsequent animal studies confirmed that it is the activity (or lack of activity) of agents against enterococci in the upper GI tract that is responsible for the establishment of colonization. Human stud-ies confirmed the association of the administration of agents with potent anti-anaerobic activity with increases in the concentrations of enterococci in the feces (Donskey et al., 2000a). As such, it is not pos-sible to come up with a “perfect” antibiotic to mini-mize resistant enterococcal colonization, although the frequent association of extended-spectrum cephalosporins in clinical studies suggests that pre-disposing to the establishment of colonization is more important than persistent high-level coloniza-tion in perpetuating vancomycin-resistant entero-coccal outbreaks in the hospital.

Concept 2b: There is always a last dose

In discussing antimicrobial dosage strategies designed to minimize the emergence of resistance, consider-able attention has been focused on developing regi-mens designed to maintain inhibitory concentrations throughout the dosing interval. In this manner, a

“mutant selection window” is avoided. As noted above, such strategies have been developed for dosing fluoroquinolones, and these strategies have proven quite effective in animal studies of infection.

Unfortunately, the translation of these studies to

the clinical setting is problematic, primarily because there will always be a last dose. After the last dose, local concentrations of antibiotic will decay at a defined rate. During that decay, the concentrations will pass through the mutant selection window, during which time first-order resistant mutants will have a selective advantage. This concept was ele-gantly demonstrated by Fantin et al. (2009), who followed the feces of volunteers administered cipro-floxacin for 42 days after a 14 day course of therapy.

They did not observe the emergence of fluoroqui-nolone-resistant strains during the treatment inter-val (when the concentrations of ciprofloxacin in the feces were at suprainhibitory levels), but did observe the emergence of these strains in the post-therapy period, during which time the fecal concen-trations of ciprofloxacin declined. Follow-up studies showed that the resistant strains that emerged were not detectable at the start of therapy, suggesting either that they were present in numbers too small to detect or that they were acquired during therapy (de Lastours et al., 2012).

Misconception 3: Preclinical