Waves of Resistance: Staphylococcus aureus in the Antibiotic Era

Introduction

Staphylococcus aureus is naturally susceptible to virtually every antibiotic that has ever been developed. Resistance is often acquired by horizontal transfer to genes from outside sources, although chromosomal mutation and antibiotic selection are also important. This exquisite susceptibility of S. aureus led to Alexander Fleming’s discovery of penicillin, ushering in the “antibiotic era.” Penicillin was truly a miracle drug: uniformly fatal infections could be cured. Yet, by the mid-1940s, only a few years after its introduction into clinical practice, penicillin resistance was encountered in hospitals and within a decade it had become a significant problem in the community. S. aureus is remarkable in its ability to acquire resistance to any antibiotic.

A fundamental biological property of S. aureus is the ability to asymptomatically colonize normal people. Approximately 30% of humans are asymptomatic nasal carriers of S. aureus^{1, 2}; i.e., S. aureus is normal flora. S. aureus carriers are at higher risk of infection and they are presumed to be an important source of spread of S. aureus strains among individuals. The primary mode of transmission of S. aureus is by direct contact, usually skin-to-skin contact with a colonized or infected individual, although contact with contaminated objects and surfaces or might also play a role^3–6. Various host factors, including loss of the normal skin barrier, presence of underlying diseases such as diabetes and acquired immunodeficiency syndrome, or defects in neutrophils function predispose to infection.

Infections caused by antibiotic-resistant strains of S. aureus have reached epidemic proportions globally⁷. The overall burden of staphylococcal disease, particularly that caused by methicillin resistant S. aureus strains (MRSA), is increasing in many countries in both healthcare and community settings^8–13. In the United States the emergence of community-associated MRSA (CA-MRSA) strains as a major cause of skin and soft-tissue infections^{14, 15} accounts for much of this increase. The rapidity and extent to which CA-MRSA strains have spread has been remarkable. In addition to the United States CA-MRSA strains have been reported from Canada, Asia, South America, Australia, and throughout Europe, including Norway, the Netherlands, Denmark, and Finland, countries with historically low prevalence of MRSA.^{12, 16–29} Globally, CA-MRSA strains have shown a remarkable diversity in the number of different clones that have been identified.

In addition to increasing prevalence and incidence CA-MRSA strains appear to be especially virulent. Overwhelming and tissue-destructive infections, such as necrotizing fasciitis and fulminant, necrotizing pneumonia^30–32, which have been associated with CA-MRSA strains, were rarely seen prior to their emergence. The factor or factors responsible for this hypervirulent behavior of CA-MRSA are not known, but PVL, which has been epidemiologically associated with severe skin infections and pneumonia caused by methicillin-susceptible S. aureus (MSSA) strains³³, has been proposed as a potential leading candidate.

Antibiotics arguably constitute the most concentrated selective pressure ever brought to bear on S. aureus in its long co-evolutionary history with mankind. The consequences of this selective pressure in conjunction with horizontal and vertical gene transfer are the subject of this review. Given their critical importance as therapeutic agents, the story will focus on resistance to penicillins and the structurally related beta-lactam antibiotics.

Epidemic Waves of Antibiotic Resistant Staphylococcus aureus

Emergence of antibiotic resistance by S. aureus can be visualized as a series of waves (Figure 1). The first wave began in the mid-1940s as the proportion of infections caused by penicillin-resistant S. aureus rose in hospitals ^{34, 35}. These strains produced a plasmid-encoded penicillinase that hydrolyzes the beta-lactam ring of penicillin essential for its antimicrobial activity. Penicillin-resistant strains then were observed to cause community infections; by the early 1950s and 1960s they had become pandemic ³⁶. These infections, both in hospitals and the community, were caused primarily by a S. aureus clone known as phage-type 80/81 ^36–39. Pandemic phage-type 80/81 S. aureus infections largely disappeared after the introduction of methicillin ⁴⁰, but the prevalence of penicillinase-producing strains of other S. aureus lineages has remained high ever since.

Introduction of methicillin marks the onset of the second wave of resistance. The first reports of a S. aureus strain that was resistant to methicillin were published in 1961 ^{41, 42}. Although the specific gene, mecA, the methicillin resistance determinant which encodes the low affinity penicillin binding protein, PBP 2a (also referred to as PBP 2′), was not identified until more than 20 years later, it was appreciated early on that the resistance mechanism was different from penicillinase-mediated resistance because there was no drug inactivation. Unlike penicillinase-mediated resistance, which is narrow in its spectrum, methicillin resistance is broad beta-lactam antibiotic class resistance to penicillins, cephalosporins, and carbapenems. Among the very earliest of MRSA clinical isolates is the archetypal strain COL, a member of the “archaic” clone of MRSA and perhaps the most studied MRSA strain, which was isolated from a patient in Colindale, United Kingdom in 1960 ⁴². COL is a member of the most successful of all MRSA lineages, which includes both hospital and community-associated strains.

These archaic clone of MRSA strains circulated in hospitals throughout Europe until the 1970s ⁴³. There were isolated reports of MRSA from hospitals in the United States^{44, 45}, but the rest of the world was largely spared and these early MRSA never gained a foothold in the community. By the 1980s for unclear reasons archaic MRSA strains had largely disappeared from European hospitals, marking the end of the second and the beginning of the third wave of antibiotic. Descendants of the archaic MRSA clone (e.g., the Iberian and Rome clones⁴⁶) and other highly successful MRSA lineages emerged (Table 1) ^47–49, constituting the third wave of antibiotic resistance. Outbreaks of infections caused by MRSA strains were reported in hospitals in the United States in late 1970s and by the mid-1980s were endemic^{50, 51}. These strains swept the globe leading to the worldwide pandemic of MRSA in hospitals that continues to the present time. Although global in distribution and impact, MRSA was still confined mainly to hospitals and other institutional healthcare settings, such as long-term care facilities. The ever increasing burden of MRSA infections in hospitals led to more usage of vancomycin, the last remaining antibiotic to which MRSA strains were reliably susceptible, and under this intensive selective pressure vancomycin intermediate S. aureus (VISA, which are not inhibited in vitro at vancomycin concentrations below 4 to 8 μg/ml)⁵² and vancomcyin-resistant S. aureus (VRSA, inhibited only at concentrations of 16 μg/ml or more)⁵³ strains of MRSA emerged.

The MRSA invasion of the community constitutes the fourth and latest wave of antibiotic resistance. Some of the earliest cases of community-associated MRSA (CA-MRSA) infections occurred in indigenous populations in Western Australia in the early 1990s^54–56. These MRSA strains were distinguishable from contemporary clones (i.e., genotypes) circulating in Australian hospitals by their pulsed field gel electrophoresis patterns and susceptibility to most antibiotics other than beta-lactams, suggesting that they were either remote, feral descendants of hospital strains or community strains that had acquired mecA by horizontal gene transfer. In the US, the first well-documented cases of MRSA infection that were truly community associated occurred in otherwise healthy children in 1997–99 ⁵⁷. These children had no risk factors for MRSA and all died with overwhelming infection, suggesting that these community MRSA strains were especially virulent. Like their Australian counterparts, these CA-MRSA isolates were unrelated to hospital clones and were susceptible to most antibiotics The CA-MRSA epidemic in the US can be traced to the early 1990s, based on retrospective data from 1993–1995 showing a dramatic increase in MRSA infections in Chicago among children lacking risk factors for hospital-associated MRSA exposure⁵⁸. CA-MRSA has since been reported in numerous populations including American Indians and Alaska natives ⁵⁹; Pacific Islanders ⁶⁰; athletes ⁴; jail and prison inmates ⁶¹; men who have sex with men ⁶²; contacts of patients with CA-MRSA infection ⁶³; military personnel ⁶¹; adult emergency room patients ¹⁴; and children in day care centers ⁶⁴. CA-MRSA clones have also gained a foothold in hospitals and are increasingly identified as a cause of hospital-onset and heathcare-associated infections ^{10, 12, 25, 65, 66}.

The epidemic wave of CA-MRSA in the United States, and Canada as well^{67, 68}, is actually two overlapping epidemics. The USA400 clone, which was isolated from the pediatric cases described above, was most prevalent prior to 2001 ^{3, 57, 69}. USA400 remains a common cause of community-onset disease in among indigenous populations in Alaska and the Pacific Northwest ⁷⁰. A second epidemic clone, USA300, which is unrelated to USA400 and has largely displaced it in most other locations, emerged between 1999 and 2001, and now causes the vast majority of CA-MRSA infections in the United States^{3, 4, 71–74}.

Outbreaks and epidemics of CA-MRSA now occur worldwide and with a similar epidemiology, although the specific clones that have emerged vary with geographical location. CA-MRSA strains are not merely escapees from healthcare facilities; their genotypes indicate that they are not closely related to endemic hospital clones and these community strains are susceptible to numerous antibiotics to which hospital strains are routinely resistant. Two molecular markers not found in typical hospital MRSA are strongly associated with emergence of CA-MRSA regardless of geographical origin: a specific cassette element encoding mecA and genes encoding Panton-Valentine leukocidin (PVL). These markers are discussed in detail below.

Skin and soft-tissue infections are the most common type of CA-MRSA infection, accounting for approximately 90% of cases, of which 90% are abscesses and/or cellulitis with purulent drainage ^{14, 15}. CA-MRSA strains also appear to be especially virulent with the capacity to cause fulminant, overwhelming infections, such as necrotizing fasciitis, necrotizing pneumonia, bone and joint infections accompanied by septic thromboembolic disease ^{31, 75–77}, purpura fulminans with or without Waterhouse-Friderichsen syndrome ⁷⁸, orbital cellulitis and endophthalmitis ⁷⁹, infections of the central nervous system ^{80, 81}, and bacteremia and endocarditis ^{66, 82}

Molecular epidemiology of Staphylococcus aureus in the antibiotic era

Epidemiology of Community-Associated MRSA

As mentioned above, the earliest reported cases of CA-MRSA infection in the US were caused by a USA400 strain, MW2 ⁵⁷. MW2 is closely related to the PVL-negative clone, WA-1, which is an important CA-MRSA in Australia and to the MSSA strain 476 in the United Kingdom.⁵⁵ USA400 has been supplanted by USA300, which is by far the most frequent cause of CA-MRSA infections in the US ¹¹³. The USA300 clone seems to be particularly well adapted to the community with reports of CA-MRSA infections caused by USA300 or its close relatives in Australia and Denmark and outbreaks of CA-MRSA in Columbia^114–116. USA300 strains can also cause healthcare-associated infections^{65, 66, 117, 118}

While there is evidence of international spread of these USA300 and USA400,^{18, 23, 119, 120} CA-MRSA strains unrelated to either have been responsible for infections outside of the United States. ST80 in is the predominant clone circulating in Europe, ST59 in Taiwan, and ST30 in Eastern Australia, demonstrating that CA-MRSA strains have evolved in separate geographical regions ^21–23. There also can be considerable strain diversity in CA-MRSA from country to country. For example in Australia 45 distinct clones of CA-MRSA have been identified²³; many of these are related to well-known MRSA lineages, but others appear to be novel. Diversity of CA-MRSA isolates has been noted by others as well^{18, 27, 114, 119, 120}. In the United Kingdom the vast majority of CA-MRSA infections are caused by EMRSA-15 (ST22) and EMRSA-16 (ST36), which are also important hospital clones¹²¹; ST80 is also present, but it accounts only for a small proportion of isolates¹²². A CA-MRSA strain, ST 398, of swine origin and transmissible to humans has also been described^{123, 124}.

The epidemiology of CA-MRSA is quite similar regardless of country of origin. Isolates tend not to be multiple drug-resistant, SCCmec types IV and V are typically present, and infections of skin and soft tissue are the most common. The presence of PVL among CA-MRSA isolates is more variable. For example in Australia and the United Kingdom most CA-MRSA clones do not produce PVL^{23, 121} and prevalence of PVL among the more common CA-MRSA isolates from Denmark ranged from 17% to 100%¹²⁰. On the other hand isolates of clones that typically do not carry PVL genes, e.g., EMRSA-15 and EMRSA-16, have been found on occasion to be PVL-positive¹²¹.

Nasal carriage of MRSA has increased in parallel with the emergence of MRSA as a community pathogen, which is not unexpected given that approximately 30% of people have asymptomatic nasal colonization with S. aureus. Between 2001 and 2004 carriage of MRSA strains in a US population based study approximately doubled from 0.8% to 1.5% ² and the percentage of community-associated MRSA genotypes increased from7% to 24.2% ⁸⁸. Although the sites of carriage (e.g., nasal versus groin versus other) and the relationship between carriage of CA-MRSA strains and disease is not entirely clear, CA-MRSA strains, especially USA300, appear to be more easily transmitted than other strains,¹²⁵ which could account for increasing carriage rates in the community. Thus, no individual or group can be considered not at risk for CA-MRSA infection.

Virulence of Community-Associated MRSA

Compared to infections caused by healthcare-associated MRSA strains and community MSSA, CA-MRSA infections have been associated with fulminant and lethal infections and worse clinical outcomes^{30, 77, 126}, giving rise to the clinical impression that CA-MRSA strains, especially USA300, are more virulent than other strains. Much of what the information about the unique virulence properties of CA-MRSA is based on studies of USA300 strains, the most extensively investigated clone. The USA300 core genome (chromosome excluding mobile genetic elements) is quite similar to that of the early MRSA strain, COL ¹²⁷. Yet, studies in animal models indicate that USA300 is more virulent than COL^{128, 129}. Expression of virulence factors by USA300 is high and USA300 ^{130, 131} and closely related strains are more lethal than more distant relatives and cause more extensive disease in animal models of infection^{129, 130}. The major difference between COL and USA300 genomes resides in mobile genetic elements, which include prophages, plasmids, pathogenicity islands, and transposons, acquired through horizontal gene transfer. These elements encode factors that may impact transmission, antibiotic resistance, and virulence. Prophages ΦSa2 and ΦSa3, which are present in USA300 strains and not in COL, could contribute to the noted differences in virulence between these two lineages. Prophage ΦSa2 contains lukS-PV and lukF-PV, which encode PVL. Prophage ΦSa3 encodes staphylokinase, staphylococcal complement inhibitor (SCIN), and S. aureus chemotaxis inhibitory protein (CHIPS), all of which are modulators of the innate immune system ^{132, 133}. ΦSa3 is present in strains other than CA-MRSA. A pathogenicity island, SaPI5, similar to the one that is in COL, is present in USA300. SaPI5 encodes two additional superantigens not present in COL, SEQ and SEK, which also are found in other MRSA and MSSA lineages. S. aureus produces many other molecules that promote host colonization, facilitate evasion of the innate immune system, and/or alter immune responses (Tables 3–6) ^{131, 134, 135}. Most of these molecules are not unique to CA-MRSA. The virulence factors more commonly found in CA-MRSA compared to other strains, those that are linked by epidemiology to CA-MRSA infections, or those that have been studied in animal models of CA-MRSA infection are discussed below.

Treatment in the Era of Community-Associated MRSA

CA-MRSA has had a profound impact on empirical therapy of suspected staphylococcal infection. Most beta-lactam antibiotics, including all orally available agents, no longer can be assumed to be effective for a variety of common staphylococcal infections and skin and soft-tissue infections in particular. In regions where CA-MRSA is prevalent antimicrobial therapy, if it is indicated for treatment of staphylococcal infection, should be active against MRSA strains. Yet, there are few clinical data to support the use of agents other than vancomycin, daptomycin, or linezolid. The oral agents that are recommended for treatment of CA-MRSA skin and soft tissue infections, despite lack of rigorous clinical studies, include clindamycin, long-acting tetracyclines (doxycycline and minocycline), TMP-SMX, and, as adjunctive agents to be used in combination, rifampin and fusidic^160–162.

Surgical incision and drainage is the treatment of choice for cutaneous abscesses; adjunctive antimicrobial therapy is of little or no benefit in most cases ^{14, 15, 163, 164}. Antibiotic therapy after drainage of CA-MRSA abscesses is not routinely recommended unless the patient has severe or extensive disease, or has rapid progression in the presence of associated cellulitis; has signs and symptoms of systemic illness; is very old or very young or has medical comorbidities or immune suppression (e.g., diabetes mellitus, HIV infections, neoplastic disease); or has an abscess in area that is difficult to drain or an abscess that is associated with septic phlebitis ¹⁶⁰.

Vancomycin is still is the preferred drug for treatment of serious MRSA infections. However, prolonged, persistent, or recurrent bacteremia during therapy ^{165, 166}, high rates of microbiological and clinical failures ¹⁶⁷, nephrotoxicity ¹⁶⁸, and increasing prevalence of non-susceptible strains ^{169, 170} limit its effectiveness. Randomized clinical trials of alterative agents such as linezolid and daptomycin show that they are comparable, or more precisely, non-inferior, but not superior, to standard therapy ^171–176. and drug toxicity remain concerns regardless the choice of agent.

One or more compounds under development are likely to become available for treatment of MRSA infections in the near future ^{177, 178}. Telavancin, dalbavancin, and oritavancin are vancomycin derivatives that rapidly kill S. aureus in a concentration-dependent manner in vitro. Whether more rapid killing will translate into improved efficacy over vancomycin for more serious infections, such as endocarditis or bacteremia, remains to be determined. Carbapenems and cephalosporins that bind PBP 2a, the penicillin-binding protein that mediates methicillin resistance, with much higher affinity than the currently available beta-lactams, have been developed ¹⁷⁹. Two cephalosporins, ceftobiprole and ceftaroline ^{180, 181}, have been shown to be clinically effective for treatment of MRSA skin and soft infections. An issue with these and the other anti-MRSA beta-lactams under development is that they are very broad spectrum for the targeted treatment of MRSA infection. Further studies are needed to define their eventual role in therapy of MRSA infections.

The vancomycin-derivatives and anti-MRSA beta-lactams, which can only be administered intravenously, do not address the need for orally active agents. Orally bioavailable oxazolidinones active against MRSA are in early stages of development ¹⁸².

Several non-traditional approaches to treatment and prevention of MRSA infections have been or are under investigation. These include lysostaphin ¹⁸³, antimicrobial peptides ¹⁸⁴ and other natural products (e.g., tea tree oil) ¹⁸⁵, and anti-staphylococcal vaccines ¹⁸⁶. There are major challenges in the development of these agents, including prohibitively expensive cost, potential for hypersensitivity with repeated administration of protein products, short half lives with systemic administration, and short-lived or partially protective immunity with vaccines, as was the case with an anticapsular vaccine that proved to be ineffective ¹⁸⁷. These are years away from the clinic, if they make it at all. Prudent use of agents that are now available is essential to avoid further erosion of the antimicrobial armamentarium.

Concluding Remarks

S. aureus is an extraordinarily adaptable pathogen with a proven ability to develop resistance. Especially concerning is the steadily erosion in the effectiveness of beta-lactam antibiotics during a relatively brief 60-year time period. Although details vary the basic themes of each successive wave of antibiotic resistance are similar. Resistance, often as a consequence of horizontal gene transfer, is initially encountered in hospitals and healthcare institutions, where the selective pressures for resistance are greatest. Resistant strains are contained within hospitals temporarily, but eventually though a series of modifications and adjustments, invariably find their way into or arise from within the community to emerge as fully fit and virulent pathogens. Understanding of the forces that direct the evolution of virulent and drug-resistant organisms is imperfect, but overuse and misuse of antibiotics is clearly a contributing factor. Discovery and development of new antimicrobials, while necessary, is unlikely to solve the problem of drug resistance for very long. New technologies leading to improved and more rapid diagnostics, a better understanding of pathogenesis of staphylococcal disease, and non-antimicrobial approaches to prevention and treatment of infection will also be needed to forestall the coming of the post-antibiotic era.