REVIEW. MRSA the tip of the iceberg P. C. Appelbaum. Pennsylvania State College of Medicine, Hershey, Pennsylvania, USA

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1 REVIEW MRSA the tip of the iceberg P. C. Appelbaum Pennsylvania State College of Medicine, Hershey, Pennsylvania, USA ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) strains cause serious nosocomial infections all over the world. Overall, approximately 20% of S. aureus isolates in Europe are reported as methicillinresistant, whereas in US hospitals the prevalence ranges from 33% to 55%. The past few years have also witnessed an increase in life-threatening community-acquired infections caused by Panton Valentine leukocidin-producing MRSA in the USA. Increasing use of glycopeptides for treatment of communityacquired MRSA infections may result in higher rates of glycopeptide resistance. Since 1996, five vancomycin-intermediate S. aureus (VISA; vancomycin MIC = 8 16 mg L) strains have been identified in Europe, Asia and the USA, and vancomycin-resistant S. aureus (VRSA) strains (vancomycin MIC 32 mg L) have also been reported in the USA between 2002 and Most infections with VISA and VRSA have occurred in a setting of heavy prior use of glycopeptides and other antimicrobial agents. Emergence of reduced vancomycin susceptibility in S. aureus increases the possibility that currently available antimicrobial agents may become ineffective for treating systemic infections, especially bacteraemia, endocarditis and osteomyelitis. Ceftobiprole is a novel broad-spectrum cephalosporin with expanded activity against Gram-positive bacteria, including MRSA. Ceftobiprole is refractory to the development of endogenous resistance both in vitro and in vivo. The additional activity of ceftobiprole against MRSA strains makes it a potentially important addition to currently available agents. Keywords Ceftobiprole, community-acquired, methicillin-resistant Staphylococcus aureus, vancomycin Clin Microbiol Infect 2006; 12 (Suppl. 2): 3 10 INTRODUCTION Staphylococcus aureus is a major cause of serious hospital-acquired infections. In the past decade, the prevalence of methicillin-resistant S. aureus (MRSA) strains has increased worldwide and they now represent a serious cause of nosocomial infections in many countries [1]. In Europe, approximately 20% of S. aureus isolates are reported as methicillin-resistant, and in the USA, the prevalence of MRSA is > 50% [2]. The past few years have also witnessed an increase in lifethreatening community-acquired infections caused by Panton Valentine leukocidin (PVL)- producing MRSA in the USA. Recent data have shown important changes in the pattern of antibiotic resistance among MRSA isolates over time [1]. Since 1996, vancomycin-intermediate S. aureus Corresponding author and reprint requests: P. C. Appelbaum, Department of Pathology, Hershey Medical Center, 500 University Drive, Hershey, PA pappelbaum@psu.edu (VISA) isolates, with an MIC for vancomycin of 8 16 mg L, have been identified in Europe, Asia, and the USA [3]. Since 2002, vancomycin-resistant S. aureus (VRSA) (vancomycin MIC 32 mg L) has been reported in the USA (J. T. Rudrick, personal communication) [4,5]. There is now a fifth report, also from Michigan (D. M. Sievert, personal communication). Infections attributed to VISA and VRSA often occur after repeated use of other antimicrobial agents. Currently, all available b-lactams are considered to be inactive against MRSA strains. Increased resistance to antimicrobial drugs is not confined to staphylococci but also has been reported with other Gram-positive and Gramnegative pathogens [6]. Further multidrug-resistant VISA strains have been identified [7], and VRSA strains are also multiresistant. The emergence of VISA and VRSA suggests that currently available antimicrobial agents may become ineffective in the future for treating systemic infections such as bacteraemia, endocarditis, and osteomyelitis. This article will discuss challenges Ó 2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 4 Clinical Microbiology and Infection, Volume 12 Supplement 2, 2006 posed by the rising incidence of MRSA, VISA, and VRSA, and address the question: Can b-lactams act against MRSA? EPIDEMIOLOGY OF MRSA The European Antimicrobial Resistance Surveillance System is an ongoing surveillance programme for documenting antimicrobial resistance. The prevalence of MRSA and changes in the prevalence over time in European Antimicrobial Resistance Surveillance System-participating countries are routinely collected and recorded [1]. Between January 1999 and December 2002, nosocomial isolates were collected from 495 hospitals in 26 countries. The prevalence of MRSA varied from < 1% in northern Europe to > 40% in southern and western Europe. During this time, the incidence of MRSA increased significantly in many countries, including Belgium, Germany, Ireland, and the UK (Fig. 1), highlighting the need for better infection control procedures. In the USA and in Asian countries (including Taiwan, Korea, and Australia), the incidence of MRSA isolates is also increasing dramatically [6,8 10]. Hsueh et al. reported an increase in the prevalence of MRSA isolates from 26% to 77% between 1986 and 2001 in Taiwan [10]. During the period , the prevalence of MRSA was Fig. 1. Percentage of Staphylococcus aureus isolates that were methicillin-resistant by country from 1999 to 2002 in Europe [1]. MRSA, methicillin-resistant S. aureus. 64% in Korean tertiary-care hospitals [8]. Thus, MRSA is increasingly a problem worldwide. COMMUNITY-ACQUIRED MRSA MRSA has historically been considered to be a problem associated with the hospital environment. However, in recent years a dramatic increase in the prevalence of MRSA has been reported that is linked to an expanding reservoir of community-acquired MRSA (CA-MRSA) [11]. The first CA-MRSA isolate was noted among intravenous drug users in Detroit, Michigan [12]. Other populations in the USA in which CA-MRSA has been found include: children without identified risks; the disadvantaged; native American Indians in Alaska, Minnesota, and Washington; prison populations; and the urban homeless [13]. In the early 1990s, CA-MRSA was reported among aboriginal populations or native islanders living in Australia and New Zealand [13]. Recently, reports of CA-MRSA include athletes participating in contact sports [14,15] and those infected with human immunodeficiency virus [16]. Lifethreatening pneumonia [17] and infections in the neonatal unit [18] involving MRSA have also been described. The rates of colonisation or infection with CA-MRSA are difficult to determine. Hidron et al. reported nasal colonisation with CA-MRSA in 7% of their population, which was higher than the 1 5% reported in other studies [16]. A recently published review reported similar levels in a variety of settings, including community-based high-risk populations [19]. A number of risk-factors for CA-MRSA have been identified. In many cases, patients have received antibiotic therapy within the previous 3 months, have been hospitalised within the previous 12 months, are residents in long-term care facilities, or have a history of chronic illness [16,20]. Looking at the secular trend of MRSA in San Francisco, a marked increase in the total number of MRSA isolates was observed between 1996 and 2002 [11]. This increase can be attributed to the increase in the number of CA-MRSA isolates, with residents in long-term care facilities and day care centres acting as a reservoir for these organisms [21]. The molecular biology of hospital-acquired MRSA (HA-MRSA) is distinct from that of CA-MRSA. HA-MRSA isolates usually contain the type I, II or III genetic element staphylococcal

3 Appelbaum MRSA the tip of the iceberg 5 cassette chromosome mec (SCCmec), which confers resistance to currently available b-lactam antibiotics and also many other non-b-lactam antibiotics [22]. HA-MRSA exhibits multidrug resistance to drugs such as clindamycin, gentamicin, and fluoroquinolones. By contrast, CA-MRSA isolates often possess a novel type IV SCCmec, as well as the PVL gene, which encodes for a toxin contributing to the virulence of infection [19,22]. Thus, MRSA represents a growing problem worldwide, and therapeutic options for these infections remain untested. DECREASING VANCOMYCIN SENSITIVITY OF S. AUREUS The emergence of VISA and VRSA has also compromised treatment options for Gram-positive infections. The first reported case of decreased sensitivity of S. aureus to vancomycin was described in 1996 [23]. The mechanism for resistance or decreased sensitivity of S. aureus to vancomycin and glycopeptides in general is thought to be due to cell wall alterations, which result in thickening of the bacterial cell wall [24] and cell wall reorganisation [25]. In addition, overproduction of penicillin-binding protein (PBP2) has been observed [26], and absent PBP4 expression has also been reported [27]. Resistance to vancomycin is mediated by the vana gene, which is specific for glycopeptides. Other genes found in VRSA strains include meca for b-lactams, erm(a) and erm(b) for macrolides, aac-aph for aminoglycosides, and tetk for tetracyclines. Mutations of GyrA S84L and GrlB E471K are associated with resistance to quinolones [5,28,29]. VISA VISA was first identified in isolates from two patients in 1996 [23]. The first VISA strain was isolated from a leg ulcer and a catheter tip of a patient who had responded poorly to vancomycin treatment. The second VISA strain was isolated from an infant who also responded poorly to vancomycin treatment. Subsequently, a case of VISA was reported from a hospital in France [30], and since then other cases of infections due to VISA have been reported in the USA and elsewhere [7,31,32]. Patients at increased risk for VISA include those having recurrent MRSA infections treated with vancomycin and those with chronic renal failure [31]. Additional strains of MRSA exhibiting reduced susceptibility to vancomycin have also been reported in France [33,34]. In total, there have been about 100 staphylococcal isolates with reduced susceptibility to vancomycin (VISA) reported in Europe, Asia, and the USA. VISA strains cannot be detected reliably by standard disk diffusion methods [7,31,32]. The current Clinical and Laboratory Standards Institute guidelines define VISA as having a vancomycin MIC of 8 16 mg L; however, in cases where other susceptibility testing methods have been used, vancomycin MICs of 4 8 mg L have been found [7,32]. Also, reports have appeared of patients with endocarditis caused by S. aureus strains with vancomycin MICs of 4 mg L, which have not responded to vancomycin [35 37]. S. aureus isolates for which vancomycin MICs are 4mg L should be sent to a public health laboratory for confirmation [31], because automated methods for detecting VISA are not acceptable. Acceptable methodology for detecting VISA includes Clinical and Laboratory Standards Institute broth microdilution, agar dilution, and Etest, although the exact MIC for defining resistance is not clear. The CDC recommends an MIC of 8 16 mg L using broth microdilution, growth of > 1 colony in 24 h using agar dilution, and an MIC 6mg L using the Etest method. Additional research is required to fully delineate optimal laboratory testing for these resistant isolates. VISA strains may, depending on the site of infection, remain susceptible to treatment with vancomycin, but often require higher doses and prolonged treatment periods, and most are susceptible to trimethoprim sulphamethoxazole [31]. Clinically, serious infections may occur with S. aureus strains exhibiting vancomycin MICs of 4 and even 2 mg L, which do not respond to vancomycin [31]. The above reports, together with documented cases of resistance to teicoplanin, especially among coagulase-negative staphylococci, indicate the growing problem of resistance among staphylococci to all classes of antimicrobial agents, including glycopeptides. VRSA In early 2002, the first VRSA strain containing a vana-resistant gene was isolated in Detroit [4].

4 6 Clinical Microbiology and Infection, Volume 12 Supplement 2, 2006 About 6 months later, a second VRSA strain was isolated from an obese elderly man in Hershey, Pennsylvania [5], and early in 2004 a third strain of VRSA was isolated from a urine specimen from a patient in a long-term care facility [38]. In early 2005, a fourth VRSA strain was isolated from a toe wound of an elderly patient who had recently been hospitalised for aortic valve replacement and then spent 5 weeks in a nursing home (J. T. Rudrick, personal communication). These four cases all occurred in elderly, debilitated patients with one or more chronic diseases, such as diabetes mellitus, chronic renal failure, or peripheral vascular disease. Obesity and or residence in a long-term care facility, as applies to three of these patients, may also be risk-factors for development of resistance. Patients with VISA or VRSA have usually been treated with multiple previous courses of antibiotic therapy, which may predispose patients to complicated, multidrugresistant infections. In addition, Fridkin et al. determined that recent vancomycin use and a positive culture for MRSA within the previous 2 3 months were risk-factors for reduced susceptibility to vancomycin [28]. Different characteristics of the first three strains of VRSA are noted in Table 1. In the Michigan case, the vancomycin MIC was 1024 mg L, whereas the other two strains had much lower MICs (32 64 mg L), despite all three strains containing the vana gene. However, the patient in Michigan had also received previous vancomycin therapy, and vancomycin-resistant Enterococcus faecalis had been isolated from the same wound in which the VRSA was initially cultured. The difference between the MICs of the Pennsylvania and New York strains and the first Michigan case could be attributed to the level of expression of the vana gene present on the 120-kb plasmid, which was present in the Pennsylvania and New York strains but not in the Detroit strain, and is not typical of S. aureus. This 120-kb Table 1. Comparison of features from the first three VRSA isolates [4,5,38] VRS1 Michigan VRS2 Pennsylvania VRS3 New York MIC 1024 mg L 32 mg L 64 mg L Vancomycin treatment Yes No? VRE Yes No Yes Plasmid 60 kb 120 kb 120 kb In-vitro transfer No No? Carrier No No? plasmid may represent either an enterococcal or co-integrated plasmid composed of enterococcal and staphylococcal plasmid sequences. For both the first Michigan VRSA strain and the NYC VRSA strain, potential vancomycin-resistant enterococcal donors were found in the same patient (F. Tenover, personal communication). All of the first three VRSA isolates remained sensitive to multiple other antibiotics on in-vitro testing, including imipenem, linezolid, minocycline, quinupristin dalfopristin, rifampin, and trimethoprim sulphamethoxazole. From the preliminary report (J. T. Rudrik, personal communication), it appears that a vancomycin-resistant E. faecalis strain was grown from a rectal culture of the patient. More information on this fourth VRSA strain is not currently available. In summary, the only patient with a VRSA strain but no vancomycin-resistant enterococci was from Hershey [5]. As with VISA, automated susceptibility testing methods have failed to reliably detect VRSA strains. Thus, it has been recommended that a non-automated method of susceptibility testing, such as a vancomycin-agar screening plate or conventional agar, microdilution MIC or Etest be used when S. aureus is isolated [5,38]. RISING GRAM-POSITIVE RESISTANCE AND THERAPEUTIC OPTIONS Unfortunately, the consequence of increasing resistance among S. aureus and other pathogens is an increase in morbidity and mortality due to bacteraemia [29,39]. Outcomes of S. aureus infections in critically ill patients with bacteraemia were assessed in a retrospective analysis [39]. Compared to patients with methicillin-sensitive S. aureus (MSSA), patients with MRSA bacteraemia experienced more acute renal failure and haemodynamic instability, had longer stays in the intensive care unit, and were more dependent on a ventilator. In-hospital and 30-day mortality were significantly (p < 0.05) higher among MRSA patients than among MSSA patients, even after adjustment for disease severity. In a prospective evaluation of patients with S. aureus bacteraemia [29], patients with MRSA were significantly more likely to develop complications including endocarditis and bacteraemia than those with MSSA (Fig. 2). Adverse outcomes were significantly greater in patients with MRSA

5 Appelbaum MRSA the tip of the iceberg 7 expected that resistance to other antibiotics used for treating MRSA will increase over time. All currently available b-lactam antibiotics are inactive against resistant staphylococci. New antimicrobial agents that overcome current resistance mechanisms are needed to treat MRSA and other resistant bacteria. Restoring the trusted activity of cephalosporins to include MecA-triggered resistance would be a great achievement in antimicrobial development. Fig. 2. The effects of increasing resistance among Staphylococcus aureus isolates on mortality [29,39]. MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus. infections. Similarly, in a surveillance programme conducted at 274 sites in Germany to examine differences in mortality and risk-factors associated with nosocomial S. aureus infections in intensive care unit patients [40], MRSA was associated with a significantly higher mortality rate. A retrospective analysis of MRSA bacteraemia in a neonatal intensive care unit [18] reported 8 17 (47%) to be due to MRSA. Isolates from six of the eight infants with MRSA carried the SCCmec type IV genes, a feature of CA-MRSA in the USA. All six isolates were resistant to macrolides and b-lactams, and one was also resistant to clindamycin. One non-typeable isolate carried the SCCmec type II gene characteristically found in HA-MRSA, and was only susceptible to vancomycin. Seven (88%) infants developed septic shock, three infants died, and three survivors required prolonged antibiotic therapy for complications. NEED FOR NEW b -LACTAMS There are now limited therapeutic options for treating serious bacterial infections, and in the face of increasing resistance, an urgent need exists for new antibiotics. Vancomycin is becoming less than optimal for treating MRSA and other resistant Gram-positive bacteria. Widespread use of vancomycin (which is already widely used orally in the treatment of pseudomembranous colitis) in the community to treat CA-MRSA infection causes an increased selective pressure that is ideal for selection of VISA and maintenance of vana plasmids in S. aureus strains. Resistance to linezolid is emerging rapidly [41], and it can be CEFTOBIPROLE Ceftobiprole is the first of a new class of parenteral cephalosporins, and is highly b-lactamasestable. Ceftobiprole has demonstrated a strong affinity for penicillin-binding proteins (PBP2, PBP2x, PBP1a) present in a wide variety of bacteria, including staphylococci, pneumococci, and other Gram-positive and Gram-negative pathogens, and especially for PBP2a, which mediates resistance of MRSA to b-lactams. Ceftobiprole medocaril, a water-soluble prodrug, is rapidly converted in human plasma to the active drug, ceftobiprole. Results from single- and multipledose pharmacokinetic studies demonstrate that the mean volume of distribution for ceftobiprole (16 20 L) approximates the extracellular compartment, suggesting extensive extracellular distribution [42,43]. Ceftobiprole is eliminated primarily by renal excretion with a half-life of 3 4 h. The in-vitro antibacterial activity of ceftobiprole and other antimicrobial agents was compared against staphylococcal isolates (Ednie L, Appelbaum PC. Antistaphylococcal activity of ceftobiprole [BAL9141] and comparators. 44th ICAAC 2004, abstract E-2021). Bacteria included 152 MSSA Table 2. Comparative activities (MIC 90 ) of various antimicrobial agents against methicillin-sensitive and -resistant Staphylococcus aureus and coagulase-negative staphylococci (CNS) [44] Staphylococcus aureus CNS Meth-R Meth-S Meth-R Meth-S Ceftobiprole Cefazolin > Linezolid Quinupristin dalfopristin Minocycline Gentamicin > 16 > 16 > Co-trimoxazole > 8 8 > 8 8 Vancomycin Teicoplanin Daptomycin

6 8 Clinical Microbiology and Infection, Volume 12 Supplement 2, 2006 Fig. 3. Ceftobiprole time-kill graph of vancomycin-resistant Staphylococcus aureus strain [45]. and MRSA strains and 151 MSSA and MRSA coagulase-negative strains (Table 2). The MIC 90 for ceftobiprole was < 4 mg L for all isolates, and 100% of isolates were susceptible to ceftobiprole at a preliminary breakpoint of 4 mg L (Ednie L, Applebaum PC. Antistaphylococcal activity of ceftobiprole [BAL9141] and comparators. 44th ICAAC 2004, abstract E-2021). Ceftobiprole MICs were in the range of mg L for VISA, VRSA, and coagulase-negative isolates. All strains were also susceptible to linezolid, quinupristin dalfopristin, daptomycin, and vancomycin, but resistant to amoxycillin clavulanate, cefazolin, gentamicin, levofloxacin, and teicoplanin. Time-kill studies comparing the activity of ceftobiprole with the activities of 18 other antimicrobial agents against drug-resistant staphylococcal isolates found that at concentrations twice the MIC, ceftobiprole was bactericidal for 11 of 12 isolates (Fig. 3) (Lin G, Appelbaum PC. Comparative time-kill determination of the antipneumococcal activity of ceftobiprole [BAL9141]. 14th ECCMID 2004, abstract E-2019). Other comparators also demonstrated bactericidal activity by 24 h, but linezolid was bacteriostatic. The potential for development of endogenous resistance in three methicillin-resistant staphylococcal isolates was examined for ceftobiprole, linezolid, and moxifloxacin (Heller S, Marrer E, Page MGP, Shapiro S, Thenoz L. Development of endogenous resistance by staphylococci to BAL9141 and comparators. 14th ECCMID 2004, abstract P675). Each isolate was serially transferred at 48-h intervals on Mueller Hinton agar containing either ceftobiprole, linezolid or moxifloxacin until the MIC exceeded 64 mg L, up to a maximum of 50 passages. The MIC of ceftobiprole reached a plateau of 32 mg L after 50 passages, whereas the MIC exceeded 64 mg L with linezolid after passages and with moxifloxacin after 4, 8 or 22 passages, depending upon the strain (Fig. 4). Another serial passage study, this Fig. 4. Development of endogenous resistance by a methicillin-resistant Staphylococcus aureus clinical isolate (Heller S, Marrer E, Page MGP, Shapiro S, Thenoz L. Development of endogenous resistance by staphylococci to BAL9141 and comparators. 14th ECCMID 2004, abstract P675). one performed at 24-h intervals in liquid medium, yielded MICs of ceftobiprole in the 1 8 mg L range by 50 passages. In comparison, the MIC of linezolid reached 64 mg L in some clones and increased from 8 to 16 mg L in others [44]. These results strongly suggest that emergence of resistance during treatment with ceftobiprole is highly unlikely. Similar experiments conducted first by Hebeisen et al. showed that the ceftobiprole MIC 90 for MRSA was 4 mg L. Ceftobiprole was bactericidal against MRSA, and the development of resistance in MRSA was not observed [45]. Favourable results from a phase II trial in patients with complicated skin and skin structure infections have been reported (Heep M, Querner S, Harsch M, O Riordan W. Ceftobiprole [BAL5788], the first of a new class of anti-mrsa cephalosporins: microbiological results from a phase II study in complicated skin and skin structure infections. 44th ICAAC 2004, abstract L-361). Phase III clinical trials have been initiated with ceftobiprole for the treatment of complicated skin and skin structure infections and hospitalacquired pneumonia. CONCLUSIONS Changing patterns of resistance have compounded and exacerbated the need for new antimicrobial agents. Ceftobiprole is a promising new cephalosporin that is refractory to the development of endogenous resistance in staphylococci. Not only is ceftobiprole active against MRSA and other Gram-positive organisms, but it also demonstrates activity comparable to that of cefepime against Enterobacteriaceae and Pseudomonas spe-

7 Appelbaum MRSA the tip of the iceberg 9 cies (Kresken M, Heep M. In vitro activities of BAL9141, the active component of BAL5788, and seven other beta-lactams against selected strains of Pseudomonas aeruginosa susceptible or resistant to ceftazidime. 14th ECCMID 2004, abstract E Jones RN, Sader HS, Fritsche TR. In vitro susceptibility testing guidelines for ceftobiprole [BAL9141] using CLSI disk diffusion and broth microdilution MIC methods. 45th ICAAC 2005, abstract D-1647) and is active and bactericidal against Haemophilus influenzae and Moraxella catarrhalis (Appelbaum PC, Smith K. MIC values of ceftobiprole and comparators towards Haemophilus influenzae and Moraxella catarrhalis. 45th ICAAC 2005, abstract E-304. Pankuch G, Lin G, Appelbaum PC. Time-kill activities of ceftobiprole and eight other agents against H. influenzae and M. catarrhalis. 45th ICAAC 2005, abstract E-305. Clark C, Bogdanovich T, Ednie LM, Appelbaum PC. 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