Microbiology of Antibiotic Resistance In Staphylococcus aureus

Transcription

1 Microbiology of Antibiotic Resistance In Staphylococcus aureus Peter Appelbaum, MD, PhD Professor, Division of Clinical Pathology Penn State College of Medicine Medical Director, Clinical Microbiology Milton S. Hershey Medical Center Hershey, Pennsylvania 1 This presentation is titled: Microbiology of Antibiotic Resistance in Staphylococcus aureus Speaker introduction: Peter Appelbaum, MD, PhD Professor, Division of Clinical Pathology Penn State College of Medicine Medical Director, Clinical Microbiology Milton S. Hershey Medical Center Hershey, Pennsylvania 1

2 Staphylococcus aureus: : Development of Penicillin and Methicillin Resistance 1940s: Penicillin used to treat infection : Penicillin-resistant Staphylococcus aureus recognized in hospitals s: 1960s: > 80% of hospital- and community- acquired Staphylococcus aureus are penicillin resistant : Introduction of methicillin, rapidly followed by reports of resistance 3 Sources: 1. Lowy FD. J Clin Invest. 2003;111: Rammelkamp CH, Maxon T. Proc Royal Soc Exper Biol Med. 1942;51: Jevons MP. Br Med J. 1961;1: The development of antimicrobial-resistant S aureus is of great concern, because of the ability of this bacterium to cause life-threatening infections and its adaptive capacity. 1 Even in this era of antibiotics, mortality due to S aureus bacteremia is 30% to 40%. 2 Pre-antibiotics, mortality was double this; the introduction of penicillin in the 1940s substantially reduced the mortality associated with infection. 3 However, this joy was short-lived, as the emergence of S aureus resistant to penicillin was first recognized in hospitals in 1942, followed by its emergence in the community. 4 By the late 1960s, more than 80% of hospital and community acquired S aureus were resistant to the effects of penicillin. 3 The introduction of methicillin in the 1960s offered an opportunity to treat S aureus isolates resistant to penicillin. However, resistance to this agent ensued rapidly. 5 Methicillin resistance is now increasingly recognized in the community. s 1. Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998;339: Mylotte JM, McDermott C, Spooner JA. Prospective study of 114 consecutive episodes of Staphylococcus aureus bacteremia. Rev Infect Dis. 1987;9: Lowy FD. Antimicrobial resistance: the example of Staphylococcus aureus. J Clin Invest. 2003;111: Rammelkamp CH, Maxon T. Resistance of Staphylococcus aureus to the action of penicillin. Proc Royal Soc Exper Biol Med. 1942;51: Jevons MP. Celbenin -resistant staphylococci. Br Med J. 1961;1:

3 The Tip of the Iceberg MRSA strains now distributed worldwide 3 pandemic clones traced to original Danish isolates 1 Generally MDR Dramatic increase in MRSA linked to expanding reservoir of CoMRSA 2 CoMRSA susceptible to most antimicrobial agents except β-lactams and macrolides 3 Carried characteristic gene complex for methicillin resistance (SCCmec type IV) and gene for PVL 3 Importance of PVL in CoMRSA has recently been called into question 4 MRSA = methicillin resistant Staphylococcus aureus; MDR = multidrug resistant; CoMRSA = community-onset MRSA. SCCmec = staphylococcal cassette chromosome; PVL = Panton-Valentine leukocidin. Sources: 1. Crisostomo MI, et al. Proc Natl Acad Sci U S A. 2001;98: Carleton HA, et al. J Infect Dis. 2004;190: Kazakova SV, et al. N Engl J Med. 2005;352: Voyich JM, et al. J Infect Dis. 2006;194: To gain information about the origin and spread of methicillin-resistant S aureus (MRSA), the genetic background of strains of methicillin-susceptible S aureus (MSSA) recovered in the early 1960s were examined. These strains may have been the original recipients of the mec element at the time when the first European isolates of MRSA were identified in Denmark and in the United Kingdom. 1 In a population dynamics study of an expanding community reservoir of MRSA, the data refuted the generally accepted view that the majority of CoMRSA isolates are attributed to the spread of hospital-associated (HA) strains into the community. Conversely, it appears that community-associated (CA) strains are migrating to the hospital setting. 2 An investigation into a cluster of skin abscesses among professional football players and other recent outbreaks of skin infection in the US showed that they were caused by an emerging MRSA clone. This community-associated clone differed from strains of MRSA circulating in healthcare settings in that it was susceptible to most antimicrobial agents other than beta-lactams and macrolides, it primarily caused skin infections in otherwise healthy persons, and it carried both a characteristic gene complex for methicillin resistance (SCCmec type IVa) and the gene for Panton-Valentine leukocidin, a cytotoxin that has been associated with severe abscesses and necrotizing pneumonia. 3 A single toxin, Panton-Valentine leukocidin (PVL), has been linked by epidemiological studies to CA-MRSA disease. Using a mouse infection model, the virulence of PVL-positive and PVL-negative CoMRSA strains were compared. Strains that lacked PVL were as virulent in mouse sepsis and abscess models as those containing the leukotoxin. While PVL may be a highly linked epidemiological marker for CoMRSA strains, it may not be the major virulence determinant of CoMRSA. 4 s 1. Crisostomo MI, Westh H, Tomasz A, Chung M, Oliveira DC, de Lencastre H. The evolution of methicillin resistance in Staphylococcus aureus: similarity of genetic backgrounds in historically early methicillin-susceptible and -resistant isolates and contemporary epidemic clones. Proc Natl Acad Sci U S A. 2001;98: Carleton HA, Diep BA, Charlebois ED, Sensabaugh GF, Perdreau-Remington F. Community-adapted methicillin-resistant Staphylococcus aureus (MRSA): population dynamics of an expanding community reservoir of MRSA. J Infect Dis. 2004;190: Kazakova SV, Hageman JC, Matava M, et al. A clone of methicillin-resistant Staphylococcus aureus among professional football players. N Engl J Med. 2005;352: Voyich JM, Otto M, Mathema B, et al. Is Panton-Valentine leukocidin the major virulence determinant in communityassociated methicillin-resistant Staphylococcus aureus disease? J Infect Dis. 2006;194:

4 Life-Threatening CoMRSA Infections Neonatal sepsis 1 8/17 S aureus bacteremia due to MRSA 6/8 carried SCCmec IV Three fatalities Three had complications requiring prolonged therapy Community-acquired pneumonia 2 Four patients, all strains PVL + with SCCmec IV Two patients with concomitant influenza A One fatality The other 3 required prolonged hospitalization due to severe complications Sources: 1. Healy CM, et al. Clin Infect Dis. 2004;39: Francis JS, et al. Clin Infect Dis. 2005;40: Methicillin resistance has moved outside the hospital environment. Comparison of the strains involved in epidemiologically unrelated outbreaks found them to be indistinguishable. 1 Life-threatening CoMRSA infections have also been documented. 2,3 Healy et al conducted a retrospective cohort study among neonatal intensive care unit (NICU) infants with bacteremia due to MRSA during 2003 in a large tertiary care center NICU in Texas. In order to determine whether the genetic traits of CoMRSA were present, the isolates were characterized by antimicrobial susceptibility testing and staphylococcal cassette chromosome mec(sccmec) typing by polymerase chain reaction (PCR). A total of 8 (47%) of 17 infants with bacteremia due to S aureus had MRSA infection. Isolates from 6 (75%) of these 8 infants carried the SCCmec genes characteristic of community MRSA. All 6 isolates were resistant to beta-lactam antibiotics and erythromycin. Additionally, 1 was also resistant to clindamycin. One isolate could not be typed, and another carried the SCCmec type II gene (typical of hospital-associated MRSA [HA-MRSA] strains) with susceptibility only to vancomycin. Seven (88%) of 8 infants presented in septic shock. Of the 6 infants that had MRSA isolates with genetic characteristics of community origin, 3 (38%) infants died despite initial treatment with vancomycin, and 3 survivors had complications requiring prolonged antimicrobial therapy. 2 According to a report by Francis et al, 4 healthy adults presented to 1 of 2 Baltimore hospitals with severe necrotizing MRSA pneumonia during a 2-month period in the winter of The typical risk factors for MRSA infection were absent in these cases. These infections were associated with a syndrome characterized by an influenza or influenza-like prodrome, high fever, severe necrotizing pneumonia, leukopenia, respiratory failure, and shock. All 4 patients' MRSA isolates carried the PVL genes, and the SCCmec type IV element, and belonged to the USA300 pulsed-field type. Two of the patients had concomitant influenza A diagnosed, which likely contributed to the severity of their presentation. Three patients recovered but had significant morbidities and required 1 to 3 months in an ICU. One patient developed progressive hypotension and hypoxia, requiring aggressive hydration and increased oxygenation and died on day 2 of hospitalization after developing pulseless electrical activity and showing no response to resuscitation. These patients represent the first reported cases of North American adults with severe CoMRSA pneumonia caused by strains carrying the PVL genes. 3 s 1. Kazakova SV, Hageman JC, Mataya M, et al. A clone of methicillin-resistant Staphylococcus aureus among professional football players. N Engl J Med. 2005;352: Healy CM, Hulten KG, Palazzi DL, Campbell JR, Baker CJ. Emergence of new strains of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit. Clin Infect Dis ;39: Francis JS, Doherty MC, Lopatin U, et al. Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes. Clin Infect Dis. 2005;40:

5 Types of Infections Caused by MRSA Folliculitis Cellulitis Abscess Impetigo Osteomyelitis Meningitis Staphylococcal scalded skin syndrome Pemphigus neonatorum Scarlet fever (rare) Endocarditis Pneumonia Bacteremia Necrotizing soft tissue infections Toxic shock syndrome Waterhouse-Friderichsen syndrome Food poisoning Breast abscess in lactating women 5 This slide outlines the types of infections caused by MRSA. 5

6 MRSA: Key Features Mechanism involves altered target site New penicillin-binding protein PBP 2' 2 (PBP 2a) Encoded by chromosomally located meca gene Confers resistance to all available β-lactams Methicillin-resistance determinant located on mobile genetic element SCCmecmec Not all meca-positive clones are resistant to methicillin Sources: Katayama Y, et al. Antimicrob Agents Chemother. 2000;44: Berger-Bachi B, Rohrer S. Arch Microbiol. 2002;178: Key features of MRSA are described here. 1,2 The beta-lactam resistance of MRSA is caused by the production of a novel penicillin-binding protein (PBP) designated PBP 2' (or PBP 2a), which, unlike the intrinsic set of PBPs (PBP 1 to 4) of S aureus, has remarkably reduced binding affinities to beta-lactam antibiotics. Despite the presence of otherwise inhibitory concentrations of beta-lactam antibiotics, MRSA can continue cell wall synthesis solely depending upon the uninhibited activity of PBP 2'. 1 The protein PBP 2' is encoded by a meca gene located on the chromosome of MRSA. The meca gene is widely distributed among S aureus as well as coagulase-negative staphylococci. Therefore, it has been suggested that the methicillin resistance determinant (mec determinant) is freely transmissible among staphylococcal species. 1 The methicillin resistance gene of MRSA is carried by a novel genetic element, SCCmec whose integration into and excision from the S aureus chromosome are mediated by a unique set of recombinase genes, ccra and ccrb (cassette chromosome recombinases A and B). The presence of these 2 genes is required for excision and integration processes of SCCmec. 1 Resistance levels in MRSA depend on efficient PBP 2' production and are modulated by chromosomal factors. Depending on the genetic background of the strain that acquired meca, resistance levels range from phenotypically susceptible to highly resistant. 2 s 1.Katayama Y, Ito T, Hiramatsu K. A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 2000;44: Berger-Bachi B, Rohrer S. Factors influencing methicillin resistance in staphylococci. Arch Microbiol. 2002;178:

7 Characteristics of SCCmec SCCmecmec type SCCmecmec size, kb Other resistant elements Origin Presence of PVL I Hospital Infrequent II 53 PUB110 ( (aadd) a Tn554 ( (erma) b Hospital Infrequent III 67 PUB110 ( (aadd) a PT181 ( (tetk) c Hospital Infrequent IV Community Frequent V Community Unknown kb = kilobase. a encodes resistance to tobramycin and kanamycin; b encodes resistance to macrolide-lincosamide-streptogramin antibiotics; c encodes resistance to tetracycline. Source: Deresinski S. Clin Infect Dis. 2005;40: This slide summarizes the characteristics of SCCmec, including size, other resistant elements, origin, and presence of PVL. The mobile SCCmec elements are classified by analysis of their cassette chromosome recombinase (ccr) and meca gene complexes. SCCmec types also differ in terms of their acquisition of resistance determinants acquired as the result of integration of plasmids and transposons. At least 5 SCCmec types (types I through V), varying in size from ~20 kb to 68 kb, have been identified. The size of the respective mec types determine the resistance to various antibiotics. HA- MRSA (mainly types II and III) are resistant to more antibiotics than CA-MRSA (mainly type IV), but the community variety may be more virulent. The smallest of these SCCmec types I, IV, and V contain only recombinase genes and the structural and regulatory genes for resistance to methicillin and lack the transposable elements and genes encoding resistance to non-beta-lactam antibiotics carried by types II and III. SCCmec types I IV contain alleles of ccra and ccrb, whereas type V, which has to date been identified in a small number of Australian CA-MRSA isolates, contains a novel ccr designated ccrc. Two possible additional SCCmec types have recently been identified among Australian CA- MRSA strains. Deresinski S. Methicillin-resistant Staphylococcus aureus: an evolutionary, epidemiologic, and therapeutic odyssey. Clin Infect Dis. 2005;40:

8 Antimicrobial Susceptibility Profiles Antibiotic CA-MRSA Number (%) Susceptible (n = 106) HA-MRSA Number (%) Susceptible (n = 211) P Value Methicillin 0 0 NA Ciprofloxacin 84 (79) 33 (16) <.001 Clindamycin 88 (83) 44 (21) <.001 Erythromycin 47 (44) 18 (9) <.001 Gentamicin 100 (94) 168 (80).001 Rifampin 102 (96) 199 (94).64 Tetracycline 98 (92) 194 (92).95 Trimethoprim- Sulfamethoxazole 101 (95) 189 (90).13 Vancomycin 106 (100) 211 (100)... CA-MRSA = community-associated methicillin resistant Staphylococcus aureus isolates; HA-MRSA = healthcare-associated methicillin resistant Staphylococcus aureus isolates. Source: Naimi TS, et al. JAMA. 2003;290: Antimicrobial susceptibility profiles for listed antimicrobials according to Naimi and colleagues are shown. The percent susceptible values were tested at the Minnesota Department of Public Health Laboratory by broth microdilution using Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) break points. The P value refers to the statistical probability that the percentage susceptible among CA isolates differed from the percentage susceptible among HA isolates. Susceptibility and pulsed field gel electrophoresis (PFGE) testing were performed on 106 CA isolates and a representative sample of 211 HA isolates. Oxacillin resistance (a surrogate for methicillin resistance) was confirmed in all MRSA isolates. CA-MRSA isolates were generally susceptible to antimicrobials other than beta-lactams and were more likely than HA isolates to be susceptible to multiple agents. Among CA isolates, susceptibilities did not differ between pediatric and adult isolates. CA isolates from skin sites were more likely to be susceptible to ciprofloxacin and clindamycin compared with CA isolates from other sites. CA-MRSA isolates were also more likely than HA-MRSA isolates to be susceptible to ciprofloxacin, clindamycin, gentamicin, and trimethoprim-sulfamethoxazole. In a logistic regression model adjusted for age, sex, surrogate income, laboratory location (Minneapolis-St Paul metropolitan versus greater Minnesota), and culture site (skin versus other), susceptibility to all 4 antimicrobials was an independent predictor of having a CA case definition. Naimi TS, LeDell KH, Como-Sabetti K, et al. Comparison of community- and healthcare-associated methicillin-resistant Staphylococcus aureus infection. JAMA. 2003;290:

9 Disk Diffusion Testing With Cefoxitin Well correlated with the presence of meca-mediated mediated oxacillin resistance in Staphylococci New cefoxitin DD breakpoints Breakpoints S aureus CoNS Sens Spec 19 mm (resistant) mm (susceptible) 24 mm (resistant) mm (susceptible) Easier to read no need for transmitted light Results equivalent to, or better than, oxacillin broth microdilution and oxacillin DD tests DD = disk diffusion; CoNS = coagulase-negative staphylococci; Sens = sensitivity; Spec = specificity. Source: Swenson JM, et al. J Clin Microbiol. 2005;43: Several detection methods for MRSA have been documented in the literature. These include: dilution methods, E-test method, breakpoint methods, agar screening method, disk diffusion, latex agglutination (PBP 2a detection), automated methods, and molecular methods (meca gene detection). 1 The cefoxitin disk diffusion (DD) test is used for predicting meca-mediated oxacillin resistance in staphylococci. Cefoxitin, a cephamycin, is a more potent inducer of the meca regulatory system than are the penicillins. 2 Several research groups have reported that results of cefoxitin DD tests correlate better with the presence of meca than do results of DD tests using oxacillin. 2 Oxacillin minimum inhibitory concentration (MIC) and DD tests and cefoxitin MIC and DD tests are essentially equivalent in performance for both sensitivity and specificity for S aureus. All 4 tests also exhibit equivalent sensitivities for CoNS. However, for CoNS, the cefoxitin MIC and DD tests showed better specificity than did tests with oxacillin. 2 All the tests that use oxacillin must be examined carefully by holding the plates up to transmitted light and examining carefully for any growth either in the wells of the MIC plate, in the zone of inhibition, or in the quadrant of the oxacillin salt agar screen plate. Therefore, cefoxitin DD test offers an advantage over oxacillin since it is easier to read. 2 The cefoxitin DD test can be used to predict the presence of meca in S aureus and CoNS with a high degree of sensitivity and specificity when compared with meca detection using PCR. The test improved on the specificity of oxacillin DD and oxacillin MIC testing without sacrificing sensitivity, especially for CoNS. 2 Some novel molecular detection methods have also been descried recently. These include: threedimensional microarray system (greater sensitivity than PCR methods) and novel multiplex PCR assay (characterization and concomitant subtyping of SCCmec types I to V in MRSA). 3,4 1. Brown DF, Edwards DI, Hawkey PM, et al. Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). J Antimicrob Chemother. 2005;56: Swenson JM, Tenover FC; Cefoxitin Disk Study Group. Results of disk diffusion testing with cefoxitin correlate with presence of meca in Staphylococcus spp. J Clin Microbiol. 2005;43: Nagaoka T, Horii T, Satoh T, et al. Use of a three-dimensional microarray system for detection of levofloxacin resistance and the mec A gene in Staphylococcus aureus. J Clin Microbiol. 2005;43: Zhang K, McClure JA, Elsayed S, Louie T, Conly JM. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2005;43:

10 Beneficial Outcomes With Rapid Detection Median reporting time decreased from 4 days to 1 day Previously unknown MRSA carriers rapidly identified Preemptive isolation and cohorting led to reduced MRSA cross-infections Time (hours) a P <.001. a Time From Admission to Screening a Time To Result Notification a Total Time Standard Culture Rapid qmrsa Test qmrsa = quick, multiplex immunocapture-coupled PCR technique to detect meca gene. Source: Harbarth S, et al. Critical Care. 2006;10:R The most efficient approach to control of endemic MRSA remains controversial. Several authorities have suggested that screening at ICU admission and subsequent patient isolation may decrease the risk for MRSA cross-infection. Rapid screening tests may further improve MRSA control, because traditional microbiologic methods for MRSA screening are slow. Delays in receiving screening results means either that negative patients remain isolated for too long or that positive patients remain a hidden reservoir for cross-infection. With the availability of rapid molecular MRSA screening methods, determining their value in daily practice is of great importance. A new molecular technique enabling quick MRSA screening (qmrsa) was used for early detection of MRSA and to decrease the time between ICU admission and notification of screening results. Median time from ICU admission to notification of test results decreased from 87 to 21 hours in the surgical ICU (P <.001) and from 106 to 23 hours in the medical ICU (P <.001). In the surgical ICU, 1227 preemptive isolation days for 245 MRSA-negative patients were saved by using qmrsa. The qmrsa test decreased median time to notification from 4 days to 1 day and helped to identify previously unknown MRSA carriers rapidly. A strategy linking the rapid screening test to preemptive isolation and cohorting of patients with MRSA substantially reduced MRSA cross-infections in the medical, but not in the surgical, ICU. Harbarth S, Masuet-Aumatell C, Schrenzel J, et al. Evaluation of rapid screening and pre-emptive contact isolation for detecting and controlling methicillin-resistant Staphylococcus aureus in critical care: an interventional cohort study. Crit Care. 2006;10:R25. 10

11 The Emergence of Vancomycin Nonsusceptible S aureus 1996: First report of hvisa in Japan Mu3 case history 64-year year-old Japanese man who had MRSA pneumonia Treatment with vancomycin for 12 days was ineffective Subsequently treated successfully with 10-day course of aminoglycoside + ampicillin/sulbactam Sputum sample revealed MRSA (Mu3) with vancomycin MIC of 4 µg/ml on BHIA plates Subpopulations grew in the presence of 5 99 mg/l vancomycin This strain is a true VISA by the latest criteria hvisa = heterogeneous vancomycin-intermediate S aureus; VISA = vancomycin intermediate S aureus; MIC = minimum inhibitory concentration; BHIA = brain heart infusion agar. Source: Hiramatsu K. et al. Lancet. 1997;350: hvisa is the precursor to the development of VISA. hvisa strains contain subpopulations of daughter cells that are vancomycin-intermediate, although the parent strain remains vancomycin-sensitive. 1 In 1996 the first hvisa strain, Mu3, was reported in Japan from a man with MRSA pneumonia unresponsive to vancomycin. 2 The MIC for the isolate was 4 μg/ml, although subpopulations were able to grow in media containing vancomycin concentrations ranging from 5 to 9 μg/ml. Treatment with vancomycin for 12 days was ineffective. The patient was subsequently treated successfully with a 10-day course of aminoglycoside plus ampicillin/sulbactam. Sputum sample revealed MRSA (Mu3) with vancomycin MIC of 4 µg/ml on BHIA plates. This strain is a true VISA according to the latest criteria (hvisa would be 1-2 µg/ml). 2 Pulse-field gel electrophoresis demonstrated that Mu3 was indistinguishable from the VISA strain, Mu50, that was later isolated in the same hospital in When Mu3 was serially passaged through increasing concentrations of vancomycin, subpopulations of strains with similar levels of resistance to Mu50 developed, 2 indicating that vancomycin exposure was exerting selection pressure on the isolates in favor of a more resistant phenotype. 3 A review of hvisa isolates from around the world demonstrated that the majority are methicillin resistant. 1 Furthermore, Hiramatsu also published the first report (1997) of VISA in Japan: Mu50 case history involving a 4-monthold Japanese infant who underwent heart surgery. Two weeks postoperatively, the infant became febrile and developed a purulent incision site discharge. The infant was treated with vancomycin for 29 days; fever and discharge persisted. Aminoglycoside was added to treatment regimen for 12 days and the wound healed. An abscess developed 12 days later and the infant was treated with aminoglycoside + ampicillin/sulbactam. Debridement sample revealed MRSA (Mu50) with vancomycin MIC of 8 μg/ml by broth microdilution method. 2 s 1. Liu C, Chambers HF. Staphylococcus aureus with heterogeneous resistance to vancomycin: epidemiology, clinical significance, and critical assessment of diagnostic methods. Antimicrob Agents Chemother. 2003;47: Hiramatsu K, Aritaka N, Hanaki H et al. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet. 1997;350: Hiramatsu K. Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect Dis. 2001;1:

12 Definitions of Vancomycin Susceptibility in S aureus Susceptibility Vancomycin susceptible S aureus (VSSA) Vancomycin intermediate S aureus (VISA) Vancomycin resistant S aureus (VRSA) Vancomycin MICs 2 μg/ml 4-8 μg/ml 16 μg/ml Source: Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing: 17th Informational Supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2007:M100-S The vancomycin MIC ranges are depicted for VSSA, VISA, and VRSA as currently specified by the CLSI. The upward drift in MICs may necessitate changes in future ranges. For example: VISA may need to be defined from 4-16 μg/ml and VRSA 32 μg/ml. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: 17th Informational Supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2007:M100-S17. 12

13 Underreporting of VISA and hvisa VISA and hvisa are underreported due to unsatisfactory detection techniques such as Disk diffusion alone (may be acceptable for VRSA) Automated methods (eg, Vitek and Microscan): neither method accurately identified Hershey and NYC VRSAs with a vancomycin MIC µg/ml A vancomycin agar screening plate must be added with a vancomycin concentration LOWER THAN 6 µg/ml (perhaps 3 µg/ml) Low frequency of subpopulations decreases the sensitivity of testing methods Routine screening for glycopeptide resistance must be done (using CLSI microdilution and 24 hour E-test) when MRSA is isolated, in order to prevent outbreaks MIC = minimum inhibitory concentration. Sources: de Lassence A, et al. Clin Infect Dis. 2006;42: Sancak B, et al. J Antimicrob Chemother. 2005;56: S aureus strains with reduced susceptibility to vancomycin are susceptible to vancomycin according to NCCLS MIC criteria (MIC 4 μg/ml) but contain subpopulations at a frequency of 10-6 or higher with MICs of vancomycin of more than 4 μg/ml. Because of the low frequency of these subpopulations, hetero-visa strains could not be detected either by DD or via standard MIC methods. As a result, differentiation of these isolates from susceptible strains presents a real problem. Alternative methods to analyze subpopulations range from the aztreonam disc method based on the antagonism of vancomycin and betalactam, to the E-test with a dense inoculum called the macro E-test. 1 In patients with S aureus infections that have reduced susceptibility to vancomycin, the low frequency ( 10-6 ) of the subpopulations decreases the sensitivity of the testing methods. Therefore, it is important to use effective methods that can detect heteroresistance of S aureus isolates. Detection of VISA is possible with standard laboratory methods, but the detection of hetero-visa remains difficult. 1 Rigorous screening must be performed post-outbreak. MRSA strains should be examined using the E-test with vancomycin and teicoplanin. Screening specimens from nostrils, axillae, and rectum should be obtained from all patients at admission, and once or twice weekly. 2 s 1. Sancak B, Ercis S, Menemenlioglu D, Coladoglu S, Hascelik G. Methicillin-resistant Staphylococcus aureus heterogeneously resistant to vancomycin in a Turkish university hospital. J Antimicrob Chemother. 2005;5: de Lassence A, Hidri N, Timsit JF, et al. Control and outcome of a large outbreak of colonization and infection with glycopeptide intermediate Staphylococcus aureus in an intensive care unit. Clin Infect Dis. 2006;42:

14 Detection of hvisa (MICs 1-21 µg/ml) Do Only If Patient Not Responding to Vancomycin hvisa cannot be detected by standard MIC methods Vancomycin agar screen test with vancomycin <6 µg/ml.. All positive isolates subject to macro E-test E Macro E-test E for vancomycin and teicoplanin on BHIA with inoculum turbidity of 2 McFarland, incubation 24/48 hours Different interpretative criteria for macro E-test; E hvisa if: 8 µg g of vancomycin per ml and 8 µg g of teicoplanin per ml 12 µg g of teicoplanin per ml Source: Walsh TR, et al. J Clin Microbiol. 2001;39: This slide summarizes detection of hvisa (MICs 1-2 μg/ml) and what to do in instances of nonresponse to vancomycin. The sensitivity and specificity of 7 methods (agar dilution, broth microdilution, E-test at 0.5 and 2.0 McFarland [McF] inocula, 2 agar screening methods, and population studies [PS]) were evaluated in a double-blind study involving 284 MRSA strains and 45 Staphylococcus strains with reduced susceptibilities to vancomycin (SRSV). The best E-test interpretative criteria for the 2.0 McF inoculum was 8 μg of vancomycin per ml and 8 μg teicoplanin per ml, or 12 μg of teicoplanin per ml. A vancomycin agar screening plate must be added with a vancomycin concentration LOWER THAN 6 µg/ml (perhaps 3 µg/ml). All positive isolates subject to macro E-test OR All strains with 24 hour E-test 1-2 mg/l subject to macro E-test (depending on the capability of the lab). The use of the E-test provides a reliable and sensitive method for the detection of subtle variations in glycopeptide resistance, including heteroresistance, without incurring too many false positives. Yet it is also recommended that all SRSV-positive results by E-test be confirmed with PAP-AUC ratios, and, as far as possible, by the clinical outcome of therapy. PAP-AUC = population analysis profile-area under the curve. Walsh TR, Bolmstrom A, Qwarnstrom A, et al. Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides. J Clin Microbiol. 2001;39:

15 Macro E-test: E Results for QC & hgisa Strains GSSA hgisa GISA VA TP VA TP VA TP QC = quality control; hgisa = heterogeneous glycopeptide-intermediate S aureus; GSSA = glycopeptide-susceptible S aureus; GISA = glycopeptide-intermediate S aureus; VA = vancomycin; TP = teicoplanin. Source: Bolmstrom A, Karlsson A, Mills K, et al. Presented at: 45th Interscience Conference on Antimicrobial Agents and Chemotherapy. December 15-19, 2005; Washington, DC. Poster C Detection of reduced susceptibility to glycopeptides in S aureus is very difficult in a routine clinical laboratory. Standard MIC values and current MIC breakpoints are not able to discriminate the hgisa/gisa phenotype from glycopeptide-susceptible S aureus. The term GISA refers to glycopeptide intermediate Staphylococcus aureus, and is synonymous with VISA, as vancomycin is the glycopeptide most physicians are familiar with. The teicoplanin susceptibility is lost in MRSA before vancomycin susceptibility. In the macro E-test, the zone sizes for vancomycin and teicoplanin are compared. If the zone sizes are both low, then it's susceptible. If the zone sizes for the teicoplanin are higher than the vancomycin, then it is hetero-visa. If they're both high, then it is a regular VISA. Bolmstrom A, Karlsson A, Mills K, et al. Presented at: 45th Interscience Conference on Antimicrobial Agents and Chemotherapy. December 15-19, 2005; Washington, DC. Poster C

16 The Emergence of VRSA 2002: First report of VRSA in the United States 40-year year-old man from Michigan with diabetes, peripheral vascular disease and chronic renal failure Gangrenous toe amputated in April 2002; then developed MRSA bacteremia caused by infected arteriovenous hemodialysis graft Treated with vancomycin, rifampicin, and removal of the graft Developed suspected catheter exit-site infection in June; catheter subsequently removed Catheter tip cultures grew Staphylococcus aureus resistant to oxacillin and vancomycin Later, chronic foot ulcer appeared infected; cultures grew VRSA, VRE, and Klebsiella oxytoca VRSA = vancomycin resistant S aureus; VRE = vancomycin resistant enterococcus. Source: Sievert DM, et al. MMWR. 2002;51: More recently, there have been reports of S aureus strains exhibiting resistance to vancomycin. 1-3 The first clinical isolate was reported in Michigan, United States, in June This strain exhibited high resistance to vancomycin, having an MIC > 128 μg/ml. 2 Resistance to vancomycin is defined by an MIC of 32 μg/ml. 4 The patient in this instance had diabetes, peripheral vascular disease, and chronic renal failure. His gangrenous toe was amputated in April 2002 and he subsequently developed MRSA bacteremia caused by infected arteriovenous hemodialysis graft. The patients was treated with vancomycin, rifampicin, and removal of the graft. He then developed suspected catheter exit-site infection in June and the catheter was subsequently removed. Catheter tip cultures grew S aureus resistant to oxacillin and vancomycin. Later, the chronic foot ulcer appeared infected; these cultures grew VRSA, VRE, and Klebsiella oxytoca. 2 This was soon followed by another report of VRSA in September 2002 in Pennsylvania, USA (MIC = 64 μg/ml). 3 s 1. Kacica M. Brief Report: Vancomycin-resistant Staphylococcus aureus: New York, MMWR 2004;53: Sievert DM, Boulton ML, Stoltman G. Staphylococcus aureus resistant to vancomycin: United States MMWR 2002;51: Miller D, Urdaneta V, Weltman A. Vancomycin-resistant Staphylococcus aureus: Pennsylvania MMWR 2002;51: Liu C, Chambers HF. Staphylococcus aureus with heterogeneous resistance to vancomycin: epidemiology, clinical significance, and critical assessment of diagnostic methods. Antimicrob Agents Chemother 2003;47:

17 Epidemiology Of Staphylococcal Resistance Andrew Shorr, MD, MPH, FCCP Associate Professor of Medicine Chief, Pulmonary Clinic Pulmonary and Critical Care Medicine Washington Hospital Center Washington, DC 17 This presentation is titled: Epidemiology of Staphylococcal Resistance Speaker introduction: Andrew Shorr, MD, MPH, FCCP Associate Professor of Medicine Chief, Pulmonary Clinic Pulmonary and Critical Care Medicine Washington Hospital Center Washington, DC 17

18 Regional MRSA Prevalence: MRSA Is Everywhere Pacific IP 51% OP 44.4% Mountain IP 57% OP 46.5% West North Central IP 53.8% OP 46.2% East North Central IP 58.9% OP 40.8% New England IP 49.9% OP 37.6% Mid-Atlantic IP 52.4% OP 36.3% South Atlantic IP 59.8% OP 50.6% West South Central IP 59.2% OP 56.8% East South Central IP 63% OP 63% IP = inpatient; OP = outpatient. Source: Styers D, et al. Ann Clin Microbiol Antimicrob. 2006;5:2. 18 The prevalence of antimicrobial resistance has been on the rise in the past decade. According to the 2004 report of the National Nosocomial Infections Surveillance System NNIS), a 12% increase in methicillin-resistant Staphylococcus aureus (MRSA) infections occurred in 2003 compared to the prevalence noted from 1998 to Data from The Surveillance Network-USA (TSN), an electronic surveillance network that collects microbiology data from 300 clinical microbiology laboratories across the United States (US), were used as the source for analysis that included prevalence of S aureus in clinical specimens, MRSA and multidrug resistance phenotype rates and trends according to patients location, geographic distributions, and specimen source. 2 Analysis of inpatient and outpatient MRSA rates demonstrated that in all regions of the US Census Bureau, except New England, inpatient MRSA rates were above 50%. The lowest MRSA rates occurred in the Mid-Atlantic (36.3%) and New England (37.6%) regions; while the highest rate (63%) occurred in the East South Central region where inpatient and outpatient MRSA rates were the same. Prevalence of MRSA among both inpatient and outpatient specimens continues to increase with multidrug resistance as a common phenotype. Continued emergence of outpatient MRSA that exhibit multidrug resistant phenotypes has important implications for developing and evolving outpatient treatment guidelines. The MRSA rates were highest (55.9%) among strains from inpatient lower respiratory specimens and lowest (37.6%) among strains from outpatient skin and soft tissue specimens. s 1. Centers for Disease Control and Prevention. National Nosocomial Infections Surveillance System Report. October Available at: Accessed April 3, Styers D, Sheehan DJ, Hogan P, Sahm DF. Laboratory-based surveillance of current antimicrobial resistance patterns and trends among Staphylococcus aureus: 2005 status in the United States. Ann Clin Microbiol Antimicrob. 2006;5:2. 18

19 MRSA Trends by Patient Location Staphylococcus aureus Is the Most Frequent Bacterial Specimen Encountered in Clinical Samples From Inpatients and ICU, and Second Most Frequent in Outpatients MRSA (%) Year All Patients ICU Patients Inpatients Outpatients ICU = intensive care unit. Sources: Styers D, et al. Ann Clin Microbiol Antimicrob. 2006;5:2. Streit JM, et al. Int J Antimicrob Agents. 2004;24: Analysis of data from TSN showed that S aureus was the most prevalent species isolated from inpatient specimens (18.7% of all bacterial isolates) and the second most prevalent (14.7%) from outpatient specimens. 1 As depicted in the graph, several resistance trends were noted in the above analysis. Overall MRSA rates have steadily increased in the US since 1998 and the rate appeared to be still on the rise as of March 2005 (53.3%). An increase was noted among each patient group including strains from ICU (intensive care unit) patients, non-icu inpatients, and outpatients with current MRSA rates of 55%, 59.2%, and 47.9% respectively. Since 2002 the lowest rate of increase in MRSA occurred among specimens from ICU patients, while the rates among S aureus from other inpatients and outpatients were higher. 1 A total of 1321 bacterial strains from blood, respiratory tract, urine, and wound sites were processed at a central laboratory, as part of the SENTRY Antimicrobial Surveillance Program (2001), to assess their occurrence rates and antimicrobial susceptibility profiles. These strains originated from 25 selected ICUs in North America (Canada and US). S aureus comprised the largest number of organisms isolated from all sources, at 24.1%. 2 s 1. Styers D, Sheehan DJ, Hogan P, Sahm DF. Laboratory-based surveillance of current antimicrobial resistance patterns and trends among Staphylococcus aureus: 2005 status in the United States. Ann Clin Microbiol Antimicrob. 2006;5:2. 2. Streit JM, Jones RN, Sader HS, Fritsche TR. Assessment of pathogen occurrences and resistance profiles among infected patients in the intensive care unit: report from the SENTRY Antimicrobial Surveillance Program (North America, 2001). Int J Antimicrob Agents. 2004;24:

20 High Prevalence of MRSA in Pneumonia Epidemiology of Staphylococcal Resistance Varies by Point of Acquisition MRSA as S aureus (%) a P <.01 a P <.05 a P <.05 0 VAP CAP HAP HCAP * P values when compared with HCAP. VAP = ventilator-associated pneumonia; CAP = community-acquired pneumonia; HAP = hospital-acquired pneumonia; HCAP = healthcare-associated pneumonia. Source: Kollef, et al. Chest. 2005;128: Kollef and colleagues retrospectively evaluated the microbiology and outcomes among 4543 patients with culture-positive pneumonia from 59 US hospitals during 2002 and 2003: CAP, HCAP, HAP, and VAP. The distribution of pathogens varied among the 4 pneumonia categories. S aureus was the dominant pathogen, and its subtypes (methicillin-susceptible S aureus [MSSA] and MRSA) were identified in large proportions of patients for all types of pneumonia. The frequency of occurrence of bacterial pathogens associated with CAP, HCAP, HAP and VAP for all MRSA as percentage of all S aureus was: 34.8 for CAP (n =2221), 56.8 for HCAP (n = 988), 48.6 for HAP (n = 835), and 34.4 for VAP (n = 499). S aureus is a major pathogen of all pneumonias, with higher rates in non-cap pneumonias. Comparable mortality rates were associated with HCAP (19.8%) and HAP (18.8%) (P >.05), and both were significantly higher than that for CAP (10%, all P <.0001) and lower than that for VAP (29.3%, all P <.0001). Mean length of stay and mean hospital charge also varied significantly with pneumonia category (in order of ascending values: CAP, HCAP, HAP, and VAP; all P <.0001). Kollef MH, Shorr A, Tabak YP, Gupta V, Liu LZ, Johannes RS. Epidemiology and outcomes of health-care-associated pneumonia: results from a large US database of culture-positive pneumonia. Chest. 2005;128:

21 Changing Epidemiology of MRSA Community-acquired MRSA Has Increased Rapidly Since Number of MRSA Positive Isolates Total MRSA Community-acquired MRSA Nosocomial MRSA Source: Crum NF, et al. Am J Med. 2006;119: The purpose of this study was to elucidate the evolutionary, microbiologic, and clinical characteristics of MRSA infections. MRSA cases from military medical facilities in San Diego, from 1990 to 2004, were evaluated and categorized as community-acquired or nosocomial. A total of 1888 MRSA isolates were found during the 15-year period; data to establish the case as community-acquired or nosocomial MRSA were available on 1846 isolates (97.8%). Of these, 1208 (65.4%) were community-acquired MRSA cases and 638 (34.6%) were nosocomial MRSA cases. Of the 1888 isolates, 1533 (81.2%) represented an MRSA infection, 348 (18.4%) represented a colonization, and 7 (0.4%) could not be classified. Community-acquired MRSA was the cause of the majority of the infections resulting from MRSA (n = 1044, 68.1%) in this cohort, and 16.4% (171/1044) of community-acquired MRSA infections required hospitalization. Community-acquired MRSA cases were more often soft-tissue and less often urinary, lung, or bloodstream infections compared with nosocomial MRSA (P <.001). Total MRSA isolates increased from 10 in 1990 to 632 in 2004 and community-acquired infections increased rapidly since Crum NF, Lee RU, Thornton SA, et al. Fifteen-year study of the changing epidemiology of methicillin-resistant Staphylococcus aureus. Am J Med. 2006;119:

22 Nosocomial Versus Community: Infection Types Infection Type CA-MRSA Number (%) (n = 131) HA-MRSA Number (%) (n = 937) P Value Skin/soft tissue 98 (75) 343 (37) <.001 Otitis media/externa 9 (7) 11 (1) <.001 Respiratory tract 8 (6) 205 (22) <.001 Bloodstream 5 (4) 83 (9) <.07 Urinary tract 1 (1) 185 (20) <.001 Others a 10 (8) 110 (12).21 CA-MRSA = Community-associated methicillin resistant Staphylococcus aureus isolates; HA-MRSA = Healthcare-associated methicillin resistant Staphylococcus aureus isolates. a Others included bone, peritoneal fluid, joint, surgical specimen, and postoperative wound. Source: Naimi TS, et al. JAMA. 2003;290: Naimi et al conducted a prospective cohort study of patients with MRSA infection identified at 12 Minnesota laboratory facilities throughout the year CA-MRSA cases were characterized with regard to their epidemiologic and microbiologic characteristics and were compared with HA-MRSA cases. The above table represents CA- and HA-MRSA cases, by infection type. If patients had > 1 type of infection, only 1 was selected for inclusion in this table. The hierarchy for selecting the type of infection for patients with multiple sources was: bacteremia, bone, pleural fluid, peritoneal fluid, joint, surgical specimen, postoperative wound, eye, ear, sputum, urine, and skin. The P value refers to the statistical probability that the type of infection among communityassociated cases differed from the percentage among healthcare-associated cases (P =.05) Among healthcare-associated isolates, some respiratory tract isolates were obtained from endotracheal tubes, and some urinary tract isolates were obtained from Foley catheters. The distribution of clinical infections differed between CA- and HA-MRSA cases. Compared with healthcare-associated cases, community-associated case infections were more likely to involve skin and soft tissue (OR [odds ratio], 4.25; 95% CI [confidence interval], ) and less likely to be respiratory tract infections (OR, 0.22; 95% CI, ) or urinary tract infections (OR, 0.04; 95% CI, ; P<.001 for all comparisons). Of the 131 community-associated cases, 24% (31) were hospitalized because of their MRSA infection and 5% (7) required intensive care treatment. Naimi TS, LeDell KH, Como-Sabetti K, et al. Comparison of community- and health-care associated methicillin-resistant Staphylococcus aureus infection. JAMA. 2003;290:

23 MRSA in the Emergency Department 97% of MRSA isolates were USA300 (community) 57% received antibiotics inappropriate for MRSA Susceptibilities of MRSA isolates Rifampin and TMP-SMX 100% Clindamycin 95% Fluoroquinolones 60% Erythromycin 6% Risk factors for MRSA infection in ED Close contact with similarly infected person OR 3.8 History of MRSA infection OR 3.4 Spider bite OR 3.0 Antibiotic use in the past month OR 2.4 Non-Hispanic black OR 1.9 TMP-SMX = trimethoprim-sulfamethoxazole; ED = emergency department; OR = odds ratio. Source: Moran GJ, et al. N Engl J Med. 2006;355: Moran et al studied temporal trends in the prevalence of MRSA among emergency department patients. Cultures were obtained from adult patients with acute, purulent skin and soft-tissue infections who presented to 11 university-affiliated emergency departments during the month of August Available S aureus isolates were characterized by antimicrobial-susceptibility testing, pulsed-field gel electrophoresis, and detection of toxin genes. Typing of the staphylococcal cassette chromosome mec (SCCmec) was also performed on MRSA isolates. S aureus was isolated from 320 of 422 patients with skin and soft-tissue infections (76%). The prevalence of MRSA was 59% overall and ranged from 15% to 74%. Pulsed-field type USA300 isolates accounted for 97% of MRSA isolates, of which 74% were a single strain (USA ). SCCmec type IV and the Panton-Valentine leukocidin toxin gene were detected in 98% of MRSA isolates. Among the MRSA isolates, 95% were susceptible to clindamycin, 6% to erythromycin, 60% to fluoroquinolones, 100% to rifampin and trimethoprim-sulfamethoxazole, and 92% to tetracycline. Of concern, 57% of patients received inappropriate antibiotic treatment for the index MRSA infection. Factors associated with the isolation of MRSA compared with the isolation of any other bacteria included the following: antibiotic use in the month before enrollment, the presence of an abscess or a lesion attributed to a spider bite at enrollment, history of MRSA infection, and a recent history of close contact with someone with a similar skin infection. Moran GJ, Krishnadasan A, Gorwitz RJ, et al, and the EMERGEncy ID Net Study Group. Methicillin-resistant S. aureus infections among patients in the emergency department. NEJM. 2006;355:

24 Antibiotic Use and MRSA Hospital Fluoroquinolone Use Associated With MRSA Development Resistant Isolates (%) r =.77, P = r =.35, P = Hospital Fluoroquinolone Use, DDD per 1000 Patient-days Resistant Isolates (%) Community Fluoroquinolone Use, DDD per 1000 Inhabitant-days DDD = defined daily doses. Source: MacDougall C, et al. Clin Infect Dis. 2005;41: Institutional as well as individual antibiotic use have been reported to correlate with the risk for MRSA colonization and infection. Multiple analyses reveal that in hospitalized patients, prior antibiotic exposure (of almost any kind) is strongly linked to subsequent infection with MRSA. The relationship between use of fluoroquinolones and the percentage of resistant S aureus using isolates from 17 US hospitals and their surrounding communities in the year 2000 were evaluated by MacDougall et al. The figure on the left depicts resistance rates in study pathogens at individual hospitals, expressed as the percentage of isolates with drug resistance, versus hospital fluoroquinolone use, expressed in DDD per 1000 patient-days. There was a significant relationship between total fluoroquinolone use in hospitals and the percentage of S aureus isolates that were MRSA (r = 0.77; P =.0003). There was also a statistically significant relationship between the percentage of S aureus isolates that were MRSA and levofloxacin use (r =.74; P =.0007). Associations between the percentage of S aureus isolates that were MRSA and ciprofloxacin use were not statistically significant (r =.24; P =.32). The figure on the right depicts resistance rates in study pathogens at individual hospitals, expressed as the percentage of isolates with drug resistance versus community fluoroquinolone use, expressed in DDD per 1000 inhabitant-days. Total fluoroquinolone use within a 16-km radius was not associated with the prevalence of MRSA in the hospital (r =.35; P =.18). Conversely, community use of levofloxacin was associated with the percentage of MRSA in the hospital (r =.72; P =.0011). Ciprofloxacin use did not correlate to the prevalence of MRSA in the hospital, even though ciprofloxacin was used in the community more often than was levofloxacin. MacDougall C, et al. Hospital and community fluoroquinolone use and resistance in Staphylococcus aureus and Escherichia coli in 17 US hospitals. Clin Infect Dis. 2005;41: