STAPHYLOCOCCI: KEY AST CHALLENGES
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1 Romney Humphries, PhD D(ABMM) Section Chief, UCLA Clinical Microbiology Los Angeles CA STAPHYLOCOCCI: KEY AST CHALLENGES
2 THE CHALLENGES detection of penicillin resistance detection of MRSA Staphylococcus pseudintermedius vancomycin and S. aureus including VISA inducible clindamycin resistance in staphylococci
3 Penicillin and Staphylococcus spp. - -lactamase testing
4 SPECIMEN: BONE DIAGNOSIS: OSTEOMYELITIS STAPHYLOCOCCUS AUREUS MIC ( g/ml) clindamycin 0.5 S erythromycin 0.5 S oxacillin 0.25 S penicillin 0.06 S?? vancomycin 0.5 S Should we do anything about penicillin?
5 STAPHYLOCOCCUS SPP. PENICILLIN > 90% of staphylococci are penicillin R Penicillin rarely considered for treatment of staphylococcal infections but might be considered for infections requiring lengthy therapy (e.g., endocarditis, osteomyelitis) IF penicillin S Some Staphylococcus spp. that test S by MIC or disk diffusion may possess a β- lactamase and may fail penicillin therapy Agent MIC (µg/ml) Zone (mm) S I R S I R Penicillin
6 S. aureus S. aureus and CoNS (including S. lugdunensis) CLSI M100-S25. Table 3D.
7 STAPHYLOCOCCUS AUREUS DISK ZONE EDGE TEST (10 U PENICILLIN DISK AND STANDARD DISK DIFFUSION METHOD NOT VALIDATED FOR MIC PURIT Y PLATES) Fuzzy beach = β-lactamase negative Penicillin - S Sharp cliff = β-lactamase positive Penicillin - R S. aureus QC: Neg - ATCC Pos - ATCC (supplemental QC) CLSI M100-S25. Table 3D.
8 STAPHYLOCOCCUS SPP. PENICILLIN STRATEGY Organism Action S. aureus Report PEN if R; Suppress PEN if S If PEN needed, perform nitrocefin β-lactamase test If nitrocefin β-lactamase test negative, perform PEN disk zone edge test (next day) CoNS Report PEN if R; Suppress PEN if S (except S. If PEN needed, perform nitrocefin β-lactamase test lugdunensis) If nitrocefin β-lactamase test negative, perform induced nitrocefin β-lactamase test All Continue to test subsequent isolates from patient
9 SPECIMEN: BONE DIAGNOSIS: OSTEOMYELITIS STAPHYLOCOCCUS AUREUS clindamycin MIC ( g/ml) 0.5 S erythromycin 0.5 S oxacillin 0.25 S vancomycin 0.5 S Contact laboratory if penicillin results needed
10 WHAT ABOUT S. LUGDUNENSIS? Usually very susceptible including penicillin-s UCLA 40-50% PCN R UCLA 3-21% oxacillin R Recommended Rx: oxacillin / nafcillin or penicillin (if β-lactamase negative) Sanford Guide, 45th ed PCN Oxa Do not suppress penicillin results!
11 DETECTION OF BETA-LACTAMASE IN S. LUGDUNENSIS 100 isolates of S. lugdunensis (n=36 blaz + by PCR) Accuracy Sensitivity Specificity Penicillin MIC 100% ( ) 100% ( ) 100% ( ) Induced-nitrocefin 100% ( ) 100% ( ) 100% ( ) Penicillin Disk Diffusion 98.91% ( ) 100% ( ) 98.18% ( ) Penicillin Zone Edge 45.65% ( ) 100% ( ) 9.09% ( ) I. McHardy, Manuscript in preparation Many false-positive results!
12 S. lugdunensis and Penicillin S. lugdunensis Report PEN if R Perform nitrocefin β-lactamase test If nitrocefin β-lactamase test negative, perform induced nitrocefin β-lactamase test Up to 50% are PEN S CLSI M100-S25. Table 3D.
13 MRSA methicillin-resistant S. aureus - Oxacillin vs. cefoxitin tests - BORSA
14 ANTIMICROBIAL AGENT OPTIONS FOR MSSA INFECTIONS Betalactams Predictable activity dicloxacillin / nafcillin cephalexin / cefazolin amoxicillin-clav ampicillin-sulbactam carbapenem vancomycin dalbavacin, oritavancin, televancin daptomycin linezolid ceftaroline (β-lactam) tigecycline Need AST clindamycin trimethoprim-sulfa macrolide (e.g. clarithromycin) tetracycline fluoroquinolone (e.g. ciprofloxacin) (gentamicin) (rifampin)
15 ANTIMICROBIAL AGENT OPTIONS FOR MSSA INFECTIONS Betalactams Predictable activity dicloxacillin / nafcillin cephalexin / cefazolin amoxicillin-clav ampicillin-sulbactam carbapenem vancomycin dalbavacin, oritavancin, televancin daptomycin linezolid ceftaroline (β-lactam) tigecycline Need AST clindamycin trimethoprim-sulfa macrolide (e.g. clarithromycin) tetracycline fluoroquinolone (e.g. ciprofloxacin) (gentamicin) (rifampin)
16 TESTS FOR meca-mediated OXACILLIN RESISTANCE IN S. AUREUS Phenotypic tests - Disk diffusion or MIC Cefoxitin as a surrogate Report results for OXACILLIN, not cefoxitin Oxacillin MIC (+ 2% NaCl) Oxacillin-salt agar screen for S. aureus Detection of gene or gene product meca PBP2a Why use cefoxitin to detect MRSA? Sensitivity Specificity Oxacillin 86 % 74 % Cefoxitin 100 % 100 % Swenson et al JCM :
17 Heteroresistant MRSA Cefoxitin (R) 10 mm zone Cefoxitin detects heteroresistant meca-mediated MRSA better than oxacillin Colonies in zone (heteroresistant) Staphylococcus aureus / S. lugdunensis Agent MIC (µg/ml) Zone (mm) S I R S I R Cefoxitin 4* - 8** 22* - 21** Oxacillin 2-4 NA * Report as oxacillin susceptible ** Report as oxacillin resistant
18 DEFINITION OF MRSA (2) MRSA are those strains of S. aureus that express meca or another mechanism of methicillin resistance, such as changes in affinity of penicillin binding proteins for oxacillin (modified S. aureus [MOD-SA] strains) MRSA = S. aureus with meca and/or oxacillin MIC >2 µg/ml and/or cefoxitin R CLSI M100-S25. Table 2C.
19 OX S. AUREUS OR S. LUGDUNENSIS TESTING BOTH OX AND CX CX Resistance mechanism Relative Prevalence Report as OX: S S None Common S R R meca Common R S R meca (low level expression) Uncommon R* R S PBP changes or hyperproduction of β-lactamase (borderline MRSA) Rare R* *most commercial system software expertise to R (3) Isolates that test resistant by oxacillin minimal inhibitory concentration (MIC), cefoxitin MIC, or cefoxitin disk test should be reported as oxacillin resistant. CLSI M100-S25. Table 2C.
20 WHAT ABOUT BORDERLINE MRSA? Oxacillin MIC slightly above R breakpoint Cefoxitin S meca negative Mechanisms: Modifications in penicillin-binding proteins (PBPs) 1,2,4 (MOD-SA) Hyperproduction of blaz-encoded penicillinase Methicillinase Infrequently encountered Limited clinical information in literature re: therapy Chambers Clin Microbiol Rev. 10:781. Skinner et al J Clin Microbiol. 47:859. Croes et al Clin Microbiol Infection. 16:979. Maalej et al J Clin Microbiol. 50:3345.
21 WHY IS IT IMPORTANT TO CORRECTLY AND QUICKLY IDENTIFY MRSA AND MSSA? MRSA infections - β-lactams (except ceftaroline) ineffective MSSA infections β-lactams better than vancomycin For bacteremia Vancomycin associated with 2 3 times the risk of morbidity and mortality vs β-lactam (e.g., oxacillin and nafcillin, cefazolin) Switching from vancomycin to β-lactam inferior to starting with β-lactam McConeghy et al Clin Infect Dis. 57:1760. If you focus on ONE thing focus on getting this right!
22 WHAT S MECC? mecc 1 Human and bovine MRSA were reported in 2011 that carry a new meca homologue, mecc Human cases retrospectively identified in 1975, 1992 but most since Harbors ~68-70% genetic similarity to meca Mostly found in veterinary isolates, all in Europe, but some human cases to date in Denmark, Germany, France, UK and Spain Currently only detected by cefoxitin resistance - Typically have low oxacillin MICs - meca PCR, PBP2a tests do not work for these 1. Additional reading for mecc: Laurent et al 2012 EID 18:1465; Garcia-Alvarez et al 2011 Lancet Infect Dis 11: Petersen et al CMI :E16 3. Cartwright et al J Clin Microbiol. 51:
23 WHAT ARE CLSI RULES FOR DETECTING MRCONS? Species Rule S. lugdunensis Use oxacillin and cefoxitin breakpoints as for S. aureus S. saprophyticus Don t test; add comment to report re: appropriate therapy for S. saprophyticus UTI S. epidermidis Use CoNS oxacillin MIC and/or cefoxitin DD breakpoints - work well Other CoNS Use caution for oxacillin MICs µg/ml; may or may not have meca! CoNS oxacillin-r more heterogeneous than S. aureus; lower breakpoint
24 REPORTING STRATEGY OXACILLIN MIC RESULTS FOR CONS* * For testing non-s. epidermidis isolates from sterile sites where CoNS is causing an infection Oxacillin MIC (µg/ml) S I R 0.25 Report Oxacillin S Negative Oxacillin MIC (µg/ml) Do meca or PBP2a or Cefoxitin disk Positive 4 Report Oxacillin R Report Oxacillin S Report Oxacillin R
25 Staphylococcus pseudintermedius 25
26 STAPHYLOCOCCUS PSEUDINTERMEDIUS Part of Staphylococcus intermedius Group (SIG) Other species in SIG Staphylococcus intermedius Staphylococcus delphini Colonizes nares and anal mucosa of healthy dogs and cats Most common cause of canine pyoderma and sometimes causes urinary tract and joint infections in dogs and cats 26
27 STAPHYLOCOCCUS PSEUDINTERMEDIUS Increasingly isolated from human specimens: wounds (bites), sinuses, and blood, etc. Identification MALDI TOF Tube coagulase positive; clumping factor (slide coagulase) negative Masquerades as S. aureus S. pseudintermedius can be misdiagnosed as S. aureus in humans with dog bite wounds Börjesson et al., Eur J Clin Microbiol Infect Dis. 34: Commercial phenotypic ID tests often call this S. intermedius Some meca positive Not detected with CLSI S. aureus cefoxitin or oxacillin breakpoints 27
28 S. PSEUDINTERMEDIUS VS. S. AUREUS S. pseudintermedius BAP 24 hr 35 C (ambient air) Test S. aureus S. pseudintermedius Hemolysis + + Tube coag + + Slide coag + - PYR - + VP + V D- Trehalose % strains negative + 90% strains positive V, variable; 11-89% strains positive Courtesy of Lars Westblade Modified from Manual of Clinical Microbiology, 11 th ed. 28
29 % Oxacillin Resistant METHICILLIN-RESISTANT S. PSEUDINTERMEDIUS ISOLATES FROM DOGS N=13 6 N=17 6 N=24 4 N=25 4 N=23 7 N=30 4 Year N=40 4 N=47 9 N=39 3 N=45 8 N=56 0 N=54 3 Courtesy of David Bemis University of Tennessee 29
30 5 labs Testing STAPHYLOCOCCUS PSEUDINTERMEDIUS CLSI MULTICENTER STUDY meca PCR; mecc PCR SCCmec typing and reppcr CLSI disk diffusion (oxacillin/cefoxitin) CLSI broth microdilution MIC (oxacillin/cefoxitin) Automated systems: BD Phoenix; MicroScan; Vitek2 Alere PBP2a (induced/uninduced) Isolates (n=111): 45 human 69 nonhuman 37 meca + 74 meca Wu et al J Clin Micro. Epub Nov
31 Number of Isolates S. pseudintermedius Oxacillin MIC Distribution (n=111) S. pseudintermedius Breakpoint S. aureus Breakpoint meca positive meca negative >16 MIC (µg/ml) 31
32 no zone Number of Isolates S. pseudintermedius Oxacillin Zone Distribution (n=111) S. pseudintermedius Breakpoint meca positive meca negative Zone Size (mm) 32
33 Number of Isolates S. pseudintermedius Cefoxitin Zone Distribution (n=111) Don t use cefoxitin! CoNS Breakpoint S. aureus Breakpoint meca positive meca negative Some false S and some false R with any breakpoint! Zone (mm) 33
34 S. pseudintermedius Performance Compared to meca Result Phenotypic Test CA VME ME Oxacillin MIC 99.1% 0 (0%) 1 (1.3%) Oxacillin Disk 99.1% 0 (0%) 1 (1.3%) PBP2a uninduced 96.5% 4 (10.8%) 0 (0.0%) PBP2a uninduced 100% 0 (0.0%) 0 (0.0%) 111 isolates; 37 meca +; 74 meca CA, category agreement (S, R agreement) VME, very major errors (false S ) ME, major errors (false R ) 34
35 Staphylococcus pseudintermedius M100S 26 th ed. p
36 AUTOMATED AST OXACILLIN VS. S. PSEUDINTERMEDIUS System BD Phoenix S. aureus Breakpoints CoNS Breakpoints CA VME ME CA VME ME 90.4% Vitek2 93.0% 11 (29.7%) 8 (21.6%) % % 4 (10.8%) 1 (2.7%) MicroScan 95.7% 5(13.5%) % 0 1 (1.3%) 1 (1.3%) 1 (1.3%) No commercial system is FDA cleared for AST of Staphylococcus pseudintermedius 36
37 AND, WAIT Forthcoming for 2018: S. schleiferi breakpoints updated to be the same as S. pseudintermedius! Other staphylococci are to be evaluated
38 Vancomycin and S. aureus See.. Howden et al Clin Microbiol Rev. 23:99.
39 SPECIMEN: BLOOD DIAGNOSIS: BACTEREMIA STAPHYLOCOCCUS AUREUS oxacillin penicillin vancomycin vancomycin MIC ( g/ml) >16 R R 1 S (automated) 1.5 S (Etest) What about vancomycin MIC?
40 S. AUREUS - VANCOMYCIN MIC INTERPRETIVE CRITERIA ( G/ML) Susceptible Intermediat e Resistant VSSA 1 vs 2 µg/ml VISA VRSA Vancomycin released 1950s; first resistance (VISA) 1990s! CLSI M100-S25. Table 2C.
41 CONTEMPORARY THOUGHTS ON TREATING MRSA BACTEREMIA WITH VANCOMYCIN Based on PK/PD, clinical outcome may vary if vancomycin treatment for isolates with susceptible MICs of 1 vs. 2 µg/ml Maintain trough serum concentration of mg/l Liu et al Clin Infect Dis. 52:285. Some say if vancomycin S MIC (µg/ml): <1.5 use vancomycin use alternative Recent study: No differences if MIC <1.5 or 1.5 µg/ml in risk of death; can t exclude increased mortality risk Kalil et al JAMA. 312:1552. Reproducibility of MIC = +/- 1 two-fold dilution!!!!
42 IDSA GUIDELINES SUGGEST USING VANCOMYCIN MICS TO GUIDE THERAPY AS FOLLOWS.. MIC (µg/ml) 2 (Susceptible) >2 (e.g., VISA or VRSA) IDSA Guidelines Recommendation The patient s clinical response should determine the continued use of vancomycin, independent of the MIC An alternative to vancomycin should be used Liu et al Clin Infect Dis. 52:1.
43 Vancomycin MIC (N=101 MRSA) Etest MICs > Reference Broth Microdilution and Agar Dilution MICs Prakash et al Antimicrob Agents Chemother. 52:4528. See also Hsu et al Intl J Antimicrob Agents. 32:378. Sader et al Antimicrob Agents Chemother. 53:3162.
44 48 hour growth on blood agar plate MRSA VISA VSSA VISA VSSA Marlowe, et al J Clin Microbiol. 39:2637. Howden et al CMR. 23:99.
45 CHARACTERISTICS SOMETIMES OBSERVED FOR VISA Colony morphology may be atypical for S. aureus (some pinpoint) Delayed growth in broth (e.g., blood culture) Weak / delayed coagulase reaction After several subcultures: Colony morphology becomes typical for S. aureus Vancomycin MIC decreases and isolate becomes vancomycin susceptible MICs for daptomycin increase MICs for -lactams decrease
46 PERSISTENT MRSA BACTEREMIA PATIENT #1 DAPTOMYCIN MIC STORY Patient receives 49 days daptomycin for MRSA Presents to ED with sepsis: MRSA in blood MRSA only tested every 5 days for AST from blood Daptomycin NS MRSA recognized on hospital day 8 Daptomycin NS isolate was found in blood bottle collected in ED (but not tested because it took longer to grow) Giltner et al J Clin Microbiol. 52:357.
47 PERSISTENT MRSA BACTEREMIA PATIENT #1 DAPTOMYCIN MIC STORY Day of Hospitalization on Which Blood Culture Drawn Routine AST done on S. aureus at time of isolation? Why? 1 Yes First S. aureus isolated from this hospitalization 0 No Recovered after MRSA from Day 1 was isolated 8 Yes More than 5 days elapsed since previous AST done No Less than 5 days elapsed since previous AST done 15 Yes More than 5 days elapsed since previous AST done Daptomycin MIC (µg/ml) 0.5 S 4 NS* 2 NS 0.5 S, 1S 2 NS* 8 NS *results obtained from retrospective testing Giltner et al J Clin Microbiol. 52:357.
48 WHAT SHOULD WE DO ABOUT TESTING VANCOMYCIN (& DAPTOMYCIN) FOR MRSA? Use MIC method verified in your laboratory; report according to your SOP Will be some differences by method Avoid repeated subcultures If use multiple methods and get conflicting results must review patient response to vancomycin! Test all isolates from blood, especially if they have atypical morphotype or take a long time to grow If MIC 3 µg/ml by any method, report VISA Most likely in patients previously treated with vancomycin Physicians should be informed about issues with vancomycin MIC testing and need to continually monitor patient response to vancomycin therapy Some refs Howden et al Clin Microbiol Rev. 23:99. Liu et al Clin Infect Dis. 52:1. Van Hal et al Clin Infect Dis. 54:755. Kalil et al JAMA 312:1552.
49 CAN CONS SHOW DECREASED SUSCEPTIBILITY TO VANCOMYCIN? May show increase in MICs following exposure to vancomycin Most common in S. haemolyticus and S. epidermidis Some suggest use alternative to vancomycin for S. haemolyticus Falcone et al Diagn Microbiol Infect Dis. 57:325. Method - Organism MIC - S. aureus MIC (µg/ml) S I R 2 * MIC - CoNS CLSI M100-S25. Table 2C.
50 Drug OTHER ANTIMICROBIAL AGENTS FOR MRSA Route PO IM IV Comments ceftaroline x FDA indications for complicated skin infxns, communityacquired pneumonia β-lactam with activity against MRSA FDA indications for complicated skin infxns, bacteremia daptomycin x Disk diffusion does not work; must do MIC S breakpoint only NOT for respiratory infxns (lung surfactant inhibits drug); do not report on respiratory specimens linezolid x x FDA indications for nosocomial pneumonia, complicated skin infxns, uncomplicated skin infxns (MSSA); communityacquired pneumonia (MSSA) For disk diffusion, examine with transmitted light
51 NEWER ANTIMICROBIAL AGENTS MRSA Drug Route PO IM IV Comments dalbavancin oritavancin telavancin x x x FDA indications for complicated skin infxns; S only breakpoint Once/week dose FDA indications for complicated skin infxns; S only breakpoint. One dose FDA indications for complicated skin infxns; community-acquired / hospital-associated pneumonia; S only breakpoint Once/day dose tedizolid x x FDA indications for complicated skin infxns; S/R breakpoint for S. aureus Once/day dose
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