STAPHYLOCOCCI:

KEY AST CHALLENGES

April Abbott
Deaconess Health System
Indiana University School of Medicine
Evansville, IN

Special thanks to Romney Humphries

 THE CHALLENGES

 Detection of penicillin resistance

 Detection of MRSA

 Staphylococcus pseudintermedius

 Vancomycin and S. aureus including VISA

 Inducible clindamycin resistance in staphylococci

Penicillin and Staphylococcus spp.
- -lactamase testing
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?

 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
MIC (µg/ml) Zone (mm)
Agent
S I R S I R
Penicillin ≤0.12 - ≥0.25 ≥29 - ≤28
 S. aureus and CoNS
S. aureus
(including S. lugdunensis)

CLSI M100-S25. Table 3D.

 INDUCED ß-LACTAMASE TEST

Oxacillin
(inducer)
- Sub isolate to agar (e.g., BAP, MHA)
- Drop ß-lactam disk (e.g., oxacillin,
cefoxitin)
- Incubate overnight
- Test cells from periphery of zone
- If β-lactamase positive (with or without
induction), report penicillin R

Pos Neg
 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 S. aureus QC:
Penicillin - S Neg - ATCC 25923
Pos - ATCC 29213
(supplemental QC)

Sharp “cliff” =
β-lactamase positive
Penicillin - R

CLSI M100-S25. Table 3D.

 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

SPECIMEN: BONE
DIAGNOSIS: OSTEOMYELITIS
STAPHYLOCOCCUS AUREUS
MIC (g/ml)
clindamycin ≤0.5 S

erythromycin ≤0.5 S
oxacillin ≤0.25 S
vancomycin ≤0.5 S
“Contact laboratory if penicillin results needed”
 WHAT ABOUT S. LUGDUNENSIS?
PCN
Usually very susceptible
…including penicillin-S
Recommended Rx:
oxacillin / nafcillin …or
penicillin (if β-lactamase
negative)
Sanford Guide, 45th ed. 2015.

Oxa

Do not suppress penicillin results!

 DETECTION OF BETA -LACTAMASE IN S.
LUGDUNENSIS

100 isolates of S. lugdunensis (n=36 blaZ + by PCR)

Penicillin Disk Penicillin Zone
Penicillin MIC Induced-nitrocefin Diffusion Edge
100% 100% 98.91% 45.65%
Accuracy
(96.07 - 100) (96.07 - 100) (94.09 - 99.97) (35.22 - 56.37)
100% 100% 100% 100%
Sensitivity
(90.51 - 100) (90.51 - 100) (90.51 - 100) (90.51 - 100)
100% 100% 98.18% 9.09%
Specificity
(93.51 - 100) (93.51 - 100) (90.28 - 99.95) (3.02 - 19.95)

Many false-positive
results!
McHardy IH, J Clin Microbiol.2017 Feb;55(2):585-595
 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

Do not use disk zone edge test for S. lugdunensis

CLSI M100-S25. Table 3D.

MRSA – methicillin-resistant S. aureus
- Oxacillin vs. cefoxitin tests
- BORSA
 ANTIMICROBIAL AGENT OPTIONS FOR
MSSA INFECTIONS
Predictable activity Need AST
dicloxacillin / nafcillin clindamycin
cephalexin / cefazolin trimethoprim-sulfa
Beta-
amoxicillin-clav macrolide
lactams ampicillin-sulbactam (e.g. clarithromycin)
carbapenem
tetracycline
vancomycin
dalbavacin, oritavancin, fluoroquinolone (e.g.
televancin ciprofloxacin)
daptomycin (gentamicin)
linezolid (rifampin)
ceftaroline (β-lactam)
tigecycline
 ANTIMICROBIAL AGENT OPTIONS FOR
MRSA INFECTIONS
Predictable activity Need AST
dicloxacillin / nafcillin clindamycin
cephalexin / cefazolin trimethoprim-sulfa
Beta-
amoxicillin-clav macrolide
lactams ampicillin-sulbactam (e.g. clarithromycin)
carbapenem
tetracycline
vancomycin
dalbavacin, oritavancin, fluoroquinolone (e.g.
televancin ciprofloxacin)
daptomycin (gentamicin)
linezolid (rifampin)
ceftaroline (β-lactam)
tigecycline
 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)
Detection of gene or gene product
 mecA Why use cefoxitin to detect MRSA?
 PBP2a Sensitivity Specificity

Oxacillin 86 % 74 %

Cefoxitin 100 % 100 %

Swenson et al JCM 2005 43:3818-23

 Heteroresistant MRSA

Cefoxitin detects
“heteroresistant”
mecA-mediated MRSA
Cefoxitin (R)
10 mm zone better than oxacillin

Colonies in zone Staphylococcus aureus / S. lugdunensis

(heteroresistant)
MIC (µg/ml) Zone (mm)
Agent
S I R S I R
Cefoxitin ≤4* - ≥8** ≥22* - ≤21**
Oxacillin ≤2 - ≥4 NA

* Report as oxacillin susceptible

** Report as oxacillin resistant
 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.

 S. AUREUS OR S. LUGDUNENSIS
TESTING BOTH OX AND CX
Resistance Relative Report
OX CX
mechanism Prevalence as OX:
S S None Common S
R R mecA Common R

S R mecA (low level expression) Uncommon R*

PBP changes or hyper-
R S production of β-lactamase Rare R*
(borderline MRSA)

*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.
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. 2013. Clin Infect Dis.
57:1760.
If you focus on ONE thing – focus on getting this right!
 WHAT’S MECC?

 mecC
 Human and bovine MRSA were reported in 2011 that carry
a new mecA homologue, mecC
 Mostly found in veterinary isolates, all in Europe, but some
human cases to date in Denmark, Germany, France, UK and
Spain
 Detected by cefoxitin resistance but have low oxacillin MICs
 Would be negative for mecA and PBP2a tests

1. Additional reading for mecC: Laurent et al 2012 EID 18:1465; Garcia-Alvarez et

al 2011 Lancet Infect Dis 11:595
2. Petersen et al CMI 2013. 19:E16
3. Cartwright et al. 2014. J Clin Microbiol. 51:2732.
22
WHAT ARE CLSI RULES FOR
DETECTING MR CONS?
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 0.5-2.0 µg/ml;

cefoxitin DD testing is better!

CoNS – oxacillin-R more heterogeneous than S. aureus;

lower breakpoint
 REPORTING STRATEGY
OXACILLIN MIC RESULTS FOR CONS*
Oxacillin MIC
*”For testing non-S. (µg/ml)
epidermidis isolates
from sterile sites ≤0.25 0.5-2.0 ≥4
where CoNS is
causing an infection”
Report Do mecA or Report
Oxacillin “S” PBP2a or Oxacillin “R”
Cefoxitin disk
Oxacillin
MIC (µg/ml)
Negative Positive
S I R
≤0.2 - ≥0.5 Report Report
5 Oxacillin “S” Oxacillin “R”
 Staphylococcus
pseudintermedius

25
 STAPHYLOCOCCUS
PSEUDINTERMEDIUS
♦ Part of Staphylococcus intermedius Group
(SIG)
♦ 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
 STAPHYLOCOCCUS
PSEUDINTERMEDIUS

 Increasingly isolated from human specimens:

wounds (bites), sinuses, and blood, etc.
 Identification
 MALDI TOF
 Tube coagulase positive; clumping factor (slide coagulase)
negative
 Commercial phenotypic ID tests often call this S. intermedius
 Some mecA positive
 Not detected with CLSI S. aureus cefoxitin or oxacillin
breakpoints

27
 S. PSEUDINTERMEDIUS VS.
S. AUREUS
S. S.
Test
aureus pseudintermedius
Hemolysis + +
Tube coag + +
Slide coag + -
PYR - +
VP + V
D-
+ +
Trehalose
S. pseudintermedius
- ≥90% strains negative
BAP (BD) @ 24 hr 35°C
(ambient air) + ≥90% strains positive
V, variable; 11-89% strains positive
Courtesy of Lars Westblade
Modified from Manual of Clinical Microbiology, 11th ed.
28
 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 un-
96.5% 4 (10.8%) 0 (0.0%)
induced
PBP2a un-
100% 0 (0.0%) 0 (0.0%)
induced
111 isolates; 37 mecA +; 74 mecA –
CA, category agreement (S, R agreement)
VME, very major errors (false “S”)
ME, major errors (false “R”)
29
Staphylococcus pseudintermedius

M100S 26th ed. p. 77. 30

 AUTOMATED AST
OXACILLIN VS. S. PSEUDINTERMEDIUS

S. aureus Breakpoints CoNS Breakpoints

System
CA VME ME CA VME ME
BD 11 4 1
90.4% 0 95.7%
Phoenix (29.7%) (10.8%) (1.3%)
8 1 1
Vitek2 93.0% 0 98.3%
(21.6%) (2.7%) (1.3%)
1
MicroScan 95.7% 5(13.5%) 0 99.1% 0
(1.3%)

No commercial system is FDA cleared for AST

of Staphylococcus pseudintermedius

31
 Vancomycin and S. aureus

See…..
Howden et al. 2010. Clin Microbiol Rev. 23:99.
SPECIMEN: BLOOD
DIAGNOSIS: BACTEREMIA
STAPHYLOCOCCUS AUREUS
MIC (g/ml)
oxacillin >16 R
penicillin R
vancomycin 2 S (automated)

What about vancomycin MIC?

 S. AUREUS - VANCOMYCIN
MIC INTERPRETIVE CRITERIA (G/ML)

Susceptible Intermediate Resistant

2 4-8 16

VSSA VISA VRSA

1 vs 2 µg/ml
Vancomycin released 1950s;
first resistance (VISA) 1990s!

CLSI M100-S25. Table 2C.

IDSA GUIDELINES SUGGEST USING
VANCOMYCIN MICS TO GUIDE THERAPY
AS FOLLOWS…..

MIC (µg/ml) Recommendation

The patient’s clinical
≤2 response should determine
the continued use of
(Susceptible) vancomycin, independent of
the MIC
>2
An alternative to
(e.g., VISA or vancomycin should be used
VRSA)

IDSA Guidelines
Liu et al. 2011. Clin Infect Dis. 52:1.
Vancomycin MIC (N=101 MRSA)
Etest MICs > Reference Broth Microdilution and Agar Dilution MICs

Prakash et al. 2008. Antimicrob Agents Chemother. 52:4528.

See also…Hsu et al. 2008. Intl J Antimicrob Agents. 32:378.
Sader et al. 2009. Antimicrob Agents Chemother. 53:3162.
 48 hour growth on
MRSA VISA VSSA
blood agar plate

VISA

VSSA
Marlowe, et al. 2001. J Clin Microbiol. 39:2637. Howden et al. 2010. CMR. 23:99.
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
 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. 2014. J Clin Microbiol. 52:357.

 PERSISTENT MRSA BACTEREMIA
PATIENT #1 – DAPTOMYCIN MIC STORY
Day of Hospitalization Routine AST done Daptomycin
on Which Blood on S. aureus at Why? MIC
Culture Drawn time of isolation? (µg/ml)
1 Yes First S. aureus isolated 0.5 S
from this hospitalization
0 No Recovered after MRSA 4 NS*
from Day 1 was isolated
8 Yes More than 5 days elapsed 2 NS
since previous AST done
10-13 No Less than 5 days elapsed 0.5 S, 1S
since previous AST done 2 NS*
15 Yes More than 5 days elapsed 8 NS
since previous AST done

*results obtained from retrospective testing

Giltner et al. 2014. J Clin Microbiol. 52:357.
 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 as phenotype may
“disappear”
 If use multiple methods and get conflicting
results…must review patient response to
vancomycin!
 Test blood isolates frequently, 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
 Staphylococcus spp.
-Inducible clindamycin resistance
SPECIMEN: PUS (L BUTTOCK LESION)
DIAGNOSIS: LOCALIZED ABSCESS
MANY STAPHYLOCOCCUS AUREUS
clindamycin S ???
erythromycin R
oxacillin R
penicillin R
vancomycin S

What should we do about clindamycin

results?
 STAPHYLOCOCCUS SPP.
ERYTHROMYCIN / CLINDAMYCIN

Mechanism Determinant Erythro Clinda

Efflux msrA R S

Ribosome modification erm R S*

Ribosome modification erm R R

constitutive

msrA = macrolide streptogramin resistance

erm = erythromycin ribosome methylase
*requires induction to show resistance
 S. AUREUS
“D ZONE TEST”
D zone test is only for
staphylococci that are:
1
Erythromycin “R” and
Clindamycin “S” or “I”

Photo 1: inducible clindamycin R

Photos 2 and 3: erythromycin R and 2
clindamycin R (D zone test NOT
needed)

3
SPECIMEN: PUS (L BUTTOCK LESION)
DIAGNOSIS: LOCALIZED ABSCESS
MANY STAPHYLOCOCCUS AUREUS
Final Report with
clindamycin R Optional Comment

erythromycin R
oxacillin R
penicillin R
vancomycin S

“This S. aureus is presumed to be clindamycin resistant

based on detection of inducible clindamycin resistance.
Clindamycin may still be effective in some patients.”
 BREAK TIME

Questions?