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ENTEROCOCCUS AND
STREPTOCOCCUS
April Abbott, PhD, D(ABMM)
Director, Microbiology
Deaconess Health System
Evansville, IN
ENTEROCOCCI
Discuss basic antimicrobial susceptibility
principles and resistance mechanisms for
Enterococcus
Describe issues surrounding AST of
enterococci
Ampicillin and penicillin resistance
Aminoglycosides and synergy testing
Vancomycin resistant enterococci
Discuss antimicrobial susceptibility testing
and reporting strategies
OBJECTIVES
ampicillin 2 S
vancomycin ≤0.5 S
gentamicin synergy S
streptomycin synergy R
SPECIMEN: BLOOD
DIAGNOSIS: PERITONITIS
MIC (µg/ml)
Is isolate imipenem “S”? What about
penicillin? Is β-lactamase testing needed?
Enterococcus faecalis
“……(5) The results of ampicillin susceptibility
tests should be used to predict the activity of
amoxicillin…amoxicillin -clavulanate,
ampicillin-sulbactam, piperacillin, and
piperacillin-tazobactam. Ampicillin
susceptibilty can be used to predict imipenem
susceptibility, providing the species is
confirmed to be E. faecalis.”
Cannot extrapolate to other carbapenems
PREDICTING BETA-LACTAM
SUSCEPTIBILITY
Altered penicillin binding proteins Mostly E. faecium
Rare reports in E. faecalis
Ono et al. 2005. Antimicrob Agents Chemother. 49:2954
Rare isolates -lactamase producers Most reports from 1980s and in E. faecalis
Murray, B. E. 1992. Antimicrob Agents Chemother. 36:2355.
Rice, L. B., et al. 1995 . Dev Biol Stand. 85:107.
Rare recent reports
1 from India in 2005; Agarwal et al. Jpn J Infect Dis. 62:158.
Enterococcal penicillinase gene same as that in staphylococcus; genes expressed differently
Usually occurs in strains that have high-level gentamicin resistance
ENTEROCOCCUS SPP. AMPICILLIN AND PENICILLIN RESISTANCE
MECHANISMS
“……(8) Penicillin or ampicillin resistance among enterococci due to -lactamase production has been reported very rarely. Penicillin or ampicillin resistance due to -lactamase production is not reliably detected with routine disk or dilution methods, but is detected using a direct, nitrocefin-based -lactamase test. Because of the rarity of -lactamase–positive enterococci, this test need not be performed routinely, but can be used in selected cases. A positive -lactamase test predicts resistance to penicillin, as well as amino- and ureidopenicillins .”
ENTEROCOCCUS SPP. Β-LACTAMASE TESTING
CLSI M100-S22. Table 2D.
SPECIMEN: BLOOD
DIAGNOSIS: ENDOCARDITIS
Should we add any comment to the report?
ampicillin 2 S
vancomycin ≤0.5 S
gent synergy S
strep synergy R
MIC (µg/ml)
Enterococcus faecalis
ampicillin 2 S
vancomycin ≤0.5 S
gent synergy S
strep synergy R
SPECIMEN: BLOOD
DIAGNOSIS: ENDOCARDITIS
“Serious enterococcal infections need combination therapy with
ampicillin or vancomycin plus an aminoglycoside. Synergy
occurs only when both drugs in the combination are
susceptible.”
Final Report with
Optional Comment
MIC (µg/ml)
Enterococcus faecalis
“Rx: Combination therapy of ampicillin,
penicillin, or vancomycin (for susceptible
strains), plus an aminoglycoside, is
usually indicated for serious enterococcal
infections, such as endocarditis…”
“enterococci with low levels of penicillin
or ampicillin resistance may be
susceptible to synergistic killing…”
ENTEROCOCCUS SPP.
CLSI “RX” COMMENT
CLSI M100-S26. Table 2D.
CLSI M100-S26. Table 3I
Disk diffusion 120 µg gentamicin; 300 µg streptomycin
10 mm = NO HLAR
6 mm = HLAR
7-9 mm = inconclusive; perform MIC
MIC screen gentamicin: 500 µg/ml
streptomycin: 1000 µg/ml (broth); 2000 µg/ml (agar)
no growth = NO HLAR
Perform testing when combination therapy is desired (e.g., endocarditis)
ENTEROCOCCUS SPP.
HLAR (SYNERGY) SCREENING
ampicillin >32 R
vancomycin 32 R
gent syn R
strep syn R
SPECIMEN: BLOOD
DIAGNOSIS: UROSEPSIS
ENTEROCOCCUS SPP.
MIC (µg/ml)
Is this a “true” VRE and
epidemiologically significant?
ENTEROCOCCUS SPP.
VANCOMYCIN TESTING1
Drug Susc Int Res
vancomycin
MIC (µg/ml)
≤4 8-16 32
vancomycin – 10 µg
zone (mm) 2
17
15-16 3 ≤14
CLSI M100S 26. Table 3F.
1 incubate 24 h before reporting “S” 2 Examine zones with transmitted light 3 Retest with MIC method
Vancomycin Screen
Agar for VRE (BHI + 6 g/ml vancomycin)
- control
+ control – E. faecalis ATCC 51299
- control – E. faecalis ATCC 29212
+ control
Result: >1 colony = presumptive
Additional testing/reporting: Perform vancomycin MIC and tests for motility and pigment production to distinguish species with acquired resistance (VanA and VanB) from those with intrinsic, intermediate-level resistance to vancomycin (VanC) such as E. gallinarum or E. casseliflavus which often grow on the vancomycin screen plate. In contrast to other enterococci, E. casseliflavus and E. gallinarum with vancomycin MICs of 8-16 g/ml (intermediate) differ from vancomycin-resistant enterococci for infection control purposes.
ENTEROCOCCUS SPP. (CLSI M100-S26 SUPPLEMENTAL TABLE 3F)
ENTEROCOCCUS SPP.
Species Genotype Motility MGP 1 Arabanose Infection Control
Significance?
E. faecalis vanA or
vanB
- - - yes
E. faecium vanA or
vanB
- - + yes
E. casseliflavus 2 vanC4 + + + no
E. gallinarum vanC4 + + + no 3
1 acidification of methyl-a-D-glucopyranoside 2 yellow pigment 3 one report (Contreras et al. 2008. J Hosp Infect. 70:346.) 4 see slide 19 for vanA information
Rapid MGP
Incubate 3 to 5 hours;
acidification of methyl-a-D-
glucopyranoside = yellow
POS NEG
E. casseliflavus
Yellow pigment
E. faecalis Usually susceptible to ampicillin and penicillin
Usually resistant to quinupristin-dalfopristin
Can acquire resistance to vancomycin – usually due to vanA or vanB genes
E. faecium Usually resistant to ampicillin and penicillin
Usually susceptible to quinupristin-dalfopristin
Can acquire resistance to vancomycin – usually due to vanA or vanB genes
WHAT ARE TYPICAL RESISTANCE PATTERNS FOR ENTEROCOCCI?
E. gallinarum and E. casseliflavus:
have intrinsic low-level vancomycin resistance due to the vanC gene
Rarely can acquire vanA or vanB genes
E. raffinosus, E. avium, and E. durans can become resistant to vancomycin by acquiring
vanA or vanB or less frequently vanD, vanE or vanG
If vancomycin MIC ≥32 g/ml for any Enterococcus spp. consider VRE (might have acquired vanA); discuss case with infection control
WHAT ARE TYPICAL RESISTANCE PATTERNS FOR ENTEROCOCCI?
AMPICILLIN OR VANCOMYCIN
RESISTANCE BY SPECIES
Garrison, MW, et al. 2009. Diagn Microbiol Infect Dis. 65:288.
ampicillin >32 R
vancomycin >32 R
gent syn R
strep syn R
SPECIMEN: BLOOD
DIAGNOSIS: UROSEPSIS
ENTEROCOCCUS FAECIUM
MIC (µg/ml)
What other drugs can we test?
Table 1A Drugs to Test/Report (1)
CLSI M100-S26.
ampicillin >32 R
daptomycin ≤0.5 S
doxycycline ≤0.5 S
linezolid 1 S
rifampin ≤0.5 S
quin-dalfopristin ≤1 S
vancomycin >32 R
gent synergy R
strep synergy R
SPECIMEN: BLOOD
DIAGNOSIS: UROSEPSIS
ENTEROCOCCUS FAECIUM
MIC (µg/ml)
“VRE isolated. Please check
infection control policies.
Rifampin should not be
used alone for antimicrobial
therapy. Infectious Diseases
consult suggested.”
Final Report with
Optional
Comment
ampicillin R
ciprofloxacin R
tetracycline S
nitrofurantoin S
vancomycin R
SPECIMEN: URINE
DIAGNOSIS: UTI
ENTEROCOCCUS FAECIUM
Final Report
WHAT IS THIS???
Vancomycin-dependent VRE Note: isolate can lose vancomycin dependence on subculture
Tambyah et al. 2004. Emerg Infect Dis. 10:1277.
STREPTOCOCCI
Discuss basic antimicrobial susceptibility
principles and resistance mechanisms for
streptococci:
Viridans group streptococci and penicillin
Group B Streptococcus and prenatal screens
S. pneumoniae newer penicillin breakpoints
Discuss antimicrobial susceptibility testing
and reporting strategies
OBJECTIVES
ceftriaxone ≤0.5 S
penicillin 0.5 I
vancomycin ≤0.5 S
SPECIMEN: BLOOD
DIAGNOSIS: ENDOCARDITIS
MIC (µg/ml)
What should we know about penicillin - “I” and viridans group streptococci?
Streptococcus anginosis
STREPTOCOCCUS SPP. VIRIDANS GROUP
PENICILLIN INTERPRETIVE CRITERIA
CLSI M100S 26. Table 2H-2
(6) Viridans streptococci isolated from normally sterile body sites (eg, CSF, blood, bone) should be tested for penicillin susceptibility using an MIC method.
(7) Rx: Penicillin- or ampicillin-intermediate isolates may require combined therapy with an aminoglycoside for bactericidal action.
Zone (mm) MIC (μg/ml)
S I R S I R
- - - ≤0.12 0.25 – 2 ≥4
Baddour et al. 2016. Circulation.
STREPTOCOCCUS SPP. VIRIDANS GROUP
ENDOCARDITIS THERAPY RECOMMENDATIONS
BASED ON PENICILLIN MIC
Baddour et al. 2015. Circulation.
*vancomycin for patients who can’t tolerate beta-lactams
Penicillin MIC (µg/ml) Therapy*
≤0.12 pen +/- gent; ceftriaxone
>0.12 - <0.5 pen or ceftriaxone + gent for 1st 2 wks
≥0.5 pen or amp + gent OR vancomycin
Significant incidence of penicillin non-susceptible isolates (MICs >0.12 µg/ml)
Moet et al. 2007. DMID. 57:333
Yap et al. 2006. Infection. 34:339.
67.5% in children with cancer (blood isolates)
Ahmed et al. 2003. Pediatric Hematol Oncol. 20:439
Most prevalent in S. mitis group
Unusual among certain species (milleri or anginosus group streptococci)
STREPTOCOCCUS SPP. VIRIDANS GROUP
PENICILLIN RESISTANCE RATES
ceftriaxone ≤0.5 S
penicillin 0.5 I
vancomycin ≤0.5 S
SPECIMEN: BLOOD
DIAGNOSIS: ENDOCARDITIS
MIC (µg/ml)
“Penicillin-intermediate Streptococcus anginosus may need combined therapy with penicillin and an aminoglycoside for bactericidal action. Infectious Diseases consult suggested”
Final Report with
Optional Comment
Streptococcus anginosis
SPECIMEN: ANOVAGINAL
TEST: PRENATAL SCREEN
Many Group B Streptococcus
Should we do more?
http://www.cdc.gov/groupbstrep/about/index.html
(13) Rx: Recommendations for intrapartum prophylaxis for Group B streptococci are penicillin or ampicillin. Although cefazolin is recommended for penicillin-allergic women at low risk for anaphylaxis, those at high risk for anaphylaxis may receive clindamycin or erythromycin. Group B streptococci are susceptible to ampicillin, penicillin, and cefazolin, but may be resistant to clindamycin and/or erythromycin. When a Group B Streptococcus is isolated from a pregnant woman with severe penicillin allergy (high risk for anaphylaxis), clindamycin and erythromycin should be tested and only clindamycin reported. See Table 3G
GROUP B STREPTOCOCCUS
CLSI M100-S26. p. 48
SPECIMEN: ANOVAGINAL
TEST: PRENATAL SCREEN
Group B Streptococcus
“Group B Streptococci are susceptible to ampicillin,
penicillin and cefazolin, but may be clindamycin
resistant. Contact laboratory if
clindamycin results necessary.”
SPECIMEN: ANOVAGINAL
TEST: PRENATAL SCREEN
Drive AST by order entry
Group B Strep Screen ordered, physician
must answer the following question
before order will be placed
Is the patient allergic to penicillin?
Yes/No
When physician answers “yes” then the
special request field is populated with
“Patient allergic to penicillin” in LIS
Check for
inducible
clindamycin R if
erythromycin-R
and clindamycin-S
http://www.cdc.gov/groupbstrep/
about/index.html
clindamycin R
penicillin 0.06 S
vancomycin ≤0.5 S
“This Group B Streptococcus is presumed to be clindamycin resistant based on detection of inducible clindamycin resistance. Clindamycin may still be effective in some patients.”
MIC (µg/ml)
Final Report with
Optional Comment SPECIMEN: ANOVAGINAL
TEST: PRENATAL SCREEN
Group B Streptococcus
STREPTOCOCCUS PNEUMONIAE PENICILLIN BREAKPOINT HISTORY
Prior to 2008 one set of breakpoints (µg/ml)
Based on treating meningitis
Had “footnote” that “I” results meant “S” when treating
pneumonia, but footnote generally not used
Resulted in underutilization of penicillins for treating
pneumococcal pneumonia for isolates with MICs ≤1
µg/ml
Breakpoints changed in 2008
Good review….Weinstein et al. 2009. Clin Infect Dis. 48:1596.
S I R
Penicillin ≤0.06 0.12-1 2
S. PNEUMONIAE PENICILLIN %S, I, R USING CURRENT AND OLD CLSI BREAKPOINTS
Syndrome / Specimen
Source
Current Old (before 2008)
BPsb %S %I %R BPsc %S %I %R
Meningitis
CSF
S ≤0.06
R ≥0.12 65.2 - 34.8
S ≤0.06
I 0.12-1
R ≥2
65.2 22.5 12.4
Nonmeningitis
Parenteral
(Blood / bronch)
S ≤2
I 4
R ≥8
92.2 5.2 2.6
Nonmeningitis
Oral
(Ear/NP)
S ≤0.06
I 0.12-1
R ≥2
49.9 26.2 23.8
a adapted from Mera et al. 2011. Microb Drug Res. 17:47 bBPs, breakpoints cone set of BPs for all syndromes / routes of administration
2008 N. America isolates, N=11,185) a
Disk
content
Zone (mm) MIC (µg/ml)
Equivalent
R I S R S
Penicillin 1 µg
oxacillin - - 20 - ≤0.06*
Use oxacillin disk as surrogate for penicillin susceptibility
*correlates with penicillin meningitis and oral penicillin breakpoints
S. PNEUMONIAE PENICILLIN DISK DIFFUSION METHOD
OXACILLIN DISK AS SURROGATE FOR PENICILLIN SUSCEPTIBILITY IN S. PNEUMONIAE
20 mm – report penicillin as S (also S to cefotaxime, ceftriaxone, meropenem, etc.) When penicillin MIC was performed on this isolate, MIC = 0.03 µg/ml
≤19 mm – perform penicillin MIC (also perform MIC for cefotaxime or ceftriaxone or meropenem) When penicillin MIC was performed on this isolate, MIC = 1 µg/ml which is “S” for non-meningitis infections (e.g., pneumonia)
OX
1
OX
1
Timeline
1967 first reported in Australia Hansman et al. 1967. Lancet. 277:264
1987 first report in USA Spika et al. 1991. J Infect Dis. 163:1273
Rapid dissemination in late 1980s and 1990s
Modified penicillin binding proteins (PBP 1a, 1b, 2a, 2b, 2x, 3) Linares et al. 1992. J Antimicrob Chemother. 30:279
Lynch et al. 2009. Semin Respir Crit Care Med. 30:210
WHAT IS THE MECHANISM OF DECREASED PENICILLIN SUSCEPTIBILITY (>0.12 µG/ml) IN
S. PNEUMONIAE?
Not due to β-lactamases!
Variable Disk Diffusion MIC
Inoculum – Pick colonies from BAP incubated 16-20 hours
– Use saline or MHB to standardize inoculum to
0.5 McFarland turbidity
– Inoculate test within 15 minutes
Medium Mueller Hinton agar
with 5% sheep blood
Mueller Hinton broth with
2.5-5% lysed horse blood
Incubation – 5% CO2
– 20-24 hours
- Ambient air
- 20-24 hours
QC − S. pneumoniae ATCC 49619
WHAT VARIABLES MUST WE STANDARDIZE WHEN PERFORMING AST ON S. PNEUMONIAE?
SPECIMEN: BLOOD DIAGNOSIS: PNEUMONIA
MIC (µg/ml)
How do we interpret ceftriaxone and
penicillin results?
ceftriaxone 0.5 ???
erythromycin >1 R
levofloxacin 1 S
meropenem ≤0.25 S
penicillin 1 ???
vancomycin 0.5 S
Streptococcus pneumoniae
STREPTOCOCCUS PNEUMONIAE
BREAKPOINTS*
Susc Int Res
Ceftriaxone (meningitis) ≤0.5 1 2
Ceftriaxone (nonmeningitis) ≤1 2 4
Penicillin parenteral (meningitis) ≤0.06 - 0.12
Penicillin parenteral (nonmeningitis) ≤2 4 8
Penicillin (oral penicillin V) ≤0.06 0.12-1 2
*MICs in μg/ml
SPECIMEN: BLOOD DIAGNOSIS: PNEUMONIA
MIC (µg/ml)
ceftriaxone
erythromycin
levofloxacin
meropenem
penicillin (meningitis)
penicillin (nonmeningitis)
penicillin (oral penicillin V)
vancomycin
Streptococcus pneumoniae
0.5 S
>1 R
1 S
≤0.25 S
1 R
1 S
1 I
0.5 S
Final Report
ceftriaxone (meningitis) 0.5 S
meropenem ≤0.25 S
penicillin (meningitis) 0.12 R
vancomycin 0.5 S
SPECIMEN: CSF DIAGNOSIS: MENINGITIS
MIC (µg/ml)
Streptococcus pneumoniae
CSF specimens Report penicillin, cefotaxime, ceftriaxone using
meningitis breakpoints ONLY
All other specimens Report penicillin, cefotaxime, ceftriaxone using both
meningitis and nonmeningitis breakpoints
APPLYING PENICILLIN, CEFTRIAXONE,
CEFOTAXIME BREAKPOINTS….
SOME Q & A
S. PNEUMONIAE AND PENICILLIN
At minimum, when should penicillin MICs be performed?
• As soon as possible for CSF isolates
• When oxacillin zone is ≤19 mm
If oxacillin zone is ≤19 mm, can we report penicillin?
• No, isolates with oxacillin zone of ≤19 mm may have penicillin MICs in S, I, or R range so a penicillin MIC is needed.
Should we ever do an oxacillin MIC?
• No, oxacillin DISK diffusion tests are used to predict penicillin susceptibility, but oxacillin MICs are not useful.
If QC with S. pneumoniae ATCC 49619 and oxacillin shows a zone much larger than the accepted range, what is the most likely problem?
• The inoculum source plate may be too old
• The inoculum may not have been properly standardized
• The inoculum may have been in liquid diluent too long resulting in autolysis and a decrease in the number of viable cells