ENTEROCOCCUS AND STREPTOCOCCUS - SWACM · ENTEROCOCCUS AND STREPTOCOCCUS April Abbott, PhD, D(ABMM) Director, Microbiology Deaconess Health System Evansville, IN ... Ampicillin …

ENTEROCOCCUS AND

STREPTOCOCCUS

April Abbott, PhD, D(ABMM)

Director, Microbiology

Deaconess Health System

Evansville, IN

[email protected]

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)


ampicillin 2 S

vancomycin ≤0.5 S

gent synergy S

strep synergy R

SPECIMEN: BLOOD


“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)


“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


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



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


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


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.


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)


PENICILLIN RESISTANCE RATES

ceftriaxone ≤0.5 S

penicillin 0.5 I

vancomycin ≤0.5 S

SPECIMEN: BLOOD


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



Group B Streptococcus

“Group B Streptococci are susceptible to ampicillin,

penicillin and cefazolin, but may be clindamycin

resistant. Contact laboratory if

clindamycin results necessary.”



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


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


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)


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

Documents

ENTEROCOCCUS AND STREPTOCOCCUS - SWACM · ENTEROCOCCUS AND STREPTOCOCCUS April Abbott, PhD, D(ABMM) Director, Microbiology Deaconess Health System Evansville, IN ... Ampicillin …