2015 Webinar Series

CLSI 2015 Antimicrobial Susceptibility Testing

2/5/2015
Speaker
Janet A. Hindler, MCLS MT(ASCP), Sr. Specialist, Clinical Microbiology, UCLA Health System, Los
Angeles, CA
Janet has worked as a clinical microbiologist with a primary focus on AST for over 40 years. She
sits on the CLSI subcommittee on AST and contributes to several other professional clinical
laboratory organizations.

Objectives
At the conclusion of this program, participants will be able to:
 Identify the major changes found in the new CLSI M100-S25.
 Design a strategy for implementing the new practice guidelines into their laboratory
practices.
 Develop a communication strategy for informing clinical staff of significant AST and
reporting changes.

Continuing Education Credit

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Self-assessment
It is important for clinical laboratories to incorporate the new recommendations into routine practice to optimize detection
and reporting of antimicrobial resistance. Please familiarize yourself with the changes in the new addition of CLSI
documents M100-S25, M02-A12 and M07-A10 before taking this self-assessment.
URL: https://www.surveymonkey.com/s/2015astselfassessment
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 What’s New in the 2015 CLSI
Standards for Antimicrobial
Susceptibility Testing (AST) ?

Janet A. Hindler, MCLS MT(ASCP)

UCLA Health System
jhindler@ucla.edu

“and consultant with the Association of

Public Health Laboratories”

Objectives
 Identify the major changes found in CLSI
document M100-S25, M02-A12, M07-A10.
 Design a strategy for implementing the new
guidelines into your laboratory practices.
 Develop a communication strategy for informing
clinical staff of significant AST and reporting
changes.
Please check supplemental
materials posted for this webinar:
• Self study program
• Implementation Checklist

CLSI AST Standards

January 2015
M100-S25 Tables (2015)1

M02-A12 Disk Diffusion Method (2015)2

M07-A10 MIC Method (2015)2
M11-A8 Anaerobe MIC Testing (2012)

1 M100 updated at least yearly

2 M02, M07 updated every 3 years
3

jhindler clsi update 2015 1

 Major Changes 2015
M100-S25
 General
– Introduced Epidemiological Cutoff Value (ECV)
 Enterobacteriaceae
– Salmonella
• Added pefloxacin disk diffusion test to differentiate isolates “S”
vs. “not susceptible” to fluoroquinolones
• Added azithromycin disk diffusion and MIC tests for S. Typhi
 Enterobacteriaceae, P. aeruginosa, Acinetobacter
– Added Carba NP test for detection of carbapenemases

Summary of
Changes

M100-S25. p. 13-15.

Major Changes 2015

M02-A12; M07-A10
 Formatting
– Labeled main sections as “chapters”
– Summarized content in “chapters”
– Added “step-action tables”
– Added “flow charts”
 Brought up to date with M100 (updated annually)
 Content
– Staphylococcus spp. – expanded discussion of tests to
detect oxacillin resistance
– Quality control
• Several new flow charts
6

jhindler clsi update 2015 2

 “Chapter” Example
Disk Diffusion
Testing Process
M02-A12. p. 15.

“Step-Action” Table
Example
Inoculate Disk
Diffusion Plates
7

Determine need for susceptibility testing

(Chapter 2)

Select antimicrobial agents

(Subchapter 2.1)
“Flow Chart” Example
MIC Susceptibility Prepare stock solutions and inoculum,
including patient and QC organisms
Testing Process (Subchapters 3.2 and 3.3)

Inoculate plates/tubes/trays depending

on method used and incubate
(Subchapters 3.4 - 3.10)

Determine end points and interpret

results
(Subchapters 3.5.9 and 3.11)
Assess QC
(Subchapters 4.2 – 4.8)

Report results
M07-A10. p. 16. (Subchapters 4.9 and 4.12)

Photos Added… MIC Testing

Trailing Endpoints
Trimeth-Sulfamethoxazole
Erythromycin
Linezolid

M07-A10. p. 31-32.
9

jhindler clsi update 2015 3

 Fosfomycin

10

Fosfomycin – Urine Isolates

M100-S25 . p. 32.

11

http://www.idsociety.org
Infectious Diseases Society of America

Recommend
(Acute Cystitis)
Nitrofurantoin
TMP-SMX
Fosfomycin

Fluoroquinolone
β-lactam (oral)

Uncomplicated UTIs (uUTIs)

IDSA Guidelines

12

jhindler clsi update 2015 4

 Fosfomycin
 Oral form in USA; single dose “sachet”
 FDA approved for uUTIs in women due to E. coli
and E. faecalis
– Active against other Enterobacteriaceae, staphylococci,
P. aeruginosa
Karageorgopoulos, et al. 2012. J Antimicrob Chemother. 67:255.

 IV form used for serious infections (MDR in

combination therapy)
Michalopoulos et al. 2011. Int J Infect Dis. 15:e732.
Keating, G. 2013. Drugs. 73:1951.
Lee et al. 2014. Infect Chemother. 46:19.
 IV form NOT FDA approved
– May be prescribed for compassionate use in USA
13

What about testing fosfomycin?

 Only agar methods (disk, agar dilution, Etest)
– Disks available in USA
 CLSI and FDA breakpoints only for E. coli and E.
faecalis
Disk diffusion (mm) MIC (µg/ml)
S I R S I R
M100-S25. p. 50 & 75.
≥16 13-15 ≤12 ≤64 128 ≥256

 EUCAST has MIC breakpoints for

“Enterobacteriaceae” (≤32 µg/ml = S; >32 µg/ml = R)
– Same for both oral (sachet) and IV form
14

ESBL Positive Isolates

% Susceptible Fosfomycin1,2
Organism N %S
E. coli 3 1657 96.8

K. pneumoniae4 748 81.3

1 Review of 17 studies
2 Various methods
3 2 clinical studies (n=80), fosfomycin effective in 93.8%
4 Interpreted with E. coli breakpoints

Falagas et al. 2010. Lancet. 10:43.

15

jhindler clsi update 2015 5

 Fosfomycin % Resistant
CRE Isolates1 (N=107, Germany)
% R Fosfomycin
Species N Range (µg/ml)
(MIC >32 µg/ml)2
K. pneumoniae 50 0.5 - >1024 32
E. coli 24 ≤0.25 - 256 16.7
E. cloacae 17 0.5 - >1024 23.5
All3 107 ≤0.25 - >1024 28.04
1 OXA-48 (n=24), OXA-162 (n=4), VIM (n=21), KPC (n=23), NDM (n=4), GIM
(n=3), VIM + KPC (n=1); carbapenemase not detected in 27 isolates
2 Agar dilution method
3 Included species in addition to those above
4 19.7% R using CLSI urine breakpoint (>64 µg/ml)

NOTE: 92.5% Etest categoric agreement (S, I, R) with agar dilution

Kaase et al. 2014. J Clin Microbiol. 52:1893. 16

Fosfomycin Disk Diffusion

 Sometimes colonies in zone
 Read inner zone; ignore
small (non dense) colonies?
 Media (MHA) differences?
Fosfomycin (200 µg)
Zone (mm)
S I R
≥16 13-15 ≤12
E. coli ATCC 25922
M100-S25. p. 50 & 75.
17

Specimen: Urine Final Report with

Optional Comment
Diagnosis: Recurrent cystitis
Klebsiella pneumoniae
MIC (µg/ml)
ampicillin >32 R
“Fosfomycin reported per Dr.
oral cephalosporins R Jones request.
ceftriaxone >32 R No standard interpretive criteria
ciprofloxacin >4 R for fosfomycin and K.
fosfomycin S pneumoniae; test interpreted
gentamicin 2S with standard criteria available
for E. coli.”
nitrofurantoin >128 R
trimeth-sulfa >4/76 R

What if asked to test fosfomycin on GNR

species other than E. coli?
18

jhindler clsi update 2015 6

 Clarification
Enterobacteriaceae - Cefazolin

19

M100-S25. p. 32.
20

Enterobacteriaceae Cefazolin
Test/ MIC Breakpoint
Report Agent (µg/ml) Comments
Group S I R
Cephems (Parenteral)
A Cefazolin ≤2 4 ≥8 based on dose of 2 g every 8 h
Cephems (Oral)
U Cefazolin ≤16 - ≥32 Footnote (20)
(20) Cefazolin - predicts results for the oral agents - cefaclor, cefdinir,
cefpodoxime, cefprozil, cefuroxime axetil, cephalexin, and loracarbef when
used for therapy of uncomplicated UTIs due to E. coli, K. pneumoniae, and
P. mirabilis.

M100-S25. p. 47 & 47.

21

jhindler clsi update 2015 7

 Prescribing Cefazolin
vs. MIC Interpretation
 IM, IV administration
For “S” E. coli, K. pneumoniae, P. mirabilis:
Uncomplicated UTIs
Bacteremia
Breakpoints (µg/ml)
Reason for Testing Dose
S I R
1 g every 12 h
Predict cefazolin use for uUTI ≤16 - ≥32
(IM or IV)
Predict cefazolin use for systemic 2 g every 8 h
≤2 4 ≥8
infections (IM or IV)
Surrogate for oral cephalosporins
≤16 - ≥32 PO (various)
to use for uUTI

M100-S25. p. 47 & 47.

22

Final Report with

Specimen: Urine Optional Comment
Diagnosis: UTI
E. coli MIC (µg/ml)
ampicillin >32 R
oral cephalosporins S
cefazolin (for uUTI) S MIC in this case
was 16 µg/ml
cefazolin (for systemic) R
ciprofloxacin >2 R
nitrofurantoin ≤16 S
trimeth-sulfa >4/76 R

“Oral cephalosporins include cephalexin and cefpodoxime.

Cefazolin reported per Dr. Jones request”
UCLA
23

Staphylococcus
• CoNS
• MRSA and mecC
• S. pseudintermedius

24

jhindler clsi update 2015 8

 Report Isolates of Staphylococcus
spp. as Oxacillin Resistant if….
They test resistant by:
– oxacillin MIC or
– cefoxitin MIC (only for S. aureus) or
– cefoxitin disk test or
– they are positive for mecA or PBP 2a

M07-A10 p. 39.
25

Methods for Detection of Oxacillin

Resistance in Staphylococci
Oxacillin Salt
Oxacillin Cefoxitin Cefoxitin
Organism Agar Screening
MIC MIC DD
Test
S. aureus Yes Yes Yes Yes
S. lugdunensis Yes Yes Yes No
CoNS (except S.
Yes* No Yes No
lugdunensis)

*The oxacillin MIC interpretive criteria listed in M100 for CoNS may
OVERCALL RESISTANCE for some species other than S. epidermidis.
These isolates display MICs in the 0.5 to 2 μg/mL range but lack mecA.
For serious infections with CoNS other than S. epidermidis, testing for
mecA or for PBP 2a or with cefoxitin disk diffusion may be appropriate for
strains for which the oxacillin MICs are 0.5 to 2 μg/ml.

M07-A10. p. 38.
26

Specimen: Shunt fluid

Diagnosis: Spina Bifida
Coagulase-negative Staphylococcus
MIC (µg/ml)
oxacillin 1
penicillin R
vancomycin 0.5 S

How do we interpret oxacillin?

….don’t want to report false “R”
27

jhindler clsi update 2015 9

 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 Report
Do mecA or
Oxacillin “S” PBP2a or Oxacillin “R”
Cefoxitin disk
Oxacillin
MIC (µg/ml)
S I R Negative Positive

≤0.25 - ≥0.5
Report Report
Oxacillin “S” Oxacillin “R”

28

Specimen: Shunt fluid

Diagnosis: Spina Bifida
Coagulase-negative Staphylococcus
MIC (µg/ml)
oxacillin 1 S
penicillin R
vancomycin 0.5 S

IF oxacillin MIC is 1 µg/ml …and cefoxitin disk is “S”

THEN report oxacillin “S”
29

MRSA Due to mecC

 “Mechanisms of oxacillin resistance other than
mecA are rare and include a novel mecA homologue, mecC “
M07-A10 p. 37.
– 2007 - found during investigation of bovine mastitis in UK
– Retrospective testing found isolates with mecC from 1975
 Subsequent isolation from various animals and humans in
Europe (none reported in USA to date!)
 NOT always OX-R and CX-R
– CX better than OX
 NOT detected by tests directed at mecA or PBP 2a
 Also in CoNS García-Álvarez et al. 2011. Lancet Infect Dis. 11:595-603.
Garcia-Garrote et al. 2014. J Antimicrob Chemother. 69:45.
Paterson et al. 2014. Trends in Microbiol. 22:42.
Skov et al. 2014. J Antimicrob Chemother. 69:133. 30

jhindler clsi update 2015 10

 Staphylococcus aureus (N=896)
Vitek 2 Results1
No. S and/or R Isolates / Total No. Isolates (%)
Identify of N Tested by Vitek 2
S. aureus (Total) OX-S OX-R OX-R OX-S
CX-R CX-R CX-S CX-S
MRSA mecC pos 62 55 (88.7) 7 (11.3) 0 0
MRSA mecA pos 455 4 (0.9) 446 (98) 5 (1.1) 0
MSSA
mecC neg 379 0 0 4 (1.1) 375 (98.9)
mecA neg
1Staph AST-P620 card; UK Isolates 2006-2012
NOTE: Vitek 2 expert (and CLSI) rule would call these “R” (call
S. aureus “R” if it tests R to either OX or CX)

Cartwright et al. 2014. J Clin Microbiol. 51:2732.

31

Staphylococcus pseudintermedius

 Veterinary pathogen; can infect humans

 Identification –
– Seeing more with MALDI
– Coagulase positive; clumping factor negative
– Masquerades as S. aureus
 Some mecA positive
– Often not detected with CLSI M100-S25 S. aureus cefoxitin or
oxacillin breakpoints
• If report as “S. aureus” and use S. aureus oxacillin MIC or
cefoxitin MIC or disk diffusion breakpoints, may report false
oxacillin susceptible
– Detected with CLSI VET01-S2 oxacillin MIC breakpoints
(veterinary standard; same as M100-S25 CoNS breakpoints)

32

Staphylococcus pseudintermedius
Oxacillin and Cefoxitin Breakpoints
Oxacillin Cefoxitin
Standard/ Guideline
S I R S I R
MIC (µg/ml)
CLSI M100-S25 - S. aureus ≤2 - ≥4 ≤4 - ≥8
CLSI M100-S25 - CoNS ≤0.25 - ≥0.5 NA
CLSI VET01-S2 – S. pseudint ≤0.25 - ≥0.5 NA
EUCAST – S. pseudint NA NA
Zone (mm)
CLSI M100-S25 - S. aureus NA ≥22 - ≤21
CLSI M100-S25 - CoNS NA ≥25 - ≤24
CLSI VET01-S2 – S. pseudint ≥18 - ≤17 NA
EUCAST – S. pseudint NA ≥35 <35

Circled worked best (recent multi-lab experience)!

33

jhindler clsi update 2015 11

 Tests for Carbapenemases

34

Carbapenem-Resistant
Enterobacteriaceae
Two mechanisms of resistance
– Carbapenemase - -lactamase that hydrolyzes
carbapenems
– Cephalosporinase + porin loss
• Some AmpC -lactamases and ESBLs have low-level
carbapenemase activity
• Porin loss limits entry of the carbapenem into the cell

35

Old vs. Current CLSI Breakpoints

Klebsiella pneumoniae
MIC (µg/ml)
CLSI Meropenem BPs (µg/ml)
amikacin >32 R Old Current
cefepime >32 R
S I R S I R
ceftriaxone >32 R
≤4 8 ≥16 ≤1 2 ≥4
ciprofloxacin >2 R
ertapenem >8 R
gentamicin >10 R
meropenem 4 Test for Test for cbp’ase,
piper-tazobactam >128/4 R cbp’ase if requested
tobramycin >10 R routinely if (eg, for
trimeth-sulfa >4/76 R screen pos epidemiology)

36

jhindler clsi update 2015 12

 Introduction to Tables 3B and 3C. Tests for
Carbapenemases in Enterobacteriaceae, Pseudomonas
aeruginosa, and Acinetobacter spp.

M100-S25. p. 112.
37

Introduction to Tables 3B and 3C. Tests for

Carbapenemases in Enterobacteriaceae, Pseudomonas
aeruginosa, and Acinetobacter spp.
MHT Carba NP Molecular
Use Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae
P. aeruginosa P. aeruginosa
Acinetobacter Acinetobacter

Strengths Simple Rapid Determines type of

carbapenemase
Limitations Some false pos (eg, Special “fresh” Special reagents
ESBL/ampC + porin) reagents

Some false neg Some invalid results Specific to targeted

(eg NDM) gene

Enterobacteriaceae False neg for OXA-

only type carbapenemase

M100-S25. p. 112.
28

Introduction to Tables 3B and 3C.

 Institutional protocols may require identification of
carbapenemase-producing Enterobacteriaceae, P. aeruginosa,
and Acinetobacter spp.
– Carbapenemase testing is not currently
recommended for routine use.
 Carbapenemase-producing isolates of Enterobacteriaceae:
– Usually test “I” or “R” to one or more carbapenems using
current breakpoints (NOTE: ertapenem nonsusceptibility is
the most sensitive indicator of carbapenemase production),
and
– Usually test “R” to one or more agents in cephalosporin
subclass III (eg, cefoperazone, cefotaxime, ceftazidime,
ceftizoxime, and ceftriaxone).
– However, some isolates that produce carbapenemases such
as SME or IMI often test “S“ to these cephalosporins.
M100-S25. p. 112.
39

jhindler clsi update 2015 13

 Carba NP Test for
Carbapenemase Production
 Isolated colonies (lyse)
 Hydrolysis of imipenem
 Detected by change in pH of
indicator (red to yellow/orange)
 Rapid <2h
 Microtube method
NO +
imipenem imipenem
Nordmann et al. 2012. Emerg Infect Dis. 18:1503.
Tijet et al. 2013. Antimicrob Agents Chemother. 57:4578.
Vasoo et al. 2013. J Clin Microbiol. 51:3092.
Dortet et al. 2014. J Med Microbiol. 63:772.
Dortet et al. 2014. Antimicrob Agents Chemother. 58:2441.
40

Invalid

+
+
+
+ M100-S25. p. 120-126.
41

Carba NP Test Materials/Reagents

Testing simple
Reagent Preparation
takes time
Reagents
Prepare solutions:
10 mM Zinc sulfate heptahydrate
Phenol red solution
0.1 N NaOH

Carba NP Solution A
(phenol red + zinc solutions)

Carba NP Solution B
(Carba NP Solution A + imipenem)

42

jhindler clsi update 2015 14

 5 Most Common Carbapenemases
Enterobac- Non-
Class Carbapenemases
teriaceae fermenters

A1 KPC2 +++ +

B (metallo) NDM3, IMP, VIM, +++ +++

D OXA-48-like +++ +/-

1also includes SME; 2most common in USA; 3increasing in USA

….but several types within 5 groups and other types of

carbapenemases
43

CLSI Carba NP Multi-lab Study

 7 laboratories
 80 isolates (tested by each lab)
– 44 had Cbp’ase gene
• 10 KPC, 14 NDM, 4 IMP, 8 VIM, 1 SPM, 2 SME, 5 OXA
– 66 Enterobacteriaceae; 14 non-fermenters
 Very good performance for all Cbp’ases but OXA
 False positives in 7 different isolates each at 1/7 sites
 8/10 KPC had low carbapenem MICs (range 0.05-2 µg/ml)
– 4/7 labs missed KPC in an Enterobacter cloacae
– 1/7 labs missed KPC in an E. coli
Limbago et al. 2014. ICAAC.
44

Carba NP Method - Some Details

False Negatives may be due to
– Weak carbapenemases (e.g., OXA-48-like, SME-1,
or GES-5)
– Carbapenemases in mucoid cells
– Source plate medium used for inoculum
Time to positive may vary by carbapenemase
– KPC quick
– Others slower (OXA slowest)

Dortet et al. 2014. Antimicrob Agents Chemother. 58:1269.

Lee et al. 2014. J Clin Microbiol. 52:4023.
Tijet et al. 2014. Antimicrob Agents Chemother. 58:1270.
45

jhindler clsi update 2015 15

 Carba NP Test

Blank Neg KPC OXA48 OXA181 NDM IMP VIM SME

Courtesy of:
UCLA Shaun Yang
P. Hemarajata
46

Modified Hodge Test

Neg Control KPC NDM OXA 232

- + False - +
Courtesy of:
UCLA Shaun Yang
P. Hemarajata
47

Commercial Test
Rapid CARB Screen Kit
 Commercial kit; similar to Carba NP
 Enterobacteriaceae and P. aeruginosa
 Tablets
– Imipenem + indicator neg
neg pos pos
– Negative control cntrl
 ≤2 hours
 CLSI study isolates – UCLA results: NOT FDA
– More difficult to read than Carba NP cleared
– Good agreement with Carba NP but more initial
invalids that required repeating
– Most problems with Acinetobacter baumannii – NDM
(not indicated for this species)
www.rosco.dk
48

jhindler clsi update 2015 16

 Enterobacteriaceae
Carbapenemase Detection
Rapid CARB
Study N Carba NP MHT
Screen Kit
97% sens 98% sens
1 235 -
100% spec 83% spec
91% sens 73% sens
2 92 -
100% spec 100% spec
98% sens 75% sens
3 150 -
100% spec 91% spec
1 Huang et al. 2014. J Clin Microbiol. 52:3060.
2 Yousef et al. 2014. Eur J Clin Microbiol Infect Dis. Jul 10 epub.
3 Simner et al. 2015. J Clin Microbiol. 53:105.

Rapid CARB Screen Kit discontinued !!!!

Reformatted Product is Neo-Rapid CARB Screen Kit 49

Molecular Tests for Carbapenemases

 Biofire *
– KPC
 Nanosphere *
– KPC, NDM, OXA, IMP, VIM
 BD Max * FDA cleared
– KPC, NDM, OXA-48
 Cepheid
– KPC, NDM, OXA-48, IMP-1, VIM
 Check-Points
– KPC, NDM, OXA-48, IMP, VIM
Others?
50

K. pneumoniae
AST MIC (µg/ml):
Carbapenem I or R Ertapenem >2 R In Pursuit of a
Imipenem 4R
Meropenem >8 R Carbapenemase!

Biological Test for Story line by…

Carbapenemase Shaun Yang
P. Hemarajata
Carba NP - MHT +

PCR for Cbp’ase genes Neg for: KPC, NDM,

IMP, VIM, OXA-48, SME

Whole Genome Sequencing OXA-232

51

jhindler clsi update 2015 17

 Salmonella
• Fluoroquinolones
• Azithromycin

52

Global Incidence - Typhoid Fever

Basnyat et al. 2005. Clin Infect Dis. 41: 1467.

“A standard for global application developed

through the CLSI consensus process”
53

WHO Background document: The diagnosis, treatment and

prevention of typhoid fever. 2003.
54

jhindler clsi update 2015 18

 What is the issue for fluoroquinolones
(FQs) and Salmonella?
 Typhoid fever a problem in developing countries
– High morbidity/mortality if untreated
 FQs good (inexpensive, PO route) for treatment of
salmonellosis, including typhoid fever
– Emerging resistance; need to test!
 Clinical response rates to ciprofloxacin are poorer
for isolates with “decreased ciprofloxacin
susceptibility” (vs. “S” isolates)
• MICs of 0.12 – 1.0 µg/ml
Crump et al. 2008. Antimicrob Agents Chemother. 52:1278.
Parry et al. 2010. Antimicrob Agents Chemother. 54:5201.

 Need easy test (disk diffusion) to differentiate

isolates that are “S” vs. “not S”!
55

Salmonella spp. - FQ Resistance

Previously used Nalidixic Acid
Phenotype
Genotype Ciprofloxacin
Nalidixic Acid
MIC (µg/ml)
Wild type (NO resistance) 0.008-0.06 Usually susceptible
*Chromosomal gyrA (single mutation) 0.12 - 2.0 Usually resistant
*Chromosomal gyrB (single mutation) 0.12 – 0.5 Usually susceptible
*Chromosomal gyrA, gyrB (multiple
≥4.0 Resistant
mutations)
PMQR (e.g. qnr or aac(6’)-lb-cr) 0.12 - 2.0 Often susceptible

*QRDR = “quinolone resistant determining region”; relates to

chromosomal mutations
PMQR, plasmid-mediated quinolone resistance; less common than
chromosomal gyrase mutations; mostly due to qnr
56

Now how can we detect Salmonella that are not

susceptible to FQs?
S I R S I R

M100-S25. p. 49.
57

jhindler clsi update 2015 19

 Salmonella spp.
Fluoroquinolone Breakpoints
Disk DD (mm) MIC (µg/ml)
Antimicrobial
Content
Agent Susc Int Res Susc Int Res
(µg)
Ciprofloxacin 5 ≥31 21-30 ≤20 ≤0.06 0.12-0.5 ≥1

Levofloxacin - - - - ≤0.12 0.25-1 ≥2

Ofloxacin - - - - ≤0.12 0.25-1 ≥2

Pefloxacin * 5 ≥24 - ≤23 - - -

Nalidixic acid ** 5 ≥19 14-18 ≤13 ≤16 - ≥32

*Surrogate test for ciprofloxacin; **also surrogate but not labeled as such
Strains of Salmonella that test “nonsusceptible” to ciprofloxacin, levofloxacin,
ofloxacin, pefloxacin, or nalidixic acid may be associated with clinical failure
or delayed response in fluoroquinolone-treated patients with salmonellosis.
58

What is pefloxacin?
 FQ introduced in early 1980s
 Used for uncomplicated gonorrhoeae, UTIs,
gastroenteritis, typhoid fever
– Dupont. 1993. Drugs. 45:119.
 Studies in Europe suggested pefloxacin disk
superior in differentiating FQ “S” vs “not
susceptible” isolates
– Some noted difficulties with using ciprofloxacin disk
– Does not detect isolates with aac(6’)-lb-cr
 Neither pefloxacin drug nor disk available in USA
 CLSI added pefloxacin to address “global needs”
59

Performance of Disk Diffusion (DD)

and Etest for Detection of FQ-R
Salmonella enterica (n=135)
No. Isolates
“R” Mechanism
(% typhoidal)
aac (6’) 1 (0)
qnr 36 (0)
QRDR mutation 45 (0)
Not characterized 29 (90)
None 24 (25)

Deak et al. 2015. J Clin Microbiol. 53:298.

UCLA
CLSI Agenda Book June 2014.
60

jhindler clsi update 2015 20

 Distribution of Ciprofloxacin MICs (n=135)

S Int (“Not S”) Res (“Not S”)

UCLA Deak et al. 2015. J Clin Microbiol. 53:298.

CLSI Agenda Book June 2014.
61

Salmonella spp. (N=135)

DD vs. Ref MIC
How well do disks identify isolates that are
“S” vs. “not susceptible” to FQs?

False “S” False “R”

Agent CA (%)
N (%)1 N (%)2
Ciprofloxacin 98.5 0 2 (8)
Nalidixic Acid 94.9 2 (1.8) 5 (20)
Pefloxacin 97.8 0 3 (12)
CA, categoric agreement (same “S “ or “not S” result)
1 based on 110 “R” isolates
2 based on 25 “S” isolates

UCLA Deak et al. 2015. J Clin Microbiol. 53:298.

CLSI Agenda Book June 2014.
64

Salmonella spp. (N=135)

Etest vs. Ref MIC
Performance [N (%)]
Agent
EA CA
131
Ciprofloxacin 135 (100)
(97.0)1

Levofloxacin 125 (92.6) 135 (100)

EA, essential agreement (within +/- 1 dilution)

CA, categoric agreement (same “S” or “not S” result)

UCLA
Deak et al. 2015. J Clin Microbiol. 53:298.
63

jhindler clsi update 2015 21

 Why azithromycin for Salmonella Typhi?
 Management of enteric fever generally includes
antimicrobial agents
 Azithromycin distribution in vivo
– Low serum concentration
– Concentrates in PMNs, monocytes, lymphocytes, alveolar
macrophages; achieves high intracellular concentrations (≈ 80-200
times > serum concentration)
– Azithromycin MICs lower than intracellular concentration
 Salmonella Typhi is an intracellular pathogen
 Successfully used for over many years; very few clinical
failures
Frenck R et al. 2004. Clin Infect Dis. 38:951–7.
Girgis et al. 1999. Antimicrob Agents Chemother. 43:1441-4.
64

Salmonella Typhi
Azithromycin Breakpoints
Disk DD (mm) MIC (µg/ml)
Antimicrobial
Content Comments
Agent S I R S I R
(µg)
(33) Salmonella Typhi
Azithromycin 15 ≥13 - ≤12 ≤16 - ≥32 only: Interpretive
criteria are based on
MIC distribution data.

M100-S25. p. 49.

EUCAST:
“Azithromycin has been used in the treatment of infections with
Salmonella typhi (MIC ≤16 mg/L for wild type isolates) and
Shigella spp.” eucast.org

65

Salmonella USA, 2012

Distribution of Azithromycin MICs

MIC µg/ml
National Antimicrobial Resistance Monitoring
System (NARMS) http://www.cdc.gov/narms/
66

jhindler clsi update 2015 22

 Salmonella Typhi
Azithromycin

MIC
MIC Method
(µg/ml)
Inner zone
Broth microdilution 8
Outer zone
Etest inner zone 4
Etest outer zone 1

Azm

Often observe “double”

zones on Etest and DD…
read inner zone
67

Salmonella spp. % Susceptible

USA 2012
Non-typhoidal
Antimicrobial Breakpoint S. Typhi S. Paratyphi A
Salmonella spp.
Agent (µg/ml) (n=326) (n=111)
(n=2236)
Ampicillin ≤8.0 91.0 100 100

Ceftriaxone ≤1.0 97.2 100 100

Ciprofloxacin ≤0.06 96.4 31.6 4.5

Trimeth-sulfa ≤2/38 98.6 89.8 100

Azithromycin ≤16.0 99.9 100 100

*National Antimicrobial Resistance Monitoring System (NARMS)

http://www.cdc.gov/narms/
68

When should we test Salmonella spp.? What drugs?

– Extraintestinal isolates
– Typhoidal Salmonella from all sources
– Other when requested (select patient populations?)
– ampicillin, a fluoroquinolone, trimethoprim-sulfamethoxazole +
3rd generation cephalosporin for extraintestinal isolates
How can we test fluoroquinolones?
– Of commercial AST systems, only Etest currently encompasses
new low MIC breakpoints for ciprofloxacin (not FDA cleared with
Salmonella breakpoints)
– Ciprofloxacin disk diffusion
Should we test azithromycin? If yes, how?
– On request only
– Disk diffusion and MIC breakpoints for S. Typhi only

69

jhindler clsi update 2015 23

 Anaerobes

70

Table 2J-1 Anaerobe MIC Testing

M100-S25. p. 102. 71

Cumulative Antibiogram - Anaerobes

M100-S25. p. 208.
72

jhindler clsi update 2015 24

 Microbiological Breakpoint ≤4 µg/ml
S. aureus (n=1000) (Hypothetical Drug X)

Wild type isolates Isolates w/ acquired

or mutational
resistance

73

Microbiological Breakpoint ≤8 µg/ml

S. aureus (n=1000) (Hypothetical Drug Y)

Wild type isolates

No S. aureus
found with R to
Drug Y!

74

Epidemiological Cutoff Values (ECVs)

 An MIC value that separates bacterial populations
into those with and without acquired and/or
mutational R mechanisms
– Based solely on in vitro data
 Used to signal the emergence of non-wild type
strains

wild type non-wild type

NO YES
“R” mechanism “R” mechanism
75

jhindler clsi update 2015 25

 Why ECVs for vancomycin and
P. acnes?
 P. acnes opportunistic pathogen
– CSF infections
– Implant-associated infections
 Vancomycin is a therapy option
 Insufficient clinical data to set “clinical breakpoints”
 Concern about emerging resistance
 MIC ≥4 µg/ml would be suspicious for emerging
resistance

Achermann et al. 2014. Clin Microbiol Rev. 27:419.

76

Table 2J-2. MIC Epidemiological Cutoff

Values (ECVs)
Propionibacterium acnes
ECV (µg/ml)
Antimicrobial Agent
WT NWT
Vancomycin ≤2 ≥4

WT, wild type; NWT, non-wild type

Clinical Breakpoints based on: ECVs based on:
•MIC distributions •MIC distributions only
•Pharmacokinetic/
pharmacodynamic data
•Clinical outcome data M100-S25. p. 106.
Appendix G. p. 220-221.
77

Vancomycin – Propionibacterium acnes

ECV = 2 µg/ml

eucast.org
78

jhindler clsi update 2015 26

 Specimen: Knee Aspirate Final Report with
Optional Comment
Diagnosis: Post surgical infection
Propionibacterium acnes
MIC (µg/ml)
clindamycin ≤0.06 S “Vancomycin reported per Dr.
penicillin ≤0.5 S Jones request following
consultation with Antibiotic
metronidazole >32 R Stewardship team. No standard
vancomycin 2 vancomycin interpretive criteria.
Vancomycin MIC for this P.
acnes falls within wild type
distribution. ”

What if asked to test vancomycin on P. acnes?

79

http://community.clsi.org/micro/2014/12/19/rangefinder/

80

Quality Control

81

jhindler clsi update 2015 27

 4 flow charts:
Weekly to Daily QC:
20-30 day
3 x 5 day
Corrective action:
Daily
Weekly

M02-A12. p. 54-57.
M07-A10. p. 64-67.
82

Maintenance of
QC Strains

F1: 1st sub from

frozen or dried
F2: sub of F1
F3: sub of F2

M02-A12. p. 69-70.
M07-A10. p. 83-84.
83

IV Agents Active Against GNR in

Advanced Clinical Development
Enterobacteriaceae P. aeruginosa
Agent
ESBL sCBP mCBP WT
Aztreonam –
Yes Yes Yes Yes
avibactam
*Ceftolozane –
Yes No No Yes
tazobactam
Ceftazidime –
Yes Yes No Yes
avibactam
Ceftaroline –
Yes Yes No No
avibactam

sCBP – serine carbapenemase (eg, KPC)

mCBP, metallo-carbapenemase (eg, NDM, VIM, IMP)
WT – wild type
*FDA approved Adapted from Boucher et al. 2013. Clin Infect Dis.
84

jhindler clsi update 2015 28

 Antimicrobial Agents
Approved in 2014
Great Resource!!

http://www.centerwatch.com/drug-information/fda-approved-
drugs/drug/100013/sivextro-tedizolid-phosphate- 85

Older β-lactamase inhibitor combinations

 Use E. coli ATCC 35218 or K. pneumoniae ATCC 700603
Newer β-lactamase inhibitor combinations
 Use K. pneumoniae ATCC 700603
M100-S25. p. 158.
86

β-lactamase Producing Strains

(to QC β-lactamase inhibitor drugs)

Plasmid encodes
QC Strain Amp Ceftaz
β-lactamase:
E. coli TEM-1
R S
ATCC® 35218 (non-ESBL)
K. pneumoniae
SHV-18 ESBL R R
ATCC® 700603

• These are tested to detect presence of “inhibitor” in combinations

• Can loose plasmid if maintained improperly
Must test with “R” β-lactam (alone) to ensure strain produces β-lactamase

87

jhindler clsi update 2015 29

 E. coli
ATCC 35218

amoxicillin- ceftazidime-
ampicillin ceftazidime
clavulanate avibactam

K. pneumoniae
ATCC 700603

88

Removal of CLSI References in

CLIA Interpretive Guidelines
(Including weekly QC option for AST)

89

AST QC “Frequency” Options 1/1/16

CMS Requirements
Follow CLIA QC Regs
– Daily QC
Develop IQCP
or
…..will be cited!

ASM, CAP, CLSI are working together to

address IQCP for Clinical Microbiology
90

jhindler clsi update 2015 30

 Some Additional Topics Under
Evaluation by CLSI AST Subcommittee
 IQCP suggestions (ASM, CAP, CLSI)
 Update M45 (fastidious organism) guideline (2015)
– Add…Aerococcus, Gemella, Lactococcus, Micrococcus, Rothia

 Refinements for testing non-Enterobacteriaceae

 Examine direct AST for positive blood cultures
 Actively develop educational materials for users
– Expand availability / user friendliness of material on CLSI website

Annual CLSI AST Update 2016

will have a new voice !!!
91

CLSI
Website

• Information from
AST meetings
• Order CLSI AST
products

CLSI AST Subcommittee welcomes new volunteers!

http://clsi.org/standards/micro/
92

….for listening today and to our program

for the past 12 years!

And thanks to:

APHL Staff (especially Denise Korzeniowski)
CLSI Staff
CLSI Subcommittee on AST

93

jhindler clsi update 2015 31

 The following summary slides will
not be discussed and are
presented for participant’s
review.

94

Summary (1)
 CLSI updates AST tables (M100) each January.
 CLSI updates documents that describe how to perform
reference disk diffusion (M02) and reference MIC (M07)
tests every 3 years.
– All 3 documents updated in 2015!
 Changes to CLSI documents are summarized in the front
of each document.
 Information listed in boldface type is new or modified
since the previous edition of M100.
 Recent interpretive criteria (breakpoint) addition/revision
dates are listed in the front of M100-S25 (pages 18-19).

95

Summary (2)
 New formatting for M02 and M07:
– Sections now called “chapters”
– Chapter content summarized in the beginning of each chapter
– “Step action tables” added to better explain processes
– More “flow charts” added to better explain processes
– Added photos of reading difficult MIC endpoints (M07)
 Fosfomycin added to Table 1A Group U to reflect it’s utility
in treating uncomplicated UTIs (uUTIs).
– One of 3 main oral agents recommended by IDSA for treating uUTIs.
– Only oral form available in USA and approved for uUTIs of E. coli
and E. faecalis.
– Tested by agar (not broth) methods (e.g., disk diffusion)
– Active against some multidrug-R organisms (including ESBL
producers)

96

jhindler clsi update 2015 32

 Summary (3)
 Fosfomycin IV used for systemic MDR infections (including
CRE), mainly outside USA; not approved in USA, but
available on compassionate plea.
 Cefazolin (IM or IV) occasionally used to treat infections
due to cefazolin-susceptible isolates of E. coli, Klebsiella
pneumoniae, or Proteus mirabilis.
– uUTI (use urine breakpoint)
– Systemic infections (use systemic breakpoint)
 Cefazolin (surrogate agent) can be tested to predict the
activity of oral cephalosporins that might be used for
therapy of uUTIs due to E. coli, Klebsiella pneumoniae, or
Proteus mirabilis.
 Any staphylococcus that tests resistant to oxacillin or
cefoxitin or both should be reported as oxacillin resistant.
– When discordant results occur between the two, however, results
should be carefully checked to rule out a testing error.
97

Summary (4)
 Coagulase-negative staphylococci (other than S.
epidermidis or S. lugdunensis) with oxacillin-resistant
MICs of 0.5–2.0 µg/ml might lack mecA.
– Test further to avoid reporting false oxacillin resistance; use mecA,
PBP2a or cefoxitin disk diffusion test
 Some MRSA have mecC (not mecA)
– Never reported in USA; sometimes seen in Europe; humans and
animals
– Not always oxacillin and cefoxitin resistant; best detected with
cefoxitin
 Staphylococcus pseudintermedius
– Tube coagulase positive; clumping factor negative (might be called
S. aureus and falsely oxacillin S)
– Veterinary pathogen but can infect humans
– Not detected reliably with cefoxitin; oxacillin MIC / CoNS
breakpoints work best
98

Summary (5)
 If using old CLSI carbapenem Enterobacteriaceae
breakpoints, testing for carbapenemases should be
performed on isolates suspicious for carbapenemases.
 Carbapenemase testing is not recommended for routine
use when using current (M100-S25) carbapenem
Enterobacteriaceae breakpoints.
 Modified Hodge, Carba NP and/or molecular assays can
be performed to detect carbapenemases.
– All of these tests have strengths and limitations
– MHT is only recommended for Enterobacteriaceae, whereas the
other 2 test types can be done on Enterobacteriaceae, P.
aeruginosa and Acinetobacter spp.
 The Carba NP test is based on a pH change following
hydrolysis of imipenem by a carbapenemase-producing
isolate and results are available within 2 h.

99

jhindler clsi update 2015 33

 Summary (6)
 Fluoroquinolones and azithromycin are often administered
when oral therapy is desirable for treating typhoid fever in
developing countries.
 CLSI efforts to find a reliable disk diffusion test to
differentiate fluoroquinolone “S” vs. “not S” Salmonella
spp. has been based, in part, on global needs.
– In a UCLA study, ciprofloxacin and pefloxacin (surrogate) disks
performed comparably
– Pefloxacin disks are not available in the USA
– Nalidixic acid (surrogate) tests are inferior
 Azithromycin intracellular concentrations are much higher
than serum concentrations
– Salmonella is an intracellular pathogen, hence explains,
azithromycin’s effectiveness

100

Summary (7)
 Epidemiological Cutoff Values (ECVs) refer to MIC values
that separate bacterial populations into those with and
without acquired and/or mutational R mechanisms.
 ECVs are based on MIC distributions only and are different
than “clinical breakpoints”.
 Newer β-lactamase inhibitor combination drugs are
becoming available for clinical use.
– Ceftolozane-tazobactam (FDA approved); also aztreonam-
avibactam; ceftazidime-avibactam; ceftaroline-avibactam
– Must QC with β-lactamase-producing K. pneumoniae ATCC 700603
 Laboratories will be required to follow CLIA regulations
precisely or introduce IQCP by 1/1/16.
– CLIA requires daily QC of AST
– ASM, CAP, CLSI are working to provide guidance for clinical
microbiology
101

Summary (8)
 Minutes of CLSI AST Subcommittee meetings and other
materials are available at www.clsi.org.
 CLSI and other groups welcome help with improving
susceptibility testing and reporting!

102

Comments/concerns about this program, contact webinar@aphl.org

jhindler clsi update 2015 34