Romney Humphries, PhD D(ABMM) Section Chief, UCLA Cl inical Microbiology

Los Angeles CA

rhumphries@mednet.ucla.edu

ENTEROBACTERIACEAE:

KEY AST CHALLENGES

Cefazolin testing to predict oral cephalosporins for

uUTI

Cefepime susceptible dose-dependent breakpoints

ESBL and AmpC

Carbapenem Resistance

Salmonella and Shigella testing

THE CHALLENGES

Cefazolin:

Surrogate Agent to Predict Oral Cephs

Infectious Diseases Society of America Practice Guideline

re: β-lactams for Acute uUTI in Women

http://www.idsociety.org

β-lactams including

amox-clav,

cefdinir,

cefaclor,

cefpodoxime

are choices when other agents (e.g.,

nitrofurantoin, trimeth-sulfa, fosfomycin,

fluoroquinolone), cannot be used

Other β-lactams (e.g., cephalexin) are

less well studied but may also be

appropriate in certain settings.

uUTI = uncomplicated urinary tract infection

UCLA EMERGENCY DEPT. CHOICES FOR

EMPIRIC TREATMENT OF UTI

2014, RRMC ED: 579 Patients with E. coli isolated from urine

05

101520253035404550

% Resistance, E. coli • >20% R, do not use

empirically1

• NN: Good choice, but

cannot use if renal

impairment (elderly): 25%

of patients

• Cefazolin was a good

choice by 2009 BP, but not

by 2010 BP

CEFAZOLIN % SUSCEPTIBLE

WITH 2010 AND PRE-2010 BREAKPOINTS

54

69

11

72

81 83

0

20

40

60

80

100

E. coli (n=581) K. pneumoniae

(n=313)

P. mirabilis

(n=169)

% S

usce

pti

ble

"New" Cefazolin <=2

"Old" Cefazolin <=8

UCLA

M100 -S25 TABLE 1 A

Test/ Report

Group Agent

MIC Breakpoint (µg/ml) Comments

Susc Int Res

Cephems (Parenteral)

A Cefazolin ≤2 4 ≥8 based on dose of 2 g every 8 h

Cephems (Oral)

U Cefazolin* ≤16 - ≥32 Footnote (20)

ENTEROBACTERIACEAE CEFAZOLIN

* “surrogate” agent

CLSI M100-S25 Table 2A

• (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.

PRESCRIBING CEFAZOLIN VS. MIC

INTERPRETATION

IM, IV administration

Introduced 1973; today, mostly used for:

MSSA

Prophylaxis for some surgical procedures

If GNR known “S” (only E. coli, K. pneumoniae, P. mirabilis): Uncomplicated UTIs; bacteremia

M100-S25. Table 2A.

Reason for Testing Breakpoints (µg/ml)

Dose S I R

Predict cefazolin use for uUTI ≤16 - ≥32 1 g every 12 h

(IM or IV)

Predict cefazolin use for systemic

infections ≤2 4 ≥8

2 g every 8 h (IM or IV)

Surrogate for oral cephalosporins to

use for uUTI ≤16 - ≥32 PO (various)

UCLA EMERGENCY DEPT. CHOICES FOR

EMPIRIC TREATMENT OF UTI

2014, RRMC ED: 579 Patients with E. coli isolated from urine

05

101520253035404550

% Resistance, E. coli • >20% R, do not use

empirically1

• NN: Good choice, but

cannot use if renal

impairment (elderly): 25%

of patients

• Oral ceph was a good

choice by 2009 BP

ampicillin >32 R

cefazolin 8

ciprofloxacin >2 R

nitrofurantoin ≤16 S

trimeth-sulfa >4/76 R

SPECIMEN: URINE

DIAGNOSIS: CYSTITIS

E. COLI

MIC (µg/ml)

How should cefazolin MIC of 8 µg/ml be

interpreted and reported for treatment

of UTI?

ampicillin >32 R

cefazolin 8 S

ciprofloxacin >2 R

nitrofurantoin ≤16 S

trimeth-sulfa >4/76 R

SPECIMEN: URINE

DIAGNOSIS: RECURRENT CYSTITIS

E. COLI

MIC (µg/ml)

“Cefazolin results should only be used to predict potential

effectiveness of oral cephalosporins (eg, cephalexin) for

treating uncomplicated urinary tract infections.”

Final Report with

Optional Comment

Example 1

ampicillin >32 R

oral cephalosporins S

ciprofloxacin >2 R

nitrofurantoin ≤16 S

trimeth-sulfa >4/76 R

MIC (µg/ml)

“Oral cephalosporins include cephalexin,

cefpodoxime, cefdinir.”

Final Report with

Optional Comment

Example 2

SPECIMEN: URINE

DIAGNOSIS: RECURRENT CYSTITIS

E. COLI

ampicillin >32 R

cefazolin 8 R

ceftriaxone ≤0.5 S

ciprofloxacin >2 R

gentamicin 1 S

piper-tazobactam ≤ 8 S

trimeth-sulfa >4/76 R

SPECIMEN: BLOOD

DIAGNOSIS: PYELONEPHRITIS

E. COLI

MIC (µg/ml)

“Cefazolin susceptible results are based on a dose of

2 g every 8 h.”

Final Report with

Optional Comment

Example 3

Test urine isolates with Vitek 2 Test other isolates with broth microdilution E. coli, Klebsiella spp. and Proteus mirabilis

Report “oral cephalosporins” on urine isolates Report “cefazolin” on special request

Commercial Systems

Most not FDA cleared for new breakpoints (BPs) Some panels do not have low dilutions for systemic

breakpoints

Verify new breakpoints? urine? systemic? Determine when testing for systemic isolates needed

CEFAZOLIN – UCLA PROTOCOL

Cefepime:

Susceptible Dose Dependent

Breakpoints

ampicillin >32 R

cefazolin >32 R

cefepime 4

ciprofloxacin >2 R

ertapenem 0.5 S

gentamicin 2 S

piperacillin-tazo >128/4 R

trimeth-sulfa >4/76 R

SPECIMEN: BLOOD

DIAGNOSIS: BACTEREMIA

ENTEROBACTER CLOACAE

MIC (µg/ml)

How should cefepime MIC of 4 µg/ml be

interpreted?

Agent

Old Breakpoints New Breakpoints

Susc Int Res Susc S-DD Res

Cefepime ≤8 16 ≥32 ≤2 4-8 ≥16

ENTEROBACTERIACEAE

CLSI CEFEPIME MIC (µG/ML) BREAKPOINTS

The interpretive criterion for susceptible is based on a dosage regimen

of 1 g every 12 h.

The interpretive criterion for SDD is based on dosing regimens that

result in higher cefepime exposure:

higher doses

more frequent doses

both, up to approved maximum dosing regimens

New breakpoints – cover all dosage ranges outside the urinary tract

CLSI M100-S24

Cefepime

Prescribing

Information

Information Sandoz, Inc. 10/2011

Dosing Options

Plasma Drug

Concentrations

With higher dose, higher

concentration – use for

higher MICs

MIC (µg / mL) Dose

≤2 1 g Q 12hr

4 1 g Q 8hr

2 g Q 12hr

8 2 g Q 8hr

≥16 Resistant

Not prescriptive

Keep in mind inherent variability of MIC is ±1 log2 dilution

Must discuss with Antibiotic Stewardship Team (or equivalent….e.g., ID, Pharmacy, Infection Control)

Performance with AST systems largely unknown

Disk dif fusion?

DOSING REGIMENS USED TO

ESTABLISH S-DD

CLS M100-S25.

Appendices E & F.

S-DD Result

Obtained on

Repeat result by BMD (%) Repeat Result by Vitek 2 (%)

S S-DD R S S-DD R

Vitek 2 (n=39)

0 27

73 15 64

21

BMD (n=19)

11 68 21 73 21 6

CEFEPIME S-DD REPRODUCIBILITY?

• Many S-DD results on Vitek2 were R by BMD

• About one third of S-DD results did not repeat,

regardless of method

ampicillin >32 R

cefazolin >32 R

cefepime 4 S-DD

ciprofloxacin >2 R

ertapenem 0.5 S

gentamicin 2 S

pip-tazo >128/4 R

trimeth-sulfa >4/76 R

MIC (µg/ml)

Final Report with

UCLA Comment

“SDD (susceptible dose dependent) indicates the isolate is susceptible to cefepime, when doses of 2g Q8 hr are used, in patients with normal renal function. Consultation with antimicrobial stewardship for dosing recommendations is strongly advised.”

SPECIMEN: BLOOD

DIAGNOSIS: BACTEREMIA

ENTEROBACTER CLOACAE

ESBLs and AmpC

β-lactamases -

Enterobacteriaceae

AmpC and ESBL

ESBL (plasmid)

E. coli Klebsiella P. mirabilis

Other organisms

AmpC (chromosome)

Induction of AmpC β-lactamase

Selection of stably derepressed AmpC-R mutants

AmpC transfer to plasmid

No CLSI

endorsed test for

AmpC!

CLSI ESBL test

for E, K, P…

Agent Old Breakpoints Current Breakpoints

Susc Int Res Susc Int Res

Cefazolin ≤8 16 ≥32 ≤2 4 ≥8

Cefotaxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftizoxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftriaxone ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftazidime ≤8 16 ≥32 ≤4 8 ≥16

Cefepime ≤8 16 ≥32 ≤2 4 ≥8

Aztreonam ≤8 16 ≥32 ≤4 8 ≥16

ENTEROBACTERIACEAE - CEPHALOSPORINS

CLSI BREAKPOINTS (MIC µG/ML)1

M100-S25 Table 2A.

1 CLSI also revised corresponding disk diffusion breakpoints

REVISED CEPHALOSPORIN /

AZTREONAM BREAKPOINTS

Minimal impact on overall %S for each agent

Considerable reluctance to eliminate ESBL testing

Still not much data for “ESBL” isolates treated with

susceptible cephalosporin

More controversial than carbapenem

breakpoint changes!

“I or R” to one or more of: cefotaxime

ceftriaxone

ceftazidime

UCLA uses “ceftriaxone R

Enterobacteriaceae”

WHAT ABOUT INFECTION CONTROL RISK?

Agent Susc Int Res

Cefotaxime ≤1 2 ≥4

Ceftazidime ≤4 8 ≥16

Ceftriaxone ≤1 2 ≥4

Revised

breakpoint

s (µg/ml)

AMPC -LACTAMASES

AmpC Hydrolyze ceftriaxone, cefotaxime, ceftazidime, cephamycins

(cefoxitin) NOT cefepime

Not inhibited by -lactamase inhibitors

Chromosomal in SPICE species

“S” isolates can become R during therapy

E. coli, Klebsiella, P. mirabilis and Salmonella can acquire

ampC on plasmid

“SPICE” BUGS*

Serratia marcescens

Pseudomonas aeruginosa

Indole positive Proteeae –

Morganella morganii & Providencia spp.

Citrobacter freundii

Enterobacter spp.

Concern – de-repression of AmpC = resistance develops on therapy

*Also Hafnia alvei, Aeromonas spp., Acinetobacter baumannii and certain non-

fermenting gram-negative rods

No testing for AmpC (or ESBL)

Use current breakpoints

Report:

ceftriaxone for E. coli / Klebsiella / P. mirabilis

cefepime for SPICE organisms (poor inducer of AmpC)

IF doctor calls, report ceftriaxone with disclaimer

“Due to the high likelihood of selecting resistant mutants, ceftriaxone for serious infections caused by Enterobacter spp. may result in high level resistance”

UCLA APPROACH

Carbapenemase Detection

UCLA, 2009

“We have a

weird-looking

Klebsiella

pneumoniae

on bloods”

32

BMD AST RESULTS: KLEBSIELLA

PNEUMONIAE

Agent

Amikacin 32 I

Aztreonam, cefepime, cetazidime, ceftriaxone

>32 R

Ciprofloxacin >2 R

Ertapenem >16 R

Gentamicin, tobramycin >10 R

Imipenem >16 R

Meropenem >16 R

Minocycline >32 R

Piperacillin-tazobactam >128 R

Trimeth-sulfa >4/76 R

Tigecycline 2 S

Colistin >16 ?

Isolate MHT+

33

“This isolate has unusual

carbapenem resistance results.

Infectious Diseases consultation

strongly suggested”

WHY ARE CRE IMPORTANT?

Clinically important

Often resistant to multiple

classes of antibiotics

Pan-resistant CRE have been

described (several cases at

UCLA)

Associated with high mortality

rates (up to 70%)1

May be > 50% in ICU patients

Combination therapy appears to

improve outcomes

Antimicrobial % susceptible

Carbapenems

Meropenem

Imipenem

Ertapenem

2

0

0

Cephalosporins

Ceftriaxone

Ceftazidime

Cefepime*

0

1

8

Piperacillin-Tazobactam 0

Aminoglycosides

Gentamicin

Amikacin

Tobramycin

58

64

2

Fluoroquinolones

Ciprofloxacin

Levofloxacin

8

8

Trimethoprim-

sulfamethoxazole

22

* Using M100-S23 breakpoint

UCLA Data, n=90 CRE (2013)

34

WHY ARE CRE IMPORTANT?

Epidemiologically important

Highly transmissible

Have spread throughout healthcare settings across the United States,

(endemic in some areas)

Potential for CRE to become widespread if not contained

PR

AK

HI

States with CRE confirmed by CDC, 2013 35

REVIEW: MECHANISMS OF BETA-LACTAM

RESISTANCE IN ENTEROBACTERIACEAE

37

CARBAPENEMASES Class Examples Produced by: Notes

A

ESBLs [TEM, SHV, CTX-M] KPC carbapenemases

SME carbapenemases

K. pneumoniae and other Enterobacteriaceae

S. marcescens

Most inhibited by clavulanic acid Usually plasmid-mediated (not SME)

B

Metallo-β-lactamases (MBL) (e.g. NDM, VIM, IMP, GIM, SPM carbapenemases)

P. aeruginosa

Enterobacteriaceae

Acinetobacter

S. maltophilia

Inhibited by EDTA Do not hydrolyze aztreonam

C AmpC Enterobacteriaceae Some non-fermenters

Inducible in some genera Not inhibited by clavulanic acid

D

OXA carbapenemases (e.g. OXA-48, -181, -232)

Acinetobacter baumannii

Enterobacteriaceae

Hydrolyze carbapenems to some degree

Adapted from Queenan & Bush. 2007. Clin Microbiol Rev. 20:440.

Bush & Jacoby. 2010. AAC. 54:969; Bush, K. 2013. Ann NY Acad Sci 1277:84. Slide from Janet Hindler

WHAT IS A CRE?

- Any Enterobacteriaceae or just Klebsiella

pneumoniae?

- Resistant to any carbapenem, all carbapenems,

select carbapenems?

- Resistant vs. not-susceptible?

- Must have a carbapenemase?

38

WHAT IS A CRE?

Current CDC Surveillance Definition

Enterobacteriaceae that are:

Resistant to one or more of the following: doripenem, ertapenem, meropenem, or imipenem* OR

Production of a carbapenemase detected by PCR. MHT, Carba-NP, metallo-beta-lactamase test

UCLA Clinical Definition

Enterobacteriaceae that are:

Intermediate OR resistant to one or more of the following: doripenem, meropenem, or imipenem*

Remember! Not all CRE

have a “carbapenemase” 39

*Proteus/Providencia/Morganella exceptions for imipenem

MHT Carba NP Molecular

Use Enterobacteriaceae Enterobacteriaceae

P. aeruginosa

Acinetobacter

Enterobacteriaceae

P. aeruginosa

Acinetobacter

Strengths Simple Rapid Determines type of

carbapenemase

Limitations Some false pos (eg,

ESBL/ampC + porin)

Some false neg

(eg NDM)

Enterobacteriaceae

only

Special “fresh”

reagents

Some invalid results

False neg for OXA-

type carbapenemase

Special reagents

Specific to targeted

gene

Introduction to Tables 3B and 3C. Tests for

Carbapenemases in Enterobacteriaceae, Pseudomonas

aeruginosa, and Acinetobacter spp.

CLSI M100-S25.

Test for suspected carbapenemase production in

Enterobacteriaceae, P. aeruginosa, Acinetobacter spp.

When to do this test:

“For epidemiological or infection control purposes. NOTE: no

change in interpretation of carbapenem susceptibility results is

required for Carba-NP positive isolates. Such testing is not

currently recommended for routine use .”

p. 120 M100 S25

CARBA NP TEST

CARBA NP TEST

Isolated colonies (lyse)

Hydrolyse imipenem if

carbapenemase present

Change in pH of indicator

(red to yellow/orange)

Rapid <2h

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.

NO

imipenem

+

imipenem

CARBA NP TEST

MATERIALS/REAGENTS

Solut ions to prepare:

10 mM Zinc sul fate heptahydrate

Phenol red solut ion

0 .1 N NaOH

Carba NP Solut ion A

(phenol red + z inc solut ions)

Carba NP Solut ion B

(Carba NP Solut ion A + imipenem)

Reagent prep

takes time!

UCLA uses current CLSI carbapenem BP, no routine carbapenemase detection per formed

Hospital epidemiology and laboratory noticed uptick in number of invasive CRE cases

Tradit ional epidemiological tools cannot identify source

UCLA postdocs per form whole genome sequencing on three CRE from UCLA patients

Questionable if unit-based transmission

PFGE results inconclusive

Two isolates are KPC… but one is an OXA-232!!

UTILITY OF CARBAPENEMASE TESTS:

UCLA STORY

Shaun Yang and Peera Hemarajata

OXA-232 PHENOTYPE

Neg KPC OXA48 OXA232

Courtesy of:

Shaun Yang

P. Hemarajata

Neg KPC OXA232

Carba-NP Negative MHT Positive

Ertapenem >16 μg/mL

Meropenem >16 μg/mL

Imipenem 2 μg/mL

- Screen all CRE for this phenotype

- Confirm with LDT PCR for OXA-232

Courtesy of:

Shaun Yang

P. Hemarajata

Screen CRE from collection

34 isolates January 2014– January

2015

Identify source patient (happened

to be patient whose isolate was

sequenced by NGS)

7 additional patients with OXA -232

isolates identified (all identical by rep -

PCR)

Duodenoscope transmission

Revise steri l ization method for

duodenoscopes

UTILITY OF CARBAPENEMASE TESTS:

UCLA STORY

0

1

2

3

4

5

6

Nu

mb

er

of

Pa

tie

nts

wit

h C

RE

KPC NDM-1/SME OXA-232 None

Cefazolin can be used as a surrogate agent to predict the

activity of the oral cephalosporins for the treatment of uUTI

caused by E. coli, Klebsiella, P. mirabilis

Cefepime susceptible dose-dependent breakpoints were

added to M100 in 2014 – implementation will depend on your

institution

ESBL testing is no longer required if current cephalosporin /

aztreonam breakpoints are used

SUMMARY (1)

Carba-NP was introduced for detection of carbapenemase

producing Enterobacteriaceae, P. aeruginosa and

Acinetobacter spp., this is for epidemiological testing

SUMMARY (2)