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GNRs

Enterobacteriaceae

Gram negatives

Pseudomonas,StenotrophomonasAcinetobacter baumannii

(ferment glucose)

(non-fermenters)

Non-enteric, fastidious

Salmonella

E.coliProteusKlebsiella

ESBL KlebsiellaESCPPMs

Enterobacter

CitrobacterSerratia…

HaemophilusHACEK, Others

>community

> hospital



Bacterial resistance mechanisms

x

x

x

x xx x

PorinOM

Cell wall

PBPs

n↓entry or active effluxBroad substrate specificity of pumps. Low level crossresistance. Overexpression of pumps -HLR

50SRibosome

DNA

nmodified target

n inactivating enzymes(acquired or mutated)

eg. β-lactamases

n Intrinsic or acquired

n Chromosomal or plasmid

30S

x



What determines antibioticsensitivity ?

ie.

The relationship between the bacterialMIC and the achievable antibiotic

concentration

orThe presence of a resistance

mechanism located on the bacterialchromosome or plasmid



What determines antibiotic sensitivity ?

MICrange:

A/b AA/b AA/b CA/b C

bacterial MIC vs. antibiotic blood/tissue levels

(eg. S.pneumoniae )

A/b BA/b B

0.10.1 1.01.0 10 mg/L10 mg/L

Chromosomal or plasmid

or

the presence of a resistance factor (eg. ESBL)



Nos (%) of isolatesSusceptibility Breakpoints

S SS R

MIC (mg/L): 1 2 4 8

Sensitive

population

Serum Concn-based

Resistantpopulation

Population -based

Resistantpopulation

R R



6.2

31.533.6

13.1

4.9

20.5 0.2 0.9 0.8

6.4

0

5

10

15

20

25

30

35

40

45

0.01 0.03 0.13 0.5 2 8

Ceftriaxone &. Ceftazidime susceptibility - AGAR 1996

3.3

24.4

40

15.7

6.1

1.5 1.4 1.4 0.9

5.3

0

5

10

15

20

25

30

35

40

45

0.01 0.03 0.13 0.5 2 8

%

K.pneumoniae

n=657

Ceftriaxone

Ceftazidime

1.9%

3.7%



Terminology

Plasmid: Extra-chromosomal usually circular DNA.Plasmids are often conjugative, can promote theirtransfer between bacterial hosts. Plasmids carry ‘R’genes, often organized into integrons or on transposons.

Transposon: A mobile unit of DNA that can jump, ortranspose, from one DNA to another - eg, from plasmidto chromosome or plasmid to plasmid, usually withoutsite specificity.

Integron: Unit of variable length DNA containing a genefor a site-specific conserved integrase (intl), genecassettes, and a recombination site (attl), into whichgene cassettes (R genes) can be integrated. Promotersensure that integrated genes are efficiently expressed.Integrons can be part of a transposon. Class 1 integrons



Narrow-spectrum Class A β-lactamases

TEM-1– Ampicillin / (Ticarcillin)-R

– E. coli , H. influenzae, N. gonorrhoeae

TEM-2 (like TEM-1) SHV-1

– Ampicillin-R in K. pneumoniae

β-lactamase inhibitors & expandedspectrum β- lactams active



Emerging Gram-negative Resistances

Extended-spectrum β-lactamases (ESBLs)

Chromosomal cephalosporinases (AmpC)

eg. Enterobacter Plasmid mediated AmpC

Carbapenem resistance

Fluoroquinolone resistance ‘paradigm’ multi-drug resistant organisms

(eg. A.baumannii, P.aeruginosa )

Beta-lactamase negative ampicillin-resistant(BLNAR) Haemophilus influenzae



0

5

10

15

20

1 9 8 9

1 9 9 0

1 9 9 1

1 9 9 2

1 9 9 3

1 9 9 4

1 9 9 5

1 9 9 6

1 9 9 7

1 9 9 8

1 9 9 9

2 0 0 0

Non-ICU

ICU

Antimicrobial Resistance - Hospital-onset Infections

ESBLK.pneumoniae

% R

National Nosocomial Infections Surveillance (NNIS) System



ESBLs Hospital – primarily TEM / SHV derived

(K.pneumoniae, E.coli , Enterobacter) Community emergence – CTX-M (E.coli ) Inhibited by clavulanate Co-resistance: genta, tmp, quinolone

– Useful in Enterobacter in predicting ESBL– Multiresistance less common in CTX-M

Carbapenems & cephamycins (cefoxitin) active

↓ detection if 3’GC MICs low - screening rules

Infection Control challenge in LTCF & hospital –isolation ? Long-term faecal carriage (LTCF) ?



ESBLs

(based on molecular phylogeny)

2a-2f

Ambler Classification:

EnterobacterSerratiaC.freundii

K.Pneumoniae E.Coli

Enterobacter sp.

A.baumannii Bacteroides,Aeromonas Flavo,Chryseobacterium

Stenotrophomonas IMP, VIM, GIM



likelihood of routine surface surveillance

swabs to detect ESBL & MRSA carriage

ESBL MRSAR

detectable not detectable

GIT, urinary tract, wounds wounds



CAZCTX CLA

AZT

ESBL Detection - Jarliers DDST

CAZ > CTX

TEM ESBL



CAZCAZ

CTXCTX

CLACLA

CROCRO

AZT AZT

CTX-M ESBL CTX > CAZ

ceftriaxone

cefotaximeceftazidime

aztreonam



Etest for ESBLs

Cefotaxime

Cefotaxime + clavulanate



E.cloacae

with ESBL

Cefoxitin

Ceftazidime

Ceftriaxone

Aztreonam

Amox/Clav



CTX CAZ

CTX+CA CAZ+CA

FOX



-1

1

3

5

7

9

11

13

15

J u l - 9

4

O

c t - 9 4

D

e c - 9 4

F e b

- 9 5

A

p r - 9 5

J u n

- 9 5

A u g

- 9 5

O

c t - 9 5

D

e c - 9 5

F e b

- 9 6

A

p r - 9 6

J u n

- 9 6

A u g

- 9 6

O

c t - 9 6

D

e c - 9 6

F e b

- 9 7

A

p r - 9 7

J u n

- 9 7

A u g

- 9 7

O

c t - 9 7

D

e c - 9 7

F e b

- 9 8

A

p r - 9 8

J u n

- 9 8

A u g

- 9 8

O

c t - 9 8

D

e c - 9 8

Presumed ESBL (Gent/Tmp res) ESBL confirmed

Enterobacter cloacae Concord Hospital 1994-1998

No.

Unrecognised ESBLs Outbreak recognised(all +ves isolated)



0

20

40

60

80

100

120

140

2000 2001 2002 2003 2004 2005 2006

E.coli

E.cloacaeK.pneumoniae

All ESBL isolates - per patient /month



Inducible cephalosporinase - AmpC

cephalosporinase (>pen) encoded by amp C cluster

natural chromosomal (Class C) enzymes, low level

not inhibited by clavulanate

controlled by repressor protein (ampR )

produced by most GNRs, except Klebsiella, P.mirabilis,Salmonella sp. Present but attenuated in E.coli

inducibility common in ‘ESCPPM” complex

inducible by ß-lactams, clavulanate, (bind ampR )reversible

“stably derepressed” (mutation) high level production– constitutive - non-reversible

– R to cefoxitin (cefotetan), unlike ESBLs



Double disc antagonism for inducible AmpC

Cefoxitin Ceftazidime



AmpC -lactamase

Inducible -lactamases

Enterobacter cloacae / E.aerogenes

Serratia marcescens

Citrobacter freundii

Providencia species Proteus vulgaris

Morganella morganii

Hafnia alvei Pseudomonas aeruginosa



Enterobacter cloacae

MER IMP CTX CAZ CPM MER IMP CTX CAZ CPM

>128

64

16

4

1

0.2

0.06

<0.01

Inducible phenotype derepressed phenotype

MICug/ml

= modal MIC

Canton R, Hosp Ramon y Cajal, Madrid



AmpC - Activity of Cephalosporinase

Activity varies. Enzyme levels 10 x less inMorganella, Proteus, Providencia

Activity of inhibitors varies, mostly R tazobactam active v. Morganella , Providencia clavulanate (not tazo) inhibits Proteus (2e) aztreonam active v. Morganella and Providencia clavulanate, cefoxitin, carbapenems AmpC

inducers sulbactam and tazobactam poor inducers inhibitor combinations affected - 3’GC

reporting: report as R vs do not report



Treatment options for ESBLs and ESCPPMs

33’’GG cephalosporinscephalosporins –– ––

CefepimeCefepime (4(4’’GC)GC) –– ++++

QuinolonesQuinolones +/+/–– ++++

Piperacillin/tazobactamPiperacillin/tazobactam ++ +/+/––

CarbapenemsCarbapenems ++++++ ++++++

CoCo--trimoxazoletrimoxazole –– ++

Antibiotic E.S.C…ESBLs



0

1

2

3

4

5

6

0 . 0 2 3

0 . 0 4 7

0 . 0 9 4

0 . 2 5

0

. 5 1 2 4 8 1 6 4 8 6 4

1 2 8

2 5 6

Cefotaxime MIC <0.5 mg/L ESBL -ve (n=16)

Cefotaxime MIC >2 mg/L ESBL-ve (n=14)

Cefotaxime MIC >2 mg/L ESBL+ve (n=21)

Cefepime E-test MICs: Enterobacter cloacae

‘UNINDUCED’

‘INDUCED’ESBL

Gottlieb T, Wolfson C. JAntimicrob Chemother. 2000



Inoculum Effect: Clinical failure ?

Cefepime, against SHV-ESBL K. pneumoniae

MIC (mg/L)

Ceftazidime Cefotaxime Cefepime

Strain 105 107 105 107 105 107

SHV-5 1024 1024 64 64 32 256

SHV-12 128 256 8 8 32 64

SHV-2+/2a 128 256 8 32 8 512

Bedenic B et al. Clin Microbiol Infect 2001;7:626-35.



Klebsiella oxytoca

K1 hyper-production

– Klebsiella oxytoca, Citrobacter diversus

– chromosomal not plasmid– ceftriaxone R, cefotaxime I/R

– ceftazidime S

susceptible to ß lactamase inhibitors

usually susceptible to cephalosporins



E.coli, Klebsiella & Enterobacter resistance

ICU multicenter study, 1999-2000

Friedland I. Inf Cont Hosp Epid 2003;

20.559.735.9Ciprofloxacin20.357.434.1Gentamicin

36.640.927.2Pip-tazo

12.635.117.3Cefepime

2.514.97.7Amikacin

1.51.21.5Imipenem

ceftazidime Rn = 601

ceftazidime Rn = 470

ESBL +n = 1120

EnterobacterE.Coli & Klebsiella sp



Plasmid mediated AmpCs

Plasmid mediated AmpCs AmpC genes mobilised from chromosomes

eg. CMY from Aeromonas, C.freundii ; MIR from E.cloacae

As mobilised to plasmid, not inducible by β-lactams In E.coli - resistance profile of β-lactam-R Enterobacter. Klebsiella, Salmonellae, P.mirabilis if 3’GC R, Fox R / Clav-

In US, 3-4% of Klebsiella sp cefoxitin R due to plasmidAmpC. Associated clinical failures with 3’GCs

If testing CTX, CAZ, co-existence of ESBLs masked byampC . Cefepime will aid detect both.

Detection - Boronic acid, AmpC disc tests; PCR



AmpC inhibition using Boronic Acid

1. Coudron PE. J Clin Microbiol 2005; 43:4163-41672. Yagi T. J Clin Microbiol 2005; 43:2551-2558

CAZ + CLA > CAZ

CTT + BOR = CTT CTT + BOR > CTT

CAZ + CLA = CAZ

CTT

CAZCAZ + CLA

CTT

CTT + BOR

CAZCAZ + CLAESBL+ AmpC+

30µg + 400µg3030µµgg + 400+ 400µµgg

CTT + BOR

A C di t t f E li i l t ith l id A C



AmpC disc test for non E.coli isolates with plasmid AmpC

1. Black JA et al. J Clin Microbiol 2005; 43:3110-3113

AmpC disc = EDTA + testisolate

Cefoxitin S E.coli



AmpC multiplex PCR

AmpC β-lactamase –

molecular detection

Perez, Hanson

J. Clin Micro 2002: 40:2153-2162



Cefepime >0.5

Cefoxitin >8g/ml

Confirmed

…Any of above

Aztreonam >8

Ceftazidime >1

Ceftriaxone >1mg/L

18.1%

Enterobacter spp.6.6%3.0%

plasmid-borne AmpC ß-lactamase production

5.4%1.2%

8.8%3.7%

6.9%2.0%

5.3%2.7%

8.0%2.2%

K.pneumoniae E.coli

AGAR 2004 GNR Survey - presumptive/confirmed3rd Gen Cephalosporin resistance



Carbapenem Resistance

2 major mechanisms1. Failure of antibiotic penetration

– Porin channel mutations– Efflux pumps

2. Carbapenem hydrolysing enzymes– Metallo-β-lactamases (MBL)– Oxacillinases (OXA)

– Other Serine carbapenemases3. Combined



Serine carbapenemases

Class A

– KPC (USA) - low level carbapenem resistance

– Poorly detected by automated methods– Inhibited by clavulanic acid

– Klebsiella, Salmonella. Outbreak in NYC

Class D– Chromosomal; +/-integron

– OXA-23, OXA-24, OXA-51, OXA-58..

– Low level carbapenem resistance

– Acinetobacter baumannii



metallo-β-lactamases (MBL)

Class B: IMP, VIM, (SPM, GIM, SIM) Worldwide, mostly SE Asia & Europe Gene cassettes in Class 1 integrons,

plasmid integration increases mobility. Hydrolyze all β-lactams exc. aztreonam Inhibited by EDTA - metal (Zn2+) chelator Expression of resistance variable

– A.baumannii , P.aeruginosa, (higher MICs)– Enterobacter, Serratia (lower MICs)

Spread parallels ESBLs in mid 1980s



MBL (Class B) E. cloacae MICs (mg/L)

Yan et al., JAC 2002, 50, 503

MEM IMP AZT CTX CTZ CFM

R947 IMP-8

TEM-1

1 2 0.03 >256 >256 32

Y580 IMP-8

TEM-1

0.5 2 0.02 16 128 32

T524 IMP-8

TEM-1

1 4 0.03 32 >256 32

N947

C. freundii

VIM-2 0.5 1 0.06 32 64 16

MEM IMP AZT CTX CTZ CFM

R947 IMP-8

TEM-1

1 2 0.03 >256 >256 32

Y580 IMP-8

TEM-1

0.5 2 0.02 16 128 32

T524 IMP-8

TEM-1

1 4 0.03 32 >256 32

N947

C. freundii

VIM-2 0.5 1 0.06 32 64 16



Problems detecting MBLs

Carbapenem MICs vary from 0.5 to 8 mg/L.Discs often borderline

Gentamicin resistance freq. present Timentin resistant, pip-tazo can be variable

Differentiating forms of resistance in

P.aeruginosa, A.baumannii Multi-R A.baumannii with OXA may show

increased ZD with EDTA and imipenem, but

is usually <4mm

MBL S i



MBL+ Serratia marcescens

Imipenem

DDST +ve

Imipenem + EDTA

Large inhibition zonearound aztreonam

Blank disc +EDTA

Imipenem



DDST positive

Blank disc

with EDTA

K.pneumoniae MBL+ve and ESBL +ve



p

DDST +ve

Sensitivity to ATMrestored byclavulanate

Increase in zone diameter

by addition of EDTA

P d i MBL



Pseudomonas aeruginosa MBL+

Intrinsic partial

resistance toaztreonam

Imipenem

Imipenem + EDTA

Synergy not visible

A i t b t b ii



Acinetobacter baumannii

Imipenem

Imipenem+ EDTA

Small increasein ZD Aztreonam

Blank disc

with EDTA

Imipenem

OXA-type carbapenemase+ve MBL-ve

E cloacae with MBL and de repressed AmpC



E.cloacae with MBL and de-repressed AmpC

Blank discwith EDTA

Imipenem

Imipenem

Imipenem +

EDTA

Aztreonam



CAZ CAZ + EDTA

IMP + EDTA

IMP

IMP + EDTA

IMP



FOX CEF

CEF

+ CLAV

CAZ

CTX

CAZ + CLAV

CTX + CLAV

=

=

FOX = R

synergy

E cloacae MBL+ ESBL+



E.cloacae MBL+ ESBL+

3 strainsMICs: 0.5, 2, 64

EDTA

IMI + EDTA IMI

MER

AZT

CLAV



Real time PCR:IMP-4 metallo-β-Lactamase detection

Patient first admitted 19/9/2007



Patient first admitted 19/9/2007

+1SCVC22/10/07 *

+4RBlood23/10/07

+2SBlood19/10/07

+<=0.25SEnvir31/10/07 **

+>=16RSputum28/10/07

+1SCVC15/10/07

Imp-4RT PCR

Vitek MeroMIC g/mlMer 1SiteDate

* First detected ** ICU dressing trolley



ESBLs

Class CClass CAmpCAmpC

33’’GG CephCeph RR

Class AClass ATEM, SHV,TEM, SHV,

VEB, PERVEB, PERKPCKPC

ESBLsESBLs

Class BClass BIMP, VIM,IMP, VIM,

SIMSIM

Class DClass DOXAOXA

CarbapenemCarbapenem RR CarbapenemCarbapenem RR

Disk tests for Gram Negative MROs



Adapted fr. Jacoby et al. J Clin Microbiol 2006. 44:1971-1976


ESC, P.aeruginosa

A.baumannii

YesS

R

NoRNo+Carbapenemase(MBL) class B

(IMP, VIM)

E.coli, K.pneumoniae

P.mirabilis,Salmonella

NoSYesRNo+(plasmid) AmpC(CMY, MOX)

E.coli, ESCPPM,

K.pneumoniae

NoSNoSYes+ESBL(TEM, SHV, CTX)

EDTAsynergy

IPMAPB(Boronic

acid)

FOXScreen

Clavulanatesynergy

>5mm

CAZor CTX

Screen

ββββ-lactamase




Jacoby et al. J Clin Microbiol 2006. 44:1971-1976


YesRNoRNo++Metalloenzymes

Yes

SNoRNo+-Carbapenemaseclass B(IMP, VIM)

NoSNoR Yes++

NoSNoS Yes+-Carbapenemaseclass A(KPC)

NoR/ SNoRNo++E.coli,K.pneumoniae

NoS YesRNo+-(plasmid) AmpC(CMY, MOX)

NoSNoR Yes++E.coli, ESCPPM,K.pneumoniae

NoSNoS Yes+-ESBL(TEM, SHV, CTX)

EDTAsynergy

IPMAPB(Boronicacid)

FOXScreen

Clavulanatesynergy>5mm

CAZor CTXScreen

Porinloss

ββββ-lactamase



multiple β-lactamases within organisms

complicates phenotypic resistance detection

– false -ves & false +ves. molecular methodologies needed to detect

ESBLs, AmpCs, & esp. metallo-β-lactamases.

Quinolone-resistant P aeruginosa



Quinolone resistant P.aeruginosa

NNIS data, 2002

0

5

10

20

30

25

15

89 90 91 92 93 94 95 96 97 98 99 00

Resistance (%)

ICU

Non-ICU

Year

Targets of Quinolone Action



Targets of Quinolone Action

QuinolonesQuinolones bind tobind to gyrasegyrase andand topoisomerasetopoisomerase,,nuclear enzymes inhibiting DNA replicationnuclear enzymes inhibiting DNA replication

Quinolone resistance (chromosomal): mutations of target enzymes — ( gyrA and parC,). mutation in regulator genes for efflux

transcriptionDNA replication

Supercoiling Decateination(segregation)

Gyrase Gyrase Topo IV

DNADNA

Plasmid mediated quinolone resistance



asm m at qu no on r s stanc

qnrA, qnrS – acquired resistance genes.

Protect bacterial DNA from quinolone binding.

Natural host: Shewanella algae .

In Enterobacteriaceae (Klebsiella, Citrobacter,E.coli, Enterobacter ) . 3-5% China, Korea, UK

Gene expression variable, supplements otherforms of quinolone resistance

Association between qnr genes and ESBL genes

Not detectable by current phenotypic methods

Other MDR Gram-Negatives:



Other MDR Gram Negatives

Acinetobacter baumannii

β-lactam resistance (non carbapenem)



β lactam res stance (non carbapenem)

Intrinsic AmpC-type cephalosporinase

– Base level does not reduce β-lactam activity

– Upstream promoter sequences (ISAba1) assoc. with ceftazidime resistance

Acquired

Overexpression multidrug efflux pumps

Reduced permeability (porin mutations)

– Also may result in carbapenem resistance ESBLs

(outbreaks: VEB-1 France; CTX-M-2 Japan)

Carbapenem resistance



p m

Intrinsic (chromosomal) Oxacillinases – OXA 51/69-like variants Low level expression; in A.baumannii Share weak identity with other oxacillinases Those with ISAba1 adjacent to bla OXA-51

carbapenem resistant

Prob. minor role in carbapenem resistanceAcquired Class D: OXA enzymes (CHDLs) Class B: MBLs:

Turton, J. FEMS Microbiology Letters 258 (1), 2007: 72–77



Brown S, Amyes S. JAC 2006:57:1-3

Subgroup 3

Subgroup 2

Subgroup 1

unrelatedCarbapenemCarbapenemresistantresistant

Serine carbapenemases - class D



Oxa-23 - Aus, UK, Polynesia, France, Brazil, Singapore …

Oxa-40, 24 - Spain, USA;

Oxa-58 - France, Italy, Greece, Singapore…..

most specific to A.baumannii (unlike MBLs). Low level resistance, Upstream IS (ISAba1 ) acts as promoter for

acquisition & expression of OXA-23. plasmid (-23, -58); chromosomal (-24) - transfer carbapenem resistance

Poirel, L, Nordmann P. Clin Microbiol Infection 2006; 12:826-36

Class B metallo-β-lactamases (MBL)



( )

Hydrolyze all β-lactams exc. aztreonam

Inhibited by EDTA - Zn2+ chelator

IMP (-1-2; -4-6;-11), VIM (-1-2), SIM (-1) Expression of resistance variable

– A.baumannii , P.aeruginosa, (higher MICs)

– Enterobacter, Serratia (lower MICs)

On class 1 integrons, on mobile plasmids

Asia, Western Europe Spread parallels ESBLs in mid 1980s

Acinetobacter baumannii - summary:



Numerous OXA enzymes confer β-lactam resistance OXA carbapenemases do not hydrolyze carbapenems robustly. In presence of promoter IS element, can result in

carbapenem R.

Outer membrane protein (OMP porin) changes reducetransport into periplasmic space. Access to PBPs is reduced,and weak β-lactamase activity is amplified.

Many OXA β-lactamases found as part of integrons

IMP and VIM type metallo–enzymes reported in A. baumannii. Many found in class 1 integrons as part of transposons Quinolone resistance may be due to mutations in efflux

regulation genes or to mutations of target enzymes—

topoisomerases II and IV (encoded by gyrA and parC ). Plasmid-mediated quinolone resistance not yet detected in

A.baumannii

Other MDR Gram-Negatives:



Pseudomonas aeruginosa Beta-Lactam resistance

– AmpC chromosomal beta-lactamase

– PSE-1, -3, -4, occasionally ESBL (e.g., OXA-enzymes), Grp. 3 metallo-enzymes

– OMP modifications - OmpD

Aminogycoside resistance - inactivatingenzymes, OMP modifications

Fluroquinolone resistance - gyrA MDR - Mex B, D, F efflux - cephs, FQ

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