PROFESSOR PETER M. HAWKEY

Health Protection Agency West Midlands Public Health Laboratory, Heart of England

NHS Foundation Trust, B5 9SS

The University of BirminghamEdgbaston, B15 2TT

p.m.hawkey@bham.ac.uk;peter.hawkey@heartsol.wmids.nhs.uk

ESBLs: From Soil to Suppuration

BSAC Spring MeetingTuesday 7th April 2009

International Convention Centre, Birmingham

• I have called this principle, by which each slight variation, if useful, is preserved, “Natural Selection”

• Resistant bugs live

• Sensitive ones die

‘The plough is one of the most ancient and valuable of man’s inventions; but long before he existed, the land was in fact regularly ploughed, and continues to be ploughed by earthworms’

Charles Darwin, 1881

Proportion of 3rd generation cephalosporins resistant to E. coli isolates, EARSS data

20062001

Taken from Canton and Coque, Curr Opin Micro 2006, 9: 466-475

Endemicity of CTX-M

The CTX‐M pandemic, how did it all start . . . ?

1989, Paris, Institut Gustave Roussy

Cancer patient isolate of E. coli

Resistant to cefotaxime, ceftazidime; sensitive to cefoxitin

Transferable 85 kb plasmid

Called MEN‐1

Bernard, et al, 1992, JAC, 29:590

The plot thickens . . .

• MEN‐1 shown to be identical to CTX‐M‐1 described in Germany from an isolate of E.coli 1989a

• CTX‐M‐2 described in S. typhimurium from Argentina in 1992b

• CTX‐M‐2 then foundc

Israel (1992)Paraguay (1994)Argentina (1994) E.coli / P. mirabilis

K. pneumoniae

a Bauernfeind, et al, 1992, Infection, 18:294b Bauernfeind et al, 1992, Infection, 20:158

c Bauernfeind, et al, 1996, AAC, 40: 509

CTX-M-1 Group

CTX-M-2 Group

CTX-M-9 Group

CTX-M-8/25/26 Group

CTX‐M like β‐lactamases in Kluyvera

K. ascorbata KLUA‐1

K. cryocrescens KLUC‐1

K. georgiana KLUG‐1

Poirel, et al, AAC, 46, 4038

By producing root hairs, plants increase

root length and surface area

Organisms no./g soil Biomass g/m2

Bacteria 108 – 109 40 – 500

Actinomycetes 107 – 107 40 – 500

Fungi 105 – 106 100 – 1,500

Algae 104 – 105 1 ‐ 50

Soil microflora

Extra-radicle hyphal development is less extensive in AM fungi than in EM fungi

The principle benefit of mycorrhizae is a large enhancement of the absorptive surface area of the root system. The extensive network of gossamer mycorrhizal hyphae can increase the root's effective surface area by several orders of magnitude. In fact, several kilometers of hyphae have been found in a liter of soil.

Diffusion shell: volume of soil around root that is depletedin nutrient concentrations due to uptake

Ions that diffuse rapidly (‘mobile nutrients’) have diffusion shells with larger radii than ions that diffuse slowly (‘immobile nutrients’)

Mobilisation of blaCTX-M

• ISEcp1 – IS1380 family mobilises by one ended transposition.blaCTX-M 1, 3, 10, 14, & 15

• Class I integron associated ISCR1 rolling circle transposition, not as a cassetteblaCTX-M 1, 14 & 9

• Bacteriophage related sequences have been found adjacent to

Genetic environment of blaKLUA-1 gene encoding the β-lactamase produced by K. ascorbata strain CIP 82.95T.

Humeniuk, C., et al, 2002, AAC, 46:3045-9

Schematic map of the potential structure in natural plasmid pILT-3 of K. pneumoniae ILT-

3

Poirel, et al, AAC, Jan 2005, 447-50

Schematic map of the complex class 1 integron carrying the blaCTX-M-14 gene

on plasmid pAJE0508

Bae, et al, AAC, Aug 2007, 3017-19

Hospital Community

00s

90s

80s

60s/70s

CTX-M

Kluyvera

CTX-ME

CTX-M EEK

TEM/SHV

TEM/SHV

E/K

EKE E.coli Klebsiellae

TEM/SHV ESBL

K E

TEM/SHV ESBL K

Mutation Gene Transfer

Unexpected things can happen with sex in bacteria!

• Evolution of ESBLs by mutation of existing β‐lactamase genes

VERSUS

• Importation of β‐lactamase gene giving ESBL spectrum from Kluyvera spp. (environmental bacteria)

17th July 2004:broadsheets discover CTX-M

0

5

10

15

20

25

90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06

Cipro

3 gen ceph

both

% R

esis

tant

E. coli from blood & CSF susceptibilities, HPA data

0

2

4

6

8

10

12

14

% positive

2001 2002 2003 2004 2005 2006year

ESBLESBL CTX-M

BSAC UK bacteraemia susceptibility surveyE. coli resistance

www.bsacsurv.org+ (226) (230) (228) (228) (227) (223)

+ number of strains

Proportion of CTX-M+ve Strains of Total Resistant to 3GC’s isolated from Hospital and

Community, Birmingham UK.

050

100150200250300350

1999

2000(Jul-Dec)

2001

2002

2003

2004

Total number ofisolates resisant to3GC

Proportion positivefor CTX-M

0

20

40

60

80

100

19992000 (Jul-Dec)

20012002

20032004

YEAR

% C

TX

-M p

ositi

ve E.coli

K pneumoniae

K. oxytoca

Enterobacter sp

Distribution of CTX-M genes in different species of Enterobacteriaceae from clinical samples from

hospital and community – Birmingham, UK

RAPD genotyping Birmingham CTX-M 15 E.coli

The ST131 E. coli clone includes epidemic strain A

Lau, et al, 2008, JAC , 62: 1241-44

Year Country % (n) ESBL Reference

1999 Pakistan 35 (200) Zaman, Pak Armed Forces Med J, 1999

1999 China, Japan, Taiwan, Singapore, Philippine

>20 (2193) Bell, Diagn Microbiol Infect Dis, 2002

1999 Hong Kong 11‐13 (1174) Ho, APMIS, 2000

2002 India 68 (678) Mathur, Ind J Med Res, 2002

2002 Pakistan 48 (400) Shah, New Microbiol, 2002

2002 Pakistan 40 (2840) Jabeen, J Pak Med Assoc, 2005

Asian Countries with High Prevalence of ESBL phenotype

Hubli

VaranasiAligarh

Characterisation of bla CTX‐M in India

130 isolates of E. coli and K. pneumoniae selected as resistant to 3GCs

95/130 (73%) carried bla CTX‐M

All PCR positive were bla CTX‐M‐15

• China is dominated by bla CTX‐M

genotypes 14, 9 & 3

• Only 4/399 genotyped isolates from China wide survey bla CTX‐M15*

*Yu, et al, J. Infect 2007, 54: 53-7

Prevalence & genotype of bla CTX‐M

from Hunan Province 2004/5

bla CTX‐M genotype

Species ESBL/Total 3 15 14 9

E. coli 50/160 6 6 35 1K. pneumoniae 47/110 8 12 13 1E. cloacae 31/98 10 2 3 3Citro. freundii 7/17 5 0 0 0

Liu, et al 2009 J.A.C. March 18th epub.

Seattle Washington

Orange,

California

Omaha, Nebraska

Denver, Colorado

Houston, Texas

Lexington, KY

Cleveland, Ohio

Little Rock,

Arkansas

New Brunswick, NJ

New York, NYSalt Lake City,

UT

CTX-M-15CTX-M-14CTX-M-3

Castanheira, et al, Micro. D. Resist., 2008

Global ESBL TrendsPer Region

Observer, 29th March 2009

Schematic map of the complex class 1 integron carrying the blaCTX-M-14 gene

on plasmid pAJE0508

Bae, et al, AAC, Aug 2007, 3017-19

Diagram of mill and reed bed system

Gaze, et al, AAC, May 2005, 1802-07

Relative resistance to DTDMAC and CTAB at 50 µg/ml

Gaze, et al, AAC, May 2005, 1802-07

Incidence of int11, qaE, and qacE∆1

Gaze, et al, AAC, May 2005, 1802-07

Gaze, et al, AAC, May 2005, 1802-07

Incidence of int11, qaE, and qacE∆1

Identification of isolates

Gaze, et al, AAC, May 2005, 1802-07

Gaze, et al, AAC, May 2005, 1802-07

Identification of isolates

WATER

SOIL

FARM ANIMALS

Xenobiotics e.g. QACs

FOOD

SEWAGEHUMANS GUT/FLORA

Veterinary Antibiotics

Clinical Antibiotics

Flow of antibiotic resistance genes in E. coli in the biosphere

Li Xu

Thomas Ling Craig Munday

Vicki Ensor Jian hui Xiong

mmm

Mohammed Shahid

Where did it all go wrong?

“I spent a lot of money on booze, birds and fast cars. The rest I just squandered”

George Best

CTX‐M‐3 & ‐15: effect of Asp240Gly

Vmax (%) MIC (mg/L)

M‐3 M‐15 M‐3 M‐15

C’tax 100 100 64 128

C’taz <0.01 1.3 1 64

C’pirome 3 80 ‐ ‐

Aztreonam 50 1 4 64

Rasmussen & Hoiby, Can J Micro 2004, 50, 137

02468

101214161820

% resistant

2001 2002 2003 2004 2005 2006

cefotaximeceftazidimeciprofloxacingentamicin

BSAC UK bacteraemia susceptibility surveyKlebsiella

www.bsacsurv.org

**

* No data available

+ number of strains

+ (146) (169) (155) (170) (164) (176)year

Meta-analysis of delay in effective therapy in in ESBL-producing versus non-ESBL-producing Enterobacteriaceae bacteraemia

Schwaber & Carmelli, 2007, J Antimicrob Chemother, 60: 913-20

Meta-analysis of mortality in ESBL-producing versus non-ESBL-producing Enterobacteriaceae bacteraemia

Schwaber & Carmelli, 2007, J Antimicrob Chemother, 60: 913-20

Estimated world populations by July 2006

China 1,313,973,713

India 1,095,351,995

World 6,525,170,264

ANTRES project

Antibiotic resistance in Peru and Bolivia

• Large scale survey of faecal E. coli in pre‐school healthy children

• 1st January 2002, reported 2005

• ESBL rate 0.1% 1.7% due to CTX‐M spread

Pallecchi, et al, 2007, AAC, 51: 2720

Pallecchi, et al, 2007, AAC, 51: 2720

Geographic distribution of CTX-M E.coli, 2005 ANTARES study

CTX‐M group

Type No Antibiotic resistance co‐transferred

Plasmid Inc group

CTX‐M‐2 CTX‐M‐2 8 GEN A/C; I1

4 GEN TET SXT F VII

2 GEN TET A/C

1 TET A/C

1 GEN SXT ‐

CTX‐M‐56 2 GEN A/C

CTX‐M‐9 CTX‐M‐14 8 NONE I1

CTX‐M‐24 2 NONE I1

CTX‐M‐1 CTX‐M‐15 6 NONE

1 TET CIP* FII

1 TET CIP* GEN FII

* aac (6')–Ib‐cr

Pallecchi, L., et al, 2007, AAC, 51: 2720‐5

“Human history has become more and more a race between education and catastrophe.”

H.G. Wells The Outline of History

The Problem with CTX-M……

• Rapid and wide dissemination – most prevalent ESBL worldwide

• Most important resistance determinant threatening use of β-lactams

• Dominance observed in both community andnosocomial settings

• Multiresistance - complicates treatment of previously uncomplicated infection

• Drives earlier and wider use of carbapenems

Mortality rates (21 day) in 97 ESBL-BSI patients treated with antimicrobial agents to which the infecting organism displayed in vitro susceptibility

Tumbarello, M., 2007, Antimicrob Agents Chemother 51: 1987-94

Structures of the studied molecules

Matagne, et al, Biochem 1993, 293, 607-11

Effect of temocillin against KPC producing bacteria from 2 states in USA

MIC Temocillin mg/L

8 16 32 64 1281 x 104 K. pneumoniae (30) 12 15 3

E. coli (3) 1 21 x 106 K. pneumoniae (30) 1 3 15 10 1

E. coli (3) 1 1 1

Adams-Haduch, et al, AAC (2009) epub 30th March

Tigecycline for Acinetobacter spp infections review

• 22 microbiological studies of 2,384 isolates (1906 Acb)

• 90% susceptibility (MIC ≤ 2mg/L)

• Efficacy 42 severely ill patients (31 resp. Infections, 8 bacteria)

• Tigecycline med in combination in 28/42 patients – effective in 32/42

Karageorgopoulos, et al, JAC (2008); 62:45-55

Colistin resistant K. pneumoniae in Greek ICUs

• Heavy empirical use of colistin

• 18 isolates from 13 patients over 16/12

• Long stay (median: 69 days), old (mean: 70 yrs)Long course colistin (median: 27 days)

• 2 bacterias, 1 VAP, 2 CSSTI

Antoniadou, et al, JAC (2007) 59: 786-90

Polymyxins (A‐E) – Colistin (A‐B)

• Mixtures of cyclic peptides –colistimethate for i.v. Use

• Variation in potency of different preperations, hydrolysed to colistin variably in patient

• Colycin (Forest Labs) 240‐480 mg colistimethate/day (Usually 2 doses)

Landman, et al, Clin Micro Revs (2008) 21: 449-65

Temocillin• Developed and marketed by Beechams in

1980’s

• 6 x methoxy derivative of ticarcillin → +++ β‐lactamase stability

• Staxxx to serine activated β‐lactamase e.g. CTX‐M, TEM, SHV, ESBLs

• No selection fro depressed AmpC production

• Probably little C. difficile selection

• Burkholderia cepacia susceptible (mode MIC 32 mg/L)

But....

• Withdrawn because of low sales

• Temocillin has no activity against Pseudomonas anaerobes or Gram positive bacteria

• Better with continuous infusion – serum levels ≥ 16 mg/L

• Most experience in UTI, but also BelgiumVAP (non Pseudomonas spp)

Polymyxins

Finch, et al, Antibiotic & Chemotherapy, 8th Edition, 2003, pp409

Use of cephalosporins to treat ESBL in China ‐ but all CTX‐M 14/3 no OXA‐1 so....

22 cases of bacteremia Survival %

ceftasidime (7) 86imipenem/cilastatin (8)

86cefoperazone/sulbactam (7) 72

Cao, et al, Diag. Micro. & ID (2006) 56:351-7

Sulbactam

• Has intrinsic activity against A. baumannii

• Early studeis showed high rates of susceptibility a

‐ declined recently b

• Has been used in pneumonia with moderate effect a CID (1996) 22:1026

b JAC (2007) 59:583

Future Approaches

• Trinem (Tricyclic carbapenem) – most discontinued but LK‐157 structureal analogue.

• Inhibits Class A & C β‐lactamase but not stable to carbapenemases.

LK-157

Paukner, et al, AAC (2009) 505-11

Odds & Sods

Nitrofuratonin

Fosfomycin

Amikacin

Mecillinam (± clavulanic acid)

CARBAPENEM ANTIBIOTICS...

Resistance mechanisms to carbapenems

• Intrinsic carbapenemases L1 • Loss of porin D2• Efflux pumps MexE-F-OprN • Class A&D carbapenemase KPC-2, OXA-23• Class B carbapenemase IMP,VIM

Endemic KPC Sporadic cases of KPC

Distribution of KPC in 2009

The future . . .

• CTX‐M dominant now– Will a new gunslinger emerge?

• In view of CTX‐M’s genetic mobility and in prevalence in China/India will rates continue to climb

• CTX‐M could assume same faecal flora/carriage rates as TEM

• The impact of ESBLs on empirical therapy of serious GNB sepsis – loss of third generation cephalosporins, quinolones, aminoglycocides,massive increase in use of carbapenems.

• New agents . . . .

Antibacterial resistance rates of genetically diverse cephalosporin-resistant E.coli from 3 geographically distinct centres in India

No and % resistant

Ensor, V.M., et al, 2006, J Antimicrob Chemother, 58:1260-3

Community acquired UTI at JNMC Hospital, Aligarh, IndiaAugust 2004‐July 2005

920 MSU tested in outpatient clinics

10.8% gave significant growth, 100 significant isolates

Resistance %

n ESBL COT NFX GEN AMK

E. coli 61 34% 76 69 64 51

K. pneumoniae 22 22% 53 47 53 35

Relative resistance to DTDMAC and CTAB at 50 ug/ml

Gaze, et al, AAC, May 2005, 1802-07

Geographical locations of the centers that have isolated Escherichia coli harboring the following ESBL: CTX-M group 1 (#), CTX-M group 9 (§ ), other CTX-M groups ( †).

Galas, et al, 2008, Antimicrob Agents Chemother, 52: 786-9

Centres (Sites)

BJ (3)

SH (3)

ZJ (5)

WH (2)

HN (4)

GZ (5)

HK (1)7 Centres ( 23 Institutions/laboratories)

Study Sites

Susceptibility of community isolates of E. coli collected in 2002-3 in China

0 0

14.4

2.7

8

17.3

4.6

29.1

7.1

50.6

2.4

39.4

0

10

20

30

40

50

Perc

ent

Ertapene

mIm

ipenem

Cefotaxim

eCefta

zidim

eCefep

ime

Cefoperazo

neCefoperz

one/sul

Amoxycilli

n/cla

Piperacilli

n/taz

Ciproflo

xacin

Amikacin

Gentam

icin

Ling, et al, 2006, Antimicrob Agents Chemother; 50: 374-8.

E. coli n=953 (% resistant)

ESBLs in UK E. coli: 2003-4

• increasing reference requests for ESBL confirmation in E.coli

– most expressing phenotype consistent with CTX-M enzymes

– including isolates from community-acquired UTIs

– little recent hospital contact

Bacterial Isolates

• Aligarh, N India, July – August 2005– 1857 samples– 143 E.coli or K. pneumoniae– 74 3GC R (51.7%)

• 4 week stored samples 2003-2004 (n=47)• 19 isolates (10 E.coli, 9 K. pneumoniae) from

Hubli and Varanasi

The arbuscular mycorrhizal structure is much less conspicuous than that of ectomycorrhizae. Both types have external hyphae, but arbuscular mycorrhizae do not form a fungal sheath around the root.

Comparison of Arbuscular mycorrhizae and Ectomycorrhizae, the two major types of mycorrhizae globally

Nutrient Mechanism of nutrient supply (% of total absorbed)

RootInterception

Mass Flow

Diffusion

Sedge tundra (Natural ecosystem)

Nitrogen - 0.5 99.5Phosphorus - 0.7 99.3Potassium - 6 94Calcium - 250 0

Magnesium - 83 17

Corn crop (Agricultural ecosystem)Nitrogen 1 79 (NO3

-) 20Phosphorus 2 4 94Potassium 2 18 80Calcium 150 413 0

Magnesium 33 244 0

Sulfur 5 95 0

Iron - 53 -

Manganese - 133 0

Zinc - 33 -

Boron - 350 0

Copper - 400 0

Molybdenum - 200 0

In both natural andagricultural ecosystems,

diffusion is the most important mechanism

for growth limiting nutrients

Mass flow isimportant for

nutrients that are

or required in smallamounts

abundant in soil,

Arbuscular mycorrhizae, typical of many herbaceous plants‐ effective in phosphorus acquisition

‐ also in mitigating water stress

Clover root infected with arbuscular mycorrhizal fungi. The intricately branched arbuscules (arb) are sites of nutrient exchange with the plant cells; the vesicles (ves) are fungal storage bodies; the swelling at the point of fungal entry into the root is termed an

appressorium (ap). Plant root hairs (rh) also are shown.

By producing root hairs, plants increase root length and surface area