ESBLs: From Soil to Suppuration

ESBLs: From Soil to Suppuration
BSAC Spring Meeting
Tuesday 7th April 2009
International Convention Centre, Birmingham
PROFESSOR PETER M. HAWKEY
The University of Birmingham
Edgbaston, B15 2TT
Health Protection Agency West Midlands
Public Health Laboratory, Heart of England
NHS Foundation Trust, B5 9SS
p.m.hawkey@bham.ac.uk;peter.hawkey@heartsol.wmids.nhs.uk
• 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
2001
2006
Endemicity of CTX-M
Taken from Canton and Coque, Curr Opin Micro 2006, 9: 466-475
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)
K. pneumoniae
Paraguay (1994)
Argentina (1994) E.coli / P. mirabilis
Bauernfeind, et al, 1992, Infection, 18:294
b Bauernfeind et al, 1992, Infection, 20:158
c Bauernfeind, et al, 1996, AAC, 40: 509
a
CTX-M-1
Group
CTX-M-2
Group
CTX-M-8/25/26
Group
CTX-M-9
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
Soil microflora
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
Organisms
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.
Extra-radicle hyphal development
is less extensive in AM fungi than
in EM fungi
Diffusion shell: volume of soil around root that is depleted
in 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 cassette
blaCTX-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 ILT3
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
TEM/SHV ESBL
K
CTX-M
Community
K
CTX-M
E
E
00s
E
90s
CTX-M
TEM/SHV
K
ESBL
E
Mutation
Gene Transfer
Kluyvera
TEM/SHV
80s
E/K
TEM/SHV
E E.coli
K Klebsiellae
E
60s/70s
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
E. coli from blood & CSF susceptibilities, HPA data
25
% Resistant
20
Cipro
3 gen ceph
15
both
10
5
0
90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 06
BSAC UK bacteraemia susceptibility survey
E. coli resistance
14
12
10
8
% positive
ESBL
ESBL CTX-M
6
4
+ number of strains
2
0
2001
+
(226)
2002
(230)
2003
(228)
2004
(228)
year
2005
(227)
2006
(223)
www.bsacsurv.org
Proportion of CTX-M+ve Strains of Total
Resistant to 3GC’s isolated from Hospital and
Community, Birmingham UK.
Total number of
isolates resisant to
3GC
350
300
250
Proportion positive
for CTX-M
200
150
100
50
0
04
20
03
20
02
20
01
20
)
ec
l-D
(Ju
00
20
99
19
% CTX-M positive
Distribution of CTX-M genes in different species of
Enterobacteriaceae from clinical samples from
hospital and community – Birmingham, UK
E.coli
100
80
K pneumoniae
60
K. oxytoca
40
Enterobacter sp
20
0
19
9
9
20
00
(J
ul
20
01
-D
ec
)
YEAR
20
02
20
03
20
04
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
Asian Countries with High Prevalence of ESBL phenotype
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
Aligarh
Hubli
Varanasi
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
K. pneumoniae
E. cloacae
Citro. freundii
50/160
47/110
31/98
7/17
6
8
10
5
6
12
2
0
35
13
3
0
1
1
3
0
Liu, et al 2009 J.A.C. March 18th epub.
Seat
Wash tle
ingto
n
Sa
lt
,
ge a
an ni
Or lifor
Ca
La
k
UT e C
i ty
,
Denver,
Colorado
Cleveland,
Ohio
Omaha,
Nebraska
ck,
o
R
le
Litt ansas
Ark
Houston, Texas
Lexington,
KY
N
Yo ew
r k,
NY
Bru New
nsw
NJ ick,
CTX-M-15
CTX-M-14
CTX-M-3
Castanheira, et al, Micro. D. Resist., 2008
Global ESBL Trends
Per 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
Incidence of int11, qaE, and qacE∆1
Gaze, et al, AAC, May 2005, 1802-07
Identification of isolates
Gaze, et al, AAC, May 2005, 1802-07
Identification of isolates
Gaze, et al, AAC, May 2005, 1802-07
Flow of antibiotic resistance genes in
E. coli in the biosphere
FARM
ANIMALS
Xenobiotics
e.g. QACs
SOIL
WATER
SEWAGE
Veterinary
Antibiotics
HUMANS
GUT/FLORA
FOOD
Clinical
Antibiotics
Li Xu
Vicki Ensor
Jian hui Xiong
mmm
Thomas Ling
Mohammed Shahid
Craig Munday
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
BSAC UK bacteraemia susceptibility survey
Klebsiella
20
18
16
14
12
cefotaxime
ceftazidime
ciprofloxacin
gentamicin
% resistant 10
8
6
4
* No data available
2
+ number of strains
0
*
*
2001 2002 2003 2004 2005 2006
+
(146)
(169)
(155)
(170)
year
(164)
(176)
www.bsacsurv.org
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-ESBLproducing 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
Geographic distribution of CTX-M E.coli, 2005 ANTARES study
Pallecchi, et al, 2007, AAC, 51: 2720
CTX‐M
group
CTX‐M‐2
CTX‐M‐9
CTX‐M‐1
Type
CTX‐M‐2
CTX‐M‐56
CTX‐M‐14
CTX‐M‐24
CTX‐M‐15
No Antibiotic resistance
co‐transferred
8 GEN
4 GEN TET SXT
2 GEN TET
1 TET
1 GEN SXT
2 GEN
8 NONE
2 NONE
6 NONE
1 TET CIP*
1 TET CIP* GEN
Plasmid Inc
group
A/C; I1
F VII
A/C
A/C
‐
A/C
I1
I1
FII
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 and
nosocomial 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
1 x 104 K. pneumoniae (30)
E. coli (3)
1 x 106 K. pneumoniae (30)
E. coli (3)
1
1
1
16
12
2
3
1
32
15
64 128
3
15
1
10
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 Belgium
VAP (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
ceftasidime (7)
imipenem/cilastatin (8)
86
cefoperazone/sulbactam (7)
Survival %
86
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
Distribution of KPC in 2009
Endemic KPC
Sporadic cases of KPC
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 cephalosporinresistant 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, India
August 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
Study Sites
Centres (Sites)
BJ
(3)
SH (3)
ZJ
(5)
WH (2)
HN (4)
GZ (5)
7 Centres
( 23 Institutions/laboratories)
HK (1)
Susceptibility of community isolates of E. coli collected in
2002-3 in China
50.6
50
E. coli n=953 (% resistant)
39.4
29.1
30
20
17.3
14.4
8
10
2.7
0
7.1
4.6
2.4
Ce
fo
ta
xi
m
e
Ce
fta
zi
di
m
e
Ce
fe
pi
Ce
m
e
fo
pe
ra
Ce
zo
ne
fo
pe
rz
on
e/
Am
su
ox
l
yc
ill
in
Pi
/c
pe
la
ra
ci
lli
n/
ta
Ci
z
pr
of
lo
xa
ci
n
Am
ik
ac
in
G
en
ta
m
ic
in
0
en
em
Im
ip
ne
m
0
Er
ta
pe
Percent
40
Ling, et al, 2006, Antimicrob Agents Chemother; 50: 374-8.
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
Comparison of Arbuscular mycorrhizae and Ectomycorrhizae,
the two major types of mycorrhizae globally
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.
Nutrient
Mechanism of nutrient supply (% of total absorbed)
Root
Interception
Mass
Flow
Diffusion
Sedge tundra (Natural ecosystem)
Nitrogen
Phosphorus
Potassium
-
0.5
-
0.7
-
6
Calcium
-
250
0
Magnesium
-
83
17
99.5
99.3
94
Corn crop (Agricultural ecosystem)
79 (NO3-)
Nitrogen
Phosphorus
Potassium
1
2
18
94
80
Calcium
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
2
4
20
Mass flow is
important for
nutrients that are
abundant in soil,
or required in small
amounts
In both natural and
agricultural ecosystems,
diffusion is the most
important mechanism
for growth limiting nutrients
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