E.coli neonatal sepsis and meningitis

Y Aujard and E Bingen Neonatal and Microbiology departments Hopital Robert Debré Paris 1

Robert Debré Hopital

• Universitary pediatric hospital • + Maternity 3000 births/year • Neonatal department with 37 beds including 25 level II and 6 level III – 780 admissions / year • Microbiology department ( Pr E Bingen) 2

Part I Clinical presentation

3

Pathophysiology

EOS, day 0 – day 4: *Blood, 10 % *Vaginal colonization , 90 %: -GBS, 40 % -

E coli, 20 %

-

Transmission rate, 50 %

-

Neonatal colonization # 10%

-

Neonatal infection #2 ‰ births

-

LOS, day 5 – day 28

-

post natal contamination

4

Early onset systemic sepsis :microbial epidemiology (all gestational ages) • • Group B

Streptococcus:

40 - 60 %

Escherichia coli

: 20 - 50 % • Group D

Streptococcus

: 5 - 10 % •

Listeria monocytogenes

1 - 2 % • Others :  10 % :

18 16 14 12 % 10 8 6 4 2 0 16,5 1000/1500 9 2,5 1501/2000 2001/2500 Birth weight (g) ) 0,8 >2500

5

Early onset systemic sepsis in BW < 1500 g Blood cultures (+) n=84/5447(1.5%) • • •

Gram negative

–

E coli :

– – –

Haemophilus influenzae : Citrobacter : others : Gram positive

–

GBS :

– –

Streptococcus viridans: other streptococci:

– – –

Listeria: Coag negative Staph: Other : Candida : 60.7% 44.4% 8.3 % 2.4% 6 % 36.9% 10.7% 3.6% 4.8% 2.4% 10.7% 4.8% 2.4%

BJ Stoll et al, NEJM, 2002 ; 347 :240

6

Childhood bacterial meningitis > 1 month. France

in France (2001-2003) 1-2 m 2m – 1y > 1 month 1-2 y 2-15 y 15-18 y

7

Bingen CID october 2005

E coli Neonatal meningitis ( 1 to 2 % of systemic sepsis ).

France 2001-2003 n 25 20 15

Strepto B E coli Nm B Sp autres

10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 jours

8

n 80 30 20 10 0 70 60 50 40

69 12

Day1Day4

5

Age distribution of neonatal E coli Meningitis (n=259) ACTIV / GPIP

42 34 21

Day5Day14

15 19

Day15Day21

2

E coli GBS Others

24 9

Day22Day28

7

Days

9

Number of cases of meningitis caused by E. coli by age at diagnosis (n=99) * 6 5 Number of 4 cases 3 2 1 8 7 Median 0 1 4 7 10 13 16 19 24 28 31 46 Age in days 60 89

* V Houdoin, E Bingen et al Lived Died 10

E coli, GBS and other BM in PT and FT newborn ACTIV / GPIP, 2001- 2004 (n= 252) GBS (n = 146) E coli* (n = 78) Others (n = 35) Preterm (n=60) %

16/60 (26.7) 31/60 (51.7)

13/60 (21.7)

E coli meningitis, 30.5 % E coli ampicillin resistance : 33,8 % E coli K1 : 93,7 %

Full-term (n =192) %

127/192 (66.1) 46/192 (23.9)

19/192 (9.9) p

0.0001 0.0001

0.02

11

Clinical characteristic and mortality of GBS, E coli and other neonatal BM Activ / Gpip GBS n = 146

E coli

n = 78 Birth weight (g) mean±SD Prematurity (n = 60) %

3222*±573 16/143 (11.2)*

Seizures (n = 85) % Seizures before treatment (n = 43) % Seizures during treatment (n = 64) % Shock (n = 55) % Coma± mechanical ventilation (n = 79) % Mortality (n = 35) % Mean delay after first LP (days)

61/142 (42.9)** 34/141 (24.1)*** 41/138 (29.7) 32/137 (23.3) 43/140 (30.7) 19/144 (13.2) 7.6±1.7 *p<0.0001 =, **p =0.001, ***p =0.009

2742*±918 31/77 (40.3)* 16/77 (20.8)** 7/75 (9.3)*** 16/76 (21.1) 18/75 (24) 23/75 (30.7) 8/76 (10.5) 10.5±3.9

Others n = 35

2623±723 13/22 (40.6) 8/35 (22.8) 2/34 (5.9) 7/34 (20.6) 5/34 (14.7) 13/35 (37.1) 8/35 (22.8) 14.5±5.1 12

E coli Neonatal meningitis : outcome • Mortality rate: 10-15 % • Short term complications – Cerebral abscess ( 16%)*, infarctus, ventriculitis • Significant sequelae : 20-50% – mental and motor disabilities – convulsive disorders – hydrocephalus – hearing loss – abnormal speech patterns – others

Y Aujard et al, 1999

13

E coli Neonatal meningitis Prognosis factors

• Immature immunity :

– cellular – IgA = 0 – IgG < 32 weeks, 1-4 g/l – CH 50 , 50 % • • • •

Initial complications High bacteria concentration in CSF Low antibiotic efficacy,Treatment delay Brain tissue localisation : abscess

•

E coli virulence factors ?

JPP 1999

14

Capacity of sera from neonates to kill

E. coli

and complement of newborn infants in comparison with adults Lassiter JID 1992

p < 0.001

Wolach Acta Paediatr 1997 15

E coli Neonatal meningitis Prognosis factors

•

Immature immunity :

– – – –

cellular IgA = 0 IgG < 32 weeks, 1-4 g/l CH 50 , 50 %

• •

• Initial complications

High bacteria concentration in CSF Low antibiotic efficacy,Treatment delay

•

Brain tissue localisation : abscess

•

E coli virulence factors ?

JPP 1999

16

E coli neonatal meningitis ; prognosis factor Variable Male Gestational age < 32 SA 32 SA - <37 SA ≥ 37 SA

Hypotension Seizures Apnea

Positive blood culture (n=71) Positive CSF 48 hours after treatment (n=76)

Normal ultrasonic initial evaluation of the brain (n=77)

Birth weight (g) (mean) Age at diagnostic (d) (mean) CSF cell count (x10 6 /L) (n=71)

CSF protein (mg/dl) (n=67) CSF glucose (mmol/l) (n=64)

Good outcome (n=59) 27 (45,7) Adverse outcome (n=30) 14 (46,7) 5 (8,5) 11 (18,5) 43 (73) 1 (1,7) 7 (12) 8 (13,6) 41 (82) 6 (11) 39 (76) 3000 18,2 5667 295 1,75 5 (16,7) 9 (30) 16 (53,3) 10 (33,3) 18 (60) 9 (30) 16 (76) 5 (23) 13 (50) 2700 14,3 8864 463 0,75 p Value p> 0,05 p> 0,05

0,0001 0,0001 0,0001

p> 0,05 p> 0,05

0,019

p> 0,05 p> 0,05 p> 0,05

0,03 0,01

17

Brain abscess in E coli neonatal meningitis

• Incidence: 16 % • Mortality rate # 50 %

( JO Klein, 2001)

• Cerebral colonisation contemporary to CSF invasion (bacteremia) • β lactamin : brain / blood < 10 % • • Stationnary phase • Aminoglycoside inactivated by pus  fluoroquinolone 18

Fluoroquinolone in E coli neonatal meningitis • High Fluoroquinolone concentration in – CSF, WBC , pus, brain / brain abscess • Low toxicity * • Hypothesis : Addition of a FQ to conventional agents for the 4 first days of treatment : - 20 mg/k/d < day 8 ; 30 mg/kg/d > day 7 - reduction of brain abscess - better prognosis *

Schaad U, Aujard, Y et al. Pediatr Infect Dis J

19

Fluoroquinolone in neonatal meningitis. Clinical study n=36

Cipro + cefotaxime + AG vs cefotaxime + AG

Ciprofloxacin (+)

n = 15

Ciprofloxacin (-)

n = 21 P* = 4 T** = 10 *P: premature ; **T : term newborn P = 11 T = 11 20

Ciprofloxacin in E coli meningitis • CSF/ blood ratio : mean 29.6 % – H2, 14 % – H8, 66 % – H12, 20% • CSF concentration / MIC E coli – x 80

21

Neonatal meningitis and cipro. Short term outcome Death Seizure Abscess / Ventriculitis/ Brain infarctus / Ventr dilatation./ Grey nuclear necrosis IV H > grade I Leukomalacia None CIPRO (+) CIPRO (-) GNB n = 14 others n = 1 1 2 1 0 1 1 12 0 0 0 0 0 0 1 Total n = 15 1 (6.6 %) 2 (13 %) 1 (6.6 %) 0 1 (6.6 %) 13 (87 %) GNB n = 5 Others n = 16 0 0 2 1 1 3 0 4 4 9 2 1 1 7 Total n = 21 5 (24 %) NS 5 (24 %) 12 (75 %) 2 (9.5 %) 1 (4.7 %) 2 (43 %) S**

** p = 0.014

22

Neonatal meningitis and cipro.

One year follow up

Motors disabilities Speech disorders Vision

Hearing loss Seizure

Normal Cipro (+) (n = 14) 0 0 2 (11 %) 0 0 12 (83 %) Cipro (-) (n = 16) 3 (16 %) 3 (16 %) 2 (14 %) 0 0 11 (61 %) [S]* * p = 0.038

23

E coli in neonatal sepsis and meningitis. Clinical conclusions • Increasing responsability – decrease of GBS with per partum ampicillin – premature infant ++ – meningitis • Severe prognosis when associated to – Prematurity – Meningitis • seizures, apnea, hypotension, low CSF glucose, high CSF protein • Ampicillin resistance 35– 40 % – CIII cephalosporin – fluoroquinolone in meningitis • Selective per partum prophylaxy ?

24

E coli Neonatal meningitis Prognosis factors

• • • •

Immature immunity :

– – – –

cellular IgA = 0 IgG < 32 weeks, 1-4 g/l CH 50 , 50 % Initial complications High bacteria concentration in CSF Low antibiotic efficacy,Treatment delay

•

Brain tissue localisation : abscess

• E coli virulence factors

?

25

Molecular epidemiology of E.coli neonatal meningitis

26

E. coli

O (173) H (56) K (80)



~700 000 Commensal Pathogens Intestinal Pathogenic (IPEC) Extra-intestinal Pathogenic (ExPEC) UTI Blood Meningitis (ECNM)

Similar profiles and genetic background 27

E. coli

neonatal meningitis isolate Special virulence factors are required for successfull penetration into CNS

of ECNM

Pathophysiology of bacterial meningitis

29

Steps involved in the pathophysiology of community bacterial meningitis

Mucosa Blood Brain Barrier BLOOD CSF Colonization Translocation High level Bacteremia BBB crossing

30

E. coli

Enteric colonization 10 9 CFU/g (Quorum sensing ?) Gut Translocation

Requires >10 9 CFU/g

Bacterial factors

•

P Fimbriae

•

Type 1 Fimbriae UTI : 20%

Prevalence of bacteremia

< 1 month 1-2 m 2-3 m 31% 21% 14% >3 m 5%

Ginsburg Pediatrics 1982 31

E. coli bacteremia in neonates

Bacterial factors

•

K1 capsule

•

O-LPS

•

Iron captation systems : iroN, fyuA (HPI), aerobactin

•

PAI I C5 / PAI II J96

Increase serum survival

Host factors

•

Serum

•

Neutrophils High level bacteremia (Neonates)

32

Persistent High Level Bacteremia BBB crossing

Dietzman J Pediatrics 1974

Higher incidence of meningitis 33

E. coli

characteristics in neonatal meningitis

34

Phylogenetic distribution of

E. coli

(ECOR collection) n=72

E.coli

species represented by ECOR collection – Tree based on MLEE with 38 enzymes fall in 4 main phylogenetic groups

Herzer. J. Bacteriol. 1990

35

Escobar-Paramo, Appl Environ Microbiol, 2004

 Most commensal strains belong to group A and B1 36

Phylogenetic distribution of virulence genes among

E. coli

of the ECOR collection

Boyd J. Bacteriol. 1998

37

Phylogenetic Analysis of 69 neonatal meningitis isolates (Ribotyping)

E.coli

neonatal meningitis belong primarly to B2 group (68%) and to lesser extend to D group (20%)

Bingen JID 1998

38

Phylogenetic Analysis of 138 neonatal E. coli meningitis isolates [North America and Europe] A=8% B1=3% D=16% B2=65%

Bonacorsi, JID, 2003)

39

Prevalence of Phylogenetic Group in

E. coli

neonatal meningitis

B2/D B2 D A B1 France (n=91)** 90% 81% 9% 9% 1% North America (n=41)** 90% 61% 29% 7,5% 2,5% Netherlands (n=70)* 88% 81% 7% 1% 10%

* Johnson JID 2002 ** Bonacorsi JID 2003

40

E. coli

Neonatal meningitis main serotypes

Netherlands Mulder (1984) n=101 083 018 26% 22% 07 014 O auto 10% 7% 22% Non typable 14% France Houdouin (2005) n=99 045 018 25% 25% 01 07 083 06 Non typable 18% 4% 6% 1% 18% North America : O18:K1:H7, O7

Johnson, JID, 2001 - Achtman, II, 1983 - Hacker, Microb Pathog, 1990

OMP6 PAI IIJ96 + OMP9 PAI IIJ96 41

Ribotyping of 134

E. coli

meningitis isolates from North America and France.

23 ribotypes •Oligoclonality : (independent of geographic origin) 4 ribotypes = 70% of isolates •Ribotype diversity : N. America (15/41)

>

France (15/91) France North America

Bonacorsi, IJMM, 2005

42

ECNM Ribotyping 134 strains (Europe, USA)

A=8% B1=3% D=16% B2=65% (Bingen-Bidois IAI 2002, Bonacorsi JID 2003, J urol 2005)

Ribotype B 2 1 :

Archetypal clone (strains C5 & RS218) O18:K1 B2 1 : 44%

Major virulent subgroup

O45:K1 « French » clone

Particular genetic background

aquisition of VF (UTI, meningitis) Male infants urosepsis (37%) UTI male infants (32%) Adult urosepsis (19%) 43

Distribution (%) of specific virulent determinants involved in the pathogenesis of ECNM among different studies

ECOR n=72 Aerobactin (

iucC / iutA

) 15 p<0.001

22 p<0.001

29 p<0.001

15 11 p=0.003

1 p<0.001

4 p<0.001

11 21 p<0.001

44

Bonacorsi IJMM 2005

Prevalence of virulence genes (%) according to the origin of the strains

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% * * * K1 14.9kb pap sfa/foc ibeA * hly * aer iroN HPI cnf1 Half of the strains are devoid of adhesins (sfa) and invasins (ibeA)

Bingen. JCM 1997, Bingen. CID 1996, Johnson JID 2000-2001, Bidet IAI 2005

Stools Blood CSF * p<0.05

45

« Pathogenicity island » (PAI)

Virulence genes are clustered in chromosomal structures known as PAI or « genetic island » • • • • •

Absent from non pathogenic strains Length (> 30kb) G+C content different from that of the core genome Located near tRNA Mobility genes (IS, integrase, transposase )

Hacker & Carniel EMBO 2001 These structures were termed « Ectochromosomal DNA » (ECDNA) 46 Welch 2002

BBB crossing B

Different ectochromosomal DNA – like domains have been involved in the virulence of neonatal E.coli meningitis isolates

Bonacorsi, JID, 2003 47 a

Prevalence and distribution of ectochromosomal DNA like domains among the different phylogenetic groups of 134 ECNM.

%

BBB crossing Bacteremia

 Specific genetic backgrounds are required for the integration, retention and expression of PAIs Bonacorsi IJMM 2005 48

Meningeal virulence in the rat neonatal model of the main meningitis clones

Newborn hematogenous meningitis rat model (

Bortolussi, Infect . Immun, 1979

) Rats 5 days old 18 hours after inoculation 5 µl of blood was obtained by tail incision.

5 µl blood

IP Injection 300 CFU

Were inoculated intraperitoneally with 300 +/- 50 CFU in physiological saline Animals were killed and 5 µl of CSF was obtained by cisterna magna punction 49

Correlation between isolates’ genetic background and capacity to induce bacteremia and of represntative E.coli meningitis isolates Rats 5 days old Meningitis in vulnerable neonates

d : p<0.05 vs strain C5

O45 same capacity as C5 but devoid of

sfa

and

ibeA other unknown mechanisms of BBB penetration in this clonal group?

sfa

+,

ibeA

+

sfa

-,

ibeA

-

Phylogenetic group A (6%) meningitis isolates vs group A ECOR isolates

K1 Meningitis (n=11) 73% ECOR (n=25) 4% HPI Aerobactin 100% 100% 32% 24% Atypical Virulent group (avirulent in animal model) Prevalence of VF was higher in group A meningitis isolates

Bacteremia BBB crossing

045:K1:H7

Serotype

iroN aer fyuA

PAI I C5

sfaS ibeA

meningitis O18:K1 + -/+ + + meningitis O45:K1 + + +

• Highly virulent clonal group (Ribotype B 2 1 ) phylogenetically related to archetypal strains C5 and RS 218

+ + -

• 30% of French isolates •Avian pathogen • Same capacity to induce bacteremia and meningitis as strain C5 although they did not harbor any of the ECDNA- like domains implicated in BBB crossing (PAI III 536 , GimA).

52

Contribution of PAI II

J96

– like domains to bacterial survival in different ecological niches « From commensalism to pathogenesis »

53

PAI II

J96

/ PAI I

C5

PATHOGENICITY ISLAND

hly cnf1 prs hra

110 kb

Swenson, IAI, 1996

UPEC J96 94’ (PheU)

1 Core :

cnf1, hly, hra

consistently colocalized (« signature »).

(Bingen-Bidois IAI 2002, Bonacorsi JID 2003) 2 Implication of PAI II J96 / PAI I C5 in bacteremia and meningitis (rat meningitis model) Strain C5 (O18) meningitis C5 D PAI I C5 (Houdouin IAI 2002) bacteremia 100% (5,7) 66% (4.67)

*

meningites 52% 26% log(blood cfu/ml)

*p < 0,05

 PAI II J96 / PAI I C5 contribute to bacterial survival in blood by inducing high level bacteremia 54

Prevalence of PAI II

J96

-like domains among isolates of different collections

(Bidet, IAI 2005 )

55

PAI II

J96

/ PAI I

C5

PATHOGENICITY ISLAND

3- Analysis of tRNA insertion sites by

hra

- tRNA long range PCR  Chromosomal insertion : in the viciny of three different tRNA

0’

hly, cnf1, pap, hra

LeuX PheU

75’ 25’

PheV

50’

(Bingen Bidois, IAI 2002, Bidet IAI 2005 )

56

B2 Group Clinical expression of E. coli carrying PAI II J96 / PAI I C5 according to the genetic background and the insertion site – (1)

0’

IS100, hly, cnf1, pap, hra

LeuX PheU

Chromosomal insertion of PAI II J96

75’

PheV

50’

B2 1

LeuX (50%)

/ PheU (50%) <-> meningitis « PAI » (Bidet IAI 2005 ) B2 6 PheU (86%) <-> U.T.I.

« PAI » Allelic exchange papGIII  papGII B2 7

PheV

<-> colonization

« Saprophytic island »

No allelic exchange

25’

acquisition PAI II J96 ↑ expression of fitness factors  survival in the gut

PAI II

J96

-like domains

Fitness Island Enhances bacterial survival Human Gut Urinary tract Blood

58

E. coli

neonatal meningitis isolate Bacteremia

Enterobactin Salmochelin Yersiniabactin Aerobactin Heme captation FepA, Fiu, Cir IroN FyuA (HPI) IutA ChuA Type 1 fimbriae P fimbriae LPS Siderophore Fe 3+ Receptor Fe 3+ ~Transferrin Fe 3+ Omp A Fe 3+

K1 capsule

Transferrin

BBB crossing 59

Conclusion

•

E. coli

• first pathogen in premature infants • in meningitis mainly found in children < 3 months old.

• ECNM mainly belong to group B2 Subgroup 1 • A highly virulent clonal group O45:K1:H7 phylogenetically related to archetypal strains C5 and RS218 was identified in France • Virulence factors identified to date alone are insufficient to fully understand the pathophysiology of neonatal meningitis. • Half of the ECNM strains are devoid of specific adhesins (

sfa

) and invasins (

ibe

A) • Development of new molecular tools, and new Sequency genome projects of

E. coli

would serve to find common pathogenic mechanisms among different ECNM clonal groups that may be used as potential target for a worldwide efficacious prevention strategy.

60

Acknowledgements and collaborations Neonatology • C Farnoux • P LeHuidoux • S Lefevre • L Maury • F Autret • O Baud • … Microbiology (EA 3105) • Ph Bidet • P Mariani- Kurdjian • S Bonacorsi • C Doit • V Houdouin • M Bingen-Bidois • ACTIV – E Cohen – F Delarocque – C Levy 61