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 : 18 20 - 50 % 16 14 16,5 • Group D Streptococcus : 12 10 5 - 10 % % 8 9 • Listeria monocytogenes : 6 1 - 2 % 4 0,8 2 2,5 • Others :  10 % 0 1000/1500 1501/2000 2001/2500 >2500 Birth weight (g))

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

• Gram negative 60.7% – E coli : 44.4% – Haemophilus influenzae : 8.3 % – Citrobacter : 2.4% – others : 6 % • Gram positive 36.9% – GBS : 10.7% – Streptococcus viridans: 3.6% – other streptococci: 4.8% – Listeria: 2.4% – Coag negative Staph: 10.7% – Other : 4.8% • Candida : 2.4% BJ Stoll et al, NEJM, 2002 ; 347 :240

6 Childhood bacterial meningitis > 1 month. France

in France (2001-2003) > 1 month

1-2 m 2m – 1y 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 Strepto B E coli Nm B Sp autres

0 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425262728jours

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

n 69 80 E coli 70 GBS 60 42 Others 50 34 40 30 24 21 19 20 15 12 10 9 5 7 0 2 Day1Day4 Day5Day14 Day15Day21 Day22Day28 Days

9 Number of cases of meningitis caused by E. coli by age at diagnosis (n=99) *

7 Median 6

5 Number of 4 cases Lived 3 Died 2

0 1 4 7 10 13 16 19 24 28 31 46 60 89 Age in days

* V Houdoin, E Bingen et al 10 E coli, GBS and other BM in PT and FT newborn ACTIV / GPIP, 2001- 2004 (n= 252)

Preterm (n=60) Full-term (n =192) p

% %

GBS (n = 146) 16/60 (26.7) 127/192 (66.1) 0.0001

E coli* (n = 78) 31/60 (51.7) 46/192 (23.9) 0.0001

Others (n = 35) 13/60 (21.7) 19/192 (9.9) 0.02

E coli meningitis, 30.5 % E coli ampicillin resistance : 33,8 % E coli K1 : 93,7 % 11 Clinical characteristic and mortality of GBS, E coli and other neonatal BM Activ / Gpip

GBS E coli Others n = 146 n = 78 n = 35

Birth weight (g) mean±SD 3222*±573 2742*±918 2623±723 Prematurity (n = 60) % 16/143 (11.2)* 31/77 (40.3)* 13/22 (40.6) Seizures (n = 85) % 61/142 (42.9)** 16/77 (20.8)** 8/35 (22.8) Seizures before treatment (n = 43) % 34/141 (24.1)*** 7/75 (9.3)*** 2/34 (5.9) Seizures during treatment (n = 64) % 41/138 (29.7) 16/76 (21.1) 7/34 (20.6) Shock (n = 55) % 32/137 (23.3) 18/75 (24) 5/34 (14.7) Coma± mechanical ventilation (n = 79) % 43/140 (30.7) 23/75 (30.7) 13/35 (37.1) Mortality (n = 35) % 19/144 (13.2) 8/76 (10.5) 8/35 (22.8) Mean delay after first LP (days) 7.6±1.7 10.5±3.9 14.5±5.1

*p<0.0001 =, **p =0.001, ***p =0.009 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

p < 0.001

neonatesneonates

Lassiter JID 1992 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 Good outcome Adverse outcome p Value (n=59) (n=30) Male 27 (45,7) 14 (46,7) p> 0,05 Gestational age < 32 SA 5 (8,5) 5 (16,7) 32 SA - <37 SA 11 (18,5) 9 (30) p> 0,05 ≥ 37 SA 43 (73) 16 (53,3) Hypotension 1 (1,7) 10 (33,3) 0,0001 Seizures 7 (12) 18 (60) 0,0001 Apnea 8 (13,6) 9 (30) 0,0001 Positive blood culture (n=71) 41 (82) 16 (76) p> 0,05 Positive CSF 48 hours after treatment 6 (11) 5 (23) p> 0,05 (n=76) Normal ultrasonic initial evaluation of 39 (76) 13 (50) 0,019 the brain (n=77) Birth weight (g) (mean) 3000 2700 p> 0,05 Age at diagnostic (d) (mean) 18,2 14,3 p> 0,05 CSF cell count (x106/L) (n=71) 5667 8864 p> 0,05 CSF protein (mg/dl) (n=67) 295 463 0,03 CSF glucose (mmol/l) (n=64) 1,75 0,75 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 (+) Ciprofloxacin (-)

n = 15 n = 21

P* = 4 T** = 10 P = 11 T = 11

*P: premature ; **T : term newborn

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

CIPRO (+) CIPRO (-) GNB others Total GNB Others Total n = 14 n = 1 n = 15 n = 5 n = 16 n = 21

Death 1 0 1 (6.6 %) 1 4 5 (24 %) NS Seizure 2 0 2 (13 %) 1 4 5 (24 %) Abscess / Ventriculitis/ Brain infarctus / 1 0 1 (6.6 %) 3 9 12 (75 %) Ventr dilatation./ Grey nuclear necrosis 0 0 0 0 2 2 (9.5 %) IV H > grade I 1 0 1 (6.6 %) 0 1 1 (4.7 %) Leukomalacia 1 0 13 0 1 2

None 12 1 (87 %) 2 7 (43 %) S**

** p = 0.014

22 Neonatal meningitis and cipro. One year follow up

Cipro (+) Cipro (-) (n = 14) (n = 16)

Motors disabilities 0 3 (16 %) Speech disorders 0 3 (16 %) Vision 2 (11 %) 2 (14 %) Hearing loss 0 0 Seizure 0 0

Normal 12 (83 %) 11 (61 %) [S]* * p = 0.038

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

• 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 Extra-intestinal Pathogenic (IPEC) (ExPEC)

UTI Blood Meningitis (ECNM)

Similar profiles and genetic background

27 E. coli neonatal meningitis isolate

Special virulence factors are required for successfull Key aspects of the molecular epidemiology 28 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 BBB crossing Bacteremia 30 E. coli

Enteric colonization Translocation 109 CFU/g Gut Requires (Quorum sensing ?) >109CFU/g

Bacterial factors •P Fimbriae •Type 1 Fimbriae

UTI : 20%

Prevalence of bacteremia < 1 month 31% 1-2 m 21% 2-3 m 14% >3 m 5% Ginsburg Pediatrics 1982 31 E. coli bacteremia in neonates

Bacterial factors Host factors •K1 capsule •Serum •O-LPS •Neutrophils

•Iron captation systems : Increase serum survival iroN, fyuA (HPI), aerobactin

•PAI IC5 / PAI IIJ96

High level bacteremia (Neonates)

32 Persistent BBB High Level crossing Bacteremia

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 40 ** Bonacorsi JID 2003 E. coli Neonatal meningitis main serotypes

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

North America : O18:K1:H7, OMP6 PAI IIJ96 + O7 OMP9 PAI IIJ96 - Johnson, JID, 2001 - Achtman, II, 1983 - Hacker, Microb Pathog, 1990 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% Archetypal clone Ribotype B21: (strains C5 & RS218) D=16% O18:K1 B21 : 44%

O45:K1 B2=65% Major virulent «French» clone subgroup

Particular genetic Male infants urosepsis (37%) background UTI male infants (32%) Adult urosepsis (19%) (Bingen-Bidois IAI 2002, aquisition of VF 43 Bonacorsi JID 2003, J urol 2005) (UTI, meningitis) Distribution (%) of speciﬁc virulent determinants involved in the pathogenesis of ECNM among different studies

ECOR n=72

15 p<0.001

22 p<0.001

Aerobactin (iucC / iutA) 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% Stools 60% Blood 50% * * CSF 40% 30% 20% 10% 0% K1 14.9kb pap sfa/foc ibeA hly aer iroN HPI* p<0.05 cnf1

Half of the strains are devoid of adhesins (sfa) and invasins (ibeA) 45 Bingen. JCM 1997, Bingen. CID 1996, Johnson JID 2000-2001, Bidet IAI 2005 « 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 Different ectochromosomal DNA –like domains have been involved in the virulence of neonatal E.coli meningitis isolates

BBB crossing

B a

Bonacorsi, JID, 2003 47 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)

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

IP Injection 300 CFU Were inoculated intraperitoneally with Animals were killed and 5 µl of CSF was 300 +/- 50 CFU in physiological saline 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

sfa +, ibeA + sfa -, ibeA -

d : p<0.05 vs strain C5 O45 same capacity as C5 but devoid of sfa and ibeA Bonacorsi, JID, 200350 other unknown mechanisms of BBB penetration in this clonal group? Phylogenetic group A (6%) meningitis isolates vs group A ECOR isolates

Meningitis ECOR (n=11) (n=25)

K1 73% 4%

HPI 100% 32%

Aerobactin 100% 24%

Atypical Prevalence of VF was higher in Virulent group group A meningitis isolates (avirulent in animal model)

(Detection of group A isolates causing Meningitis may point to a immune deficiency)51 Bacteremia BBB crossing

Serotype 045:K1:H7 iroN aer fyuA PAI IC5

meningitis O18:K1 +-/+++ ++ sfaS ibeA

meningitis O45:K1 +++- --

• Highly virulent clonal group (Ribotype B 21) 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 III536, GimA).

52 Contribution of PAI IIJ96 – like domains to bacterial survival in different ecological niches

« From commensalism to pathogenesis »

53 PAI IIJ96 / PAI IC5 PATHOGENICITY ISLAND

hly cnf1 prs hra Swenson, IAI, 1996 UPEC J96 110 kb 94’ (PheU)

1- Core : cnf1, hly, hra consistently colocalized (« signature »). (Bingen-Bidois IAI 2002, Bonacorsi JID 2003)

2- Implication of PAI IIJ96 / PAI IC5 in bacteremia and meningitis (rat meningitis model)

Strain bacteremia meningites C5 (O18) meningitis 100% (5,7) 52%

C5  PAI IC5 66% (4.67) * 26%

(Houdouin IAI 2002) log(blood cfu/ml) *p < 0,05

 PAI IIJ96 / PAI IC5 contribute to bacterial survival in blood by inducing high level bacteremia 54 Prevalence of PAI IIJ96-like domains among isolates of different collections

(Bidet, IAI 2005) 55 PAI IIJ96 / PAI IC5 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 Clinical expression of E. coli carrying

PAI IIJ96 / PAI IC5 according to the genetic background and the insertion site – (1) 0’ IS100, hly, cnf1, pap, hra LeuX PheU 75’ 25’ PheV Chromosomal insertion of PAI IIJ96 50’ B21 LeuX (50%) / PheU (50%) <-> meningitis «PAI»

B26 PheU (86%) <-> U.T.I. acquisition B2 «PAI» PAI IIJ96 Group Allelic exchange papGIII  papGII

B27 PheV <-> colonization « Saprophytic island » ↑ expression of No allelic exchange fitness factors  57 survival in the gut (Bidet IAI 2005) PAI IIJ96-like domains

Fitness Island Enhances bacterial survival

Human Gut Urinary tract Blood

58 E. coli neonatal meningitis isolate Bacteremia

Enterobactin FepA, Fiu, Cir Salmochelin IroN Yersiniabactin FyuA (HPI) Aerobactin IutA Heme captation ChuA

Siderophore Receptor P fimbriae Fe 3+ Fe 3+~Transferrin LPS

Type 1 fimbriae Fe 3+

Fe 3+ Transferrin K1 capsule

Omp A

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 (ibeA)

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