ASSOCIATION OF CLASSICAL MICROBIOLOGY AND TARGETED METAGENOMIC ANALYSIS TO EVALUATE

THE PRESENCE OF CLOSTRIDIUM DIFFICILEIN A BELGIAN

NURSING HOME

B. Taminiau, C. Rodriguez, N. Korsak, V. Avesani, J. Van Broeck, M. Delmee, G. Daube

Gut Day 20137th november 2013

Groningen, the Netherlands

Background• Since toxigenic C. difficile was recognized as the major cause of antibiotic-associated

diarrhea and pseudomembranous colitis in 1978, many outbreaks have been

documented

• enhanced virulence and increased antibiotic resistance of C. difficile strains (PCR-

ribotype 078/NAP-1/B1) has been observed for the last years

• Patients with serious illnesses and prolonged hospitalizations are at particular risk, as

people above 65 years of age . The increased risk of acquiring C. difficile in the elderly

may be de to age-related changes in intestinal flora, immune senescence or the

presence of underlying diseases

• There are emerging data on the occurrence of C. difficile infection in the community:

non-hospitalized and younger patients with absence of other traditional risk factors

Hypothesis about a potential risk of foodborne infections linked to C. difficile

@google images 2013

Clostridium difficile Presence in Nursing homes

@google images 2013

15%

2.1-8.1%

39%

10% 0.03%

8.9% 4.6%

There is not much data describing the prevalence and molecular epidemiology of C.difficile in nursing homes in absence of an epidemic situation

Objectives

• To evaluate and follow the prevalence of C.

difficile in a Belgian nursing home

• To establish a relationship between other

intestinal bacterial populations and C. difficile

colonization

• To evaluate the global evolutions of the total

microflora and the relation with the C. difficile

presence

• During a 4-month period, stool samples from a

group of 23 elderly care home residents were

collected weekly (From March to June 2013)

Study design

• Direct and enrichment culture

Home-made cycloserine cefoxitin

fructose taurocholate

• Detection of a species-specific internal fragment of tpi, detection of genes for

toxin B, toxin A and binary toxin (cdtA) by PCR and Genotype Cdiff test system

• PCR-ribotyping

• Targeted Metagenomic analysis : 16S V1-V3 libraries

• Home-made bioinformatic pipeline

Results: Prevalence of C. difficile in nursing home residents

• 7/23 (30.4%) residents were (at least one

week) positive for C. difficile

• C. difficile was detected in 13/30 (43.3%)

episodes of diarrhea

• 4/13 (30.7%) residents positives for

C. difficile had previously received an

antibiotic therapy

C. difficile recovery:

Patient Week

N° 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 171 D D/AB/P AB

2

3

4 D D

5 AB/P D

6 AB

7 AB AB

8 H H H H P

9

10

11

12 « D » « D » « D » « D » « D » « D »

13

14

15 D D D D D D D D D/P D/P P P

16 D/AB AB AB X X X X X X X X X X X

17 D/AB AB AB AB

18 D

19 D D

20

21 D D D D P

22 X X X X X X X X

23

24 AB/P AB

Positive results after 3 days of enrichmentPositive after direct cultureNegativeSample not available

D diarrhea AB antibiotic P probiotic H hospitalization X death or resident outside of the study

Analyzed in metagenomicsAnalyzed in metagenomics

Resident Week

N° 01 02 03 05 06 07 08 09 10 11 12 13 14 15 16 171 020 020 020 020 020 020 020

2

3

4

5

6

7

8

9

10

11

12

13 UCL36 UCL36

14

15 027 027 027 027 027 027 027 027 027 027 027 027 027

16

17

18 027 027 027 027 027 027

19 UCL46 UCL46 027 027 027 UCL36

20

21

22

23 UCL36

24 UCL36 UCL36UCL36

Positive results after 3 days of enrichmentPositive after direct cultureNegativeSample not available

C. difficile abundance

13 15 18 19 24

residentresident

• Proportion of sequences of C. difficile detected for each resident each week• Residents positive for C. difficile by classical microbiology showed an

important proportion of C. difficile sequences

The results so far:

80 samples sequenced and analyzed: 6300 OTUs

Positive detection of Clostridium difficile :

n Microbiology Amplicon sequencing

20 + +

36 - -

19 + -

5 - +

C. difficile detection

0,000%

10,000%

20,000%

30,000%

40,000%

50,000%

60,000%

70,000%

80,000%

90,000%

100,000%

P1

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P1

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VictivallaceaeVibrionaceaeVerrucomicrobiaceaeVeillonellaceaeunclassifiedSynergistaceaeStreptococcaceaeStaphylococcaceaeSphingomonadaceaeRuminococcaceaeRikenellaceaeRhizobiaceaePseudomonadaceaePseudoalteromonadaceaePropionibacteriaceaePrevotellaceaePorphyromonadaceaePeptostreptococcaceaePasteurellaceaeOxalobacteraceaeMoraxellaceaeMicrococcaceaeMicrobacteriaceaeLactobacillaceaeLachnospiraceaeFusobacteriaceaeFlavobacteriaceaeFamily_XIII_Incertae_SedisFamily_XII_Incertae_SedisFamily_XI_Incertae_SedisEubacteriaceaeErysipelotrichaceaeEnterococcaceaeEnterobacteriaceaeDesulfovibrionaceaeCorynebacteriaceaeCoriobacteriaceaeComamonadaceaeClostridiaceaeCarnobacteriaceaeCampylobacteraceaeBifidobacteriaceaeBacteroidaceaeAlcaligenaceaeActinomycetaceae

Relative proportions of the differentbacterialfamiliesThere isa bacterialcomposition thatappearsstable alongthe weeks

Relative proportions of the differentbacterialfamiliesThere isa bacterialcomposition thatappearsstable alongthe weeks

0.05

P12_week_10

P5_week_12

P5_week_03

P1_week_08

P13_week_04

P19_week_13

P19_week_1

1

P18_w

eek_07

P18_week_17

P16_week_06

P15_week_08

P13_week_06

P1_week_12

P17_week_08

P17_week_06

P12_week_07

P2_week_15

P15_week_14

P19_week_03

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P1_week_04

P24_week_14

P13_week_08

P15_week_12

P24_week_10

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Phylotype tree based on population distribution – Braycurtis dissimilarity index

• Study the phylotype composition of the samples

• This tree reflects how many samples have the same bacterial content or not

• Almost all the samples are clustered in a sub-tree corresponding to a single resident

• Each resident has his own bacterial imprint which is stable during the entire study

Different bacteria in case of positive (blue) and negative (orange) residents in relation with C. difficile

Statistical differences in genera abundance between

Cdiff positive (blue)Cdiff negative (orange)Cdiff positive (blue)Cdiff negative (orange)

Diarrheic (orange)Non diarrheic (blue)Diarrheic (orange)Non diarrheic (blue)

The story so far

• C. difficile prevalence of 30.4% in a Belgian nursing home

• The most common PCR-ribotype identified was 027

• Residents have all their microbiota print

• Metagenomics analysis can’t substitute targeted procotocols

• But It offers a global picture of the microbiota context:– With correlations– Identifications– Follow up

Prof. Michel Delmée

Véronique Avesani

Johan Van Broeck

Eléonore Lyeza

Prof. Georges Daube

Dr. Cristina Rodriguez

Dr. Nicolas Korsak

ACKNOWLEDGEMENTS

Nursing Home Sainte-Joséphine(Theux-Belgium)