ASSOCIATION OF CLASSICAL MICROBIOLOGY AND
TARGETED METAGENOMIC ANALYSIS TO EVALUATE
THE PRESENCE OF CLOSTRIDIUM DIFFICILE
IN A BELGIAN
NURSING HOME
B. Taminiau, C. Rodriguez, N. Korsak, V. Avesani, J. Van Broeck, M. Delmée, G. Daube
Gut Day 2013
7th november 2013
Background
•
Since toxigenic C. difficile was recognized as the major cause of antibiotic-associateddiarrhea 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, aspeople 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
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.
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 cultureHome-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 librariesResults: 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
Patient
Week
N° 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 1 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 ABPositive results after 3 days of enrichment Positive after direct culture
Negative Sample not available
D diarrhea AB antibiotic P probiotic H hospitalization X death or resident outside of the study
Analyzed in metagenomics
Analyzed in metagenomics
Resident
Week
N° 01 02 03 05 06 07 08 09 10 11 12 13 14 15 16 17 1 020 020 020 020 020 020 020 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 02719 UCL46 UCL46 027 027 027 UCL36
20 21 22 23 UCL36 24 UCL36 UCL36 UCL36
Positive results after 3 days of enrichment Positive after direct culture
Negative Sample not available
C. difficile abundance
13
15
18
19
24
resident
resident
• Proportion of sequences of C. difficile detected for each resident each week • Residents positive for C. difficile by classical microbiology showed an
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% P 1_ w e ek01 P 1_ w e ek02 P 1_ w e ek04 P 1_ w e ek05 P 1_ w e ek07 P 1_ w e ek08 P 1_ w e ek10 P 2_ w e ek01 P 2_ w e ek03 P 2_ w e ek05 P2_ w e ek07 P 2_ w e ek09 P 13_ w e ek0 1 P 13_ w e ek0 2 P 13_ w e ek0 3 P 13_ w e ek0 4 P 13_ w e ek0 6 P 13_ w e ek0 8 P 13_ w e ek1 0 P 15_ w e ek0 1 P 15_ w e ek0 2 P 15_ w e ek0 3 P 15_ w e ek0 4 P 15_ w e ek0 6 P15_ w e ek0 7 P 15_ w e ek0 8 P 15_ w e ek0 9 P 18_ w e ek0 5 P 18_ w e ek0 7 P 18_ w e ek0 9 P 19_ w e ek0 1 P 19_ w e ek0 2 P 19_ w e ek0 3 P 19_ w e ek0 7 P 19_ w e ek0 9 P19_ w e ek1 0 P 24_ w e ek0 3 P 24_ w e ek0 5 P 24_ w e ek0 6 P 24_ w e ek0 8 Victivallaceae Vibrionaceae Verrucomicrobiaceae Veillonellaceae unclassified Synergistaceae Streptococcaceae Staphylococcaceae Sphingomonadaceae Ruminococcaceae Rikenellaceae Rhizobiaceae Pseudomonadaceae Pseudoalteromonadaceae Propionibacteriaceae Prevotellaceae Porphyromonadaceae Peptostreptococcaceae Pasteurellaceae Oxalobacteraceae Moraxellaceae Micrococcaceae Microbacteriaceae Lactobacillaceae Lachnospiraceae Fusobacteriaceae Flavobacteriaceae Family_XIII_Incertae_Sedis Family_XII_Incertae_Sedis Family_XI_Incertae_Sedis Eubacteriaceae Erysipelotrichaceae Enterococcaceae Enterobacteriaceae Desulfovibrionaceae Corynebacteriaceae Coriobacteriaceae Comamonadaceae Clostridiaceae Carnobacteriaceae Campylobacteraceae Bifidobacteriaceae Bacteroidaceae Alcaligenaceae Actinomycetaceae
Relative proportions of the differentbacterialfamilies
There isa bacterialcomposition thatappearsstable alongthe weeks Relative proportions of the differentbacterialfamilies
0.05 P 12 _w ee k_ 10 P5_wee k_12 P5_ wee k_03 P1_ wee k_08 P13 _w eek _04 P19_w eek_13 P19_week_11 P1 8_w ee k_ 07 P18_w eek_17 P 16_ w ee k_0 6 P15_week_08 P1 3_w eek_ 06 P1_ wee k_12 P17_week_08 P17_w eek_06 P 12 _w ee k_ 07 P2_w eek_ 15 P15_ week _14 P19_week_03 P15_week_0 4 P1_ wee k_04 P24 _wee k_14 P1 3 _w eek_ 08 P15_ week _12 P24_week_10 P17_week_07 P2 _w ee k_ 11 P15_week_09 P2 _w eek_ 13 P 2_w ee k_ 09 P24_w eek_05 P15_we ek_03 P1 8_we ek_0 9 P 18 _w ee k_ 05 P 12 _w ee k_ 11 P24_week_06 P15_ week _13 P24_week_08 P19_ wee k_02 P1_w eek_ 01 P1_w eek_ 16 P24 _we ek_12 P 12 _w ee k_ 03 P13 _wee k_01 P24_week_17 P18_ we ek_12 P19_we ek_07 P15_w eek_06 P 2_w ee k_ 01 P13 _w eek_ 02 P13 _w eek_ 03 P5_w eek_16 P1_w eek_ 14 P15_ week _11 P19_ week _15 P 13 _w e ek _1 4 P 2_w ee k_ 05 P19_week _09 P24_ week_03 P1 8_w eek _15 P 5 _w ee k_0 1 P15_week_07 P15_week_0 2 P1 _w eek_ 10 P1_ we ek_ 07 P1_w eek_ 05 P19_we ek_10 P 2_ w ee k_ 07 P15_ wee k_17 P19_week_01 P2_w eek_1 7 P1_ wee k_02 P 13 _w ee k_1 2 P15_week_01 P1_ wee k_11 P 1 3_ w e e k_1 0 P 2_w ee k_ 03 P17_ week_05 P 1 _w e e k_ 17
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
