Characterisation of vancomycin-
resistance vanD-like genes from human
intestinal microbiota by metagenomics
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4.1. Avant-propos
Statut d’auteur de l’étudiant
Eliel Brochu est le premier auteur de l’article présenté.
Rôle de l’étudiant dans la préparation de l’article
Eliel Brochu a réalisé les analyses bio-informatiques nécessaires à la recherche des gènes
vanD dans les données de séquençage métagénomique et culturomique, a effectué la
caractérisation de ces gènes ainsi que de leur contexte et a comparé les deux approches utilisées dans cet article afin d’étudier la présence des gènes vanD dans le résistome provenant du microbiote intestinal humain. Eliel Brochu a aussi rédigé cet article et a intégré les commentaires des coauteurs.
Coauteurs
Les coauteurs de l’article sont Ann Huletsky, Maurice Boissinot, Dominique K. Boudreau, Frédéric Raymond, Ève Bérubé et Michel G. Bergeron. Ann Huletsky, Maurice Boissinot, Jacques Corbeil et Michel G. Bergeron ont participé à l’élaboration et à la supervision du projet de recherche. Ann Huletsky a effectué un suivi technique de l’avancée des travaux de même que participer à l’analyse globale des résultats. Dominique K. Boudreau a participé au suivi technique de l’avancée des travaux et a contribué à certaines analyses bio- informatiques. Frédéric Raymond a réalisé le séquençage métagénomique et culturomique. Ève Bérubé a réalisé la culture des échantillons pour la culturomique. Amin Ahmed Ouameur a réalisé l’extraction d’ADN pour la métagénomique. Les coauteurs ont lu et accepté la version présentée de l’article.
Statut de l’article
Le manuscrit : «Characterisation of vancomycin-resistance vanD-like genes from human intestinal microbiota by metagenomics and culturomics» est en rédaction pour publication dans un futur rapproché.
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Commentaires sur l’article inséré dans ce mémoire
Cet article a été présenté par Eliel Brochu sous forme d’affiche au Cell symposia : Human Immunity and the Microbiome in Health and Disease 2015, Montréal, Canada; ainsi que sous forme de présentation orale à la 3e Journée de Médecine Moléculaire de l’Université Laval 2015, Québec, Canada.
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4.2. Résumé
Le microbiote intestinal humain est un important réservoir de gènes de résistance aux antibiotiques encore méconnu. Dans cette étude, nous avons exploré le microbiote de 24 volontaires sains avant et après une exposition de 7 jours à l’antibiotique cefprozil. Les approches métagénomiques et culturomiques ont été utilisées pour évaluer la présence de gènes de résistance à la vancomycine de type vanD dans les microbiotes des 24 participants, puis pour caractériser ces gènes, tout en examinant l’altération produite par les antimicrobiens. La culturomique a permis une détection de gènes vanD chez un plus grand nombre de participants (46%) comparativement à la métagénomique (8%). L’analyse des résultats a aussi montré que la prise d’un antibiotique de la famille des β-lactamines communément utilisé, tel que le cefprozil, a fortement augmenté l’abondance des bactéries contenant des gènes de résistance à un antibiotique d’une autre classe, comme la vancomycine, un antibiotique de dernier recours.
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Characterisation of vancomycin-resistance vanD-like
genes
from
human
intestinal
microbiota
by
metagenomics and culturomics
Eliel Brochu 1, 2, Ann Huletsky 1, 2, Dominique K. Boudreau 1, Frédéric Raymond 1, 2, Ève Bérubé 1, Amin Ahmed Ouameur 1, 2, Maurice Boissinot 1, 2, Jacques Corbeil 1, 2, Michel G. Bergeron 1, 2
Centre de recherche en infectiologie de l’Université Laval, Centre hospitalier universitaire (CHU) de Québec, Québec, Canada 1
Département de microbiologie-infectiologie et d’immunologie, Faculté de médecine, Université Laval, Québec, Québec, Canada 2
4.3. Abstract
The human intestinal microbiota is an important yet poorly known antibiotic resistance genes reservoir. In this study, we explored the microbiota of 24 healthy volunteers: 18 participants with stool samples collected before and after seven days of cefprozil β-lactam antibiotic exposure and 6 control participants with stool samples collected at both time points, but who were not exposed to the antibiotic. The presence of vancomycin resistance
vanD-like genes was then evaluated for the 24 healthy volunteers. Metagenomic and
culturomic approaches were used to characterise the vanD-like genes in the human intestinal microbiota and examine alteration by antimicrobials. For metagenomics, the DNA from the stool samples of the participants was directly sequenced by high-throughput sequencing. For culturomics, the bacteria from the stool samples were cultured under four different conditions, then the DNA was extracted and sequenced by high-throughput sequencing. The culturomic approach allowed detection of vanD-like genes in a large number of participants (46%) compared to the metagenomic approach (8%). Analysis of results also showed that the intake of a commonly used β-lactam antibiotic such as cefprozil increased the abundance of bacteria containing resistance genes to another class of antibiotic such as vancomycin, a last resort antibiotic.
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4.4. Introduction
The human intestinal microbiota contains a high density and diversity of bacteria. Actually, the intestine is the host of about 1013-1014 bacteria which is 1 to 10 times more than the number of human nucleated cells in all the body (Clemente et al., 2012; Sender et al., 2016; Xu & Gordon, 2003). Normal intestinal microbiota is dominated by anaerobic bacteria which are 100 to 1000 times more abundant than aerobic and facultative aerobic bacteria (Quigley, 2010). Furthermore, the intestinal microbiota is composed of about 1000 different bacterial species from only a few phyla (Qin et al., 2010; Xu & Gordon, 2003). The two principal phyla are the Bacteroidetes and the Firmicutes (Eckburg et al., 2005; Qin et al., 2010). The human intestinal microbiota also harbors a diverse reservoir of antibiotic resistance genes (resistome) (D'Costa et al., 2006; Fouhy et al., 2014). With the increasing emergence of antibiotic resistance in bacteria, the study of the intestinal resistome is essential for a better understanding of the origin and evolution of resistance.
Several approaches can be used to study the resistome of the human intestinal microbiota. Metagenomics is a powerful tool to study the high diversity of both cultivable and non- cultivable bacteria and their resistance genes (Wang et al., 2015). Yet, it allows only the characterisation of abundant bacteria due to the limitation of the depth bias (Lagier et al., 2012). Culturomics or bacterial culture allows the detection of antibiotic resistant genes from the less abundant resistant bacteria (Lagier et al., 2012). However, it detects only cultivable bacteria (Wang et al., 2015). The combination of metagenomics and culturomics can thus allow a better complete view of the resistome.
Vancomycin is a member of the glycopeptide family of antibiotics. It inhibits the synthesis of the cell wall of Gram positive bacteria by preventing the synthesis of peptidoglycan chain. It binds to the D-Ala-D-Ala terminal dipeptide of the precursor pentapeptide of the peptidoglycan chain (Yao & Moellering, 2011). Vancomycin is a last resort antibiotic used for treatment of severe infections caused by Gram positive bacteria (Domingo et al., 2005a; Sass et al., 2012). However, bacteria have acquired nine different vancomycin resistance clusters providing resistance by synthetizing two alternatives to the D-Ala-D-Ala terminal dipeptide (Courvalin, 2006). The vanA, vanB, vanD, vanF, and vanM clusters synthetize a
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D-Ala-D-Lac substitute peptide whereas the vanC, vanE, vanG, vanL, and vanN clusters synthetize a D-Ala-D-Ser substitute peptide (Depardieu et al., 2015; Fraimow et al., 2005; Rubinstein & Keynan, 2014). It has been shown that a proportion of hospitalized patients are carriers of different vancomycin resistance van genes, with a highest prevalence (26- 40%) of vanD-like genes (Domingo et al., 2005b). VanD resistance type is characterised by a constitutive expression in enterococci (Depardieu et al., 2004). Seven vanD subtypes are currently described in enterococci: vanD1-vanD7 (Boyd et al., 2000; Boyd et al., 2004; Boyd et al., 2016; Boyd et al., 2006; Casadewall & Courvalin, 1999; Dalla Costa et al., 2000; Ostrowsky et al., 1999). However, the presence of vanD genes has not been evaluated yet in healthy people.
The objective of this study was to determine the presence of vanD genes and characterise their genetic context in the human intestinal microbiota of healthy participants prior and following antibiotic exposure using metagenomic and culturomic sequencing of the stool samples. Healthy volunteers were recruited and exposed during seven days to cefprozil, a commonly used β-lactam (2nd-generation cephalosporin). A β-lactam was used for this study because antibiotics of this family are the most widely used in prophylaxis and antibiotherapy. Stool samples were collected before and after antibiotic exposure and analyzed.
4.5. Material and methods
Ethics approval
The clinical protocol was approved by the ethical committee of the «Centre Hospitalier Universitaire (CHU) de Québec-Université Laval». Informed written consent was obtained from participants enrolled in this study.
Participants’s recruitment and samples collection
The 24 healthy volunteers enrolled in this study were young adults from the Quebec City region (Canada) between 21 and 35 years old, had a normal intestinal transit, and met several inclusion, and exclusion criteria (Raymond et al., 2015). Among the 24 healthy volunteers, 18 were treated twice a day with a 500 mg oral dose of cefprozil (CPR), a
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second-generation cephalosporin, during seven days. The stool specimens were collected at two time points: before the beginning of the treatment (D0) and seven days later at the end of the treatment (D7), as described in Raymond et al. 2015. The 6 participants unexposed to the antibiotic were used as controls. Their stool specimens were also collected at D0 and D7. All samples were brought to the laboratory within 2 hours of collection and placed immediately in an anaerobic chamber for processing. Stools were aliquoted and stored at - 80°C.
DNA extraction for metagenomics
Genomic DNA was extracted from 500 mg of the 48 stool samples (24 participants at D0 and D7) using the PowerMax Soil DNA Extraction Kit (MO BIO Laboratories, Carlsbad, CA, USA) as described previously (Raymond et al., 2015).
Sample culture for culturomics
Four culture conditions were tested in the study: (1) anaerobic culture without selection (ANA), (2) anaerobic culture with 32 µg/mL cefoxitin (ANA-FOX), (3) aerobic culture with 5% CO2 without selection (CO2) and (4) aerobic culture with 5% CO2 and 32 µg/mL
cefoxitin (CO2-FOX). The broth medium used was an enriched brain-heart infusion (BHI)
broth medium supplemented with haemin, lactate, L-cystine, pyruvate and vitamin K (Domingo et al., 2007). Stool sample (1 g) was suspended in 15 mL of PBS with 0.1% cysteine (PBSc) and homogenized by vortexing during 5 minutes. A supernatant phase was obtained after a 5-minute sedimentation step (Goodman et al., 2011). Two hundred (200) µL of the supernatant was used to inoculate 10 mL of the broth with or without cefoxitin depending on the culture condition to test. The inoculated broths were incubated during 7 days at 35°C under anaerobic or aerobic (5% CO2) conditions depending on the culture
condition to test. Aliquots of cultures were stored at -80°C without glycerol.
DNA extraction for culturomics
Bacterial DNA was extracted from the 192 stool cultures (culturomics samples) (24 participants at D0 and D7 each under 4 conditions). DNA was extract from 1 mL of the stool culture using the BD GeneOhm Lysis Kit (BD Diagnostics-GeneOhm, Québec,
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Canada) and purified using the Biosprint 15 DNA Blood Kit (Qiagen, Mississauga, Ontario, Canada) and the KingFisher device (Thermo Fisher Scientific, Waltham, MA, USA) following the instructions of the manufacturers.
Quality verification of the purified DNA
DNA extracted for sequencing was quantified using the NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and by QuantiFluor dsDNA System (Promega, Madison, WI, USA) following the instructions of the manufacturers. DNA integrity was assessed by electrophoresis on agarose gel (Raymond et al., 2015).
Sequencing
Sequencing libraries were constructed with 50 ng of purified DNA from the 240 samples (48 metagenomic samples and 192 culturomic samples) and sequencing was performed by shotgun sequencing using the HiSeq 1000 high-throughput sequencer (Illumina, San Diego, CA, USA) as described previously (Raymond et al., 2015). Two metagenomic libraries were pooled per HiSeq lane while eight culturomic libraries were pooled per HiSeq lane, for a total of 24 lanes used for each approach. An average of 15 Gb was sequenced per metagenomic sample and 2.5 Gb per culturomic sample (Raymond et al., 2015).
Reads assembly
The assembly of the genomic reads produced by sequencing of metagenomic and culturomic samples was performed using the Ray Meta 2.0 Assembler software (Boisvert et al., 2010; Boisvert et al., 2012) as described previously (Raymond et al., 2015). Contigs obtained were filtrated for a length >500 nucleotides.
Search of vanD-like genes in sequencing data
Homology search was performed between sequences of vanD genes listed in the curated MERGEM database (http://www.mergem.genome.ulaval.ca) and the metagenomic and culturomic sequencing data using BLAT alignment software (http://genome.ucsc.edu/). Genes were identified as potential vanD-like genes if they had nucleic-acid identity >50%
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and an alignment length of >300 nucleotides. Open Reading Frame Finder tool (ORF finder) from NCBI (http://www.ncbi.nlm.nih.gov/projects/gorf/) was used to find ORFs of potential vanD-like genes. Nucleotides homology searches were made against the sequences listed in the «Whole-genome shotgun contigs» (WGS) NCBI database whereas amino acid homology searches were made against the sequences listed in the «Non- redundant protein sequences» (NR) NCBI database using the BLAST tool to confirm the identity of the vanD-like genes.
Comparison with reference sequences
A protein phylogenetic neighbor-joining tree was constructed using the Mega6 software (Tamura et al., 2013) to compare the translated sequences of the complete vanD1 of
Enterococcus faecium BM4339 (NG_048359.1), vanD2 of E. faecium A902
(NG_048358.1), vanD3 of E. faecium N97-330 (NG_048360.1), vanD4 of E. faecium 10/96A (NG_048361.1), vanD5 of E. faecium N03-0072 (NG_048362.1), vanD6 of
Enterococcus gallinarum N04-0414 (NG_048364.1), vanD7 of E. faecium N15-508
(KT825491.1), vanA of E. faecium BM4147 (NG_048323.1), and vanB of Enterococcus
faecalis V583 (U35369.1) genes reference sequences, the translated sequence of a vanD
gene found in Ruminococcus gauvreauii CCRI-16110 (NG_048365.1), two translated sequences of vanD gene found in Clostridium clostridioforme CM201 and 2_1_49FAA (AGYS01000016.1 and ADLL01000098.1), and a VanD sequence found in Bariatricus
massiliensis (WP_029467253.1) (Atarashi et al., 2013). The vanA (NG_048323.1) and vanB (U35369) translated reference sequences and the translated sequence of a vanB gene
found in C. clostridioforme CIP 110249 (JF313105.1) were used as an outgroup. All reference sequences are available in the NCBI database (https://www.ncbi.nlm.nih.gov).
Comparison of metagenomic and culturomic approaches and the
different culture conditions at different sampling times
The presence of complete or partial vanD genes was evaluated in metagenomic and culturomic sequencing data of the 24 participants at D0 and D7. The detection of complete or partial vanD genes was also compared for the four culture conditions used in the culturomic approach (anaerobic (ANA), aerobic (CO2), anaerobic with cefoxitin (ANA-
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FOX), and aerobic with cefoxitin (CO2-FOX)) at the the two different times of sampling
(D0 and D7). The detection depth was determined for the two approaches by calculating the number of participants for which vanD-like genes were detected.
Characterisation of genetic contexts of vanD-like genes
ORF finder tool was used to explore the genetic context of the complete vanD genes. ORFs longer than 50 amino acids located inside and outside the vanD locus were subjected to homology searches against the sequences listed in the NCBI database using the BLAST tool. The bacterial identity consensus obtained for the ORFs outside the vanD locus in a same contig represents the bacterial original host of the vanD-like gene.
4.6. Results
Comparison of metagenomic and culturomic approaches and the
different culture conditions at different sampling times
The metagenomic and culturomic approaches allowed detection of complete or partial
vanD-like genes in the microbiotas of 2 and 11 out of the 24 participants, respectively
(Figure 4.1; Table 4.1). At D0, prior to in vivo antibiotic exposure, vanD-like genes were not detected under anaerobic (ANA) and aerobic (CO2) culture conditions. vanD-like genes
were detected at D0 only with in vitro antibiotic selection with cefoxitin either under anaerobic (ANA-FOX) or aerobic (CO2-FOX) culture conditions (Figure 4.2). However, at
D7, following in vivo antibiotic exposure, vanD-like genes were detected with and without
in vitro antibiotic selection, in all four culture conditions (Figure 4.2). In some cases, vanD-
like genes were found at D0, with in vitro antibiotic exposure, but were not found at D7 for the same participants and conditions. However, the opposite was more common (Table 4.1).
Phylogenetic comparison of the VanD proteins
The complete VanD proteins found in the microbiotas of participants are presented in a phylogenetic neighbor-joining tree (Figure 4.3). These VanD proteins can be divided in six groups. The VanD of groups 1, 2, and 3 (Figure 4.3) identified with metagenomics and
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mostly with culturomics were genetically different from the seven known VanD subtypes found in enterococci (VanD1-VanD7), but exhibited 100% identity with their closest VanD homologs in the NCBI database (Table 4.1). The VanD of groups 1 and 2 were identical to VanD of different strains of C. clostridioforme. The five VanD of group 1 were identical to VanD of strains CM201 (AGYS01000016.1), 90A1 (AGYP01000043.1), 90A3 (AGYQ01000049.1), 90A4 (AGYM01000086.1), 90A6 (AGYL01000071.1), 90B1 (AGYO01000028.1), and WAL-7855 (ADLM01000013.1) whereas the six VanD of group 2 were identical to the VanD of strains 2_1_49FAA (ADLL01000098.1) and CAG:511 (CBKN010000201.1) (Figure 4.3; Table 4.1). VanD of group 1 were found in two participants (P3 and P22) at D7 with both metagenomics and culturomics in different culture conditions. VanD of group 2 were found in two participants (P2 and P17) at D7 with culturomics only in different culture conditions. The four VanD of group 3 were found in the same participant (P22) at different time points and in different culture conditions. They were all identical to the VanD found in B. massiliensis AT12 and Clostridium
scindens VE202-05 (WP_029467253.1) (Figure 4.3; Table 4.1). The two VanD of group 4
was 100% identical with VanD4 from E. faecium 10/96A and were found at D7 in a same participant (P13) by culturomics in different culture conditions (Figure 4.3; Table 4.1). The unique VanD proteins of groups 5 and 6 were most closely related to, respectively, VanD7 and VanD6 of enterococci. The VanD of group 5 showed an identity of 98% with the known VanD7 subtype from E. faecium N15-508 and was found at D0 in one participant (P20) by culturomics with cefoxitin selection. The VanD of group 6 was more phylogenetically distinct from all known VanD. It exhibited 91% identity with its closest VanD homolog, i.e. VanD6 from E. gallinarum N04-0414 and was found in one participant (P1) at D0 by culturomics with cefoxitin selection. The genes located on each side of the
vanD locus were, most of the time, associated with the same bacterial species as that of the
closest known VanD homolog in NCBI (Table 4.1).
Analysis of the different vanD loci
Six different vanD loci were identified with metagenomic and culturomic sequencing (Figure 4.4). Locus 1 was related to the vanD loci of C. clostridioforme strains CM201, 90A1, 90A3, 90A4, 90A6, 90B1, and WAL-7855, sharing 100% nucleic acid identity along
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all the length of the locus (Figure 4.4). Locus 2 was related to the vanD loci of C.
clostridioforme strains 2_1_49FAA and CAG:511, sharing 100% identity along all the
length of the locus (Figure 4.4). The genes located outside of loci 1 and 2 were also related to C. clostridioforme (Table 4.1). The closest enterococci Van proteins identified in loci 1 and 2 were those from the vanD4 locus (Table 4.2). The VanD ligases of loci 1 and 2 had a deletion of one amino acid (Figure 4.4). The VanHD proteins of loci 1 and 2 were deleted of
the N-terminal 241 amino acids and of one amino acid close to the C-terminal region (Figure 4.4).
Locus 3 was related to the vanD loci of B. massiliensis AT12 and C. scindens VE202-05, showing 100% identity along all the length of the locus of B. massiliensis including an integrase gene (Figure 4.4), but the the integrase gene was absent in C. scindens. The genes located outside of locus 3 were also related to B. massiliensis or C. scindens (Table 4.1). The closest enterococci VanRD, VanSD, VanYD, and VanXD proteins identified in locus 3
were those from the vanD6 locus while the closest enterococci VanHD and VanD proteins
of this locus were those from the vanD7 locus (Table 4.2). The VanD protein identified in locus 3 shared an identity of 93,6% with VanD7, but was also very close to VanD6 with 92,7% identity (Table 4.2). The VanYD and VanHD proteins from locus 3 were more distant
from their closest homologs in the vanD6 and vanD7 loci. VanYD shared 88,3% identity
with its homolog in the vanD6 locus whereas VanHD showed 87,3% identity with its
homolog in the vanD7 locus (Table 4.2).
Locus 4 was highly related to the vanD4 locus of E. faecium 10/96A along all the length of the locus, except for VanHD which showed 99,7% identity with its homolog of the vanD4
locus (Table 4.2) and the absence of the 640 amino acids ISEfa4 insertion sequence observed in the VanSD of E. faecium 10/96A (Depardieu et al., 2003). All other Van
proteins were 100% identical with their homologs of the E. faecium vanD4 locus. An integrase gene was also identified with 100% identity with that of E. faecium 10/96A. The genes located outside of locus 4 were also related to E. faecium (Table 4.1).
Locus 5 was related to the vanD7 locus of E. faecium N15-508, showing about 99% identity along all the length of the locus (Figure 4.4). The presence of VanRD could not be
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homolog of the vanD7 locus (Table 4.2). All other Van proteins identified in the locus showed between 98,3% and 99,5% identity with their homologs of the vanD7 locus (Table 4.2). Locus 5 was found in only one contig and was too short to identify the genes located