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A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes
Infection, Genetics and Evolution xxx (2015) xxx–xxx
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes Yolande Hauck a, Charles Soler b, Patrick Gérôme c, Rithy Vong b, Christine Macnab b, Géraldine Appere b, Gilles Vergnaud a,d, Christine Pourcel a,⇑ a
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris Sud, Université Paris-Saclay, 91405 Orsay cedex, France Laboratoire de biologie clinique, HIA Percy, Clamart, France Service de biologie médicale, HIA Desgenettes, 69275 Lyon cedex 03, France d ENSTA ParisTech, Université Paris-Saclay, 91762 Palaiseau cedex, France b c
a r t i c l e
i n f o
Article history: Received 22 January 2015 Received in revised form 4 May 2015 Accepted 8 May 2015 Available online xxxx Keywords: VNTR Genotyping In silico MLVA CRISPR Post-surgery infection
a b s t r a c t Propionibacterium acnes plays a central role in the pathogenesis of acne and is responsible for severe opportunistic infections. Numerous typing schemes have been developed that allow the identification of phylotypes, but they are often insufficient to differentiate subtypes. To better understand the genetic diversity of this species and to perform epidemiological analyses, high throughput discriminant genotyping techniques are needed. Here we describe the development of a multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) method. Thirteen VNTRs were identified in the genome of P. acnes and were used to genotype a collection of clinical isolates. In addition, publically available sequencing data for 102 genomes were analyzed in silico, providing an MLVA genotype. The clustering of MLVA data was in perfect congruence with whole genome based clustering. Analysis of the clustered regularly interspaced short palindromic repeat (CRISPR) element uncovered new spacers, a supplementary source of genotypic information. The present MLVA13 scheme and associated internet database represents a first line genotyping assay to investigate large number of isolates. Particular strains may then be submitted to full genome sequencing in order to better analyze their pathogenic potential. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Propionibacterium acnes is an anaerobic, Gram-positive bacillus, which can survive for long periods of time in aerobic conditions. It is a major part of the skin microbiome, an aggravating factor in the pathogenesis of acne vulgaris (Fitz-Gibbon et al., 2013; Tomida et al., 2013), and also an opportunistic pathogen responsible for infections of different gravities. In the hospital it is frequently associated with infection of implants, prostheses and other devices on which it can form biofilms (for a review see (Achermann et al., 2014; Aubin et al., 2014)). The species has been classified into three major phylogenetic clusters or lineages, also called phylotypes or types. Type I and type II members were distinguished based on serological agglutination tests, cell wall sugar analysis, and monoclonal antibody (mab) typing (Johnson and Cummins, 1972; McDowell et al., 2005). Later, type I was further subdivided into subtypes IA and IB, and a third lineage, type III, was discovered ⇑ Corresponding author at: I2BC, Bat. 400, Université Paris Sud, 91405 Orsay cedex, France. E-mail address: [email protected] (C. Pourcel).
(McDowell et al., 2008). This was based on polymorphism of recA, and mab typing. Recently MALDI-TOF MS fingerprinting was shown to discriminate phylotypes I, II and III of P. acnes (Nagy et al., 2013). A study by Rollason and co-workers suggested that the different phylotypes presented different pathogenicity and were associated to various clinical situations (Rollason et al., 2013). It also demonstrated that patients frequently carried multiple phylogroups. Type III was recently shown to be associated with skin, similarly to the other lineages (Dekio et al., 2012). In order to better characterize strains and further study their association with particular infections, genotyping methods were developed and their advantages and drawbacks have been compared (Yu et al., 2015). Random amplification of polymorphic DNA (RAPD) (Perry et al., 2003) and pulse field gel electrophoresis (PFGE) (Oprica et al., 2004) were among early typing methods but they were time consuming and suffered from problems of reproducibility. They were replaced by Multi Locus Sequence Typing (MLST), a sequence-based method making use of housekeeping and virulence genes. Whereas a total of nine (MLST9) genes were selected for the Aarhus scheme (Kilian et al., 2012; Lomholt and Kilian, 2010), seven (MLST7) or eight (MLST8) genes were
http://dx.doi.org/10.1016/j.meegid.2015.05.009 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
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Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
subsequently used in the Belfast scheme (McDowell et al., 2011, 2012), later simplified with a selection of four genes (MLST4) (McDowell et al., 2013). MLST correctly defines the different lineages and, in addition, it separates type IA into two distinct groups, IA1 and IA2 (McDowell et al., 2011). A comparison with repetitive-sequence-based typing (DiversiLab) showed that this technique was less discriminating than MLST (Davidsson et al., 2012). Typing based upon whole-genome single nucleotide polymorphism (SNP) analysis allowed the most correct definition of phylogenetic trees and phylotypes. It currently represents the gold standard for describing P. acnes population and provides a reference to which clustering achieved by typing methods needs to be compared (Fitz-Gibbon et al., 2013; Tomida et al., 2013). This method, however, is not yet suitable as a first-line assay for large scale investigations because of cost issues. Recently, a novel sequence-based technique, Single Locus Sequence Typing (SLST), has been proposed to replace MLST (Scholz et al., 2014). It is capable of distinguishing the main phylotypes previously defined by whole-genome sequence analysis, but its resolution may be insufficient for epidemiological analyses. Along the same line but with a lower discriminatory power, a rapid pre-screening multiplex touchdown PCR assay able to assign a strain to one of the six main phylotypes IA1, IA2, IB, IC, II, and III was proposed (Barnard et al., 2015). In summary, currently available low cost assays have a limited discriminatory power, and MLST assays are progressively being replaced by a whole genome sequence analysis approach. There is a need for a first-line assay with a sufficient discriminatory power. Two additional sources of polymorphism have proved to be highly valuable for typing a number of pathogens. Intra-species variation can be found in clustered regularly interspaced short palindromic repeat (CRISPR) elements, which accumulate short stretches of unique sequences derived from invading DNA. Their polymorphism provides phylogenetic information, and in some species constitutes an informative genotyping tool (Drevet and Pourcel, 2012). CRISPRs and CRISPR-associated genes (cas) of the type I-E subfamily were described in some type II and type III P. acnes genomes and showed strain diversity (Brüggemann et al., 2012; Marinelli et al., 2012). The system was absent in all tested type I strains. The second source is polymorphic tandem repeats. The PCR analysis of several variable number of tandem repeats (VNTR) sequences, called multiple locus VNTR analysis (MLVA), provides a genotype in the form of a numeric code, corresponding to the number of repeats at each analyzed locus. This code can be stored in databases and easily compared between laboratories (Grissa et al., 2008). MLVA has been used successfully to genotype a large variety of bacterial species (Lindstedt et al., 2013; Vergnaud and Pourcel, 2009) and, for several, has become a routine typing technique (Garofolo, 2015; Hyytia-Trees et al., 2006; Li et al., 2009; Thierry et al., 2014; Wuyts et al., 2013). Here we evaluate the potential of MLVA for the typing of P. acnes. We have developed an MLVA assay based on the polymorphism of 13 VNTRs, subsequently called MLVA13. We determined the MLVA13 profile of 120 clinical P. acnes strains, and show that this technique compares favorably with sequence-based typing assays (MLST and SLST) for epidemiological analyses.
was collected from different wards in two military hospitals (from soldiers and civilian patients) from 2010 to 2013: 28 P. acnes, six Propionibacterium avidum and three Propionibacterium sp were from Hopital d’Instruction des Armées (HIA) Percy in the Paris area, France, and 20 P. acnes and four P. avidum were from HIA Desgenettes located in Lyon, France. Nine were isolated from healthy skin, eleven were from blood and the others were associated with different infection sites (Table 1). The bacteria were
Table 1 Propionibacterium strains from two French hospitals.
2. Materials and methods 2.1. Strains The isolates were obtained from specimens of patients, as part of the patients’ usual care. A total of 61 Propionibacterium isolates
Strain ID
Species
Hospital
Year
Site
Pacn01 Pacn02 Pacn03 Psp04a Psp05 Pacn06 Pacn07 Pacn08 Pav09 Pacn10 Pacn11 Pacn12a Pav13 Pav14 Pacn15 Psp16 Pav17 Pacn18 Pacn19 Pacn20 Pacn21 Pav22 Pav23 Pacn24 Pacn25 Pacn26 Pacn27 Pacn28 Pacn29 Pacn30 Pacn31 Pacn33 Pacn34 Pacn35 Pacn36 Pav37 Pacn38 Pacn39 Pacn40 Pacn41 Pacn42 Pacn43b Pacn44b Pav45 Pacn46 Pacn47 Pacn48 Pav49 Pacn50c Pav51c Pacn52 Pacn53 Pacn54 Pacn55 Pacn56 Pacn57 Pacn58 Pacn59 Pacn60 Pacn61 Pacn63
P. acnes P. acnes P. acnes UN UN P. acnes P. acnes P. acnes P. avidum P. acnes P. acnes P. acnes P. avidum P. avidum P. acnes UN P. avidum P. acnes P. acnes P. acnes P. acnes P. avidum P. avidum P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. avidum P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. avidum P. acnes P. acnes P. acnes P. avidum P. acnes P. avidum P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes
Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Percy Percy Percy Percy Percy Percy Desgenettes
2010 2012 2010 2010 2010 2011 2012 2012 2008 2008 2008 2009 2012 2013 2010 2012 2012 2012 2012 2012 2011 2011 2011 2011 2011 2010 2011 UN 2010 2013 2010 2010 2013 2010 2013 2010 2011 2011 2011 2012 2012 2012 2012 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013
Abscess, thigh Bone, graft Skin Synovial fluid Blood Bone marrow Abscess, shoulder Joint, shoulder Abscess, armpit Skin Bone, tibia Prosthesis, knee Abscess, armpit Abscess, pubis Skin Muscle, shoulder Abscess, groin Blood Adenoma, neck Bone, tibia Bone, ankle Abscess, arm Abscess, parotide Skin Blood Skin Skin Skin Abscess, vertebra Prosthesis, thighbone Bone, thighbone LN, neck Abscess, back LN, aorta Blood Sinus Bone, tibia Blood Pleural liquid Blood Blood Spinal disc, liquid Spinal disc, biopsy Abscess, armpit CRL Lung biopsy Parotide gland LN Joint, thighbone Bone, thighbone Bone Spinal disc, liquid Blood Bone, tibia Blood Skin Skin Joint, hip Blood LN, neck Joint, thighbone
Abbreviations: CRL, cephalo rachidian liquid; LN, lymph node; UN, unknown. Two strains were isolated from the same patient.
a, b, c
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
grown on Schaedler agar with 5% sheep blood, in anaerobic condition, using a GENbox and GENbox generators (bioMérieux, France). Bacteria were identified as P. acnes or P. avidum using standard automated biochemical testing methods and by PCR-RFLP on 16S DNA. For this, universal primers U1 50 -CCAGCAGCCGCGGTAATAC G-30 and U2 50 -ATCGGYTACCTTGTTACGACTTC-30 (Lu et al., 2000) were used to amplify a 1001 bp fragment which was then digested with HaeIII. The digestion pattern was analyzed by electrophoresis on a 2% agarose gel. In silico digestion of 10 P. acnes amplicons produced five fragments of 517 bp, 226 bp, 154 bp, 71 bp and 33 bp, whereas P. avidum strain 44067 produced five fragments of 380 bp, 317 bp, 201 bp, 71 bp and 33 bp, and this was confirmed experimentally with our collection of strains. RecA sequencing was performed to identify the lineage of selected strains, using primers PAR-1 5’-AGCTCGGTGGGGTTCTCT CATC-30 and PAR-2 50 -GCTTCCTCATACCACTGGTCATC-30 as described by McDowell et al. (2005). The genome sequence of 102 well characterized strains was recovered from the NCBI resources at http://www.ncbi.nlm.nih. gov/. Most of these strains (82) were isolated from acne or healthy skin and their diversity has been described in detail by Tomida et al. (Tomida et al., 2013). Some are reference genomes for the Human Microbiome Project ‘‘HMP’’. (http://www.ncbi.nlm.nih. gov/bioproject/51439). Accession numbers are listed in Table S1. 2.2. VNTR markers The annotated genome sequences of P. acnes KPA171202 (Brüggemann et al., 2004), Genbank (NC_006085.1) and SK137 (NC_014039.1) were inspected for the presence of potential VNTR loci by using the Microbial Tandem Repeats Database (http://tandemrepeat.u-psud.fr/) (Le Flèche et al., 2001). VNTR loci with a repeat unit size above nine bp were named large (L)-repeat VNTRs, whereas VNTR loci with a repeat size up to nine bp were named small (S)-repeat VNTRs. Each VNTR locus was designated according to the position (expressed in kilobases) of the primers designed for PCR amplification, relative to the genome sequence of P. acnes KPA171202 (ie. Pacnes-0232, Pacnes-0320, Pacnes-0440, Pacnes-0866, Pacnes-1614, Pacnes-1815, Pacnes-1868, Pacnes-1902, Pacnes-1938, Pacnes-2303, Pacnes-2320, Pacnes-2333, Pacnes-2393). The in silico MLVA genotype was determined using tools available on the MLVAbank website at http://mlva.u-psud.fr/. A public MLVA database which can be queried via internet has been created on MLVAbank at http:// mlva.u-psud.fr. 2.3. DNA extraction, PCR amplification and electrophoresis Thermolysates were prepared by incubating bacteria suspension with 40 mM NaOH for 45 min at 60 °C, followed by neutralization by 100 mM Tris pH 8.0 according to (Brüggemann et al., 2012). DNA was purified in some cases when PCR amplification failed using thermolysates. For this, bacteria were resuspended in PBS and treated by lysozyme (0.4 mg/ml) and mutanolysin (8 lg/ml) (Sigma, France), for 48 h at 37 °C. Subsequently, one volume of a lysis buffer containing 10% SDS, 20 mM Tris HCl, 20 mM EDTA and 20 mM NaCl was added to the bacterial suspension with 50 lg/ml of proteinase K. After 3 h at 50 °C, DNA was extracted using phenol and chloroform. Oligonucleotide primers targeting the 50 and 30 flanking regions of each VNTR locus are listed in Table S2. Primers annealing on published P. acnes genome sequences were selected in silico with the help of the PCR primers BLAST tool from the Microbial Tandem Repeats Database. Primers targeting the cas1 gene and the CRISPR locus were as described in (Brüggemann et al., 2012). When the complete cas1 gene was present, a 790 bp product was
3
obtained, whereas a 302 bp amplicon was obtained in type I strain with a partially deleted locus. When the system was totally absent, as in some type II strains, no PCR product was observed. PCRs were performed in 15 ll final volume containing one to five ng of genomic DNA, 1 reaction buffer (Euromedex), 1.5 mM MgCl2, 1 U of Taq DNA polymerase (Euromedex), 200 lM of each deoxynucleoside triphosphate (dNTP) (Roche), 0.3 lM of each primer (synthesized by Eurogentec). Amplification was performed with a PTC-200 DNA Engine (Bio-Rad) applying initial denaturation for 5 min at 94 °C followed by 36 cycles including denaturation for 30 s at 94 °C, annealing for 30 s at 60 °C, and elongation at 72 °C for 30 s, with final elongation at 72 °C for 7 min. An appropriate volume (generally 2 ll) of each PCR product was loaded in a 2% regular agarose (Qbiogene) gel for L-VNTRs or in a 3% agarose gel, composed of 1.5% regular agarose plus 1.5% Metaphor (FMC BioProducts), for S-VNTRs. Electrophoresis was performed in 25 cm-long gels and run at 4 V/cm in 0.5 Tris–borate–EDTA buffer for about 5 h. The 100-bp (Euromedex) or 20-bp (Biolabs) ladders, or a mixture of both, were used as DNA size markers. The gels were stained in 0.5 lg/ml ethidium bromide for 1 h, rinsed with water, and photographed under UV illumination.
2.4. Agarose gel image analysis The band size was determined using the BioNumerics software v. 6.6 (Applied Maths, Sint-Martens-Latem, Belgium). Minor adjustments were made taking into account the amount of DNA present in each band, as large amounts of DNA usually result in slightly faster apparent migration.
2.5. Nomenclature and description of MLVA profiles The expected amplicon size, repeat length, number of repetitions, were determined in reference genomes of strains available at the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/sites/genome), using the In silico MLVA genotyping tool from MLVAbank (Grissa et al., 2008). The number of repeats in VNTR alleles for isolates with unknown genome was estimated using an alleles chart. In loci containing a non integer number of repeats, the number of repeats was rounded-up as explained in (Vergnaud and Pourcel, 2006). The allelic profile (MLVA code) was defined as the number of repeats at each VNTR locus included in the MLVA scheme. Typeability of a given VNTR was defined as the fraction of samples for which an allele could be defined. Clustering analyses were performed using the categorical coefficient and the Unweighted Pair Group Method with Arithmetic mean (UPGMA) algorithm, producing a dendrogram. For comparison in BioNumerics, alleles which repeat number was estimated to be 0.5 were rounded-up to 1. Alternatively a minimum spanning tree (MST) analysis was performed, allowing the observation of clusters in a more condensed form.
2.6. Nomenclature of MLST and whole-genome phylotypes Throughout the text, tables and figures, we have used the Belfast MLST8 phylogroup terminology as extracted from the public database at http://pacnes.mlst.net/ and where the last numerical designation is superscript as in IA1 (Lomholt and Kilian, 2010), or the whole-genome phylotype designation which includes dashes as in IA-1 (Fitz-Gibbon et al., 2013; Tomida et al., 2013).
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
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3. Results 3.1. Selection of VNTRs and in silico analysis By comparing strain KPA171202 (type IB) and strain SK137 (type IA1), 13 polymorphic tandem repeats were identified (Fig. S1), and their polymorphism was analyzed in silico on additional genomes. The repeat units of 10 VNTR loci with size ranging from 14 to 31 bp were classified as large (L-VNTR), whereas three had 6 bp small size repeats (S-VNTR). Four VNTR loci (Pacnes-0232, Pacnes-0440, Pacnes-1815, Pacnes-1902) were localized in an intergenic region and the others were part of putative open reading frames (Table 2). The three 6 bp-repeats VNTRs encoded stretches of proline/threonine or proline/alanine inside putative membrane proteins (similar to fibronectin-binding protein I for Pacnes-1815 and to adhesion or S-layer proteins for Pacnes-2303 and Pacnes-2210). Pacnes-2333 encoded a seven amino-acids repeat into a putative penicillin-binding protein. In total, genotyping data was retrieved for 102 P. acnes strains, of which eight were full genome sequences and the rest were draft. The VNTR polymorphism of strains belonging to six different phylotypes is shown on Table 2, and the complete results are shown on Table S3. The three S-VNTRs, Pacnes-1868, Pacnes-2303 and Pacnes-2393 could not be measured for 39, nine and 29 draft genomes, respectively, because the loci were split into two portions present on two different contigs. We also analyzed the genome of non-acnes Propiobacterium strains, including Propiobacterium humerusii, and could not generate an in silico profile. The MLST8, MLST9, SLST and SNP codes of P. acnes sequenced genomes were recovered from the associated publications and from public MLST databases. Clustering analysis based on 13 VNTRs MLVA (MLVA13) codes showed a good overall concordance with the other sequence-based genotyping data (Figs. 1A and S2) suggesting that the MLVA13 code could be deduced correctly from the available genome assemblies, and that it was relevant for strain comparison. Some discrepancies were observed in the distribution of several strains by the different methods. Three IB-2 strains, P.acn17, P.acn31 and P.acn33, originating from French patients with ophthalmic infections (McDowell et al., 2011) were clustered with IA-1. The S-VNTRs provided a high discriminatory power to MLVA13, and on several occasions allowed to distinguish strains with the same sequence type (ST). The number of observed alleles varied from 2 to 20. We found that a panel of 10 VNTRs (MLVA10) not-including the S-VNTRs, clustered correctly the members of a given phylogroup (Fig. 1B), although the full collection of VNTRs provided the highest discriminatory power. The stability of the S-repeats was tested by performing PCR on 100 colonies recovered from three different strains after four or five replatings. No modification of the repeat copy number was observed for any of the VNTRs. 3.2. MLVA genotyping of clinical isolates A total of 61 Propionibacterium isolates were provided by two hospitals, of which 48 were P. acnes, 10 were P. avidum and three were Propionibacterium sp (Table 1). They originated mainly from burn patients, and from patients who underwent orthopedic surgery, from 2008 to 2013. Three patients provided two strains, and in two of them one strain was P. acnes and the other was P. avidum or Propionibacterium sp. Upon genotyping with the 13 VNTRs, a high typeability was observed for the P. acnes samples (Figs. 2 and S3), whereas only faint amplification was observed with a few VNTRs for the other species (not shown). VNTR amplification was similarly unsuccessful on several Staphylococcus isolates recovered on the skin of the same patients.
Full typeability (T = 1.00) was achieved for all VNTRs except Pacnes-1868 being unamplified in three samples (T = 0.96) and Pacnes-2303 in one sample (T = 0.98). Discriminatory power was evaluated using the full collection of unrelated clinical isolates. The Hunter-Gaston discriminatory index (HGDI) was close to one, as only two isolates showed the same MLVA code (Tables 3 and S3). Upon clustering analysis using all MLVA data (shown by MST representation on Fig. S4), most of the French isolates showed MLVA profiles similar to those of sequenced strains, allowing a putative assignation to a phylogroup by genotype comparison. Type II strains clustered into a single group, and this was also the case of type IB and type III strains. Type IA isolates were distributed into several subgroups that did not always match with the MLST subtyping but fit with whole-genome SNP-based subgrouping. Pacn10, Pacn35, Pacn54 and Pacn63 appeared to form a specific group related to type II strains, and this was confirmed by recA sequencing. Pacn28 which clustered with the two type III strains, JCM18909 and HL201PA1, was confirmed to be a type III strain by recA analysis. 3.3. CRISPR/cas analysis In order to further characterize the new isolates, they were all searched for the presence of the cas1 gene and, when positive, for the CRISPR locus by PCR analysis. Five isolates (Pacn38, Pacn41, Pacn43, Pacn46, Pacn54) clustering by MLVA13 with type II strains, and strain Pacn28 clustering with type III strains, were positive for the cas1 gene. Polymorphism in the spacers number and sequence was observed (Table S4). Six new 33 bp spacers numbered 51 to 56 were found, in addition to some described previously in other strains (Brüggemann et al., 2012; Marinelli et al., 2012). Two spacers targeted phages and two spacers targeted a plasmid. The CRISPR locus in Pacn28 had no spacer, similarly to the related strain JCM18909. 4. Discussion We report the identification of a collection of VNTRs suitable for MLVA genotyping of P. acnes. The present MLVA13 assay is specific for P. acnes, as demonstrated by failure to amplify the VNTR loci in 10 P. avidum isolates, and in other closely related species or skin commensals. MLVA13 provides a very good resolution, much higher than MLST, brought by the S-VNTRs with a high polymorphism level. Such VNTRs with small repeats allow to discriminate among epidemiologically related isolates from different patients. MLVA13 allows clustering of isolates of similar type as revealed by the analysis of strains which genome was sequenced. Similarly to SNP-based typing, MLVA13 separates type IA1 strains into two groups, although there are a few strains which do not cluster according to their MLST subtype. This is related to polymorphism at VNTR locus Pacnes-0320, present in an hypothetical coding region. Three VNTRs display only two alleles, and consequently do not contribute much to the resolution of the assay but they may have a phylogenetic value and are therefore useful in the present multiple locus assay. MLVA is routinely used for the typing of several bacterial species, and databases have been constructed that allow sharing of data (Grissa et al., 2008; Vergnaud and Pourcel, 2009). Validation of informative markers and calibration of fragments analysis is possible through common use of reference strains which genome has been sequenced, and definition of conventions for allele calling (Pourcel et al., 2007, 2011; Riehm et al., 2012). MLVA13 can be performed manually at low cost using simple agarose gels but this requires more hands-on time. Multiplexing the PCR reactions using fluorescent primers and capillary electrophoresis will be of great
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
VNTR marker
Pacnes-0232_14bp_238bp_2U Pacnes-0320_27bp_172bp_4U Pacnes-0440_31bp_325bp_3U Pacnes-0866_18bp_154bp_4U Pacnes-1614_15bp_293bp_2U Pacnes-1815_18bp_129bp_2U Pacnes-1868_6bp_161bp_18U
Pacnes-1902_25bp_275bp_5U Pacnes-1938_15bp_332bp_3U Pacnes-2303_6bp_222bp_19U Pacnes-2320_15bp_138bp_3U Pacnes-2333_21bp_158bp_3U
Pacnes-2393_6bp_345bp_28U a b c d
The Not Not Not
Position in KPA171202
232518–232549 320927–321034 440364–440448 866074–866145 1614945–1614975 1815554–1815589 1868929–1869036
1902255–1902364 1938299–1938344 2303280–2303393 2320922–2320963 2333722–2333784
2393901–2394061
ORF
Intergenic Hypothetical Intergenic Putative HtaA Domain protein Hypothetical Intergenic Putative Fibronectin-binding protein Intergenic Hypothetical Hypothetical Putative Adhesion protein Putative Penicillin-binding protein Putative Adhesion protein
Expected amplicon size (bp)a IB KPA171202
IA-1 SK137
IA-2 17 Type IA2
238 2 172 4 325 3 154 4 293 2 129 2 161 18
237 2 145 3 263 1 226 8 323 4 111 1 137 14
238 2 118 2 294 2 226 8 293 2 111 1 107 9
275 5 332 3 222 19 138 3 158 3
200 2 317 2 324 36 153 4 137 2
225 3 317 2 222 19 153 4 137 2
345 28
357 30
351 29
IC HL097PA1 2 2 3 8 2 1 9
II ATCC 11828 238 2 117 2 263 1 171 5 293 2 129 2 107 9
1
200 2 332 3 222 19 138 3 137 2
32
302 21
5 2 31 4
Number of alleles in study
Number of alleles in silico
3
4
5
5
3
4
6
6
2
4
3
3
26
17b
4
4
2
2
17
17c
2
2
3
4
22
20d
III HL201PA1 1 2 3 2 1 1
2 2 19 4 2
Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
20
number of repeats is indicated under the allele size. measurable in 39 genomes. measurable in nine genomes. measurable in 29 genomes.
5
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
Table 2 Characteristics of the 13 VNTRs.
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Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
IC IA1 IA1
A
IA1 IA1
IA1 IA1 IA1
IA1
IA1 IA1 IA1 IA1
IA1
IA2
II IA1 IA1 IA1
IA1
IA2
II
IA1
IA1
IA2 IA1
II
IA1
IA1
IA1 IA1 IA1
IA1
IA1
IA1 IA1
IA1 IA1
IA1 IA2 IA2
IA1
IA1 IA1 IA1
IA1
IA1 IA1
IA2
IA2 IA2
IA2
IA1
IB IA2
IB
IB
IA1 IA1
IA2
IA2
IB
II
IA2
IA2 IA2
IA1
IA2 IA2
II
IA1 IA1
IA2
II II
IA1
II II
IA1
IA1
II
IB III
III
IB
B
Fig. 1. MLVA-based minimum spanning tree clustering analysis of the 102 P. acnes isolates, produced in silico from genomic data. Isolates classified into the same cluster are indicated using colored circles. The size of the circle and the number of bars correspond to samples with the same genotype. Phylogroup identification derived from MLST (Belfast scheme) is indicated near each sample. (A) MLVA13 analysis, (B) MLVA10 analysis. The same color code is used in both analyses. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
importance for the automation and accuracy of the assay as illustrated for other MLVA assays (Sobral et al., 2012a,b). MLVA is a fast technique which allows the typing of large amount of samples and the systematic investigation of clinical isolates, whereas MLST typing is often performed only on selected isolates (Giannopoulos et al., 2014). The cost can be in the order of 1 to a few dollars per sample depending on the methodology and the number of samples analyzed in a single assay. The recently described SLST scheme is more simple and allows a correct identification of phylotypes, although its resolution may be insufficient to differentiate some subtypes (Scholz et al., 2014). It will be interesting to see in
the future if one assay is preferred, or if they are complementary and used in parallel. We show that a large proportion of the strains in a French hospital display very similar genotypes, differing only at VNTRs with a high level of discriminatory power. According to their MLVA type, the larger group of French isolates belongs to phylogroup IA, and particularly to IA1. This type is the most frequently found on skin and associated with acne, as shown in different studies, which may be due to the preferential skin infection by certain strains (Table S1 and Fig. S1) (Lomholt and Kilian, 2010; McDowell et al., 2005, 2011). The proportion of type II and IB strains was more
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
7
Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
IA2
IA1 IA2 III
II
IB
Pacnes-2303
Pacnes-1868
Pacnes-1815
Pacnes-2333
Pacnes-1938
Pacnes-2320
Pacnes-1902
Pacnes-1614
Pacnes-0866
Pacnes-0440
Pacnes-0320
100
Pacnes-2393
IA1
MLVA13
Pacnes-0232
50
MLVA13
Strain
2
3
2
8
2
2
2
4
2
1
47
28
34
Pacn15
2
3
2
8
2
2
2
4
2
1
34
32
35
Pacn60
2013
Percy
blood
2
3
2
8
2
2
2
4
2
1
30
31
25
Pacn55
2013
Desgenettes
bone, tibia
2
3
2
8
2
3
2
4
2
1
31
28
34
Pacn02
2012
Percy
bone, graft
2
3
2
8
2
3
2
4
2
1
46
35
34
Pacn48
2013
Desgenettes
parotide gland
2
3
2
8
2
3
2
4
2
1
37
27
34
Pacn07
2012
Percy
abcess, shoulder
2
3
2
8
2
3
2
4
2
1
55
32
26
Pacn11
2008
Percy
bone, tibia
26
Pacn12
2009
Percy
prosthesis, knee
34
Pacn53
2013
Desgenettes
spinal disc, liquid
34
Pacn24
2011
Percy
skin
2010
Percy
skin
2
3
2
8
2
3
2
4
2
1
55
2
3
2
8
2
3
2
4
2
1
55
2
3
2
8
2
3
2
4
2
1
29
32 32
Year
Hospital
Site
2010
Percy
skin
2
3
2
8
2
3
2
4
2
1
44
32
38
Pacn26
2
3
2
8
2
3
2
4
2
1
44
29
36
Pacn27
2011
Percy
skin
2
3
2
8
2
3
2
4
2
1
36
31
32
Pacn31
2010
Percy
bone, thighbone
2
3
2
8
2
3
2
4
2
1
27
31
24
Pacn40
2011
Desgenettes
pleural liquid
2
3
2
8
2
3
2
4
2
1
48
31
37
Pacn50
2013
Desgenettes
joint, thighbone
2
2
2
8
2
3
2
4
2
1
35
23
28
Pacn25
2011
Percy
blood
2
2
2
8
2
3
2
4
2
1
30
30
34
Pacn44
2012
Desgenettes
spinal disc biopsy
2
2
2
8
2
3
2
4
2
1
31
22
32
Pacn59
2013
Percy
joint, hip
2
1
2
8
2
3
2
4
2
1
28
28
36
Pacn61
2013
Percy
LN, neck
2
2
2
8
1
2
2
4
2
1
18
34
32
Pacn29
2010
Percy
abcess, vertebra
2
3
1
8
2
2
3
4
2
1
30
27
18
Pacn08
2012
Percy
joint, shoulder
2
3
1
8
2
2
3
4
2
1
21
23
26
Pacn33
2010
Desgenettes
LN, neck
2
2
1
8
2
2
3
4
2
1
15
27
27
Pacn52
2013
Desgenettes
bone
2010
Percy
skin
2
3
1
8
2
2
2
4
2
1
23
36
36
Pacn03
2
3
1
8
2
2
2
4
2
1
19
23
29
Pacn39
2011
Desgenettes
blood
2
4
2
8
2
2
3
4
2
1
12
14
26
Pacn34
2013
Desgenettes
abcess, back
2012
Desgenettes
blood
2011
Percy
bone, ankle
Percy
skin
2
4
2
8
2
2
3
4
2
1
20
31
30
Pacn42
2
5
2
8
2
2
3
4
2
1
28
36
32
Pacn21
1
2
1
3
2
2
2
4
2
1
14
19
23
Pacn28
2
2
1
5
2
3
3
4
2
2
9
19
22
Pacn19
2012
Percy
adenoma, neck
2
2
1
13
2
3
3
4
2
2
12
19
25
Pacn36
2013
Desgenettes
blood
2
2
1
5
2
2
3
4
2
2
14
17
Pacn38
2011
Desgenettes
bone, tibia
2
2
1
5
2
2
3
4
2
2
14
20
Pacn46
2012
Desgenettes
CRL
2
2
1
5
2
2
3
3
2
2
9
19
20
Pacn41
2012
Desgenettes
blood
2
2
1
7
2
2
3
3
2
2
9
18
22
Pacn43
2012
Desgenettes
spinal disc, liquid
2
2
2
7
2
5
3
4
2
2
21
20
23
Pacn35
2010
Desgenettes
LN, aorta
2
2
1
7
2
5
3
4
2
2
21
20
24
Pacn63
2013
Desgenettes
joint, thighbone
2
2
3
7
2
4
3
4
2
2
10
22
16
Pacn10
2008
Percy
skin
13
2
1
5
2
3
3
4
3
2
11
14
21
Pacn54
2013
Desgenettes
blood
2012
Percy
bone, tibia
2
4
2
4
2
3
3
4
2
2
23
15
17
Pacn20
2
4
2
4
2
3
3
4
2
2
10
22
33
Pacn56
2013
Percy
blood
2
6
2
4
2
3
3
4
2
2
6
19
13
Pacn18
2012
Percy
blood
2013
Percy
prosthesis, thighbone
2
4
2
8
2
3
3
4
2
2
14
17
25
Pacn30
2
5
2
4
2
3
3
4
3
2
21
22
20
Pacn06
2011
Percy
bone marrow
2
5
2
4
2
3
3
4
3
2
15
14
33
Pacn57
2013
Percy
skin
2
3
2
4
2
3
3
4
2
2
12
14
29
Pacn47
2012
Desgenettes
lung biopsy
2
3
2
4
2
2
3
4
2
2
15
14
18
Pacn58
2013
Percy
skin
2
3
1
5
2
2
3
4
1
2
14
18
Pacn01
2010
Percy
abcess, thigh
Fig. 2. Clustering analysis of MLVA13 data for 48 French isolates. The dendrogram was constructed from MLVA analysis using 13 VNTRs. On the right are shown the strain ID, the isolation year, the hospital and the isolation site. On the left is indicated the phylogroup, by comparison with published strains.
elevated in the present study as compared to others investigating healthy skin and acne. Similarly, such strains were found to be enriched in non-skin infections in another study (McDowell et al., 2013). We did not detect a significant link between the site of infection and the strain genotype, likely due to the small sample size. In Desgenettes hospital, several isolates with a very similar
genotype were recovered from different patients over a period of three years suggesting the existence of a reservoir for these infections. Two different Propionibacterium strains were isolated from three patients, and one of them had two P. acnes with different genotypes. Indeed, two spinal disc samples from the same patient led to the isolation of Pacn43 and Pacn44, of type II and type IA
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
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Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
Table 3 Hunter-Gaston discriminatory index for individual or combined VNTR loci (MLVA13) calculated from 47 unrelated P. acnes isolates. Locus
Allele number
Total n = 47
MLVA10
Pacnes-0232 Pacnes-0320 Pacnes-0440 Pacnes-0866 Pacnes-1614 Pacnes-1815 Pacnes-1868 Pacnes-1902 Pacnes-1938 Pacnes-2303 Pacnes-2320 Pacnes-2333
3 6 3 6 2 2 27 4 2 18 2 3
0.0842 0.6772 0.4570 0.5883 0.0426 0.4829 0.9750 0.5412 0.5106 0.9315 0.0833 0.1619
X X X X X X
Pacnes-2393 Global index for MLVA13
22 Total genotype nbr 46
0.9537 Index for all strains 0.9991
X X X X
respectively. This was previously observed in patients suffering from lumber disc herniation (Rollason et al., 2013). Similarly, in acne patients and control carriers in Denmark, it was shown that different MLST clones were carried by some subjects (Lomholt and Kilian, 2014). It was also observed using whole-genome sequencing that most individuals harbored multiple P. acnes strains from the same or different lineages (Fitz-Gibbon et al., 2013; Tomida et al., 2013). The described P. acnes MLVA13 scheme should allow, not only to differentiate between clones, but also to identify differences between strains belonging to the same clones, thanks to the high polymorphism of the S-VNTRs. In the present study we also investigated the polymorphism of the CRISPR element present in some P. acnes strains. A study by Marinelli and co-workers suggested that the CRISPR-Cas system plays an active role in protecting the bacteria from phage infection (Marinelli et al., 2012). Strains with the most spacers are those that show extensive phage resistance, implicating CRISPR in the phenotype. It is interesting to note that strain Pacn38 possesses four novel spacers of which two target plasmid HL096PA1 (Fitz-Gibbon et al., 2013), while Pacn41 possesses two novel spacers targeting two genes from a single phage family. Phages in P. acnes display limited diversity as all the presently described phages belong to a single group found in sebaceous follicles with healthy skin or acne (Marinelli et al., 2012). This suggests that these strains that are genetically related to other strains with a different CRISPR composition, have acquired the spacers recently. 5. Conclusions We believe that the present MLVA assay will represent a first line genotyping assay, which can help investigate nosocomial infections, but also the systematic analysis of strains present on healthy individuals and on patients in order to better understand the diversity and pathogenicity of this species. Acknowledgement The authors are grateful to the laboratory staff of Percy and Desgenettes hospitals for carrying out the microbiological analyses. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2015.05. 009.
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A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes Yolande Hauck a, Charles Soler b, Patrick Gérôme c, Rithy Vong b, Christine Macnab b, Géraldine Appere b, Gilles Vergnaud a,d, Christine Pourcel a,⇑ a
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris Sud, Université Paris-Saclay, 91405 Orsay cedex, France Laboratoire de biologie clinique, HIA Percy, Clamart, France Service de biologie médicale, HIA Desgenettes, 69275 Lyon cedex 03, France d ENSTA ParisTech, Université Paris-Saclay, 91762 Palaiseau cedex, France b c
a r t i c l e
i n f o
Article history: Received 22 January 2015 Received in revised form 4 May 2015 Accepted 8 May 2015 Available online xxxx Keywords: VNTR Genotyping In silico MLVA CRISPR Post-surgery infection
a b s t r a c t Propionibacterium acnes plays a central role in the pathogenesis of acne and is responsible for severe opportunistic infections. Numerous typing schemes have been developed that allow the identification of phylotypes, but they are often insufficient to differentiate subtypes. To better understand the genetic diversity of this species and to perform epidemiological analyses, high throughput discriminant genotyping techniques are needed. Here we describe the development of a multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) method. Thirteen VNTRs were identified in the genome of P. acnes and were used to genotype a collection of clinical isolates. In addition, publically available sequencing data for 102 genomes were analyzed in silico, providing an MLVA genotype. The clustering of MLVA data was in perfect congruence with whole genome based clustering. Analysis of the clustered regularly interspaced short palindromic repeat (CRISPR) element uncovered new spacers, a supplementary source of genotypic information. The present MLVA13 scheme and associated internet database represents a first line genotyping assay to investigate large number of isolates. Particular strains may then be submitted to full genome sequencing in order to better analyze their pathogenic potential. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Propionibacterium acnes is an anaerobic, Gram-positive bacillus, which can survive for long periods of time in aerobic conditions. It is a major part of the skin microbiome, an aggravating factor in the pathogenesis of acne vulgaris (Fitz-Gibbon et al., 2013; Tomida et al., 2013), and also an opportunistic pathogen responsible for infections of different gravities. In the hospital it is frequently associated with infection of implants, prostheses and other devices on which it can form biofilms (for a review see (Achermann et al., 2014; Aubin et al., 2014)). The species has been classified into three major phylogenetic clusters or lineages, also called phylotypes or types. Type I and type II members were distinguished based on serological agglutination tests, cell wall sugar analysis, and monoclonal antibody (mab) typing (Johnson and Cummins, 1972; McDowell et al., 2005). Later, type I was further subdivided into subtypes IA and IB, and a third lineage, type III, was discovered ⇑ Corresponding author at: I2BC, Bat. 400, Université Paris Sud, 91405 Orsay cedex, France. E-mail address: [email protected] (C. Pourcel).
(McDowell et al., 2008). This was based on polymorphism of recA, and mab typing. Recently MALDI-TOF MS fingerprinting was shown to discriminate phylotypes I, II and III of P. acnes (Nagy et al., 2013). A study by Rollason and co-workers suggested that the different phylotypes presented different pathogenicity and were associated to various clinical situations (Rollason et al., 2013). It also demonstrated that patients frequently carried multiple phylogroups. Type III was recently shown to be associated with skin, similarly to the other lineages (Dekio et al., 2012). In order to better characterize strains and further study their association with particular infections, genotyping methods were developed and their advantages and drawbacks have been compared (Yu et al., 2015). Random amplification of polymorphic DNA (RAPD) (Perry et al., 2003) and pulse field gel electrophoresis (PFGE) (Oprica et al., 2004) were among early typing methods but they were time consuming and suffered from problems of reproducibility. They were replaced by Multi Locus Sequence Typing (MLST), a sequence-based method making use of housekeeping and virulence genes. Whereas a total of nine (MLST9) genes were selected for the Aarhus scheme (Kilian et al., 2012; Lomholt and Kilian, 2010), seven (MLST7) or eight (MLST8) genes were
http://dx.doi.org/10.1016/j.meegid.2015.05.009 1567-1348/Ó 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
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Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
subsequently used in the Belfast scheme (McDowell et al., 2011, 2012), later simplified with a selection of four genes (MLST4) (McDowell et al., 2013). MLST correctly defines the different lineages and, in addition, it separates type IA into two distinct groups, IA1 and IA2 (McDowell et al., 2011). A comparison with repetitive-sequence-based typing (DiversiLab) showed that this technique was less discriminating than MLST (Davidsson et al., 2012). Typing based upon whole-genome single nucleotide polymorphism (SNP) analysis allowed the most correct definition of phylogenetic trees and phylotypes. It currently represents the gold standard for describing P. acnes population and provides a reference to which clustering achieved by typing methods needs to be compared (Fitz-Gibbon et al., 2013; Tomida et al., 2013). This method, however, is not yet suitable as a first-line assay for large scale investigations because of cost issues. Recently, a novel sequence-based technique, Single Locus Sequence Typing (SLST), has been proposed to replace MLST (Scholz et al., 2014). It is capable of distinguishing the main phylotypes previously defined by whole-genome sequence analysis, but its resolution may be insufficient for epidemiological analyses. Along the same line but with a lower discriminatory power, a rapid pre-screening multiplex touchdown PCR assay able to assign a strain to one of the six main phylotypes IA1, IA2, IB, IC, II, and III was proposed (Barnard et al., 2015). In summary, currently available low cost assays have a limited discriminatory power, and MLST assays are progressively being replaced by a whole genome sequence analysis approach. There is a need for a first-line assay with a sufficient discriminatory power. Two additional sources of polymorphism have proved to be highly valuable for typing a number of pathogens. Intra-species variation can be found in clustered regularly interspaced short palindromic repeat (CRISPR) elements, which accumulate short stretches of unique sequences derived from invading DNA. Their polymorphism provides phylogenetic information, and in some species constitutes an informative genotyping tool (Drevet and Pourcel, 2012). CRISPRs and CRISPR-associated genes (cas) of the type I-E subfamily were described in some type II and type III P. acnes genomes and showed strain diversity (Brüggemann et al., 2012; Marinelli et al., 2012). The system was absent in all tested type I strains. The second source is polymorphic tandem repeats. The PCR analysis of several variable number of tandem repeats (VNTR) sequences, called multiple locus VNTR analysis (MLVA), provides a genotype in the form of a numeric code, corresponding to the number of repeats at each analyzed locus. This code can be stored in databases and easily compared between laboratories (Grissa et al., 2008). MLVA has been used successfully to genotype a large variety of bacterial species (Lindstedt et al., 2013; Vergnaud and Pourcel, 2009) and, for several, has become a routine typing technique (Garofolo, 2015; Hyytia-Trees et al., 2006; Li et al., 2009; Thierry et al., 2014; Wuyts et al., 2013). Here we evaluate the potential of MLVA for the typing of P. acnes. We have developed an MLVA assay based on the polymorphism of 13 VNTRs, subsequently called MLVA13. We determined the MLVA13 profile of 120 clinical P. acnes strains, and show that this technique compares favorably with sequence-based typing assays (MLST and SLST) for epidemiological analyses.
was collected from different wards in two military hospitals (from soldiers and civilian patients) from 2010 to 2013: 28 P. acnes, six Propionibacterium avidum and three Propionibacterium sp were from Hopital d’Instruction des Armées (HIA) Percy in the Paris area, France, and 20 P. acnes and four P. avidum were from HIA Desgenettes located in Lyon, France. Nine were isolated from healthy skin, eleven were from blood and the others were associated with different infection sites (Table 1). The bacteria were
Table 1 Propionibacterium strains from two French hospitals.
2. Materials and methods 2.1. Strains The isolates were obtained from specimens of patients, as part of the patients’ usual care. A total of 61 Propionibacterium isolates
Strain ID
Species
Hospital
Year
Site
Pacn01 Pacn02 Pacn03 Psp04a Psp05 Pacn06 Pacn07 Pacn08 Pav09 Pacn10 Pacn11 Pacn12a Pav13 Pav14 Pacn15 Psp16 Pav17 Pacn18 Pacn19 Pacn20 Pacn21 Pav22 Pav23 Pacn24 Pacn25 Pacn26 Pacn27 Pacn28 Pacn29 Pacn30 Pacn31 Pacn33 Pacn34 Pacn35 Pacn36 Pav37 Pacn38 Pacn39 Pacn40 Pacn41 Pacn42 Pacn43b Pacn44b Pav45 Pacn46 Pacn47 Pacn48 Pav49 Pacn50c Pav51c Pacn52 Pacn53 Pacn54 Pacn55 Pacn56 Pacn57 Pacn58 Pacn59 Pacn60 Pacn61 Pacn63
P. acnes P. acnes P. acnes UN UN P. acnes P. acnes P. acnes P. avidum P. acnes P. acnes P. acnes P. avidum P. avidum P. acnes UN P. avidum P. acnes P. acnes P. acnes P. acnes P. avidum P. avidum P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. avidum P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. avidum P. acnes P. acnes P. acnes P. avidum P. acnes P. avidum P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes P. acnes
Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Percy Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Desgenettes Percy Percy Percy Percy Percy Percy Desgenettes
2010 2012 2010 2010 2010 2011 2012 2012 2008 2008 2008 2009 2012 2013 2010 2012 2012 2012 2012 2012 2011 2011 2011 2011 2011 2010 2011 UN 2010 2013 2010 2010 2013 2010 2013 2010 2011 2011 2011 2012 2012 2012 2012 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013
Abscess, thigh Bone, graft Skin Synovial fluid Blood Bone marrow Abscess, shoulder Joint, shoulder Abscess, armpit Skin Bone, tibia Prosthesis, knee Abscess, armpit Abscess, pubis Skin Muscle, shoulder Abscess, groin Blood Adenoma, neck Bone, tibia Bone, ankle Abscess, arm Abscess, parotide Skin Blood Skin Skin Skin Abscess, vertebra Prosthesis, thighbone Bone, thighbone LN, neck Abscess, back LN, aorta Blood Sinus Bone, tibia Blood Pleural liquid Blood Blood Spinal disc, liquid Spinal disc, biopsy Abscess, armpit CRL Lung biopsy Parotide gland LN Joint, thighbone Bone, thighbone Bone Spinal disc, liquid Blood Bone, tibia Blood Skin Skin Joint, hip Blood LN, neck Joint, thighbone
Abbreviations: CRL, cephalo rachidian liquid; LN, lymph node; UN, unknown. Two strains were isolated from the same patient.
a, b, c
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
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grown on Schaedler agar with 5% sheep blood, in anaerobic condition, using a GENbox and GENbox generators (bioMérieux, France). Bacteria were identified as P. acnes or P. avidum using standard automated biochemical testing methods and by PCR-RFLP on 16S DNA. For this, universal primers U1 50 -CCAGCAGCCGCGGTAATAC G-30 and U2 50 -ATCGGYTACCTTGTTACGACTTC-30 (Lu et al., 2000) were used to amplify a 1001 bp fragment which was then digested with HaeIII. The digestion pattern was analyzed by electrophoresis on a 2% agarose gel. In silico digestion of 10 P. acnes amplicons produced five fragments of 517 bp, 226 bp, 154 bp, 71 bp and 33 bp, whereas P. avidum strain 44067 produced five fragments of 380 bp, 317 bp, 201 bp, 71 bp and 33 bp, and this was confirmed experimentally with our collection of strains. RecA sequencing was performed to identify the lineage of selected strains, using primers PAR-1 5’-AGCTCGGTGGGGTTCTCT CATC-30 and PAR-2 50 -GCTTCCTCATACCACTGGTCATC-30 as described by McDowell et al. (2005). The genome sequence of 102 well characterized strains was recovered from the NCBI resources at http://www.ncbi.nlm.nih. gov/. Most of these strains (82) were isolated from acne or healthy skin and their diversity has been described in detail by Tomida et al. (Tomida et al., 2013). Some are reference genomes for the Human Microbiome Project ‘‘HMP’’. (http://www.ncbi.nlm.nih. gov/bioproject/51439). Accession numbers are listed in Table S1. 2.2. VNTR markers The annotated genome sequences of P. acnes KPA171202 (Brüggemann et al., 2004), Genbank (NC_006085.1) and SK137 (NC_014039.1) were inspected for the presence of potential VNTR loci by using the Microbial Tandem Repeats Database (http://tandemrepeat.u-psud.fr/) (Le Flèche et al., 2001). VNTR loci with a repeat unit size above nine bp were named large (L)-repeat VNTRs, whereas VNTR loci with a repeat size up to nine bp were named small (S)-repeat VNTRs. Each VNTR locus was designated according to the position (expressed in kilobases) of the primers designed for PCR amplification, relative to the genome sequence of P. acnes KPA171202 (ie. Pacnes-0232, Pacnes-0320, Pacnes-0440, Pacnes-0866, Pacnes-1614, Pacnes-1815, Pacnes-1868, Pacnes-1902, Pacnes-1938, Pacnes-2303, Pacnes-2320, Pacnes-2333, Pacnes-2393). The in silico MLVA genotype was determined using tools available on the MLVAbank website at http://mlva.u-psud.fr/. A public MLVA database which can be queried via internet has been created on MLVAbank at http:// mlva.u-psud.fr. 2.3. DNA extraction, PCR amplification and electrophoresis Thermolysates were prepared by incubating bacteria suspension with 40 mM NaOH for 45 min at 60 °C, followed by neutralization by 100 mM Tris pH 8.0 according to (Brüggemann et al., 2012). DNA was purified in some cases when PCR amplification failed using thermolysates. For this, bacteria were resuspended in PBS and treated by lysozyme (0.4 mg/ml) and mutanolysin (8 lg/ml) (Sigma, France), for 48 h at 37 °C. Subsequently, one volume of a lysis buffer containing 10% SDS, 20 mM Tris HCl, 20 mM EDTA and 20 mM NaCl was added to the bacterial suspension with 50 lg/ml of proteinase K. After 3 h at 50 °C, DNA was extracted using phenol and chloroform. Oligonucleotide primers targeting the 50 and 30 flanking regions of each VNTR locus are listed in Table S2. Primers annealing on published P. acnes genome sequences were selected in silico with the help of the PCR primers BLAST tool from the Microbial Tandem Repeats Database. Primers targeting the cas1 gene and the CRISPR locus were as described in (Brüggemann et al., 2012). When the complete cas1 gene was present, a 790 bp product was
3
obtained, whereas a 302 bp amplicon was obtained in type I strain with a partially deleted locus. When the system was totally absent, as in some type II strains, no PCR product was observed. PCRs were performed in 15 ll final volume containing one to five ng of genomic DNA, 1 reaction buffer (Euromedex), 1.5 mM MgCl2, 1 U of Taq DNA polymerase (Euromedex), 200 lM of each deoxynucleoside triphosphate (dNTP) (Roche), 0.3 lM of each primer (synthesized by Eurogentec). Amplification was performed with a PTC-200 DNA Engine (Bio-Rad) applying initial denaturation for 5 min at 94 °C followed by 36 cycles including denaturation for 30 s at 94 °C, annealing for 30 s at 60 °C, and elongation at 72 °C for 30 s, with final elongation at 72 °C for 7 min. An appropriate volume (generally 2 ll) of each PCR product was loaded in a 2% regular agarose (Qbiogene) gel for L-VNTRs or in a 3% agarose gel, composed of 1.5% regular agarose plus 1.5% Metaphor (FMC BioProducts), for S-VNTRs. Electrophoresis was performed in 25 cm-long gels and run at 4 V/cm in 0.5 Tris–borate–EDTA buffer for about 5 h. The 100-bp (Euromedex) or 20-bp (Biolabs) ladders, or a mixture of both, were used as DNA size markers. The gels were stained in 0.5 lg/ml ethidium bromide for 1 h, rinsed with water, and photographed under UV illumination.
2.4. Agarose gel image analysis The band size was determined using the BioNumerics software v. 6.6 (Applied Maths, Sint-Martens-Latem, Belgium). Minor adjustments were made taking into account the amount of DNA present in each band, as large amounts of DNA usually result in slightly faster apparent migration.
2.5. Nomenclature and description of MLVA profiles The expected amplicon size, repeat length, number of repetitions, were determined in reference genomes of strains available at the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/sites/genome), using the In silico MLVA genotyping tool from MLVAbank (Grissa et al., 2008). The number of repeats in VNTR alleles for isolates with unknown genome was estimated using an alleles chart. In loci containing a non integer number of repeats, the number of repeats was rounded-up as explained in (Vergnaud and Pourcel, 2006). The allelic profile (MLVA code) was defined as the number of repeats at each VNTR locus included in the MLVA scheme. Typeability of a given VNTR was defined as the fraction of samples for which an allele could be defined. Clustering analyses were performed using the categorical coefficient and the Unweighted Pair Group Method with Arithmetic mean (UPGMA) algorithm, producing a dendrogram. For comparison in BioNumerics, alleles which repeat number was estimated to be 0.5 were rounded-up to 1. Alternatively a minimum spanning tree (MST) analysis was performed, allowing the observation of clusters in a more condensed form.
2.6. Nomenclature of MLST and whole-genome phylotypes Throughout the text, tables and figures, we have used the Belfast MLST8 phylogroup terminology as extracted from the public database at http://pacnes.mlst.net/ and where the last numerical designation is superscript as in IA1 (Lomholt and Kilian, 2010), or the whole-genome phylotype designation which includes dashes as in IA-1 (Fitz-Gibbon et al., 2013; Tomida et al., 2013).
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
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3. Results 3.1. Selection of VNTRs and in silico analysis By comparing strain KPA171202 (type IB) and strain SK137 (type IA1), 13 polymorphic tandem repeats were identified (Fig. S1), and their polymorphism was analyzed in silico on additional genomes. The repeat units of 10 VNTR loci with size ranging from 14 to 31 bp were classified as large (L-VNTR), whereas three had 6 bp small size repeats (S-VNTR). Four VNTR loci (Pacnes-0232, Pacnes-0440, Pacnes-1815, Pacnes-1902) were localized in an intergenic region and the others were part of putative open reading frames (Table 2). The three 6 bp-repeats VNTRs encoded stretches of proline/threonine or proline/alanine inside putative membrane proteins (similar to fibronectin-binding protein I for Pacnes-1815 and to adhesion or S-layer proteins for Pacnes-2303 and Pacnes-2210). Pacnes-2333 encoded a seven amino-acids repeat into a putative penicillin-binding protein. In total, genotyping data was retrieved for 102 P. acnes strains, of which eight were full genome sequences and the rest were draft. The VNTR polymorphism of strains belonging to six different phylotypes is shown on Table 2, and the complete results are shown on Table S3. The three S-VNTRs, Pacnes-1868, Pacnes-2303 and Pacnes-2393 could not be measured for 39, nine and 29 draft genomes, respectively, because the loci were split into two portions present on two different contigs. We also analyzed the genome of non-acnes Propiobacterium strains, including Propiobacterium humerusii, and could not generate an in silico profile. The MLST8, MLST9, SLST and SNP codes of P. acnes sequenced genomes were recovered from the associated publications and from public MLST databases. Clustering analysis based on 13 VNTRs MLVA (MLVA13) codes showed a good overall concordance with the other sequence-based genotyping data (Figs. 1A and S2) suggesting that the MLVA13 code could be deduced correctly from the available genome assemblies, and that it was relevant for strain comparison. Some discrepancies were observed in the distribution of several strains by the different methods. Three IB-2 strains, P.acn17, P.acn31 and P.acn33, originating from French patients with ophthalmic infections (McDowell et al., 2011) were clustered with IA-1. The S-VNTRs provided a high discriminatory power to MLVA13, and on several occasions allowed to distinguish strains with the same sequence type (ST). The number of observed alleles varied from 2 to 20. We found that a panel of 10 VNTRs (MLVA10) not-including the S-VNTRs, clustered correctly the members of a given phylogroup (Fig. 1B), although the full collection of VNTRs provided the highest discriminatory power. The stability of the S-repeats was tested by performing PCR on 100 colonies recovered from three different strains after four or five replatings. No modification of the repeat copy number was observed for any of the VNTRs. 3.2. MLVA genotyping of clinical isolates A total of 61 Propionibacterium isolates were provided by two hospitals, of which 48 were P. acnes, 10 were P. avidum and three were Propionibacterium sp (Table 1). They originated mainly from burn patients, and from patients who underwent orthopedic surgery, from 2008 to 2013. Three patients provided two strains, and in two of them one strain was P. acnes and the other was P. avidum or Propionibacterium sp. Upon genotyping with the 13 VNTRs, a high typeability was observed for the P. acnes samples (Figs. 2 and S3), whereas only faint amplification was observed with a few VNTRs for the other species (not shown). VNTR amplification was similarly unsuccessful on several Staphylococcus isolates recovered on the skin of the same patients.
Full typeability (T = 1.00) was achieved for all VNTRs except Pacnes-1868 being unamplified in three samples (T = 0.96) and Pacnes-2303 in one sample (T = 0.98). Discriminatory power was evaluated using the full collection of unrelated clinical isolates. The Hunter-Gaston discriminatory index (HGDI) was close to one, as only two isolates showed the same MLVA code (Tables 3 and S3). Upon clustering analysis using all MLVA data (shown by MST representation on Fig. S4), most of the French isolates showed MLVA profiles similar to those of sequenced strains, allowing a putative assignation to a phylogroup by genotype comparison. Type II strains clustered into a single group, and this was also the case of type IB and type III strains. Type IA isolates were distributed into several subgroups that did not always match with the MLST subtyping but fit with whole-genome SNP-based subgrouping. Pacn10, Pacn35, Pacn54 and Pacn63 appeared to form a specific group related to type II strains, and this was confirmed by recA sequencing. Pacn28 which clustered with the two type III strains, JCM18909 and HL201PA1, was confirmed to be a type III strain by recA analysis. 3.3. CRISPR/cas analysis In order to further characterize the new isolates, they were all searched for the presence of the cas1 gene and, when positive, for the CRISPR locus by PCR analysis. Five isolates (Pacn38, Pacn41, Pacn43, Pacn46, Pacn54) clustering by MLVA13 with type II strains, and strain Pacn28 clustering with type III strains, were positive for the cas1 gene. Polymorphism in the spacers number and sequence was observed (Table S4). Six new 33 bp spacers numbered 51 to 56 were found, in addition to some described previously in other strains (Brüggemann et al., 2012; Marinelli et al., 2012). Two spacers targeted phages and two spacers targeted a plasmid. The CRISPR locus in Pacn28 had no spacer, similarly to the related strain JCM18909. 4. Discussion We report the identification of a collection of VNTRs suitable for MLVA genotyping of P. acnes. The present MLVA13 assay is specific for P. acnes, as demonstrated by failure to amplify the VNTR loci in 10 P. avidum isolates, and in other closely related species or skin commensals. MLVA13 provides a very good resolution, much higher than MLST, brought by the S-VNTRs with a high polymorphism level. Such VNTRs with small repeats allow to discriminate among epidemiologically related isolates from different patients. MLVA13 allows clustering of isolates of similar type as revealed by the analysis of strains which genome was sequenced. Similarly to SNP-based typing, MLVA13 separates type IA1 strains into two groups, although there are a few strains which do not cluster according to their MLST subtype. This is related to polymorphism at VNTR locus Pacnes-0320, present in an hypothetical coding region. Three VNTRs display only two alleles, and consequently do not contribute much to the resolution of the assay but they may have a phylogenetic value and are therefore useful in the present multiple locus assay. MLVA is routinely used for the typing of several bacterial species, and databases have been constructed that allow sharing of data (Grissa et al., 2008; Vergnaud and Pourcel, 2009). Validation of informative markers and calibration of fragments analysis is possible through common use of reference strains which genome has been sequenced, and definition of conventions for allele calling (Pourcel et al., 2007, 2011; Riehm et al., 2012). MLVA13 can be performed manually at low cost using simple agarose gels but this requires more hands-on time. Multiplexing the PCR reactions using fluorescent primers and capillary electrophoresis will be of great
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
VNTR marker
Pacnes-0232_14bp_238bp_2U Pacnes-0320_27bp_172bp_4U Pacnes-0440_31bp_325bp_3U Pacnes-0866_18bp_154bp_4U Pacnes-1614_15bp_293bp_2U Pacnes-1815_18bp_129bp_2U Pacnes-1868_6bp_161bp_18U
Pacnes-1902_25bp_275bp_5U Pacnes-1938_15bp_332bp_3U Pacnes-2303_6bp_222bp_19U Pacnes-2320_15bp_138bp_3U Pacnes-2333_21bp_158bp_3U
Pacnes-2393_6bp_345bp_28U a b c d
The Not Not Not
Position in KPA171202
232518–232549 320927–321034 440364–440448 866074–866145 1614945–1614975 1815554–1815589 1868929–1869036
1902255–1902364 1938299–1938344 2303280–2303393 2320922–2320963 2333722–2333784
2393901–2394061
ORF
Intergenic Hypothetical Intergenic Putative HtaA Domain protein Hypothetical Intergenic Putative Fibronectin-binding protein Intergenic Hypothetical Hypothetical Putative Adhesion protein Putative Penicillin-binding protein Putative Adhesion protein
Expected amplicon size (bp)a IB KPA171202
IA-1 SK137
IA-2 17 Type IA2
238 2 172 4 325 3 154 4 293 2 129 2 161 18
237 2 145 3 263 1 226 8 323 4 111 1 137 14
238 2 118 2 294 2 226 8 293 2 111 1 107 9
275 5 332 3 222 19 138 3 158 3
200 2 317 2 324 36 153 4 137 2
225 3 317 2 222 19 153 4 137 2
345 28
357 30
351 29
IC HL097PA1 2 2 3 8 2 1 9
II ATCC 11828 238 2 117 2 263 1 171 5 293 2 129 2 107 9
1
200 2 332 3 222 19 138 3 137 2
32
302 21
5 2 31 4
Number of alleles in study
Number of alleles in silico
3
4
5
5
3
4
6
6
2
4
3
3
26
17b
4
4
2
2
17
17c
2
2
3
4
22
20d
III HL201PA1 1 2 3 2 1 1
2 2 19 4 2
Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
20
number of repeats is indicated under the allele size. measurable in 39 genomes. measurable in nine genomes. measurable in 29 genomes.
5
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
Table 2 Characteristics of the 13 VNTRs.
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Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
IC IA1 IA1
A
IA1 IA1
IA1 IA1 IA1
IA1
IA1 IA1 IA1 IA1
IA1
IA2
II IA1 IA1 IA1
IA1
IA2
II
IA1
IA1
IA2 IA1
II
IA1
IA1
IA1 IA1 IA1
IA1
IA1
IA1 IA1
IA1 IA1
IA1 IA2 IA2
IA1
IA1 IA1 IA1
IA1
IA1 IA1
IA2
IA2 IA2
IA2
IA1
IB IA2
IB
IB
IA1 IA1
IA2
IA2
IB
II
IA2
IA2 IA2
IA1
IA2 IA2
II
IA1 IA1
IA2
II II
IA1
II II
IA1
IA1
II
IB III
III
IB
B
Fig. 1. MLVA-based minimum spanning tree clustering analysis of the 102 P. acnes isolates, produced in silico from genomic data. Isolates classified into the same cluster are indicated using colored circles. The size of the circle and the number of bars correspond to samples with the same genotype. Phylogroup identification derived from MLST (Belfast scheme) is indicated near each sample. (A) MLVA13 analysis, (B) MLVA10 analysis. The same color code is used in both analyses. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
importance for the automation and accuracy of the assay as illustrated for other MLVA assays (Sobral et al., 2012a,b). MLVA is a fast technique which allows the typing of large amount of samples and the systematic investigation of clinical isolates, whereas MLST typing is often performed only on selected isolates (Giannopoulos et al., 2014). The cost can be in the order of 1 to a few dollars per sample depending on the methodology and the number of samples analyzed in a single assay. The recently described SLST scheme is more simple and allows a correct identification of phylotypes, although its resolution may be insufficient to differentiate some subtypes (Scholz et al., 2014). It will be interesting to see in
the future if one assay is preferred, or if they are complementary and used in parallel. We show that a large proportion of the strains in a French hospital display very similar genotypes, differing only at VNTRs with a high level of discriminatory power. According to their MLVA type, the larger group of French isolates belongs to phylogroup IA, and particularly to IA1. This type is the most frequently found on skin and associated with acne, as shown in different studies, which may be due to the preferential skin infection by certain strains (Table S1 and Fig. S1) (Lomholt and Kilian, 2010; McDowell et al., 2005, 2011). The proportion of type II and IB strains was more
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
7
Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
IA2
IA1 IA2 III
II
IB
Pacnes-2303
Pacnes-1868
Pacnes-1815
Pacnes-2333
Pacnes-1938
Pacnes-2320
Pacnes-1902
Pacnes-1614
Pacnes-0866
Pacnes-0440
Pacnes-0320
100
Pacnes-2393
IA1
MLVA13
Pacnes-0232
50
MLVA13
Strain
2
3
2
8
2
2
2
4
2
1
47
28
34
Pacn15
2
3
2
8
2
2
2
4
2
1
34
32
35
Pacn60
2013
Percy
blood
2
3
2
8
2
2
2
4
2
1
30
31
25
Pacn55
2013
Desgenettes
bone, tibia
2
3
2
8
2
3
2
4
2
1
31
28
34
Pacn02
2012
Percy
bone, graft
2
3
2
8
2
3
2
4
2
1
46
35
34
Pacn48
2013
Desgenettes
parotide gland
2
3
2
8
2
3
2
4
2
1
37
27
34
Pacn07
2012
Percy
abcess, shoulder
2
3
2
8
2
3
2
4
2
1
55
32
26
Pacn11
2008
Percy
bone, tibia
26
Pacn12
2009
Percy
prosthesis, knee
34
Pacn53
2013
Desgenettes
spinal disc, liquid
34
Pacn24
2011
Percy
skin
2010
Percy
skin
2
3
2
8
2
3
2
4
2
1
55
2
3
2
8
2
3
2
4
2
1
55
2
3
2
8
2
3
2
4
2
1
29
32 32
Year
Hospital
Site
2010
Percy
skin
2
3
2
8
2
3
2
4
2
1
44
32
38
Pacn26
2
3
2
8
2
3
2
4
2
1
44
29
36
Pacn27
2011
Percy
skin
2
3
2
8
2
3
2
4
2
1
36
31
32
Pacn31
2010
Percy
bone, thighbone
2
3
2
8
2
3
2
4
2
1
27
31
24
Pacn40
2011
Desgenettes
pleural liquid
2
3
2
8
2
3
2
4
2
1
48
31
37
Pacn50
2013
Desgenettes
joint, thighbone
2
2
2
8
2
3
2
4
2
1
35
23
28
Pacn25
2011
Percy
blood
2
2
2
8
2
3
2
4
2
1
30
30
34
Pacn44
2012
Desgenettes
spinal disc biopsy
2
2
2
8
2
3
2
4
2
1
31
22
32
Pacn59
2013
Percy
joint, hip
2
1
2
8
2
3
2
4
2
1
28
28
36
Pacn61
2013
Percy
LN, neck
2
2
2
8
1
2
2
4
2
1
18
34
32
Pacn29
2010
Percy
abcess, vertebra
2
3
1
8
2
2
3
4
2
1
30
27
18
Pacn08
2012
Percy
joint, shoulder
2
3
1
8
2
2
3
4
2
1
21
23
26
Pacn33
2010
Desgenettes
LN, neck
2
2
1
8
2
2
3
4
2
1
15
27
27
Pacn52
2013
Desgenettes
bone
2010
Percy
skin
2
3
1
8
2
2
2
4
2
1
23
36
36
Pacn03
2
3
1
8
2
2
2
4
2
1
19
23
29
Pacn39
2011
Desgenettes
blood
2
4
2
8
2
2
3
4
2
1
12
14
26
Pacn34
2013
Desgenettes
abcess, back
2012
Desgenettes
blood
2011
Percy
bone, ankle
Percy
skin
2
4
2
8
2
2
3
4
2
1
20
31
30
Pacn42
2
5
2
8
2
2
3
4
2
1
28
36
32
Pacn21
1
2
1
3
2
2
2
4
2
1
14
19
23
Pacn28
2
2
1
5
2
3
3
4
2
2
9
19
22
Pacn19
2012
Percy
adenoma, neck
2
2
1
13
2
3
3
4
2
2
12
19
25
Pacn36
2013
Desgenettes
blood
2
2
1
5
2
2
3
4
2
2
14
17
Pacn38
2011
Desgenettes
bone, tibia
2
2
1
5
2
2
3
4
2
2
14
20
Pacn46
2012
Desgenettes
CRL
2
2
1
5
2
2
3
3
2
2
9
19
20
Pacn41
2012
Desgenettes
blood
2
2
1
7
2
2
3
3
2
2
9
18
22
Pacn43
2012
Desgenettes
spinal disc, liquid
2
2
2
7
2
5
3
4
2
2
21
20
23
Pacn35
2010
Desgenettes
LN, aorta
2
2
1
7
2
5
3
4
2
2
21
20
24
Pacn63
2013
Desgenettes
joint, thighbone
2
2
3
7
2
4
3
4
2
2
10
22
16
Pacn10
2008
Percy
skin
13
2
1
5
2
3
3
4
3
2
11
14
21
Pacn54
2013
Desgenettes
blood
2012
Percy
bone, tibia
2
4
2
4
2
3
3
4
2
2
23
15
17
Pacn20
2
4
2
4
2
3
3
4
2
2
10
22
33
Pacn56
2013
Percy
blood
2
6
2
4
2
3
3
4
2
2
6
19
13
Pacn18
2012
Percy
blood
2013
Percy
prosthesis, thighbone
2
4
2
8
2
3
3
4
2
2
14
17
25
Pacn30
2
5
2
4
2
3
3
4
3
2
21
22
20
Pacn06
2011
Percy
bone marrow
2
5
2
4
2
3
3
4
3
2
15
14
33
Pacn57
2013
Percy
skin
2
3
2
4
2
3
3
4
2
2
12
14
29
Pacn47
2012
Desgenettes
lung biopsy
2
3
2
4
2
2
3
4
2
2
15
14
18
Pacn58
2013
Percy
skin
2
3
1
5
2
2
3
4
1
2
14
18
Pacn01
2010
Percy
abcess, thigh
Fig. 2. Clustering analysis of MLVA13 data for 48 French isolates. The dendrogram was constructed from MLVA analysis using 13 VNTRs. On the right are shown the strain ID, the isolation year, the hospital and the isolation site. On the left is indicated the phylogroup, by comparison with published strains.
elevated in the present study as compared to others investigating healthy skin and acne. Similarly, such strains were found to be enriched in non-skin infections in another study (McDowell et al., 2013). We did not detect a significant link between the site of infection and the strain genotype, likely due to the small sample size. In Desgenettes hospital, several isolates with a very similar
genotype were recovered from different patients over a period of three years suggesting the existence of a reservoir for these infections. Two different Propionibacterium strains were isolated from three patients, and one of them had two P. acnes with different genotypes. Indeed, two spinal disc samples from the same patient led to the isolation of Pacn43 and Pacn44, of type II and type IA
Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009
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Y. Hauck et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx
Table 3 Hunter-Gaston discriminatory index for individual or combined VNTR loci (MLVA13) calculated from 47 unrelated P. acnes isolates. Locus
Allele number
Total n = 47
MLVA10
Pacnes-0232 Pacnes-0320 Pacnes-0440 Pacnes-0866 Pacnes-1614 Pacnes-1815 Pacnes-1868 Pacnes-1902 Pacnes-1938 Pacnes-2303 Pacnes-2320 Pacnes-2333
3 6 3 6 2 2 27 4 2 18 2 3
0.0842 0.6772 0.4570 0.5883 0.0426 0.4829 0.9750 0.5412 0.5106 0.9315 0.0833 0.1619
X X X X X X
Pacnes-2393 Global index for MLVA13
22 Total genotype nbr 46
0.9537 Index for all strains 0.9991
X X X X
respectively. This was previously observed in patients suffering from lumber disc herniation (Rollason et al., 2013). Similarly, in acne patients and control carriers in Denmark, it was shown that different MLST clones were carried by some subjects (Lomholt and Kilian, 2014). It was also observed using whole-genome sequencing that most individuals harbored multiple P. acnes strains from the same or different lineages (Fitz-Gibbon et al., 2013; Tomida et al., 2013). The described P. acnes MLVA13 scheme should allow, not only to differentiate between clones, but also to identify differences between strains belonging to the same clones, thanks to the high polymorphism of the S-VNTRs. In the present study we also investigated the polymorphism of the CRISPR element present in some P. acnes strains. A study by Marinelli and co-workers suggested that the CRISPR-Cas system plays an active role in protecting the bacteria from phage infection (Marinelli et al., 2012). Strains with the most spacers are those that show extensive phage resistance, implicating CRISPR in the phenotype. It is interesting to note that strain Pacn38 possesses four novel spacers of which two target plasmid HL096PA1 (Fitz-Gibbon et al., 2013), while Pacn41 possesses two novel spacers targeting two genes from a single phage family. Phages in P. acnes display limited diversity as all the presently described phages belong to a single group found in sebaceous follicles with healthy skin or acne (Marinelli et al., 2012). This suggests that these strains that are genetically related to other strains with a different CRISPR composition, have acquired the spacers recently. 5. Conclusions We believe that the present MLVA assay will represent a first line genotyping assay, which can help investigate nosocomial infections, but also the systematic analysis of strains present on healthy individuals and on patients in order to better understand the diversity and pathogenicity of this species. Acknowledgement The authors are grateful to the laboratory staff of Percy and Desgenettes hospitals for carrying out the microbiological analyses. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2015.05. 009.
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Please cite this article in press as: Hauck, Y., et al. A novel multiple locus variable number of tandem repeat (VNTR) analysis (MLVA) method for Propionibacterium acnes. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.009