Optimization of genotypic and biochemical methods to profile P. acnes isolates from a patient population

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Highlights

  • The MALDI-TOF is a suitable method for P. acnes identification.

  • 24 clinical strains have been clustered into phylogroups I (n = 21) and II (n = 3).

  • Combination of PAtig gene and multiplex touchdown PCRs provides a fast typing.

  • No clear relation was observed between antibiotic susceptibility and virulence.

Abstract

Propionibacterium acnes is a key factor in the pathogenesis of acne vulgaris, although currently it is also being associated with medical-device infections. The aim of this work was to validate a safe and quick identification and typing of 24 clinical isolates of Propionibacterium acnes, applying a range of biochemical as well as genetic methods, and investigating the pathogenic potential to associate the different types with human health. RAPD-PCRs revealed the existence of two discernible clusters in correspondence with the phylogroups I and II, according to the PAtig gene polymorphism, leading them to be assigned as P. acnes subsp. acnes subsp. nov. Biotyping according to the pattern of sugar fermentation evidenced that all the isolates from acne and the majority from opportunistic infections fit the biotype I-B3. Consistent with the multiplex touchdown analysis, nearly all the isolates included in this biotype belonged to the subgroups IA1 (the exception being four strains classified as IB). The remaining ones were assigned to phylogroup II, considered to be part of the normal cutaneous microbiota. The susceptibility to three antibiotics was also investigated to explore the relations with the virulence, although no clear trend was identified.

Introduction

The high GC Gram-positive anaerobe Propionibacterium acnes is a member of the resident microbiota of virtually every human. It is an exclusive bacterium that colonizes the hostile environment of sebaceous follicles, facilitating its involvement in the pathogenesis of acne vulgaris, but it is also found among the nasal, oral, and gastrointestinal microbiota (Dessinioti and Katsambas, 2010, Bek-Thomsen et al., 2014). Although its role in human infections is likely to have been significantly underestimated (Pan et al., 2005, Nisbet et al., 2007, Levy et al., 2013), an increasing number of studies have implicated P. acnes as an opportunistic pathogen, frequently isolated from blood, skin, and soft tissues, in biofilm-associated infections of medical devices, in serious infections (endocarditis, osteoarthritis, endophthalmitis, sarcoidosis., etc.) and even in the metastasis of the prostate gland and related to neurodegenerative disorders (Oprica and Nord, 2005, Perry and Lambert, 2011, Holland et al., 2010, Achermann et al., 2014, Aubin et al., 2014, Leheste et al., 2017).

Current approaches to identify differences in the pathogenic potential of P. acnes have provided data on the existence of assorted subpopulations in association with health and/or diseases (Nagy et al., 2005, McDowell et al., 2005, McDowell et al., 2011, Lomholt and Kilian, 2010, Kilian et al., 2012). Classical methods for differentiating those groups are based on immunofluorescence with polyclonal antisera (Holmberg and Forsum, 1973), bacteriophage (Webster and Cummins, 1978), fermentation typing (Kishishita et al., 1979), as well as mass spectrometry typing (Nagy et al., 2013). However, the major advance in phylogenetic reconstruction and genetic typing was achieved by the introduction of multilocus sequence typing (MLST), a complex genetic analysis of concatenated sequences of several housekeeping genes, which displays excellent resolution and differentiation of different sequence types (McDowell et al., 2005, McDowell et al., 2011, Lomholt and Kilian, 2010, Kilian et al., 2012). A recently developed multiplex touchdown PCR assay allows, in a single reaction, the confirmation of the species identity and phylogeny of isolates based on their pattern of reaction with a six primer sets (Barnard et al., 2015). Genetic approaches based on individual or multiple genes have classified P. acnes into three major genetic divisions (I, II, and the filamentous type III). Besides, group I was further separated into different subdivisions (IA1, IA2, IB, and IC). Moreover, the phylotypes had been recently recognized as subspecies, based on their intraspecies heterogeneity. This has resulted in the proposal of designing phylotypes I/II such as P. acnes subsp. acnes subsp. nov., and phylotype III as P. acnes subsp. elongatum subsp. nov. (Dekio et al., 2015). There is growing evidence, although not full agreement, about their associations with specific infections (potential production of putative virulence factors, inflammation, biochemical, aggregative, and morphological characteristics) or being compatible with health (McDowell et al., 2005, McDowell et al., 2008, McDowell et al., 2011, McDowell et al., 2012, Lomholt and Kilian, 2010, Yu et al., 2015). Therefore, phylogroups IA-1 and IC are commonly associated with acne, while IA-2, II, and III are associated mainly with infections of the prosthetic joint, spinal disc, macular hypomelanosis, and other tissue (Yu et al., 2015, Barnard et al., 2016). Nevertheless, some subgroups of phylotypes II and III associated with healthy skin, have even been proposed as a topical probiotic for treating acne (Yu et al., 2015).

For a safe and quick identification and typing of the 24 Propionibacterium acnes clinical isolates, several molecular tools have been applied in the present study. We used a range of methods based in fermentation profile (biotyping) and DNA bases analysis consisting in random amplification of polymorphic DNA (RAPD), PAtig gene polymorphism, and multiplex touchdown. The results confirmed that all isolates were distributed within of two discernible clusters in broad correspondence with the phylogroups I (n = 21) and II (n = 3), thus belonging to P. acnes subsp. acnes subsp. nov. and that of those from acne and the majority from opportunistic infections were predominantly classified into biotype IB3. Moreover, according to the results of multiplex touchdown, most the strains of this biotype fit the phylogroup IA1, which is involved in the most common form of acne lesions (McDowell et al., 2012, Yu et al., 2015), with the exception of four isolates identified as subgroup IB (P10, P12, P19 and P20) for the presence of sodD amplicon (145 bp). Remarkably, the highly conserved P7, P9, and P23 isolates were identified in all assays as belonging to phylotype II, which is considered as part of the normal cutaneous microbiota (Horváth et al., 2012). Finally, the susceptibility of these clinical isolates to three antibiotics has been also investigated.

Section snippets

Origin and identification of the bacterial isolates

The clinical isolates of P. acnes (n = 23) examined in this study were obtained from different patients of two different hospitals: 15 were from the Microbiology Service of Virgen de las Nieves Hospital of Granada (Spain) (P3, P4, P11, P12, P13, and P24 from inflammatory acne; P1, P10, P14, and P25 from wound exudates; P15, P17, and P18 from abscesses and P26 from aspirate contamination) and 8 from the Regional University Hospital of Málaga (Spain) (P6 from a bone biopsy; P22 from synovial fluid;

Identification of the clinical samples as Propionibacterium acnes

A total of 23 fresh clinical isolates were identified as P. acnes in this work, 7 of which were from inflammatory acne (P0, P3, P4, P11, P12, P13, and P24), 9 from opportunistic infections: wound exudates (P1, P10, P14, and P25), abscesses (P15, P17, P18, and P27), or bone (P6), and the remaining 8 were contaminants (Table 2). The MALDI-TOF method was used for species determination in the routine microbiological hospital laboratories (Clark et al., 2013).

Genotyping by RAPD-PCR

We investigated the potential of RAPD to

Discussion

The aim of this work was to validate the less labour-intensive and inexpensive methods for an accurate identification and typing of Propionibacterium acnes isolates. For this, we have examined the correlation between the clusters deduced from four RAPDs, the polymorphism of PAtig gene (Furukawa et al., 2009), the existing biotypes established according to the pattern of sugar fermentations (Kishishita et al., 1979), and the phylogroups and subgroups recognized by the multiplex touchdown

Conclusions

These results show the importance of applying different methods (Fig. 4) to distinguish some features of P. acnes subpopulations with different pathogenic potential, and to understand the role of different types in the pathogenesis of this bacterium. The differences found in the analyses performed demonstrate that the subgroups within a phylotype are genetically distinct, and it is desirable to have a rapid typing scheme capable of distinguishing them. In this sense, although all methods worked

Ethical approval

The study protocol was carried out in accordance with the Declaration of Helsinki. This was a non-interventional study with no additional investigation to routine procedures. Biological material was only used for standard diagnostics following physicians' prescriptions. No additional sampling or modification of the routine sampling protocol was performed. Data analyses were carried out using an anonymous database.

Acknowledgements

We acknowledge the work of David Nesbitt in editing the text. This work was funded by a Grant (Project SAF2013-48971-C2-1-R) from the Spanish Ministry of Economy and Competitiveness that included funds from European Regional Development (ERDF) and by the Research Group General (BIO160, UGR).

Conflict of interest

The authors declare that no competing interests exist.

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