A novel combinatorial approach for the Identification of Cutibacterium namnetense and Cutibacterium modestum from Facial Acne Samples

Full Length Research Article 

A novel combinatorial approach for the Identification of Cutibacterium namnetense and Cutibacterium modestum from Facial Acne Samples

Lalita Ambigai Sivasamugham1*, Geetha Subramaniam1, Wong Ling Shing1, Preamala Gunabalasingam2, Nithiya Visayaragawan2, Nurfara Ain Ramli2, Gan Li-Lian3, Ravindran Vythilingam4, Anshoo Agarwal5

Adv. life sci., vol. 12, no. 1, pp. 120-126, February 2025
*- Corresponding Author: Lalita Ambigai Sivasamugham (Email: lalitaa.sivasamugham@newinti.edu.my)
Authors' Affiliations

 1. Sciences, INTI International University, Persiaran Perdana BBN, Putra Nilai, 71800 Nilai, Negeri Sembilan – Malaysia
2. Dermatology Department, Hospital Tuanku Jaafar, Seremban, Negeri Sembilan, Ministry of Health – Malaysia
3. Clinical Research Center, Hospital Tuanku Jaafar, Seremban, Negeri Sembilan, Ministry of Health – Malaysia
4. International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Federal Territory of Kuala Lumpur – Malaysia
5. Northern Border University, Arar Kingdom – Saudi Arabia

[Date Received: 20/03/2024; Date Revised: 28/05/2024; Date Published: 31/12/2024]

Abstractaa download_button
Introduction
Methods
Results
Discussion
References 

Abstract

Background:  Cutibacterium spp. is one of the most understudied bacteria and this is owed to its slow growing nature and its stringent requirement for anoxic conditions. To date, shortgun metagenomic sequencing and MALDI-TOF MS are widely used for species detection but, the latter is not able to distinguish C. acnes from C. modestum and C. namnetense. Our study has innovatively combined colony morphology, biochemical assays and16s rRNA gene sequencing to identify C. acnes as well as the underreported C. namnetense and C. modestum from facial clinical acne samples.

Methods: The clinical samples were obtained using a non-invasive method from acne patients at the Dermatology Clinic of Hospital Tuanku Jaafar, Seremban, Malaysia between January 2022 to December 2022. Colonies of Cutibacterium spp. were screened on BHI agar followed by subjecting them to the catalase and indole tests. The isolates were verified as Cutibacterium spp. using API20A and 16s rRNA Sanger gene sequencing.

Result: Out of 68 Cutibacterium spp. isolates, 3 were identified as C. modestum and 1 as C. namnetense while the rest were C. acnes. All isolates were present as raised, white colonies with 0.03 to 1mm in diameter on BHI agar. 89.71% of these isolates were indole producers. All isolates were identified as C. acnes in API20A but, the 16srRNA gene sequencing revealed 4 isolates as C. modestum and C. namnetense.

Conclusion: This study is the first to report the isolation of C. namnetense and C. modestum in clinical facial acne samples from Malaysia and across Asia, employing a modified combination of morphological, biochemical, and 16srRNA gene analyses. This methodical yet straightforward approach serves as a viable alternative in research settings lacking access to advanced techniques like MALDI-TOF and shotgun metagenomic sequencing. Moreover, this conventional isolation approach is valuable in assessing the sensitivity of the isolates to inhibitory agents apart from antibiotics, expanding researchers' abilities to develop potent antibacterial agents required for human health and wellbeing.

Keywords:  Acne; Clinical Samples; Combinatorial; Identification 

Introduction6th button-01

Cutibacterium spp. are aerotolerant anaerobes and are comprised of C. acnes, C. granulosum, C. avidum, C. modestum and C. namnetense [1]. While Cutibacterium acnes is long known to cause acne vulgaris (AV), less is known of the role of C. modestum and C. namnetense in the pathophysiology of AV [1,2]. Cutibacterium modestum previously known as Propionibacterium humerusii, was first isolated from human humeral bone in 2011 [3]. They are also found in the pilosebaceous unit of the skin and have been isolated together with C. acnes and thus, the human skin is predicted to be the main reservoir for this bacterium. However, this is yet to be verified [1]. Cutibacterium namnetense was first identified in 2016 and was found in diverse locations of the human body including the skin and bones [1].

Unlike other skin bacteria, Cutibacterium spp. remains a challenge for identification using conventional techniques. This is because, they require anoxic conditions and at least 5 days or more to grow on a solid growth medium [2,4]. Presence of oxygen retards their growth even though they are aerotolerant cells. In addition, enriched media such as BHI, TSA, Blood agar, Brucella Blood Agar, SLM, Reinforced Clostridial Agar are needed for cultivation. Some of these media are not only expensive but also have a shorter shelf-life making the conventional isolation cost intensive. A similar study even used furazolidone in the growth medium to inhibit the growth of other skin bacteria to prevent them from deterring the detection of C. acnes in the clinical samples [5]. Altogether, these challenges have caused Cutibacterium spp. to be less commonly studied compared to other skin bacteria such as Staphylococcus aureus and S. epidermidis [2,6,7]. Due to this, MALDI-TOF is widely used as an alternative in the detection of C. acnes mainly in clinical samples. However, this robust system, is unable to discriminate C. modestum and C. namnetense leading to the misidentification of these species as C. acnes [1]. More recent studies have resorted the use of shortgun metagenomic sequencing to identify C. modestum and C. namnetense in clinical samples which is essential for rapid disease diagnosis [8-10]. However, this system cannot be used if progressive work is required to study these clinical isolates for their sensitivity towards potent antibacterial agents apart from antibiotics.

Thus, this study was aimed to devise a cost-effective systematic approach to identify Cutibacterium spp. from clinical acne samples using a combination of colony characteristics, biochemical tests and 16s rRNA gene Sanger sequencing enabling researchers to further investigate this understudied bacterial species.

Methods6th button-01

Ethical Clearance

Ethical approval was acquired from the National Medical Research Register, Malaysia (NMRR-21-1891-61558) and the Research & ethical Committee of INTI International University (INTI/UEC/2018/001) before the commencement of the study. The Patient Information Sheet (PIS) or the informed consent form as approved by the Medical Research Ethics Committee (MREC) was signed by the patients who took part in this study. Two language versions of the PIS were available: English and Bahasa Malaysia. The Case Report Form was used to record patients brief demographic details along with the sample number.

Sample Collection

138 acne samples were obtained from acne patients at the Dermatology Clinic of Hospital Tuanku Jaafar Seremban (HTJS) between January 2022 to December 2022. Patients who were pregnant, below 18 years old, receiving topical antibiotic therapy, or had used antibiotics in the past were excluded from this study. A panel of dermatologists at HTJS evaluated the severity of the acne (mild, moderate, and severe) using the Comprehensive Acne Severity Scale (CASS) before sample collection. Samples were taken from the patient's forehead, cheeks, jaw, and areas of papules, pustules, and nodules. The skin area above the acne lesions was gently swabbed with an alcohol pad (70%) followed by swabbing with the Amies transport media swab (Microscience) with charcoal. The swabs were transported to the Molecular Biology Laboratory 1 of INTI International University within 24-48 h upon collection for processing.

Colony Screening on BHI Agar

The Amies swabs were streaked on BHI agar (Oxoid) and incubated for five days at 37 °C in anoxic conditions (Gaspak EZ, BD). Pure cultures were obtained by subculturing single colonies with morphology typical of the positive control Cutibacterium acnes ATCC 11827 (raised, smooth, pearly-white, 0.03 mm to 1 mm diameter) on BHI agar under the same growth conditions. The pure cultures were subjected to indole and catalase tests as well as gram staining prior to identifying with Biomerieux's API20A [11–13].                                               

Molecular Analyses

DNA Extraction

Single colonies were cultivated in BHI broth (Oxoid) and grown in anoxic conditions. After centrifuging the bacterial culture for 5 min at 4000 rpm, the pellet was resuspended in 200 µL of cell lysis buffer (Tween20, TritonX, 0.5M EDTA pH 8.0, 1M Tris-HCL, pH). The cell lysate was vortexed vigorously for 2-3 min and placed in a dry heat block at 95 ℃ for 15 min [14]. The cell lysate was re-centrifuged for 5 min at 4000 rpm, and the supernatant containing the DNA was electrophoresed in a 1% agarose gel that had been prestained with ViSafe Red Gel dye (Vivantis).

Amplification of 16S rRNA gene and Phylogenetic Analysis

The DNA of the isolates and C. acnes ATCC 11827 were amplified using universal primers; forward primer, 50- AGAGTTTGATCCTGGCTCA-30 (27), and reverse primer, 50- AAGGAGGTGATCCAGCCGCA-30 (1525) corresponding to bases 27 and 1525 of the 16s rRNA gene [15]. The PCR mixture was done using the GoTaq Green Master Mix, 2X, Promega in a total volume of 25 µL adapted from the study by Alnabati et al (2021) [15]. The thermocyling conditions were applied as described by Alnabati et al (2021) [15]. The PCR products were sent to Apical Scientific Sdn Bhd to determine the sequence. The data obtained was analysed using Bioedit.Ink and MEGA11 (version 11) and compared with the sequences in the NCBI database. The confirmed isolates of Cutibacterium spp., were aligned with several reference sequences that served as ingroup and outgroup species using the ClustalW method in MEGA11. The reference taxa were chosen based on the study by Dekio et al (2019) [16]. The Bayesian information criterion (BIC), the HKY model was chosen to generate the tree. The reliability of the internal branches was assessed using standard bootstrap (SH-aLRT), aBayes test and the ultrafast boot-strap (UFBoot) tests for 1000 replicates respectively. The phylogenetic tree obtained through IQ-Tree was reconstructed in FigTree V1.4.4 and MEGA11. 

Results6th button-01

Colony Identification on BHI Agar             

A total of 138 facial acne swabs were obtained from the acne patients who participated in this study from January 2022 to December 2022. 68 Cutibacterium spp. isolates were obtained with 94% (n=64) of these isolates were identified as C. acnes while, 4% (n=3) isolates were identified as C. modestum. 2% (n=1) of the isolate was identified as C. namnetense. The facial acne swabs streaked on BHI agar produced colonies with heterogenous appearance. However, the colonies of Cutibacterium spp. were distinct from the other colonies that were mostly pale yellow, convex with irregular morphologies and a diameter of more than 1mm (Figure 1). All Cutibacterium spp. isolates were observed as raised, pearly-white and smooth colonies ranging from 0.03 to 1.00 mm in diameter within 5 days of incubation in anoxic conditions. Interestingly, no distinct differences were observed between the C. acnes, C. namnetense and C. modestum colonies (Table 1). This colonies were subcultured on BHI agar to obtain pure cultures and were further identified using gram staining and other biochemical tests as well as 16s rRNA gene sequencing.

Biochemical Identification of Cutibacterium spp.

All of the Cutibacterium spp. colonies identified on BHI agar were Gram positive pleomorphic rods with either single or pair wise arrangements (Table 2). All isolates were also catalase-producers.

89.71% (N=61) of the 68 Cutibacterium spp. isolates and C. acnes ATCC11827 were indole positive while the rest were indole-negative. Out of the 3 isolates of C. modestum, 2 were negative for indole production while, the C. namnetense were indole positive. All 68 isolates were identified as C. acnes with the percentage of identity of more 99% using the API20A kit.

16srRNA Gene Sequencing

The PCR products for the 16srRNA gene in all isolates were separated by electrophoresis in a 1.5 % agarose gel and were observed at 1500 bp in size (Figure 2).

The partial sequencing of the 16srRNA gene which were analyzed using Bioedit.Ink followed by BLAST revealed more than 99% of nucleotide similarities with the Cutibacterium sequences deposited in the GenBank database. The phylogenetic analysis using MEGA11, FigTree and NCBI database revealed that the some of the isolates of C. acnes along with the positive control C. acnes ATCC11827 were clustered with reference strain LC7522328.1 C. acnes ATCC6919 and C. acnes elangotum NR145912.1 (Figure 3). C. namnetense (Isolate J22/78 (5)) were clustered with strains KM507346.1 C. namnetense whereas, C. modestum isolates M22/57(4) and J22/67(5) were clustered with strains LC466959.1 (C. modestum), LC637867.1 (C. modestum) and LR118646.1 (P. humerusii – now, known as C. modestum) (Figure 3). The support values at the main branches showed more than 91% of confidence level for SH-aLRT (Figure 3). Although the UFBoot analysis showed a varying degree of confidence ranging between 85%-100%, the additional bootstrapping support of 0.91 to 1 for the Bayesian analysis (aBayes test) indicates the overall reliability of the phylogenetic tree. Accession numbers NR115826.1 and NR114803.1 were used as the outgroup (Figure 3).

Figures & Tables

 

Discussion6th button-01

While many studies of similar nature have used invasive methods to obtain cultures of C. acnes, our study has shown that even the isolation of C. namnetense and C. modestum as well as C. acnes is possible using a non-invasive approach [15-17]. A similar finding was reported in a previous study in Malaysia but no studies have been documented in Malaysia or in Asia on the use of non-invasive approach to isolate C. namnetense and C. modestum from clinical acne samples [17]. The isolation of Cutibacterium spp. colonies on BHI agar was crucial in this study as it led to successful identification of Cutibacterium spp. thereafter. This was done by thorough screening of colonies that showed similar morphology with the positive control C. acnes ATCC11827. These colonies always appeared raised and pearly-white in colour with a diameter ranging from 0.03 to 1.00 mm making them easily distinct from other colonies which were significantly larger, with a convex elevation and irregular morphologies. No distinct differences were observed between the colonies of C. acnes, C. modestum and C. namnetense making it relatively easy to identify these bacteria if this method is reproduced for future work using clinical and non-clinical samples. Such detailed descriptions on the colony morphology of C. acnes, C. modestum and C. namnetense on BHI agar to our best knowledge, is the first. C. acnes is generally grown in media specific for anaerobic or fastidious bacteria such as Schaedler agar, chocolate agar, Brucella blood agar, Wilkins-Chalgren agar, reinforced clostridial medium, blood agar as well as brain heart infusion agar [18-21]. In our study, the brain heart infusion agar proved to be a cost- effective medium with an extended shelf life enabling the growth of not only C. acnes but also C. namnetense and C. modestum. Although the number of Cutibacterium spp. colonies were significantly lower than the larger colonies which are either Staphylococcus or Corynebacterium, the other common bacteria in acne samples, the stark differences in the elevation, the colony colour and consistency eased the screening process [22]. In fact, the colonies screened to be similar or with the same colony morphology with the positive control were all identified as Cutibacterium spp. This is noteworthy as the identification of colony morphology is the pivotal step in the identification of the Cutibacterium spp. We have also shown that the isolation of Cutibacterium spp. from clinical isolates can be done without the incorporation of furazolidone in the growth medium in contrast with the study by Sheffer-Levi et al. (2020) [5].  However, the bacterial cultures must be incubated at 37°C in an anoxic condition for 5 to 7 days [15]. Cutibacterium spp. including the positive control were initially attempted to be grown using Candle jars as they are aerotolerant anaerobes but, no growth was visible even after 7 days (data not included). Hence, future isolation of Cutibacterium sp must be done in anoxic conditions using anaerobic growth systems such as GasPak (BD BBL) and Anaerogen (Merck, Millipore) [4].

The indole production using the Kovac’s indole reagent showed mixed results. While most of the previous studies have shown indole production as a main biochemical property for the isolation of Cutibacterium spp. from clinical or non-clinical samples, we report that a small percentage of the isolates of C. acnes as well as C. modestum and C. namnetense were indole-negative [23-25] . Puhvel (1968) has pointed out that the differences in the production of indole is mainly due to the differences in the strains of C. acnes [26]. Thus, the variation in indole production must be considered when Cutibacterium spp. including C. modestum and C. namnetense are being identified to prevent the indole-negative strains from being disregarded for further analysis.

API20A was used in this study as only C. acnes was anticipated to be isolated from the clinical acne samples due to the limited studies reported on C. namnetense and C. modestum during the research period. However, similar studies in the future should use API rapid 32A to enable rapid discrimination of C. acnes from C. modestum, C. namnetense and possibly other species of Cutibacterium [23]. The 16S rRNA gene is widely used for the identification of bacteria as the presence of hypervariable regions provide significant sequences to distinguish bacterial species [27]. The stretch of the conserved regions which flank the hypervariable regions enables the design of universal primers for the identification of bacterial species [27]. The primers used in this study were forward primer, 5’-AGAGTTTGATCCTGGCTCA-3’(27), and reverse primer, 5’-AAGGAGGTGATCCAGCCGCA-3’(1525) used by Alnabati et al (2021) was able to distinguish C. acnes and the closely related C. modestum and C. namnetense and this was not reported elsewhere at the time of writing [15]. This is noteworthy, as it shows that a relatively simple and cost effective 16srRNA gene sequencing is discriminative enough to differentiate species of Cutibacterium unlike the limitations reported by Ruffier d’Epenoux et al. (2020) which showed the misidentification of C. namnetense as C. acnes when MALDI-TOF was used (Ruffier d’Epenoux et al., 2020) [28]. Similar limitation was also reported by Goldenberger et al (2021) when MALDI-TOF is widely used in clinical settings in the detection of C. acnes from clinical samples [7].

The phylogenetic analysis was also consensus with the study by Dekio et al (2021) as the isolates obtained in this study were closely clustered with C. acnes, C. namenetense, C. modestum and C. avidum [1]. In addition, all C. acnes isolates including C. acnes ATCC11827 were grouped with the reference strain LC752328.1 C. acnes ATCC6919 while, some isolates of C. acnes were grouped closer to NR145912.1 C. acnes subs elangotum. In general, the maximum-likelihood tree indicated reasonably accepted relationships between the isolates and the reference strains. This is evidenced by the support values at the main branches showing more than 97% of confidence level for SH-aLRT [29]. Although the UFBoot analysis showed a varying degree of confidence ranging between 67%-100%, the additional bootstraping support of 0.7 to 1 for the Bayesian analysis (aBayes test) indicates a reliable phylogenetic tree. SH-alRT, UFBoot and Bayesian analysis are bootstrapping methods incorporated in IQ-Tree. IQ-Tree is a fast and efficient in constructing phylogenetic tree [31]. It is also widely used for its rapid analysis. The inclusion of 3 branch support analyses in IQ-Tree which are the standard bootstrap (SH-aLRT), aBayes test and the ultrafast boot-strap (UFBoot) in a single run makes it a robust system [30-32].

The isolation and identification of Cutibacterium species succeeded with the screening of colony morphology on a cost-effective brain heart infusion agar. The morphological identification proved to be a crucial step in isolating Cutibacterium species, complemented by 16srRNA gene analysis that overcame limitations observed with the MALDI-TOF system. While shotgun metagenomic analysis is currently favored for its rapid and accurate identification of Cutibacterium species, our methodical conventional approach offers a viable alternative, particularly in regions lacking access to advanced analytical methods. In conclusion, our study presents an accessible method for the isolation and identification of Cutibacterium species from clinical samples and possibly even from non-clinical samples advancing research efforts in various global settings. The systematic and cost-effective approach which starts with the colony morphology screening on BHI agar, biochemical tests, API 20A and 16s rRNA gene sequencing gave a 100% success rate in the identification of C. acnes isolates from facial acne clinical samples and is expected to do the same when this methodical approach is replicated using other clinical samples for Cutibacterium spp. isolation.

Acknowledgements

We would like to express our gratitude to the Director General of Health Malaysia for his permission to publish this article and also INTI International University, for funding this project; grant numbers (INTI-FHLS-11-02-2021) and (INTI-FHLS-01-17-2022).

Author Contributions

Lalita Ambigai Sivasamugham: Planned and designed the study, experimental conduct, data analysis, manuscript generation

Geetha Subramaniam: Planned the research, supervised the study, reviewed the data, manuscript revision

Wong Ling Shing1: Reviewed the data, supervised the study, manuscript revision

Preamala Gunabalasingam: Sample collection

Nithiya Visayaragawan: Sample collection

Nurfara Ain Ramli: Sample collection

Gan Li-Lian: Clinical advisor

Ravindran Vythilingam: Sample collection

Anshoo Agarwal: Advisor

Conflict of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

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