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

Lalita Ambigai Sivasamugham, Geetha Subramaniam, Wong Ling Shing, Preamala Gunabalasingam, Nithiya Visayaragawan, Nurfara Ain Ramli, Gan Li-Lian, Ravindran Vythilingam, Anshoo Agarwal

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 


Full Text:

PDF

References


Dekio I, Asahina A, Shah HN. Unravelling the eco-specificity and pathophysiological properties of Cutibacterium species in the light of recent taxonomic changes. Anaerobe, (2021); 71(102411): 1-9.

Loh KC, Chan LC, Phang LF. Perceptions and psychosocial judgement of patients with acne vulgaris. The Medical Journal of Malaysia, (2020); 75(1): 18-23.

Butler-Wu SM, Sengupta DJ, Kittichotirat W, Matsen III FA, Bumgarner RE. Genome sequence of a novel species, Propionibacterium humerusii. Journal of Bacteriology, (2011); 193(14): 3678.

Kadler BK, Mehta SS, Funk L. Propionibacterium acnes infection after shoulder surgery. International Journal of Shoulder Surgery, (2015); 9(4): 139-144.

Sheffer-Levi S, Rimon A, Lerer V, Shlomov T, Coppenhagen-Glazer S, et al. Antibiotic susceptibility of Cutibacterium acnes strains isolated from Israeli Acne Patients. Acta Dermato-venereologica, (2020); 100(17): 1–8.

Bokshan SL, Ramirez GJ, Chapin KC, Green A, Paxton ES. Reduced time to positive Cutibacterium acnes culture utilizing a novel incubation technique: a retrospective cohort study. Journal of Shoulder and Elbow Arthroplasty, (2019); 3: 1–6.

Goldenberger D, Søgaard KK, Cuénod A, Seth-Smith H, de Menezes D, Vandamme P, Egli A. Cutibacterium modestum and “Propionibacterium humerusii” represent the same species that is commonly misidentified as Cutibacterium acnes. Antonie Van Leeuwenhoek, (2021); 114(8): 1315-1320.

Chen H, Li J, Yan S, Sun H, Tan C, et al. Identification of pathogen(s) in infectious diseases using shotgun metagenomic sequencing and conventional culture: a comparative study. PeerJ, (2021); 9:e11699: 1-20

Huang YT, Yang MY, Mao YC, Lee DY, Kuo YL, et al. Identification of Cutibacterium modestum in Spondylitis by Metagenomics Analysis. In Vivo, (2023); 37(3): 1384-1388.

Wei Q, Li Z, Gu Z, Liu X, Krutmann J, et al. Shotgun metagenomic sequencing reveals skin microbial variability from different facial sites. Frontiers in Microbiology, (2022); 13:933189: 1-11.

MacWilliams MP. Indole Test Protocol. American Society for Microbiology for Microbiology, (2009); 1–9, (Accessed: 10.9.2024).

Reiner K. Catalase Test Protocol, (2013); 1–9. http://www.microbelibrary.org/library/laboratory-test/3226-catalase-test-protocol, (Accessed: 10.9.2024).

Smith AC, Hussey MA. Gram stain protocols. American Society for Microbiology, (2019); 1: 14.

Dimitrakopoulou ME, Stavrou V, Kotsalou C, Vantarakis A. Boiling extraction method vs commercial kits for bacterial DNA isolation from food samples. Journal of Food Science and Nutrition Research, (2020); 3(4): 311-319.

Alnabati NA, Al-Hejin AM, Noor SO, Ahmed MM, Abu-Zeid M, Mleeh NT. The antibacterial activity of four Saudi medicinal plants against clinical isolates of Propionibacterium acnes. Biotechnology and Biotechnological Equipment, (2021); 35(1): 415-424.

Dekio I, McDowell A, Sakamoto M, Tomida S, Ohkuma M. Proposal of new combination, Cutibacterium acnes subspp. elongatum comb. nov., and emended descriptions of the genus Cutibacterium, Cutibacterium acnes subspp. acnes and Cutibacterium acnes subspp. defendens. International Journal of Systematic and Evolutionary Microbiology, (2019); 69(4):1087-1092.

Tang J, Heng A, Chan L, Tang M, Roshidah B. Antibiotic sensitivity of Propionibacterium acnes isolated from patients with acne vulgaris in Hospital Kuala Lumpur, Malaysia. Malaysian Journal of Dermatology, (2012); 28: 1511–5356.

Alkhawaja E, Hammadi S, Abdelmalek M, Mahasneh N, Alkhawaja B, Abdelmalek SM. Antibiotic resistant Cutibacterium acnes among acne patients in Jordan: a cross sectional study. BMC Dermatology, (2020); 20: 1-9.

Borrel V, Gannesen AV, Barreau M, Gaviard C, Duclairoir‐Poc C, et al. Adaptation of acneic and non-acneic strains of Cutibacterium acnes to sebum‐like environment. Microbiology Open, (2019); 8(9): e00841 1-10.

Jeverica S, El Sayed F, Čamernik P, Kocjančič B, Sluga B, et al. Growth detection of Cutibacterium acnes from orthopaedic implant-associated infections in anaerobic bottles from BACTEC and BacT/ALERT blood culture systems and comparison with conventional culture media. Anaerobe, (2020); 61:102133 1-6.

Zhang N, Yuan R, Xin KZ, Lu Z, Ma Y. Antimicrobial susceptibility, biotypes and phylotypes of clinical Cutibacterium (formerly Propionibacterium) acnes strains isolated from acne patients: an observational study. Dermatology and Therapy, (2019); 9: 735-746.

Ederveen TH, Smits JP, Boekhorst J, Schalkwijk J, van den Bogaard EH, Zeeuwen PL. Skin microbiota in health and disease: From sequencing to biology. The Journal of Dermatology, (2020); 47(10):1110-1118.

Dekio, I., Sakamoto, M., Suzuki, T., Yuki, M., Kinoshita, S., et al. Cutibacterium modestum spp. nov., isolated from meibum of human meibomian glands, and emended descriptions of Cutibacterium granulosum and Cutibacterium namnetense. International Journal of Systematic and Evolutionary Microbiology, (2020); 70(4): 2457–2462.

Polugari R, Maria SR, Shailaja D. Isolation and Molecular Characterization of acne causing Propionibacterium acnes. International Journal of Scientific and Research Publications, (2016); 6(6): 809-814.

Sowmiya M, Malathi J, Swarnali S, Priya JP, Therese KL, Madhavan HN. A study on the characterization of Propionibacterium acnes isolated from ocular clinical specimens. Indian Journal of Medical Research, (2015); 142(4): 438-449.

Puhvel SM. Characterization of Corynebacterium acnes. Journal of General Microbiology, (1968); 50: 313–320.

Chakravorty S, Helb D, Burday M, Connell N, Alland D. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. Journal of Microbiological Methods, (2007); 69(2): 330-339.

Ruffier d’Epenoux L, Arshad N, Bémer P, Juvin ME, Le Gargasson G, et al. Misidentification of Cutibacterium namnetense as Cutibacterium acnes among clinical isolates by MALDI-TOF VitekMS: usefulness of gyrB sequencing and new player in bone infections. European Journal of Clinical Microbiology and Infectious Diseases, (2020); 39: 1605-1610.

Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, (2016); 44(W1): W232-W235.

Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology, (2010); 59(3): 307-321.

Anisimova M, Gil M, Dufayard JF, Dessimoz C, Gascuel O. Survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihood-based approximation schemes. Systematic Biology, (2011); 60(5): 685-699.

Hoang DT, Chernomor O, Von Haeseler A, Minh BQ, Vinh LS. UFBoot2: Improving the ultrafast bootstrap approximation. Molecular Biology and Evolution, (2018); 35(2): 518-22.




DOI: http://dx.doi.org/10.62940/als.v12i1.3250

Refbacks

  • There are currently no refbacks.