Complete nucleotide sequence of the 16S rRNA from Lactobacillus paracasei HS-05 isolated from women’s hands
© Choi and Lee. 2015
Received: 5 September 2015
Accepted: 31 October 2015
Published: 10 December 2015
We determined the complete nucleotide sequence of the 16S rRNA from a new bacterium collected from the surfaces of women’s hands. We also compared the presence of various bacteria based on the subjects’ sex and age. The number of colonies isolated from the hand surface was larger for women than men, and the largest number of isolates was confirmed to be present for the women in their 30 s and men in their 40 s (147 and 34 isolates, respectively). The morphology of an isolated bacterial strain was found to be rod type, and the bacterium was identified as Lactobacillaceae species based on the GenBank database, through a phylogenetic analysis using the 16S rRNA sequence. Based on the results of a homology search, the isolated strain was 99 % identical to Lactobacillus paracasei, so it was designated Lactobacillus paracasei HS-05 and was registered in the Korea Culture Center of Microorganisms (KCCM) database as [KCCM11349P].
In addition to their use in fermenting milk products or natural substances, lactic acid bacteria have been reported to be able to perform other special activities, such as producing antibiotics (Lim et al. 2008). In addition, lactic acid bacteria are introduced into the intestines to improve the properties of the intestinal microflora, where they play beneficial roles in host animals, such as stabilization of the intestinal microflora, disease prevention by suppressing the settlement of harmful bacteria, immune activation, anticancer activity, and lowering the LDL cholesterol level (Cotter et al. 2005; Jack et al. 1995; Kojic et al. 1991; Maeng et al. 1997; Marie et al. 1996). Among the substances produced by lactic acid bacteria, bacteriocin is a natural antibiotic protein or protein-based substance and is known to have effective germicidal activity against pathogenic microorganisms (Holo et al. 1991; Petersen et al. 2006; Schillinger and Lucke 1989; Todorov and Dicks 2005).
In addition to their applications in fermentation, lactic acid bacteria are being increasingly utilized in the cosmetics industry. Lactic acid bacteria fragments have shown antioxidant effects and whitening effects, and they increase the activity of cosmetic agents (Choi et al. 2013). However, to date, the lactic acid bacteria that have been used for cosmetics were those that were already in use in food fermentation, such as Lactobacillus rhamnosus, Lactobacillus paracasei, and Lactobacillus casei (Frederic and Vuyst 2004). Therefore, lactic acid bacteria that better fit the characteristics needed for the cosmetics industry should be identified to provide more effective activities.
The human skin contains numerous microorganisms, with the Propionibacterium, Streptococcaceae, Staphylococcaceae, and Lactobacillaceae known to exist in the largest numbers. Among these, Propionibacterium and Streptococcaceae have been reported to be predominant, with the Lactobacillaceae comprising a smaller population (Fierera et al. 2008). However, among the various human body parts, lactic acid bacteria (especially Lactobacillaceae) are found in large numbers on the hands, and it has previously been shown that more lactic acid bacteria are present on women’s hands than on men’s hands (Fierera et al. 2008; Costello et al. 2009; Dong et al. 2011). If new human skin-derived lactic acid bacteria can be isolated and identified, they can be utilized in the development of natural food preservatives in the current probiotics market and as alternative medicines (to provide antibiotics), and moreover, these bacteria may be more appropriate for use in the cosmetics industry. Therefore, in the present study, a new human skin-derived Lactococcus paracasei strain, HS-05, was isolated, and its morphological characteristics were investigated. In addition, 16S rRNA sequencing was conducted to identify the microorganisms isolated from human hands.
Materials and methods
The isolation of a new bacterial strain from human hands
To isolate lactic acid bacteria from human skin, approximately 40 men and women in their teens to their 40 s were asked to participate in an experiment 7 days prior to the experiment being performed. The experiment was conducted on an unspecified day so the participants would not wash or treat their hands differently before the experiment (Fierera et al. 2008). The right palm of each participant was pressed on MRS Agar (288210, Difco, USA), which was made in advance to inoculate the culture medium with initial microorganisms, and the inoculated initial samples were cultivated for 48 h in 37 °C incubators using anaerobic gas packs (Gas Pak, BBL, USA). After the culture, the obtained microorganisms were dispensed by streaking with platinum loops on MRS Agar and were cultured under anaerobic conditions for 48 h in 37 °C incubators to obtain individual strains. Using the isolated lactic acid bacteria, the phylogenetic tree of the 16S rRNA sequences was analyzed (Baek et al. 2010). For more detail characterization of the bacterium, the changes of cell growth and pH in the medium were also measured according to the culture temperature since the temperature of the hands was mostly affected under conventional environments. First, the cultures of the bacterium were incubated at 25, 30, 37, 40 and 45 °C for 24 h. At the end of each incubation time, the counts of surviving cells were determined by plating on MRS agar. In addition, the pH (initial pH of the medium was 6.5) was also measured for 24 h cultivation by a pH meter (Sentron Titan pH meter) (Tomas et al. 2003).
Analysis of the phylogenetic tree of the 16S rRNA sequences
To compare the phylogenetic trees of the isolated bacteria, a 16S rRNA analysis was conducted using the iQ5 real-time PCR detection system (Bio-Rad Laboratories). The phylogenetic tree was generated through a molecular phylogenic analysis based on the 16S rRNA gene base sequences of the isolated strains. The primers used to amplify the16S rRNA genes were the 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) primer and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) primer synthesized based on the conserved sequence of the E. coli 16S rRNA gene. The RT-PCR was conducted by performing 33 cycles of denaturation (94 °C, 30 s), annealing (60 °C, 30 s), and elongation (72 °C, 45 s), followed by incubation for 15 min at 72 °C (Baek et al. 2010). After determining the 16S rRNA gene nucleotide sequences, each base sequence was compared with the base sequences of similar strains in the GenBank database to determine the phylogenetic locations of the strains (Baek et al. 2010).
Scanning electron microscopy (SEM)
To photograph the newly isolated bacterial strain, a low-vacuum scanning electron microscope (SEM) (XL 30, Philips, The Netherlands) was used at 400× magnification to observe the morphology of the bacteria. Sample slices were immersed in an 8 % paraformaldehyde and 2.5 % glutaraldehyde solution made using 0.05 M sodium cacodylate buffer (pH 7.2) and were fixed four 2 h at 4 °C. The sample slices were washed three times for 2 min each time using 0.05 M sodium cacodylate buffer (pH 7.2) and were then fixed by treatment in a 1 % osmium tetroxide solution for 2 h at 4 °C, followed by two washes with tertiary distilled water at room temperature. The fixed samples were dehydrated in 30, 50, 70, 80, and 90 % ethanol for 10 min each and in 100 % ethanol three times for 10 min each time. After the dehydration, the samples were mounted on metal stubs by two transitions for 15 min using 100 % propylene oxide and were then coated with gold using a sputter coater (Agar Scientific Ltd. SC502, USA). The samples were observed using a scanning electron microscope (Philips XL30E, USA) (Williams and Davies 1967).
Isolation and characteristics of a new bacterial strain
Analysis of the phylogenetic tree of the 16S rRNA sequence of the new strain
The complete nucleotide sequence of the 16S rRNA from Lactobacillus paracasei HS-05
From this work, a human hand-derived lactic acid bacterium was successfully isolated. Although Proprionibacteria and Streptococcaceae are known to be present in large numbers on human skin, the Lactobacillaceae were found to be distributed in large numbers on the human hands (Fierera et al. 2008). In addition, more colonies were cultured from women’s hands than from men’s hands, and this is considered to be attributable to the fact that unlike the other types of common skin bacteria, the number of Lactobacillaceae are more variable and are not evenly distributed between men and women (Fierera et al. 2008). In particular, large numbers of colonies were observed for the hands of women in their 30 s, which was considered to be due to the fact that their frequency of contact with cosmetics and/or foods is high (Costello et al. 2009; Dong et al. 2011). From the result of this work, Lactobacillus paracasei HS-05 was not found on men’s hands but was found on women’s hands. Moreover, it was also first confirmed that the bacterium profile and existence of Lactobacillus paracasei HS-05 were not much affected by the personal histories and their job careers by having consistently high numbers of the colonies in most groups of women hands, not any kinds of men’s hands. It is very interesting that the dominant existence of specific lactic acid bacterium was observed on the women and specially for young age women, regardless of their jobs, which could be further utilized for isolating other specific purpose bacterium from human body.
Lactobacillus paracasei HS-05 bacteria could be identified through a 16S rRNA analysis, as shown in Table 1, and no strain that has the same specific base sequence has been reported yet (Janda and Abbott 2007). Therefore, based on the 16S rRNA analysis and the molecular phylogenic analysis, the isolated strain was considered to fall under the phylogenic group that includes Lactobacillus, and had 99 % homology with Lactobacillus paracasei subsp. Paracasei D79212. This is the first report of the existence of this strain on the hand surface of women, and this bacterium was thus named Lactobacillus paracasei HS-05 and has been registered in the Korea Culture Center of Microorganisms (KCCM) as [KCCM11349P].
WYC carried out the studies of analyzing microorgnisms and other related experiments, and HYL carried out the studies of identifying new microorganisms and drafted the manuscript. All authors read and approved the final manuscript.
This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. HN12C0060).
The authors declare that they have no competing interests.
Ethics and consent
All of the persons involved in this experiments were notified about this study and particiapted with their consent.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Adamberg K, Kask S, Laht TM, Paalme T (2003) The effect of temperature and pH on the growth of lactic acid bacteria: a pH-auxostat study. Int J Food Microbiol 85:171–183View ArticlePubMedGoogle Scholar
- Baek H, Ahn HR, Cho YS, Oh KH (2010) Antibacterial effects of Lactococcus lactis HK-9 isolated from feces of a new born infant. Korean J Microbiol 46:127–133Google Scholar
- Choi WS, Kwon HS, Lim HW, No RW, Lee HY (2013) Whitening effects of Lactobacillus rhamnosus associated with its antioxidative activities. Korean J Microbiol Biotechnol 41:183–189View ArticleGoogle Scholar
- Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R (2009) Bacterial community variation in human body habitats across space and time. Sci 326:1694–1697View ArticleGoogle Scholar
- Cotter PD, Hill C, Ross RP (2005) Bacteriocins: developing innate immunity for food. Nat Rev Microbiol 3:777–788View ArticlePubMedGoogle Scholar
- Dong Q, Brulc JM, Iovieno A, Bates B, Garoutte A, Miller D, Revanna KV, Gao X, Antonopoulos DA, Slepak VZ, Shestopalov VI (2011) Diversity of bacteria at healthy human conjunctiva. Invest Ophthalmol Vis Sci 52:5408–5413PubMed CentralView ArticlePubMedGoogle Scholar
- Fierera N, Hamady M, Lauber CL, Knight R (2008) The influence of sex, handedness, and washing on the diversity of hand surface bacteria. PNAS 105:17994–17999View ArticleGoogle Scholar
- Frederic L, Vuyst L (2004) Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci Technol 15:67–78View ArticleGoogle Scholar
- Holo H, Nilssen Q, Nes IF (1991) Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene. J Bacteriol 173:3879–3887PubMed CentralPubMedGoogle Scholar
- Jack RW, Tagg JR, Ray B (1995) Bacteriocins of grampositive bacteria. Microbiol Rev 59:171–200PubMed CentralPubMedGoogle Scholar
- Janda JM, Abbott SL (2007) 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J Clin Microbiol 45:2761–2764PubMed CentralView ArticlePubMedGoogle Scholar
- Kojic M, Svircevic J, Banina A, Topisirovic L (1991) Bacteriocin-producing strain of Lactococcus lactis subsp. Diacitilactis S50. Appl Environ Microbiol 57:1835–1837PubMed CentralPubMedGoogle Scholar
- Lim YS, Kim SY, Lee SK (2008) Characteristics of lactic acid bacteria isolated from kefir made of goat milk. Korean J Food Sci Ani Resour 28:82–90View ArticleGoogle Scholar
- Maeng KJ, Kim JS, Ji GE, Kim JH (1997) Isolation of bacteriocin-producing lactic acid bacteria from human intestines and the characteristics or their bacteriocins. J Kor Food Sci Nutr 26:1228–1236Google Scholar
- Marie A, Junelles R, Lefebvre G (1996) Purification and N-terminal amino acid sequence of dextranicin 24, a bacteriocin of Leuconostoc sp. Curr Microbiol 33:136–137View ArticleGoogle Scholar
- Petersen FC, Fimland G, Scheie AA (2006) Purification and functional studies of a potent modified quorum-sensing peptide and a two-peptide bacteriocin in Streptococcus mutans. Mol Microbiol 61:1322–1334View ArticlePubMedGoogle Scholar
- Schillinger U, Lucke FK (1989) Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol 55:1901–1906PubMed CentralPubMedGoogle Scholar
- Todorov SD, Dicks LMT (2005) Characterization of bacteriocins produced by lactic acid bacteria isolated from spoiled black olives. J Basic Microbiol 45:312–322View ArticlePubMedGoogle Scholar
- Tomas MSJ, Ocana VS, Wiese B, Nader-Macıas ME (2003) Growth and lactic acid production by vaginal Lactobacillus acidophilus CRL 1259, and inhibition of uropathogenic Escherichia coli. J Med Microbiol 52:1117–1124View ArticleGoogle Scholar
- Williams ST, Davies FL (1967) Use of a scanning electron microscope for the examination of actinomycetes. Microbiol 48:171–177Google Scholar