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INTRODUCTIONThe human skin microflora is unique and multifaceted and is made up of mixture of diverse groups of microorganisms including aerobic, anaerobic, and facultative bacteria such as Staphylococci spp., fungi such as Malassezia spp., viruses, and bacteriophages.[1,2] Based on the metagenomic and cultural studies, it has been investigated that propionibacteria take over the sebaceous sites and Staphylococci mostly colonize moist areas of skin.[1-3]

Among the Staphylococcus spp., the species Staphylococcus epidermidis plays an important role in the skin microbiota. S. epidermidis is also known by synonyms: S. epidermidis albus, Micrococcus epidermidis, and Albococcus epidermidis as per taxonomy database and available at https://www.ncbi.nlm.nih.gov/Taxonomy.[4-6] The genome of S. epidermidis encodes protein using the genetic code translated as per translation Table 11.[7]

Bacteriocins are becoming more popular due to its ability to kill bacteria and also reported in maintaining the target population number by reducing them through Bacteriocin specific activity. Unlike antibiotics which are secondary metabolites, bacteriocins are ribosomally synthesized proteins/peptides and sensitive to proteases, also being harmless to humans and linked environment, which makes it better candidate to investigate in detail.[8-10]

In the present study, the interaction of bacteriocin produced by S. epidermidis on the Staphylococcus aureus has been investigated with reference to growth inhibitory action. Keeping in a view, mechanism of bacteriocins, its purification and identification studies has been carried out during the present investigations.

MATERIALS AND METHODSIsolation of S. epidermidisTo isolate the S. epidermidis from human skin samples, 10 subjects were taken into consideration and swabs were collected from hands and legs. The swabs were aseptically inoculated on the selective media mannitol salt agar and incubated at 37°C for 24 h to obtain

Exploring innovative approach to combat multiple drug resistance of Staphylococcus aureusAbhishek Mokase1, Arti Shanware1*, Archana Kulkarni2

Research Article

ABSTRACT

Aim: Bacteriocins are ribosomally synthesized antimicrobial peptides which are active against bacteria either of same or closely related species. The protection against the pathogen using bacteriocin was considered as an innovative approach, especially against the multidrug-resistant bacteria. Materials and Methods: Human skin sample has been swabbed and inoculated on Mannitol salt agar to isolate Staphylococcus epidermidis and later on they have been identified by biochemically and by 16S rRNA gene sequencing. Cell-free bacteriocin produced by the S. epidermidis has been checked against the multidrug-resistant Staphylococcus aureus by well diffusion assay. Result: S. epidermidis SE2 identified by 16S rRNA gene sequencing found to be producing the bacteriocin which is inhibiting multiple drug resistant S. aureus with the 13 mm zone of inhibition proving its medical potential. Conclusion: Human skin possesses S. epidermidis having bacteriocin producing ability and that can also be used to control multidrug-resistant S. aureus and proving its potential in medical biotechnology.

KEY WORDS: Antibacterial activity, Bacteriocin, Staphylococcus aureus, Staphylococcus epidermidis, Well diffusion assay

1Department of  Biotechnology, Rajiv Gandhi Biotechnology Center, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India, 2Department of Microbiology, Dharampeth M.P. Deo Memorial Science College, Nagpur, Maharashtra, India

*Corresponding author: Arti Shanware, Rajiv Gandhi Biotechnology Center, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur - 440033, Maharashtra, India. E-mail: artishanware@gmail.com

Received on: 20-10-2017; Revised on: 25-11-2017; Accepted on: 21-12-2017

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Website: jprsolutions.info ISSN: 0974-6943

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colonies. Further, isolate’s Gram staining, biochemical tests such as coagulase, esculinase, ornithine decarboxylase test, urease test, and hydrogen sulfide test, and motility was also carried out to confirm the identity of the S. epidermidis.

Selection of S. epidermidisThe isolates showing coagulase - negative, esculinase - negative, ornithine decarboxylase - negative, tryptophanase - negative, urease - positive, citrate - negative, hydrogen sulfide - positive, methyl red - negative, and Voges Proskauer - positive were identified as S. epidermidis[11-15]

Antibiotic Sensitivity Assay of S. aureusThe ability to record S. epidermidis bacteriocin-based inhibition has been checked against the standard strain S. aureus RG13. The strain RG13 has been first checked for the antibiotic sensitivity by well diffusion assay on Mueller-Hinton agar. Standard antibiotics with concentration as Chloramphenicol (30 µg), Amikacin (30 µg), Clindamycin (2 µg), Cefalothin (30 µg), Linezolid (30 µg), Minocycline (30 µg), Erythromycin (15 µg), Tetracycline (30 µg), Gentamicin (10 µg), Oxacillin (1 µg), Penicillin (10 µg), and Vancomycin (30 µg) has been used to record the sensitivity as per Clinical Laboratory Standards Institute (CLSI) chart.

Screening for Bacteriocin ProductionBased on prominent biochemical features, isolates were recognized as S. epidermidis and they were used to purify the bacteriocin in liquid culture medium. Inoculated 50 ml nutrient broth was kept in rotatory shaker for 24 h at 37°C. After incubation, developed growth has been made cell free by centrifugation and obtained suspension was stored in the refrigerator as a bacteriocins supernatant.

In the next step, standard strain S. aureus RG13 was grown to the optical density of 1 in a nutrient broth. In process, first in a Petri plate molten 15 ml of the nutrient agar was allowed to solidify. After ensuring the complete solidification, 100 µl of S. aureus was spread plated and allowed to absorb in medium. Later on using the sterile steel borer, wells were formed on agar, and then, the 100 µl of bacteriocin supernatant was introduced and plates were kept for complete absorption for 10–15 min. Thereafter, all plates were kept for incubation for 24 h at 37°C to record the zone of inhibition formed by S. epidermidis bacteriocin around the well along with the control taken as a supernatant of nutrient broth.

Novobiocin Sensitivity TestIsolated S. epidermidis strains were tested for the antibiotic sensitivity by checking its growth in the presence of novobiocin by a similar protocol mentioned earlier in section of antibiotic assay.

Identification of S. epidermidis by 16S rRNA sequencingWith bacteriocin production efficiency, the isolate was identified as per 16S rRNA gene sequencing using standard protocol [Tables 1-3].

RESULTSIsolation of S. epidermidisTotal 10 samples were inoculated on the mannitol salt agar, after incubation, all probable colonies of non-mannitol fermenting S. epidermidis appeared on the plates were recorded [Table 4]. The colonies were observed as small pink, raised, and cohesive about 1–2 mm without changing color of the medium was considered positive, i.e., S. epidermidis [Figure 1] and those having yellow colonies and changed the medium color to yellow from pink identified as S. aureus [Figure 1].

Table 1: PCR mixture set up

PCR reaction mixture

PCR components Volume (μl)Nuclease-free water 10.7510×reaction buffer with MgCl2 (1.5 mM)

2.00

dNTP mix (2.5 mM) 2.00Primer 16S F (10 picomoles/μl) 2.00Primer 16S R (10picomoles/μl) 2.00Taq DNA polymerase (5 U) 0.25Template DNA (50 ng/μl) 1.00Total volume 20.0PCR: Polymerase chain reaction

Table 2: Conditions of PCR set up

PCR temperature profileInitial denaturation 94°C for 2 minDenaturation 94°C for 50 sAnnealing 48°C for 30 s 30 cyclesExtension 72°C for 1 min 30 sFinal extension 72°C for 6 min

Figure 1: (a) Typical S. aureus appeared as pink color colonies without changing the medium color (pink). (b) Isolated Staphylococcus epidermidis fermenting mannitol with yellow color colonies changing the medium color (yellow)

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Gram StainingAll the typical and presumptive S. epidermidis colonies were appeared Gram-positive cocci as shown in Figure 2 and recorded as non-motile [Table 5].

Biochemical CharacterizationThe sampling was done from the surface of human skin, wherein a high density of S. epidermidis was initially explored. Keeping in consideration,

the scope of research in further studies, randomly selected 10 S. epidermidis strains named as SE 1-10 were characterized further for standard biochemical features and that also allowed further to select few of these strains up to genus and species level by 16S rRNA gene identification.

Table 3: 16S rRNA universal primers used in amplification

Oligonucleotide Universal primers

Oligonucleotide Sequences (5’‑ 3’) GC % Tm value Length Product size 16S FP AGA GTT TGA TCC TGG CTC AG 50 51.0°C 20 1500 bp16S RP AAG GAG GTG ATC CAG CCG CA 60 56.0°C 20

Table 4: Colony count for the presumptive S. epidermidis on the human skin surface developed on mannitol salt agar

Number of Colonies Human subjects

1 2 3 4 5 6 7 8 9 10

H L H L H L H L H L H L H L H L H L H LS. epidermidis 3 4 5 5 2 2 7 5 5 2 0 3 6 6 2 6 6 6 6 8Non typical 2 3 4 6 3 7 8 3 4 1 8 4 5 4 2 2 2 3 7 5H: Sampling of hands, L: Sampling of legs, +R Gram-positive rod, N: Non-motile

Table 5: Typical biochemical features along with morphology recorded for the 10 presumptive S. epidermidis SE1‑10

Tests Presumptive S. epidermidis SE1‑10

1 2 3 4 5 6 7 8 9 10Gram nature +R +R +R +R +R +R +R +R +R +RMotility − − − − − − − − − −Coagulase − − − − − − − − − −Esculinase − − + − + − − − − −Ornithine decarboxylase − − − − − − − − − +Tryptophanase − − − − + − − − − −Urease + + + + + + + + + +Citrate − − − + + + − − − −Hydrogen sulfide + + + + + + + + + +Methyl red − − − − − − − − − −Voges Proskauer + + + + + + + + + ++R: Gram-positive cocci, +: Positive, −: Negative;

Figure 2: Gram-positive cocci observed under oil immersion lens (×100) typical of Staphylococcus epidermidis

Figure 3: Staphylococcus epidermidis SE2 recorded to be sensitive for the antibiotic novobiocin

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Based on the biochemical features of said presumptive S. epidermidis isolates, it has been found that as per ABIS encyclopedia available at http://www.tgw1916.net/Staphylococcus/epidermidis.html coagulase negative

four isolates SE 1, SE 2, SE 8, and SE 9 were suspected to be featuring like S. epidermidis and were further tested fe bacteriocin production.

Antibiotic Sensitivity of Strain SE2 Against NovobiocinThe bacteriocin-producing S. epidermidis strain SE2 was found to be sensitive for the antibiotic novobiocin and further confirming it as S. epidermidis amongst other Coagulase Negetive Staphylococci. Based on the zone formed, it has been measured that the 12 mm of inhibition was recorded [Figure 3] after 24 h of incubation.

Antibiotic Sensitivity of S. aureus RG13As per CLSI chart, it has been recorded that pathogen strain S. aureus RG13 was resistant to half of the tested antibiotics, namely, Amikacin, Cefalothin, Erythromycin, Gentamicin, Oxacillin, and Penicillin. On the other hand, Clindamycin, Linezolid, Minocycline, Tetracycline, and Vancomycin found to be highly inhibiting the culture growth and strain remained sensitive to them. In contrast, only one antibiotic Chloramphenicol showed the pattern on intermediate inhibition. Overall, results suggested that given strain RG13 was multidrug-resistant, and hence, susceptibility of this strain against bacteriocin produced by S. epidermidis SE2 may prove to be beneficial [Table 6] and Figure 4.

Screening of Bacteriocin ProductionBiochemically determined S. epidermidis strains SE 1, SE 2, SE 8, and SE 9 when screened for the extracellular bacteriocin production under in vitro conditions; it has been evident that the isolate SE2 got inhibited with bacteriocin of S. epidermidis SE2 as observed with zone of inhibition (13 mm) formed around the well [Figure 5] [Table 7]. In comparison, SE 1, SE 8, and SE 9 found to be marginally inhibiting the S. aureus (<4 mm) which is not significant at all to consider the said isolates for further studies. In decision, strain SE2 was identified up to molecular level to confirm its species level.

Bacteria identification by 16S rRNA Identification bacteriocin-producing strain SE2 was identified by 16S rRNA gene sequencing. With the help of universal primers, successful targeting of the 16S rRNA gene locus was achieved when the PCR amplicon of 1500 base pairs has been resolved on the 1.2% gel along with molecular marker [Figure 6].

The amplicon when sequenced with the forward and reverse primers about 566 base pair sequence was obtained. Based on the BLASTN homology, it has been observed that this sequence showed defined homology with the S. epidermidis rRNA gene (Acc. No. LT571449.1)which confirms the identity of

Figure 4: Antibiogram of Staphylococcus aureus RG13 showcased both resistant and sensitivity pattern against antibiotics tested

Figure 5: Bacteriocin producing Staphylococcus epidermidis SE2

Figure 6: 16S rRNA gene amplicon of 1500 base pairs resolved on 1.2% agarose gel for the strain SE2 (Lane 1) along with molecular marker (Lane 2)

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SE2 strain as S. epidermidis [Figure 7]. Moreover, the phylogenetic analysis also depicted the close homology of SE2 with S. epidermidis (Acc. No. LT571449.1) [Figure 8] further confirms the species level identification.

DISCUSSIONIn the present study, human skin found to possess the bacteriocin-producing S. epidermidis isolates which have been confirmed to showcase antibacterial activity against the multidrug resistance S. aureus.

The study highlighted that S. aureus carrying multidrug resistance is getting inhibited by the strain S. epidermidis SE2 with minimum zone of inhibition of 13 mm, whereas other isolates found to be less effective.

As per the report, microbiome of human body plays an important role as commensal microbiota. Among them, S. epidermidis is predominant in human epithelia. S. epidermidis function in alliance with host and keeps the pathogens at bay by producing several antimicrobial compounds.[16]

In Brazilian, dairy herds about 188 coagulase-negative Staphylococcus strains were isolated, out of which six were identified as S. epidermidis produced a bacteriocin either identical or similar to aureocin A70 having antimicrobial activity.[17]

S. epidermidis accessory gene regulator (agr) quorum sensing system in controls the expression of surface proteins and exoproteins and also produces lantibiotic epidermin.[18] Not only staphylococcus species but also lactobacillus such as Lactococcus lactis produce Bacteriocin KU24 and found to be inhibitory to methicillin-resistant S. aureus. Bacteriocin KU24 got inactivated by protease XIV owing to its proteinaceous nature on S. aureus ATCC 33591. The cell membranes of S. aureus ATCC 33591 reported to be damaged with the treatment of 500 AU/mL of bacteriocin KU24 which confirms the potential of such bacteriocin in multidrug resistance.[19] The present study highlighted S. epidermidis strain SE2 also capable of producing the bacteriocin and capable of controlling the multiple drug resistance S. aureus effectively.

Table 6: Antibiotics sensitivity pattern of S. aureus RG13

Antibiotics Sensitivity patternAmikacin - (30 μg) ResistantCefalothin - (30 μg) ResistantGentamicin - (10 μg) ResistantErythromycin - (15 μg) ResistantOxacillin - (1 μg) ResistantPenicillin - (10 μg) ResistantClindamycin - (2 μg) SensitiveLinezolid - (30 μg) SensitiveMinocycline - (30 μg) SensitiveVancomycin - (30μg) SensitiveTetracycline - (30 μg) SensitiveChloramphenicol - (30 μg) IntermediateS. aureus: Staphylococcus aureus

Table 7: Activity of bacteriocin against S. aureus recorded in the supernatant of S. epidermidis SE 1,2,8,9

Strains SE1 SE2 SE8 SE9Zone of inhibition in millimeter

02±01 13±02 3±01 3.8±01

Figure 7: 16S rRNA gene (556 base pairs) recorded homology with the Staphylococcus epidermidis (Acc. No. LT571449.1) genome region encoding 16S rRNA

Figure 8: Phylogram of SE2 16S rRNA gene showcased best-scored homology with the Staphylococcus epidermidis (Acc. No. LT571449.1) 16S r RNA gene sequence

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CONCLUSIONIn this study, it has been noted that human skin possesses microbiota which has the ability to fight against the pathogens, and hence keeps the feature to maintain homeostasis. Identified S. epidermidis SE2 isolate found to produce bacteriocin which could be purified and may use in further research by identifying its exact nature which will be useful and recommended in medical biotechnology research. Not only is that bacteriocin successfully controlled the growth of human pathogen S. aureus which marks the success of the protein synthesized by S. epidermidis and put forward another bacteriocin to investigate in detail for medicinal therapies.

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