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Abstract

Traditionally, preservatives have been used in cosmetic products to minimize bacterial contamination. Some opportunistic Corynebacterium spp. have become resistant to these preservatives and other alternatives are required. A potential candidate is Conifer Green Needle Complex (CGNC), a pharmaceutical-grade complex substance from the green verdure of Pinus sylvestris and Picea abies with antibacterial, antimycotic, and antitrichomonal activity. The susceptibility of Corynebacterium xerosis and Corynebacterium flavescens to CGNC (3.5, 7, 15, 30, 60, 125, 250, and 500 mg/mL) was evaluated using broth dilution and agar methods. The antibacterial effect of CGNC was also evaluated after exposure for 30 minutes and 1, 3, and 24 hours at concentrations of 0, 3.5, 7, 30, 125, and 500 mg/mL. Corynebacteria xerosis was inhibited when exposed to low levels of CGNC (1560 mg/mL), whereas an antibacterial effect on C. flavescens was observed at slightly higher levels (60 and 125 mg/mL). CGNC also inhibited the growth of C. xerosis and C. flavescens at various incubation time points. The most prominent effect was observed after 24 hours where all growth was inhibited at all concentrations. However, CGNC inhibited or decreased the growth of Corynebacterium spp. even at lower exposure times. The results obtained in this study demonstrated that CGNC is an effective bactericidal agent against C. xerosis and C. flavescens isolated from clinical samples and may have potential as an alternative to preservatives currently used in cosmetic products.

Corynebacterium isolated from clinical specimens, with the exception of Corynebacterium diphtheriae, were considered contaminants, but recent studies have shown that some strains such as Corynebacterium xerosis are implicated in diseases. Corynebacterium xerosis has been found to cause problems in people who are immunocompromised and was isolated from people with endocarditis.^1

-4 Studies have demonstrated the presence of C. xerosis in wounds,^5,6 in breast abscesses,⁷ and prosthetic devices.^8,9 Further, this bacterium has affected people who have spent long periods in hospitals and those with conjunctivitis^8,9 or septicemia.^2,10,11 Indeed, non-diphtherial corynebacteria have been implicated in central venous catheter infections in pediatric oncology patients¹² and from catheter sites in other patients.⁶ Other coryneforms such as Corynebacterium flavescens have also been implicated in skin infections^13
-15 and, in older studies, in adults with a history of intravenous drug use.¹⁶

Antibiotic susceptibility studies have shown that corynebacteria, including C. xerosis, develop antibiotic resistance. Corynebacterium xerosis isolates from breast abscesses were shown to be resistant to antibiotics such as clindamycin.⁷ In older studies, C. xerosis was found to be resistant to clindamycin, erythromycin, azithromycin, ciprofloxacin,¹⁷ and gentamicin,^6,17 while other studies found erythromycin¹⁸ and penicillin^6,18 had poor antibacterial activity against this strain.

Plants and their parts have been shown to exhibit antimicrobial, antifungal, antiviral, and anti-inflammatory properties.^19

-22 Indeed, pine and spruce needle preparations have been used as treatments for colds and iron and vitamin deficiencies.²³ Interestingly, there have been studies investigating the antibacterial properties of natural substances in cosmetic products such as deoderants^24

-27 and mouthwashes.²⁸ Cosmetics products that are used repeatedly, such as face creams and mascara, may become contaminated by bacteria^29

-32 and preservatives used in these products may be toxic or cause hypersensitivity in people. In response to these issues, a push away from using synthetic chemicals as preservatives in cosmetic products has resulted in researchers investigating natural substances as alternatives.^33,34

Conifer Green Needle Complex (CGNC; also known as Bioeffective® A) is a complex substance obtained from green verdure of Pinus sylvestris and Picea abies (L) Karst. CGNC contains chlorophyll derivatives, carotenoids, vitamins A, E, and K, phytosterols, polyphenols, squalene, sodium salts of fatty and resin acids, and essential oils including natural antibiotics (phytoncides).³⁵

A component of CGNC, diterpene resin acids, has been shown to exhibit antimicrobial, antimycotic,^19,36,37 and antitrichomonal activities.^38,39 CGNC is a strong antioxidant³⁵ with antimicrobial properties, and it suppresses Helicobacter pylori in vitro and in vivo,^40

-43 Candida spp.³⁵ and Trichomonas vaginalis in vitro.^44,45

This study presents results demonstrating the antibacterial properties of CGNC on strains of C. xerosis and C. flavescens isolated from clinical samples obtained from patients.

The antibacterial effect of CGNC against С. xerosis and С. flavescens was evaluated using a broth dilution test and an agar method. When the broth dilution test was used, the minimal inhibitory concentration (MIC) for CGNC that inhibited the growth of C. xerosis ranged between 15 and 60 mg/mL, while the MIC that inhibited the growth of C. flavescens was 60 mg/mL.

In comparison, when the agar method was used, the MIC that inhibited the growth of C. xerosis ranged between 60 and 125 mg/mL, while the MIC that inhibited the growth of C. flavescens was 125 mg/mL.

The antibacterial effect of CGNC against С. xerosis and С. flavescens after various exposure times (30 minutes, 1, 3, and 24 hours) was also evaluated.

After 30 minutes exposure to 500 mg/mL of CGNC, the growth of all C. xerosis strains was completely inhibited. CGNC did not inhibit the growth of either C. xerosis (isolate 1) or C. xerosis (isolate 3) at 3.5, 7, 30, and 125 mg/mL, but there was a decrease in the number of colonies when compared with the control. These results were significant (P < 0.01). For C. xerosis (isolate 1) at the highest concentration (125 mg/mL), the decrease compared with the control was 0.6 log. For C. xerosis (isolate 3), the decrease was even greater, namely, 1.9 log. C. xerosis (isolate 2) was inhibited at 125 mg/mL and there was a decrease (P < 0.01) in the number of colonies at 3.5, 7, and 30 mg/mL. The greatest decrease (0.8 log) was observed at 30 mg/mL when compared with the control.

Exposure to CGNC for 30 minutes did not inhibit the growth of C. flavescens at any of the concentrations, although there was a decrease (P < 0.01) in the number of colonies when compared with the control. The greatest decrease (0.7 log) was observed at 500 mg/mL when compared with the control.

The growth of all C. xerosis strains was inhibited when the bacteria were exposed to 125 and 500 mg/mL CGNC for 1 hour. At the lower concentrations (3.5, 7, and 30 mg/mL), there was a decrease (P < 0.01) in the number of colonies when compared with the control. The decrease was between 1 and 2 log for the C. xerosis (isolate 1) and C. xerosis (isolate 3) compared with their controls. The decrease observed was slightly less (between 0.5 and 1.3 log) for C. xerosis (isolate 2) when compared with the control.

The growth of C. flavescens was inhibited at 500 mg/mL CGNC after 1 hour, and there was also a substantial decrease (1.6 log) in the number of C. flavescens colonies at 125 mg/mL CGNC when compared with the control.

After a 3-hour exposure to CGNC, the growth of all strains of C. xerosis and C. flavescens was completely inhibited at 125 and 500 mg/mL. Growth of 1 C. xerosis strain (isolate 3) was also inhibited at 30 mg/mL, and there was a reduction in colony numbers for this bacterium at 7 and 3.5 mg/mL when compared with the control. The decrease was 2.3 log at 7 mg/mL and 1.3 log at 3.5 mg/mL compared with the control. The growth of C. flavescens was not inhibited at the lower concentrations (3.5, 7 and 30 mg/mL), although there was a decrease in the number of colonies when compared with the control. The decrease was 0.8 log at 3.5 mg/mL, 0.9 log at 7 mg/mL, and 1.1 log at 30 mg/mL.

Exposure to CGNC for 24 hours resulted in the inhibition of growth at all concentrations (3.5, 7, 30, 125, and 500 mg/mL) for all strains of C. xerosis and C. flavescens.

Reducing bacterial contamination in commonly used cosmetic products without the addition of substances that confer bacterial resistance is an important goal as previous studies have shown that antibiotic resistance in Corynebacterium spp. is an ongoing concern.^26,31,32 Indeed, researchers have found that some Corynebacterium spp. exhibit simultaneous resistance to 3 groups of antibiotics.⁴⁶ Opportunistic Corynebacterium spp. showed resistance to macrolides, lincosamides, and streptogramins B (MLSB) and resistance to tetracyclines and fluoroquinolones.⁴⁷

In other studies, CGNC was shown to have antiprotozoal effects against T. vaginalis,⁴⁵ antibacterial effects against H. pylori, ^40

-43 and antimycotic activity against clinical strains of Candida albicans.³⁵ It was proposed that resin acids (including diterpene resin acids such as abietic and pimaric acids) found in CGNC may contribute to the antibacterial, antiprotozoal, and antimycotic activities exhibited by this substance.^35,45 As well as the ability to reduce bacterial contamination, CGNC offers the additional advantage of no observed toxicity in humans.⁴³ The results from these studies and those from the study described in this manuscript suggest that CGNC may potentially be used to provide an alternative to preservatives currently used in cosmetic products.

Experimental

Ethics

The ethics committee of The Pasteur Research Institute (Saint Petersburg, Russia) approved the experiments described in this manuscript. Experiments outlined in this manuscript also complied with the National Standard of the Russian Federation GOST R 33044-2014 Principles of Good Laboratory Practice (Order of the Federal Agency for Technical Regulation and Metrology of the 20 November 2014 No 1700 Article interstate standard GOST 33044–2014, enacted as a national standard of the Russian Federation on 1 August 2015). This standard is identical to the The OECD Principles of Good Laboratory Practice, No. 1, revised in 1997 and published in 1998.

Conifer Green Needle Complex

The preparation of CGNC has been previously described.³⁵

CGNC is a dark green, almost black, slightly alkaline (pH 8-9), viscous, aqueous-based paste comprising several hundred components. CGNC (as provided by Prenolica Limited, formerly Solagran Limited) is available in Australia, where it is approved by the TGA for use as a therapeutically active ingredient in listed oral and topical medicines.

The CGNC used in this study (Batch No. B140109 CHEMICAL (SPFTA), December 2009) was provided by Prenolica Limited and manufactured by Catalent (Australia).

Preparation of CGNC for the Microbiological Studies

CGNC was provided as a sterile substance and diluted in sterile, distilled water at 56°C. The concentration of the CGNC solution was 500 mg/mL. To test the susceptibility of bacteria to CGNC, the macrobroth dilution method (involving standard serial broth dilutions) and an agar dilution method were used in this study.

Microbiological Strains

Clinical strains of С. xerosis and C. flavescens were used in the experiments described in this manuscript. Сorynebacterium xerosis (isolate 1) was isolated in September 2008 from the blood of a patient with endocarditis. Сorynebacterium xerosis (isolate 2) was isolated in November 2008 from the conjunctiva of a patient with bacterial conjunctivitis. Сorynebacterium xerosis (isolate 3) was isolated in June 2009 from a punctate boil. Сorynebacterium flavescens was isolated in February 2009 from discharge from a patient with otitis media.

Susceptibility Testing of Corynebacterium spp. to CGNC—Broth Dilution Method

To test the susceptibility of Corynebacteria spp. to CGNC, serial dilutions of CGNC were made in test tubes containing Muller–Hinton broth and 5% defibrinated lamb blood. The final volume of the assay was 3.5 mg/mL.

The initial concentration of CGNC was 500 mg/mL. This solution was diluted (by double dilution) to provide working solutions with concentrations of 250, 125, 60, 30, 15, 7, and 3.5 mg/mL. The concentrations of CGNC used for testing the susceptibility of C. xerosis and C. flavescens were 3.5, 7, 30, 60, 125, 250, and 500 mg/mL.

The 3 clinical strains of C. xerosis and 1 strain of C. flavescens used in this study were isolated in a bacteriology laboratory at the Paster Medical Center (St Petersburg, Russia). The cultures were grown on horse blood agar and incubated at 36.6°C.

The inoculum density of each strain was standardized with a 0.5 McFarland turbidity standard. The suspension had a final inoculum of approximately 5 × 10⁵ colony-forming unit/mL. A negative control (inoculum, but no test substance) was also included. This procedure was carried out for each of the isolates.

One milliliter of the inoculum was added to the test tubes containing CGNC. Tubes containing C. xerosis was incubated at 35°C for 16-20 hours, while those containing C. flavescens were incubated for 20-24 hours. The negative control was incubated at 4°C. The experiment was carried out in triplicate.

The lowest concentration of CGNC that completely inhibited the visual growth of bacteria (no turbidity) was recorded as the MIC.

Susceptibility Testing of Corynebacterium spp. to CGNC—Agar Method

Serial dilutions of CGNC were made in test tubes; serial dilutions of CGNC were made in test tubes containing Muller–Hinton broth and 5% defibrinated lamb blood (as described in the section ‘Susceptibility Testing of Corynebacterium spp. to CGNC–Broth Dilution Method’).

CGNC was used at concentrations of 3.5, 15, 60, 125, 250, and 500 mg/mL. A test tube without CGNC was also included as a control.

Three strains of C. xerosis and 1 strain of C. flavescens were used in this experiment. A bacterial suspension (at a concentration of 1 × 10⁶) was made of each strain and this was distributed over the surface of an agar plate. Three microliters of each concentration of the test substance were then applied on the dried agar surface. The agar plates were placed in an incubator for 24 hours. The experiment was carried out in triplicate.

The lowest concentration of CGNC that completely inhibited the visual growth of bacteria on the surface of the agar was the MIC.

Susceptibility Testing of Corynebacterium spp. to CGNC Using a Range of Incubation Times

Dilutions of CGNC (0, 3.5, 7, 30, 125, and 500 mg/mL) were prepared in broth, and each tube was inoculated with 0.5 mL of bacterial suspension (C. xerosis and C. flavescens were prepared at a concentration of 1 × 10⁴). The bacteria were exposed to CGNC for 30 minutes, 1, 3, and 24 hours. A test tube containing no CGNC was also included as a control.

At the end of each time point, 3 µL of inoculum was spread on a nutrient agar plate and the plate was incubated for 48 hours. The number of colonies that grew at each dilution and time point was recorded.

Footnotes

Acknowledgments

The authors are grateful to Prenolica Ltd for providing the CGNC. We would also like to thank Dr Vicky Vallas for providing editorial support.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Dr Vagif Soultanov is an academic scientist involved in decades of research into substances from conifer needles in Russia. He is a director and shareholder of Prenolica Limited, which is the company that is commercializing the technology. Prenolica Limited supplied the CGNC used in the experiments described in this manuscript.

The remaining author declares that there is no conflict of interest in this research.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article. This study was performed at The Pasteur Insitute in Saint Petersburg, Russia, funded by the Russian Government’s Ministry of Healthcare and Social Development under general funding provided for the Institute. Prenolica Limited supplied the CGNC and provided funding to Dr Vallas for editorial support.

ORCID ID

Vagif S. Soultanov

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Antibacterial Activity of Conifer Green Needle Complex Against Corynebacteria

Abstract

Experimental

Ethics

Conifer Green Needle Complex

Preparation of CGNC for the Microbiological Studies

Microbiological Strains

Susceptibility Testing of Corynebacterium spp. to CGNC—Broth Dilution Method

Susceptibility Testing of Corynebacterium spp. to CGNC—Agar Method

Susceptibility Testing of Corynebacterium spp. to CGNC Using a Range of Incubation Times

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

ORCID ID

References