Assessing the Susceptibilty of Some Gut Bacteria to the Extract from Needles of Turkish Pine

Plant extracts have the potential to be safe alternatives to antibiotics that disrupt the gut flora. The aim of the present study was to assess the susceptibility of some gut bacteria to the extract from needles of Turkish pine (Pinus brutia Ten.) using microdilution method in an anaerobic chamber. Turkish pine needle extract promoted the growth of Bifidobacterium bifidum, Bifidobacterium infantis, and Lactobacillus acidophilus from gut commensals at 0.2-6.25 mg/mL, 0.4-6.25 mg/mL, and 0.4-1.6 mg/mL dose ranges, respectively (P<0.05). However, the extract had a potential inhibitory activity on Bifidobacterium species starting from 12.5 mg/mL, on L. acidophilus starting from 6.25 mg/mL, and on L. casei starting from 3.13 mg/mL concentrations (P<0.05). Minimal inhibitory concentration (MIC) was 25 mg/mL for all commensal species (P<0.05). Turkish pine needle extract also showed a potential inhibitory activity against gut pathogens Escherichia coli and Clostridium perfringens from 0.4 mg/mL dose and against Staphylococcus aureus and Fusobacterium nucleatum from 0.8 mg/mL dose (P<0.05). The MICs were 6.25, 12.5, 25, and 50 mg/mL for S. aureus, F. nucleatum, E. coli, and C. perfringens, respectively (P<0.05). It was concluded that using the Turkish pine needle extract in a dose range of 0.2-6.25 mg/mL, where it protected most of the commensal bacteria and was toxic against some of the pathogens, might produce desirable impacts in the gut.


Introduction
The gut flora is a large and dynamic bacterial community that participates in normal physiological functions, but also protects against pathogens by forming a defensive barrier and competing for available substrates (Ahn et al., 1998;Canny and McCormick, 2008). Balance between commensal and pathogenic species has great importance in terms of maintaining the gut health. Conventional antibiotics can prevent the growth of both commensal and pathogenic species and decrease diversity of the gut flora (Bäumler and Sperandio, 2016). In recent years, many studies have been focused on plant extracts as natural alternatives for antibiotics.
Turkish pine or Turkish red pine (Pinus brutia Ten.) is the most common pine species in Turkey which has ability to grow on a wide range of Mediterranean-and Black Sea regions (Balaban Ucar et al., 2013). The use of Turkish pine in the forest products industry has been widely accepted because of its suitability for the manufacture of desirable products (Üner et al., 2011). In a previous study, we observed that the extract from barks of Turkish pine, which containing phenolic compounds, had a potential to inhibit pathogenic bacteria in the gut while protect commensal ones (Demirtaş, 2020). Furthermore, Pinus densiflora (Japanese pine) leaf derived components, (1R)-(+)-α-pinene and limonene, strongly inhibited the growth of Staphylococcus aureus, Escherichia coli, and Clostridium perfringens without adverse effects on the growth of five commensal bacteria (Bifidobacterium bifidum, B. longum, B. adolescentis, Lactobacillus acidophilus, and L. casei) (Hwang and Lee, 2002). The needle of Turkish pine also contains several flavonoids (Kaundun et al., 1997) and essential oil components (Yener et al., 2014) with antioxidant and antibacterial capacity. However, the effects of extract from Turkish pine needle on gut bacteria have not been evaluated previously. Therefore, the aim of the present study was to assess the susceptibility of some gut bacteria to the extract from needles of Turkish pine.

Antibacterial screening
The effect of Turkish pine needle extract on the growth of gut bacteria was tested by a broth dilution method in the anaerobic chamber (CLSI, 2016). A stock solution was prepared by dissolving pine needle extract in 50% ethanol. Ten serial dilutions of the extract starting at a concentration of 50 mg/mL were prepared from the stock solution in the bacterial strain specific growth media. Two hundred microliters of each dilution were added to wells of a 96-well plate. Next, 20 μL of the test bacteria suspension was inoculated into each well. Each bacterium was tested in triplicate wells. Plates were incubated for 24 h at 37°C in the anaerobic chamber. Bacterial growth was detected with a microplate reader at 600 nm (Epoch, BioTek, USA). The minimal inhibitory concentration (MIC) is defined as the lowest concentration of added extract at which no significant bacterial cell growth was observed. A significantly lower OD600 value compared to control dose (0 mg/mL) was accepted as potential inhibitory activity (Ko et al., 2018) while significantly higher value was accepted as stimulatory effect (Das et al., 2015).

Statistical analyses
Statistical analysis was carried out by the use of one-way ANOVA followed by Dunnett's test. Each well of a 96-well plate was an experimental unit. A probability value at P<0.05 was considered statistically significant.

Results
Effects of Turkish pine needle extract on gut bacteria are showed in Figure 1 and Figure 2. Turkish pine needle extract promoted the growth of B. bifidum, B. infantis, and L. acidophilus from gut commensals at 0.2-6.25 mg/mL, 0.4-6.25 mg/mL, and 0.4-1.6 mg/mL dose ranges, respectively (P<0.05). That effect was more obvious for B. infantis. However, the extract had a potential inhibitory activity on Bifidobacterium species starting from 12.5 mg/mL, on L. acidophilus starting from 6.25 mg/mL, and on L. casei starting from 3.13 mg/mL concentrations (P<0.05). The MIC was 25 mg/mL for all commensal species (P<0.05) (

Discussion
The presence of a diverse and balanced bacterial community in the gut is of great importance for host physiology. Disruption of commensal flora in the gut is one of the major complications encountered in the treatment of infections with antibiotics (Bäumler and Sperandio, 2016). Accordingly, to determine the safe dose range of therapeutic agents that protects commensal bacteria while suppressing pathogens has great importance in terms of gut health. Turkish pine needle extract at low concentrations stimulated the growth of B. bifidum, B. infantis, and L. acidophilus from commensals, more prominently for B. bifidum. However, this stimulatory effect turned into a potential inhibitory effect on Bifidobacterium species starting from 12.5 mg/mL and on L. acidophilus starting from 6.25 mg/mL concentrations. The extract completely inhibited all commensal species at 25 mg/mL. Although there is no literature on the effects of Turkish pine needle extract on commensal gut bacteria, it was reported that low doses of several plant metabolites could stimulate bacterial growth in the gastrointestinal tract while high doses induced inhibition (Patra et al., 2012;Demirtas et al., 2019;Goker and Demirtas, 2020). Aldehydes, one of the plant secondary metabolites from the green leaf volatiles family, moderately promote the growth of L. acidophilus and B. bifidum at lower concentrations while had inhibitory effects at higher concentrations in a previous study (Goker and Demirtas, 2020). Trans-2-decenal, also an aldehyde from green leaf volatiles, stimulated the growth of Fibrobacter succinogenes, which is a fibrolytic bacterium from the rumen, at low doses Escherichia coli and S. aureus are common foodborne pathogens that can cause severe gastro-intestinal illness (Ørskov and Ørskov, 1992;Rajkovic, 2014 et al., 2012). The difference in MIC values is probably due to the difference in bacterial strains and also due to active ingredients in needles of the pine trees from the different origins. The dominant flavonoids found in the needles of P. brutia were reported as quercetin (41%), kaempferol (29%), and isorhamnetin (%23) (Kaundun et al., 1997) while the main essential oil component was reported as β-pinene (Yener et al., 2014). In this study, one or more of these active ingredients, that were likely to be contained in the extract, might be responsible for the antibacterial effects.
Fusobacterium nucleatum, which is obviously associated with colorectal cancer (Shang and Liu, 2018), was more sensitive to Turkish pine needle extract than C. perfringens in the present study. Extract from the barks of P. brutia also had an inhibitory potential on this bacterium from 150 μg/mL concentration in a previous study (Demirtas, 2020). On the other hand, C.
perfringens, which is generally linked to gastrointestinal symptoms such as vomiting and diarrhea (Keeratirathawat et al., 2013), was the most resistant species to the used extract in this study. Turkish pine needle extract inhibited the growth of this bacterium at 50 mg/mL. Keeratirathawat et al. (2013) also reported that oils from the needles of four different Pinus species (Pinus radiata, P. pinaster, P. sylvestris, and P. nigra) did not exhibit any antibacterial activity against C. perfringens.
It was concluded that using the Turkish pine needle extract in a dose range of 0.2-6.25 mg/mL, where it protected most of the commensal bacteria and was toxic against some of the pathogens, might produce desirable impacts in the gut. Further in vitro and in vivo studies required to clarify its beneficial effects on the gut health. Demirtaş, A., 2020. Influence of Pinus brutia bark extract containing phenolic compounds on some commensal and pathogenic bacteria from the intestinal