Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression

The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.


Results
The antibacterial and antifungal activity of ACV against E. coli, S. aureus and C. albicans. In order to determine the anti-microbial activity of ACV, E. coli, S. aureus and C. albicans were directly cultured with different concentrations of ACV. Figure 1 represents the experimental results. The minimum dose required to restrict growth for C. albicans was neat, undiluted ACV (5% acidity), for S. aureus it was a 1/2 dilution (2.5% acidity) and for E-coli, growth was restricted at a significantly lower dilution of 1/50 (equivalent to 0.1% acidity). We also measured the equivalent zones of inhibition (in mm) for each of the microbes at varying dilutions of ACV which is depicted in the photographs of the culture plates (Fig. 2). To translate the MIC into supplementary tablet dosages required, we tested concentration ranges from 400 µg/ml in doubling microdilutions to the lowest of 3.1 µg/ml against each microbe. The MIC for ACV tablets at which no growth was visible was 62 µg/ml for E. coli; 125 µg/ml for S. aureus and 250 µg/ml for C. albicans respectively. Both sets of results were also confirmed further by microdilution. We used the Braggs ACV for all future experiments at the minimum inhibitory dilution required for each organism.
Downregulation of pro-inflammatory cytokine secretion by ACV in monocytes exposed to microbes. The microbes utilised in our study have been extensively studied and are known to cause inflammation through their capacity to stimulate the leucocyte pro-inflammatory cytokine cascades 19,20 . Hence, we proceeded to measure mononuclear derived TNF-α and IL-6 cytokines as indicators of inflammation which are also the markers of choice for clinical diagnosis of septic infections 21 . Figure 3 depicts the effects of a dose dependent reduction in TNFα and IL-6 release from monocytes which have been co-cultured with ACV, together with either C. albicans, E-coli or S. aureus for 24 h.
Consistent with the microbial growth inhibition data depicted in Fig. 2, the effect of ACV at the minimum inhibitory concentration of 1/50 resulted in a significant reduction in monocyte derived TNFα (p = 0.008) and IL-6 release (P = 0.001) in monocytes cultured with E-coli. For S. aureus the minimum inhibitory concentration for ACV was found to be 1/10 in terms of reduction of TNFα (p = 0.011) and IL-6 (p = 0.03). For C.albicans the minimum inhibitory dilution was lower at 1/2 dilution, for TNFα (p = 0.003) and IL-6 (p = 0.008). It was imperative to ascertain whether the monocytes were alive during inoculation with the various microbes especially after incubation for 24 h at 37 °C. We added Trypan blue directly to monocytes which had been co-cultured with microbes after 2, 6 and 24 h. Light microscopy revealed that greater than 90% of cells were alive after 24 h in all co-cultures as demonstrated in (Fig. 4a,b,c) which represents the light microscopic images of monocytes and the microbes in co-cultures.
Upregulation of phagocytic capacity. We also investigated whether ACV could have an effect on the phagocytic function of monocytes alone and also after a 4 h exposure to microbes with or without ACV treatment. A 14.2, 13.7 and 20.4% increase in monocyte phagocytic capacity was observed after E. coli, S. aureus and C. albicans co-culture with ACV respectively and in comparison to the resting unstimulated monocytes. Results are expressed as the mean and SD of 3 similar experiments (Table 1). This suggests that ACV can increase phagocytic potential in monocytes which is significant as microbial phagocytosis is a key effector function of innate immunity 22 . Proteomic results of E. coli, S. aureus and C. albicans after exposure to ACV. The bottom-up proteomic study of ACV treated E-coli cultures revealed the absence in detection of key enzymes; citrate synthase, isocitrate dehydrogenase, deoxyribose-phosphate idolase, malate dehydrogenase, aminomethyltransferase and formate acetyltransferasesuccinyl-CoA ligase ( Table 2). An absence in acyl carrier protein, DNA protein including DNA protection during starvation protein, integration host factor subunit alpha and ribosome associated inhibitor A was evident. Following ACV treatment S. aureus cultures failed to express 50 s ribosomal proteins L2, L15, L23, L24, enzymes; alcohol dehydrogenase, catalase, formate acetyltransferase, L-lactate dehydrogenase-2, ornithine aminotransferase and serine hydroxymethyl transferase (Table 3). Cell division protein ftsZ and chaperone protein Dnak were also not detected. However an important pentose phosphate pathway enzyme: 6 phosphogluconate dehydrogenase decarboxylating was displayed. Table 4 demonstrates that key enzymes required for glycolysis and candida immunogenicity were undetected after 24 h of exposure to ACV in C. albicans. These incorporated fructose bisphosphate aldolase, phosphogluconate dehydrogenase, pyruvate kinase and peptidyl-propyl cis-trans isomerase.

Discussion
ACV has multiple antimicrobial properties on different microbial species, affecting microbe growth, suppressing mononuclear cytokine and phagocytic responses. The tandem mass spectroscopy results are in cohesion with The minimum inhibitory dilution of ACV required for significant pro-inflammatory downregulation varied with each microbe. For TNFα, a 1/50 ACV minimum inhibitory dilution was required for EC, p = 0.0008; 1/10 for SA, p = 0.01; 1/2 for CA, p = 0.0003 respectively. For IL-6, 1/50 ACV dilution was required for EC, p = 0.0008; 1/10 for SA, p = 0.03; 1/2 for CA, p = 0.008 respectively. Results represented are mean +/− SD of 3 experiments. We used student's paired t-tests for statistical evaluation (Excel 2017) with statistical significance taken when p < 0.05. EC = E-coli, SA = S. aureus, CA = C. albicans, ACV = Apple cider vinegar.
ScieNTific RepoRTS | (2018) 8:1732 | DOI:10.1038/s41598-017-18618-x these observations. The microbes underwent significant impairment following ACV addition which damaged cell integrity, structural and metabolic proteins as well as nuclear material. Indeed the enzymes citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, aminomethyltransferase and formate acetyltransferasesuccinyl-CoA ligase are crucial for E-coli growth, gene regulation and central, intracellular carbon metabolism. An absence of these enzymes would affect glycolytic, tricarboxylic acid cycle, pentose phosphate, glycoxlate shunt and oxidative phosphorylation pathways in E. coli 23 . Furthermore, the absence of DNA binding protein from starved cells, is significant as it protects E-coli and functions to control gene regulation during cell starvation 24 . With respect to other proteins, we observed the presence of ribosomal proteins (L1, L6 and 30 s ribosomal proteins (S1, S4, S6,   Table 1. Effect of ACV on human monocyte phagocytic capacity. In vitro differentiated monocytes were incubated with microbes for 4 h at 37 °C. Cells cultured with microbes with or without ACV were washed and processed for detection on the Beckton Dickinson flow cytometer. An analysis of changes in regional gated profiles and % shift in side scatter was measured. Data is presented as mean ± SD of 3 similar experiments.  Table 3. List of S. aureus proteins identified following ACV treatment. S. aureus were cultured with 1/10 dilution of ACV or alone in broth for 24 hours at 37 °C in a shaking incubator. After which mass spectroscopy analysis was carried out. The "*" indicates the presence of protein whilst the blank region denotes no detection of that particular protein.  25 . Collectively these results support the cytotoxic effects of ACV we observed on E-coli. ACV treated S. aureus cultures did not express the chaperone protein Dnak and the cell division protein ftsz. This is significant as previous studies have shown that a non-functional DnaK system can cause a reduced tolerance to heat, oxidative, antibiotic stresses and lowered carotenoid production 26 . There was also an absence of gateway enzymes involved in multiple pathways such as ornithine aminotransferase 27 . 6 phosphogluconate dehydrogenase decarboxylating was expressed which is not surprising as it plays a critical role in protecting cells from oxidative stress 28 . The effect of ACV on C.albican protein moieties was less dramatic, nevertheless we did detect the absence of key enzymes which are fundamental in maintaining cell integrity and biosynthetic pathways 29 . There could be a strong possibility that ACV acts like other anti-pathogenic compounds in diverting monocyte responses through toll receptor signalling pathways. There is evidence that E-coli in particular can induce a typical M1 monocyte profile through mechanisms involving NF-kB activation. This results in upregulation of inflammatory cytokines TNF-α and PI3 kinase stimulation 30 . The in vitro M1 monocyte phenotype is also prominent in severe sepsis. This was shown in a study using baboons where a substantial mortality rate correlated with high serum levels of TNFα and IL-6 following induced sepsis infection 31 . Unchecked high levels of circulating M1 cytokines can rapidly lead to cardiac arrest and death hence any factor capable of lowering pro-inflammatory cytokine concentrations is essential in therapy 32 . It has been reported that ACV consists of acetic acid, flavonoids such as gallic acid, tyrosol catechin, epicatechin, benzoic acid, vaninilin, caftaric acid, coutaric acid, caffeic acid, acid and ferrulic acid These constituents have been reported to affect immune defence and oxidative responses 18,33 . Furthermore, the mechanism of ACV activity could be attributed in part to the apple polyphenol content. Yang et al. (2010) reported on the cellular protective effects of apple polyphenols on induced liver damage whereby histopathological tissue destruction was limited and liver activity maintained in mice that received the polyphenols 34 . The mechanisms involved were free radical scavenger action, lipid peroxidation modulation and the antioxidant upregulation capacity of ACV. Interestingly, a study by Denis et al. demonstrated the anti-inflammatory potential of apple phenols on gastrointestinal cell inflammation which involved downregulation of TNF and IL-6 cytokines 35 . Another means of action could involve the acetic acid component of ACV which is able to reduce the cell hydrogen potential hence could potentially facilitate diffusion across the plasma membrane of microbes. Furthermore, there is evidence that organic acids can alter immune responses by binding to GPR3, a G protein coupled receptor which is mostly expressed on inflammatory leukocytes 36 . Also, an investigation reported on upregulated blood and plasma antioxidant enzyme release after apple consumption which would encourage immune protection 16 .
The positive benefits of dietary ACV supplementation have been highlighted in vivo. ACV decreased the serum lipid profile in mice fed a high cholesterol diet over 28 days. Intragastric ACV addition induced a protective effect against erythrocyte, kidney and liver oxidative injury as well as lowering cholesterol levels 18 Table 4. List of C. albicans proteins identified following ACV treatment. C. albicans were cultured with 1/2 dilution of ACV or alone in broth for 24 hours at 37 °C in a shaking incubator. After which mass spectroscopy analysis was carried out. The "*" indicates the presence of protein whilst the blank region denotes no detection of that particular protein.
ScieNTific RepoRTS | (2018) 8:1732 | DOI:10.1038/s41598-017-18618-x also decreased blood triglyceride and very low density lipoprotein levels in rats which had induced cholesterol induced hepatic steatosis 33 . In an infection induced model of denture stomatitis, ACV addition resulted in anti-fungal activity against Candida Spp which was comparable to nystatin in terms of reducing microbial adherence and destruction 37 . Severe infections, autoimmunity or transplantation can inevitably lead to ineffective immunity in patients. An analysis of macrophages from Crohn's disease patients revealed that they had defective responses to E-coli due to Crohn's related systemic immunosuppression 38 . A recent report showed that co-administration of ACV with L-casei boosted systemic and mucosal immune responses, antioxidant enzyme and growth genes in fish 39 . Equally, perhaps additive dietary supplementation with ACV could be of benefit in acute infections, autoimmune induced immune dysregulation or antibiotic redundancy in humans. Future studies would establish whether ACV could be used as a potential therapeutic, In vivo models of infection could be induced by infusing microbes systemically into mice followed by treatment with or without intraperitoneal ACV. Intragastric ACV has been fed to animals used as models of obesity and infection in the past 18,37,39 . ACV efficacy could be evaluated by measuring microbial burden, serum cytokine levels, leukocyte counts and tissue pathology. Side effects could include acid reflux, nausea or delayed digestion as ACV has a pH of 4.2. However, the acidity could be neutralised by the addition of sodium bicarbonate to preparations. The results of this study could have clinical implications as ACV could be used as an additive component of an antimicrobial therapeutic regimen especially in immunocompromised patients presenting with infections of the aforementioned microbes.
We conclude that ACV can have multiple antimicrobial effects directly on E-coli, S. aureus and C. albicans. ACV addition can also decrease induced inflammatory cytokine release during mononuclear leukocyte infection and increases monocyte phagocytic capacity. Mechanisms include alteration of the microbial protein physiology destroying structural pathogenic proteins and metabolic enzymes. Collectively our results highlight the potent antimicrobial and therefore beneficial actions of ACV. This preliminary study encourages further work on dietary ACV supplementation investigating its antimicrobial role and the constituents which could be responsible for this activity.

Materials and Methods
Chemical reagents, microorganisms, media and culture conditions. A selection of microbial specimens which represented a typical gram positive, a gram negative and a yeast species were chosen for initial investigation. Microbial strains: E-coli strain NCTC 10418 and S. aureus strains NCTC 6571 were purchased from Health Protection Agency (Colindale, U.K.). C. albicans strain 90828 was purchased from the American Type Culture Collection (LGC Promochem).

Inoculum preparation and measurement of anti-microbial activity of ACV. Cultures of E. coli and
S. aureus were grown in nutrient media whereas C. albicans was grown in Sabourand media. All cultures were cultivated in a shaking incubator at 37 °C for 24 h overnight prior to use. Mueller hinton agar (MHA) was prepared by dissolving 38 g in 1 litre of distilled water, boiling the mixture for 1 min, after cooling and autoclaving, the media was poured into petri dishes. The plates were left to dry and subsequently stored at 37 °C. All microbial cultures were adjusted to 0.5 McFarland's standard 1.5 × 10 8 CFU/ml and 4 × 10 6 CFU/ml of each organism used in experiments. Each microbe was swabbed evenly onto plates containing MHA. For sample addition, 100 µL of ACV at varying concentrations was added to the wells which were punched into the agar. The plates were then incubated at 37 °C for 24 h. Zones of inhibition surrounding samples were identified, photographed and measured in mm 40 . Experiments were repeated at least 5 times.
Ethical Approval and Informed Consent. All experimental protocols were approved by the Middlesex University Natural Sciences Ethics Committee number 2323. Further the methods were carried out in accordance to the relevant guidelines and regulations. Informed consent was received when applicable.
Human mononuclear cell isolation procedure from whole peripheral blood. Human leucocyte rich cones and serum were obtained from volunteer donors collected from NHS Cord blood and transplant bank at Colindale, London and treated as described previously 41 . Briefly the cones were washed with phosphate buffer saline to harvest the leucocyte rich cells. These were then spun on histopaque density gradient at 1200 RPM for 20 min. Monocytes were purified using the CD14 positive mononuclear portion which was isolated according to manufacturer's instruction provided with the pan monocyte isolation kit. Cells washed with HANKS balanced solution, counted and cultured into 24 well plates at 4 × 10 5 cells per mL. Cells were allowed to adhere for an hour at after which non-adherent cells were washed away and full media replenished with Dulbecco's media containing 10% human serum. Monocytes were determined using light microscopy and flow cytometry phenotypic analysis of differentiation markers as described previously using CD14 41 . Freshly isolated monocytes were cultured with varying concentrations of ACV and either C. albicans, E. coli or S. aureus at counts of 4 × 10 6 CFU/ml respectively ScieNTific RepoRTS | (2018) 8:1732 | DOI:10.1038/s41598-017-18618-x for 24 h at 37 °C and 5% CO 2 after which supernatants were collected and analysed for TNF-α or IL-6 secretion using ELISA kits following manufacturer's protocols.

Monocyte phagocytic capacity measurement by flow cytometry. Isolated human mononuclear
cells were cultured at 4 × 10 5 /mL in 24 well plates over a period of two days after which they were incubated with microbes (4 × 10 6 CFU/ml) for 4 h at 37 °C and 5 CO 2 . Cells were then scraped replenished in ice cold PBS containing 1 mM EDTA, washed and removed from plates. The resultant pellets were fixed in 400 µL of 4% paraformaldehyde and analysed using a FACS Calibur flow cytometer (Beckton Dickinson Immunocytometry Systems, UK and Cell Quest software).
Preparation of microbial tryptic digests for mass spectroscopy analysis. An aliquot of microbial suspension was collected and treated over 24 h with 1/100 ACV, washed with PBS, resuspended in PBS and then treated with 1 mL of ice-cold acetone. The bacterial cells were harvested after centrifugation 13,000 g for 5 min. The pellet was dried and then reconstituted in 50 mM ammonium bicarbonate. Cells were lysed using a Soniprep 150 Plus (MSE, U.K.) for 10 s with the amplitude set at 13. Subsequently proteins were denatured and reduced with 3 μL of 100 mM dithiothreitol in 50 mM ammonium bicarbonate at 95 °C for 5 min followed by alkylation with 6 μL of 100 mM iodoacetamide in the dark at room temperature for 20 min. Proteins were then digested with 2 μL of trypsin (0.1 μg/μL dissolved in 50 mM ammonium bicarbonate) at 37 °C for 3 h. A further 2 μL of trypsin was added to the sample and the mixture was incubated at 37 °C for an additional 2 h. The samples were diluted in 150 µL 50 mM ammonium bicarbonate and passed through Mini-Uni Prep filter devices.
Liquid Chromatography-Electrospray Ionisation Tandem Mass Spectrometry. The tryptic peptides were analysed using a Shimadzu Prominence HPLC system hyphenated to an electrospray ionisation hybrid ion-trap time-of-flight (IT-TOF) mass spectrometer (Shimadzu, U.K.) operated in tandem mass spectrometry mode. Peptides were separated using an Ascentis Express 150 × 2.1 mm, 2.7 µm C18 column (Sigma-Aldrich, Poole, U.K.) using a flow rate of 0.21 mL/min. The column oven temperature was set to 40 °C. Data was acquired and processed using LabSolutions ® software (version 3.50.348, Shimadzu, UK). A linear gradient elution profile composed of ' A'−0.1% formic acid in water and 'B'-0.1% formic acid in acetonitrile was used. The gradient profile was 0-40% B, 70 min; 40-90% B, 1 min; maintained at 90% B, for 3 min; 90-0% B, 1 min; and 15 min re-equilibration at 0% B. An injection volume of 40 µL was used. Samples were kept in the auto sampler set to 4 °C. Mass spectrometry analysis was performed in MS/MS mode using positive ions electrospray. The precursors' acquisition range was set to 400-1,800 m/z while the fragments acquisition range was set to 200-1,500 m/z. For both precursors and fragments the ion accumulation time was set to 30 msec. The other instrument conditions were set as follows: detector voltage 1.6 kV, CID energy 70%, nebulising gas flow 1.5 L/min, CDL temperature 200 °C, heat block temperature 200 °C, interface voltage 4.5 V, detector voltage 2 kV. The data acquisition was performed in a 37.5 min interval.

LC-MS/MS Data Processing. MS/MS data were extracted from the resulting instrument files using Mascot
Distiller software (version 2.5.1.0, Matrix Science, London, UK). For precursors peak picking the following parameters were used: correlation threshold -0.7, minimum signal to noise ratio -5, minimum peak m/z -50, maximum peak m/z -100,000, minimum peak width -0.02 Da, expected peak width -0.2 Da, maximum peak width -2 Da. The MS/MS ion list was searched using Mascot search engine against all entries in Swiss-Prot database (2016_2). For database search the following parameters were used: two missed cleavages, carbamidomethylation of cysteine (as fixed modification) and oxidation of methionine (as variable modification). The tolerance for precursor peptides was set to 10 ppm and for fragments to 0.3 Da. Peptide charges used for peak picking was +2, +3 and +4. Statistical analysis. All experimental results are expressed as the mean ± standard deviation (SD). Statistical analyses was carried out using one way ANOVA or students t-test, outcomes were considered significant where p < 0.05 (when comparing apple cider vinegar treated microbes to the untreated groups in all experiments). All experiments were repeated at least 3-5 times. Analysis was carried out using Excel software version 2016. Data availability. The datasets generated and analyzed during the current study reside with the corresponding author and can be made available upon request.