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UNIVERSITY OF BELGRADE FACULTY OF BIOLOGY

Microtitre plate-based antibacterial assay with resazurin for detection of antibacterial agents Master thesis

					Student

Salahaldin Abdulkarim Omar Alfurjany

Belgrade, Serbia, 2016

Acknowledgement

This work was done at Chair of Microbiology, University of Belgrade - Faculty of Biology, Serbia.Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.Nutrient media In this study the following nutrient media were used: o MHB - Mueller-Hinton Broth: MHB powder (21 g), distilled water (1000 ml); o MHA - Mueller-Hinton Agar: MHB powder (21g), Agar "Difco" (15g), distilled water (1000ml); o BHI - Brain-Heart Infusion Broth: BHI powder (37g) , distilled water (1000 ml); o BHA - Brain-Heart Infusion Agar: BHI powder (37g) , Agar "Difco" (15g), distilled water (1000 ml); Nutrient media were sterilized in an autoclave for 20 minutes at a temperature of 121?C and a pressure of 101.3 kPa. 5.1.2. Antibiotics and other reagents used in the MIC assay o Streptomicin (Str): antibiotic solution in MHB; concentration of working stock 500 ug/ml o Rifampicin (Rif): antibiotic solution in BHI, concentration of working stock 200 ug/ml o Dimethyl sulfoxide (DMSO): solvent, used for EO o Resazurin Sodium Salt: distilled water solution, concentration of working stock 0,675 mg/mL 5.1.3. Bacterial strains The following bacterial strains were used to determine antibacterial activity of the EO and PDW: Gram - positive bacteria: o Staphylococcus aureus ATCC25923 o Listeria innocua ATCC33090 Gram - negative bacteria: o Salmonella enteritidis ATCC13076 o Pseudomonas aeruginosa ATCC15442

BACTERIAL RESISTANCE TO ANTIBIOTICS Resistence mechanism Antibiotic example Genetic basis of resistance Mechanism presented in Reduced permeability Penicillin Chromosomal Salmonella enteritidis Pseudomonas aeruginosa Chloramphenicol Erytrommycin Lincomycin Plasmid Listerya innocua Inactivation of antibiotic Penicillin Plasmid and chromosomal Staphylococcus aureus Salmonella enteritidis Chloramphenicol Plasmid and chromosomal Staphylococcus aureus Salmonella enteritidis Alteration of target Eritromycin Rifamycin Streptomycin Norfloxacin Chromosomal Staphylococcus aureus Salmonella enteritidis Development of resistant biochemical pathaway Sulfonamides Chromosomal Staphylococcus aureus Salmonella enteritidis Efflux(pumping out of cell) Tetracycline Chloramphenicol Plasmid Chromosomal Salmonella enteritidis Staphylococcus aureus

Table 1.As advised, these guidelines provide a uniform procedure for testing that is practical to perform in most clinical microbiology laboratories and bioassay to be performed in a standardized approach in order to evaluate the clinical relevance of results Nevertheless if dilution is prepared in agar or in broth, the range of antibiotic concentrations used for determining MICs is universally accepted to be in doubling dilution steps up and down from 1 mg/mL, as required (Balouiri, 2016).Studying of biological properties indicates that Juniperus species are endowed with numerous activities including antioxidant, antiseptic, diuretic, anticancer, antirheumatic, antihelmintic, anti-inflammatory, immunomodulatory, analgesic, antituberculotic and abortifacient activities (Glisic et al. 2007; Orphan et al., 2011; Swanston-Flatt et al. 1990).MIC and MBC values* Bacterial strains EO PDW Str Rif MIC MBC MIC MBC MIC MBC MIC MBC Staphylococcus aureus 6.25 12.5 3.125 6.25 12.5 25 nt nt Listeria innocua 6.25 6.25 1.56 1.56 nt nt / 0.19 Salmonella enteritidis 50 50 6.25 6.25 3.125 6.25 nt nt Pseudomonas aeruginosa / / 12.5 25 25 50 nt nt / - not determined in used concentration range nt - not tested *MIC and MBC in mg/mL for EO and PDW, and in ug/mL for Str and Rif.In such environments the multi-drug resistant pathogens appear, making huge difficulties in curing of infections caused by them (Figure 3.) The mechanisms of bacterial resistance to antibiotics include reduced permeability, inactivation of antibiotics, alteration of target, development of specific biochemical pathways, as well as the efflux of antibiotics (Table 1).Interestingly, although the antibacterial effect is not high, the importance is the fact that Juniperus communis extracts possess antibacterial effect against important patogenic bacteria Mycobacterium tuberculosis, a causative agent of tuberculosis disesase (Carpenter et al., 2012; Jimenez-Arellanes et al., 2003).In vitro methods for antimicrobial evaluation Two parameters are important to determine by antimicrobial susceptibility assays - minimal inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC).The interpretative standards for this method are published by different national organizations such as the Clinical and Laboratory Standards Institute (CLSI) in the USA and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (Wiegand et al. 2008).Evaluate and quantitatively determine the antibacterial potential of J. communis essential oil and post-distillation waste against Staphylococcus aureus, Listeria innocua, Salmonella enteritidis and Pseudomonas aeruginosa.RESULTS AND DISCUSSION 21 CONCLUSIONS 25 REFERENCES 26

INTRODUCTION High mortality and morbidity worldwide, especially in developing countries, are frequently caused by microbial infections that occur as a result of poor sanitation, unhygienic and overcrowded living conditions.One variant of diffusion method is followed by placing of filter paper discs (about 6 mm in diameter), containing the test compound at a desired concentration (ug/disk), on the agar surface (Balouiri, 2016).The principal disadvantages of the macrodilution method are the tedious, manual task of preparing the antibiotic solutions for each test, the possibility of errors in preparation of the antibiotic solutions, and the relatively large amount of reagents and space required for each test (Jorgensen, 2009).Although agar dilution method is significantly more expensive and laborious to perform, it is especially suitable for testing the compounds (or extracts) which masks the detection of microbial growth in the liquid medium by their coloring (Balouiri, 2016).Essential oils (EOs) of many aromatic plants, such as Origanum vulgare, Thumus capitatus and Ocimum basilicum, are proved as potent antibacterial agents (Dzamic et al., 2015; Sokovic et al., 2010).If colonization of P. aeruginosa occurs in critical body organs, such as lungs, urinary tract and kidneys, the results could be fatal (https://en.wikipedia.org/wiki/Pseudomonas aeruginosa).Low antibacterial effect of J. communis derivatives is in line with previously published data, indicating moderate and low antibacterial effect of Juniperus species (Andogan et al., 2002; Angioni et al., 2003; Glisic et al., 2007; Karaman et al., 2003; Nikolic et al, 2016; Lesjak et al., 2014).Nevertheless, an approximate MIC can be calculated for some microorganisms and antibiotics by comparing the inhibition zones with stored algorithms (Jorgensen and Ferraro, 2009).Microtitre plate-based antibacterial assay incorporating resazurin (Figure 2.) is a method that is generally accepted for application in the in vitro antibacterial screening of different antibiotics and phytochemicals (Sarker et al., 2007).Taking into account the widespread occurrence of this genus, as well as the richness in the active substances, it is not surprising to find extensive use of EO and differently prepared extracts of many Juniperus species (Adams, 2014).Furthermore, study of Khan et al. (2012) provides a pharmacological basis for traditional use of Juniperus excelsa against respiratory disorders and gut hyperactivity, such as asthma, diarrhoea and colic.For that purpose, additional plating of suspension from all the wells without visible growth onto the corresponding agar medium (BHA for L. innocua, and MHA for all the rest bacteria) was performed.Judged by the dominant constituents of EO, obtained antibacterial effect could be at least partially attributed to ?-pinene and sabinene (Arunkumar et al., 2014; Da Silva et al. 2012).Comparing to antibiotic controls (Str or Rif ), the antibacterial potential of EO and PDW was several times lower, indicating that J. communis derivatives could be defined as substances with low antibacterial potential.Moreover, the agar diffusion method is not appropriate to determine the minimum inhibitory concentration (MIC), as it is impossible to quantify the amount of the antimicrobial agent diffused into the agar medium.1.2 Dilution methods

Although disk-diffusion method is standardized variant of testing, another variant of diffusion method is also frequently applied.Diffusion methods provides qualitative results, by categorizing bacteria as susceptible, intermediate or resistant (Qi et al 2006).1.1.2 Antimicrobial gradient method (E-test) The E-test is a commercial version of antimicrobial gradient method that combines the principle of dilution methods with diffusion methods, in order to determine the MIC value by this technique.In the procedure, a strip impregnated with an increasing concentration gradient of the antimicrobial agent from one end to the other is deposited on the agar surface, previously inoculated with the microorganism tested (Balouiri, 2016).Microdilution assay reduced the sample volumes from milliliters, used in test tubes in macrodilution assay, to microliters in 96-wells microtiter plates, and even to nanoliters in plates with thousands wells.He was the first to have the notion to apply calibrated spiral wire loops for multiple simultaneous serial dilutions in plastic multiwell strips (Manns, 1999 ommited in the list).Juniperus derivatives have been used for fragrance and flavoring in alcoholic beverage industry, in food preparation, as well as for insecticidal and cosmetic purposes (Lawless, 2013).The chosen bacterial indicator strains were Staphylococcus aureus, Listeria innocua, Salmonella enteritidis and Pseudomonas aeruginosa.Although it is not always pathogenic, it could be causative agent of some skin and respiratory infections, and also could be food poisoning (https://en.wikipedia.org/wiki/ Staphylococcus_aureus).The two main clinical manifestations of listeriosis are sepsis and meningitis (https://en.wikipedia.org/wiki/Listeria).Indicator bacterial strains: A. Staphylococcus aureus; B. Listeria innocua; C. Salmonella enteritidis; D. Pseudomonas aeruginosa

4.Chemical compositions of EO and PDW were determined by GC-MS and LC-MS/MS analysis, respectively, as previously described (Lesjak et al., 2013; Orcic et al., 2014).Bacterial suspension preparation Bacterial cultures were freshly prepared for every experiment in corresponding medium (BHI for L. innocua, MHB for all the rest bacteria).GC-MS analysis determined 93.95 % of total EO composition and revealed exclusively monoterpene (40.7 %) and sesquiterpene (59.3 %) hydrocarbon as dominant constituents.This could be attributed to the high proportion of the hydrocarbon monoterpenes, which possess the lowest effect compared to other terpenoid compounds, including oxigenated ones (Griffin et al., 1999).Quantitative determination of plant phenolics in Urtica dioica extracts by high-performance liquid chromatography coupled with tandem mass spectrometric detection.I owe a great debt of gratitude to Prof. Dr. Jelena Knezevic-Vukcevic, Prof. Dr. Slavisa Stankovic, Prof. Dr Tanja Beric , Dr Ivica Dimkic, Dr Stoimir Kolarevic, Dr Karolina Sunjog, Ivan Nikolic and Jovana Kostic.Most often, the antimicrobial susceptibility tests complements the Gram staining and assessment of cultural properties, the results of which are obtained previously.Agar dilution method The agar dilution method is based on the incorporation of variable concentrations of the antimicrobial agent into molten agar medium, using serial two-fold dilutions.Plant derivatives as potent antimicrobial agents

Due to the growing occurrence of microbial resistance to currently available antimicrobials, there is a continuous need for new agents, which can serve as a potential alternative (Davies and Davies, 2010).Consider if microtitre plate-based antibacterial assay with resazurin is a reliable and accurate method to detect antibacterial properties of agents from plant origin.Briefly, air-dried and finely ground needles and seed cones of the J. communis plant sample were submitted to hydro-distillation using an apparatus of Clevenger type.Bacterial suspensions were centrifuged at 4000 rpm for 10 min and resuspended in 0.01M MgSO4 to achieve 106 CFU/mL.Regardless to J. communis derivative that was used, the analysis of bacterial susceptibility revealed the same pattern: Gram-positive bacteria were more sensitive than Gram-negative ones, against both test substances.The microtitre plate-based antibacterial assay with resazurin is a sensitive, reliable and accurate method, which could be used to detect antibacterial properties of agents from plant origin.Antibacterial effects of theaflavin and synergy with epicatechin against clinical isolates of Acinetobacter baumannii and Stenotrophomonas maltophilia.Levy, S. B., O'Brien, T. F., Davey, P. G., McEwen, S. A., Barrett, J. F., Avorn, J., World Health Organisation., Alliance for the Prudent Use of Antibiotics., 2001.Screening of the antibacterial effect of Juniperus sibirica and Juniperus sabina essential oils in a microtitre plate-based MIC assay.On the other hand, the MBC/MFC concentration is defined as a minimal concentration of antibacterial/antifungal agent that completely (>=99.9%) killed particular microorganism (Levinson, 2010).This variant is so-called 'disk-diffusion method', since test compounds are applied by filter paper discs as carriers (Figure 1).Generally, antimicrobial agent diffuses into the agar and inhibits the growth of the test microorganism, leading to the formation of growth zones inhibition.Based on obtained results, i.e. inhibition zones sizes, bacteria used as test organisms are then interpreted into susceptibility categories.There are many approved guidelines for dilution antimicrobial susceptibility testing of bacteria, yeast and filamentous fungi.In addition, MBC value is determined by taking a small sample (0.01 or 0.1 mL) from the tubes used for the MIC assay and spreading it over the surface of appropriate agar plate (the plate does not contain the antimicrobial agent).Following overnight incubation at appropriate temperature, optimized for every particular bacterial species, the tubes are examined for visible bacterial growth, as evidenced by turbidity.In the environments with high presence of antibiotics, the prevalence of resistance increases rapidly as a result of mutation, genetic exchange and natural selection (Aminov, 2007 omitted in the list).saxatilis derivatives against chosen bacterial species in resazurin-incorporated microtitre plate-based antibacterial assay.After distillation of EO, residual aqueous solution was evaporated in vacuum at 45?C in order to prepare dried PDW extract.o Columns with EO and PDW: In the first well, 160 uL of MHB (or BHI for L. innocua) was added, while 100 uL of medium was added in all the remaining wells.The final volume of samples in the wells was 200 uL. Prepared concentration ranges were 50-0.39 mg/mL and 25-0.19 mg/mL for EO and PDW, respectively.o Positive controls (antibiotics Str and Rif): The columns with gradient concentrations of antibiotics were prepared as previously explained, but instead of test-substance solution, antibiotic stock solution was used.Among the quantified constituents, rutin and quinic acid were the most abundant, determined at 12.2 mg/g and 11.1 mg/g, respectively, and followed by catechin (5.53 mg/g) and epicatechin (1.74 mg/g).saxatilis EO and PDW were tested against two Gram-positive (S. aureus and L. innocua) and two Gram-negative (S. enteritidis and P. aeruginosa) bacteria.The stronger activity against Gram-positive bacteria could be attributed to the cell wall structure and is in accordance with numerous literature data (Burt, 2004; Dzamic et al., 2015; Lesjak et al, 2014).The most sensitive was Gram-positive L. innocua, with equal MIC and MBC values determined at 6.25 mg/mL and 1.56, for EO and PDW, respectively.Furthermore, in the case of the most resistant P. aeruginosa, it was not possible to determine MIC and MBC values of EO in applied concentration range.In the case of PDW, literature data indicate that catechin and epicatechin could contribute to its antibacterial effect (Betts et al., 2011; Taylor et al., 2005).Phytochemical composition and antioxidant, anti-inflammatory and antimicrobial activities of Juniperus macrocarpa Sibth.Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals.saxatilis sensitizes lung cancer cells to the anticancer effects of doxorubicin in vitro, in press Wiegand, I., Hilpert, K., Hancock, R.E., 2008.https://en.wikipedia.org/wiki/ Staphylococcus_aureus https://en.wikipedia.org/wiki/Listeria https://en.wikipedia.org/wiki/Salmonella https://en.wikipedia.org/wiki/Pseudomonas_aeruginosaPreviously to the antibiotic therapy practice, it is necessary to determine correctly the antimicrobial potential of antibiotics, since it is the only way to define doses of antibiotics that should be use in therapy.The MIC value is defined as the lowest concentration of a drug that will inhibit the visible growth of a microorganism after overnight incubation.In general, assessment of antimicrobial activity is performed by three basic types of tests: diffusion, dilution and bioautography (Fennell et al. 2004).Agar diffusion method In general, in agar diffusion methods agar plates are inoculated with a standardized inoculum of the test microorganism.Since bacterial growth inhibition does not obligatory mean the bacterial death, this method cannot distinguish bactericidal and bacteriostatic effects (Balouiri, 2016).When applied to the surface of an inoculated agar plate, the gradient is transferred from the strip to the agar plate and remains stable for a period that covers the wide variation of critical times associated with the growth characteristics of different microorganisms.The MIC value in ug/ml can be read as the point where the ellipse edge intersects the precalibrated E-test strip, providing a precise MIC value (Balouiri, 2016).1, 2, 4, 8, and 16 ug/mL) in a liquid growth medium dispensed in test tubes (macrodilution) or in the microtitre-plate wells (microdilution).Bactericidal antibiotics usually have an MBC equal or very similar to the MIC, whereas bacteriostatic antibiotics usually have an MBC significantly higher than the MIC (Levinson, 2010).In the broth microdilution assay, the microorganisms are grown in the plate wells, to which various concentrations of the tested compound are added.Resazurin is an oxidation-reduction indicator used for the evaluation of cell growth, particularly in various cytotoxicity assays.It is a blue non-fuorescent and non-toxic dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells.Listeria species are gram-positive, rod-shaped, and facultative anaerobic bacteria, and do not produce endospores (Figure 4B).1.1.1.2.1.1.2.1.1.1.2.1.2.2.2.5.5.2.5.2.1.5.2.2.5.2.3.6.2.3.4.1806.40.2, 71.

Original text

UNIVERSITY OF BELGRADE
FACULTY OF BIOLOGY

Microtitre plate-based antibacterial assay with resazurin for detection of antibacterial agents
Master thesis

					Student

Salahaldin Abdulkarim Omar Alfurjany

Belgrade, Serbia, 2016

Acknowledgement

This work was done at Chair of Microbiology, University of Belgrade - Faculty of Biology, Serbia. I would like to thank to my mentor Doc. Dr Biljana Nikolić, and also to Bojana Vasilijević for the continuous support of my M.Sc study and research, for their patience, motivation, enthusiasm, and immense knowledge. Their advices and guidance helped me in all the time of research and writing of this thesis. I also thank to Prof Dr. Branka Vuković-Gačić for her immense help during study, as well as for her final advices which improved the quality of the Master Thesis.
I owe a great debt of gratitude to Prof. Dr. Jelena Knežević-Vukčević, Prof. Dr. Slaviša Stanković, Prof. Dr Tanja Berić , Dr Ivica Dimkić, Dr Stoimir Kolarević, Dr Karolina Sunjog, Ivan Nikolić and Jovana Kostić.
I would like to thank to my family for all their love, support and encouragement.

Table of Contents
Acknowledgement 2
INTRODUCTION 4

In vitro methods for antimicrobial evaluation 4

1 Diffusion methods 5
1.1.1. Agar diffusion method 5
1.1.2 Antimicrobial gradient method (E-test) 6
1.2 Dilution methods 7
1.2.1. Broth dilution method 7
1.2.2. Agar dilution method 10

Antimicrobial resistance 10

Plant derivatives as potent antimicrobial agents 13

AIM OF THE WORK 16

MATERIAL AND METHODS 17
5.1. Material 17
5.1.1. Nutrient media 17
5.1.2. Antibiotics and other reagents used in the MIC assay 17
5.1.3. Bacterial strains 17
5.1.4. Plant material and preparation of EO and PDW 18
5.2. Method - MIC assay 18
5.2.1. Bacterial suspension preparation 18
5.2.2. Preparation of the plates: 19
5.2.3. MIC and MBC determination 20

RESULTS AND DISCUSSION 21
CONCLUSIONS 25
REFERENCES 26

INTRODUCTION
High mortality and morbidity worldwide, especially in developing countries, are frequently caused by microbial infections that occur as a result of poor sanitation, unhygienic and overcrowded living conditions. In order to combat with microbial infections, different antibiotic cures have been developed. One of the most important achievements of the medicine in the twentieth century is surely the use of antibiotics. It has revolutionary changed the medical treatment of infective diseases. Antibiotic therapy is widely practiced for the treatment of various infections (Davies and Davies, 2010). Previously to the antibiotic therapy practice, it is necessary to determine correctly the antimicrobial potential of antibiotics, since it is the only way to define doses of antibiotics that should be use in therapy. For that purposes, different methods have been developed to measure antimicrobial potential. Most often, the antimicrobial susceptibility tests complements the Gram staining and assessment of cultural properties, the results of which are obtained previously. Antimicrobial susceptibility tests can guide the physician in drug choice and dosage for difficult-to-treat infections (Levinson, 2010).

In vitro methods for antimicrobial evaluation
Two parameters are important to determine by antimicrobial susceptibility assays - minimal inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC). The MIC value is defined as the lowest concentration of a drug that will inhibit the visible growth of a microorganism after overnight incubation. Especially for some microorganisms, such as anaerobes, which require prolonged incubation for growth, incubation period is extended (Andrews, 2001). On the other hand, the MBC/MFC concentration is defined as a minimal concentration of antibacterial/antifungal agent that completely (≥99.9%) killed particular microorganism (Levinson, 2010).

In general, assessment of antimicrobial activity is performed by three basic types of tests: diffusion, dilution and bioautography (Fennell et al. 2004). Although many common facts are characteristic for both antibacterial and antifungal testing, in further text, focus will be placed at assessment of antibacterial potential.

1 Diffusion methods
1.1.1. Agar diffusion method
In general, in agar diffusion methods agar plates are inoculated with a standardized inoculum of the test microorganism. The test is performed by applying a bacterial inoculum of approximately (1-2) x 108 CFU/mL to the surface of a large (100 mm diameter) Mueller-Hinton agar plate. One variant of diffusion method is followed by placing of filter paper discs (about 6 mm in diameter), containing the test compound at a desired concentration (µg/disk), on the agar surface (Balouiri, 2016). This variant is so-called ‘disk-diffusion method’, since test compounds are applied by filter paper discs as carriers (Figure 1). The standard agar disc-diffusion test is also known as Kirby–Bauer method, Kirby–Bauer antibiotic testing, KB testing, disc-diffusion antibiotic sensitivity testing, or antibiogram method (Qi et al., 2006). Up to 12 commercially-prepared paper antibiotic disks with fixed concentrations are placed on the inoculated agar surface (Jorgensen, 2009).

Figure 1. Agar disc diffusion test (from Madigan and Martinko, 2006)

Although disk-diffusion method is standardized variant of testing, another variant of diffusion method is also frequently applied. It differs from the previously explained in the terms of the test substances applying. In this variant, the solution of potential antimicrobial substance is added to a test-system in well, previously prepared in agar plate, rather than onto disc. For that reason it is so-called ‘well-diffusion method’.
In both variants, Petri dishes are incubated under suitable conditions. Generally, antimicrobial agent diffuses into the agar and inhibits the growth of the test microorganism, leading to the formation of growth zones inhibition. After specified period of incubation, the diameter of the inhibition zone around each disk/well is measured. The diameter of the zone is related to the susceptibility of the isolate and to the diffusion rate of the drug through the agar medium. Based on obtained results, i.e. inhibition zones sizes, bacteria used as test organisms are then interpreted into susceptibility categories. Diffusion methods provides qualitative results, by categorizing bacteria as susceptible, intermediate or resistant (Qi et al 2006).
Since bacterial growth inhibition does not obligatory mean the bacterial death, this method cannot distinguish bactericidal and bacteriostatic effects (Balouiri, 2016). Moreover, the agar diffusion method is not appropriate to determine the minimum inhibitory concentration (MIC), as it is impossible to quantify the amount of the antimicrobial agent diffused into the agar medium. Nevertheless, an approximate MIC can be calculated for some microorganisms and antibiotics by comparing the inhibition zones with stored algorithms (Jorgensen and Ferraro, 2009).
1.1.2 Antimicrobial gradient method (E-test)
The E-test is a commercial version of antimicrobial gradient method that combines the principle of dilution methods with diffusion methods, in order to determine the MIC value by this technique. E-test is a commercially available test that utilizes a plastic test strip impregnated with a gradually decreasing concentration of a particular antibiotic. The strip also displays a numerical scale that corresponds to the antibiotic concentration contained therein. This method provides for a convenient quantitative test of antibiotic resistance of a clinical isolate. However, a separate strip is needed for each antibiotic, and therefore the cost of this method can be high. In the procedure, a strip impregnated with an increasing concentration gradient of the antimicrobial agent from one end to the other is deposited on the agar surface, previously inoculated with the microorganism tested (Balouiri, 2016).
E-test is simple to implement; thus, it is routinely used to meet the demands of clinicians. It is technique that employs a preformed and predefined gradient of an antimicrobial agent immobilized on a plastic strip. The concentration gradient covers a MIC range across 15 two-fold dilutions of the conventional dilution method. When applied to the surface of an inoculated agar plate, the gradient is transferred from the strip to the agar plate and remains stable for a period that covers the wide variation of critical times associated with the growth characteristics of different microorganisms. After overnight incubation or longer, an elliptical zone of inhibition centered along the axis of the strip develops. The MIC value in µg/ml can be read as the point where the ellipse edge intersects the precalibrated E-test strip, providing a precise MIC value (Balouiri, 2016).
1.2 Dilution methods

Dilution methods are the most appropriate ones for the determination of MIC values, since they offer the possibility to estimate the concentration of the tested antimicrobial agent in the agar (agar dilution) or broth medium (macrodilution or microdilution). MIC value is usually expressed in µg/mL (or mg/L). There are many approved guidelines for dilution antimicrobial susceptibility testing of bacteria, yeast and filamentous fungi. The most recognized standards are provided by the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). As advised, these guidelines provide a uniform procedure for testing that is practical to perform in most clinical microbiology laboratories and bioassay to be performed in a standardized approach in order to evaluate the clinical relevance of results Nevertheless if dilution is prepared in agar or in broth, the range of antibiotic concentrations used for determining MICs is universally accepted to be in doubling dilution steps up and down from 1 mg/mL, as required (Balouiri, 2016).
1.2.1. Broth dilution method
Broth dilution method can be performed in two variants - as a macrodilution and as a microdilution procedure. In both cases, procedure involves preparing of two-fold dilutions of test substances (eg. 1, 2, 4, 8, and 16 µg/mL) in a liquid growth medium dispensed in test tubes (macrodilution) or in the microtitre-plate wells (microdilution). Broth dilution method is suitable to determine MIC, but also MBC value. MIC is determined as the lowest concentration of antibiotic that prevented bacterial growth. In addition, MBC value is determined by taking a small sample (0.01 or 0.1 mL) from the tubes used for the MIC assay and spreading it over the surface of appropriate agar plate (the plate does not contain the antimicrobial agent). If bacterial growth was only inhibited, but viable cells remain in the sample, they could reproduce and form the colonies on the agar plate. After a standard incubation, the lowest concentration that has reduced the number of colonies by 99.9% is the MBC. Bactericidal antibiotics usually have an MBC equal or very similar to the MIC, whereas bacteriostatic antibiotics usually have an MBC significantly higher than the MIC (Levinson, 2010).
1.2.1.1. Broth macrodilution method
The antibiotic-containing tubes are inoculated with a standardized bacterial suspension of 1–5×105CFU/mL. Following overnight incubation at appropriate temperature, optimized for every particular bacterial species, the tubes are examined for visible bacterial growth, as evidenced by turbidity. In some cases, in order to facilitate the results reading, specific bacterial growth bioindicator could be added in the test system. The advantage of this technique is the generation of a quantitative result (ie, determination of numerical MIC value). The principal disadvantages of the macrodilution method are the tedious, manual task of preparing the antibiotic solutions for each test, the possibility of errors in preparation of the antibiotic solutions, and the relatively large amount of reagents and space required for each test (Jorgensen, 2009). The interpretative standards for this method are published by different national organizations such as the Clinical and Laboratory Standards Institute (CLSI) in the USA and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (Wiegand et al. 2008).
1.2.1.2. Broth microdilution method
Microtitre plate based antibacterial assay is the dilution method, consisted of series of small test tubes arranged in a regular matrix pattern on a plastic plate, usually made from transparent polystyrene. The little plate makes it possible to handle many samples in parallel. Mostly used size of plate for testing is 96 wells, but there are also plates with several thousand wells. In the case of 96-wells microtiter plates, result can be read off visually, but in the case of the plate with thousands wells, the results must be read in an automated plate reader. Microdilution assay reduced the sample volumes from milliliters, used in test tubes in macrodilution assay, to microliters in 96-wells microtiter plates, and even to nanoliters in plates with thousands wells. This significantly saves money spent on reagents.
The first microtiter plate was hand-made in 1951 by Hungarian Dr. Takácsy, who developed spiral loop instead of pipette and glass-plates with wells instead of tubes. He used the term micromethod in his manuscript explaining the method, published in 1952 in Hungarian, and in 1955 in English. He was the first to have the notion to apply calibrated spiral wire loops for multiple simultaneous serial dilutions in plastic multiwell strips (Manns, 1999 ommited in the list).
In the broth microdilution assay, the microorganisms are grown in the plate wells, to which various concentrations of the tested compound are added. As in other dilution methods, the antibacterial effect was monitored for the serial two-fold dilutions of the test supstances. The growth of the microorganisms could be indicated by the presence of turbidity in the wells. However, since small amounts of culture medium are presented in the wells, it is not always easy to visualize the change of turbidity. For that purposes, different bacterial growth indicators could be added. Microtitre plate-based antibacterial assay incorporating resazurin (Figure 2.) is a method that is generally accepted for application in the in vitro antibacterial screening of different antibiotics and phytochemicals (Sarker et al., 2007).
Resazurin is an oxidation–reduction indicator used for the evaluation of cell growth, particularly in various cytotoxicity assays. It is a blue non-fuorescent and non-toxic dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases within viable cells. Resorufin could be further reduced to hydroresorufin (uncoloured and nonfluorescent). In resazurin-incorporated microtitre plate-based antibacterial assay, MIC value is determines as the lowest concentration that induces no color change, i.e. well remains blue (Sarker et al., 2007).

Figure 2. Microdilution Method (modified Figure from Madigan and Martinko, 2006)
1.2.2. Agar dilution method
The agar dilution method is based on the incorporation of variable concentrations of the antimicrobial agent into molten agar medium, using serial two-fold dilutions. Next step is inoculation of a defined microbial inoculum onto the agar plate surface. This method also reveals the determination of the MIC values. Although agar dilution method is significantly more expensive and laborious to perform, it is especially suitable for testing the compounds (or extracts) which masks the detection of microbial growth in the liquid medium by their coloring (Balouiri, 2016).
2. Antimicrobial resistance

The prevalence of antimicrobial resistance among key microbial pathogens is increasing worldwide and reaching an alarming rate. Antibiotic resistance is a huge problem of presence health systems and it often occurs in the environment enriched with antibiotics, such as hospitals and other medical institutions. In order to facilitate the curing of hospital-acquired infections in the future, the international, national, and local antibiotic stewardship campaigns have been developed to encourage prudent use and limit unnecessary exposure to antibiotics. The ultimate goal of such campaigns is preserving of antibiotic effectiveness for serious and life-threatening infections (Belongia et al., 2005; Levy et al., 2001).
Antibiotic resistance is a result of the fact that antibiotic-producing microorganisms developed resistance mechanisms to neutralize the effects of their own antibiotics. However, genes encoding the resistance mechanisms can by transferred to other, usually related, organisms. In the environments with high presence of antibiotics, the prevalence of resistance increases rapidly as a result of mutation, genetic exchange and natural selection (Aminov, 2007 omitted in the list). In such environments the multi-drug resistant pathogens appear, making huge difficulties in curing of infections caused by them (Figure 3.) The mechanisms of bacterial resistance to antibiotics include reduced permeability, inactivation of antibiotics, alteration of target, development of specific biochemical pathways, as well as the efflux of antibiotics (Table 1).

Figure 3: Apperance of multi-drug resistant pathogens (from Madigan and Martinko, 2006)

BACTERIAL RESISTANCE TO ANTIBIOTICS
Resistence mechanism Antibiotic example Genetic basis of resistance Mechanism presented in
Reduced permeability Penicillin
Chromosomal
Salmonella enteritidis
Pseudomonas aeruginosa
Chloramphenicol
Erytrommycin
Lincomycin Plasmid Listerya innocua
Inactivation of antibiotic Penicillin
Plasmid and chromosomal
Staphylococcus aureus
Salmonella enteritidis
Chloramphenicol Plasmid and chromosomal Staphylococcus aureus
Salmonella enteritidis
Alteration of target Eritromycin
Rifamycin
Streptomycin
Norfloxacin
Chromosomal Staphylococcus aureus
Salmonella enteritidis
Development of resistant biochemical pathaway Sulfonamides Chromosomal Staphylococcus aureus
Salmonella enteritidis
Efflux(pumping out of cell) Tetracycline
Chloramphenicol Plasmid
Chromosomal Salmonella enteritidis
Staphylococcus aureus

Table 1. Bacterial resistance to antibiotics

Plant derivatives as potent antimicrobial agents

Due to the growing occurrence of microbial resistance to currently available antimicrobials, there is a continuous need for new agents, which can serve as a potential alternative (Davies and Davies, 2010). Different compounds of plant origin possess significant antimicrobial potential and they could be good sources for the development of new antimicrobial chemotherapeutics (Chin et al. 2006; Newman et al. 2000). Essential oils (EOs) of many aromatic plants, such as Origanum vulgare, Thumus capitatus and Ocimum basilicum, are proved as potent antibacterial agents (Džamić et al., 2015; Soković et al., 2010). Some literature data indicate that Juniperus species also possess antibacterial properties (Glišić et al., 2007; Nikolić et al., 2016).
Genus Juniperus belongs to the family of Cupressaceae and is widely distributed in the Northern Hemisphere. It is one of the most diverse genera of the conifers, comprised of about 70 species. Juniperus derivatives have been used for fragrance and flavoring in alcoholic beverage industry, in food preparation, as well as for insecticidal and cosmetic purposes (Lawless, 2013). Taking into account the widespread occurrence of this genus, as well as the richness in the active substances, it is not surprising to find extensive use of EO and differently prepared extracts of many Juniperus species (Adams, 2014). Studying of biological properties indicates that Juniperus species are endowed with numerous activities including antioxidant, antiseptic, diuretic, anticancer, antirheumatic, antihelmintic, anti-inflammatory, immunomodulatory, analgesic, antituberculotic and abortifacient activities (Glišić et al. 2007; Orphan et al., 2011; Swanston-Flatt et al. 1990). Furthermore, study of Khan et al. (2012) provides a pharmacological basis for traditional use of Juniperus excelsa against respiratory disorders and gut hyperactivity, such as asthma, diarrhoea and colic. In this work we evaluated the antibacterial potential of two derivatives of Juniperus communis. We tested its EO, but also the post-distillation waste (PDW), which remained after EO distillation.
The chosen bacterial indicator strains were Staphylococcus aureus, Listeria innocua, Salmonella enteritidis and Pseudomonas aeruginosa. S. aureus (Figure 4A) is a gram-positive coccal bacterium and is frequently found in the nose, respiratory tract, and on the skin. Although it is not always pathogenic, it could be causative agent of some skin and respiratory infections, and also could be food poisoning (https://en.wikipedia.org/wiki/ Staphylococcus_aureus).
Listeria species are gram-positive, rod-shaped, and facultative anaerobic bacteria, and do not produce endospores (Figure 4B). The major human pathogen is L. monocytogenes, being the causative agent of listeriosis, a serious infection caused by consummation of contaminated food. The two main clinical manifestations of listeriosis are sepsis and meningitis (https://en.wikipedia.org/wiki/Listeria).
Salmonella is rod-shaped gram-negative genus of the Enterobacteriaceae family (Figure 4C). It is facultative anaerobic bacteria, predominantly motile, with peritrichous flagella. They are facultative intracellular pathogens that induced infections due to ingestion of contaminated food. They can infect a range of animals, and are zoonotic, meaning they can be transferred between humans and other animals (https://en.wikipedia.org/wiki/Salmonella).
P. aeruginosa is rod-shaped Gram-negative bacterium (Figure 4D) that is pathogen inducing disease in plants and animals, including humans. However, P. aeruginosa is considered as opportunistic pathogens in humans. It has considerable medical importance as a prototypical multidrug resistant pathogen. Advanced antibiotic resistance mechanisms could be associated with serious illnesses – especially nosocomial infections such as pneumonia and various sepsis syndromes. If colonization of P. aeruginosa occurs in critical body organs, such as lungs, urinary tract and kidneys, the results could be fatal (https://en.wikipedia.org/wiki/Pseudomonas aeruginosa).

Figure 4. Indicator bacterial strains: A. Staphylococcus aureus; B. Listeria innocua; C. Salmonella enteritidis; D. Pseudomonas aeruginosa

THE AIM OF THE WORK
The aim of this research was to monitor the antibacterial potential of Juniperus communis var. saxatilis derivatives against chosen bacterial species in resazurin-incorporated microtitre plate-based antibacterial assay. The research was performed in order to:

Consider if microtitre plate-based antibacterial assay with resazurin is a reliable and accurate method to detect antibacterial properties of agents from plant origin.

Evaluate and quantitatively determine the antibacterial potential of J. communis essential oil and post-distillation waste against Staphylococcus aureus, Listeria innocua, Salmonella enteritidis and Pseudomonas aeruginosa.

MATERIAL AND METHODS
5.1. Material
5.1.1. Nutrient media
In this study the following nutrient media were used:
• MHB - Müeller-Hinton Broth: MHB powder (21 g), distilled water (1000 ml);
• MHA - Müeller-Hinton Agar: MHB powder (21g), Agar “Difco” (15g), distilled water (1000ml);
• BHI - Brain-Heart Infusion Broth: BHI powder (37g) , distilled water (1000 ml);
• BHA - Brain-Heart Infusion Agar: BHI powder (37g) , Agar “Difco” (15g), distilled water (1000 ml);
Nutrient media were sterilized in an autoclave for 20 minutes at a temperature of 121°C and a pressure of 101.3 kPa.

5.1.2. Antibiotics and other reagents used in the MIC assay
• Streptomicin (Str): antibiotic solution in MHB; concentration of working stock 500 µg/ml
• Rifampicin (Rif): antibiotic solution in BHI, concentration of working stock 200 µg/ml
• Dimethyl sulfoxide (DMSO): solvent, used for EO
• Resazurin Sodium Salt: distilled water solution, concentration of working stock 0,675 mg/mL
5.1.3. Bacterial strains
The following bacterial strains were used to determine antibacterial activity of the EO and PDW:
Gram – positive bacteria:
• Staphylococcus aureus ATCC25923
• Listeria innocua ATCC33090
Gram – negative bacteria:
• Salmonella enteritidis ATCC13076
• Pseudomonas aeruginosa ATCC15442

5.1.4. Plant material and preparation of EO and PDW
Plant material was collected in July 2011 at Stara Planina Mountain, Serbia. The voucher specimen (Juniperus communis var. saxatilis, No. 16693) was prepared, identified and deposited at the Herbarium of University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden "Jevremovac” (BEOU Herbarium).
Preparation of EO and PDW, and determination of their chemical compositions were done in previous work (Vasilijević et al., 2016). Briefly, air-dried and finely ground needles and seed cones of the J. communis plant sample were submitted to hydro-distillation using an apparatus of Clevenger type. After distillation of EO, residual aqueous solution was evaporated in vacuum at 45˚C in order to prepare dried PDW extract. Chemical compositions of EO and PDW were determined by GC-MS and LC-MS/MS analysis, respectively, as previously described (Lesjak et al., 2013; Orčić et al., 2014).
5.2. Method - MIC assay

The MIC assay was performed in 96-well microtitre plates, by making the serial two-fold dilutions of test substances.
5.2.1. Bacterial suspension preparation
Bacterial cultures were freshly prepared for every experiment in corresponding medium (BHI for L. innocua, MHB for all the rest bacteria). The bacteria were growen until appropriate concentration (108 CFU/mL) was obtained. Bacterial concentration was setted by optical densitiy (OD600) measurements. Appropriate OD600 values, determined by previous calibration experiments (data not shown), were: 0.2 for L. innocua, 0.3 for S. aureus and S. enteritidis, and 0.4 for P. aeruginosa. Bacterial suspensions were centrifuged at 4000 rpm for 10 min and resuspended in 0.01M MgSO4 to achieve 106 CFU/mL.
5.2.2. Preparation of the plates:
• Blank (control of sterility): In four wells 200 µl of corresponding media (MHB, BHI) was added.
• Negative control (control of bacteria growth): In four wells 180 µl of corresponding media and 20 µl of bacteria were added.
• Columns with EO and PDW: In the first well, 160 μL of MHB (or BHI for L. innocua) was added, while 100 μL of medium was added in all the remaining wells. In the first well 40 µl of appropriate dilution of test substance was added. The serial dilutions were performed by pipetting 100 μL of test substance solution from row to row and removing of 100 μL of solution from the last well. Such preparation led to the two-fold dilutions of test-substances. Additional 80 µl of corresponding medium and 20 µL of bacterial suspension were added in each well in order to obtain 1.0 x 104 CFU per well. The final volume of samples in the wells was 200 µL. Prepared concentration ranges were 50–0.39 mg/mL and 25–0.19 mg/mL for EO and PDW, respectively.
• Solvent control (DMSO): The column with gradient concentration of solvent was prepared as previously explained, but instead of test-substance solution, DMSO was used.
• Positive controls (antibiotics Str and Rif): The columns with gradient concentrations of antibiotics were prepared as previously explained, but instead of test-substance solution, antibiotic stock solution was used. Tested concentration ranges were 50– 0.39 µg/mL and 25 – 0.19 µg/mL, for Str and Rif, respectively. Rif was used as positive control only for L. innocua, while Str was used for all the rest bacteria.
All test-substances were examined in triplicate. Plates were incubated at 37 °C for 18–24 h. After incubation, 22 µL of resazurin solution was added to each well. The microtitre plates were wrapped with vapor film and incubated for an additional 3 h. After that plates were monitored.

5.2.3. MIC and MBC determination
The lowest concentration of the test substance without visible color change was determined as the MIC value. To investigate if detected antibacterial effect was bactericidal or bacteriostatic, further screening was performed. For that purpose, additional plating of suspension from all the wells without visible growth onto the corresponding agar medium (BHA for L. innocua, and MHA for all the rest bacteria) was performed. After incubation for 24 h at 37°C, MBC was determined as the lowest concentration without visible growth (≥99.9% colonies were killed).

RESULTS AND DISCUSSION

In pursuing for new antibacterial agents of plant origin different extracts of less studied species from our region were screened. Preliminary results directed our study to Juniperus species. In this work we monitored antibacterial effect of J. communis var. saxatilis EO and PDW. It is based on the previous research concerning chemical composition of J. communis EO and PDW (Vasilijević et al., 2016). GC-MS analysis determined 93.95 % of total EO composition and revealed exclusively monoterpene (40.7 %) and sesquiterpene (59.3 %) hydrocarbon as dominant constituents. Remaining 6.05% of the EO belongs to the sesquiterpenes that could not be identified. The main constituents of EO were α-pinene (23.61%), followed by δ-cadinene (10.71%), sabinene (9.53%), germacrene D (7.25%), α-muurolene (6.58%) and γ-cadinene (5.87%) (Figure 5A). The high abundance of α-pinene and sabinene was consistent with previous studies regarding J. communis (Adams, 2014; Seca and Silva, 2006; Shahmir et al., 2003). LC-MS/MS analysis of PDW identified only 3.2% of its total content. Among the quantified constituents, rutin and quinic acid were the most abundant, determined at 12.2 mg/g and 11.1 mg/g, respectively, and followed by catechin (5.53 mg/g) and epicatechin (1.74 mg/g). The identified composition of PDW is shown in Figure 5B.

Figure 5. Chemical composition of EO (A) and PDW (B) of J. communis var.saxatilis

Antibacterial potential of J. communis var. saxatilis EO and PDW were tested against two Gram-positive (S. aureus and L. innocua) and two Gram-negative (S. enteritidis and P. aeruginosa) bacteria. The microtitre plate-based antibacterial assay using resazurin as an indicator of the cell growth was performed.
The results of MIC and MBC values for EO and PDW are summarized in Table 2. Comparison of effects of both EO and PDW indicates that antibacterial potential of PDW was slightly higher. Regardless to J. communis derivative that was used, the analysis of bacterial susceptibility revealed the same pattern: Gram-positive bacteria were more sensitive than Gram-negative ones, against both test substances. The stronger activity against Gram-positive bacteria could be attributed to the cell wall structure and is in accordance with numerous literature data (Burt, 2004; Džamić et al., 2015; Lesjak et al, 2014).

Table 2. MIC and MBC values*
Bacterial strains EO PDW Str Rif
MIC MBC MIC MBC MIC MBC MIC MBC
Staphylococcus aureus 6.25 12.5 3.125 6.25 12.5 25 nt nt
Listeria innocua 6.25 6.25 1.56 1.56 nt nt / 0.19
Salmonella enteritidis 50 50 6.25 6.25 3.125 6.25 nt nt
Pseudomonas aeruginosa ∕ ∕ 12.5 25 25 50 nt nt
/ - not determined in used concentration range
nt – not tested
*MIC and MBC in mg/mL for EO and PDW, and in µg/mL for Str and Rif.

The most sensitive was Gram-positive L. innocua, with equal MIC and MBC values determined at 6.25 mg/mL and 1.56, for EO and PDW, respectively. Comparing to L. innocua, the susceptibility of S. aureus to EO was approximately the same, while its sensitivity to PDW was lower. On the other hand, sensitivity of Gram-negative strains was considerably lower. Bactericidal effect of PDW against S. enteritidis and P. aeruginosa was obtained at 6.25 and 25 mg/mL, respectively. The effect of EO was negligible since the highest tested concentration (50 mg/mL) was needed in order to both inhibit and completely killed S. enteritidis. Furthermore, in the case of the most resistant P. aeruginosa, it was not possible to determine MIC and MBC values of EO in applied concentration range.
Judged by the dominant constituents of EO, obtained antibacterial effect could be at least partially attributed to α-pinene and sabinene (Arunkumar et al., 2014; Da Silva et al. 2012). In the case of PDW, literature data indicate that catechin and epicatechin could contribute to its antibacterial effect (Betts et al., 2011; Taylor et al., 2005). However, the contribution of other constituents of both EO and PDW could not be excluded.
Comparing to antibiotic controls (Str or Rif ), the antibacterial potential of EO and PDW was several times lower, indicating that J. communis derivatives could be defined as substances with low antibacterial potential. This could be attributed to the high proportion of the hydrocarbon monoterpenes, which possess the lowest effect compared to other terpenoid compounds, including oxigenated ones (Griffin et al., 1999). Low antibacterial effect of J. communis derivatives is in line with previously published data, indicating moderate and low antibacterial effect of Juniperus species (Andogan et al., 2002; Angioni et al., 2003; Glišić et al., 2007; Karaman et al., 2003; Nikolić et al, 2016; Lesjak et al., 2014). Interestingly, although the antibacterial effect is not high, the importance is the fact that Juniperus communis extracts possess antibacterial effect against important patogenic bacteria Mycobacterium tuberculosis, a causative agent of tuberculosis disesase (Carpenter et al., 2012; Jimenez-Arellanes et al., 2003).

CONCLUSIONS

Based on the results of this work, following statements could be concluded:

The microtitre plate-based antibacterial assay with resazurin is a sensitive, reliable and accurate method, which could be used to detect antibacterial properties of agents from plant origin.

Both essential oil and post-distillation waste of Juniperus communis var. saxatilis possess weak antibacterial potential. Compared to EO, antibacterial effect of PDW was slightly higher.

The sensitivity of Gram-positive bacteria (S. aureus, L. innocua) was higher than the sensitivity of Gram-negative ones (S. enteritidis, P. aeruginosa). The most sensitive was L. innocua, while the list sensitive was P. aeruginosa, which showed even complete resistance to applied concentration range of EO (up to 50 mg/mL).

Taking into account that antibacterial effect of EO and PDW was weak, the J. communis derivatives could be recommended only as auxiliary antibacterial remedies. Further research should be directed to investigate the antibacterial potential of J. communis and other medicinal plants in combinations.

REFERENCES

Adams, R.P., 2014. Junipers of the world: the genus Juniperus, fourth ed. Trafford Publishing, Waco, USA.
Andoğan, B.C., Baydar, H., Kaya, S., Demirci, M., Özbaşar, D., Mumcu, E., 2002. Antimicrobial activity and chemical composition of some essential oils. Arch. Pharm. Res. 25, 860-864.
Andrews, J.M., 2001. Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother. 48, 5-16.
Angioni, A., Barra, A., Russo, M.T., Coroneo, V., Dessí, S., Cabras, P., 2003. Chemical composition of the essential oils of Juniperus from ripe and unripe berries and leaves and their antimicrobial activity. J. Agric. Food Chem. 51, 3073-3078.
Arunkumar, R., Nair, S.A., Rameshkumar, K.B., Subramoniam, A., 2014. The essential oil constituents of Zornia diphylla (L.) Pers, and anti-inflammatory and antimicrobial activities of the oil. Rec. Nat. Prod. 8, 385-393.
Balouiri, M., Sadiki, M., Ibnsouda, S. K., 2016. Methods for in vitro evaluating antimicrobial activity: A review. J. Pharm. Anal. 6, 71-79.
Belongia, E.A., Knobloch, M.J., Kieke, B.A., Davis, J.P., Janette, C., Besser, R.E., 2005. Impact of statewide program to promote appropriate antimicrobial drug use. Emerg. Infect. Dis. 11, 912-920.
Betts, J.W., Kelly, S.M., Haswell, S.J., 2011. Antibacterial effects of theaflavin and synergy with epicatechin against clinical isolates of Acinetobacter baumannii and Stenotrophomonas maltophilia. Int. J. Antimicrob. Agents 38, 421-425.
Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in foods-a review. Int. J. Food Microbiol. 94, 223-253.
Carpenter, C.D., O'Neill, T., Picot, N., Johnson, J.A., Robichaud, G.A., Webster, D., Gray, C.A., 2012. Anti-mycobacterial natural products from the Canadian medicinal plant Juniperus communis. J. Ethnopharmacol. 143, 695-700.
Chin, Y.W., Balunas, M.J., Chai, H.B., Kinghorn, A.D., 2006. Drug discovery from natural sources. Aaps. J. 8, 239-253.
Davies, J., Davies, D., 2010. Origins and evolution of antibiotic resistance. Microbiol. Mol. Biol. R. 74, 417-433.
Džamić, A.M., Nikolić, B.J., Giweli, A.A., Mitić‐Ćulafić, D.S., Soković, M.D., Ristić, M.S., Knežević-Vukčević, J.B., Marin, P.D., 2015. Libyan Thymus capitatus essential oil: antioxidant, antimicrobial, cytotoxic and colon pathogen adhesion‐inhibition properties. J. Appl. Microbiol. 119, 389-399.
Fennell, C.W., Lindsey, K.L., Mcgaw, L.J, Sparg, S.G., Stafford, G.I., Elgorashi, E.E., Grace, O.M., Van Staden, J., 2004. Assessing African medicinal plants for efficacy and safety: pharmacological screening and toxicology. J. Ethnopharmacol. 94, 205-217.
Glišić, S.B., Milojević, S.Ž., Dimitrijević, S.I., Orlović, A.M., Skala, D.U., 2007. Antimicrobial activity of the essential oil and different fractions of Juniperus communis L. and a comparison with some commercial antibiotics. J. Serb. Chem. Soc.72, 311-320.
Griffin, S.G., Wyllie S.G., Markham, J.L., Leach D.N., 1999. The role of structure and molecular properties of terpenoids in determining their antimicrobial activity. Flavour. Frag. J. 14, 322-332.
Jimenez‐Arellanes, A., Meckes, M., Ramirez, R., Torres, J., Luna‐Herrera, J., 2003. Activity against multidrug‐resistant Mycobacterium tuberculosis in Mexican plants used to treat respiratory diseases. Phytother. Res. 17, 903-908.
Karaman, I., Şahin, F., Güllüce, M., Öǧütçü, H., Şengül, M., Adıgüzel, A., 2003. Antimicrobial activity of aqueous and methanol extracts of Juniperus oxycedrus L. J. Ethnopharmacol. 85, 231-235.
Khan, M., Khan, A.U., Gilani, A.H., 2012. Pharmacological explanation for the medicinal use of Juniperus excelsa in hyperactive gastrointestinal and respiratory disorders. J. Nat. Med. 66, 292-301.
Lawless, J., 2013. The Encyclopedia of Essential Oils: The Complete Guide to the Use of Aromatic Oils In Aromatherapy, Herbalism, Health, and Well Being. Conari Press.
Lesjak, M.M., Beara, I.N., Orčić, D.Z., Petar, K.N., Simin, N.Đ., Emilija, S.Đ., Mimica-Dukić, N.M., 2014. Phytochemical composition and antioxidant, anti-inflammatory and antimicrobial activities of Juniperus macrocarpa Sibth. et Sm. J. Funct. Foods 7, 257-268.
Lesjak, M.M., Beara, I.N., Orčić, D.Z., Ristić, J.D., Anačkov, G.T., Božin, B. N., Mimica-Dukić, N.M., 2013. Chemical characterisation and biological effects of Juniperus foetidissima Willd. 1806. LWT-Food Sci. Technol. 53, 530-539.
Levison, W., 2010. Review of Medical Microbiology and Immunology, eleventh ed. McGraw-Hill, USA.
Levy, S. B., O'Brien, T. F., Davey, P. G., McEwen, S. A., Barrett, J. F., Avorn, J., World Health Organisation., Alliance for the Prudent Use of Antibiotics., 2001. Antibiotic resistance: synthesis of recommendations by expert policy groups.
Madigan, M.T., Martinko, M. J., 2006. Brock Biology of Microorganisms, eleventh ed. Pearson Prentice Hall, New York.
Newman, D.J., Cragg, G.M., Snader, K.M., 2000. The influence of natural products upon drug discovery. Nat. Prod. Rep. 17, 215-234.
Nikolić, B., Vasilijević, B., Mitić-Ćulafić, D., Lesjak, M., Vuković-Gačić, B., Dukić, N. M., Knežević-Vukčević, J., 2016. Screening of the antibacterial effect of Juniperus sibirica and Juniperus sabina essential oils in a microtitre plate-based MIC assay. Bot. Serb. 40.
Orčić, D., Francišković, M., Bekvalac, K., Svirčev, E., Beara, I., Lesjak, M., Mimica-Dukić, N., 2014. Quantitative determination of plant phenolics in Urtica dioica extracts by high-performance liquid chromatography coupled with tandem mass spectrometric detection. Food Chem.143, 48–53.
Orhan, N., Orhan, I.E., Ergun, F., 2011. Insights into cholinesterase inhibitory and antioxidant activities of five Juniperus species. Food Chem. Toxicol. 49, 2305-2312.
Reller, L.B., Weinstein, M., Jorgensen, J.H., Ferraro, M. J., 2009. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin. Infect. Dis. 49, 1749-1755.
Sarker, S. D., Nahar, L., Kumarasamy, Y., 2007. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 42, 321-324.
Seca, A.M., Silva, A.M., 2006. The chemical composition of the Juniperus genus (1970–2004). Recent progress in medicinal plants 16, 401-522.
Shahmir, F., Ahmadi, L., Mirza, M., Korori, S.A.A., 2003. Secretory elements of needles and berries of Juniperus communis L. ssp. communis and its volatile constituents. Flavour Frag. J. 18, 425-428.
Silva, A.C.R.D., Lopes, P.M., Azevedo, M.M.B.D., Costa, D.C.M., Alviano, C.S., Alviano, D.S., 2012. Biological activities of a-pinene and β-pinene enantiomers. Molecules 17, 6305-6316.
Soković, M., Tzakou, O., Pitarokili, D., Couladis, M., 2002. Antifungal activities of selected aromatic plants growing wild in Greece. Nahrung-Food 46, 317-320.
Swanston-Flatt, S.K., Day, C., Bailey, C.J., Flatt, P.R., 1990. Traditional plant treatments for diabetes. Studies in normal and streptozotocin diabetic mice. Diabetologia 33, 462-464.
Taylor, P.W., Hamilton-Miller, J.M., Stapleton, P.D., 2005. Antimicrobial properties of green tea catechins. Food Sci. Tech. Bull. 2, 71.
Vasilijević, B., Knežević-Vukčević, J., Orčić, D., Francišković, M., Mitić-Ćulafić, D., Jovanović, M., Nikolić, B., 2016. Essential oil and post-distillation waste of Juniperus communis var. saxatilis sensitizes lung cancer cells to the anticancer effects of doxorubicin in vitro, in press
Wiegand, I., Hilpert, K., Hancock, R.E., 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc.3, 163-175.
https://en.wikipedia.org/wiki/ Staphylococcus_aureus
https://en.wikipedia.org/wiki/Listeria
https://en.wikipedia.org/wiki/Salmonella
https://en.wikipedia.org/wiki/Pseudomonas_aeruginosa

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