Antimicrobial Potential of Caffeic Acid against

Research ArticleAntimicrobial Potential of Caffeic Acid againstStaphylococcus aureus Clinical Strains

MaBgorzata Kwpa Maria MiklasiNska-Majdanik Robert D Wojtyczka Danuta IdzikKonrad Korzeniowski Joanna SmoleN-Dzirba and Tomasz J Wdsik

Department of Microbiology and Virology School of Pharmacy with the Division of Laboratory Medicine in SosnowiecMedical University of Silesia in Katowice ul Jagiellonska 4 41-200 Sosnowiec Poland

Correspondence should be addressed to Tomasz J Wąsik twasiksumedupl

Received 6 February 2018 Accepted 3 July 2018 Published 15 July 2018

Academic Editor Gail B Mahady

Copyright copy 2018 MałgorzataKępa et alThis is an open access article distributed under theCreativeCommonsAttributionLicensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Phenolic compounds constitute one of the most promising and ubiquitous groups with many biological activities Synergisticinteractions between natural phenolic compounds and antibiotics could offer a desired alternative approach to the therapies againstmultidrug-resistant bacteriaThe objective of the presented study was to assess the antibacterial potential of caffeic acid (CA) aloneand in antibiotic-phytochemical combination against Staphylococcus aureus reference and clinical strains isolated from infectedwounds The caffeic acid tested in the presented study showed diverse effects on S aureus strains with the minimum inhibitoryconcentration (MIC) varied from 256 120583gmL to 1024 120583gmLThe supplementation of Mueller-Hinton agar (MHA) with 14 MIC ofCA resulted in augmented antibacterial effect of erythromycin clindamycin and cefoxitin and to the lesser extent of vancomycinThe observed antimicrobial action of CA seemed to be rather strain than antibiotic dependent Our data support the notion that CAalone exerts antibacterial activity against S aureus clinical strains and has capacity to potentiate antimicrobial effect in combinationwith antibiotics The synergy between CA and antibiotics demonstrates its potential as a novel antibacterial tool which couldimprove the treatment of intractable infections caused by multidrug-resistant strains

1 Introduction

Antimicrobial drug resistance is currently one of the majorpublic health problems worldwide Infections caused bymultidrug-resistant (MDR) strains are hard to treat and oftenturn out to be fatal especially among hospitalized patientswith diminished immunity [1ndash3] Due to the steady risein the incidence of intractable infections with multidrug-resistant strains there is an immediate need to search forthe alternative antimicrobial therapies and new antibacte-rial agents It has been documented that many naturallyoccurring polyphenolic compounds have the capacity toinhibit bacterial growth and to sensitize MDR strains tothe bactericidal or bacteriostatic action of wide range ofantibiotics [4]

Skin is the largest human organ which can be colonizedwith antibiotic resistant bacterial strains and these bacteriamay cause infections for which limited therapeutic optionsare available [5ndash7] Among them Staphylococcus aureus is

one of the most common pathogens in both community andhospital associated superficial and deep skin infections Thewidespread emergence of multidrug-resistant staphylococcistrains compromises common therapeutic strategies basedon the broad-spectrum antibiotics thus worsening infectioncontrol What is more such therapies greatly affect skinmicrobiome andmay result in further selection ofmultidrug-resistant nonstaphylococci bacteria [8ndash10]

Rapidly growing bacterial resistance to antibiotics dic-tates the ongoing search for an alternative approach to thetreatment of intractable infections [11ndash20] Our previousstudies have shown that phytochemical compounds such ascatechin hydrate and protocatechuic acid ethyl ester demon-strate antimicrobial properties against Staphylococcus aureusstrains [21 22] Caffeic acid a plant phenylpropanoid pathwaysecondary metabolite is classified as a hydroxycinnamicacid containing both phenolic and acrylic functional groupsand its derivatives include amides esters sugar esters andglycosides Caffeic acid can be found in many plant products

HindawiBioMed Research InternationalVolume 2018 Article ID 7413504 9 pageshttpsdoiorg10115520187413504

2 BioMed Research International

coffee beverages argan oil oats wheat rice and olive oil[3 23] It has been reported that CA showed antimicrobialpotential andor synergistic effects with antibiotics againstS aureus S epidermidis K pneumoniae S marcescensP mirabilis E coli P aeruginosa B cereus M luteus Lmonocytogenes and C albicans strains [20 24ndash27]

The majority of studies on antibacterial action of CA orits derivatives have been focused on the reference bacterialstrains Studies on antibacterial potential of CA againstclinical isolates are scarce either with respect to the CA aloneor in CA-antibiotic combination In the face of the observedsteady increase in the incidence of nosocomial skin infectionscaused by bacteria resistant to broad spectrum of antibioticsthe objective of the presented study was to assess in vitroantibacterial potential of caffeic acid alone and in antibiotic-phytochemical combination using a panel of Staphylococcusaureus clinical strains isolated from intractable infectedwounds

2 Materials and Methods

21 Examined StrainsMedia and Reagents Twenty S aureusclinical strains were isolated from infected wounds of hospi-talized patients and 3 S aureus reference strains S aureusATCC 25923 S aureus ATCC 43300 and S aureus ATCC6538 were obtained directly from ATCC (American TypeCulture Collections) To ensure the homogeneity of clinicalstrains all isolates were derived from the surgical woundsleading to the relatively small sample size All examinedstrains were stored in Tryptic Soy Broth (TSB) medium with20 of glycerol at -80∘C until further use Caffeic acid waspurchased from Sigma Chemical Co (St Louis MO USA)and dissolved in DMSO (Sigma Chemical Co St Louis MOUSA) before use

22 Molecular Identification and Characteristic of ClinicalStrains Detection of dnaJ gene fragment was performedby restriction fragment length polymorphism (PCR-RFLP)For PCR-RFLP method bacterial DNA was extracted withthe GeneMATRIX Tissue amp Bacterial DNA Purification KIT(EURx Ltd Gdansk Poland) according to themanufacturerrsquosrecommendations with the modification described by Shahet al [28] Briefly 883 bp region of dnaJ gene coding forthe N-terminal domain of the receptor was amplified withSA-(F) 51015840-GCC AAA AGA GAC TAT TAT GA-31015840 and SA-(R) 51015840-ATT GTT TAC CTG TTT GTG TAC C-31015840 as forwardand reverse primers followed by digestion with 10 U ofrestriction enzymes XapI and Bsp143I (Fermentas VilniusLithuania) [28] Digestion patterns were checked against 1Kb HypeLadderIV (BLIRT SA Gdansk Poland) molecularweight marker and visualized under the UV light

Detection of mecA gene linked to methicillin resistancewas done by polymerase chain reaction (PCR)with sequence-specific primers as described previously by Murakami et al[29] Briefly 533 bp coding region ofmecA genewas amplifiedwith sequence-specific primers (F) 51015840-AAA ATC GAT GGTAAA GGT TGG C-31015840 and (R) 51015840-AGT TCT GCA GTA CCGGAT TTG C-31015840 The PCR amplifications were carried outusing 10 times PCR RED master mix kit (BLIRT SA Gdansk

Poland) in a MJ Mini Personal Thermal Cycler (Bio-RadHercules CA USA) PCR products were electrophoresedin 15 agarose gel containing ethidium bromide (PromegaMadison WI USA) PCR products size was checked against1 Kb HypeLadder IV (BLIRT SA Gdansk Poland) molecularweight marker and visualized under the UV light

23 Phenotypic Drug Resistance Evaluation The resistancephenotypes to methicillin were determined according tothe disc-diffusion method according to the EUCAST rec-ommendation [30] Briefly a bacterial colony suspensionequivalent to 05 McFarland units was inoculated to Mueller-Hinton agar (MHA BTL Łodz Poland) with a 30 120583gcefoxitin disk (EMAPOL Gdansk Poland) and interpretedafter 20 h of incubation at 35∘C All strains were classified asMRSA (methicillin-resistant Staphylococcus aureus) orMSSA(methicillin-sensitive Staphylococcus aureus) by analysis ofgrowth inhibition zone diameter size (lt22 and ge22 respec-tively) according to the EUCAST recommendation [30]

All examined strainswere tested for antimicrobial suscep-tibility to macrolides and lincosamides by the disk diffusionmethod according to the EUCAST recommendations [31]For disk diffusion method 90 mm plates with the agarmedium were inoculated by swabbing the agar with a swabsoaked in a bacterial suspension of 1 times 108 cellsmLThe anal-ysis of antimicrobial susceptibility was done with the disks(EMAPOL Gdansk Poland) containing 2 120583g clindamycin(DA) and 15 120583g erythromycin (E) with distance betweendisksrsquo edges of 15-16 mm [31] The growth inhibition zonediameter size was interpreted after 18 h of incubation at 35∘CThe isolates were classified as resistant or sensitive based onthe zone diameter size and shape

Vancomycin resistance for all strains was determinedwith the use of E-test method according to the EUCASTrecommendation [32]

24 Minimum Inhibitory Concentrations Determination withthe MicrodilutionMethod Theminimum inhibitory concen-trations (MICs) of caffeic acid towards the staphylococcalstrains were measured using the standard microdilution liq-uid method in sterile 96-well polystyrene plates (FL MedicalTorreglia Italy) in a final volume of 200 120583L [33] The cellconcentrations were estimated from the optical densities at600 nm wavelength with the formula CFUmL = A600 (38times 108) where CFU was the number of colony-forming unitsOne hundred microliters of mid-logarithmic-phase bacterialcultures (5 times 105 CFUmL) in TSB was added to 100 120583Lof serially diluted CA (1 2 4 8 16 32 64 128 256 and1024 120583gmL) The stock solution of CA at 4096 120583gl wasprepared from CA powder Serial dilutions were made asfollows 11 wells of 96-well polystyrene plate were filled withTSB in the next step 100 120583l of CA stock solution was addedto the first well and mixed thoroughly subsequently 100 120583lwas transferred to the next and remaining wells in the samemanner and finally from the last well 100 120583l was removedWells containing TSB with bacterial inoculum only served asa bacterial growth control (GC) Additional controls includedTSB alone as a medium sterility control and TSB withdifferent concentrations of CA as a blank Microplates were

BioMed Research International 3

incubated at 37∘C for 24 h and the bacterial cell growthwas assessed by measuring the optical density of culturesat 600 nm wavelength with a Multiskan EX microplatereader (Thermo Electron Corp Vantoa Finland) [34 35]The MIC is defined as the lowest compound concentrationthat yields no visible microorganism growth and it indicatesthe resistance of bacteria to an antimicrobial agent anddetermines the potency of new antimicrobial agents [33] Allexperiments were carried out in triplicate

25 CombinedEffect of CAandAntibiotics on S aureus StrainsAll strains were tested for antimicrobial susceptibility accord-ing to the EUCAST recommendations by the E-test methodusing commercially available MIC Test Strips (LiofilchemItaly) with antibiotics concentration gradient [32] For E-test method 90 mm plates with the MHA were inoculatedby swabbing the agar with a swab soaked in a bacterialsuspension of 1 times 108 cellsmL MIC Test Strips containingconcentration gradient of erythromycin (E) clindamycin(DA) cefoxitin (FOX) and vancomycin (VA) were used forthe analysis of S aureus antimicrobial susceptibility

The combined effects of CA and antibiotics were evalu-ated using plates with MHA with a subinhibitory concentra-tion of CA (one-fourth of the MIC of CA) added [36 37]The test strips were placed onto the agar surface and gentlypressed to ensure contact using the sterile forceps Plates wereincubated at 35∘C for 20 h under aerobic condition Afterincubation MIC values were read The susceptibility testingof each antibiotic for all clinical and reference strains wasperformed in triplicate and the median MIC values werecalculated

In order to assess combined effect of CA and antibioticsMICs changeswere expressed asΔand calculated accordingto the following formula (MIC of antibiotic - MIC of antibi-otic with CA)MIC of antibiotic x 100 Obtained Δ valueswere presented with the opposite sign (-Δ) to indicatethe reduction or increase of MIC value for antibiotic afteraddition of CA in comparison with the MIC for antibioticonly

26 Statistical Analysis To compareMICs andMICs changesacross MRSA and MSSA U MannndashWhitney test was usedand the Kruskal-Wallis tests were used to compare MICs andMICs changes across strains negative for MLSB (macrolidelincosamide and type B streptogramin mechanism) cMLSB(constitutiveMLSB mechanism) and iMLSB (inducible MLSBmechanism)

The MIC changes were expressed as a difference betweenMIC of antibiotic alone and MIC of antibiotic after CAsupplementation [Δ= MIC of antibiotic - MIC of antibioticwith CA]

Bonferroni test was used in a post hoc analysisThe resultsof combined effect of CA and antibiotics were submitted tothe Wilcoxon Signed-Rank Test For all tests p le 005 wasconsidered as statistically significant Data were analyzed byuse of STATISTICA v 100 software (StatSoft Polska) onWindows platform (Microsoft Corp USA)

3 Results and Discussion

31 Identification and Drug Resistance of Examined StrainsAll tested isolates were classified as members of Staphylo-coccus aureus species by both classic microbiological andmolecular methods Detection of mecA gene and the pheno-typic resistance profiles to methicillin MLSB antibiotics andvancomycin were performed for all analyzed strains (Table 1)

Fifteen out of 23 (65) examined staphylococci strainswere resistant to methicillin according to the presence ofmecA gene and 11 (48) demonstrated the constitutivemechanism of resistance to MLSB antibiotics Nine strains(39) exhibited bothmethicillin andMLSB resistance profileand all strains were sensitive to vancomycin

32 Anti-Staphylococcus Action of Caffeic Acid The antibac-terial activity of CA against S aureus strains was observed inall analyzed strains and its magnitude was strain-dependentThe MIC values for CA ranged from 256 to 1024 120583gmLwith a median (Me) of 512 120583gmL lower quartile (LQ) 512120583gmL and upper quartile (UQ) 1024 120583gmL (Table 2) Thelowest MIC value of 256 120583gmL was detected for S aureusstrains number 7 and 20 and all reference strains CA at con-centration of 512 120583gmL inhibited growth of 11 of examinedstaphylococci while the concentration of 1024 120583gmL provedto be active against 7 examined strains (Table 2)

There were no significant differences in CA MICs valuesacross MRSA versus MSSA strains (p = 0463) as wellas between strains sensitive to MLSB antibiotics and withdifferent phenotypes ofMLSB resistance (p = 0949) (Table 2)

33 Effects of Interaction of Caffeic Acid and AntibacterialAgents against S aureus Strains Subsequently we examinedthe effect of CA in the presence of selected antibioticsCombined in vitro interactions of CA and erythromycin(E) clindamycin (DA) cefoxitin (FOX) or vancomycin (VA)are shown in Table 2 A synergistic effect of suppressionof examined strainsrsquo growth was noted when CA was usedin combination with one of three antibiotics erythromycin(p = 00004) clindamycin (p = 00003) and cefoxitin (p= 00003) The addition of one-fourth of the MIC of CAto the MHA medium generally increased sensitivity of theexamined strains to vancomycin but this effect did not reachthe level of statistical significance (p = 0091)

The diminished MICs of E in the presence of CA wasobserved for 16 of examined strains Synergistic effect of CAand E was the most visible for S aureus 3 13 14 17 and 20strains which under the influence of CA showed high level ofsensitivity to E with substantial reduction of MICs by almost100 The level of resistance to E was not affected by the CApresence for S aureus ATCC 25923 S aureus ATCC433008 12 16 and 19 strains The increase of E MIC values afterCA supplementation was noted for S aureus ATCC 6538(Table 2)

The most noticeable decreases (near 100) of DA MICsafter CA supplementation were observed for S aureus strains13 17 and 20 For seven S aureus strains (S aureus ATCC43300 2 7 12 14 16 and 19) we have not observed DAMICschanges in the presence of CA Analyzing the other S aureus


Table 1 Antibiotic resistance profiles for tested Staphylococcus aureus strains

Strain

CefoxitinDiameter of theInhibition Zone

[mm]

Methicillinresistance

according tomecA presence

ErythromycinDiameter of theInhibition Zone

[mm]

ClindamycinDiameter of theInhibition Zone

[mm]

Mechanism ofResistance to

MLSBAntibiotics

The MIC ofVancomycin[120583gmL]

Vancomycinresistanceprofile

S aureus ATCC25923 35 MSSAb 25 25 - 075 Sf

S aureus ATCC43300 21 MRSAc 0 0 cMLSB

d 038 S

S aureus ATCC6538 31 MRSA 30 30 - 050 S

S aureus 1119886 34 MSSA 25 25 - 075 SS aureus 2 32 MSSA 23 25 - 038 SS aureus 3 31 MSSA 0 25 iMLSB

e 050 SS aureus 4 32 MRSA 25 27 - 050 SS aureus 5 13 MRSA 0 30 iMLSB 075 SS aureus 6 31 MSSA 30 35 - 038 SS aureus 7 32 MRSA 35 33 - 050 SS aureus 8 31 MSSA 30 35 - 038 SS aureus 9 30 MRSA 35 25 - 038 SS aureus 10 31 MSSA 10 22 iMLSB 038 SS aureus 11 31 MSSA 21 22 - 038 SS aureus 12 8 MRSA 0 0 cMLSB 075 SS aureus 13 14 MRSA 0 0 cMLSB 075 SS aureus 14 0 MRSA 0 0 cMLSB 075 SS aureus 15 21 MRSA 25 30 - 038 SS aureus 16 18 MRSA 0 0 cMLSB 050 SS aureus 17 11 MRSA 0 0 cMLSB 038 SS aureus 18 19 MRSA 25 30 - 050 SS aureus 19 14 MRSA 0 0 cMLSB 050 SS aureus 20 19 MRSA 0 0 cMLSB 038 S119886119878taphylococcus aureus 1 divide 20 strains from intractable surgical wounds

bMSSA methicillin-susceptible S aureuscMRSA methicillin-resistant S aureusdcMLSB constitutive macrolide lincosamide and type B streptogramin mechanismeiMLSB inducible macrolide lincosamide and type B streptogramin mechanismf S sensitive

strains we noted substantial reduction of DAMICs from 27to 75 (Table 2)

The strongest augmented effect of FOX and CA wasnoted for S aureus 14 17 and 20 strains which showedsensitivity to the lowest concentrations of this antibioticunder the influence of CA with substantial reduction ofMICs at almost 100 For the other examined strains thediminished MICs of FOX after CA supplementation rangedfrom 25 to 91 For some strains the CA-FOX combinationhas not influenced the susceptibility to FOX The MICschanges after supplementation with CA were not observed inthe case of S aureus ATCC 25923 2 6 7 9 and 12 strains(Table 2)

Analysis of the susceptibility of S aureus strains to VA-CAcombination revealed substantial reduction ofMICs from24 to 98 in comparison with MICs of VA alone for 8

staphylococci strains For S aureusATCC 43300 2 and 6 theincrease of MICs was noted in the presence of CA The levelof resistance to VA was not affected by the presence of CAfor twelve S aureus strains (S aureus ATCC 25923 S aureusATCC 6538 3 4 8 9 10 12 15 16 18 and 19) (Table 2)

Statistical analysis revealed no significant differencesbetween MICs changes for MRSA versus MSSA strains (E minusp =0974 DA minus p = 0922 FOX minus p =0089 VA minus p =0264)as well as for MLSB negative versus cMLSB and iMLSB strainswith respect to E minus p =0112 DA minus p =0943 VA minus p = 0368For cefoxitin statistical analysis revealed differences amongstrains with diverse susceptibility to MLSB antibiotics (p =0045) but this was not confirmed in the post hoc analysiswith Bonferroni test (Table 2)

The presented study showed significant synergistic effectof CA in association with E DA and FOX The synergism


Table2

Antibacteria

lpotentia

lofcaffeica

cid(C

A)alone

andinantib

iotic

-phytochem

icalcombinatio

nagainstStaphylo

coccus

aureusTh

ecom

binedeffectofC

Aandantib

iotic

swasevaluated

usingplates

with

MHAenric

hedwith

thes

ubinhibitory

concentrationof

CA

Strain119886

CAbMIC

c(120583gmL)

EdE+

CA-Δ

eDA

fDA+C

A-Δ

FOXg

FOX+

CA-Δ

VA

hVA

+CA

-Δ

Saureus

ATCC

25923

256

038

038

0006

40047

-27

11

0075

075

0Saureus

ATCC

43300

256

256

256

0256

256

012

8-33

038

050

-32

Saureus

ATCC

6538

256

006

40125

950023lt0016

-30

21

-50

050

050

0Saureus

11024

050

025

-50

006

40032

-50

215

-25

075

050

-33

Saureus

2512

050

038

-24

006

4006

40

075

075

0038

075

97Saureus

3512

256

019

lt-999

90023lt0016

-30

15038

-75

050

050

0Saureus

4512

038

019

-50

006

40047

-27

21

-50

050

050

0Saureus

5512

256

4-98

0094

006

4-32

256

24-91

075

050

-33

Saureus

6512

050

0125

-75

006

4lt0016

-75

1515

0038

050

32Saureus

7256

038

019

-50

0032

0032

01

10

050

038

-24

Saureus

81024

019

019

00032lt0016

-50

15050

-67

038

038

0Saureus

9512

038

019

-50

006

4lt0016

-75

11

0038

038

0Saureus

10512

323

-91

0047lt0016

-66

2075

-63

038

038

0Saureus

111024

038

019

-50

0047

0032

-32

15075

-50

038

019

-50

Saureus

12512

256

256

0256

256

0256

256

0075

075

0Saureus

13512

256

0125lt-999

9256

0094lt-999

932

12-63

075

050

-33

Saureus

141024

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9075

lt0016

-98

Saureus

151024

025

019

-24

006

40023

-64

815

-81

038

038

0Saureus

161024

256

256

0256

256

0256

32-88

050

050

0Saureus

17512

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9038

lt0016

-96

Saureus

18512

038

0094

-75

0047lt0016

-66

6075

-88

050

050

0Saureus

191024

256

256

0256

256

0256

64-75

050

050

0Saureus

20256

256lt0016lt-999

9256lt0016lt-999

912

lt0016lt-999

9038

lt0016

-96

Median

512

05

019

-50

006

40032

-32

21

-63

050

05

0pi

00004

00003

00003

0091

a 119878taphylococcusa

ureus1divide20strains

from

intractables

urgicalw

ound

sb C

Acaffeica

cid

c MIC

minim

uminhibitory

concentration

d Eerythromycin

e MICsc

hanges

accordingto

theformula[

(MIC

ofantib

iotic

-MIC

ofantib

iotic

with

CA)

MIC

ofantib

iotic

]x100

f D

Aclin

damycin

g FOX

cefoxitin

h V

Avancomycin

i Wilcoxon

Sign

ed-RankTest


between CA and VAwas also noted but it did not prove to bestatistically significant

34 Discussion The emergence of multidrug-resistant bacte-rial strains results in part from widespread and inappropri-ate use of broad-spectrum antibiotics for treatment of skinand soft tissue infections In consequence the steady rise inthe incidence of intractable wounds infections in the hospitalenvironment has been widely noticed Thus the intensivesearch for new antibacterial compounds and the alternativesto the therapies based on common antibiotics is ongoingAmong many natural sources the polyphenolic plant secondmetabolites have been explored in this content [38ndash43] Ithas been reported that polyphenols show wide range ofpromising anti-inflammatory antimicrobial antiviral andantioxidant activities with low toxicity towards human cells[11]

Our study investigated the antibacterial effect of caffeicacid and evaluated whether the supplementation of this nat-ural compound augments the biological action of commonlyused antibiotics Although the antibacterial properties ofcaffeic acid have been assessed by several authors [3 9 11 1425 44ndash46] the present study to the best of our knowledgeis the first to report activity of CA alone and in antibiotic-phytochemical combination against panel of S aureus clinicalstrains isolated from intractable wounds infections What ismore contrary to present study where pure caffeic acid hasbeen used inmajority of previous experiments various caffeicacid derivatives have been evaluated [38ndash43]

Previous studies on the antimicrobial properties of CAagainst S aureus reference strains in some cases yieldedambiguous results with the different MIC values assessed forthe same strains For example Fu et al explored antibacterialactivity of CA and 23 caffeic acid amides against S aureusATCC 6538 The obtained CA MIC values were lower thannoted in our study and ranged from41 to 50 120583gmL indicatingthat caffeic acid amides had stronger antibacterial activitythan pure CA which suggests that amide groups couldenhance the action of CA [3] Higher value of MIC for caffeicacid (625 120583gmL) was obtained for the same S aureus ATCC6538 strain by Pinho et al [11] In our work the MIC value ofS aureus ATCC 6538 (256 120583gmL Table 2) was one of thesmallest compared to the other analyzed strains Howeverthis strain was relatively insensitive to the synergistic effectsof CA and antibiotics (Table 2) The inconsistency in the CAMIC values between authors could be due to the differencesin the experiment setup

The antibacterial effect of CA against other reference Saureus strains ATCC 29213 and ATCC 25923 was assessedby Vaquero et al [25] who observed antibacterial activity ofCA against S aureus ATCC 29213 while S aureus ATCC25293 turned out to be resistant to this compound [25]Interestingly in our work S aureus ATCC 25293 was oneof the most susceptible strains to CA with MIC value of256 120583gmL However it must be noted that Vaquero inhis research used the disc-diffusion method to evaluatethe sensitivity of the examined strains In turn Luis et alexamined antibacterial activity of CA against S aureusATCC25923 and two clinical MRSA isolates [9] In their study all

examined strains were sensitive to CA (MIC lt 250 120583gmL)In our work MIC values for clinical strains ranged from256 120583gmL to 1024 120583gmL indicating higher resistance toCA Since clinical strains in our work have been isolatedfrom the intractable infections it is possible that they wereless susceptible to wide range of antimicrobials than theseexamined by Luis and coauthors In our study S aureusATCC 43300 was sensitive to CA action (MIC = 256 120583gmL)(Table 2) but in the experiment presented by Kyaw et al [14]CA MIC value against this strain was sixteen times higherthan in our study while MIC values for other tested MRSAstrains ranged from 512 120583gmL to 1024 120583gmL which is inaccordance with data obtained in our study

Lima et al examined antibacterial activity of CA aloneand in combination with selected antibiotics against S aureusclinical strain isolated from rectal swab The MIC valueobtained for CA in their study was ge1024 120583gmL From thewide spectrum of antibiotics which they examined only nor-floxacin exhibited synergistic effect with CA while combinedeffect of CA and cefoxitin clindamycin or erythromycin wasnot observed [44]

According to Luis et al the molecular mechanism ofCA antimicrobial action is associated with polyphenol-membrane interaction Using flow cytometry authors showedincreased membrane permeability depolarization of cellmembrane and reduction of respiratory activity in S aureusATCC 25923 strain in the presence of CA The authorssuggested that CA mechanism of action may be associatedwith damage of cell membrane integrity and interferes withaerobic metabolism of S aureus cells [9] Similar conclusionsabout CA antibacterial activity were drawn by Nguefack et aland Hayouni et al [45 46] Furthermore CA as a phenolicacid shows relatively strong nucleophilic properties [25] thusit can donate an electron pair to electrophile functionalgroups of plasma membrane proteins andor lipids probablyleading to membrane function impairment which is inaccordance with the flow cytometry data [45 46]This notionis also supported by the observation that CA inhibited 120572-hemolysin secretion of in S aureus the process which ismembrane dependent [25]

It has been shown that among many polyphenolic com-pounds caffeic acid could be considered as one of the mostpotent and promising antimicrobial agents Vaquero et alnoted that CA possesses stronger antibacterial activity thanother examined polyphenols gallic acid vanillic acid andprotocatechuic acid [25] Stojkovic et al examined CA activ-ity as a food preservative against S aureus contaminationTheauthors concluded that CA had better antibacterial activitycompared to other tested compounds p-coumaric acid andrutin According to the authors the higher antimicrobialeffect observed for CA could be associated with one morehydroxyl group substituted at the CA phenol ring [23] Basedon our previous studies we can compare antibacterial andsynergistic effects of CA to other natural compounds proto-catechuic acid ethyl ester (EDHB) and catechin hydrate (CH)[21 22] We demonstrated that CA exhibits stronger antibac-terial action against staphylococcal strains than EDHB andCH What is more CA exhibited greater synergistic effectwith antibiotics than other compounds [21 22] Caffeic


acid as a hydroxycinnamic acid has propenoic side chainwhich makes it much less polar than the protocatechuicacid which is hydroxybenzoic acid What is more caffeicacid as less polar compound also exhibits lipophilicity whichmay contribute to interfering with the permeability of thecell membrane Andrade et al in their work proved that 120572-tocopherol as lipophilic compound may damage the phos-pholipid and protein membrane components essential for itsintegrity which consequently leads to increased membranepermeability [47] The experiments with catechins have alsoproven that antibacterial properties of these compounds areincreasingwith the number of carbons in the alkyl chainThisCA property can thus facilitate its transport across the cellmembrane which in turn might be related to the strongerantibacterial action [9 45 46] Therefore the differences inthe antibacterial action of CA CH and EDHB observed inour works can be explained by the mechanisms describedabove [21 25]

Unfortunately the magnitude of CA antibacterial prop-erties are not species dependent but as our data show differamong S aureus strains It is highly probable that the diversesensitivity of the tested staphylococci strains to CA was dueto the ontogenetic diversity within the species The statisticalanalysis excluded that the differences between MIC valueswere affected by methicillin resistance profile or phenotypeof resistance to MLSB antibiotics The CA susceptibility doesnot seem to be influenced by the presence of the mecA geneIn the presented experimental setup the influence of the otherresistance genes on the observed resultsrsquo dispersion couldnot be excluded However since CA molecular mechanismis not fully understood yet we can only speculate on director indirect factors engaged in observed differentiation Thatstrongly points to the necessity for future research on CA-bacterial cell interactions

Our study on antibacterial potential of caffeic acidshowed that CA augments antimicrobial effect of com-mon antibiotics We showed that CA diminishes MIC val-ues for erythromycin clindamycin and cefoxitin in theCA-antibiotic combination (Table 2) while the synergismbetween CA and vancomycin though noted did not reachthe level of statistical significance (Table 2) The resistanceto MLSB antibiotics did not affect MICs changes after CAaddition and the presence ofmecA gene was also irrelevant

As in the case of studies on combined effects of CA andantibiotics which are limited to one work of Lima et al [44]the number of reports addressing the combined effects ofother natural compounds such as ethanol extract of propolisand selected antibiotics [15] baicalein and ciprofloxacin[16] flavones and 120573-lactam antibiotics [48] berberine andazithromycin ampicillin levofloxacin or cefazolin [49]towards S aureus strains are relatively small Published datapoint out to a promising effect of such phenol-antibioticcombination on clinical staphylococci strains In the previousstudies we have analyzed antibacterial and synergistic effectsof other natural compounds protocatechuic acid ethyl ester(EDHB) and catechin hydrate (CH) [21 22] Our resultssupport the notion that CA is one of the most active plantsecond metabolites and show stronger antibacterial actionagainst staphylococcal strains than EDHB and CH What is

more CA exhibited greater synergistic effect with antibioticsthan other analyzed compounds Our previous study onantibacterial and synergistic activity of protocatechuic acidethyl ester on S aureus strains proved significant synergisticeffects between EDHB and DA only The synergism betweenEDHB-E and EDHB-VA combinations was also noted butit did not reach the level of statistical significance while forFOX and EDHB the opposite trend was observed [22] Incase of CH the most noticeable synergistic effect was notedin combination with E and DAThe synergism between CH-VA and CH-FOX combinations was also observed withoutreaching the level of statistical significance [21] On the otherhand the influence of caffeic acid on the antibacterial effectof the above antibiotics showed a substantial reduction of theMICs for three of them (E DA and FOX) The synergismbetween CA and VA was also noted but it did not prove tobe statistically significant

Our study has some limitations The number of S aureusstrains was relatively small and additional antimicrobials incombination with caffeic acid could be evaluated Howeverthis work was planned as a pilot screening aimed to assessantibacterial potential of caffeic acid against staphylococciclinical strains

As we mentioned earlier there is a strong and stillgrowing necessity to find an alternative to the standardantimicrobial therapies Infections caused by multidrug-resistant strains pose serious and rapidly growing medicalproblem and so far no satisfying solution has been found [50]New antibacterial drug discovery and implementation are anextended-in-time and very expensive process It seems thatwe have approached the solid wall in finding new classes ofantibiotics andor their chemical derivatives we can base thenew therapies on In this light the plant secondarymetabolitescould offer a promising alternative [51 52] We and theabove-mentioned studies showed that natural compoundshave lesser antibacterial potential than common antibioticsThe relatively high CAMICs against S aureus clinical strainsexclude its application as a single agent to treat infectionsbut on the other hand subinhibitory CA concentration effec-tively augmented antibacterial activity of common antibioticsallowing for its use in polyphenol-antibiotic combinationSuch synergistic effect of CA and antibiotics is especiallydesired in treatment of intractable wound infections andpossibly may contribute to the reduction of the antibioticsside effects

4 Conclusions

Our study indicates that S aureus susceptibility to CA aloneor in the phenolic acid-antibiotic combination is strain-dependent and is not associated with MRSA and MLSBresistance The combination of caffeic acid with commonantibiotic shows promising synergistic activity against Saureus strains isolated from intractable wound infectionswhich implies the necessity for further research focusedon the mechanisms of antimicrobial action of antibioticCA interactions Such a research would contribute to thedevelopment of the new therapies effective against MDR Saureus clinical strains


Data Availability

The data supporting the results reported in the presentedmanuscript are available at Department of Microbiology andVirology School of Pharmacy with the Division of Labora-tory Medicine in Sosnowiec Medical University of Silesia inKatowice ul Jagiellonska 4 41-200 Sosnowiec Poland andunder Tomasz J Wąsikrsquos e-mail twasiksumedupl

Conflicts of Interest

The authors declares that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by the research grant fromMedicalUniversity of Silesia [nos KNW-2-Z67D6K and KNW-1-026N6l]

References

[1] S Leekha C L Terrell and R S Edson ldquoGeneral principles ofantimicrobial therapyrdquo Mayo Clinic Proceedings vol 86 no 2pp 156ndash167 2011

[2] J A Patzer ldquoStrategia antybiotykoterapii zakazen bakteryjnychw srodowisku szpitalnym w oparciu o wyniki badan mikrobi-ologicznychrdquo Standardy MedycznePediatria vol 6 no 2 pp232ndash240 2009

[3] J Fu K Cheng Z-M Zhang R-Q Fang and H-L ZhuldquoSynthesis structure and structure-activity relationship analysisof caffeic acid amides as potential antimicrobialsrdquo EuropeanJournal of Medicinal Chemistry vol 45 no 6 pp 2638ndash26432010

[4] P D Stapleton S Shah J C Anderson Y Hara J M THamilton-Miller and P W Taylor ldquoModulation of 120573-lactamresistance in Staphylococcus aureus by catechins and gallatesrdquoInternational Journal of Antimicrobial Agents vol 23 no 5 pp462ndash467 2004

[5] A D Metcalfe and M W J Ferguson ldquoMolecular and cellularbasis of regeneration and tissue repair Skin stem and progen-itor cells Using regeneration as a tissue-engineering strategyrdquoCellular and Molecular Life Sciences vol 65 no 1 pp 24ndash322008

[6] N Boucard C Viton D Agay et al ldquoThe use of physicalhydrogels of chitosan for skin regeneration following third-degree burnsrdquoBiomaterials vol 28 no 24 pp 3478ndash3488 2007

[7] M Song Q Zeng Y Xiang et al ldquoThe antibacterial effectof topical ozone on the treatment of MRSA skin infectionrdquoMolecular Medicine Reports 2017

[8] M S Dryden ldquoSkin and soft tissue infection microbiology andepidemiologyrdquo International Journal of Antimicrobial Agentsvol 34 supplement 1 pp S2ndashS7 2009

[9] A Luıs F Silva S Sousa A P Duarte and F DominguesldquoAntistaphylococcal and biofilm inhibitory activities of galliccaffeic and chlorogenic acidsrdquo Biofouling vol 30 no 1 pp 69ndash79 2014

[10] B A Lipsky and C Hoey ldquoTopical antimicrobial therapy fortreating chronic woundsrdquo Clinical Infectious Diseases vol 49no 10 pp 1541ndash1549 2009

[11] E Pinho I C F R Ferreira L Barros A M Carvalho GSoares and M Henriques ldquoAntibacterial potential of north-eastern portugal wild plant extracts and respective phenoliccompoundsrdquo BioMed Research International vol 2014 ArticleID 814590 pp 1ndash8 2014

[12] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[13] B C Aridogan H Baydar S KayaMDemirci D Ozbasar andEMumcu ldquoAntimicrobial activity and chemical composition ofsome essential oilsrdquo Archives of Pharmacal Research vol 25 no6 pp 860ndash864 2002

[14] B M Kyaw S Arora and C S Lim ldquoBactericidal antibiotic-phytochemical combinations against methicillin resistantStaphylococcus aureusrdquo Brazilian Journal of Microbiology vol43 no 3 pp 938ndash945 2012

[15] R D Wojtyczka A Dziedzic D Idzik et al ldquoSusceptibility ofStaphylococcus aureus clinical isolates to propolis extract aloneor in combination with antimicrobial drugsrdquoMolecules vol 18no 8 pp 9623ndash9640 2013

[16] B C L Chan M Ip C B S Lau et al ldquoSynergistic effectsof baicalein with ciprofloxacin against NorA over-expressedmethicillin-resistant Staphylococcus aureus (MRSA) and inhibi-tion of MRSA pyruvate kinaserdquo Journal of Ethnopharmacologyvol 137 no 1 pp 767ndash773 2011

[17] T P T Cushnie and A J Lamb ldquoRecent advances in under-standing the antibacterial properties of flavonoidsrdquo Interna-tional Journal of Antimicrobial Agents vol 38 no 2 pp 99ndash1072011

[18] J Qiu Y Jiang L Xia et al ldquoSubinhibitory concentrationsof licochalcone A decrease alpha-toxin production in bothmethicillin-sensitive and methicillin-resistant Staphylococcusaureus isolatesrdquo Letters in Applied Microbiology vol 50 no 2pp 223ndash229 2010

[19] A Borges C Ferreira M J Saavedra andM Simoes ldquoAntibac-terial activity and mode of action of ferulic and gallic acidsagainst pathogenic bacteriardquoMicrobial Drug Resistance vol 19no 4 pp 256ndash265 2013

[20] T P T Cushnie and A J Lamb ldquoAntimicrobial activity offlavonoidsrdquo International Journal of Antimicrobial Agents vol26 no 5 pp 343ndash356 2005

[21] M Miklasinska M Kepa R D Wojtyczka D Idzik ADziedzic and T J Wąsik ldquoCatechin hydrate augments theantibacterial action of selected antibiotics against staphylococ-cus aureus clinical strainsrdquo Molecules vol 21 no 2 article no244 2016

[22] M Miklasinska M Kepa R D Wojtyczka et al ldquoAntibacterialactivity of protocatechuic acid ethyl ester on staphylococcusaureus clinical strains alone and in combination with anti-staphylococcal drugsrdquoMolecules vol 20 no 8 pp 13536ndash135492015

[23] D Stojkovic J Petrovic M Sokovic J Glamoclija J Kukic-Markovic and S Petrovic ldquoIn situ antioxidant and antimicro-bial activities of naturally occurring caffeic acid p-coumaricacid and rutin using food systemsrdquo Journal of the Science ofFood and Agriculture vol 93 no 13 pp 3205ndash3208 2013

[24] A N Loes L Ruyle M Arvizu K E Gresko A L Wilson andC E Deutch ldquoInhibition of urease activity in the urinary tractpathogen Staphylococcus saprophyticusrdquo Letters in AppliedMicrobiology vol 58 no 1 pp 31ndash41 2014

[25] M J R Vaquero M R Alberto and M C M de NadraldquoAntibacterial effect of phenolic compounds from differentwinesrdquo Food Control vol 18 no 2 pp 93ndash101 2007


[26] M P Almajano R Carbo M E Delgado and M H GordonldquoEffect of pH on the antimicrobial activity and oxidative stabil-ity of oil-in-water emulsions containing caffeic acidrdquo Journal ofFood Science vol 72 no 5 pp C258ndashC263 2007

[27] J F dos Santos S R Tintino T S de Freitas et al ldquoInvitro e in silico evaluation of the inhibition of Staphylococcusaureus efflux pumps by caffeic and gallic acidrdquo ComparativeImmunology Microbiology amp Infectious Diseases vol 57 pp 22ndash28 2018

[28] M M Shah H Iihara M Noda et al ldquodnaJ gene sequence-based assay for species identification and phylogenetic groupingin the genus Staphylococcusrdquo International Journal of Systematicand Evolutionary Microbiology vol 57 no 1 pp 25ndash30 2007

[29] K Murakami W Minamide K Wada E Nakamura HTeraoka and S Watanabe ldquoIdentification of methicillin-resistant strains of staphylococci by polymerase chain reactionrdquoJournal of Clinical Microbiology vol 29 no 10 pp 2240ndash22441991

[30] ldquoEUCAST guidelines for detection of resistance mechanismsand specific resistance of clinical andor epidemiological impor-tancerdquo European Committee on Antimicrobial SusceptibilityTesting version 201 July 2017 httpwwweucastorgresistancemechanisms

[31] ldquoBreakpoint tables for interpretation of MICs and zonesdiametersrdquo European Committee on Antimicrobial SusceptibilityTesting 2015 httpwwweucastorgast of bacteriapreviousversions of documents

[32] ldquoBreakpoint tables for interpretation of MICs and zonediametersrdquo European Committee on Antimicrobial SusceptibilityTesting 2017 httpwwweucastorgast of bacteriapreviousversions of document

[33] D Amsterdam ldquoSusceptibility testing of antimicrobials inliquid mediardquo in Antibiotics in Laboratory Medicine V LorianEd pp 61ndash143 LippincootWilliams andWilkins PhiladelphiaPA USA 5th edition 2005

[34] M Cudic B A Condie D J Weiner et al ldquoDevelopment ofnovel antibacterial peptides that kill resistant isolatesrdquo Peptidesvol 23 no 12 pp 2071ndash2083 2002

[35] K F Devienne andM S G Raddi ldquoScreening for antimicrobialactivity of natural products using a microplate photometerrdquoBrazilian Journal of Microbiology vol 33 no 2 pp 166ndash1682002

[36] A Fernandes Jr E C Balestrin J E C Betoni R D O OrsiM D L R D S da Cunha and A C Montelli ldquoPropolis Anti-Staphylococcus aureus activity and synergism with antimicro-bial drugsrdquoMemorias do Instituto Oswaldo Cruz vol 100 no 5pp 563ndash566 2005

[37] C R Mahon D C Lehman and G Manuselis Textbook ofDiagnostic Microbiology Saunders WB Philadelphia PA USA5th edition 2014

[38] A Ghasemzadeh H Z E Jaafar S Ashkani et al ldquoVariationin secondary metabolite production as well as antioxidantand antibacterial activities of Zingiber zerumbet (L) at dif-ferent stages of growthrdquo BMC Complementary and AlternativeMedicine vol 16 no 1 article no 104 2016

[39] S-S Wang D-M Wang W-J Pu and D-W Li ldquoPhyto-chemical profiles antioxidant and antimicrobial activities ofthree potentilla speciesrdquo BMC Complementary and AlternativeMedicine vol 13 p 321 2013

[40] L J McGaw V P Bagla P A Steenkamp et al ldquoAntifungaland antibacterial activity and chemical composition of polar

and non-polar extracts of Athrixia phylicoides determinedusing bioautography and HPLCrdquo BMC Complementary andAlternative Medicine vol 13 p 356 2013

[41] A Ghasemzadeh H Z E Jaafar A Rahmat and S AshkanildquoSecondary metabolites constituents and antioxidant anti-cancer and antibacterial activities of Etlingera elatior (Jack)RMSm grown in different locations of Malaysiardquo BMC Com-plementary and Alternative Medicine vol 15 no 1 p 335 2015

[42] A Shabbir M Rashid and H N Tipu ldquoPropolis a hope for thefuture in treating resistant periodontal pathogensrdquo Cureus vol8 no 7 p 682 2016

[43] M J Rodrıguez Vaquero P A Aredes Fernandez M C Mancade Nadra and A M Strasser de Saad ldquoPhenolic compoundcombinations on Escherichia coli viability in a meat systemrdquoJournal of Agricultural and Food Chemistry vol 58 no 10 pp6048ndash6052 2010

[44] V N Lima C D M Oliveira-Tintino E S Santos et alldquoAntimicrobial and enhancement of the antibiotic activity byphenolic compounds Gallic acid caffeic acid and pyrogallolrdquoMicrobial Pathogenesis vol 99 pp 56ndash61 2016

[45] J Nguefack B B Budde and M Jakobsen ldquoFive essentialoils from aromatic plants of Cameroon Their antibacterialactivity and ability to permeabilize the cytoplasmic membraneof Listeria innocua examined by flow cytometryrdquo Letters inApplied Microbiology vol 39 no 5 pp 395ndash400 2004

[46] E A Hayouni M Bouix M Abedrabba J-Y Leveau andM Hamdi ldquoMechanism of action of Melaleuca armillaris (SolEx Gaertu) Sm essential oil on six LAB strains as assessedby multiparametric flow cytometry and automated microtiter-based assayrdquo Food Chemistry vol 111 no 3 pp 707ndash718 2008

[47] J C Andrade M F B Morais-Braga G M M Guedes et alldquoEnhancement of the antibiotic activity of aminoglycosides byalpha-tocopherol and other cholesterol derivatesrdquo Biomedicineamp Pharmacotherapy vol 68 no 8 pp 1065ndash1069 2014

[48] Y SatoH Shibata TArai et al ldquoVariation in synergistic activityby flavone and its related compounds on the increased suscepti-bility of various strains of methicillin-resistant Staphylococcusaureus to beta-lactam antibioticsrdquo International Journal ofAntimicrobial Agents vol 24 no 3 pp 226ndash233 2004

[49] G-Y Zuo Y Li J Han G-C Wang Y-L Zhang andZ-Q Bian ldquoAntibacterial and synergy of berberines withantibacterial agents against clinical multi-drug resistant iso-lates of methicillin-resistant Staphylococcus aureus (MRSA)rdquoMolecules vol 17 no 9 pp 10322ndash10330 2012

[50] F D Lowy ldquoAntimicrobial resistance the example of Staphylo-coccus aureusrdquoThe Journal of Clinical Investigation vol 111 no9 pp 1265ndash1273 2003

[51] R Qin K Xiao B Li et al ldquoThe combination of catechin andepicatechin gallate from fructus crataegi potentiates 120573-lactamantibiotics against Methicillin-Resistant Staphylococcus aureus(MRSA) in vitro and in vivordquo International Journal of MolecularSciences vol 14 no 1 pp 1802ndash1821 2013

[52] P W Taylor P D Stapleton and J Paul Luzio ldquoNew ways totreat bacterial infectionsrdquoDrug DiscoveryTherapy vol 7 no 21pp 1086ndash1091 2002

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


coffee beverages argan oil oats wheat rice and olive oil[3 23] It has been reported that CA showed antimicrobialpotential andor synergistic effects with antibiotics againstS aureus S epidermidis K pneumoniae S marcescensP mirabilis E coli P aeruginosa B cereus M luteus Lmonocytogenes and C albicans strains [20 24ndash27]

The majority of studies on antibacterial action of CA orits derivatives have been focused on the reference bacterialstrains Studies on antibacterial potential of CA againstclinical isolates are scarce either with respect to the CA aloneor in CA-antibiotic combination In the face of the observedsteady increase in the incidence of nosocomial skin infectionscaused by bacteria resistant to broad spectrum of antibioticsthe objective of the presented study was to assess in vitroantibacterial potential of caffeic acid alone and in antibiotic-phytochemical combination using a panel of Staphylococcusaureus clinical strains isolated from intractable infectedwounds

2 Materials and Methods

21 Examined StrainsMedia and Reagents Twenty S aureusclinical strains were isolated from infected wounds of hospi-talized patients and 3 S aureus reference strains S aureusATCC 25923 S aureus ATCC 43300 and S aureus ATCC6538 were obtained directly from ATCC (American TypeCulture Collections) To ensure the homogeneity of clinicalstrains all isolates were derived from the surgical woundsleading to the relatively small sample size All examinedstrains were stored in Tryptic Soy Broth (TSB) medium with20 of glycerol at -80∘C until further use Caffeic acid waspurchased from Sigma Chemical Co (St Louis MO USA)and dissolved in DMSO (Sigma Chemical Co St Louis MOUSA) before use

22 Molecular Identification and Characteristic of ClinicalStrains Detection of dnaJ gene fragment was performedby restriction fragment length polymorphism (PCR-RFLP)For PCR-RFLP method bacterial DNA was extracted withthe GeneMATRIX Tissue amp Bacterial DNA Purification KIT(EURx Ltd Gdansk Poland) according to themanufacturerrsquosrecommendations with the modification described by Shahet al [28] Briefly 883 bp region of dnaJ gene coding forthe N-terminal domain of the receptor was amplified withSA-(F) 51015840-GCC AAA AGA GAC TAT TAT GA-31015840 and SA-(R) 51015840-ATT GTT TAC CTG TTT GTG TAC C-31015840 as forwardand reverse primers followed by digestion with 10 U ofrestriction enzymes XapI and Bsp143I (Fermentas VilniusLithuania) [28] Digestion patterns were checked against 1Kb HypeLadderIV (BLIRT SA Gdansk Poland) molecularweight marker and visualized under the UV light

Detection of mecA gene linked to methicillin resistancewas done by polymerase chain reaction (PCR)with sequence-specific primers as described previously by Murakami et al[29] Briefly 533 bp coding region ofmecA genewas amplifiedwith sequence-specific primers (F) 51015840-AAA ATC GAT GGTAAA GGT TGG C-31015840 and (R) 51015840-AGT TCT GCA GTA CCGGAT TTG C-31015840 The PCR amplifications were carried outusing 10 times PCR RED master mix kit (BLIRT SA Gdansk

Poland) in a MJ Mini Personal Thermal Cycler (Bio-RadHercules CA USA) PCR products were electrophoresedin 15 agarose gel containing ethidium bromide (PromegaMadison WI USA) PCR products size was checked against1 Kb HypeLadder IV (BLIRT SA Gdansk Poland) molecularweight marker and visualized under the UV light

23 Phenotypic Drug Resistance Evaluation The resistancephenotypes to methicillin were determined according tothe disc-diffusion method according to the EUCAST rec-ommendation [30] Briefly a bacterial colony suspensionequivalent to 05 McFarland units was inoculated to Mueller-Hinton agar (MHA BTL Łodz Poland) with a 30 120583gcefoxitin disk (EMAPOL Gdansk Poland) and interpretedafter 20 h of incubation at 35∘C All strains were classified asMRSA (methicillin-resistant Staphylococcus aureus) orMSSA(methicillin-sensitive Staphylococcus aureus) by analysis ofgrowth inhibition zone diameter size (lt22 and ge22 respec-tively) according to the EUCAST recommendation [30]

All examined strainswere tested for antimicrobial suscep-tibility to macrolides and lincosamides by the disk diffusionmethod according to the EUCAST recommendations [31]For disk diffusion method 90 mm plates with the agarmedium were inoculated by swabbing the agar with a swabsoaked in a bacterial suspension of 1 times 108 cellsmLThe anal-ysis of antimicrobial susceptibility was done with the disks(EMAPOL Gdansk Poland) containing 2 120583g clindamycin(DA) and 15 120583g erythromycin (E) with distance betweendisksrsquo edges of 15-16 mm [31] The growth inhibition zonediameter size was interpreted after 18 h of incubation at 35∘CThe isolates were classified as resistant or sensitive based onthe zone diameter size and shape

Vancomycin resistance for all strains was determinedwith the use of E-test method according to the EUCASTrecommendation [32]

24 Minimum Inhibitory Concentrations Determination withthe MicrodilutionMethod Theminimum inhibitory concen-trations (MICs) of caffeic acid towards the staphylococcalstrains were measured using the standard microdilution liq-uid method in sterile 96-well polystyrene plates (FL MedicalTorreglia Italy) in a final volume of 200 120583L [33] The cellconcentrations were estimated from the optical densities at600 nm wavelength with the formula CFUmL = A600 (38times 108) where CFU was the number of colony-forming unitsOne hundred microliters of mid-logarithmic-phase bacterialcultures (5 times 105 CFUmL) in TSB was added to 100 120583Lof serially diluted CA (1 2 4 8 16 32 64 128 256 and1024 120583gmL) The stock solution of CA at 4096 120583gl wasprepared from CA powder Serial dilutions were made asfollows 11 wells of 96-well polystyrene plate were filled withTSB in the next step 100 120583l of CA stock solution was addedto the first well and mixed thoroughly subsequently 100 120583lwas transferred to the next and remaining wells in the samemanner and finally from the last well 100 120583l was removedWells containing TSB with bacterial inoculum only served asa bacterial growth control (GC) Additional controls includedTSB alone as a medium sterility control and TSB withdifferent concentrations of CA as a blank Microplates were



















Strain


[mm]




[mm]


[mm]


MLSBAntibiotics





d 038 S












Table2

Antibacteria

lpotentia

lofcaffeica

cid(C

A)alone

andinantib

iotic

-phytochem

icalcombinatio

nagainstStaphylo

coccus

aureusTh

ecom

binedeffectofC

Aandantib

iotic

swasevaluated

usingplates

with

MHAenric

hedwith

thes

ubinhibitory

concentrationof

CA

Strain119886

CAbMIC

c(120583gmL)

EdE+

CA-Δ

eDA

fDA+C

A-Δ

FOXg

FOX+

CA-Δ

VA

hVA

+CA

-Δ

Saureus

ATCC

25923

256

038

038

0006

40047

-27

11

0075

075

0Saureus

ATCC

43300

256

256

256

0256

256

012

8-33

038

050

-32

Saureus

ATCC

6538

256

006

40125

950023lt0016

-30

21

-50

050

050

0Saureus

11024

050

025

-50

006

40032

-50

215

-25

075

050

-33

Saureus

2512

050

038

-24

006

4006

40

075

075

0038

075

97Saureus

3512

256

019

lt-999

90023lt0016

-30

15038

-75

050

050

0Saureus

4512

038

019

-50

006

40047

-27

21

-50

050

050

0Saureus

5512

256

4-98

0094

006

4-32

256

24-91

075

050

-33

Saureus

6512

050

0125

-75

006

4lt0016

-75

1515

0038

050

32Saureus

7256

038

019

-50

0032

0032

01

10

050

038

-24

Saureus

81024

019

019

00032lt0016

-50

15050

-67

038

038

0Saureus

9512

038

019

-50

006

4lt0016

-75

11

0038

038

0Saureus

10512

323

-91

0047lt0016

-66

2075

-63

038

038

0Saureus

111024

038

019

-50

0047

0032

-32

15075

-50

038

019

-50

Saureus

12512

256

256

0256

256

0256

256

0075

075

0Saureus

13512

256

0125lt-999

9256

0094lt-999

932

12-63

075

050

-33

Saureus

141024

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9075

lt0016

-98

Saureus

151024

025

019

-24

006

40023

-64

815

-81

038

038

0Saureus

161024

256

256

0256

256

0256

32-88

050

050

0Saureus

17512

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9038

lt0016

-96

Saureus

18512

038

0094

-75

0047lt0016

-66

6075

-88

050

050

0Saureus

191024

256

256

0256

256

0256

64-75

050

050

0Saureus

20256

256lt0016lt-999

9256lt0016lt-999

912

lt0016lt-999

9038

lt0016

-96

Median

512

05

019

-50

006

40032

-32

21

-63

050

05

0pi

00004

00003

00003

0091



from

intractables

urgicalw

ound

sb C

Acaffeica

cid

c MIC

minim

uminhibitory

concentration

d Eerythromycin

e MICsc

hanges

accordingto

theformula[

(MIC

ofantib

iotic

-MIC

ofantib

iotic

with

CA)

MIC

ofantib

iotic

]x100

f D

Aclin

damycin

g FOX

cefoxitin

h V

Avancomycin

i Wilcoxon

Sign

ed-RankTest



















4 Conclusions



Data Availability




Acknowledgments


References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




















Strain


[mm]




[mm]


[mm]


MLSBAntibiotics





d 038 S












Table2

Antibacteria

lpotentia

lofcaffeica

cid(C

A)alone

andinantib

iotic

-phytochem

icalcombinatio

nagainstStaphylo

coccus

aureusTh

ecom

binedeffectofC

Aandantib

iotic

swasevaluated

usingplates

with

MHAenric

hedwith

thes

ubinhibitory

concentrationof

CA

Strain119886

CAbMIC

c(120583gmL)

EdE+

CA-Δ

eDA

fDA+C

A-Δ

FOXg

FOX+

CA-Δ

VA

hVA

+CA

-Δ

Saureus

ATCC

25923

256

038

038

0006

40047

-27

11

0075

075

0Saureus

ATCC

43300

256

256

256

0256

256

012

8-33

038

050

-32

Saureus

ATCC

6538

256

006

40125

950023lt0016

-30

21

-50

050

050

0Saureus

11024

050

025

-50

006

40032

-50

215

-25

075

050

-33

Saureus

2512

050

038

-24

006

4006

40

075

075

0038

075

97Saureus

3512

256

019

lt-999

90023lt0016

-30

15038

-75

050

050

0Saureus

4512

038

019

-50

006

40047

-27

21

-50

050

050

0Saureus

5512

256

4-98

0094

006

4-32

256

24-91

075

050

-33

Saureus

6512

050

0125

-75

006

4lt0016

-75

1515

0038

050

32Saureus

7256

038

019

-50

0032

0032

01

10

050

038

-24

Saureus

81024

019

019

00032lt0016

-50

15050

-67

038

038

0Saureus

9512

038

019

-50

006

4lt0016

-75

11

0038

038

0Saureus

10512

323

-91

0047lt0016

-66

2075

-63

038

038

0Saureus

111024

038

019

-50

0047

0032

-32

15075

-50

038

019

-50

Saureus

12512

256

256

0256

256

0256

256

0075

075

0Saureus

13512

256

0125lt-999

9256

0094lt-999

932

12-63

075

050

-33

Saureus

141024

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9075

lt0016

-98

Saureus

151024

025

019

-24

006

40023

-64

815

-81

038

038

0Saureus

161024

256

256

0256

256

0256

32-88

050

050

0Saureus

17512

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9038

lt0016

-96

Saureus

18512

038

0094

-75

0047lt0016

-66

6075

-88

050

050

0Saureus

191024

256

256

0256

256

0256

64-75

050

050

0Saureus

20256

256lt0016lt-999

9256lt0016lt-999

912

lt0016lt-999

9038

lt0016

-96

Median

512

05

019

-50

006

40032

-32

21

-63

050

05

0pi

00004

00003

00003

0091



from

intractables

urgicalw

ound

sb C

Acaffeica

cid

c MIC

minim

uminhibitory

concentration

d Eerythromycin

e MICsc

hanges

accordingto

theformula[

(MIC

ofantib

iotic

-MIC

ofantib

iotic

with

CA)

MIC

ofantib

iotic

]x100

f D

Aclin

damycin

g FOX

cefoxitin

h V

Avancomycin

i Wilcoxon

Sign

ed-RankTest



















4 Conclusions



Data Availability




Acknowledgments


References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Strain


[mm]




[mm]


[mm]


MLSBAntibiotics





d 038 S












Table2

Antibacteria

lpotentia

lofcaffeica

cid(C

A)alone

andinantib

iotic

-phytochem

icalcombinatio

nagainstStaphylo

coccus

aureusTh

ecom

binedeffectofC

Aandantib

iotic

swasevaluated

usingplates

with

MHAenric

hedwith

thes

ubinhibitory

concentrationof

CA

Strain119886

CAbMIC

c(120583gmL)

EdE+

CA-Δ

eDA

fDA+C

A-Δ

FOXg

FOX+

CA-Δ

VA

hVA

+CA

-Δ

Saureus

ATCC

25923

256

038

038

0006

40047

-27

11

0075

075

0Saureus

ATCC

43300

256

256

256

0256

256

012

8-33

038

050

-32

Saureus

ATCC

6538

256

006

40125

950023lt0016

-30

21

-50

050

050

0Saureus

11024

050

025

-50

006

40032

-50

215

-25

075

050

-33

Saureus

2512

050

038

-24

006

4006

40

075

075

0038

075

97Saureus

3512

256

019

lt-999

90023lt0016

-30

15038

-75

050

050

0Saureus

4512

038

019

-50

006

40047

-27

21

-50

050

050

0Saureus

5512

256

4-98

0094

006

4-32

256

24-91

075

050

-33

Saureus

6512

050

0125

-75

006

4lt0016

-75

1515

0038

050

32Saureus

7256

038

019

-50

0032

0032

01

10

050

038

-24

Saureus

81024

019

019

00032lt0016

-50

15050

-67

038

038

0Saureus

9512

038

019

-50

006

4lt0016

-75

11

0038

038

0Saureus

10512

323

-91

0047lt0016

-66

2075

-63

038

038

0Saureus

111024

038

019

-50

0047

0032

-32

15075

-50

038

019

-50

Saureus

12512

256

256

0256

256

0256

256

0075

075

0Saureus

13512

256

0125lt-999

9256

0094lt-999

932

12-63

075

050

-33

Saureus

141024

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9075

lt0016

-98

Saureus

151024

025

019

-24

006

40023

-64

815

-81

038

038

0Saureus

161024

256

256

0256

256

0256

32-88

050

050

0Saureus

17512

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9038

lt0016

-96

Saureus

18512

038

0094

-75

0047lt0016

-66

6075

-88

050

050

0Saureus

191024

256

256

0256

256

0256

64-75

050

050

0Saureus

20256

256lt0016lt-999

9256lt0016lt-999

912

lt0016lt-999

9038

lt0016

-96

Median

512

05

019

-50

006

40032

-32

21

-63

050

05

0pi

00004

00003

00003

0091



from

intractables

urgicalw

ound

sb C

Acaffeica

cid

c MIC

minim

uminhibitory

concentration

d Eerythromycin

e MICsc

hanges

accordingto

theformula[

(MIC

ofantib

iotic

-MIC

ofantib

iotic

with

CA)

MIC

ofantib

iotic

]x100

f D

Aclin

damycin

g FOX

cefoxitin

h V

Avancomycin

i Wilcoxon

Sign

ed-RankTest



















4 Conclusions



Data Availability




Acknowledgments


References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Table2

Antibacteria

lpotentia

lofcaffeica

cid(C

A)alone

andinantib

iotic

-phytochem

icalcombinatio

nagainstStaphylo

coccus

aureusTh

ecom

binedeffectofC

Aandantib

iotic

swasevaluated

usingplates

with

MHAenric

hedwith

thes

ubinhibitory

concentrationof

CA

Strain119886

CAbMIC

c(120583gmL)

EdE+

CA-Δ

eDA

fDA+C

A-Δ

FOXg

FOX+

CA-Δ

VA

hVA

+CA

-Δ

Saureus

ATCC

25923

256

038

038

0006

40047

-27

11

0075

075

0Saureus

ATCC

43300

256

256

256

0256

256

012

8-33

038

050

-32

Saureus

ATCC

6538

256

006

40125

950023lt0016

-30

21

-50

050

050

0Saureus

11024

050

025

-50

006

40032

-50

215

-25

075

050

-33

Saureus

2512

050

038

-24

006

4006

40

075

075

0038

075

97Saureus

3512

256

019

lt-999

90023lt0016

-30

15038

-75

050

050

0Saureus

4512

038

019

-50

006

40047

-27

21

-50

050

050

0Saureus

5512

256

4-98

0094

006

4-32

256

24-91

075

050

-33

Saureus

6512

050

0125

-75

006

4lt0016

-75

1515

0038

050

32Saureus

7256

038

019

-50

0032

0032

01

10

050

038

-24

Saureus

81024

019

019

00032lt0016

-50

15050

-67

038

038

0Saureus

9512

038

019

-50

006

4lt0016

-75

11

0038

038

0Saureus

10512

323

-91

0047lt0016

-66

2075

-63

038

038

0Saureus

111024

038

019

-50

0047

0032

-32

15075

-50

038

019

-50

Saureus

12512

256

256

0256

256

0256

256

0075

075

0Saureus

13512

256

0125lt-999

9256

0094lt-999

932

12-63

075

050

-33

Saureus

141024

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9075

lt0016

-98

Saureus

151024

025

019

-24

006

40023

-64

815

-81

038

038

0Saureus

161024

256

256

0256

256

0256

32-88

050

050

0Saureus

17512

256lt0016lt-999

9256lt0016lt-999

9256

lt0016lt-999

9038

lt0016

-96

Saureus

18512

038

0094

-75

0047lt0016

-66

6075

-88

050

050

0Saureus

191024

256

256

0256

256

0256

64-75

050

050

0Saureus

20256

256lt0016lt-999

9256lt0016lt-999

912

lt0016lt-999

9038

lt0016

-96

Median

512

05

019

-50

006

40032

-32

21

-63

050

05

0pi

00004

00003

00003

0091



from

intractables

urgicalw

ound

sb C

Acaffeica

cid

c MIC

minim

uminhibitory

concentration

d Eerythromycin

e MICsc

hanges

accordingto

theformula[

(MIC

ofantib

iotic

-MIC

ofantib

iotic

with

CA)

MIC

ofantib

iotic

]x100

f D

Aclin

damycin

g FOX

cefoxitin

h V

Avancomycin

i Wilcoxon

Sign

ed-RankTest



















4 Conclusions



Data Availability




Acknowledgments


References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




















4 Conclusions



Data Availability




Acknowledgments


References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018










4 Conclusions



Data Availability




Acknowledgments


References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



Data Availability




Acknowledgments


References

























































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018

































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

Antimicrobial Potential of Caffeic Acid against