research article antibacterial and antibiofilm...

Research ArticleAntibacterial and Antibiofilm Activity of Methanolic PlantExtracts against Nosocomial Microorganisms

Eduardo Saacutenchez1 Catalina Rivas Morales1 Sandra Castillo2 Catalina Leos-Rivas1

Ledy Garciacutea-Becerra1 and David Mizael Ortiz Martiacutenez1

1Departamento de Quımica Facultad de Ciencias Biologicas Universidad Autonoma de Nuevo LeonCiudad Universitaria 66451 San Nicolas de los Garza NL Mexico2Departamento de Alimentos Facultad de Ciencias Biologicas Universidad Autonoma de Nuevo LeonCiudad Universitaria 66451 San Nicolas de los Garza NL Mexico

Correspondence should be addressed to Eduardo Sanchez eduardosanchezguanlmx

Received 23 January 2016 Revised 16 May 2016 Accepted 9 June 2016

Academic Editor Serkan Selli

Copyright copy 2016 Eduardo Sanchez et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Biofilm is a complexmicrobial community highly resistant to antimicrobialsThe formation of biofilms in biotic and abiotic surfacesis associated with high rates of morbidity and mortality in hospitalized patients New alternatives for controlling infections havebeen proposed focusing on the therapeutic properties of medicinal plants and their antimicrobial effects In the present studythe antimicrobial and antibiofilm activities of 8 methanolic plant extracts were evaluated against clinical isolated microorganismsPreliminary screening by diffusion well assay showed the antimicrobial activity of Prosopis laevigata Opuntia ficus-indica andGutierrezia microcephala The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) weredetermined ranging from 07 to gt15mgmLThe specific biofilm formation index (SBF) was evaluated before and after the additionof plant extracts (MBC times 075) Opuntia ficus-indica caused the major reduction on SBF in dose-dependent manner Cytotoxicactivity of plant extracts was determined using brine shrimp lethality test (Artemia salina L) Lethal Dose concentration (LD

50

values) of the plant extractswas calculated LD50values forP laevigata and Gmicrocephalawere 1416 and 3233 120583gmL respectively

while O ficus-indica showed a slight lethality with 9392120583gmL Phytochemical analyses reveal the presence of flavonoids tanninsand coumarines

1 Introduction

Microbial biofilms are communities of bacteria embeddedin a self-producing matrix forming on living and nonlivingsolid surfaces [1] Biofilm-associated cells have the ability toadhere irreversibly on a wide variety of surfaces includingliving tissues and indwelling medical devices as cathetersvalves prosthesis and so forth [2]

They are considered an important virulence factor thatcauses persistent chronic and recurrent infections they arehighly resistant to antibiotics and host immune defenses[3] Bacteria protected within biofilm exopolysaccharidesare up to 1000 times more resistant to antibiotics thanplanktonic cells (free-floating) [4] which generates seriousconsequences for therapy and severely complicates treatment

options [5] An estimated 75 of bacterial infections involvebiofilms that are protected by an extracellular matrix [6]

Biofilm resistance is due to several reasons like restricteddiffusion of antibiotics into biofilm matrix expression ofmultidrug efflux pumps type IV secretion systems decreasedpermeability and the action of antibiotic-modifying enzymes[7] The increased biofilm resistance to conventional treat-ments enhances the need to develop new control strategies[8]

Biofilm inhibition is considered as major drug target forthe treatment of various bacterial and fungal infections andpharmacological development of this drugs is now exten-sively studied [9] In recent years several green nonlethalstrategies for biofilm control have been developed becausethe mode of action of these novel antibiofilm agents is

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 1572697 8 pageshttpdxdoiorg10115520161572697

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Overview of the collected plants used in this investigation

Scientific name Common name Family Part used Voucher numberSophora secundiflora (Ortega) Lag ExDC Mountain laurel Fabaceae Aerial parts 027770

Sphaeralcea ambigua A Gray Desert globemallow Malvaceae Bark 027771Prosopis laevigata (Humb et Bonpl exWilld) MC Johnston Smooth mesquite Fabaceae Bark and leaves 027772

Opuntia ficus-indicaMill Nopal cactus Cactaceae Cladode 027773Marrubium vulgare L White horehound Lamiaceae Aerial parts 027774Scutellaria drummondii Benth Drummondrsquos skullcap Lamiaceae Aerial parts 027775Nothoscordum bivalve Britton Crowpoison Alliaceae Bulb 027776Gutierrezia microcephala (DC) Gray Sticky snakeweed Asteraceae Aerial parts 027777

much less susceptible to the emergence of resistance [10]However although they are promising strategies they havedisadvantages because none have been totally effective [5]

One promising alternative is the search for naturallyoccurring compounds of plant origin capable of blockingbiofilm formation [11] Historically plant extracts and theirbiologically active compounds have been a valuable sourceof natural products which have played a central role in theprevention and treatment of diseases helping to maintainhuman health [12] Furthermore they are widely accepteddue to the perception that they are safe and have a long historyof use in folk medicine to cure diseases and illnesses sinceancient times [13]

Considering the above and based on previous resultsobtained in our laboratory in the present study we proposeto evaluate the antibiofilm effect of 8 extracts plants against 5clinical isolated pathogens

2 Material and Methods

21 Plant Material Fresh and healthy plants growing wildaround theCasablanca community located in SantaCatarinaNuevo Leon Mexico (25∘391015840113310158401015840N 100∘421015840410910158401015840W) werecollected between March and April 2014 Voucher sampleswere deposited at the herbariumof the Botanical Departmentof Biological Sciences School Universidad Autonoma deNuevo Leon for identification purposes Collected plants(Table 1) were washed thoroughly in tap water followed bysuccessive washing in distilled water Washed plants were cutinto small pieces and air-dried at room temperature (25plusmn2∘C)under shade Finally dried material was grounded to coarsepowder in amanual grainmill and stored in plastic containersfor further analysis

22 Bacterial Strains and Culture Conditions The microor-ganisms used in this study were 5 nosocomial pathogens4 Gram-negative (Klebsiella pneumoniae Enterococcus fae-calis Escherichia coli and Stenotrophomonas maltophilia)and 1 Gram-positive (Staphylococcus aureus) All strain werekindly provided by Dra Elvira Garza Gomzalez School ofMedicineUANL Strainsweremaintained inMueller-Hinton

(MH) agar (Difco) at 4∘C Active cultures were obtained byinoculation of a loopful of each strain into separated 5mLMH broth (Difco) and incubated for 18 h at 37∘C

23 Preparation of Plant Extracts Extracts were preparedfollowing the methodology proposed by Sanchez et al [14]withminormodifications Briefly one hundred grams (100 g)of dried plant material was soaked with 500mL of methanolfor 24 h at room temperature (25 plusmn 2∘C) under occasionalshaking Extractionwas repeated three times and the extractsobtained were combined and filtered throughWhatman filterpaper number 1 After that theywere concentrated to drynessunder reduced pressure using a rotary evaporator at 45∘CStock solutions (200mgmL) were prepared in methanol andstored at 4∘C in the dark for further experiments

24 Qualitative Phytochemical Screening The extracts weresubjected to standard phytochemical tests in order to evaluatetheir chemical composition for different active constituentsfor this extracts (3ndash5mgmL) they were separately suspendedin 1mL of absolute ethanol or distilled water (carbohydratedetermination) using clean test tubes

25 Bayerrsquos Test for Unsaturation In this case aqueous 1KMnO

4was added dropwise to the extract solution A

positive test was evidenced by the disappearance of thepurple color of KMnO

4and the appearance of a brown solid

precipitate (MnO2) [15]

26 Detection of TriterpenesSteroids (Liebermann-BurchardReagent) One mL of acetic anhydride and 5 drops of con-centrated sulfuric acid (H

2SO4) were added to the extract

A color change from violet to blue confirms the presence ofsteroids [16] and formation of blue-green ring indicated thepresence of terpenoids [17]

27 Coumarins Three mL of 2N NaOH was added to 2mLof aqueous extract Formation of yellow color indicated thepresence of coumarins Confirmation test was performed byadding 1mL of 5N HCl in this case a colorless solutionformed at the upper layer is considered positive [18]

Evidence-Based Complementary and Alternative Medicine 3

28 Alkaloids Ethanolic extracts (20 120583L) were applied onTLC plates (Silica Gel 60G 5 times 10 cm) and eluted usingtoluene-ethyl acetate-diethylamine (70 20 10) as solventsystem Alkaloids were detected after spraying Dragendorff rsquosreagent as orange-brown spots on TLC plates [19]

29 Screening for Sesquiterpene Lactones The Baljet reaction(1 Picric acid in 10 sodium hydroxide) was used to detectsesquiterpene lactones in the extracts Reagents were mixedat a 1 1 ratio and added to 1mL of extracts (2-3mg) Thetransformation of the sodium picrate solutionrsquos yellow colorto orange-red color confirmed the positive reaction [20]

210 Test for Quinones Extracts suspended in ethanol (1mL)were treated with 1mL of concentrated sulfuric acid Forma-tion of red color shows the presence of quinones [21]

211 Carboxyl Group The presence of carboxyl groups wasevidenced by adding 10 drops of 10 sodium bicarbonatesolution visible bubbles of carbon dioxide were considereda positive reaction [21]

212 Test for Tannins Extracts were treated with 1mL of 5ferric chloride which was addedThe presence of tannins wasindicated by the formation of bluish black or greenish blackprecipitate [22]

213 Shinoda Test Few fragments of magnesium metal rib-bon (3-4 pieces) were added to 1mL of ethanolic extractfollowed by dropwise addition of concentrated hydrochloricacid Formation of pink or red color indicated the presence offlavonoids [23]

214 Saponin Two mL of distilled water was added toextracts suspended in ethanol andwas shaken vigorouslyTheformation of copious foam layer indicates the presence ofsaponins [23]

215 Carbohydrates For carbohydrates test extracts (10mg)were suspended in 1mL of distilled water afterward 2mL of02 anthrone reagent and 5 drops of concentrated sulfuricacid were added Dark green color showed the presence ofcarbohydrates [21]

216 Evaluation of Antimicrobial Activity Antimicrobialactivity of plant extracts was performed using the agar-welldiffusion bioassay Briefly 100 120583L of fresh culture (approx-imately 106 CFUmL) was uniformly spread onto Mueller-Hinton agar (MHA) plates by sterile Driglasky loop Theninoculated plates were allowed to dry at room temperaturefor 20min After that wells of 6mm in diameter were madein the agar using a sterilized cup-borer and 100 120583L of eachextract was poured in thewellsMethanolwas used as controlPlates were incubated at 37∘C for 18 h Antibacterial activitywas evidenced by the presence of clear inhibition zone aroundeach well The diameter of this zone was measured andrecorded [14]

217 Assessment of Minimum Inhibitory Concentration (MIC)and Minimum Bactericidal Concentration (MBC) The MICand MBC were determined on plant extracts that showedantimicrobial activity by a broth microdilution methodproposed by Novy et al [24] with minor modificationsBriefly 100 120583L ofMueller-Hinton Broth (Difco) plus differentconcentrations of plant extracts was prepared and transferredto each microplate well to obtain dilutions of the activeextract ranging from 10 to 25mgmL Then 10 120583L of afresh culture (final concentration of 1 times 106 CFUmL) of testorganisms was addedMicroplates were incubated at 37∘C for24 h [25] MIC was defined as the lowest concentration of theextract that restricted the visible growth of microorganismtested

To determineMBC 100 120583L from eachwell that showednovisible growth was reinoculated on MH agar plates then theplates were incubated at 37∘C for 24 h MBC was defined asthe lowest extract concentration showingnobacterial growthMethanol was used as blank and tetracycline (Sigma AldrichMexico City Mexico) as positive control Once the MBCwas recorded the sublethal activity on bacterial growth wasdetermined for this concentrations of 75 50 and 25 ofMBC were tested in a 96-well microplate and the countsof microbial cells were done by plate count technique aspreviously mentioned

218 Biofilm Formation Inhibition The effect of extracts onbiofilm formation was evaluated in 96-well polystyrene flat-bottom plates [26] Briefly 300 120583L of inoculated fresh trypti-case soy yeast broth (TSY) (final concentration 106 CFUmL)was aliquoted into each well of microplate and cultured inpresence of sublethal concentrations (75 50 and 25 ofMBC) previously determined Wells containing medium andthose without extracts and only with methanol were used ascontrols Plates were incubated at 37∘C for 48 h After incu-bation supernatant was removed and each well was washedthoroughly with sterile distilled water to remove free-floatingcells thereafter plates were air-dried for 30min and thebiofilm formed was stained during 15min at room temper-ature with 01 aqueous solution of crystal violet Followingincubation the excess of stain was removed washing the platethree times with sterile distilled water Finally the dye boundto the cells was solubilized by adding 250 120583L of 95 ethanolto each well and after 15min of incubation absorbance wasmeasured using microplate reader at a wavelength of 570 nmBiofilm determination was made using the formula SBF =(AB minus CW)G where SBF is the specific biofilm formationAB is theOD570 nmof the attached and stained bacteria CWis the OD570 nm of the stained control wells containing onlybacteria-free medium and G is the OD630 nm of cell growthin broth [27]

219 Toxicity Bioassay Brine shrimp (Artemia salina) lethal-ity bioassay was carried out in accordance with methodologyproposed by Meyer et al [28] to determine the toxicity ofextract plants For this brine shrimps cysts were hatched ina shallow rectangular container which was divided into twounequal compartments filled with sterile artificial seawater


Table 2 Diameter of inhibition zone of methanolic extracts against clinical isolated bacteria

Plant Inhibition zone (cm)K pneumoniae E faecalis E coli S maltophilia S aureus

S secundiflora NI NI NI NI 21 plusmn 03S ambigua NI NI NI NI 12 plusmn 01P laevigata 14 plusmn 03 17 plusmn 03 15 plusmn 03 NI 26 plusmn 03O ficus-indica 17 plusmn 01 15 plusmn 01 16 plusmn 03 NI 16 plusmn 03M vulgare NI 07 plusmn 001 NI NI 18 plusmn 02S drummondii NI 06 plusmn 001 NI NI 17 plusmn 02N bivalve NI NI NI NI NIG microcephala NI NI 16 plusmn 01 NI 23 plusmn 02Values are means plusmn standard deviations NI no inhibition

(prepared by dissolving sea salt 38 gL and adjusted to pH 85using 1N NaOH) under constant aeration and proper lightCyst (ca 50mg) was sprinkled into the larger compartmentwhich was darkened while the smaller was illuminated Yeastsolution 006was added to the hatching chamber to feed thelarvae After 48 h the phototropic free nauplii were collectedfrom the lighted side

Lethality bioassay was performed using 10 collected nau-plii which were transferred into vials contained tested crudeplant extract at 10 100 and 1000 120583gmL and artificial seawa-ter Appropriate quantities of methanol were used as negativecontrol

After 24 h of incubation live nauplii were counted andthe LC

50values were estimated using a Probit regression

analysis Extracts giving LC50values above 1000120583gmL were

considered nontoxic

220 Statistical Analysis All experimental results wereexpressed as mean plusmn standard deviation (SD) for analysisperformed in duplicate at least three times Statistical analysisof the data was performed by Analysis of Variance (ANOVA)and mean comparison using Studentrsquos 119905-test using SPSSsoftware version 170 The LC

50for bioassay with A salina

was determined according to the Probit statistical method119875 lt 005 was considered statistically significant

3 Results and Discussion

A total of 8 methanolic plant extracts were tested against 5clinical bacterial isolatesMethanol was selected as extractionsolvent because it is one of the best solvents used for theextraction of antimicrobial substances [29 30] Moreovermethanol polarity ensured the extraction of polar and mod-erately polar active compounds from plants against microor-ganisms like terpenoids tannins flavones and polyphenols[31]

Results of preliminary antimicrobial tests performed bythe well diffusion method were quite variable between eachplant extract ranging from 0 to 28 cm (Table 2) P laevigataextract was active against all the clinical isolates while Nbivalve bulb did not show activity against anymicroorganismThe highest diameter of inhibition was obtained with Plaevigata extract (28 plusmn 05 cm) against S aureus strain

followed byG microcephala (23plusmn02 cm) andO ficus-indica(16 plusmn 03 cm) also against S aureus Meanwhile E coli wasless susceptible to these extracts showing diameters of 17 plusmn03 14 plusmn 01 and 16 plusmn 01 cm respectively K pneumoniaeand E faecalis were more resistant to the extracts onlyinhibited by P laevigata and O ficus-indica with inhibitionzones ranging from 07 plusmn 008 to 13 plusmn 02 on the otherhand S maltophiliawas the onlymicroorganism that was notinhibited by the extracts

However the well diffusion assay is considered a qual-itative technique and is mainly used for selecting extractswith antimicrobial activity mostly when diameters zonesof inhibition are ge10mm [32] It is important to recognizethat the size of inhibition zones of different extracts couldbe due to the compounds polarity obtained since a morediffusible but less active extract could give a bigger diameterof inhibition than a nondiffusible butmore active extract [33]

Minimum inhibitory concentration (MIC) results arecomparable to those obtained in the agar-well diffusiontechnique because the lowest MIC were obtained usingthe extracts showing the best antimicrobial activity (datanot shown) Meanwhile results of minimum bactericidalconcentrations (MBC) are listed in Table 3 where P laevigataextract had the lowest MBC with a value of 2mgmL for Ecoli 28mgmL for E faecalis 38mgmL for K pneumoniaeand 07mgmL for S aureus Extracts and O ficus-indica gotthe highest CMB ranging from 10 to ge15mgmL CMBs ofG microcephala were 28 and 83mgmL against S aureusand E coli respectively MBC results show that S aureuswas the more sensitive microorganism being inhibited for 8methanolic extracts while S maltophilia was not inhibitedby any extract Broadly our results agree with previousreports which mention greater activity of extracts towardsGram-positive microorganisms compared to Gram-negativemicroorganisms [34] These differences can probably beattributed to the structural and compositional differencesin the cell wall and membranes [25] The Gram-negativebacteria have an outer membrane that serves as barrier formany molecules also the presence of efflux pump systemhas been demonstrated which can mediate the resistanceto natural compounds [35] Escherichia coli was the mostsusceptible of the Gram-negative bacteria this finding alsoagrees with previous reports [36]


Table 3 Minimum bactericidal concentration (MBC) of methanolic extracts against clinical isolated bacteria

Plant MBC (mgmL)K pneumoniae E faecalis E coli S maltophilia S aureus

S secundiflora NE NE NE NE 91 plusmn 04S ambigua NE NE NE NE gt15P laevigata 38 plusmn 01 27 plusmn 01 15 plusmn 02 NE 07 plusmn 001O ficus-indica gt15 gt15 40 plusmn 03 NE 10 plusmn 02M vulgare NE 07 plusmn 001 NE NE 39 plusmn 03S drummondii NE 06 plusmn 001 NE NE 73 plusmn 02N bivalve NE NE NE NE NEG microcephala NE NE 83 plusmn 02 NE 28 plusmn 03Values are means plusmn standard deviations NE not evaluated

Table 4 Phytochemical screening results of selected methanolic extracts

Compounds P laevigata O ficus-indica G microcephalaUnsaturation mdash ++ ++Triterpenessteroids ++Steroids ++Triterpenes +++TriterpenesCoumarins +++ + +++Alkaloids +++ mdash mdashSesquiterpene lactones mdash mdash ++Quinones mdash + +Carboxyl group mdash mdash mdashTannins +++ ++ +++Saponins mdash mdash mdashCarbohydrates ++ +++ mdashFlavonoids ++ + +++ low intensity reaction ++ medium intensity reaction and +++ strong intensity reaction

According to the previously mentioned results it wasdecided to select 3 plant extracts (P laevigata O ficus-indicaandGmicrocephala)whichwere active against E coli (Gram-negative) and S aureus (Gram-positive) moreover theseextracts showed the lowest MBC

Phytochemical screening results of selected plant extractsare summarized in Table 4 and show the presence ofdifferent functional groups Coumarins alkaloids tanninsand flavonoids were found in P laevigata extract Similarcompounds have been reported in different species of thisplant like P juliflora where the presence of tannins phe-nolics flavonoids steroids terpenes and alkaloids has beenreported [37] Likewise reports of Prosopis spp mentionedthat this plant contains harmine prosopine which is analkaloid reported in several papers tyramine prosopinineand juliflorine which are alkaloids that intercalate into DNAand could explain the antimicrobial activity of this extract[31 38]

In case of O ficus-indica results indicate the presenceof triterpenes coumarins quinones tannins carbohydratesand flavonoids flavonoids cause bacterial death by inhibitingDNA or RNA synthesis and tannins including possibleinhibition of extracellular microbial enzymes [39 40]

Meanwhile triterpenes coumarins quinones tanninsflavonoids and sesquiterpene lactones were found in Gmicrocephala According to Goren et al [41] sesquiterpene

lactones are the main secondary metabolite responsiblefor the antimicrobial activity in Asteraceae family WhileMcDaniel and Ross [42] report the presence of alkaloids andsaponins conferring some toxicity at this plant

Biofilm formation inhibition results by addition of subin-hibitory concentrations (75 50 and 25 of MBC) of plantextracts against E coli and S aureus indicated that theobtained effect was dose-dependent The best biofilm reduc-tion is observed in higher concentrations of the extracts (75of WBC) Similar results were reported by Issac Abrahamet al [43] who reported that methanolic caper extractsignificantly inhibited biofilm formation and EPS productionin E coli Serratia marcescens Pseudomonas aeruginosa andProteus mirabilis As well Ravichandiran et al [44] reportedthat ethanolic extract of the bark of Melia dubia causeda strong suppression of hemolysis swarming motility andbiofilm formation in E coli Results of the effect of concentra-tions corresponding to 75 and 50 ofMBC caused significant(119875 lt 005) reduction of the specific biofilm formation (SBF)of E coli (Figure 1) from approximately 3 (strong biofilm) tolevels of 02 (weak biofilm 75 MBC) and 12 (moderatedbiofilm 50 MBC) The SBF classification categories werementioned by Mittal et al [45] who mention that strongbiofilm producers (SBF index gt 200) intermediate biofilmproducers (SBF index between 1 and 2) andweak biofilmpro-ducers (SBF index lt 100) Similar results were obtained with


000

050

100

150

200

250

300

350

Spec

ific b

iofil

m fo

rmat

ion

(SBF

)

Extract concentration ( MBC)

Ctr MeOHP laevigata

O ficus-indicaG microcephala

0 75 50 25

Figure 1 Inhibition of biofilm formation by different concentrationsof plant extracts against E coli

S aureus (data not shown) Inhibition of biofilm formationcan be explained by the presence of flavonoids previouslyreported such as quercetin kaempferol naringenin andapigenin which are capable of reducing biofilm synthesisbecause they can suppress the activity of the autoinducer-2responsible for cell-to-cell communication [46]

A salina bioassay is used to evaluate the toxicity of plantextracts and has the advantage of being inexpensive reliableand reproducible [47] In a previous study Ahmed et al[48] determined the toxicity of methanol extract of Prosopisspicigera reporting 60 survived nauplii at 100 120583gmL whichis consistent with the results obtained in this work becauseLD50

obtained of P laevigata was 1416 120583gmL indicatingthat the extract is moderately toxic this may be due tothe presence of certain bioactive compounds which may berelated to the antibacterial activity For G microcephala wasmoderately toxic with LD

50of 3233 120583gmL some studies

mentioned that this toxicity may be due to the presence ofsaponins essential oils mono- and sesquiterpenes tanninsand alkaloids [42 49] Results of O ficus-indica indicateslight toxicity (9392 120583gmL) this is consistent as reported byDeciga-Campos et al [50] Low toxicity could be explainedwith the common use of this plant in traditional medicineFurthermore in vivo and in vitro experiments of cladodesand fruits show a beneficial effect on health due to thepresence of flavonoids which have health-related propertieswhich are based in their antioxidant activity [51 52]

4 Conclusions

Some of the plant extracts evaluated in present researchhad potential antimicrobial and antibiofilm activities againstisolated nosocomial bacteria which can be an alternative tocontrol the formation of microbial biofilms or can be used asmodel to the search for new drugs

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by Programa de InvestigacionCientıfica y Tecnologica (PAICyT 2012) UniversidadAutonoma de Nuevo Leon The authors wish to express theirgratitude to Dr Jorge Armando Verduzco Martınez for hisexcellent work in the identification of collected plants

References

[1] R Vasudevan ldquoBiofilms microbial cities of scientific signifi-cancerdquo Journal of Microbiology amp Experimentation vol 1 no 3pp 1ndash16 2014

[2] M R Parsek and P K Singh ldquoBacterial biofilms an emerginglink to disease pathogenesisrdquo Annual Review of Microbiologyvol 57 pp 677ndash701 2003

[3] S S Grant and D T Hung ldquoPersistent bacterial infectionsantibiotic tolerance and the oxidative stress responserdquo Viru-lence vol 4 no 4 pp 273ndash283 2013

[4] T B Rasmussen and M Givskov ldquoQuorum-sensing inhibitorsas anti-pathogenic drugsrdquo International Journal of MedicalMicrobiology vol 296 no 2-3 pp 149ndash161 2006

[5] F Sun F Qu Y Ling et al ldquoBiofilm-associated infectionsantibiotic resistance and novel therapeutic strategiesrdquo FutureMicrobiology vol 8 no 7 pp 877ndash886 2013

[6] D J Musk D A Banko and P J Hergenrother ldquoIron saltsperturb biofilm formation and disrupt existing biofilms ofPseudomonas aeruginosardquo Chemistry and Biology vol 12 no 7pp 789ndash796 2005

[7] M N Alekshun and S B Levy ldquoMolecular mechanisms ofantibacterialmultidrug resistancerdquoCell vol 128 no 6 pp 1037ndash1050 2007

[8] L C Simoes M Simoes and M J Vieira ldquoBiofilm interactionsbetween distinct bacterial genera isolated from drinking waterrdquoApplied and Environmental Microbiology vol 73 no 19 pp6192ndash6200 2007

[9] S K R Namasivayam B Beninton B Christo S M KarthigaiK Arun Muthu Kumar and K Deepak ldquoAnti-biofilm effectof biogenic silver nanoparticles coated medical devices againstbiofilm of clinical isolate of Staphylococcus aureusrdquo GlobalJournal of Medical Research vol 13 no 3 pp 1ndash7 2013

[10] M Simoes L C Simoes and M J Vieira ldquoA review of currentand emergent biofilm control strategiesrdquo LWTmdashFood Scienceand Technology vol 43 no 4 pp 573ndash583 2010

[11] J A Lizana S Lopez A Marchal U Serrano D Velasco andM Espinosa-Urgel ldquoUse of plant extracts to block bacterialbiofilm formationrdquo in High School Students for AgriculturalScience Research Proceedings of the 3rd Congress PIIISA pp 43ndash50 2013

[12] P Karuppiah and M Mustaffa ldquoAntibacterial and antioxidantactivities of Musa sp leaf extracts against multidrug resistantclinical pathogens causing nosocomial infectionrdquo Asian PacificJournal of Tropical Biomedicine vol 3 no 9 pp 737ndash742 2013

[13] T Rasamiravaka Q Labtani P Duez and M El Jaziri ldquoTheformation of biofilms by Pseudomonas aeruginosa a review ofthe natural and synthetic compounds interfering with controlmechanismsrdquo BioMed Research International vol 2015 ArticleID 759348 17 pages 2015

[14] E Sanchez N Heredia and S Garcıa ldquoExtracts of edibleand medicinal plants damage membranes of Vibrio choleraerdquoApplied and Environmental Microbiology vol 76 no 20 pp6888ndash6894 2010


[15] A Ghosh A Bandyopadhyay P Ghosh and P ChatterjeeldquoEvaluation of antibacterial potentiality of a Cyclopenta Naph-thalene tetraol terpenoid isolated from Curcuma caesia RoxbrdquoJournal of Botanical Science vol 3 no 1 pp 27ndash34 2013

[16] M Boxi Y Rajesh V R Kumar B Praveen and KMangammaldquoExtraction phytochemical screening and in-vitro evaluation ofanti-oxidant properties of Commicarpus chinesis (aqueous leafextract)rdquo International Journal of Pharma and Bio Science vol1 no 4 p 547 2010

[17] A B Fawehinmi H Lawal S O Etatuvie and F O OyedejildquoPreliminary phytochemical screening and antimicrobial eval-uation of four medicinal plants traditionally used in Nigeria forskin infectionrdquo African Journal of Pure and Applied Chemistryvol 7 no 2 pp 44ndash49 2013

[18] S J P Jacob and S Shenbagaraman ldquoEvaluation of antioxidantand antimicrobial activities of the selected green leafy vegeta-blesrdquo International Journal of PharmTech Research vol 3 no 1pp 148ndash152 2011

[19] H Wagner and S Bladt Plant Drug Analysis A Thin LayerChromatography Atlas Springer New York NY USA 2ndedition 2001

[20] M A B Aziz ldquoQualitative phytochemical screening and evalu-ation of anti-inflammatory analgesic and antipyretic activitiesof Microcos paniculata barks and fruitsrdquo Journal of IntegrativeMedicine vol 13 no 3 pp 173ndash184 2015

[21] X A Dominguez Metodos en Investigacion FitoquımicaLIMUSA 1973

[22] S Firdouse andPAlam ldquoPhytochemical investigation of extractof Amorphophallus campanulatus tubersrdquo International Journalof Phytomedicine vol 3 no 1 pp 32ndash35 2011

[23] S K Rathore S Bhatt S Dhyani andA Jain ldquoPreliminary phy-tochemical screening of medicinal plant Ziziphus mauritianaLam fruitsrdquo International Journal of Current PahrmaceuticalResearch vol 4 no 3 pp 160ndash162 2012

[24] P Novy H Davidova C S Serrano-Rojero J RondevaldovaJ Pulkrabek and L Kokoska ldquoComposition and antimicrobialactivity of Euphrasia rostkovianaHayne essential oilrdquo Evidence-Based Complementary and Alternative Medicine vol 2015Article ID 734101 5 pages 2015

[25] P A Lambert ldquoCellular impermeability and uptake of biocidesand antibiotics in gram-positive bacteria and mycobacteriardquoJournal of Applied Microbiology vol 92 supplement pp 46Sndash54S 2002

[26] A L S Antunes D S Trentin J W Bonfanti et al ldquoApplicationof a feasible method for determination of biofilm antimicrobialsusceptibility in staphylococcirdquo Acta Patologica Microbiologicaet Immunologica Scandinavica vol 118 no 11 pp 873ndash877 2010

[27] C Niu and E S Gilbert ldquoColorimetric method for identifyingplant essential oil components that affect biofilm formation andstructurerdquoApplied and Environmental Microbiology vol 70 no12 pp 6951ndash6956 2004

[28] B N Meyer N R Ferrigni J E Putnam L B Jacobsen DE Nichols and J L McLaughlin ldquoBrine shrimp a convenientgeneral bioassay for active plants constituentsrdquo Journal ofMedicinal Plants Research vol 45 pp 31ndash34 1982

[29] V C Perez-Najera E C Lugo-Cervantes M Gutierrez-Lomelı and C L Del-Toro-Sanchez ldquoExtraccion de com-puestos fenolicos de la cascara de lima (Citrus limetta risso) ydeterminacion de su actividad antioxidanterdquo Biotecnia vol 15no 3 pp 18ndash22 2015

[30] Y Vaghasiya and S V Chanda ldquoScreening of methanol andacetone extracts of fourteen Indianmedicinal plants for antimi-crobial activityrdquo Turkish Journal of Biology vol 31 no 4 pp243ndash248 2007

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

[32] A Usman F I Abdulrahman and A Usman ldquoQualitativephytochemical screening and in vitro antimicrobial effects ofmethanol stem bark extract of Ficus thonningii (Moraceae)rdquoAfrican Journal of Traditional Complementary and AlternativeMedicines vol 6 no 3 pp 289ndash295 2009

[33] F Savaroglu S Ilhan and C Filik-Iscen ldquoAn evaluation ofthe antimicrobial activity of some Turkish mossesrdquo Journal ofMedicinal Plants Research vol 5 no 14 pp 3286ndash3292 2011

[34] I C Zampini S Cuello M R Alberto et al ldquoAntimicro-bial activity of selected plant species from lsquothe ArgentinePunarsquo against sensitive and multi-resistant bacteriardquo Journal ofEthnopharmacology vol 124 no 3 pp 499ndash505 2009

[35] C Wendakoon P Calderon and D Gagnon ldquoEvaluation ofselected medicinal plants extracted in different ethanol con-centrations for antibacterial activity against human pathogensrdquoJournal of Medicinally Medicinal Plants vol 1 no 2 pp 60ndash682012

[36] D L Njimoh J C N Assob S E Mokake D J NyhalahC K Yinda and B Sandjon ldquoAntimicrobial activities of aplethora of medicinal plant extracts and hydrolates againsthuman pathogens and their potential to reverse antibioticresistancerdquo International Journal of Microbiology vol 2015Article ID 547156 15 pages 2015

[37] S Singh ldquoPhytochemical analysis of different parts of Prosopisjuliflorardquo International Journal of Current PharmacologyResearch vol 4 no 3 pp 59ndash61 2012

[38] A Aqeel A K Khursheed A Viqaruddin and Q SabihaldquoAntimicrobial activity of julifloricine isolated from Prosopisjuliflorardquo Arzneimittel-Forschung vol 39 no 6 pp 652ndash6551989

[39] K Akiyama H Matsuoka and H Hayashi ldquoIsolation andidentification of a phosphate deficiency-induced C-glycosyl-flavonoid that stimulates arbuscular mycorrhiza formation inmelon rootsrdquo Molecular Plant-Microbe Interactions vol 15 no4 pp 334ndash340 2002

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

[41] N Goren H J Woerdenbag and C Bozok-Johansson ldquoCyto-toxic and antibacterial activities of sesquiterpene lactones iso-lated from Tanacetum praeteritum subsp praeteritumrdquo PlantaMedica vol 62 no 5 pp 419ndash422 1996

[42] K C McDaniel and T T Ross ldquoSnakeweed poisonous proper-ties livestock losses and management considerationsrdquo Journalof Range Management vol 55 no 3 pp 277ndash284 2002

[43] S V P Issac Abraham A Palani B R Ramaswamy K PShunmugiah and V R Arumugam ldquoAntiquorum sensing andantibiofilm potential of Capparis spinosardquo Archives of MedicalResearch vol 42 no 8 pp 658ndash668 2011

[44] V Ravichandiran K ShanmugamKAnupama SThomas andA Princy ldquoStructure-based virtual screening for plant-derivedSdiA-selective ligands as potential antivirulent agents againsturopathogenic Escherichia colirdquo European Journal of MedicinalChemistry vol 48 pp 200ndash205 2012


[45] R Mittal S Sharma S Chhibber S Aggarwal V Gupta andK Harjai ldquoCorrelation between serogroup in vitro biofilm for-mation and elaboration of virulence factors by uropathogenicPseudomonas aeruginosardquo FEMS Immunology and MedicalMicrobiology vol 58 no 2 pp 237ndash243 2010

[46] A Vikram G K Jayaprakasha P R Jesudhasan S D Pillai andB S Patil ldquoSuppression of bacterial cell-cell signalling biofilmformation and type III secretion system by citrus flavonoidsrdquoJournal of AppliedMicrobiology vol 109 no 2 pp 515ndash527 2010

[47] J L McLaughlin Ch Chang and D L Smith Simple Bioassayfor the Detection and Isolation of Bioactive Natural ProductsDepartament of Medicinal Chemistry and PharmacognosySchool of Pharmacy and Pharmacal Sciences Pardue Univer-sity West Lafayerre Ind USA 1988

[48] W Ahmed M Ahmed A Rauf S Feroz Z Khan and ZU Haq ldquoPhytochemistry and cytotoxicity of Prosopis spicigerausing Brine Shrimp lethality assayrdquo Topclass Journal of HerbalMedicine vol 3 no 3 pp 14ndash16 2014

[49] J M Kingsbury Poisonous Plants of the United States andCanada Prentice-Hall Englewood Cliffs NJ USA 1964

[50] M Deciga-Campos I Rivero-Cruz M Arriaga-Alba et alldquoAcute toxicity and mutagenic activity of Mexican plants usedin traditional medicinerdquo Journal of Ethnopharmacology vol 110no 2 pp 334ndash342 2007

[51] O Benavente-Garcıa J Castillo F R Marin A Ortuno and JA Del Rıo ldquoUses and properties ofCitrus flavonoidsrdquo Journal ofAgricultural and Food Chemistry vol 45 no 12 pp 4505ndash45151997

[52] G Ginestra M L Parker R N Bennett et al ldquoAnatomicalchemical and biochemical characterization of cladodes fromprickly pear [Opuntia ficus-indica (L)Mill]rdquo Journal of Agricul-tural and Food Chemistry vol 57 no 21 pp 10323ndash10330 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Overview of the collected plants used in this investigation

Scientific name Common name Family Part used Voucher numberSophora secundiflora (Ortega) Lag ExDC Mountain laurel Fabaceae Aerial parts 027770

Sphaeralcea ambigua A Gray Desert globemallow Malvaceae Bark 027771Prosopis laevigata (Humb et Bonpl exWilld) MC Johnston Smooth mesquite Fabaceae Bark and leaves 027772

Opuntia ficus-indicaMill Nopal cactus Cactaceae Cladode 027773Marrubium vulgare L White horehound Lamiaceae Aerial parts 027774Scutellaria drummondii Benth Drummondrsquos skullcap Lamiaceae Aerial parts 027775Nothoscordum bivalve Britton Crowpoison Alliaceae Bulb 027776Gutierrezia microcephala (DC) Gray Sticky snakeweed Asteraceae Aerial parts 027777

much less susceptible to the emergence of resistance [10]However although they are promising strategies they havedisadvantages because none have been totally effective [5]

One promising alternative is the search for naturallyoccurring compounds of plant origin capable of blockingbiofilm formation [11] Historically plant extracts and theirbiologically active compounds have been a valuable sourceof natural products which have played a central role in theprevention and treatment of diseases helping to maintainhuman health [12] Furthermore they are widely accepteddue to the perception that they are safe and have a long historyof use in folk medicine to cure diseases and illnesses sinceancient times [13]

Considering the above and based on previous resultsobtained in our laboratory in the present study we proposeto evaluate the antibiofilm effect of 8 extracts plants against 5clinical isolated pathogens

2 Material and Methods

21 Plant Material Fresh and healthy plants growing wildaround theCasablanca community located in SantaCatarinaNuevo Leon Mexico (25∘391015840113310158401015840N 100∘421015840410910158401015840W) werecollected between March and April 2014 Voucher sampleswere deposited at the herbariumof the Botanical Departmentof Biological Sciences School Universidad Autonoma deNuevo Leon for identification purposes Collected plants(Table 1) were washed thoroughly in tap water followed bysuccessive washing in distilled water Washed plants were cutinto small pieces and air-dried at room temperature (25plusmn2∘C)under shade Finally dried material was grounded to coarsepowder in amanual grainmill and stored in plastic containersfor further analysis

22 Bacterial Strains and Culture Conditions The microor-ganisms used in this study were 5 nosocomial pathogens4 Gram-negative (Klebsiella pneumoniae Enterococcus fae-calis Escherichia coli and Stenotrophomonas maltophilia)and 1 Gram-positive (Staphylococcus aureus) All strain werekindly provided by Dra Elvira Garza Gomzalez School ofMedicineUANL Strainsweremaintained inMueller-Hinton

(MH) agar (Difco) at 4∘C Active cultures were obtained byinoculation of a loopful of each strain into separated 5mLMH broth (Difco) and incubated for 18 h at 37∘C

23 Preparation of Plant Extracts Extracts were preparedfollowing the methodology proposed by Sanchez et al [14]withminormodifications Briefly one hundred grams (100 g)of dried plant material was soaked with 500mL of methanolfor 24 h at room temperature (25 plusmn 2∘C) under occasionalshaking Extractionwas repeated three times and the extractsobtained were combined and filtered throughWhatman filterpaper number 1 After that theywere concentrated to drynessunder reduced pressure using a rotary evaporator at 45∘CStock solutions (200mgmL) were prepared in methanol andstored at 4∘C in the dark for further experiments

24 Qualitative Phytochemical Screening The extracts weresubjected to standard phytochemical tests in order to evaluatetheir chemical composition for different active constituentsfor this extracts (3ndash5mgmL) they were separately suspendedin 1mL of absolute ethanol or distilled water (carbohydratedetermination) using clean test tubes

25 Bayerrsquos Test for Unsaturation In this case aqueous 1KMnO

4was added dropwise to the extract solution A

positive test was evidenced by the disappearance of thepurple color of KMnO

4and the appearance of a brown solid

precipitate (MnO2) [15]

26 Detection of TriterpenesSteroids (Liebermann-BurchardReagent) One mL of acetic anhydride and 5 drops of con-centrated sulfuric acid (H

2SO4) were added to the extract

A color change from violet to blue confirms the presence ofsteroids [16] and formation of blue-green ring indicated thepresence of terpenoids [17]

27 Coumarins Three mL of 2N NaOH was added to 2mLof aqueous extract Formation of yellow color indicated thepresence of coumarins Confirmation test was performed byadding 1mL of 5N HCl in this case a colorless solutionformed at the upper layer is considered positive [18]
























considered nontoxic























000

050

100

150

200

250

300

350

Spec

ific b

iofil

m fo

rmat

ion

(SBF

)


Ctr MeOHP laevigata


0 75 50 25







4 Conclusions


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































considered nontoxic























000

050

100

150

200

250

300

350

Spec

ific b

iofil

m fo

rmat

ion

(SBF

)


Ctr MeOHP laevigata


0 75 50 25







4 Conclusions


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























000

050

100

150

200

250

300

350

Spec

ific b

iofil

m fo

rmat

ion

(SBF

)


Ctr MeOHP laevigata


0 75 50 25







4 Conclusions


Competing Interests


Acknowledgments


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article antibacterial and antibiofilm...

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