Research Article Methicillin-Resistant Staphylococcus ...downloads.hindawi.com/journals/bmri/2016/4708425.pdf · Research Article Methicillin-Resistant Staphylococcus aureus Biofilms

Research ArticleMethicillin-Resistant Staphylococcus aureus Biofilms andTheir Influence on Bacterial Adhesion and Cohesion

Khulood Hamid Dakheel1 Raha Abdul Rahim23 Vasantha Kumari Neela4

Jameel R Al-Obaidi5 Tan Geok Hun6 and Khatijah Yusoff13

1Department of Microbiology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia 43400 SerdangSelangor Darul Ehsan Malaysia2Department of Cell and Molecular Biology Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra Malaysia 43400 Serdang Selangor Darul Ehsan Malaysia3Institute of Bioscience Universiti Putra Malaysia 43400 Serdang Selangor Darul Ehsan Malaysia4Department of Medical Microbiology and Parasitology Faculty of Medicine and Health Sciences Universiti Putra Malaysia43400 Serdang Selangor Darul Ehsan Malaysia5Agro-Biotechnology Institute Malaysia (ABI) co MARDI Headquarters 43400 Serdang Selangor Malaysia6Department of Agriculture Technology Faculty of Agriculture Universiti Putra Malaysia 43400 Serdang Selangor Malaysia

Correspondence should be addressed to Khatijah Yusoff kyusoffupmedumy

Received 14 October 2016 Revised 8 November 2016 Accepted 13 November 2016

Academic Editor Carla R Arciola

Copyright copy 2016 Khulood Hamid Dakheel et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Twenty-fivemethicillin-resistant Staphylococcus aureus (MRSA) isolateswere characterized by staphylococcal proteinAgene typingand the ability to form biofilms The presence of exopolysaccharides proteins and extracellular DNA and RNA in biofilms wasassessed by a dispersal assay In addition cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead methodandmechanical disruption respectivelyThe predominant genotype was spa type t127 (22 out of 25 isolates) the majority of isolateswere categorized asmoderate biofilm producers Twelve isolates displayed PIA-independent biofilm formation while the remaining13 isolates were PIA-dependent Both groups showed strong dispersal in response to RNase and DNase digestion followed byproteinase K treatment PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment whereas PIA-independent biofilms showed enhanced biofilm formation There was no correlation between the extent of biofilm formation orbiofilm components and the adhesion or cohesion abilities of the bacteria but the efficiency of adherence to glass beads increasedafter biofilm depletion In conclusion nucleic acids and proteins formed the main components of the MRSA clone t127 biofilmmatrix and there seems to be an association between adhesion and cohesion in the biofilms tested

1 Introduction

Since it was first identified in 1961 methicillin-resistantStaphylococcus aureus (MRSA) has been implicated in noso-comial infections worldwide [1]These infections can compli-cate treatments involving in-dwelling catheters and medicalimplants through biofilm formation [2]

Biofilms can be graded based on the activities of thebacteria within them Distinct subpopulations of the bacte-ria are located within the biofilm based on their differentmetabolic states [3] The cells on the surface of the biofilm

are aerobic whereas those located deeper where the oxygenconcentration is low are fermentative and dormant [4 5]Therefore distinct matrix layers representing subpopulationsof bacteria are found in biofilms resulting in differentselective pressures and the emergence of antibiotic-resistantstrains [6ndash8] In most cases biofilm-associated infections aredetected after the biofilms are already formed and can nolonger be eliminated because of the tolerance of the biofilmto most antimicrobial treatments [4]

The biofilm matrix components comprising polysac-charides proteins and DNA play a major role in its

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4708425 14 pageshttpdxdoiorg10115520164708425

2 BioMed Research International

general structure and contribute to its conservation andresistance phenotype [9] In general two biofilm phenotypeshave been identified Polysaccharide intercellular adhesion-(PIA-) dependent biofilms are composed of poly-120573-16-N-acetylglucosamine- (PNAG-) based matrices PIA is synthe-sized from the products of genes located at the ica locus [10]The other type PIA-independent biofilm is composed of cellsurface components such as teichoic acid [11] fibronectin-binding proteins FnBpA and FnBpB [12ndash15] and autolysinextracellular DNA (eDNA) [16 17]

The synthesis of biofilms is influenced by a number offactors Biofilm production however does not appear tobe linked to the ica locus OrsquoNeill et al [18] observed thatalthough the ica locus is present and expressed in PIA-independent biofilms the genes do not appear to be involvedin their formation Houston et al [19] found that deletionof the major autolysin (atl) gene in MRSA strains impairedtheir ability to form FnBP-dependent biofilms Some MRSAclinical isolates even produce biofilms of both phenotypesMRSA strain 132 is able to switch from PIA-dependent toPIA-independent proteinaceous biofilm matrices dependingon environmental conditions [15]

The biofilm dispersion is investigated in vitro usingenzymatic detachment methods [20] and treatment withchemicals such as periodate (HIO

4or NaIO

4)These conven-

tional methods are used to identify the biofilm matrix com-ponents of both Gram-negative and Gram-positive bacteria[21 22] Moreover bacteria within biofilms are significantlyaffected by matrix components that influence adhesion ofthe cells to solid substrata and cohesion between bacterialcells [23] Specificmatrix components can increase the abilityof bacteria to aggregate [24] The structure of extracellularpolymeric substances (EPS) is complex and variable and itsprecise role in cell adhesion and cohesion is not completelyunderstood [17]

The aims of this study were to examine the ability ofa collection of MRSA isolates with spa type t127 to formbiofilms to determine the extracellular matrix componentsin the biofilms formed by these strains and to elucidate theinfluence of biofilms on the ability of these bacteria to adhereand aggregate

2 Material and Methods

21 Identification and Genotyping of MRSA Strains A totalof 25 MRSA clinical isolates were obtained from the MedicalMicrobiology Laboratory at the Universiti Putra MalaysiaThese isolates were obtained from different systemic in-fection sites and their identity was confirmed by Gramstaining growth on mannitol-salt agar (Oxoid UK) andCHROMagarMRSA (Paris France) Kirby-Bauer testing wasperformed for oxacillin (1 120583g) (Oxoid UK) and cefoxitin(30 120583g) (Oxoid UK) on Muller-Hinton agar (Oxoid UK)[25] The MRSA strain ATCC33591 and clinical methicillin-sensitive Staphylococcus aureus (MSSA) strain were used asstandards in every test which were performed in triplicateThe isolates were confirmed to be S aureus by detectionof the Sa442 fragment and MRSA by detection of the

mecA gene A single polymerase chain reaction (PCR) wasused to detect the Sa442 fragment with the Sa442 forwardprimer 51015840-AATCTTTGTCGGTACACGATATTCTTCACG-31015840 and Sa442 reverse primer 51015840-CGTAATGAGATTTCA-GTAAATACAACA-31015840 PCR conditions were the follow-ing an initial temperature of 96∘C (3min) followed bydenaturation at 95∘C (1min) annealing at 55∘C (30 s) andelongation at 72∘C (3min) and a final elongation step at72∘C (4min) Amplicons of the expected size (108 bp) wereobtained [26] The mecA gene was detected using mecA for-ward primer 51015840-ACCAGATTACAACTTCACCAGG-31015840 andmecA reverse primer 51015840-CCACTTCATATCTTGTAACG-31015840initial temperature of 95∘C (1min) denaturation 95∘C (15 s)annealing 45∘C (15 s) and elongation 72∘C (30 s) with afinal extension at 72∘C (4min) Amplicons of the expectedsize (162 bp) existed [27] All isolates were subjected to spatyping according to Christensen et al [28] The polymor-phic X region of the protein A gene (spa) was amplifiedwith primer designed from an S aureus sequence in Gen-Bank (accession number J01786) 1079 F [1079ndash1099] 51015840-TCATCCAAAGCCTTAAAGACC-31015840 and 1516R [1536ndash1516]51015840-GTCAGCAGTAGTGCCGTTTG-31015840 The PCR reactionwas performed using a KOD FX Neo Kit from Toyobo CoLtd (Osaka Japan) as recommended by the manufacturerPCR conditions were 94∘C for 2min 35 cycles each of94∘C for 30 s 50∘C for 30 s and 72∘C for 60 s and afinal extension at 72∘C for 5min The expected productsize was between 300 bp and 600 bp with the size varyingby the number of spa repeats All PCR products weresequenced using 1st BASE (BioSyntech Inc) after purifi-cation with the GeneJET PCR Purification Kit (ThermoFisher Scientific) Sequence assembly was performed inClone Manager Basic 9 (SciEd) followed by analysis ofthe spa tandem repeats using spa typing online software(httpspatyperfortinbrasus) and the Ridom Spa Serverdatabase (httpwwwspaserverridomde) [29]

22 Biofilm Semiquantification with Crystal Violet (CV) Stain-ing Biofilm formation byMRSA strains was quantified usingthe microwell plate method described by Christensen et aland Manago et al [28 29] All MRSA isolates were grownin tryptone soya broth with 1 glucose (TSBG) and then250 120583L of each bacterial strain culture was diluted to an 119860

600

of 005 and incubated in 96-well flat-bottomed polystyrenemicrowell plates (MWP) at 37∘C for 48 h without shakingThe well contents were removed by flipping the plates andthe wells were washed with phosphate buffered saline (PBSpH 72) heat-fixed by exposing the plate to hot air at 60∘Cin a hybridization oven (model HS-101 Amerex USA) for1 h and then stained with 250120583L of 01 (wv) CV solutionfor 15min at room temperature to allow the dye to penetratethe biofilm and be washed with tap water The plates wereemptied and left to dry overnight Biofilmswere quantified byeluting the CV stainwith 250120583L of 33 glacial acetic acid andmeasuring the absorbance of the solution at 570 nm (119860

570)

using a BioTek Synergy 2 plate reader The biofilm assay wasperformed for each strain in triplicate using amicrowell plateand the background was determined by using noninoculated

BioMed Research International 3

media as a control The amount of biofilm produced wasquantified by comparing the experimental values betweenthe inoculated and noninoculatedmediaThe cut-off value ofnoninoculated media at an optical density at 570 nm (OD

570)

was recorded as 131 This value was considered the deadlinepoint to define biofilm quantities The biofilm formationabilities of isolates were categorized based on this valueThe isolates were considered strong biofilm producers anddenoted as ldquo+++rdquo when the absorbance was more than 524(119860570gt 524) moderate biofilm producers as ldquo++rdquo when the

absorbance was between 262 and 524 (119860570

= 262ndash524)weak biofilm producers as ldquo+rdquo (131 lt 119860

570lt 262) and

biofilm nonproducers as ldquominusrdquo (119860570lt 131) These criteria

were established by Stepanovic et al [30]

23 Phenotypic Evaluation of Colony Morphotypes Colonymorphologieswere assessed using a spot test on tryptone soyaagar (Oxoid UK) supplemented with 1 glucose (TSAG)whereas Congo red agar [brain heart infusion agar (OxoidUK) supplemented with 5 sucrose and 40 120583gmL Congored dye (BDH Chemicals Ltd UK)] was used to differentiatebetween PNAG-producing (black colony) and non-PNAG-producing (red colony) phenotypes as described previously[18] In brief strains were cultured on TSAG (1 glucose)plates at 37∘C for 16 h Cells were resuspended in tryp-tone soya broth (TSB) medium and the concentration wasadjusted to an OD

600of 2 Five microliters of the suspension

was spotted on TSAG and Congo agar plates The phenotypewas observed after 48 h

24 Biochemical Composition of Biofilms Biofilms were pre-pared in 96-well plates of MWP as described above andthen treated with 250120583L of 40mM NaIO

4in 50mM sodium

acetate buffer (pH 55) for exopolysaccharides degradationproteinase K (01 and 1mgmL) in 20mM Tris-HCl (pH 75)with 100mMNaCl and trypsin (01 and 1mgmL) for proteindegradation 140UmL DNase I in 5mM MgCl

2for DNA

degradation and RNase 100 120583gmL for RNA degradation Allplates were incubated for 16 h at 37∘C except for plates withNaIO4and its control which were incubated at 37∘C in the

dark for 16 h [22 31 32] In addition deoxyribonuclease witha final concentration of 140UmL in magnesium peptonewater buffer (01 peptone and 5mM MgCl

2) which was

incubated at 37∘C for 16 h and proteinase K with a finalconcentration of 100120583gmL in Tris-peptone buffer (01peptone 20mM Tris-HCl [pH 75] and 100mM NaCl)which was incubated at 37∘C for 16 h were added successivelyto the established biofilm in MWP Control wells were filledwith appropriate buffers without enzymes The biofilms wererinsed twice with PBS (pH 72) dried for 1 h at 60∘C andstained with 01 CV as described above Biofilm dispersionwas calculated as the absorbance of the CV-stained biofilm at570 nm For each sample three replicates were used and eachexperiment was repeated at least three times independently

25 Role of Biofilms in MRSA Adhesiveness and CohesivenessTwo preparations of bacterial cells ldquounwashed cellsrdquo andldquowashed cellsrdquo were prepared for each MRSA isolate After

Figure 1 The packed-bead method was used to test cell adhesive-ness

an overnight incubation in TSB supplemented with 1glucose each bacterial culture was diluted to OD

660= 08

in TSB without glucose Then 80mL from each samplewas centrifuged at 8000timesg for 10min The pellet formedwas dissolved in 80mL PBS (pH 72) These cells wereconsidered ldquounwashed cellsrdquo a substantial amount of biofilmmatrix was left on their cell walls Mechanical disruptionwas used to prepare ldquowashed cellsrdquo by repeatedly dissolvingcell pellets in 80mL PBS (pH 72) followed by sonication(Sonic Ruptor 400 OMNI International GA USA) for 5min(1min sonication power output 5 pulses 5 with 30 s rest) andcentrifugation The supernatant was discarded and the cellpellet was resuspended in PBS by vortexingThis process wasrepeated five timesWashed and unwashed cells of each of the25 bacterial isolates were used to determine cell adhesivenessby the packed-bead method as shown in Figure 1 accordingto [24]

MRSA biofilm cohesiveness (aggregation) was assessedusing the washed cells Total culture turbidity was measuredat 660 nm with the initial turbidity designated OD

119905and the

culture after the fifth round of sonication designated OD119904

The percentage of cells that were aggregated was estimatedas follows aggregation = [(OD

119905minus OD

119904) times 100]OD

119905

as described previously [33 34] These experiments wereperformed three times independently in a sterilized laminarflow cabinet

3 Statistical Analysis

All statistical analyses were performed using SPSS Statistics21 for windows (IBM) Data were expressed as mean valuesplusmn standard error of mean (SEM) Comparison of OD

570and

OD660

between groups was carried out using Studentrsquos 119905-testAll results were considered statistically significant at the 119901 lt005 level

4 Results

41 Confirmation of S aureus Identity All isolates studiedproduced golden-yellow round smooth raised and mucoidcolonies surrounded by a large yellow zone on mannitol-salt


81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

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81

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82

66

77

t12711

t12725

t22469

t1277

t1278

t1275

t12716

t79019

t22320

t12721

t12722

t12724

t12718

t1273

t1274

t12713

t12714

t1271

t12710

t12723

t12717

t1272

t12712

t1276

t12715

Figure 2 Phylogenetic tree based on the spa type ofMRSA isolatesThe phylogenetic tree was constructed based on the nucleotide sequencesof spa gene using MEGA7 Molecular Evolutionary Genetics Analysis version 70 by using neighbour-joining method

agar and changed in colour from rose to mauve on CHRO-Magar MRSA These isolates were confirmed to be S aureusby the presence of the specific glutamate synthetase (Sa442)fragment and to be methicillin-resistant by the presence ofthe mecA gene All isolates were completely resistant (100)to oxacillin and cefoxitin Isolates were classified into fourclones with the majority (2225) belonging to clone t127and the others belonging to t2246 (125) t790 (125) andt223 (125) Phylogenetic tree analysis for these clones wasshown in Figure 2 Furthermore the Ridom Spa Server-Spa-MLSTmapping shows that clone t127 related to sequence type(ST-1)

42 Biofilm Formation Of the 25 MRSA isolates 22 (88)exhibited moderate biofilms with an average OD

570ranging

from 2696 to 3257 whereas three (12) exhibited weakbiofilms with an average OD

570between 1916 and 2590 The

vastmajority of the isolates (1922) belonged to clone t127 andexhibited moderate biofilm formation (Table 1)

43 Morphology of MRSA MRSA biofilms on TSA with 1glucose developed complex architectural features as shown inFigure 3(a) including a layer of highly autoaggregated cellsat the centre of each colony mounted on transparent layersof adherent cells with irregular margins along the edges


Table 1 Quantification of biofilms formed by methicillin-resistantStaphylococcus aureus isolates by microwell plate assay Biofilmswere stainedwith 01 crystal violet solution after 48 h of incubationat 37∘CThe values represent mean plusmn standard error of mean (SEM)for three independent replicates

Isolates Biofilm formationMean plusmn SEM Type of Biofilm

t1271 3246 plusmn 0099 Moderate (++)t1272 3248 plusmn 0134 Moderate (++)t1273 3071 plusmn 0352 Moderate (++)t1274 3245 plusmn 0055 Moderate (++)t1275 3226 plusmn 0115 Moderate (++)t1276 3256 plusmn 0070 Moderate (++)t1277 3121 plusmn 0067 Moderate (++)t1278 2942 plusmn 0282 Moderate (++)t22469 2771 plusmn 0425 Moderate (++)t12710 2761 plusmn 0438 Moderate (++)t12711 2590 plusmn 0448 Weak (+)t12712 3114 plusmn 0330 Moderate (++)t12713 2409 plusmn 0440 Weak (+)t12714 2575 plusmn 0729 Weak (+)t12715 3166 plusmn 0110 Moderate (++)t12716 2696 plusmn 0740 Moderate (++)t12717 2616 plusmn 0951 Weak (+)t12718 2083 plusmn 0617 Moderate (++)t79019 2879 plusmn 0618 Moderate (++)t22320 2735 plusmn 0750 Weak (+)t12721 1916 plusmn 0970 Weak (+)t12722 1219 plusmn 0406 Moderate (++)t12723 2884 plusmn 0548 Moderate (++)t12724 2696 plusmn 0533 Moderate (++)t12725 3257 plusmn 0095 Moderate (++)

Some colonies had circular or vertical lines radiating fromthe centre giving the colonies a bloom-shaped appearanceSome of these colonies were black because of the presence ofexopolysaccharides or red because of the presence of proteinson Congo red agar (Figure 3(b))

44 Biofilm Components The mature MRSA biofilms wereexamined for interactions with NaIO

4 proteinase K trypsin

DNase I and RNase A Figure 4 shows 48 h MRSA biofilmsformed inmicrowell plates that were subsequently exposed toNaIO4for 16 h Some isolates showed significant detachment

of biofilms and displayed reductions in biofilms of 76(t79019) 67 (t12717) and 42ndash52 in the rest of the isolatesIn contrast isolates t22320 t22469 t1277 t12725 andt1271 showed only a slight reduction in biofilm formation inthe presence of NaIO

4 The remaining isolates (t1273 t1275

t12710 t12716 t12723 and 12724) showed an increase inbiofilm formation when treated with NaIO

4of up to twofold

compared to that of the control

Biofilm formed from all of the isolates displayed a rangeof sensitivities to proteinase K (100120583gmL) (Figure 5) Isolatet1276 showed only a 14 reduction in biofilm biomasswhereas isolate t12722 showed strong dispersal of the biofilm(a 75 reduction) No significant biofilm dispersal wasobserved for isolates t1272 t1273 t1274 t1275 t1276t12712 and t12716 however these isolates displayed areduction in biomass of up to 30 In contrast isolatest12711 t12713 and t22320 exhibited significant differencesbetween their replicates with a 29 reduction in biofilmformation by isolates t12713 and t22320 whereas isolatet12711 showed only a 27 reduction relative to that of thecontrol

Because proteinase K (100120583gmL) did not completely dis-perse the established biofilms the experiments were repeatedwith a higher concentration of proteinase K (1mgmL)Interestingly as shown in Figure 6 proteinase K at thisconcentration enhanced biofilm formation in the majorityof the isolates tested except for isolates t12722 and t12725which showed reductions in biofilmbiomass of 56 and 48respectively Isolates t12715 t12718 and t12723 seemed notto be affected by proteinase K at this concentration in spite ofshowing sensitivity to proteinase K at the lower concentrationof 100120583gmL

When trypsin (100 120583gmL) was added to a 48 h estab-lished biofilm some of the isolates displayed biofilm dis-persion whereas others displayed biofilm enhancement Asseen in Figure 7 isolates t12715 t12718 t22319 t12721t12722 and t12725 showed a significant reduction in biofilmbiomass (up to 60) when compared to isolates t12714t12716 t22320 and t12723 which displayed a reduction ofno more than 23 The remaining isolates showed biofilmenhancement in the presence of trypsin (100 120583gmL) Theexperiments when repeated with a higher concentration oftrypsin (1mgmL) (Figure 8) and isolates t1271 t12715t12718 t22319 t12321 t12722 and t12725 showed reduc-tions in biofilm biomass of up to 57 However isolatest1272 and t12710 showed a noticeable but not significantincrease in biofilm biomass compared with isolates t1273and t12724 Interestingly biofilm biomass increased with anincrease in enzyme concentration for isolate t1273 from 17with 100 120583gmL trypsin to 26with 1mgmL trypsin and forisolate t12710 which increased from 216 to 42

Figure 9 shows that DNase reduced biofilm for themajority of isolates tested with a loss in biofilm biomass of upto 84 except for isolates t12721 and t12722 which showedless sensitivity to DNase with 19 and 10 reductions inbiofilm biomass with 119901 values of 009 and 02 respectivelySimilarly to this effect biofilm biomass was moderately tohighly sensitive to dispersal by RNase as shown in Figure 10The majority of isolates were highly sensitive with biofilmreductions of up to 78 (119901 le 0009) On the otherhand isolates t1271 t1273 and t1276 showed minimalreductions in biofilm biomass (26 15 and 6 resp)Thisindicated that both eDNA and extracellular RNA (eRNA)were components of the biofilm matrix produced by all ofthese isolates

Many previous studies have shown that eDNA andproteins are main components of MRSA biofilms Our study


t1271 t1272 t1273 t1274 t1275

t1276 t1277 t1278 t22469 t12710

t12711 t12712 t12713 t12714 t12715

t12716 t12717 t12718 t79019 t22320

t12721 t12722 t12723 t12724 t12725(a)

t1274 t12711 t12714 t79019(b)

Figure 3 Colonymorphologies as distinguishing features of methicillin-resistant Staphylococcus aureus biofilms (a)Morphology of coloniesproduced on TSA supplemented with 1 glucose Most colonies had the same structure in the middle with a wide circular and smoothappearance (t12714 t12717 t79019 t22320 and t12722) whereas other isolates (t1271 t1272 t1273 t1275 t1276 t1277 t1238 t12710t12711 t12712 t12713 t12715 t12716 t12716 t12718 t12721 t12723 t12724 and t12725) showed net-like structures with small raisednodules in transparent layers with irregular margins Clones t1272 t22469 and t22320 showed unique structures with large cavities in themiddle surrounded by highly autoaggregated transparent cell layers Isolate t1244 formed colonies that appeared like transparent flowerswith circular and vertical lines radiating from the centres of the colonies (b) Morphology of colonies produced on Congo red agar (CRA)medium differences based on biofilm components can be seenThe interaction of proteins withCongo red dye produced a red colour whereasa black colour resulted from the interaction of the dye with exopolysaccharides Images were captured by a digital camera (Canon IXUS265HS)


lowastlowast

lowast lowastlowast

lowast

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lowastlowast lowast lowast

lowast lowast

BufferSodium metaperiodate

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

3

A570nm

Figure 4 Dispersal of 48 h mature methicillin-resistant Staphylo-coccus aureus (MRSA) biofilms by NaIO

4 The MRSA isolates are

indicated on the 119909-axis the biofilmsmatured for 16 hwere treated bybuffer alone (blue bar) or buffer containing 40mMmL NaIO

4(red

bar) Bars represent the mean values plusmn standard error of the meanof at least three independent experiments Asterisk (lowast) indicates a 119901value of less than 005 between the treated group and correspondingcontrol

lowast

lowast lowast lowast lowast lowast lowast lowast

lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 100120583g

005

115

225

3

A570nm

Figure 5 Dispersal of 48 h mature methicillin-resistant Staphylo-coccus aureus (MRSA) biofilms by proteinase K The MRSA isolatesare indicated on the 119909-axis the biofilms matured for 16 h weretreated by buffer alone (blue bar) or buffer containing 100 120583gmLproteinase K (red bar) Bars represent the mean values plusmn standarderror of themean of at least three independent experiments Asterisk(lowast) indicates a 119901 value of less than 005 between the treated groupand corresponding control

found thatDNase Iwas amore effective biofilm inhibitor thanproteinase K but that neither dispersed biofilms completelyThe maximum percentage biofilm dispersal by DNase was84 whereas with proteinase K this was 75 To investigatewhether DNase and proteinase K could complement eachother to eliminate biofilms 48 h established biofilms weretreated consecutively with DNase and proteinase K treat-ment As shown in Figure 11 the majority of isolates showeda significantly greater (119901 = 0001) reduction in biofilmscompared to that withDNase or proteinaseK aloneHoweverisolates t12714 t79019 t22320 and t12724 showed moreeffective biofilm dispersal when treated with DNase alonecompared with either treatment with proteinase K alone ortreatment with DNase followed by proteinase K

45 Biofilm Adhesiveness and Cohesiveness In previousexperiments in this study the emphasis was on detecting

lowastlowast

lowast

lowastlowast

lowastlowast lowast

lowast lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 1mg

005

115

225

335

A570nm

Figure 6 Dispersal of 48 h mature methicillin-resistant Staphylo-coccus aureus (MRSA) biofilms by proteinase K The MRSA isolatesare indicated on the 119909-axis the biofilms matured for 16 h weretreated by buffer alone (blue bar) or buffer containing 1mgmLproteinase K (red bar) Bars represent the mean values plusmn standarderror of themean of at least three independent experiments Asterisk(lowast) indicates a 119901 value of less than 005 between the treated groupand corresponding control

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowastlowastlowast lowast lowast

lowastlowast lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 100120583g

005

115

225

3A

570nm

Figure 7 Dispersal of 48 h mature methicillin-resistant Staphylo-coccus aureus (MRSA) biofilms by trypsin The MRSA isolates areindicated on the 119909-axis biofilms matured for 16 h were treated bybuffer alone (blue bar) or buffer containing 100 120583gmL trypsin (redbar) Bars represent the mean values plusmn standard error of the meanof at least three independent experiments Asterisk (lowast) indicates a 119901value of less than 005 between the treated group and correspondingcontrol

biofilm components To investigate whether cell-to-surfaceadhesion and cell-to-cell cohesion can be affected by biofilmsthe MRSA isolates were tested for these abilities The isolatescould be classified into two categories depending on biofilmcomponents found in this studyThe first category comprisedthose isolates that formed PIA-independent biofilms whichincluded t1272 t1273 t1275 t12710 t12711 t12712 t12713t12715 t12716 t12721 t12723 and t12724 The secondcategory comprised isolates that formed PIA-dependentbiofilms which included t1271 t1274 t1276 t1277 t1278t22469 t12714 t12717 t12718 t79019 t22320 t12722and t12725 regardless of the exopolysaccharide quantity orwhether the isolates possessed weak or moderate biofilm-forming abilities

In the adhesion assay the impact of biofilms on celladhesion to the surface of glass beads was investigated using


lowast lowast

lowastlowast

lowast

lowastlowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 1mg

005

115

225

3

A570nm

Figure 8 Dispersal of 48 h mature methicillin-resistant Staphylo-coccus aureus (MRSA) biofilms by trypsin The MRSA isolates areindicated on the 119909-axis biofilms matured for 16 h were treated bybuffer alone (blue bar) or buffer containing 1mgmL trypsin (redbar) Bars represent the mean values plusmn standard error of the meanof at least three independent experiments Asterisk (lowast) indicates a 119901value of less than 005 between the treated group and correspondingcontrol

lowastlowast lowast lowast lowast

lowast lowast

lowast

lowastlowast

lowastlowast lowast lowast lowast lowast

lowast

lowast

lowast lowast

lowastlowastlowast

BufferDNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm

Figure 9 Dispersal of 48 h mature methicillin-resistant Staphylo-coccus aureus (MRSA) biofilms by DNase The MRSA isolates areindicated on the 119909-axis the biofilms matured for 16 h were treatedby buffer alone (blue bar) or buffer containing 140UmLDNase (redbar) Bars represent the mean values plusmn standard error of the meanof at least three independent experiments Asterisk (lowast) indicates a 119901value of less than 005 between the treated group and correspondingcontrol

unwashed and washed bacteria As shown in Figure 12isolates t1273 t1275 t12710 t12713 t12716 and t12723in the PIA-independent biofilm category exhibited increasedadhesion to glass beads Similarly the PIA-dependent isolatest1271 t1276 t1277 t1278 t22499 t12717 and t12722 alsoshowed increased adhesion to glass beads There appearedto be no correlation between biofilm components and celladhesiveness as the washed cells of isolates t1272 t12711t12712 t12715 and t12721 which formed PIA-independentbiofilms and t1274 t12718 t79019 t22320 and t12725which formed PIA-dependent biofilms appeared to haveincreased abilities to adhere to glass beads compared to thoseof unwashed cells Figure 13 shows that the EPS from cells thatwere only partially removed by the rinsing procedure did notalways exhibit increased abilities of MRSA spa type t127 cellsto adhere to surfaces


lowastlowast

lowastlowast


lowastlowast

lowast lowast

lowastlowast

lowast lowast lowast

BufferRNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm

Figure 10 Dispersal of 48 h mature methicillin-resistant Staphylo-coccus aureus (MRSA) biofilms by RNase digestion The MRSA iso-lates are indicated on the 119909-axis the biofilms matured for 16 h weretreated by buffer alone (blue bar) or buffer containing 100120583gmLRNase (red bar) Bars represent the mean values plusmn standard errorof the mean of at least three independent experiments Asterisk (lowast)indicates a 119901 value of less than 005 between the treated group andcorresponding control

lowastlowast lowast

lowast lowast lowastlowast lowast lowast

lowast

lowast lowast

lowastlowast lowast

lowast

lowast lowastlowast lowast lowast

lowast

lowastlowast

lowast

BufferDNase amp proteinase K

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm

Figure 11 Dispersal of 48 h mature methicillin-resistant Staphy-lococcus aureus (MRSA) biofilms by DNase and proteinase KThe MRSA isolates are indicated on the 119909-axis mature biofilmswere treated by buffer alone (blue bar) or by consecutive DNaseand proteinase K treatment (red bar) Bars represent the meanvalues plusmn standard error of the mean of at least three independentexperiments Asterisk (lowast) indicates a 119901 value of less than 005between the treated group and corresponding control

Bacterial cohesiveness is shown in Table 2 The isolatet127 which formed a PIA-independent biofilm showedaggregation of 13 to 47 compared to those isolates thatformed PIA-dependent biofilms which showed 6 to 54aggregates The isolates t2246 t790 and t223 displayed cellaggregation percentages of 38 23 and 17 respectively

These results indicated no correlation between biofilmcomponents and cell-to-cell associations within biofilmsInterestingly isolates t1273 t1275 t12710 t12713 t12716and t12723 which formed PIA-independent biofilms andisolates t1271 t1276 t1277 t1278 t22469 t12717 andt12722 which formed PIA-dependent biofilms showed ahigh percentage of adhesiveness in unwashed cells comparedto percentage of cell aggregation as their ability to adhereonto glass beads after the washing process is reduced Incontrast isolates t1272 t12711 t12712 t12715 and t12721


Before washingAfter washing

PIA-independent biofilm PIA-dependent biofilm

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)

Figure 12 Biofilm adhesiveness assay for PIA-independent andPIA-dependent biofilms The 119909-axis shows methicillin-resistantStaphylococcus aureus isolates before (blue bars) and after washing(red bars) the 119910-axis shows the percentage of cells that adhered toglass beads Bars represent the mean values plusmn standard error of themean of at least three independent experiments

which formed PIA-independent biofilms and isolates t1274t12718 t79019 t22320 and t12725 with PIA-dependentbiofilms showed increased adhesion to glass beads afterthe washing process Both the washed and unwashed PIA-independent biofilm of isolate t12724 and PIA-dependentbiofilm of isolate t12714 showed similar cell adhesivenessand cohesiveness The relationship between cell-to-surfaceadhesion and cell-to-cell cohesion within biofilms of MRSAisolates shall be addressed in a more intensive study

5 Discussion

MRSA biofilms play a significant role in numerous chronicinfections [35 36] To improve MRSA diagnostics it isnecessary to understand the biofilms that lead to chronicinfections [37] Although there have been many studies onthe components of MRSA biofilms very few of these studieshave addressed the impact of biofilms on the adhesivenessand cohesiveness of bacterial cells [13 14 38ndash40]

The gene spa type t127 is frequently present community-acquired MRSA in the UK [41] as well as in the US [42]Similarly in this study we found that the majority of MRSAisolates tested had spa type t127 with a small number havingspa types t2246 t790 and t223 Based on a semiquantitativemicrowell plate assay the majority of these isolates showed amoderate ability to produce biofilmsThe production of slimeon TSAG (Figure 3(a)) however did not seem to be related tothe adhesion strength of these biofilms on microwell plates

Assessing biofilm dispersal is considered the mainmethod to determine the components involved in biofilmformation In our study antibiofilm agents such as NaIO

4

and extracellular enzymes were used to try to dispersemature biofilms of isolates t127 t2246 t790 and t223 Theseantibiofilm agents have been shown to eliminate biofilmsfrom nonliving and living surfaces [43 44] However it isimportant to consider the structures of the biofilms that are

Table 2 Percentage of methicillin-resistant Staphylococcus aureusisolates that aggregated after mechanical disruption of the biofilms

Isolates Aggregationt1271 36t1272 17t1273 28t1274 47t1275 41t1276 32t1277 6t1278 12t22469 38t12710 13t12711 24t12712 20t12713 27t12714 34t12715 34t12716 20t12717 19t12718 34t79019 23t22320 17t12721 47t12722 18t12723 47t12724 34t12725 58

being targeted [45] as many of these agents differ in theireffects on the various forms of biofilms produced by differentbacterial species [14 46 47]

PIAPNAG polymeric chains appear to be major con-stituents of many biofilms in both Gram-positive and Gram-negative pathogens [48] NaIO

4can modify these polymeric

chains by splitting the C3-C4 bonds on exopolysaccharideresidues and oxidizing the carbons to yield vicinal hydroxylgroups [45] Our study showed that NaIO

4had varying

effects from high to low levels of biofilm reduction forMRSAisolates related to clone t127 This could be of a result of theeffects of NaIO

4on exopolysaccharides that are chemically

identical in structure but that have some differences inboth the amount of acetates O-linked with succinate andacetylation levels of amino groups [32 49] In biofilms thepolysaccharides do not exist alone but appear either in asso-ciation or segregated interacting with a broad range of othermolecular species including DNA proteins and lipids [50]As a consequence depolymerisation of exopolysaccharides inresponse to NaIO

4varies depending on biofilm components

In our study the colony morphologies of MRSA isolatesobserved on Congo red agar revealed different patterns ofinteraction between the exopolysaccharides (black colour)and proteins (red colour) some isolates produced smoothblack and red colonies and others produced mucoid red-black colonies with a red pellet that appeared to have melted


20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)

Figure 13 Images of methicillin-resistant Staphylococcus aureus biofilms stained with crystal violet and examined by light microscopy at amagnification 100x (a) Unwashed and (b) washed cells of isolate t1272 (c) unwashed and (d) washed cells of isolate t12718 (e) unwashedand (f) washed cells of isolate t12721 ((a) and (b)) Biofilms prevented crystal violet from penetrating cell walls ((a) (b) and (c)) Cell clusterswere completely enveloped by the biofilm matrix (d) Large batches of biofilm that covered some cells Scale bar = 20 120583m

inside (Figure 3(b)) Sager et al [51] showed that NaIO4had

a stimulating influence on established biofilms of Pasteurellapneumotropica

The exopolysaccharides present in bacterial capsulesseemed to have a negative effect on biofilm production Forexample mutations in the capsule genes of S haemolyticusVibrio vulnificus and Porphyromonas gingivalis resulted inan increase in biofilm formation compared to the wild-typestrains because of decreased capsular exopolysaccharide pro-duction [52ndash54] NaIO

4seemed to enhance the production of

biofilms as indicated in Figure 4 by increasing the ability ofsomeMRSA isolates related to clone t127 to produce biofilmsThis could be the result of exopolysaccharides present in thecapsules of bacteria being eliminated

Protease treatment is known to disperse mature MRSAbiofilms Kumar Shukla and Rao [55] showed that proteinaseK treatment impaired biofilm formation because of theabsence of biofilm-associated protein (encoded by Bap) onthe surface of S aureus strain V329 but that it did not haveany effect on strain M556 which lacked Bap In this studyproteinase K and trypsin were used to determine whetherproteins were components of mature biofilms ProteinaseK (100 120583gmL) caused preformed biofilms to detach butwith dispersal percentages that were comparatively low forall 25 MRSA isolates tested However the majority of our

isolates appeared to be sensitive to proteinase K (100 120583gmL)consistent with the findings of previous studies that showedthe high sensitivity of S aureus biofilms to proteinase K[13 14 40 45 47] Our results showed that in 48 h establishedbiofilms treatment with a high concentration of proteinase K(1mgmL) promoted biofilm formation by all of the isolatesexcept t12722 and t12725

Additionally trypsin (100 120583gmL) showed a variety ofeffects In half of the isolates studied including isolates relatedto clones t127 and t2246 trypsin treatment increased biofilmformationwhereas in the other half including isolates relatedto clones t127 t790 and t223 it decreased biofilm biomassto varying degrees Interestingly trypsin (1mgmL) wasable to partially remove biofilms of some isolates Howeverthe reason behind these inconsistent observations in theinteractions between the two common proteases trypsin andproteinaseK is not clearThe biofilms of some of isolates wereefficiently removed by both proteases According to Boles andHorswill [44] proteinase K inhibited biofilm formation andpromoted the dispersal of established biofilms Our resultsagreed with findings by Gilan and Sivan [56] who showedthat proteinase K (1mgmL) treatment doubled the size ofa Rhodococcus ruber C208 biofilm Moreover the biofilmseemed to be multilayered mucoid and more robust thanthat before treatment However the established biofilm was


decreased by trypsin with a monolayered sparser structureresultingWe propose that a high concentration of proteinaseK enhances autolysis of bacterial cells thereby releasingextracellular DNA [57 58]

eDNA is an important part of biofilm structure [59] Thiswas first discovered in Pseudomonas aeruginosa and then inother bacterial species [17 60ndash63] eDNA is released mainlythrough cell lysis [64ndash68] or is secreted from cells [63 69 70]Biofilm formation has been reported to be blocked or itsmorphology altered by DNase I treatment of Gram-negativecells such as Pseudomonas aeruginosa and Escherichia coli aswell as Gram-positive cells such as S aureus S pneumoniaand L monocytogenes [59 71 72] Our data shows that DNaseI significantly affected the dispersal of biofilms in themajorityof isolates tested Consistent with this Rice et al [17] foundthat the structural stability of S aureus biofilms dependedon eDNA Moreover DNase I-induced degradation of eDNAresulted in a reduction in the biofilm

Mulcahy et al [73] suggested that eDNA not onlyincreased biofilm stability but also its resistance to antibioticsOur study showed that eRNA is also an important partof biofilms as similar effects on established biofilms wereobserved in response to DNase I and RNase treatment (Fig-ure 10) Nishimura et al [74] showed the presence of eRNA inbiofilms of the marine bacterium Rhodovulum sulfidophilumSimilarly Gilan and Sivan [75] showed that applying RNaseto cultures of Rhodococcus ruber strain C208 reduced biofilmformation They also showed that the formation of biofilmswas not increased by the addition of short fragments ofDNA (ca 300 and 500 bp) in C208 culture Izano et al [62]suggested that the size of the eDNA in S aureus is importantto the formation of biofilms as different forms of nucleicacids play different roles in this process eDNA seems to beimportant structural component of biofilms whereas eRNAmay be involved in regulating biofilm formation because ofthe significant size difference between these molecules

To confirm the role of protein in biofilm formation48 h biofilms were first treated with DNase I and then byproteinase K in microwell plates The results shown inFigure 11 confirmed the significant roles played by bothDNAand proteins in biofilm matrix formation Our findings areconsistent with an earlier report showing thatMRSA biofilmsdecreased significantly in the presence of the two enzymesas compared to treatment with the individual enzymesalone [31] This is further supported by the observation thatautolysin (encoded by Atl) and fibronectin-binding proteins(encoded by FnBP) expression is a basic feature of the MRSAbiofilm phenotype [13 19]

Many studies have shown that biofilms are sessile com-munities of bacteria that precipitate and adhere to all surfaces[76 77] The architecture of a biofilm is dependent on cell-to-surface and cell-to-cell interactions [24 78ndash80] Figure 12shows that biofilms of some MRSA isolates only weaklyadhered to glass beads whereas these same isolates stronglyadhered to glass beads after extensive washing

We speculate that slime layers on biofilms reduced theability of the biofilms to adhere to glass beads As shownin Figure 13 the washing process reduced the amount ofslime present on the biofilms and increased the percentage

of cells that aggregated It is probable that after the washingprocess some clusters of bacteria were still covered orsurrounded by remnants of the polymer matrix therebyincreasing the adhesiveness of cells to glass beads Thesefindings are consistent with those of Gomez-Suarez et al [81]who reported that the ability to adhere to solid surfaces wasgreater for nonbiofilmed Pseudomonas aeruginosa SG81R1than for biofilmed P aeruginosa SG81

Our data showed a specific relationship between adhe-siveness and cohesiveness of theMRSAbiofilm isolates testedWhen the percentage of cell-to-cell aggregates (Table 2) washigher than that of cell-to-surface aggregates in biofilmsthe cells seemed to have an increased ability to attach toglass beads after washing However when the percentage ofcell-to-cell aggregates was lower than that of cell-to-surfaceaggregates the ability of the cells to attach to glass beads wasreduced afterwashing (Figure 12)MRSA isolates in this studydid not depend on static electricity and polymeric interac-tions to adhere to glass surfaces as proposed by Tsuneda et al[24] as there was no correlation between the amount of EPSin the biofilms and cell adhesiveness This could be becausethe majority of our isolates produced a moderate amountof biofilm Moreover there was no correlation between celladhesiveness and PIA independence or dependence of thebiofilms

6 Conclusion

Based on the comparative analysis of biofilm extracellu-lar matrices it can be concluded that the tested biofilmsconsisted of nucleic acid-protein complexes with or with-out exopolysaccharides Different biofilm phenotypes wereobserved for the same MRSA clone In addition thereseemed to be an association between cellular adhesivenessand cohesiveness of MRSA biofilms

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Khulood Hamid Dakheel and Khatijah Yusoff designed thestudy Khulood Hamid Dakheel performed the experimentsand analyzed the data KhuloodHamidDakheel andKhatijahYusoff drafted themanuscript Jameel R Al-Obaidi VasanthaKumari Neela Raha Abdul Rahim and Tan Geok Hun readand revised the manuscript and provided critical commentsAll authors approved the final version of the manuscript forpublication

Acknowledgments

This study was supported by the Ministry of Higher Edu-cation Fundamental Research Grant Scheme (FRGS12015SGOSUPM012)


References

[1] M Z David and R S Daum ldquoCommunity-associated meth-icillin-resistant Staphylococcus aureus epidemiology and clin-ical consequences of an emerging epidemicrdquoClinical Microbiol-ogy Reviews vol 23 no 3 pp 616ndash687 2010

[2] M Lazaro-Dıez S Remuzgo-Martinez C Rodriguez-Mironeset al ldquoEffects of subinhibitory concentrations of ceftaroline onmethicillin-resistant Staphylococcus aureus (MRSA) biofilmsrdquoPLoS ONE vol 11 no 1 Article ID e0147569 2016

[3] L Yang J A J Haagensen L Jelsbak et al ldquoIn situ growthrates and biofilm development of Pseudomonas aeruginosapopulations in chronic lung infectionsrdquo Journal of Bacteriologyvol 190 no 8 pp 2767ndash2776 2008

[4] T Bjarnsholt O Ciofu S Molin M Givskov and N HoslashibyldquoApplying insights from biofilm biology to drug developmentmdashcan a new approach be developedrdquo Nature Reviews DrugDiscovery vol 12 no 10 pp 791ndash808 2013

[5] S A Rani B Pitts H Beyenal et al ldquoSpatial patterns of DNAreplication protein synthesis and oxygen concentration withinbacterial biofilms reveal diverse physiological statesrdquo Journal ofBacteriology vol 189 no 11 pp 4223ndash4233 2007

[6] P Sharma and G Vishwanath ldquoStudy of vancomycin suscep-tibility in methicillin-resistant Staphylococcus aureus isolatedfrom clinical samplesrdquo Annals of Tropical Medicine and PublicHealth vol 5 no 3 p 178 2012

[7] R Singh P Ray A Das and M Sharma ldquoPenetration ofantibiotics through Staphylococcus aureus and Staphylococcusepidermidis biofilmsrdquo Journal of Antimicrobial Chemotherapyvol 65 no 9 Article ID dkq257 pp 1955ndash1958 2010

[8] KD XuGAMcFeters and P S Stewart ldquoBiofilm resistance toantimicrobial agentsrdquoMicrobiology vol 146 no 3 pp 547ndash5492000

[9] V A Ray A R Morris and K L Visick ldquoA semi-quantitativeapproach to assess biofilm formation using wrinkled colonydevelopmentrdquo Journal of Visualized Experiments no 64 ArticleID e4035 2012

[10] S E Cramton C Gerke N F Schnell W W Nichols andF Gotz ldquoThe intercellular adhesion (ica) locus is present inStaphylococcus aureus and is required for biofilm formationrdquoInfection and Immunity vol 67 no 10 pp 5427ndash5433 1999

[11] I Sadovskaya E Vinogradov S Flahaut G Kogan and SJabbouri ldquoExtracellular carbohydrate-containing polymers ofa model biofilm-producing strain Staphylococcus epidermidisRP62Ardquo Infection and Immunity vol 73 no 5 pp 3007ndash30172005

[12] J McCourt D P OrsquoHalloran H Mccarthy J P OrsquoGara andJ A Geoghegan ldquoFibronectin-binding proteins are requiredfor biofilm formation by community-associated methicillin-resistant Staphylococcus aureus strain LACrdquo FEMSMicrobiologyLetters vol 353 no 2 pp 157ndash164 2014

[13] E OrsquoNeill H Humphreys and J P OrsquoGara ldquoCarriage of boththe fnbA and fnbB genes and growth at 37 ∘C promote FnBP-mediated biofilm development in meticillin-resistant Staphylo-coccus aureus clinical isolatesrdquo Journal of Medical Microbiologyvol 58 no 4 pp 399ndash402 2009

[14] E OrsquoNeill C Pozzi P Houston et al ldquoA novel Staphylococcusaureus biofilm phenotype mediated by the fibronectin-bindingproteins FnBPA and FnBPBrdquo Journal of Bacteriology vol 190no 11 pp 3835ndash3850 2008

[15] M Vergara-Irigaray J Valle N Merino et al ldquoRelevant role offibronectin-binding proteins in Staphylococcus aureus biofilm-associated foreign-body infectionsrdquo Infection and Immunityvol 77 no 9 pp 3978ndash3991 2009

[16] D K Ranjit J L Endres and K W Bayles ldquoStaphylococcusaureus CidA and LrgA proteins exhibit holin-like propertiesrdquoJournal of Bacteriology vol 193 no 10 pp 2468ndash2476 2011

[17] K C Rice E E Mann J L Endres et al ldquoThe cidA mureinhydrolase regulator contributes to DNA release and biofilmdevelopment in Staphylococcus aureusrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 104 no 19 pp 8113ndash8118 2007

[18] E OrsquoNeill C Pozzi P Houston et al ldquoAssociation betweenmethicillin susceptibility and biofilm regulation in Staphylococ-cus aureus isolates from device-related infectionsrdquo Journal ofClinical Microbiology vol 45 no 5 pp 1379ndash1388 2007

[19] P Houston S E Rowe C Pozzi E M Waters and J P OrsquoGaraldquoEssential role for the major autolysin in the fibronectin-binding protein-mediated Staphylococcus aureus biofilm phe-notyperdquo Infection and Immunity vol 79 no 3 pp 1153ndash11652011

[20] J B Kaplan ldquoBiofilm dispersal mechanisms clinical implica-tions and potential therapeutic usesrdquo Journal of dental researchvol 89 no 3 pp 205ndash218 2010

[21] H Rohde C Burdelski K Bartscht et al ldquoInduction ofStaphylococcus epidermidis biofilm formation via proteolyticprocessing of the accumulation-associated protein by staphylo-coccal and host proteasesrdquo Molecular Microbiology vol 55 no6 pp 1883ndash1895 2005

[22] X Wang J F Preston III and T Romeo ldquoThe pgaABCD locusof Escherichia coli promotes the synthesis of a polysaccharideadhesin required for biofilm formationrdquo Journal of Bacteriologyvol 186 no 9 pp 2724ndash2734 2004

[23] T R GarrettM Bhakoo and Z Zhang ldquoBacterial adhesion andbiofilms on surfacesrdquo Progress in Natural Science vol 18 no 9pp 1049ndash1056 2008

[24] S Tsuneda H Aikawa H Hayashi A Yuasa and A HirataldquoExtracellular polymeric substances responsible for bacterialadhesion onto solid surfacerdquo FEMS Microbiology Letters vol223 no 2 pp 287ndash292 2003

[25] J B Patel F R Cockerill J Alder PA Bradford G M Eliopou-los and D J Hardy ldquoPerformance standards for antimicro-bial susceptibility testing twenty-fourth informational supple-mentrdquo CLSI Standards for Antimicrobial Susceptibility Testingvol 34 no 1 pp 1ndash226 2014

[26] F Martineau F J Picard P H Roy M Ouellette and M GBergeron ldquoSpecies-specific and ubiquitous-DNA-based assaysfor rapid identification of Staphylococcus aureusrdquo Journal ofClinical Microbiology vol 36 no 3 pp 618ndash623 1998

[27] C Milheirico D C Oliveira and H de Lencastre ldquoMulti-plex PCR strategy for subtyping the staphylococcal cassettechromosome mec type IV in methicillin-resistant Staphylococ-cus aureus rsquoSCCmec IV multiplexrsquordquo Journal of AntimicrobialChemotherapy vol 60 no 1 pp 42ndash48 2007

[28] G D Christensen W A Simpson J J Younger et al ldquoAdher-ence of coagulase-negative Staphylococci to plastic tissue cultureplates a quantitative model for the adherence of Staphylococcito medical devicesrdquo Journal of Clinical Microbiology vol 22 no6 pp 996ndash1006 1985

[29] K Manago J Nishi N Wakimoto et al ldquoBiofilm formationby and accessory gene regulator typing of methicillin-resistant


Staphylococcus aureus strains recovered from patients withnosocomial infectionsrdquo Infection Control and Hospital Epidemi-ology vol 27 no 2 pp 188ndash190 2006

[30] S Stepanovic D Vukovic I Dakic B Savic and M Svabic-Vlahovic ldquoA modified microtiter-plate test for quantificationof staphylococcal biofilm formationrdquo Journal of MicrobiologicalMethods vol 40 no 2 pp 175ndash179 2000

[31] I V Chebotar A G Pogorelov V A Yashin E L Guryev andG G Lominadze ldquoModern technologies of bacterial biofilmstudyrdquoModern Technologies in Medicine vol 5 no 1 pp 14ndash202013

[32] A I Spiliopoulou M I Krevvata F Kolonitsiou et al ldquoAnextracellular Staphylococcus epidermidis polysaccharide rela-tion to polysaccharide intercellular adhesin and its implicationin phagocytosisrdquo BMCMicrobiology vol 12 article 76 2012

[33] D Kadouri E Jurkevitch and Y Okon ldquoInvolvement ofthe reserve material poly-120573-hydroxybutyrate in Azospirillumbrasilense stress endurance and root colonizationrdquo Applied andEnvironmentalMicrobiology vol 69 no 6 pp 3244ndash3250 2003

[34] D Kadouri N C Venzon and G A OrsquoToole ldquoVulnerabilityof pathogenic biofilms to Micavibrio aeruginosavorusrdquo Appliedand Environmental Microbiology vol 73 no 2 pp 605ndash6142007

[35] J Esteban D Molina-Manso I Spiliopoulou et al ldquoBiofilmdevelopment by clinical isolates of Staphylococcus spp fromretrieved orthopedic prosthesesrdquoActa Orthopaedica vol 81 no6 pp 674ndash679 2010

[36] P Stoodley S F Conti P J Demeo et al ldquoCharacterizationof a mixed MRSAMRSE biofilm in an explanted total anklearthroplastyrdquo FEMS Immunology andMedicalMicrobiology vol62 no 1 pp 66ndash74 2011

[37] L Hall-Stoodley P Stoodley S Kathju et al ldquoTowardsdiagnostic guidelines for biofilm-associated infectionsrdquo FEMSImmunology and Medical Microbiology vol 65 no 2 pp 127ndash145 2012

[38] S Chopra K Harjai and S Chhibber ldquoAntibiotic susceptibilityof ica-positive and ica-negative MRSA in different phases ofbiofilm growthrdquo Journal of Antibiotics vol 68 no 1 pp 15ndash222015

[39] T L Nicholson S M Shore T C Smith and T SFraena ldquoLivestock-associatedmethicillin-resistant Staphylococ-cus aureus (LA-MRSA) isolates of swine origin form robustbiofilmsrdquo PLoS ONE vol 8 no 8 Article ID e73376 2013

[40] K Seidl C Goerke C Wolz D Mack B Berger-Bachiand M Bischoff ldquoStaphylococcus aureus CcpA affects biofilmformationrdquo Infection and Immunity vol 76 no 5 pp 2044ndash2050 2008

[41] J A Otter N L Havill J M Boyce and G L French ldquoCom-parison of community-associated meticillin-resistant Staphylo-coccus aureus from teaching hospitals in London and the USA2004ndash2006 where is USA300 in the UKrdquo European Journal ofClinical Microbiology and Infectious Diseases vol 28 no 7 pp835ndash839 2009

[42] F C Tenover I A Tickler R V Goering B N Kreiswirth J RMediavilla and D H Persinga ldquoCharacterization of nasal andblood culture isolates of methicillin-resistant Staphylococcusaureus from patients in United States hospitalsrdquo AntimicrobialAgents and Chemotherapy vol 56 no 3 pp 1324ndash1330 2012

[43] C Pozzi EMWaters J K Rudkin et al ldquoMethicillin resistancealters the biofilm phenotype and attenuates virulence in Staphy-lococcus aureus device-associated infectionsrdquo PLoS Pathogensvol 8 no 4 Article ID e1002626 2012

[44] B R Boles and A R Horswill ldquoAgr-mediated dispersal ofStaphylococcus aureus biofilmsrdquo PLoS Pathogens vol 4 no 4Article ID e1000052 2008

[45] P Chaignon I Sadovskaya C Ragunah N Ramasubbu JB Kaplan and S Jabbouri ldquoSusceptibility of staphylococcalbiofilms to enzymatic treatments depends on their chemicalcompositionrdquo Applied Microbiology and Biotechnology vol 75no 1 pp 125ndash132 2007

[46] B R Boles and A R Horswill ldquoStaphylococcal biofilm disas-semblyrdquo Trends inMicrobiology vol 19 no 9 pp 449ndash455 2011

[47] H Rohde E C Burandt N Siemssen et al ldquoPolysaccharideintercellular adhesin or protein factors in biofilm accumulationof Staphylococcus epidermidis and Staphylococcus aureus iso-lated from prosthetic hip and knee joint infectionsrdquo Biomate-rials vol 28 no 9 pp 1711ndash1720 2007

[48] M Otto ldquoStaphylococcal biofilmsrdquo in Bacterial Biofilms pp207ndash228 Springer Berlin Germany 2008

[49] D Mack W Fischer A Krokotsch et al ldquoThe intercellularadhesin involved in biofilm accumulation of Staphylococcusepidermidis is a linear 120573-16-linked glucosaminoglycan purifi-cation and structural analysisrdquo Journal of Bacteriology vol 178no 1 pp 175ndash183 1996

[50] I W Sutherland ldquoBiofilm exopolysaccharides a strong andsticky frameworkrdquoMicrobiology vol 147 no 1 pp 3ndash9 2001

[51] M Sager W P M Benten E Engelhardt C Gougoula and LBenga ldquoCharacterization of biofilm formation in [Pasteurella]pneumotropica and [Actinobacillus] muris isolates of mouseoriginrdquo PLoS ONE vol 10 no 10 Article ID e0138778 2015

[52] M E Davey and M J Duncan ldquoEnhanced biofilm formationand loss of capsule synthesis deletion of a putative glycosyl-transferase in Porphyromonas gingivalisrdquo Journal of Bacteriol-ogy vol 188 no 15 pp 5510ndash5523 2006

[53] S Flahaut E Vinogradov K A Kelley S Brennan K Hira-matsu and J C Lee ldquoStructural and biological characteriza-tion of a capsular polysaccharide produced by Staphylococcushaemolyticusrdquo Journal of Bacteriology vol 190 no 5 pp 1649ndash1657 2008

[54] L A Joseph and A C Wright ldquoExpression of vibrio vulnificuscapsular polysaccharide inhibits biofilm formationrdquo Journal ofBacteriology vol 186 no 3 pp 889ndash893 2004

[55] S Kumar Shukla and T S Rao ldquoDispersal of Bap-mediatedStaphylococcus aureus biofilm by proteinase Krdquo Journal ofAntibiotics vol 66 no 2 pp 55ndash60 2013

[56] I Gilan and A Sivan ldquoEffect of proteases on biofilm forma-tion of the plastic-degrading actinomycete Rhodococcus ruberC208rdquo FEMS Microbiology Letters vol 342 no 1 pp 18ndash232013

[57] P S Guiton C S Hung K A Kline et al ldquoContributionof autolysin and sortase A during Enterococcus faecalis DNA-dependent biofilm developmentrdquo Infection and Immunity vol77 no 9 pp 3626ndash3638 2009

[58] V C Thomas Y Hiromasa N Harms L Thurlow J Tomichand L E Hancock ldquoA fratricidal mechanism is responsiblefor eDNA release and contributes to biofilm development ofEnterococcus faecalisrdquoMolecular Microbiology vol 72 no 4 pp1022ndash1036 2009

[59] C B Whitchurch T Tolker-Nielsen P C Ragas and J SMattick ldquoExtracellular DNA required for bacterial biofilmformationrdquo Science vol 295 no 5559 p 1487 2002

[60] M Allesen-Holm K B Barken L Yang et al ldquoA characteriza-tion of DNA release in Pseudomonas aeruginosa cultures and


biofilmsrdquo Molecular Microbiology vol 59 no 4 pp 1114ndash11282006

[61] U Bockelmann A Janke R Kuhn et al ldquoBacterial extracellularDNA forming a defined network-like structurerdquo FEMS Micro-biology Letters vol 262 no 1 pp 31ndash38 2006

[62] E A Izano M A Amarante W B Kher and J B Kaplan ldquoDif-ferential roles of poly-N-acetylglucosamine surface polysac-charide and extracellular DNA in Staphylococcus aureus andStaphylococcus epidermidis biofilmsrdquo Applied and Environmen-tal Microbiology vol 74 no 2 pp 470ndash476 2008

[63] V CThomas L RThurlow D Boyle and L E Hancock ldquoReg-ulation of autolysis-dependent extracellular DNA release byEnterococcus faecalis extracellular proteases influences biofilmdevelopmentrdquo Journal of Bacteriology vol 190 no 16 pp 5690ndash5698 2008

[64] H L Hamilton NMDomınguez K J Schwartz K T Hackettand J P Dillard ldquoNeisseria gonorrhoeae secretes chromosomalDNA via a novel type IV secretion systemrdquoMolecular Microbi-ology vol 55 no 6 pp 1704ndash1721 2005

[65] T G Patton K C Rice M K Foster and K W Bayles ldquoTheStaphylococcus aureus cidC gene encodes a pyruvate oxidasethat affects acetate metabolism and cell death in stationaryphaserdquo Molecular Microbiology vol 56 no 6 pp 1664ndash16742005

[66] Z Qin Y Ou L Yang et al ldquoRole of autolysin-mediated DNArelease in biofilm formation of Staphylococcus epidermidisrdquoMicrobiology vol 153 no 7 pp 2083ndash2092 2007

[67] K C Rice J B Nelson T G Patton S-J Yang andKW BaylesldquoAcetic acid induces expression of the Staphylococcus aureuscidABC and lrgABmurein hydrolase regulator operonsrdquo Journalof Bacteriology vol 187 no 3 pp 813ndash821 2005

[68] S-J Yang K C Rice R J Brown et al ldquoA LysR-type regulatorCidR is required for induction of the Staphylococcus aureuscidABC operonrdquo Journal of Bacteriology vol 187 no 17 pp5893ndash5900 2005

[69] M Y Abajy J Kopec K Schiwon et al ldquoA type IV-secretion-like system is required for conjugative DNA transport of broad-host-range plasmid pIP501 in gram-positive bacteriardquo Journalof Bacteriology vol 189 no 6 pp 2487ndash2496 2007

[70] J A Draghi and P E Turner ldquoDNA secretion and gene-levelselection in bacteriardquo Microbiology vol 152 no 9 pp 2683ndash2688 2006

[71] M Moscoso E Garcıa and R Lopez ldquoBiofilm formation byStreptococcus pneumoniae role of choline extracellular DNAand capsular polysaccharide in microbial accretionrdquo Journal ofBacteriology vol 188 no 22 pp 7785ndash7795 2006

[72] G V Tetz N K Artemenko and V V Tetz ldquoEffect of DNaseand antibiotics on biofilm characteristicsrdquo Antimicrobial Agentsand Chemotherapy vol 53 no 3 pp 1204ndash1209 2009

[73] H Mulcahy L Charron-Mazenod and S Lewenza ldquoExtracel-lular DNA chelates cations and induces antibiotic resistance inPseudomonas aeruginosa biofilmsrdquo PLoS Pathogens vol 4 no11 Article ID e1000213 2008

[74] S Nishimura T Tanaka K Fujita M Itaya A Hiraishi and YKikuchi ldquoExtracellular DNA and RNA produced by a marinephotosynthetic bacteriumRhodovulum sulfidophilumrdquoNucleicAcids Research no 3 pp 279ndash280 2003

[75] I Gilan and A Sivan ldquoExtracellular DNA plays an importantstructural role in the biofilm of the plastic degrading actino-myceteRhodococcus ruberrdquoAdvances inMicrobiology vol 3 no8 pp 543ndash551 2013

[76] R M Donlan and J W Costerton ldquoBiofilms survival mecha-nisms of clinically relevant microorganismsrdquo Clinical Microbi-ology Reviews vol 15 no 2 pp 167ndash193 2002

[77] S M Soto ldquoImportance of biofilms in urinary tract infectionsnew therapeutic approachesrdquo Advances in Biology vol 2014Article ID 543974 13 pages 2014

[78] J Azeredo and R Oliveira ldquoThe role of exopolymers in theattachment of Sphingomonas paucimobilisrdquo Biofouling vol 16no 1 pp 59ndash67 2000

[79] C Formosa-Dague C Feuillie A Beaussart et al ldquoStickymatrix adhesion mechanism of the staphylococcal polysaccha-ride intercellular adhesinrdquo ACS Nano vol 10 no 3 pp 3443ndash3452 2016

[80] M C M van Loosdrecht W Norde J Lyklema and AJ B Zehnder ldquoHydrophobic and electrostatic parameters inbacterial adhesionmdashdedicated to Werner Stumm for his 65thbirthdayrdquo Aquatic Sciences vol 52 no 1 pp 103ndash114 1990

[81] C Gomez-Suarez J Pasma A J van der Borden et alldquoInfluence of extracellular polymeric substances on depositionand redeposition of Pseudomonas aeruginosa to surfacesrdquoMicrobiology vol 148 no 4 pp 1161ndash1169 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


general structure and contribute to its conservation andresistance phenotype [9] In general two biofilm phenotypeshave been identified Polysaccharide intercellular adhesion-(PIA-) dependent biofilms are composed of poly-120573-16-N-acetylglucosamine- (PNAG-) based matrices PIA is synthe-sized from the products of genes located at the ica locus [10]The other type PIA-independent biofilm is composed of cellsurface components such as teichoic acid [11] fibronectin-binding proteins FnBpA and FnBpB [12ndash15] and autolysinextracellular DNA (eDNA) [16 17]

The synthesis of biofilms is influenced by a number offactors Biofilm production however does not appear tobe linked to the ica locus OrsquoNeill et al [18] observed thatalthough the ica locus is present and expressed in PIA-independent biofilms the genes do not appear to be involvedin their formation Houston et al [19] found that deletionof the major autolysin (atl) gene in MRSA strains impairedtheir ability to form FnBP-dependent biofilms Some MRSAclinical isolates even produce biofilms of both phenotypesMRSA strain 132 is able to switch from PIA-dependent toPIA-independent proteinaceous biofilm matrices dependingon environmental conditions [15]

The biofilm dispersion is investigated in vitro usingenzymatic detachment methods [20] and treatment withchemicals such as periodate (HIO

4or NaIO

4)These conven-

tional methods are used to identify the biofilm matrix com-ponents of both Gram-negative and Gram-positive bacteria[21 22] Moreover bacteria within biofilms are significantlyaffected by matrix components that influence adhesion ofthe cells to solid substrata and cohesion between bacterialcells [23] Specificmatrix components can increase the abilityof bacteria to aggregate [24] The structure of extracellularpolymeric substances (EPS) is complex and variable and itsprecise role in cell adhesion and cohesion is not completelyunderstood [17]

The aims of this study were to examine the ability ofa collection of MRSA isolates with spa type t127 to formbiofilms to determine the extracellular matrix componentsin the biofilms formed by these strains and to elucidate theinfluence of biofilms on the ability of these bacteria to adhereand aggregate

2 Material and Methods

21 Identification and Genotyping of MRSA Strains A totalof 25 MRSA clinical isolates were obtained from the MedicalMicrobiology Laboratory at the Universiti Putra MalaysiaThese isolates were obtained from different systemic in-fection sites and their identity was confirmed by Gramstaining growth on mannitol-salt agar (Oxoid UK) andCHROMagarMRSA (Paris France) Kirby-Bauer testing wasperformed for oxacillin (1 120583g) (Oxoid UK) and cefoxitin(30 120583g) (Oxoid UK) on Muller-Hinton agar (Oxoid UK)[25] The MRSA strain ATCC33591 and clinical methicillin-sensitive Staphylococcus aureus (MSSA) strain were used asstandards in every test which were performed in triplicateThe isolates were confirmed to be S aureus by detectionof the Sa442 fragment and MRSA by detection of the

mecA gene A single polymerase chain reaction (PCR) wasused to detect the Sa442 fragment with the Sa442 forwardprimer 51015840-AATCTTTGTCGGTACACGATATTCTTCACG-31015840 and Sa442 reverse primer 51015840-CGTAATGAGATTTCA-GTAAATACAACA-31015840 PCR conditions were the follow-ing an initial temperature of 96∘C (3min) followed bydenaturation at 95∘C (1min) annealing at 55∘C (30 s) andelongation at 72∘C (3min) and a final elongation step at72∘C (4min) Amplicons of the expected size (108 bp) wereobtained [26] The mecA gene was detected using mecA for-ward primer 51015840-ACCAGATTACAACTTCACCAGG-31015840 andmecA reverse primer 51015840-CCACTTCATATCTTGTAACG-31015840initial temperature of 95∘C (1min) denaturation 95∘C (15 s)annealing 45∘C (15 s) and elongation 72∘C (30 s) with afinal extension at 72∘C (4min) Amplicons of the expectedsize (162 bp) existed [27] All isolates were subjected to spatyping according to Christensen et al [28] The polymor-phic X region of the protein A gene (spa) was amplifiedwith primer designed from an S aureus sequence in Gen-Bank (accession number J01786) 1079 F [1079ndash1099] 51015840-TCATCCAAAGCCTTAAAGACC-31015840 and 1516R [1536ndash1516]51015840-GTCAGCAGTAGTGCCGTTTG-31015840 The PCR reactionwas performed using a KOD FX Neo Kit from Toyobo CoLtd (Osaka Japan) as recommended by the manufacturerPCR conditions were 94∘C for 2min 35 cycles each of94∘C for 30 s 50∘C for 30 s and 72∘C for 60 s and afinal extension at 72∘C for 5min The expected productsize was between 300 bp and 600 bp with the size varyingby the number of spa repeats All PCR products weresequenced using 1st BASE (BioSyntech Inc) after purifi-cation with the GeneJET PCR Purification Kit (ThermoFisher Scientific) Sequence assembly was performed inClone Manager Basic 9 (SciEd) followed by analysis ofthe spa tandem repeats using spa typing online software(httpspatyperfortinbrasus) and the Ridom Spa Serverdatabase (httpwwwspaserverridomde) [29]

22 Biofilm Semiquantification with Crystal Violet (CV) Stain-ing Biofilm formation byMRSA strains was quantified usingthe microwell plate method described by Christensen et aland Manago et al [28 29] All MRSA isolates were grownin tryptone soya broth with 1 glucose (TSBG) and then250 120583L of each bacterial strain culture was diluted to an 119860

600

of 005 and incubated in 96-well flat-bottomed polystyrenemicrowell plates (MWP) at 37∘C for 48 h without shakingThe well contents were removed by flipping the plates andthe wells were washed with phosphate buffered saline (PBSpH 72) heat-fixed by exposing the plate to hot air at 60∘Cin a hybridization oven (model HS-101 Amerex USA) for1 h and then stained with 250120583L of 01 (wv) CV solutionfor 15min at room temperature to allow the dye to penetratethe biofilm and be washed with tap water The plates wereemptied and left to dry overnight Biofilmswere quantified byeluting the CV stainwith 250120583L of 33 glacial acetic acid andmeasuring the absorbance of the solution at 570 nm (119860

570)

using a BioTek Synergy 2 plate reader The biofilm assay wasperformed for each strain in triplicate using amicrowell plateand the background was determined by using noninoculated



570)




570lt 262) and







4in 50mM sodium


2for DNA



2) which was





660= 08



119905and the



119905minus OD

119904) times 100]OD

119905




570and

OD660


4 Results



81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

82

66

77

t12711

t12725

t22469

t1277

t1278

t1275

t12716

t79019

t22320

t12721

t12722

t12724

t12718

t1273

t1274

t12713

t12714

t1271

t12710

t12723

t12717

t1272

t12712

t1276

t12715




570ranging
















4of up to twofold








t1271 t1272 t1273 t1274 t1275

t1276 t1277 t1278 t22469 t12710

t12711 t12712 t12713 t12714 t12715

t12716 t12717 t12718 t79019 t22320

t12721 t12722 t12723 t12724 t12725(a)

t1274 t12711 t12714 t79019(b)



lowastlowast

lowast lowastlowast

lowast

lowastlowast


lowast lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

3

A570nm




4(red


lowast


lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 100120583g

005

115

225

3

A570nm




lowastlowast

lowast

lowastlowast

lowastlowast lowast

lowast lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 1mg

005

115

225

335

A570nm


lowast

lowast

lowastlowast

lowast

lowast

lowast


lowastlowast lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 100120583g

005

115

225

3A

570nm





lowast lowast

lowastlowast

lowast

lowastlowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 1mg

005

115

225

3

A570nm



lowast lowast

lowast

lowastlowast


lowast

lowast

lowast lowast

lowastlowastlowast

BufferDNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm




lowastlowast

lowastlowast


lowastlowast

lowast lowast

lowastlowast


BufferRNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm


lowastlowast lowast


lowast

lowast lowast

lowastlowast lowast

lowast


lowast

lowastlowast

lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm







t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)



5 Discussion




4








4had varying







20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



570)




570lt 262) and







4in 50mM sodium


2for DNA



2) which was





660= 08



119905and the



119905minus OD

119904) times 100]OD

119905




570and

OD660


4 Results



81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

82

66

77

t12711

t12725

t22469

t1277

t1278

t1275

t12716

t79019

t22320

t12721

t12722

t12724

t12718

t1273

t1274

t12713

t12714

t1271

t12710

t12723

t12717

t1272

t12712

t1276

t12715




570ranging
















4of up to twofold








t1271 t1272 t1273 t1274 t1275

t1276 t1277 t1278 t22469 t12710

t12711 t12712 t12713 t12714 t12715

t12716 t12717 t12718 t79019 t22320

t12721 t12722 t12723 t12724 t12725(a)

t1274 t12711 t12714 t79019(b)



lowastlowast

lowast lowastlowast

lowast

lowastlowast


lowast lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

3

A570nm




4(red


lowast


lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 100120583g

005

115

225

3

A570nm




lowastlowast

lowast

lowastlowast

lowastlowast lowast

lowast lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 1mg

005

115

225

335

A570nm


lowast

lowast

lowastlowast

lowast

lowast

lowast


lowastlowast lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 100120583g

005

115

225

3A

570nm





lowast lowast

lowastlowast

lowast

lowastlowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 1mg

005

115

225

3

A570nm



lowast lowast

lowast

lowastlowast


lowast

lowast

lowast lowast

lowastlowastlowast

BufferDNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm




lowastlowast

lowastlowast


lowastlowast

lowast lowast

lowastlowast


BufferRNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm


lowastlowast lowast


lowast

lowast lowast

lowastlowast lowast

lowast


lowast

lowastlowast

lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm







t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)



5 Discussion




4








4had varying







20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

81

82

66

77

t12711

t12725

t22469

t1277

t1278

t1275

t12716

t79019

t22320

t12721

t12722

t12724

t12718

t1273

t1274

t12713

t12714

t1271

t12710

t12723

t12717

t1272

t12712

t1276

t12715




570ranging
















4of up to twofold








t1271 t1272 t1273 t1274 t1275

t1276 t1277 t1278 t22469 t12710

t12711 t12712 t12713 t12714 t12715

t12716 t12717 t12718 t79019 t22320

t12721 t12722 t12723 t12724 t12725(a)

t1274 t12711 t12714 t79019(b)



lowastlowast

lowast lowastlowast

lowast

lowastlowast


lowast lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

3

A570nm




4(red


lowast


lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 100120583g

005

115

225

3

A570nm




lowastlowast

lowast

lowastlowast

lowastlowast lowast

lowast lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 1mg

005

115

225

335

A570nm


lowast

lowast

lowastlowast

lowast

lowast

lowast


lowastlowast lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 100120583g

005

115

225

3A

570nm





lowast lowast

lowastlowast

lowast

lowastlowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 1mg

005

115

225

3

A570nm



lowast lowast

lowast

lowastlowast


lowast

lowast

lowast lowast

lowastlowastlowast

BufferDNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm




lowastlowast

lowastlowast


lowastlowast

lowast lowast

lowastlowast


BufferRNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm


lowastlowast lowast


lowast

lowast lowast

lowastlowast lowast

lowast


lowast

lowastlowast

lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm







t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)



5 Discussion




4








4had varying







20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












4of up to twofold








t1271 t1272 t1273 t1274 t1275

t1276 t1277 t1278 t22469 t12710

t12711 t12712 t12713 t12714 t12715

t12716 t12717 t12718 t79019 t22320

t12721 t12722 t12723 t12724 t12725(a)

t1274 t12711 t12714 t79019(b)



lowastlowast

lowast lowastlowast

lowast

lowastlowast


lowast lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

3

A570nm




4(red


lowast


lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 100120583g

005

115

225

3

A570nm




lowastlowast

lowast

lowastlowast

lowastlowast lowast

lowast lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 1mg

005

115

225

335

A570nm


lowast

lowast

lowastlowast

lowast

lowast

lowast


lowastlowast lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 100120583g

005

115

225

3A

570nm





lowast lowast

lowastlowast

lowast

lowastlowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 1mg

005

115

225

3

A570nm



lowast lowast

lowast

lowastlowast


lowast

lowast

lowast lowast

lowastlowastlowast

BufferDNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm




lowastlowast

lowastlowast


lowastlowast

lowast lowast

lowastlowast


BufferRNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm


lowastlowast lowast


lowast

lowast lowast

lowastlowast lowast

lowast


lowast

lowastlowast

lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm







t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)



5 Discussion




4








4had varying







20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


t1271 t1272 t1273 t1274 t1275

t1276 t1277 t1278 t22469 t12710

t12711 t12712 t12713 t12714 t12715

t12716 t12717 t12718 t79019 t22320

t12721 t12722 t12723 t12724 t12725(a)

t1274 t12711 t12714 t79019(b)



lowastlowast

lowast lowastlowast

lowast

lowastlowast


lowast lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

3

A570nm




4(red


lowast


lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 100120583g

005

115

225

3

A570nm




lowastlowast

lowast

lowastlowast

lowastlowast lowast

lowast lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 1mg

005

115

225

335

A570nm


lowast

lowast

lowastlowast

lowast

lowast

lowast


lowastlowast lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 100120583g

005

115

225

3A

570nm





lowast lowast

lowastlowast

lowast

lowastlowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 1mg

005

115

225

3

A570nm



lowast lowast

lowast

lowastlowast


lowast

lowast

lowast lowast

lowastlowastlowast

BufferDNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm




lowastlowast

lowastlowast


lowastlowast

lowast lowast

lowastlowast


BufferRNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm


lowastlowast lowast


lowast

lowast lowast

lowastlowast lowast

lowast


lowast

lowastlowast

lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm







t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)



5 Discussion




4








4had varying







20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowastlowast

lowast lowastlowast

lowast

lowastlowast


lowast lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

3

A570nm




4(red


lowast


lowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 100120583g

005

115

225

3

A570nm




lowastlowast

lowast

lowastlowast

lowastlowast lowast

lowast lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Prot K 1mg

005

115

225

335

A570nm


lowast

lowast

lowastlowast

lowast

lowast

lowast


lowastlowast lowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 100120583g

005

115

225

3A

570nm





lowast lowast

lowastlowast

lowast

lowastlowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 1mg

005

115

225

3

A570nm



lowast lowast

lowast

lowastlowast


lowast

lowast

lowast lowast

lowastlowastlowast

BufferDNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm




lowastlowast

lowastlowast


lowastlowast

lowast lowast

lowastlowast


BufferRNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm


lowastlowast lowast


lowast

lowast lowast

lowastlowast lowast

lowast


lowast

lowastlowast

lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm







t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)



5 Discussion




4








4had varying







20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowast lowast

lowastlowast

lowast

lowastlowastlowast

lowast

Buffer

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

Trypsin 1mg

005

115

225

3

A570nm



lowast lowast

lowast

lowastlowast


lowast

lowast

lowast lowast

lowastlowastlowast

BufferDNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm




lowastlowast

lowastlowast


lowastlowast

lowast lowast

lowastlowast


BufferRNase

t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm


lowastlowast lowast


lowast

lowast lowast

lowastlowast lowast

lowast


lowast

lowastlowast

lowast


t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

005

115

225

335

A570nm







t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)



5 Discussion




4








4had varying







20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




t127

25

t127

7t127

8t2246

9

t127

5

t127

16

t127

21

t127

22

t127

24

t127

18

t790

19

t223

20

t127

3

t127

4

t127

13

t127

14

t127

1

t127

10

t127

11

t127

23

t127

17

t127

2

t127

12

t127

6

t127

15

0102030405060708090

100

Cel

l (

)



5 Discussion




4








4had varying







20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


20120583m 20120583m

20120583m 20120583m 20120583m

20120583m

(f)

(e)

(d)

(c)

(b)

(a)



















6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










6 Conclusion


Competing Interests




Acknowledgments



References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References





























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Methicillin-Resistant Staphylococcus ...downloads.hindawi.com/journals/bmri/2016/4708425.pdf · Research Article Methicillin-Resistant Staphylococcus aureus Biofilms