Buya Anti-quorumsensing Rosemary Sage Journal 2016

7/26/2019 Buya Anti-quorumsensing Rosemary Sage Journal 2016

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ANTI-QUORUM SENSING ACTIVITY OF ROSEMARY (Rosmarinus officinalis) AND

SAGE (Salvia officinalis) LEAF EXTRACTS AGAINST METHICILLIN-RESISTANT

Staphylococcus aureus ANDPseudomonas aeruginosa

A Research

Submitted to the Faculty of the

School of Natural Sciences

Saint Louis University

In Partial Fulfillment of the Requirements

for the Degree

Bachelor in Medical Laboratory Science

!uya "ennifer #$Asistin Ral%h "ustine A$

&hio' "erelyne #$Feliciano Reynold ($

Navarro Angelica )ae !$Reyes *dgar "r$ S$

#errado )elvin "ethro *$+entere, (eena Lindsay F$

)ay -./0

/


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Certificate f Ori!i"a#it$

#his manuscri%t hereby submitted by the undersigned researchers serves to %resent the

results of a study that the undersigned researchers %ersonally conducted$ #o the best of our

'no1ledge and belief it has no material %reviously %ublished or 1ritten by another %erson that is

not %ro%erly documented or %ermission for its inclusion and use not sought 1hen necessary$

2e certify further that a co%y of the PlagScan evaluationis attached to this manuscri%t 3see

attached4

#he discourse is my o1n intellectual e5ercise although assistance 1as received from

others on style %hraseology %resentation and organi,ation$

S1: ASISTIN, RALPH JUSTINE A. S5: REYES, EDGAR JR. S.(signature over printed name) (signature over printed name)

S2: CHIOK, JERELYNE T. S6:TERRADO, MELVIN JETHRO E.(signature over printed name) (signature over printed name)

S3: FELICIANO, REYNOLD G. S7: VENTEREZ, GEENA LINDSAY F.

(signature over printed name) (signature over printedname)

S4: NAVARRO, ANGELICA MAE B. S8: ______________________________ (signature over printed name) (signature over printed name)

Date6 "une 7 -./0

"*NNIF*R #$ !U8A R)# FA&UL#8 R*S*AR&9 PR:):#*R

Date6 "une 7 -./0

-


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ENDORSEMENT

In %artial fulfillment of the requirements for the degree

Bachelor in Medical Laboratory Science

this research entitled

%ANTI- QUORUM SENSING ACTIVITY OF ROSEMARY (Rosmarinus officinalis) AND


Staphylococcus aureus ANDPseudomonas aeruginosa&

%re%ared and submitted by6



S3: FELICIANO, REYNOLD G. S7: VENTEREZ, GEENA LINDSAY F. (signature over printed name) (signature over printedname)


is hereby endorsed for evaluation and a%%roval$

"*NNIF*R #$ !U8A R)# Faculty Research Promoter

Date6 "une 7 -./0

;


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A''ROVAL SHEET

This is to certify that this Research entitled,

%ANTI- QUORUM SENSING ACTIVITY OF ROSEMARY (Rosmarinus officinalis) AND


Staphylococcus aureus ANDPseudomonas aeruginosa&

%re%ared and submitted by



S3: FELICIANO, REYNOLD G. S7: VENTEREZ, GEENA LINDSAY F. (signature over printed name) (signature over printedname)


has been a%%roved and acce%ted as %artial fulfillment of the requirements for the degree

Bachelor in Medical Laboratory Scienceon


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.

A##a" a$ C* E+,irit. RMT. M*Sc*

Undergraduate Research &oordinator


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/

I"tr/cti"

&enters for Disease &ontrol and Prevention 3&D& -.//4 studies sho1 that about one inthree 3;;@4 %eo%le carry Staphylococcus aureusin their nose usually 1ithout any illness$ #1oin one hundred %eo%le carry )ethicillinresistant S. aureus 3)RSA4$Immunocom%romised%atients may acquire the bacteria 1hich may lead to more serious illnesses$

Increase in morbidity mortality and healthcare costs are re%orted due to multidrug resistantorganism 3)DR:4 acquired infections$ )RSA is 'no1n for its %ersistence in clinical settingsdue to increasing resistance to antimicrobial agents ma'ing it the maBor %athogen for nosocomialinfection$ !iofilm formation a%%ears as its survival strategy3Ando )onden )itsuhataCariyama Cumon -..?E #ay 8e1 -./;4$

#he develo%ment of ne1 antibacterial drugs for the treatment of )RSA infections is ofincreasing interest 3Schito -..04$ #he resistance has been emerging not only to methicillin butalso to other many antibiotics including vancomycin leading to further restriction on availableantibiotic o%tions 3!a'ri Al9ommos Shehabi Naffa &ui 9iramitsu -..E )ohammad-./.4$

Another arising resistant %athogen isPseudomonas aeruginosa$ Strains 1ith resistance are

due to biofilm formation 1hich %rotects them from antibiotics$ In addition it uses a large arsenalof %athogenicity factors to interfere 1ith host defenses 39auser :,er -.//4$Study conducted by )endo,a and So 3-./G4 in the Phili%%ines sho1ed that the most

common antibiotics used 1ere ce%halos%orin betalactamHbetalactamase inhibitors thenfluoroquinolones$ Antibiotics such as %i%eracillinta,obactam ceftria5onolones and tetra%enemincreased significantly decreasing the %revalence of P. aeruginosa 1hile )RSA remainsunchanged$

!acteria have certain mechanisms 1hich allo1 them to survive$ :ne is called quorumsensing$ =uorum sensing 3=S4 is a signalling %henomenon used by bacteria in order to e5%ressvirulence 3+attem )ihali' &ri5ell )cLean -..4$ )echanisms of =S include6 genee5%ression virulence e5%ression biofilm formation s%orulation bioluminescence and mating3Na,arro Fratianni &o%%ola -./;4$ Coh Sam 8in #an Crishnan &hong and &han 3-./;4defined it as a system of stimuli and res%onses in relation to bacterial cell %o%ulation density thatregulates gene e5%ression including virulence determinants$

Since several %rocesses res%onsible for successful establishment of bacterial infection aremediated by quorum sensing 3+attem et al$ -..4 it has been an attractive target for thedevelo%ment of novel antiinfective measures that do not rely on the use of antibiotics 3Coh etal$ -./;4$

=S is the communication mechanism bet1een bacterial cells that enables them to control the1ay genes are e5%ressed$ #his ty%e of communication of microorganisms is only achieved athigher cell densities$ #he bacteria release autoinducers various molecules that act as mediatorsof quorum sensing 3Raina De +i,io :dell &lements +anhulle -..74$ #he molecules that areim%licated in quorum sensing should satisfy four essential characteristics$ First a quorumsensing molecule must be able to regulate its o1n %roduction$ Second is that it should be%roduced throughout the gro1th of the organism since it is de%endent to cell density$ #hird itmust be able to elicit a similar res%onse in a mutant strain that has a deficiency in %roducing theautoinducers 1ith the e5ogenous addition of the quorum sensing molecule$ Lastly a quorumsensing molecule must be able to res%ond not only related to metaboli,ing or deto5ifying themolecule itself$ It should res%ond %hysiologically in the organism in 1hich it is %roduced 3Rainaet al$ -..74$


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#he quorum sensing molecules can be degraded or inhibited in order to disru%t quorumsensing$ =uorum sensing antagonists are being investigated in targeting the signal rece%tor3Raina et al$ -..74$

#1o other mechanisms 1ere suggested by basic researchers aside from targeting signalgeneration dissemination and rece%tion$ :ne e5am%le is to use the quorum sensing molecules

not to 'ill infectious bacteria but to have an effect in the modification of the behaviour such asturning off of virulence 39ent,er Riedel Rasmussen 9eydorn Andersen -..;4$!oth P. aeruginosa and )RSA e5hibit quorum sensing activities$ #hese ma'e them

a%%ro%riate test organisms in searching for com%ounds that could inhibit quorum sensingactivity$ 2orld 9ealth :rgani,ation encouraged health systems in different countries since /7.sto interact 1ith herbal medicine for identifying and assessing means that build u% bases for ne1and safe herbal agents 1hich can be used for treatment of infectious and noninfectious diseases329: /74$

&ertain bacteria can become resistant to certain antibiotics 1hen surviving strains are left tore%roduce 3&D& -.//4$ #he increasing amounts of antibiotics develo%ed over the %ast 0. years

have %rovo'ed bacteria to res%ond by %ro%agating %rogeny no longer susce%tible to theseantibiotics ma'ing the develo%ment of ne1er generations of antibiotics increasingly obsolete as amethod of combating %athogenic bacteria 3Levy -...4$ Drugs that disru%t quorum sensing onthe other hand 1ould s%are the microorganism sim%ly %reventing them from causing disease orcausing antibiotic resistance 32enner -..74$

Several %lants and natural %roducts 1ere initially e5amined for their traditional medicine useand recent studies suggest that these can also be a source of natural inhibitors for =S activity3Coh et al$ -./;E Jahin 9asan Aqil Chan 9usain Ahmad -./.E and #olmachevaRogo,hin Deryabin -./?4$ According to S,abK et al$ 3-./.4 rosemary oil is a %otent quorumsensing inhibitor against Chromobacterium violaceum$ Another study by Nagy 3-./.4 sho1edseveral %lant e5tracts including rosemary e5hibiting anti quorum sensing activity against C.violaceum andP. aeruginosa$ #hus rosemary can be a %otent source for antiquorum sensingactivity of microorganism$ Sage(Salvia officinalis) is of the same family as that of rosemary(Rosmarinus officinalis) 1hich is the Labiatae family$ According to researches done by9i%%olyte )arin !accou "onard 3/77-4 a natural antio5idant called rosmarinic acid is%roduced by the cell sus%ension cultures of sage$ 2ith the same chemical com%onent utili,ed ina study conducted by Coh et al$ 3-./;4 and Nagy 3-./.4rosmarinic acid e5tracted from s1eetbasil decreased the e5%ression of the elastase and %rotease as 1ell as biofilm formation in P.aeruginosa.

Assessment of the antiquorum sensing %ro%erties of these %lant e5tracts 1hich are locallyavailable offers an alternative to the reduction of antibiotic resistance develo%ment$ S%ecific%roblems addressed by the study include6 first the determination of significant difference inbiofilm formation bet1een the e5tracttreated and untreated cultures of P. aeroginosa 1ithdifferent e5tract concentrationsE determination of significant difference in biofilm formationbet1een e5tracttreated and untreated cultures of mecAnegative S. aureus1ith different e5tractconcentrations and determination of the significant difference in antibiotic resistance bet1eene5tracttreated and untreated cultures of mecA%ositive S. aureus as measured by quantity ofbacterial cells as e5%ressed through o%tical density and ,one of inhibition in the cefo5itindiffusion method$


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Re+earc0 1et0/ a"/ /e+i!"

#his research utili,ed the e5%erimental method of research follo1ing the factoriale5%erimental design$ #he inde%endent variables are the %resenceHabsence of the antiquorumsensing com%ounds that 1ould be e5tracted from Rosemary 3R. officinalis4 and Sage 3S.officinalis4 and the concentrations of these e5tracts in the treatment grou%s$ #he de%endent

variables 1ould be the biosensor indicators identified for each assay$

A"ti-2r1 Se"+i"! C1,"/ E3tract 're,arati"

)ature leaves of Rosemary and Sage 1ere collected from Ala%ang La #rinidad !enguet$*5traction 1as done by initially 1ashing the collected leaves sun drying them and finally%lacing them in the oven to eliminate their moisture content$ #he oven %rocessed leaves 1erethen soa'ed in alcohol 37G@ ethanol4 for ? hours$ &oncentration %rocess 1as done thru So5hletmethod for ? hours or more to obtain the leaf e5tract$ #he crude e5tract 1as %laced in a 1aterbath to remove the e5cess alcohol to obtain a crude e5tract$ It 1as then dissolved 1ith dimethylsulfo5ide 3D)S:4 to %roduce a stoc' solution having a concentration of /.. gHmL$

Different concentrations of e5tracts 1ere %re%ared from the stoc' solution and %laced in an

amber bottle then autoclaved at /-.M& to remove im%urities$ #he concentrations %re%ared 1ere asfollo1s6 /. gHmL -. gHmL ;. gHmL ?. gHmL G. gHmL 0. gHmL . gHmL .gHmL 7. gHmL and /.. gHmL$ Additional concentration of e5tract at /... gHmL 1as also%re%ared for the antibiotic resistance assay$

ifi#1 I"0i4iti" A++a$ +i"!P. aeruginosa'A56 a"/7r Met0ici##i"-re+i+ta"tS. aureus

#his assay 1as ada%ted from Ando et al$ 3-..?4$ P. aeruginosaPA./ 1as gro1n overnight at;o& in a nutrient broth$ #he culture 1as diluted /6/.. in medium and G. L of the cellsus%ension 1as inoculated into sterile 701ell %olystyrene microtiter %lates %reviously added1ith G. L of /.fold increasing concentrations of the e5tracts from .@ vHv to /..@ vHv diluted1ith distilled 1ater$ #he %lates 1ere incubated for ? hours at ;o& 1ithout sha'ing$ Afterincubation the 1ells 1ere gently 1ashed three times 1ith -.. L of distilled 1ater dried in aninverted %osition and stained 1ith -.. L of -@ crystal violet solution in 1ater for ?G minutes$#he 1ells 1ere destained 1ith -.. L ethanolacetic acid 37G6G vHv4$ #1o hundred microlitersfrom each 1ell 1as transferred into a cuvette containing .. L of distilled 1ater and the o%ticaldensity of crystal violet %resent in the destaining solution 1as measured at G?GG. nm using as%ectro%hotometer$ #he same %rocedures 1ere carried out 1ith )RSA$

As a control an uninoculated medium 1as used to determine bac'ground :D$ It 1asused to set the s%ectro%hotometer at ,ero absorbance reading$ A negative control containingtri%ticase soy broth 1as %re%ared 1hich has the lo1est absorbance reading$ A %ositive controlcontaining a bacterial sus%ension 3P. aeruginosa and )RSA4 1as also %re%ared and has thehighest absorbance reading$

#he %resence of antiquorum sensing activity 3biofilm formation inhibition4 is a sho1n bya decrease in absorbance readings of the solutions 1ith e5tract 1hen com%ared to the %ositivecontrol$


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A"ti4itic Re+i+ta"ce A++a$ i" 1ecA(8) Met0ici##i"-Re+i+ta"tS. aureusQuantitative tube method

A culture tube containing nutrient broth 1as inoculated 1ith mecA%ositive methicillinresistant S. aureus 3)RSA4 A#&& ?;;.. and incubated at ;o& for si5 hours 1ith /.. Lsu%%lementation 1ith sterile test e5tract every t1o hours$ Another tube 1as also inoculated and

incubated follo1ing the same conditions as the first but 1ithout su%%lementation$ :ne hundredmicroliters of direct culture sus%ensions equivalent to .$G )cFarland standard from the t1o tubes1as se%arately subcultured onto a4 #ry%tic Soy !roth culture tube 1ith ?@ Na&l and 0gHmLo5acillin and b4 #ry%tic Soy !roth culture tube 1ithout su%%lements$ #he four resulting tubes1ere incubated at ;o& for /0/ hours and gro1th 1as assessed s%ectro%hotometrically at G.nm$

mecA-mediated !acillin Resistance using "efo!itin #$is% $iffusion Method&

A culture tube containing nutrient broth 1as inoculated 1ith mecA%ositive methicillinresistant S. aureus3)RSA4 A#&& ?;;.. and 1as incubated at ;o& for si5 hours 1ith /.. Lsu%%lementation 1ith sterile test e5tract every t1o hours$ Another tube 1as also inoculated and

incubated follo1ing the same conditions as the first but 1ithout su%%lementation$ Direct colonysus%ensions equivalent to .$G )cFarland standard from the t1o tubes 1ere se%arately %lated onto)ueller 9inton Agar %late 1ith ?@ Na&l and 0gHmL o5acillin then three ;. g cefo5itin dis's1ere introduced equidistantly in each %late$ #he t1o resulting %lates 1ere incubated at ;o& for/0/ hours$

Treat1e"t f Data

All measured values in each assay 1ere subBected to Analysis of +ariance to determinesignificant differences$ Statistically significant sets 1ere further analy,ed using #u'eyOs 9SD todetermine 1here the significant differences lie$ &onfidence levels 1ere uniformly set at .$.Gal%ha$

Re+#t+ a"/ Di+c++i"

ifi#1 I"0i4iti" A++a$ +i"!P. aeruginosa 'A56

Screening of antiquorum sensing activity of e5tracts of rosemary and sage 1as done byusing P. aeruginosa as biosensor$ #he treatment includes /. different concentrations of thee5tract tri%ticase soy broth as the negative control and P. aeruginosaovernight culture as the%ositive control$

Table no. 1 Tests of et!een"Sub#ects $ffects

&oncentrations (rou%s &oncentrations(rou%s

Rosemary e5tract $... $... $...Sage e5tract $... $../ $//-

%esser than &.&' indicates significance#he concentrations refer to the /. different concentrations of e5tract used$ #he grou%s

refer to the com%arison bet1een grou% / and -$ (rou% / contained the e5tract andmicroorganism 1hile grou% - contained the e5tract only$


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Rosemary e5tract sho1s significant interaction of the different concentrations 1hich meansthat the mean absorbances are statistically different as affected by the different concentrations$#he mean absorbance of the grou%s sho1s significant difference 1hich means that 1ith themicroorganism biofilm is %roduced and 1ithout the microorganism no biofilm is %roduced$ #hissho1s that the microorganism 1as not 'illed entirely corres%onding to its antiquorum sensing

activity rather than antibiotic activity$ #he interaction bet1een concentration and grou% sho1significant difference 1hich means that the different concentration of the e5tract used to treat thegrou%s significant decrease in biofilm is observed$

Sage e5tract sho1s significant interaction of the different concentrations 1hich means thatthe mean absorbances are statistically different as affected by the different concentrations$ #hemean absorbance of the grou%s sho1s significant difference 1hich means that 1ith or 1ithoutthe microorganism there is no biofilm %roduced$ #his sho1s that the microorganism 1as 'illedcorres%onding to its antibiotic activity rather than antiquorum sensing activity$ #he interactionbet1een concentration and grou% sho1 no significant difference 1hich means that regardless ofthe concentration of the e5tract used to treat the grou%s no significant decrease in biofilm isobserved$

Table no. eans for groups in *omogenous Subsets for Rosemary

Tu+ey *Sa,b,c

&oncentrations N Subset/ - ;

/ 0 $.?;0// 0 $.G;/- 0 $.0GG.

Sig$ $/7G /$... $-

Same subset indicates no significant difference

&oncentrations / and /- are significant$ #his means that the microorganism decreased itsbiofilm formation at the lo1est concentration of the e5tracts$ &oncentration /- 3%ositive control4is significantly different from the other 1ells$ &oncentrations - to /. sho1 no significantdifference 1hich means that biofilm formation is reduced regardless of the differentconcentrations$

Table no.- eans for groups in *omogenous Subsets for Sage

&oncentrations

N Subset/ - ;

// 0 $.;G;; $.;G;;/ G $.?.. $.?..

/- 0 $.0;;Sig$ $;/. $.? $0;


&oncentrations / and /- are not significant$ #his means that the biofilm formation at thelo1est concentration of the e5tract 1as not significantly reduced$ &oncentrations // 3negativecontrol4 and /- 3%ositive control4 have a significant difference 1hich means that the %ositive


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control %roduced biofilm 1hile the negative control did not$ &oncentrations - to // sho1 nosignificant difference this means that biofilm formation is reduced regardless of the differentconcentrations of the e5tracts$

ifi#1 I"0i4iti" A++a$ +i"!S. aureus

Second screening of antiquorum sensing %ro%erties of e5tracts of rosemary and sage 1asdone by using S. aureusas biosensor$ #he treatment includes /. different concentrations of thee5tract tri%ticase soy broth as the negative control and S. aureus overnight culture as the%ositive control$

Table no. Tests of et!een"Sub#ects $ffects

&oncentrations (rou%s &oncentrations(rou%s

Rosemary e5tract $/-- $G? $7?0Sage e5tract $.7? $77/ $/

%esser than &.&' indicates significance

#he concentrations refer to the /. different concentrations of e5tract used$ #he grou%s referto the com%arison bet1een grou% / and -$ (rou% / contained the e5tract and microorganism1hile grou% - contained the e5tract only$

Rosemary e5tract sho1s no significant interaction of the different concentrations 1hichmeans that the mean absorbances are not statistically different as affected by the differentconcentrations$ #he mean absorbance of the grou%s sho1s no significant difference 1hichmeans that 1ith or 1ithout the microorganism there is no biofilm %roduced$ #his sho1s that themicroorganism 1as 'illed corres%onding to its antibiotic activity rather than anti quorum sensingactivity$ #he interaction bet1een concentration and grou% sho1 no significant difference 1hichmeans that regardless of the concentration of the e5tract used to treat the grou%s no significantdecrease in biofilm is observed$

Sage e5tract sho1s no significant interaction of the different concentrations 1hich meansthat the mean absorbances are not statistically different as affected by the differentconcentrations$ #he mean absorbance of the grou%s sho1s no significant difference 1hichmeans that 1ith or 1ithout the microorganism there is no biofilm %roduced$ #his sho1s that themicroorganism 1as 'illed corres%onding to its antibiotic activity rather than anti quorum sensingactivity$ #he interaction bet1een concentration and grou% sho1 no significant difference 1hichmeans that regardless of the concentration of the e5tract used to treat the grou%s no significantdecrease in biofilm is observed$

Table no.' eans for groups in *omogenous Subsets for Rosemary

Tu+ey *S

a,b,c

&oncentrations NSubset

// 0 $.?;;// 0 $.0;../- 0 $/G0Sig$ $.G



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#he /. concentrations do not differ significantly$ #his means that the use of the differente5tract concentrations do not significantly reduce biofilm formation$

Table no./ eans for groups in *omogenous Subsets for Sage

Tu+ey *Sa,b

&oncentrations NSubset

// 0 $.?;;// 0 $.G?0/- 0 $/0//Sig$ $.G7


#he /. concentrations do not differ significantly$ #his means that the use of the differente5tracts do not significantly reduce biofilm formation$ #he results sho1 that concentration / of

the test sho1s antiquorum sensing activity 1hile concentrations - to /. sho1 antibiotic%ro%erty$ According to Coh et al$ 3-./;4 rosemary e5hibited antiquorum sensing activity on P.aeruginosa and S. aureusalthough concentrations 1ere not determined$ According to S,abo and9ohmann 3-./.4 rosemary essential oils e5hibit %otent antiquorum sensing activity on C.violaceum 1hich is also one of the bacterium used as biosensor in determining antiquorumsensing activity of %lants$

A"ti4itic re+i+ta"ce a++a$

Antiquorum sensing activity against )RSA 1as evaluated using the quantitative tubemethod and mecAmediated o5acillin resistance using cefo5itin 3dis' diffusion method4$Positiveand negative controls controls 1ere used to evaluate the results$ RNA III Inhibiting Pe%tide

3RIP4 a he%ta%e%tide that inhibits S. aureus %athogenesis by disru%ting quorumsensingmechanisms 1as used as a su%%lement for %ositive control 3!alaban (iacometti &irioni (ov(hiselli )occhegiani +iticchi Del Prete Saba Scalise and DellOAcqua -..-4$

Qa"titati9e t4e 1et0/

#he test of bet1een subBects of re%licates sho1 that the e5tract for rosemary sho1s a.$... significance 1hile for sage there is .$.07 significance for concentration and .$./ forconcentrationsu%%lement$

Table no. 0 ntibiotic resistance assay2 3uantitative tube method

)edium+olume and

&oncentration

Sage

*5tract

Rosemary

*5tract

Positive

&ontrol

Negative

&ontrol

#S!

0 mL of /.. LHmL .$?/;;; .$00-00 .$G;;; .$-..0 mL of /. LHmL .$G... .$0--... .$G/;;; .$-..0.. L of /...LHmL

.$?G;;; .$-?... .$?/;;; .$-..

0.. L of /.. LHmL .$//00 .$G..... .$;;;;; .$-..#S! Q:5acillin

0 mL of /.. LHmL .$.G0;;; .$./0... .$/;00 .$00-000 mL of /. LHmL .$.7G... .$/0/... .$0/;00 .$00-00


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0.. L of /...LHmL

.$.?;;; .$.;;;;; .$.GG... .$00-00

0.. L of /.. LHmL .$.-0... .$..;000 .$.0-;;; .$00-00

#his table sho1s that from the initial gro1th in the #S! medium there are decreasing

volumes and concentrations in both solutions$ #his may %rove the %ossibility that themicroorganism is 'illed by :5acillin$ #hus antibiotic activity may indirectly decrease thegro1th observed$ Similar observations 1ere noted by Nagy 3-./.4 on the activity of the bothe5tract on C. voilaceum$

1ecA-1e/iate/ O3aci##i" Re+i+ta"ce +i"! Cef3iti" (Di+: Diff+i" Met0/)

Table no.4 ean iameter of the 5one of 6nhibition in mm of the Replicates

)edium+olume and&oncentration

Sage*5tract

Rosemary*5tract

Positive&ontrol

Negative&ontrol

#S!

0 mL of /.. gHmL -.$;;;; -.$.... /- /70 mL of /. gHmL /7$;;;; /$.... // /7

0.. mL of /...gHmL /$00. /$00. / /7

0.. mL of /..gHmL

/$.... --$.... -. /7

#he /.. gHmL su%%lemented in 0 mL sho1s to have the highest diameter of the differentvolume and concentration of sage and the 0.. mL of /.. gHmL sho1s to be the highest inrosemary$ For the 0 mL of /. gHmL and 0.. mL of /... gHmL of the sage e5tract sho1s tohave an inhibition of /7$ #hese concentrations are believed to have antiquorum sensing activityof the e5tract$

A value of / diameters in 0.. mL of /.. gHmL in sage e5tract and 0 mL of /. gHmL of

rosemary sho1ed to be the lo1est$ For the 0 mL of /. gHmL and 0.. mL of /... gHmL of thesage e5tract have an inhibition of /7 and those 0 mL of /.. gHmL and 0.. mL of /... gHmLof the rosemary e5tract sho1ed -. and /$00. res%ectively$ #hese concentrations do not sho1antiquorum sensing activity instead it can be inter%reted as antibacterial$ Antibacterial activityof sage and rosemary 1as also observed by Al,oubi Ibrahim Alsbou and Aqel 3-./?4$According to #olmacheva et al$ 3-./?4 sage has antimicrobial activity against C. violaceum$

#he activity of the t1o e5tracts is more of antibiotic rather than antiquorum sensing$9o1ever 1hen drugs are used so 1idely and for so long the antibiotics designed to 'illinfectious microorganisms have ada%ted to them ma'ing the drugs less effective 3&D& -./04$

C"c#+i" a"/ Rec11e"/ati"

#he researchers therefore conclude thatR. officinalis leaf e5tract has antiquorum sensingactivity against P. aeruginosa and S. aureusat the concentration of /.gHmL as e5em%lified inthe inhibition of biofilm formation$ &oncentrations greater than /.gHmL have antibiotic activity$

S. officinalisleaf e5tract has no antiquorum sensing activity againstP. aeruginosa and S.aureusat the concentration of /.gHmL antibiotic activity 1as rather observed$

In the antibiotic resistance assays all volumes and concentrations of the rosemary andsage e5tract %re%arations e5hibited antibiotic %ro%erties$


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It can therefore be said that the antibiotic and antiquorum sensing activity of rosemary andsage e5tracts is due to inde%endent activity of its com%onents$

#he researchers recommend the study of lo1er concentrations of both rosemary and sage inorder to determine the minimum concentration of the solutions 1hich allo1 them to e5hibit antiquorum sensing activity$ Isolation and evaluation for antiquorum sensing activity of the

rosmarinic acid com%onent 1ill also ma'e the study more s%ecific$


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R*F*R*N&*S6Al,oubi 9$ )$ Ibrahim A$ I$ Alsbou )$ S$ Aqel A$ A$ 3-./? Se%tember4$ Inhibitory *ffect

of )editerranean Sage and Rosemary on &linical and &ommunity Isolates of )ethicillinResistant Sta%hylococcus aureus$ "ordan "ournal of !iological Sciences 3;4 /0//0?$

Ando *$ )onden C$ )itsuhata R$ Cariyama R$ Cumon 9$ 3-..?4$ !iofilm Formation

among )ethicillinResistant Sta%hylococcus aureus Isolates from Patients 1ith Urinary #ractInfection$ Retrieved from htt%6HH111$lib$o'ayamau$ac$B%H111HactaH%dfHG


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Raina S$ De +i,io D$ :dell )$ &lements )$ +anhulle S$ Ceshavar,#$ 3-..74$ )icrobialquorum sensing6 a tool or a target for antimicrobial thera%y Retrieved fromhtt%6HH111$researchgate$netH%ublicationH-000;;.


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/-

APP*NDIT A6 &:)PL*#* DA#A

!I:FIL) F:R)A#I:N ASSA8 USIN(Pseudomonas aeruginosaR:S*)AR86* Te+t+ f et=ee"-S4>ect+ Effect+

De%endent +ariable6 Absorbances

Source #y%e III Sum ofSquares df )ean Square F Sig$

&orrected )odel $.-7a -; $../ -G$;- $...Interce%t $.0. / $.0. /-/7$/; $...&oncentrations $.-. // $..- ;0$?G $...(rou% $..? / $..? -$?-7 $...&oncentrations (rou% $..0 // $../ /.$?; $...*rror $..- ? ?$7/.*G#otal $.7/ -&orrected #otal $.;/ /

a$ R Squared $7-G 3AdBusted R Squared $74

?* M#ti,#e C1,ari+"De%endent +ariable6 Absorbances#u'ey 9SD

3I4 &oncentrations 3"4 &oncentrations

)eanDifference 3I

"4 Std$ *rror Sig$

7G@ &onfidence Interval

Lo1er !ound U%%er !ound

/ - $./; $..?.?G $../ $..?7? $.;--

; $.--0 $..?.?G $... $.. $.;0G0

? $.-.G. $..?.?G $... $..00/ $.;?;7

G $.--;; $..?.?G $... $..?? $.;0--

0 $.-;G. $..?.?G $... $..70/ $.;;7

$.-;/ $..?.?G $... $..7- $.;.0

$.-G;;

$..?.?G $... $.//?? $.;7--7 $.;.;; $..?.?G $... $./0?? $.??--

/. $.-;; $..?.?G $... $./??? $.?---

// $./G/ $..?.?G $.-- $.-7.0 $../-

/- $.-/; $..?.?G $... $.;G- $..7?- / $./; $..?.?G $../ $.;-- $..?7?

; $..;; $..?.?G $77 $./..0 $./-? $../0 $..?.?G /$... $./--- $./GG0G $..;G. $..?.?G $777 $./.;7 $./;70 $..?0 $..?.?G $77. $..7-- $./G0 $..?;; $..?.?G $77G $..7G0 $./-- $..0G. $..?.?G $7 $..;7 $.-.;7

7 $.//G. $..?.?G $/7G $..-;7 $.-G;7/. $..7G. $..?.?G $?G7 $..?;7 $.-;;7// $.;?.. $..?.?G $... $.?7 $.-.///- $.?.0 $..?.?G $... $.G?G0 $.-0

; / $.--0 $..?.?G $... $.;0G0 $..- $..;; $..?.?G $77 $./- $./..0? $..-/ $..?.?G /$... $./0.0 $.//-G $...;; $..?.?G /$... $./?-- $./;G00 $...; $..?.?G /$... $./;.0 $./?-


18/45

/;

$...G. $..?.?G /$... $./;;7 $./?;7 $..-0 $..?.?G /$... $.//-- $./0G07 $..0 $..?.?G $G0 $..0-- $.-/G0/. $..G0 $..?.?G $7G $..-- $./7G0// $.;; $..?.?G $... $.G/- $.-;7?/- $.??G. $..?.?G $... $.G;7 $.;.0/

? / $.-.G. $..?.?G $... $.;?;7 $..00/- $../0 $..?.?G /$... $./GG0 $./---; $..-/ $..?.?G /$... $.//- $./0.0G $../; $..?.?G /$... $./-.0 $./G-0 $..;.. $..?.?G /$... $./.7 $./07 $..-0 $..?.?G /$... $.//-- $./0G0 $..?; $..?.?G $7 $..7.0 $./-7 $..7; $..?.?G $?.0 $..?.0 $.-;-/. $..; $..?.?G $;/ $..0.0 $.-/-// $.;G0 $..?.?G $... $.?7G0 $.-//- $.?-;; $..?.?G $... $.G0-- $.-??

G / $.--;; $..?.?G $... $.;0-- $..??

- $..;G. $..?.?G $777 $./;7 $./.;7; $...;; $..?.?G /$... $./;G0 $./?--? $../; $..?.?G /$... $./G- $./-.00 $..// $..?.?G /$... $./-- $./G.0 $...; $..?.?G /$... $./;.0 $./?- $..;.. $..?.?G /$... $./.7 $./077 $.... $..?.?G $.G $..G7 $.-/7/. $..0.. $..?.?G $7; $..7 $./77// $.;G. $..?.?G $... $.G/;7 $.-;0//- $.??/ $..?.?G $... $.G.0 $.;.-

0 / $.-;G. $..?.?G $... $.;;7 $..70/- $..?0 $..?.?G $77. $./G0 $..7--

; $...; $..?.?G /$... $./?- $./;.0? $..;.. $..?.?G /$... $./07 $./.7G $..// $..?.?G /$... $./G.0 $./-- $...;; $..?.?G /$... $./?-- $./;G0 $../; $..?.?G /$... $./-.0 $./G-7 $..0; $..?.?G $0? $...0 $.-.-/. $..?; $..?.?G $7 $..7.0 $./-// $.;0 $..?.?G $... $.G-G0 $.-?/- $.?G;; $..?.?G $... $.G7-- $.;/??

/ $.-;/ $..?.?G $... $.;.0 $..7-- $..?;; $..?.?G $77G $./-- $..7G0; $...G. $..?.?G /$... $./?;7 $./;;7? $..-0 $..?.?G /$... $./0G0 $.//--G $...; $..?.?G /$... $./?- $./;.00 $...;; $..?.?G /$... $./;G0 $./?-- $..-/ $..?.?G /$... $.//- $./0.07 $../ $..?.?G $-G $..0- $.-/.0/. $..G/ $..?.?G $7 $..- $./7.0// $.;;; $..?.?G $... $.G--- $.-???/- $.?G.. $..?.?G $... $.G7 $.;///

/ $.-G;; $..?.?G $... $.;7-- $.//??


19/45

/?

- $..0G. $..?.?G $7 $.-.;7 $..;7; $..-0 $..?.?G /$... $./0G0 $.//--? $..?; $..?.?G $7 $./- $..7.0G $..;.. $..?.?G /$... $./07 $./.70 $../; $..?.?G /$... $./G- $./-.0 $..-/ $..?.?G /$... $./0.0 $.//-

7 $..G.. $..?.?G $7; $..7 $./7/. $..;.. $..?.?G /$... $./.7 $./07// $.?.G. $..?.?G $... $.G?;7 $.-00//- $.?/ $..?.?G $... $.0/.0 $.;;-

7 / $.;.;; $..?.?G $... $.??-- $./0??- $.//G. $..?.?G $/7G $.-G;7 $..-;7; $..0 $..?.?G $G0 $.-/G0 $..0--? $..7; $..?.?G $?.0 $.-;- $..?.0G $.... $..?.?G $.G $.-/7 $..G70 $..0; $..?.?G $0? $.-.- $...0 $../ $..?.?G $-G $.-/.0 $..0- $..G.. $..?.?G $7; $./7 $..7

/. $..-.. $..?.?G /$... $./G7 $.//7// $.?GG. $..?.?G $... $.G7;7 $.;/0//- $.G-/ $..?.?G $... $.00.0 $.;-

/. / $.-;; $..?.?G $... $.?--- $./???- $..7G. $..?.?G $?G7 $.-;;7 $..?;7; $..G0 $..?.?G $7G $./7G0 $..--? $..; $..?.?G $;/ $.-/- $..0.0G $..0.. $..?.?G $7; $./77 $..70 $..?; $..?.?G $7 $./- $..7.0 $..G/ $..?.?G $7 $./7.0 $..- $..;.. $..?.?G /$... $./07 $./.77 $..-.. $..?.?G /$... $.//7 $./G7

// $.?;G.

$..?.?G $... $.G;7 $.-70//- $.G./ $..?.?G $... $.0?.0 $.;0-// / $./G/ $..?.?G $.-- $../- $.-7.0

- $.;?.. $..?.?G $... $.-.// $.?7; $.;; $..?.?G $... $.-;7? $.G/-? $.;G0 $..?.?G $... $.-/ $.?7G0G $.;G. $..?.?G $... $.-;0/ $.G/;70 $.;0 $..?.?G $... $.-? $.G-G0 $.;;; $..?.?G $... $.-??? $.G--- $.?.G. $..?.?G $... $.-00/ $.G?;77 $.?GG. $..?.?G $... $.;/0/ $.G7;7/. $.?;G. $..?.?G $... $.-70/ $.G;7/- $..00 $..?.?G $- $.-.G0 $..--

/- / $.-/; $..?.?G $... $..7? $.;G-

- $.?.0 $..?.?G $... $.-0 $.G?G0

; $.??G. $..?.?G $... $.;.0/ $.G;7

? $.?-;; $..?.?G $... $.-?? $.G0--

G $.??/ $..?.?G $... $.;.- $.G.0

0 $.?G;; $..?.?G $... $.;/?? $.G7--

$.?G.. $..?.?G $... $.;/// $.G7

$.?/ $..?.?G $... $.;;- $.0/.0


20/45

/G

7 $.G-/ $..?.?G $... $.;- $.00.0

/. $.G./ $..?.?G $... $.;0- $.0?.0

// $..00 $..?.?G $- $..-- $.-.G0

!ased on observed means$#he error term is )ean Square3*rror4 ?$7/.*G$$ #he mean difference is significant at the $.G level$

;* H1!e"e+ +4+et+

#u'ey 9SDab

&oncentrations N

Subset

/ - ;

7 0 $./;;;/. 0 $./G;; 0 $./;;0 0 $.-./ 0 $.-.G.; 0 $.-/..G 0 $.-/;;? 0 $.-;/- 0 $.-?;/ 0 $.?;0// 0 $.G;/- 0 $.0GG.Sig$ $/7G /$... $-

)eans for grou%s in homogeneous subsets are dis%layed$!ased on observed means$#he error term is )ean Square3*rror4 ?$7/.*G$a$ Uses 9armonic )ean Sam%le Si,e 0$...$b$ Al%ha $.G$

SA(*@* Te+t+ f et=ee"-S4>ect+ Effect+


Source#y%e III Sum of

Squares df )ean Square F Sig$

&orrected )odel $.-.a -; $../ ?$G00 $...Interce%t $.G? / $.G? -.$-?0 $...&oncentrations $./? // $../ 0$GG? $...(rou% $..- / $..- //$0 $../&oncentrations (rou% $..? // $... /$00; $//-*rror $..7 ? $...#otal $.? /&orrected #otal $.-7 .

a$ R Squared $07/ 3AdBusted R Squared $G?.4

* M#ti,#e C1,ari+"

M#ti,#e C1,ari+"+

De%endent +ariable6 Absorbances#u'ey 9SD


)eanDifference 3I

"4 Std$ *rror Sig$




21/45

/0

/ - $./0 $..?// $G?G $./.-? $.?G

; $.-G;; $..?// $/; $..;G $.G?-?

? $.-;;; $..?// $--? $..GG $.G--?

G $.-0G. $..?// $/.. $..-?/ $.GG?/

0 $./-G. $..?// $7; $./0?/ $.?/?/

$.;.;;

$..?// $.;- $../?- $.G7-? $.;/0 $..?// $.-/ $..-0 $.0.G

7 $.;-0 $..?// $./G $..;0 $.0/G

/. $.;-.. $..?// $./7 $..;.7 $.0.7/

// $.//0 $..?// $70. $./-? $.?.G

/- $./0; $..?// $07. $.?G? $./-.- / $./0 $..?// $G?G $.?G $./.-?

; $..00 $.../7 $777 $.-.7. $.;?-;? $..?0 $.../7 /$... $.--7. $.;--;G $..; $.../7 $77 $./7; $.;G?.0 $..0/ $.../7 /$... $.;;; $.-/?. $.//0 $.../7 $7?G $./G7. $.;7-;

$./;.. $.../7 $7- $./?G $.?.G7 $./?.. $.../7 $; $./;G $.?/G/. $./;;; $.../7 $0 $./?-; $.?.7.// $.... $.../7 $777 $.;?G $.-.G/- $.;GG. $.../7 $..; $.0;. $..7;

; / $.-G;; $..?// $/; $.G?-? $..;G- $..00 $.../7 $777 $.;?-; $.-.7.? $..-.. $.../7 /$... $.-7G $.-GGG $..// $.../7 /$... $.-0?. $.-;0 $./-; $.../7 $7.. $.?.?. $./?; $..G.. $.../7 /$... $.--G $.;-G $..0;; $.../7 /$... $.-/-; $.;;7.7 $..;; $.../7 $777 $.-.-; $.;?7./. $..00 $.../7 $777 $.-.7. $.;?-;// $./;0 $.../7 $G $.?/-; $./;7./- $.?-/ $.../7 $... $.07; $./?0.

? / $.-;;; $..?// $--? $.G--? $..GG- $..?0 $.../7 /$... $.;--; $.--7.; $..-.. $.../7 /$... $.-GG $.-7GG $..;/ $.../7 /$... $.-??. $.;.;0 $./.; $.../7 $70 $.;?. $./0; $.... $.../7 $777 $.-.G $.;?G $..;; $.../7 $770 $./7-; $.;G7.7 $..7;; $.../7 $77 $./-; $.;07./. $..0 $.../7 $77? $./7. $.;0-;

// $.//0 $.../7 $7?G $.;7-; $./G7./- $.?./ $.../7 $... $.0; $./-0.

G / $.-0G. $..?// $/.. $.GG?/ $..-?/- $..; $.../7 $77 $.;G?. $./7;; $..// $.../7 /$... $.-; $.-0?.? $..;/ $.../7 /$... $.;.; $.-??.0 $./?.. $.../7 $; $.?/G $./;G $..;; $.../7 /$... $.-;; $.;/?.


22/45

/

$..G/ $.../7 /$... $.--?. $.;-;7 $..0/ $.../7 /$... $.-/?. $.;;;/. $..GG. $.../7 /$... $.--. $.;;.// $./?; $.../7 $- $.?-?. $./-;/- $.?;;; $.../7 $... $..7. $./G

0 / $./-G. $..?// $7; $.?/?/ $./0?/

- $..0/ $.../7 /$... $.-/?. $.;;;; $./-; $.../7 $7.. $./?; $.?.?.? $./.; $.../7 $70 $./0; $.;?.G $./?.. $.../7 $; $./;G $.?/G $./; $.../7 $G?- $..7; $.?G?. $./7/ $.../7 $?;- $..?. $.?0;7 $.-./ $.../7 $;G0 $..?. $.?;/. $./7G. $.../7 $?.0 $... $.?.// $...; $.../7 /$... $.-?. $.-0;/- $.-7;; $.../7 $.- $.G07. $../

/ $.;.;; $..?// $.;- $.G7-? $../?-- $.//0 $.../7 $7?G $.;7-; $./G7.

; $..G.. $.../7 /$... $.;-G $.--G? $.... $.../7 $777 $.;?G $.-.GG $..;; $.../7 /$... $.;/?. $.-;;0 $./; $.../7 $G?- $.?G?. $..7; $../;; $.../7 /$... $.-0-; $.-7.7 $..-;; $.../7 /$... $.-G-; $.-77./. $../0 $.../7 /$... $.-G7. $.-7-;// $./0 $.../7 $?; $.?0-; $..7./- $.?/ $.../7 $... $.?; $./70.

/ $.;/0 $..?// $.-/ $.0.G $..-0- $./;.. $.../7 $7- $.?.G $./?G; $..0;; $.../7 /$... $.;;7. $.-/-;

? $..;; $.../7 $770 $.;G7. $./7-;G $..G/ $.../7 /$... $.;-; $.--?.0 $./7/ $.../7 $?;- $.?0; $..?. $../;; $.../7 /$... $.-7. $.-0-;7 $../.. $.../7 /$... $.-0G $.-G/. $...;; $.../7 /$... $.--; $.-7.// $.-... $.../7 $;07 $.?G $..G/- $.?G. $.../7 $... $.0. $.-.7;

7 / $.;-0 $..?// $./G $.0/G $..;0- $./?.. $.../7 $; $.?/G $./;G; $..;; $.../7 $777 $.;?7. $.-.-;? $..7;; $.../7 $77 $.;07. $./-;G $..0/ $.../7 /$... $.;;; $.-/?.0 $.-./ $.../7 $;G0 $.?; $..?. $..-;; $.../7 /$... $.-77. $.-G-; $../.. $.../7 /$... $.-G $.-0G/. $...0 $.../7 /$... $.--; $.-07.// $.-/.. $.../7 $-77 $.?G $..0G/- $.?7G. $.../7 $... $.. $.-/7;

/. / $.;-.. $..?// $./7 $.0.7/ $..;.7- $./;;; $.../7 $0 $.?.7. $./?-;


23/45

/

; $..00 $.../7 $777 $.;?-; $.-.7.? $..0 $.../7 $77? $.;0-; $./7.G $..GG. $.../7 /$... $.;;. $.--.0 $./7G. $.../7 $?.0 $.?. $... $../0 $.../7 /$... $.-7-; $.-G7. $...;; $.../7 /$... $.-7. $.--;

7 $...0 $.../7 /$... $.-07. $.--;// $.-.;; $.../7 $;?? $.?7. $..-;/- $.?; $.../7 $... $.0?. $.-/-

// / $.//0 $..?// $70. $.?.G $./-?- $.... $.../7 $777 $.-.G $.;?G; $./;0 $.../7 $G $./;7. $.?/-;? $.//0 $.../7 $7?G $./G7. $.;7-;G $./?; $.../7 $- $./-; $.?-?.0 $...; $.../7 /$... $.-0; $.-?. $./0 $.../7 $?; $..7. $.?0-; $.-... $.../7 $;07 $..G $.?G7 $.-/.. $.../7 $-77 $..0G $.?G

/. $.-.;; $.../7 $;?? $..-; $.?7./- $.-G. $.../7 $.; $.G0. $...7;

/- / $./0; $..?// $07. $./-. $.?G?

- $.;GG. $.../7 $..; $..7; $.0;.

; $.?-/ $.../7 $... $./?0. $.07;

? $.?./ $.../7 $... $./-0. $.0;

G $.?;;; $.../7 $... $./G $..7.

0 $.-7;; $.../7 $.- $../ $.G07.

$.?/ $.../7 $... $./70. $.?;

$.?G. $.../7 $... $.-.7; $.0.

7 $.?7G. $.../7 $... $.-/7; $..

/. $.?; $.../7 $... $.-/- $.0?.

// $.-G. $.../7 $.; $...7; $.G0.


B* H1!e"e+ +4+et+

A4+r4a"ce+

#u'ey 9SDabc

&oncentrations N

Subset

/ - ;

7 0 $./?;;/. 0 $./G..

0 $./G;; 0 $./00G 0 $.-.G. $.-.G.; 0 $.-/0 $.-/0? 0 $.-;0 $.-;0- 0 $.-;; $.-;;0 0 $.;?G. $.;?G.// 0 $.;G;; $.;G;;/ G $.?.. $.?../- 0 $.0;;


24/45

/7

Sig$ $;/. $.? $0;

)eans for grou%s in homogeneous subsets are dis%layed$!ased on observed means$#he error term is )ean Square3*rror4 $...$a$ Uses 9armonic )ean Sam%le Si,e G$7.-$

b$ #he grou% si,es are unequal$ #he harmonic mean of the grou%si,es is used$ #y%e I error levels are not guaranteed$c$ Al%ha $.G$

!I:FIL) F:R)A#I:N ASSA8 USIN( Staphylococcus aureusR:S*)AR86* Te+t+ f et=ee"-S4>ect+ Effect+




&orrected )odel $.Ga -; $..? $7- $G/?Interce%t $;.0 / $;.0 .$/;0 $...&oncentrations $.0 // $..0 /$0-; $/--

(rou% $... / $... $.;? $G?&oncentrations (rou% $./ // $..- $?. $7?0*rror $/; ? $..?#otal $G? -&orrected #otal $-0 /

a$ R Squared $;? 3AdBusted R Squared $.;04

?* M#ti,#e C1,ari+"



)eanDifference 3I

"4 Std$ *rror Sig$


Lo1er !ound U%%er !ound/ - $..;G. $.;G0GG /$... $/-G7; $//7;

; $.;;; $.;G0GG $77 $/G0-0 $.0.

? $..-G. $.;G0GG /$... $/-?7; $//77;

G $..-;; $.;G0GG /$... $/-?0 $/-./.

0 $.;-; $.;G0GG $777 $/GG-0 $.70.

$..G/ $.;G0GG /$... $/-0. $//-0

$./.G. $.;G0GG /$... $/;-7; $///7;

7 $../; $.;G0GG /$... $/-?-0 $/-.0.

/. $.-7;; $.;G0GG $777 $/G/0 $.7;/.

// $./7/ $.;G0GG /$... $/?/0. $/.;-0

/- $//?; $.;G0GG $.G $-;-0 $..0.- / $..;G. $.;G0GG /$... $//7; $/-G7;

; $.;.;; $.;G0GG $777 $/G-0 $.7-/.? $../.. $.;G0GG /$... $/-/?; $/-;?;G $..// $.;G0GG /$... $/-/-0 $/-;0.0 $.-7;; $.;G0GG $777 $/G/0 $.7;/. $../0 $.;G0GG /$... $/-?/. $/-.0 $.... $.;G0GG /$... $/-7?; $//G?;7 $../0 $.;G0GG /$... $/-.0 $/-?/.


25/45

-.

/. $.-G; $.;G0GG /$... $/?-0 $.700.// $./G0 $.;G0GG /$... $/;/. $/.00/- $///;; $.;G0GG $/. $-;;0 $.///.

; / $.;;; $.;G0GG $77 $.0. $/G0-0- $.;.;; $.;G0GG $777 $.7-/. $/G-0? $.;/;; $.;G0GG $777 $.7//. $/G;0

G $.;/G. $.;G0GG $777 $.7.7; $/G;7;0 $../.. $.;G0GG /$... $/-/?; $/-;?; $.-0 $.;G0GG /$... $.7;0 $/G//. $.-;;; $.;G0GG /$... $.77/. $/?G07 $.;-.. $.;G0GG $777 $.7.?; $/G??;/. $..?G. $.;G0GG /$... $//7; $/-07;// $./?0 $.;G0GG /$... $/.0 $/;/./- $./.. $.;G0GG $G.7 $-.;?; $.?/?;

? / $..-G. $.;G0GG /$... $//77; $/-?7;- $../.. $.;G0GG /$... $/-;?; $/-/?;; $.;/;; $.;G0GG $777 $/G;0 $.7//.G $.../ $.;G0GG /$... $/---0 $/--0.

0 $.;.;; $.;G0GG $777 $/G-0 $.7-/. $..-0 $.;G0GG /$... $/-G/. $//70 $.... $.;G0GG /$... $/;.?; $//??;7 $...0 $.;G0GG /$... $/-/0 $/-;/./. $.-0; $.;G0GG /$... $/?7-0 $.7G0.// $./00 $.;G0GG /$... $/;7/. $/.G0/- $//-;; $.;G0GG $/.. $-;?0 $././.

G / $..-;; $.;G0GG /$... $/-./. $/-?0- $..// $.;G0GG /$... $/-;0. $/-/-0; $.;/G. $.;G0GG $777 $/G;7; $.7.7;? $.../ $.;G0GG /$... $/--0. $/---00 $.;.G. $.;G0GG $777 $/G-7; $.7/7;

$..-; $.;G0GG /$... $/-G-0 $//70. $../ $.;G0GG /$... $/;.0. $//?-07 $...G. $.;G0GG /$... $/-/7; $/--7;/. $.-.. $.;G0GG /$... $/?7?; $.7G?;// $./0; $.;G0GG /$... $/;7-0 $/.G0./- $//-G. $.;G0GG $.77 $-;?7; $..77;

0 / $.;-; $.;G0GG $777 $.70. $/GG-0- $.-7;; $.;G0GG $777 $.7;/. $/G/0; $../.. $.;G0GG /$... $/-;?; $/-/?;? $.;.;; $.;G0GG $777 $.7-/. $/G-0G $.;.G. $.;G0GG $777 $.7/7; $/G-7; $.-0 $.;G0GG /$... $.7?0 $/G./. $.--;; $.;G0GG /$... $/../. $/??07 $.;/.. $.;G0GG $777 $.7/?; $/G;?;/. $..;G. $.;G0GG /$... $//7; $/-G7;// $./;0 $.;G0GG /$... $/.0 $/;0/./- $.-.. $.;G0GG $?7/ $-.??; $.?.?;

/ $..G/ $.;G0GG /$... $//-0 $/-0.- $../0 $.;G0GG /$... $/-.0 $/-?/.; $.-0 $.;G0GG /$... $/G//. $.7;0? $..-0 $.;G0GG /$... $//70 $/-G/.


26/45

-/

G $..-; $.;G0GG /$... $//70. $/-G-00 $.-0 $.;G0GG /$... $/G./. $.7?0 $..G;; $.;G0GG /$... $/-0 $///.7 $..;;; $.;G0GG /$... $//7/. $/-G0/. $.-?/ $.;G0GG /$... $/?00. $.7-0// $./?.. $.;G0GG /$... $/;0?; $/.?;

/- $/.70 $.;G0GG $//7 $-;-/. $./-0 / $./.G. $.;G0GG /$... $///7; $/;-7;

- $.... $.;G0GG /$... $//G?; $/-7?;; $.-;;; $.;G0GG /$... $/?G0 $.77/.? $.... $.;G0GG /$... $//??; $/;.?;G $../ $.;G0GG /$... $//?-0 $/;.0.0 $.--;; $.;G0GG /$... $/??0 $/../. $..G;; $.;G0GG /$... $///. $/-07 $..0 $.;G0GG /$... $//;0 $/;//./. $./; $.;G0GG /$... $/?/-0 $/.;0.// $..0 $.;G0GG /$... $/;//. $//;0/- $/.?;; $.;G0GG $/0? $--00 $.//.

7 / $../; $.;G0GG /$... $/-.0. $/-?-0- $../0 $.;G0GG /$... $/-?/. $/-.0; $.;-.. $.;G0GG $777 $/G??; $.7.?;? $...0 $.;G0GG /$... $/-;/. $/-/0G $...G. $.;G0GG /$... $/--7; $/-/7;0 $.;/.. $.;G0GG $777 $/G;?; $.7/?; $..;;; $.;G0GG /$... $/-G0 $//7/. $..0 $.;G0GG /$... $/;//. $//;0/. $.-G. $.;G0GG /$... $/?77; $.7?7;// $./;; $.;G0GG /$... $/;70 $/.G/./- $//;.. $.;G0GG $.70 $-;G?; $..7?;

/. / $.-7;; $.;G0GG $777 $.7;/. $/G/0

- $.-G; $.;G0GG /$... $.700. $/?-0; $..?G. $.;G0GG /$... $/-07; $//7;? $.-0; $.;G0GG /$... $.7G0. $/?7-0G $.-.. $.;G0GG /$... $.7G?; $/?7?;0 $..;G. $.;G0GG /$... $/-G7; $//7; $.-?/ $.;G0GG /$... $.7-0 $/?00. $./; $.;G0GG /$... $/.;0. $/?/-07 $.-G. $.;G0GG /$... $.7?7; $/?77;// $././ $.;G0GG /$... $//--0 $/;-0./- $.GG. $.;G0GG $?- $-.7; $.;07;

// / $./7/ $.;G0GG /$... $/.;-0 $/?/0.- $./G0 $.;G0GG /$... $/.00 $/;/.; $./?0 $.;G0GG /$... $/;/. $/.0? $./00 $.;G0GG /$... $/.G0 $/;7/.G $./0; $.;G0GG /$... $/.G0. $/;7-00 $./;0 $.;G0GG /$... $/;0/. $/.0 $./?.. $.;G0GG /$... $/.?; $/;0?; $..0 $.;G0GG /$... $//;0 $/;//.7 $./;; $.;G0GG /$... $/.G/. $/;70/. $././ $.;G0GG /$... $/;-0. $//--0/- $.7G0 $.;G0GG $-00 $-//. $.-00


27/45

--

/- / $//?; $.;G0GG $.G $..0. $-;-0

- $///;; $.;G0GG $/. $.///. $-;;0

; $./.. $.;G0GG $G.7 $.?/?; $-.;?;

? $//-;; $.;G0GG $/.. $././. $-;?0

G $//-G. $.;G0GG $.77 $..77; $-;?7;

0 $.-.. $.;G0GG $?7/ $.?.?; $-.??;

$/.70 $.;G0GG $//7 $./-0 $-;-/.

$/.?;; $.;G0GG $/0? $.//. $--00

7 $//;.. $.;G0GG $.70 $..7?; $-;G?;

/. $.GG. $.;G0GG $?- $.;07; $-.7;

// $.7G0 $.;G0GG $-00 $.-00 $-//.


;* H1!e"e+ +4+et+

&oncentrations N

Subset

// 0 $.?;;7 0 $.?G0G 0 $.?0/? 0 $.?0;;- 0 $.?;; 0 $.?7.. 0 $.G?;;// 0 $.0;../. 0 $.;/0 0 $.00; 0 $.0/- 0 $/G0Sig$ $.G

)eans for grou%s in homogeneous subsets are dis%layed$!ased on observed means$#he error term is )ean Square3*rror4 ?$7/.*G$a$ Uses 9armonic )ean Sam%le Si,e 0$...$b$ Al%ha $.G$

SA(*@* Te+t+ f et=ee"-S4>ect+ Effect+




&orrected )odel $.7?a

-; $..? /$//0 $;0?Interce%t $-7 / $-7 0$;- $...&oncentrations $.07 // $..0 /$;; $.7?(rou% G$...* / G$...* $... $77/&oncentrations (rou% $.-? // $..- $0./ $/*rror $/G ? $..?#otal $G? -&orrected #otal $-07 /

a$ R Squared $07/ 3AdBusted R Squared $G?.4


28/45

-;

* M#ti,#e C1,ari+"

M#ti,#e C1,ari+"+



)ean

Difference 3I"4 Std$ *rror Sig$



/ - $..;G. $.;?0; /$... $/-;-/ $//0-/

; $./; $.;?0; /$... $/;G? $/..

? $..-G. $.;?0; /$... $/---/ $//-/

G $../;; $.;?0; /$... $/-/.? $//;

0 $./7/ $.;?0; /$... $/; $/..G?

$..G/ $.;?0; /$... $/-? $//?G?

$./.G. $.;?0; /$... $/;.-/ $/.7-/

7 $../; $.;?0; /$... $/-/G? $//

/. $.-7;; $.;?0; $777 $/?7.? $.7.;

// $./.; $.;?0; /$... $/;.G? $/.

/- $//;; $.;?0; $.G7 $-;.? $..-;- / $..;G. $.;?0; /$... $//0-/ $/-;-/

; $./G;; $.;?0; /$... $/;G.? $/.?;? $../.. $.;?0; /$... $/// $/-./G $..-/ $.;?0; /$... $//G? $/-/0 $./G0 $.;?0; /$... $/;G; $/.?.? $../0 $.;?0; /$... $/-/; $//.? $.... $.;?0; /$... $/-0/ $//-/7 $../0 $.;?0; /$... $//.? $/-/;/. $.-G; $.;?0; /$... $/?GG? $.7;// $..;; $.;?0; /$... $/-.? $//-;/- $//;; $.;?0; $.0 $-;;G? $..G

; / $./; $.;?0; /$... $/.. $/;G?- $./G;; $.;?0; /$... $/.?; $/;G.?? $./0;; $.;?0; /$... $/.;; $/;0.?G $./G. $.;?0; /$... $/.--/ $/;-/0 $...;; $.;?0; /$... $/-..? $//7; $./;0 $.;?0; /$... $/.0.? $/;;; $..;; $.;?0; /$... $///; $/-.?7 $./.. $.;?0; /$... $/.-/ $/;0//. $./.G. $.;?0; /$... $/;.-/ $/.7-/// $.... $.;?0; /$... $//// $/-//- $.7G. $.;?0; $-.- $-/-/ $.-/-/

? / $..-G. $.;?0; /$... $//-/ $/---/

- $../.. $.;?0; /$... $/-./ $///; $./0;; $.;?0; /$... $/;0.? $/.;;G $..// $.;?0; /$... $//G? $/-.0 $./00 $.;?0; /$... $/;0; $/.;.? $..-0 $.;?0; /$... $/--; $//.? $.... $.;?0; /$... $/-/ $////7 $...0 $.;?0; /$... $//7.? $/-.;/. $.-0; $.;?0; /$... $/?0G? $.7-// $..;; $.;?0; /$... $/-.? $///;


29/45

-?

/- $//?; $.;?0; $./ $-;?G? $..?G / $../;; $.;?0; /$... $//; $/-/.?

- $..-/ $.;?0; /$... $/-/ $//G?; $./G. $.;?0; /$... $/;-/ $/.--/? $..// $.;?0; /$... $/-. $//G?0 $./; $.;?0; /$... $/;G? $/./

$..;; $.;?0; /$... $/-;G? $//G $..7/ $.;?0; /$... $/- $//.G?7 $...G. $.;?0; /$... $/-.-/ $//7-//. $.-.. $.;?0; /$... $/?/ $.7//// $..7G. $.;?0; /$... $/-7-/ $//.-//- $//0.. $.;?0; $.0G $-;G/ $..;/

0 / $./7/ $.;?0; /$... $/..G? $/;- $./G0 $.;?0; /$... $/.?.? $/;G;; $...;; $.;?0; /$... $//7; $/-..?? $./00 $.;?0; /$... $/.;.? $/;0;G $./; $.;?0; /$... $/./ $/;G? $./?.. $.;?0; /$... $/.G/ $/;;/

$..0 $.;?0; /$... $///.? $/-;7 $./;; $.;?0; /$... $/.-; $/;.?/. $././ $.;?0; /$... $/-7 $/.7G?// $..;; $.;?0; /$... $///; $/-.?/- $.7/ $.;?0; $-.0 $-/ $.-/G?

/ $..G/ $.;?0; /$... $//?G? $/-?- $../0 $.;?0; /$... $//.? $/-/;; $./;0 $.;?0; /$... $/;;; $/.0.?? $..-0 $.;?0; /$... $//.? $/--;G $..;; $.;?0; /$... $//G $/-;G?0 $./?.. $.;?0; /$... $/;;/ $/.G/ $..G;; $.;?0; /$... $/-G.? $//?;

7 $..;;; $.;?0; /$... $//0; $/-;.?/. $.-?/ $.;?0; /$... $/?; $.7GG?// $..G0 $.;?0; /$... $/-G; $//?.?/- $//-/ $.;?0; $.G $-;/ $..G?

/ $./.G. $.;?0; /$... $/.7-/ $/;.-/- $.... $.;?0; /$... $//-/ $/-0/; $..;; $.;?0; /$... $/-.? $///;? $.... $.;?0; /$... $//// $/-/G $..7/ $.;?0; /$... $//.G? $/-0 $..0 $.;?0; /$... $/-; $///.? $..G;; $.;?0; /$... $//?; $/-G.?7 $..0 $.;?0; /$... $///.? $/-;/. $./; $.;?0; /$... $/;G? $/..// $...;; $.;?0; /$... $/-..? $//7;/- $/.0; $.;?0; $/-- $--0G? $./-

7 / $../; $.;?0; /$... $// $/-/G?- $../0 $.;?0; /$... $/-/; $//.?; $./.. $.;?0; /$... $/;0/ $/.-/? $...0 $.;?0; /$... $/-.; $//7.?G $...G. $.;?0; /$... $//7-/ $/-.-/0 $./;; $.;?0; /$... $/;.? $/.-;


30/45

-G

$..;;; $.;?0; /$... $/-;.? $//0; $..0 $.;?0; /$... $/-; $///.?/. $.-G. $.;?0; /$... $/?-/ $.7--/// $..7.. $.;?0; /$... $/-/ $//.//- $//GG. $.;?0; $.0 $-;G-/ $..?-/

/. / $.-7;; $.;?0; $777 $.7.; $/?7.?

- $.-G; $.;?0; /$... $.7; $/?GG?; $./.G. $.;?0; /$... $/.7-/ $/;.-/? $.-0; $.;?0; /$... $.7- $/?0G?G $.-.. $.;?0; /$... $.7// $/?/0 $././ $.;?0; /$... $/.7G? $/-7 $.-?/ $.;?0; /$... $.7GG? $/?; $./; $.;?0; /$... $/.. $/;G?7 $.-G. $.;?0; /$... $.7--/ $/?-/// $./G. $.;?0; /$... $/./-/ $/;-//- $... $.;?0; $;G. $-./ $.;//

// / $./.; $.;?0; /$... $/. $/;.G?- $..;; $.;?0; /$... $//-; $/-.?

; $.... $.;?0; /$... $/-/ $////? $..;; $.;?0; /$... $///; $/-.?G $..7G. $.;?0; /$... $//.-/ $/-7-/0 $..;; $.;?0; /$... $/-.? $///; $..G0 $.;?0; /$... $//?.? $/-G; $...;; $.;?0; /$... $//7; $/-..?7 $..7.. $.;?0; /$... $//./ $/-//. $./G. $.;?0; /$... $/;-/ $/./-//- $/.0G. $.;?0; $/-? $--0-/ $./;-/

/- / $//;; $.;?0; $.G7 $..-; $-;.?

- $//;; $.;?0; $.0 $..G $-;;G?

; $.7G. $.;?0; $-.- $.-/-/ $-/-/

? $//?; $.;?0; $./ $..? $-;?G?G $//0.. $.;?0; $.0G $..;/ $-;G/

0 $.7/ $.;?0; $-.0 $.-/G? $-/

$//-/ $.;?0; $.G $..G? $-;/

$/.0; $.;?0; $/-- $./- $--0G?

7 $//GG. $.;?0; $.0 $..?-/ $-;G-/

/. $... $.;?0; $;G. $.;// $-./

// $/.0G. $.;?0; $/-? $./;-/ $--0-/


B* H1!e"e+ +4+et+

A4+r4a"ce+

#u'ey 9SDabc

&oncentrations N

Subset

/

/ 0 $.?;;G 0 $.?G/7 0 $.?G0


31/45

-0

? 0 $.?0;;- 0 $.?;; 0 $.?7.. 0 $.G?;;// 0 $.G?0; 0 $.0-00 0 $.0;../. 0 $.;//- 0 $/0//Sig$ $.G7

)eans for grou%s in homogeneous subsets are dis%layed$!ased on observed means$#he error term is )ean Square3*rror4 $...$a$ Uses 9armonic )ean Sam%le Si,e G$7.-$b$ #he grou% si,es are unequal$ #he harmonic mean of the grou%si,es is used$ #y%e I error levels are not guaranteed$c$ Al%ha $.G$

ANTIIOTIC RESISTANCE ASSAY

CEFOXITIN

ROSEMARY

)RSA/ )RSA- )RSA; )RSA? SA/ SA- SA; SA?

Rosemary

/7 / /7 -- -7 -7 -? ?.

-/ / / -- ;. ;/ -G ?/

-. / /7 -- ;. ;. -G ?.

Pos /- // / -. - -7 -. -

/- // / -. - -7 -G -

/- // / -. - - -G -

Neg -/ 0 -/ 0 ;- 0 ;- 0

-- 0 -- 0 ;. 0 ;. 0

-; 0 -; 0 ;. 0 ;. 0

AN:+A6 #1oFactor 1ith Re%licationSU))AR8 )RSA/ )RSA- )RSA; )RSA? SA/ SA- SA; SA? #otal

Rosemary

&ount ; ; ; ; ; ; ; ; -?

Sum 0. G? G0 00 7 7. ? /-/ 0/.

Average -. / /$0000 -- -7$0000 ;. -?$0000 ?.$;;;;; -G$?/00

+ariance / . .$;;;;;; . .$;;;;;; / .$;;;;;; .$;;;;;; G-$7?7-

Pos

&ount ; ; ; ; ; ; ; ; -?

Sum ;0 ;; G? 0. ? 0 . ? G.

Average /- // / -. - -$0000 -;$;;;;; - -/$/-G

+ariance . . . . . .$;;;;;; $;;;;;; . ?0$.7

9eg

&ount ; ; ; ; ; ; ; ; -?

Sum 00 / 00 / 7- / 7- / ;

Average -- 0 -- 0 ;.$0000 0 ;.$0000 0 /0$/000


32/45

-

+ariance / . / . /$;;;;;; . /$;;;;;; . //$.G

Total

&ount 7 7 7 7 7 7 7 7

Sum /0- /.G /0 /?? -0G /7? -;0 --;

Average / //$0000 /7$GGGG0 /0 -7$????? -/$GGGG0 -0$----- -?$

+ariance -/$G -$-G ;$ G /$ /;0$ /;$7???? --0$7???

AN:+ASource of

:ariation

SS df S 8 P"value 8 crit

Sam%le /.-$G- - G/?$-0;7 ?.$G? $;*; ;$/7.-

&olumns -/-0$G?- ;.;$7/ ?;$?0 ;$-G*?/ -$-.?;0

Interaction -?7$7/ /? -.;$G0GG -7;$/;?; /$*?/ /$7.;0G;

2ithin ;;$;;;;; ? .$07????

#otal 0.;$;/7 /

SAGE

)RSA/

)RSA-

)RSA;

)RSA? SA/ SA- SA; SA?

Sage -/ -. /7 / -7 ;/ ;. --. /7 /7 /7 -7 ;/ ;. ;.-. /7 / / -7 ;/ ;. ;.

Pos /- // / -. - -7 -. -/- // / -. - -7 -G -/- // / -. - - -G -

Neg -/ 0 -/ 0 ;- 0 ;- 0

-- 0 -- 0 ;. 0 ;. 0-; 0 -; 0 ;. 0 ;. 0

AN:+A6 #1oFactor 1ith Re%lication

SU))AR8 )RSA/ )RSA- )RSA; )RSA? SA/ SA- SA; SA? #otal

Sage

&ount ; ; ; ; ; ; ; ; -?

Sum 0/ G G0 G? 7; 7. G

Average -.$;;;;; /7$;;;;; /$0000 / -7 ;/ ;. -7$;;;;; -?$?G;;

+ariance .$;;;;;; .$;;;;;; .$;;;;;; / . . . /$;;;;;; ;/$/-0-

Pos&ount ; ; ; ; ; ; ; ; -?

Sum ;0 ;; G? 0. ? 0 . ? G.

Average /- // / -. - -$0000 -;$;;;;; - -/$/-G

+ariance . . . . . .$;;;;;; $;;;;;; . ?0$.7

9eg

&ount ; ; ; ; ; ; ; ; -?

Sum 00 / 00 / 7- / 7- / ;


33/45

-

Average -- 0 -- 0 ;.$0000 0 ;.$0000 0 /0$/000

+ariance / . / . /$;;;;;; . /$;;;;;; . //$.G

Total

&ount 7 7 7 7 7 7 7 7

Sum /0; /.7 /0 /;- -0; /7 -G- /7.

Average /$///// /-$///// /7$GGGG0 /?$0000 -7$----- -/$7 - -/$/////+ariance -/$0/// ;?$///// ;$ ?;$-G /$07???? /?;$//// /?$G /-7$////

AN:+A

Source of

:ariation


Sam%le ;G$G;; - ?/$7/ 0./$0- /$.;*;?

;$/7.-

&olumns --/.$/0 ;/G$;/ ?G?$00-7 /$;/*?/

-$-.?;0

Interaction --0?$?/ /? /0/$?? -;-$7//? ?$/-*

;7

/$7.;0G;

2ithin ;;$;;;;; ? .$07????#otal G;?;$G /

OXACILLIN

ROSEMARY

#S!/ #S!- #S!; #S!? #S!Q:5a/

#S!Q:5a-

#S!Q:5a;

#S!Q:5a?

Rosemary

.$0? .$0- .$- .$G?- . .$/./ .$.- .$..?.$0?0 .$0- .$070 .$??G .$./G .$//0 .$.-G ..$.- .$0/7 .$.? .$G/; .$.;; .$-00 .$.? .$..

Pos .$G? .$G- .$- .$0/ . .$0/ .$.0 .$.-.$0 .$?77 .$- .$?? .$/? .$0;G .$.?- .$.

.$G .$G-G .$ .$G .$; .$G7 .$.0; .$.;Neg .$? .$? .$? .$? .$0G .$0G .$0G .$0G

.$G .$G .$G .$G .$0-G .$0-G .$0-G .$0-G

.$G .$G .$G .$G .$0 .$0 .$0 .$0

AN:+A6 #2:FA:R 2I#9 R*PLI&A#I:NSU))AR8

#S!/ #S!- #S!; #S!? #S!Q:5a/

#S!Q:5a-

#S!Q:5a;

#S!Q:5a?

#otal

Rosemary

&ount ; ; ; ; ; ; ; ;Sum /$7 /$00 -$/- /$G .$.? .$?; .$/ .$.//

Average .$00-00

.$0-- .$-? .$G .$./0 .$/0/ .$.;;;;; .$..;00 .$;?

+ariance .$..//07

.$..../7

.$../??

.$..-?7

.$...-; .$..;-G .$.../?- /$-;*.G .$.7

Pos

&ount ; ; ; ; ; ; ; ;


34/45

-7

Sum /$0- /$GG- -$--? -$- .$GG/ /$?/ .$/0G .$/ /.

Average .$G;;;

.$G/;;;

.$?/;;;

.$;;;;;

.$/;00 .$0/;00 .$.GG .$.0-;;; .$?;

+ariance .$.../07

.$...-G?

.$...700

.$..--7?

.$.;G0.- .$...G; .$.../-7 .$...7?G .$.

9eg

&ount ; ; ; ; ; ; ; ;Sum -$;/0 -$;/0 -$;/0 -$;/0 /$7 /$7 /$7 /$7 /

Average .$- .$- .$- .$- .$00-00 .$00-00 .$00-00 .$00-00 .$

+ariance .$.../7

.$.../7

.$.../7

.$.../7

.$../.0 .$../.0 .$../.0 .$../.0 .$..

Total

&ount 7 7 7 7 7 7 7 7

Sum 0$.00 G$;? 0$/- 0$./0 -$G ?$;/- -$-G; -$/0

Average .$0? .$0;/// .$?G

.$00???

.$-??? .$?7/// .$-G.;;; .$-?-7

+ariance .$..0?

.$./-?.?

.$..//0

.$./?/

.$.7;.? .$.G7G .$.70.0/ .$/..-?

AN:+ASource of :ariation SS df S 8 P"value 8 crit

Sam%le /$?/-/ - .$7-.0.7 ;0$?0/ $7?*;. ;$/7.-

&olumns -$.07G? .$?..77; /0.$.0G ?$*;/ -$-.?;0

Interaction /$/?.7/- /? .$./?7? ;-$G;.?; /$.0*/7 /$7.;0G;

2ithin .$/-.-? ? .$..-G.G

#otal G$7.7;;/ /

SAGE#S!/ #S!- #S!; #S!? #S!Q:5a

/#S!Q:5a-

#S!Q:5a;

#S!Q:5a?

Sage .$0; .$0.- .$;0 .$/7 .$./- .$.-; .$/;- .$../

.$.; .$0. .$;G .$-? .$.G .$..- .$.? .$.;/

.$.? .$GG- .$0G .$77- .$.77 .$-0 .$./ .$.?0

Pos .$G? .$G- .$- .$0/ . .$0/ .$.0 .$.-

.$0 .$?77 .$- .$?? .$/? .$0;G .$.?- .$.

.$G .$G-G .$ .$G .$; .$G7 .$.0; .$.;

Neg .$? .$? .$? .$? .$0G .$0G .$0G .$0G

.$G .$G .$G .$G .$0-G .$0-G .$0-G .$0-G

.$G .$G .$G .$G .$0 .$0 .$0 .$0

AN:+A6 #1oFactor 1ith Re%licationSU))AR8

#S!/ #S!- #S!; #S!? #S!Q:5a/

#S!Q:5a-

#S!Q:5a;

#S!Q:5a?

#otal

Sage

&ount ; ; ; ; ; ; ; ; -?

Sum /$?/? /$0/ -$-;0 -$?;G .$/07 .$-G .$--; .$. $0./


35/45

;.

Average .$?/;;;

.$G .$?G;;;

.$//00

.$.G0;;; .$.7G .$.?;;; .$.-0 .$;G;G

+ariance .$/-./-?

.$...7-G

.$...-7 .$.-?;70

.$../7? .$.-.G-7 .$..;;.0 .$...G-G .$//G0G

Pos

&ount ; ; ; ; ; ; ; ; -?

Sum /$0- /$GG- -$--? -$- .$GG/ /$?/ .$/0G .$/ /.$?-Average .$G;;

;.$G/;;

;.$?/;;

;.$;;;;

;.$/;00 .$0/;00 .$.GG .$.0-;;; .$?;0G

+ariance .$.../07

.$...-G?

.$...700

.$..--7?

.$.;G0.- .$...G; .$.../-7 .$...7?G .$..000

9eg

&ount ; ; ; ; ; ; ; ; -?

Sum -$;/0 -$;/0 -$;/0 -$;/0 /$7 /$7 /$7 /$7 /$-/0

Average .$- .$- .$- .$- .$00-00 .$00-00 .$00-00 .$00-00 .$/;;;

+ariance .$.../7

.$.../7

.$.../7

.$.../7

.$../.0 .$../.0 .$../.0 .$../.0 .$..;GG

Total&ount 7 7 7 7 7 7 7 7

Sum G$?7- G$0-7 0$0 0$7G/ -$. ?$//? -$;0 -$-G;

Average .$0/.---

.$0-G???

.$G-7

.$-;;;

.$;..7 .$?G/// .$-0? .$-G.;;;

+ariance .$.?;0G

.$./;;;?

.$...G .$../

.$.0;.G .$.7?; .$.7.G77 .$.70G-

AN:+A

Source of:ariation


Sam%le /$.700

- .$G?;

7?$/; -$??*/

;$/7.-

&olumns -$7-?/

.$?/;?G

?0$.000

;$0?*/7

-$-.?;0

Interaction /$-;-

/? .$..G0

7$070?

/$.*.7

/$7.;0G;

2ithin .$?;G7./

? .$..7./

#otal 0$;.0/

/


36/45

;/

A''ENDIX

IOFILM FORMATION ASSAY

After i!"#$ti% $t &'%C

After i!"#$ti% $t &'%C

De!$ti( %f tr)*ti!$+e +%)

#r%t -it r"i( -$ter


37/45

;-

Tree tie+ -$+i( -it /00

12 %f 3i+ti22e3 -$ter

St$ii( -it /00 12 %f !r)+t$2

4i%2et $3 +t$3 f%r 56

i"te+

De!$ti( %f +t$i -it

r"i( -$ter $fter +t$3i(

f%r 56 i"te+


38/45

;;

B2%t 3r)i( $fter +t$ii(

After #2%t 3r)i(

Di++%24i( +t$i -it /00 12 %f

7686 et$%2 $!eti! $!i3


39/45

;?

Mi9i( te +$*2e $3 te

7686 et$%2 $!eti! $!i3 :6

tie+;

Tr$+ferri( te +%2"ti% t%

t"#e+

Re$3i(

+*e!tr%*%t%etri!$22) $t 6'0

$3 re!%r3i( %f re+"2t %f


40/45

;G

ANTIIOTIC RESISTANCE ASSSAY

Re$3i(+*e!tr%*%t%etri!$22) $t 656

, 66


41/45

;0

Getti( />& 2%%*f"22 %fi%!"2" t% #e i%!"2$te3 t%

M"e22er Hit% A($r

I%!"2$ti% it% M"e22er Hit%

A($r

Perf%ri( $+e*ti! te!i@"e

$fter i%!"2$ti%


42/45

;

Getti( i%!"2" t% #e

+-$##e3 t% M"e22er Hit%

A($r

S-$##i( t% M"e22er Hit%

A($r


f%r 3i+! 3i="+i% et%3


43/45

;

Pr%*er te!i@"e f%r (etti(

3i+!+

Pr%*er *2$!i( %f 3i+!+ t% te

!"2t"re e3i" -it

i!r%%r($i++


$fter *2$!i( te 3i+!+


44/45

;7

A,,e"/i3 C GAANT C0art (Ti1eta4#e)

Activities Aug Se% :ct Nov Dec "an Feb )ar A%r )ay

Researchto%ic

(athering ofdata for

%ro%osalIntroduction

Revision ofIntroduction

)ethodology

Revision ofmethodology

Pro%osal

&onducting*5%eriment

(athering ofResults

#reatment ofData

Finali,ing ofResearchDefense


45/45

?.

A,,e"/i3 D /!etar$ Re2ire1e"t

Item Particulars Price

mecA%ositive methicillinresistant Staphylococcus

aureus3)RSA4 A#&& ?;;..

/ lot lyo%hili,ed bacterialcells

PhP /7..$..

mecAnegativeStaphylococcus aureusA#&&

-G7-;


PhP /7..$..

Pseudomonas aeruginosaPA./


PhP /7..$..

C. violaceum &+.-0 / lot lyo%hili,ed bacterialcells

PhP /7..$..

C. violaceumA#&& ;/G;- / lot lyo%hili,ed bacterialcells

PhP /7..$..

)ueller 9inton Agar / G..g%ac' PhP 7.$..

#ry%tic Soy !roth / G..g%ac' PhP 7.$..

&efo5itin discs - G.%cs%ac'age PhP 0..$..

)icrotiter %lates /. %ieces PhP G..$..

)iscellaneous reagents andsu%%lies

V PhP /G..$..

#:#AL PhP /?.0.$..

Documents

Buya Anti-quorumsensing Rosemary Sage Journal 2016