Research Article. EFFECT OF ph ON BIOFILM FORMATION AND MOTILITIES OF Pseudomonas aeruginosa, Escherichia coli AND Staphylococcus aureus ATCC STRAINS

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1 BMR Microiology Open ccess Scientific Pulisher Reserch rticle EFFECT OF ph ON BIOFILM FORMTION ND MOTILITIES OF Pseudomons eruginos, Escherichi coli ND Stphylococcus ureus TCC STRINS BSTRCT Tsiry Rsmirvk 1, ndo Llniin Rndrinierenn 1, Mrson Rherimmdimy 1, Blndine ndrinriso 1 1 Lortoire de Biotechnologie et Microiologie, Déprtement de Biochimie fondmentle et ppliquée, Fculté des Sciences, Université d ntnnrivo (U), BP 9, ntnnrivo 11, Mdgscr Correspondence should e ddressed to Tsiry Rsmirvk Received Jnury 11, 1; ccepted Ferury, 1; Pulished Mrch 9, 1; Copyright: 1 Tsiry Rsmirvk et l. This is n open ccess rticle distriuted under the Cretive Commons ttriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. Cite This rticle: Rsmirvk, T., Rndrinierenn,., Rherimmdimy, M., ndrinriso, B.(1). Effect of ph on iofilm formtion nd motilities of Pseudomons eruginos, Escherichi coli nd Stphylococcus ureus TCC strins. BMR Microiology, (1). 1- Biofilms re communities of microorgnism enclosed in n extrcellulr mtrix ttched to vrious surfces. Biofilms nd motilities my represent negtive impcts in helth nd industril sectors, however they lso ply crucil ecologicl role. The min ojective of this study ws to estimte iofilm formtion nd motilities ilities of three common cteri under different culture condition (medi of growth nd ph). Results show tht ph vrition (from to ) nd composition of medi growth (minimum nd complex medi) hve little influence on iofilm formtion y Stphylococcus ureus TCC 93 nd Escherichi coli TCC 9. However, iofilm formtion y Pseudomons eruginos TCC 73 is inhiited t ph nd in oth complex nd minimum medi. P. eruginos swimming, swrming nd twitching motilities re not ffected y ph vrition wheres S. ureus nd E. coli re only le to twitch in complex medi. This preliminry study demonstrted tht ph my influence cteril ehvior formtion prticulrly for P. eruginos. KEYWORDS: Biofilm, Motilities, ph, Pseudomons, Stphylococcus, Escherichi. INTRODUCTION Bcteril infection is minly relted to the ility of cteri to colonize nd disseminte through their hosts y using different types of motility, y uilding structured iofilm nd y relesing myrid of virulence fctors which led to host cell nd tissue dmges [1]. Biofilms re surfce-ssocited communities enclosed within n extrcellulr mtrix minly composed of polyscchrides, proteins, nucleic cids, lipids nd other mcromolecules nd chemicls [] which protect cteri cells ginst environmentl ssults nd predtion, including ntimicroils nd host defenses. s orgnism re present vrious ph t the exmple of long the digestive system tht present ph rnge from to [3], it s importnt to understnd the influence of environment ph tht promote the expression of these cteril ehviors prticulrly iofilm formtion nd motilities. Indeed, comprehension of these ehviors is importnt to elorte dequte strtegy in order to struggle ginst cteril infection or to exploit their iotechnologicl potentil. The present study evlute the ilities of Escherichi coli TCC 9, Stphylococcus ureus TCC 93 nd P. eruginos TCC 73 strins to move nd to develop iofilm under complex nd minimum medi nd different ph condition from to. BM Volume Issue 1 1 1

2 p H S w rm in g m o tility ( m m ) p H B io film fo r m tio n (C ris t l v io le t 9 n m ) B io film fo r m tio n (C ris t l v io le t 9 n m ) p H S w rm in g m o tility ( m m ) p H MTERILS ND METHODS Bcteril strins nd culture conditions BMR Microiology performed y mesuring the formed iofilm stined y crystl violet t 9nm with spectrophotometer BK- UV1 device (Biose, Chin). Motility ssys Reference strins Escherichi coli TCC 9, Stphylococcus ureus TCC 93 nd P. eruginos TCC 73 were purchsed from OXOID nd grown in LB-MOPS roth. Biofilm Formtion nd Quntifiction The formtion of iofilm ws studied on 9-well micropltes nd quntified y crystl violet stining protocol s previously descried []. Briefly, n overnight culture of Escherichi coli TCC 9, Stphylococcus ureus TCC 93 or P. eruginos TCC 73 ws wshed twice nd diluted in fresh iofilm roth (BB) medium (NHPO, 1. g/l; FeSO.7HO. g/l; glucose,. g/l; (NH)SO,.1 g/l; MgSO.7HO,. g/l nd KHPO,. g/l) or LB-MOPS roth medium. Experiments were relized using BB or LB t four different ph (,, 7 nd ) in 9-well polystyrene pltes, μl of the diluted culture ( etween.1 nd.1) ws dded to 1 μl of BB medium or LB roth. Escherichi coli TCC 9, Stphylococcus ureus TCC 93 nd P. eruginos TCC 73 cultures were incuted stticlly for h t 37 C nd iofilm quntifiction ws RESULTS Swimming, swrming nd twitching motilities were exmined y using LB gr t different ph,, 7 nd (.3%,.% nd 1%, respectively) s descried y Rsmirvk et l. [] nd H et l. []. fter steriliztion nd cooling (- C) of LB gr, five μl of cteril culture ( = 1) were inoculted t the center of ech comprtment of the Petri dishes nd incuted t 37 C for (for swimming nd swrming) or h (for twitching motility). Bcteri spreding from the inocultion spot were mesured with sliding cliper fter dequte incution time. The inocultion spot is generlly round mm nd spreding less thn mm ws considered s non-motile mechnism. Sttistics ll experiments were performed in triplicte nd repeted in 3 independent ssys. The dt were sttisticlly nlyzed y conducting the one-wy NOV with Tukey s multiple comprison tests nd P-vlue <.1 ws considered s eing significnt. Biofilm formtion y Stphylococcus ureus is not influenced y vrition of cidity medium. Indeed, there is no significnt difference etween iofilm formed in ph,, 7 nd (Figure 1). Moreover, S. ureus form iofilm in oth minimum nd complex medi. In regrd of motilities, S. ureus ws unle to spred in ll BB medium gr nd unle to swim nd swrm in complex medium (Figure 1). However, S. ureus ws le to twitch under ph7 nd. ( ) 1.. S t p h y lo c o c c u s u re u s S t p h y lo c o c c u s u re u s S t p h y lo c o c c u s u re u s 1 S t p h y lo c o c c u s u re u s BM Volume Issue 1 1

3 p H p H B io film fo r m tio n (C ris t l v io le t 9 n m ) B io film fo r m tio n (C ris t l v io le t 9 n m ) p H S w rm in g m o tility ( m m ) p H S w rm in g m o tility ( m m ) BMR Microiology Figure 1: Effect of ph vrition (from to ) nd growth medium (complex nd minimum medi) on iofilm development nd motilities y Stphylococcus ureus TCC 93. () Biofilm formtion y S. ureus grown under different ph (from to ) nd medium. (B) Swimming motility of S. ureus grown under different ph (from to ) nd medium. (C) Swrming motility of S. ureus grown under different ph (from to ) nd medium. (D) Twitching motility of S. ureus grown under different ph (from to ) nd medium. ll experiments were performed in triplicte with three independent ssys. Different letters ( or nd B or ) ove the rs indicte dt tht re sttisticlly different from ech other ccording to the one-wy NOV with Tukey s multiple comprison test (p.1). Biofilm formtion y Escherichi coli is similr to S. ureus; indeed, Escherichi coli form iofilm t ll tested ph when grown in complex nd minimum medi (Figure ). However, they develop more consistent iofilm when grown in minimum medi. In regrd of motilities, Escherichi coli ws unle to spred in ll BB medium gr nd unle to swim in complex medium (Figure ). However, slight swrming (1mm) which is not influenced y ph vrition is oserved in complex medium nd interestingly, Escherichi coli twitch more under cidic ph thn neutrl nd lkline ph condition. ( ) 1.. E s c h e r ic h i c o li E s c h e r ic h i c o li E s c h e r ic h i c o li 1 E s c h e r ic h i c o li Figure : Effect of ph vrition (from to ) nd growth medium (complex nd minimum medi) on iofilm development nd motilities y Escherichi coli TCC 9. () Biofilm formtion y E. coli grown under different ph (from to ) nd medium. (B) Swimming motility of E. coli grown under different ph (from to ) nd medium. (C) Swrming motility of E. coli grown under different ph (from to ) nd medium. (D) Twitching motility of E. coli grown under different ph (from to ) nd medium. ll experiments were performed in triplicte with three independent ssys. Different letters ( or nd B or ) ove the rs indicte dt tht re sttisticlly different from ech other ccording to the one-wy NOV with Tukey s multiple comprison test (p.1). Biofilm formtion y P. eruginos is influence y cidity of medium. Indeed, ph7 represent the optimum cidity for iofilm formtion s in cidic environment (ph nd ) P. eruginos fil to form iofilm under oth complex nd minimum medi. t ph P. eruginos form iofilm ut significntly reduced compred to tht of iofilm iomss t ph7 (Figure 3). Interestingly, swimming, swrming nd twitching motilities re not ffected y ph vrition in complex medium suggesting tht regultion of flgellr movement nd pili retrction re not influenced y ph vrition, wheres P. eruginos ws not le to spred in BB medium gr (Figure 3). 3 BM Volume Issue 1 1

4 p H S w rm in g m o tility ( m m ) p H B io film fo r m tio n (C ris t l v io le t 9 n m ) B io film fo r m tio n (C ris t l v io le t 9 n m ) p H S w rm in g m o tility ( m m ) p H BMR Microiology ( ) 1... P se udom on s eruginos B C c P se udom on s eruginos P se udom on s eruginos 1 3 P se udom on s eruginos Figure 3: Effect of ph vrition (from to ) nd growth medium (complex nd minimum medi) on iofilm development nd motilities y Pseudomons eruginos TCC 73. () Biofilm formtion y P. eruginos grown under different ph (from to ) nd medium. (B) Swimming motility of P. eruginos grown under different ph (from to ) nd medium. (C) Swrming motility of P. eruginos grown under different ph (from to ) nd medium. (D) Twitching motility of P. eruginos grown under different ph (from to ) nd medium. ll experiments were performed in triplicte with three independent ssys. Different letters ( or nd B or ) ove the rs indicte dt tht re sttisticlly different from ech other ccording to the one-wy NOV with Tukey s multiple comprison test (p.1). DISCUSSION This study indictes tht the ph vlue will ffect iofilm nd motility ehviors differently, depending upon the tested orgnism. S. ureus nd E. coli iofilm formtion seems to e not influenced y ph vrition suggesting tht the mechnism regulting iofilm formtion in oth strins is not ph dependent. s non-motile cterium, S. ureus ws expected to not le to swim nd swrm. However, the presence of twitching motilities is intriguing. The twitching motilities oserved in ph 7 nd my e not linked to twitching mechnism ut rther sliding motilities. Indeed, S. ureus re le to slide which is sliding is kind of surfce trnsloction produced y the expnsive forces in growing culture in comintion with specil surfce properties []. The sliding is pssive motility nd the spreding depends on cteril growth nd QS-regulted surfctnt phenol solule modulin (PSM) so it s suggested tht S. ureus grown nd produce well the surfctnt in ph 7 nd condition. E. coli twitch well prticulrly under cidic BM Volume Issue 1 1 condition suggesting the full ctivtion of pili t this condition. s E. coli do not swim nd slightly swrm the flgell in not ctivte under LB medium. For P. eruginos Biofilm formtion is influenced y ph vrition, further studies should e ddressed which of the steps for iofilm formtion is relly ph dependent. Moreover, influence of ph vrition should e lso investigted for mechnisms considered to regulte iofilm formtion such s quorum sensing, two component system GcS/Gc nd the c-di-gmp pool concentrtion. One point tht is lredy cler is tht swrming nd twitching motilities, which re importnt for iofilm formtion [7] seems not to e ffected y ph vrition. Finlly, BB medium re not proly dequte for motilities testing of P. eruginos, E. coli nd S. ureus strins s well s LB medium is not pproprite for testing swimming n swrming of E. coli nd S. ureus strins. Thus, use of other medium should e investigted to evlute which components re relly necessry for iofilm formtion.

5 BMR Microiology CONCLUSION This preliminry study demonstrted tht ph my influence cteril ehvior formtion prticulrly for Pseudomons eruginos. Further investigtion is necessry to shed light the mechnism of this phenomenon. REFERENCES [1]. Wu H.-J, Wng HJ, Jennings, MP: Discovery of virulence fctors of pthogenic cteri. Current Opinion in Chemicl Biology, 1: []. O Toole G: To uild iofilm. Journl of Bcteriology 3, 1: 7 9. [3]. Fllingorg J : Intrluminl ph of the humn gstrointestinl trct. Dnish medicl ulletin 1999, (3), []. Rsmirvk T, Vndeputte OM, Stévigny C, Duez P, ElJziri M : Pseudomons eruginos iofilm formtion nd persistence, long with the production of quorum sensing- dependent virulence fctors, re disrupted y triterpenoid coumrte ester isolted from Dlergi trichocrp, tropicl legume, PloS One 1, 1(7): e []. H DG, Kuchm SL, O Toole G.: Plte-sed ssy for swimming motility in Pseudomons eruginos, Pseudomons Methods nd Protocols, pp. 9-, Edited y Springer New York, 1. []. Pollitt EJG, Diggle SP: Defining motility in Stphylococci. Cellulr nd Moleculr Life Sciences 17, 7: 93 [7]. Rsmirvk T, Ltni Q, Duez P, El Jziri M: The formtion of iofilms y Pseudomons eruginos: review of the nturl nd synthetic compounds interfering with control mechnisms. BioMed Reserch Interntionl 1, rticle ID 793. BM Volume Issue 1 1