1 Effct of Elctrical Stimulation on Bactrial Growth By Jrrold Ptrofsky Ph D Michal Laymon DPTSc* Wndy Chung DPTSc Klly Collins BS* Tin-Ning Yang BS* Dpts. of Physical Thrapy Loma Linda Univrsity Loma Linda, California and Azusa PACific Univrsity* Azusa California Snd rprint rqusts to: Dr. Jrrold Ptrofsky Profssor and Dirctor of Rsarch Dpartmnt of Physical Thrapy Loma Linda Univrsity Loma Linda California, (909)
2 Abstract Background; Elctrical stimulation has bn usd as a modality for many yars for wound haling. On of th mchanisms proposd for why it works is that lctrical stimulation is blivd to b bactria static. Howvr, arly studis making this claim usd much gratr than normal stimulation voltags and currnts. Mthods and procdurs; In th prsnt invstigation, 3 typs of bactria, Eschrichia coli, Psudomonas aruginosa, and Staphylococcus aurus wr xamind with physiological lvls of AC (5 and 20 ma) and DC lctrical stimulation to s if ths sam currnts that travrs through tissu during normal lctrical stimulation for wound tratmnt could impair bactrial growth. Rsults; DC micro currnt did not altr bactria growth for any of th thr bactria studid. Psudomonas aruginosa showd significantly rducd growth with both (5 and 20 ma) AC currnt intnsitis (p<0.05) whras th othr bactria showd no consistnt rductions in growth with AC currnt; ach showing som snsitivity to AC stimulation. Whil thr wr significant diffrncs btwn th ph of th culturs with AC stimulation in th xprimntal vs. th control groups in som conditions, thr was no consistncy in any corrlation btwn ph and growth. Thus, whil psudomonas was inhibitd with AC stimulation, thr was no vidnc from ths studis that lctrical stimulation had a consistnt bactria static ffct on th othr bactria. Evn in psudomonas, bactria growth was only slowd and not stoppd with 30 minuts of stimulation. Conclusions; whil thr was som snsitivity at last in on bactrium to AC stimulation, largly th bactria static ffct of lctrical stimulation undr th conditions usd hr was d minimis. Ky words: wounds, lctrical stimulation, bactria, haling
3 Introduction Wound haling, spcially in popl with diabtic nuropathis, can b xtrmly difficult to accomplish. 1 For xampl, of popl who hav diabtic ulcrs on thir ft, inability of wounds to hal causs an avrag amputation rat to b about 25% 2,3. Many 2, 3, 4 diabtic ulcrs and vn dcubitus ulcrs can tak months, yars, or nvr hal at all. This inability of convntional mdical practic to hal wounds has bn trmd a failur of th halth car systm. 5 Evn nwr thrapis such as myocutanous flaps, watr dbridmnt, and othr tchniqus hav not incrasd th haling rat apprciably in th past fifty yars or th rat of roccurrnc. 5 On modality that has bn studid is lctrical stimulation. 6 Many paprs point to th us of lctrical stimulation across wounds to incras wound haling. 7, 8 Howvr, vn this tchnology dos not always work. Som paprs show wound haling with DC micro currnts whil othrs show only haling with strong AC currnts. 9, 10, 11 Som paprs rport that in th arly stags of wound haling th polarity nds to b positiv nar th wound and ngativ away from th wound and othr paprs rport th opposit. 12, 13, 14 In fact, in a summary of diffrnt studis on wound haling, Yarkony rportd nothing conclusiv in stting th intnsity of currnts, whthr it was DC or AC stimulation or th frquncy of lctrical stimulation in th ability of lctrical stimulation to hal wounds. 12 Rcnt vidnc, howvr, links this disparity to possibly bing linkd to room tmpratur. Vasoconstriction of th circulation during tratmnt du to a cool or cold xamination room blocks th blood flow incras du to lctrical stimulation of th wound. If th room is warmd, blood flow incrass and wounds hal 15, 16 wll with lctrical stimulation.
4 Numrous mchanisms hav bn suggstd on why lctrical stimulation promots wound haling including incrasd circulation, 1,17 18, 19, 20 incrasd angiognsis incrasd prolifration of pidrmal tissu, and a bactria static ffct of lctrical stimulation. 21 Th possibl bactria static ffct of lctrical stimulation was first rportd ovr 30 yars ago by Rowly & McKnna. 22, 23 Using high voltag lctrical stimulation (300 volts), bactria (E. coli) did aftr a brif sssion of lctrical stimulation. Whil a fw studis hav usd similar voltags for clinical lctrical stimulation of wounds 11, 24 most studis that rport haling us a fraction of ths voltags and currnts. 12, 13, 25, 17 Th stimulation voltags usd by Rowly t al. would b xtrmly painful for th patint. Furthr, sinc DC micro currnt has bn shown, in som studis, to aid in haling of wounds, whn th voltags usd by Rowly and collagus ar compard to DC micro currnts, thy ar thousands of tims highr. Thus, th currnts usd in ths studis wr not clinically rlvant and, thrfor, th application of ths studis to ral lif situations is qustionabl. Th prsnt invstigation was conductd to study th ffct of lctrical stimulation on bactrial growth in thr common bactria that ar sn in wounds, Eschrichia coli, Psudomonas aruginosa, and Staphylococcus Aurus. 23, 26 Ths bactria wr grown in cultur and thn xposd to ithr DC microcurrnt (100 µamps) or biphasic sin wav stimulation at 5 or 20 ma for 30 minuts. Ths currnts ar normal currnts usd in wound haling. 16, 15 Bactrial growth was thn assssd aftr 24 hours of incubation to s if th normal currnts that ar usd with lctrical stimulation clinically to hal wounds can altr bactrial growth.
5 Mthods Bactria- Bactria wr grown in a soy broth (tryptic soy broth, soyban- casin digst) from Hardy Diagnostics (Cat # C7141, Santa Monica, CA). Th bactria usd wr Eschrichia coli, Psudomonas aruginosa, and Staphylococcus aurus. Ths bactria wr obtaind from stock concntrations (Micro biologics, ST Cloud, Minnsota). Bactria wr grown in broth at a tmpratur of 37 o C until th concntration of bactria was btwn 4-5 on th McFarland Scal. 27 All bactria wr grown and tstd for activity by standard microbiological light microscopy. Th broth was tstd for growth in an absorption spctromtr at a wavlngth of 580 nanomtrs; th rlationship btwn bactrial dnsity and broth absorption was stablishd. Onc this was stablishd, stock broth culturs wr placd btwn two 2 X 2 cm carbonizd rubbr lctrods, so that thy could b subjctd to lctrical stimulation for a priod of thirty minuts. Th sparation distanc btwn th lctrods was 10.3cm. Th covrd cultur siz was 10.3 X 8.5 cm and 6.75 cm dp. Th volum was 400ml. Using ths lctrods and lctrod sizs, th impdanc of th broth was 1280 ohms during lctrical stimulation, a valv similar to that rcordd across wounds. 15 Th siz of th broth holdr was slctd so that if currnt was masurd ovr a 0.5 cm distanc in th broth, th rcordd currnt was 15, 16 similar to that rcordd in human wounds.
6 Elctrical Stimulation- AC lctrical stimulation was providd by a challng 8000 powrd muscl stimulator (MPTS Tustin, CA). This stimulator providd 4 channls of currnt controlld output with a biphasic sin wav at 250 µsc puls width and a frquncy of 30 Hz. DC stimulation was providd at a currnt of 1 ma by a battry with a rgulatd currnt controlld output. ph- Th ph was masurd with an Accrumnt Basic AB15 ph mtr (Fishr Scintific, Singapor).
7 Procdurs Using th stock broth bactrial culturs dscribd abov, ach of th 3 bactria wr placd in a bactrial broth in th rctangular culturs. Aftr th broth had bn culturd, lctrical stimulation was applid for thirty minuts. AC currnt was dlivrd with biphasic lctrical stimulation (sin wav) at a frquncy of 30 Hz and a puls width of 250 microsconds at intnsitis of ithr 5 or 20 ma for a priod of 30 minuts. Th stimulation output was currnt controlld. DC stimulation was also applid for 30 minuts. Th bactrial cultur along with culturs whr no stimulation was applid, wr thn placd back into th incubator for anothr 24 hours and growth was assssd. Th procss was rpatd on 8 culturs for ach bactrium. Statistical Analysis- Statistical analysis involvd th calculation of mans, standard dviations, and t tsts. Valus in th txt ar shown as th man+/-sd. Th lvl of significanc was p < 0.05.
8 Rsults Initially, th McFarland Scal was calculatd at a frquncy of 580 nanomtrs for ach of th bactria. Absorbncy at this frquncy was plottd against st concntrations of bactria to obtain a calibration curv. Th corrlation btwn th concntrations of bactria to th absorbncy was This yildd an R 2 of and, th quation rlatd concntration to absorbncy was Y = x. Givn th calibration on th McFarland Scal, broth was mixd and th studis wr compltd as dscribd undr procdurs. Th rsults for ach bactrium ar listd blow. Eschrichia coli - Th rsults showing growth in Eschrichia coli ar shown in figur 1, panl A and B. Panl A, in figur 1, shows th bactrial colony growth from th bginning to th nd of th study (24 hours aftr th initial incubation). As shown in panl A of figur 1, with a currnt of 5 milliamps, th incras in bactrial colony growth ovr 24 hours was 365 +/- 57 x 10 6 bactria pr millilitr whras for 20 milliamps stimulation th incras in growth was 366 +/- 16 x 10 6 bactria pr millilitr. For th controls, whr no stimulation was accomplishd, bactrial colony growth incrasd by 401 +/- 7.5 X 10 6 bactria pr millilitr ovr th 24 hours. Thr was no statistical diffrnc btwn growth in th 5 and 20 ma and control groups (p < 0.01). Bactrial colony growth for DC stimulation was 460 +/- 74 x 10 6 bactria pr millilitr. Th only significant growth rduction compard to th controls was for stimulation at 20 ma (p<0.01). Hr, howvr, th rduction in growth was only 9.8%.
9 ph was also masurd at th bginning and at th nd of studis. For th control bactria colonis (no stimulation) ph startd at / At th nd of th 24 hour priod, ph avragd /- 0.18, a rduction of an avrag of 1.22 ph units. For bactrial stimulation at 5 and 20 milliamps, thr was a significantly gratr rduction in ph ovr th 24 hours compard to th control colonis (p > 0.01). Th chang in ph was small, avraging 0.4 ph units. For DC stimulation, th chang in ph, ovr th 24 hour priod was not significantly diffrnt than that of th controls (p > 0.05). Thr was no corrlation for any group in ph and bactrial growth (p>0.05). Psudomonas aruginosa- As shown in th top panl of figur 2, undr all four conditions, thr was an incras in bactrial growth ovr th 24 hour priod for ths colonis. As shown in panl A of figur 2, th incras in bactrial growth with 5 milliamps of stimulation avragd 204 +/- 116 x 10 6 pr millilitr, for 20 milliamps stimulation avragd 228 +/- 121 x 10 6 bactria pr millilitr and aftr DC stimulation avragd 331 +/- 161 x 10 6 bactria pr millilitr ovr th 24 hour priod. Th growth for all conditions was significantly lowr than growth for E. coli (p< 0.05). Compard to th control group, th bactrial growth ovr th 24 hour priod was significantly lss for th AC stimulation than for th control group (p<0.05). Bactrial growth avragd a 38% rduction in growth with AC stimulation. For DC stimulation, thr was no significant diffrnc to th growth of th control group (p>0.05). As shown in th bottom panl of figur 2, th ovrall chang in ph was lss than that obsrvd for E. coli (p < 0.01). Howvr, th rduction in ph, which avragd /- 0.5 in th control group, was only statistically lss with stimulation at DC and 20 ma
10 AC (p<0.05). Thr was a significant corrlation btwn ph and growth only for stimulation at 20ma. (p<0.01) Staphylococcus aurus- Figur 3 shows th rsults of th Staphylococcus xprimnts. As shown in th top panl of figur 3, th avrag chang in growth for ths bactria was 358 +/- 8 x 10 6 bactria pr millilitr aftr stimulation with currnts of 5 milliamps, 376 +/- 20 x 10 6 bactria pr millilitr aftr stimulation at 20 milliamps and 405 +/- 69 x 10 6 aftr DC stimulation. Only th growth aftr 5ma AC stimulation was diffrnt than that of th controls (p < 0.01). Howvr, th rduction in growth was only 3.9% compard to th control group. As shown in th bottom of th panl, th ph was diffrnt in th 5ma group compard to th control group (p<0.01). Thr was no diffrnc btwn th DC and AC 20 ma groups compard to th control groups (p>0.05). Thr was a significant corrlation btwn ph and growth in th 20ma and DC groups (p<0.01).
11 Discussion On of th mchanisms causing wounds to not hal is bactrial invasion. 21 Bactria ruptur cll mmbrans and maintain chronic inflammation to prvnt wound haling 27 Many studis show that ithr DC microcurrnt or low currnt AC stimulation has th ability to incras wound haling. 6, 9, 10, 17 In arlir studis, Rowly and collagus publishd data showing that lctrical stimulation has a bactria static ffct on Eschrichia coli and svral othr bactria. Barranco t al. 28 showd inhibition in Staphylococcus aurus growth with 400µA DC stimulation. But th lctrods wr mtal and at 400 µamps, corrosion was notd. Othr in vitro studis with 100 µa DC stimulation also showd a bactria static ffct but hr silvr lctrods wr also usd. 29, 30 Othr studis showd a bactria static ffct of DC at 1, 5, or 10 ma but not AC 22, 23 stimulation. 31 Ths studis showd larg changs in ph with DC stimulation, probably du to th fact that, unlik clinical situations whr stimulation is a 30 minut modality, th currnt was lft on for hours. 32 This ph chang was sn also with AC stimulation at 500 volts. 33 But ths studis wr conductd in vitro with a small sparation distanc btwn th lctrods. Thus th currnt pr mm of distanc on th bactrial culturs wr larg. Furthr, mtal ions lik silvr can kill bactria in thmslvs. In addition, ths studis wr conductd at vry high stimulation voltags (ovr 300 volts). In wounds, nrv ndings ar snsitizd by th rlas of cytokins. 34 Evn mild lctrical stimulation can thrfor b painful. In prvious studis, 16, 1, 15 th currnt usd to hal wounds was lss than 20 ma. Thrfor this was th currnt usd in ths studis.
12 In th prsnt invstigations, th dnsity of th broth, siz of th chambr and siz of lctrods wr adjustd so that th lctrods wr sparatd by th distanc sn in a typical wound tratmnt. 15, 16 Th impdanc of th broth matchd that of a human wound, about 1200 ohms btwn th lctrods. This, in itslf, is diffrnt than prvious studis whr larg currnt dnsitis wr sn du to high voltags bing applid to small bactria culturs. Whil w usd a larg chambr for th broth, th currnt dnsity may hav still bn somwhat highr thn in a limb du to th highr volum in a limb. Howvr, whil currnt dnsity may hav bn somwhat highr than in a wound hr, arlir in vitro studis usd a fraction of th volum usd hr btwn th lctrods and lctrods wr closr togthr making currnt dnsity in.g. Rowly t al 22, 23 much highr than in wounds. Whn currnts wr applid in th prsnt xprimnts for a 30 minut stimulation priod, similar to a standard tratmnt priod for wound haling, DC stimulation had no ffct on bactria growth. AC had som ffct on ach of th bactria but th only ral ffct on growth with lctrical stimulation was on psudomonas. Any ffct sn on Eschrichia coli and Staphylococcus aurus was small. For psudomonas, th growth rduction at ithr AC currnt was larg. Howvr, bactria can still b dstroyd by lctrical stimulation through anothr mchanism in wounds. Elctrical stimulation and, lctrical stimulation of wounds causs th rlas of prostaglandins and othr cytokins. 35, 36, 37, 38 Ths prostaglandins and othr cytokins attract macrophags to th ara whr lctrical stimulation is applid. 31 Thrfor, although lctrical stimulation may not hav a dirct ffct on bactria in wounds, thr is som vidnc that whit blood clls such as macrophags
13 ar attractd as a scondary ffct of th lctrical stimulation, this may xplain why lctrical stimulation may b associatd with bactrial dath. Th prsnt studis still rmain in dirct contradiction to th studis by Rowly t al showing a dirct ffct of lctrical stimulation on inhibiting growth in Eschrichia coli. Howvr, whil Eschrichia coli and Staphylococcus aurus showd ithr a statistically insignificant chang in growth or an insignificant clinical chang in growth with lctrical stimulation, this is not to say that lctrical currnt dos not altr bactrial bhavior. Bactrial motility and attachmnt to substrats appars to b impaird by stimulation with lctric currnts (39). This is spcially tru for psudomonas whr flaglla ar ndd for mobility (40). Th fact that psudomonas hr did hav som impaird growth may b du to th fact that thr ar two flaglla protins involvd in its mobility and attachmnt- if ithr is impaird, growth is rducd 41. Hr, ths flaglla, sinc thy us contractil protins and ion channls for movmnt, may b impaird with lctric currnt. This maks ths bactria diffrnt from th othr 2 which do not mov by this mchanism. 22, 23 Howvr, thr ar conditions whr lctrical stimulation will inhibit growth in all 3 bactria. Whil currnts of up to 20ma in thmslvs with ths sam bactria did not rduc bactrial growth 39,42, in combination with antibiotics, th rducd ability of th bactria to attach to substrats mad thm mor suscptibl to attack by th antibiotics. Thus whil antibiotics alon or lctrical stimulation alon did not kill ths bactria, th combination of th two did 39,42. Thus, lctrical stimulation with ithr DC or wak AC currnts may hav an unsn ffct which may kill bactria in vivo but not undr ths circumstancs in vitro. Furthr invstigation is warrantd. Hr, for xampl, currnt
14 was usd for 30 minuts, a standard tratmnt modality. Prhaps longr xposur to currnt would hav a mor dramatic ffct in vivo.
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19 A B
20 Figur 2 A B
21 Figur 3 A B