Influence of Culture Media on Microbial Fingerprints Using Raman Spectroscopy

Senso s 2015, 15, 29635-29647; doi:10.3390/s151129635 senso s ISSN 1424-8220 www.mdpi.com/jou nal/senso s Technical No e In luence o Cul u e Media on Mic obial Finge p in s Using Raman Spec oscopy Ka a ína Mlyná iko á 1, O a Samek 2, Sil ie Be na o á 2, Filip Růžička 1,*, Jan Ježek 2, And ea Há oniko á 3, Ma in Šile 2, Pa el Zemánek 2 and Ve onika Holá 1 1 Depa men o Mic obiology, Facul y o Medicine and S . Anne’s Facul y Hospi al, B no 65691, Czech Republic; E-Mails: k.mlyna iko [email protected] (K.M.); [email p o ec ed] (V.H.) 2 Ins i u e o Scien i ic Ins umen s o he Academy o Sciences o he Czech Republic, . .i., K álo opolská 147, B no 61264, Czech Republic; E-Mails: osamek@isib no.cz (O.S.); [email p o ec ed] (S.B.); jezek@isib no.cz (J.J.); sile @isib no.cz (M.Š.); pa [email p o ec ed] (P.Z.) 3 Cen e o Ma e ial Resea ch, B no Uni e si y o Technology, Pu kyňo a 118, B no 61200, Czech Republic; E-Mail: [email p o ec ed] * Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed]; Tel.: +420-543-183-090. Academic Edi o : Bayden Wood Recei ed: 3 Sep embe 2015 / Accep ed: 19 No embe 2015 / Published: 24 No embe 2015 Abs ac : Raman spec oscopy has a b oad ange o applica ions ac oss nume ous scien i ic ields, including mic obiology. Ou wo k he e moni o s he in luence o cul u e media on he Raman spec a o clinically impo an mic oo ganisms (Esche ichia coli, S aphylococcus au eus, S aphylococcus epide midis and Candida albicans). Choosing an adequa e medium may enhance he ep oducibili y o he me hod as well as simpli ying he da a p ocessing and he e alua ion. We es ed ou di e en media pe o ganism depending on he nu i ional equi emen s and clinical usage di ec ly on a Pe i dish. Some o he media ha e a signi ican in luence on he mic obial inge p in (Roos el -Pa k Ins i u e Medium, CHROMaga ) and should no be used o he acquisi ion o Raman spec a. I was ound ha he mos sui able medium o mic obiological expe imen s ega ding hese o ganisms was Muelle -Hin on aga . Keywo ds: Raman spec oscopy; bac e ia; yeas s; cul u e media OPEN ACCESS Senso s 2015, 15 29636 1. In oduc ion Raman spec oscopy is a physical me hod wi h a b oad ange o applica ions—i is sui able o he iden i ica ion o subs ances, hei s uc u e and composi ion. The me hod is based on a non-des uc i e measu emen o inelas ic sca e ing o ligh (Raman sca e ing) ha allows iden i ica ion o a b oad spec um o molecules o cha ac e iza ion o molecula composi ion o a biological/non-biological sample by p o iding i s spec oscopic inge p in [1,2]. In las ew yea s has been used o iden i ica ion and desc ip ion o biological sys ems anging om biomolecules o whole cells and o ganisms [3–20]. The mos equen use o he Raman spec oscopy in mic obiology is a pheno ype measu emen o s ain iden i ica ion (including bac e ia, yeas s and algae) [2,3,11,13], bu he me hod can be used o de e mine a p esence o i ulence ac o s in a ce ain s ain, oo [10,16,21]. Mic oo ganisms can be analyzed di ec ly om colonies g own on an aga pla e [10,16], mic ocolonies on solid cul u e media [22] o single-cells ob ained a e a cul i a ion in liquid media [23]. As he Raman spec oscopy measu es he molecula composi ion o he cells, spec a migh be in luenced by cul u e condi ions. Cul u e media di e in hei composi ion o sa is y he nu i i e equi emen s o a pa icula g oup o o ganisms as well as o se e as a selec i e and/o diagnos ic ool. The aim o ou s udy is o ind sui able cul u e media ( om commonly used media in he labo a o ies o clinical mic obiology) o selec ed o ganisms by assessing limi a ions caused by he in luence o cul u e media on he Raman inge p in o he gi en s ain. Thus ep oducible spec a o mic oo ganisms can be ob ained which, in u n, simpli ies he da a p ocessing and e alua ion. The in es iga ion p esen ed he e shows he in luence o commonly used cul u e media on he Raman inge p in o selec ed mic oo ganisms belonging o he g oups o G am-posi i e bac e ia, G am-nega i e bac e ia and yeas s—namely, S aphylococcus au eus, S aphylococcus epide midis, Esche ichia coli, and Candida albicans. S aphylococci a e G am-posi i e (wi h a compac cell wall con aining a hick pep idoglycan laye ) cocci ha end o o m i egula clus e s. Mos o he species belonging o he genus S aphylococcus colonize he skin plus mucous memb anes o humans [24,25] and unde ce ain condi ions can cause in ec ions [26]. The mos clinically ele an species o he genus is S aphylococcus au eus, which can cause in ec ious diseases like impe igo, os ei is, celluli is, boils, scalded skin synd ome, and sep icemia, as well as a oxin-media ed ood poisoning and a oxic shock synd ome [27]. When S. au eus en e s he bloods eam, li e- h ea ening in asi e diseases, such as sepsis, os eomyeli is, endoca di is and meningi is can a ise. These in ec ions o en ha e a nosocomial cha ac e [28] and a e di icul o ea [28,29]. S aphylococcus epide midis, he second mos equen ly isola ed ep esen a i e o s aphylococci, is an impo an sap ophy ic o ganism ound on he human skin. Howe e , i can signi ican ly con ibu e o e e -inc easing mo bidi y and mo ali y o hospi al-acqui ed in ec ions [30]. Majo in ec ions caused by S. epide midis a e bio ilm-associa ed medical-de ice- ela ed in ec ions. These include p os he ic al e endoca di is, u ina y ac in ec ions, cen al ne ous sys em in ec ions, eye in ec ions as well as in a ascula -ca he e associa ed in ec ions and sep ic loosening o join p os heses a e o al join a h oplas y [25,31–37]. Esche ichia coli is a G am-nega i e (ha ing a mo e complica ed cell-wall s uc u e han he G am-posi i e bac e ia: he pep idoglycan laye is hin and sandwiched be ween he cy oplasmic memb ane and bac e ial ou e memb ane, and i con ains phospholipids, lipopolysaccha ides and p o eins) od ha occu s in he in es inal ac o mammals as a common pa o no mal mic o lo a. Howe e , ce ain Senso s 2015, 15 29637 s ains may cause in ec ion in he human in es inal ac (e.g., oxin-p oducing s ains). Mo eo e , E. coli is he mos equen ly isola ed pa hogen om communi y-acqui ed u ina y ac in ec ions as well as he mos equen ly isola ed G am-nega i e species om bloods eam in ec ions [38]. Candida albicans s ill emains he mos equen ly isola ed yeas om clinical specimens [39,40]. I is a polymo phic ungus ha can swi ch pheno ype be ween he unicellula yeas o m and he ilamen ous o m (hypha, pseudohypha) [41,42] ha usually inhabi s he mucosal memb anes in humans. Howe e , when he balance be ween C. albicans and hos is dis up ed, his yeas can cause a ious in ec ions anging om o al/supe icial candidiasis and in ec ions o he ep oduc i e sys em o se ious in ec ions—in asi e candidiasis and candidaemia, which a e o en li e- h ea ening [43]. In he p esen s udy, we exploi poin -by-poin eco ding o signi ican pa s o he mic obial colonies g own on ou di e en cul u e media (di ec ly on he Pe i dish) o each s ain (selec ed media a e commonly used in a mic obiological labo a o y o cul i a ion o he gi en o ganism). Speci ically, we es ic ed he spo measu emen s o he cen al, middle and uppe pe iphe y o he mic obial colony su aces. We engaged he app op ia e e ocusing on a sample o each Raman spec a o he pu pose o s aying wi hin he ocal dep h o he lase exci a ion and imaging op ics. 2. Resul s and Discussion As was men ioned abo e, in o de o assess he in luence o ce ain media on Raman spec a o mic oo ganisms conce ning he epea abili y as well as he quali y o speci ic inge p in s, we analyzed ou o ganisms (S aphylococcus au eus, S aphylococcus epide midis, Esche ichia coli and Candida albicans), each on ou cul u e media, as desc ibed u he in Ma e ials and Me hods ion. All cul u e media con ain biomolecules so hey can p o ide an adequa e nu ien supply o he mic oo ganisms o g ow and ep oduce. As mic obial colonies a e ela i ely small, a signal acqui ed om a mic obial colony can also include a signal om cul u e medium when measu ing he edges plus he molecules om he medium can be ound in he olume o a colony (see Expe imen al Se up). The Raman spec a o selec ed cul u e media a e shown in Figu e 1. Majo i y o es ed media (Endo aga —ENDO, blood aga —BA, blood aga wi h 10% NaCl—BA-NaCl, Muelle -Hin on aga —MH) show weak in ensi y o peaks ( anging up o 2500–3000 a.u.). The o he wo media (CHROMaga Candida—CHROM and Roos el -Pa k Ins i u e Medium 1640—RPMI) show s ong signals wi h nume ous de ec able peaks wi h in ensi y up o 5000 a.u. o CHROM and 12,000 a.u. o RPMI. The in ensi y o he peaks de ec able in mic obial spec a anges om app oxima ely 2000 a.u. up o 20,000 a.u. As we showed in ou p e ious wo k, he o e all epea abili y o he speci ic Raman spec a associa ed wi h a ce ain mic obial s ain a e epea ed cul i a ion is e y high, e en o samples p epa ed mo e han a yea apa [16]. To assess he epea abili y o each o ganism-medium combina ion wi hin he analysis we an a p incipal componen analysis (PCA) o all measu emen s o one o ganism g own on all co esponding media (Figu e 2). All o ganisms we e measu ed a e iden ical cul i a ion condi ions using he same equipmen and exposi ion ime. Senso s 2015, 15 29638 Figu e 1. Raman spec a ob ained om di e en cul u e media (ENDO—Endo aga , BA—blood aga ; BA-NaCl—blood aga wi h 10% NaCl; MH—Muelle -Hin on aga ; CHROM—CHROMaga Candida; RPMI—Roos el -Pa k Ins i u e Medium 1640). Figu e 2. Repea abili y o Raman spec a o he gi en o ganism a e g ow h on di e en cul u e media (ENDO—Endo aga , BA—blood aga ; BA-NaCl—blood aga wi h 10% NaCl; MH—Muelle -Hin on aga ; CHROM—CHROMaga Candida; RPMI—Roos el -Pa k Ins i u e Medium 1640). Plo s ma ked “Wi hou RPMI” and “Wi hou CHROM” a e displayed o simpli y he compa ison o a iabili y o Raman spec a measu emen on all o he media. The lowe he a iabili y wi hin he species is, he smalle he a ea co e ed by an ellipsoid. Clus e ing o S. au eus spec a indica es ha he mos sui able medium o Raman spec al acquisi ion o his o ganism is MH medium. BA and BA-NaCl can also be used, bu he epea abili y o he inge p in is lowe . BA-NaCl is a selec i e medium, which could easily induce a dissocia ion o he s ain (in a-s ain a ia ions) and he e o e i migh cause he a iabili y in Raman spec a. A chemically Senso s 2015, 15 29639 well-de ined medium con aining many supplemen s, RPMI, induces a high a iabili y in Raman spec a o S. au eus and he e o e should no be used o analyses o his o ganism. Resul o S aphylococcus epide midis show he e y same pa e n. Repea abili y o C. albicans Raman spec a is high when using MH as well as SAB medium. On he o he hand, cul i a ion on CHROM aga and RPMI medium induces a high a iabili y o spec a. This migh by in luenced by di e en colony mo phology induced by nu ien s in a medium. Fo example, RPMI medium induces he hyphal o m de elopmen leading o domina ion o his o m o e he yeas o m [44]. The e is a low a iabili y o Raman spec a ob ained om E. coli colonies g own on MH aga , oo. Cul i a ion on BA aga is shown o be compa able o MH, conce ning he a iabili y o spec a. E. coli inge p in epea abili y on ENDO aga is, howe e , lowe han on p e iously men ioned media. This migh be caused by he p esence o a dye ( uchsine) in he medium. Fuchsine is ela i ely oxic o bac e ia and can induce a dissocia ion o he gi en s ain. Equally as in s aphylococci, RPMI medium induces he high a iabili y o Raman spec a in E. coli and he e o e he epea abili y o measu emen on his medium is no su icien . Figu e 3 shows he compa ison o a iabili y in E. coli spec a om RPMI and MH as an example o bes -case scena io and wo s -case scena io cul i a ion media o Raman spec al acquisi ion om bac e ial colonies. Fo compa ison, Figu e 3c includes a plo o loading o he bes -case scena io o all es ed mic oo ganisms—cul i a ion on MH aga . Mo e de ailed analysis o Raman spec a co esponding o he selec ed o ganisms show ha he inge p in o RPMI medium (he e RPMI appea ed as a wo s -case scena io medium) is isible in he spec um o mic obial colonies (de ec able peaks wi h high in ensi y a app ox. 820 cm−1, 1050 cm−1, 1310 cm−1 and 1450 cm−1) (Figu e 4). Consequen ly, such a high in luence o RPMI medium on he Raman spec a o selec ed mic oo ganisms ansla es o he la ge size o he a ea gi en by he Mahalanobis dis ance (Figu es 2 and 3). Also high in luence o hese peaks can be a ace in dedica ed PC-loadings (each PCA can be u he analyzed using he PC-loadings) when using PCA analysis o he selec ed samples (Figu e 3). This is in con a y o MH aga (see Figu e 3c) which con ibu ion o PCA analysis o gi en mic oo ganisms is e y small—i is also ansla ed o small a ea o ellipsoids calcula ed using Mahalanobis dis ance. This p oblem (as wi h RPMI medium) is also obse ed when using CHROMaga (de ec able peaks a app ox. 1040 cm−1, 1360 cm−1 and 1600 cm−1). Thus, de ec ion o medium-induced peaks in Raman spec a o mic oo ganisms impai s he associa ion o he spec a wi h he ce ain co esponding o ganism as well as he speci ici y o mic obial inge p in and he e o e should be a oided. In addi ion, he spec a o each o ganism sligh ly change wi h he used cul u e medium. This migh be a esul o di e en nu ien s in ake leading o he di e ences in cell and colony composi ion. Acco ding o he esul s p esen ed abo e, he mos sui able medium o analyses o all es ed bac e ial s ains is MH aga . Mo eo e , analyses o colonies g own o MH aga show a e y good epea abili y also o he yeas s ains. This wi hin-analysis epea abili y o MH aga used o yeas s ains a e consis en wi h ou ecen wo k employing Candida pa apsilosis s ains [16]. In addi ion o MH aga , SAB aga is shown o p o ide he compa able epea abili y o he yeas s ain measu emen . Senso s 2015, 15 29640 (a) (b) (c) Figu e 3. (a) Sco es plo o he i s wo p incipal componen s ela ion o E. coli cul u ed on wo di e en media— op clus e RPMI (down- iangle), bo om clus e MH (up- iangle). Using wo p inciple componen s, one can clea ly sepa a e he clus e s o spec a ela ed o cul i a ion o he same mic oo ganism on wo di e en media. This sugges s ha du ing he g ow h o bac e ia cul i a ed on RPMI aga , some subs ances om aga we e abso bed in o he olume o he colony ansla ing o he inal bac e ial spec a; (b) Plo o loading o PC2 co esponding o Figu e 3a suppo ing he con ibu ion o spec al bands ela ed o RPMI medium. Because o ocusing condi ions his is no due o he con ibu ion om he unde lying aga ; (c) Plo o loading o PC2 co esponding o ou bac e ia included in ou s udy cul u ed on he Muelle -Hin on aga suppo ing he con ibu ion o spec al bands ela ed o gi en mic oo ganisms ( h ee bands o be a-ca o ene). This is in con as o Figu e 3b whe e spec al bands ela ed o RPMI medium we e obse ed and shows ha he Muelle -Hin on aga should be he medium o choice o mic obiological expe imen s employing Raman spec oscopy measu emen s. Senso s 2015, 15 29641 Figu e 4. No malized ep esen a i e Raman spec a o he mic oo ganisms on all used cul u e media (ENDO—Endo aga , BA—blood aga ; BA-NaCl—blood aga wi h 10% NaCl; MH—Muelle -Hin on aga ; CHROM—CHROMaga Candida; RPMI—Roos el -Pa k Ins i u e Medium 1640). 3. Ma e ials and Me hods 3.1. Mic oo ganisms and Sample P epa a ion Selec ed o ganisms ep esen ing G am-posi i e bac e ia (S aphylococcus au eus D 47, S aphylococcus epide midis, STO 60), G am-nega i e bac e ia (Esche ichia coli, CCM 3988) and yeas s (Candida albicans, CCM 8261) we e used in he s udy. The CCM s ains we e ob ained om he Czech Collec ion o Mic oo ganisms. Bo h s aphylococcal s ains we e isola ed om clinical ma e ial and s o ed in he Cul u e Collec ion o he Depa men o Mic obiology, S . Anne’s Facul y Hospi al in B no, Czech Republic. All o hese s ains we e s o ed a –70 °C. Be o e he expe imen , he s ains we e hawed quickly a he oom empe a u e and cul i a ed on he Muelle -Hin on aga (MH, Oxoid, Basings oke, UK) a 37 °C o 24 h. G own colonies we e consequen ly ans e ed on o ce ain solid media in Pe i dishes: MH, Blood aga wi h sheep e y h ocy es (BA), Blood aga wi h sheep e y h ocy es and 10% NaCl (BA-NaCl), Sabou aud aga (SAB, Me ck, Ge many), Roos el -Pa k Ins i u e Medium 1640 wi h L-glu amine (RPMI, Sigma Ald ich, Munich, Ge many) and CHROMaga Candida (CHROM, CHROMaga Company, Pa is, F ance), Endo aga (ENDO, Oxoid, Basings oke, UK), wi h each o ganism cul u ed on ou media (see Table 1). Selec ed media a e commonly used in clinical Senso s 2015, 15 29642 labo a o ies. Howe e , selec ed o ganisms ha e di e en nu i i e demands and he e o e no all o he media a e sui able o all o he o ganisms and can inhibi hei g ow h (Table 1). The cul i a ion on selec ed media be o e he expe imen an o 24 h (excep o Candida albicans on CHROMaga , 48 h) a 37 °C. Table 1. Cul i a ion o mic oo ganisms using di e en cul u e media (x = used o he gi en o ganism). Medium/O ganism S aphylococcus au eus S aphylococcus epide midis Esche ichia coli Candida albicans Muelle -Hin on aga x x x x Blood aga x x x Blood aga -NaCl x x CHROMaga Candida x Roos el -Pa k Ins i u e Medium 1640 wi h L-glu amine x x x x Sabou aud aga x Endo aga x 3.2. Expe imen al Se up G own mic obial colonies we e examined using a comme cial Renishaw Raman spec ome e (Renishaw inVia Raman Spec ome e , Renishaw plc., Wo on-unde -Edge, UK), wi h 785 nm single-mode diode lase as he exci a ion sou ce. A e cul i a ion o 24 h on MH aga he colony size was app oxima ely 100 µm so ha spec a could be aken easily wi h ocused lase (diame e o lase is app ox. 2 μm × 10 μm). The s eep dec ease in colony heigh ou side he la cen e o a g owing colony (he e he diame e is app ox. 40 μm). This is isualized by un ocused colony su ace a he pe iphe y o he colony (Figu e 5). No e ha he displayed colony is ela i ely small and was chosen jus o demons a e he shape o a bac e ial colony. Ma u e colonies can be as la ge as a ew mm in diame e wi h es ima ed hicknesses o hund eds o mic ome e s. We es ima ed he heigh o he displayed colony a he dis ance o 15 μm om he bo om o he colony by e ocusing lase spo on he sample a 30 μm and he la cen e on he op o he colony a 110 μm. Figu e 5. Image o a colony o S aphylococcus epide midis. Senso s 2015, 15 29643 A lase beam was ocused on o a sample by he mic oscope objec i e (Leica, We zla , Ge many, 50×, NA (Nume ical ape u e) 0.5) wi h he lase spo diame e o app oxima ely 2 μm × 10 μm (no e ha such lase spo shape is cha ac e is ic o he Renishaw inVia ins umen ), wi h ull axial dep h o he exci a ion egion a 8 µm [45]. As he lase was ocused on o a su ace o he pa icula colony, we measu ed he esponse o a small ac ion o a colony di ec ly g own on he cul u e medium in he pe i dish as ecen ly desc ibed in he wo k o Samek e al. [16]. To eliably apply he Raman echnique, he lase was no ocused a he a ea nea he pe iphe y whe e he heigh o he colony alls s eeply. In his way, he signal con ibu ion om he unde lying aga can be minimized— he signal which o igina es om he cul u e medium is e y low and no in luencing he Raman spec a om a colony. O e iew spec a we e acqui ed in he ange o 600–1700 cm−1. As he in e e ing signal om he cul u e medium may a y depending on ac ual colony p opo ions, each spec um was measu ed o 15 s om di e en pa s o a colony in a o al o 10 measu emen s o one s ain (spec a we e ob ained om a leas 3 colonies pe s ain). 3.3. Da a Analysis The Raman spec a we e ea ed wi h he Sa i zky-Golay coupled ad anced olling il e backg ound emo al ou ine (see [13]), and subsequen ly analyzed using he s anda d mul i a ia e p inciple componen p og am w i en in-house using Ma Lab so wa e (Ma hWo ks, Na ick, MA, USA). Spec a shown a Figu e 2 we e no malized o he peak assigned o he amino acid phenylalanine [12] a app oxima ely 1004 cm−1. Phenylalanine is p esen in all o he es ed mic oo ganisms and was used o s anda diza ion ollowing nume ous es s o use his peak in ensi y. This is no ideal, howe e , his p ocedu e could be used in ins ances whe e no p ope s anda ds a ailable [12]. The g oups we e ma ked by ellipsoids wi h Mahalanobis dis ance o 3 [46]. 4. Conclusions We analyzed ou mic oo ganisms (S aphylococcus au eus, S aphylococcus epide midis, Esche ichia coli and Candida albicans), each g own on ou di e en cul u e media, by Raman spec oscopy o ind which medium in luences he Raman spec um inge p in o each o ganism less. We showed ha Muelle -Hin on aga is he mos sui able medium o analyses o all es ed mic oo ganisms (compa ed wi h blood aga , blood aga wi h 10% NaCl, Roos el -Pa k Ins i u e Medium 1640, Endo aga and CHROMaga Candida) p o iding a good epea abili y o he mic obial inge p in as well as a low a iabili y o spec a wi hin he analysis. Con a iwise, he Roos el -Pa k Ins i u e Medium 1640 and he CHROMaga Candida we e shown o impai he speci ic mic obial inge p in by in oducing he medium-speci ic peaks in o i and subsequen ly esul ing in a iable wi hin-analysis spec a and low epea abili y o he measu emen .