JK-Practitiner vlume 17 Ns (1-3)January-September 2012 riginal article WHOLE CELL PROTEIN PROFILES OF PSEUDOMONAS AERUGINOSA STRAINS ISOLATED AT SHER-I-KASHMIR INSTITUTE OF MEDICAL SCIENCES, SRINAGAR J&K (SKIMS) Syed Arshi, Deeba Bashir. Thkur Manzr, Abdul Rashid, Syed Khurshid, Rubeena Lne, Syed Shafiq Syed Arshi, Deeba Bashir, Thkur Manzr,Abdul Rashid, Syed Khurshid, Rubeena Lne Department f Micrbilgy Syed Shafiq Department f Neursurgery SKIMS, Sura,Srinagar. Received July 2010 Accepted September 20 Crrespndence Dr. Syed Arshi,MDt Department f Micrbilgy,SKIMS Medical Cllege, Bemina Kashmir IndiaSrinagar 190 0, Kashmir (J&K) India. E mail deebabashir@gmail.cm Abstract Whle cell prtein analysis (WCPA) by SDS-PAGE divided 60 islates int 16 grups (S1 S16) n the basis f banding patterns. The patters were stable and reprducible. All the strains were als examined by cnventinal methds and antibitic sensitivity patterns were determined Gel electrphresis permitted subdivisin f the strains int grups, in a manner which suggests that the technique may be valuable in typing strains islated during utrbreaks f infectin in hspital. JK-Practitiner 2012;17(1-3): 39-43 Key wrds: Pseudmnas aeruginsa, SDS-PAGE. 39
Intrductin assciated with immunsuppressin, chrnic abscesses, iatrgenic meningitis and resistant urinary tract In recent years, utbreaks f infectin caused by infectins. pseudmnas aeruginsa have becme a grwing 1-4 cncern in hspitals. Pseudmnas aeruginsa shws Out f 120 strains f pseudmnas aeruginsa, 38 a high degree f resistance t disinfectants and were frm pus, 26 frm urine, 24 frm sputum, 20 frm 5,6 antimicrbial agents. It is widely distributed in bld, 6 frm CSF and 6 frm catheter tips. Clinical hspital envirnment and has been islated frm samples were prcessed and identified as per the varius inanimate surces such as ventilatry 13 standard prcedures. The strains were als tested fr equipment, bld cllectin tubes, mattresses and their sensitivity t the antibitics by the Stkes disc plastic urinals and als frm shwers, sink drains, tilets 14 diffusin methd with discs prcured frm Hi-media in ICU's. Pseudmnas aeruginsa is the mst cmmn Mumbai. gram negative bacterium fund in nscmial infectins.1-4 This rganism is capable f causing life Culture media and grwth cnditins 7 threatening infectins including septicemia, Strains were grwn n MacCnkey's agar fr 48 hurs 8 pneumnia and meningitis. at 37 C befre bichemical and sensitivity testing and There are varius methds fr typing Pseudmnas befre harvesting fr electrphresis. 9 aeruginsa namely bityping, bactericin typing, Preparatin f prtein extracts sertyping, phage typing, determinatin f antibitic Apprximately 0.3g net weight f cells was harvested susceptibility patterns, whle cell and cell envelpe and washed by centrifugatin in 4ml f distilled water. prtein electrphresis, plasmid analysis, ribtyping, The pellets were resuspended in equal vlumes f plymerase chain reactin fingerprinting and DNA sample treatment buffer (0.0625 M Tris-HCl Ph 6.8, restrictin fragment length plymrphism determined SDS 2%, glycerl 10%, 2 mercaptethanl 5%), heated by pulsed field gel electrphresis (PFGE). at 100 C in a water bath fr 10 minutes and then Numerical taxnmy10 based n the pattern f whle centrifuged at 3,8000 fr 30 minutes at 4 C in an HS 18 cell prteins separated in plyacrylamide gel is centrifuge (MSE). The supernates were stred at -20 C. becming an established methd f classifying bacteria. SDS-PAGE Relatinships revealed by electrphretic patterns shw a gd crrelatin with thse measured by DNA-DNA Out f 120 strains f P. aeruginsa SDS-PAGE f hybridizatin. Statistical indices btained by using sixty strains, was dne in the Department f pycin typing, ribtyping and whle cell prtein Micrbilgy, AIIMS, New Delhi by the methd analysis (WCPA) shws better discriminatry pwer described by Laemmli15 with an acrylamide gradient f f WCPA as cmpared t pycin typing and ribcin 10-20% in the running gel. Biled samples (20l) were typing. applied t the gel apparatus (Bi rad) at a vltage f 60V t run the sample thrugh the stacking gel, after which Prtein patterns f cell membranes f gram negative the vltage was increased t 200 V. Gels were stained bacteria btained by sdium ddecyl sulphate with Cmassie Brilliant Blue. The mlecular weight plyacrylanmide gel electrphresis (SDS PAGE) can 12 standards were phsphrylase B (97.4 kda), bvine be used t identify strains and clnes (Overbeeke and albumin (66.2kDa), va albumin (45 kda), carbnic Lutenberg, 1980; Achtman et al, 1983) and this anhydrase (31 kda), trypsin inhibitr (21.5 kda) and apprach has been used in epidemilgical studies lyszyme (14.4 kda) (Bi Rad, Richmnd, CA, USA). (Leb and Smith, 1980; Frasch and Mcca, 1982). The stained gels were phtgraphed and examined fr The purpse f this study was t investigate whether the presence r absence f bands. The mlecular weight SDS-PAGE prtein patterns culd be used t classify f individual bands was determined. strains f P. aeruginsa. We investigated: (i) whether Results strains f this species have characteristic and reprducible prtein patterns when the cell prteins are Separatin f whle cell prteins f 60 strains frm s u b j e c t e d t S D S - PA G E ; ( i i ) w h e t h e r different surces by SDS-PAGE prduced patterns epidemilgically related strains have similar and cntaining upt 16 visibly detectable bands S1 t S16 unrelated strains dissimilar patterns and (iii) t see any (the bands had mlecular weight in the range f 290.0 crrelatin between whle cell prtein analysis and KD t 97.4 KD), based n the similarity f matrix at drug resistance pattern. abve 91% level, divided strains in 7 grups. Grups 1, 2, 3, 4, 5, 6 and 7 frm sputum, CSF, bld, urine, Material and Methds catheter tips pus and endtracheal aspirate cntained 5, A ttal f ne hundred and twenty strains f P. 3, 8, 10, 6, 21 and 7 strains in that rder respectively. aeruginsa were islated frm a selected grup f Strains in different grups were bichemically similar. hspitalized patients frm June 1999 t June 2001. Grup-1 (Sputum): Patients included in the study were thse wh had catheter assciated urinary tract infectins, mechanical Cnsisted f 5 strains f pseudmnas aeruginsa ventilatr assciated pneumnias, septicemia with 4 islates having S1 and ne islate with S2 40
Whle cell prtein SDS-PAGE pattern. Mlecular weight standards are shwn n the right. W C P B a n d P a t t e r n n S D S - P a g e T t a l N. Lane M = Mlecular weight markers, Lane 1 = Strain (59) S15, Lane 2 = Strain (48) S 10, Lane3 = Strain (49) S10, Lane 4 = Strain (60) S16, Lane 5 = Strain (50) S10 TABLE: WHOLE CELL PROTEIN PROFILE OF PS. AERUGINOSA (N=60) FROM DIFFERENT CLINICAL SPECIMENS. P u s ( 2 1 ) U r in e ( 1 0 ) C. T ip s ( 6 ) E n d - t ra c h e a l S e c r - e t i n s ( 7 ) S p u tu m ( 5 ) B l d ( 8 ) C S F ( 3 ) S 1 - - - - 4 - - S 2 - - - - 1 - - S 3 - - - - - - 1 S 4 - - - - - - 1 S 5 - - - - - - - S 6 - - - - - 5 - S 7-9 - - - 2 - S 8 - - 1 - - - - S 9 - - 5 - - - - S 1 0 2 1 - - - - - - S 1 1 - - - - - - - S 1 2 - - - 5 - - - S 1 3 - - - - - 1 - S 1 4-1 - - - - - S 1 5 - - - - - - - S 1 6 - - - - - - 1 41
pattern n SDS-PAGE. 80% f these strains shared P. aeruginsa is less discriminatry and is nt being antibitic resistance t gentamicin, ciprflxacin and cnsidered an efficient tl any mre fr typing the carbencillin. They were universally sensitive t strains. tbramycin, amikacin, ceftaxime, cefazlin, The main purpse f this study was t analyse P. ceftaziduim, ceflxacin, flxacin, piperacillin, aeruginsa islates frm different specimens fr whle deftriaxne and cefperazne. cell prtein prfile and antibigram. Antibigrams Grup-2 (CSF): appear t relate significantly t sme f the electrphretic grups. Hwever, acquisitin r lss f Cntained 3 strains having visible band patterns S3, S4 resistance plasmids wuld cnfuse an epidemilgical and S16. All the strains were sensitive t ceftaxime, study based n antibigrams alne. Nevertheless, ceftazidime and shared resistance t gentamicin, prtein patterns are useful fr differentiating strains with tbramycin, amikacin, carbencillin and ciprflxacin. 16 identical antibigram. Grup-3 (Bld): SDS-PAGE has been described previusly als fr Cnsisted f 8 strains with visible band patterns S5 (5 typing P. aeruginsa strains. Whle cell prtein islates), S6 (2 islates) and S12 (ne islate). 71% f electrphresis has been used t trace specific strains the strains were resistant t tbramycin, carbencillin and during endemic episdes and utbreaks in hspitals ciprflxacin. (Dykshrn et al, 1987) 17. Grup-4 (Urine): Cmprised f 10 P. aeruginsa strains with 9 strains having S7 pattern and ne strain had S14 pattern. 66.6% f these strains were resistant t gentamycin, ceftaxime, ceftazidime and tbramycin. Grup-5 (Catheter tips) Included 6 P. aeruginsa strains with all 6 strains having S8 pattern. 66.6% f these strains were resistant t gentamicin, tbramycin, carbencillin, ciprflxacin, deftazidine and the remaining 33.4% were sensitive t amikacin and ceftaxime. Grup-6 (Pus): Cnsisted f 21 P. aeruginsa strains with all 21 strains having S10 band pattern. 61.9% f these strains were reistant t ciprflxacin, carbencillin, ceftaxime, ceftazidime and the rest were sensitive t amikacin, tbramycin and gentamicin. Grup-7 This grup cntained 7 P. aeruginsa strains with five strains having S band pattern and tw strains having S13 and S15 band pattern n the gel. 60% f the strains f this grup were resistant t gentamicin, tbramycin, carbencillin, ciprflxacin and ceftazidime and 40% were sensitive t amikacin and ceftaxime. Discussin All typing systems fr epidemilgical investigatin shuld have established typeability and reprducibility, be sensitive enugh t distinguish rganisms that are similar but nt identical and be f prven value n the basis f previus testing in an epidemilgical investigatin. Bityping is an easy methd fr screening strains. Hwever, cell envelpe prtein typing is a mre sensitive methd t detect differences, even thugh the methd is mre labrius t interpret. It is still preferred because individually it has better discriminatry pwer than ribtyping. Pycin typing fr A cllectin f clinical islates and 4 islates frm hspital envirnment were examined t discver if they wuld reslve int grups and whether such grups were assciated with any particular characteristic e.g. surce f islatin, antibigram r bichemical characteristics. Five f the eight strains frm bld exhibited S5 band pattern and the islates frm bld shwed universal resistance fr tbramycin, ciprflxacin and carbencillin. Nine islates f P. aeruginsa frm urine exhibited S7 band pattern and the strains were mstly resistant t gentamicin, tbramycin, ciprflxacin and carbencillin. This study has shwn that subdivisins can be made which appear meaningful in epidemilgical terms. All the strains islated frm pus appeared in ne cluster r grup (with all islates shwing S10 band pattern) and a clse relatinship was demnstrated fr strains islated frm sputum (with all 4 ut f 5 islates shwing S1 band pattern. The purpse f ding typing by WCPA was t study the relatedness f bacterial strains and als t identify thse types which are cmmn in ur hspital envirnments. The types which were fund infrequently culd be either cmmunity islates r smehw nt able t establish themselves in hspital envirnment whereas the cmmnly ccurring types culd be the nes which cntinue t crss infect in a hspital and hence need special attentin regarding their cntrl. The use f SDS-PAGE wuld thus seem ptentially t have a very imprtant rle fr epidemilgical 18 investigatins and may als be useful fr cnfirming identificatins. In the frmer case, visual cmparisns f gel tracks might be adequate. Further studies will cncentrate n the investigatin f hspital utbreaks f infectin t determine the value f SDS-PAGE fingerprinting as an epidemilgical tl. References 1 Wiblin RT. Nscmial pneumnia. In: Wenzel RP, editr preventin and cntrl f nscmial infectins 3rd ed. Baltimre : Wilkias and Wilkisn; 1997; 42
p 807-19. Khan FG, Ashk Rattan. Khan IA, Kalia A. 2 Pllack M. Pseudmnas aeruginsa. In: Study f pseudmnas aeruginsa causing ventilatr Mandell GL, Benett JE, Dlin R, editrs. Principles and assciated pneumnia. Ind J Med Res 1998; 107: 68-74. practice f infectius diseases 4th ed. New Yrk 12 Dijkshrn, Michel MF and Degener JE. Cell Churchill Livingstne 1995; p 1980-2003. envelpe prtein prfiles f Acinetbacter 3 Kluytmans J. Surgical infectins including calcaceticus strains islated in hspitals. J Med burns. In : Wenzel RP, editr. Preventin and cntrl f Micrbil 1987; 23: 313-19. nscmial infectins 3rd ed. Baltimre : Williams and 13 Pllack M. Pseudmnas aeruginsa. In : Wilkins 1997; P 841-65. Mandell GL, Bennett JE, Dlin R, editrs Principles and 4 Grdn SM, Serkey JM, Keys TF, Rayan T, practice f infectius diseases 4th ed. New Yrk Factica CA, Schmitt SK, et al. Secular trends in Churchill Livingstne 1995; p 1980-2003. nscmial bld stream infectins in a 55 bed 14 Stkes EJ and Ridgway GL. Clinical cardithracic intensive care unit. Ann Thracic Surg Bacterilgy 1980; 5th ed. Lndn Arnld. 1998; 65: 95-100. 15 Laemmli UK. Cleavage f structural prteins 5 Nnai A, Chulasiri MV and Skkawi N. during the assembly f head f bacteriphage T4. N Antibitic and antiseptic resistance f pseudmnas ature 1970; 227: 680-85. aeruginsa islated frm patients and hspital 16 Marcs MA, Jimenez MT, De Anta, Vila J. envirnments. Thai J Pharmaceutical Sci 1985; 10: 1-7. Crrelatin f six methds fr typing nscmial 6 Shrinwas. Antimicrbial agents in causatin islates f Acinetbactr baumannii, Department f and treatment f P. aeruginsa infectin : Micrbilgy, Faculty f Medicine, Hspital Clinic sympsium antibitica 1979 published by Ikatrin Abli Univ ersity f Barcelnia. Farmaclgi Indnesia Carbaug Surabaya (1980). 17 Dykshrn L, Michel MF and Degener JE. 7 Mrrisn AJ, Wenzd RP. Epidemilgy f Cell envelpe prtein prfiles f acinetbacter infectins due t pseudmnas aeruginsa. Rev Infect calcaceticus strains islated in hspitals. J Med Dis 1984; 6: 627-42. Micrbil 1987' 23: 313-19. 8 Dunn M, Wunderink RG. Ventilatr assciated 18 Alexander M, Ismail F. Jackman PJH, Nble pneumnia caused by pseudmnas infectin (review). WC. Fingerprinting Acinetbacter strains frm clinical Clinics in Chest medicine 1995; 16: 95-109. surces by numerical analysis f electrphretic prtein 9 Dminique S Blanc, Hans H. Siegrist, Rland patterns. J Med Micrbil 1984; 18: 56-64. Sahli, et al. Ribtyping f pseudmnas aeruginsa : Acknwledgement Discriminatry pwer and usefulness as a tl fr We are grateful t Prf. Pradeep Seth, Head, epidemilgical studies. J Clin Micrbil 1993; 31: 71- Department f Micrbilgy, AIIMS, New Delhi and 77. Prf. Arti Kapil, Department f Micrbilgy, AIIMS, 10 Jackman PJH (1982). Classificatin f New Delhi fr their guidance and supprt while crynebacterium species frm axillary skin by perfrming the whle cell prtein prfile f these numerical analysis f electrphretic prtein patterns. pseudmnas aeruginsa strains at AIIMS, New Delhi. Jurnal f Med Micrbil 1982; 15: 485-91. 43