Last printed 9/21/2004 08:25:00 AM

Manual on Antimicrobial Susceptibility Testing

(Under the auspices of Indian Association of Medical Micro iolo!ists"

#r$ M$%$ Lalitha &rofessor of Micro iolo!' #epart(ent of Micro iolo!' )hristian Medical )olle!e *ellore+ ,a(il -adu

CONTENTS
1. Introduction !. Principle . #actors In$luencing Antimicrobial Susceptibility Testing ". Met&ods o$ Antimicrobial Susceptibility Testing ".1 (is) (i$$usion ".! (ilution ". (ilution And (i$$usion. %. Susceptibility Testing O$ #astidious ,acteria '. Errors in Interpretation and reporting -esults *. /uality Control in Antimicrobial Susceptibility Testing .. Standard Met&ods $or t&e (etection o$ Antimicrobial -esistance. 0. Application o$ Computers in Antimicrobial Susceptibility Testing 1+. Selected ,ibliograp&y Anne1ure I. Guide lines $or Antimicrobial Susceptibility Testing I. Suggested (ilution -anges $or MIC Testing II. Sol2ents and (iluents $or Antibiotics

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1. Introduction
.esistance to anti(icro ial a!ents (AM." has resulted in (or idit' and (ortalit' fro( treat(ent failures and increased health care costs$ Althou!h definin! the precise pu lic health ris/ and esti(atin! the increase in costs is not a si(ple underta/in!+ there is little dou t that e(er!ent anti iotic resistance is a serious !lo al pro le($ Appropriate anti(icro ial dru! use has un0uestiona le enefit+ ut ph'sicians and the pu lic fre0uentl' use these a!ents inappropriatel'$ Inappropriate use results fro( ph'sicians pro1idin! anti(icro ial dru!s to treat 1iral infections+ usin! inade0uate criteria for dia!nosis of infections that potentiall' ha1e a acterial aetiolo!'+ unnecessaril' prescri in! e2pensi1e+ road3spectru( a!ents+ and not follo4in! esta lished reco((endations for usin! che(o proph'la2is$ ,he a1aila ilit' of anti iotics o1er the counter+ despite re!ulations to the contrar'+ also fuel inappropriate usa!e of anti(icro ial dru!s in India$ ,he eas' a1aila ilit' of anti(icro ial dru!s leads to their incorporation into her al or 5fol/5 re(edies+ 4hich also increases inappropriate use of these a!ents$ 6idespread anti iotic usa!e e2erts a selecti1e pressure that acts as a dri1in! force in the de1elop(ent of anti iotic resistance$ ,he association et4een increased rates of anti(icro ial use and resistance has een docu(ented for nosoco(ial infections as 4ell as for resistant co((unit' ac0uired infections$ As resistance de1elops to 5first3line5 anti iotics+ therap' 4ith ne4+ roader spectru(+ (ore e2pensi1e anti iotics increases+ ut is follo4ed ' de1elop(ent of resistance to the ne4 class of dru!s$ .esistance factors+ particularl' those carried on (o ile ele(ents+ can spread rapidl' 4ithin hu(an and ani(al populations$ Multidru!3resistant patho!ens tra1el not onl' locall' ut also !lo all'+ 4ith ne4l' introduced patho!ens spreadin! rapidl' in suscepti le hosts$ Anti iotic resistance patterns (a' 1ar' locall' and re!ionall'+ so sur1eillance data needs to e collected fro( selected sentinel sources$ &atterns can chan!e rapidl' and the' need to e (onitored closel' ecause of their i(plications for pu lic health and as an indicator of appropriate or inappropriate anti iotic usa!e ' ph'sicians in that area$

,he results of in31itro anti iotic suscepti ilit' testin!+ !uide clinicians in the appropriate selection of initial e(piric re!i(ens and+ dru!s used for indi1idual patients in specific situations$ ,he selection of an anti iotic panel for suscepti ilit' testin! is ased on the co((onl' o ser1ed suscepti ilit' patterns+ and is re1ised periodicall'$

!. Principle
,he principles of deter(inin! the effecti1it' of a no2ious a!ent to a acteriu( 4ere 4ell enu(erated ' .ideal +6al/er and others at the turn of the centur'+ the disco1er' of anti iotics (ade these tests(or their (odification"too cu( erso(e for the lar!e nu( ers of tests necessar' to e put up as a routine$ ,he ditch plate (ethod of a!ar diffusion used ' Ale2ander 7le(in! 4as the forerunner of a 1ariet' of a!ar diffusion (ethods de1ised ' 4or/ers in this field $,he 82ford !roup used these (ethods initiall' to assa' the anti iotic contained in lood ' allo4in! the anti iotics to diffuse out of reser1oirs in the (ediu( in containers placed on the surface$ 6ith the introduction of a 1ariet' of anti(icro ials it eca(e necessar' to perfor( the anti(icro ial suscepti ilit' test as a routine$ 7or this+ the anti(icro ial contained in a reser1oir 4as allo4ed to diffuse out into the (ediu( and interact in a plate freshl' seeded 4ith the test or!anis(s$ 91en no4 a 1ariet' of anti(icro ial containin! reser1oirs are used ut the anti(icro ial i(pre!nated a sor ent paper disc is ' far the co((onest t'pe used$ ,he disc diffusion (ethod of A:, is the (ost practical (ethod and is still the (ethod of choice for the a1era!e la orator'$ Auto(ation (a' force the (ethod out of the dia!nostic la orator' ut in this countr' as 4ell as in the s(aller la oratories of e1en ad1anced countries+ it 4ill certainl' e the (ost co((onl' carried out (icro iolo!ical test for (an' 'ears to co(e$ It is+ therefore+ i(perati1e that (icro iolo!ists understand the principles of the test 4ell and /eep updatin! the infor(ation as and 4hen necessar'$ All techni0ues in1ol1e either diffusion of anti(icro ial a!ent in a!ar or dilution of anti iotic in a!ar or roth$ 91en auto(ated techni0ues are 1ariations of the a o1e (ethods$

.
#actors In$luencing Antimicrobial Susceptibility Testing
p3 ,he p; of each atch of Meller3;inton a!ar should e chec/ed 4hen the (ediu( is prepared$ ,he e2act (ethod used 4ill depend lar!el' on the t'pe of e0uip(ent a1aila le in the la orator'$ ,he a!ar (ediu( should ha1e a p; et4een <$2 and <$4 at roo( te(perature after !ellin!$ If the p; is too lo4+ certain dru!s 4ill appear to lose potenc' (e$!$+ a(ino!l'cosides+ 0uinolones+ and (acrolides"+ 4hile other a!ents (a' appear to ha1e e2cessi1e acti1it' (e$!$+ tetrac'clines"$ If the p; is too hi!h+ the opposite effects can e e2pected$ ,he p; can e chec/ed ' one of the follo4in! (eans: = = = Macerate a sufficient a(ount of a!ar to su (er!e the tip of a p; electrode$ Allo4 a s(all a(ount of a!ar to solidif' around the tip of a p; electrode in a ea/er or cup$ Use a properl' cali rated surface electrode$

Moisture If+ >ust efore use+ e2cess surface (oisture is present+ the plates should e placed in an incu ator (?5)" or a la(inar flo4 hood at roo( te(perature 4ith lids a>ar until e2cess surface (oisture is lost ' e1aporation (usuall' 10 to ?0 (inutes"$ ,he surface should e (oist+ ut no droplets of (oisture should e apparent on the surface of the (ediu( or on the petri dish co1ers 4hen the plates are inoculated$ E$$ects o$ T&ymidine or T&ymine Media containin! e2cessi1e a(ounts of th'(idine or th'(ine can re1erse the inhi itor' effect of sulfona(ides and tri(ethopri(+ thus 'ieldin! s(aller and less distinct @ones+ or e1en no @one at all+ 4hich (a' result in false3resistance reports$ Meller3;inton a!ar that is as lo4 in th'(idine content as possi le should e used$ ,o e1aluate a ne4 lot of MAeller3 ;inton a!ar, Enterococcus faecalis A,)) 29212+ or alternati1el'+ E. faecalis A,)) ??18B+ should e tested 4ith tri(ethopri(/sulfa(etho2a@ole dis/s$ :atisfactor' (edia 4ill pro1ide essentiall' clear+ distinct @ones of inhi ition 20 (( or !reater in dia(eter$ Unsatisfactor'

(edia 4ill produce no @one of inhi ition+ !ro4th 4ithin the @one+ or a @one of less than 20 (($ E$$ects o$ 4ariation in (i2alent Cations *ariation in di1alent cations+ principall' (a!nesiu( and calciu(+ 4ill affect results of a(ino!l'coside and tetrac'cline tests 4ith P. aeruginosa strains$ 92cessi1e cation content 4ill reduce @one si@es+ 4hereas lo4 cation content (a' result in unaccepta l' lar!e @ones of inhi ition$ 92cess @inc ions (a' reduce @one si@es of car apene(s$ &erfor(ance tests 4ith each lot of Meller3;inton a!ar (ust confor( to the control li(its$ Testing strains t&at $ail to gro5 satis$actorily 8nl' aero ic or facultati1e acteria that !ro4 4ell on unsupple(ented MAeller3;inton a!ar should e tested on that (ediu($ )ertain fastidious acteria such as Haemophilus spp., N. gonorrhoeae+ S. pneumoniae+ and 1iridans and C3hae(ol'tic streptococci do not !ro4 sufficientl' on unsupple(ented MAeller3;inton a!ar$ ,hese or!anis(s re0uire supple(ents or different (edia to !ro4+ and the' should e tested on the (edia descri ed in separate sections$

". Met&ods o$ Antimicrobial Susceptibility Testing

Anti(icro ial suscepti ilit' testin! (ethods are di1ided into t'pes ased on the principle applied in each s'ste($ ,he' include: (i$$usion :to/es (ethod %ir '3Dauer (ethod (ilution Mini(u( Inhi itor' )oncentration i" Droth dilution ii"A!ar #ilution (i$$usion6(ilution 93,est (ethod

".1 (is) (i$$usion

-eagents $or t&e (is) (i$$usion Test 1. M7eller83inton Agar Medium 8f the (an' (edia a1aila le+ MAeller3;inton a!ar is considered to e the est for routine suscepti ilit' testin! of nonfastidious acteria for the follo4in! reasons: = = = = It sho4s accepta le atch3to3 atch reproduci ilit' for suscepti ilit' testin!$ It is lo4 in sulphona(ide+ tri(ethopri(+ and tetrac'cline inhi itors$ It !i1es satisfactor' !ro4th of (ost nonfastidious patho!ens$ A lar!e od' of data and e2perience has een collected concernin! suscepti ilit' tests perfor(ed 4ith this (ediu($ Althou!h MAeller3;inton a!ar is relia le !enerall' for suscepti ilit' testin!+ results o tained 4ith so(e atches (a'+ on occasion+ 1ar' si!nificantl'$ If a atch of (ediu( does not support ade0uate !ro4th of a test or!anis(+ @ones o tained in a dis/ diffusion test 4ill usuall' e lar!er than e2pected and (a' e2ceed the accepta le 0ualit' control li(its$ 8nl' MAeller3;inton (ediu( for(ulations that ha1e een tested accordin! to+ and that (eet the acceptance li(its descri ed in+ -))L: docu(ent MB23A<3 &rotocols for 91aluatin! #eh'drated MAeller3;inton A!ar should e used$ Preparation o$ M7eller83inton Agar MAeller3;inton a!ar preparation includes the follo4in! steps$ 1$ 2$ ?$ MAeller3;inton a!ar should e prepared fro( a co((erciall' a1aila le deh'drated ase accordin! to the (anufacturerEs instructions$ I((ediatel' after autocla1in!+ allo4 it to cool in a 45 to 50) 4ater ath$ &our the freshl' prepared and cooled (ediu( into !lass or plastic+ flat3 otto(ed petri dishes on a le1el+ hori@ontal surface to !i1e a unifor( depth of appro2i(atel' 4 (($ ,his corresponds to B0 to <0 (l of (ediu( for plates 4ith dia(eters of 150 (( and 25 to ?0 (l for plates 4ith a dia(eter of 100 (($ 4$ ,he a!ar (ediu( should e allo4ed to cool to roo( te(perature and+ unless the plate is used the sa(e da'+ stored in a refri!erator (2 to 8)"$

5$

&lates should a!ar$

e used 4ithin se1en da's after preparation unless ade0uate

precautions+ such as 4rappin! in plastic+ ha1e een ta/en to (ini(i@e dr'in! of the B$ A representati1e sa(ple of each atch of plates should e e2a(ined for sterilit' ' incu atin! at ?0 to ?5) for 24 hours or lon!er$ !. Preparation o$ antibiotic stoc) solutions Anti itiotics (a' e recei1ed as po4ders or ta lets$ It is reco((ended to o tain pure anti iotics fro( co((ercial sources+ and not use in>ecta le solutions$ &o4ders (ust e accuratel' 4ei!hed and dissol1ed in the appropriate diluents (Anne2ure III" to 'ield the re0uired concentration+ usin! sterile !lass4are$ :tandard strains of stoc/ cultures should e used to e1aluate the anti iotic stoc/ solution$ If satisfactor'+ the stoc/ can e ali0uoted in 5 (l 1olu(es and fro@en at 320F) or 3B0F)$ :toc/ solutions are prepared usin! the for(ula (1000/&" G * G )H6+ 4here &Ipotenc' of the anit iotic ase+ *H1olu(e in (l re0uired+ )Hfinal concentration of solution and 6H4ei!ht of the anti(icro ial to e dissol1ed in *$ Preparation o$ dried $ilter paper discs 6hat(an filter paper no$ 1 is used to prepare discs appro2i(atel' B (( in dia(eter+ 4hich are placed in a &etri dish and sterili@ed in a hot air o1en$ ,he loop used for deli1erin! the anti iotics is (ade of 20 !au!e 4ire and has a dia(eter of 2 (($ ,his deli1ers 0$005 (l of anti iotics to each disc$ Storage o$ commercial antimicrobial discs )artrid!es containin! co((erciall' prepared paper dis/s specificall' for suscepti ilit' testin! are !enerall' pac/a!ed to ensure appropriate anh'drous conditions$ #iscs should e stored as follo4s: = .efri!erate the containers at 8) or elo4+ or free@e at 314 ) or elo4+ in a nonfrost3free free@er until needed$ :ealed pac/a!es of dis/s that contain dru!s fro( the C3lacta( class should e stored fro@en+ e2cept for a s(all 4or/in! suppl'+ 4hich

(a' e refri!erated for at (ost one 4ee/$ :o(e la ile a!ents (e$!$+ i(ipene(+ cefaclor+ and cla1ulanic acid co( inations" (a' retain !reater sta ilit' if stored fro@en until the da' of use$ = ,he unopened disc containers should e re(o1ed fro( the refri!erator or free@er one to t4o hours efore use+ so the' (a' e0uili rate to roo( te(perature efore openin!$ ,his procedure (ini(i@es the a(ount of condensation that occurs 4hen 4ar( air contacts cold dis/s$ = 8nce a cartrid!e of discs has een re(o1ed fro( its sealed pac/a!e+ it should e placed in a ti!htl' sealed+ desiccated container$ 6hen usin! a disc3dispensin! apparatus+ it should e fitted 4ith a ti!ht co1er and supplied 4ith an ade0uate desiccant$ ,he dispenser should e allo4ed to 4ar( to roo( te(perature efore openin!$ 92cessi1e (oisture should e a1oided ' replacin! the desiccant 4hen the indicator chan!es color$ = = 6hen not in use+ the dispensin! apparatus containin! the discs should al4a's e refri!erated$ 8nl' those discs that ha1e not reached the (anufacturerEs e2piration date stated on the la el (a' e used$ #iscs should e discarded on the e2piration date$ Turbidity standard $or inoculum preparation ,o standardi@e the inoculu( densit' for a suscepti ilit' test+ a Da:84 tur idit' standard+ e0ui1alent to a 0$5 Mc7arland standard or its optical e0ui1alent (e$!$+ late2 particle suspension"+ should e used$ A Da:84 0$5 Mc7arland standard (a' e prepared as follo4s: 1$ A 0$53(l ali0uot of 0$048 (ol/L Da)l 2 (1$1<5J 4/1 Da)l2 $ 2;28" is added to 99$5 (l of 0$18 (ol/L ;2:84 (1J 1/1" 4ith constant stirrin! to (aintain a suspension$ 2$ ,he correct densit' of the tur idit' standard should a sor ance$ e 1erified ' usin! a spectrophoto(eter 4ith a 13c( li!ht path and (atched cu1ette to deter(ine the ,he a sor ance at B25 n( should e 0$008 to 0$10 for the 0$5 Mc7arland standard$ ?$ ,he Dariu( :ulfate suspension should e transferred in 4 to B (l ali0uots into scre43cap tu es of the sa(e si@e as those used in !ro4in! or dilutin! the acterial inoculu($

4$ ,hese tu es should e ti!htl' sealed and stored in the dar/ at roo( te(perature$ 5$ ,he ariu( sulfate tur idit' standard should e 1i!orousl' a!itated on a (echanical 1orte2 (i2er efore each use and inspected for a unifor(l' tur id appearance$ If lar!e particles appear+ the standard should e replaced$ Late2 particle suspensions should e (i2ed ' in1ertin! !entl'+ not on a 1orte2 (i2er B$ ,he ariu( sulfate standards should e replaced or their densities 1erified (onthl'$

(isc di$$usion met&ods

,he %ir '3Dauer and :to/esE (ethods are usuall' used for anti(icro ial suscepti ilit' testin!+ 4ith the %ir '3Dauer (ethod ein! reco((ended ' the -))L:$ ,he accurac' and reproduci ilit' of this test are dependent on (aintainin! a standard set of procedures as descri ed here$ -))L: is an international+ interdisciplinar'+ non3profit+ non3!o1ern(ental or!ani@ation co(posed of (edical professionals+ !o1ern(ent+ industr'+ healthcare pro1iders+ educators etc$ It pro(otes accurate anti(icro ial suscepti ilit' testin! (A:," and appropriate reportin! ' de1elopin! standard reference (ethods+ interpretati1e criteria for the results of standard A:, (ethods+ esta lishin! 0ualit' control para(eters for standard test (ethods+ pro1ides testin! and reportin! strate!ies that are clinicall' rele1ant and cost3effecti1e Interpretati1e criteria of -))L: are de1eloped ased on international colla orati1e studies and 4ell correlated 4ith MI)Ks and the results ha1e corro orated 4ith clinical data$ Dased on stud' results -))L: interpretati1e criteria are re1ised fre0uentl'$ -))L: is appro1ed ' 7#A3U:A and reco((ended ' 6;8$ Procedure $or Per$orming t&e (isc (i$$usion Test Inoculum Preparation Gro5t& Met&od ,he !ro4th (ethod is perfor(ed as follo4s 1$ At least three to fi1e 4ell3isolated colonies of the sa(e (orpholo!ical t'pe are selected fro( an a!ar plate culture$ ,he top of each colon' is touched 4ith a loop+

10

and the !ro4th is transferred into a tu e containin! 4 to 5 (l of a suita le roth (ediu(+ such as tr'ptic so' roth$ 2$ ?$ ,he roth culture is incu ated at ?5) until it achie1es or e2ceeds the tur idit' of the 0$5 Mc7arland standard (usuall' 2 to B hours" ,he tur idit' of the acti1el' !ro4in! roth culture is ad>usted 4ith sterile saline or roth to o tain a tur idit' opticall' co(para le to that of the 0$5 Mc7arland standard$ ,his results in a suspension containin! appro2i(atel' 1 to 2 2 10 8 )7U/(l for E.coli A,)) 25922$ ,o perfor( this step properl'+ either a photo(etric de1ice can e used or+ if done 1isuall'+ ade0uate li!ht is needed to 1isuall' co(pare the inoculu( tu e and the 0$5 Mc7arland standard a!ainst a card 4ith a 4hite ac/!round and contrastin! lac/ lines$ (irect Colony Suspension Met&od 1$ As a con1enient alternati1e to the !ro4th (ethod+ the inoculu( can e prepared ' (a/in! a direct roth or saline suspension of isolated colonies selected fro( a 183 to 243hour a!ar plate (a nonselecti1e (ediu(+ such as lood a!ar+ should e used"$ ,he suspension is ad>usted to (atch the 0$5 Mc7arland tur idit' standard+ usin! saline and a 1orte2 (i2er$ 2$ ,his approach is the reco((ended (ethod for testin! the fastidious or!anis(s+ Haemophilus spp$+ N. gonorrhoeae+ and streptococci+ and for testin! staph'lococci for potential (ethicillin or o2acillin resistance$

Inoculation o$ Test Plates

1$ 8pti(all'+ 4ithin 15 (inutes after ad>ustin! the tur idit' of the inoculu( suspension+ a sterile cotton s4a is dipped into the ad>usted suspension$ ,he s4a should e rotated se1eral ti(es and pressed fir(l' on the inside 4all of the tu e a o1e the fluid le1el$ ,his 4ill re(o1e e2cess inoculu( fro( the s4a $ 2$ ,he dried surface of a MAeller3;inton a!ar plate is inoculated ' strea/in! the s4a o1er the entire sterile a!ar surface$ ,his procedure is repeated ' strea/in! t4o (ore ti(es+ rotatin! the plate appro2i(atel' B0 each ti(e to ensure an e1en distri ution of inoculu($ As a final step+ the ri( of the a!ar is s4a ed$ 11

?$

,he lid (a' e left a>ar for ? to 5 (inutes+ ut no (ore than 15 (inutes+ to allo4 for an' e2cess surface (oisture to e a sor ed efore appl'in! the dru! i(pre!nated dis/s$

-8,9: 92tre(es in inoculu( densit' (ust e a1oided$ -e1er use undiluted o1erni!ht roth cultures or other unstandardi@ed inocula for strea/in! plates$

Application o$ (iscs to Inoculated Agar Plates

1$ ,he predeter(ined atter' of anti(icro ial discs is dispensed onto the surface of the inoculated a!ar plate$ 9ach disc (ust e pressed do4n to ensure co(plete contact 4ith the a!ar surface$ 6hether the discs are placed indi1iduall' or 4ith a dispensin! apparatus+ the' (ust e distri uted e1enl' so that the' are no closer than 24 (( fro( center to center$ 8rdinaril'+ no (ore than 12 discs should e placed on one 150 (( plate or (ore than 5 discs on a 100 (( plate$ Decause so(e of the dru! diffuses al(ost instantaneousl'+ a disc should not e relocated once it has co(e into contact 4ith the a!ar surface$ Instead+ place a ne4 disc in another location on the a!ar$ 2$ ,he plates are in1erted and placed in an incu ator set to ?5 ) 4ithin 15 (inutes after the discs are applied$ 6ith the e2ception of Haemophilus spp$+ streptococci and N. gonorrhoeae + the plates should not e incu ated in an increased )82 at(osphere+ ecause the interpreti1e standards 4ere de1eloped ' usin! a( ient air incu ation+ and )82 4ill si!nificantl' alter the si@e of the inhi itor' @ones of so(e a!ents$

-eading Plates and Interpreting -esults

1$ After 1B to 18 hours of incu ation+ each plate is e2a(ined$ If the plate 4as satisfactoril' strea/ed+ and the inoculu( 4as correct+ the resultin! @ones of inhi ition 4ill e unifor(l' circular and there 4ill e a confluent la4n of !ro4th$ If indi1idual colonies are apparent+ the inoculu( 4as too li!ht and the test (ust e repeated$ ,he dia(eters of the @ones of co(plete inhi ition (as >ud!ed ' the unaided e'e" are (easured+ includin! the dia(eter of the disc$ Lones are (easured 12

to the nearest 4hole (illi(eter+ usin! slidin! calipers or a ruler+ 4hich is held on the ac/ of the in1erted petri plate$ ,he petri plate is held a fe4 inches a o1e a lac/+ nonreflectin! ac/!round and illu(inated 4ith reflected li!ht$ If lood 4as added to the a!ar ase (as 4ith streptococci"+ the @ones are (easured fro( the upper surface of the a!ar illu(inated 4ith reflected li!ht+ 4ith the co1er re(o1ed$ If the test or!anis( is a Staphylococcus or Enterococcus spp$+ 24 hours of incu ation are re0uired for 1anco('cin and o2acillin+ ut other a!ents can e read at 1B to 18 hours$ ,rans(itted li!ht (plate held up to li!ht" is used to e2a(ine the o2acillin and 1anco('cin @ones for li!ht !ro4th of (ethicillin3 or 1anco('cin3 resistant colonies+ respecti1el'+ 4ithin apparent @ones of inhi ition$ An' discerna le !ro4th 4ithin @one of inhi ition is indicati1e of (ethicillin or 1anco('cin resistance$ 2$ ,he @one (ar!in should e ta/en as the area sho4in! no o 1ious+ 1isi le !ro4th that can e detected 4ith the unaided e'e$ 7aint !ro4th of tin' colonies+ 4hich can e detected onl' 4ith a (a!nif'in! lens at the ed!e of the @one of inhi ited !ro4th+ is i!nored$ ;o4e1er+ discrete colonies !ro4in! 4ithin a clear @one of inhi ition should e su cultured+ re3identified+ and retested$ :trains of Proteus spp$ (a' s4ar( into areas of inhi ited !ro4th around certain anti(icro ial a!ents$ 6ith Proteus spp$+ the thin 1eil of s4ar(in! !ro4th in an other4ise o 1ious @one of inhi ition should e i!nored$ 6hen usin! lood3supple(ented (ediu( for testin! streptococci+ the @one of !ro4th inhi ition should e (easured+ not the @one of inhi ition of he(ol'sis$ 6ith tri(ethopri( and the sulfona(ides+ anta!onists in the (ediu( (a' allo4 so(e sli!ht !ro4thM therefore+ disre!ard sli!ht !ro4th (20J or less of the la4n of !ro4th"+ and (easure the (ore o 1ious (ar!in to deter(ine the @one dia(eter$ ?$ ,he si@es of the @ones of inhi ition are interpreted ' referrin! to ,a les 2A throu!h 2I (Lone #ia(eter Interpretati1e :tandards and e0ui1alent Mini(u( Inhi itor' )oncentration Drea/points" of the -))L: M1003:12: &erfor(ance :tandards for Anti(icro ial :uscepti ilit' ,estin!: ,4elfth Infor(ational :upple(ent+ and the or!anis(s are reported as either suscepti le+ inter(ediate+ or resistant to the a!ents

13

that ha1e een tested$ :o(e a!ents (a' onl' e reported as suscepti le+ since onl' suscepti le rea/points are !i1en$

".! (ilution Met&ods

#ilution suscepti ilit' testin! (ethods are used to deter(ine the (ini(al concentration of anti(icro ial to inhi it or /ill the (icroor!anis($ ,his can e achie1ed ' dilution of anti(icro ial in either a!ar or dilutions (t4o fold"$ Minimum In&ibitory Concentration 9MIC: #iffusion tests 4idel' used to deter(ine the suscepti ilit' of or!anis(s isolated fro( clinical speci(ens ha1e their li(itationsM 4hen e0ui1ocal results are o tained or in prolon!ed serious infection e$!$ acterial endocarditis+ the 0uantitation of anti iotic action 1is3a31is the patho!en needs to e (ore precise$ Also the ter(s N:uscepti leK and N.esistantK can ha1e a realistic interpretation$ ,hus 4hen in dou t+ the 4a' to a precise assess(ent is to deter(ine the MI) of the anti iotic to the or!anis(s concerned$ ,here are t4o (ethods of testin! for MI): (a" Droth dilution (ethod ( " A!ar dilution (ethod$ ,rot& (ilution Met&od ,he Droth #ilution (ethod is a si(ple procedure for testin! a s(all nu( er of isolates+ e1en sin!le isolate$ It has the added ad1anta!e that the sa(e tu es can e ta/en for MD) tests also: Materials :terile !raduated pipettes of 10(l+ 5(l+ 2(l and 1(l :terile capped <$5 2 1$? c( tu es / s(all scre43capped ottles+ &asteur pipettes+ o1erni!ht roth culture of test and control or!anis(s ( sa(e as for disc diffusion tests"+ re0uired anti iotic in po4der for( (either fro( the (anufacturer or standard la orator' acco(panied ' a state(ent of its acti1it' in roth (edia$ Anti(icro ials are tested in lo! 2 serial

14

(!/unit or per (l$ )linical preparations should not e used for reference techni0ue$"+ re0uired sol1ent for the anti iotic+ sterile #istilled 6ater 3 500(l and suita le nutrient roth (ediu($ ,ri(ethopri( and sulphona(ide testin! re0uires th'(idine free (edia or addition of 4J l'sed horse lood to the (edia A suita le rac/ to hold 22 tu es in t4o ro4s i3e 11 tu es in each ro4$ Stoc) solution :toc/ solution can e prepared usin! the for(ula 1000 3333333 2 * 2 )H 6 & 6here &H&otenc' !i1en ' the (anufacturer in relation to the ase *H *olu(e in (l re0uired )H7inal concentration of solution ((ultiples of 1000" 6H 6ei!ht of the anti(icro ial to e dissol1ed in the 1olu(e * 92a(ple: 7or (a/in! 10 (l solution of the stren!th 10+000(!/l fro( po4der ase 4hose potenc' is 980 (! per !ra(+the 0uantities of the anti(icro ials re0uired is 6H 1000 3333333 2 10 2 10H102$04(! 980 -ote:the stoc/ solutions are (ade in hi!her concentrations to (aintain their /eepin! 0ualities and stored in suita le ali0uots at 320 o) $8nce ta/en out+the' should not e refro@en or reused$ :u!!ested dilution ran!es of so(e anti(icro ials are sho4n in Anne2ure II$ Met&od &repare stoc/ dilutions of the anti iotic of concentrations 1000 and 100 O!/L as re0uired fro( ori!inal stoc/ solution (10+000(!/L"$ Arran!e t4o ro4s of 12 sterile <$5 21$? c(

15

capped tu es in the rac/$ In a sterile ?0(l (uni1ersal" scre4 capped ottle+ prepare 8(l of roth containin! the concentration of anti iotic re0uired for the first tu e in each ro4 fro( the appropriate stoc/ solution alread' (ade$ Mi2 the contents of the uni1ersal ottle usin! a pipette and transfer 2(l to the first tu e in each ro4$ Usin! a fresh pipette +add 4 (l of roth to the re(ainin! 4 (l in the uni1ersal ottle (i2 and transfer 2(l to the second tu e in each ro4$ )ontinue preparin! dilutions in this 4a' ut 4here as (an' as 10 or (ore are re0uired the series should e started a!ain half the 4a' do4n$ &lace 2(l of anti iotic free roth to the last tu e in each ro4$ Inoculate one ro4 4ith one drop of an o1erni!ht roth culture of the test or!anis( diluted appro2i(atel' to 1 in 1000 in a suita le roth and the second ro4 4ith the control or!anis( of /no4n sensiti1it' ' the si@e of the si(ilarl' diluted$ ,he result of the test is si!nificantl' affected

inoculu($,he test (i2ture should contain 10 B or!anis(/(l$If the roth culture used has !ro4n poorl'+it (a' e necessar' to use this undiluted$ Incu ate tu es for 18 hours at ?<o)$ Inoculate a tu e containin! 2(l roth 4ith the or!anis( and /eep at I4o) in a refri!erator o1erni!ht to e used as standard for the deter(ination of co(plete inhi ition$ Calculations $or t&e preparation o$ t&e original dilution. ,his often presents pro le(s to those unaccusto(ed to perfor(in! these tests$ ,he follo4in! (ethod ad1ocated ' &a(ela M 6ater4orth is presented$ )alculate the total 1olu(e re0uired for the first dilution$ ,4o sets of dilution are ein! prepared (one for the test and one for the control"+ each in 2(l 1olu(es i3e a total of 4 (l for each concentration as 4(l is re0uired to (a/e the second dilution+ the total re0uire(ent is 8(l$ -o4 calculate the total a(ount of the anti iotic re0uired for 8(l$ 7or B4 !/l concentration+ 82B4(!/l H512O! in 8 (l$ &lace a deci(al point after the first fi!ure (5$12" and ta/e this 1olu(e in (l (i$e 5$12 (l" of the dilution elo4 512(!/l and (a/e upto 8(l 4ith roth$ In this e2a(ple !i1en a o1e+ the series has to e started a!ain (id 4a' at 2 (!/l 4hich 4ould e o tained in the sa(e 4a': 8(l of 2(!/lH1BO! in 8(l$ 1$B (l of 10 (!/ l I B$4 (l of roth$

16

-eading o$ result MI) is e2pressed as the lo4est dilution+ 4hich inhi ited !ro4th >ud!ed ' lac/ of tur idit' in the tu e$ Decause 1er' faint tur idit' (a' e !i1en ' the inoculu( itself+ the inoculated tu e /ept in the refri!erator o1erni!ht (a' co(plete inhi ition$ :tandard strain of /no4n MI) 1alue run 4ith the test is used as the control to chec/ the rea!ents and conditions$ e used as the standard for the deter(ination of

Minimum ,actericidal Concentrations9M,C:

,he (ain ad1anta!e of the NDroth dilutionK (ethod for the MI) deter(ination lies in the fact that it can readil' e con1erted to deter(ine the MD) as 4ell$ Met&od #ilutions and inoculations are prepared in the sa(e (anner as descri ed for the deter(ination of MI)$ ,he control tu e containin! no anti iotic is i((ediatel' su cultured (Defore incu ation" ' spreadin! a loopful e1enl' o1er a 0uarter of the plate on a (ediu( suita le for the !ro4th of the test or!anis( and incu ated at ?< o) o1erni!ht$ ,he tu es are also incu ated o1erni!ht at ?< o)$ .ead the MI) of the control or!anis( to chec/ that the dru! concentrations are correct$ -ote the lo4est concentration inhi itin! !ro4th of the or!anis(s and record this as the MI)$ :u culture all tu es not sho4in! 1isi le !ro4th in the sa(e (anner as the control tu e descri ed a o1e and incu ate at ?<o) o1erni!ht$ )o(pare the a(ount of !ro4th fro( the control tu e efore incu ation+4hich represents the ori!inal inoculu($ ,he test (ust include a second set of the sa(e dilutions inoculated 4ith an or!anis( of /no4n sensiti1it' $,hese tu es are not su culturedM the purpose of the control is to confir( ' its MI) that the dru! le1el is correct+4hether or not this or!anis( is /illed is i((aterial$

17

-eading o$ result
,hese su cultures (a' sho4 :i(ilar nu( er of colonies3 indicatin! acteriostasis onl'$ A reduced nu( er of colonies3indicatin! a partial or slo4 actericidal acti1it'$ -o !ro4th3 if the 4hole inoculu( has een /illed ,he hi!hest dilution sho4in! at least 99J inhi ition is ta/en as MD)

Micro8brot& dilution test

,his test uses dou le3stren!th MAeller3;inton roth+ 4G stren!th anti iotic solutions prepared as serial t4o3fold dilutions and the test or!anis( at a concentration of 2210 B/(l$ In a 9B 4ell plate+ 100 l of dou le3stren!th M;D+ 50 l each of the anti iotic dilutions and the or!anis( suspension are (i2ed and incu ated at ?5) for 18324 hours$ ,he lo4est concentration sho4in! inhi ition of !ro4th 4ill e considered the MI) of the or!anis($ -eading o$ result MI) is e2pressed as the hi!hest dilution 4hich inhi ited !ro4th >ud!ed ' lac/ of tur idit' in the tu e$ Decause 1er' faint tur idit' (a' e !i1en ' the inoculu( itself+the inoculated tu e /ept in the refri!erator o1erni!ht (a' e used as the standard for the deter(ination of co(plete inhi ition$ :tandard strain of /no4n MI)+ run 4ith the test is used as the control to chec/ the rea!ents and conditions$

T&e Agar dilution Met&od

A!ar dilutions are (ost often prepared in petri dishes and ha1e ad1anta!e that it is possi le to test se1eral or!anis(s on each plate $If onl' one or!anis( is to e tested e$! M.tuberculosis+the dilutions can e prepared in a!ar slopes ut it 4ill then e necessar' to prepare a second identical set to e inoculated 4ith the control or!anis($,he dilutions are (ade in a s(all 1olu(e of 4ater and added to a!ar 4hich has een (elted and cooled to not (ore than B0o)$Dlood (a' e added and if Nchocolate a!arK is re0uired+the (ediu( (ust e heated efore the anti iotic is added$

18

It 4ould e con1enient to use 90 (( dia(eter petri dishes and add one (l of desired dru! dilutions to 19 (l of roth$,he factor of a!ar dilution (ust e allo4ed for in the first calculation as follo4s$ final 1olu(e of (ediu( in plate ,op anti iotic concentrations ,otal a(ount of dru! 2(l of 1280 O! /(l 4ill e re0uired to start the dilution H 20 (l H B4(!/l H 1280O! to e added to 1 (l of 4ater H 25B0O! in 2 (l H 1$28(l of 2000O! /(l P 0$<2 (l of 1 (l of this 4ill e added to 19 (l a!ar$ (-ote stoc/ dilution of 2000O! /(l is re0uired for this ran!e of MI)" T&e ;uic)est 5ay to prepare a range o$ dilutions in agar is as $ollo5s< La el a sterile petri dish on the ase for each concentration re0uired$ &repare the dilutions in 4ater placin! 1 (l of each in the appropriate dish$ 8ne (l 4ater is added to a control plate$ &ipette 19 (l (elted a!ar+ cooled to 55 o) to each plate and (i2 thorou!hl'$ Ade0uate (i2in! is essential and if sufficient technical e2pertise is not a1aila le for the s/illed (anipulation+ it is stron!l' reco((ended that the a!ar is first (easured into stoppered tu es or uni1ersal containers and the dru! dilution added to these and (i2ed ' in1ersion efore pourin! into petri dishes$ After the plates ha1e set the' should e 4ell dried at ?<o) 4ith their lids tipped for 20 to ?0 (inutes in an incu ator$ ,he' are then inoculated either 4ith a (ultiple inoculator as spots or 4ith a 4ire loop or a platinu( loop cali rated to deli1er 0$001(l spread o1er a s(all area$ In either case the culture should e diluted to contain 105 to 10B or!anis(s per (l$ 6ith ordinar' fast !ro4in! or!anis(s+ this can e o tained appro2i(atel' ' addin! 5 Ol of an o1erni!ht roth culture to 5(l roth or peptone 4ater$ It is possi le to test spreadin! or!anis( such as P.mirabilis ' this (ethod either ' cuttin! ditches in the a!ar et4een the inocula+ or ' confinin! each 4ith s(all !lass or porcelain c'linders pressed into the a!ar$ Althou!h s4ar(in! of P.mirabilis can pre1ented e ' the use of hi!her concentration of a!ar in the (ediu(+ this is not 4ater$

19

reco((ended for deter(ination of MI) ecause of the difficult' of ensurin! ade0uate (i2in! of the dru! 4ith this 1er' 1iscous (ediu($ :electi1e (edia should not e used and electrol'te deficient (edia 4ill !i1e false results ecause of the effect of 1ariation in the salt content on the action of (an' anti iotics$ -eading o$ results ,he anti iotic concentration of the first plate sho4in! 99J inhi ition is ta/en as the MI) for the or!anis($

". (ilution and (i$$usion

9 test also /no4n as the epsilo(eter test is an Ne2ponential !radientK testin! (ethodolo!' 4here N9K in 9 test refers to the Qree/ s'( ol epsilon ( "$,he 9 test(AD Diodis/" 4hich is a 0uantitati1e (ethod for anti(icro ial suscepti ilit' testin! applies oth the dilution of anti iotic and diffusion of anti iotic into the (ediu($$ A predefined sta le anti(icro ial !radient is present on a thin inert carrier strip$ 6hen this 9 test strip is applied onto an inoculated a!ar plate+ there is an i((ediate release of the dru!$ 7ollo4in! incu ation + a s'((etrical inhi ition ellipse is produced$ ,he intersection of the inhi itor' @one ed!e and the cali rated carrier strip indicates the MI) 1alue o1er a 4ide concentration ran!e (R10 dilutions" 4ith inherent precision and accurac' $ 9 test can e used to deter(ine MI) for fastidious or!anis(s li/e S. pneumoniae+

C3he(ol'tic streptococci+ N.gonorrhoeae+ Haemophilus sp$ and anaero es$ It can also e used for -onfer(entin! Qra( -e!ati1e acilli (-7Q-D" for e!3 Pseudomonas sp$ and Burkholderia pseudomallei$ .esistance of (a>or conse0uence (a' e detected for e$!$+ the test is 1er' useful in detectin! !l'copeptide resistant 9nterococci (Q.9" and !l'copeptide inter(ediate S.aureus (QI:A" and slo4 !ro4in! patho!ens such as Mycobacterium tuberculosis. 7urther it can e used for detection of e2tended spectru( eta lacta(ases (9:DL"$ In conclusion 9 test is a si(ple+ accurate and relia le (ethod to deter(ine the MI) for a 4ide spectru( of infectious a!ents$ 20

%. Susceptibility o$ #astidious ,acteria

(ISC (I##=SION #O- #ASTI(IO=S O-GANISMS Antibiotic susceptibility testing o$ S.pneumoniae Media $or disc di$$usion MAeller 3;inton :heep lood a!ar Standardi>ation o$ inoculum. ,he inocula for seedin! the suscepti ilit' (edia 4ith S.pneumoniae is prepared fro( fresh pure cultures (!ro4n o1erni!ht on )hocolate a!ar"$ )ell suspensions of the acteria to e tested are prepared in sterile saline or MAeller3;inton roth$ ,he cell suspension is prepared ' transferrin! a portion of the fresh !ro4th 4ith a s4a or inoculatin! loop to the suspendin! (ediu(+ usin! caution 4hen (i2in! the cells 4ith the suspendin! (ediu( so as not to for( u les$ ,he suspension is then co(pared to the Mc7arland standard ' holdin! the suspension and Mc7arland standard in front of a li!ht a!ainst a 4hite ac/!round 4ith contrastin! lac/ lines and co(parin! the tur idit'$ If the tur idit' is too hea1'+ the suspension should e diluted 4ith additional suspendin! (ediu($ If the tur idit' is too li!ht additional cells should e added to the suspension$ 7or S.pneumoniae S #irect colon' suspension is (ade in nor(al saline and tur idit' ad>usted to 0$5 Mc7arland standard$ 6ithin 15 (inutes after ad>ustin! the tur idit' of the suspension the plate should e inoculated$ Inoculation o$ t&e susceptibility test media After proper tur idit' is achie1ed+ a ne4 sterile s4a (cotton or dacron" is su (er!ed in the suspension+ lifted out of the roth+ and the e2cess fluid is re(o1ed ' pressin! and rotatin! the s4a a!ainst the 4all of the tu e$ ,he s4a is then used to inoculate the entire surface of the supple(ented MAeller ;inton a!ar plate three ti(es+ rotatin! the plate B0 de!rees et4een each inoculation$ ,he inoculu( is allo4ed to dr' (usuall' ta/in! onl' a fe4 (inutes ut no lon!er than 15 (inutes" efore the discs are placed on

21

the plates$ ,he discs should

e placed on the a!ar 4ith sterile forceps and tapped

!entl' to ensure the adherence to the a!ar$ ,he plates containin! the dis/s are incu ated at ?5o) for 1B to 18 h in an in1erted position in a 5J )8 2 incu ator$ A candle e2tinction >ar (a' e used if a )82 incu ator is not a1aila le$ Estimating t&e susceptibility o$ t&e strains After o1erni!ht incu ation+ the dia(eter of each @one of inhi ition is (easured 4ith a ruler or calipers$ ,he @ones of inhi ition on the (edia containin! lood are (easured fro( the top surface of the plate 4ith the top re(o1ed$ It is con1enient to use a ruler 4ith a handle attached for these (easure(ents+ holdin! the ruler o1er the surface of the dis/ 4hen (easurin! the inhi ition @one$ )are should e ta/en not to touch the dis/ or surface of the a!ar$ :terili@e the ruler occasionall' to pre1ent trans(ission of acteria$ In all (easure(ents+ the @ones of inhi ition are (easured fro( the ed!es of the last 1isi le colon'3for(in! !ro4th$ ,he ruler should e positioned across the center of the disc to (a/e these (easure(ents$ ,he results are recorded in (illi(eters (((" and interpretation of suscepti ilit' is o tained ' co(parin! the results to the standard @one si@es$ 7or S.pneumoniae the @one (easure(ent is fro( top of plate 4ith the lid re(o1ed$ 7aint !ro4th of tin' colonies that (a' appear to fade fro( the (ore o 1ious @one should e i!nored in the (easure(ent$ Interpretation 9ach @one si@e is interpreted ' reference to the ,a le 2Q (Lone #ia(eter Interpretati1e :tandards and e0ui1alent Mini(u( Inhi itor' )oncentration Drea/points for S.pneumoniae" of the -))L: M1003:12: &erfor(ance :tandards for Anti(icro ial :uscepti ilit' ,estin!: ,4elfth Infor(ational :upple(ent as suscepti le+ inter(ediate and resistant$

22

Antibiotic susceptibility o$ Haemophilus species ,he (ediu( of choice for disc diffusion testin! of Haemophilus sp$ is ;ae(ophilus ,est Mediu( (;,M"$ MAeller3;inton chocolate a!ar is not reco((ended for routine testin! of Haemophilus spp$ In its a!ar for(+ ;ae(ophilus ,est (ediu( consists of the follo4in! in!redients$ = = = = MAeller3;inton a!ar+ 15 !/(l C3-A#+ 15 !/(l o1ine he(atin+ and 53(!/(l 'east e2tract$

,o (a/e ;,M+ first a fresh he(atin stoc/ solution is prepared ' dissol1in! 50 (! of o1ine he(atin po4der in 100 (l of 0$01 (ol/L -a8; 4ith heat and stirrin! until the po4der is thorou!hl' dissol1ed$ ,hirt' (l of the he(atin stoc/ solution are added to 1 L of M;A 4ith 5 ! of 'east e2tract$ After autocla1in! and coolin! to 45 to 50 )+ ? (l of an -A# stoc/ solution (50 (! of -A# dissol1ed in 10 (l of distilled 4ater and filter sterili@ed" are also asepticall' added$ ,he p; should e <$2 to <$4$ Test Procedure 1$ ,he direct colon' suspension procedure should e used 4hen testin! Haemophilus sp$ Usin! colonies ta/en directl' fro( an o1erni!ht (prefera l' 20 to 24 hour" chocolate a!ar culture plate+ a suspension of the test or!anis( is prepared in MAeller3;inton roth or 0$9J saline$ ,he suspension should e ad>usted to a tur idit' e0ui1alent to a 0$5 Mc7arland standard usin! a photo(etric de1ice$ ,his suspension 4ill contain appro2i(atel' 1 to 4 2 108 )7U/(l$ )are (ust e e2ercised in preparin! this suspension+ ecause hi!her inoculu( concentrations (a' lead to false3resistant results 4ith so(e C3lacta( anti iotics+ particularl' 4hen C3lacta(ase

23

producin! strains of H. influenzae are tested$ 6ithin 15 (inutes after ad>ustin! the tur idit' of the inoculu( suspension+ it should e used for plate inoculation$ 2$ ,he procedure for the disc test should e follo4ed as descri ed for nonfastidious acteria+ e2cept that+ in !eneral+ no (ore than 9 discs should e applied to the surface of a 1503(( plate or no (ore than 4 discs on a 1003(( plate$ ?$ &lates are incu ated at ?5) in an at(osphere of 5J )8 2 for 1B to 18 hours efore (easurin! the @ones of inhi ition$ 4$ ,he @one (ar!in should e considered as the area sho4in! no o 1ious !ro4th 1isi le 4ith the unaided e'e$ 7aint !ro4th of tin' colonies that (a' appear to fade fro( the (ore o 1ious @one should e i!nored in the (easure(ent$

?one (iameter Interpreti2e Criteria ,he anti(icro ial a!ents su!!ested for routine testin! of Haemophilus sp$ are indicated in Anne2ure I$ 9ach @one si@e is interpreted ' reference to the ,a le 29 (Lone #ia(eter Interpretati1e :tandards and e0ui1alent Mini(u( Inhi itor' )oncentration Drea/points for Haemophilus sp$" of the -))L: M1003:12: &erfor(ance :tandards for Anti(icro ial :uscepti ilit' ,estin!: ,4elfth Infor(ational :upple(ent as suscepti le+ inter(ediate and resistant$ #isc diffusion testin! of Haemophilus spp$ 4ith other a!ents is not reco((ended$

24

Antibiotic susceptibility testing $or Neisseria gonorrhoeae ,he reco((ended (ediu( for testin! N. gonorrhoeae consists of Q) a!ar to 4hich a 1J defined !ro4th supple(ent is added after autocla1in!$ )'steine3free !ro4th supple(ent is not re0uired for disc testin!$ 9nriched chocolate a!ar is not reco((ended for suscepti ilit' testin! of N.gonorrhoeae$ Test Procedure 1$ ,he direct colon' suspension procedure should e used 4hen testin!

N. gonorrhoeae. Usin! colonies ta/en directl' fro( an o1erni!ht chocolate a!ar culture plate+ a suspension e0ui1alent to that of the 0$5 Mc7arland standard is prepared in either MAeller3;inton roth or 0$9J saline$ 6ithin 15 (inutes after ad>ustin! the tur idit' of the inoculu( suspension+ it should e used for plate inoculation$ 2$ ,he disc diffusion test procedure steps+ as descri ed for nonfastidious acteria+ should e follo4ed$ -o (ore than 9 anti(icro ial discs should e placed onto the a!ar surface of a 1503(( a!ar plate not (ore than 4 discs onto a 1003(( plate$ ;o4e1er+ 4hen testin! so(e a!ents (e$!$+ 0uinolones" 4hich produce e2tre(el' lar!e @ones+ fe4er discs (a' need to e tested per plate$ ?$ ,he plates are incu ated at ?5) in an at(osphere of 5J )82 for 20 to 24 hours efore (easurin! the @ones of inhi ition$ ?one (iameter Interpreti2e Criteria ,he anti(icro ial a!ents su!!ested for routine testin! of N. gonorrhoeae are indicated in Anne2ure I$ 9ach @one si@e is interpreted ' reference to the ,a le 27 (Lone #ia(eter Interpretati1e :tandards and e0ui1alent Mini(u( Inhi itor' )oncentration Drea/points for N. gonorrhoeae" of the -))L: M1003:12: &erfor(ance :tandards for Anti(icro ial :uscepti ilit' ,estin!: ,4elfth Infor(ational :upple(ent as suscepti le+ inter(ediate

25

and resistant$ #isc diffusion testin! of N. gonorrhoeae 4ith other a!ents is not reco((ended$ -8,9: 8r!anis(s 4ith 103! penicillin disc @one dia(eters of T 19 (( !enerall' produce C3lacta(ase$ ;o4e1er+ C3lacta(ase tests are faster and are therefore preferred for 8r!anis(s 4ith plas(id3(ediated reco!nition of this plas(id3(ediated resistance$

resistance to tetrac'cline also ha1e @ones of inhi ition (?03! tetrac'cline discs" of T 19 (($ )hro(oso(al (echanis(s of resistance to penicillin and tetrac'cline produce lar!er @one dia(eters+ 4hich can e accuratel' reco!ni@ed usin! the interpreti1e criteria indicated in ,a le 27 (Lone #ia(eter Interpretati1e :tandards and e0ui1alent Mini(u( Inhi itor' )oncentration Drea/points for N. gonorrhoeae" of the -))L: M1003:12: &erfor(ance :tandards for Anti(icro ial :uscepti ilit' ,estin!: ,4elfth Infor(ational :upple(ent$

(etermination o$ MIC $or #astidious organisms

T&e Agar dilution met&od Standardi>ation o$ inoculum$ ,he inoculu( should e an acti1el' !ro4in! culture diluted in saline to 10 4 to 105 (icroor!anis( per (l$ 7or S.pneumoniae S #irect colon' suspension fro( a 12315 hour culture fro( ,:DA (ediu( is to e used$ ,he colonies are suspended in 0$5(l of nor(al saline and the opacit' ad>usted to Mc7arland 0$5$ A 1/10 dilution of this suspension is (ade and 4ithin 15 (inutes of (a/in! the diluted suspension the test plates should e inoculated 4ith either a platinu( loop cali rated to deli1er 0$001(l or (ultipoint inoculator$ 7or N.gonorrhoeae and H. influenzae3 :i(ilar to the procedure descri ed a o1e for S.pneumoniae Inoculation o$ test plate In !eneral the inoculu( should e applied as a spot that co1ers a circle a out 538(( in dia(eter $A platinu( loop cali rated to deli1er 0$001(l of the inoculu( is used to spot 26

inoculate the cultures$ Appropriate A,)) 0ualit' control or!anis((s" should e included alon!4ith each test$ Inoculated plates are left undistur ed until the spots of inoculu( ha1e dried$ Incubation After the spots of inoculu( ha1e dried+ the plates are incu ated at ?5o) for 1B to 18 h in an in1erted position in a 5J )8 2 incu ator$ A candle e2tinction >ar (a' e used if a )82 incu ator is not a1aila le$ -eading ,he control plate should sho4 the !ro4th of the U) test or!anis($ ,he MI) of the 0ualit' control strain should e in the e2pected 0ualit' control ran!e$ ,he end point is the lo4est concentration of anti iotic that co(pletel' inhi its !ro4th$ A arel' 1isi le ha@iness or sin!le colon' should e disre!arded$ .esults are reported as the MI) in (icro!ra(s or units/(l$ Interpretation is (ade in accordance to the !uidelines laid do4n in the -))L: M1003:12: &erfor(ance :tandards for Anti(icro ial :uscepti ilit' ,estin!: ,4elfth Infor(ational :upple(ent (MI) Interpretati1e :tandards" as suscepti le+ inter(ediate and resistant$

27

'.Errors in Interpretation and reporting results

In the interpretation of test results there are possi ilities for errors to occur$ Dased on i(pact of errors in treat(ent of patient the' are classified as (inor errors+ (a>or errors and 1er' (a>or errors$ ,his is achie1ed ' co(parin! dis/ diffusion+ 4hich is 4idel' used to report 4ith the MI)+ 4hich is reference (ethod$ ,he follo4in! flo4 chart sho4s the errors inreportin!$(adopted fro( Manual of )linical Micro iolo!'+ <th edition"

-esistant

Minor 9rror

*er' Ma>or 9rror

MIC 9 g@ml:

Minor 9rror

Intermediate

Minor 9rror

Ma>or 9rror

Minor 9rror

Susceptible

(is) (i$$usion (iameter 9mm:

28

*. /uality Control in Antibiotic Susceptibility Testing

U) is perfor(ed to chec/ the 0ualit' of (ediu(+ the potenc' of the anti iotic+ to chec/ (anual errors$ Uualit' control strains should e included dail' 4ith the test$ -ot (ore than 1 in 20 results should e outside accurac' li(its$ -o @one should e (ore than 4 standard de1iations a4a' fro( (idpoint et4een the stated li(its$ If+ for reasons of e2pense or (anpo4er constraints+ it is not possi le to include all strains on a dail' asis+ then the follo4in! !uidelines should e follo4ed$ ,he fre0uenc' can e decreased to once 4ee/l' if proficienc' has een de(onstrated ' 1$ &erfor(in! U) dail' for ?0 da's 4ith less than 10J inaccurac' for each dru! 2$ &roficienc' testin! is repeated for each ne4 dru! included in the testin! ?$ All docu(entation is (aintained indefinitel' 4$ &roficienc' testin! is repeated for each ne4 atch of (edia or rea!ents All tests (ust e 4ithin accurac' li(its if U) is done once 4ee/l'$ -e$erence strains $or ;uality control Escherichia coli A,)) 25922 ( eta3lacta(ase ne!ati1e" Escherichia coli A,)) ?5218 ( eta3lacta(ase positi1e" Staphylococccus aureus A,)) 2592? ( eta3lact(ase ne!ati1e+ o2acillin suscepti le" Staphylococccus aureus A,)) ?8591 ( eta3lact(ase positi1e" Pseudomonas aeruginosa A,)) 2<85? (for a(ino!l'cosides" Enterococcus faecalis A,)) 29212 (for chec/in! of th'(idine or th'(ine le1el of M;A" Haemophilus influenzae A,)) 49<BB (for cephalosporins" Haemophilus influenzae A,)) 10211 (for (ediu( control" Neissseria gonorrheae A,)) 4922B :toc/ cultures should e /ept at 3<0) in Drucella roth 4ith 10J !l'cerol for up to ? 'ears$ Defore use as a U) strain+ the strain should e su cultured at least t4ice and retested for

29

characteristic features$ 6or/in! cultures are (aintained on ,:A slants at 238 ) for up to 2 4ee/s$

.. Standard Met&ods #or T&e (etection O$ Antibacterial -esistance.

(etection o$ Oxacillin/Methicillin-resistant Staphylococcus aureus 9M-SA: :trains that are o2acillin and (ethicillin resistant+ historicall' ter(ed (ethicillin3resistant S. aureus (M.:A"+ are resistant to all eta3lacta( a!ents+ includin! cephalosporins and car apene(s$ M.:A isolates often are (ultipl' resistant to co((onl' used anti(icro ial a!ents+ includin! er'thro('cin+ clinda('cin+ and tetrac'cline$ :ince 199B+ reports of M.:A strains 4ith decreased suscepti ilit' to 1anco('cin ((ini(u( inhi itor' concentration VMI)W+ R8 !/(l" ha1e een pu lished$ Ql'copeptides+ 1anco('cin and ,eicoplanin are the onl' dru! of choice for treat(ent of se1ere M.:A infections+ althou!h so(e strains re(ain suscepti le to fluoro0uinolones+ tri(ethopri(/sulfa(etho2a@ole+ !enta(icin+ or rifa(pin$ Decause of the rapid e(er!ence of rifa(pin resistance+ this dru! should ne1er e used as a sin!le a!ent to treat M.:A infections$ ,he -ational )o((ittee for )linical La orator' :tandards (-))L:" has reco((ended 5:creenin! ,est for 82acillin3resistant S. aureusX and uses an a!ar plate containin! B (icro!/(l of o2acillin and MAeller3;inton a!ar supple(ented 4ith -a)l (4J 4/1M 0$B8 (ol/L"$ ,hese plates can e stored refri!erated for up to 2 4ee/s$ ,he inoculu( is prepared ' (atchin! a 0$5 Mc7arland tu e$ ,4o (ethods can e follo4ed for inoculation: 1$ #ilute the supension 1:100+ and inoculate 10 (icroL on the plate+ to !et an inoculu( of 104 )7U$ 2$ #ip a s4a in the suspension and e2press e2cess fluid ' pressin! s4a a!ainst the 4all of the tu e$ :trea/ s4a o1er a 131$5 inch area$ 30

In oth (ethods+ an' !ro4th after 24 hours incu ation at ?5 ) denotes o2acillin resistance+ if controls are satisfactor'$ Accurate detection of o2acillin/(ethicillin resistance can e difficult due to the presence of t4o su populations (one suscepti le and the other resistant" that (a' coe2ist 4ithin a culture$ All cells in a culture (a' carr' the !enetic infor(ation for resistance ut onl' a s(all nu( er can e2press the resistance in 1itro$ ,his pheno(enon is ter(ed heteroresistance and occurs in staph'lococci resistant to penicillinase3sta le penicillins+ such as o2acillin$ ;eteroresistance is a pro le( for clinical la orator' personnel ecause cells e2pressin! resistance (a' !ro4 (ore slo4l' than the suscepti le population$ ,his is 4h' isolates ein! tested a!ainst o2acillin+ (ethicillin+ or nafcillin should e incu ated at ?5 ) for a full 24 hours efore readin!$ ,he rea/points for S. aureus are different fro( those for coa!ulase3 ne!ati1e staph'lococci ()o-:"$

MICs S. aureus )o-:

O1acillin Susceptible T2 !/(l T0$25! /(l

O1acillin Intermediate no inter(ediate MI) no inter(ediate MI)

O1acillin -esistant MI)R4 ! /(l MI)R0$5 ! /(l

?one si>es S. aureus )o-:

O1acillin Susceptible O1acillin Intermediate R1? (( R18 (( 11312 (( no inter(ediate @one

O1acillin -esistant T10 (( T1< ((

31

6hen used correctl'+ roth3 ased and a!ar3 ased tests usuall' can detect M.:A$ 82acillin screen plates can e used in addition to routine suscepti ilit' test (ethods or as a ac/3up (ethod$ A(plification tests li/e those ased on the pol'(erase chain reaction (&)." detect the mec !ene$ ,hese tests confir( o2acillin/(ethicillin resistance caused ' mec in Staphylococcus species$ (etection o$ Oxacillin-resistant Coagulase-negati e Staphylococcus sp! Althou!h there are a out 20 )o-: species+ the' often are considered to e a sin!le !roup$ :o(e species are (ore resistant to co((onl' used anti(icro ial a!ents than others$ Identification to species le1el can aid in the reco!nition of out rea/s and in trac/in! resistance trends$ S. epidermidis is the (ost co((on )o-: isolated in clinical la oratories$ Usuall', S. epidermidis, S. haemolyticus and S. hominis are (ore li/el' to e (ultipl' resistant to anti(icro ial a!ents than are other )o-: species$ ;o4e1er+ resistance patterns of )o-: (a' differ et4een hospitals and 4ards$ 82acillin3resistant )o-: isolates are resistant to all eta3lacta( a!ents+ includin!

penicillins+ cephalosporins+ and car apene(s$ In addition+ o2acillin3resistant )o-: isolates are often resistant to other co((onl' used anti(icro ial a!ents+ so 1anco('cin is fre0uentl' the dru! of choice for treat(ent of clinicall' si!nificant infections$ Accurate detection of o2acillin resistance can e difficult$ )olon' si@es of )o-: are often s(aller than those of S. aureus+ (a/in! !ro4th (ore difficult to read$ In addition+ li/e S. aureus+ t4o su populations (one suscepti le and the other resistant" (a' coe2ist 4ithin a culture$ 6hen studies 4ere perfor(ed to e1aluate o2acillin rea/points for )o-:+ the current rea/points for S. aureus failed to detect (an' )o-: that contained the mec !ene$ In !eneral+ the ne4 rea/points for )o-: correlate etter 4ith mec production for )o-:$

32

(etection o$ E1tended8Spectrum ,eta8Aactamases 9ES,As: 9:DLs are en@'(es that (ediate resistance to e2tended3spectru( (third !eneration" cephalosporins (e$!$+ cefta@idi(e+ cefota2i(e+ and ceftria2one" and (ono acta(s (e$!$+ a@treona(" ut do not affect cepha('cins (e$!$+ cefo2itin and cefotetan" or car apene(s (e$!$+ (eropene( or i(ipene("$ 9:DLs can e difficult to detect ecause the' ha1e different le1els of acti1it' a!ainst 1arious cephalosporins$ ,hus+ the choice of 4hich anti(icro ial a!ents to test is critical$ 7or e2a(ple+ one en@'(e (a' acti1el' h'drol'@e cefta@idi(e+ resultin! in cefta@idi(e (ini(u( inhi itor' concentrations (MI)s" of 25B (icro!/(l+ ut ha1e poor acti1it' on cefota2i(e+ producin! MI)s of onl' 4 (icro!/(l$ If an 9:DL is detected+ all penicillins+ cephalosporins+ and a@treona( should e reported as resistant+ e1en if in 1itro test results indicate suscepti ilit'$ ,here are standard roth (icrodilution and disc diffusion screenin! tests usin! selected anti(icro ial a!ents$ 9ach !. pneumoniae+ !. o"ytoca+ or Escherichia coli isolate should e considered a potential 9:DL3producer if the test results are as follo4s: (is) di$$usion cefpodo2i(e T 22 (( cefta@idi(e a@treona( cefota2i(e ceftria2one T 22 (( T 2< (( T 2< (( T 25 (( MICs cefpodo2i(e R 2 ! /(l cefta@idi(e a@treona( cefota2i(e ceftria2one R 2 ! /(l R 2 ! /(l R 2 ! /(l R 2 ! /(l

,he sensiti1it' of screenin! for 9:DLs in enteric or!anis(s can 1ar' dependin! on 4hich anti(icro ial a!ents are tested$ ,he use of (ore than one of the fi1e anti(icro ial a!ents su!!ested for screenin! 4ill i(pro1e the sensiti1it' of detection$ )efpodo2i(e and cefta@idi(e sho4 the hi!hest sensiti1it' for 9:DL detection$ &henot'pic confir(ation of potential 9:DL3producin! isolates of !. pneumoniae+ !. o"ytoca+ or E. coli can e done ' testin! oth cefota2i(e and cefta@idi(e+ alone and in co( ination 4ith cla1ulanic acid$ ,estin! can e perfor(ed ' the roth (icrodilution

33

(ethod or ' dis/ diffusion$ 7or MI) testin!+ a decrease of R ? dou lin! dilutions in an MI) for either cefota2i(e or cefta@idi(e tested in co( ination 4ith 4 ! /(l cla1ulanic acid+ 1ersus its MI) 4hen tested alone+ confir(s an 9:DL3producin! or!anis($ 7or disc diffusion testin!+ a R 5 (( increase in a @one dia(eter for either anti(icro ial a!ent tested in co( ination 4ith cla1ulanic acid 1ersus its @one 4hen tested alone confir(s an 9:DL3 producin! or!anis($ #iscs can e (ade ' addin! 10 l of a 1000 ! /(l stoc/ solution of cla1ulanic acid to cefota2i(e and cefta@idi(e dis/s each da' of testin!$ In future+ co((ercial (anufacturers of anti(icro ial discs (a' produce discs containin! cefota2i(e and cefta@idi(e 4ith cla1ulanic acid$ Until co((ercial discs are a1aila le+ :(ith%line Deecha( can pro1ide clinical la oratories 4ith cla1ulanic acid po4der for routine use$ !. pneumoniae A,)) <00B0? (positi1e control" and E. coli A,)) 25922 (ne!ati1e control" should e used for 0ualit' control of 9:DL tests$ :o(e or!anis(s 4ith 9:DLs contain other eta3lacta(ases that can (as/ 9:DL production in the phenot'pic test+ resultin! in a false3ne!ati1e test$ ,hese eta3lacta(ases include A(p)s and inhi itor3resistant ,9Ms (I.,s"$ ;'per3production of ,9M and/or :;* eta3 lacta(ases in or!anis(s 4ith 9:DLs also (a' cause false3ne!ati1e phenot'pic confir(ator' test results$ )urrentl'+ detection of or!anis(s 4ith (ultiple eta3lacta(ases that (a' interfere 4ith the phenot'pic confir(ator' test can onl' e acco(plished usin! isoelectric focusin! and #-A se0uencin!+ (ethods that are not usuall' a1aila le in clinical la oratories$ If an isolate is confir(ed as an 9:DL3producer ' the -))L:3reco((ended phenot'pic confir(ator' test procedure+ all penicillins+ cephalosporins+ and a@treona( should 4hich should e reported accordin! to their routine test results$ e reported as resistant$ ,his list does not include the cepha('cins (cefotetan and cefo2itin"+ If an isolate is not confir(ed as an 9:DL3producer+ current reco((endations su!!est reportin! results as for

34

routine testin!$ #o not chan!e interpretations of penicillins+ cephalosporins+ and a@treona( for isolates not confir(ed as 9:DLs$ 8ther isolates of 9ntero acteriaceae+ such as Salmonella species and Proteus mirabilis+ and isolates of Pseudomonas aeruginosa also produce 9:DLs$ ;o4e1er+ at this ti(e+ (ethods for screenin! and phenot'pic confir(ator' testin! of these isolates ha1e not deter(ined$ (etection o$ Imipenem or Meropenem -esistance in Gram8negati2e Organisms 6ithin a health care settin!+ increases in species3specific car apene( resistance should e (onitored and sudden increases in1esti!ated to rule out an out rea/ of resistant or!anis(s or spurious test results$ &u lished reports indicate so(e resistance in a 1ariet' of clinical Qra(3ne!ati1e or!anis(s+ includin! Pseudomonas aeruginosa, Burkholderia cepacia+ cinetobacter species+ Proteus species, Serratia marcescens+ Enterobacter species+ and !. pneumoniae. Stenotrophomonas maltophilia isolates are intrinsicall' resistant to i(ipene($ 8r!anis(s can produce (ore than one h'drol'@in! en@'(e and (a' sho4 (odifications in (ore than one porin+ producin! hi!h3le1el resistance to the car apene(s ((ini(u( inhi itor' concentration VMI)W R1B !/(l"$ 8r!anis(s 4ith decreased suscepti ilit' produced ' porin chan!es alone often ha1e lo4er MI)s (238 (icro!/(l"$ 8r!anis(s 4ith MI)s near interpretation rea/points ha1e !reater potential for reportin! errors$ 7or e2a(ple+ isolates of P. aeruginosa often ha1e MI)s that are at or near the car apene( inter(ediate (8 !/(l" and resistant (R1B !/(l" rea/points$ :o(e species+ such as P. mirabilis, P. #ulgaris, and Morganella morganii, often ha1e MI)s (134 !/(l" >ust elo4 the car apene( inter(ediate rea/point of 8 (!/(l$ Most other species of 9ntero acteriaceae are 1er' suscepti le (T0$5 !/(l"$ Droth (icrodilution (ethods usuall' detect car apene( resistance 4hen the tests are perfor(ed properl'$ ;o4e1er+ studies ha1e sho4n false resistance to i(ipene( in 35 een

co((erciall' prepared test panels due to de!radation of the dru! or to a (anufacturin! pro le( 4here concentrations of i(ipene( 4ere too lo4$ 6hen perfor(ed properl'+ disc diffusion and a!ar !radient diffusion also are accepta le (ethods for car apene( testin!$ I(ipene( de!rades easil'$ :tudies su!!est that (eropene( (a' e (ore sta le than i(ipene($ ;o4e1er+ for either anti(icro ial a!ent+ stora!e conditions of suscepti ilit' panels+ cards+ and discs (ust e (onitored carefull' and 0ualit' control results chec/ed fre0uentl'$ If possi le+ store supplies containin! car apene(s at the coldest te(perature ran!e stated in the (anufacturerEs directions$ An additional test (ethod+ such as a!ar !radient diffusion (i$e$+ 9 test"+ can e used to 1erif' inter(ediate or resistant results$ /uinolones and resistance ,he nu( er and location of (utations affectin! critical sites deter(ine the le1el of resistance$ 8r!anis(s (a' ha1e alterations in (ore than one en@'(e tar!et site and+ in !ra(3ne!ati1e or!anis(s+ (a' contain (ore than one porin chan!e$ Man' resistant or!anis(s ha1e (ultiple en@'(e tar!et site+ porin+ and efflu2 (utations+ producin! hi!h3 le1el resistance to 0uinolones$ In contrast+ or!anis(s 4ith decreased suscepti ilit' produced onl' ' porin chan!es usuall' ha1e lo4er (ini(u( inhi itor' concentrations (MI)s"$ ,he fluoro0uinolone suscepti ilit' profile for each clinical isolate is deter(ined ' the nu( er and location of (utational chan!es in specific en@'(e tar!et sites+ porin proteins+ and efflu2 (echanis(s$ ,he effect of each (utation in an isolate is not e0ui1alent for all fluoro0uinolones+ due to 1ariations of the che(ical structures a(on! this class of a!ents$ ,herefore+ an or!anis( 4ith one or (ore (utations (a' ha1e resistant MI)s/@one si@es to one 0uinolone ut ha1e inter(ediate or suscepti le MI)s/@one si@es to another 0uinolone$ #urin! therap'+ the potential e2ists for an or!anis( 4ith a sin!le (utation to ac0uire a second (utation+ leadin! to hi!h3le1el resistance$ After (ultiple (utations occur+ an or!anis( is !enerall' hi!hl' resistant to all 0uinolones$ .esistance to 0uinolones has een reported in a 1ariet' of i(portant acterial patho!ens+ includin! E. coli, !. pneumoniae and other enteric or!anis(sM P. aeruginosaM $hlamydia

36

trachomatis+ Mycoplasma pneumoniaeM $ampylobacter %e%uni+ B. cepaciaM S. maltophilia+ N. gonorrhoeae+ S. aureus (especiall' o2acillin3resistant strains"+ Enterococcus faecium and S. pneumoniae$

37

(etection o$ -esistant Enterococci Penicillin@Ampicillin -esistance 9nterococci (a' e resistant to penicillin and a(picillin ecause of production of lo43 affinit'+ penicillin3 indin! proteins (&D&s" or+ less co((onl'+ ecause of the production of C3lacta(ase$ ,he disc diffusion test can accuratel' detect isolates 4ith altered &D&s+ ut it 4ill not relia l' detect C3lacta(ase producin! strains$ ,he rare C3lacta(ase3 producin! strains are detected est ' usin! a direct+ nitrocefin3 ased+ C3lacta(ase test$ )ertain penicillin3 a(picillin3 resistant enterococci (a' possess hi!h3le1el resistance (i$e$+ penicillin MI)s R 128 !/(l or a(picillin MI)s R B4 !/(l"$ ,he disc test 4ill not differentiate those 4ith nor(al resistance fro( this hi!h3le1el resistance$ 7or enterococci reco1ered fro( lood and ):7+ the la orator' should consider deter(inin! the actual MI) for penicillin or a(picillin since E. faecium strains 4ith nor(al lo4er le1el resistance (penicillin MI)s T B4 !/(l and a(picillin T ?2 !/(l" should e considered potentiall' suscepti le to s'ner!' 4ith an a(ino!l'coside (in the a sence of hi!h3le1el a(ino!l'coside resistance" 4hereas strains 4ith hi!her le1el resistance (a' e resistant to such s'ner!'$

4ancomycin -esistance Accurate detection of 1anco('cin3resistant enterococci ' the disc diffusion test re0uires that plates e incu ated for a full 24 hours (rather than 1B to 18 hours" and that an' @one surroundin! the 1anco('cin disc e e2a(ined carefull' 4ith trans(itted li!ht for e1idence of s(all colonies or a li!ht fil( !ro4in! 4ithin the @one$ An inter(ediate cate!or' result ' the disc diffusion test should e 1erified ' deter(inin! the 1anco('cin MI)$ 3ig&8le2el Aminoglycoside -esistance ;i!h3le1el resistance to a(ino!l'cosides is an indication that an enterococcal isolate 4ill not e affected s'ner!isticall' ' a co( ination of a penicillin or !l'copeptide plus an a(ino!l'coside$ :pecial+ hi!h3content !enta(icin (120 !" and strepto('cin (?00 !" discs can e used to screen for this t'pe of resistance$ -o @one of inhi ition indicates resistance+ and @ones of R 10 (( indicate a lac/ of hi!h3le1el resistance$ :trains that 'ield 38

@ones of < to 9 (( should e e2a(ined usin! a dilution screen test$ 8ther a(ino!l'cosides need not e tested+ ecause their acti1ities a!ainst enterococci are not superior to !enta(icin or strepto('cin$ Aminoglycoside -esistance in Enterobacteriaceae and Pseudomonas aeruginosa .esistance to one a(ino!l'coside (a' not predict resistance to the others$ In !eneral+ resistance is relati1el' co((on in P. aeruginosa ut less co((on in 9ntero acteriaceae$ 9ntero acteriaceae resistant to !enta(icin and to ra('cin can e suscepti le to a(i/acin or netil(icin ecause these dru!s are not affected ' (an' of the a(ino!l'coside (odif'in! en@'(es (AM9s"$ ,herefore+ the pre1alence of a(i/acin resistance can e lo4er than the pre1alence of resistance to !enta(icin and to ra('cin+ dependin! upon the resistance (echanis(s present at a healthcare facilit'$ A(ino!l'cosides are not clinicall' effecti1e a!ainst Salmonella species and Shigella species+ althou!h the' (a' appear suscepti le+ a(ino!l'cosides should not e tested or reported$

0. Application o$ Computers in Antibacterial Susceptibility Testing

Anti(icro ial resistance is a !lo al pro le($ 9(er!ence of (ultidru! resistance has li(ited the therapeutic options+ hence (onitorin! resistance is of para(ount i(portance$ Anti(icro ial resistance (onitorin! 4ill help to re1ie4 the current status of anti(icro ial resistance locall'+ nationall' and !lo all' and helpful in (ini(i@in! the conse0uence of dru! resistance+ li(it the e(er!ence and spread of dru! resistant patho!ens$ ,housands of la oratories are distri uted 4orld34ide and need to e lin/ed to inte!rate data on (ost clinicall' rele1ant or!anis( on a dail' asis to o tain an accurate picture of Yreal resistanceX$ 7or this purpose a soft4are 4as de1eloped for the (ana!e(ent of routine la orator' results ' 6;8 called N6;8-9,K$ ,his soft4are has focused on data anal'sis particularl' on the results of A:,$

39

6;8-9, 5$1 is a data ase soft4are co(pati le 4ith+ 6indo4s 95+ 6indo4s 98+ 6indo4s -, and later 1ersions of 6indo4s$ Installation capacit' of the soft4are is 9$?4 ( + this soft4are is a1aila le in 4e site http://444$4ho$int/e(c/6;8-9,/Instructions$ht(l$ ,his pro!ra((e is useful in suppl'in! current !uidelines+ protocols to local la oratories+ in identif'in! the clusters of resistant isolates and e(er!in! out rea/s$ Use of 6;8-9, soft4are (a' aid the local la orator' in net4or/in! efficientl' 4ith the national reference la orator'+ in de1elopin! !uidelines for use of anti(icro ial a!ents and re1ie4 the sa(e periodicall'+ in stud'in! the effect of anti(icro ial control pro!ra((es on resistant acteria+ to net4or/ efficientl' 4ith international odies such as 6;8+ -))L: etc$ ,his is also used for research studies$

40

1+. ,ibliograp&y
#oern Q$*$ :uscepti ilit' tests of fastidious acteria$ Manual of )linical Micro iolo!'+ Bth edition+ Murra' &$.+ Daron 9$Z+ &faller M$A+ ,eno1er 7$)+ [ol/en .+ A(erican :ociet' for Micro iolo!'+ 6ashin!ton #)+ 1995+ &$ 1?423 1?49$ Ira .$ Dacteriolo!'+ :tandard 8perati1e procedure (anual for (icro iolo!' la oratories+ -ational Institute of Diolo!icals$ 1995+ &<?39< Zohn #$, and Za(es ;$Z Anti(icro ial :uscepti ilit' testin!: Qeneral )onsiderations$ Manual of )linical Micro iolo!' <th edition+ Murra' &$.+ Daron 9$Z+ &faller M$A+ ,eno1er 7$)+ [ol/en .+ A(erican :ociet' for Micro iolo!'+ 6ashin!ton #)+ 1999+ &$ 14B9314<?$ -ational )o((ittee for )linical La orator' :tandards$ Performance Standards for antimicrobial susceptibility testing$ 8th Infor(ational :upple(ent$ M100 :12$ -ational )o((ittee for )linical La orator' :tandards+ 2002$ *illano1a+ &a$ ,hrupp L$#$ :uscepti ilit' ,estin! of Anti iotic in Li0uid Media$ Anti iotics In La orator' Medcine 2nd 9dition+ *ictor Lorian+ 6illia(s and 6il/ins+ Daliti(ore$ 198BM &$ 9?3150$ 6ater 4orth &$M$ Uuantitati1e (ethods for acterial sensiti1it' testin!$ La orator' (ethods in anti(icro ial che(otherap'$ .ee1es #$:+ &hilips I+ 6illia(s Z$#+ 6ise .$ )hurchill Li1in!stone+ Dalti(ore$ 19<8M &$ ?1341$ %ra(er Z$ and %irsh au( A$ 9ffect of paper on the perfor(ance assa' in the control of anti iotic sensiti1it' discs$ ppl Microbiol 19B1: 9M &$ ??43??B$ .ippere .$A$ 9ffects of paper on the perfor(ance of anti iotic3i(pre!nated discs$ & Pharma Sci 19<8: B<M &$ ?B<3?<1$ 41

Lalitha M$%+ $Mana'ani #$Z+ &ri'a L+ Zesudason M$* + ,ho(as %+ :teinhoff M$)$ 9 test as an alternati1e to con1entional MI) deter(ination for sur1eillance of dru! resistant S.pneumoniae$ 'ndian &. Med (es. 199<: 10BM &$ 500350?$

Morle' #$)$ A si(ple (ethod for testin! the sensiti1it' of 4ound acteria to penicillin and sulphathia@ole ' use of i(pre!nated lottin! paper disc$ &. Pathol Bacteriol. 1945: 5<M &$ ?<93?82$

A !uide to sensiti1it' testin!: .eport of 4or/in! part' on anti iotic sensiti1it' testin! of the Dritish :ociet' for Anti(icro ial )he(otherap'$ &. ntimicrob $hemothrap 1991M 2<: :upple(ent #+ &$ 1350$

42

Anne1ure I.
Guidelines #or Antimicrobial Susceptibility Testing.
Suggested ,attery O$ Antibiotics #or Susceptibility Testing. Staphylococcus sp. &enicillin 82acillin )ephalothin Qenta(icin -etil(icin A(i/acin )hlora(phenicol ,etrac'cline 9r'thro('cin )o3tri(o2a@ole )linda('cin 8flo2acin .ifa(picin *anco('cin ,eicoplanin Gram negati2e Streptococcus bacilli "Enterococcus# Pneumococcus$ A(picillin &enicillin &iperacillin 82acillin )ephalothin )efota2i(e )efta@idi(e Qenta(icin -etil(icin A(i/acin )hlora(phenicol ,etrac'cline )o3tri(o2a@ole -alidi2ic Acid )iproflo2acin 8flo2acin -itrofurantoin I(ipene( Meropene( Haemophilus sp A(picillin A(o2'cillin/ N! gonorrhoeae &enicillin )efa@olin

)la1ulanic acid A(picillin )efuro2i(e )eftria2one )efota2i(e )efota2i(e )hlora(phenicol 9r'thro('cin ,etrac'cline )iproflo2acin )hlora(phenicol 9r'thro('cin ,etrac'cline )hlora(phenicol *anco('cin

-ote: ,he choice of anti iotic depends on the pattern e2hi ited locall'$ ,he selection of anti iotics 1aries ased on speci(en and the isolates under consideration$

43

Anne1ure II.
Suggested Antibiotic (ilution -anges #or MIC Testing ANTIMIC-O,IAA AGENT &enicillin Q Methicillin -aficillin/82acillin A(picillin(Qra( I1e" A(picillin(Qra(31e" )ephalosporin(Qra( I1e" )ephalosporin I !eneration (Qra( 31e" )ephalosporin II\III !eneration (Qra(31e" )ephalosporin III !eneration CONCENT-ATION -ANGE9Bg@ml: 0$0153?2 0$00?3B4 0$00?3B4 0$153?2 0$1?325B 0$0153?2 0$1?325B 0$0?3B4 0$1?325B

(Pseudomonas sp$" *anco('cin 0$01531B A(i/acin 0$0B3128 Qenta(icin/,o ra('cin 0$0?3B4 9r'thro('cin 0$0153?2 )linda('cin 0$0153?2 .ifa(picin 0$0153?2 )hlora(phenicol 0$0B325B ,etrac'cline 0$0B325B ,ri(ethopri( 0$0?3B4 :ulpha(etho2a@ole 0$B3121B ,ri(ethopri((U,I+Qra( 3 1e" 0$53B4 :ource3A !uide to sensti1it' testin!: .eport of 4or/in! part' on anti iotic sensiti1it' testin! of the Dritish societ' for Anti(icro ial )he(otherap'$Z$Anti(icro )he(otherap 1991M(2<"::upple(ent #+1350$

44

Anne1ure III. Sol2ents and (iluents $or Antibiotics

45

#ootNote< a$ )ephalosporins and cephe(s not listed a o1e or in footnote d are solu li@ed (unless the (anufacturer indicates other4ise" in phosphate uffer+ p; B$0+ 0$1 (ol/L+ and further diluted in sterile distilled 4ater$ $ ,he dia((oniu( salt of (o2alacta( is 1er' sta le+ ut it is al(ost pure . iso(er$ Ma2olacta( for clinical use is a 1:1 (i2ture of . and : iso(ers$ ,herefore+ the salt is dissol1ed in 0$04 (ol/L ;)L and allo4ed to react for 1$5 to 2$0 hours to con1ert it to e0ual parts of oth iso(ers$ c$ Alternati1el'+ nitrofurantoin is dissol1ed in di(eth'l sulfo2ide$ d$ ,hese sol1ents and diluents are for (a/in! stoc/ solutions of anti(icro ial a!ents that re0uire sol1ents other than 4ater$ ,he' should e diluted further+ as necessar'+ in 4ater or roth$ ,he products /no4n to e suita le for 4ater sol1ents and diluents are a(i/acin+ a@locillin+ car encillin+ cefaclor+ cefe(andole+ cefonicid+ cefota2i(e+ cefopera@one+ cefo2itin+ cefti@o2i(e+ ceftria2@one+ ciproflo2acin+ clinda('cin+ !atiflo2acin (4ith stirrin!"+ !e(iflo2acin+ !enta(icin+ /ana('cin+ line@olid+ (ecillina(+ (eropene(+ (ethicillin+ (etronida@ole+ (e@locillin+ (inoc'clin+ (o2iflo2acin+ nafcillin+ netil('cin+ o2acillin+ penicillin+ piperacillin+ 0uinupristin3dalfopristin+ sparflo2acin+ sul acta(+ ta@o acta(+ teicoplanin+ tetrac'clines+ to ra('cin+ tri(ethopri( (if lactate"+ trospecto('cin and 1anco('cin$ e$ Anh'drous sodiu( car onate is used at a 4ei!ht of e2actl' 10J of the cefta@id(e to e used$ ,he sodiu( car onate is dissol1ed in solution in (ost of the re0uired 4ater$ ,he anti iotic is dissol1ed in this sodiu( car onate solution and 4ater is added to desired 1olu(e$ ,he solution is to e used as soon as possi le+ ut can e stored up to si2 hours at not (ore than 25)$

46

f$ ,hese co(pounds are potentiall' to2ic$ )onsult the (aterial safet' datasheets (M:#:" a1aila le fro( the product (anufacturer (aterials$ !$ Use ] 1olu(e of 4ater+ then add !lacial acetic acid drop4ise until dissol1ed not to e2ceed 2$5l/(l$ efore usin! an' of these

Anne1ure

III< -eproduced 5it& permissionC $rom NCCAS publication

M1++8S1! Per$ormance Standards $or Antimicrobial Testing< T5el$t& In$ormational Supplement 9IS,N 18%'! .8 "%" 8':. Copies o$ t&e current edition may be obtained $rom NCCASC 0"+ Dest 4alley -oadC Suite 1"++C DayneC Pennsyl2ania 10+.*81.0.C =SA.

47