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

Manual on Antimicrobial Susceptibility Testing

(Under the auspices of Indian Association of Medical Microiolo!ists"
#r$ M$%$ Lalitha
&rofessor of Microiolo!'
#epart(ent of Microiolo!'
)hristian Medical )olle!e
*ellore+ ,a(il -adu
CONTENTS PAGE No.
1. Introduction
!. Principle "
. #actors In$luencing Antimicrobial Susceptibility Testing %
". Met&ods o$ Antimicrobial Susceptibility Testing '
".1 (is) (i$$usion *
".! (ilution 1"
". (ilution And (i$$usion. !+
%. Susceptibility Testing O$ #astidious ,acteria !1
'. Errors in Interpretation and reporting -esults !.
*. /uality Control in Antimicrobial Susceptibility Testing !0
.. Standard Met&ods $or t&e (etection o$ Antimicrobial -esistance. +
0. Application o$ Computers in Antimicrobial Susceptibility Testing 0
1+. Selected ,ibliograp&y "1
Anne1ure "
I. Guide lines $or Antimicrobial Susceptibility Testing
ii. Suggested (ilution
-anges $or MIC
Testing
III. Sol2ents and (iluents $or Antibiotics
2
1. Introduction
.esistance to anti(icroial a!ents (AM." has resulted in (oridit' and (ortalit' fro(
treat(ent failures and increased health care costs$ Althou!h definin! the precise pulic
health ris/ and esti(atin! the increase in costs is not a si(ple underta/in!+ there is little
dout that e(er!ent antiiotic resistance is a serious !loal prole($
Appropriate anti(icroial dru! use has un0uestionale enefit+ ut ph'sicians and the
pulic fre0uentl' use these a!ents inappropriatel'$ Inappropriate use results fro( ph'sicians
pro1idin! anti(icroial dru!s to treat 1iral infections+ usin! inade0uate criteria for dia!nosis
of infections that potentiall' ha1e a acterial aetiolo!'+ unnecessaril' prescriin! e2pensi1e+
road3spectru( a!ents+ and not follo4in! estalished reco((endations for usin! che(o
proph'la2is$ ,he a1ailailit' of antiiotics o1er the counter+ despite re!ulations to the
contrar'+ also fuel inappropriate usa!e of anti(icroial dru!s in India$ ,he eas' a1ailailit'
of anti(icroial dru!s leads to their incorporation into heral or 5fol/5 re(edies+ 4hich also
increases inappropriate use of these a!ents$
6idespread antiiotic usa!e e2erts a selecti1e pressure that acts as a dri1in! force in the
de1elop(ent of antiiotic resistance$ ,he association et4een increased rates of
anti(icroial 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
antiiotics+ therap' 4ith ne4+ roader spectru(+ (ore e2pensi1e antiiotics increases+ ut is
follo4ed ' de1elop(ent of resistance to the ne4 class of dru!s$
.esistance factors+ particularl' those carried on (oile ele(ents+ can spread rapidl' 4ithin
hu(an and ani(al populations$ Multidru!3resistant patho!ens tra1el not onl' locall' ut
also !loall'+ 4ith ne4l' introduced patho!ens spreadin! rapidl' in susceptile hosts$
Antiiotic 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 pulic health and as an indicator of
appropriate or inappropriate antiiotic usa!e ' ph'sicians in that area$
3
,he results of in31itro antiiotic susceptiilit' 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 antiiotic panel for susceptiilit' testin! is ased on the
co((onl' oser1ed susceptiilit' 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
antiiotics (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 $,he 82ford !roup used these (ethods initiall' to assa' the
antiiotic contained in lood ' allo4in! the antiiotics to diffuse out of reser1oirs in the
(ediu( in containers placed on the surface$
6ith the introduction of a 1ariet' of anti(icroials it eca(e necessar' to perfor( the
anti(icroial susceptiilit' test as a routine$ 7or this+ the anti(icroial 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(icroial containin! reser1oirs are
used ut the anti(icroial i(pre!nated asorent 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 laorator'$ Auto(ation (a' force the (ethod out of
the dia!nostic laorator' ut in this countr' as 4ell as in the s(aller laoratories of e1en
ad1anced countries+ it 4ill certainl' e the (ost co((onl' carried out (icroiolo!ical
test for (an' 'ears to co(e$ It is+ therefore+ i(perati1e that (icroiolo!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(icroial a!ent in a!ar or dilution of
antiiotic in a!ar or roth$
91en auto(ated techni0ues are 1ariations of the ao1e (ethods$
4
.
#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 a1ailale in
the laorator'$ ,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' calirated surface electrode$
Moisture
If+ >ust efore use+ e2cess surface (oisture is present+ the plates should e placed in an
incuator (?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$$ects o$ T&ymidine or T&ymine
Media containin! e2cessi1e a(ounts of th'(idine or th'(ine can re1erse the inhiitor'
effect of sulfona(ides and tri(ethopri(+ thus 'ieldin! s(aller and less distinct @ones+ or
e1en no @one at all+ 4hich (a' result in false3resistance reports$ Meller3;inton a!ar that is
as lo4 in th'(idine content as possile should e used$ ,o e1aluate a ne4 lot of MAeller3
;inton a!ar, Enterococcus faecalis A,)) 29212+ or alternati1el'+ E. faecalis A,)) ??18B+
should e tested 4ith tri(ethopri(/sulfa(etho2a@ole dis/s$ :atisfactor' (edia 4ill pro1ide
essentiall' clear+ distinct @ones of inhiition 20 (( or !reater in dia(eter$ Unsatisfactor'
5
(edia 4ill produce no @one of inhiition+ !ro4th 4ithin the @one+ or a @one of less than 20
(($
E$$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 unacceptal' lar!e @ones of
inhiition$ 92cess @inc ions (a' reduce @one si@es of carapene(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' aeroic 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 descried in separate
sections$
". Met&ods o$ Antimicrobial Susceptibility Testing
Anti(icroial susceptiilit' testin! (ethods are di1ided into t'pes ased on the principle
applied in each s'ste($ ,he' include:
(i$$usion (ilution (i$$usion6(ilution
:to/es (ethod Mini(u( Inhiitor' )oncentration 93,est (ethod
%ir'3Dauer (ethod i" Droth dilution
ii"A!ar #ilution
6
".1 (is) (i$$usion
-eagents $or t&e (is) (i$$usion Test
1. M7eller83inton Agar Medium
8f the (an' (edia a1ailale+ MAeller3;inton a!ar is considered to e the est for routine
susceptiilit' testin! of nonfastidious acteria for the follo4in! reasons:
= It sho4s acceptale atch3to3atch reproduciilit' for susceptiilit' testin!$
= It is lo4 in sulphona(ide+ tri(ethopri(+ and tetrac'cline inhiitors$
= It !i1es satisfactor' !ro4th of (ost nonfastidious patho!ens$
= A lar!e od' of data and e2perience has een collected concernin! susceptiilit'
tests perfor(ed 4ith this (ediu($
Althou!h MAeller3;inton a!ar is reliale !enerall' for susceptiilit' testin!+ results otained
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 otained in a dis/ diffusion test 4ill
usuall' e lar!er than e2pected and (a' e2ceed the acceptale 0ualit' control li(its$ 8nl'
MAeller3;inton (ediu( for(ulations that ha1e een tested accordin! to+ and that (eet the
acceptance li(its descried 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$ MAeller3;inton a!ar should e prepared fro( a co((erciall' a1ailale deh'drated
ase accordin! to the (anufacturerEs instructions$
2$ 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+ flat3otto(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)"$
7
5$ &lates should 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
a!ar$
B$ A representati1e sa(ple of each atch of plates should e e2a(ined for sterilit' '
incuatin! at ?0 to ?5) for 24 hours or lon!er$
!. Preparation o$ antibiotic stoc) solutions
Antiitiotics (a' e recei1ed as po4ders or talets$ It is reco((ended to otain pure
antiiotics fro( co((ercial sources+ and not use in>ectale 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 antiiotic 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 anitiotic ase+ *H1olu(e in (l re0uired+ )Hfinal concentration of solution and
6H4ei!ht of the anti(icroial 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 antiiotics is (ade of 20 !au!e 4ire and has a dia(eter of
2 (($ ,his deli1ers 0$005 (l of antiiotics to each disc$
Storage o$ commercial antimicrobial discs
)artrid!es containin! co((erciall' prepared paper dis/s specificall' for susceptiilit'
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
8
(a' e refri!erated for at (ost one 4ee/$ :o(e laile a!ents (e$!$+ i(ipene(+
cefaclor+ and cla1ulanic acid co(inations" (a' retain !reater stailit' 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' e0uilirate 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 lael (a' e used$ #iscs should e discarded on the e2piration date$
Turbidity standard $or inoculum preparation
,o standardi@e the inoculu( densit' for a susceptiilit' test+ a Da:84 turidit' 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)l2 (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 turidit' standard should e 1erified ' usin! a
spectrophoto(eter 4ith a 13c( li!ht path and (atched cu1ette to deter(ine the
asorance$ ,he asorance 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 tues of the sa(e si@e as those used in !ro4in! or dilutin! the acterial
inoculu($
9
4$ ,hese tues should e ti!htl' sealed and stored in the dar/ at roo( te(perature$
5$ ,he ariu( sulfate turidit' standard should e 1i!orousl' a!itated on a (echanical
1orte2 (i2er efore each use and inspected for a unifor(l' turid 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(icroial susceptiilit'
testin!+ 4ith the %ir'3Dauer (ethod ein! reco((ended ' the -))L:$ ,he accurac'
and reproduciilit' of this test are dependent on (aintainin! a standard set of procedures as
descried 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(icroial susceptiilit' testin! (A:," and appropriate reportin!
' de1elopin! standard reference (ethods+ interpretati1e criteria for the results of standard
A:, (ethods+ estalishin! 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 collaorati1e
studies and 4ell correlated 4ith MI)Ks and the results ha1e corroorated 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 tue containin! 4 to 5 (l of a suitale roth
(ediu(+ such as tr'ptic so' roth$
2$ ,he roth culture is incuated at ?5) until it achie1es or e2ceeds the turidit' of
the 0$5 Mc7arland standard (usuall' 2 to B hours"
?$ ,he turidit' of the acti1el' !ro4in! roth culture is ad>usted 4ith sterile saline or
roth to otain a turidit' opticall' co(parale 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( tue 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 turidit' 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 turidit' 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 tue
ao1e 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
distriution of inoculu($ As a final step+ the ri( of the a!ar is s4aed$
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 asored 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(icroial 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 distriuted 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 incuator 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 incuated in an increased )82 at(osphere+
ecause the interpreti1e standards 4ere de1eloped ' usin! a(ient air incuation+
and )82 4ill si!nificantl' alter the si@e of the inhiitor' @ones of so(e a!ents$
-eading Plates and Interpreting -esults
1$ After 1B to 18 hours of incuation+ each plate is e2a(ined$ If the plate 4as
satisfactoril' strea/ed+ and the inoculu( 4as correct+ the resultin! @ones of
inhiition 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 inhiition (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 ao1e 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 incuation 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 inhiition$ An' discernale !ro4th 4ithin
@one of inhiition is indicati1e of (ethicillin or 1anco('cin resistance$
2$ ,he @one (ar!in should e ta/en as the area sho4in! no o1ious+ 1isile !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 inhiited !ro4th+
is i!nored$ ;o4e1er+ discrete colonies !ro4in! 4ithin a clear @one of inhiition
should e sucultured+ re3identified+ and retested$ :trains of Proteus spp$ (a'
s4ar( into areas of inhiited !ro4th around certain anti(icroial a!ents$ 6ith
Proteus spp$+ the thin 1eil of s4ar(in! !ro4th in an other4ise o1ious @one of
inhiition should e i!nored$ 6hen usin! lood3supple(ented (ediu( for testin!
streptococci+ the @one of !ro4th inhiition should e (easured+ not the @one of
inhiition 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 o1ious (ar!in to deter(ine the
@one dia(eter$
?$ ,he si@es of the @ones of inhiition are interpreted ' referrin! to ,ales 2A throu!h
2I (Lone #ia(eter Interpretati1e :tandards and e0ui1alent Mini(u( Inhiitor'
)oncentration Drea/points" of the -))L: M1003:12: &erfor(ance :tandards for
Anti(icroial :usceptiilit' ,estin!: ,4elfth Infor(ational :upple(ent+ and the
or!anis(s are reported as either susceptile+ inter(ediate+ or resistant to the a!ents
13
that ha1e een tested$ :o(e a!ents (a' onl' e reported as susceptile+ since onl'
susceptile rea/points are !i1en$
".! (ilution Met&ods
#ilution susceptiilit' testin! (ethods are used to deter(ine the (ini(al concentration
of anti(icroial to inhiit or /ill the (icroor!anis($ ,his can e achie1ed ' dilution of
anti(icroial in either a!ar or roth (edia$ Anti(icroials are tested in lo!2 serial
dilutions (t4o fold"$
Minimum In&ibitory Concentration 9MIC:
#iffusion tests 4idel' used to deter(ine the susceptiilit' of or!anis(s isolated fro(
clinical speci(ens ha1e their li(itationsM 4hen e0ui1ocal results are otained or in
prolon!ed serious infection e$!$ acterial endocarditis+ the 0uantitation of antiiotic
action 1is3a31is the patho!en needs to e (ore precise$ Also the ter(s N:usceptileK and
N.esistantK can ha1e a realistic interpretation$ ,hus 4hen in dout+ the 4a' to a precise
assess(ent is to deter(ine the MI) of the antiiotic 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 tues 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( tues /
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 antiiotic in po4der for( (either fro(
the (anufacturer or standard laorator' acco(panied ' a state(ent of its acti1it' in
14
(!/unit or per (l$ )linical preparations should not e used for reference techni0ue$"+
re0uired sol1ent for the antiiotic+ sterile #istilled 6ater 3 500(l and suitale 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 suitale rac/ to hold 22 tues in t4o ro4s i3e 11 tues 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(icroial 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(icroials re0uired is
6 H 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 suitale 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(icroials are sho4n in Anne2ure II$
Met&od
&repare stoc/ dilutions of the antiiotic 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 tues in the rac/$ In a sterile ?0(l (uni1ersal" scre4 capped ottle+ prepare 8(l of
roth containin! the concentration of antiiotic re0uired for the first tue 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 tue 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 tue 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 antiiotic free roth to the last tue 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
suitale roth and the second ro4 4ith the control or!anis( of /no4n sensiti1it'
si(ilarl' diluted$ ,he result of the test is si!nificantl' affected ' the si@e of the
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$ Incuate tues for 18 hours at
?<
o
)$ Inoculate a tue containin! 2(l roth 4ith the or!anis( and /eep at I4
o
) in a
refri!erator o1erni!ht to e used as standard for the deter(ination of co(plete inhiition$
Calculations $or t&e preparation o$ t&e original dilution.
,his often presents prole(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 antiiotic 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 ao1e+ the series has to e started a!ain (id
4a' at 2 (!/l 4hich 4ould e otained 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 inhiited !ro4th >ud!ed ' lac/ of
turidit' in the tue$
Decause 1er' faint turidit' (a' e !i1en ' the inoculu( itself+ the inoculated tue /ept
in the refri!erator o1erni!ht (a' e used as the standard for the deter(ination of
co(plete inhiition$
:tandard strain of /no4n MI) 1alue run 4ith the test is used as the control to chec/ the
rea!ents and conditions$
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 descried for the
deter(ination of MI)$ ,he control tue containin! no antiiotic is i((ediatel'
sucultured (Defore incuation" ' spreadin! a loopful e1enl' o1er a 0uarter of the plate
on a (ediu( suitale for the !ro4th of the test or!anis( and incuated at ?<
o
)
o1erni!ht$ ,he tues are also incuated o1erni!ht at ?<
o
)$ .ead the MI) of the control
or!anis( to chec/ that the dru! concentrations are correct$ -ote the lo4est concentration
inhiitin! !ro4th of the or!anis(s and record this as the MI)$ :uculture all tues not
sho4in! 1isile !ro4th in the sa(e (anner as the control tue descried ao1e and
incuate at ?<
o
) o1erni!ht$ )o(pare the a(ount of !ro4th fro( the control tue efore
incuation+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 tues are not
suculturedM 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 sucultures (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 inhiition is ta/en as MD)
Micro8brot& dilution test
,his test uses doule3stren!th MAeller3;inton roth+ 4G stren!th antiiotic 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 doule3stren!th M;D+ 50 l each of the antiiotic dilutions and
the or!anis( suspension are (i2ed and incuated at ?5) for 18324 hours$ ,he lo4est
concentration sho4in! inhiition of !ro4th 4ill e considered the MI) of the or!anis($
-eading o$ result
MI) is e2pressed as the hi!hest dilution 4hich inhiited !ro4th >ud!ed ' lac/ of
turidit' in the tue$ Decause 1er' faint turidit' (a' e !i1en ' the inoculu( itself+the
inoculated tue /ept in the refri!erator o1erni!ht (a' e used as the standard for the
deter(ination of co(plete inhiition$ :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
possile 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 B0
o
)$Dlood (a' e added and if Nchocolate a!arK is re0uired+the (ediu(
(ust e heated efore the antiiotic 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 H 20 (l
,op antiiotic concentrations H B4(!/l
,otal a(ount of dru! H 1280O! to e added to
1 (l of 4ater
2(l of 1280 O! /(l 4ill e re0uired to start the dilution H 25B0O! in 2 (l
H 1$28(l of 2000O! /(l
P 0$<2 (l of 4ater$
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<
Lael 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 a1ailale for the
s/illed (anipulation+ it is stron!l' reco((ended that the a!ar is first (easured into
stoppered tues 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 incuator$ ,he' are then
inoculated either 4ith a (ultiple inoculator as spots or 4ith a 4ire loop or a platinu(
loop calirated to deli1er 0$001(l spread o1er a s(all area$ In either case the culture
should e diluted to contain 10
5
to 10
B
or!anis(s per (l$ 6ith ordinar' fast !ro4in!
or!anis(s+ this can e otained appro2i(atel' ' addin! 5 Ol of an o1erni!ht roth
culture to 5(l roth or peptone 4ater$
It is possile 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 e
pre1ented ' the use of hi!her concentration of a!ar in the (ediu(+ this is not
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' antiiotics$
-eading o$ results
,he antiiotic concentration of the first plate sho4in! 99J inhiition 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(icroial susceptiilit' testin! applies oth the dilution
of antiiotic and diffusion of antiiotic into the (ediu($$ A predefined stale
anti(icroial !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! incuation + a s'((etrical inhiition ellipse is produced$ ,he intersection of
the inhiitor' @one ed!e and the calirated 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 anaeroes$ It can also e
used for -onfer(entin! Qra( -e!ati1e acilli (-7Q-D" for e!3Pseudomonas 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 reliale (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 susceptiilit' (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( ules$ ,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 turidit'$ If the turidit' is too hea1'+ the suspension should e diluted
4ith additional suspendin! (ediu($ If the turidit' 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 turidit'
ad>usted to 0$5 Mc7arland standard$ 6ithin 15 (inutes after ad>ustin! the turidit' of the
suspension the plate should e inoculated$
Inoculation o$ t&e susceptibility test media
After proper turidit' 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 tue$ ,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 incuated
at ?5
o
) for 1B to 18 h in an in1erted position in a 5J )82 incuator$ A candle e2tinction
>ar (a' e used if a )82 incuator is not a1ailale$
Estimating t&e susceptibility o$ t&e strains
After o1erni!ht incuation+ the dia(eter of each @one of inhiition is (easured 4ith a
ruler or calipers$ ,he @ones of inhiition 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 inhiition @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 inhiition are (easured fro( the ed!es of the last
1isile 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 susceptiilit' is otained ' 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 o1ious @one should
e i!nored in the (easure(ent$
Interpretation
9ach @one si@e is interpreted ' reference to the ,ale 2Q (Lone #ia(eter Interpretati1e
:tandards and e0ui1alent Mini(u( Inhiitor' )oncentration Drea/points for
S.pneumoniae" of the -))L: M1003:12: &erfor(ance :tandards for Anti(icroial
:usceptiilit' ,estin!: ,4elfth Infor(ational :upple(ent as susceptile+ 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 (preferal' 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
turidit' e0ui1alent to a 0$5 Mc7arland standard usin! a photo(etric de1ice$ ,his
suspension 4ill contain appro2i(atel' 1 to 4 2 10
8
)7U/(l$ )are (ust e e2ercised
in preparin! this suspension+ ecause hi!her inoculu( concentrations (a' lead to
false3resistant results 4ith so(e C3lacta( antiiotics+ particularl' 4hen C3lacta(ase
23
producin! strains of H. influenzae are tested$ 6ithin 15 (inutes after ad>ustin! the
turidit' of the inoculu( suspension+ it should e used for plate inoculation$
2$ ,he procedure for the disc test should e follo4ed as descried 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 incuated at ?5) in an at(osphere of 5J )82 for 1B to 18 hours efore
(easurin! the @ones of inhiition$
4$ ,he @one (ar!in should e considered as the area sho4in! no o1ious !ro4th
1isile 4ith the unaided e'e$ 7aint !ro4th of tin' colonies that (a' appear to fade
fro( the (ore o1ious @one should e i!nored in the (easure(ent$
?one (iameter Interpreti2e Criteria
,he anti(icroial a!ents su!!ested for routine testin! of Haemophilus sp$ are indicated in
Anne2ure I$ 9ach @one si@e is interpreted ' reference to the ,ale 29 (Lone #ia(eter
Interpretati1e :tandards and e0ui1alent Mini(u( Inhiitor' )oncentration Drea/points for
Haemophilus sp$" of the -))L: M1003:12: &erfor(ance :tandards for Anti(icroial
:usceptiilit' ,estin!: ,4elfth Infor(ational :upple(ent as susceptile+ inter(ediate and
resistant$ #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 susceptiilit'
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 turidit' of the inoculu( suspension+ it should e used for plate
inoculation$
2$ ,he disc diffusion test procedure steps+ as descried for nonfastidious acteria+
should e follo4ed$ -o (ore than 9 anti(icroial 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 incuated at ?5) in an at(osphere of 5J )82 for 20 to 24 hours
efore (easurin! the @ones of inhiition$
?one (iameter Interpreti2e Criteria
,he anti(icroial a!ents su!!ested for routine testin! of N. gonorrhoeae are indicated in
Anne2ure I$ 9ach @one si@e is interpreted ' reference to the ,ale 27 (Lone #ia(eter
Interpretati1e :tandards and e0ui1alent Mini(u( Inhiitor' )oncentration Drea/points
for N. gonorrhoeae" of the -))L: M1003:12: &erfor(ance :tandards for Anti(icroial
:usceptiilit' ,estin!: ,4elfth Infor(ational :upple(ent as susceptile+ 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
reco!nition of this plas(id3(ediated resistance$ 8r!anis(s 4ith plas(id3(ediated
resistance to tetrac'cline also ha1e @ones of inhiition (?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 ,ale 27 (Lone #ia(eter Interpretati1e :tandards and e0ui1alent Mini(u( Inhiitor'
)oncentration Drea/points for N. gonorrhoeae" of the -))L: M1003:12: &erfor(ance
:tandards for Anti(icroial :usceptiilit' ,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 10
5
(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 calirated to deli1er 0$001(l or (ultipoint inoculator$
7or N.gonorrhoeae and H. influenzae3 :i(ilar to the procedure descried ao1e for
S.pneumoniae
Inoculation o$ test plate
In !eneral the inoculu( should e applied as a spot that co1ers a circle aout 538(( in
dia(eter $A platinu( loop calirated 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 undistured until the spots of inoculu(
ha1e dried$
Incubation
After the spots of inoculu( ha1e dried+ the plates are incuated at ?5
o
) for 1B to 18 h in
an in1erted position in a 5J )82 incuator$ A candle e2tinction >ar (a' e used if a )82
incuator is not a1ailale$
-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 antiiotic that co(pletel' inhiits !ro4th$ A arel' 1isile
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(icroial :usceptiilit'
,estin!: ,4elfth Infor(ational :upple(ent (MI) Interpretati1e :tandards" as susceptile+
inter(ediate and resistant$
27
'.Errors in Interpretation and reporting results
In the interpretation of test results there are possiilities 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 Microiolo!'+ <
th
edition"
-esistant Minor 9rror *er' Ma>or 9rror

Minor 9rror Intermediate Minor 9rror
Ma>or 9rror Minor 9rror Susceptible
(is) (i$$usion (iameter 9mm:
28
MIC
9g@ml:
*. /uality Control in Antibiotic Susceptibility Testing
U) is perfor(ed to chec/ the 0ualit' of (ediu(+ the potenc' of the antiiotic+ 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 possile 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 susceptile"
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 sucultured 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
carapene(s$ M.:A isolates often are (ultipl' resistant to co((onl' used anti(icroial
a!ents+ includin! er'thro('cin+ clinda('cin+ and tetrac'cline$ :ince 199B+ reports of
M.:A strains 4ith decreased susceptiilit' to 1anco('cin ((ini(u( inhiitor'
concentration VMI)W+ R8 !/(l" ha1e een pulished$
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 susceptile 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 Laorator' :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 tue$ ,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
10
4
)7U$
2$ #ip a s4a in the suspension and e2press e2cess fluid ' pressin! s4a a!ainst the 4all
of the tue$ :trea/ s4a o1er a 131$5 inch area$
30
In oth (ethods+ an' !ro4th after 24 hours incuation at ?5) denotes o2acillin resistance+
if controls are satisfactor'$
Accurate detection of o2acillin/(ethicillin resistance can e difficult due to the presence of
t4o supopulations (one susceptile 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 penicillinase3stale penicillins+ such
as o2acillin$
;eteroresistance is a prole( for clinical laorator' personnel ecause cells e2pressin!
resistance (a' !ro4 (ore slo4l' than the susceptile population$ ,his is 4h' isolates ein!
tested a!ainst o2acillin+ (ethicillin+ or nafcillin should e incuated 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 O1acillin Susceptible O1acillin Intermediate O1acillin -esistant
S. aureus T2 !/(l no inter(ediate MI) MI)R4 ! /(l
)o-: T0$25! /(l no inter(ediate MI) MI)R0$5 ! /(l
?one si>es O1acillin Susceptible O1acillin Intermediate O1acillin -esistant
S. aureus R1? (( 11312 (( T10 ((
)o-: R18 (( no inter(ediate @one T1< ((
31
6hen used correctl'+ roth3ased and a!ar3ased tests usuall' can detect M.:A$ 82acillin
screen plates can e used in addition to routine susceptiilit' 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-negatie Staphylococcus sp!
Althou!h there are aout 20 )o-: species+ the' often are considered to e a sin!le !roup$
:o(e species are (ore resistant to co((onl' used anti(icroial a!ents than others$
Identification to species le1el can aid in the reco!nition of outrea/s and in trac/in!
resistance trends$ S. epidermidis is the (ost co((on )o-: isolated in clinical laoratories$
Usuall', S. epidermidis, S. haemolyticus and S. hominis are (ore li/el' to e (ultipl'
resistant to anti(icroial 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 carapene(s$ In addition+ o2acillin3resistant )o-: isolates
are often resistant to other co((onl' used anti(icroial 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 supopulations (one susceptile 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 (onoacta(s (e$!$+
a@treona(" ut do not affect cepha('cins (e$!$+ cefo2itin and cefotetan" or carapene(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(icroial 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( inhiitor' 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 susceptiilit'$
,here are standard roth (icrodilution and disc diffusion screenin! tests usin! selected
anti(icroial 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 MICs
cefpodo2i(e T 22 (( cefpodo2i(e R 2 ! /(l
cefta@idi(e T 22 (( cefta@idi(e R 2 ! /(l
a@treona( T 2< (( a@treona( R 2 ! /(l
cefota2i(e T 2< (( cefota2i(e R 2 ! /(l
ceftria2one T 25 (( ceftria2one R 2 ! /(l
,he sensiti1it' of screenin! for 9:DLs in enteric or!anis(s can 1ar' dependin! on 4hich
anti(icroial a!ents are tested$ ,he use of (ore than one of the fi1e anti(icroial 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 ? doulin! 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(icroial 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(icroial discs (a' produce discs containin! cefota2i(e and cefta@idi(e 4ith
cla1ulanic acid$ Until co((ercial discs are a1ailale+ :(ith%line Deecha( can pro1ide
clinical laoratories 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 inhiitor3resistant ,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' a1ailale in clinical
laoratories$
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 e
reported as resistant$ ,his list does not include the cepha('cins (cefotetan and cefo2itin"+
4hich should e reported accordin! to their routine test results$ 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 9nteroacteriaceae+ 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 een
deter(ined$
(etection o$ Imipenem or Meropenem -esistance in Gram8negati2e Organisms
6ithin a health care settin!+ increases in species3specific carapene( resistance should e
(onitored and sudden increases in1esti!ated to rule out an outrea/ of resistant or!anis(s
or spurious test results$
&ulished 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 carapene(s ((ini(u( inhiitor' concentration
VMI)W R1B !/(l"$ 8r!anis(s 4ith decreased susceptiilit' 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
carapene( 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 carapene( inter(ediate rea/point of 8 (!/(l$ Most other species of
9nteroacteriaceae are 1er' susceptile (T0$5 !/(l"$
Droth (icrodilution (ethods usuall' detect carapene( resistance 4hen the tests are
perfor(ed properl'$ ;o4e1er+ studies ha1e sho4n false resistance to i(ipene( in
35
co((erciall' prepared test panels due to de!radation of the dru! or to a (anufacturin!
prole( 4here concentrations of i(ipene( 4ere too lo4$ 6hen perfor(ed properl'+ disc
diffusion and a!ar !radient diffusion also are acceptale (ethods for carapene( testin!$
I(ipene( de!rades easil'$ :tudies su!!est that (eropene( (a' e (ore stale than
i(ipene($ ;o4e1er+ for either anti(icroial a!ent+ stora!e conditions of susceptiilit'
panels+ cards+ and discs (ust e (onitored carefull' and 0ualit' control results chec/ed
fre0uentl'$ If possile+ store supplies containin! carapene(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 susceptiilit' produced
onl' ' porin chan!es usuall' ha1e lo4er (ini(u( inhiitor' concentrations (MI)s"$
,he fluoro0uinolone susceptiilit' 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 susceptile 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'+ penicillin3indin! 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 relial' detect C3lacta(ase producin! strains$ ,he rare C3lacta(ase3 producin!
strains are detected est ' usin! a direct+ nitrocefin3ased+ 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 laorator' 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' susceptile to s'ner!'
4ith an a(ino!l'coside (in the asence 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 incuated 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 inhiition 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 9nteroacteriaceae$
9nteroacteriaceae resistant to !enta(icin and tora('cin can e susceptile 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 tora('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 susceptile+ a(ino!l'cosides should not e tested or
reported$
0. Application o$ Computers in Antibacterial Susceptibility Testing
Anti(icroial resistance is a !loal prole($
9(er!ence of (ultidru! resistance has li(ited the therapeutic options+
hence (onitorin! resistance is of para(ount i(portance$
Anti(icroial resistance (onitorin! 4ill help to re1ie4 the current status of
anti(icroial resistance locall'+ nationall' and !loall' 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 laoratories are distriuted 4orld34ide and need to e lin/ed to inte!rate
data on (ost clinicall' rele1ant or!anis( on a dail' asis to otain an accurate picture of
Yreal resistanceX$ 7or this purpose a soft4are 4as de1eloped for the (ana!e(ent of
routine laorator' 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$1 is a data ase soft4are co(patile 4ith+ 6indo4s 95+
6indo4s 98+
6indo4s -, and later 1ersions of 6indo4s$ Installation capacit' of the soft4are is
9$?4 (+ this soft4are is a1ailale in 4esite
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 laoratories+
in identif'in! the clusters of resistant isolates and e(er!in! outrea/s$ Use of 6;8-9,
soft4are (a' aid the local laorator' in net4or/in! efficientl' 4ith the national
reference laorator'+ in de1elopin! !uidelines for use of anti(icroial a!ents and re1ie4
the sa(e periodicall'+ in stud'in! the effect of anti(icroial 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$*$ :usceptiilit' tests of fastidious acteria$ Manual of )linical
Microiolo!'+ B
th
edition+ Murra' &$.+ Daron 9$Z+ &faller M$A+ ,eno1er 7$)+
[ol/en .+ A(erican :ociet' for Microiolo!'+ 6ashin!ton #)+ 1995+ &$ 1?423
1?49$
Ira .$ Dacteriolo!'+ :tandard 8perati1e procedure (anual for (icroiolo!'
laoratories+ -ational Institute of Diolo!icals$ 1995+ &<?39<
Zohn #$, and Za(es ;$Z Anti(icroial :usceptiilit' testin!: Qeneral
)onsiderations$ Manual of )linical Microiolo!' <
th
edition+ Murra' &$.+ Daron
9$Z+ &faller M$A+ ,eno1er 7$)+ [ol/en .+ A(erican :ociet' for Microiolo!'+
6ashin!ton #)+ 1999+ &$ 14B9314<?$
-ational )o((ittee for )linical Laorator' :tandards$ Performance Standards
for antimicrobial susceptibility testing$ 8
th
Infor(ational :upple(ent$ M100 :12$
-ational )o((ittee for )linical Laorator' :tandards+ 2002$ *illano1a+ &a$
,hrupp L$#$ :usceptiilit' ,estin! of Antiiotic in Li0uid Media$ Antiiotics In
Laorator' Medcine 2
nd
9dition+ *ictor Lorian+ 6illia(s and 6il/ins+ Daliti(ore$
198BM &$ 9?3150$
6ater 4orth &$M$ Uuantitati1e (ethods for acterial sensiti1it' testin!$
Laorator' (ethods in anti(icroial che(otherap'$ .ee1es #$:+ &hilips I+
6illia(s Z$#+ 6ise .$ )hurchill Li1in!stone+ Dalti(ore$ 19<8M &$ ?1341$
%ra(er Z$ and %irshau( A$ 9ffect of paper on the perfor(ance assa' in the
control of antiiotic sensiti1it' discs$ ppl Microbiol 19B1: 9M &$ ??43??B$
.ippere .$A$ 9ffects of paper on the perfor(ance of antiiotic3i(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 antiiotic sensiti1it'
testin! of the Dritish :ociet' for Anti(icroial )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.
Gram negati2e
bacilli
Streptococcus
"Enterococcus#
Pneumococcus$
Haemophilus sp N! gonorrhoeae
&enicillin A(picillin &enicillin A(picillin &enicillin
82acillin &iperacillin 82acillin A(o2'cillin/
)la1ulanic acid
)efa@olin
)ephalothin )ephalothin A(picillin )efuro2i(e )eftria2one
Qenta(icin )efota2i(e )efota2i(e )efota2i(e )hlora(phenicol
-etil(icin )efta@idi(e 9r'thro('cin ,etrac'cline )iproflo2acin
A(i/acin Qenta(icin )hlora(phenicol 9r'thro('cin
)hlora(phenicol -etil(icin ,etrac'cline )hlora(phenicol
,etrac'cline A(i/acin *anco('cin
9r'thro('cin )hlora(phenicol
)o3tri(o2a@ole ,etrac'cline
)linda('cin )o3tri(o2a@ole
8flo2acin -alidi2ic Acid
.ifa(picin )iproflo2acin
*anco('cin 8flo2acin
,eicoplanin -itrofurantoin
I(ipene(
Meropene(
-ote:
,he choice of antiiotic depends on the pattern e2hiited locall'$
,he selection of antiiotics 1aries ased on speci(en and the isolates under
consideration$
43
Anne1ure II.
Suggested Antibiotic (ilution -anges #or MIC Testing
ANTIMIC-O,IAA AGENT CONCENT-ATION -ANGE9Bg@ml:
&enicillin Q 0$0153?2
Methicillin 0$00?3B4
-aficillin/82acillin 0$00?3B4
A(picillin(Qra( I1e" 0$153?2
A(picillin(Qra(31e" 0$1?325B
)ephalosporin(Qra( I1e" 0$0153?2
)ephalosporin I !eneration (Qra( 31e" 0$1?325B
)ephalosporin II\III !eneration (Qra(31e" 0$0?3B4
)ephalosporin III !eneration
(Pseudomonas sp$"
0$1?325B
*anco('cin 0$01531B
A(i/acin 0$0B3128
Qenta(icin/,ora('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$53B4
:ource3A !uide to sensti1it' testin!: .eport of 4or/in! part' on antiiotic sensiti1it'
testin! of the Dritish societ' for Anti(icroial )he(otherap'$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 ao1e or in footnote d are soluli@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' stale+ 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(icroial a!ents
that re0uire sol1ents other than 4ater$ ,he' should e diluted further+ as
necessar'+ in 4ater or roth$ ,he products /no4n to e suitale for 4ater sol1ents
and diluents are a(i/acin+ a@locillin+ carencillin+ 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+ sulacta(+ ta@oacta(+
teicoplanin+ tetrac'clines+ tora('cin+ tri(ethopri( (if lactate"+ trospecto('cin
and 1anco('cin$
e$ Anh'drous sodiu( caronate is used at a 4ei!ht of e2actl' 10J of the cefta@id(e
to e used$ ,he sodiu( caronate is dissol1ed in solution in (ost of the re0uired
4ater$ ,he antiiotic is dissol1ed in this sodiu( caronate solution and 4ater is
added to desired 1olu(e$ ,he solution is to e used as soon as possile+ 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:#:" a1ailale fro( the product (anufacturer efore usin! an' of these
(aterials$
!$ Use ] 1olu(e of 4ater+ then add !lacial acetic acid drop4ise until dissol1ed not
to e2ceed 2$5l/(l$
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