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Manual on Antimicrobial Susceptibility Testing 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 "1 * 1" !+ !1 !. !0 + 0 ' " % PAGE No.

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 Resistance to antimicrobial agents (AMR) has resulted in morbidity and mortality from treatment failures and increased health care costs. Although defining the precise public health risk and estimating the increase in costs is not a simple undertaking, there is little doubt that emergent antibiotic resistance is a serious global problem. Appropriate antimicrobial drug use has unquestionable benefit, but physicians and the public frequently use these agents inappropriately. Inappropriate use results from physicians pro iding antimicrobial drugs to treat iral infections, using inadequate criteria for diagnosis of infections that potentially ha e a bacterial an etiology, unnecessarily prescribing e!pensi e, broad"spectrum agents, and not follo#ing established recommendations for using chemo prophyla!is. $he a ailability of antibiotics o er the counter, despite regulations to the contrary, also fuel inappropriate usage of antimicrobial drugs in India. $he easy a ailability of antimicrobial drugs leads to their incorporation into herbal or %folk% remedies, #hich also increases inappropriate use of these agents.

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&idespread antibiotic usage e!erts a selecti e pressure that acts as a dri ing force in the de elopment of antibiotic resistance. $he association bet#een increased rates of antimicrobial use and resistance has been documented for nosocomial infections as #ell as for resistant community acquired infections. As resistance de elops to %first"line% antibiotics, therapy #ith ne#, broader spectrum, more e!pensi e antibiotics increases, but is follo#ed by de elopment of resistance to the ne# class of drugs. Resistance factors, particularly those carried on mobile elements, can spread rapidly #ithin human and animal populations. Multidrug"resistant pathogens tra el not only locally but also globally, #ith ne#ly introduced pathogens spreading rapidly in susceptible hosts. Antibiotic resistance patterns may ary locally and regionally, so sur eillance data needs to be collected from selected sentinel sources. 'atterns can change rapidly and they need to be monitored closely because of their implications for public health and as an indicator of appropriate or inappropriate antibiotic usage by physicians in that area.

$he results of in" itro antibiotic susceptibility testing, guide clinicians in the appropriate selection of initial empiric regimens and, drugs used for indi idual patients in specific situations. $he selection of an antibiotic panel for susceptibility testing is based on the commonly obser ed susceptibility patterns, and is re ised periodically.

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&alker and others at the turn of the century. $he disc diffusion method of A. (or this. the antimicrobial contained in a reser oir #as allo#ed to diffuse out into the medium and interact in a plate freshly seeded #ith the test organisms. $he ditch plate method of agar diffusion used by Ale!ander (leming #as the forerunner of a ariety of agar diffusion methods de ised by #orkers in this field . All techniques in ol e either diffusion of antimicrobial agent in agar or dilution of antibiotic in agar or broth. It is.!.$he )!ford *roup used these methods initially to assay the antibiotic contained in blood by allo#ing the antibiotics to diffuse out of reser oirs in the medium in containers placed on the surface. &ith the introduction of a ariety of antimicrobials it became necessary to perform the antimicrobial susceptibility test as a routine. therefore. it #ill certainly be the most commonly carried out microbiological test for many years to come. Principle $he principles of determining the effecti ity of a no!ious agent to a bacterium #ere #ell enumerated by Rideal . the disco ery of antibiotics made these tests(or their modification)too cumbersome for the large numbers of tests necessary to be put up as a routine. Automation may force the method out of the diagnostic laboratory but in this country as #ell as in the smaller laboratories of e en ad anced countries. + en no# a ariety of antimicrobial containing reser oirs are used but the antimicrobial impregnated absorbent paper disc is by far the commonest type used. imperati e that microbiologists understand the principles of the test #ell and keep updating the information as and #hen necessary.$ is the most practical method and is still the method of choice for the a erage laboratory. 4 .

#actors In$luencing Antimicrobial Susceptibility Testing p3 $he p. certain drugs #ill appear to lose potency (e.is too high. $he e!act method used #ill depend largely on the type of equipment a ailable in the laboratory. e!cess surface moisture is present. Moisture If. $he surface should be moist. #hile other agents may appear to ha e e!cessi e acti ity (e. quinolones.g.. the opposite effects can be e!pected. Allo# a small amount of agar to solidify around the tip of a p.. $he p. If the p./ and .. If the p. but no droplets of moisture should be apparent on the surface of the medium or on the 'etri dish co ers #hen the plates are inoculated. 5 . and macrolides).electrode. the plates should be placed in an incubator (56°7) or a laminar flo# hood at room temperature #ith lids a4ar until e!cess surface moisture is lost by e aporation (usually 89 to 59 minutes).can be checked by one of the follo#ing means1 2 2 2 Macerate a sufficient amount of agar to submerge the tip of a p. 4ust before use.of each batch of Mueller"-inton agar should be checked #hen the medium is prepared. $he agar medium should ha e a p.0 at room temperature after gelling.. 3se a properly calibrated surface electrode.is too lo#. amino glycosides.+ en automated techniques are ariations of the abo e methods.electrode in a beaker or cup.bet#een . tetracycline). .g.

+!cess :inc ions may reduce :one si:es of carbapenems. +!cessi e cations content #ill reduce :one si:es. #ill affect results of amino glycoside and tetracycline tests #ith P. distinct :ones of inhibition /9 mm or greater in diameter.E$$ects o$ T&ymidine or T&ymine Media containing e!cessi e amounts of thymidine or thymine can re erse the inhibitory effect of sulfonamides and trimethoprim. E.atisfactory media #ill pro ide essentially clear. 'erformance tests #ith each lot of M ueller"-inton agar must conform to the control limits. thus yielding smaller and less distinct :ones. 6 ./8/. or a :one of less than /9 mm. faecalis A$77 558<=. 3nsatisfactory media #ill produce no :one of inhibition. aeruginosa strains. gro#th #ithin the :one. Enterococcus faecalis A$77 /. or alternati ely. principally magnesium and calcium. M ueller"-inton agar that is as lo# in thymidine content as possible should be used. $o e aluate a ne# lot of Mueller"-inton agar. should be tested #ith trimethoprim>sulfametho!a:ole disks. #hereas lo# cations content may result in unacceptably large :ones of inhibition. #hich may result in false"resistance reports. . or e en no :one at all. E$$ects o$ 4ariation in (i2alent Cations ?ariation in di alent cations.

Met&ods o$ Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing methods are di ided into types based on the principle applied in each system. 7ertain fastidious bacteria such as Haemophilus sp. and iridans and @"hemolytic streptococci do not gro# sufficiently on unsupplemented Mueller"-inton agar.tokes method Airby"Bauer method (ilution i) Broth dilution ii )Agar Cilution (i$$usion 6 (ilution +"$est method Minimum Inhibitory 7oncentration $hese organisms require supplements or different media to gro#. ". and they should be tested on the media described 7 . in separate sections.Testing strains t&at $ail to gro5 satis$actorily )nly aerobic or facultati e bacteria that gro# #ell on unsupplemented Mueller"-inton agar should be tested on that medium. gonorrhoeae. N. $hey include1 (i$$usion . S. pneumoniae.

Preparation o$ Mueller73inton Agar Mueller"-inton agar preparation includes the follo#ing steps.1 (is) (i$$usion -eagents $or t&e (is) (i$$usion Test 1. on occasion. Although Mueller"-inton agar is reliable generally for susceptibility testing. /. Mueller"-inton agar should be prepared from a commercially a ailable dehydrated base according to the manufacturerEs instructions. It gi es satisfactory gro#th of most nonfastidious pathogens. ary significantly. and that meet the acceptance limits described in. trimethoprim. Mueller"-inton agar is considered to be the best for routine susceptibility testing of non fastidious bacteria for the follo#ing reasons1 2 2 2 2 It sho#s acceptable batch"to"batch reproducibility for susceptibility testing. Immediately after autocla ing. 8. document M=/"A. It is lo# in sulphonamide. )nly Mueller"-inton medium formulations that ha e been tested according to. D77L. 8 . Mueller73inton Agar Medium )f the many media a ailable."." 'rotocols for + aluating Cehydrated Mueller"-inton Agar should be used. and tetracycline inhibitors. A large body of data and e!perience has been collected concerning susceptibility tests performed #ith this medium. :ones obtained in a disk diffusion test #ill usually be larger than e!pected and may e!ceed the acceptable quality control limits. results obtained #ith some batches may. If a batch of medium does not support adequate gro#th of a test organism. allo# it to cool in a 06 to 69°7 #ater bath.

6. A representati e sample of each batch of plates should be e!amined for sterility by incubating at 59 to 56°7 for /0 hours or longer. $his corresponds to =9 to . using sterile glass#are. !. unless the plate is used the same day. the stock can be aliquoted in 6 ml olumes and fro:en at "/9F7 or "=9F7. 'lates should be used #ithin se en days after preparation unless adequate precautions. Preparation o$ antibiotic stoc) solutions Antibitiotics may be recei ed as po#ders or tablets. 'our the freshly prepared and cooled medium into glass or plastic. 0. such as #rapping in plastic. It is recommended to obtain pure antibiotics from commercial sources. $he agar medium should be allo#ed to cool to room temperature and.5. flat"bottomed 'etri dishes on a le el. hori:ontal surface to gi e a uniform depth of appro!imately 0 mm.tandard strains of stock cultures should be used to e aluate the antibiotic stock solution. . If satisfactory. ha e been taken to minimi:e drying of the agar.9 ml of medium for plates #ith diameters of 869 mm and /6 to 59 ml for plates #ith a diameter of 899 mm. stored in a refrigerator (/ to <°7). =. 9 . 'o#ders must be accurately #eighed and dissol ed in the appropriate diluents (Anne!ure III) to yield the required concentration. and not use in4ectable solutions.

#here 'Ipotency of the anitbiotic base. $he loop used for deli ering the antibiotics is made of /9 gauge #ire and has a diameter of / mm. or free:e at "80 °7 or belo#. #hich are placed in a 'etri dish and sterili:ed in a hot air o en.ealed packages of disks that contain drugs from the @"lactam class should be stored fro:en. ?H olume in ml required. . 2 $he unopened disc containers should be remo ed from the refrigerator or free:er one to t#o hours before use. and cla ulanic acid combinations) may retain greater stability if stored fro:en until the day of use. imipenem. Ciscs should be stored as follo#s1 2 Refrigerate the containers at <°7 or belo#. #hich may be refrigerated for at most one #eek.ome labile agents (e. cefaclor. so they may equilibrate to room temperature before 10 . . 7Hfinal concentration of solution and &H#eight of the antimicrobial to be dissol ed in ?. $his deli ers 9...g. Preparation o$ dried $ilter paper discs &hatman filter paper no. e!cept for a small #orking supply.tock solutions are prepared using the formula (8999>') G ? G 7H&.996 ml of antibiotics to each disc. 8 is used to prepare discs appro!imately = mm in diameter. in a nonfrost"free free:er until needed. Storage o$ commercial antimicrobial discs 7artridges containing commercially prepared paper disks specifically for susceptibility testing are generally packaged to ensure appropriate anhydrous conditions.

g.6 Mc(arland standard or its optical equi alent (e. $his procedure minimi:es the amount of condensation that occurs #hen #arm air contacts cold disks.6 ml of 9. )nly those discs that ha e not reached the manufacturerEs e!piration date stated on the label may be used. equi alent to a 9. 2 2 &hen not in use.) 0 turbidity standard. it should be fitted #ith a tight co er and supplied #ith an adequate desiccant.. +!cessi e moisture should be a oided by replacing the desiccant #hen the indicator changes color.6"ml aliquot of 9. A Ba.opening. a Ba.)0 9.8. it should be placed in a tightly sealed.8< mol>L -/. /..)0 (8J > ) #ith constant stirring to maintain a suspension. Turbidity standard $or inoculum preparation $o standardi:e the inoculum density for a susceptibility test. desiccated container. Ciscs should be discarded on the e!piration date. 2 )nce a cartridge of discs has been remo ed from its sealed package.6J #> Ba7l/ . $he dispenser should be allo#ed to #arm to room temperature before opening. /-/)) is added to . late! particle suspension).6 Mc(arland standard may be prepared as follo#s1 8. &hen using a disc"dispensing apparatus. A 9.90< mol>L Ba7l/ (8. should be used. the dispensing apparatus containing the discs should al#ays be refrigerated. $he correct density of the turbidity standard should be erified by using a spectrophotometer #ith a 8"cm light path and matched cu ette to determine the 11 ..

the standard should be replaced. interpretati e criteria for the results of standard A. Late! particle suspensions should be mi!ed by in erting gently. non"go ernmental organi:ation composed of medical professionals. If large particles appear. $he Barium .ulfate suspension should be transferred in 0 to = ml aliquots into scre#"cap tubes of the same si:e as those used in gro#ing or diluting the bacterial inoculum. $he barium sulfate standards should be replaced or their densities erified monthly. educators etc. is an international. 0. $hese tubes should be tightly sealed and stored in the dark at room temperature. D77L. 5.tokesE methods are usually used for antimicrobial susceptibility testing. $he absorbance at =/6 nm should be 9.$ methods. 6.89 for the 9. industry. $he barium sulfate turbidity standard should be igorously agitated on a mechanical orte! mi!er before each use and inspected for a uniformly turbid appearance. #ith the Airby"Bauer method being recommended by the D77L. not on a orte! mi!er =. go ernment. $he accuracy and reproducibility of this test are dependent on maintaining a standard set of procedures as described here. interdisciplinary. It promotes accurate antimicrobial susceptibility testing (A.absorbance.$) and appropriate reporting by de eloping standard reference methods.99< to 9. (isc di$$usion met&ods $he Airby"Bauer and ..6 Mc(arland standard. healthcare pro iders. non"profit. establishing quality control parameters for standard test 12 .

//. At least three to fi e #ell"isolated colonies of the same morphological type are selected from an agar plate culture. either a 7(3>ml for E. /. pro ides testing and reporting strategies that are clinically rele ant and cost" effecti e Interpretati e criteria of D77L. $he top of each colony is touched #ith a loop.6 Mc(arland standard (usually / to = hours) $he turbidity of the acti ely gro#ing broth culture is ad4usted #ith sterile saline or broth to obtain a turbidity optically comparable to that of the 9. $his results in a suspension containing appro!imately 8 to / ! 89 < $o perform this step properly. is appro ed by (CA"3.6 Mc(arland standard. 5. Based on study results D77L. interpretati e criteria are re ised frequently.coli A$77 /6. and the gro#th is transferred into a tube containing 0 to 6 ml of a suitable broth medium. adequate light is needed to 13 . such as tryptic soy broth. Procedure $or Per$orming t&e (isc (i$$usion Test Inoculum Preparation Gro5t& Met&od $he gro#th method is performed as follo#s 8. photometric de ice can be used or. if done isually. $he broth culture is incubated at 56°7 until it achie es or e!ceeds the turbidity of the 9. D77L.A and recommended by &-). are de eloped based on international collaborati e studies and #ell correlated #ith MI7Ks and the results ha e corroborated #ith clinical data.methods.

)ptimally. and streptococci.. $his procedure is repeated by streaking t#o more times. rotating the plate appro!imately =9 ° each time to ensure an e en distribution of inoculum. $he s#ab should be rotated se eral times and pressed firmly on the inside #all of the tube abo e the fluid le el. and for testing staphylococci for potential methicillin or o!acillin resistance. $his #ill remo e e!cess inoculum from the s#ab. #ithin 86 minutes after ad4usting the turbidity of the inoculum suspension. N. (irect Colony Suspension Met&od 8.6 Mc(arland turbidity standard. 14 . using saline and a orte! mi!er. such as blood agar. should be used). a sterile cotton s#ab is dipped into the ad4usted suspension.isually compare the inoculum tube and the 9. /. the inoculum can be prepared by making a direct broth or saline suspension of isolated colonies selected from a 8<" to /0"hour agar plate (a nonselecti e medium. $he dried surface of a MLeller"-inton agar plate is inoculated by streaking the s#ab o er the entire sterile agar surface. the rim of the agar is s#abbed. Haemophilus spp.6 Mc(arland standard against a card #ith a #hite background and contrasting black lines. $he suspension is ad4usted to match the 9. gonorrhoeae. As a con enient alternati e to the gro#th method. $his approach is the recommended method for testing the fastidious organisms. Inoculation o$ Test Plates 8. /. As a final step.

but no more than 86 minutes. &ith the e!ception of Haemophilus spp.. Instead. streptococci and N. +ach disc must be pressed do#n to ensure complete contact #ith the agar surface. $he plates are in erted and placed in an incubator set to 56 °7 #ithin 86 minutes after the discs are applied. $he lid may be left a4ar for 5 to 6 minutes. and 7)/ #ill significantly alter the si:e of the inhibitory :ones of some agents. gonorrhoeae . D)$+1 +!tremes in inoculum density must be a oided. a disc should not be relocated once it has come into contact #ith the agar surface. /. $he predetermined battery of antimicrobial discs is dispensed onto the surface of the inoculated agar plate. the plates should not be incubated in an increased 7) / atmosphere. no more than 8/ discs should be placed on one 869 mm plate or more than 6 discs on a 899 mm plate. )rdinarily. De er use undiluted o ernight broth cultures or other unstandardi:ed inocula for streaking plates. Application o$ (iscs to Inoculated Agar Plates 8.5. because the interpreti e standards #ere de eloped by using ambient air incubation. they must be distributed e enly so that they are no closer than /0 mm from center to center. 15 . place a ne# disc in another location on the agar. to allo# for any e!cess surface moisture to be absorbed before applying the drug impregnated disks. &hether the discs are placed indi idually or #ith a dispensing apparatus. Because some of the drug diffuses almost instantaneously.

$he diameters of the :ones of complete inhibition (as 4udged by the unaided eye) are measured. Any discernable gro#th #ithin :one of inhibition is indicati e of methicillin or ancomycin resistance. After 8= to 8< hours of incubation. If indi idual colonies are apparent. the inoculum #as too light and the test must be repeated. If blood #as added to the agar base (as #ith streptococci). including the diameter of the disc. $he :one margin should be taken as the area sho#ing no ob ious. nonreflecting background and illuminated #ith reflected light. respecti ely. $he petri plate is held a fe# inches abo e a black. $ransmitted light (plate held up to light) is used to e!amine the o!acillin and ancomycin :ones for light gro#th of methicillin" or ancomycin" resistant colonies. the :ones are measured from the upper surface of the agar illuminated #ith reflected light.-eading Plates and Interpreting -esults 8. If the plate #as satisfactorily streaked. the resulting :ones of inhibition #ill be uniformly circular and there #ill be a confluent la#n of gro#th.. (aint gro#th of tiny colonies. If the test organism is a Staphylococcus or Enterococcus spp. and the inoculum #as correct. isible gro#th that can be detected #ith the unaided eye. /0 hours of incubation are required for ancomycin and o!acillin. #hich is held on the back of the in erted petri plate. #hich can 16 . #ithin apparent :ones of inhibition. /. #ith the co er remo ed. using sliding calipers or a ruler. each plate is e!amined. but other agents can be read at 8= to 8< hours. Mones are measured to the nearest #hole millimeter.

disregard slight gro#th (/9J or less of the la#n of gro#th). is ignored. may s#arm into areas of inhibited gro#th around certain antimicrobial agents. &ith trimethoprim and the sulfonamides.8/1 'erformance . M899".! (ilution Met&ods 17 . since only susceptible breakpoints are gi en. .be detected only #ith a magnifying lens at the edge of the :one of inhibited gro#th.tandards for Antimicrobial . and measure the more ob ious margin to determine the :one diameter. or resistant to the agents that ha e been tested.usceptibility $esting1 $#elfth Informational . re"identified. 5.ome agents may only be reported as susceptible. the thin eil of s#arming gro#th in an other#ise ob ious :one of inhibition should be ignored. &ith Proteus spp. $he si:es of the :ones of inhibition are interpreted by referring to $ables /A through /I (Mone Ciameter Interpretati e ..upplement. and the organisms are reported as either susceptible.trains of Proteus spp. intermediate. discrete colonies gro#ing #ithin a clear :one of inhibition should be subcultured. not the :one of inhibition of hemolysis.tandards and equi alent Minimum Inhibitory 7oncentration Breakpoints) of the D77L. . &hen using blood"supplemented medium for testing streptococci. the :one of gro#th inhibition should be measured. ". -o#e er. and retested. antagonists in the medium may allo# some slight gro#thN therefore.

the #ay to a precise assessment is to determine the MI7 of the antibiotic to the organisms concerned. $here are t#o methods of testing for MI71 (a) Broth dilution method (b) Agar dilution method. Minimum In&ibitory Concentration 8MIC9 Ciffusion tests #idely used to determine the susceptibility of organisms isolated from clinical specimens ha e their limitationsN #hen equi ocal results are obtained or in prolonged serious infection e.rot& (ilution Met&od $he Broth Cilution method is a simple procedure for testing a small number of isolates. Also the terms O. the quantitation of antibiotic action is"a" is the pathogen needs to be more precise. $his can be achie ed by dilution of antimicrobial in either agar or broth media. bacterial endocarditis.usceptibleK and OResistantK can ha e a realistic interpretation. Antimicrobials are tested in log / serial dilutions (t#o fold). It has the added ad antage that the same tubes can be taken for MB7 tests also1 18 . e en single isolate.g. .Cilution susceptibility testing methods are used to determine the minimal concentration of antimicrobial to inhibit or kill the microorganism. $hus #hen in doubt.

5 cm tubes > small scre#"capped bottles. sterile Cistilled &ater " 699ml and suitable nutrient broth medium.tock solution can be prepared using the formula 8999 """"""" ! ? ! 7H & ' &here 'H'otency gi en by the manufacturer in relation to the base ?H ?olume in ml required 7H(inal concentration of solution (multiples of 8999) &H &eight of the antimicrobial to be dissol ed in the olume ? 19 .Materials . /ml and 8ml . required antibiotic in po#der form (either from the manufacturer or standard laboratory accompanied by a statement of its acti ity in mg>unit or per ml. 7linical preparations should not be used for reference technique. Stoc) solution .. $rimethoprim and sulphonamide testing requires thymidine free media or addition of 0J lysed horse blood to the media A suitable rack to hold // tubes in t#o ro#s i"e 88 tubes in each ro#.6 ! 8. required sol ent for the antibiotic.). 'asteur pipettes. o ernight broth culture of test and control organisms ( same as for disc diffusion tests).terile graduated pipettes of 89ml.terile capped . 6ml.

<9 mg per gram. Arrange t#o ro#s of 8/ sterile .they should not be refro:en or reused. 'lace /ml of antibiotic free broth to the last tube in each ro#. 3sing a fresh pipette . Inoculate one ro# #ith one drop of an o ernight broth culture of the test organism diluted appro!imately 20 .uggested dilution ranges of some antimicrobials are sho#n in Anne!ure II.add 0 ml of broth to the remaining 0 ml in the uni ersal bottle mi! and transfer /ml to the second tube in each ro#. .999mg>l from po#der base #hose potency is .90mg . Met&od 'repare stock dilutions of the antibiotic of concentrations 8999 and 899 Pg>L as required from original stock solution (89. prepare <ml of broth containing the concentration of antibiotic required for the first tube in each ro# from the appropriate stock solution already made..+!ample1 (or making 89 ml solution of the strength 89.6 !8.the quantities of the antimicrobials required is &H 8999 """"""" ! 89 ! 89H89/.<9 Dote1the stock solutions are made in higher concentrations to maintain their keeping qualities and stored in suitable aliquots at "/9 o7 .5 cm capped tubes in the rack. 7ontinue preparing dilutions in this #ay but #here as many as 89 or more are required the series should be started again half the #ay do#n.999mg>L). Mi! the contents of the uni ersal bottle using a pipette and transfer /ml to the first tube in each ro#.)nce taken out. In a sterile 59ml (uni ersal) scre# capped bottle.

the total requirement is <ml. $he result of the test is significantly affected by the si:e of the inoculum. $his often presents problems to those unaccustomed to performing these tests.0 ml of broth. each in /ml olumes i"e a total of 0 ml for each concentration as 0ml is required to make the second dilution.to 8 in 8999 in a suitable broth and the second ro# #ith the control organism of kno#n sensiti ity similarly diluted. In this e!ample gi en abo e. Inoculate a tube containing /ml broth #ith the organism and keep at I0 o7 in a refrigerator o ernight to be used as standard for the determination of complete inhibition. 8. $he follo#ing method ad ocated by 'amela M &ater#orth is presented. (or =0 g>l concentration. $#o sets of dilution are being prepared (one for the test and one for the control).= ml of 89 mg> l I =. 21 . <!=0mg>l H68/Pg in < ml. 7alculate the total olume required for the first dilution.If the broth culture used has gro#n poorly.8/) and take this olume in ml (i. the series has to be started again mid #ay at / mg>l #hich #ould be obtained in the same #ay1 <ml of /mg>lH8=Pg in <ml.$he test mi!ture should contain 89= organism>ml. Do# calculate the total amount of the antibiotic required for <ml. Calculations $or t&e preparation o$ t&e original dilution.8/ ml) of the dilution belo# 68/mg>l and make upto <ml #ith broth.o7.it may be necessary to use this undiluted. 'lace a decimal point after the first figure (6.e 6. Incubate tubes for 8< hours at 5.

Dote the lo#est concentration inhibiting gro#th of the organisms and record this as the MI7. o7 o ernight.-eading o$ result MI7 is e!pressed as the lo#est dilution. Minimum . $he control tube containing no antibiotic is immediately sub cultured (Before incubation) by spreading a loopful e enly o er a quarter of the plate on a medium suitable for the gro#th of the test organism and incubated at 5. #hich inhibited gro#th 4udged by lack of turbidity in the tube.C9 $he main ad antage of the OBroth dilutionK method for the MI7 determination lies in the fact that it can readily be con erted to determine the MB7 as #ell. Because ery faint turbidity may be gi en by the inoculum itself.actericidal Concentrations 8M.ubculture all tubes not 22 . . Met&od Cilutions and inoculations are prepared in the same manner as described for the determination of MI7. the inoculated tube kept in the refrigerator o ernight may be used as the standard for the determination of complete inhibition. $he tubes are also incubated o ernight at 5.o7.tandard strain of kno#n MI7 alue run #ith the test is used as the control to check the reagents and conditions. . Read the MI7 of the control organism to check that the drug concentrations are correct.

#hich represents the original inoculum. $he test must include a second set of the same dilutions inoculated #ith an organism of kno#n sensiti ity .sho#ing isible gro#th in the same manner as the control tube described abo e and incubate at 5.o7 o ernight.$hese tubes are not sub culturedN the purpose of the control is to confirm by its MI7 that the drug le el is correct. #hether or not this organism is killed is immaterial. 7ompare the amount of gro#th from the control tube before incubation. 23 .

tandard strain of kno#n MI7. -eading o$ result MI7 is e!pressed as the highest dilution #hich inhibited gro#th 4udged by lack of turbidity in the tube. 24 . • Do gro#th" if the #hole inoculum has been killed • $he highest dilution sho#ing at least .the inoculated tube kept in the refrigerator o ernight may be used as the standard for the determination of complete inhibition. run #ith the test is used as the control to check the reagents and conditions. • A reduced number of colonies"indicating a partial or slo# bactericidal acti ity.imilar number of colonies" indicating bacteriostasis only. Because ery faint turbidity may be gi en by the inoculum itself. 899 µl of double"strength M-B. .. In a .J inhibition is taken as MB7 Micro7brot& dilution test $his test uses double"strength MLeller"-inton broth. $he lo#est concentration sho#ing inhibition of gro#th #ill be considered the MI7 of the organism.-eading o$ result $hese subcultures may sho# • . 0G strength antibiotic solutions prepared as serial t#o"fold dilutions and the test organism at a concentration of /!89 =>ml. 69 µl each of the antibiotic dilutions and the organism suspension are mi!ed and incubated at 56°7 for 8<"/0 hours.= #ell plate.

.Blood may be added and if Ochocolate agarK is required.the dilutions can be prepared in agar slopes but it #ill then be necessary to prepare a second identical set to be inoculated #ith the control organism. 8 ml of this #ill be added to 8./<ml of /999Pg >ml Q 9. final olume of medium in plate $op antibiotic concentrations $otal amount of drug 8 ml of #ater /ml of 8/<9 Pg >ml #ill be required to start the dilution H /6=9Pg in / ml H 8.T&e Agar dilution Met&od Agar dilutions are most often prepared in petri dishes and ha e ad antage that it is possible to test se eral organisms on each plate . (Dote stock dilution of /999Pg >ml is required for this range of MI7) T&e :uic)est 5ay to prepare a range o$ dilutions in agar is as $ollo5s.If only one organism is to be tested e.tuberculosis.9 mm diameter petri dishes and add one ml of desired drug dilutions to 8.g M. It #ould be con enient to use .$he dilutions are made in a small olume of #ater and added to agar #hich has been melted and cooled to not more than =9o7. ml agar.$he factor of agar dilution must be allo#ed for in the first calculation as follo#s.the medium must be heated before the antibiotic is added. H /9 ml H =0mg>l H 8/<9Pg to be added to 25 ./ ml of #ater. ml of broth.

it is strongly recommended that the agar is first measured into stoppered tubes or uni ersal containers and the drug dilution added to these and mi!ed by in ersion before pouring into 'etri dishes. cooled to 66 o7 to each plate and mi! thoroughly. Although s#arming of P. $hey are then inoculated either #ith a multiple inoculators as spots or #ith a #ire loop or a platinum loop calibrated to deli er 9.Label a sterile petri dish on the base for each concentration required. &ith ordinary fast gro#ing organisms.mirabilis by this method either by cutting ditches in the agar bet#een the inocula. or by confining each #ith small glass or porcelain cylinders pressed into the agar.998ml spread o er a small area. It is possible to test spreading organism such as P. Adequate mi!ing is essential and if sufficient technical e!pertise is not a ailable for the skilled manipulation. this is not recommended for determination of MI7 because of the difficulty of ensuring adequate mi!ing of the drug #ith this ery iscous medium. ml melted agar. In either case the culture should be diluted to contain 89 6 to 89= organisms per ml. 26 . After the plates ha e set they should be #ell dried at 5.electi e media should not be used and electrolyte deficient media #ill gi e false results because of the effect of ariation in the salt content on the action of many antibiotics. )ne ml #ater is added to a control plate.o7 #ith their lids tipped for /9 to 59 minutes in an incubator. .mirabilis can be pre ented by the use of higher concentration of agar in the medium. 'repare the dilutions in #ater placing 8 ml of each in the appropriate dish. 'ipette 8. this can be obtained appro!imately by adding 6 Pl of an o ernight broth culture to 6ml broth or peptone #ater.

(ollo#ing incubation. It can also be used for Donfermenting *ram Degati e bacilli (D(*DB) for eg" Pseudomonas sp. Resistance of ma4or consequence may be detected for e.g. A predefined stable antimicrobial gradient is present on a thin inert carrier strip. and Burkholderia pseudomallei. N.gonorrhoeae. Haemophilus sp.. there is an immediate release of the drug. a symmetrical inhibition ellipse is produced.. ". (ilution and (i$$usion + test also kno#n as the epsilometer test is an Oe!ponential gradientK testing methodology #here O+K in + test refers to the *reek symbol epsilon ( ε). pneumoniae.J inhibition is taken as the MI7 for the organism. the test is ery useful in detecting glycopeptide resistant +nterococci (*R+) and glycopeptide intermediate 27 . $he intersection of the inhibitory :one edge and the calibrated carrier strip indicates the MI7 alue o er a #ide concentration range (R89 dilutions) #ith inherent precision and accuracy .. @"hemolytic streptococci. + test can be used to determine MI7 for fastidious organisms like S.-eading o$ results $he antibiotic concentration of the first plate sho#ing ≥ .$he + test(AB Biodisk) #hich is a quantitati e method for antimicrobial susceptibility testing applies both the dilution of antibiotic and diffusion of antibiotic into the medium. and anaerobes. &hen this + test strip is applied onto an inoculated agar plate.

(urther it can be used for detection of e!tended spectrum beta lactamases (+.acteria (ISC (I##<SION #O. $he suspension is then compared to the Mc(arland standard by holding the suspension and Mc(arland standard in front of a light against a #hite background #ith contrasting black lines and comparing the turbidity. %.pneumoniae Media $or disc di$$usion MLeller "-inton . If the turbidity is too hea y. $he inocula for seeding the susceptibility media #ith S.S. 7ell suspensions of the bacteria to be tested are prepared in sterile saline or Mueller"-inton broth. Susceptibility o$ #astidious . $he cell suspension is prepared by transferring a portion of the fresh gro#th #ith a s#ab or inoculating loop to the suspending medium.A) and slo# gro#ing pathogens such as Mycobacterium tuberculosis. the suspension should be diluted 28 . In conclusion + test is a simple.pneumoniae is prepared from fresh pure cultures (gro#n o ernight on 7hocolate agar). using caution #hen mi!ing the cells #ith the suspending medium so as not to form bubbles.heep blood agar Standardi=ation o$ inoculum.BL).aureus (*I. accurate and reliable method to determine the MI7 for a #ide spectrum of infectious agents.#ASTI(IO<S O-GANISMS Antibiotic susceptibility testing o$ S.

6 Mc(arland standards. lifted out of the broth. rotating the plate =9 degrees bet#een each inoculation. a ne# sterile s#ab (cotton or Cacron) is submerged in the suspension. Inoculation o$ t&e susceptibility test media After proper turbidity is achie ed. $he plates containing the disks are incubated at 56o7 for 8= to 8< h in an in erted position in a 6J 7) / incubator. the diameter of each :one of inhibition is measured #ith a ruler or calipers. holding the ruler o er the surface of the 29 . and the e!cess fluid is remo ed by pressing and rotating the s#ab against the #all of the tube. If the turbidity is too light additional cells should be added to the suspension.pneumoniae S Cirect colony suspension is made in normal saline and turbidity ad4usted to 9. $he s#ab is then used to inoculate the entire surface of the supplemented Mueller -inton agar plate three times. Estimating t&e susceptibility o$ t&e strains After o ernight incubation. $he inoculum is allo#ed to dry (usually taking only a fe# minutes but no longer than 86 minutes) before the discs are placed on the plates. $he :ones of inhibition on the media containing blood are measured from the top surface of the plate #ith the top remo ed. (or S. $he discs should be placed on the agar #ith sterile forceps and tapped gently to ensure the adherence to the agar. A candle e!tinction 4ar may be used if a 7)/ incubator is not a ailable. It is con enient to use a ruler #ith a handle attached for these measurements. &ithin 86 minutes after ad4usting the turbidity of the suspension the plate should be inoculated.#ith additional suspending medium.

terili:e the ruler occasionally to pre ent transmission of bacteria. M899". In all measurements. $he ruler should be positioned across the center of the disc to make these measurements. .usceptibility $esting1 $#elfth Informational . 7are should be taken not to touch the disk or surface of the agar.disk #hen measuring the inhibition :one.pneumoniae) of the D77L. the :ones of inhibition are measured from the edges of the last isible colony"forming gro#th. Antibiotic susceptibility o$ Haemophilus species 30 . intermediate and resistant.pneumoniae the :one measurement is from top of plate #ith the lid remo ed.tandards and equi alent Minimum Inhibitory 7oncentration Breakpoints for S. (or S.8/1 'erformance . (aint gro#th of tiny colonies that may appear to fade from the more ob ious :one should be ignored in the measurement.tandards for Antimicrobial . Interpretation +ach :one si:e is interpreted by reference to the $able /* (Mone Ciameter Interpretati e . $he results are recorded in millimeters (mm) and interpretation of susceptibility is obtained by comparing the results to the standard :one si:es.upplement as susceptible.

Test Procedure 8. 2 2 2 2 Mueller"-inton agar./ to . 5 ml of an DAC stock solution (69 mg of DAC dissol ed in 89 ml of distilled #ater and filter sterili:ed) are also aseptically added.. first a fresh hematin stock solution is prepared by dissol ing 69 mg of bo ine hematin po#der in 899 ml of 9. $he direct colony suspension procedure should be used #hen testing Haemophilus sp. After autocla ing and cooling to 06 to 69 °7. a suspension of the test organism is prepared in 31 . -aemophilus $est medium consists of the follo#ing ingredients. MLeller"-inton chocolate agar is not recommended for routine testing of Haemophilus spp. $hirty ml of the hematin stock solution are added to 8 L of M-A #ith 6 g of yeast e!tract. 86 µg>ml @"DAC.. and 6"mg>ml yeast e!tract. 3sing colonies taken directly from an o ernight (preferably /9 to /0 hour) chocolate agar culture plate.98 mol>L Da). $he p. $o make -$M. In its agar form.#ith heat and stirring until the po#der is thoroughly dissol ed. 86 µg>ml bo ine hematin.should be .$he medium of choice for disc diffusion testing of Haemophilus sp. is -aemophilus $est Medium (-$M).0.

$he :one margin should be considered as the area sho#ing no ob ious gro#th isible #ith the unaided eye. e!cept that. &ithin 86 minutes after ad4usting the turbidity of the inoculum suspension. it should be used for plate inoculation. influenzae are tested. 'lates are incubated at 56°7 in an atmosphere of 6J 7)/ for 8= to 8< hours before measuring the :ones of inhibition.J saline. 0. in general.. $he procedure for the disc test should be follo#ed as described for nonfastidious bacteria. discs should be applied to the surface of a 869"mm plate or no more than 0 discs on a 899"mm plate. 7are must be e!ercised in preparing this suspension. no more than . 5.MLeller"-inton broth or 9. /. $he suspension should be ad4usted to a turbidity equi alent to a 9. (aint gro#th of tiny colonies that may appear to fade from the more ob ious :one should be ignored in the measurement. $his suspension #ill contain appro!imately 8 to 0 ! 89< 7(3>ml. particularly #hen @"lactamase producing strains of H.6 Mc(arland standard using a photometric de ice. >one (iameter Interpreti2e Criteria 32 . because higher inoculum concentrations may lead to false"resistant results #ith some @"lactam antibiotics.

$he antimicrobial agents suggested for routine testing of Haemophilus sp.usceptibility $esting1 $#elfth Informational . 33 .) of the D77L. intermediate and resistant.tandards for Antimicrobial . #ith other agents is not recommended. are indicated in Anne!ure I. Cisc diffusion testing of Haemophilus spp.8/1 'erformance .tandards and equi alent Minimum Inhibitory 7oncentration Breakpoints for Haemophilus sp. +ach :one si:e is interpreted by reference to the $able /+ (Mone Ciameter Interpretati e . M899".upplement as susceptible.

.Antibiotic susceptibility testing $or Neisseria gonorrhoeae $he recommended medium for testing N. &ithin 86 minutes after ad4usting the turbidity of the inoculum suspension.J saline.gonorrhoeae. fe#er discs may need to be tested per plate. $he direct colony suspension procedure should be used #hen testing N. a suspension equi alent to that of the 9. 7ysteine"free gro#th supplement is not required for disc testing. Test Procedure 8. Do more than . 3sing colonies taken directly from an o ernight chocolate agar culture plate. antimicrobial discs should be placed onto the agar surface of a 869"mm agar plate not more than 0 discs onto a 899"mm plate. $he disc diffusion test procedure steps. +nriched chocolate agar is not recommended for susceptibility testing of N. as described for nonfastidious bacteria. #hen testing some agents (e.6 Mc(arland standard is prepared in either Mueller"-inton broth or 9.. it should be used for plate inoculation. /. gonorrhea. gonorrhoeae consists of *7 agar to #hich a 8J defined gro#th supplement is added after autocla ing. should be follo#ed. -o#e er. quinolones) #hich produce e!tremely large :ones. 34 .g.

8/1 'erformance .tandards for Antimicrobial .upplement.tandards and equi alent Minimum Inhibitory 7oncentration Breakpoints for N.usceptibility $esting1 $#elfth Informational . )rganisms #ith plasmid"mediated resistance to tetracycline also ha e :ones of inhibition (59" µg tetracycline discs) of T 8. @"lactamase tests are faster and are therefore preferred for recognition of this plasmid"mediated resistance. 7hromosomal mechanisms of resistance to penicillin and tetracycline produce larger :one diameters.usceptibility $esting1 $#elfth Informational . gonorrhoeae are indicated in Anne!ure I. mm generally produce @"lactamase.5. >one (iameter Interpreti2e Criteria $he antimicrobial agents suggested for routine testing of N. #hich can be accurately recogni:ed using the interpreti e criteria indicated in $able /( (Mone Ciameter Interpretati e .tandards and equi alent Minimum Inhibitory 7oncentration Breakpoints for N.8/1 'erformance . Cisc diffusion testing of N. intermediate and resistant. M899". D)$+1 )rganisms #ith 89"µg penicillin disc :one diameters of T 8. M899". gonorrhoeae) of the D77L. gonorrhoeae) of the D77L. -o#e er. mm.upplement as susceptible. 35 . gonorrhoeae #ith other agents is not recommended. $he plates are incubated at 56°7 in an atmosphere of 6J 7)/ for /9 to /0 hours before measuring the :ones of inhibition.tandards for Antimicrobial . +ach :one si:e is interpreted by reference to the $able /( (Mone Ciameter Interpretati e .

influenzae" .998ml of the inoculum is used to spot inoculate the cultures.pneumoniae Inoculation o$ test plate In general the inoculum should be applied as a spot that co ers a circle about 6"<mm in diameter . 36 . A 8>89 dilution of this suspension is made and #ithin 86 minutes of making the diluted suspension the test plates should be inoculated #ith either a platinum loop calibrated to deli er 9.(etermination o$ MIC $or #astidious organisms T&e Agar dilution met&od Standardi=ation o$ inoculum.BA medium is to be used. (or N. (or S.6ml of normal saline and the opacity ad4usted to Mc(arland 9. Appropriate A$77 quality control organism(s) should be included along #ith each test.998ml or multipoint inoculator. $he inoculum should be an acti ely gro#ing culture diluted in saline to 89 0 to 896 microorganisms per ml. Inoculated plates are left undisturbed until the spots of inoculum ha e dried.A platinum loop calibrated to deli er 9. $he colonies are suspended in 9.imilar to the procedure described abo e for S.gonorrhoeae and H.pneumoniae S Cirect colony suspension from a 8/"86 hour culture from $.6.

tandards for Antimicrobial .tandards) as susceptible. intermediate and resistant. $he end point is the lo#est concentration of antibiotic that completely inhibits gro#th. $he MI7 of the quality control strain should be in the e!pected quality control range. 37 . Results are reported as the MI7 in micrograms or units>ml. -eading $he control plate should sho# the gro#th of the U7 test organism.8/1 'erformance . Interpretation is made in accordance to the guidelines laid do#n in the D77L.upplement (MI7 Interpretati e . M899". the plates are incubated at 56 o7 for 8= to 8< h in an in erted position in a 6J 7)/ incubator. A candle e!tinction 4ar may be used if a 7)/ incubator is not a ailable.usceptibility $esting1 $#elfth Informational . A barely isible ha:iness or single colony should be disregarded.Incubation After the spots of inoculum ha e dried.

#hich is reference method.th edition) -esistant Minor +rror ?ery Ma4or +rror MIC 8µ g?ml9 Minor +rror Intermediate Minor +rror Ma4or +rror Minor +rror Susceptible (is) (i$$usion (iameter 8mm9 38 . ma4or errors and ery ma4or errors. #hich is #idely used to report #ith the MI7.(adopted from Manual of 7linical Microbiology. $his is achie ed by comparing disk diffusion.'. Errors in Interpretation and reporting results In the interpretation of test results there are possibilities for errors to occur. . Based on impact of errors in treatment of patient they are classified as minor errors. $he follo#ing flo# chart sho#s the errors inreporting.

Dot more than 8 in /9 results should be outside accuracy limits. to check manual errors. Uuality control strains should be included daily #ith the test. the potency of the antibiotic.*. All documentation is maintained indefinitely 0. Do :one should be more than 0 standard de iations a#ay from midpoint bet#een the stated limits. it is not possible to include all strains on a daily basis. -e$erence strains $or :uality control Escherichia coli A$77 /6. /uality Control in Antibiotic Susceptibility Testing U7 is performed to check the quality of medium. for reasons of e!pense or manpo#er constraints.// (beta"lactamase negati e) Escherichia coli A$77 56/8< (beta"lactamase positi e) Staphylococccus aureus A$77 /6. o!acillin susceptible) Staphylococccus aureus A$77 5<6. 'roficiency testing is repeated for each ne# batch of media or reagents All tests must be #ithin accuracy limits if U7 is done once #eekly. 'roficiency testing is repeated for each ne# drug included in the testing 5. then the follo#ing guidelines should be follo#ed. If./5 (beta"lactmase negati e. 'erforming U7 daily for 59 days #ith less than 89J inaccuracy for each drug /.8 (beta"lactmase positi e) 39 . $he frequency can be decreased to once #eekly if proficiency has been demonstrated by 8.

tock cultures should be kept at ".A).. (etection o$ Oxacillin/Methicillin-resistant Staphylococcus aureus 8M-SA9 .A slants at /" <°7 for up to / #eeks. .. including cephalosporins and carbapenems. historically termed methicillin"resistant S. R< µg>ml) ha e been published. Before use as a U7 strain. including erythromycin.<65 (for aminoglycosides) Enterococcus faecalis A$77 /. the strain should be subcultured at least t#ice and retested for characteristic features.//= .A strains #ith decreased susceptibility to inhibitory concentration VMI7W.ince 8.== (for cephalosporins) Haemophilus influenzae A$77 89/88 (for medium control) Neissseria gonorrheae A$77 0. Standard Met&ods #or T&e (etection O$ Antibacterial -esistance. and tetracycline./8/ (for checking of thymidine or thymine le el of M-A) Haemophilus influenzae A$77 0.A isolates often are multiply resistant to commonly used ancomycin (minimum antimicrobial agents.. 40 . reports of MR. .trains that are o!acillin and methicillin resistant. aureus (MR. MR.9°7 in Brucella broth #ith 89J glycerol for up to 5 years.=. clindamycin.Pseudomonas aeruginosa A$77 /. &orking cultures are maintained on $. are resistant to all beta"lactam agents.

6 Mc(arland tube. Accurate detection of o!acillin>methicillin resistance can be difficult due to the presence of t#o subpopulations (one susceptible and the other resistant) that may coe!ist #ithin a 41 . gentamicin. $he inoculum is prepared by matching a 9. and inoculate 89 microL on the plate. if controls are satisfactory. although some strains remain susceptible to fluoroquinolones.tandards (D77L.6 inch area.=< mol>L). Because of the rapid emergence of rifampin resistance. $he Dational 7ommittee for 7linical Laboratory . $#o methods can be follo#ed for inoculation1 8. this drug should ne er be used as a single agent to treat MR. to get an inoculum of 890 7(3.creening $est for )!acillin"resistant S.treak s#ab o er a 8"8.*lycopeptides. aureusX and uses an agar plate containing = microg>ml of o!acillin and MLeller"-inton agar supplemented #ith Da7l (0J #> N 9. or rifampin. $hese plates can be stored refrigerated for up to / #eeks. trimethoprim>sulfametho!a:ole. any gro#th after /0 hours incubation at 56°7 denotes o!acillin resistance. .A infections. /. In both methods. Cilute the supension 81899. ancomycin and $eicoplanin are the only drug of choice for treatment of se ere MR.) has recommended %.A infections. Cip a s#ab in the suspension and e!press e!cess fluid by pressing s#ab against the #all of the tube.

T9. or nafcillin should be incubated at 56 ° 7 for a full /0 hours before reading. MICs O1acillin Susceptible O1acillin Intermediate O1acillin -esistant S. aureus 7oD./6µg >ml no intermediate MI7 MI7R0 µg >ml MI7R9. All cells in a culture may carry the genetic information for resistance but only a small number can e!press the resistance in such as o!acillin. $his is #hy isolates being tested against o!acillin. R85 mm R8< mm O1acillin Intermediate O1acillin -esistant T89 mm T8. $he breakpoints for S.culture. $his phenomenon is termed heteroresistance and occurs in staphylococci resistant to penicillinase"stable penicillins.). methicillin. -eteroresistance is a problem for clinical laboratory personnel because cells e!pressing resistance may gro# more slo#ly than the susceptible population. aureus T/ µg>ml 7oD. aureus are different from those for coagulase"negati e staphylococci (7oD. itro.6 µg >ml no intermediate MI7 >one si=es O1acillin Susceptible S. mm 88"8/ mm no intermediate :one 42 .

including penicillins. species.ome species are more resistant to commonly used antimicrobial agents than others. S. species. Identification to species le el can aid in the recognition of outbreaks and in tracking resistance trends. isolates are often resistant to other commonly used antimicrobial agents. epidermidis is the most common 7oD.&hen used correctly. broth"based and agar"based tests usually can detect MR. haemolyticus and S. . $hese tests confirm o!acillin>methicillin resistance caused by mec in Staphylococcus species. Amplification tests like those based on the polymerase chain reaction ('7R) detect the mec gene. epidermidis. S. 43 . cephalosporins. isolated in clinical laboratories. hominis are more likely to be multiply resistant to antimicrobial agents than are other 7oD. )!acillin"resistant 7oD. S. In addition. they often are considered to be a single group. o!acillin"resistant 7oD.A. and carbapenems. 3sually. may differ bet#een hospitals and #ards. resistance patterns of 7oD. isolates are resistant to all beta"lactam agents. )!acillin screen plates can be used in addition to routine susceptibility test methods or as a back"up method. -o#e er. so ancomycin is frequently the drug of choice for treatment of clinically significant infections. (etection o$ Oxacillin-resistant Coagulase-negati e Staphylococcus sp! Although there are about /9 7oD.

In general. that contained the mec gene. are often smaller than those of S. making gro#th more difficult to read. the current breakpoints for S.. like S. aureus. aureus failed to detect many 7oD.Accurate detection of o!acillin resistance can be difficult. 44 . t#o subpopulations (one susceptible and the other resistant) may coe!ist #ithin a culture. aureus. the ne# breakpoints for 7oD. 7olony si:es of 7oD. In addition.. &hen studies #ere performed to e aluate o!acillin breakpoints for 7oD. correlate better #ith mec production for 7oD.

one en:yme may acti ely hydroly:e cefta:idime. !. pneumoniae. o"ytoca. mm T /6 mm 45 .BLs can be difficult to detect because they ha e different le els of acti ity against arious cephalosporins. e en if in itro test results indicate susceptibility. (or e!ample.(etection o$ E1tended7Spectrum . cefo!itin and cefotetan) or carbapenems (e. +.eta7@actamases 8ES.BL"producer if the test results are as follo#s1 (is) di$$usion cefpodo!ime cefta:idime T // mm T // mm MICs cefpodo!ime cefta:idime a:treonam R / µg >ml cefota!ime ceftria!one R / µg >ml R / µg >ml R / µg >ml R / µg >ml a:treonam T /... cefota!ime.g. If an +. producing MI7s of only 0 microg>ml. mm cefota!ime ceftria!one T /. and a:treonam should be reported as resistant.g. the choice of #hich antimicrobial agents to test is critical.. a:treonam) but do not affect cephamycins (e. cefta:idime. meropenem or imipenem).BLs are en:ymes that mediate resistance to e!tended"spectrum (third generation) cephalosporins (e..g. and ceftria!one) and monobactams (e.BL is detected. resulting in cefta:idime minimum inhibitory concentrations (MI7s) of /6= microg>ml. +ach !.@s9 +. but ha e poor acti ity on cefota!ime. cephalosporins. $here are standard broth microdilution and disc diffusion screening tests using selected antimicrobial agents. all penicillins. or Escherichia coli isolate should be considered a potential +.g. $hus.

BL"producing organism. !. 46 .BLs in enteric organisms can ary depending on #hich antimicrobial agents are tested. coli can be done by testing both cefota!ime and cefta:idime. $esting can be performed by the broth microdilution method or by disk diffusion. (or MI7 testing. ersus its MI7 #hen tested alone. 7efpodo!ime and cefta:idime sho# the highest sensiti ity for +. 'henotypic confirmation of potential +. pneumoniae A$77 . a R 6 mm increase in a :one diameter for either antimicrobial agent tested in combination #ith cla ulanic acid ersus its :one #hen tested alone confirms an +.BL tests. o"ytoca.// (negati e control) should be used for quality control of +. or E. commercial manufacturers of antimicrobial discs may produce discs containing cefota!ime and cefta:idime #ith cla ulanic acid. 3ntil commercial discs are a ailable.BL"producing isolates of !. coli A$77 /6. confirms an +. a decrease of R 5 doubling dilutions in an MI7 for either cefota!ime or cefta:idime tested in combination #ith 0 µg >ml cla ulanic acid. .$he sensiti ity of screening for +.BL detection. !. pneumoniae.BL"producing organism. Ciscs can be made by adding 89 µl of a 8999 µg >ml stock solution of cla ulanic acid to cefota!ime and cefta:idime disks each day of testing.mithAline Beecham can pro ide clinical laboratories #ith cla ulanic acid po#der for routine use. In future. (or disc diffusion testing. alone and in combination #ith cla ulanic acid. $he use of more than one of the fi e antimicrobial agents suggested for screening #ill impro e the sensiti ity of detection.99=95 (positi e control) and E.

BL"producer by the D77L. detection of organisms #ith multiple beta"lactamases that may interfere #ith the phenotypic confirmatory test can only be accomplished using isoelectric focusing and CDA sequencing.. #hich should be reported according to their routine test results. resulting in a false"negati e test. at this time.BLs.-? beta"lactamases in organisms #ith +. 47 . -o#e er. If an isolate is confirmed as an +.BLs contain other beta"lactamases that can mask +.BL"producer. If an isolate is not confirmed as an +. methods for screening and phenotypic confirmatory testing of these isolates ha e not been determined. and isolates of Pseudomonas aeruginosa also produce +."recommended phenotypic confirmatory test procedure. and a:treonam should be reported as resistant. )ther isolates of +nterobacteriaceae. -yper"production of $+M and>or . $hese beta"lactamases include Amp7s and inhibitor"resistant $+Ms (IR$s).ome organisms #ith +.BLs also may cause false"negati e phenotypic confirmatory test results. such as Salmonella species and Proteus mirabilis.BL production in the phenotypic test. cephalosporins. cephalosporins. methods that are not usually a ailable in clinical laboratories. all penicillins.BLs. and a:treonam for isolates not confirmed as +. Co not change interpretations of penicillins. current recommendations suggest reporting results as for routine testing. 7urrently. $his list does not include the cephamycins (cefotetan and cefo!itin).

(etection o$ Imipenem or Meropenem -esistance in Gram7negati2e Organisms &ithin a health care setting, increases in species"specific carbapenem resistance should be monitored and sudden increases in estigated to rule out an outbreak of resistant organisms or spurious test results. 'ublished reports indicate some resistance in a ariety of clinical *ram"negati e cinetobacter

organisms, including Pseudomonas aeruginosa, Burkholderia cepacia,

species, Proteus species, Serratia marcescens, Enterobacter species, and !. pneumoniae. Stenotrophomonas maltophilia isolates are intrinsically resistant to imipenem. )rganisms can produce more than one hydroly:ing en:yme and may sho# modifications in more than one porin, producing high"le el resistance to the carbapenems (minimum inhibitory concentration VMI7W R8= µg>ml). )rganisms #ith decreased susceptibility produced by porin changes alone often ha e lo#er MI7s (/"< microg>ml). )rganisms #ith MI7s near interpretation breakpoints ha e greater potential for reporting errors. (or e!ample, isolates of P. aeruginosa often ha e MI7s that are at or near the carbapenem intermediate (< µg>ml) and resistant (R8= µg>ml) breakpoints. ,ome species, such as P. mirabilis, P. #ulgaris, and Morganella morganii, often ha e MI7s (8"0 µg>ml) 4ust belo# the carbapenem intermediate breakpoint of < mg>ml. Most other species of +nterobacteriaceae are ery susceptible (T9.6 µg>ml). Broth microdilution methods usually detect carbapenem resistance #hen the tests are performed properly. -o#e er, studies ha e sho#n false resistance to imipenem in
48

commercially prepared test panels due to degradation of the drug or to a manufacturing problem #here concentrations of imipenem #ere too lo#. &hen performed properly, disc diffusion and agar gradient diffusion also are acceptable methods for carbapenem testing. Imipenem degrades easily. ,tudies suggest that meropenem may be more stable than imipenem. -o#e er, for either antimicrobial agent, storage conditions of susceptibility panels, cards, and discs must be monitored carefully and quality control results checked frequently. If possible, store supplies containing carbapenems at the coldest temperature range stated in the manufacturerEs directions. An additional test method, such as agar gradient diffusion (i.e., + test), can be used to erify intermediate or resistant results. /uinolones and resistance $he number and location of mutations affecting critical sites determine the le el of resistance. )rganisms may ha e alterations in more than one en:yme target site and, in gram"negati e organisms, may contain more than one porin change. Many resistant organisms ha e multiple en:yme target site, porin, and efflu! mutations, producing high" le el resistance to quinolones. In contrast, organisms #ith decreased susceptibility produced only by porin changes usually ha e lo#er minimum inhibitory concentrations (MI7s). $he fluoroquinolone susceptibility profile for each clinical isolate is determined by the number and location of mutational changes in specific en:yme target sites, porin proteins, and efflu! mechanisms. $he effect of each mutation in an isolate is not equi alent for all

49

fluoroquinolones, due to ariations of the chemical structures among this class of agents. $herefore, an organism #ith one or more mutations may ha e resistant MI7s>:one si:es to one quinolone but ha e intermediate or susceptible MI7s>:one si:es to another quinolone. Curing therapy, the potential e!ists for an organism #ith a single mutation to acquire a second mutation, leading to high"le el resistance. After multiple mutations occur, an organism is generally highly resistant to all quinolones. Resistance to quinolones has been reported in a ariety of important bacterial pathogens, including E. coli, !. pneumoniae and other enteric organismsN P. aeruginosaN $hlamydia trachomatis, Mycoplasma pneumoniaeN $ampylobacter %e%uni, B. cepaciaN S. maltophilia, N. gonorrhoeae, S. aureus (especially o!acillin"resistant strains), Enterococcus faecium and S. pneumoniae.

50

(or enterococci reco ered from blood and 7. less commonly. $he rare @"lactamase" producing strains are detected best by using a direct. faecium strains #ith normal lo#er le el resistance (penicillin MI7s T =0 µg>ml and ampicillin T 5/ µg>ml) should be considered potentially susceptible to synergy #ith an aminoglycoside (in the absence of high"le el aminoglycoside resistance) #hereas strains #ith higher le el resistance may be resistant to such synergy.e. penicillin MI7s R 8/< µg>ml or ampicillin MI7s R =0 µg>ml). 4ancomycin -esistance Accurate detection of ancomycin"resistant enterococci by the disc diffusion test requires that plates be incubated for a full /0 hours (rather than 8= to 8< hours) and that any :one surrounding the ancomycin disc be e!amined carefully #ith transmitted light for 51 .(. but it #ill not reliably detect @"lactamase producing strains. 7ertain penicillin" ampicillin" resistant enterococci may possess high"le el resistance (i. $he disc test #ill not differentiate those #ith normal resistance from this high"le el resistance.. @"lactamase test. $he disc diffusion test can accurately detect isolates #ith altered 'B's. penicillin"binding proteins ('B's) or. because of the production of @"lactamase.(etection o$ -esistant Enterococci Penicillin?Ampicillin -esistance +nterococci may be resistant to penicillin and ampicillin because of production of lo#" affinity. nitrocefin"based. the laboratory should consider determining the actual MI7 for penicillin or ampicillin since E.

depending upon the resistance mechanisms present at a healthcare facility. Aminoglycoside -esistance in Enterobacteriaceae and Pseudomonas aeruginosa Resistance to one aminoglycoside may not predict resistance to the others. In general. $herefore.pecial. . Do :one of inhibition indicates resistance. erified by determining the 52 . the pre alence of amikacin resistance can be lo#er than the pre alence of resistance to gentamicin and tobramycin.trains that yield :ones of . resistance is relati ely common in P. aeruginosa but less common in +nterobacteriaceae.e idence of small colonies or a light film gro#ing #ithin the :one. +nterobacteriaceae resistant to gentamicin and tobramycin can be susceptible to amikacin or netilmicin because these drugs are not affected by many of the aminoglycoside modifying en:ymes (AM+s). because their acti ities against enterococci are not superior to gentamicin or streptomycin. 3ig&7le2el Aminoglycoside -esistance -igh"le el resistance to aminoglycosides is an indication that an enterococcal isolate #ill not be affected synergistically by a combination of a penicillin or glycopeptide plus an aminoglycoside. to . high"content gentamicin (8/9 µg) and streptomycin (599 µg) discs can be used to screen for this type of resistance. )ther aminoglycosides need not be tested. An intermediate category result by the disc diffusion test should be ancomycin MI7. mm should be e!amined using a dilution screen test. and :ones of R 89 mm indicate a lack of high"le el resistance. .

<. nationally and globally and helpful in minimi:ing the consequence of drug resistance.$. +mergence of multidrug resistance has limited the therapeutic options. although they may appear susceptible. 0. hence monitoring resistance is of paramount importance. &indo#s . limit the emergence and spread of drug resistant pathogens.Aminoglycosides are not clinically effecti e against Salmonella species and Shigella species. &indo#s .6. 53 . aminoglycosides should not be tested or reported. &-)D+$ 6. Application o$ Computers in Antibacterial Susceptibility Testing Antimicrobial resistance is a global problem.8 is a data base soft#are compatible #ith. Antimicrobial resistance monitoring #ill help to re ie# the current status of antimicrobial resistance locally. $housands of laboratories are distributed #orld"#ide and need to be linked to integrate data on most clinically rele ant organism on a daily basis to obtain an accurate picture of Yreal resistanceX. (or this purpose a soft#are #as de eloped for the management of routine laboratory results by &-) called O&-)D+$K. $his soft#are has focused on data analysis particularly on the results of A.

$his is also used for research studies.int>emc>&-)D+$>Instructions. etc. D77L. 3se of &-)D+$ soft#are may aid the local laboratory in net#orking efficiently #ith the national reference laboratory. $his programme is useful in supplying current guidelines. to net#ork efficiently #ith international bodies such as &-). in de eloping guidelines for use of antimicrobial agents and re ie# the same periodically. in identifying the clusters of resistant isolates and emerging outbreaks.50 mb. this soft#are is a ailable in #ebsite http1>>###. protocols to local laboratories.#ho.. 54 .&indo#s D$ and later ersions of &indo#s.html. Installation capacity of the soft#are is . in studying the effect of antimicrobial control programmes on resistant bacteria.

Baron +. &ashington C7.. 8.8/. ?ictor Lorian. 8. 'faller M.5"869.?.7.5".R. . $eno er (. Manual of 7linical Microbiology . • Ira R.. =th edition.Z.<=N '. Bacteriology. • Zohn C. 850/" 850.tandard )perati e procedure manual for microbiology laboratories.ociety for Microbiology. '.ibliograp&y • Coern *.upplement. • $hrupp L.A. Murray '. .A. . &illiams and &ilkins. $eno er (.. 'faller M.7. [olken R.R."80. • Dational 7ommittee for 7linical Laboratory . American .Z Antimicrobial . 80=.. American .tandards. /99/. Dational 7ommittee for 7linical Laboratory . M899 . .Z. Baron +. Dational Institute of Biologicals. '. .1+. Manual of 7linical Microbiology.6. Antibiotics In Laboratory Medcine /nd +dition.C..ociety for Microbiology. 8..th edition.. 8.5. Performance Standards for antimicrobial susceptibility testing. [olken R. '.usceptibility testing1 *eneral 7onsiderations. <th Informational .usceptibility tests of fastidious bacteria.6. 'a. &ashington C7. 55 .usceptibility $esting of Antibiotic in Liquid Media. Murray '.tandards. Balitimore. ?illano a.$ and Zames -.

Zesudason M. 8"69. Laboratory methods in antimicrobial chemotherapy..<1 =. 'hilips I.ociety for Antimicrobial 7hemotherapy. 58"08.1 .7. 5=.. 7hurchill Li ingstone.A.C. & Pharma Sci 8. • Morley C. ... &illiams Z. +ffects of paper on the performance of antibiotic"impregnated discs.1 89=N '. 56 . &ise R. &. +ffect of paper on the performance assay in the control of antibiotic sensiti ity discs. 8. 699"695."5</.Z.. • Rippere R. 'riya L. 'ndian &.<N '.Manayani C. Uuantitati e methods for bacterial sensiti ity testing.7. A simple method for testing the sensiti ity of #ound bacteria to penicillin and sulphathia:ole by use of impregnated blotting paper disc.N '.pneumoniae. '.N '. and Airshbaum A. ntimicrob $hemothrap 8. Baltimore.. 8.teinhoff M.8N /. Ree es C. Med (es. • Lalitha M.M.? .8.• &ater #orth '. .A. 8.N '.=81 . • Aramer Z. 550"55=. &.upplement C. $homas A. Pathol Bacteriol. + test as an alternati e to con entional MI7 determination for sur eillance of drug resistant S.. 5. ppl Microbiol 8. • A guide to sensiti ity testing1 Report of #orking party on antibiotic sensiti ity testing of the British ."5.061 6..

Staphylococcus sp. 57 .attery O$ Antibiotics #or Susceptibility Testing. Suggested .Anne1ure I. 'enicillin )!acillin 7ephalothin *entamicin Detilmicin Amikacin 7hloramphenicol $etracycline +rythromycin 7o"trimo!a:ole 7lindamycin )flo!acin Rifampicin ?ancomycin $eicoplanin Gram negati2e Streptococcus bacilli "Enterococcus# Pneumococcus$ Ampicillin 'enicillin 'iperacillin )!acillin 7ephalothin 7efota!ime 7efta:idime *entamicin Detilmicin Amikacin 7hloramphenicol $etracycline 7o"trimo!a:ole Dalidi!ic Acid 7iproflo!acin )flo!acin Ditrofurantoin Imipenem Meropenem Haemophilus sp N! gonorrhoeae Ampicillin Amo!ycillin> 'enicillin 7efa:olin 7la ulanic acid Ampicillin 7efuro!ime 7eftria!one 7efota!ime 7efota!ime 7hloramphenic +rythromycin $etracycline 7iproflo!acin 7hloramphenicol +rythromycin $etracycline 7hloramphenicol ?ancomycin Dote1 • $he choice of antibiotic depends on the pattern e!hibited locally. Guidelines #or Antimicrobial Susceptibility Testing.

58 .• $he selection of antibiotics aries based on specimen and the isolates under consideration.

Suggested Antibiotic (ilution -anges #or MIC Testing ANTIMIC-O.ulphametho!a:ole $rimethoprim(3$I.986"5/ 9.Anne1ure II.986"5/ 9.85"/6= (*ram " e) 7ephalosporin II\III generation 9.9="/6= 9.986"8= 9.6"=0 59 .986"5/ 9.*ram " e) 9.995"=0 9.995"=0 9.9="8/< 9.IA@ AGENT 'enicillin * Methicillin Daficillin>)!acillin Ampicillin(*ram I e) Ampicillin(*ram" e) 7ephalosporin(*ram I e) 7ephalosporin I generation CONCENT-ATION -ANGE8Ag?ml9 9.95"=0 9.95"=0 9.86"5/ 9.9="/6= 9.986"5/ 9.95"=0 (*ram" e) 7ephalosporin III generation (Pseudomonas sp.85"/6= 9.85"/6= 9.="8/8= 9.986"5/ 9.) ?ancomycin Amikacin *entamicin>$obramycin +rythromycin 7lindamycin Rifampicin 7hloramphenicol $etracycline $rimethoprim .

8"69.Z...Antimicrob 7hemotherap 8.upplement C.8N(/.ource"A guide to sensti ity testing1 Report of #orking party on antibiotic sensiti ity testing of the British society for Antimicrobial 7hemotherapy. 60 .)1.

Anne1ure III. Sol2ents and (iluents $or Antibiotics 61 .

quinupristin"dalfopristin. ta:obactam. in #ater or broth. netilmycin. clindamycin. gentamicin. cefopera:one. 9. cefemandole.9. 7ephalosporins and cephems not listed abo e or in footnote d are solubli:ed (unless the manufacturer indicates other#ise) in phosphate buffer. a. a:locillin. $hese sol ents and diluents are for making stock solutions of antimicrobial agents that require sol ents other than #ater. metronida:ole. trimethoprim (if lactate). gemiflo!acin. p. kanamycin.8 mol>L. line:olid. piperacillin. nitrofurantoin is dissol ed in dimethyl sulfo!ide. $hey should be diluted further. and further diluted in sterile distilled #ater. nafcillin.6 to /. Alternati ely. mecillinam. teicoplanin. penicillin. meropenem.9 hours to con ert it to equal parts of both isomers. carbencillin. sulbactam. cefo!itin. the salt is dissol ed in 9. ceftria!:one. cefonicid. gatiflo!acin (#ith stirring). tetracyclines. $herefore. d. cefota!ime.=. methicillin. cefti:o!ime.#ootNote. b. trospectomycin and ancomycin. sparflo!acin. mo!iflo!acin. o!acillin. tobramycin. cefaclor. Ma!olactam for clinical use is a 818 mi!ture of R and . $he diammonium salt of mo!alactam is ery stable. but it is almost pure R isomer. minocyclin. as necessary. c. me:locillin.90 mol>L -7L and allo#ed to react for 8. 62 . isomers. ciproflo!acin. $he products kno#n to be suitable for #ater sol ents and diluents are amikacin.

$he sodium carbonate is dissol ed in solution in most of the required #ater.e. g.C. 7onsult the material safety datasheets (M. 63 . f. Anhydrous sodium carbonate is used at a #eight of e!actly 89J of the cefta:idme to be used. $he antibiotic is dissol ed in this sodium carbonate solution and #ater is added to desired olume. then add glacial acetic acid drop#ise until dissol ed not to e!ceed /. 3se ] olume of #ater.0. -eproduced 5it& permissionB $rom NCC@S publication M1++7S1! Per$ormance Standards $or Antimicrobial Testing. but can be stored up to si! hours at not more than /6°7. Anne1ure III.*71.N 17%'! . T5el$t& In$ormational Supplement 8IS.7 "%" 7'9.B <SA. $he solution is to be used as soon as possible. Copies o$ t&e current edition may be obtained $rom NCC@SB 0"+ Cest 4alley -oadB Suite 1"++B CayneB Pennsyl2ania 10+.6µl>ml. $hese compounds are potentially to!ic.) a ailable from the product manufacturer before using any of these materials.