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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.

&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.

!. Principle $he principles of determining the effecti ity of a no!ious agent to a bacterium #ere #ell enumerated by Rideal ,&alker and others at the turn of the century, 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. $he ditch plate method of agar diffusion used by Ale!ander (leming #as the forerunner of a ariety of agar diffusion methods de ised by #orkers in this field .$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. (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. + 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. $he disc diffusion method of A,$ is the most practical method and is still the method of choice for the a erage laboratory. 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, it #ill certainly be the most commonly carried out microbiological test for many years to come. It is, therefore, imperati e that microbiologists understand the principles of the test #ell and keep updating the information as and #hen necessary. All techniques in ol e either diffusion of antimicrobial agent in agar or dilution of antibiotic in agar or broth.

+ en automated techniques are ariations of the abo e methods. . #actors In$luencing Antimicrobial Susceptibility Testing p3 $he p- of each batch of Mueller"-inton agar should be checked #hen the medium is prepared. $he e!act method used #ill depend largely on the type of equipment a ailable in the laboratory. $he agar medium should ha e a p- bet#een ../ and ..0 at room temperature after gelling. If the p- is too lo#, certain drugs #ill appear to lose potency (e.g., amino glycosides, quinolones, and macrolides), #hile other agents may appear to ha e e!cessi e acti ity (e.g., tetracycline). If the p- is too high, the opposite effects can be e!pected. $he p- 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- electrode. Allo# a small amount of agar to solidify around the tip of a p- electrode in a beaker or cup. 3se a properly calibrated surface electrode.

Moisture If, 4ust before use, e!cess surface moisture is present, the plates should be placed in an incubator (567) 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). $he surface should be moist, 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.

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

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. 7ertain fastidious bacteria such as Haemophilus sp. N. gonorrhoeae, S. pneumoniae, and iridans and @"hemolytic streptococci do not gro# sufficiently on unsupplemented Mueller"-inton agar. in separate sections. ". Met&ods o$ Antimicrobial Susceptibility Testing Antimicrobial susceptibility testing methods are di ided into types based on the principle applied in each system. $hey include1 (i$$usion ,tokes method Airby"Bauer method (ilution i) Broth dilution ii )Agar Cilution (i$$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

".1 (is) (i$$usion -eagents $or t&e (is) (i$$usion Test 1. Mueller73inton Agar Medium )f the many media a ailable, 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. It is lo# in sulphonamide, trimethoprim, and tetracycline inhibitors. It gi es satisfactory gro#th of most nonfastidious pathogens. A large body of data and e!perience has been collected concerning susceptibility tests performed #ith this medium. Although Mueller"-inton agar is reliable generally for susceptibility testing, results obtained #ith some batches may, on occasion, ary significantly. If a batch of medium does not support adequate gro#th of a test organism, :ones obtained in a disk diffusion test #ill usually be larger than e!pected and may e!ceed the acceptable quality control limits. )nly Mueller"-inton medium formulations that ha e been tested according to, and that meet the acceptance limits described in, D77L, document M=/"A." 'rotocols for + aluating Cehydrated Mueller"-inton Agar should be used. Preparation o$ Mueller73inton Agar Mueller"-inton agar preparation includes the follo#ing steps. 8. /. Mueller"-inton agar should be prepared from a commercially a ailable dehydrated base according to the manufacturerEs instructions. Immediately after autocla ing, allo# it to cool in a 06 to 697 #ater bath.

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'our the freshly prepared and cooled medium into glass or plastic, flat"bottomed 'etri dishes on a le el, hori:ontal surface to gi e a uniform depth of appro!imately 0 mm. $his corresponds to =9 to .9 ml of medium for plates #ith diameters of 869 mm and /6 to 59 ml for plates #ith a diameter of 899 mm.

0.

$he agar medium should be allo#ed to cool to room temperature and, unless the plate is used the same day, stored in a refrigerator (/ to <7).

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'lates should be used #ithin se en days after preparation unless adequate precautions, such as #rapping in plastic, ha e been taken to minimi:e drying of the agar.

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A representati e sample of each batch of plates should be e!amined for sterility by incubating at 59 to 567 for /0 hours or longer.

!. Preparation o$ antibiotic stoc) solutions Antibitiotics may be recei ed as po#ders or tablets. It is recommended to obtain pure antibiotics from commercial sources, and not use in4ectable solutions. 'o#ders must be accurately #eighed and dissol ed in the appropriate diluents (Anne!ure III) to yield the required concentration, using sterile glass#are. ,tandard strains of stock cultures should be used to e aluate the antibiotic stock solution. If satisfactory, the stock can be aliquoted in 6 ml olumes and fro:en at "/9F7 or "=9F7.

,tock solutions are prepared using the formula (8999>') G ? G 7H&, #here 'Ipotency of the anitbiotic base, ?H olume in ml required, 7Hfinal concentration of solution and &H#eight of the antimicrobial to be dissol ed in ?. Preparation o$ dried $ilter paper discs &hatman filter paper no. 8 is used to prepare discs appro!imately = mm in diameter, #hich are placed in a 'etri dish and sterili:ed in a hot air o en. $he loop used for deli ering the antibiotics is made of /9 gauge #ire and has a diameter of / mm. $his deli ers 9.996 ml of antibiotics to each disc. Storage o$ commercial antimicrobial discs 7artridges containing commercially prepared paper disks specifically for susceptibility testing are generally packaged to ensure appropriate anhydrous conditions. Ciscs should be stored as follo#s1 2 Refrigerate the containers at <7 or belo#, or free:e at "80 7 or belo#, in a nonfrost"free free:er until needed. ,ealed packages of disks that contain drugs from the @"lactam class should be stored fro:en, e!cept for a small #orking supply, #hich may be refrigerated for at most one #eek. ,ome labile agents (e.g., imipenem, cefaclor, and cla ulanic acid combinations) may retain greater stability if stored fro:en until the day of use. 2 $he unopened disc containers should be remo ed from the refrigerator or free:er one to t#o hours before use, so they may equilibrate to room temperature before

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opening. $his procedure minimi:es the amount of condensation that occurs #hen #arm air contacts cold disks. 2 )nce a cartridge of discs has been remo ed from its sealed package, it should be placed in a tightly sealed, desiccated container. &hen using a disc"dispensing apparatus, it should be fitted #ith a tight co er and supplied #ith an adequate desiccant. $he dispenser should be allo#ed to #arm to room temperature before opening. +!cessi e moisture should be a oided by replacing the desiccant #hen the indicator changes color. 2 2 &hen not in use, the dispensing apparatus containing the discs should al#ays be refrigerated. )nly those discs that ha e not reached the manufacturerEs e!piration date stated on the label may be used. Ciscs should be discarded on the e!piration date. Turbidity standard $or inoculum preparation $o standardi:e the inoculum density for a susceptibility test, a Ba,) 0 turbidity standard, equi alent to a 9.6 Mc(arland standard or its optical equi alent (e.g., late! particle suspension), should be used. A Ba,)0 9.6 Mc(arland standard may be prepared as follo#s1 8. A 9.6"ml aliquot of 9.90< mol>L Ba7l/ (8.8.6J #> Ba7l/ . /-/)) is added to ;;.6 ml of 9.8< mol>L -/,)0 (8J > ) #ith constant stirring to maintain a suspension. /. $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

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absorbance.

$he absorbance at =/6 nm should be 9.99< to 9.89 for the 9.6

Mc(arland standard. 5. $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. 0. $hese tubes should be tightly sealed and stored in the dark at room temperature. 6. $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. If large particles appear, the standard should be replaced. Late! particle suspensions should be mi!ed by in erting gently, not on a orte! mi!er =. $he barium sulfate standards should be replaced or their densities erified monthly. (isc di$$usion met&ods $he Airby"Bauer and ,tokesE methods are usually used for antimicrobial susceptibility testing, #ith the Airby"Bauer method being recommended by the D77L,. $he accuracy and reproducibility of this test are dependent on maintaining a standard set of procedures as described here. D77L, is an international, interdisciplinary, non"profit, non"go ernmental organi:ation composed of medical professionals, go ernment, industry, healthcare pro iders, educators etc. It promotes accurate antimicrobial susceptibility testing (A,$) and appropriate reporting by de eloping standard reference methods, interpretati e criteria for the results of standard A,$ methods, establishing quality control parameters for standard test

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methods, pro ides testing and reporting strategies that are clinically rele ant and cost" effecti e Interpretati e criteria of D77L, are de eloped based on international collaborati e studies and #ell correlated #ith MI7Ks and the results ha e corroborated #ith clinical data. Based on study results D77L, interpretati e criteria are re ised frequently. D77L, is appro ed by (CA"3,A and recommended by &-). 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. At least three to fi e #ell"isolated colonies of the same morphological type are selected from an agar plate culture. $he top of each colony is touched #ith a loop, and the gro#th is transferred into a tube containing 0 to 6 ml of a suitable broth medium, such as tryptic soy broth. /. 5. $he broth culture is incubated at 567 until it achie es or e!ceeds the turbidity of the 9.6 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 9.6 Mc(arland standard. $his results in a suspension containing appro!imately 8 to / ! 89 < $o perform this step properly, either a 7(3>ml for E.coli A$77 /6;//.

photometric de ice can be used or, if done isually, adequate light is needed to

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isually compare the inoculum tube and the 9.6 Mc(arland standard against a card #ith a #hite background and contrasting black lines. (irect Colony Suspension Met&od 8. As a con enient alternati e to the gro#th method, 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, such as blood agar, should be used). $he suspension is ad4usted to match the 9.6 Mc(arland turbidity standard, using saline and a orte! mi!er. /. $his approach is the recommended method for testing the fastidious organisms, Haemophilus spp., N. gonorrhoeae, and streptococci, and for testing staphylococci for potential methicillin or o!acillin resistance. Inoculation o$ Test Plates 8. )ptimally, #ithin 86 minutes after ad4usting the turbidity of the inoculum suspension, a sterile cotton s#ab is dipped into the ad4usted suspension. $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. $his #ill remo e e!cess inoculum from the s#ab. /. $he dried surface of a MLeller"-inton agar plate is inoculated by streaking the s#ab o er the entire sterile agar surface. $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. As a final step, the rim of the agar is s#abbed.

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5.

$he lid may be left a4ar for 5 to 6 minutes, but no more than 86 minutes, to allo# for any e!cess surface moisture to be absorbed before applying the drug impregnated disks.

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

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

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

".! (ilution Met&ods

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Cilution susceptibility testing methods are used to determine the minimal concentration of antimicrobial to inhibit or kill the microorganism. $his can be achie ed by dilution of antimicrobial in either agar or broth media. Antimicrobials are tested in log / serial dilutions (t#o fold). 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.g. bacterial endocarditis, the quantitation of antibiotic action is"a" is the pathogen needs to be more precise. Also the terms O,usceptibleK and OResistantK can ha e a realistic interpretation. $hus #hen in doubt, 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. ,rot& (ilution Met&od $he Broth Cilution method is a simple procedure for testing a small number of isolates, e en single isolate. It has the added ad antage that the same tubes can be taken for MB7 tests also1

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Materials ,terile graduated pipettes of 89ml, 6ml, /ml and 8ml ,terile capped ..6 ! 8.5 cm tubes > small scre#"capped bottles, 'asteur pipettes, o ernight broth culture of test and control organisms ( same as for disc diffusion tests), 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.), required sol ent for the antibiotic, sterile Cistilled &ater " 699ml and suitable nutrient broth medium. $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#. Stoc) solution ,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 ?

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+!ample1 (or making 89 ml solution of the strength 89,999mg>l from po#der base #hose potency is ;<9 mg per gram,the quantities of the antimicrobials required is &H 8999 """"""" ! 89 ! 89H89/.90mg ;<9 Dote1the stock solutions are made in higher concentrations to maintain their keeping qualities and stored in suitable aliquots at "/9 o7 .)nce taken out,they should not be refro:en or reused. ,uggested dilution ranges of some antimicrobials are sho#n in Anne!ure II. Met&od 'repare stock dilutions of the antibiotic of concentrations 8999 and 899 Pg>L as required from original stock solution (89,999mg>L). Arrange t#o ro#s of 8/ sterile ..6 !8.5 cm capped tubes in the rack. In a sterile 59ml (uni ersal) scre# capped bottle, prepare <ml of broth containing the concentration of antibiotic required for the first tube in each ro# from the appropriate stock solution already made. Mi! the contents of the uni ersal bottle using a pipette and transfer /ml to the first tube in each ro#. 3sing a fresh pipette ,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#. 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. 'lace /ml of antibiotic free broth to the last tube in each ro#. Inoculate one ro# #ith one drop of an o ernight broth culture of the test organism diluted appro!imately
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to 8 in 8999 in a suitable broth and the second ro# #ith the control organism of kno#n sensiti ity similarly diluted. $he result of the test is significantly affected by the si:e of the inoculum.$he test mi!ture should contain 89= organism>ml.If the broth culture used has gro#n poorly,it may be necessary to use this undiluted. Incubate tubes for 8< hours at 5.o7. 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. Calculations $or t&e preparation o$ t&e original dilution. $his often presents problems to those unaccustomed to performing these tests. $he follo#ing method ad ocated by 'amela M &ater#orth is presented. 7alculate the total olume required for the first dilution. $#o sets of dilution are being prepared (one for the test and one for the control), each in /ml olumes i"e a total of 0 ml for each concentration as 0ml is required to make the second dilution, the total requirement is <ml. Do# calculate the total amount of the antibiotic required for <ml. (or =0 g>l concentration, <!=0mg>l H68/Pg in < ml. 'lace a decimal point after the first figure (6.8/) and take this olume in ml (i.e 6.8/ ml) of the dilution belo# 68/mg>l and make upto <ml #ith broth. In this e!ample gi en abo e, 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. 8.= ml of 89 mg> l I =.0 ml of broth.

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-eading o$ result MI7 is e!pressed as the lo#est dilution, #hich inhibited gro#th 4udged by lack of turbidity in the tube. Because ery faint turbidity may be gi en by the inoculum itself, the inoculated tube kept in the refrigerator o ernight may be used as the standard for the determination of complete inhibition. ,tandard strain of kno#n MI7 alue run #ith the test is used as the control to check the reagents and conditions. Minimum ,actericidal Concentrations 8M,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. Met&od Cilutions and inoculations are prepared in the same manner as described for the determination of MI7. $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. o7 o ernight. $he tubes are also incubated o ernight at 5.o7. Read the MI7 of the control organism to check that the drug concentrations are correct. Dote the lo#est concentration inhibiting gro#th of the organisms and record this as the MI7. ,ubculture all tubes not

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sho#ing isible gro#th in the same manner as the control tube described abo e and incubate at 5.o7 o ernight. 7ompare the amount of gro#th from the control tube before incubation, #hich represents the original inoculum. $he test must include a second set of the same dilutions inoculated #ith an organism of kno#n sensiti ity .$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.

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-eading o$ result $hese subcultures may sho# ,imilar number of colonies" indicating bacteriostasis only. A reduced number of colonies"indicating a partial or slo# bactericidal acti ity. Do gro#th" if the #hole inoculum has been killed $he highest dilution sho#ing at least ;;J inhibition is taken as MB7 Micro7brot& dilution test $his test uses double"strength MLeller"-inton broth, 0G strength antibiotic solutions prepared as serial t#o"fold dilutions and the test organism at a concentration of /!89 =>ml. In a ;= #ell plate, 899 l of double"strength M-B, 69 l each of the antibiotic dilutions and the organism suspension are mi!ed and incubated at 567 for 8<"/0 hours. $he lo#est concentration sho#ing inhibition of gro#th #ill be considered the MI7 of the organism. -eading o$ result MI7 is e!pressed as the highest dilution #hich inhibited gro#th 4udged by lack of turbidity in the tube. Because ery faint turbidity may be gi en by the inoculum itself,the inoculated tube kept in the refrigerator o ernight may be used as the standard for the determination of complete inhibition. ,tandard strain of kno#n MI7, run #ith the test is used as the control to check the reagents and conditions.

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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 .If only one organism is to be tested e.g M.tuberculosis,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.$he dilutions are made in a small olume of #ater and added to agar #hich has been melted and cooled to not more than =9o7.Blood may be added and if Ochocolate agarK is required,the medium must be heated before the antibiotic is added. It #ould be con enient to use ;9 mm diameter petri dishes and add one ml of desired drug dilutions to 8; ml of broth.$he factor of agar dilution must be allo#ed for in the first calculation as follo#s. final olume of medium in plate $op antibiotic concentrations $otal amount of drug 8 ml of #ater /ml of 8/<9 Pg >ml #ill be required to start the dilution H /6=9Pg in / ml H 8./<ml of /999Pg >ml Q 9../ ml of #ater. 8 ml of this #ill be added to 8; ml agar. (Dote stock dilution of /999Pg >ml is required for this range of MI7) T&e :uic)est 5ay to prepare a range o$ dilutions in agar is as $ollo5s; H /9 ml H =0mg>l H 8/<9Pg to be added to

25

Label a sterile petri dish on the base for each concentration required. 'repare the dilutions in #ater placing 8 ml of each in the appropriate dish. )ne ml #ater is added to a control plate. 'ipette 8; ml melted agar, cooled to 66 o7 to each plate and mi! thoroughly. Adequate mi!ing is essential and if sufficient technical e!pertise is not a ailable for the skilled manipulation, 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. After the plates ha e set they should be #ell dried at 5.o7 #ith their lids tipped for /9 to 59 minutes in an incubator. $hey are then inoculated either #ith a multiple inoculators as spots or #ith a #ire loop or a platinum loop calibrated to deli er 9.998ml spread o er a small area. In either case the culture should be diluted to contain 89 6 to 89= organisms per ml. &ith ordinary fast gro#ing organisms, this can be obtained appro!imately by adding 6 Pl of an o ernight broth culture to 6ml broth or peptone #ater. It is possible to test spreading organism such as P.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. Although s#arming of P.mirabilis can be pre ented by the use of higher concentration of agar in the medium, 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. ,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.

26

-eading o$ results $he antibiotic concentration of the first plate sho#ing ;;J inhibition is taken as the MI7 for the organism. ". (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 ( ).$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.. A predefined stable antimicrobial gradient is present on a thin inert carrier strip. &hen this + test strip is applied onto an inoculated agar plate, there is an immediate release of the drug. (ollo#ing incubation, a symmetrical inhibition ellipse is produced. $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 . + test can be used to determine MI7 for fastidious organisms like S. pneumoniae, @"hemolytic streptococci, N.gonorrhoeae, Haemophilus sp. and anaerobes. It can also be used for Donfermenting *ram Degati e bacilli (D(*DB) for eg" Pseudomonas sp. and Burkholderia pseudomallei. Resistance of ma4or consequence may be detected for e.g., the test is ery useful in detecting glycopeptide resistant +nterococci (*R+) and glycopeptide intermediate
27

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

28

#ith additional suspending medium. If the turbidity is too light additional cells should be added to the suspension. (or S.pneumoniae S Cirect colony suspension is made in normal saline and turbidity ad4usted to 9.6 Mc(arland standards. &ithin 86 minutes after ad4usting the turbidity of the suspension the plate should be inoculated. Inoculation o$ t&e susceptibility test media After proper turbidity is achie ed, a ne# sterile s#ab (cotton or Cacron) is submerged in the suspension, lifted out of the broth, and the e!cess fluid is remo ed by pressing and rotating the s#ab against the #all of the tube. $he s#ab is then used to inoculate the entire surface of the supplemented Mueller -inton agar plate three times, rotating the plate =9 degrees bet#een each inoculation. $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 discs should be placed on the agar #ith sterile forceps and tapped gently to ensure the adherence to the agar. $he plates containing the disks are incubated at 56o7 for 8= to 8< h in an in erted position in a 6J 7) / incubator. A candle e!tinction 4ar may be used if a 7)/ incubator is not a ailable. Estimating t&e susceptibility o$ t&e strains After o ernight incubation, the diameter of each :one of inhibition is measured #ith a ruler or calipers. $he :ones of inhibition on the media containing blood are measured from the top surface of the plate #ith the top remo ed. It is con enient to use a ruler #ith a handle attached for these measurements, holding the ruler o er the surface of the

29

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

Antibiotic susceptibility o$ Haemophilus species

30

$he medium of choice for disc diffusion testing of Haemophilus sp. is -aemophilus $est Medium (-$M). MLeller"-inton chocolate agar is not recommended for routine testing of Haemophilus spp. In its agar form, -aemophilus $est medium consists of the follo#ing ingredients. 2 2 2 2 Mueller"-inton agar, 86 g>ml @"DAC, 86 g>ml bo ine hematin, and 6"mg>ml yeast e!tract.

$o make -$M, first a fresh hematin stock solution is prepared by dissol ing 69 mg of bo ine hematin po#der in 899 ml of 9.98 mol>L Da)- #ith heat and stirring until the po#der is thoroughly dissol ed. $hirty ml of the hematin stock solution are added to 8 L of M-A #ith 6 g of yeast e!tract. After autocla ing and cooling to 06 to 69 7, 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. $he p- should be ../ to ..0. Test Procedure 8. $he direct colony suspension procedure should be used #hen testing Haemophilus sp. 3sing colonies taken directly from an o ernight (preferably /9 to /0 hour) chocolate agar culture plate, a suspension of the test organism is prepared in
31

MLeller"-inton broth or 9.;J saline. $he suspension should be ad4usted to a turbidity equi alent to a 9.6 Mc(arland standard using a photometric de ice. $his suspension #ill contain appro!imately 8 to 0 ! 89< 7(3>ml. 7are must be e!ercised in preparing this suspension, because higher inoculum concentrations may lead to false"resistant results #ith some @"lactam antibiotics, particularly #hen @"lactamase producing strains of H. influenzae are tested. &ithin 86 minutes after ad4usting the turbidity of the inoculum suspension, it should be used for plate inoculation. /. $he procedure for the disc test should be follo#ed as described for nonfastidious bacteria, e!cept that, in general, no more than ; discs should be applied to the surface of a 869"mm plate or no more than 0 discs on a 899"mm plate. 5. 'lates are incubated at 567 in an atmosphere of 6J 7)/ for 8= to 8< hours before measuring the :ones of inhibition. 0. $he :one margin should be considered as the area sho#ing no ob ious gro#th isible #ith the unaided eye. (aint gro#th of tiny colonies that may appear to fade from the more ob ious :one should be ignored in the measurement.

>one (iameter Interpreti2e Criteria

32

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

33

Antibiotic susceptibility testing $or Neisseria gonorrhoeae $he recommended medium for testing N. gonorrhoeae consists of *7 agar to #hich a 8J defined gro#th supplement is added after autocla ing. 7ysteine"free gro#th supplement is not required for disc testing. +nriched chocolate agar is not recommended for susceptibility testing of N.gonorrhoeae. Test Procedure 8. $he direct colony suspension procedure should be used #hen testing N. gonorrhea. 3sing colonies taken directly from an o ernight chocolate agar culture plate, a suspension equi alent to that of the 9.6 Mc(arland standard is prepared in either Mueller"-inton broth or 9.;J saline. &ithin 86 minutes after ad4usting the turbidity of the inoculum suspension, it should be used for plate inoculation. /. $he disc diffusion test procedure steps, as described for nonfastidious bacteria, should be follo#ed. Do more than ; 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. -o#e er, #hen testing some agents (e.g., quinolones) #hich produce e!tremely large :ones, fe#er discs may need to be tested per plate.

34

5.

$he plates are incubated at 567 in an atmosphere of 6J 7)/ for /9 to /0 hours before measuring the :ones of inhibition.

>one (iameter Interpreti2e Criteria $he antimicrobial agents suggested for routine testing of N. gonorrhoeae are indicated in Anne!ure I. +ach :one si:e is interpreted by reference to the $able /( (Mone Ciameter Interpretati e ,tandards and equi alent Minimum Inhibitory 7oncentration Breakpoints for N. gonorrhoeae) of the D77L, M899",8/1 'erformance ,tandards for Antimicrobial ,usceptibility $esting1 $#elfth Informational ,upplement as susceptible, intermediate and resistant. Cisc diffusion testing of N. gonorrhoeae #ith other agents is not recommended. D)$+1 )rganisms #ith 89"g penicillin disc :one diameters of T 8; mm generally produce @"lactamase. -o#e er, @"lactamase tests are faster and are therefore preferred for recognition of this plasmid"mediated resistance. )rganisms #ith plasmid"mediated resistance to tetracycline also ha e :ones of inhibition (59" g tetracycline discs) of T 8; mm. 7hromosomal mechanisms of resistance to penicillin and tetracycline produce larger :one diameters, #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. gonorrhoeae) of the D77L, M899",8/1 'erformance ,tandards for Antimicrobial ,usceptibility $esting1 $#elfth Informational ,upplement.

35

(etermination o$ MIC $or #astidious organisms T&e Agar dilution met&od Standardi=ation o$ inoculum. $he inoculum should be an acti ely gro#ing culture diluted in saline to 89 0 to 896 microorganisms per ml. (or S.pneumoniae S Cirect colony suspension from a 8/"86 hour culture from $,BA medium is to be used. $he colonies are suspended in 9.6ml of normal saline and the opacity ad4usted to Mc(arland 9.6. A 8>89 dilution of this suspension is made and #ithin 86 minutes of making the diluted suspension the test plates should be inoculated #ith either a platinum loop calibrated to deli er 9.998ml or multipoint inoculator. (or N.gonorrhoeae and H. influenzae" ,imilar to the procedure described abo e for S.pneumoniae Inoculation o$ test plate In general the inoculum should be applied as a spot that co ers a circle about 6"<mm in diameter .A platinum loop calibrated to deli er 9.998ml of the inoculum is used to spot inoculate the cultures. Appropriate A$77 quality control organism(s) should be included along #ith each test. Inoculated plates are left undisturbed until the spots of inoculum ha e dried.

36

Incubation After the spots of inoculum ha e dried, 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. -eading $he control plate should sho# the gro#th of the U7 test organism. $he MI7 of the quality control strain should be in the e!pected quality control range. $he end point is the lo#est concentration of antibiotic that completely inhibits gro#th. A barely isible ha:iness or single colony should be disregarded. Results are reported as the MI7 in micrograms or units>ml. Interpretation is made in accordance to the guidelines laid do#n in the D77L, M899",8/1 'erformance ,tandards for Antimicrobial ,usceptibility $esting1 $#elfth Informational ,upplement (MI7 Interpretati e ,tandards) as susceptible, intermediate and resistant.

37

'. 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, ma4or errors and ery ma4or errors. $his is achie ed by comparing disk diffusion, #hich is #idely used to report #ith the MI7, #hich is reference method. $he follo#ing flo# chart sho#s the errors inreporting.(adopted from Manual of 7linical Microbiology, .th edition)

-esistant

Minor +rror

?ery Ma4or +rror

MIC 8 g?ml9

Minor +rror

Intermediate

Minor +rror

Ma4or +rror

Minor +rror

Susceptible

(is) (i$$usion (iameter 8mm9

38

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

39

Pseudomonas aeruginosa A$77 /.<65 (for aminoglycosides) Enterococcus faecalis A$77 /;/8/ (for checking of thymidine or thymine le el of M-A) Haemophilus influenzae A$77 0;.== (for cephalosporins) Haemophilus influenzae A$77 89/88 (for medium control) Neissseria gonorrheae A$77 0;//= ,tock cultures should be kept at ".97 in Brucella broth #ith 89J glycerol for up to 5 years. Before use as a U7 strain, the strain should be subcultured at least t#ice and retested for characteristic features. &orking cultures are maintained on $,A slants at /" <7 for up to / #eeks.

.. Standard Met&ods #or T&e (etection O$ Antibacterial -esistance. (etection o$ Oxacillin/Methicillin-resistant Staphylococcus aureus 8M-SA9 ,trains that are o!acillin and methicillin resistant, historically termed methicillin"resistant S. aureus (MR,A), are resistant to all beta"lactam agents, including cephalosporins and carbapenems. MR,A isolates often are multiply resistant to commonly used ancomycin (minimum antimicrobial agents, including erythromycin, clindamycin, and tetracycline. ,ince 8;;=, reports of MR,A strains #ith decreased susceptibility to inhibitory concentration VMI7W, R< g>ml) ha e been published.

40

*lycopeptides, ancomycin and $eicoplanin are the only drug of choice for treatment of se ere MR,A infections, although some strains remain susceptible to fluoroquinolones, trimethoprim>sulfametho!a:ole, gentamicin, or rifampin. Because of the rapid emergence of rifampin resistance, this drug should ne er be used as a single agent to treat MR,A infections. $he Dational 7ommittee for 7linical Laboratory ,tandards (D77L,) has recommended %,creening $est for )!acillin"resistant S. aureusX and uses an agar plate containing = microg>ml of o!acillin and MLeller"-inton agar supplemented #ith Da7l (0J #> N 9.=< mol>L). $hese plates can be stored refrigerated for up to / #eeks. $he inoculum is prepared by matching a 9.6 Mc(arland tube. $#o methods can be follo#ed for inoculation1 8. Cilute the supension 81899, and inoculate 89 microL on the plate, to get an inoculum of 890 7(3. /. Cip a s#ab in the suspension and e!press e!cess fluid by pressing s#ab against the #all of the tube. ,treak s#ab o er a 8"8.6 inch area. In both methods, any gro#th after /0 hours incubation at 567 denotes o!acillin resistance, if controls are satisfactory. 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

culture. 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. -eteroresistance is a problem for clinical laboratory personnel because cells e!pressing resistance may gro# more slo#ly than the susceptible population. $his is #hy isolates being tested against o!acillin, methicillin, or nafcillin should be incubated at 56 7 for a full /0 hours before reading. $he breakpoints for S. aureus are different from those for coagulase"negati e staphylococci (7oD,). itro. $his phenomenon is termed heteroresistance and occurs in staphylococci resistant to penicillinase"stable penicillins,

MICs

O1acillin Susceptible

O1acillin Intermediate

O1acillin -esistant

S. aureus T/ g>ml 7oD, T9./6g >ml

no intermediate MI7

MI7R0 g >ml MI7R9.6 g >ml

no intermediate MI7

>one si=es O1acillin Susceptible S. aureus 7oD, R85 mm R8< mm

O1acillin Intermediate

O1acillin -esistant T89 mm T8. mm

88"8/ mm no intermediate :one

42

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

43

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

44

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

a:treonam T /. mm cefota!ime ceftria!one T /. mm T /6 mm

45

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

46

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

47

(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= g>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 (< g>ml) and resistant (R8= g>ml) breakpoints. ,ome species, such as P. mirabilis, P. #ulgaris, and Morganella morganii, often ha e MI7s (8"0 g>ml) 4ust belo# the carbapenem intermediate breakpoint of < mg>ml. Most other species of +nterobacteriaceae are ery susceptible (T9.6 g>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

(etection o$ -esistant Enterococci Penicillin?Ampicillin -esistance +nterococci may be resistant to penicillin and ampicillin because of production of lo#" affinity, penicillin"binding proteins ('B's) or, less commonly, because of the production of @"lactamase. $he disc diffusion test can accurately detect isolates #ith altered 'B's, but it #ill not reliably detect @"lactamase producing strains. $he rare @"lactamase" producing strains are detected best by using a direct, nitrocefin"based, @"lactamase test. 7ertain penicillin" ampicillin" resistant enterococci may possess high"le el resistance (i.e., penicillin MI7s R 8/< g>ml or ampicillin MI7s R =0 g>ml). $he disc test #ill not differentiate those #ith normal resistance from this high"le el resistance. (or enterococci reco ered from blood and 7,(, the laboratory should consider determining the actual MI7 for penicillin or ampicillin since E. faecium strains #ith normal lo#er le el resistance (penicillin MI7s T =0 g>ml and ampicillin T 5/ g>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.

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

e idence of small colonies or a light film gro#ing #ithin the :one. An intermediate category result by the disc diffusion test should be ancomycin MI7. 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. ,pecial, high"content gentamicin (8/9 g) and streptomycin (599 g) discs can be used to screen for this type of resistance. Do :one of inhibition indicates resistance, and :ones of R 89 mm indicate a lack of high"le el resistance. ,trains that yield :ones of . to ; mm should be e!amined using a dilution screen test. )ther aminoglycosides need not be tested, because their acti ities against enterococci are not superior to gentamicin or streptomycin. Aminoglycoside -esistance in Enterobacteriaceae and Pseudomonas aeruginosa Resistance to one aminoglycoside may not predict resistance to the others. In general, resistance is relati ely common in P. aeruginosa but less common in +nterobacteriaceae. +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). $herefore, the pre alence of amikacin resistance can be lo#er than the pre alence of resistance to gentamicin and tobramycin, depending upon the resistance mechanisms present at a healthcare facility. erified by determining the

52

Aminoglycosides are not clinically effecti e against Salmonella species and Shigella species, although they may appear susceptible, aminoglycosides should not be tested or reported.

0. Application o$ Computers in Antibacterial Susceptibility Testing Antimicrobial resistance is a global problem. +mergence of multidrug resistance has limited the therapeutic options, hence monitoring resistance is of paramount importance. Antimicrobial resistance monitoring #ill help to re ie# the current status of antimicrobial resistance locally, nationally and globally and helpful in minimi:ing the consequence of drug resistance, limit the emergence and spread of drug resistant pathogens. $housands of laboratories are distributed #orld"#ide and need to be linked to integrate data on most clinically rele ant organism on a daily basis to obtain an accurate picture of Yreal resistanceX. (or this purpose a soft#are #as de eloped for the management of routine laboratory results by &-) called O&-)D+$K. $his soft#are has focused on data analysis particularly on the results of A,$.

&-)D+$ 6.8 is a data base soft#are compatible #ith, &indo#s ;6, &indo#s ;<,

53

&indo#s D$ and later

ersions of &indo#s. Installation capacity of the soft#are

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

54

1+. ,ibliograp&y Coern *.?. ,usceptibility tests of fastidious bacteria. Manual of 7linical Microbiology, =th edition, Murray '.R, Baron +.Z, 'faller M.A, $eno er (.7, [olken R, American ,ociety for Microbiology, &ashington C7, 8;;6, '. 850/" 850;. Ira R. Bacteriology, ,tandard )perati e procedure manual for microbiology laboratories, Dational Institute of Biologicals. 8;;6, '.5";. Zohn C.$ and Zames -.Z Antimicrobial ,usceptibility testing1 *eneral 7onsiderations. Manual of 7linical Microbiology .th edition, Murray '.R, Baron +.Z, 'faller M.A, $eno er (.7, [olken R, American ,ociety for Microbiology, &ashington C7, 8;;;, '. 80=;"80.5. Dational 7ommittee for 7linical Laboratory ,tandards. Performance Standards for antimicrobial susceptibility testing. <th Informational ,upplement. M899 ,8/. Dational 7ommittee for 7linical Laboratory ,tandards, /99/. ?illano a, 'a. $hrupp L.C. ,usceptibility $esting of Antibiotic in Liquid Media. Antibiotics In Laboratory Medcine /nd +dition, ?ictor Lorian, &illiams and &ilkins, Balitimore. 8;<=N '. ;5"869.

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 &ater #orth '.M. Uuantitati e methods for bacterial sensiti ity testing. Laboratory methods in antimicrobial chemotherapy. Ree es C.,, 'hilips I, &illiams Z.C, &ise R. 7hurchill Li ingstone, Baltimore. 8;.<N '. 58"08. Aramer Z. and Airshbaum A. +ffect of paper on the performance assay in the control of antibiotic sensiti ity discs. ppl Microbiol 8;=81 ;N '. 550"55=. Rippere R.A. +ffects of paper on the performance of antibiotic"impregnated discs. & Pharma Sci 8;.<1 =.N '. 5=."5.8. Lalitha M.A, .Manayani C.Z, 'riya L, Zesudason M.? , $homas A, ,teinhoff M.7. + test as an alternati e to con entional MI7 determination for sur eillance of drug resistant S.pneumoniae. 'ndian &. Med (es. 8;;.1 89=N '. 699"695. Morley C.7. A simple method for testing the sensiti ity of #ound bacteria to penicillin and sulphathia:ole by use of impregnated blotting paper disc. &. Pathol Bacteriol. 8;061 6.N '. 5.;"5</. A guide to sensiti ity testing1 Report of #orking party on antibiotic sensiti ity testing of the British ,ociety for Antimicrobial 7hemotherapy. &. ntimicrob $hemothrap 8;;8N /.1 ,upplement C, '. 8"69.

56

Anne1ure I. Guidelines #or Antimicrobial Susceptibility Testing. Suggested ,attery O$ Antibiotics #or Susceptibility Testing. Staphylococcus sp. '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.

57

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

58

Anne1ure II. Suggested Antibiotic (ilution -anges #or MIC Testing ANTIMIC-O,IA@ AGENT 'enicillin * Methicillin Daficillin>)!acillin Ampicillin(*ram I e) Ampicillin(*ram" e) 7ephalosporin(*ram I e) 7ephalosporin I generation CONCENT-ATION -ANGE8Ag?ml9 9.986"5/ 9.995"=0 9.995"=0 9.86"5/ 9.85"/6= 9.986"5/ 9.85"/6=

(*ram " e) 7ephalosporin II\III generation 9.95"=0 (*ram" e) 7ephalosporin III generation (Pseudomonas sp.) ?ancomycin Amikacin *entamicin>$obramycin +rythromycin 7lindamycin Rifampicin 7hloramphenicol $etracycline $rimethoprim ,ulphametho!a:ole $rimethoprim(3$I,*ram " e) 9.85"/6= 9.986"8= 9.9="8/< 9.95"=0 9.986"5/ 9.986"5/ 9.986"5/ 9.9="/6= 9.9="/6= 9.95"=0 9.="8/8= 9.6"=0

59

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

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Anne1ure III. Sol2ents and (iluents $or Antibiotics

61

#ootNote; a. 7ephalosporins and cephems not listed abo e or in footnote d are solubli:ed (unless the manufacturer indicates other#ise) in phosphate buffer, p- =.9, 9.8 mol>L, and further diluted in sterile distilled #ater. b. $he diammonium salt of mo!alactam is ery stable, but it is almost pure R isomer. Ma!olactam for clinical use is a 818 mi!ture of R and , isomers. $herefore, the salt is dissol ed in 9.90 mol>L -7L and allo#ed to react for 8.6 to /.9 hours to con ert it to equal parts of both isomers. c. Alternati ely, nitrofurantoin is dissol ed in dimethyl sulfo!ide. d. $hese sol ents and diluents are for making stock solutions of antimicrobial agents that require sol ents other than #ater. $hey should be diluted further, as necessary, in #ater or broth. $he products kno#n to be suitable for #ater sol ents and diluents are amikacin, a:locillin, carbencillin, cefaclor, cefemandole, cefonicid, cefota!ime, cefopera:one, cefo!itin, cefti:o!ime, ceftria!:one, ciproflo!acin, clindamycin, gatiflo!acin (#ith stirring), gemiflo!acin, gentamicin, kanamycin, line:olid, mecillinam, meropenem, methicillin, metronida:ole, me:locillin, minocyclin, mo!iflo!acin, nafcillin, netilmycin, o!acillin, penicillin, piperacillin, quinupristin"dalfopristin, sparflo!acin, sulbactam, ta:obactam, teicoplanin, tetracyclines, tobramycin, trimethoprim (if lactate), trospectomycin and ancomycin.

62

e. Anhydrous sodium carbonate is used at a #eight of e!actly 89J of the cefta:idme to be used. $he sodium carbonate is dissol ed in solution in most of the required #ater. $he antibiotic is dissol ed in this sodium carbonate solution and #ater is added to desired olume. $he solution is to be used as soon as possible, but can be stored up to si! hours at not more than /67. f. $hese compounds are potentially to!ic. 7onsult the material safety datasheets (M,C,) a ailable from the product manufacturer before using any of these materials. g. 3se ] olume of #ater, then add glacial acetic acid drop#ise until dissol ed not to e!ceed /.6l>ml.

Anne1ure

III; -eproduced 5it& permissionB $rom NCC@S publication

M1++7S1! Per$ormance Standards $or Antimicrobial Testing; T5el$t& In$ormational Supplement 8IS,N 17%'! .7 "%" 7'9. Copies o$ t&e current edition may be obtained $rom NCC@SB 0"+ Cest 4alley -oadB Suite 1"++B CayneB Pennsyl2ania 10+.*71.0.B <SA.

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