Evaluation of an automatic gas chromatographic system ...downloads.hindawi.com/journals/jamc/1989/452512.pdfrecommendation, trypticase soybroth agar (TSBA) as a secondary medium, onto

Journal of Automatic Chemistry Vol. 11, No. 5 (September-October 1989), pp. 191-200

Evaluation of an automatic gaschromatographic system for theidentification of bacterial infective agents

C. Arcelloni, A. Griffini,* R. Paroni and P. A. BoniniIstituto Scientijqco H. S. Raffaele, Via Olgettina 60, 20132 Milan, Italy

The potential clinical application of gas chromatography tomicrobial identifcation was evaluated. A completely automatedsystem, the MIS (Microbial Identij3cation System; Hewlett-Packard) can analyse and identify pure strains by comparison oftheir cellular fatty acids patterns (C9-C2o) with the referenceparameters stored in a library. Three hundred and sixty-sevenstrains were tested, comparing the gas chromatographic resultswith those obtained by the traditional microbiological methods inthe bacteriology laboratory of our Institute. A standardizedextractive procedure wasfollowed to obtain thefatty acid methylesters (FAMEs), but some modifications to the recommendedprocedure were introduced in the bacterial growth procedures:colonies harvested not onlyfrom the recommendedgrowth media butalso from selective media routinely used in the bacteriologylaboratory were successfully examined. These modifications did not

influence the results but improved the ease for the user; goodagreement with the comparison method was observed as far as

identications ofgenus and species are concernedfor 238 cases.The major advantages of this computerized system are a reductionin the time required to obtain thejnal results, the elimination ofhuman errors by using the autosampler and a better inter-laboratorycomparability ofresults owing to a higher degree ofobjectivity. Onthe other hand, the limited throughput ofMIS (only 40 samples in24 h) prevents its use in a large routine laboratory; this technologyis appropriate in emergency cases, in taxonomic studies and as a

conjqrmatory method.

Introduction

Traditional techniques for the identification and classifi-cation ofmicrobial infective agents are based on morpho-logical, inmmuno-biochemical and physiological charac-teristics. Sometimes these parameters are insufficient to

classify some strains and, according to Kreig ], some ofthe routine methods so far used, especially for anaerobiccultures, are expensive and time consuming. Further,biochemical methods have a very variable discriminatorypower, giving poorly comparable results. Moss [2]showed that the gas chromatographic analysis of meta-bolic products or ofbacterial cell components offer a goodtool in clinical microbiological laboratories for identifyingthe infective agents and for studying the taxonomicclassification of bacteria. Goodfellow and Minnikin [3]and Brondz and Olsen [4] recently introduced newcriteria for classifying the microorganisms on the basis of

* Istituto di Biochimica e di Chimica, FacoltS di Agraria,Universitt di Milano, Milan, Italy.

the proteic, lipidic and saccharidic composition of thebacterial cell.

The lipidic components of the bacterial envelope were

particularly studied as specific markers for many strains:in Gram-positive bacteria, the cell lipids are concentratedin the plasma membrane whereas in Gram-negativebacteria lipoproteins and polar and non-polar lipids arelocated in the plasma or in the outer membrane. Thechemotaxonomic classification of Gram-positive bacteriacould be based only on the cellular fatty acid patternbecause the metabolic products of these bacteria(ketones, alcohols and amines) are not specific enough[3]. However, Brondz and Olsen [4] and Drucker [5]reported that short-chain (1-7 carbon atoms) andnon-hydroxylated fatty acids are specific components ofthe structure of anaerobic bacteria.

According to Asselineau and Asselineau [6], the intro-duction of fused-silica capillary columns with polar andnon-polar stationary phases in gas chromatographicanalysis has facilitated the identification of a largenumber of fatty acids and improved the resolution of thismethod for microbial identification.

The MIS (Microbial Identification System; Hewlett-Packard, Avondale, PA, USA) is a computerized andcompletely automated gas chromatographic apparatusfor the identification of aerobic and anaerobic bacteriabased on their cellular fatty acids composition (C9-C20).A pattern recognition program compares the fatty acidsofan unknown sample with those ofthe reference bacteriastored in a computer library. The unknown strain isidentified only if its fatty acids pattern has characteristicsclose to some of the patterns present in the library.

So far, the library contains the fatty acid patterns ofmanyGram-positive cocci, Gram-positive rods, Gram-negativecocci and Gram-negative fermenters and non-fermenters;it is expected to be updated for other anaerobic bacteria,yeasts, moulds and other fungi and nycobacteria.

In this paper, the potential application of the gaschromatographic MIS in clinical laboratories as a

support for and/or alternative tool to traditional micro-biological analyses is examined.

Abbreviations

BA blood agar; BHI brain heart infusion agar; ECLequivalent chain length; FAME fatty acid tnethyl

ester; FID flame-ionization detector; GC gas

0142-0453/89 $3.00 () 1989 Taylor & Francis Ltd.

C. Arcelloni et al. Automatic GC system for bacterial intkctive agents

chromatography; HPLC high-performance liquidchromatography; MCK MacConkey agar; MISMicrobial Identification System; MH Miiller-Hinton;MSA mannitol salt agar; PEA phenyl ethyl agar; RT

retention time; SI similarity index; KV Schdler-KV agar; TSB trypticase soy broth; TSBAtrypticase soy broth agar.

Materials and methods

Description of the microbial identification system

The MIS consists of the following: HP 5890A gaschromatograph (Hewlett-Packard), with methylphenyl-silicone fused-silica capillary column (25 m 0"2mm i.d.); flame ionization detector (FID); HP 3392Aintegrator; HP 9816S computer equipped with a 9133Hdisk drive; HP 2225A printer; and HP 7673A automatedinjector with sampler controller and sample tray for 100vials.

Operating conditions

Ultra-high purity hydrogen (SIO-ALPHAGAZ) wasutilized as the carrier gas. The column head pressure was10 lb in-2, the injector temperature was 250C and thedetector temperature was 300C. Other operatingparameters were: FID air, 400 ml min-1; FID H2, 30ml min-1; FID N2, 30 ml min-1; trap purge,40 ml min-1; septum purge, 5 ml min; splitting ratio,100:1; and splitter, 50 ml min-1.

At an initial oven temperature of 170 C, a temperatureprogram of 50 C min-1 was activated at injection andcontinued to a final temperature of 270C, which washeld isothermal for 2 min. The time required for each runwas 22 min and the re-equilibration of the columnrequired 3 min.

Extraction procedure

All the reagents were of HPLC grade. The cellular fattyacids were extracted and derivatized following a stan-dardized procedure. Reagent was 45 g of sodiumhydroxide (Merck, Darmstadt, FRG), 150 ml ofmethanol (Fluka, Buchs, Switzerland) and 150 ml ofdoubly distilled water. Reagent 2 was 325 ml of 6 M

hydrochloric acid (Carlo Erba, Milan, Italy) and 275 mlof methanol. Reagent 3 was 200 ml of hexane (Merck)and 200 ml of diethyl ether stabilized with 2% ethanol(Merck). Reagent 4 was 10"8 g of sodium hydroxide in900 ml of doubly distilled water.

Bacterial colonies were harvested with a 4-mm inoculat-ing loop and coated at the bottom of the glass tubes(Pyrex, 14 x 100 mm) provided with Teflon-linedscrew-caps. The amount of bacteria harvested with adouble collection was sufficient for processing. A 1-mlvolume of reagent was pipetted into each tube, mixedfor 5-10 s, heated at 100C in a block heater (Supelco,Bellefonte, PA, USA) for 5 min, mixed again and kept at100 C for 25 min.

The methylation of fatty acids was achieved by adding 2ml of reagent 2 to the cooled uncapped tubes, which were

192

then mixed for 5-10 s and heated at 80 C for 10 min. Thefatty acid methyl esters (FAMEs) were extracted byadding 1-25 ml ofreagent 3 and shaking gently for 10 minon a laboratory rotator. The lower aqueous phase wasremoved with a Pasteur pipette and discarded. The upperphase was washed with 3 ml of reagent 4 and shakengently for 10 min. Two thirds ofeach organic extract weretransferred with a Pasteur pipette to the autosamplervials (Teflon caps) for the gas chromatographic analysis.

Cultures

In the bacteriology laboratory of our Institute, differentaliquots of the same biological specimen were streaked asusual on four different media: blood agar (a non-specificmedium for a quantitative evaluation of the bacteria),mannitol salt agar (MSA) (specific for the growth ofStaphylococci, MacConkey Agar (MCK) for the identifi-cation of Gram-negative bacteria and Sabouraudmedium for growth offungi. These plates were incubatedat 37 C for 24 h. To detect anaerobic bacteria, specimenswere streaked both on Schidler’s medium, for quantita-tive evaluation, on Sch/idlar-KV agar (KV) for identifi-cation of Bacteriodes and on phenyl ethyl agar (PEA)Gram-negative cocci growth. The plates were thenincubated at 37C in an anaerobic atmosphere until asuitable growth was obtained (2-5 days). After theprimary isolation, both the aerobic and anaerobic strainswere further characterized with microscopic, biochemicaland serological tests. For the biochemical analyses API(Ayerst) and Enterotube (Roche) strips were used. Thepure strains were subsequently transplanted on Mtiller-Hinton (MH) medium for the antibodies sensitivity test,according to Bauer et al. [7]. For our gas chromatographicstudy, the aerobic colonies were harvested directly fromthe specific media or from Miiller-Hinton medium. Forsome aerobic strains, we used, according to the MISrecommendation, trypticase soy broth agar (TSBA) as a

secondary medium, onto which the previously isolatedcolonies were transferred. This medium consists of30 g oftrypticase soy broth (BBL, Becton Dickinson), 15 g ofBacto agar (Difco, Detroit, MI, USA) and of distilledwater. The ingredients were combined, boiled until theagar melted, autoclaved for 15 min at 121 C (15 lb in-)and cooled to 60C, then dispensed into sterile Petridishes. The cultures were incubated for 24 h at 28 C. Allthe anaerobic bacteria examined in this study wereharvested directly from KV or from PEA, without a

secondary isolation on the media suggested by Hewlett-Packard.

Samples analysed

We studied 367 strains isolated ti’om routine specimens ofthe bacteriology laboratory of our Institute. Biologicalspecimens were represented by blood, catheter points,pus from deep wounds, swabs (from pharynx, rectum,vagina) and urine. All the samples were analysed both bythe gas chromatographic technique and by classicalmicrobiological procedures used as a reference methodand the results were compared.

Calibration for MISFor quantitative calibration, the FAMEs mixture for a

C. Arcelloni et al. Automatic GC system for bacterial infective agents

Ft re: I)lTA:Ft5821401kttleIIh

bate eL r,ortt 13-SEP-a7 13J33:210ate ,L runJ 23-U6-87 11:51:02latt HitHt 23US-Y 17:40:13

1.496 272L3004) 0.0542.4432.9263.5413.5903.7343.9734.4ql

5.616

6.959

I.II

I,I

I.0

I.I

ECL I Area Cot

7.039 SOLW.JI INC enk6114 0.02 1.218 . 9t0 .H142 0.026 1.147 10. 1010 10.tl Pel Mtcl42 0.02 t0.$l

30777 0.02 l.3 lt.l$2 100 2W 2.07 Peak MLcN 0.15 0.0 1.070 11.4 i0t0 1.05 PNk Mt .21571 0.031 1.0 12. 1210 10.23 Peek BLck77 0.0 1.013 13. 1310 5. Pe6k Mtck13 0.047 1.I

1320 0.037 .7 14. 1410 10.21 Pllk Mtck

1 0,02 0,%5 15.2 140 2 2.11 Peak Mtck 0.363 0.043 0.9 5. 14:0 3 [.01 Peak tck .19i72 0.043 0.4 1. lk:0 10.2 Pek Mtck .7705 0.045 0.943 17,0 1710 5.03 Peak Mtck.

0,047 0.fqt 17.23i t:0 2.15 -Peak Mtck 0.3174 0, 0.9 1l. 10 10.1l Peak tch17 0,07 0, 1. 1:0 5. Peak Mtck 0.1

Ct

27263000 |71/ 1’1,052 P.N IM7050

tlTL’IIIItl IIIITI011RITCIIII ERROR (1t115) IS 0.003.

Figure 1. Gas chromatographic projqle of aquantitative standard calibration mixture con-taining 17fatty acid methyl esters (FAMEs).The upper part shows the GC profile and inthe lower part the printed report is shown.Section A: Bottle, position ofthe sample in thesample-tray; ID, identification number of thevarious samples programmed in the SampleTable; Name, name of the sampleprogrammed in the Sample Table. Section B:RT, peak retention time (in minutes); Area,peak absolute area; Ar/Ht, peak width athalf-height; Respon, correction factor of thepeak absolute areas obtainedfrom the compari-son with the peak area of an ideal standardmixture; ECL, equivalent chain length of theideal standard calibration mixture (see text);Name, name of thefatty acid identifed by thesystem; Area %, peak area as a percentage ofthe NamedArea (see below); Comment 1, thissection shows if the eluted peak has an RT inthe range C9-Co (peak match) or not (>RTor <RT); in the latter case the peak is notidentife& the number reported on the rightside of Peak match +... indicates theshifting of the ECL obtained from the idealone stored in the computer memory. Section C:Solvent Area, area of the hexane/ether peak;Total Area, amount of the peak areas elutedbetween C9 and Co; Named Area, size of theidentiofiedpeak area, % Named, percentage ofthe total area; Total Amnt, product betweenthe Area Named and the Respon factor; NbrRef, number of the peaks assumed as refer-ences; if present, these peaks are listed inComment 2; obviously, this indication is usedonly for the unknown samples and not forstandards.

capillary column (Supelco) contains 12 straight-chainfatty acids (C9 0-C20 0) and 5 hydroxy acids (C 10 020H, C 10 0 30H, C 14 0 20H, C 14 0 30H, C 16 0 30H).The straight-chain fatty acids are used as references forthe identification of the FAMEs in the bacterial samples,while the hydroxy acids are added to detect the columndegradation, which is evident from tailed peaks. Theinjection of the quantitative calibration mixture wasprogrammed in the Sequence Table ofthe M|S computerat the beginning of each batch of samples and after every15 analyses.

Every 350 samples, three different qualitative calibrationmixtures were injected. These mixtures are combinedextracts of selected bacteria containing a different assort-

ment of fatty acids (about 80 in each mixture). Some ofthese fatty acids are not available commercially and arespecific for particular microorganisms only. The patternrecognition program could identify up to 150 differentfatty acids.

System programmingAfter identification by the reference method, the sampleschosen for the extraction, methylation and subsequentGC analysis were programmed by the user in a ’SampleTable,’ according to the MIS computer software, andthen analysed.

The qualitative and quantitative calibration mixturesinjections were also programmed in the Sequence Table.

193


( r,,al(: cnWyial Identihcation Svste (Uer: I,I) (Sof(are /n: 2611RI00(02 Co.poter /n: 2510#12111078

Figure 2. Gas chromatographic profile andanalysis report of a qualitative calibrationmixture. For the description of the analysisreport, see legend ofjgure 1. In addition:Section B: in Comment 1 column, ECLDeviates, shifting of the obtained ECLfromthe ideal one; in Comment 2 column, Reference+ peaks used as reference to adjust theRT of the other peaks. The value (+_) showsthe direction of the adjustment given by thesystem. Section C: RefECL Shift, root meansquare (RMS) ofthe adjustments made on theRT of the peaks listed in Comment 2;Deviation, standard deviation of the mean ofthe ECL shift listed in Comment 1.

hle: OIi:F,B0.!00427Bottle:ID:H: (JlILlIRll C@L

Oate of report: 00-tI-98 12:24:10Date of run: 0t1-it-88 12:24:10

RT ree BrAf( Respon

1,g 379909 0.91. 5 0.027I.I lle 0.0II. 57 0.6I,2 I9 0.?I, 6 0.0232.09 I 0.0252.21 0.?2. I 0.030 1.28Z.612 I79 0.02 1,182

2.6 I131 0.026 1.172.?29 0.2.

15.%016.04116,419

IG.BI?17.B06.18,74

ECL H Rrea Cogent Comment

7.059 SOlMl PERK <mn7.575 in rt7,705 zn rt8.007 (mn rt8.2 (mnrt

8.184 mn rt8.]00 (mnrt

8.675 mn rt9.000 9:0 i.40 ECL deviate 0.000 Reference 0.0009.527 unkmn 9.521 O. 12 [CL devzate50.OOG9.604 I0:0 ISO 0.76. ECL deviates -0.001g, 777

19182 0.026 1.139 I0,000 I0:0 1.25 ECL deviates-0.000 Reference 0.0001763 0.045 I0.43

1!8 0.043 19.4%9323 0.053 0.946 19.549 18:0 30H 0.50 ECL deviates -0.002

15492 0.047 0,850 19.769 20:I CIS 11 0.84 ECL devzates -0. 00124335 0.048 0.555 20.0180 20:0 1.32 ECL deviates 0.000 Reference48 0.044 20.575 )xrt

59 0.053 20.905 >xrt

Slent r Total #Irea Nd flrea Haed To #in( Nbr lef Ref ECL Shzft [CL 1)eation

2448111) 180296 7).65 1754%2 t2 0.001 C

Results

In figures and 2 examples of chromatograms andanalysis reports of the standard mixtures used forquantitative and qualitative calibration are shown. TheGC profile is given during the run, while the analysisreport is printed at the end. For an explanation of all theGC parameters reported, see the legends of the figures.The equivalent chain length (ECL) is a mathematical

parameter calculated by the MIS software, which is veryimportant for the interpretation of an unknown peak.This value corresponds to the number ofcarbon atoms inthe fatty acid chain and allows the determination of thechemical structure of the unknown fatty acids eluting inthe analysis. By convention, the C-C0 straight-chainfatty acids were taken as reference points in the calcula-tion of the ECLs of all the other fatty acids contained inthe sample. The ECLs of the C-C0 straight-chain acids

194


Figure 3. Example ofchromatograph, analy-sis report and comparison chart ofpure strain0fStaphylococcus aureus analysis. For thereport see legends of jqgures 1 and 2. Inaddition: TSBA [Rev 2.0], identificationname of the library in which the sample wassearched; Staphylococcus, genus; aureus,species; aureus GC subgroup A, sub-species;0"225 SI, the similarity index in this exampleis 0"225 (see text). The comparison chart givesthefollowing information: on the left side allfatty acidsfound in both the unknown sampleand in a library entry are listed in the elutionorder while a scale of% is printed across thetop of the chart. Symbols: x percentage ofthe fatty acid found in the sample;

’.range ofthepercentage composition ofthe samele ,,’Icl, I..llT,,arlu, SI (e: .,), un: 2711011) ( /n: gll l?-Jl-S? : acid in the r@rence bacteria; * appears whenthe percentages of thefat acid in the sampleI0: IIS1 SIIL *O,C 9 te 0 r: l?-J-87 li::

ttl: S S[ [] and in the rrence bacteria are identical.

RT Area flr/Ht Respon EL ,e Comment Cogent

H, ,[A[ n r.818 O.O 1041 12.092 11:0 "3 deviates 0.002G.721 ’3S O.o5 585 I.G,7 I:0 ISO 0.45 [CL dvta-0.I frence 0.2

O.SGS ’". [SO .GO [CL das 0. Rfrr 0.I8. 72312 0.0I 0. 14"’I IS:OH,[O % devzaLe nu.nnnuuu Referee 0.19.9 2527 0.042 0,949 Ir GO, r’ deuzatas 0.I Reference 0.I0.4 8 O. 1.v O.S ur’ deflates 0.0 Reference-0.I11.S73 TT90 0.045 0.535 T. ?:0 [SOt1. 4003 0.045 0.939 IG.,., ,"’",. H],,or 2S, I0 [CL dezates -0.I Reference13.331 i2T 0.048 0.9 17.632 I:0 ISO 0.8o, r’ deusae .nnnn.uuu Reference-0.13. ,n, O.OB 0.53 1.0 1:0 S,, uurr’ deflates-0.000 Eeferere-0.21S.O SO O.T 0.93 T.3ff 15:0 [SO ." rr’ devzates ,.n ,,,an’ Reference-0.I15.5 ’0%3 0330.048 I., 15:0 RHT[ISO deuzates17.181 I 0 0.I . :0:0 !.S [C[ eu;ates 0. Eeferee

luent llr Total 8tea Hed rea )Led ’,,, Rent Hbr Ref [CL Oeu’atzon Ref [CL Shzft

4131J00 ISG9 ’%589 I00,00 149477 11

TSBR [Rev Staph1ococcus 0.225S. aureus O.2Z; S.I., a. aureus GC sabgroap 0.225

Coeartson wzLh TSBll [Reu aph,ococcu-aureus-aureu GC sroup Ots: 5.I5

11:0 ISO 3 "+-xI:01 ". x-+--.IS:O ISO ----15:0 RIIT[I ---t-X-.15:0 I -nt--

16:Q x"+-.17:9 ISO xt’"17:0 RIIT[ISO -’--"-i---" x18:0 I18:O +----.1:0 I -ix-.;; T[ln --+-, X20:0 ,<,.-+----.

195


Figure 4. In the upperpart a chromatogram ofone strain of Escherichia coli (A) andKluyvera cryocrescens (B) obtained in ourlaboratory is reported. In the lower part, the"ideal’ (reported in the library) pattern ofthesemicroorganisms is reported.

0.93 0.90 0.00 2.833.86 0.88 2.33 6.430.04 0.14 0.00 0.690.03 0.09 0.00 0.316.69 0.91 .31 8.900.gs 0.32 0.00 1.48

0.0! 0.04 0.00 0.24

unlmo 1Q.8

19 12:028 13:037 1:0 ISO39 14:046 unkno 14.503

? :I ClS II.7 .68 4. 21.5868 16:1 0.02 0.07 0.00 0.069 6:0 .24 2. 24. )6.668) 17:1 0.0 0. 0.00 0.6486 :0 .59 .2 5. 28.9)88 17:0 0.)5 0.0 0.00 1.6520 8:0 0. 0.2 0.000 9:0 12 .94 2.80 0.00 2.2

28 2L67 4.90 12.5

unknovn 10.928 1. 0.3419 12:0 4.6328 :0 0.2537 1.:0 ISI) 2 0.9 1:0 9.27no 1.50 O.

54 15:0 1.67 :1 CIS 14.668 16:1 0.0169 6:0

8 :1 0.0186 7:0 14. .88 :0 0.5 0.01 18:0 0.20 0.07 1:0 I 0.0 0.18110 :0 Cl1-2 1,90 1.4912 0.10 0.2512 7.0 1.28128 . .

0.98 1.960,77 3.52 6.260.40 O.OO 1.890.00 O.OO 0.2)0. 7.58 11.60.4 O.O0 1.191. O.O04.97 6.60 21.780. 0.0! 1.17

0.05 0.00 I.).70.0 0.0| 0.18

O.O| 1.51O.OI 1.9}0.0| 1.280.00 4.60.00 1.685.87 9.875.32 16.65

Table 1. Comparison between two different preparative methods.

Similarity index

Hewlett-PackardOrganism Our method method

Enterobacter cloacae 0"807 0’547Hafnia 0- 760 0-513Proteus mirabilis 0"316 0" 159Providentia stuartii 0"493 0"217Pseudomonas aeruginosa 0"621 0" 786Salmonella choleraesuis 0"345 0"300Staphylococcus aureus 0" 120 0" 190

The similarity indices of the same strains analysed both afteradditional cultivation on TSBA of the previously isolatedcolonies (Hewlett-Packard method) and directly from theroutine isolation media (our method) were compared to

quantify the influence of the growth media on the FAMEpattern and consequently on the MIS identification.

were assumed to be whole numbers between 9000 and20000. The relationship between the ECL and theretention time of an unknown peak is expressed by theequation

(Rtx- Rt)ECL

(Rtn + Rtn)

where Rtx is the retention time of the FAME x, Rtn is theretention time of the C(n:0), i.e. the straight-chain fattyacid that elutes before the FAME x and Rt + is the

retention time ofC(n + 1)"0, i.e. the straight-chain fattyacid eluting just after the FAME x.

The identification of the fatty acid structure is performedusing the ECL in a ’family plot’ in which various fattyacid families are represented in relation to the straight-chain length (x axes) and the retention time (y axes). TheECL reported in the qualitative and quantitative stan-dard mixture reports are those calculated under theoptimum analytical conditions. The identification of theunknown peaks during the sample analysis is performedon the basis of these ideal parameters. In figure 3 thechromatogram, the analysis report and the comparisonchart for the analysis of a pure strain of Staphylococcusaureus are shown. At the bottom of the report, in additionto the parameters reported for the calibration mixture,the genus and species and sub-species of the identifiedbacteria and the similarity index (SI) are given. The SI isa parameter that quantifies the reliability of MISidentification by measuring the overlapping ofthe FAMEpatterns ofthe sample and one ofthe various microorgan-isms stored in the computer library. The SI is a numberwhich indicates how closely the FAME composition ofanunknown sample compares with that of the libraryreference bacteria selected by the MIS. A value ofSImeans perfect overlapping and values less than indicatethat the patterns are not identical with a consequenthigher inaccuracy of the result. The unknown strain isusually identified with the reference bacteria which givesthe higher SI.

When the GC analysis is not good enough or the sample istoo dilute, concentrated or contaminated, the identifica-

196


Table 2. Day-to-@ precision of the gas chromatic analysis.

Day 4 5 6 8

MIS identification Similarity index M + SD CV%

Pseudomonasaeruginosa 0"012 0"010 0"009 0"010 0"010 0"012 0"006 0"006 0"010 + 0"001 10"0Staphylococcushominis 0"010 0"010 0"009 0"012 0"010 0"009 0"010 0"010 0"010 + 0"0009 9"0Kluyveracryocrescens 0"446 0"335 0"402 0"387 0"381 0"401 0"392 0"386 0"391 + 0"030 7-3Proteusmirabilis 0"407 0"402 0"416 0"339 0"409 0"396 0"410 0"398 0"397 + 0"022 5"7Shigelladysenteriae 0"498 0"425 0"433 0’492 0"523 0"489 0’421 0"428 0"458 + 0"037 8"0Streptococcusepidermidis 0.156 0"182 0"203 0"187 0"198 0"189 0’204 0"210 0"191 +_ 0"016 8"3Klebsiellaozenae 0"483 0"492 0"482 0"473 0"480 0"462 0"478 0"475 0"478_+ 0"008 1"7

Table 3. Bacterial identcation obtained by reference method (microbiological identifica-tion) and gas chromatographic analyses.

Bacteriology laboratoryidentification MIS identification Tribe Family

GRAM NEGATIVE AEROBES

Acinetobactercalcoaceticus (n 7)Citrobacterfreundii(n=9)

Escherichia coli(n 63)

Enterobacter cloacae(n 8)

Klebsiella ozaenae(n 16)

Morganella morganii(n= 4)

Proteus mirabilis(n 23)

Providentia stuartii

(n 3)Salmonella choleraesuis(n= 7)Pseudomonas aeruginosa(n 45)Serratia liquefaciens(n= l)

GRAM POSITIVE AEROBES

Staphylococcus aureus(n=83)

Staphylococcus hominis(n 46)

Staphylococcus epidermidis(n=9)

Streptococcuspyogenes(n= 7)Streptococcus agalactiae(n=9)

Acinetobacter calcoac. (7)

Citrobacterfreundii (5) SalmonelleaeEnterobacter cloacae (2) KlebsielleaeErwinia herbicola (1) ErwinieaeShigella dysenteriae (1) EscherichieaeEscherichia coli (21) EscherichieaeShigella dysenteriae (4) EscherichieaeKluivera cryocrescens (19)Citrobacterfreundii (12) SalrnonelleaeMorganella rnorganii (5) ProteeaeEnterobacter cloacae (2) Klebsielleae

Enterobacter cloacae (2) KlebsielleaeSerratia (1) KlebsielleaeEscherichia coli (2) EscherichieaeKluyvera cryocrescens (1)Proteus rnirabilis (1) ProteeaeMorganella rnorganii (1) Proteeae

Klebsiella ozaenae (6) KlebsielleaeEnterobacter cloacae (1) KlebsielleaeSerratia (2) KlebsielleaeKluyvera cryocrescens (2)Shigella dysenteriae (2) EscherichieaeMorganella rnorganii (1) ProteeaeSalmonella choleraesuis (2) Salrnonelleae

Morganella morganii (1) ProteeaeSerratia (2) Klebsielleae

Hafnia alvei (1) Klebsielleae

Proteus mirabilis (20) ProteeaeKluyvera cryocrescens (1)Enterobacter cloacae (2) Klebsielleae

Providentia stuartii (3) Proteeae

Salmonella choleraesuis (6) SalmonelleaeErwinia herbicola (1) Erwinieae

Pseudomonas aeruginosa (45)

Acinetobacter calcoac. (1)

Staphylococcus aureus (75)Staphylococcus horninis (8)

Staphylococcus hominis (16)Staphylococcus kloosii (15)Staphylococcus (14)Acinetobacter calcoac. (1)

Staphylococcus epidermidis (5)Staphylococcus (2)Staphilococcus kloosii (2)Streptococcuspyogenes (7)

Streptococcus agalactiae (2)Streptococcuspyogenes (4)Streptococcusjaecalis (3)

Klebsielleae

Neisseriaceae

Enterobacteriaceae

P eudomonadaceae

EnterobacteriaceaeNeisseriaceae

Micrococcaceae

Neisseriaceae

Streptococcaceae

197


Table 3- continued.

Bacteriology laboratoryidentification MIS identification Tribe Family

Streptococcus equisimilis(n 2) Streptococcuspyogenes (2)Streptococcusfaecalis Streptococcusfaecalis (1 O)(n tO)

Streptococcus Streptococcusl-hemoliticus (n 2) [l-hemoliticus (2)

GRAM NEGATIVE ANAEROBES

Bacteroidesfragilis Bacteroidesfragilis (3)(n 7) Bacteroides oris (3)

Fusobacterium necrosis (1)Fusobacterium necrosis Fusobacterium necrosis (2)

RA.M eoslrlVE ANAEROBES

Clostridium difficile Clostridium difficile (1)(n= 1)

Streptococcus intermedius(n 2) Streptococcuspyogenes (2)

Propionibacterium acnes Propionibacterium acnes (I)(n= I)

Bacteroidaceae

Bacillaceae

Streptococcaceae

Propionibacteriaceae

In the first column (on the left) the results of the microbiological identification (Genusand species) and the number of the samples tested are reported. In the second columnthe results of the MIS identification of the same strains are reported: results identicalwith the reference method are reported in the first line for each group in bold, whiledifferent results are listed below. The number of the respective cases is written inbrackets. Under ’Tribe’ and ’Family’ both the cases of agreement (in bold) anddisagreement between the two methods are indicated.

Table 4. Distribution of the percentage of disagreement between microbiological and GCbacterial identification.

Family Genus Species

Gram negative aerobes

1/186

(0.5%)70/186 70/186(37%) (37%)

Gram positive aerobes

1/168(0.6%)

1/168 51/168(0.6%) (30.3%)

Gram positive anaerobes

0/4 0/4 2/4(50%)

Gram negative anaerobes

0/9 1/9 4/9(11%) (44.5%)

tion cannot be carried out and a comment is reported atthe bottom of the sample report. A graphical representa-tion of the library search by means of the comparisonchart (figure 3) is printed if requested by the operator.The computer program can compare the unknownsample pattern with those of 1, 2, 3 or 4 bacteria stored inthe library and print the relative comparison chart

explaining the degree of similarity between the differentbacteria. In the comparison chart all the fatty acids foundin the unknown sample and the typical lipidic com-ponents of the reference bacteria stored in the library arelisted. For each fatty acid listed, the percentage present inthe unknown extract is compared with that of the sameacid in the reference bacteria.

198


The distance between each FAME percentage found inthe two compared bacteria is indicated visually in thecomparison chart and an asterisk is printed when the twopercentages are identical.

To verify the absence of interfering peaks, at thebeginning of this study, reagents 1, 2, 3 and 4 used for theextraction and methylation were processed as microbialsamples and injected into the gas chromatograph. Noneof them gave interfering peaks in the chromatogram.

In order to make the recommended standardized proce-dure easier, we analysed some strains by harvesting thepure colonies directly from MSA, MCK, MH, KV andPEA (the selective media commonly used in a bacteriol-ogy laboratory) and compared the results with thoseobtained after an additional passage of the previouslyisolated colonies on TSBA medium (as recommended byHewlett-Packard).

As shown in Table 1, with the recommended procedurefor some bacteria (Proteus mirabilis, Providencia stuartii) theSis were lower than those found with our method. For theother strains the Sis were similar. In both cases theidentification obtained with MIS agreed well with thereference method, so we decided to perform the extractiondirectly from the selective media with obvious advan-tages.

The day-to-day precision ofthe GC analysis was tested byinjecting daily, for 8 days, the extracts of 7 differentstrains stored at +4C in tubes capped with a Teflonseptum. The coefficient of variation (CV) of the Sisranged from 1" 7 to 10%. The results are shown in Table 2.

The results obtained from the GC analyses of367 samplesand the comparison with the reference method aresummarized in Table 3.

According to Lennette et al. [8], the bacteria wereclassified into family, tribe, genus and species. Tribe isalso considered for the Enterobacteriaceae family as agroup including genus and species. All the bacteria testedwere divided into four groups (aerobe Gram positive,aerobe Gram negative, anaerobe Gram positive andanaerobe Gram negative). As can be seen in Table 3, weobtained identical results in 65% of cases; all themicroorganisms belonging to the families ofNesseriaceae,Pseudomonadaceae, Bacillaceae and Propionibacteriaceae were identified with the two methods. A lowerpercentage of agreement was obtained in the otherfamilies.

In Table 4 a detailed distribution of the percentage ofdisagreement in the bacterial identification is shown. Thehighest number of.disagreements was found in the groupof the aerobe Gram negative, especially within theEnterobacteriaceae family. Seventy cases (37%) of thetotal aerobe Gram negative tested (186) showed adifferent genus and species identification and conse-quently a different tribe attribution. Only one case wasascribed to a different family. In the aerobe Gram positivegroup, a very low percentage of disagreement was found

for the family and genus classification (0"6%) whereas30% of the identified samples disagreed for the species.For the anaerobe (Gram positive and Gram negative) theresults are also reported even though the number ofsamples tested was low.

The greater disagreement in the identification of theEnterobacteriaceae family is probably due to the verysimilar lipidic compositions of the bacterial cell walls andto the similar metabolic behaviour of the bacteriaincluded in this family. It must be emphasized that, forsome bacteria, the MIS has a higher discriminatorypower than the reference method; for example, thedifferentiation between Kluyvera cryocrescens and Escherichiacoli is only possible with MIS, whereas with our referencemethod they are both classified as Escherichia coli.

Discussion and Conclusion

The following points need some discussion: (1) theagreement between the MIS and the reference method;(2) the reproductibility of the MIS; (3) the need of anadditional transplate of isolated colonies; and (4) somepractical aspects concerning routine applications ofMIS.

It is evident from our results that there is a certain degreeofdisagreement between the MIS and reference methods.This is variable within the different families studied. Asexpected, the highest degree ofdisagreement concerns thespecies identification, whereas it is lower for genusidentification and almost absent for families identifica-tion. Even if chemical identification tests for bacteria are.widely accepted, it is well known that most of them arebased on very questionable chemical reactions (subjec-tive detection, low sensitivity, etc.); in addition, thebiological variability of bacteria accounts for the lowreproducibility and accuracy. In some cases they have a

discriminatory power much lower than MIS.

Hence the disagreement observed in our research is notsurprising; more sensitive, reproducible and standar-dized methods for identification of bacteria are needed asstandards or reference methods, with which new proce-dures should be compared. It must be stressed that thehigh discriminatory power of MIS, which is able toidentify 150 different fatty acids, makes this system usefulin taxonomic studies.

The following conclusions are suggested:

1. The reproducibility of the MIS is very good.

2. There is no need for an additional culture of isolatedstrains before injecting them into the gas chromato-graph.

3. The system is easy to use because sample handling inthe preparation of the extracts is minimized.

4. To obtain the final results and the identification of thebacterium, a shorter time is required when comparedwith traditional methods, which makes this systemvery suitable for emergencies. The total analysis timeis about 2 h, including extraction, methylation and thegas chromatographic analysis, whereas with tradi-

199


tional tests 24-48 h are needed to contirm theidentification of a pure strain.

5. The current methods require human interpretation,while the automated analysis improves the objectivityofthe results and allows an easier comparison betweendifferent laboratories.

Acknowledgement

This work was supported by Grant No. 561 of theRegione Lombardia: ’Precocious Diagnosis of BacterialInfections By Gas Chromatographic and Mass Spec-trometric Techniques: Development and Evaluation ofMethodologies’.

6. The autosampler allows the elimination of analysterrors, and thus reduces the waiting time betweeninjections.

7. The cost of a gas chromatographic analysis (about $3in Italy) is lower than that one for Enterotube II(Roche) or API (Ayerst) (about $5, $4 or $8"5)although the capital cost of the instrument is high.

8. The major practical disadvantage of this technique isits limited throughput (not more than 40 samples per24 h), which prevents its use in a large routinelaboratory, unless two or more instruments areinstalled.

Methodological and instrumental improvements to thistechnique will hopefully improve the quality ofmicrobio-logical tests in routine laboratories and the classificationof bacteria.

References

1. KREIC,, N. R., in Bergey’s Manual of Systematic Bacteriology,Kreig, N. R. (Ed.) (William and Wilkins, Baltimore, 1984),p. 1.

2. Moss, C. W., Journal of Chromatography, 203 981), 337.3. GOOOFELLOW, M. and MINNIKIN, D. E., in Chemical Methods in

Bacteriology Systematics, Goodfellow, M. and Minnikin, D. E.(Eds) (Academic Press, London, 1985), p. 1.

4. BaonDz, I. and OLs.n, I., Journal of Chromatography, 379(1986), 367.

5. Davcia, D. B., in Microbiological Applications ofGas Chromat-ography, Drucker, D. B. (Ed.) (Cambridge University Press,Cambridge, 1981), p. 251.

6. ASSELInEAV, C. and ASSELInEAV, J., in Gas Chromatography-Mass Spectrometry. Applications in Microbiology, Odham, G.,Larsson, L. and Mardh, P. A. (Eds) (Plenum Press, NewYork, London, 1984), p. 57.

7. Bau,jA. W., Perry, D. M. and Kirby, W. M., Archives ofInternal Medicine, 104 (1959), 208.

8. LENNETTE, E. H., BALLOWS, A., HAUSLER, W. J., JR., andTRUANT, H. P., in Manual of Clinical Microbiology (AmericanSociety for Microbiology, Washington, DC, 1980).

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