Rapid diagnosis of anaerobic infections bydirect gas-liquid chromatography of clinicalspeciments.

S L Gorbach, … , H Thadepalli, A B Onderdonk

J Clin Invest. 1976;57(2):478-484. https://doi.org/10.1172/JCI108300.

Current methods to isolate and identify anaerobic bacteria are laborious and timeconsuming. It was postulated that the short-chain fatty acids (SCFA) produced by theseorganisms might serve as microbial markers in clinical material. 98 specimens of pus orserous fluid were analyzed by gas-liquid chromatography, and findings were compared withculture results. Good correlations were found for the recovery of anaerobic Gram-negativebacilli and the presence of isobutyric, butyric, and succinic acids. 19 of 20 specimens withsignificant amounts of these acids (greater than 0.01 mumol/ml) yielded bacteroides orfusobacteria. Culture of the single "false-positive" specimen failed to grow anaerobic Gram-negative bacilli, although clinical data and Gram-stain suggested their presence. 77 of 78specimens which has insignificant concentrations of the marker acids failed to yieldanaerobic, Gram-negative bacilli in culture. The single "false-negative" specimen yieldedBacteroides pneumosintes, an organism which does not ferment carbohydrates. It isconcluded that direct gas-liquid chromatographic analysis of clinical specimens provides arapid presumptive test for the presence of anaerobic, Gram-negative bacilli.

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Rapid Diagnosis of Anaerobic Infections by

Direct Gas-Liquid Chromatography of Clinical Specimens

SHERWOODL. GORBACH,JAMESW. MAYHEW,JoHN G. BARU'rr,HARAGOPALTHADEPALLI, and ANDREWB. ONDERDoNK

From the Infectious Disease Section, Veterans Administration Hospital,Sepulveda, California 91343, and The Department of Medicine, University ofCalifornia Medical Center, Los Angeles, California 90024

A B S T R A C T Current methods to isolate and identifyanaerobic bacteria are laborious and time consuming.It was postulated that the short-chain fatty acids(SCFA) produced by these organisms might serve asmicrobial markers in clinical material. 98 specimens ofpus or serous fluid were analyzed by gas-liquid chro-matography, and findings were compared with cultureresults. Good correlations were found for the recoveryof anaerobic Gram-negative bacilli and the presence ofisobutyric, butyric, and succinic acids. 19 of 20 speci-mens with significant amounts of these acids (> 0.1ismol/ml) yielded bacteroides or fusobacteria. Cultureof the single "false-positive" specimen failed to growanaerobic Gram-negative bacilli, although clinical dataand Gram-stain suggested their presence. 77 of 78specimens which had insignificant concentrations of themarker acids failed to yield anaerobic, Gram-negativebacilli in culture. The single "false-negative" specimenyielded Bacteroides pneumosintes, an organism whichdoes not ferment carbohydrates. It is concluded thatdirect gas-liquid chromatographic analysis of clinicalspecimens provides a rapid presumptive test for thepresence of anaerobic, Gram-negative bacilli.

INTRODUCTIONComprehensive anaerobic bacteriology of clinical speci-mens is an expensive and time-consuming operation.Laudable pus from an intra-abdominal abscess, for ex-ample, may harbor multiple aerobic and anaerobic mi-croorganisms, and it may require 1-2 wk for completebacteriologic identification of these strains (1, 2).

Reprint requests should be addressed to Dr. SherwoodGorbach, Infectious Disease Section, Tufts-New EnglandMedical Center, Boston, Mass. 02111.

Received for publication 11 September 1974 and in revisedform 8 October 1975.

Bacterioides fragilis is particularly important to recog-nize since this organism appears to be more virulentthan other anaerobes. In addition, B. fragilis has un-usual antimicrobial susceptibility patterns since it isrelatively resistent to penicillins, cephalosporins, andtetracyclines. Hence, an early indication of B. fragilis ininfected material would be a useful guide to the choiceof appropriate antibiotics.

We have attempted to develop a rapid test for thepresence of anaerobic bacteria in clinical specimensbased on their metabolic activities. These microorgan-isms, particularly bacteroides, clostridia, and fusobacteria,produce short-chain fatty acids (SCFA) ' from metabo-lism of carbohydrate and protein. Such end products areso regularly produced that they can be used for taxo-nomic purposes (3). It was hoped that these acids couldbe detected in purulent material and thereby serve asfingerprints for the presence of putrefactive anaerobes.

METHODSSpecimens of pus and body fluids were rapidly deliveredto our laboratory after collection. Care was exercised tocollect fluid samples from the infected site and to avoidcontamination with sources of normal flora such as skin,vaginal mucosa, or intestinal effluent. The specimens werecollected in tubes rendered oxygen-free by gas exchangewith CO or in syringes that had been sealed to prevententrance of air. Samples were divided for aerobic andanaerobic bacteriology and for gas-liquid chromatography(GLC) analysis.

Anaerobic bacteriology was performed with prereducedmedia in suitable anaerobic conditions, as previously de-scribed (3, 4). A Gram-stained slide was prepared of theinitial specimen for interpretation by three experiencedmicrobiologists. Culture results, Gram-stain findings, andGLC determinations were recorded for each specimen with-

'Abbreviations used in this paper: GLC, gas-liquid chro-matography; PYG, peptone-yeast glucose; SCFA, short-chain fatty acids; VFA, volatile fatty acid.

The Journal of Clinioal Investigation Volume 57 February 1976- 478-484478

out knowledge of results of the other studies. The codewas broken at the conclusion of the study.

GLC. SCFA are resolved into two broad classes on thebasis of susceptibility to steam distillation. The volatilecompounds can be chromatographed directly. i.e., acetic,propionic, isobutyric, butyric, isovaleric, and valeric acids.Nonvolatille compounds such as lactic and succinic acidsmust be derivatized to encourage chromatographic vola-tility.

Specimens were prepared for GLC by dilution of 1 volof purulent material with 1 vol of 5% aqueous H2SO4.After chilling in an ice bath, a small amount of NaCl and1 vol of diethylether were added. The complete specimenwas agitated, centrifuged at 50C, and 2.0 jul of the organicphase was injected onto the chromatographic column forvolatile fatty acid (VFA) determination. Relatively poorethereal extraction of acetic (< 10%) and propionic (<50%) acids are obtained with these procedures. Longerchain acids, including butyric acid, isobutyric acid, andsuccinic acid, those of primary interest to this study, areextracted well (> 90%o).

For methyl esters, an additional acid-diluted sample wasprepared. A volume of boron trifluoride methanol equiva-lent to the pus, and an acid diluent was added. The finalproduct was incubated at 55°C in a water bath for 30min, and extracted with 1 vol of chloroform. 2 IAI wasinjected into the GLC for methyl ester analysis. If thevolume of the specimen was very small, the ether phasewas evaporated after VFA analysis at 350C with a gentlenitrogen stream. The subsequent steps of methylation andinjection were the same as described above. Parallel analy-ses with known acid concentrations revealed uniform lac-tic and succinic recoveries from identical portions whethermethylated directly or shaken with ether, subjected togentle organic-phase evaporation, and then methylated.

Packard 419 (Packard Instruments Co., Inc., Down-ers Grove, Ill.) and Shimadzu GC4BMPFdual-column gaschromatographs (Shimadzu, Seisakusho, Ltd., Kyoto, Ja-pan) equipped with flame ionization detectors were em-ployed. Chromatographic deflections were visualized onHoneywell (Honeywell Inc., Minneapolis, Minn.) andShimadzu 1.0-mV span recorders, and peak areas wereinterrupted as uV s on a Shimadzu ITG-2A electronic digi-tal integrator. 2.0-,ul samples were injected with a 5-i1Hamilton syringe (Hamilton Company Inc., Whittier,Calif.) directly onto coiled, silylated Pyrex columns, 6feet long X 2 mminternal diameter. The analytical columnscontained 6% Carbowax 20M-terephthalic acid on 80/100mesh Gas-Chrom Q (Applied Science Labs, Inc., StateCollege, Pa.). Peak identity was verified by chromatographyon an alternate phase (10% Carbowax 20M, unconjugatedat elevated column temperature, and carrier flow (150'Cand 30 ml/min). The specimens were supplemented with an-hexanoate (volatile analysis) and malonyl dimethyl ester(derivatized analysis) as relative retention times standardsfor both phases. Routine analysis conditions included: in-jection and detector blocks, 200° C: oven isothermal at125°C: nitrogen, hydrogen, and air flows, respectively, at30, 30, and 300 ml/min. Detector sensitivity was customarilyset at 2.5 X 10"1 A while 2.5 X 10' A was utilized to de-tect and confirm trace amounts of metabolites.

Integration values compiled at the lower sensitivity wererelated to standard curves for conversion to micromoles permilliliter. Titrated aqueous solutions of SCFA were dilutedin sterile human ascitic fluid, extracted, and chromato-graphed to obtain the standard values. The chromatogramdevelopment times were 9 min for valeric acid and 7 min

for succinyl dimethyl ester. Thus, total time for submissionof the pus specimen to the conclusion of chromatographicanalysis was approximately 30 min.

To ascertain the accuracy of the method, ascitic fluid wassupplemented with known organic acid concentrations atlevels likely to be encountered in purulent specimens. Noacids were detected in the ascitic fluid at 2.5 X 10'1 Abefore supplementation. Mean recoveries for triplicate in-jections of all acids had an average relative error of 9%from the true concentrations. The relative SD (precision)encountered was 10.2% based on replicate analyses of iso-valeric acid. Acetic and propionic acids could be reliablydetected to the 1 ,umol/ml level, and all other acids to 0.1,tmol/ml.

SCFA production in vitro. All of the anaerobes recov-ered in this study were isolated in pure culture, and weregrown for 48 h under anaerobic conditions in prereducedpeptone-yeast glucose (PYG) broth, according to themethod of the Virginia Polytechnic Institute, Blacksburg,Va. (3). SCFA were analyzed by GLC, and the resultsformed the basis of the taxonomic designation of the par-ticular strain.

In an effort to more closely simulate the actual metabolicevents in vivo, we incubated bacteria in human ascites fluidfor prolonged periods of time. The chromatographic pat-terns of bacterial end products under these conditions werecompared with those obtained in PYG after 48 h ofgrowth. Five strains of B. fragilis and one strain of Esche-richia coli were employed in these analyses. In each in-stance the isolate was recovered from a clinical specimenused in the study. For the anaerobes, approximately 0.1 mlof an 18-h PYG broth culture was inoculated into 10ml of sterile, human ascitic fluid. This was incubatedat 370C in the anaerobic chamber with an atmosphere of5%o CO2, 10% H2, and 85%o N2. 0.5-ml aliquots were re-moved for GLC analysis at intervals of 0, 24, 48, 72, 168,336, and 672 h. The E. coli strain was incubated in atmos-pheric air and was processed in a similar manner.

RESULTS

Comparison of bacteriological data and GLC determi-nations revealed that the acids that most reliably indi-cated the presence of putrefactive anaerobes were iso-butyric or butyric acid in the volatile series, and suc-cinic acid in the methylated group. Concentrations of0.1 imol/ml or greater of these acids were considereda significant concentration by the techniques of ouranalysis.

Purulent material writi significant concentrations ofisobutyric, butyric, or succinic acid. 20 clinical speci-mens of pus or body fluids had significant quantities ofeither isobutyric, butyric, or succinic acid (Table I).The sources of the infections were: abdominal abscessesor wounds (nine cases), perirectal abscess (n = 2),empyema (n = 3), decubitus abscess (n = 2), subman-dibular abscess (n = 2), prostatic abscess (n = 1), andbrain abscess (n = 1). Isobutyric acid was detected in12 samples, butyric acid in 14, and succinic acid in all20. Small concentrations of acetic and lactic acid werepresent in many of these specimens, but these acidsappeared in the noninfected body fluids as well (seebelow).

Diagnosis of Anaerobic Infections by Direct Gas-Liquid Chromatography 479

TABLE I

Purulent Material with Isobutyric, Butyric, or Succinic Acids

SCFA CulturesCase Site ofno. infection iB B S Aerobes Anaerobes

1 Intra-abdominalabscess

2 Intra-abdominalabscess

3 Intra-abdominalabscess

4 Intra-abdominalabscess

5 Intra-abdominalabscess

6 Abdominalwound

7 Perirectalabscess

8 Perirectalabscess

9 Subphrenicabscess

10 Appendicealabscess

11 Pelvic abscess

12 Prostaticabscess

13 Decubitisabscess

14 Decubitisabscess

15 Submandibularabscess

16 Submandibularabscess

17 Empyema

18 Empyema

19 Empyema

20 Brain abscess

Mmol/ml2.6 14.4 E. coli

Pr. mirabilisEnterococcus

1.4 E. coliPr. mirabilisEnterococcus

0.1 0.7 0.2 E. coli

0.1 0.3 Enterococcus

0.1 E. coliKlebsiellaEnterococcus

- 0.1 0.3

0.1 0.1 1.8

- 0.4 24.0

0.1 0.1

- - 0.1

0.2 4.0 5.1

0.7 8.2 31.0

6.5 - 30.0

6.1 - 27.0

0.5 0.4 2.5

0.5 2.1 2.8

1.1 0.9 13.6

1.1 4.3 26.0

0.2 0.8 15.2

E. coliPr. mirabilis

Pr. mirabilis

E. coliEnterobacterKlebsiella

EnterococcusP. aeruginosa

Pr. mirabilisPr. rettgeri

Pr. mirabilis

KlebsiellaPr. mirabilis

N. catarrhalis

E. coli

N. catarrhalis

E. coliEnterococcus

0.1 2.2 S. viridans

B. fragilis, Cl. ramosumF. nucleatum, Ps. intermediusPc. prevotii, V. parvula

F. nucleatum

B. fragilis, F. nucleatum

B. fragilis, B. melaninogenicusE. lentum, F. symbiosumB. fragilis, Cl. sporogenes,Cl. ramosum, clostridium species

B. fragilis

B. fragslts, B. melaninogenicusE. aerofaciens, F. gonidiaformansPs. anaerobius

B. fragilis, Cl. paraputrificumPc. prevotii

B. fragilis, clostridium species

B. fragilis, F. symbiosum

B. fragilis, Pc. prevotiiPs. micros

B. fragilis, Pc. prevotiiPs. micros

B. fragilis, Pc. microsPc. asaccharolyticus

B. fragilis, Ps. micros

B. fragilis, B. melaninogenicusE. lentum, Ps. intermedius

F. nucleatum

B. fragilis, E. lentum

B. melaninogenicus

Ps. intermedius, V. parvula

bacteroides species

E, Escherichia; B, Bacteroides; F, Fusobacterium; P, Pseudomonas, Cl, Clostridium; E, Eubacterium; Pr, Proteus; Ps,Peptostreptococcus; Pc, Peptococcus; N, Neisseria; V, Veillonella; S, Streptococcus.

480 Gorbach, Mayhew, Bartlett, Thadepalli, and Onderdonk

s

FIGURE 1 Chromatographic pattern of exudate from a pel-vic abscess. VFA (pattern on left) were acetic (A), pro-pionic (P), isobutyric (iB), butyric (B), and isovaleric(iV); methyl esters (pattern on right) were lactic (L) andsuccinic (S) acids. Culture yielded B. fragilis, Ps. inter-medius, and Peptococcus prevotii.

Examination of Gram-stained slides of these 20 puru-lent specimens revealed Gram-negative bacilli in 14,as determined by three observers in double-blind read-ings. However, the morphologic features in only fiveslides were sufficiently distinctive to suggest anaerobes.

Cultures generally yielded a complex miscellany ofaerobic and anaerobic forms. B. fragilis was present in15 specimens, usually associated with other anaerobicbacteria. In only one specimen, a suppurative abdomi-nal wound (case 6) was B. fragilis recovered in pureculture. Bacteroides melaninogenicus was isolated inone case of putrid empyema, associated with a neisseriaspecies. An unclassified bacteroides species was re-covered from a case of brain abscess. Fusobacteria werefound in 7 cases; in 5 instances a bacteroides was co-existing, but in 2 patients (cases 2 and 15), Fusobac-terium nucleatum was the only anaerobic isolate. Otheranaerobes commonly found in combination with eitherbacteroides or fusobacteria were Gram-positive cocci(peptostreptococci or peptococci) in nine cases, clos-tridia in four, and eubacteria in four.

There was one false-positive specimen that containedisobutyric, butyric, and succinic acids (case 19). Thiswas a sample of foul-smelling pleural fluid from a caseof empyema associated with a subphrenic abscess. Cul-ture of the empyema fluid yielded E. coli, enterococcus,Peptostreptococcus intermedius, and Veilonela parvula,but no bacteroides or fusobacteria. The Gram stain ofthe pus, however, showed thin pleomorphic Gram-negative bacilli. Furthermore, culture of the adjacentsubphrenic abscess yielded B. fragilis in addition to theorganisms recovered in the pleural fluid specimen. Inthis instance, there may have been a failure of culture

techniques with the empyema specimen since otherparameters suggested the presence of Gram-negativeanaerobic bacilli.

Fig. 1 shows the chromatographic pattern of pusobtained from a pelvic abscess (case 11). This speci-men yielded acetic, propionic, isobutyric, butyric, andisovaleric acids in the volatile fraction, and lactic andsuccinic acids in the methylated series. Our culturesrevealed B. fragilis, peptococcus, and peptostreptococcusfrom the same specimen.

Another example is purulent discharge from a peri-rectal abscess (case 8) which showed large concentra-tions of butyric and succinic acids by GLC analysis(Fig. 2). The culture of this specimen produced B.

fragilis, anaerobic cocci, a clostridia species, and coli-forms.

Purulent material without significant concentrationsof isobutyric, butyric, or succinic acids. There were18 specimens of infected material that yielded micro-organisms on culture but failed to demonstrate sig-nificant concentrations of isobutyric, butyric, or suc-cinic acid (Table II). All samples were grossly puru-lent, and were obtained by direct aspiration from em-pyema (six cases), intra-abdominal abscess (n = 4),perirectal abscess (n = 2), liver abscess (n = 2), septicjoint (n =2), breast abscess, (n = 1), and stump ab-scess (n = 1). A variety of enteric Gram-negativerods, pseudomonas, staphylococci, and streptococci wererecovered. In addition, there was one fungal infectionwith Coccidioides immitis, and one case of an amebicliver abscess. Anaerobic bacteria were recovered in sixspecimens including four with peptostreptococci. An-

AB

FIGURE 2 Chromatographic pattern of purulent drainagefrom a perirectal abscess showing a butyric acid (B) peak,and in the methylated series, succinic acid (S), Other acidpeaks are acetic (A), propionic (P), isobutyric (iB), andisovaleric (iV). Culture of this specimen yielded B. fragilis,Clostridium paraputrificum, Peptococcus prevotii, and Pro-teus mirabilis.

Diagnosis of Anaerobic Infections by Direct Gas-Liquid Chromatography

s

481

TABLE IIPurulent Material without Detectable Isobutyric,

Butyric, or Succinic Acids

Clinical conditions

EmpyemaIntra-abdominal abscessPerirectal abscessLiver abscessSeptic jointBreast abscessStump abscess

Total

Types of microorganismsAerobes

E. coli (4)*S. aureus (4)Proteus (3)Pseudomonas (3)S. faecalis (2)Enterobacter (1)Coccidioides (1)Entamoeba

histolytica (1)Anaerobes

Peptostreptococcus (4)Cl. perfringens (1)B. pneumosintes (1)

No. of cases

6422211

18

* Number of specimens.

other patient had clinical gas gangrene of the thoracicwall associated with empyema. The specimen submittedfor GLC analysis had no detectable SCFA, althoughtypical Gram-positive rods were noted on Gram stainand Clostridium perfringens was grown in heavy cul-ture. Another specimen of empyema fluid grew B. pneu-mosintes and Enterobacter aerogenes; GLC determina-tion revealed trace concentrations of isobutyric andlactic acids (< 0.1 ,umol/ml), but no other SCFA. Itshould be noted, parenthetically, that this unusual bac-teroides species does not ferment carbohydrates andfails to produce SCFA, in vitro.

Sterile body fluids. There were 60 specimens ofsterile body fluids including pleural effusion (30 cases),ascites (n = 9), bile (n = 7), joint aspirate (n = 7),urine (n = 6), and pericardial fluid (n = 1). Signifi-cant succinic, isobutyric, or butyric acids were not de-tected in any of these samples. Some samples showedtrace deflections in the isobutyrate-butyrate region onroutine analysis. However, this tentative identificationwas not verified by cochromatography on unconjugatedCarbowax 20M. In addition, trace concentrations ofacetic, propionic, and lactic acids were found in somesamples.

Specimens cultured in outside laboratories that failedto grow anaerobes. During the period of our study,six specimens of pus that were cultured in other labora-tories not possessing optimal anaerobic capabilitieswere submitted to us for GLC analysis. These weresamples collected from cases that clinically suggestedanaerobes i.e., three cases of abdominal abscess, onepelvic abscess, one rectal abscess, and one gluteal ab-scess. The outside laboratory was unable to isolateanaerobes from these specimens, although coliforms andenterococci were present. A considerable period of timeelapsed (1-3 days) before the specimens reached ourlaboratory, and we were also unable to recover anae-robes. All six specimens yielded significant concentra-tions of succinic acid, and three also showed isobutyricor butyric acids. It appears that these metabolic markersmay have persisted in clinical specimens longer thanthe anaerobes themselves. Thus, these specimens tech-nically represented false-positives, although it couldreasonably be argued that the delay in culture pre-cluded the isolation of fastidious anaerobic bacteria fromsuch specimens.

Effect of growth media on SCFA production. Thepresence of C4 volatile acids, particularly in cases 6,14, and 17, led us to examine in vitro organic acidproduction by B. fragilis. This organism was the onlylikely source of the isobutyrate and butyrate in thesecases. However, C4 acids are seldom recovered withthe conventional chromatographic methods employedfor laboratory identification of B. fragilis. Using humanascitic fluid for culture medium and extended periods ofobservation, all five strains of B. fragilis producedisobutyrate and butyrate (Table III). Both compoundswere detected in limited quantities, but isobutyrate pro-duction was clearly favored. In a similar study withE. coli, abundant succinic acid was produced in vitro.The patient with an abdominal wound abscess involv-ing only this strain of E. coli paradoxically had nodetectable succinic acid in the purulent exudate. Theseresults indicate that there is not necessarily a directcorrelation between in vitro and in vivo acidic endproducts of bacterial metabolism.

DISCUSSION

Anaerobic bacteria are now considered significant path-ogens in human infections, particularly those relatedto contamination by the normal flora of the gastro-entestinal tract, oropharynx, female genital tract, andskin (2). Reports of increased isolation of anaerobesprobably reflect greater awareness and improved lab-oratory techniques, rather than a shift in infectiousagents. At the present time it is reckoned that anaerobesare associated with 80-90% of intra-abdominal sepsis,70-80% of female pelvic infections, 90% of pulmonary

482 Gorbach, Mayhew, Bartlett, Thadepalli, and Onderdonk

TABLE II I

Concentrations of Isobutyric and Butyric Acids in Ascitic Fluid over 672 hof Anaerobic Incubationf~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Hours

Acids 0 24 48 72 168 336 672

iB 0 <0.1 0.140.10* 0.1 I0.10 0.4±0.18 0.5+0.09 0.640.13B 0 0 0 0 0.1 I0.04 0.1±i0.02 0.1 ±t0.02

* Mean concentration from five strains of B. fragilis expressed as micromoles permilliliter + SD.

infections caused by aspiration, and 90% of brain ab-scesses, to mention the major forms of anaerobicinfections.

Rapid bacteriologic diagnosis would be desirable sinceB. fragilis, the pre-eminent pathogen, is relatively re-sistant to many antibiotics used in clinical practice.Unfortunately, clinical laboratories are unable to pro-duce results in an expeditious manner for several rea-sons: (a) anaerobes are slow growing, particularlyafter oxygen exposure during transport and processingof the clinical specimen; (b) these organisms are oftenpresent in a complex culture, on the average involvingfive different bacterial species in each specimen of puru-lent material; (c) isolating and identifying each micro-organism is time-consuming since the taxonomy ofanaerobes is more complex than that of facultativespecies, and several procedures, including GLC analy-sis may be required for identification; and (d) cultureplates of these organisms are incubated in anaerobicjars that must be opened even to look at the plates.As a rule, the jars are exposed to room air only after48-72 h of growth, rendering quick bacteriologic de-terminations rather impractical.

Shortcuts to the diagnosis of anaerobic infection areuseful in certain settings, but do not have widespreadapplication. A Gram-stained slide of the clinical speci-men should always be examined. Distinctive anaerobessuch as clostridia and fusobacteria can be recognized.Anaerobic streptococci, on the other hand, are indistin-guishable from facultative strains; coliforms may bepleomorphic in purulent exudate making it difficult todifferentiate these organisms from B. fragilis. Thus,three experienced bacteriologists in our laboratory couldonly identify 5 of 20 positive specimens as havingGram-negative anaerobes when read in a blind ex-periment.

The distinctive odor of anaerobes is apparent to alllaboratory workers, although many clinicians fail toappreciate this smell. Although it is true that all in-fections exhibiting this odor of excrement are infectedwith anaerobes, we have found that even experiencedanaerobists with a highly developed olfactory sense

can only detect the stench in approximately one-halfof clinical cases. Furthermore, at the bedside, our noseshave been unable to differentiate infections caused byanaerobic cocci and clostridia, strains sensitive to peni-cillin, from those caused by resistant B. fragilis.

Our studies suggest that direct gas chromatographyof clinical specimens offers a rapid and reliable methodof identifying putrefactive anaerobes in clinical speci-mens. All purulent samples containing B. fragilis hadsignificant quantities of succinic acid, and many alsohad isobutyric and butyric acids, as measured by thesetechniques. It should be noted that these acids werenot specific for B. fragilis since there were specimenswith fusobacteria and B. melaninogenicus that showedthese products. However, this pattern was not seen insterile specimens or in infections caused by aerobic or-ganisms or anaerobic organisms such as Gram-positivecocci and Gram-positive rods. Although GLC is notabsolutely diagnostic of B. fragilis, in that other Gram-negative anaerobes may be responsible, there is enoughevidence, in our view, from this rapid diagnostic tech-nique to initiate specific antibiotic treatment for thisvirulent pathogen. Therapy should be modified byresults of culture which will eventually become avail-able.

In the 20 yr since GLC has been developed, manyworkers have used this sensitive technique for microbialtaxonomy and the diagnosis of infectious diseases (5).The classification of microorganisms have been basedeither on analysis of metabolites in spent culture media(6-10) or on the structural components of the bacterialcell itself (11-15). Variations of these basic themeshave been used to classify clostridia, neisseria, coli-forms, bacillus, mycobacteria, and pseudomonas.

Several investigators have attempted to identify spe-cific infections in laboratory animals and in patients byGLC analysis of blood, stool, or urine. Mitruka andco-workers (16-20) have analyzed serum samples withGLC to diagnose experimental viral and bacterial in-fections. A recent report by Brooks et al. (21) hassuggested that GLC might be useful in the differentialdiagnosis of arthritis.

Diagnosis of Anaerobic Infections by Direct Gas-Liquid Chromatography 483

The use of GLC for detecting bacterial infectionsrepresents a departure from classic bacteriology inthat we are not as concerned in the first instance withgrowth of the microorganisms in artificial culture mediaas we are in detecting metabolic fingerprints of itspresence in pathologic tissue. Our demonstration ofSCFA is only one approach that may be useful withanaerobes. Microorganisms have other metabolic path-ways that are distinct from their host, and these mayprove susceptible to analysis with the gas-liquid chro-matograph for rapid diagnostic purposes.

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