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JORNAL OF CLINICAL MICROBIOLOGY, OCt. 1991, p. 23234 951137/91/1235$2./ Vol. 29, No.

1 JORNAL OF CLINICAL MICROBIOLOGY, OCt. 1991, p /91/1235$2./ Vol. 29, No. 1 Enterosistem 18R: Desription and Comparative Evaluation with Conventional Methods for Identifiation of Members of the Family Enterobateriaeae RAFFAELE PICCOLOMINI,l* ARTRO DI GIROLAMO,1 GIOVANNI CATAMO,1 LIGINA CELLINI,1 NERINO ALLOCATI,1 AND GIAMPIETRO RAVAGNAN2 Istituto di Mediina Sperimentale, Cattedra di Mirobiologia, Faoltad di Mediina e Chirurgia, niversita "G. D'Annunzio," Chieti,' and Dipartimento di Biologia, niversita di Roma "Tor Vergata," Rome,2 Italy Reeived 1 May 1991/Aepted 23 July 1991 The effiieny and auray of Enterosistem 18R (Liofilhem s.r.l., Roseto degli Abruzzi, Teramo, Italy) were ompared with those of onventional biohemial methods to identify 36 members (38 speies) of the family Enterobateriaeae. Overall, 329 strains (91.3%) were orretly identified (perentage of identifiation,.9.), with 37 (11.2%) requiring additional tests for omplete identifiation. For 11 isolates (3.1%), Enterosistem 18R gave only genus identifiations, and for 14 (3.9%), the strains did not orrespond to any key in the odebook and ould not be identified by the manufaturer's omputer servie. Only six isolates (1.7%) were misidentified. The new system aurately identified ommon and several newly desribed isolates of the family Enterobateriaeae, suh as Enterobater gergoviae, Providenia rustigianii, Serratia odorifera, and Serratia rubidaea. The system is highly reproduible, simple to perform, easy to handle, and inexpensive. With adjustments in supplementary ode numbers for some strains, Enterosistem 18R is a suitable alternative for identifiation of members of the Enterobateriaeae in linial laboratories. Identifiation of members of the family Enterobateriaeae is a major feature of linial bateriology laboratories sine these bateria, alone, are the etiologial agents of more than 5% of hospital infetions (17). With inreased government attention to health osts (2), today the linial mirobiologist is more interested than ever in rapid reporting and redutions in laboratory osts (1). Therefore, it is essential to develop new, simple, and eonomi systems for rapid and aurate identifiation of this baterial group, and many ommerial multitest systems are now available for this purpose (4, 1, 16). Computerassisted identifiation systems are already available in miniaturized test kits suh as MiroID, Minitek, API 2E, and Enterotube (11, 12, 15, 18). Enterosistem 18R (Liofilhem s.r.l., Roseto degli Abruzzi, Teramo, Italy) is a new system designed to identify members of the Enterobateriaeae to the genus and speies levels in 18 h. The system, at present available only in Europe, onsists of a disposable tray with 18 wells ontaining the dehydrated biohemial substrates. With inoulation of a baterial suspension in eah well, a sixdigit otal number an be generated from 18 different biohemial reations. From this otal number, an identifiation is derived from a odebook furnished to laboratories. To evaluate the auray and utility of Enterosistem 18R, we have ompared this system with onventional biohemial methods in identifying 36 isolates of members of the family Enterobateriaeae. MATERIALS AND METHODS Baterial strains. A total of 36 strains were tested. Of these, 213 were fresh linial isolates from our linial bateriology laboratory, 19 were stok ultures from our olletion that have been kept frozen (8 in 2% gly * Corresponding author. 23 erol, and 38 were referene strains from different international ulture olletions. Before the experiment, the 19 stok ultures and the 38 referene strains were subultured three times into sheep blood agar (Liofilhem s.r.l.) to raise their levels of enzymati ativity. Before testing, all of the 36 isolates were grown in brain heart infusion broth (Oxoid Italiana S.p.A., Garbagnate Milanese, Milan, Italy) and then subultured in a sheep blood agar to ensure purity and viability. To mask the identity of all miroorganisms throughout the experiment, we adopted the use of a progressive numbering system for eah miroorganism (from 1 to 36). Two study groups were rossemployed in this work. The first identified all isolates and revealed the results to the seond group only at the end of the study. The seond group, employed as a ontrol for the first group, onduted the work on 72 randomized isolates and used the 38 referene strains as a quality ontrol. Furthermore, to evaluate the possible effet of the growth medium on Enterosistem 18R, the same referene strains were grown on sheep blood and MaConkey agar plates (Oxoid Italiana S.p.A.). To asertain the reproduibility of results from the system, growth from both media was employed as an inoulum on three separate oasions, eah time by a different study group. Enterosistem 18R identifiation method. The Enterosistem 18R identifiation method onsists of a plasti tray ontaining 18 different reation wells overed with a transparent plasti over (Fig. 1). The 18 biohemial tests inluded in the system are listed in the legend to Fig. 1. The tray was inoulated aording to the manufaturer's instrutions with some modifiations, suh as the inoulum onditions and the inubation time. One to three wellisolated olonies were emulsified in 4.5 ml of physiologial sterile solution to reah an opaity equal to.5 MaFarland standard. The reation wells were inoulated with 2 RI of the baterial suspension by using a multihannel pipette (Titertek; Flow Laboratories, Milan, Italy). Wells for lysine Downloaded from on November 3, 218 by guest

$VOL. 29, 1991 IDENTIFYING ENTEROBACTERIACEAE WITH ENTEROSISTEM 18R 231 ONPG I 2 ilc ID * 1 ADCt 5j 7 8 9 11 1 12 [;LR MAL VP IND GL 13 14 15 16 17 is,, OANJI~\JOjSORjj,,M, 1,,,,INO 1 SAC A ARA RAF$ Test wells: 1, ONPG; 2, lysine dearboxylase; 3, ornithine dearboxylase; 4, arginine dihydrolase; 5, phenylalanine deamination; 6, itrate; 7, urea hydrolysis; 8, H2S prodution; 9, malonate

Test wells: 1, ONPG; 2, lysine dearboxylase; 3, ornithine dearboxylase; 4, arginine dihydrolase; 5, phenylalanine deamination; 6, itrate; 7, urea hydrolysis; 8, H2S prodution; 9, malonate

2 VOL. 29, 1991 IDENTIFYING ENTEROBACTERIACEAE WITH ENTEROSISTEM 18R 231 ONPG I 2 ilc ID * 1 ADCt 5j [;LR MAL VP IND GL is,, OANJI~\JOjSORjj,,M, 1,,,,INO 1 SAC A ARA RAF NOME I."I. i ENTEROSISTEM 18R PATENT PENDING PD DATA FIG. 1. Enterosistem 18R. Test wells: 1, ONPG; 2, lysine dearboxylase; 3, ornithine dearboxylase; 4, arginine dihydrolase; 5, phenylalanine deamination; 6, itrate; 7, urea hydrolysis; 8, H2S prodution; 9, malonate utilization; 1, VP; 11, indole prodution; 12 through 18, fermentation of gluose, mannitol, inositol, sorbitol, surose, arabinose, and raffinose, respetively. dearboxylase, ornithine dearboxylase, arginine dihydrolase, urease, and H2S tests were overed with sterile mineral oil. The tray was losed with the plasti over and then inubated for 18 h at 37 C in an aerobi atmosphere. After inubation, 2 drops of a 1% ferri hloride solution were added to the phenylalanine deaminase well and an immediate green reation was evident. At the same time, 3 drops of anaphthol plus 1 drop of 4% NaOH solution (VogesProskauer [VP] reagents) and 2 drops of Kovas reagent were added, respetively, to the VP test and indole prodution wells. The VP reation was evident before 12 to 15 min, while an immediate redring appearane in the indole prodution well demonstrated a positive tryptophan metabolism. All of the reations were read by a olor hart provided with the kit. The biohemial reations were reorded on data sheets provided with the kit, and a sixdigit otal number was generated for eah miroorganism, whih was then identified as a single speies or as one of several possible speies by using the Enterosistem 18R odebook index. nlisted profiles were interpreted by referring to the manufaturer's omputer. In the Enterosistem 18R odebook, identifiations are lassified aording to the perentage of identifiation (% ID) (19) as follows: exellent (% ID,.99.9), good (% ID,.9.), aeptable (% ID,.8.), and low onfidene (% ID, <8., but with the % ID sum of the first two or three taxa greater than or equal to 8). In this paper we have onsidered a orret identifiation as % ID > 9.. Conventional biohemial tests. The 36 Enterobateriaeae isolates employed in this study were identified aording to proedures desribed by Ewing (13). A few strains whih ould not be aurately identified with this proedure were fully haraterized by using the following additional tests as reported by Farmer et al. (14): growth in KCN and tyrosine learing (Citrobater amalonatius); fer 6 CIT mentation of Larabinose (Edwardsiella tarda); fermentation of amethyldgluoside and Darabitol (Enterobater gergoviae); saliin fermentation, esulin hydrolysis, and hlorampheniol suseptibility (Proteus penneri); fermentation of Dgalatose (Providenia rustigianii); and fermentation of muate and gelatin hydrolysis by rapid film method at 36 C (Salmonella arizonae). Enterobater hormaehei (enteri group 75) was identified aording to the sheme proposed by O'Hara et al. (2). Yersinia spp. were haraterized as reported by several authors (3, 5, 6, 8, 21). Final identifiation was determined aording to the table from the work of Ewing (13) and Farmer et al. (14). All fresh linial isolates of Salmonella or Shigella speies were onfirmed by serologial tests. RESLTS The results obtained with Enterosistem 18R and onventional methods in identifying the 36 strains belonging to 38 different speies of Enterobateriaeae are shown in Table 1. Enterosistem 18R agreed with the onventional methods in the identifiation of the 329 of 36 isolates (91.3%) at the speies level (% ID,.9.). Among these 329 strains, the system provided an exellent identifiation (% ID,.99.9) for most speies, in partiular those often isolated in bateriologial laboratories, suh as Citrobater diversus (8 of 9 strains tested), Citrobater freundii (9 of 12), Edwardsiella tarda (3 of 3), Enterobater aerogenes (8 of 1), Enterobater loaae (24 of 27), Esherihia oli (59 of 66), Klebsiella oxytoa (12 of 13), Klebsiella pneumoniae (25 of 33), Morganella morganii (8 of 11), Proteus mirabilis (41 of 45), P. penneri (8 of 1), and Providenia alalifaiens (2 of 2). All the Salmonella and Shigella speies were orretly identified (% ID,.9.) to the genus and speies level. However, serologial onfirmation was made throughout the evaluation of the system. Most of the unommon or newly desribed miroorganisms, suh as E. gergoviae (two of two strains tested), Providenia rustigianii (three of three), one strain of Serratia odorifera, and one isolate of Serratia rubidaea, were orretly identified (% ID,.99.9) by the system. In 11 ases (3.1%), Enterosistem 18R provided only genus identifiation. Of the abovementioned 329 strains, 54 were not diretly identified: 37 needed additional tests, and 17 were identified by the manufaturer's omputer servie. Fourteen miroorganisms (3.9%), inluding the newly desribed strains, suh as E. hormaehei and Yersinia frederiksenii, ould not be identified beause the generated sixdigit otal numbers were inluded neither in the odebook whih is furnished to laboratories nor in the data base available in the manufaturer's omputer. However, these 14 isolates produed biohemial reation patterns idential to those obtained with the onventional methods. The majority (1 of 14 strains tested) of these isolates were from the Klebsiella spp. (4 of 6), Proteus vulgaris (2 of 17), Providenia spp. (2 of 17), and Yersinia pseudotuberulosis (2 of 3). Enterosistem 18R disagreed with onventional methods for only 6 of the 36 isolates (1.7%) at both genus and speies levels (omplete disagreement). One C. diversus strain was identified as lysinenegative E. oli; one atypial strain of E. oli (onitrophenyl,3dgalatopyranoside [ONPG] negative, H2S positive, and sorbitol negative) was alled Edwardsiella tarda; one Enterobater agglomerans strain was identified Downloaded from on November 3, 218 by guest

3 232 PICCOLOMINI ET AL. J. CLIN. MICROBIOL. o4 I r r r enr4rz Ct.. N CA E ~~CO. C = z C. C. C4 C " 14 r 6 z.3 )4 '4 "~'4 r efc1. oc) C. ) E I.. O C,) CC 4.. 4o, o (A t (A '4 a. Ns C N l e 'IC N Cl4 r4 I., '4 'I ml, CO 4l, r W el Cl CO Cl It m el,t N Clr Cl4 ml Cl4 Cl CO C Cl, m r4 " 1 [ '/1. 1 <42o C OCl )1 ON '4t e t en ~e4n 1iCOOCI 1 4ON CD O C4 C NCiO\ C C n OCN \C C',M.9. tn., ClL 7NC r4 O'N L. qr r4r4w 14M r4c~" C IC ql C r 8 4, E l, *..2 :. t I Cu Cl4 ua et II u Q) n Cl,. 8 : ;.. C 4v C s C m 6. z. H:) Ot O ln en 4 P j LI.. S C o CeC 11 u: C O~ Cl el O N COe O = N Cl Nrl m el, Ill oo " C" n 4 C C * C,. Cl,.... *. C;. u C ) 4.Q N CO)..L. C LA C.. ' r C. i 4) $ H l r Downloaded from on November 3, 218 by guest (, L r C *~ > ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~k L. k) 4 L) i o *... g3a E t g ta E ff a} g 4 3t3f z 4 ~C~ 9~ *~ 4) ~ ~ ~ ~ ~ C ~ N LXL~ Z4LZ L~ Z (Z)Q Z a)amp

4 VOL. 29, 1991 IDENTIFYING ENTEROBACTERIACEAE WITH ENTEROSISTEM 18R 233 as S. rubidaea; one Yersinia enteroolitia strain that showed unusual features, suh as ornithine, urease, and sorbitol negativity, was identified as Enterobater agglomerans; finally, one ONPGnegative and H2S, arginine, and ornithinepositive C. freundii strain was misidentified by Enterosistem 18R as Salmonella spp., and one Y pseudotuberulosis was identified as ONPGpositive and indoleand sorbitolnegative Shigella spp. Instrutions for Enterosistem 18R suggest the use of the sheep blood agar as the primary medium plate from whih to prepare the tray inoulum. In onsideration of the relatively high ost and storage of this medium, we ompared the effets of the growth media (sheep blood versus MaConkey agar) on the identifiation. With the exeption of the muoid strains, suh as Klebsiella rhinosleromatis (insuffiient growth), we found that MaConkey agar an be used as a substitute for sheep blood agar for the preparation of the inoulum. Reproduibility of the tests with the quality ontrol strains was 1% suessful. DISCSSION Our results demonstrate that the Enterosistem 18R identifiation kit produed a good level of identifiation auray (% ID,.9.) for members of the Enterobateriaeae as ompared with onventional methods. Of the 329 orretly identified isolates that were reognized at speies level, 54 needed further tests or the aid of the manufaturer's omputer servie. Among these strains were of all the Hafnia alvei strains. This might be due to the inubation temperature (37 that we used, whih is not the optimum growth temperature for these speies (7). Similar problems were reported by Izard et al. with the API 2E system (16). At present, this is not mentioned in the user's instrutions for Enterosistem 18R. Although Enterosistem 18R orretly identified 91.3% of the 36 isolates tested, the profiles of 14 isolates, inluding the newly desribed strains (E. hormaehei and Y frederiksenii), did not orrespond to any key in the omputer data base or the odebook furnished to laboratories. Klebsiella spp., P. vulgaris, Providenia spp., and Y. pseudotuberulosis are the most diffiult speies to identify for Enterosistem 18R. If these 14 unlisted strains and respetive ode numbers had been inorporated in the odebook and in the manufaturer's data base, the perentage of miroorganisms orretly identified by the system would have risen from 91.3% to 95.3%. Only six strains were really misidentified by the Enterosistem 18R method beause they were atypial in a ertain harater or they gave one or two negative results with Enterosistem 18R and positive results when tested onventionally (espeially by ONPG, sorbitol fermentation, and ornithine dearboxylase tests). One strain of C. freundii and one strain of Y pseudotuberulosis were inorretly identified as Salmonella and Shigella speies, respetively. This is not a real diffiulty beause in any ase, identifiation of Salmonella and Shigella speies must inlude serologial onfirmation; in this ase, then, it is unlikely that the two misidentified speies will be onfused. Although it was misidentified, the Y enteroolitia strain was differentiated from E. agglomerans by motility, apart from the additional tests. Sine no relative indiation was given by the manufaturer, we assumed a temperature of 35 C, at whih the Y enteroolitia is nonmotile, whereas both of the speies should be motile at 18 to 22 C. It has been reported previously (11) that stok ultures would be less suitable to evaluate the performane of a baterial identifiation system than fresh linial isolates. After multiple in vitro passages or storage, bateria may undergo genotypi and phenotypi variations and somehow lose one or more biohemial harateristis. Beause Enterosistem 18R reveals onstitutive baterial enzymes, it is possible that the storage of Enterobateriaeae strains hanges enzyme systems, resulting in negative or weak reations, while fresh isolates may produe a positive reation. We did not find any differene with fresh linial isolates or frozen ultures (inluding those from the international ulture olletions). In fat, of the 213 fresh linial isolates, 92% (196 strains) were orretly identified by Enterosistem 18R, a figure whih is omparable to the 91.8% (135 of 147) of frozen ultures and referene strains orretly identified. With the lak of instrutions relative to the preparation of the tray inoulum, we suggest that the turbidity of the baterial suspension be equivalent to.5 MaFarland standard. This is espeially important beause for some strains with an inoulum density not arefully ontrolled, their identifiation by Enterosistem 18R is diffiult beause of the appearane of falsepositive or falsenegative biohemial reations. Moreover, the requirement for 4.5 ml of a baterial suspension equivalent to.5 MaFarland standard might prelude the use of this system for many primary ulture isolates after an overnight inubation. However, we disovered that if insuffiient miroorganisms are available, it is possible to use the suspension of a 6h ulture in brain heart infusion broth. In onlusion, we have evidene that Enterosistem 18R, in its present form, aurately identifies ommon and several unommon and newly desribed members of the family Enterobateniaeae. From a pratial point of view, the system was simple to use, easy to handle, and inexpensive. With adjustments in supplementary ode numbers for some strains, the auray and utility of Enterosistem 18R would be omparable to the qualities of other ommerial systems. REFERENCES 1. Bale, M. J., and J. M. Matsen Timemotion and ost omparison study of MiroID, API 2E, and onventional biohemial testing in identifiation of Enterobateriaeae. J. Clin. Mirobiol. 14: Bartlett, R. C Medial mirobiology: quality, ost and linial relevane, p John Wiley & Sons, In., New York. 3. Berovier, H., H. H. Mollaret, J. M. Alonso, J. Brault, G. R. Fanning, A. G. Steigerwalt, and D. J. Brenner Intra and interspeies relatedness of Yersinia pestis by DNA hybridization and its relationship to Yersinia pseudotuberulosis. Curr. Mirobiol. 4: Blazevi, D. J., D. L. Makay, and N. M. Warwood Comparison of MiroID and API 2E systems for identifiation of Enterobateriaeae. J. Clin. Mirobiol. 9: Bottone, E. J Atypial Yersinia enteroolitia: linial and epidemiologial parameters. J. Clin. Mirobiol. 7: Bottone, E. J. (ed.) Yersinia enteroolitia. CRC Press, In., Boa Raton, Fla. 7. Brenner, D. J Family I. Enterobatenaeae Rahn 1937, p In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systemati bateriology, vol. 1. The Williams & Wilkins Co., Baltimore. 8. Brenner, D. J., H. Berovier, J. rsing, J. M. Alonso, A. G. Steigerwalt, G. R. Fanning, G. P. Carter, and H. H. Mollaret. Downloaded from on November 3, 218 by guest

5 234 PICCOLOMINI ET AL Yersinia intermedia: a new speies of Enterobateriaeae omposed of rhamnosepositive, melibiosepositive, raffinosepositive strains (formerly alled atypial Yersinia enteroolitia or Yersinia enteroolitialike). Curr. Mirobiol. 4: Butler, T Yersinia speies (inluding plague), p In G. L. Mandel, R. G. Douglas, and J. E. Bennet (ed.), Priniples and pratie of infetious disease, 3rd ed. Churhill Livingstone, In., New York. 1. Davis, J. R., C. E. Stager, R. D. Wende, and S. M. Hussain Qadri Clinial laboratory evaluation of the AutoMirobi System Enterobateriaeae biohemial ard. J. Clin. Mirobiol. 14: Edberg, S. C., B. Atkinson, C. Chambers, M. H. Moore, L. Palumbo, C. F. Zorzon, and J. M. Singer Clinial evaluation of the MICROID, API 2E, and onventional media systems for identifiation of Enterobateriaeae. J. Clin. Mirobiol. 1: Edberg, S. C., and L. S. Konowue A systemati means to ondut a mirobiology evaluation, p In V. Lorian (ed.), Signifiane of medial mirobiology in the are of patients, 2nd ed. The Williams & Wilkins Co., Baltimore. 13. Ewing, H. E Edwards and Ewing's identifiation of Enterobateriaeae, 4th ed. Burgess Publishing Co., Minneapolis. 14. Farmer, J. J., III, B. R. Davis, F. W. HikmanBrenner, A. MWhorter, G. P. HuntleyCarter, M. A. Asbury, C. Riddle, H. G. WathenGrady, C. Elias, G. R. Fanning, A. G. Steigerwalt, C. M. O'Hara, G. K. Morris, P. B. Smith, and D. J. Brenner Biohemial identifiation of new speies and biogroups of Enterobateriaeae isolated from linial speimens. J. Clin. Mirobiol. 21:4676. J. CLIN. MICROBIOL. 15. Gooh, W. M., III, and G. A. Hill Comparison of MiroID and API 2E in rapid identifiation of Enterobateriaeae. J. Clin. Mirobiol. 15: Izard, D., M.. Husson, P. Vinent, H. Leler, D. Monget, and J. M. Boeufgras Evaluation of the fourhour Rapid 2E system for identifiation of members of the family Enterobateriaeae. J. Clin. Mirobiol. 2: Kelly, M. T., D. J. Brenner, and J. J. Farmer III Enterobateriaeae, p In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of linial mirobiology, 4th ed. Amerian Soiety for Mirobiology, Washington, D.C. 18. Kelly, M. T., and J. M. Latimer Comparison of the AutoMirobi System with API, Enterotube, MiroID, Miro Media Systems, and onventional methods for identifiation of Enterobateriaeae. J. Clin. Mirobiol. 12: Lapage, S. P., S. Basomb, W. R. Willox, and M. A. Curtis Identifiation of bateria by omputer: general aspets and perspetives. J. Gen. Mirobiol. 77: O'Hara, C. M., A. G. Steigerwalt, B. C. Hill, J. J. Farmer III, G. R. Fanning, and D. J. Brenner Enterobater hormaehei, a new speies of the family Enterobateriaeae formerly known as enteri group 75. J. Clin. Mirobiol. 27: rsing, J., D. J. Brenner, H. Berovier, G. R. Fanning, A. G. Steigerwalt, J. Brault, and H. H. Mollaret Yersinia frederiksenii: a new speies of Enterobateriaeae omposed of rhamnosepositive strains (formerly alled atypial Yersinia enteroolitia or Yersinia enteroolitialike). Curr. Mirobiol. 4: Downloaded from on November 3, 218 by guest

Simple Bacterial Preservation Medium and Its Application to

Simple Bacterial Preservation Medium and Its Application to APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1978, p. 487491 0099-2240/78/0035-0487$02.00/0 Copyright i 1978 Amerian Soiety for Mirobiology Vol. 35, No. 3 Printed in U.S.A. Simple Baterial Preservation