microbiology,', - jcm.asm.orgjcm.asm.org/content/12/4/509.full.pdf ·...

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JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1980, p. 509-516 0095-1137/80/10-0509/08$02.00/0 Vol. 12, No. 4 Evaluation of the Minitek System for Identification of Nonfermentative and Nonenteric Fermentative Gram-Negative Bacteria BRENT CHESTER" * AND TIMOTHY J. CLEARY' Laboratory Service, Veterans Administration Hospital,' and Departments of Pathology and Microbiology,"', University of Miami School of Medicine, Miami, Florida 33125 The Minitek identification system (MT) was compared with a conventional testing battery for the characterization of 735 isolates which included 57 species and groups of nonfermentative (NF) and nonenteric fermentative (NEF) gram- negative bacteria. The MT correctly identified 585 of 616 NF (94.96%) and 115 of 119 NEF (96.65%) bacteria and 700 of 735 strains (95.24%) overall. A total of 31 NF and NEF (4.22%) bacteria were misidentified, and no identification was determined for four strains (0.69%). All strains of Acinetobacter anitratus, Pseu- domonas maltophilia, P. fluorescens, and P. putida and all but one strain of P. aeruginosa were correctly identified. The most frequently misidentified taxa were CDC group Va-1, P. pickettii (Va-2), P. mendocina, and Moraxella urethralis (M-4). Supplemental tests were needed for the complete identification of 214 strains (29.11%). An average of 1.54 supplemental tests were used with each of these strains. A total of 134 strains (18.23%) had their identification delayed by 1 day due to supplemental testing. We recommend the use of the 42°C growth test with the MT. When used in accord with the manufacturer' instructions and with the MT code book, the MT was found to be a valuable system for the identification of a wide variety of common and infrequently encountered NF and NEF bacteria. Although most clinical isolates of gram-nega- tive bacteria are enterics, nonfermentative (NF) and nonenteric fermentative (NEF) bacteria are encountered daily in most laboratories. At one of our laboratories, perhaps as a consequence of the large number of chronic and debilitated pa- tients, NF and NEF bacteria account for more than 10% of all gram-negative isolates. The identification of NF and NEF bacteria has been a continuing problem for most labora- tories. Although conventional methodology de- signed to deal with this problem has been avail- able for many years, it has nevertheless failed to gain widespread acceptance. Recently, a variety of commercial, packaged systems for the identi- fication of these two bacterial groups have be- come available to the microbiologist. These sys- tems offer an attractive alternative to conven- tional methodology through such features as rapid (24 to 48 h) and computer-assisted identi- fication, convenience, ease of use, and cost-effec- tiveness. This study is the first to evaluate one such system, the Minitek (MT; BBL Microbiology Systems, Cockeysviile, Md.) with strict adher- ance to the manufacturer's instructions and with the aid of a computerized identification code book. Although three previous studies of the MT system for the identification of NF and NEF bacteria have been published (2, 15; M. Slifkin and G. R. Pouchet, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, C88, p. 50), none had the benefit of the code book nor several recent modifications of the MT methodology. Importantly, this study represents the most comprehensive evaluation, to date, of any com- mercial packaged system designed for the iden- tification of NF and NEF bacteria. MATERIALS AND METHODS Bacteria. The 735 bacteria tested contained rep- resentatives of 57 species and groups of NF and NEF bacteria (Table 1). Of the total bact ria, 306 strains were reference cultures, primarily of the less fre- quently encountered members of these two bacterial groups (courtesy of R. E. Weaver, Special Bacteriology Section, Center for Disease Control, Atlanta, Ga. and G. L. Gilardi, Hospital for Joint Diseases and Medical Center, New York City, N.Y.). The remainder, 429 strains, were fresh clinical isolates (not transferred more than two times) from the microbiology labora- tories of seven hospitals. With the exception of Pseu- domonas mallei and Moraxella lacunata, each of the 56 species and groups for which the MT system makes an identification claim was tested. One isolate of Vi- brio vulnificus (6) (Vibrio species, lactose positive), 509 on August 25, 2018 by guest http://jcm.asm.org/ Downloaded from

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Page 1: Microbiology,', - jcm.asm.orgjcm.asm.org/content/12/4/509.full.pdf · Microbiology,"', UniversityofMiamiSchoolofMedicine,Miami,Florida33125 ... book. Although three previous studies

JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1980, p. 509-5160095-1137/80/10-0509/08$02.00/0

Vol. 12, No. 4

Evaluation of the Minitek System for Identification ofNonfermentative and Nonenteric Fermentative

Gram-Negative BacteriaBRENT CHESTER"* AND TIMOTHY J. CLEARY'

Laboratory Service, Veterans Administration Hospital,' and Departments ofPathology andMicrobiology,"', University ofMiami School ofMedicine, Miami, Florida 33125

The Minitek identification system (MT) was compared with a conventionaltesting battery for the characterization of 735 isolates which included 57 speciesand groups of nonfermentative (NF) and nonenteric fermentative (NEF) gram-negative bacteria. The MT correctly identified 585 of 616 NF (94.96%) and 115 of119 NEF (96.65%) bacteria and 700 of 735 strains (95.24%) overall. A total of 31NF and NEF (4.22%) bacteria were misidentified, and no identification wasdetermined for four strains (0.69%). All strains of Acinetobacter anitratus, Pseu-domonas maltophilia, P. fluorescens, and P. putida and all but one strain of P.aeruginosa were correctly identified. The most frequently misidentified taxa wereCDC group Va-1, P. pickettii (Va-2), P. mendocina, and Moraxella urethralis(M-4). Supplemental tests were needed for the complete identification of 214strains (29.11%). An average of 1.54 supplemental tests were used with each ofthese strains. A total of 134 strains (18.23%) had their identification delayed by 1day due to supplemental testing. We recommend the use of the 42°C growth testwith the MT. When used in accord with the manufacturer' instructions and withthe MT code book, theMT was found to be a valuable system for the identificationof a wide variety ofcommon and infrequently encountered NF and NEF bacteria.

Although most clinical isolates of gram-nega-tive bacteria are enterics, nonfermentative (NF)and nonenteric fermentative (NEF) bacteria areencountered daily in most laboratories. At oneof our laboratories, perhaps as a consequence ofthe large number of chronic and debilitated pa-tients, NF and NEF bacteria account for morethan 10% of all gram-negative isolates.The identification of NF and NEF bacteria

has been a continuing problem for most labora-tories. Although conventional methodology de-signed to deal with this problem has been avail-able for many years, it has nevertheless failed togain widespread acceptance. Recently, a varietyof commercial, packaged systems for the identi-fication of these two bacterial groups have be-come available to the microbiologist. These sys-tems offer an attractive alternative to conven-tional methodology through such features asrapid (24 to 48 h) and computer-assisted identi-fication, convenience, ease of use, and cost-effec-tiveness.This study is the first to evaluate one such

system, the Minitek (MT; BBL MicrobiologySystems, Cockeysviile, Md.) with strict adher-ance to the manufacturer's instructions and withthe aid of a computerized identification codebook.

Although three previous studies of the MTsystem for the identification of NF and NEFbacteria have been published (2, 15; M. Slifkinand G. R. Pouchet, Abstr. Annu. Meet. Am. Soc.Microbiol. 1977, C88, p. 50), none had the benefitofthe code book nor several recent modificationsof the MT methodology.

Importantly, this study represents the mostcomprehensive evaluation, to date, of any com-mercial packaged system designed for the iden-tification of NF and NEF bacteria.

MATERIALS AND METHODS

Bacteria. The 735 bacteria tested contained rep-resentatives of 57 species and groups of NF and NEFbacteria (Table 1). Of the total bact ria, 306 strainswere reference cultures, primarily of the less fre-quently encountered members of these two bacterialgroups (courtesy of R. E. Weaver, Special BacteriologySection, Center for Disease Control, Atlanta, Ga. andG. L. Gilardi, Hospital for Joint Diseases and MedicalCenter, New York City, N.Y.). The remainder, 429strains, were fresh clinical isolates (not transferredmore than two times) from the microbiology labora-tories of seven hospitals. With the exception of Pseu-domonas mallei and Moraxella lacunata, each of the56 species and groups for which the MT system makesan identification claim was tested. One isolate of Vi-brio vulnificus (6) (Vibrio species, lactose positive),

509

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510 CHESTER AND CLEARY

TABLE 1. NF and NEF gram-negative bacteriaexamined

Taxon

Acinetobacter anitratusA. lwoffiAchromobacter species (Vd)A. xylosoxidansAeromonas hydrophilaAlcaligenes denitrificansA. faecalisA. odoransActinobacillus lignieresiiBordetella bronchisepticaB. parapertussisCardiobacterium hominisChromobacterium violaceumCDC groupEF-41IfIIjIIk-2IVc-2IVeVa-1Ve-1Ve-2

Flavobacterium odoratum (M-4f)Flavobacterium species IIbF. meningosepticumHaemophilus aphrophilusMoraxella bovisM. nonliquefaciensM. osloensisM. phenylpyruvicaM. urethralis (M-4)Pasteurella haemolyticaP. multocidaP. pneumotropicaP. ureaePlesiomonas shigelloidesPseudomonas acidovoransP. alcaligenesP. aeruginosa

PyocyanogenicApyocyanogenicMucoid

P. cepaciaP. diminutaP. fluorescensP. maltophiliaP. mendocina (Vb-2)P. paucimobilis (IIk-1)P. pickettii (Va-2)P. pseudoalcaligenesP. pseudomalleiP. putidaP. putrefaciensP. stutzeri (Vb-1, Vb-3)P. testosteroneP. vesicularisVibrio alginolyticusV. choleraeV. parahaemolyticusV. vulnificus

Freshclinicalisolates

36il161810o6

20O2

14

O3O42Oo1o19il72oO2oOo4Oo131

7419677

162911o12o

278183o4151

Refer-ence

strains

153183525228322

1797488109716113O845837896

i3o348447682685510647O

the results.Conventional methodology. All strains were

identified using criteria and procedures recommendedby the American Society for Microbiology (8), theCenter for Disease Control (14), and G. L. Gilardi (7).In addition, a modified silver stain for flagella (16), asemi-quantitative catalase test (3), and a distilled wa-ter immobilization test for vibrios (4) were employed.MT system for NF and NEF bacteria. The MT

system for the identification of NF and NEF bacteriaconsists of paper disks impregnated with appropriatebiochemicals: dextrose, maltose, sucrose, xylose, urea,citrate, nitrate (reduction and denitrification), phenyl-alanine, ornithine, arginine, lysine, o-nitrophenyl-,f-D-galactopyranoside (ONPG), and starch. The sus-pending broth serves as substrate for the indole test.The disks are dispensed into a plastic plate containingwells. Into each well containing a disk, 0.05 ml oforganism suspended in broth (MT enteric and nonfer-menter broth, BBL Microbiology Systems) is pipetted.Into the well containing the arginine disk, 0.10 ml ofinoculum is pipetted. For indole detection, 0.15 ml ofinoculum is placed into an empty well. After overlay-ing with oil those disks containing dextrose (a seconddextrose disk is not overlaid), urea, ornithine, arginine,and lysine, the plates are incubated in a humidor at35°C for 48 h (ONPG and urea are read at 24 h). Afterthe addition of appropriate reagents for indole, nitratereduction and denitrification, phenylalanine deami-nation, and starch hydrolysis, the observed reactionsare interpreted with tables and a code book providedby the manufacturer.MT code book. The MT NF and NEF bacteria

code book contains the MT numerical identificationsystem. Based on the reactions of a test isolate withthe 16 MT substrates, as well as the indophenol oxi-dase reaction and the ability of the isolate to grow onMacConkey agar within 48 h, a six-digit profile numberis generated. When listed in the code book, the profilenumber is accompanied by three possible identifica-tions. The first selection identification has the highestconfidence value (100 is perfect), followed by the sec-ond and third selection. Also listed for each selectionis its biotype validity value (1 is a perfect "fit").Supplemental tests are frequently listed to aid in thedifferentiation of the three selections. When a profilenumber is not listed in the code book, a toll-free cailis made to the BBL Technical Service Department toobtain any information in the master code pertainingto the profile number in question.

Criteria for correct identifications. In thisstudy, a correct identification as "system only" wasone in which the first selection had a confidence valueof 90% or greater and matched the identification ob-tained with conventional methodology.An identification was scored as "correct with the

MT system and supplemental tests" when the conven-tional identification matched one of the three selec-tions, none of which had a confidence value of at least90%, so that supplemental testing was needed.

Correct identifications obtained from the BBLTechnical Service Department were "correct with tel-ephone assistance."

not included in the MT coding system, was also ex-amined. No single taxon represented more than 14%of the total examined, to prevent any one group orspecies from asserting a disproportionate influence on

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MINITEK FOR NONFERMENTERS AND FERMENTERS 511

RESULTSThe MT system correctly identified 700 of 735

(95.24%) strains of NF and NEF bacteria.Thirty-one (4.22%) were misidentified, and four(0.69%) were not given a MT identification (Ta-ble 2).NF bacteria. Of the 616 strains of NF bacte-

ria tested the MT system correctly identified585 (94.96%), misidentified 28 (4.55%), and hadno identification for 3 (0.49%) isolates (Table 3).Among the 585 strains correctly identified, 371(63.42%) were identified by the MT systemalone, 203 (34.70%) required one or more supple-mental tests, and 11 (1.88%) needed the aid ofthe MT telephone assistance service. A 100%accuracy of identification level was reached for30 of the 40 NF bacterial taxa examined, includ-ing strains of P. aeruginosa, A. anitratus, P.maltophilia, P. putida, and P. fluorescens, butwith the exception of one pyocyanogenic P.aeruginosa isolate. Among the 28 misidentifi-cations, 16 (57.10%) were assigned to the wronggenus and the remaining 12 (42.90%) were incor-rect at the species level. The poorest perform-ance ofthe MT system was seen with P. pickettii(Va-2), CDC group Va-1, P. mendocina (Vb-2),and M. urethralis (M-4). Due to a code bookmisprint, five of six strains of CDC group Va-1were misidentified as A. xylosoxidans or P.pseudoalcaligenes. Four of six strains ofP. pick-ettii (Va-2) were misidentified as A. xylosoxi-dans. Only two of five P. mendocina (Vb-2)isolates were correctly identified, although thistaxon is not included in the code book. All fourstrains ofM. urethralis (M-4) were misidentified(Table 5).NEF bacteria. Of the 119 strains of NEF

bacteria examined, the MT system correctlyidentified 115 (96.65%), misidentified 3 (2.52%),and had no identification for 1 (0.86%) (Table 4).Among the 115 strains correctly identified, 103(90.19%) were identified by the MT systemalone, 10 (8.25%) required one or more supple-mental tests, and 2 (1.65%) needed telephoneassistance. Thirteen of the 17 NEF species andgroups had all strains correctly identified (Table4). One Pasteurella haemolytica isolate was

misidentified as P. multocida, one strain of Vi-brio alginolyticus was incorrectly identified asAeromonas hydrophila, and a strain of V. vul-nificus, not included in the MT coding system,was misidentified as V. cholerae (Table 5).Supplemental testing. Slightly more than

half of the species and groups (29 of 57) requiredno supplemental testing for correct identificationwith the MT system. Of the 28 taxa containingstrains which required supplemental testing, an

average of 1.54 tests per strain were needed.However, the majority of strains belonging tothese 28 taxa (56.77%) required no supplementaltests (Table 6).

DISCUSSIONThe MT system was used to identify 735

isolates representing 57 taxa of NF and NEFbacteria. This study is the most comprehensiveevaluation, to date, ofthe ability of a commercialsystem to identify members of these two bacte-rial groups. Overall, the MT system correctlyidentified 95.4% of these isolates (Table 2) andreached a slightly higher level with the NEFbacteria, 96.65%, than with the NF bacteria,94.96% (Tables 3 and 4). The performance of theMT system is especially impressive in view ofthe variety, number, and distribution of bacteriainvolved. Previous studies (1, 2, 5, 7, 10-13, 15)which used commercial systems for the identi-fication of NF and NEF bacteria were limited inthe number of taxa studied. In the present studyno taxon comprises more than 14% of the total.The above evaluations were unbalanced in termsof bacterial distribution, with P. aeruginosa iso-lates accounting for as much as 75% of the totalexamined. The identification rates reported inthe above studies ranged from 43 to 95.3% (1, 2,5, 7, 10-13, 15).

It was significant that only 1 isolate of 176 P.aeruginosa, P. fluorescens, P. putida, P. mal-tophilia, and A. anitratus was misidentified(Table 3). In a recent MT system evaluation,only 2 of 137 isolates of these same five groupswere misidentified (2). The fact that these fivespecies are the most common NF bacteria en-countered in significant clinical situations is re-flected in the 1979 blood culture statistics from

TABLE 2. Overall identification ability of the MT system for NF and NEF gram-negative bacteriaCorrect identifications Misidentifications

Group ~~~~~~System Tl No iden-Group Strains System with sup- Tele Wrong Wrong tificationphone as-tested only plemental sistance genus species

tests

NF 616 371 203 11 16 12 3NEF 119 101 12 2 1 2 1

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TABLE 3. Ability of theMT system to identify NF gram-negative bacteria

Correct identifications Misidentifica-tions

Taxon System Tele- No iden-Strains System with phone Wrong Wrong tificationtested only mental assist- genus species

test ance

Acinetobacter anitratus 51 36 15 0 0 0 0A. lwoffi 14 14 0 0 0 0 0Achromobacter species (Vd) 34 30 1 1 2 0 0A. xylosoxidans 21 12 9 0 0 0 0Alcaligenes denitrificans 2 0 0 0 0 0 0A. faecalis il 0 il 0 0 0 0A. odorans 22 0 22 0 0 0 0Bordetella bronchiseptica 10 10 0 0 0 0 0B. parapertussis 4 4 0 0 0 0 0CDC group

IIf 10 6 0 2 0 2 0IIj 9 7 2 0 0 0 0IIk-2 il il 0 0 0 0 0IVc-2 6 0 6 0 0 0 0IVe 8 3 4 0 1 0 0Va-i 8 0 3 0 2 3 0Ve-1 il 9 2 0 0 0 0Ve-2 9 2 7 0 0 0 0

Flavobacterium odoratum (M-4f) 26 22 0 2 2 0 0Moraxella bovis 1 0 1 0 0 0 0M. nonliquefaciens 3 0 3 0 0 0 0M. osloensis 2 0 2 0 0 0 0M. phenylpyruvica 8 2 4 1 0 0 1M. urethralis (M-4) 4 0 0 0 3 1 0Pseudomonas acidovorans 12 0 12 0 0 0 0P. alcaligenes 7 0 7 0 0 0 0P. aeruginosa

Pyocyanogenic 75 64 10 0 0 1 0Apyocyanogenic 22 16 4 0 0 0 2Mucoid 6 3 3 0 0 0 0

P. cepacia 10 10 0 0 0 0 0P. diminuta il il 0 0 0 0 0P. fluorescens 24 0 24 0 0 0 0P. maltophilia 33 32 1 0 0 0 0P. mendocina (Vb-2) 5 0 0 1 2 2 0P. paucimobilis (IIk-1) 8 6 2 0 0 0 0P. pickettii (Va-2) 6 0 1 1 4 0 0P. pseudoalcaligenes 20 12 7 1 0 0 0P. pseudomallei 2 2 0 0 0 0 0P. putida 33 O 33 O O O OP. putrefaciens 16 15 0 1 0 0 0P. stutzeri (Vb-1, Vb-3) 23 21 0 1 0 1 0P. testosteroni 8 0 8 0 0 0 0P. vesicularis 10 10 0 0 0 0 0

one of our laboratories (T.J.C.). It was found M. urethralis (M-4) (4 isolates) (Table 5). Thethat, of 130 NF isolates from blood cultures of remaining 14 misidentifications were scattered130 patients, 115 belonged to these five species among eight taxa of NF bacteria and three spe-of NF bacteria. cies of NEF bacteria. Each of five isolates ofAmong the 31 isolates misidentified, 17 be- CDC group Va-i was misidentified due to an

longed to one of four taxa of NF bacteria: CDC MT coding error which programmed positivegroup Va-i (5 isolates); P. pickettii (Va-2) (4 maltose reactions for members of this group.isolates); P. mendocina (Vb-2) (4 isolates); and These bacteria are uniformly negative for mal-

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MINITEK FOR NONFERMENTERS AND FERMENTERS 513

TABLE 4. Ability of the MT system to identify NEF gram-negative bacteria

Correct identifications Misidentifica-tions

Taxon System Tele- No iden-Strains System with hone Wrong Wrong tificationsupple-tested only mental assist- genus species

tests ance

Aeromonas hydrophila 15 14 0 1 0 0 0Actinobacillus lignieresii 2 2 0 0 0 0 0Cardiobacterium hominis 3 3 0 0 0 0 0Chromobacterium violaceum 6 6 0 0 0 0 0CDC group EF-4 1 1 0 0 0 0 0Flavobacterium species IIb 12 10 2 0 0 0 0F. meningosepticum 13 9 3 1 0 0 0Haemophilus aphrophilus 3 3 0 0 0 0 0Pasteurella haemolytica 5 4 0 0 0 1 0P. multocida 12 6 5 0 0 0 1P. pneumotropica 3 3 0 0 0 0 0P. ureae 7 7 0 0 0 0 0Plesiomonas shigelloides 9 9 0 0 0 0 0Vibrio alginolyticus 10 9 0 0 1 0 0V. cholerae 5 5 0 0 0 0 0V. parahaemolyticus 12 12 0 0 0 0 0V. vulnificus 1 0 0 0 0 1 0

tose utilization in the MT system. Each of fourisolates of P. pickettii (Va-2) was misidentifiedas A. xylosoxidans. Both of these bacteria gen-erate the same profile number, but the MTsystem is programmed to select A. xylosoxidansas the first choice (confidence value greater than90%) in this situation. To avoid this error, bac-teria identified by the MT system as A. xylosox-idans should be shown to demonstrate a rapidlypositive catalase test, whereas a delayed positivereaction would indicate further testing with P.pickettii as a possible identification (3).

In a recent evaluation of the MT system,93.4% of the NF bacteria tested were correctlyidentified (2). This result strongly supports the94.96% accuracy rate obtained in the presentstudy.

In another published report (15) using the MTsystem for the identification of NF and NEFbacteria, 92.6% of the isolates tested were iden-tified to the genus level and 88.3% were correctat the species level. These lower identificationrates, in comparison with our 95.2% rate, can beattributed to three factors. First, at the time ofthe prior evaluation, the MT coding system wasnot available. Secondly, certain improvementsin the MT system methodology, e.g., increasedinoculum size for arginine, were not utiized.Finally, the author did not use the manufac-turer's recommended battery of substrate disksbut chose to delete starch, ONPG, phenylala-nine, and sucrose while adding lactose and man-nitol. Also, only the nitrate reduction reaction

but not denitrification results were recorded.Had the MT code book, improved methodology,and the recommended disks been used in theprior evaluation, 12 of the 27 misidentificationswould have been avoided. The correspondingincrease in correct identification would havebeen 95.23% at the genus level and 93.48% at thespecies level.

It was necessary to use the BBL TechnicalService Department's telephone assistance withonly 13 (1.8%) of the isolates examined. This lowusage was in marked contrast to the 25 and 36%rates for telephone-assisted identifications re-ported for another commercial system (5, 10).Of the 57 taxa, 29 contained no strains requir-

ing supplemental tests and were identified bythe basic MT system. Among the remaining 28groups and species, an average of 1.54 supple-mental tests were needed for those strains re-quiring additional testing. When same-day sup-plemental tests such as catalase, pigmentation,and motility are discounted, only 134 (18.23%)of the 735 isolates in the study had their identi-fications delayed, usually by 1 day (Table 6).

In view of the need for testing for growth at420C to differentiate apyocyanogenic strains ofP. aeruginosa, especially denitrification-nega-tive isolates, from P. fluorescens and P. putida,we recommend the routine use of this test inaddition to the MT system to avoid an unduedelay in identification. A similar suggestion wasproposed in a recent evaluation of the MT sys-

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TABLE 5. MT system misidentifications ofNF and NEF gram-negative bacteria

Correct identification MT identification MT pro- MT discrepancies"file no.

Achromobacter species (Vd) Pseudomonas pseudoalcaligenes

Achromobacter species (Vd)Alcaligenes dentrificansA. denitrificansCDC group I1f

CDC group IIf

CDC group IVeFlavobacterium odoratum (M-4f)

F. odoratum (M-4f)

Moraxella urethralis (M-4)M. urethralis (M-4)M. urethralis (M-4)M. urethralis (M-4)Pasteurella haemolyticaPseudomonas aeruginosaPseudomonas mendocina (Vb-2)P. mendocina (Vb-2)P. mendocina (Vb-2)

P. mendocina (Vb-2)P. pickettii (Va-2)P. pickettii (Va-2)P. pickettii (Va-2)P. pickettii (Va-2)CDC group Va-1

CDC group Va-1

CDC group Va-1

CDC group Va-1

CDC group Va-i

Pseudomonas stutzeri (Vb-1,Vb-3)

Vibrio alginolyticus

Vibrio vulnificus

Moraxella phenylpyruvicaAlcaligenes faecalisA. faecalisCDC group IIj

CDC group IIj

M. phenylpyruvicaP. paucimobilis (IIk-1)

P. paucimobilis (IIk-1)

Alcaligenes/PseudomonasPseudomonas diminutaMoraxella atlantae (M-3)Alcaligenes/PseudomonasPasteurella multocidaP. pseudoalcaligenesAchromobacter species (Vd)P. aeruginosaAchromobacter species (Vd)

P. aeruginosaAchromobacter xylosoxidansA. xylosoxidansA. xylosoxidansA. xylosoxidansA. xylosoxidans

P. pseudoalcaligenes

P. pseudoalcaligenes

A. xylosoxidans

P. pseudoalcaligenes

P. aeruginosa

Aeromonas hydrophila

Vibrio choleraea -, Negative; +, positive; Dex, dextrose; Malt, maltose; Suc, sucrose; Xyl, xylose.

644022 Urea-, denitrifica-tion-

604222 Denitrification-600021 Denitrification-600021 Denitrification-474042 Dex+, Malt', Suc+,

Xyl+474042 Dex+, Malt', Suc+,

Xyl+600223 Phenylalanine+674202 Dex+, Malt', Suc+,

Xyl+674202 Dex+, Malt', Suc+,

Xyl+600001 Phenylalanine-600002 Citrate-600000 Phenylalanine-600001 Phenylalanine-574420 Ornithine+642021 Xylose-644013 Arginine-646011 Not in code book646013 Misprint in code

book, correctidentification viatelephone

646011 Not in code book604021 Urea-604021 Urea-604021 Urea-604021 Urea-604021 Code book mis-

print604023 Code book mis-

print604023 Code book mis-

print604001 Code book mis-

print604021 Code book mis-

print646211 Urea+, starch+

772161 Not in code bookdue to arginine+,ONPG+

770560 Not in code book

tem (2). Another helpful addition to the MTsystem would be an acetamide disk. Acetamideutilization can be used to provide the differen-tiation described in regard to the 42°C growthtest. In addition, an acetamide disk would negatesupplemental testing now required for the MTidentification of P. acidovorans. The acetamidedisk would also serve to distinguish A. xylosox-idans (positive) from P. pickettii (Va-2) (nega-tive) and therefore strengthen a weak identifi-cation capabiity of the MT system (Table 5).

The importance of adhering strictly to theMT procedure, as is the case with any commer-cial system, cannot be overemphasized. Thisadherance applied particularly to the use ofMTreagents and to color interpretations of the sub-strate disks. It is also important to consider themorphology of the test isolate in relation to theMT identification offered. In our study, the MTsystem was found to be a valuable system forthe identification of a wide variety of commonand infrequently encountered NF and NEF bac-

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MINITEK FOR NONFERMENTERS AND FERMENTERS 515

TABLE 6. Taxa ofNF and NEF gram-negative bacteria requiring supplemental tests with the MT system

Strains No. ofStrin requiring tests

Taxon tested supple- Alternative identification Supplemental tests Noomentary strain

tests

Acinetobacter anitratus 51 15 CDC group Ve-1, Ve-2

Achromobacter species (Vd)A. xylosoxidansAlcaligenes faecalis

A. odorans

CDC group IIj

CDC group IVc-2CDC group IVeCDC group Va-i

CDC group Ve-1

CDC group Ve-2

Flavobacterium species IIbF. meningosepticumMoraxella bovisM. nonliquefaciensM. osloensis

M. phenylpyruvicaPasteurella multocidaPseudomonas acidovorans

P. alcaligenes

P. aeruginosaPyocyanogenicApocyanogenicMucoid

P. fluorescensP. maltophiliaP. paucimobilisP. pickettii (Va-2)

P. pseudoalcaligenes

P. putida

P. testosteroni

34 1 Pseudomonaspaucimobilis21 9 P. pickettii (Va-2)il 1 Alcaligenes, Pseudomonas

acidovorans, P. testos-teroni, P. alcaligenes,P. pseudoalcaligenes

22 22 Alcaligenes, P. acidovorans,P. testosteroni, P. alcalig-enes, P. pseudoal-caligenes

9 2 Cardiobacterium hominis,Pasteurella multocida

6 6 CDC group IVe8 4 Moraxella phenylpyruvica8 3 P. pseudoalcaligenes, A.

xylosoxidans

11 2 A. anitratus, P. pauci-mobilis

9 7 A. anitratus

1213132

81212

7

123132

4512

C. hominisFlavobacterium species IIbM. osloensisM. osloensis, M. lacunataM. bovis, M. lacunata

CDC group IVeC. hominisAlcaligenes, P. testosteroni

7 Alcaligenes, P. pseudo-alcaligenes

75 10 P. fluorescens/P. putida22 4 P. fluorescens/P. putida6 3 P. fluorescens/P. putida24 24 P. aeruginosa, P. putida33 1 Pseudomonas cepacia8 2 Pasteurella haemolytica6 1 A. xylosoxidans, P.

pseudoalcaligenes20 7 Pseudomonas putrefaciens,

Alcaligenes, P. alcalig-enes

33 33 P. aeruginosa, P. fluores-cens

8 8 Alcaligenes, P. acidovorans

Mannitol or yellowpigment

Mannitol42°C or catalaseFlagella stain,

denitrification,fruity odor

Fruity odor

Catalase, gelatin

Rapid ureaMotilityFlagella stain or cat-

alase, denitrifica-tion

Mannitol or yellowpigment

Mannitol or yellowpigment

CatalaseMannitolBeta-hemolysisGelatin, Loeffler'sBeta-hemolysis,

Loeffler'sMotilityCatalaseFlagella stain,acetamide

Flagella stain,fructose

420C420C420C42°C, lecithinaseDeoxyribonucleaseMannitol420C or catalase,

denitrificationGelatin, flagella

stain, fructose

42°C, lecithinase

Flagella stain, acet-amide

teria. It was accurate and easy to use, provided-rapid identifications with minimal supplementaltesting, and was cost-effective.

LITERATURE CITED

1. Barnishan, J., and L W. Ayers. 1979. Rapid identifi-

cation of nonfermentative gram-negative rods by the

Corning N/F system. J. Clin. Microbiol. 9:239-243.2. Burdash, N. M., E. R. Bannister, J. P. Manos, and M.

E. West. 1980. A comparison of four commercial sys-tems for the identification of nonfermentative bacilli.Am. J. Clin. Pathol. 73:564-569.

3. Chester, B. 1979. Semiquantitative catalase test as an aid

in the identification of oxidative and nonsaccharolyticgram-negative bacteria. J. Clin. Microbiol. 10:525-528.

1

1i2.27

1

2

111.66

i

i

ii122

112

2

1111.831i2

1.57

1.90

2

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516 CHESTER AND CLEARY

4. Chester, B., and E. G. Poulos. 1980. Rapid, presumptiveidentification of vibrios by immobilization in distilledwater. J. Clin. Microbiol. 11:543-545.

5. Dowda, H. 1977. Evaluation of two rapid methods foridentification of commonly encountered nonfermentingor oxidase-positive, gram-negative rods. J. Clin. Micro-biol. 6:605-609.

6. Farmer, J. J. III. 1979. Vibrio ("Beneckea") vulnificus,the bacterium associated with sepsis, septicemia, andthe sea. Lancet ii:903.

7. Gilardi, G. L. 1980. Identification of nonfermentativegram-negative bacteria. Hospital for Joint Diseases andMedical Center, New York City.

8. Lennette, E. H., E. H. Spaulding, and J. P. Truant(ed.). 1974. Manual of clinical microbiology, 2nd ed.American Society for Microbiology, Washington, D.C.

9. Nadler, H., H. George, and J. Barr. 1979. Accuracy andreproducibility of the Oxi/Ferm system in identifying aselect group of unusual gram-negative bacilli. J. Clin.Microbiol. 9:180-185.

10. Oberhofer, T. 1979. Comparison of the API-20E and Oxi/Ferm systems in identification of nonfermentative andoxidase-positive fermentative bacteria. J. Clin. Micro-biol. 9:220-226.

J. CLIN. MICROBIOL.

11. Oberhofer, T. R., J. W. Rowen, G. F. Cunningham,and J. W. Higbee. 1977. Evaluation of the Oxi/Fermtube system with selected gram-negative bacteria. J.Clin. Microbiol. 6:559-566.

12. Shayegani, M., A. M. Lee, and D. M. McGlynn. 1978.Evaluation of the Oxi/Ferm tube system for identifica-tion of nonfermentative gram-negative bacteria. J. Clin.Microbiol. 7:533-538.

13. Shayegani, M., P. G. Maupin, and D. M. McGlynn.1978. Evaluation of the API-20E system for identifica-tion of nonfermentative gram-negative bacteria. J. Clin.Microbiol. 7:539-545.

14. Weaver, R. E., H. W. Tatum, and D. G. Hollis. 1972.The identification of unusual pathogenic gram-negativebacteria (Elizabeth O. King). Center for Disease Con-trol, Atlanta, Ga.

15. Wellstood-Neusse, S. 1979. Comparison of thé Miniteksystem with conventional methods for identification ofnonfermentative and oxidase-positive fermentativegram-negative bacilli. J. Clin. Microbiol. 9:511-516.

16. West, M., N. M. Burdash, and F. Freimuth. 1977.Simplified silver-plating stain for flagella. J. Clin. Mi-crobiol. 6:414-419.

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