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JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1991, p. 2300-2304 0095-1137/91/102300-05$02.00/0 |
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Vol. 29, No.
10 |
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Enterosistem
18-R: Description and Comparative Evaluation with Conventional Methods for
Identification of Members of the Family Enterobacteriaceae |
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RAFFAELE
PICCOLOMINI,1* ARTURO
DI GIROLAMO,1 GIOVANNI
CATAMO,1 LUIGINA
CELLINI,1 NERINO
ALLOCATI,1 AND GIAMPIETRO RAVAGNAN2 |
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Istituto
di Medicina Sperimentale, Cattedra di Microbiologia, Facolty di Medicina e
Chirurgia, Universita "G. D'Annunzio," Chieti,l and Dipartimento di Biologia,
University di Roma "Tor Vergata," Rome,2 Italy |
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Received 1
May 1991/Accepted 23 July 1991 |
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The
efficiency and accuracy of Enterosistem 18-R (Liofilchem s.r.l., Roseto
degli Abruzzi, Teramo, Italy) were compared with those of conventional
biochemical methods to identify 360 members (38 species) of the family
Enterobacteriaceae. Overall, 329 strains (91.3%) were correctly identified (percentage
of identification, 2_90.0), with 37 (11.2%) requiring additional tests for
complete identification. For 11 isolates (3.1%), Enterosistem 18-R gave
only genus identifications, and for 14 (3.9%), the strains did not
correspond to any key in the codebook and could not be identified by the
manufacturer's computer service. Only six isolates (1.7%) were
misidentified. The new system accurately identified common and several
newly described isolates of the family Enterobacteriaceae, such as
Enterobacter gergoviae, Providencia rustigianii,
Serratia odorifera, and Serratia rubidaea. The system is
highly reproducible, simple to perform, easy to handle, and inexpensive.
With adjustments in supplementary code numbers for some strains,
Enterosistem 18-R is a suitable alternative for identification of members
of the Enterobacteriaceae in clinical laboratories. |
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| Identification of members of the family Enterobacteriaceae is a major feature of clinical bacteriology laboratories since
these bacteria, alone, are the etiological agents of more than 50% of
hospital infections (17). With increased government
attention to health costs (2), today the clinical microbiologist is more interested than ever in rapid reporting and
reductions in laboratory costs (1). Therefore, it is essential to develop
new, simple, and economic systems for rapid and accurate identification of
this bacterial group, and many commercial multitest systems are now
available for this purpose (4, 10, 16). |
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erol, and 38
were reference strains from different international
culture collections. |
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Before the experiment, the 109 stock cultures and the 38 reference
strains were subcultured three times into sheep blood agar (Liofilchem
s.r.l.) to raise their levels of enzymatic activity.
Before testing, all of the 360 isolates were grown in brain heart infusion
broth (Oxoid Italiana S.p.A., Garbagnate Milanese, Milan, Italy) and then
subcultured in a sheep blood agar to ensure purity and viability. To mask
the identity of all microorganisms throughout the experiment, we adopted
the use of a progressive numbering system for each microorganism (from 1
to 360). Two study groups were cross-employed in this work. The first
identified all isolates and revealed the results to the second group only
at the end of the study. The second group, employed as a control for the
first group, conducted the work on 72 randomized isolates and used the 38
reference strains as a quality control. Furthermore, to evaluate the
possible effect of the growth medium on Enterosistem 18-R, the same
reference strains were grown on sheep blood and MacConkey agar plates
(Oxoid Italiana S.p.A.). To ascertain the reproducibility of results from the system, growth from both media was employed
as an inoculum on three separate occasions, each time by a different study
group. |
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| Computer-assisted identification systems are already available in
miniaturized test kits such as Micro-ID, Minitek, API 20E, and Enterotube
(11, 12, 15, 18). Enterosistem 18-R (Liofilchem s.r.l., Roseto degli
Abruzzi, Teramo, Italy) is a new system designed to identify members of
the Enterobacteriaceae
to the genus and species levels in 18 h. The
system, at present available only in Europe, consists of a disposable tray
with 18 wells containing the dehydrated biochemical substrates. With
inoculation of a bacterial suspension in each well, a six-digit octal
number can be generated from 18 different biochemical reactions. From this
octal number, an identification is derived from a codebook furnished to
laboratories. |
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| To
evaluate the accuracy and utility of Enterosistem 18-R, we have compared
this system with conventional biochemical methods in
identifying 360 isolates of members of the family Enterobacteriaceae. |
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Enterosistem 18-R identification method. The Enterosistem 18-R
identification method consists of a plastic tray containing 18 different reaction wells covered with a transparent plastic
cover (Fig. 1). The 18 biochemical tests included in the system are listed
in the legend to Fig. 1. |
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MATERIALS AND
METHODS |
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The
tray was inoculated according to the manufacturer's instructions with some
modifications, such as the inoculum conditions and the incubation time.
One to three wellisolated colonies were emulsified in
4.5 ml of physiological sterile solution to reach an opacity equal to 0.5
MacFarland standard. The reaction wells were inoculated with 200 ld of the
bacterial suspension by using a multichannel pipette (Titertek; Flow
Laboratories, Milan, Italy). Wells for lysine |
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| Bacterial
strains. A total of 360 strains were tested. Of these, 213 were fresh
clinical isolates from our clinical bacteriology laboratory, 109 were
stock cultures from our collection that have been kept frozen (-80°C) in
20% glyc |
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*
Corresponding author. |
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2300
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VOA. 29, 1991 |
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IDENTIFYING
ENTEROBACTERIACEAE WITH ENTEROSISTEM 18-R |
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2301
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mentation of L-arabinose (Edwardsiella
tarda); fermentation of a-methyl-D-gluco side and
D-arabitol (Enterobacter gergoviae); salicin fermentation, esculin
hydrolysis, and chloramphenicol susceptibility
(Proteus penneri); fermentation of D-galactose (Providencia
rustigianii); and fermentation of mucate and
gelatin hydrolysis by rapid film method at 36°C (Salmonella arizonae). Enterobacter hormaechei (enteric group 75) was identified according to the scheme proposed
by O'Hara et al. (20). Yersinia spp. were characterized as reported by several authors (3, 5, 6, 8,
21). |
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Final identification was determined according to the table from the
work of Ewing (13) and Farmer et al. (14). All fresh clinical isolates of
Salmonella or Shigella species were confirmed by
serological tests. |
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RESULTS |
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The
results obtained with Enterosistem 18-R and conventional methods in identifying the 360 strains belonging to 38
different species of Enterobacteriaceae
are shown in Table 1. |
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| FIG. 1. Enterosistem 18-R. Test wells: 1, ONPG; 2, lysine decarboxylase; 3,
ornithine decarboxylase; 4, arginine dihydrolase; 5, phenylalanine deamination; 6, citrate; 7,
urea hydrolysis; 8, HZS
production; 9, malonate
utilization; 10, VP; 11, indole production; 12
through 18, fermentation of glucose, mannitol,
inositol, sorbitol, sucrose, arabinose, and raffinose,respectively,
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Enterosistem 18-R agreed with the conventional methods in the
identification of the 329 of 360 isolates (91.3%) at the species level (%
ID, ?90.0). Among these 329 strains, the system provided an excellent
identification (% ID, >_99.9) for most species, in particular those
often isolated in bacteriological laboratories, such
as Citrobacter diversus (8
of 9 strains tested), Citrobacter freundii
(9 of 12), Edwardsiella
tarda (3 of 3), Enterobacter aerogenes (8 of 10),
Enterobacter cloacae
(24 of 27), Escherichia
coli (59 of 66), Klebsiella
oxytoca (12 of 13), Klebsiella pneumoniae (25 of 33),
Morganella morgand
(8 of 11), Proteus
mirabilis (41 of 45), P. penneri (8 of 10), and Providencia alcalifaciens (2 of 2). All
the Salmonella and
Shigella species were
correctly identified (% ID, >90.0) to the genus and species level.
However, serological confirmation was made throughout the
evaluation of the system. Most of the uncommon or
newly described microorganisms, such as E. gergoviae (two of two strains
tested), Providencia rustigianii (three of three), one strain of Serratia
odorifera, and one isolate of Serratia rubidaea, were correctly
identified (% ID, >_99.9) by the system. |
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| decarboxylase, ornithine decarboxylase, arginine
dihydrolase, urease, and HZS tests were covered with sterile
mineral oil. The tray was closed with the plastic cover and then incubated
for 18 h at 37°C in an
aerobic atmosphere. |
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| After
incubation, 2 drops of a 10% ferric chloride solution were added to the
phenylalanine deaminase well and an immediate green reaction was evident.
At the same time, 3 drops of a-naphthol plus 1 drop of 40% NaOH solution
(Voges-Proskauer [VP] reagents) and 2 drops of Kovacs reagent were added,
respectively, to the VP test and indole production wells. The VP reaction
was evident before 12 to 15 min, while an immediate red-ring appearance in
the indole production well demonstrated a positive tryptophan
metabolism. |
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| All of the reactions were read by a color chart provided with the
kit. The biochemical reactions were recorded on data sheets provided with
the kit, and a six-digit octal number was generated for each
microorganism, which was then identified as a single species or as one of
several possible species by using the Enterosistem 18-R codebook index.
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In 11 cases
(3.1%), Enterosistem 18-R provided only genus identification. |
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Of the
above-mentioned 329 strains, 54 were not directly identified: 37 needed
additional tests, and 17 were identified by the manufacturer's computer
service. |
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Fourteen microorganisms (3.9%), including the newly described
strains, such as E. hormaechei and Yersinia frederiksenii, could not be identified
because the generated six-digit octal numbers were included neither in the
codebook which is furnished to laboratories nor in the
data base available in the manufacturer's computer. However, these 14
isolates produced biochemical reaction patterns identical to those
obtained with the conventional methods. The majority
(10 of 14 strains tested) of these isolates were from the Klebsiella spp. (4 of 60), Proteus vulgaris (2 of 17), Providencia spp. (2 of 17), and Yersinia
pseudotuberculosis (2 of 3). |
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| Unlisted
profiles were interpreted by referring to the manufacturer's computer.
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| In the
Enterosistem 18-R codebook, identifications are classified according to
the percentage of identification (% ID) (19) as follows: excellent (% ID,
>99.9), good (% ID, >90.0), acceptable (% ID, >80.0), and low
confidence (% ID, <80.0, but with the % ID sum of the first two or
three taxa greater than or equal to 80). |
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| In this
paper we have considered a correct identification as % ID ? 90.0.
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| Conventional biochemical tests. The 360 Enterobacteriaceae isolates employed in this study were identified according to
procedures described by Ewing (13). A few strains which could not be
accurately identified with this procedure were fully characterized by
using the following additional tests as reported by Farmer et al. (14):
growth in KCN and tyrosine clearing (Citrobacter
amalonaticus); fer |
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TABLE 1. Comparison of Enterosistem 18-R system with
conventional methods for identification of members of the
Enterobacteriaceae o
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collection'
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tified° |
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b (~ C O O z |
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ATCC 24405
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ATCC 27156
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ATCC 15947
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NCTC 10006 |
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1 |
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D
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1 |
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1 |
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ATCC 33028
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Enterobacter honnaechei
(enteric group 75)
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ATCC 49162
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ATCC 29544
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ATCC 25922
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3 |
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10 |
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1 |
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ATCC 11297
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1 |
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ATCC 13883
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2 |
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Klebsiella
rhinoscleromatis | |
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1 |
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ATCC 25830
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1 |
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ATCC 29906
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3 |
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ATCC 33519
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2 |
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ATCC 13315
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Providencia
alcalifaciens | |
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ATCC 29944
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1 |
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ATCC 33673
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ATCC 29914
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1 |
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ATCC 12323
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