Biochemical Characterization of a Haemophilus influenzae Periplasmic Iron Transport Operon
Descripción
Vol. 14, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1981, p. 579-581 0095-1137/81/110579-03$02.00/0
Biochemical Characterization of Haemophilus Species with the Minitek Differentiation System YOSHIYUKI KAWAKAMI,* YUKIE OKIMURA, AND MASAMITSU KANAI Bacteriological Division, Central Clinical Laboratories, Shinshu University Hospital, Matsumoto 390,
Japan Received 8 January 1981/Accepted 15 June 1981
Characterization of 102 Haemophilus strains by the Minitek differentiation system (BBL Microbiology Systems, Cockeysville, Md.) was compared with that by the conventional method. The results obtained by the two methods were in good agreement (P < 0.001; test of independence). Therefore, this system was found to be applicable for the identification of Haemophilus species in routine clinical bacteriology. Identification of strains of the genus Haemophilus has long been carried out by testing for their requirement of X factor and V factor (9). The disk method has been widely applied for this purpose. However, as many as 18% of Haemophilus species were reported to be misidentified (5) when the X and V disk test alone was applied. An exact identification of Haemophilus species obviously requires some additional tests not based on growth stimulation. Kilian (4) proposed the subdivision of H. influenza into five biotypes and H. parainfluenzae into three biotypes on the basis of selected biochemical reactions. This biotyping system has been successfully adopted for investigation of the distribution of this species from clinical materials, demonstrating a particular association between biotypes and sites of isolation (1, 3, 5-8). The detailed biochemical characterization of Haemophilus strains requires various kinds of media supplemented with X factor and V factor and is time-consuming; e.g., the final recording of acid production from carbohydrates takes 7 days (4). Therefore, it is desirable to develop a simple kit method for this genus, such as those widely used in modern clinical laboratories. However, in practice, such a method should be evaluated before being used in place of the traditional method. The Minitek differentiation system (MDS) (BBL Microbiology Systems, Cockeysville, Md.) was first applied for the characterization of Haemophilus strains by Back and Oberhofer (2). However, they did not compare their results with those of the conventional method. Our report focused on the evaluation of the MDS by comparing it with the conventional method of Kilian (4). A total of 102 clinical strains of Haemophilus species, isolated and stocked in our laboratory,
were used in this study. They included 27 H. influenza, 70 H. parainfluenzae, 1 H. haemolyticus, 3 H. paraphrophilus, and 1 H. aphrophilus strain. They were examined in parallel by the two methods: the conventional method as described by Kilian (4) and the MDS test. The MDS test was performed as follows: a suspension of each Haemophilus strain was prepared at the density of a McFarland no. 5 standard in Neisseria inoculum broth (BBL Microbiology Systems) as previously described (2). Approximately 50 Au of the suspension was dropped in 1 well of the plastic tray (BBL) for indole production tests and in the other 11 wells for disk tests of urease, ornithine, arginine, H2S, glucose, sucrose, lactose, xylose, mannitol, maltose, and arabinose. The wells of ornithine, arginine, and urease were overlaid with sterile mineral oil immediately after inoculation. The results were read after incubation at 37°C for 48 h in candle jars. This incubation procedure may not necessarily be required, but we found this makes the individual color reactions much clearer than overnight incubation at 35°C in the Minitek humidor under ambient air (2). No effect of C02 was observed. The overall coincidence between the two methods is shown in Table 1. Complete agreements in indole, urease, and ornithine tests were very favorable, since they are the key reactions for the biotyping of H. influenza and H. parainfluenzae, the major species of clinical origin. The disagreements were not biotype- or speciesspecific, but occurred in several particular tests. The arabinose test gave an outstandingly low coincidence (46.1%) for all of the biotypes and the species. The tests for H2S, lactose, and xylose showed minor discordance. Judgement of the H2S test was always troublesome in the MDS
579
TABLE 1. Coincidence of biochemical reactivity between the MDS and conventional methods No. of strains Test
Indole
Urease
MDS
Arginine
Sucrose, acid
Lactose, acid
Xylose, acid
Maltose, acid
Arabinose, acid
(27) (2)
H. para- H. par-
H.
aphro-
aphro-
cus
influenzae
philus
philus
(1)
(70)
(3)
(1)
1 O O O
0
0
0
7 70
3
o
3
1
-
+ +
+ + -
19 O O 8
1 O O 0
55 O O 15
o o o
o o o
3
1
+ + -
17 O O 10
0 O O
22 O O
o o o
o o o
1
48
3
1
O
O
O
O
+
+
-
o
+ -
-
+
+
-
+ -
O 27
O 1
O 70
-
+ + -
17 5 O 5
1 0 O 0
+
+
-
27
O
1
70
3
+
+ -
O
O
-
-
O
O
o o o
o o o
+
+
+ -
+
O O
O O
-
27
+ -
+
+ +
-
-
+ -
+ -
+ Mannitol, acid
haeH. molyti-
' 21 O O 6
+
Glucose, acid
H. influ H
+
+ -
H2S
tional
+ _ + _
-
Ornithine
Conven-
O
O
O
O
O
o o
o o
3
1
70 0
0 3
1 0
o o
o o
o o 1
3
1
1
o o o
o o o
o o o
0 O
O O
3
O
O
1 1
O
0
O
1 O
-
27
1
68
0
O
+ +
22 1
1 0
0
2 0
O
-
O
O
O
1
100
100
100
91.2
100
100
99
O O
O
O
-
-
4
0
69
1
+ -
+ +
O O
O O
O O
O O
o
+ -
-
O 27
O 1
O 70
O 3
1
+ + -
+ + -
4 O O 23
1 O O 0
70 O O 0
3 O O 0
O
100
o o o
70
-
Ageement (%)
97.1
1
1
O
o o
+ + 1 4 0 0 + 13 41 0 0 1 + O O O O o 1 13 25 3 o a Total number of strains is shown within parentheses. b Not significant (P < 0.001; test of independence); all others were significant (P > 0.1). 580
100
100
NOTES
VOL. 14, 1981
because blackening of the disk was usually very weak. This contrasted strikingly with the obvious reaction in the conventional method. However, all of the disk tests except for the arabinose reactions demonstrated a significant contingency (P < 0.001; test of independence) as shown in Table 1. An interesting finding was that all of the discordances, with the exception of the uniquely positive reaction of xylose by one H. aphrophilus strain, were exclusively ascribed to the negative results in the MDS. The disagreement in the arabinose test seems to be unimportant since the test was not essential for the identification of Haemophilus strains. Therefore, the disk test with arabinose can be left out of the test battery. This may be also the case with the disk test for H2S, considering the faint and ambiguous coloration of the disk. The results of our comparative study indicated that the MDS provides a reliable substitute for the conventional method of Kilian (4) and is useful for the characterization of Haemophilus species in routine clinical bacteriology. LITERATURE CrTED 1. Albritton, W. L., S. Penner, L. Slaney, and J. Brun-
2. 3.
4. 5.
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ton. 1978. Biochemical characterization of Haemophilus influenza in relationship to source of isolation and antibiotic resistance. J. Clin. Microbiol. 7:519-523. Back, A. E., and T. R. Oberhofer. 1978. Use of the Minitek system for biotyping Haemophilus species. J. Clin. Microbiol. 7:312-313. Golberg, R., and J. A. Washington H. 1978. The tasonomy and antimicrobial susceptibility of Haemophilus species in clinical specimens. Am. J. Clin. Pathol. 70: 899-904. Kilian, M. 1976. A taxonomie study of the genus Haemophilus, with the proposal of a new species. J. Gen. Microbiol. 93:9-62. Kilian, M., I. S0rensen, and W. Frederiksen. 1979. Biochemical characteristics of 130 recent isolates from Haemophilus influenza meningitis. J. Clin. Microbiol.
9:409-412. 6. Kilian, M., J. H. Jein, and P. Bulow. 1972. Haemophilus in the upper respiratory tract of children. Acta Pathol. Microbiol. Scand. Sect. B 80:571-578. 7. Kilian, M., G. H. Mordhorst, C. R. Dawson, and H. Lautrop. 1976. The taxonomy of Haemophilus isolated from conjunctivae. Acta Pathol. Microbiol. Scand. Sect. B 84:132-138. 8. Oberhofer, T. R., and A. E. Back. 1979. Biotypes of Haemophilus encountered in clinical laboratories. J. Clin. Microbiol. 10:168-174. 9. Turk, D. C, and J. R. May. 1969. Haemophilus influenzae; its clinical importance. The English University Press Ltd., London.
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