JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1979, P. 800-804 0095-1 137/79/12-0800/05$02.00/0
Vol. 10, No. 6
Growth of Nonfermentative Bacteria at 42°C THOMAS R. OBERHOFER
Microbiology Section, Department of Pathology, Madigan Army Medical Center, Tacoma, Washington 98431
Received for publication 25 September 1979
Growth at 42°C is advocated to differentiate species of the fluorescent pseudomonas group as well as to differentiate other nonfermentative bacteria. Methodologies vary in the performance of the test, resulting in differing and often discrepant results between investigators. During this evaluation, the test was performed by inoculating 3 ml of Trypticase soy broth with a loopful of an overnight broth culture. Growth in the 42°C tube was judged as heavy or slight after 24 and 48 h incubation at 41.5 ± 0.50C. Pseudomonas aeruginosa grew abundantly after overnight incubation, whereas 16 of 74 isolates of P. putida (22%) showed slight turbidity in the broth after 24 or 48 h which could not be regarded as an inoculum effect. Trypticase soy agar was used in conjunction with Trypticase soy broth, with the growth again judged as heavy or slight. Growth of P. putida on slants was still seen in some cases (6%) although the number of strains showing growth had declined. Triphenyltetrazolium chloride was added to Trypticase soy broth (0.005%) as a color indicator of growth. Strains of P. putida, although showing visible evidence of growth, gave no color change when compared with the 35°C control. The constancy in test results using nonfermentative bacteria is not only method dependent but also strain dependent. Although the test for growth at 42°C is important as a taxonomic tool when used under controlled conditions, other tests such as acetamide are preferred as a substitute for use in the clinical laboratory.
The differentiation of apyocyanogenic Pseudomonas aeruginosa from the other fluorescent pseudomonads is clinically important due to the seriousness of infections caused by P. aeruginosa. Tests common to the two groups have been oxidation of potassium gluconate and tolerance to cetrimide, whereas those used for differentiation have included flagella arrangements, gelatin liquefaction, slime production, lecithinase activity, peptonization of litmus milk, and other less reliable characteristics. Historically, growth at 42'C has been considered to be a key differential test for separating nonpigmented P. aeruginosa and the other species of fluorescent pseudomonads. Introduction of commercial test systems (Oxi/ Ferm, API 20-E, and Corning N/F test systems) for identification of nonfermentative bacteria (NFB) has resulted in a renewed interest in the more precise identification of these organisms on a routine basis. Use of these systems has placed a requirement upon laboratories to perform temperature growth studies as part of a supplementary battery of tests for identification. Recently, however, acetamide utilization has been shown to correlate well with identification of P. aeruginosa, although direct studies com800
paring acetamide utilization and growth at 42°C had not been done (13). As such, a reexamination of the performance of the growth test seemed warranted. This report presents the findings of 42°C growth studies, describes the comparative methods used for obtaining growth, and assesses the usefulness of the test to differentiate the NFB in relation to their ability to alkalinize acetamide.
MATERIALS AND METHODS Organisms tested. The organisms included in this evaluation were fresh clinical isolates recovered from clinical materials over a 30-month period at the Madigan Army Medical Center. The isolates were not subjected to repeated subculture before testing at 42°C. Temperature-growth studies constituted part of the battery of tests used for definitive identification of each isolate (13). The data for strains of P. alcaligenes, P. pseudoalcaligenes, and P. putrefaciens, and for most strains of P. stutzeri, were collected during previous studies (unpublished data). Test media. A 3-ml amount of Trypticase soy broth (TSB) originally was used as the growth medium. Tests for growth comparisons included parallel inoculation of 3 ml of TSB and 3 ml of Trypticase soy agar (TSA) as slants. Further studies employed 2 ml of
GROWTH OF NONFERMENTATIVE BACTERIA AT 420C
VOL. 10, 1979
TSB containing 0.005% triphenyltetrazolium chloride (TTC) as an indicator of growth. In all cases, the media were brought to room temperature before inoculation. Alkalinization of acetamide was determined as reported previously (13). Test conditions. The inoculum used in all tests was an overnight broth culture of each isolate. One tube each of TSA and TSB and two tubes of TSB + TTC were inoculated with a loopful of the overnight culture. The TSA, TSB, and one TSB + TTC test culture were placed in an incubator adjusted to 41.5 ± 0.50C (hereafter referred to as 420C) and examined for growth after 18 to 24 h. The second tube of TSB + TTC was incubated at 350C and served as the growth and TTC tolerance control. Growth at 420C was subjectively scored according to turbidity and was recorded by degrees, with 4+ growth equivalent to a duplicate tube incubated at 35°C. Growth density of 1+ or less (±) was considered poor growth. Cultures showing no growth or poor growth were reincubated for an additional 24 h. Each tube of TSA to be reincubated was tilted to allow the water of syneresis to flow over the slant to remoisten it. RESULTS
Tables 1 and 2 show the results of testing 740 isolates of NFB for the ability to grow at 420C. The P. aeruginosa and unidentified fluorescent pseudomonads grew well on both TSA and TSB after overnight incubation (Table 1), although mucoid strains grew less well in TSB. All strains of P. fluorescens failed to grow at 420C. Sixteen strains of P. putida (22%) showed poor but definite growth in TSB, whereas only three strains (6%) showed poor growth on the agar slants. The other oxidative bacteria demonstrated an ability to grow on both media, although A. xy-
801
losoxidans grew somewhat more rapidly in broth and A. anitratus overall grew better on agar. In general, the broth supported growth of the oxidative NFB only slightly better than agar. The yellow-pigmented NFB, on the other hand, showed greater variability in growth. All strains grew better in broth than on agar, although a larger proportion of strains growing in broth did so poorly. The nonoxidative bacteria (Table 2) showed little difference in growth abilities on agar or in broth, and differences that did exist were attributed mainly to the number of isolates tested with the two media. There was greater tendency to have poor but definite growth in broth in contrast to more clear-cut growth on agar slants. Prompted by the unexpected results obtained with the P. putida strains, an attempt was made to ascertain the source of discrepancy between the two test media by incorporating an indicator of growth into the broth medium (Table 3). The rates of growth differed for each organism tested. P. aeruginosa (not shown) and A. anitratus grew rapidly at 420C, whereas P. maltophilia often required 48 h to exhibit good growth in both solid and liquid media. It is seen again that broth was better suited for growth of P. maltophilia than was agar. Growth of P. putida in broth was valid and not attributed to inoculum effect, since the turbidity in the TTC cultures paralleled that seen in plain broth, although the growth manifested a disappointing color change in the indicator reaction. Of the tests used to differentiate NFB, utilization of acetamide correlated best with growth
TABLE 1. Growth of oxidative NFB at 420C0 Growth on TSA
Growth in TSB
Organism
P. aeruginosa
No. tested 141 2
+ 141
"+ 0 0 0 16 0 2 2 10 0
-
0 0 24 58
No. tested
100 118 118 0 0 100 2 2 100 0 0 100 0 24 15 0 0 15 0 22 52 74 0 3 49 6 1 23 96 16 11 5 0 100 2 1 4 75 4 0 2 50 1 7 7 4 86 2 1 86 11 152 102 6 93 90 6 94 2 17 3 2 88 0 1 67 1 2 12 87 15 NTC 74 22 29 23 14 8 69 60 37 38 2 12 19 5 37 15 1 2 12 20 7 0 3 4 4 43 0 0 4 0 4 7 0 3 43 6 0 0 6 0 0 1 1 3 2 1 100 0 0 100 CDC VE-1 14 1 0 13 7 8 1 0 7 13 CDC VE-2 a +, Number of strains showing good growth in 48 h; (+), poor growth in 48 h; -, no growth in 48 h. b UFP, Unidentified fluorescent pseudomonad. c NT, Not tested.
UFPb P. fluorescens P. putida A. xylosoxidans Pseudomonas sp. 1 Pseudomonas sp. 2 A. anitratus P. stutzeri group P. pseudoalcaligenes P. maltophilia Flavobacterium IIb P. paucimobilis CDC IIk-2
2 0 0 22 1 4 131 15
802
OBERHOFER
J. CLIN. MICROBIOL.
TABLE 2. Growth of nonoxidative NFB at 42,oCa Growth in TSB
Growth on TSA
OrganismNo.
No.
tested
P. aeruginosa (non-
tested
+
(
-
3
0
0
100
3
3
0
0
100
0 7 2 2 0 0 6 0 12 3 2 9 0 5 0 0 16 1.
2 0 0 0 0 0 1 0 1 1 0 0 1 1 0 0 16
11 4 0 0 6 2 2 3 2 0 2 0 2 9 7 3 14
15 64 100 100 0 0 78 0 87 100 50 100 33 40 0 0 69
6 2 NT NT NT 2 2 3 5 2 NT 9 3 7 7 NT 45
0 2
0 0
6 0
0 100
0 0 0 5 1
0 0 0 0 1
2 2 3 0 0
0 0 0 100 100
9 0 1 0
0 0 0 0
0 3 6 7
100 0 14 0
20
4
21
53
3
glucolytic) P. acidovorans 13 P. testosteroni 11 P. putrefaciens 2 P. alcaligenes 2 P. diminuta 6 2 Alcaligenes sp. A. faecalis 9 A. denitrificans 3 A. odorans 15 4 CDC IVc B. bronchiseptica 4 Flavobacterium IIf 9 F. odoratum 3 15 M. osloensis M. nonliquefaciens 7 Moraxella M-3 3 A. Iwoffi 46 a See footnotes a and c to Table
TABLE 3. Growth of select NFB at 42°C in three growth mediaa TSB
TSA Organism
No. tested
54 A. anitratus 35 P. maltophilia P. putida 37 25 A. Iwoffi a See footnote a to Table 1.
24 h +
(+)
45 6 0 11
4 8 4 2
48 h -
+
5 46 21 10 33 0 12 11
24 h
TTC 48 h
24 h
48 h
(+)
-
3 5 4 2
5 42 5 7 44 3 7 44 3 7 47 1 6 20 7 15 13 13 10 12 10 12 13 15 11 9 33 0 5 32 0 7 30 0 6 31 0 7 30 12 11 3 11 11 3 11 9 2 14 11 1 13
on TSA at 42°C, especially that by the fluorescent pseudomonads (Table 4). Use of acetamide slants obviated the need for selective temperature studies and provided greater reliability in the identification of P. aeruginosa, A. xylosoxidans, P. acidovorans, and A. odorans.
DISCUSSION The identification and characterization of NFB usually are accomplished by conventional means or by use of one or more of the commercially available test systems. Often, confirmation of identity of biochemically similar organisms is arbitrated by selection of a single promising test. Since the fluorescent group constitutes a large part of clinical isolates (14), the test for growth at 42°C has assumed a major role during differentiation between members of this group. Various methods of performing temperature-growth studies have been reported with variable degrees of success attendant with each. Tryptone-glu-
+
(+)
-
+
(+)
-
+
(+)
-
+
(+)
-
cose-yeast slants were used during earlier studies to identify P. aeruginosa (6). Subsequently, tryptone-glucose-yeast (7, 21) and nutrient agar slants (20) were used for P. aeruginosa, and yeast extract (17) was used for P. fluorescens. Other media used for testing NFB have included TSA, (4), heart infusion agar (2, 19), Brucella agar and broth (16), brain heart infusion broth (10), TSB (11), and nutrient broth (12). Cetrimide agar has been suggested for use as well (3), although most NFB do not grow on cetrimide even at 35°C, rendering this test selective for the fluorescent pseudomonads. Incubation of the test media has been for 24 h (4, 21), for 24 or 48 h (6, 16, 19), and for as long as 10 days (2, 18), with some tests scored as positive only after the organisms had survived two or three consecutive subcultures at 42°C (2, 7, 20). In some instances, the conditions of incubation were not described fully (3, 10, 11, 12, 17). Data supporting the use of broth or solid media for temperature growth studies, based on
GROWTH OF NONFERMENTATIVE BACTERIA AT 420C
VOL. 10, 1979
TABLE 4. Growth at 42°C and alkalinization of acetamide by NFB Organism P. aeruginosa Pyomelanine Delayed pyocyanine Apyocyanine Mucoid Nonglucolytic UFPd P. fluorescens P. putida A. xylosoxidans CDC Vd Pseudomonas sp. 1 Pseudomonas sp. 2 A. anitratus P. stutzeri group P. pseudoalealigenes
P. cepacia P. maltophilia F. meningosepticum Flavobacterium Hb P. paucimobilis CDC IIk-2 CDC Ve-1 CDC Ve-2
P. P. P. P.
acidovorans testosteroni alcaligenes dininuta
Alcaligenes sp. A. faecalis A. denitrificans A. odorans CDC IVc B. bronchiseptica
42°C grovsrtha No. %+b tested
Acetamide No. %+b (+)C
tested
100
22
100
27
21
100
46
98
2
78
100
128
96
3
17 3 2 15 52 16
NT 4 7
102 17'
15'
NT
60
23 3
100 0
00
32
0
6 100 50 86 94 8887
104 26 4 7 170 44 15 9 140
100 100
37
NT
15 4 6 3' 8 13'
0 0 100
13 15
CA
2
100 0
2
0 78 0
6f
9'
3
5 2 4'
100
100 50
8
18 11 6 21 20 13 8 9
2 9 5 16 7 4
33
3 1 46 42 0 0 0 00 2 78 0 5 2
0
13
0 0 0
0 95 0
5
0 0 0
33
94
6
0 0
0 0
0 100 9 9 Flavobacterium Ilf 0 0 3 3 F. odoratum 21 0 14 7 M. osloensis 7 Moraxella sp. 91 1 53 45 A. Iwoffi a Growth test results with TSA lants. b+, Positive reaction in 1 to 2 d ' (+), Positive reaction in 3 to 7 'days. d UFP, Unidentified fluorescent pseudomonads. 'NT, Not tested. f Results with TSB.
9
careful evaluation, are rem;arkably scarce. Difficulty in the use of broth xroutinely arises with the size of the inoculum sel ected, allied with the degree of turbidity acceptei!d as the criterion for "growth." Results of the te«sts using broth are at variance with those of Kliinge (12), who advocated the use of broth meclium because of uniform distribution of the inc culum, better distribution of bacterial clumps,, and more rapid attainment of the desired temperature. Klinge
803
used a loopful of a 1:100 dilution of the test organism as inoculum, although the volume of media used was not specified. During the current study, an overnight broth culture was selected as inoculum to insure uniformity of growth phase and cell volume of each species tested. Hence, placing approximately 0.005 ml into 3 ml of broth was adequate to eliminate an inoculum efct effect. There seems to be considerable heterogeneity among the organisms designated as P. putida because of variable reactions in a host of tests. Indeed, it was reported (2) that only 11 strains of P. putida and 4 strains of P. fluorescens were isolated in a 2-year period, since they reportedly had a predilection for growth at lower than body temperature. Moreover, in the description of P. fluorescens advanced by Rhodes (17), P. fluorescens failed to grow at 370C. Today these account for 10% of clinical isolates organisms (14). The P. putida, then, may represent a continuum of activities, including strains resembling P. fluorescens as well as those resembling P.
aeruginosa in growth characteristics.
It seems clear that some organisms conforning to the description of P. putida multiplied in broth sufficiently for that medium to be judged unsuitable for use in a differential test. The choice of Trypticase as a broth or agar vehicle may be questioned, although it should be acknowledged that a more nutritious medium would support even better growth. That warmning of the medium to 420C before inoculation is not critical is attested to by the fact that the generation time of the fluorescent pseudomonads is too great to influence replication. Haynes and Rhodes (7) proposed that a test organism should survive at least three serial transfers at a given temperature before being characterized for taxonomic purposes. They observed that some strains which could initiate good growth at 420C showed increasing relucat that transfer with each serial to do soInterestingly, tance other proreports temperature. claiming multiple passages did not clearly specify the species tested or the outcome of those tests (2). The implication is that growth was necessarily present during the initial test (2, 7), fulfilling the requirement for "growth" at 420C. Wahba and Darrell (20) recorded intriguing results with organisms designated at P. fluorescens, whereby almost half of the strains tested grew at 420C during the initial growth attempt. It was recognized, however, that repeated transfer had little practical use as a routine procedure. Adaptation of P. putida was not attempted during the current study. Three isolates of non-glucose-oxidizing P. aeruginosa were unusual in their failure to al-
804
OBERHOFER
kalinize acetamide. This departure from the expected correlation (13, 14) posed little difficulty in identification of these particular isolates, since many characteristics typical of P. aeruginosa were prominent. Two isolates of unidentified fluorescent pseudomonads also failed to correlate in the tests for growth and alkalinization. Several groups of fluorescent pseudomonads capable of growing at 420C have been recovered from surface waters (8) and swimming pools (9), as well as from human sources (19). Similar to these other isolates, the apyocyanogenic strains of unidentified fluorescent pseudomonads resembled P. aeruginosa in hemolytic activity and ability to liquefy gelatin, but differed in their failure to denitrify, to attack mannitol and acetamide (1, 8), and to oxidize gluconate (1, 9). The monopolar characteristics of both groups of organisms, however, clearly distinguished them from P. putida and P. fluorescens, although other characteristics such as susceptibility to kanamycin bore considerable similarity to P. putida. The results of this study and others (5, 15) indicate that the test for growth at 420C may have little discriminating value for the differentiation of NFB usually encountered in the clinical laboratory. Growth at the selective temperature is not constant within species, which imposes severe limitations for separating closely related species. The growth ofP. putida in broth and on agar, although light, reduces the effectiveness of the test when the criterion of growth or no growth is placed on test interpretation. Furthermore, multiple passages at 420C is not practical for use on a routine basis. The test is fraught with technical difficulties. Variables include the selection of medium, the size of inoculum, the age of the inoculum, and perhaps even the apparatus used for incubation. Nevertheless, the test may have a place in a comprehensive identification protocol. The method of choice is inoculation of a slanted agar medium with a loopful of an overnight broth culture with the slant incubated for 18 to 24 h. Any evidence of growth should be noted, the extent of growth is recorded, and the test is then discarded. LITERATURE CITED Hoadley. 1976. Fluorescent pseudomonads capable of growth at 41°C but distinct
1. Ajello, G. W., and A. W.
from Pseudomonas aeruginosa. J. Clin. Microbiol. 4: 443-449.
J. CLIN. MICROBIOL. 2. Blazevic, D. J., M. H. Koepcke, and J. M. Matsen. 1973. Incidence and identification of Pseudomonas fluorescens and Pseudomonas putida in the clinical laboratory. Appl. Microbiol. 25:107-110. 3. Gilardi, G. L. 1968. Differentiation of oxidative and nonglucolytic gram-negative bacteria. Am. J. Med. Technol. 34:334-345. 4. Gilardi, G. L. 1971. Characterization of nonfermentative nonfastidious gram negative bacteria encountered in medical bacteriology. J. Appl. Bacteriol. 34:623-644. 5. Gilardi, G. L. 1976. Pseudomonas species in clinical microbiology. Mount Sinai J. Med. 43:710-726. 6. Haynes, W. C. 1951. Pseudomonas aeruginosa-its characterization and identification. J. Gen. Microbiol. 5: 939-950. 7. Haynes, W. C., and L. J. Rhodes. 1962. Comparative taxonomy of crystallogenic strains of Pseudomonas aeruginosa and Pseudomonas chlororaphis. J. Bacteriol. 84:1080-1084. 8. Hoadley, A. W., and G. Ajello. 1972. Some characteristics offluorescent pseudomonads isolated from surface waters and capable of growth at 41°C. Can. J. Microbiol. 18:1769-1773. 9. Hoadley, A. W., G. Ajello, and N. Masterson. 1975. Preliminary studies of fluorescent pseudomonads capable of growth at 41 C in swimming pool waters. Appl. Microbiol. 29:527-531. 10. Hugh, R. 1970. A practical approach to the identification of certain nonfermentative gram-negative rods encountered in clinical specimens. Publ. Health Lab. 28:168187. 11. Kantor, L T., S. D. Kominos, and R. B. Yee. 1975. Identification of nonfermentative gram-negative bacteria in the clinical laboratory. Am. J. Med. Technol. 41: 3-9. 12. Klinge, K. 1960. Differential techniques and methods of isolation of Pseudomonas. J. Appl. Bacteriol. 23:442462. 13. Oberhofer, T. R., J. W. Rowen, and G. F. Cunningham. 1977. Characterization and identification of gram-negative, nonfermentative bacteria. J. Clin. Microbiol. 5:208-220. 14. Oberhofer, T. R. 1979. Comparison of the API 20E and Oxi/Ferm systems in identification of nonfermentative and oxidase-positive fermentative bacteria. J. Clin. Microbiol. 9:220-226. 15. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159-271. 16. Pickett, M. J., and M. M. Pedersen. 1970. Characterization of saccharolytic nonfermentative bacteria associated with man. Can. J. Microbiol. 16:351-362. 17. Rhodes, M. E. 1959. The characterization of Pseudomonas fluorescens. J. Gen. Microbiol. 21:221-263. 18. Sands, D. C., M. N. Schroth, and D. C. Hildebrand. 1970. Taxonomy of phytopathogenic pseudomonads. J. Bacteriol. 101:9-23. 19. Sutter, V. L. 1968. Identification of Pseudomonas species isolated from hospital environment and human sources. Appl. Microbiol. 16:1532-1538. 20. Wabba, A. H., and J. H. Darrell. 1965. The identification of atypical strains of Pseudomonas aeruginosa. J. Gen. Microbiol. 38:329-342. 21. Weaver, R. E., H. W. Tatum, and D. G. Hoffis. 1972. The identification of unusual pathogenic gram-negative bacteria. Preliminary revision. Center for Disease Control, Atlanta, Ga.
Vol. 10, No. 6
Growth of Nonfermentative Bacteria at 42°C THOMAS R. OBERHOFER
Microbiology Section, Department of Pathology, Madigan Army Medical Center, Tacoma, Washington 98431
Received for publication 25 September 1979
Growth at 42°C is advocated to differentiate species of the fluorescent pseudomonas group as well as to differentiate other nonfermentative bacteria. Methodologies vary in the performance of the test, resulting in differing and often discrepant results between investigators. During this evaluation, the test was performed by inoculating 3 ml of Trypticase soy broth with a loopful of an overnight broth culture. Growth in the 42°C tube was judged as heavy or slight after 24 and 48 h incubation at 41.5 ± 0.50C. Pseudomonas aeruginosa grew abundantly after overnight incubation, whereas 16 of 74 isolates of P. putida (22%) showed slight turbidity in the broth after 24 or 48 h which could not be regarded as an inoculum effect. Trypticase soy agar was used in conjunction with Trypticase soy broth, with the growth again judged as heavy or slight. Growth of P. putida on slants was still seen in some cases (6%) although the number of strains showing growth had declined. Triphenyltetrazolium chloride was added to Trypticase soy broth (0.005%) as a color indicator of growth. Strains of P. putida, although showing visible evidence of growth, gave no color change when compared with the 35°C control. The constancy in test results using nonfermentative bacteria is not only method dependent but also strain dependent. Although the test for growth at 42°C is important as a taxonomic tool when used under controlled conditions, other tests such as acetamide are preferred as a substitute for use in the clinical laboratory.
The differentiation of apyocyanogenic Pseudomonas aeruginosa from the other fluorescent pseudomonads is clinically important due to the seriousness of infections caused by P. aeruginosa. Tests common to the two groups have been oxidation of potassium gluconate and tolerance to cetrimide, whereas those used for differentiation have included flagella arrangements, gelatin liquefaction, slime production, lecithinase activity, peptonization of litmus milk, and other less reliable characteristics. Historically, growth at 42'C has been considered to be a key differential test for separating nonpigmented P. aeruginosa and the other species of fluorescent pseudomonads. Introduction of commercial test systems (Oxi/ Ferm, API 20-E, and Corning N/F test systems) for identification of nonfermentative bacteria (NFB) has resulted in a renewed interest in the more precise identification of these organisms on a routine basis. Use of these systems has placed a requirement upon laboratories to perform temperature growth studies as part of a supplementary battery of tests for identification. Recently, however, acetamide utilization has been shown to correlate well with identification of P. aeruginosa, although direct studies com800
paring acetamide utilization and growth at 42°C had not been done (13). As such, a reexamination of the performance of the growth test seemed warranted. This report presents the findings of 42°C growth studies, describes the comparative methods used for obtaining growth, and assesses the usefulness of the test to differentiate the NFB in relation to their ability to alkalinize acetamide.
MATERIALS AND METHODS Organisms tested. The organisms included in this evaluation were fresh clinical isolates recovered from clinical materials over a 30-month period at the Madigan Army Medical Center. The isolates were not subjected to repeated subculture before testing at 42°C. Temperature-growth studies constituted part of the battery of tests used for definitive identification of each isolate (13). The data for strains of P. alcaligenes, P. pseudoalcaligenes, and P. putrefaciens, and for most strains of P. stutzeri, were collected during previous studies (unpublished data). Test media. A 3-ml amount of Trypticase soy broth (TSB) originally was used as the growth medium. Tests for growth comparisons included parallel inoculation of 3 ml of TSB and 3 ml of Trypticase soy agar (TSA) as slants. Further studies employed 2 ml of
GROWTH OF NONFERMENTATIVE BACTERIA AT 420C
VOL. 10, 1979
TSB containing 0.005% triphenyltetrazolium chloride (TTC) as an indicator of growth. In all cases, the media were brought to room temperature before inoculation. Alkalinization of acetamide was determined as reported previously (13). Test conditions. The inoculum used in all tests was an overnight broth culture of each isolate. One tube each of TSA and TSB and two tubes of TSB + TTC were inoculated with a loopful of the overnight culture. The TSA, TSB, and one TSB + TTC test culture were placed in an incubator adjusted to 41.5 ± 0.50C (hereafter referred to as 420C) and examined for growth after 18 to 24 h. The second tube of TSB + TTC was incubated at 350C and served as the growth and TTC tolerance control. Growth at 420C was subjectively scored according to turbidity and was recorded by degrees, with 4+ growth equivalent to a duplicate tube incubated at 35°C. Growth density of 1+ or less (±) was considered poor growth. Cultures showing no growth or poor growth were reincubated for an additional 24 h. Each tube of TSA to be reincubated was tilted to allow the water of syneresis to flow over the slant to remoisten it. RESULTS
Tables 1 and 2 show the results of testing 740 isolates of NFB for the ability to grow at 420C. The P. aeruginosa and unidentified fluorescent pseudomonads grew well on both TSA and TSB after overnight incubation (Table 1), although mucoid strains grew less well in TSB. All strains of P. fluorescens failed to grow at 420C. Sixteen strains of P. putida (22%) showed poor but definite growth in TSB, whereas only three strains (6%) showed poor growth on the agar slants. The other oxidative bacteria demonstrated an ability to grow on both media, although A. xy-
801
losoxidans grew somewhat more rapidly in broth and A. anitratus overall grew better on agar. In general, the broth supported growth of the oxidative NFB only slightly better than agar. The yellow-pigmented NFB, on the other hand, showed greater variability in growth. All strains grew better in broth than on agar, although a larger proportion of strains growing in broth did so poorly. The nonoxidative bacteria (Table 2) showed little difference in growth abilities on agar or in broth, and differences that did exist were attributed mainly to the number of isolates tested with the two media. There was greater tendency to have poor but definite growth in broth in contrast to more clear-cut growth on agar slants. Prompted by the unexpected results obtained with the P. putida strains, an attempt was made to ascertain the source of discrepancy between the two test media by incorporating an indicator of growth into the broth medium (Table 3). The rates of growth differed for each organism tested. P. aeruginosa (not shown) and A. anitratus grew rapidly at 420C, whereas P. maltophilia often required 48 h to exhibit good growth in both solid and liquid media. It is seen again that broth was better suited for growth of P. maltophilia than was agar. Growth of P. putida in broth was valid and not attributed to inoculum effect, since the turbidity in the TTC cultures paralleled that seen in plain broth, although the growth manifested a disappointing color change in the indicator reaction. Of the tests used to differentiate NFB, utilization of acetamide correlated best with growth
TABLE 1. Growth of oxidative NFB at 420C0 Growth on TSA
Growth in TSB
Organism
P. aeruginosa
No. tested 141 2
+ 141
"+ 0 0 0 16 0 2 2 10 0
-
0 0 24 58
No. tested
100 118 118 0 0 100 2 2 100 0 0 100 0 24 15 0 0 15 0 22 52 74 0 3 49 6 1 23 96 16 11 5 0 100 2 1 4 75 4 0 2 50 1 7 7 4 86 2 1 86 11 152 102 6 93 90 6 94 2 17 3 2 88 0 1 67 1 2 12 87 15 NTC 74 22 29 23 14 8 69 60 37 38 2 12 19 5 37 15 1 2 12 20 7 0 3 4 4 43 0 0 4 0 4 7 0 3 43 6 0 0 6 0 0 1 1 3 2 1 100 0 0 100 CDC VE-1 14 1 0 13 7 8 1 0 7 13 CDC VE-2 a +, Number of strains showing good growth in 48 h; (+), poor growth in 48 h; -, no growth in 48 h. b UFP, Unidentified fluorescent pseudomonad. c NT, Not tested.
UFPb P. fluorescens P. putida A. xylosoxidans Pseudomonas sp. 1 Pseudomonas sp. 2 A. anitratus P. stutzeri group P. pseudoalcaligenes P. maltophilia Flavobacterium IIb P. paucimobilis CDC IIk-2
2 0 0 22 1 4 131 15
802
OBERHOFER
J. CLIN. MICROBIOL.
TABLE 2. Growth of nonoxidative NFB at 42,oCa Growth in TSB
Growth on TSA
OrganismNo.
No.
tested
P. aeruginosa (non-
tested
+
(
-
3
0
0
100
3
3
0
0
100
0 7 2 2 0 0 6 0 12 3 2 9 0 5 0 0 16 1.
2 0 0 0 0 0 1 0 1 1 0 0 1 1 0 0 16
11 4 0 0 6 2 2 3 2 0 2 0 2 9 7 3 14
15 64 100 100 0 0 78 0 87 100 50 100 33 40 0 0 69
6 2 NT NT NT 2 2 3 5 2 NT 9 3 7 7 NT 45
0 2
0 0
6 0
0 100
0 0 0 5 1
0 0 0 0 1
2 2 3 0 0
0 0 0 100 100
9 0 1 0
0 0 0 0
0 3 6 7
100 0 14 0
20
4
21
53
3
glucolytic) P. acidovorans 13 P. testosteroni 11 P. putrefaciens 2 P. alcaligenes 2 P. diminuta 6 2 Alcaligenes sp. A. faecalis 9 A. denitrificans 3 A. odorans 15 4 CDC IVc B. bronchiseptica 4 Flavobacterium IIf 9 F. odoratum 3 15 M. osloensis M. nonliquefaciens 7 Moraxella M-3 3 A. Iwoffi 46 a See footnotes a and c to Table
TABLE 3. Growth of select NFB at 42°C in three growth mediaa TSB
TSA Organism
No. tested
54 A. anitratus 35 P. maltophilia P. putida 37 25 A. Iwoffi a See footnote a to Table 1.
24 h +
(+)
45 6 0 11
4 8 4 2
48 h -
+
5 46 21 10 33 0 12 11
24 h
TTC 48 h
24 h
48 h
(+)
-
3 5 4 2
5 42 5 7 44 3 7 44 3 7 47 1 6 20 7 15 13 13 10 12 10 12 13 15 11 9 33 0 5 32 0 7 30 0 6 31 0 7 30 12 11 3 11 11 3 11 9 2 14 11 1 13
on TSA at 42°C, especially that by the fluorescent pseudomonads (Table 4). Use of acetamide slants obviated the need for selective temperature studies and provided greater reliability in the identification of P. aeruginosa, A. xylosoxidans, P. acidovorans, and A. odorans.
DISCUSSION The identification and characterization of NFB usually are accomplished by conventional means or by use of one or more of the commercially available test systems. Often, confirmation of identity of biochemically similar organisms is arbitrated by selection of a single promising test. Since the fluorescent group constitutes a large part of clinical isolates (14), the test for growth at 42°C has assumed a major role during differentiation between members of this group. Various methods of performing temperature-growth studies have been reported with variable degrees of success attendant with each. Tryptone-glu-
+
(+)
-
+
(+)
-
+
(+)
-
+
(+)
-
cose-yeast slants were used during earlier studies to identify P. aeruginosa (6). Subsequently, tryptone-glucose-yeast (7, 21) and nutrient agar slants (20) were used for P. aeruginosa, and yeast extract (17) was used for P. fluorescens. Other media used for testing NFB have included TSA, (4), heart infusion agar (2, 19), Brucella agar and broth (16), brain heart infusion broth (10), TSB (11), and nutrient broth (12). Cetrimide agar has been suggested for use as well (3), although most NFB do not grow on cetrimide even at 35°C, rendering this test selective for the fluorescent pseudomonads. Incubation of the test media has been for 24 h (4, 21), for 24 or 48 h (6, 16, 19), and for as long as 10 days (2, 18), with some tests scored as positive only after the organisms had survived two or three consecutive subcultures at 42°C (2, 7, 20). In some instances, the conditions of incubation were not described fully (3, 10, 11, 12, 17). Data supporting the use of broth or solid media for temperature growth studies, based on
GROWTH OF NONFERMENTATIVE BACTERIA AT 420C
VOL. 10, 1979
TABLE 4. Growth at 42°C and alkalinization of acetamide by NFB Organism P. aeruginosa Pyomelanine Delayed pyocyanine Apyocyanine Mucoid Nonglucolytic UFPd P. fluorescens P. putida A. xylosoxidans CDC Vd Pseudomonas sp. 1 Pseudomonas sp. 2 A. anitratus P. stutzeri group P. pseudoalealigenes
P. cepacia P. maltophilia F. meningosepticum Flavobacterium Hb P. paucimobilis CDC IIk-2 CDC Ve-1 CDC Ve-2
P. P. P. P.
acidovorans testosteroni alcaligenes dininuta
Alcaligenes sp. A. faecalis A. denitrificans A. odorans CDC IVc B. bronchiseptica
42°C grovsrtha No. %+b tested
Acetamide No. %+b (+)C
tested
100
22
100
27
21
100
46
98
2
78
100
128
96
3
17 3 2 15 52 16
NT 4 7
102 17'
15'
NT
60
23 3
100 0
00
32
0
6 100 50 86 94 8887
104 26 4 7 170 44 15 9 140
100 100
37
NT
15 4 6 3' 8 13'
0 0 100
13 15
CA
2
100 0
2
0 78 0
6f
9'
3
5 2 4'
100
100 50
8
18 11 6 21 20 13 8 9
2 9 5 16 7 4
33
3 1 46 42 0 0 0 00 2 78 0 5 2
0
13
0 0 0
0 95 0
5
0 0 0
33
94
6
0 0
0 0
0 100 9 9 Flavobacterium Ilf 0 0 3 3 F. odoratum 21 0 14 7 M. osloensis 7 Moraxella sp. 91 1 53 45 A. Iwoffi a Growth test results with TSA lants. b+, Positive reaction in 1 to 2 d ' (+), Positive reaction in 3 to 7 'days. d UFP, Unidentified fluorescent pseudomonads. 'NT, Not tested. f Results with TSB.
9
careful evaluation, are rem;arkably scarce. Difficulty in the use of broth xroutinely arises with the size of the inoculum sel ected, allied with the degree of turbidity acceptei!d as the criterion for "growth." Results of the te«sts using broth are at variance with those of Kliinge (12), who advocated the use of broth meclium because of uniform distribution of the inc culum, better distribution of bacterial clumps,, and more rapid attainment of the desired temperature. Klinge
803
used a loopful of a 1:100 dilution of the test organism as inoculum, although the volume of media used was not specified. During the current study, an overnight broth culture was selected as inoculum to insure uniformity of growth phase and cell volume of each species tested. Hence, placing approximately 0.005 ml into 3 ml of broth was adequate to eliminate an inoculum efct effect. There seems to be considerable heterogeneity among the organisms designated as P. putida because of variable reactions in a host of tests. Indeed, it was reported (2) that only 11 strains of P. putida and 4 strains of P. fluorescens were isolated in a 2-year period, since they reportedly had a predilection for growth at lower than body temperature. Moreover, in the description of P. fluorescens advanced by Rhodes (17), P. fluorescens failed to grow at 370C. Today these account for 10% of clinical isolates organisms (14). The P. putida, then, may represent a continuum of activities, including strains resembling P. fluorescens as well as those resembling P.
aeruginosa in growth characteristics.
It seems clear that some organisms conforning to the description of P. putida multiplied in broth sufficiently for that medium to be judged unsuitable for use in a differential test. The choice of Trypticase as a broth or agar vehicle may be questioned, although it should be acknowledged that a more nutritious medium would support even better growth. That warmning of the medium to 420C before inoculation is not critical is attested to by the fact that the generation time of the fluorescent pseudomonads is too great to influence replication. Haynes and Rhodes (7) proposed that a test organism should survive at least three serial transfers at a given temperature before being characterized for taxonomic purposes. They observed that some strains which could initiate good growth at 420C showed increasing relucat that transfer with each serial to do soInterestingly, tance other proreports temperature. claiming multiple passages did not clearly specify the species tested or the outcome of those tests (2). The implication is that growth was necessarily present during the initial test (2, 7), fulfilling the requirement for "growth" at 420C. Wahba and Darrell (20) recorded intriguing results with organisms designated at P. fluorescens, whereby almost half of the strains tested grew at 420C during the initial growth attempt. It was recognized, however, that repeated transfer had little practical use as a routine procedure. Adaptation of P. putida was not attempted during the current study. Three isolates of non-glucose-oxidizing P. aeruginosa were unusual in their failure to al-
804
OBERHOFER
kalinize acetamide. This departure from the expected correlation (13, 14) posed little difficulty in identification of these particular isolates, since many characteristics typical of P. aeruginosa were prominent. Two isolates of unidentified fluorescent pseudomonads also failed to correlate in the tests for growth and alkalinization. Several groups of fluorescent pseudomonads capable of growing at 420C have been recovered from surface waters (8) and swimming pools (9), as well as from human sources (19). Similar to these other isolates, the apyocyanogenic strains of unidentified fluorescent pseudomonads resembled P. aeruginosa in hemolytic activity and ability to liquefy gelatin, but differed in their failure to denitrify, to attack mannitol and acetamide (1, 8), and to oxidize gluconate (1, 9). The monopolar characteristics of both groups of organisms, however, clearly distinguished them from P. putida and P. fluorescens, although other characteristics such as susceptibility to kanamycin bore considerable similarity to P. putida. The results of this study and others (5, 15) indicate that the test for growth at 420C may have little discriminating value for the differentiation of NFB usually encountered in the clinical laboratory. Growth at the selective temperature is not constant within species, which imposes severe limitations for separating closely related species. The growth ofP. putida in broth and on agar, although light, reduces the effectiveness of the test when the criterion of growth or no growth is placed on test interpretation. Furthermore, multiple passages at 420C is not practical for use on a routine basis. The test is fraught with technical difficulties. Variables include the selection of medium, the size of inoculum, the age of the inoculum, and perhaps even the apparatus used for incubation. Nevertheless, the test may have a place in a comprehensive identification protocol. The method of choice is inoculation of a slanted agar medium with a loopful of an overnight broth culture with the slant incubated for 18 to 24 h. Any evidence of growth should be noted, the extent of growth is recorded, and the test is then discarded. LITERATURE CITED Hoadley. 1976. Fluorescent pseudomonads capable of growth at 41°C but distinct
1. Ajello, G. W., and A. W.
from Pseudomonas aeruginosa. J. Clin. Microbiol. 4: 443-449.
J. CLIN. MICROBIOL. 2. Blazevic, D. J., M. H. Koepcke, and J. M. Matsen. 1973. Incidence and identification of Pseudomonas fluorescens and Pseudomonas putida in the clinical laboratory. Appl. Microbiol. 25:107-110. 3. Gilardi, G. L. 1968. Differentiation of oxidative and nonglucolytic gram-negative bacteria. Am. J. Med. Technol. 34:334-345. 4. Gilardi, G. L. 1971. Characterization of nonfermentative nonfastidious gram negative bacteria encountered in medical bacteriology. J. Appl. Bacteriol. 34:623-644. 5. Gilardi, G. L. 1976. Pseudomonas species in clinical microbiology. Mount Sinai J. Med. 43:710-726. 6. Haynes, W. C. 1951. Pseudomonas aeruginosa-its characterization and identification. J. Gen. Microbiol. 5: 939-950. 7. Haynes, W. C., and L. J. Rhodes. 1962. Comparative taxonomy of crystallogenic strains of Pseudomonas aeruginosa and Pseudomonas chlororaphis. J. Bacteriol. 84:1080-1084. 8. Hoadley, A. W., and G. Ajello. 1972. Some characteristics offluorescent pseudomonads isolated from surface waters and capable of growth at 41°C. Can. J. Microbiol. 18:1769-1773. 9. Hoadley, A. W., G. Ajello, and N. Masterson. 1975. Preliminary studies of fluorescent pseudomonads capable of growth at 41 C in swimming pool waters. Appl. Microbiol. 29:527-531. 10. Hugh, R. 1970. A practical approach to the identification of certain nonfermentative gram-negative rods encountered in clinical specimens. Publ. Health Lab. 28:168187. 11. Kantor, L T., S. D. Kominos, and R. B. Yee. 1975. Identification of nonfermentative gram-negative bacteria in the clinical laboratory. Am. J. Med. Technol. 41: 3-9. 12. Klinge, K. 1960. Differential techniques and methods of isolation of Pseudomonas. J. Appl. Bacteriol. 23:442462. 13. Oberhofer, T. R., J. W. Rowen, and G. F. Cunningham. 1977. Characterization and identification of gram-negative, nonfermentative bacteria. J. Clin. Microbiol. 5:208-220. 14. Oberhofer, T. R. 1979. Comparison of the API 20E and Oxi/Ferm systems in identification of nonfermentative and oxidase-positive fermentative bacteria. J. Clin. Microbiol. 9:220-226. 15. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159-271. 16. Pickett, M. J., and M. M. Pedersen. 1970. Characterization of saccharolytic nonfermentative bacteria associated with man. Can. J. Microbiol. 16:351-362. 17. Rhodes, M. E. 1959. The characterization of Pseudomonas fluorescens. J. Gen. Microbiol. 21:221-263. 18. Sands, D. C., M. N. Schroth, and D. C. Hildebrand. 1970. Taxonomy of phytopathogenic pseudomonads. J. Bacteriol. 101:9-23. 19. Sutter, V. L. 1968. Identification of Pseudomonas species isolated from hospital environment and human sources. Appl. Microbiol. 16:1532-1538. 20. Wabba, A. H., and J. H. Darrell. 1965. The identification of atypical strains of Pseudomonas aeruginosa. J. Gen. Microbiol. 38:329-342. 21. Weaver, R. E., H. W. Tatum, and D. G. Hoffis. 1972. The identification of unusual pathogenic gram-negative bacteria. Preliminary revision. Center for Disease Control, Atlanta, Ga.
