JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1975, p. 339-344 Copyright 0 1975 American Society for Microbiology
Vol. 1, No. 4 Printed in U.S.A.
Cultural and Biochemical Characteristics and Fatty Acid Composition of Pseudomonas dim inuta and Pseudomonas vesiculare CHARLES M. KALTENBACH,' C. WAYNE MOSS,*
AND
ROBERT E. WEAVER
Center for Disease Control, Atlanta, Georgia 30333
Received for publication 25 November 1974
The cultural characteristics, biochemical activity, and cellular fatty acid composition of Pseudomonas diminuta and Pseudomonas vesiculare provided means for differentiation of these closely related species. Broth cultures of P. diminuta showed turbid growth and a distinct surface pellicle after 24 h at 35 C. P. vesiculare had no pellicle, and only light, diffuse growth was observed. All strains of P. vesiculare oxidized maltose and hydrolyzed esculin to varying degrees; P. diminuta was negative in these tests. The cellular fatty acids of these two species were similar, except that P. diminuta possessed a C,9 cyclopropane acid which was not detected in P. vesiculare.
Ballard et al. confirmed an earlier observation of R. Hugh that Pseudomonas diminuta and P. vesiculare were closely related species (1). In addition, they indicated that the two species were unique among the aerobic pseudomonads and formed a subgroup, the P. diminuta group, within the Pseudomonas genus. The principal characteristics noted for this subgroup were the inability to produce acid from most carbohydrates, the negative responses to other routine biochemical tests, and the inability to grow in simple defined media (1). More recent reports demonstrated the relative inactivity in routine diagnostic tests (5, 8, 17). Consequently, the identification and differentiation of these two organisms has remained an arduous task. In the present study we thoroughly characterized several reference strains and clinical isolates of each species by use of a battery of routine diagnostic tests and procedures. We also determined the cellular fatty acid composition in the hope that this information would provide additional criteria for distinguishing these closely related organisms. MATERIALS AND METHODS Cultural. Bacterial cultures were obtained from: the American Type Culture Collection; Hans Lautrop, Statens Seruminstitut, Copenhagen, Denmark; N.J. Palleroni, University of California, Berkeley; and the culture collection of the Special Bacteriology Section, Center for Disease Control, Atlanta. Although a number of strains of each species were studied (32 of P. diminuta and 9 of P. vesiculare), to I Present address: Navajo Health Authority, P.O. Box 643, Window Rock, Ariz. 86515.
conserve space only carefully selected strains entirely representative of the majority are presented. Stock cultures were maintained in a semisolid motility medium. After initial incubation at 35 C for 24 h, the cultures were stored at 4 C. Each strain was thoroughly characterized by use of conventional bacteriological procedures (10, 18). Fatty acid analysis. Bacteria for fatty acid analysis were obtained from 24-h growth (35 C) on plates of Trypticase soy agar (BBL) supplemented with 0.1% (wt/vol) yeast extract (Difco). Washed bacteria were saponified with 5% NaOH in 50% methanol (vol/vol) for 1 h in a 100 C water bath. The fatty acids were extracted and methylated by methods previously described (13). Methyl esters were analyzed on a Barber-Colman model 5000 gas chromatograph (Barber-Colman, Rockford, Ill.) equipped with a hydrogen flame detector and a disk integrator recorder (Series 8000). Operating parameters were: injector temperature, 250 C; detector temperature, 275 C; column temperature, 150 to 260 C at 3 C/min. Samples were analyzed with both polar and nonpolar liquid phase materials contained in 1.8-m (6 feet) U-tube glass columns. The polar phase was 12% ethylene glycol adipate coated on Chromosorb P (80/100 mesh), and the nonpolar phase was 3% OV-1 methyl silicone coated on 80/100 mesh, acid-washed, dimethylchlorosilane-treated, high performance Chromosorb W (Applied Science Laboratories, State College, Pa.). For routine analysis, 2 to 4 pl of the methyl ester sample was analyzed for 35 min after injection. Eluted compounds were tentatively identified by comparing retention times on each column with retention times of methyl ester standards. Final identification was confirmed by a combination of techniques, including hydrogenation of unsaturated compounds (2), bromination of cyclopropane fatty acids (3), acylation of hydroxy acids (7), and the use of mass spectrometry (14). Combined gas-liquid
339
340
KALTENBACH, MOSS, AND WEAVER
chromatography-mass spectrometry was carried out on an LKB 900 instrument (LKB Instruments, Inc., Rockville, Md.). The mass spectra were recorded at an electron energy of 70 eV and at a trap current of 60 AA, an ion source temperature of 190 C, and a molecular separator temperature of 250 C. The esters were separated on a coiled glass column (4.8 m by 6.35 mm [16 feet by 0.25 inch]) packed with 3% OV-1. The column was conditioned for 72 h before use. Peak areas were determined by integration, and the percentage of each acid was calculated from the ratio of the area of its peak to the total area of all peaks.
RESULTS Microscopic. Microscopically, strains of P. diminuta and P. vesiculare appeared quite similar. All were small gram-negative rods of varying length (1 to 5 ,Am). The diameter of the cells ranged between 0.5 and 1.0 Am. Cells cultivated on heart infusion agar appeared as single cells or in pairs; from heart infusion broth the cells were frequently arranged in chains. In addition, each strain possessed one (or on rare occasions, two) polar flagella with an average length of 3.0 ,um and an average wavelength of 0.6 ,im, the typical flagellum of short wavelength which is characteristic of these two species (11). Colonial characteristics. Colonies of P. vesiculare and P. diminuta were similar on blood agar (5% sheep erythrocytes). No hemolysis was observed for any strain. With few exceptions, colonies were punctate, circular, and convex with an entire edge. The colony surface was smooth, glistening, and opaque. Cultural and biochemical characteristics. Unless otherwise indicated, all biochemical and cultural results reported in this paper are based upon observations after 48 h of incubation at 35 C. All strains of the P. diminuta group grew well at 25 and 35 C on tryptone glucose yeast agar slants. At 42 C, growth varied from very light to moderate, depending upon the individual strain. Distinct differences were noted between P. diminuta and P. vesiculare in heart infusion broth. At 24 h, P. diminuta developed moderate to heavy (usually diffuse) growth with a predominant surface pellicle. In contrast, P. vesiculare produced very light growth and no pellicle. Continued incubation of all P. vesiculare strains for periods up to 1 week in heart infusion broth failed to produce a surface pellicle. On heart infusion agar and on several other media, five of eight P. vesiculare strains consistently produced an orange-red pigment; pigment was absent in all P. diminuta strains. On the basis of routine biochemical tests, P. diminuta and P. vesiculare formed a rather homogenous group; however, differences were noted in some
J . C LIN . M ICROBIOL .
carbohydrate oxidations and in the ability of the two species to hydrolyze esculin. Results obtained with representative strains of each species are presented in Table 1. All strains of P. vesiculare oxidized maltose, glucose, and ethyl alcohol in the oxidative-fermentative base medium (18); cellobiose and L-arabinose were oxidized by at least 50% of the strains tested. All strains of P. vesiculare hydrolyzed esculin to varying degrees, as indicated by the appearance of a distinct jet-black color in the esculin agar butt and slant. Hydrolysis was confirmed by viewing the medium under ultraviolet light at 366 nm using a Wood's lamp. At this wavelength, unhydrolyzed esculin fluoresces brightly; no fluorescence is observed when esculin is hydrolyzed. In contrast, ethyl alcohol was the only carbohydrate oxidized with any consistency by strains of P. diminuta. Many strains oxidized glucose, but only slightly, producing a very weak acid condition in the medium. No strain hydrolyzed maltose. None of the P. diminuta hydrolyzed esculin or produced any macroscopically detectable color change in this medium after continued incubation for 1 week. On the basis of results in this laboratory, salient features that distinguish P. diminuta and P. vesiculare are summarized in Table 2. Fatty acid composition. The fatty acids of P. diminuta and P. vesiculare were similar with one distinguishing exception: the presence of a C1g cyclopropane acid in P. diminuta. Results from several determinations of representative strains of these two species are presented in Table 3, and the actual chromatograms can be compared in Fig. 1. The major fatty acids present in cells of P. vesiculare after 24 h of incubation were octadecenoic (C,18:,), palmitic (C16: 0), and 3-hydroxy lauric (3-OH,12o) acids. The same acids as well as a C1g cyclopropane acid (C,1) were present in P. diminuta. Other acids found in both species, but in comparatively small (less than 10%) or trace (less than 2%) amounts included: nonadecanoic (Clg:o), octadecanoic (C,18:0), heptadecanoic (C7: 0), pentadecanoic (C 15:0), myristic (C14: 0), 2hydroxy lauric (2-OH,2o), and two unidentified acids that appeared inconsistently. The predominant acid among strains of P. vesiculare was Ci9:l, followed in decreasing order of concentration by either C16:0 or 3-OH12:o. In contrast, C16:0 was the most abundant acid in strains of P. diminuta, followed by either C18:1 or 3-OH12:0. In general, the fourth most abundant acid in P. diminuta was the C1g cyclopropane acid. Culture age had only a minor effect on fatty
VOL. 1, 1975
CHARACTERISTICS OF P. DIMINUTA AND P. VESICULARE +
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Vol. 1, No. 4 Printed in U.S.A.
Cultural and Biochemical Characteristics and Fatty Acid Composition of Pseudomonas dim inuta and Pseudomonas vesiculare CHARLES M. KALTENBACH,' C. WAYNE MOSS,*
AND
ROBERT E. WEAVER
Center for Disease Control, Atlanta, Georgia 30333
Received for publication 25 November 1974
The cultural characteristics, biochemical activity, and cellular fatty acid composition of Pseudomonas diminuta and Pseudomonas vesiculare provided means for differentiation of these closely related species. Broth cultures of P. diminuta showed turbid growth and a distinct surface pellicle after 24 h at 35 C. P. vesiculare had no pellicle, and only light, diffuse growth was observed. All strains of P. vesiculare oxidized maltose and hydrolyzed esculin to varying degrees; P. diminuta was negative in these tests. The cellular fatty acids of these two species were similar, except that P. diminuta possessed a C,9 cyclopropane acid which was not detected in P. vesiculare.
Ballard et al. confirmed an earlier observation of R. Hugh that Pseudomonas diminuta and P. vesiculare were closely related species (1). In addition, they indicated that the two species were unique among the aerobic pseudomonads and formed a subgroup, the P. diminuta group, within the Pseudomonas genus. The principal characteristics noted for this subgroup were the inability to produce acid from most carbohydrates, the negative responses to other routine biochemical tests, and the inability to grow in simple defined media (1). More recent reports demonstrated the relative inactivity in routine diagnostic tests (5, 8, 17). Consequently, the identification and differentiation of these two organisms has remained an arduous task. In the present study we thoroughly characterized several reference strains and clinical isolates of each species by use of a battery of routine diagnostic tests and procedures. We also determined the cellular fatty acid composition in the hope that this information would provide additional criteria for distinguishing these closely related organisms. MATERIALS AND METHODS Cultural. Bacterial cultures were obtained from: the American Type Culture Collection; Hans Lautrop, Statens Seruminstitut, Copenhagen, Denmark; N.J. Palleroni, University of California, Berkeley; and the culture collection of the Special Bacteriology Section, Center for Disease Control, Atlanta. Although a number of strains of each species were studied (32 of P. diminuta and 9 of P. vesiculare), to I Present address: Navajo Health Authority, P.O. Box 643, Window Rock, Ariz. 86515.
conserve space only carefully selected strains entirely representative of the majority are presented. Stock cultures were maintained in a semisolid motility medium. After initial incubation at 35 C for 24 h, the cultures were stored at 4 C. Each strain was thoroughly characterized by use of conventional bacteriological procedures (10, 18). Fatty acid analysis. Bacteria for fatty acid analysis were obtained from 24-h growth (35 C) on plates of Trypticase soy agar (BBL) supplemented with 0.1% (wt/vol) yeast extract (Difco). Washed bacteria were saponified with 5% NaOH in 50% methanol (vol/vol) for 1 h in a 100 C water bath. The fatty acids were extracted and methylated by methods previously described (13). Methyl esters were analyzed on a Barber-Colman model 5000 gas chromatograph (Barber-Colman, Rockford, Ill.) equipped with a hydrogen flame detector and a disk integrator recorder (Series 8000). Operating parameters were: injector temperature, 250 C; detector temperature, 275 C; column temperature, 150 to 260 C at 3 C/min. Samples were analyzed with both polar and nonpolar liquid phase materials contained in 1.8-m (6 feet) U-tube glass columns. The polar phase was 12% ethylene glycol adipate coated on Chromosorb P (80/100 mesh), and the nonpolar phase was 3% OV-1 methyl silicone coated on 80/100 mesh, acid-washed, dimethylchlorosilane-treated, high performance Chromosorb W (Applied Science Laboratories, State College, Pa.). For routine analysis, 2 to 4 pl of the methyl ester sample was analyzed for 35 min after injection. Eluted compounds were tentatively identified by comparing retention times on each column with retention times of methyl ester standards. Final identification was confirmed by a combination of techniques, including hydrogenation of unsaturated compounds (2), bromination of cyclopropane fatty acids (3), acylation of hydroxy acids (7), and the use of mass spectrometry (14). Combined gas-liquid
339
340
KALTENBACH, MOSS, AND WEAVER
chromatography-mass spectrometry was carried out on an LKB 900 instrument (LKB Instruments, Inc., Rockville, Md.). The mass spectra were recorded at an electron energy of 70 eV and at a trap current of 60 AA, an ion source temperature of 190 C, and a molecular separator temperature of 250 C. The esters were separated on a coiled glass column (4.8 m by 6.35 mm [16 feet by 0.25 inch]) packed with 3% OV-1. The column was conditioned for 72 h before use. Peak areas were determined by integration, and the percentage of each acid was calculated from the ratio of the area of its peak to the total area of all peaks.
RESULTS Microscopic. Microscopically, strains of P. diminuta and P. vesiculare appeared quite similar. All were small gram-negative rods of varying length (1 to 5 ,Am). The diameter of the cells ranged between 0.5 and 1.0 Am. Cells cultivated on heart infusion agar appeared as single cells or in pairs; from heart infusion broth the cells were frequently arranged in chains. In addition, each strain possessed one (or on rare occasions, two) polar flagella with an average length of 3.0 ,um and an average wavelength of 0.6 ,im, the typical flagellum of short wavelength which is characteristic of these two species (11). Colonial characteristics. Colonies of P. vesiculare and P. diminuta were similar on blood agar (5% sheep erythrocytes). No hemolysis was observed for any strain. With few exceptions, colonies were punctate, circular, and convex with an entire edge. The colony surface was smooth, glistening, and opaque. Cultural and biochemical characteristics. Unless otherwise indicated, all biochemical and cultural results reported in this paper are based upon observations after 48 h of incubation at 35 C. All strains of the P. diminuta group grew well at 25 and 35 C on tryptone glucose yeast agar slants. At 42 C, growth varied from very light to moderate, depending upon the individual strain. Distinct differences were noted between P. diminuta and P. vesiculare in heart infusion broth. At 24 h, P. diminuta developed moderate to heavy (usually diffuse) growth with a predominant surface pellicle. In contrast, P. vesiculare produced very light growth and no pellicle. Continued incubation of all P. vesiculare strains for periods up to 1 week in heart infusion broth failed to produce a surface pellicle. On heart infusion agar and on several other media, five of eight P. vesiculare strains consistently produced an orange-red pigment; pigment was absent in all P. diminuta strains. On the basis of routine biochemical tests, P. diminuta and P. vesiculare formed a rather homogenous group; however, differences were noted in some
J . C LIN . M ICROBIOL .
carbohydrate oxidations and in the ability of the two species to hydrolyze esculin. Results obtained with representative strains of each species are presented in Table 1. All strains of P. vesiculare oxidized maltose, glucose, and ethyl alcohol in the oxidative-fermentative base medium (18); cellobiose and L-arabinose were oxidized by at least 50% of the strains tested. All strains of P. vesiculare hydrolyzed esculin to varying degrees, as indicated by the appearance of a distinct jet-black color in the esculin agar butt and slant. Hydrolysis was confirmed by viewing the medium under ultraviolet light at 366 nm using a Wood's lamp. At this wavelength, unhydrolyzed esculin fluoresces brightly; no fluorescence is observed when esculin is hydrolyzed. In contrast, ethyl alcohol was the only carbohydrate oxidized with any consistency by strains of P. diminuta. Many strains oxidized glucose, but only slightly, producing a very weak acid condition in the medium. No strain hydrolyzed maltose. None of the P. diminuta hydrolyzed esculin or produced any macroscopically detectable color change in this medium after continued incubation for 1 week. On the basis of results in this laboratory, salient features that distinguish P. diminuta and P. vesiculare are summarized in Table 2. Fatty acid composition. The fatty acids of P. diminuta and P. vesiculare were similar with one distinguishing exception: the presence of a C1g cyclopropane acid in P. diminuta. Results from several determinations of representative strains of these two species are presented in Table 3, and the actual chromatograms can be compared in Fig. 1. The major fatty acids present in cells of P. vesiculare after 24 h of incubation were octadecenoic (C,18:,), palmitic (C16: 0), and 3-hydroxy lauric (3-OH,12o) acids. The same acids as well as a C1g cyclopropane acid (C,1) were present in P. diminuta. Other acids found in both species, but in comparatively small (less than 10%) or trace (less than 2%) amounts included: nonadecanoic (Clg:o), octadecanoic (C,18:0), heptadecanoic (C7: 0), pentadecanoic (C 15:0), myristic (C14: 0), 2hydroxy lauric (2-OH,2o), and two unidentified acids that appeared inconsistently. The predominant acid among strains of P. vesiculare was Ci9:l, followed in decreasing order of concentration by either C16:0 or 3-OH12:o. In contrast, C16:0 was the most abundant acid in strains of P. diminuta, followed by either C18:1 or 3-OH12:0. In general, the fourth most abundant acid in P. diminuta was the C1g cyclopropane acid. Culture age had only a minor effect on fatty
VOL. 1, 1975
CHARACTERISTICS OF P. DIMINUTA AND P. VESICULARE +
-4
_
Cl.
t _
+-
4
rCS
-
CL) -6i
t+ -
co
1
0
._)
+ _T
+ t ^l- "T
+
+
I"
"I
t + + t CV 'T I'l cl
+ Cl CZ
-) Co 4 _
u!ulnsd
+
+
Cl
Ce- C'
F
+
Cl-C
+ +
D
"0
-m
-00
>0
+
++
+
+
+ +
E.
