Sep 23, 1986 - multocida Somatic Serotype and Actinobacillus lignieresii Strains ... Pasteurella multocida possesses a characteristically gram-negative ...
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1987, p. 67-71
Vol. 25, No. 1
0095-1137/87/010067-05$02.00/0 Copyright © 1987, American Society for Microbiology
Variability of Cell Surface Hydrophobicity among Pasteurella multocida Somatic Serotype and Actinobacillus lignieresii Strains KIMBRELL R. DARNELL, MARK E. HART, AND FRANKLIN R. CHAMPLIN*
Sciences, Mississippi State University, Mississippi State, Mississippi 39762 Received 26 June 1986/Accepted 23 September 1986
Pasteurella multocida possesses a characteristically gram-negative ultrastructure, yet its inability to grow in the presence of hydrophobic compounds and the general penicillin susceptibility of genera making up the family Pasteurellaceae suggest a cell envelope having atypical permeability properties. The cell surface hydrophobicity properties of strains representing 15 of the 16 somatic serotypes of P. miltocida and three strains of ActinobaciUlus lignieresii were assessed with hydrocarbon adherence and hydrophobic interaction chromatographic assays. These methods revealed surface hydrophobicity to vary dramatically among strains in both species. No direct correlation was observed with species, growth rate, or susceptibility to the antibiotics oxytetracycline (polar), polymyxin B (amphiphilic), or novobiocin (nonpolar) as measured with MIC determinations. All strains were susceptible to the antibiotics, although A. lignieresii was significantly less susceptible than P. multocida to novobiocin. These data suggest that cell surface hydrophobicity in P. multocida may be influenced by the type of lipopolysaccharide present but is not directly related to permeability of the antibiotics examined. The wide diversity of hydrophobic properties exhibited by strains of both P. multocida and A. lignieresii precludes the use of this parameter as a taxonomic aid.
The genera Pasteurella, Actinobacillus, and Haemophilus make up the family Pasteurellaceae (22, 35). They contain species pathogenic in humans and animals and can be collectively described as nonmotile, facultative, and gramnegative coccobacilli (22). Whereas Haemophilus species exhibit distinctive X and V growth requirements (35), members of the genera Pasteurella and Actinobacillus have proven difficult to differentiate (7, 27), and strong evidence supporting their inclusion as a single genus based on the analysis of 134 phenotypic characteristics has been presented (35). The phenotypic similarity of their type species, Pasteurella multocida and Actinobacillus lignieresii, has been recognized previously (34). Generic separation is in further doubt in view of recent studies showing that various species from the two genera exhibit similar respiratory quinone (23) and cellular fatty acid patterns (17). The extensive physiological variability exhibited by P. multocida strains (13, 15, 26, 29) limits the diagnostic value of conventional biochemical parameters, resulting in poor commercial test system reliability (26, 29) and frequent misidentification (3, 11, 19). From a structural standpoint, P. multocida appears to possess a characteristically gram-negative cell envelope (6, 30), yet the organism is susceptible to hydrophobic bile salts and crystal violet present in MacConkey agar (4, 13, 26). In light of the characteristic penicillin susceptibility of members of the Pasteurellaceae (22), a structurally typical cell envelope having atypical permeability properties may be presumed. That the atypical permeability properties are due to chemical composition rather than structural anomolies is implicit. The gel diffusion precipitin test developed by Heddleston et al. (14) has been used to demonstrate 16 distinguishable serotypes (2, 5) on the basis of the heatstable somatic antigenicity of lipopolysaccharide (LPS) (24, 31). Lugtenberg et al. (20) have reported the LPS of P. multocida to be of lower molecular weight than that found in certain smooth members of the family Enterobacteriaceae, *
suggesting fewer repeating sugars and subsequently shorter 0-side-chain length. This suggests a less hydrophilic nature analogous to that of rough enteric bacteria (21, 32). Rimler et al. (31) found that LPS from 13 of the 16 somatic serotype strains of P. multocida is extractable with phenolchloroform-petroleum ether, a solvent mixture which extracts the relatively hydrophobic LPS of rough enteric mutants more efficiently than the usual phenol-water method. LPS from serotypes 3, 9, and 13 was extractable only with phenol-water, suggesting that these serotypes are hydrophilic due presumably to their having longer O side chains. The present study was undertaken to investigate the relationship between somatic serotype specificity in P. multocida and cell surface parameters such as relative hydrophobicity and permeability for antibiotics differing in overall polarity. Three strains of A. lignieresii were included in an attempt to either support the genotypic relatedness or, conversely, to aid in the differentiation of these bacterial pathogens. MATERIALS AND METHODS Bacterial strains. A collection of 15 P. multocida somatic serotype reference strains was obtained in lyophilized form from R. B. Rimler of the National Animal Disease Center, U.S. Department of Agriculture, Ames, Iowa. They represent 15 of the 16 known somatic serotypes as determined with the gel diffusion precipitin test of Heddleston et al. (14). Three A. lignieresii reference strains were obtained in lyophilized form from the American Type Culture Collection, Rockville, Md. Pseudomonas aeruginosa PA01 (16) is maintained as a stock culture in this laboratory and was included in the present study for the purpose of comparison. Lyophilized cultures were routinely rehydrated in brain heart infusion (BHI; Difco Laboratories, Detroit, Mich.) at 37°C. Culture maintenance and growth conditions. Concentrated suspensions of all organisms were stored as triplicate stock cultures under cryoprotective conditions (BHI plus 10% glycerol) in sterile polyethylene Provials (2 ml; Dynatech
Corresponding author. 67
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J. CLIN. MICROBIOL.
TABLE 1. Generation times and cell surface hydrophobicity values for P. multocida, A. lignieresii, and P. aeruginosa reference strains % Adherenceb + SD Organism
Generation time (min)' Hydrocarbon adherence
P. multocida X-73 (1)C P-1059 (3) P-1662 (4) P-1702 (5) P-2192 (6) P-1997 (7) P-1581 (8) P-2095 (9) P-2100 (10) P-903 (11) P-1573 (12) P-1591 (13) P-2225 (14) P-2237 (15) P-2723 (16) A. lignieresii ATCC 19393 ATCC 13372 ATCC 13369 P. aeruginosa PAO1
34 36 32 40 33 38 32 34 36 44 61 67 37 47 44
26.1 ± 12.2 ± 12.4 ± 45.3 ± 54.3 ± 60.2 ± 70.6 ± 1.3 ± -2.3 ± 59.5 ± 0.3 ± 62.0 ± 1.7 ± 53.6 ± 50.3 ±
40 42 40
58.6 4.4 1.3 -1.7
3.5 6.6 2.5 12.3 6.2 3.8 2.7 1.0 1.0 3.6 1.1 3.0 0.7 1.2 1.1
± 1.1 ± 1.0 ± 2.7 ± 0.9
chromatography 15.5 11.9 22.3 43.8 100.0 84.4 99.5 4.8 3.9 99.0 2.2 95.8 3.7 96.5 57.8
100.0 95.5 97.3 44.5
± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.2 4.6 21.4 0.0 8.6 0.9 1.5 2.9 0.9 1.9 1.7 2.0 3.6 5.8
± 0.0 ± 2.9 ± 3.4 ± 1.9
Estimates obtained from turbidimetric growth curves during exponential growth in BHI broth. b Percent decrease in turbidity of cell suspension after mixing with 1,000 ,i of n-hexadecane (hydrocarbon adherence method) or eluting through an octyl-Sepharose column (hydrophobic interaction chromatography). Each value represents the mean of three to four independent determinations. c P. multocida somatic serotype designations are included in parentheses. a
Laboratories, Inc., Alexandria, Va.) at -80°C. To obtain starter culture inocula, surfaces of frozen stock preparations were scraped with a sterile loop and transferred to appropriate media. Storage under these conditions for as long as 2 years has resulted in no detectable loss of viability and has proven superior to lyophilization, since it allows routine removal of cells without sacrificing entire storage preparations. Batch cultures were grown in either 125- or 250-ml flasks containing 50 or 100 ml of BHI or Mueller-Hinton broth (MHB; Difco), respectively (flask/volume ratio of 2.5). An overnight starter culture (approximately 15 h) consisting of stationary-phase cells was used to inoculate each batch culture to an initial optical density of approximately 0.05 at 550 nm (Spectronic 20 optical spectrophotometer; Bausch & Lomb, Inc., Rochester, N.Y.). Cultures were incubated at 37°C with rotary aeration at 180 rpm in a model G24 Environmental Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, N.J.). Examination of negatively stained preparations with light microscopy before surface hydrophobicity determinations revealed capsules on less than 0.1% of cells. The growth kinetics for each strain were determined turbidimetrically by monitoring optical densities (wavelength of 550 nm) of triplicate batch cultures at 30-min intervals. Optical density measurements were related to viable cell density with the aid of a standard curve. Hydrocarbon adherence assay. Cell surface hydrophobicity was measured by assessing the degree to which bacterial
strains were able to associate with n-hexadecane by using a slight modification of the hydrocarbon adherence method developed by Rosenberg et al. (32). Cells from lateexponential-phase BHI cultures were harvested by centrifugation at 10,000 x g and 4°C for 10 min, washed in 1 volume of cold buffer (6.97 g of K2HPO4, 2.99 g of KH2PO4, and 0.2 g of MgSO4 7H20 per liter of glass-distilled water; pH 7.12), and suspended in room temperature buffer to an optical density of 0.50 at 550 nm (approximately 2.9 x 108 CFU/ml). Each cell suspension was divided into six 4.0-ml samples in test tubes (18 by 150 mm, Pyrex) to which various volumes (0.2, 0.4, 0.6, 0.8, or 1.0 ml) of n-hexadecane were added. One tube was designated a control and received no hydrocarbon. After rapid Vortex agitation for 1 min each, tubes were allowed to stand for 15 min to permit complete phase separation. Aqueous phases were transferred to colorimeter tubes (0.5-in. [ca. 1.25-cm] diameter; Bausch and Lomb), and adherence to the hydrocarbon was assessed on the basis of decreases in turbidity relative to the untreated control as measured spectrophotometrically at 550 nm. Two techniques were employed to verify that decreases in aqueous-phase turbidity were due in fact to cellular adhesion rather than bacteriolysis: (i) cells were observed adhering to oil droplets in numbers commensurate with turbidity decreases under phase contrast microscopy, and (ài) the addition of isopropanol (5% final concentration) (32) caused aqueous phase turbidity to increase significantly or be fully restored after vortex agitation for 20 s. Hydrophobic interaction chromatography. Cell surface hydrophobicity assessments were substantiated with hydrophobic interaction chromatography data obtained with columns prepared in Pasteur pipettes as described by Smyth et al. (33). Each column was packed to a final bed volume of 0.8 to 1.0 ml with octyl-Sepharose CL-4B (Sigma Chemical Co., St. Louis, Mo.) and washed sequentially with 10.0 ml of glass-distilled water and 5.0 ml of buffer. Cells from lateexponential-phase BHI cultures were harvested and washed as described above and suspended to an optical density of 1.0 at 550 nm (approximately 5.8 x 108 CFU/ml). Each column was loaded with 0.2 ml of a standardized cell suspension and washed with buffer until 4.0 ml of eluate was collected. An additional 0.2 ml of each suspension was concomitantly diluted to 4.0 ml in buffer to provide a reference control. Column eluates and reference samples were transferred to colorimeter tubes, and adherence to the hydrophobic gel bed was assessed on the basis of differences in turbidity between eluate and reference samples as measured spectrophotometrically at 550 nm. Determination of MICs. The MICs of three antibiotics for bacterial strains cultured in MHB were determined by using the macro tube dilution method of Finegold and Martin (10). All antibiotics were obtained from Sigma. Stock solutions of oxytetracycline hydrochloride (204.8 ,ug/ml), polymyxin B sulfate (2,000 ,ug/ml), and novobiocin sodium salt (204.8 ,ug/ml) were prepared in MHB, filter sterilized (0.22-ptm Acrodisc disposable filter assemblies; Gelman Sciences, Inc., Ann Arbor, Mich.), and stored at 4 or -20°C for no longer than 7 days. Serial twofold dilutions of antibiotic solutions were performed in screw-cap culture tubes (13 by 100 mm, Pyrex) with MHB as the diluent. Each tube was inoculated with 1.0 ml of an exponential-phase MHB cell suspension containing 5 x 105 CFU and incubated at 37°C and 180 rpm for 24 h. After three to five independent replicate dilutions with appropriate controls for each isolate were performed, the lowest antibiotic concentration resulting in complete growth inhibition was designated the MIC.
HYDROPHOBICITY OF P. MULTOCIDA AND A. LIGNIERESII
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RESULTS Generation times in BHI. Growth rates were determined for each strain by estimating the time required for cell densities to double during exponential growth under standard experimental conditions in BHI (Table 1). The diversity of growth rates observed for P. multocida somatic serotype strains (32 to 67 min) is clearly in contrast with the uniformity exhibited by A. lignieresii strains (40 to 42 min) and precludes the use of this parameter as a differential criterion for these organisms. Cell surface hydrophobicity. The hydrocarbon adherence method (32) was employed to determine relative cell surface hydrophobicity values by assessing the affinities of strains for n-hexadecane in an aqueous-hydrocarbon biphasic system. Results obtained for the P. multocida somatic serotype reference strains are depicted as plots of aqueous-phase turbidity remaining as a function of hydrocarbon volume in Fig. 1. These data clearly indicate that surface hydrophobicity is a highly variable property among P. multocida strains possessing serologically distinguishable LPS. The wide diversity of values obtained with the hydrocarbon adherence method was substantiated by comparison with data obtained with octyl-Sepharose hydrophobic interaction chromatography (33) (Table 1). The hydrocarbon adherence method also revealed marked differences in surface hydrophobicity among three A. lignieresii strains and no measurable hydrophobicity for P. aeruginosa PAO1 (Fig. 2). However, a comparison of hydrocarbon adherence and hydrophobic interaction chromatography data (Table 1) reveals a lack of agreement between
0.2 0.8 1.0 Hexadcane (ml)
FIG. 1. Adherence of P. multocida somatic serotype reference strains to n-hexadecane. Each value represents the percentage of cells remaining in aqueous suspension after mixing with the hydrocarbon volume indicated and is the mean of three to four independent determinations. (A) Symbols: *, X-73 (serotype 1); *, P-1059 (serotype 3); M, P-1662 (serotype 4); A, P-1702 (serotype 5). (B) Symbols: *, P-2192 (serotype 6); P-1997 (serotype 7); a, P-1581 (serotype 8); A, P-2095 (serotype 9). (C) Symbols: *, P-2100 (serotype 10); *, P-903 (serotype 11); M, P-1573 (serotype 12); A, P-1591 (serotype 13). (D) Symbols: *, P-2225 (serotype 14); , P-2237 (serotype 15); M, P-2723 (serotype 16). *,
Hexadecane (mi) FIG. 2. Adherence of A. Iignieresii and P. aeruginosa reference strains to n-hexadecane. Each value represents the percentage of cells remaining in aqueous suspension after mixing with the hydrocarbon volume indicated and is the mean of three to four independent determinations. Symbols: *, A. lignieresii ATCC 19393; M, A. lignieresii ATCC 13372; A, A. lignieresii ATCC 13369; and *, P. aeruginosa PAO1.
the methods for these strains. In view of the amphiphilic nature of octyl-Sepharose (33), control experiments were performed by eluting standardized cell suspensions through columns containing Sepharose CL-4B lacking the nonpolar octyl ligand. All four strains were found to adhere significantly in the absence of the hydrophobic octyl group (data not shown). This is supportive of the fact that A. lignieresii ATCC 13372, A. lignieresii ATCC 13369, and P. aeruginosa PAO1 are in fact relatively hydrophilic as indicated by hydrocarbon adherence data in Fig. 2. The hydrophobicity indicated by the hydrophobic interaction chromatography data for these strains (Table 1) most likely reflects adsorption to the polar Sepharose gel matrix. Susceptibility to antibiotics. The results of antibiotic susceptibility testing by the macro tube dilution method (10) are shown in Table 2. All P. multocida somatic reference strains were uniformly susceptible to oxytetracycline, with MICs of 0.80 to 6.40 ,ug/ml, to polymyxin B with MICs of 0.48 to 7.81 ,ug/ml, and to novobiocin with MICs of 0.80 to 6.40 ,ug/ml. A. lignieresii ATCC 13372 was also susceptible to oxytetracycline and polymyxin B (MICs of 1.60 and 0.48 ,utg/ml, respectively) but less susceptible to novobiocin (25.60 ptg/ml). It was not possible to obtain MICs for A. lignieresii ATCC 19393 and ATCC 13369 due to an inability to obtain uniformly turbid suspensions during growth in MHB. These results are consistent with previous studies that have established the in vitro and in vivo susceptibility of P. multocida to these compounds (1, 8, 9, 12, 36). P. aeruginosa was included for reference purposes and was found to be susceptible to oxytetracycline (12.80 ,ug/ml) and polymyxin B (1.95 ,ug/ml) but resistant to novobiocin
(>102.4 ,ug/ml). DISCUSSION One important functional role of the gram-negative outer membrane is thought to be that of a permeability barrier to potentially harmful molecules. It has been shown to be particularly impermeable to hydrophobic compounds in smooth Enterobacteriaceae having LPS with intact O side chains (18, 25). The observations that P. multocida pos-
J. CLIN. MICROBIOL.
DARNELL ET AL.
TABLE 2. Oxytetracycline, polymyxin B, and novobiocin MIC
values for P. multocida, A. lignieresii, and P. aeruginosa reference strains MIC (p.g/ml)Y
P. multocida X-73 (1)b P-1059 (3) P-1662 (4) P-1702 (5) P-2192 (6) P-1997 (7) P-1581 (8) P-2095 (9) P-2100(10) P-903 (11) P-1573 (12) P-1591 (13) P-2225 (14) P-2237 (15) P-2723 (16)
1.60 1.60 3.20 1.60 1.60 3.20 0.80 1.60 1.60 1.60 6.40 6.40 1.60 1.60 1.60
7.81 1.95 7.81 7.81 0.98 0.48 3.91 1.95 1.95 0.48 1.95 1.95 0.98 1.95 1.95
3.20 3.20 1.60 1.60 1.60 1.60 1.60 3.20 6.40 6.40 6.40 0.80 6.40 3.20 3.20
A. lignieresii ATCC 19393c ATCC 13372 ATCC 13369C
P. aeruginosa PAO1
Each value was obtained from three to five replicate twofold serial dilutions with a macro-tube dilution assay. b p. multocida somatic serotype designations are included in parentheses. c Failure of these strains to grow as uniformly turbid suspensions in MHB resulted in heavy dumping, thereby precluding valid MIC determinations. a
sesses a typically gram-negative cell envelope ultrastructure (6, 30) with atypical permeability properties manifested in the form of susceptibility to bile salts and crystal violet (4, 13, 26) and penicillin (22) present an intriguing contradiction. Although P. multocida LPS resembles that of smooth enteric bacteria in that they have major chemical components in common (24, 31), it is also similar to rough enteric LPS with regard to having lower molecular weight (20) and, in most cases, greater hydrophobicity (31). Increased outer membrane permeability in P. multocida may be due to the LPS being structurally similar to that of rough enteric mutants (31). These bacteria are known to be more hydrophobic than smooth wild-type strains (21, 32) and therefore more susceptible to hydrophobic compounds (25). The overall aim of the present study was to investigate the relationship between somatic serotype specificity (i.e., LPS structure) and other surface parameters such as cell surface hydrophobicity and cell envelope permeability for antibiotics of different polarities. Three strains of A. lignieresii were examined to further study the genotypic relatedness of the genera Pasteurella and Actinobacillus. P. aeruginosa was included for reference purposes since it has been shown to be extremely hydrophilic (32) and is known to exhibit intrinsic resistance to many antibiotics due to outer membrane impermeability. The cell surface hydrophobicity properties of strains representing 15 of the 16 known somatic serotypes of P. multocida and three strains of A. Iignieresii were assessed with hydrocarbon adherence and hydrophobic interaction chromatographic assays. These methods revealed surface hydrophobicity to vary dramatically among strains in both species. P. aeruginosa exhibited no measurable surface
hydrophobicity in a manner consistent with the previous report (32). P. multocida and A. lignieresii are important veterinary and human pathogens which represent the type species of two closely related genera within the family Pasteurellaceae. Routine differentiation of these organisms with conventional biochemical parameters has proven extremely difficult (7, 11, 15, 26, 27, 29), resulting in inefficient clinical diagnostic procedures (11, 26, 29). A considerable body of evidence exists questioning their genetic segregation (28, 35). The wide diversity of hydrophobic properties exhibited by both P. multocida somatic serotype and A. lignieresii reference strains precludes the use of this parameter as a means for differentiation, but is supportive of their taxonomic relatedness. The P. multocida somatic serotype strains exhibited diverse growth rates, whereas the A. lignieresii reference strains grew at essentially identical rates, all independent of relative surface hydrophobicity. This suggests that factors contributing to overall surface nonpolarity in P. multocida do not directly affect the rates by which nutrients permeate the cell envelope. This conclusion is supported by the uniformity of MICs for oxytetracycline, polymyxin B, and novobiocin among P. multocida strains differing in overall surface hydrophobicity, since antibiotic susceptibility can be used as a measure of cell envelope permeability (18, 25). The susceptibility of P. multocida strains to novobiocin, a hydrophobic antibiotic, further substantiates the conclusion that this organism possesses a cell envelope with atypical permeability properties. It should be noted that A. lignieresii, which grows in the presence of bile salts and crystal violet (22), appears to be significantly less susceptible to the hydrophobic novobiocin. Based on these data, we conclude that whereas cell surface hydrophobicity in P. multocida is not directly related to either growth rate or permeability for the antibiotics examined, it may be influenced by LPS composition. If so, other surface macromolecules such as proteins or phospholipids may also be involved. This appears likely since serotype strains 3, 9, and 13, which Rimler et al. (31) found to possess relatively hydrophilic LPS, exhibited disparate overall surface hydrophobicity properties in the present study. ACKNOWLEDGMENTS This study was supported by a research grant to F.R.C. from Pfizer International, Inc. We gratefully acknowledge R. B. Rimler for kindly providing P. multocida somatic serotype strains and helpful suggestions, J. Ainsworth for providing Sepharose CL-4B-200, and D. N. Downer for critically reviewing the manuscript. LITERATURE CITED 1. Bierer, B. W. 1962. Treatment of avian pasteurellosis with injectable antibiotics. J. Am. Vet. Med. Assoc. 141:1344-1346. 2. Blackburn, B. O., K. L. Heddleston, and C. J. Pfow. 1975. Pasteurella multocida serotyping results (1971-1973). Avian Dis. 19:353-356. 3. Branson, D., and F. Bunkfeldt, Jr. 1967. Pasteurella multocida in animal bites of humans. Am. J. Clin. Pathol. 48:552-555. 4. Brogden, K. A. 1980. Physiological and serological characteristics of 48 Pasteurella multocida cultures from rabbits. J. Clin.
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