Alexandria, Va.). S. minnesota Rc LPS (List Biological ..... Catherine L. Hall for advice regarding fatty acid analysis. This work was supported by U.S. Public ...
Vol. 27, No. 4
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1989, p. 762-767
0095-1137/89/040762-06$02.00/0 copyright © 19,89, American Society for Microbiology
Comparison of Lipopolysaccharides from Brazilian Purpuric Fever Isolates and Conjunctivitis Isolates of Haemophilus influenza Biogroup Aegyptius STUDY GROUPt Departments of Microbiologyl and Internal Medicine,2 University of Texas Southwestern Medical Center, Dallas, Texas 75235
ALICE L. ERWIN,' ROBERT S. MUNFORDl.2*
AND THE BRAZILIAN PURPURIC FEVER
Received 16 August 1988/Accepted 8 December 1988
Haemophilus influenza biogroup aegyptius (H. aegyptius) has been identified as the etiologic agent of the recently described. disease Brazilian purpuric fever (BPF). Although there is heterogeneity among the strains associated with conjunctivitis, isolates from patients with BPF appear to be derived from a single clone. The çlinical presentation of BPF suggests that bacterial lipopolysaccharides (LPS) are involved in its pathogenesis. We prepared LPS frgm H. inflqenzae biogroup aegyptius and found them to be similar to H. influenzae type b LPS in apparent size (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), biological activities, and f¶Itty acid composition. We compared LPS from BPF clone isolates with LPS from non-BPF clone isolates in tests of Limulus lysate activation, spleen cell mitogenesis, promotion of neutrophil adherence to LPS-treated endot1elial cells, and thç dermal Shwartzman reaction. In none of these activities were LPS from the BPF clone isolates more potent. BRcause LPS shed from growing bacteria may be involved in the pathogenesis of purpura, we also Measured the rate at which LPS were released into culture medium during bacterial growth and found no significant difference between BPF clone and non-BPF clone isolates. sess LPS that are more bioactive than LPS from non-BPF clone isolates and (ii) that BPF clone isolates release more LPS as they grow in vitro.
Haemophilus influenzae biogroup aegyptius (H. aegyptius), first described in 1883 by Robert Koch, is the etiologic agent of the epidemic purulent conjunctivitis described by Weeks (reviewed in reference 21). Association of this organism with systemic disease was not demonstrated before 1986, when' H. influenza biogroup aegyptius was cultured from the blood of several patients wit4 the recently described disease Brazilian purpuric fever (BPF) (5, 6). Comparative studies of BPF-associated isolates and strains from epidemiologically unrelated cases of conjunctivitis established that the BPF isolates have distinctive features and may be derived froM a single clone (7, 8). The clinical manifestations of BPF suggest that bacterial lipopolys4ccharties (LPS; endotoxins) may be involved in the pathogenesis of the disease. The distinctive clinical feature of BPF has been cutaneous hemorrhage, or purpura, similar to that seen with fulminant meningococcemia (5). LPS, which can produce dermal necrosis and hemorrhage in experimental animals in the dermal Shwartzman reaction (23), are obvious candidates for provoking cutaneous purpura in meningococcemia and BPF. In the experiments reported here, we examined the hypotheses (i) that isolates of the BPF clone of H. influenza biogroup aegyptius pos-
MATÉRIALS AND METHODS Bacterial strains and cultivation of bacteria. H. influenzae biogroup aegyptius isolates F1946, F3028, F3029, F3031, and F3037 were recovered from patients with BPF; all have the case clone phenotype (7). Isolates F3043, F3052, F3055, and F3065 were obtained from epidemiologically unrelated cases of conjunctivitis; none of these isolates has the case clone phenotype.- With the exception of F3065 (NCTC 8502), which was isolated in Texas (M. Pittman), all of the strains were isolated in Brazil. H. influenzae type b, strain DL42, was obtained from E. J. Hansen, Dallas, Tex. The strains were passed once on receipt and were stored at -70°C as suspensions in skim milk. Bacteria were grown at 37°C in brain heart infusion (Difco Laboratories, Detroit, Mich.) supplemented with 10 p.g of NAD per ml and 10 jxg of hemin per ml. In some cases, the broth was further supplemented with 0.1 mCi of [3H]acetate (Dupont, NEN Research Products, Boston, Mass.) per ml for labeling of LPS fatty acids. For assay of LPS release, bacteria were grown in medium made in pyrogen-free water and sterilized by filtration; the glassware was heated at 180°C for at least 4 h. Preparation of LPS. LPS from H. influenza type b were prepared by hot phenol-water extraction following digestion with lysozyme and nucleases, as described by Johnson and Perry (14). LPS from H. influenza biogroup aegyptius were recovered from the phenol phase after extraction of 68°C with 45% phenol, pH 7.0 (14). The phenol was removed by dialysis against deionized water; a brown fibrous material which formed during dialysis was discarded. LPS were precipitated from the remaining liquid by addition of 5 mg of sodium acetate per ml and 2 volumes of acetone. All LPS preparations were extracted several times with ether to remove residual phospholipids. Phenol-chloroform-petroleum ether extraction of H. influ-
* Corresponding author. t The Brazilian Purpuric Fever Study Group includes Gloria Ajello, Robert J. Arko, William Bibb, Kristin Birkness, Donald J. Brenner, Claire V. Broome, George M. Carlone, Robert C. Cooksey, Linda Gheesling, Lee Harrison, Leonard W. Mayer, Roger M. McKinney, Steven P. O'Connor, Michaél W. Reeves, Frances O. Sottnek, Arnold G. Steigerwalt, Balasubramanian Swaminathan, Jana Swenson, and Robbin Weyant; Centers for Disease Control, Atlanta, GA 30333; Carmo Elias Andrade Melles, Maria Cristina de Cunto Brandileone, Kinue Irino, Mituka Kaku, Maria Lucia Cecconi Tondella, and Eliseu Alves Waldman, Instituto Adolfo Lutz, Saq Paulo, S.P., Brazil; Ligia Regina Kerr Pontes, Escritorio Regional De Saude, Ribeirao Preto, S.P., Brazil; and David W. Pleming, State Health Division, Department of Human Resources, Portland, OR 97201.
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VOL. 27, 1989
enzae biogroup aegyptius was as described by Galanos et al. (12), except that following the extraction, LPS were precipitated by addition of 2 volumes of ethanol rather than by dropwise addition of water. LPS from Salmonella minnesota Rc, used in the dermal Shwartzman reaction, were prepared by the method of Galanos et al. (12). SDS-PAGE. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), LPS preparations (1 ,ug per lane) were electrophoresed through a 10 to 15% linear gradient of acrylamide in the system described by Kimura and Hansen (15) and silver stained by the method of Tsai and Frasch (24). Analysis of fatty acids. LPS were hydrolyzed with 4 N HCl at 100°C for 90 min, and total fatty acids were extracted into chloroform as previously described (13). For mild alkaline hydrolysis, LPS were incubated in 1 N NaOH for 15 h at room temperature. Hydrolysis by neutrophil acyloxyacyl hydrolase was as previously described (17). For separation of nonhydroxylated and hydroxylated fatty acids, samples were analyzed by one-dimensional thin-layer chromatography (TLC) as previously described (13). The locations of the resolved fatty acids were identified by fluorography; sections of silica gel were scraped from the plates, scintillation cocktail was added (Safety-Solve; Research Products International, Mount Prospect, Ill.), and the associated radioactivity was counted with a model 2425 scintillation counter (Packard Instrument Co., Inc., Rockville, Md.). For identification and quantitation of individual fatty acids, phenacyl ester derivatives of the LPS fatty acids were prepared (11; p-bromophenacyl ester derivatization kit; Alltech Associates, Inc., Applied Science Div., State College, Pa.). The derivatization products were separated from unreacted fatty acids with a Sep-Pak C18 cartridge (Waters Associates, Inc., Milford, Mass.) and analyzed by reversephase high-pressure liquid chromatography (HPLC) with a C8 column (Ultrasphere Octyl; Beckman Instruments, Inc., Fullerton, Calif.); the mobile phase was 89% methanol-11% water. Fatty acids were identified and quantitated by comparison of retention times and peak areas with those of fatty acid standards. Heptadecanoic acid added to all samples before derivatization served as an internal standard. Fractions were collected from the HPLC column, and their 3H contents were counted as described above. Assays of LPS activity. (i) Spleen cell mitogenicity. Spleen cells from C3H/HeN or C3H/HeJ mice were incubated with various concentrations of LPS; after 24 h, [3H]thymidine was added. Eighteen hours later, the cells were harvested and the extent of 3H incorporation was determined (17). (ài) Adherence of leukocytes to LPS-treated endothelial cells. The adherence of leukocytes to LPS-treated endothelial cells was determined as previously described (22), except that cultured HL60 cells, a human promyelocyte cell line, were substituted for peripheral blood neutrophils (4). Briefly, cultured human umbilical vein endothelial cells were incubated for 4 h with LPS; the cells were washed, and 51Crlabeled HL60 cells were added. After 30 min, the endothelial cells were again washed; the cells remaining in each well were lysed with 1 N NH40H, and the radioactivity in the lysates was counted (Gamma 4000; Beckman). (iii) Dermal Shwartzman reaction. The experimental method for the dermal Shwartzman reaction was similar to that described by Nowotny (20). New Zealand White rabbits weighing 2 to 3 kg were obtained from Hickory Hill, Flint, Tex., or Myrtle's Rabbitry, Thompson Station, Tenn., and maintained in our institutional animal facility. On day 1 of the experiment, the backs of the animals were shaved; 5 or
LPS FROM H. INFLUENZAE BIOGROUP AEGYPTIUS
763
10 ,ug of LPS suspended in 0.15 ml of pyrogen-free 0.9% NaCi was injected intradermally at several sites. Eighteen to twenty hours later, the rabbits were injected intravenously with 4 ,ug of S. minnesota Rc LPS per kg suspended in 1 ml of pyrogen-free 0.9% NaCI. Immediately following the intravenous injection of LPS, 5 ml of pyrogen-free 0.9% NaCI containing 1% glucose was infused intravenously. Lesions developed over 4 to 6 h following the provocative injection; the diameter of the indurated area at each injection site was measured at 6 to 7 h. The test preparations were coded before injection; the resulting lesions were measured by three observers before the code was broken. To facilitate measurement, residual hair was removed from the backs of the rabbits with a chemical depilatory (Nair; Carter Products, New York, N.Y.). Limulus assay. The activities of purified LPS or bacterial culture supernatants were measured in a chromogenic Limulus assay. Limulus amebocyte lysate (Pyrotell; Associates of Cape Code, Inc., Woods Hole, Mass.) was reconstituted in 8 ml of water. Four milligrams of the chromogenic substrate N-benzoyl-L-vaiylglycyl-L-arginine p-nitroanilide hydrochloride (Vega Biotechnologies, Inc., Tucson, Ariz.) was mixed with 3 ml of lysate, and 4 ml of H20 was added. LPS-containing samples were diluted in water; 50-tIi portions were added to wells (Linbro/Titertek EIA microtitration plates; Flow Laboratories, Inc., McLean, Va.) containing 100 ,ul of the lysate-chromogen mixture. The plates and all solutions were kept on ice to this point. The plates were then incubated at 37°C to allow color development; optical densities (ODs) at 410 nm were measured at 60 min with a microplate reader (MR 700; Dynatech Laboratories, Inc., Alexandria, Va.). S. minnesota Rc LPS (List Biological Laboratories, Campbell, Calif.) was used as the standard. For measurement of the Limulus activity released into the medium, bacteria grown on plates were washed twice with brain heart infusion and then suspended in supplemented brain heart infusion in polystyrene tubes (16 by 125 mm) to an OD at 540 nm of 0.05 to 0.15. The tubes were placed at an angle to maximize the broth surface area and incubated at 37°C without agitation (3) for 8 h. Before samples were taken, the cultures were mixed by five end-over-end rotations of the tubes and the ODs were determined. Samples were filtered slowly through a 0.45-jxm-pore-size filter (Millex-HA; Millipore Corp., Bedford, Mass.) and stored at 4°C until assayed (within 48 h). In a preliminary experiment, we found less than 6% variation in the Limulus activities measured in eight replicate culture filtrates. The ratio of the final OD to the initial OD was used to determine the number of times the bacterial mass doubled during incubation (16): number of doublings = In (final OD/initial OD)/ln 2. The change in LPS concentration in culture filtrates was divided by the number of doublings to determine the amount of LPS released per milliliter per generation during the 8 h of incubation. Preliminary experiments determined that under these culture conditions, the logarithmic phase of growth was longer than 8 h. Statistics. For the LPS release assay, the data were analyzed by using the Mann-Whitney test (25). RESULTS Preparation of LPS from H. aegyptius. Unlike the LPS of
H. influenza type b, the LPS of H. influenza biogroup aegyptius partitioned primarily into the phenol phase following hot phenol-water extraction. The aqueous phase contained material that was precipitable with ethanol; this
764
ERWIN ET AL.
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J. CLIN. MICROBIOL.
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FIG. 1. SDS-PAGE of LPS preparations. Lanes: 1 to 6, H. influenza biogroup aegyptius (1 and 3, F3065 [non-BPF clone]; 2 and 4, F3031 [BPF clone]; 5, F3029 [BPF clone]; 6, F3052 [non-BPF clone]); 7, H. influenza type b DL42; 8, E. coli J5; 9, N. meningitidis 8693; 10 and 11, S. minnesota Ra and Rc (List Biological Laboratories), respectively. H. influenza biogroup aegyptius LPS in lanes 1 and 2 were prepared by a modification of the method of Galanos et al. (12), and LPS in lanes 3 to 6 were recovered from the phenol phase following hot phenol-water extraction. Only the rele-
a
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vant portion of the gel is shown.
precipitate contained nucleic acid, as indicated by a UV absorption peak at 260 nm. After nuclease digestion, no further material could be recovered from this fraction. In contrast, material precipitated from the phenol phase had no absorption maxima between 230 and 300 nm. SDS-PAGE analysis of this material showed it to be similar in apparent size to the LPS of H. influenza type b, migrating as one or two bands with mobilities characteristic of rough LPS (Fig. 1). The material recovered from the phenol phase possessed Limulus activity and was mitogenic for spleen cells of C3H/HeN (LPS-responsive) mice; the activity toward spleen cells of C3H/HeJ (LPS-hyporesponsive) mice was less by 2.5 to 3 orders of magnitude (Fig. 2). On the basis of these observations and the fatty acid analysis (see below), we concluded that this material was LPS. A similar material was prepared from two H. influenza biogroup aegyptius isolates by a modification of the extraction procedure of Galanos et al. When analyzed by SDSPAGE, LPS prepared by the latter method appeared to be a subset of the phenol-water-extracted LPS (Fig. 1). Only phenol-water-extracted LPS were used in subsequent experiments. Fatty acid analysis. We determined the fatty acid composition of LPS from two isolates of H. influenza biogroup aegyptius and, for comparison, one strain of H. influenza type b (Table 1; Fig. 3). For these studies, LPS were prepared from bacteria grown in medium containing [3H]acetate. Acid hydrolysis of the labeled LPS, followed by chloroform-methanol extraction, showed that virtually all of the 3H was in the fatty acids. HPLC analysis showed that each LPS preparation contained only two species of fatty acid: 3-OH myristate and myristate (Fig. 3). TLC of fatty acids released by mild alkaline hydrolysis and subsequent acid hydrolysis indicated that, in each case, all of the myristate and about half of the 3-OH myristate residues were attached by relatively labile bonds; the remaining 3-OH myristate was removed by acid hydrolysis (Table 1). Treatment with acyloxyacyl hydrolase released only the myristate residues (Table 1). These data suggest that LPS of H. influenza biogroup aegyptius, as well as LPS of H. influenzae type b, have fatty acyl linkages that resemble those in lipid A of Salmonella spp. and Escherichia coli: 3-OH myristate residues are attached to the glucosamine backbone by both ester and amide linkages, and some of the hydroxyl groups on these acyl chains are substituted by ester-linked, nonhydroxylated fatty acids.
.0005
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[LPS], /Lg/mt FIG. 2. Incorporation of [3H]thymidine by spleen cells treated with LPS. For each point, the stimulation index was calculated by dividing 3H incorporated into LPS-treated cells by 3H incorporated into untreated cells. Each point represents the mean of four replicate samples. -, Spleen cells from C3H/HeN (LPS-responsive) mice; - -, spleen cells from C3H/HeJ (LPS-hyporesponsive) mice. The symbols indicate the bacterial isolate from which the LPS was prepared: E, F3029 (BPF clone); O, F3031 (BPF clone); A, F3052
(non-BPF clone); *, F3065 (non-BPF clone); V, H. influenza type b DL42.
Biological activities of LPS. Experiments on the biological activities of LPS were performed with LPS from two isolates of the BPF clone of H. influenza biogroup aegyptius (F3029 and F3031), two non-BPF clone strains (F3052 and F3065), and H. influenzae type b (DL42). (i) Spleen cell mitogenesis. As noted above, all LPS were mitogenic for spleen cells from C3H/HeN mice (Fig. 2). The activities of LPS from the different strains were similar; LPS from one BPF clone isolate, F3031, was slightly more active. (ii) Adherence of leukocytes to LPS-treated endothelial cells. This assay reflects the ability of vascular endothelium to respond to injury by promoting leukocyte adherence. All of the LPS had similar activities, although LPS from one non-BPF clone isolate, F3052, was more active than the others (Fig. 4). (iii) Dermal Shwartzman reaction. We compared the abilities of H. influenza biogroup aegyptius LPS to prepare the skin of rabbits for the dermal Shwartzman reaction. Each LPS preparation was tested at two concentrations in two or more rabbits; each injection was in duplicate. Differences among the LPS were small, and LPS from the BPF clone isolates were not more potent than LPS from control strains (Fig. 5). Release of LPS into culture medium. To establish that LPS from various isolates of H. influenza biogroup aegyptius could be quantitated with a single LPS as the standard, we determined in preliminary experiments (data not shown) that purified LPS from four isolates were equally reactive in the Limulus assay. Previous observations in this laboratory (18) and those of Anderson and Solberg (2) showed that release of LPS begins slightly after the start of the logarithmic phase and ceases shortly after entry into the stationary phase. We determined the amount of Limulus reactivity in culture
LPS FROM H. INFLUENZAE BIOGROUP AEGYPTIUS
VOL. 27, 1989
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TABLE 1. TLC analysis of LPS fatty acidsa % of LPS 3H recovered following complete or partial hydrolysis Hydrolysis
F3031
Acid Alkaline
F3065
DL42
OH-FAb
NFA
OH-FA
NFA
OH-FA
NFA
63.7 28.9 (21.6) 2.6 (57.9)
36.3 43.8 (5.6) 30.1 (9.4)
67.2 29.1 (28.3) 1.0 (62.3)
32.8 39.3 (3.3) 22.5 (14.2)
67.6 23.3 (28.9) 0.7 (73.0)
32.4 41.2 (6.6) 19.9 (6.4)
Neutrophil acyloxyacyl hydrolase a After the indicated hydrolysis, the fatty acids released
were extracted into chloroform and separated into hydroxylated fatty acids (OH-FA) and nonhydroxylated fatty acids (NFA) by using TLC. Acid hydrolysis released 100% of LPS 3H into a chloroform-extractable form. Following partial hydrolysis (alkaline or neutrophil acyloxyacyl hydrolase treatment) and chloroform extraction, the fatty acids remaining on the LPS were released by acid hydrolysis, extracted into chloroform, and separated on TLC. For each LPS, the data are percentages of total recovered 3H that separated as OH-FA or NFA on TLC. For alkaline or enzymatic hydrolysis, the percent released by the partial hydrolysis is given first; the percent released by subsequent acid hydrolysis is given in parentheses. F3031, BPF clone isolate of H. influenzae biogroup aegyptius; F3065, non-BPF clone isolate of H. influenza biogroup aegyptius; DL42, H. influenza type b. b Radioactive spots comigrating with unlabeled fatty acid standards (3-OH myristate and oleate) were scraped from the plate, and the 3H was counted. The LPS fatty acids were identified by HPLC analysis (Fig. 3) as 3-OH myristate and myristate. A small proportion of 3-OH myristate undergoes an elimination reaction during alkaline hydrolysis; the unsaturated product of this reaction comigrates with NFA in this TLC system.
filtrates of nine isolates of H. influenza biogroup aegyptius at the initiation of culture and in the late logarithmic phase, after 8 h of incubation. Because the isolates varied in growth rate, we calculated the amount of Limulus reactivity released into the culture medium per generation during the first 8 h of culture (Table 2). There was substantial variation
A.
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16:1
16:0
17:0
among the isolates; although there was a slight tendency for isolates of the BPF clone to produce more LPS than control isolates, there was considerable overlap between the two groups and the differences between the groups were not statistically significant.
DISCUSSION The pathogenesis of bacterial purpura is uncertain, although there is evidence that LPS may be involved. It was shown in the 1930s that hemorrhagic skin lesions similar to those seen in fulminant sepsis may be induced experimentally in rabbits by sequential injections of culture filtrates
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25 30 35 20 40 Retention Time, min. FIG. 3. HPLC of LPS fatty acids. (A) Fatty acid standards (120 pmol each): 3-OH C14:0, 3-OH-myristate; C12:0, laurate; C14:0, myristate; CI6,:, palmitoleate; Cl6,0, palmitate; C17:0, heptadecanoic acid. (B) Fatty acids from H. influenza biogroup aegyptius BPF clone isolate F3031 LPS (heptadecanoic acid added). The profiles for LPS fatty acids from isolate F3065 and H. influenza type b DL42 were very similar. (C) 3H recovered from the column, in the analysis shown in panel B. Fractions were collected at 0.5-min intervals (1-min intervals after elution of myristate), the solvent was allowed to evaporate, and the 3H was counted. Of the 3H recovered (88% of injected 3H), 63.9% comigrated with 3-OH myristate, 33.1% comigrated with myristate, and 3.0% comigrated with palmitate. 10
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[LPS] ,ng/ml FIG. 4. Adherence of HL60 cells to LPS-treated endothelial cells. Human umbilical vein endothelial cells were incubated with various concentrations of LPS; after removal of the LPS, 51Crlabeled HL60 cells were added. After removal of unbound HL60 cells, the cells remaining in the wells were lysed and a sample of the lysate was counted. Each point represents the mean of four replicate wells +±1 standard deviation. The symbols indicate the bacterial isolates from which the LPS were prepared: O, F3029 (BPF clone); O, F3031 (BPF clone); A, F3052 (non-BPF clone); *, F3065 (non-BPF clone); V, H. influenza type b DL42.
J. CLIN. MICROBIOL.
ERWIN ET AL.
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10MFg
5 E
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64,000 5,500 +3,000 9,600 +4,000 a The concentrationiof LPS in culture filtrates was determined by Limulus assay; bacterial growth h was monitored by measurements of OD at 540 nm (see Materials and Methodis). Initial LPS concentrations (under 50 ng/ml) were subtracted from conccentrations at 8 h to determine the LPS released. Calculation of LPS relLeased per generation was as described in Materials and Methods. The results are means ±1 standard deviation of three replicate .....................................
.....................................
.....................................
.....................................
cultures.
We compared nine isolates of H. influenzae biogroup using conditions similar to those in which Andersen and Solberg had distinguished endotoxin-liberating and nonliberating strains of N. meningitidis (2, 3). LPS was measured with the Limulus assay, which measures a bioactivity of the LPS and not necessarily the amount of LPS present. However, previous studies have established that Limulus results correlate reasonably well with the results of a radioimmunoassay for LPS, both in vitro and in vivo (18, 19). In preliminary experiments, we also found that purified LPS from two BPF clone and two non-BPF clone isolates of H. clone significantly and two didnonnot differ sifatly in in from tw oBFone biogroup aegyptius influenza potency in the Limulus test. We saw substantial variation aegyptius as to the release of LPS into culture medium by
VOL. 27, 1989 among nine isolates of H. influenza biogroup aegyptius in the amount of Limulus-reactive material released during growth. There was some tendency for the BPF clone isolates to release more LPS, but this difference was not statistically significant. The results of our experiments suggest that bacterial purpura may result from bacterium-host interactions that are not evident from a study of the purified LPS. The physical state of the purified LPS used in this study is probably quite different from that of the native LPS which occurs as part of a bacterial membrane. In addition, the LPS of bacteria grown in culture might differ from the LPS of bacteria grown in vivo. For example, Kimura and Hansen have reported that for a weakly virulent strain of H. influenza type b, bacteria recovered from experimentally infected infant rats possessed increased virulence; the increased virulence was associated with antigenic and structural alteration of the LPS (15). Although we were not able to demonstrate increased potency of LPS from isolates of the BPF clone, it is possible that such an increase occurs only in vivo. Indeed, passage of strains through animals increases the virulence of H. influenzae biogroup aegyptius for infant rats (8). Further use of animal-passed strains and animal and cell culture infection models may further understanding of the role of LPS and other potential virulence factors. It is possible, for instance, that these bacteria adhere to the vascular endothelium, perhaps even invading the endothelial cells, presenting an increased opportunity for stimulation of these cells by LPS. Finally, isolates of the BPF clone of H. influenza biogroup aegyptius may differ from non-BPF clone isolates in factors unrelated to LPS which allow invasion of the bloodstream and evasion of host defenses. Some of these factors may be host specific; others may be amenable to study in animals.
ACKNOWLEDGMENTS We thank Ahmed Adu-Oppong for technical assistance and Catherine L. Hall for advice regarding fatty acid analysis. This work was supported by U.S. Public Health Service grants AI 18188 and HD 22766 from the National Institutes of Health. LITERATURE CITED 1. Andersen, B. M., and O. Solberg. 1984. The virulence in mice of Neisseria meningitidis variants differing in free endotoxin activities and cell envelope properties. NIPH (Natl. Inst. Public Health) Ann. 7:47-59. 2. Andersen, B. M., and O. Solberg. 1988. Endotoxin liberation associated with growth, encapsulation and virulence of Neisseria meningitidis. Scand. J. Infect. Dis. 20:21-31. 3. Andersen, B. M., O. Solberg, K. Bryn, L. O. Froholm, P. Gaustad, E. A. Hoiby, B.-E. Kristiansen, and K. Bovre. 1987. Endotoxin liberation from Neisseria meningitidis isolated from carriers and clinical cases. Scand. J. Infect. Dis. 19:409-419. 4. Bevilacqua, M. P., J. S. Pober, M. E. Wheeler, R. S. Cotran, and M. A. Gimbrone. 1985. Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines. J. Clin. Invest. 76:2003-2011. 5. Brazilian Purpuric Fever Study Group. 1987. Brazilian purpuric fever: epidemic purpura fulminans associated with antecedent purulent conjunctivitis. Lancet ii:757-761. 6. Brazilian Purpuric Fever Study Group. 1987. Haemophilus aegyptius bacteremia in Brazilian purpuric fever. Lancet ii:761-763. 7. Brenner, D. J., L. W. Mayer, G. M. Carlone, L. H. Harrison, W. F. Bibb, M. C. de Canto Brandileone, F. O. Sottnek, K. Irino, M. W. Reeves, J. M. Swenson, K. A. Birkness, R. S. Weyant,
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