JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1989, p. 762-7670095-1137/89/040762-06$02.00/0copyright © 19,89, American Society for Microbiology

Comparison of Lipopolysaccharides from Brazilian Purpuric FeverIsolates and Conjunctivitis Isolates of Haemophilus influenza

Biogroup AegyptiusALICE L. ERWIN,' ROBERT S. MUNFORDl.2* AND THE BRAZILIAN PURPURIC FEVER STUDY GROUPt

Departments of Microbiologyl and Internal Medicine,2 University of Texas Southwestern Medical Center,Dallas, Texas 75235

Received 16 August 1988/Accepted 8 December 1988

Haemophilus influenza biogroup aegyptius (H. aegyptius) has been identified as the etiologic agent of therecently described. disease Brazilian purpuric fever (BPF). Although there is heterogeneity among the strainsassociated with conjunctivitis, isolates from patients with BPF appear to be derived from a single clone. Theçlinical 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 typeb LPS in apparent size (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), biological activities, andf¶Itty acid composition. We compared LPS from BPF clone isolates with LPS from non-BPF clone isolates intests of Limulus lysate activation, spleen cell mitogenesis, promotion of neutrophil adherence to LPS-treatedendot1elial cells, and thç dermal Shwartzman reaction. In none of these activities were LPS from the BPF cloneisolates 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 foundno significant difference between BPF clone and non-BPF clone isolates.

Haemophilus influenzae biogroup aegyptius (H. aegyp-

tius), first described in 1883 by Robert Koch, is the etiologicagent of the epidemic purulent conjunctivitis described byWeeks (reviewed in reference 21). Association of this organ-

ism with systemic disease was not demonstrated before1986, when' H. influenza biogroup aegyptius was culturedfrom the blood of several patients wit4 the recently de-scribed disease Brazilian purpuric fever (BPF) (5, 6). Com-parative studies of BPF-associated isolates and strains fromepidemiologically unrelated cases of conjunctivitis estab-lished that the BPF isolates have distinctive features andmay be derived froM a single clone (7, 8).The clinical manifestations of BPF suggest that bacterial

lipopolys4ccharties (LPS; endotoxins) may be involved inthe pathogenesis of the disease. The distinctive clinicalfeature of BPF has been cutaneous hemorrhage, or purpura,similar to that seen with fulminant meningococcemia (5).LPS, which can produce dermal necrosis and hemorrhage inexperimental animals in the dermal Shwartzman reaction(23), are obvious candidates for provoking cutaneous pur-pura in meningococcemia and BPF. In the experimentsreported here, we examined the hypotheses (i) that isolatesof the BPF clone of H. influenza biogroup aegyptius pos-

* 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. Cook-sey, 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 deCunto Brandileone, Kinue Irino, Mituka Kaku, Maria Lucia Cec-coni Tondella, and Eliseu Alves Waldman, Instituto Adolfo Lutz,Saq Paulo, S.P., Brazil; Ligia Regina Kerr Pontes, EscritorioRegional De Saude, Ribeirao Preto, S.P., Brazil; and David W.Pleming, State Health Division, Department of Human Resources,Portland, OR 97201.

sess LPS that are more bioactive than LPS from non-BPFclone isolates and (ii) that BPF clone isolates release moreLPS as they grow in vitro.

MATÉRIALS AND METHODS

Bacterial strains and cultivation of bacteria. H. influenzaebiogroup aegyptius isolates F1946, F3028, F3029, F3031, andF3037 were recovered from patients with BPF; all have thecase clone phenotype (7). Isolates F3043, F3052, F3055, andF3065 were obtained from epidemiologically unrelated cases

of conjunctivitis; none of these isolates has the case clonephenotype.- With the exception of F3065 (NCTC 8502),which was isolated in Texas (M. Pittman), all of the strainswere isolated in Brazil. H. influenzae type b, strain DL42,was obtained from E. J. Hansen, Dallas, Tex. The strainswere passed once on receipt and were stored at -70°C as

suspensions in skim milk. Bacteria were grown at 37°C inbrain heart infusion (Difco Laboratories, Detroit, Mich.)supplemented with 10 p.g ofNAD per ml and 10 jxg of heminper ml. In some cases, the broth was further supplementedwith 0.1 mCi of [3H]acetate (Dupont, NEN Research Prod-ucts, Boston, Mass.) per ml for labeling of LPS fatty acids.For assay of LPS release, bacteria were grown in mediummade in pyrogen-free water and sterilized by filtration; theglassware was heated at 180°C for at least 4 h.

Preparation of LPS. LPS from H. influenza type b wereprepared by hot phenol-water extraction following digestionwith lysozyme and nucleases, as described by Johnson andPerry (14). LPS from H. influenza biogroup aegyptius wererecovered from the phenol phase after extraction of 68°Cwith 45% phenol, pH 7.0 (14). The phenol was removed bydialysis against deionized water; a brown fibrous materialwhich formed during dialysis was discarded. LPS were

precipitated from the remaining liquid by addition of 5 mg ofsodium acetate per ml and 2 volumes of acetone. All LPSpreparations were extracted several times with ether toremove residual phospholipids.

Phenol-chloroform-petroleum ether extraction of H. influ-

762

Vol. 27, No. 4

on April 21, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

LPS FROM H. INFLUENZAE BIOGROUP AEGYPTIUS 763

enzae biogroup aegyptius was as described by Galanos et al.(12), except that following the extraction, LPS were precip-itated by addition of 2 volumes of ethanol rather than bydropwise addition of water.LPS from Salmonella minnesota Rc, used in the dermal

Shwartzman reaction, were prepared by the method ofGalanos et al. (12).SDS-PAGE. For sodium dodecyl sulfate-polyacrylamide

gel electrophoresis (SDS-PAGE), LPS preparations (1 ,ugper lane) were electrophoresed through a 10 to 15% lineargradient of acrylamide in the system described by Kimuraand Hansen (15) and silver stained by the method of Tsai andFrasch (24).

Analysis of fatty acids. LPS were hydrolyzed with 4 N HClat 100°C for 90 min, and total fatty acids were extracted intochloroform as previously described (13). For mild alkalinehydrolysis, LPS were incubated in 1 N NaOH for 15 h atroom temperature. Hydrolysis by neutrophil acyloxyacylhydrolase was as previously described (17). For separationof nonhydroxylated and hydroxylated fatty acids, sampleswere analyzed by one-dimensional thin-layer chromatogra-phy (TLC) as previously described (13). The locations of theresolved fatty acids were identified by fluorography; sectionsof silica gel were scraped from the plates, scintillationcocktail was added (Safety-Solve; Research Products Inter-national, Mount Prospect, Ill.), and the associated radioac-tivity was counted with a model 2425 scintillation counter(Packard Instrument Co., Inc., Rockville, Md.).For identification and quantitation of individual fatty ac-

ids, phenacyl ester derivatives of the LPS fatty acids wereprepared (11; p-bromophenacyl ester derivatization kit; All-tech Associates, Inc., Applied Science Div., State College,Pa.). The derivatization products were separated from unre-acted fatty acids with a Sep-Pak C18 cartridge (WatersAssociates, Inc., Milford, Mass.) and analyzed by reverse-phase high-pressure liquid chromatography (HPLC) with aC8 column (Ultrasphere Octyl; Beckman Instruments, Inc.,Fullerton, Calif.); the mobile phase was 89% methanol-11%water. Fatty acids were identified and quantitated by com-parison of retention times and peak areas with those of fattyacid standards. Heptadecanoic acid added to all samplesbefore derivatization served as an internal standard. Frac-tions were collected from the HPLC column, and their 3Hcontents were counted as described above.

Assays of LPS activity. (i) Spleen cell mitogenicity. Spleencells from C3H/HeN or C3H/HeJ mice were incubated withvarious concentrations of LPS; after 24 h, [3H]thymidinewas added. Eighteen hours later, the cells were harvestedand 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 cellswas determined as previously described (22), except thatcultured HL60 cells, a human promyelocyte cell line, weresubstituted for peripheral blood neutrophils (4). Briefly,cultured human umbilical vein endothelial cells were incu-bated for 4 h with LPS; the cells were washed, and 51Cr-labeled HL60 cells were added. After 30 min, the endothelialcells were again washed; the cells remaining in each wellwere lysed with 1 N NH40H, and the radioactivity in thelysates was counted (Gamma 4000; Beckman).

(iii) Dermal Shwartzman reaction. The experimentalmethod for the dermal Shwartzman reaction was similar tothat described by Nowotny (20). New Zealand White rabbitsweighing 2 to 3 kg were obtained from Hickory Hill, Flint,Tex., or Myrtle's Rabbitry, Thompson Station, Tenn., andmaintained in our institutional animal facility. On day 1 ofthe experiment, the backs of the animals were shaved; 5 or

10 ,ug of LPS suspended in 0.15 ml of pyrogen-free 0.9%NaCi was injected intradermally at several sites. Eighteen totwenty hours later, the rabbits were injected intravenouslywith 4 ,ug of S. minnesota Rc LPS per kg suspended in 1 mlof pyrogen-free 0.9% NaCI. Immediately following the intra-venous injection of LPS, 5 ml of pyrogen-free 0.9% NaCIcontaining 1% glucose was infused intravenously. Lesionsdeveloped over 4 to 6 h following the provocative injection;the diameter of the indurated area at each injection site wasmeasured at 6 to 7 h. The test preparations were codedbefore injection; the resulting lesions were measured bythree observers before the code was broken. To facilitatemeasurement, residual hair was removed from the backs ofthe rabbits with a chemical depilatory (Nair; Carter Prod-ucts, New York, N.Y.).Limulus assay. The activities of purified LPS or bacterial

culture supernatants were measured in a chromogenic Lim-ulus assay. Limulus amebocyte lysate (Pyrotell; Associatesof Cape Code, Inc., Woods Hole, Mass.) was reconstitutedin 8 ml of water. Four milligrams of the chromogenicsubstrate N-benzoyl-L-vaiylglycyl-L-arginine p-nitroanilidehydrochloride (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 por-tions were added to wells (Linbro/Titertek EIA microtitra-tion plates; Flow Laboratories, Inc., McLean, Va.) contain-ing 100 ,ul of the lysate-chromogen mixture. The plates andall solutions were kept on ice to this point. The plates werethen incubated at 37°C to allow color development; opticaldensities (ODs) at 410 nm were measured at 60 min with amicroplate reader (MR 700; Dynatech Laboratories, Inc.,Alexandria, Va.). S. minnesota Rc LPS (List BiologicalLaboratories, Campbell, Calif.) was used as the standard.For measurement of the Limulus activity released into the

medium, bacteria grown on plates were washed twice withbrain heart infusion and then suspended in supplementedbrain heart infusion in polystyrene tubes (16 by 125 mm) toan OD at 540 nm of 0.05 to 0.15. The tubes were placed at anangle to maximize the broth surface area and incubated at37°C without agitation (3) for 8 h. Before samples weretaken, the cultures were mixed by five end-over-end rota-tions of the tubes and the ODs were determined. Sampleswere filtered slowly through a 0.45-jxm-pore-size filter(Millex-HA; Millipore Corp., Bedford, Mass.) and stored at4°C until assayed (within 48 h). In a preliminary experiment,we found less than 6% variation in the Limulus activitiesmeasured 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 doubledduring incubation (16): number of doublings = In (finalOD/initial OD)/ln 2.The change in LPS concentration in culture filtrates was

divided by the number of doublings to determine the amountof LPS released per milliliter per generation during the 8 h ofincubation. Preliminary experiments determined that underthese culture conditions, the logarithmic phase of growthwas longer than 8 h.

Statistics. For the LPS release assay, the data wereanalyzed by using the Mann-Whitney test (25).

RESULTS

Preparation of LPS from H. aegyptius. Unlike the LPS ofH. influenza type b, the LPS of H. influenza biogroupaegyptius partitioned primarily into the phenol phase follow-ing hot phenol-water extraction. The aqueous phase con-tained material that was precipitable with ethanol; this

VOL. 27, 1989

on April 21, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

764 ERWIN ET AL.

l 2 3 4 5 6 7 8 9 10 Il

sPx

'w~~'c

co._

FIG. 1. SDS-PAGE of LPS preparations. Lanes: 1 to 6, H.influenza biogroup aegyptius (1 and 3, F3065 [non-BPF clone]; 2and 4, F3031 [BPF clone]; 5, F3029 [BPF clone]; 6, F3052 [non-BPFclone]); 7, H. influenza type b DL42; 8, E. coli J5; 9, N. menin-gitidis 8693; 10 and 11, S. minnesota Ra and Rc (List BiologicalLaboratories), respectively. H. influenza biogroup aegyptius LPSin lanes 1 and 2 were prepared by a modification of the method ofGalanos et al. (12), and LPS in lanes 3 to 6 were recovered from thephenol phase following hot phenol-water extraction. Only the rele-vant portion of the gel is shown.

precipitate contained nucleic acid, as indicated by a UVabsorption peak at 260 nm. After nuclease digestion, nofurther material could be recovered from this fraction. Incontrast, material precipitated from the phenol phase had noabsorption maxima between 230 and 300 nm. SDS-PAGEanalysis of this material showed it to be similar in apparentsize to the LPS of H. influenza type b, migrating as one ortwo bands with mobilities characteristic of rough LPS (Fig.1). The material recovered from the phenol phase possessedLimulus activity and was mitogenic for spleen cells ofC3H/HeN (LPS-responsive) mice; the activity toward spleencells of C3H/HeJ (LPS-hyporesponsive) mice was less by 2.5to 3 orders of magnitude (Fig. 2). On the basis of theseobservations and the fatty acid analysis (see below), weconcluded that this material was LPS.A similar material was prepared from two H. influenza

biogroup aegyptius isolates by a modification of the extrac-tion procedure of Galanos et al. When analyzed by SDS-PAGE, LPS prepared by the latter method appeared to be asubset of the phenol-water-extracted LPS (Fig. 1). Onlyphenol-water-extracted LPS were used in subsequent exper-iments.

Fatty acid analysis. We determined the fatty acid compo-sition of LPS from two isolates of H. influenza biogroupaegyptius and, for comparison, one strain of H. influenzatype b (Table 1; Fig. 3). For these studies, LPS wereprepared from bacteria grown in medium containing[3H]acetate. Acid hydrolysis of the labeled LPS, followed bychloroform-methanol extraction, showed that virtually all ofthe 3H was in the fatty acids. HPLC analysis showed thateach LPS preparation contained only two species of fattyacid: 3-OH myristate and myristate (Fig. 3). TLC of fattyacids released by mild alkaline hydrolysis and subsequentacid hydrolysis indicated that, in each case, all of themyristate and about half of the 3-OH myristate residues wereattached by relatively labile bonds; the remaining 3-OHmyristate was removed by acid hydrolysis (Table 1). Treat-ment with acyloxyacyl hydrolase released only the myristateresidues (Table 1). These data suggest that LPS of H.influenza biogroup aegyptius, as well as LPS of H. influ-enzae type b, have fatty acyl linkages that resemble those inlipid A of Salmonella spp. and Escherichia coli: 3-OHmyristate residues are attached to the glucosamine backboneby both ester and amide linkages, and some of the hydroxylgroups on these acyl chains are substituted by ester-linked,nonhydroxylated fatty acids.

a

E

.0005 .005 .05 .5 5 50

[LPS], /Lg/mtFIG. 2. Incorporation of [3H]thymidine by spleen cells treated

with LPS. For each point, the stimulation index was calculated bydividing 3H incorporated into LPS-treated cells by 3H incorporatedinto untreated cells. Each point represents the mean of four replicatesamples. -, Spleen cells from C3H/HeN (LPS-responsive) mice;- -, spleen cells from C3H/HeJ (LPS-hyporesponsive) mice. Thesymbols indicate the bacterial isolate from which the LPS wasprepared: E, F3029 (BPF clone); O, F3031 (BPF clone); A, F3052(non-BPF clone); *, F3065 (non-BPF clone); V, H. influenza typeb DL42.

Biological activities of LPS. Experiments on the biologicalactivities of LPS were performed with LPS from two isolatesof the BPF clone of H. influenza biogroup aegyptius (F3029and F3031), two non-BPF clone strains (F3052 and F3065),and H. influenzae type b (DL42).

(i) Spleen cell mitogenesis. As noted above, all LPS weremitogenic for spleen cells from C3H/HeN mice (Fig. 2). Theactivities of LPS from the different strains were similar; LPSfrom 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 torespond to injury by promoting leukocyte adherence. All ofthe LPS had similar activities, although LPS from onenon-BPF clone isolate, F3052, was more active than theothers (Fig. 4).

(iii) Dermal Shwartzman reaction. We compared the abil-ities of H. influenza biogroup aegyptius LPS to prepare theskin of rabbits for the dermal Shwartzman reaction. EachLPS preparation was tested at two concentrations in two ormore rabbits; each injection was in duplicate. Differencesamong the LPS were small, and LPS from the BPF cloneisolates were not more potent than LPS from control strains(Fig. 5).

Release of LPS into culture medium. To establish that LPSfrom various isolates of H. influenza biogroup aegyptiuscould be quantitated with a single LPS as the standard, wedetermined in preliminary experiments (data not shown) thatpurified LPS from four isolates were equally reactive in theLimulus assay. Previous observations in this laboratory (18)and those of Anderson and Solberg (2) showed that releaseof LPS begins slightly after the start of the logarithmic phaseand ceases shortly after entry into the stationary phase. Wedetermined the amount of Limulus reactivity in culture

J. CLIN. MICROBIOL.

on April 21, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

LPS FROM H. INFLUENZAE BIOGROUP AEGYPTIUS 765

TABLE 1. TLC analysis of LPS fatty acidsa

% of LPS 3H recovered following complete or partial hydrolysis

Hydrolysis F3031 F3065 DL42

OH-FAb NFA OH-FA NFA OH-FA NFA

Acid 63.7 36.3 67.2 32.8 67.6 32.4Alkaline 28.9 (21.6) 43.8 (5.6) 29.1 (28.3) 39.3 (3.3) 23.3 (28.9) 41.2 (6.6)Neutrophil acyloxyacyl hydrolase 2.6 (57.9) 30.1 (9.4) 1.0 (62.3) 22.5 (14.2) 0.7 (73.0) 19.9 (6.4)

a After the indicated hydrolysis, the fatty acids released were extracted into chloroform and separated into hydroxylated fatty acids (OH-FA) andnonhydroxylated 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. Foralkaline or enzymatic hydrolysis, the percent released by the partial hydrolysis is given first; the percent released by subsequent acid hydrolysis is given inparentheses. 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. TheLPS fatty acids were identified by HPLC analysis (Fig. 3) as 3-OH myristate and myristate. A small proportion of 3-OH myristate undergoes an eliminationreaction during alkaline hydrolysis; the unsaturated product of this reaction comigrates with NFA in this TLC system.

filtrates of nine isolates of H. influenza biogroup aegyptiusat the initiation of culture and in the late logarithmic phase,after 8 h of incubation. Because the isolates varied in growthrate, we calculated the amount of Limulus reactivity re-leased into the culture medium per generation during the first8 h of culture (Table 2). There was substantial variation

among the isolates; although there was a slight tendency forisolates of the BPF clone to produce more LPS than controlisolates, there was considerable overlap between the twogroups and the differences between the groups were notstatistically significant.

Ec

on

N

CDoc>

a 2t 1

cx

4--

u 02015

4) 10

5

o n

A. 3-OH-14:012:0

'14:016:1 16:0 17:0

B.B.

10 15 20 25 30 35 40Retention Time, min.

FIG. 3. HPLC of LPS fatty acids. (A) Fatty acid standards (120pmol each): 3-OH C14:0, 3-OH-myristate; C12:0, laurate; C14:0, my-ristate; CI6,:, palmitoleate; Cl6,0, palmitate; C17:0, heptadecanoicacid. (B) Fatty acids from H. influenza biogroup aegyptius BPFclone isolate F3031 LPS (heptadecanoic acid added). The profilesfor LPS fatty acids from isolate F3065 and H. influenza type bDL42 were very similar. (C) 3H recovered from the column, in theanalysis shown in panel B. Fractions were collected at 0.5-minintervals (1-min intervals after elution of myristate), the solvent wasallowed 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.

DISCUSSION

The pathogenesis of bacterial purpura is uncertain, al-though there is evidence that LPS may be involved. It wasshown in the 1930s that hemorrhagic skin lesions similar tothose seen in fulminant sepsis may be induced experimen-tally in rabbits by sequential injections of culture filtrates

6000 13:

Eo.

CI

c-

z

0

~0CD

50001

4000 F

30001

2000 h

1000

oO 0.001 0.01 0.1 1.0

[LPS] ,ng/ml

FIG. 4. Adherence of HL60 cells to LPS-treated endothelialcells. Human umbilical vein endothelial cells were incubated withvarious concentrations of LPS; after removal of the LPS, 51Cr-labeled HL60 cells were added. After removal of unbound HL60cells, the cells remaining in the wells were lysed and a sample of thelysate was counted. Each point represents the mean of four replicatewells +±1 standard deviation. The symbols indicate the bacterialisolates 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.

1 1 1

hi

VOL. 27, 1989

on April 21, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

766 ERWIN ET AL.

E

<D

._.E

où._

c

en

-J

15

10

5

opIIsolate #: 65BPFcase:

FIG. 5. Dermal'biogroup aegyptius lmal injections wereresulting from eaclcomparison betweenwhere LPS from an

second at a site whclone isolate was iniF3031 and F3065 LI(no. 31; BPF cloncproduced by the se

clone). Although foiisolate produced larpair, F3029 (no. 29;

from gram-negati)tion; 23). The actbeen shown to bewhich are shed (abacteria grow. Rcstrated several aproduction of bacinclude the primingand stimulation othelial cells, as N

secrete procoagulthat promote neuties, including theteristic of LPS gcurred characterira few bacterial spc

TABLE 2. Ré

Isolate

BPF cloneF1946 ...............

F3028 ...............

F3029 ...............

F3031...............F3037 ...............

Non-BPF cloneF3043 ...............

F3052 ...............

F3055 ...............

F3065 ...............

a The concentrationassay; bacterial growthMaterials and Methodsubtracted from concCalculation of LPS relMethods. The resultscultures.

It has been suggested that bacteria associated with pur-10MFg pura have LPS of increased toxicity, and a comparison ofmeningococcal LPS with LPS from S. typhimurium and E.

'A coli showed increased potency of meningococcal LPS in thedermal Shwartzman reaction (10). This study did not takeinto account, however, the larger molecular size of smoothLPS from enteric bacteria; a comparison based on LPSweight may have been misleading. A second hypothesis forthe association of particular bacteria with purpura implicatesthe LPS released from the bacterial surface. Andersen et al.have shown that strains of meningococci isolated from

31 29 52 65 31 29 52 patients with systemic meningococcal infections tend to+ + - - + + - shed more LPS than do strains isolated from carriers (3);

LPS Injected they have also reported that variants which release less LPSShwartzman reaction results with H. influenza have decreased virulence for mice (1).LPS in preparative injections. Multiple intrader- The recent description of BPF allowed us to test thesegiven to each rabbit; the size of the lesion hypotheses in a different system. It appears that BPF is

i injection is plotted. Each line represents a associated with a particular strain of H. influenza biogroupn two lesions on the same rabbit, one at a site aegyptius (7); this suggests a comparison of purpura-associ-isolate of the BPF clone was in ected and theisolathe ofathe BPFmloune wasL njecdanndF ated and control strains that is not possible for N. meningiti-iere the same amount of LPS from a non-BPF dis. We thus undertook the examination of a number of

ejected. Thus, in repeated comparisons between is.lWes undro the amination oa thePS, most injections of 5 or 10 Fig of F3031 LPS isolates of H. influenza biogroup aegyptius to study thee) produced lesions slightly larger than those potency of their LPS in various bioassays and to compareame amount of F3065 LPS (no. 65; non-BPF the amounts of LPS released.r this pair of isolates, LPS from the BPF clone The LPS from BPF clone and non-BPF clone isolatesger lesions, the reverse was true for the second differed from the LPS of H. influenza type b and N.BPF clone) and F3052 (no. 52; non-BPF clone). meningitidis in separating into the phenol phase rather than

the water phase following hot phenol-water extraction. Suchpartitioning has been described for some strains of Pseudo-

tive competent in such filtrates has since monas aeruginosa and may reflect increased hydrophobicityLPve componentsin shefactesiassel of the LPS (9). Nevertheless, the LPS from H. influenza

sLPS,componen oembratefriagment wasl biogroup aegyptius were similar to those of H. influenzais blebs or outer membrane fragments) as type b in apparent size (judged by migration on SDS-PAGE),,cent work with purified LPS has demon- biological activities, and fatty acid composition. The simi-ictivities which might contribute to the larity of these LPS to those of H. influenza type b has alsotrial purpura in an infected animal. These been noted by Gheesling et al., who noted variation amongg of neutrophils to respond to other stimuli isolates in the migration of LPS and the number of bandsf neutrophils to adhere to vascular endo- resolved on SDS-PAGE and demonstrated reactivity of H.vell as activation of endothelial ce-Ils to influenza biogroup aegyptius with monoclonal antibodies toants and to express cell surface molecules LPS of H. influenzae type b (L. L. Gheesling, G. M.trophil adherence. However, these activi- Carlone, F. 0. Sottnek, W. F. Bibb, C. W. Moss, and thedermal Shwartzman reaction, arecharac-'''dermalSwrmaratBrazilian Purpuric Fever Study Group, Abstr. Annu. Meet.

generally, while bacterial purpura has oc- Am. Soc. Microbiol. 1988, B-121, p. 49).

.ticallywith bloodstream infection by only We compared LPS from BPF clone and non-BPF cloneecies, most notably Neisseria meningitidis. isolates in two bioassays that may relate to tissue toxicity:

the preparative phase of the dermal Shwartzman reaction

lease of LPS from bacteria during growth and the ability to promote neutrophil adherence to thesurface of endothelial cells. In neither of these assays were

(ng/ml pergeneration)' LPS from isolates of the BPF clone more active. Twoadditional assays, the Limulus reaction and spleen cellmitogenesis, not thought to relate to tissue toxicity, also

..................................... 4,700 ± 1,900 failed to distinguish LPS from BPF clone and non-BPF clone

..................................... 7,600 1,700 isolates.

..................................... 1,4000 ± 3,300 We compared nine isolates of H. influenzae biogroup

..................................... 23000 ± 8,000 aegyptius as to the release of LPS into culture medium byusing conditions similar to those in which Andersen andSolberg had distinguished endotoxin-liberating and nonliber-

..................................... 4,700 +1,500 ating strains of N. meningitidis (2, 3). LPS was measured

..................................... >64,000 with the Limulus assay, which measures a bioactivity of the

..................................... 5,500 +3,000 LPS and not necessarily the amount of LPS present. How-

..................................... 9,600 +4,000 ever, previous studies have established that Limulus resultsiof LPS in culture filtrates was determined by Limulus correlate reasonably well with the results of a radioimmuno-h was monitored by measurements of OD at 540 nm (see assay for LPS, both in vitro and in vivo (18, 19). Inis). Initial LPS concentrations (under 50 ng/ml) were preliminary experiments, we also found that purified LPScentrations at 8 h to determine the LPS released. from two BPF clone and two non-BPF clone isolates of H.Leased per generation was as described in Materials and from tw oBFone and two non clone sifatly inare means ±1 standard deviation of three replicate influenza biogroup aegyptius did not differ significantly in

potency in the Limulus test. We saw substantial variation

5

J. CLIN. MICROBIOL.

on April 21, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

LPS FROM H. INFLUENZAE BIOGROUP AEGYPTIUS 767

among nine isolates of H. influenza biogroup aegyptius inthe amount of Limulus-reactive material released duringgrowth. There was some tendency for the BPF clone isolatesto release more LPS, but this difference was not statisticallysignificant.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 physicalstate of the purified LPS used in this study is probably quitedifferent from that of the native LPS which occurs as part ofa bacterial membrane. In addition, the LPS of bacteriagrown in culture might differ from the LPS of bacteria grownin vivo. For example, Kimura and Hansen have reportedthat for a weakly virulent strain of H. influenza type b,bacteria recovered from experimentally infected infant ratspossessed increased virulence; the increased virulence was

associated with antigenic and structural alteration of the LPS(15). Although we were not able to demonstrate increasedpotency of LPS from isolates of the BPF clone, it is possiblethat such an increase occurs only in vivo. Indeed, passage ofstrains through animals increases the virulence of H. influ-enzae biogroup aegyptius for infant rats (8). Further use ofanimal-passed strains and animal and cell culture infectionmodels may further understanding of the role of LPS andother 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 biogroupaegyptius may differ from non-BPF clone isolates in factorsunrelated to LPS which allow invasion of the bloodstreamand evasion of host defenses. Some of these factors may behost specific; others may be amenable to study in animals.

ACKNOWLEDGMENTS

We thank Ahmed Adu-Oppong for technical assistance andCatherine L. Hall for advice regarding fatty acid analysis.

This work was supported by U.S. Public Health Service grants AI18188 and HD 22766 from the National Institutes of Health.

LITERATURE CITED1. Andersen, B. M., and O. Solberg. 1984. The virulence in mice of

Neisseria meningitidis variants differing in free endotoxin activ-ities and cell envelope properties. NIPH (Natl. Inst. PublicHealth) Ann. 7:47-59.

2. Andersen, B. M., and O. Solberg. 1988. Endotoxin liberationassociated with growth, encapsulation and virulence of Neis-seria 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 fromcarriers and clinical cases. Scand. J. Infect. Dis. 19:409-419.

4. Bevilacqua, M. P., J. S. Pober, M. E. Wheeler, R. S. Cotran, andM. A. Gimbrone. 1985. Interleukin 1 acts on cultured humanvascular endothelium to increase the adhesion of polymorpho-nuclear leukocytes, monocytes, and related leukocyte cell lines.J. Clin. Invest. 76:2003-2011.

5. Brazilian Purpuric Fever Study Group. 1987. Brazilian purpuricfever: epidemic purpura fulminans associated with antecedentpurulent conjunctivitis. Lancet ii:757-761.

6. Brazilian Purpuric Fever Study Group. 1987. Haemophilus ae-

gyptius 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,

S. F. Berkley, T. C. Woods, A. G. Steigerwalt, P. A. D. Gri-mont, R. M. McKinney, D. W. Fleming, L. L. Gheesling, R. C.Cooksey, R. J. Arko, C. V. Broome, and the Brazilian PurpuricFever Study Group. 1988. Biochemical, genetic, and epidemio-logic characterization of Haemophilus influenza biogroup ae-gyptius (Haemophilus aegyptius) strains associated with Brazil-ian purpuric fever. J. Clin. Microbiol. 26:1524-1534.

8. Carlone, G. M., L. Gorelkin, L. L. Gheesling, A. L. Erwin, S. K.Hoiseth, M. H. Mulks, S. P. O'Connor, R. S. Weyant, J. Myrick,L. Rubin, R. S. Munford IIl, E. H. White, R. J. Arko, B.Swaminathan, L. M. Graves, L. W. Mayer, M. K. Robinson, S.P. Caudill, and the Brazilian Purpuric Fever Study Group. 1989.Potential virulence-associated factors in Brazilian purpuric fe-ver. J. Clin. Microbiol. 27:609-614.

9. Cryz, S. J., T. L. Pitt, E. Furer, and R. Germanier. 1984. Roleof lipopolysaccharide in virulence of Pseudomonas aeruginosa.Infect. Immun. 44:508-513.

10. Davis, C. E., and K. Arnold. 1974. Role of meningococcalendotoxin in meningococcal purpura. J. Exp. Med. 140:159-171.

11. Durst, H. D., M. Milano, E. J. Kitka, S. A. Connelly, and E.Grushka. 1975. Phenacyl esters of fatty acids via crown ethercatalysts for enhanced ultraviolet detection in liquid chromatog-raphy. Anal. Chem. 47:1797-1801.

12. Galanos, C., O. Luderitz, and O. Westphal. 1969. A new methodfor the extraction of R lipopolysaccharides. Eur. J. Biochem.9:245-249.

13. Hall, C. L., and R. S. Munford. 1983. Enzymatic deacylation ofthe lipid A moiety of Salmonella typhimurium lipopolysaccha-rides by human neutrophils. Proc. Natl. Acad. Sci. USA 80:6671-6675.

14. Johnson, K. G., and M. B. Perry. 1976. Improved techniques forthe preparation of bacterial lipopolysaccharides. Can. J. Micro-biol. 22:29-34.

15. Kimura, A., and E. J. Hansen. 1986. Antigenic and phenotypicvariations of Haemophilus influenza type b lipopolysaccharideand their relationship to virulence. Infect. Immun. 47:253-259.

16. Koch, A. L. 1981. Growth measurement, p. 179-207. In P.Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester,W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual ofmethods for general bacteriology. American Society for Micro-biology, Washington, D.C.

17. Munford, R. S., and C. L. Hall. 1986. Detoxification of bacteriallipopolysaccharides (endotoxins) by a human neutrophil en-zyme. Science 234:203-205.

18. Munford, R. S., and C. L. Hall. 1987. A comparison of twoquantitative assays for bacterial lipopolysaccharides, p. 93-102.In S. W. Watson, J. Levin, and T. J. Novitsky (ed.), Detectionof bacterial endotoxins with the Limulus amebocyte test. AlanR. Liss, Inc., New York.

19. Munford, R. S., C. L. Hall, and L. Grimm. 1984. Detection offree endotoxin in cerebrospinal fluid by the Limulus lysate test.Infect. Immun. 45:531-533.

20. Nowotny, A. 1969. Basic exercises in immunochemistry, p.178-180. Springer-Verlag KG, New York.

21. Pittman, M., and D. J. Davis. 1950. Identification of the Koch-Weeks bacillus (Hemophilus aegyptius). J. Bacteriol. 59:413-426.

22. Pohlman, T. H., R. S. Munford, and J. H. Harlan. 1987.Deacylated lipopolysaccharide inhibits neutrophil adherence toendothelium induced by lipopolysaccharide in vitro. J. Exp.Med. 165:1393-1402.

23. Shwartzman, G. 1937. Phenomenon of local tissue reactivity andits immunological, pathological, and clinical significance. Hoe-ber, New York.

24. Tsai, C.-M., and C. E. Frasch. 1982. A sensitive silver stain fordetecting lipopolysaccharides in polyacrylamide gels. Anal.Biochem. 119:115-119.

25. Zar, J. R. 1969. Biostatistical analysis, 2nd ed., p. 139. Prentice-Hall, Inc., Englewood Cliffs, N.J.

VOL. 27, 1989

on April 21, 2021 by guest

http://jcm.asm

.org/D

ownloaded from