Vol. 55, No. 11INFECTION AND IMMUNITY, Nov. 1987, p. 2554-25610019-9567/87/1 12554-08$02.00/0Copyright ©D 1987, American Society for Microbiology

Role of Cell-Bound Hemolysin as a Pathogenicity Factor forSerratia Infections

W. KONIG,1* Y. FALTIN,' J. SCHEFFER,1 H. SCHOFFLER,2 AND V. BRAUN'Lehr-stihlfiir Medizinisuhe Mikrobiologie iand Iminiunologie, Arbeitsgruiippe fir Infetabbwe/hrinechanismen,

Ruhr-Univ,ersitat Bo(hum, 4630 Bo(hum I,' ind Lehrstul/lfi' Mikr-obioogie H, UnihersitiithTibingen, Tibingen,2FederalRepubl/h of Germanysl}

Received 10 April 1987/Accepted 20 July 1987

The hemolytic activities of clinical isolates of Serratia marcescens, of Serratia liquefaciens, and of Escherichiacoli strains containing a cloned hemolysin gene of S. marcescens were determined. Hemolysis was induced onlyby cells and not by spent media. The hemolytically active bacteria induced the release of the leukotriene C4 andof much less leukotriene B4 from polymorphonuclear leukocytes, the release of histamine from rat mast cells,and chemoluminescence of neutrophils. The hemolytic activity was correlated with the response of theleukocytes, but quantitative differences were recorded with regard to the release of the inflammatorymediators. Therefore, other factors in addition to the hemolysin contribute to the stimulation of leukotrienegeneration and histamine release. It is concluded that the hemolysin via these inflammatory mediators can

increase vascular permeability, edema formation, and granulocyte accumulation and thus contributes to thepathogenicity of Serratia species.

Serratia marcescens is becoming widely recognized as animportant opportunistic pathogen causing respiratory andurinary tract infections, bacteremia, endocarditis, keratitis,arthritis, and meningitis (31, 32). Patients with significantSerratia infections may have a variety of debilitating dis-eases such as diabetes mellitus, heart disease, renal insuffi-ciency, or malignant diseases. Relatively little is knownabout the virulence determinants of this bacterial species(17, 23, 25, 42). Clinical isolates of S. inaircescens werepreviously shown to produce various exoenzymes (18, 33).A metalloprotease was described that induces pneumonia inlaboratory animals, enhances vascular permeability throughthe activation of a Hageman factor-dependent kallikreinpathway in vitro, and causes fibrinolysis (24, 34, 43).

Recently, Braun et al. (8) noticed that all S. inarcescensstrains rapidly lysed human erythrocytes in solution. Theseobservations stood in contrast to the narrow lysis zonearound Sei-rritia colonies on blood agar. The cell-boundhemolysin activity required metabolizing Serratia cells. Nohemolysin activity was found in the culture supernatants.Hemolysis might initiate human invasive nosocomial infec-tions with subsequent spread and bloodstream invasion (41).We recently showed that the secreted hemolysin from

Escherichia (coli (16, 20, 30, 44, 45) triggers the release ofinflammatory mediators from various cells under non-cytotoxic conditions (28, 39, 40). In the past it has beenestablished that histamine and leukotrienes are importantmediators of inflammatory reactions induced by immunolog-ical and nonimmunological stimuli. Leukotriene B4 (LTB4),unlike its isomers, is chemotactic for human neutrophils andeosinophils (10, 29). LTC4, LTD4, and LTE4 were identifiedas the slow-reacting substances of anaphylaxis which lead tothe increase in vascular permeability and are involved inbronchoconstriction and mucus production. Human poly-morphonuclear leukocytes (PMNs) release high amounts ofleukotrienes upon activation with various stimuli, e.g., thecalcium ionophore A23187 (6. 36, 38), bacterial exo- andendotoxins (11-14, 39, 40), as well as during phagocytosis

* Corresponding author.

(12, 36). Since the abovementioned target cells can beobtained without difficulties, rat mast cells were used tostudy histamine release, and human PMNs were used tomeasure oxygen radical production and leukotriene genera-tion.

In this study we investigated the release of inflammatorymediators such as oxygen radicals, histamine, and leuko-trienes induced by cell-bound hemolysin from S. inarces-cens. Further studies analyzed the extent that hemolysinfrom S. inarcesceens cloned in E. coli was expressed in itsbiologically active form and whether it was able to triggercells for mediator release.

MATERIALS AND METHODSBacterial strains. The SerrCatia strains were isolated in the

Department of Medical Microbiology and Immunology,Ruhr-University Bochum. The strains were identified withthe API system. Seirratia /iquefJiciens strain 749611 (API5306563) was isolated from the human mammary gland, S.inarcesceens 5817 and 5842 (API 5307721) were cultured fromwound swabs of two patients with leg ulcers, S. inarcescens5241 was derived from tracheal secretions, and S. marces-cenis W1436 is an exoprotease- and exolipase-deficient strainof human origin isolated in several steps by mutation (U.Winkler, Ruhr-University Bochum). The growth rates of thevarious Serrdtiti strains (W1436, 5241, 5817, 5852, and749611) were determined by optical density analysis at 600nm; bacterial cell counting was done by microscopy. Thelogarithmic phase started after 40 to 60 min of culture; thestationary phase started after 200 min.To study the expression of the Seratia hemolysin in a

defined genetic background, we constructed the following E.co/i clones, which are described in detail elsewhere (9).Briefly, DNA from S. inarcescens W1436 was partiallydigested with the restriction endonuclease Sau3A1. The40-kilobase fragment was isolated by centrifugation throughan NaCI gradient and ligated into the cosmid vector pHC79with the in vitro packaging system of Amersham-Buchler(Braunschweig, Federal Republic of Germany). E. coli 5KlisdIR lisdAM lo( i-ps-I ser tlii thi was transformed with the

2554

on July 8, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

CELL-BOUND HEMOLYSIN IN SERRATIA INFECTIONS 2555

pHC79 derivatives, and hemolysin-positive clones werescored on blood agar plates. A hemolysin-positive clone wasinfected with bacteriophage X::TnIO(ts) which coded for atemperature-sensitive repressor. The cosmid was trans-ferred after induction at 42°C into E. coli MC4100 ara lacrpsL. It was isolated from E. coli MC4100 HSH 33/1 (Hlyt )and partially digested with Sai,3A1, and the restrictionfragments were ligated into the BamHI site of the plasmidpUC19. A hemolysin-producing clone of the transformant E.coli HB101 recA hsdM hsdR siupE lacY leia tlii pro wasdesignated 19/1 and used in this study. Only the genesresponsible for the expression of Serraitia hemolysin werecloned into E. coli.

E. coli 536/31(pANN5311) was obtained from J. Hackerand W. Goebel. This strain expresses Mrh Vb (20) andcarries a recombinant plasmid for cx-hemolysin expression(21, 22).

Bacterial growth. Brain heart infusion broth (10 ml) wasinoculated with 0.1 ml of an overnight culture. After 3 to 3.5h of cultivation at 37°C on a shaker (150 rpm), the bacteriawere centrifuged at 4,000 x g for 15 min at 4°C and washedtwo times in phosphate-buffered saline (PBS). Bacteria in thelate logarithmic phase were tested.

Analysis of protease activity. Bacteria suspended in 0.15 mlof 0.9% NaCI or culture supernatants were incubated with250 [LI of 2% azocasein for 30 min at 37°C. Incubation wasstopped by the addition of 1 ml of 7% perchloric acid. Aftercentrifugation, 0.15 ml of 1 N NaOH was added to thesupernatant, and the optical density was measured at 430nm.Hemolysin assay. Brain heart infusion broth (25 ml) was

inoculated with 0.25 ml of an overnight culture and subse-quently incubated on a shaker as described previously (40).At various times 2 ml of the culture was removed andanalyzed by optical density to determine the number ofbacterial cells. Bacteria were separated by centrifugationfrom the supernatant, which was then kept on ice. Subse-quently, the bacteria were suspended in 0.5 ml of physiolog-ical saline. Hemolysin activity was determined as follows.Bacteria (5 x 108 cells in 0.1 ml) or the culture supernatantif required (0.1 ml) were incubated for 30 min at 37°C with0.9 ml of a suspension that contained 2% sheep erythrocytes,20 mM CaCl2, 10 mM Tris, and 160 mM NaCl adjusted to pH7.4 with hydrochloric acid. Then, the mixture was kept for20 min on ice, and centrifuged, and the released hemoglobinin the supernatant was determined at 530 nm. The resultswere expressed as percent lysis compared with erythrocyteslysed in distilled water. Washed bacteria (S. inarcescens.cloned E. (oli strains) as well as the bacterial culturesupernatant were evaluated for hemolysin activity.

Hemagglutination. Hemagglutination was analyzed byslightly shaking 25 pLI of a bacterial cell suspension (5 x 109cells per ml) and 25 pA of 3% erythrocytes (human and sheepas well as guinea pig) in 0.9% NaCl for 29 min on ahemagglutination plate (1).Adherence and phagocytosis. [3H]thymidine (55 kBq) was

added to a 10-ml bacterial culture after 1 h of incubation: theculture was incubated for an additional 2.5 h. Bacteria werewashed twice in TCM (Tris [25 mM. pH 7.35], NaCl [120mM], KCI [4 mM]) (15 min, 4,000 x g); 5 x 108 bacteria werepresent in 1 ml. For the detection of phagocytosis, bacteria(50 [L) were added to 500 p1 of PMNs and incubated at 37°C.Subsequently, the reaction mixture was placed on ice, andthe cells were separated by centrifugation at 300 x g for 15min. Adherent bacteria were removed from the granulocytesby incubating them on ice with Tris buffer (pH 7.35. 25 mM)

containing NaCl (120 mM), KCI (4 mM), EDTA (40 mM),and lysozyme (100 pg/ml). By repeated washing in TCM andsubsequent lysis in distilled water, the percentage ofphagocytosed bacteria was determined. Labeled bacterialsuspension (50 [LI) served as a control. For the analysis ofadherent bacteria, lysozyme treatment was omitted. Thepercent adherence was calculated as the difference betweenthe lysozyme-treated and untreated granulocyte suspen-sions. A radioactivity of 100% represents the amount com-bined with 2.5 x 107 washed bacteria.

Buffers. The medium used for washing the peripheralleukocytes and for mediator release was PBS (0.2 M phos-phate, 0.1% NaCl, pH 7.4). CaCl, (0.6 mM) and MgCl2 (1mM) were added shortly before the cells were stimulated tolower the spontaneous release of mediators.

Chemiluminescence. Neutrophils (106 cells) and luminol(20 ,ul of a 0.25 mM solution) were incubated for 15 min at37°C in 0.2 ml of PBS containing calcium and magnesium.Then, 50 [lI of bacterial cell suspension (5 x 109 cells per ml)was added. Chemiluminescence was monitored at 5-minintervals for 10 s. The results are expressed as counts perminute.

Release of enzymes. Release of lactate dehydrogenase wasmeasured as described previously (40). PMNs (107 cells in0.5 ml of buffer) were incubated with 50 pLI of washedbacterial cell suspension of various times up to 30 min.

Preparation of human cells. Human leukocytes were ob-tained from heparinized blood of healthy donors and sepa-rated on a Ficoll-metrizoate gradient, followed by dextransedimentation (7, 15). This method leads to >97% purePMNs. The cells were then washed three times at low speed(600 rpm), after which less than 2% of the platelets remained.the purity of the PMN fraction was determined by lightmicroscopy. The erythrocytes were removed by hypotonicexposure of the cell suspension.

Leukotriene release from human PMNs. Human PMNs (1X 107 or 2 x 107) were suspended in 1 ml of PBS. Thesupernatant of stimulated cells (0.5 ml) was assayed forleukotrienes by high-pressure liquid chromatography(HPLC) and radioimmunoassay (RIA).

Analysis of leukotriene release. For analysis of leukotrienerelease, the supernatant of the stimulated cells wasdeproteinized by the addition of 2 volumes of acidifiedmethanol (methanol-acetic acid [1,000:1, vol/vol]), overlaidwith argon, and frozen at -70°C for 12 h. After centrifuga-tion at 3,000 x g, the supernatants were evaporated todryness under a stream of nitrogen and suspended in 500 [Lof methanol-water (30:70, vol/vol) for reverse-phase HPLC(26, 28); HPLC analysis was performed with a Nucleosil C18 column (5-[tm pore size, 4 by 200 mm; Machery Nagel.Duren, Federal Republic of Germany) with methanol-water-acetic acid (64:36:0.98. vol/vol/vol/! pH 5.9); the titer wasdetermined with ammonia as the eluent. Consta Metricpumps I and III (LDC Laboratory Water Control, MiltonRoy, Hasselroth, Federal Republic of Germany) and theautomatic sample injection module WISP 710 B (WatersAssociates, Inc.. Milford, Mass.) were used. The A28(0 of thevolume effluent was monitored with a variable UV detector(LDC Spectromonitor III 1204 A). The peak area or heightwas calculated with an LDC Computing Integrator 301. Thechromatograms were recorded with a printer plotter (LDC).Under these conditions, the retention times were 19.50 to20.00 min for LTB4. 11.30 to 11.70 min for LTC4, 17.20 to17.60 min for LTD4 and 6-trans LTB4, and 20.60 to 21.50 minfor LTE4. Identification of leukotrienes was assessed bydetermining the retention time and comparing it with those

VOL. 55, 1987

on July 8, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

2556 KONIG ET AL.

TABLE 1. Relationship of hemolysin and protease production by strains of S. mnaves(cens to inductionof inflammatory mediators and phagocytosis"

Protet_ se" Hemolvsin leLikotriene (ng) Histamine LDH (';/r r Adherence PhagocytosisStralin C SLIpSup LTC4 LATB34 (ng)d C Sup ('7

S. InlturcesccnsW1436 - - 86 7.7 2.8 278.2 4.2 6.9 1.8 13.65241 - - 61 1.3 1.4 154.5 4.2 4.2 ND4' ND5817 - + + 47.5 5.6 1.3 117.5 4.2 2.8 NtD ND5852 - + + + 51 4.4 2.3 140.9 2.8 6.9 2.8 13.9

S. Iiqueftwicets 749611 - - 66 4.2 3.0 87.3 6.9 9.( 3.9 9.2

The experiments were perfor-med five times in tr-iplic.tte.Protease aind hemolysin were determined as described in Materials and Methods. The standaird hemolysin determination wvas 2 to 5'%c.LTB4 and LTC4 were induiced with bacteria aifter 10 min of incubation. PMNs (2 x 1(7/ml) werc incuibated with washed bacter-iai (5 x 10() for 10 min at 37TC.

Leukotrienes were detected by RIA; standard deviation = 3 to 7%/,.d Histamine release was determined alter 60 min of stimulation with washed bacteria. Waished ba.cteriai (5 x 10() wcre incubaited with rat peritonealil cells (5 x

104 mast cells). The standard deviation of histatmine atnatlysis was 3 to 8X'4.Lactate dehydrogenaise (LDH) release was determined alter 3(1 min of incuLba.tioni at 37CC. PMNs (1 X wc incLuba.ted with waished baictelia (2.5 x 10()

or culture supernatant (5o ,ul) (basal lalctitte dehydrogenatse release. 9.7/4 )./'Adherence and phagocytosis were meaisuIred aIfter- 10nmin. PMNs (1 x 1()7) w'Cre inncibated with radiolabelcd bactcria (2.5 x 1(7') at 37GC.ND. Not done.

of external standards of synthetic leukotrienes (gift from J.R. Rokach, Merck Frosst, Quebec, Quebec, Canada) andcomparing it with standards by RIA and by UV absorbance(see below). The area integration of the adsorption peaksallows quantitative analysis of the substances. By using thedescribed extraction proced,ure, the recovery rates ofleukotrienes from 250 pld of cell supernatant were determinedto be 80 to 85%c for LTC4, LTD4, and LTE4 and 90 to 95% forLTB4 (26). The standard curve for the individualleukotrienes was obtained with five different concentrations(5 to 125 ng) and showed the fQllowing correlations: LTC4,0.985; LTD4, 0.995; LTE4, 0.985; LTB4, 0.999; and 6-transLTB4, 0.998. The minimum detectable quantity was 1 ng forthe various leukotrienes.RIA for LTC4 and LTB4. In addition to HPLC analysis, the

cell supernatants were studied by RIA for LTC4 and LTB4(2, 28, 38). A 50-p.d sample of the supernatants was sus-pended in 30% methanol and evaporated to dryness undernitrogen. The material was then suspended in 100 itl of Trisbuffer (10 mM) containing 140 mM NaCI and 0.1% 'gelatin.An appropriate antiplasma dilution, as well as syntheticLTC4 and LTB4 at concentrations from 10 ng to 20 pg, orunknown samples, were added to tubes containing [3H]LTC4in a total volume of 0.6 ml. After incubation at 4°C overnight,antibody-bound and free ligands were separated with 0.5 mlof charcoal suspension (20 mg/ml) for 2 h at 37°C. Afterprecipitation of the charcoal by centrifugation, 0.9 ml of thesupernatants was added to 9 ml of Scintigel (Roth. Karls-ruhe, Federal Republic of Germany). The radioactivity wasdetermined in a liquid scintillation counter. The minimailquantity detected was approximately 20 pg for LTB4 andLTC4. For the LTC4 determination, the cross-reactivity withLTD4 was <35%; for LTB4 and LTE4, the cross-reactivitywas <2%. The RIA for LTB4 was obtained from WellcomeResearch Laboratories (Beckenham, England). The antise-rum reacted with the isolated LTB4 isomers. The correlationof the results obtained by HPLC analysis and RIA wasdetermined to be r = 0.97 + 0.02 for LTC4 and r = 0.96 ±0.03 for LTB4. In addition to the biochemical and radioim-munological analysis, the leukotrienes in selected fractionswere identified by their characteristic UV spectra (Lambda 5UV Vis spectrophotometer; The Perkin-Elmer Corp., Nor-walk, Conn.).

Histamine release. Rat peritoneal cells (5 to 10% mastcells) were suspended in 0.5 ml of PBS. and 0.1 ml of

bacterial suspension was added. Incubation proceeded for 60min at 37°C. Cells were centrifuged for 10 min at 300 x ,

and the supernatant was removed and deproteinized by theaddition of 2 ml of 2% HCl04. The supernatant was subse-quently centrifuged at 1,000 x g for 10 min; the histaminecontent was analyzed by the fluorophotometric analyzertechnique (27). Cells in the presence of buffer and bacterialsupernatant at the appropriate dilutions served as controls.

RESULTS

Analysis of cell-bound hemolysin during bacterial growth.Since S. imaircescens expresses an extracellular protease,washed bacteria as well as their culture supernatants wereanalyzed for the presence of a protease. It becaute apparentthat only strains 5817 and 5852 released such a protease intothe culture medium (after 3 h) at the late logarithmic phase(Table 1). However, with regard to the cell-derived param-eters under study (chemiluminescence, inflammatory medi-ator release), these strains revealed similar activities ascompared to the non-protease-producing bacteria (Table 1).The hemolysin activity of the various Se-rrtit strains wasanalyzed and compared with the bacterial growth. For S.mnarcescenis 5852, cell-bound hemolysin activity increased atthe beginning of the logarithmic phase. An optimum wasobtained at the late logarithmic phase after approximately 3h. Hemolysin activity decreased with the onset of thestationary phase (8). Hemolysin activity of S. liqueftlciens749611 appeared in the late logarithmic phase and showed aplateau in the stationary phase (data not shown). Thus, theproduction of hemolysin correlated with actively growingbacteria. I'he maximal amount of the total hemogloblinreleased from erythrocytes by the different strains was 86,61, 47. 51, and 66% for the W1436, 5241, 5817, 5852, and7496 strains, respectively (Table 1). No hemolysin activitywaIs present in the bacterial culture supernatant. E. coliJM101(pUC19/1) and E. coli MC4100 HSH 33/1 expressedcell-bound hemolysin activity at the mid-logarithmic growthphase (Table 2). The culture supernatants of these strainswere also hemolysin negative. In addition, strain MC4100HSH 33/1 showed a delayed lag phase which is probably dueto the induction of resistance toward ampicillin. The controlstrains E. coli JMIOI as well as E. coli MC4100 expressed nohemolysin activity when washed bacteria or the superna-tants were studied (Taible 2).

INFE-CT. IMMUN.

on July 8, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

CELL-BOUND HEMOLYSIN IN SERRATIA INFECTIONS 2557

TABLE 2. Induction of chemiluminescence and histamine andleukotriene release by parental E. (oli and E. (oli strains

bearing the Serratia hemolysin gene [E. (oli JM101(pUC19/1):E. (coli MC4100 HSH 33/11'"

Hemo- CL (103 Histamine Leukotrienes (ng)'E. co/i strain Iysin cpm)l (±SD) (ng) LTC4 LTB4

JM101 - 143.9 52.3 ± 3.5 2.0 0.1JM1O1(pUC19/1) + 336.6 91.9 ± 9.7 5.8 0.6

MC4100 - 7.5 99.3 + 1.6 0.1 0.1MC4100 HSH 33/1 + 35.1 259.3 + 59.6 3.9 0.4

" Summary of five independent experiments performed in triplicate. Arepresentative experiment is shown."Determination of chemiluminescence (CL) was done by incubating 2.5 x

10' bacteria with 1 x 10' PMNs. The results were monitored after 15 min.For histamine release. 5 x 10' bacteria were incubated with 2 x 107 rat

peritoneal cells (5 x 104 mast cells) for 60 min.d Leukotriene release was performed with 5 x 10' bacteria and 2 x 107

human PMNs. Incubation proceeded for 15 min (standard deviation = 5 to10%). Analysis was performed by RIA.

Induction of chemiluminescence from PMNs. Hemaggluti-nation of the various strains was analyzed with human,sheep, and guinea pig erythrocytes as target cells. Onlystrains 5817 and 5852 expressed mannose-sensitive hemag-glutinating activity for human, sheep, and guinea pig eryth-rocytes. In contrast, S. mnar escens W1436 and 5241 and S.liquefacnies 749611 revealed mannose-resistant hemagglu-tination, as shown with guinea pig erythrocytes (data notpresented). Studies were done to analyze the degree ofadherence and phagocytosis. For this purpose purified gran-ulocytes were incubated with radiolabeled bacteria. It be-came apparent that strains 5825, W1436, and 7496 showed anadherence ranging from 1.8 to 3.9%, while the phagocytosisrate was 9.2 to 13.6%. No correlation existed between thetype of hemagglutination and the above parameters. Sincethe Serratia strains express cell-bound hemolysin activitywhich most likely represents a pathogenicity factor, theircapacity to induce chemiluminescence from human PMNswas studied. Six separate experiments with neutrophils fromdifferent donors were performed. Although the absolute datavaried from experiment to experiment, a similar profile (Fig.1) was obtained. Strains 5241 (61% hemolysin) and 5817(47.5% hemolysin) proved to be more active than strain749611 (66% hemolysin), which showed a low chemilumine-scence. An optimal response was observed after 5 to 20 minof incubation. W1436 showed an early increase in chemilu-minescence after 5 min of incubation and stayed constantthereafter. The chemiluminescence response of strain 5852steadily increased over the time analyzed. The cloned E. colistrains [E. coli JM101(pUC19/1); E. (coli MC4100 HSH 33/11which reveal mannose-sensitive hemagglutination expressedhigher chemiluminescence than the parental strains (Table2).

Generation of leukotrienes from PMNs. Experiments werethen performed to analyze the generation of leukotrienes.Human PMNs (2 x 107/ml) were incubated with washedbacteria (5 x 108). After 10 min of incubation. the superna-tants were analyzed for LTB4 and LTC4 release. All Serr-atiastrains were able to generate leukotrienes (Table 1). Theamount of LTB4 ranged from 1.4 to 3.0 ng and that of LTC4ranged from 1.3 to 7.7 ng. To study the role of cell-boundSeirratia hemolysin more precisely, we performed kineticexperiments. The washed bacterial strains [W1436. 5817, E.co/i JM101, and E. coli JM101(pUC19/1)] were used asstimuli to activate the granulocytes. An sample of the

bacteria was analyzed for the cell-bound hemolysin activity.In addition, washed E. (oli 536/31(pANN5311) bacteriawhich express o-hemolysin and type Vb MR hemagglutina-tion were included in the studies. The hemolysin titeramounted to 25.3%. Human PMNs in PBS served as thecontrol. After various times (1, 5, 10, 30 min) of incubation,PMNs were analyzed to determine their LTC4 and LTB4release. Five separate experiments in triplicate were done.Although the absolute data varied among the donor cells, asimilar pattern was obtained in each experiment (Fig. 2). Theamount of LTC4 exceeded by two- to fourfold that of LTB4when strains expressing Serratia hemolysin were studied.An optimal release was obtained as early as 10 to 15 min ofincubation. In the described experiment S. inarcescensW1436, which showed a hemolysin titer of 45.7%, released7.7 ng of LTC4 at optimal incubation, while S. marcescens5817 with a hemolysin titer of 27.4% released 5.6 ng of LTC4.E. coli JM101(pUC19/1), expressing the cell-bound hemoly-sin from S. inarcescens (26.2% hemolysin), was more activein generating LTC4 (5.8 ng) and LTB4 (3.9 ng) than E. coliJM1O1: the latter strain induced low amounts of LTC4 (2 ng)and less than 0.1 ng of LTB4. In contrast, E. coli536/31(pANN5311) at the optimal time point induced 31 ng ofLTB4 and 21 ng of LTC4. LTB4 levels rapidly declined, incontrast to LTC4 levels, which showed a rather slow decline.Thus, it is apparent that unlike the E. coli cx-hemolysin, theSerratia hemolysin favors the release of LTC4 and not that ofLTB4. In addition, the Serratia hemolysin was less activethan the x-hemolysin of E. coli. The cellbound hemolysinactivity of S. tna'rcescens decayed more rapidly than thea-hemolysin of E. coli (data not shown).

Experiments were then done to study the hemolysin-dependent induction of leukotriene release. Washed bacteriarW1436. 5817, E. coli JM101(pUC19/1)] at various concen-trations (5 x 108, 2.5 x 108, 1.25 x 108, and 0.6 x 108) wereused as stimuli to trigger human PMNs (2 x 107). Incubationproceeded for 10 min at 37°C. In addition, the bacteria wereanalyzed for their hemolysin content. Leukotriene genera-

EQ.

u

do-,400c

ao

E200.ciC

0 10 20 30

t/minFIG. 1. Induction of chemiluminescence by various Serratia

strains. Human PMNs (1 x 106) were stimulated with 2.5 x 108washed bacteria. A single experiment is shown (nt = 6). Thestandard deviation of each point varied between 3 and 10%.

VOL.55,1987

on July 8, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

2558 KONIG ET AL.

E.coliJM 101 pUC 19/1

ng t

86

4

2

10 20 30min.

Jimlong30

_ 20

10

10 20 30 min.

E.coli 536131pANN 5311

1 5 10 15 30min. (time)S. marcescens

5817ng

_

6

22

10 20 30 mim

-*- LT CL

_ LTB4

10 20 30min. (time)

FIG. 2. Kinetics of LTC4 and LTB4 release from human granulocytes (2 x 107); 5 x 108 bacteria were applied as stimuli. The standarddeviation was 5 to 10%. E. coli JM101(pUC19/1) carries the cloned Serr-atia hemolysin gene which was cloned into the parental strain E. coliJM101. E. coli 536/31(pANN5311) expresses co-hemolysin. Hemolysin (Hly) titers were as follows: E. coli JM101(pUC19/1) (Serratia Hly+),26.2%; E. coli JM101 (Se-rratia Hly-), 0%: E. coli 536/36(pANN5311) (E. coli Hly+), 25.3%; S. mnarcescens W1437 (Hly+), 45.7%; S.inarcescens 5817 (Hly+), 27.4%. A single experiment performed in triplicate of five total experiments is shown. Leukotrienes were detectedby RIA and for E. coli 536/31(pANN5311) by RIA and HPLC.

tion was clearly dependent on the amount of hemolysinformation (Fig. 3). LTC4 release exceeded that of LTB4. Ata given hemolysin concentration (20%), strain W1436 provedto be more active than strain 5817, while E. coli JM101(pUC19/1) was inactive. However, at higher hemolysinactivity (40%), LTC4 release induced by E. coli JM101(pUC19/1) exceeded the amount obtained with W1436. In-terestingly, more LTC4 than LTB4 was released.

Induction of histamine release from rat mast cells. Experi-ments were then done to study histamine release from mastcells after their stimulation with bacteria (Fig. 4). For thispurpose rat peritoneal cells (5 x 104 mast cells) were

incubated with various concentrations (5 x 108 to 5 x 109) ofthe washed clinical isolates (S. marcescens W1436, 5241,5852, 5817; S. /iquiefaciens 749611) for 60 min at 37°C. It wasapparent that strain W1436, which exhibited the highesthemolysin activity, induced the strongest histamine release.

S. marcescensW 1436

No lactate dehydrogenase was obtained in the cellularsupernatant, indicating that the release occurred undernoncytotoxic conditions (data not shown). In comparison,strain 7496, which was the second most active strain inhemnolysin production (66%), induced less histamine thanstrain 5817 (47.5% hemolysin production) (Fig. 4; Table 1).Experiments were then performed in which histamine re-

lease was related to hemolysin production (Fig. 5). Washedbacteria were incubated at various dilutions with mast cells(5 x 104) for 60 min at 37°C. The bacteria-bound hemolysinand the induction of histamine release were determined.Also, this experiment demonstrated that strain 7496 express-ing 66% hemolysin is less active than strains 5241 or 5817showing 61 and 47.5% hemolysin activity, respectively. Ifone compares the capacity of the bacteria to induce hista-mine release referring to a hemolysin activity of 5% thefollowing results were obtained. Strains W1436 (86% hemo-

E. coliJMlOl pUC19/15817

ng ng ng

_u 6 12

0 4 4 8

'3~2k2 4

20 40 60%Hly 20 40 60%Hly 20 40 60%HlyFIG. 3. Hemolysin-dependent induction of leukotriene release. The experiment was performed four times in triplicate. A representative

experiment is shown. The standard deviation was 5 to 10%. Washed bacteria at various concentrations (5 x 10' to 0.6 x 108) were used totrigger human granulocytes (2 x 107). Incubation proceeded for 10 min at 370C. In addition, the bacteria were analyzed for their hemolysincontent. Leukotrienes were detected by RIA. Symbols: 0. LTC4; *, LTB4.

nga)

0-XL6±4

cu2

W 1436nga)C 8.-6-0

4

a, 2

INFECT. IMMUN.

I

50

2

on July 8, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

CELL-BOUND HEMOLYSIN IN SERRATIA INFECTIONS 2559

300

cmC

a. 200c

v

100

00 1 2 3 4 5

109cells (bacteria)

FIG. 4. Induction of histamine release from rat peritoneal maistcells by washed Serr/atial strains. Data represent mean values ofthree independent measurements performed in triplicate. The stan-dard deviation (bars) was 6 to 12%: cells in the absence of stimulusshowed -26.0 + 1.4 ng of histamine release (basal histaminerelease): total histamine content within the cells was -599.6 ± 31.9ng. Symbols: C]. W1436; 0. 5241; A: 5852: x. 5817 *0. 749611.

cell-bound hemolysin no clear correlation was obtained. S.Inarcescens W1436 (86% hemolysin) was less active thanstrain 5817. which expressed only 47.5% of cell-boundhemolysin activity. The latter strain also released a proteaseduring bacterial growth which may well contribute to thegeneration of oxygen radicals. In fact, strain W1436 is amutant which is exoprotease and lipase negative. Interest-ingly, W1436 is the most potent strain in inducing histaminerelease from rat mast cells, while strain 5817 is by far lessactive. However, S. liqu,efaciens 749611 expressing 66% ofcell-bound hemolysin activity only induced 87.3 ng of hista-mine, whereas strain 5241 with 61% hemolysin activitygenerated about twice as much histamine. From these data itappears likely that the process leading to the hemolysis oferythrocytes is not identical to the one releasing histaminefrom mast cells. Furthermore, one might suggest that dif-ferent hemolysins exist expressing a varying degree ofactivity for rat mast cells. It has been suggested that theexistence of ox-hemolysin complexed with lipopolysaccha-ride has important implications in the understanding of itsbiological effects (5). These data suggest that the complexedlipopolysaccharide is important in pathogenicity or hemoly-sin activity. The E. coli strains expressing cell-bound hemo-lysin activity from S. marcescens were potent inducers forthe generation of oxygen radicals. The parental strains E.(oli JM101 and MC4100 expressed type 1 fimbriae, whichhave been shown to induce a chemiluminescence responsefrom human polymorphonuclear granulocytes (3, 4, 37).However, the presence of Serratia hemolysin enhanced thisresponse by 2.5- to 5-fold. In addition, a two- to threefoldincrease in histamine release was obtained with the (Serraati

lysin), 7496 (66% hemolysin), 5852 (51% hemolysin), and5817 (47.5% hemolysin) induced 182.5, 49.3, 122.7, 82.5. and107.5 ng of histamine release. These data suggest that inaddition to the hemolysin, either other factors contribute tothe release of histamine or various hemolysins occur inSerratia strains. The Serriatia hemolysin cloned into E. (olistrains induced significant amounts of histamine. Withwashed E. coli JM101(pUC19/1) and MC4100 HSH 33/1bacteria, a two- to threefold increase in histamine release ascompared with that of the parental strains was observed(Table 2). When the bacterial culture supernatants wereanalyzed, the parental as well as the cloned strains showedno differences in their capacity to induce histamine release(data not shown).

DISCUSSION

It was demonstrated above that among the Sera-titiu strainsanalyzed, the expression of hemolysin activity is dependenton active cell growth and reaches its optimum at the mid-logarithmic phase up to the onset of the lag phase. Further-more, the data also show the expression of cell-boundhemolysin activity in E. coli strains that contained a clonedhemolysin gene from S. marcescens. Evidence had beenpresented elsewhere that only the genes required for theexpression of the S. inar/cesceens hemolysin had been clonedinto E. coli (9). All Serlaltia strains were capable of inducinga time-dependent chemiluminescence response from humanPMNs. The cellular response could not be correlated to thehemagglutination pattern of the different erythrocytes, sug-gesting that the receptor on erythrocytes is either not presentor not involved in the activation process of human polymor-phonuclear neutrophils or rat peritoneal mast cells. Whenthe maximal values were compared with the expression of

20 r5852

1 5 k

C0-

Ec

10L

5

o

W 1436

0 60 120 180 240 300 360

ng histamine

FIG. 5. Hemolysin-dependent induction of histamine releasewith washed clinical isolates of Se-rratita strains. Washed bacteriawere used at various concentrations (5 x 108 to 0.6 x 108).Incubation proceeded for 60 min. Data represent mean values ofthree independent measurements performed in triplicate. The stan-dard deviation was 6 to 12%: cells in the absence of stimulus showed-20.8 ± 2.6 ng of histamine (basal histamine release): total hista-mine content was -424.1 + 11.5 ng.

VOL. 55, 1987

on July 8, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

2560 KONIG ET AL.

hemolysin-positive) E. coli clones as compared with theparental strains.The latter strains also triggered cells for histamine release.

However, our recent studies with genetically cloned E. colistrains suggested that type 1 fimbriae are poor inducers ofhistamine release, unlike those strains expressing S-Mrh(28). These differences only became apparent when hemo-lysin-negative strains were studied. In the presence ofhemolysin, histamine release exceeded by far the amountsinduced by adhesins (S-Mrh). In addition to adhesins, addi-tional structures of the bacterial surface may account formast cell triggering. A nonimmunological (lectin-mediated)mechanism has been recently suggested which can occurwith the cell-bound immunoglobulin E without the personbeing sensitized to the microorganism in question (35).Again, the absolute quantities are much lower as comparedwith the hemolysin-induced histamine release.With regard to the release of leukotrienes, all Serratia

strains including the E. coli clones induced significantamounts of LTC4 and low amounts of LTB4; in comparisonwith the results obtained from PMNs after stimulation withthe calcium ionophore A23187, these values were, however,8- to 20-fold less (6, 36). In addition, the results for leuko-triene release induced either with the S. marcescens strainsor with the E. coli strains containing a hemolysin gene fromS. marcescens are in general lower than those obtained withthe a-hemolysin-producing E. coli 536/31 (pANN5311). Ingeneral, LTC4 levels exceeded those of LTB4. These dataare in contrast to those obtained for the a-hemolysin of E.coli (39), for the calcium ionophore A23187, and duringphagocytosis (38). Quite recently, we presented evidencethat various bacterial toxins, e.g., the thiol-activatable tox-ins, led to pronounced LTC4 release from human PMNswhich exceeded by far that of LTB4 (13). In addition,evidence was obtained that toxin-pretreated cells metabo-lized exogenous LTB4 into the biologically inactive w-oxidation products 20-OH-LTB4 and 20-COOH-LTB4 byincreasing LTB4 hydrolase activity of human PMNs (datanot shown). Such a mechanism may contribute to the factthat the amount of LTC4 generated exceeds that of LTB4. Atpresent, it can only be speculated why the ox-hemolysin of E.coli is more active in inducing leukotriene release than thehemolysin of S. inarcescens although similar amounts ofhemolysis as well as histamine release are obtained. Incontrast to E. coli o-hemolysin, the cell-bound Serratiahemolysin shows a rapid decay of hemolysin activity innongrowing bacteria (data not shown).

It is well established that activation of mast cells forhistamine release and of granulocytes for leukotriene gener-ation proceeds via phospholipase activation of the cells (6,29). We suggest that in addition to structural differences ofthe hemolysin, the susceptibility of the various cell types forthe E. coli and Serratia hemolysins as well as the biochem-ical pathways which are involved in mediator release aredifferent; one also has to consider differences as to thepotential receptive sites (receptors) for the hemolysin onhuman PMNs. In any event, the hemolysin of S. marcescensappears to be a potent inducer of histamine release whichoccurs under noncytotoxic conditions in a concentration-dependent manner for each bacterial strain studied. A he-molysin-negative strain of S. marcescens was not availableas a control despite extensive mutation experiments. There-fore, the hemolysin of S. marcescens was cloned into E. coli.Both strains (Serratia hemolysin producing) were moreactive in inducing histamine and leukotriene release from thetarget cells than were their parental strains (E. coli JM101

and E. co/i MC4100). Thus, our data clearly provide evi-dence for the role of cell-bound hemolysin of S. marcescensas a stimulus for histamine release as well as leukotrienegeneration.

In addition to the known pathogenicity factors, the pres-ence of cell-bound hemolysin may contribute to Serratiainfections. The release of inflammatory mediators may ac-count for the increase in vascular permeability, edemaformation, and granulocyte accumulation, which are pre-dominant features in bacterial inflammation.

ACKNOWLEDGMENT

This study was supported by Deutsche Forschungsgemeinschaft-''Pathogenitatsmechanismen."

LITERATURE CITED1. Adeybola, R. A., and D. C. Old. 1982. New fimbrial hemagglu-

tination in Serratia species. Infect. lmmun. 38:306-315.2. Aehringhaus, U., R. H. Wolbling, W. Konig, C. Patrono, B. M.

Peskar, and B. A. Peskar. 1982. Release of leukotriene C4 fromhuman polymorphonuclear leukocytes as determined by radio-immunoassay. FEBS Lett. 146:111-114.

3. Bjorksten, B., and T. Wasstrom. 1982. Interaction of Esche-richia coli with different fimbriae and polymorphonuclear leu-kocytes. Infect. Immun. 38:298-305.

4. Blumenstock, E., and K. Jann. 1982. Adhesion of piliatedEscherichia coli strains to phagocytes; differences betweenbacteria with mannose-sensitive pili and those with mannose-resistant pili. Infect. Immun. 35:264-269.

5. Bohach, G. A., and I. S. Snyder. 1985. Chemical and immuno-logical analysis of the complex structure of Escherichia colioL-hemolysin. J. Bacteriol. 164:1071-1080.

6. Borgeat, P., V. Fruteau de Laclos, H. Rabinovitch, S. Picard, P.Braquet, J. Hebert, and M. Laviolette. 1984. Generation andstructure of the lipoxygenase products. Eosinophil-rich humanpolymorphonuclear leukocyte preparations characteristicallyrelease leukotriene C4 on ionophore A 23187 challenge. J.Allergy Clin. Immunol. 74:310-315.

7. Boyum, A. 1968. A one stage procedure for isolation of granu-locytes and lymphocytes from human blood. General sedimen-tation properties of white blood cells in lg gravity field. Scand.J. Clin. Lab. Invest. 21(Suppl. 97):51-76.

8. Braun, V., H. Gunther, B. NeuBl, and C. Tantz. 1985. Hemolyticactivity of Serratia mnarcescens. Arch. Microbiol. 141:371-376.

9. Braun, V., B. NeuB3, Y. Ruan, E. Scheibel, A Schoffler, and G.Jander. 1987. Identification of the Serratia marcescens hemo-lysin determinant by cloning into Escherichia coli. J. Bacteriol.169:2113-2120.

10. Bray, M. A., F. M. Cunningham, A. W. Ford-Hutchinson, andM. R. H. Smith. 1981. Leukotriene B4: a mediator of vascularpermeability. Br. J. Pharmacol. 72:483-486.

11. Bremm, K. D., J. Brom, J. E. Alouf, W. Konig, B. Spur, A.Crea, and W. Peters. 1984. Generation of leukotrienes fromhuman granulocytes by alveolysin from Bacillus ailvei. Infect.Immun. 44:188-193.

12. Bremm, K. D., J. Brom, W. Konig, B. Spur, A. Crea, S. Bhakdi,F. Lutz, and J. Fehrenbach. 1983. Generation of human leu-kotrienes and lipoxygenase factors from human polymorpho-nuclear granulocytes during bacterial phagocytosis and interac-tion with bacterial exotoxins. Zentralbl. Bakteriol. Parasitenkd.Infektionskr. Hyg. Abt. 1 Orig. Reihe B 254:500-514.

13. Bremm, K. D., W. Konig, P. Pfeiffer, I. Rauschen, K. Theobald,M. Thelestam, and J. E. Alouf. 1985. Effect of thiol-activatedtoxins (streptolysin 0, alveolysin, and theta-toxin) on the gen-eration of leukotrienes and leukotriene-inducing and -metabo-lizing enzymes from human polymorphonuclear granulocytes.Infect. Immun. 50:844-851.

14. Bremm, K. D., W. Konig, B. Spur, A. Crea, and C. Galanos.1984. Generation of slow reacting substance (leukotrienes) byendotoxin and lipid A. Immunology 53:299-305.

15. Bretz, U., and M. Baggiolini. 1974. Biochemical and morpho-

INFECT. IMMUN.

on July 8, 2018 by guesthttp://iai.asm

.org/D

ownloaded from

CELL-BOUND HEMOLYSIN IN SERRATIA INFECTIONS 2561

logical characterization of azurophilic and specific granules ofhuman polymorphonuclear leukocytes. J. Cell Biol. 63:251-256.

16. Cavalieri, S. J., and I. S. Snyder. 1982. Cytotoxic activity ofpartially purified Escheric/ija coli x-hemolysin. J. Med. Micro-biol. 15:11-21.

17. Clegg, S., S. Hull, R. Hull, and J. Pruckler. 1985. Constructionand comparison of recombinant plasmids encoding type 1 fim-briae of members of the family Enterobacteriaceae. Infect.Immun. 48:275-279.

18. Doerr, M., and W. H. Traub. 1984. Purification and character-ization of two Serrlatia marcescens proteases. Zentralbl.Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A257:6-19.

19. Evans, 0. J., Jr., 0. G. Evans, L. S. Young, and J. Pitt. 1980.Hemagglutination typing of Eschlerichia coli: definition of sevenhemagglutination types. J. Clin. Microbiol. 12:235-242.

20. Gadeberg, 0. V., I. 0rskov, and J. M. Rhodes. 1983. Cytotoxiceffect of alpha hemolytic Escherichia coli strains on humanblood monocytes and granulocytes in vitro. Infect. Immun.41:358-364.

21. Hacker, J., G. Schroter, A. Schrettenbrunner, C. Hughes, andW. Goebel. 1983. Hemolytic Esclierichiiia c oli strains in thehuman fecal flora as potential urinary pathogens. Zentralbl.Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A254:370-378.

22. Hughes, C., D. Muller, J. Hacker, and W. Goebel. 1982. Genet-ics and pathogenic role of Eschericlia coli hemolysin. Toxicon20:247-252.

23. Jessop, H. J., and P. A. Lambert. 1985. Immunochemicalcharacterization of the outer membrane complex of Seriratiamnarcescens and identification of the antigens accessible to anti-bodies on the cell surface. J. Gen. Microbiol. 131:2343-2348.

24. Kamata, R., H. Yamamoto, K. Matsumoto, and H. Maeda. 1985.A Serraatia protease causes vascular permeability reaction byactivation of the Hageman factor-dependent pathway in guineapig. Infect. Immun. 48:747-753.

25. Kohno, K., T. Yamamoto, A. Kuroiwa, and K. Amako. 1984.Purification and characterization of Serratia inarcescens US5pili. Infect. Immun. 46:295-300.

26. Koller, M., W. Schonfeld, J. Knoller, K. D. Bremm, W. Konig,B. Spur, A. Crea, and W. Peters. 1985. The metabolism ofleukotrienes in blood plasma studied by high performance liquidchromatography. Biochim. Biophys. Acta 883:128-134.

27. Konig, W., and K. Ishizaka. 1976. Binding of rat IgE with thesubcellular components of normal rat mast cells. Immunochem-istry 13:345-353.

28. Konig, B., W. Konig, J. Scheffer, J. Hacker, and W. Goebel.1986. Role of Escherichia coli alpha-hemolysin and bacterialadherence in infection: requirement for release of inflammatorymediators from granulocytes and mast cells. Infect. Immun.54:886-892.

29. Konig, W., W. Kunau, and P. Borgeat. 1982. Induction andcomparison of the eosinophil chemotactic factor with endoge-nous hydroxyeicosatetraenoic acids: its inhibition by arachi-donic acid analogs, p. 301-314. In B. Samuelsson and R.Paoletti (ed.), Leukotrienes and other lipoxygenase products,vol. 9. Raven Press, New York.

30. Linggood, M. A., and P. L. Ingram. 1982. The role of alpha-

hemolysin in the virulence of Escherichia coli for mice. J. Med.Microbiol. 15:23-30.

31. Lyerly, D. M., and A. S. Kreger. 1983. Importance of Serratiaprotease in the pathogenesis of experimental Seirlatia inarces-cens pneumonia. Infect. Immun. 40:113-119.

32. Maki, D. G., C. G. Hennekens, C. W. Philips, W. V. Shaw, andJ. V. Bennet. 1973. Nosocomial urinary tract infection withSerri-atia Inarcescens. J. Infect. Dis. 128:579-587.

33. Matsumoto, K., H. Maeda, K. Takata, R. Kamata, and R.Okamura. 1984. Purification and characterization of four prote-ases from a clinical isolate of Serratia marcescens kums 3958. J.Bacteriol. 157:225-232.

34. Matsumoto, K., T. Yamamoto, R. Kamata, and H. Maeda. 1984.Pathogenesis of serr-atial infection: activation of the Hagemanfactor-prekallikrein cascade by serratial protease. J. Biochem.96:739-749.

35. Norn, S., P. Stakl Skov, C. Jensen, 0. Jarl0v, and F. Espersen.1987. Histamine release induced by bacteria. A new mechanismin asthma. Agents Action 20:29-34.

36. Palmer, R. M. J., and J. A. Salmon. 1983. Release of leukotrieneB4 from human neutrophils and its relationship to degranulationby N-formyl-methionyl-leucyl-phenylalanine, serum treatedzymosan and the ionophore A 23187. Immunology 50:65-73.

37. Perry, A., I. Ofek, and J. F. Silverblatt. 1983. Enhancement ofmannose-mediated stimulation of human granulocytes by type 1fimbriae aggregated with antibodies on Escherichia coli sur-faces. Infect. Immun. 39:1334-1345.

38. Raulf, M., M. Stuning, and W. Konig. 1985. Metabolism ofleukotrienes by L-y-glutamyltranspeptidase and dipeptidasefrom human polymorphonuclear granulocytes. Immunology55: 135-147.

39. Scheffer, J., W. Konig, J. Hacker, and W. Goebel. 1985. Bacte-rial adherence and hemolysin production from Escherichia coliinduces histamine and leukotriene release from various cells.Infect. Immun. 50:271-278.

40. Scheffer, J., K. Vosbeck, and W. Konig. 1986. Induction ofinflammatory mediators from human polymorphonuclear gran-ulocytes and rat mast cells by hemolysin-positive and -negativeE. coli strains with different adhesins. Immunology 59:541-548.

41. Traub, W. H. 1982. Virulence of nosocomial isolates of Serr-atiamnarcescens for NMRI mice: correlation with 0-antigens 06 and014. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt.1 Orig. Reihe A 252:360-369.

42. Traub, W. H. 1983. Passive protection of NMRI mice againstSerraltia marcescens: correlative efficacy of commercial humanIgG immunoglobulin preparations and rabbit anti-O -H, -K,-life cell and -protease immune sera. Zentralbl. Bakteriol. Par-asitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 254:480-488.

43. Traub, W. H., and D. Bauer. 1985. Fibrinolytic activity ofpurified Se-rratia itnarcescens metalloproteases. Zentralbl. Bak-teriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A260:35-40.

44. Waalwijk, C., D. M. MacLaren, and J. de Graaff. 1983. In vivofunction of hemolysin in the nephropathogenicity of Escherichiacoli. Infect. Immun. 42:235-249.

45. Welch, R. A., E. Patchen Dellinger, B. Minshew, and S. Falkow.1981. Hemolysin contributes to virulence of extra-intestinal E./oli infections. Nature (London) 294:665-667.

VOL. 55, 1987

on July 8, 2018 by guesthttp://iai.asm

.org/D

ownloaded from