Bactericidal substance produced by Haemophilus injluenzae b' Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIVERSITY OF MICHIGAN on 11/10/14 For personal use only.

R. A. VENEZIAA N D R. G. ROBERTSON Depnrtmertt oj'Microbiology, University yfRoclle.rtcr Scl~oolof Medicine and Dentistry, Roc/tc,ster, Nett, York 14642 Accepted June 16, 1975 VENEZIA, R. A., and R. G. ROBERTSON. 1975. Bactericidal substance produced by Hnemopl~il~ts inflrrenzne b. Can. J. Microbiol.21: 1587-1594. During bacteriophage studies on Haentophil~rsit$rren;ne, it was observed that encapsulated type b and unencapsulated Rb strains released a bactericidal substance active against types a, c.. d, e, a n d f H . injlrerlzne, non-typable H. ir~jrrenznestrains, other Hae~nophilrrsspecies, and certain members of the Enterobacteriaceae. The bactericidal activity was assayed by a plaque test utilizing an Rd strain as an Indicator lawn and was also demonstrated in mixed broth cultures ofaproducer strain and an indicator strain. Immediate lysis of sensitive bacteria by the factor was not evident. The factor is sensitive to trypsin but resistant to deoxyribonuclease, treatment with 2-mercaptoethanol, lipase, @-amylase,and heating in a 100 "C water bath for 20 min. The activity is not dependent upon increased Ca2+or Mg2+concentration as is necessary for HPlCl and S2 phage propagation. The bactericidal factor is not pelleted by high-speed centrifugation at 150000 x g for 6 h. Treatment with ultraviolet light or rnitornycin C does not result in observablephage. phage-l~keparticles, or increased bactericidal activity. The bactericidal factor is not a typical small molecular weight "colicin-like'' bacteriocin in that it is not inducible, has a wider range of activity, and does not kill by "single-hit" kinetics. On preliminary characterization, it is a thermostable protein toxic to certain bacterial strains. VENEZIA, R. A., et R. G. ROBERTSON. 1975. Bactericidal substance produced by Hnemophilrrs itflrre~zzcieb. Can. J . Microbial. 21: 1587-1594. Au cours d'etudes sur les phages d'Hcrernophilrrs ir~Jrrenzne, i l a ett observe que les souches capsul6es du type b et les souches non-capsultes Rbproduisaient une substance activecontre les types rr, c, d, e e t f d'H. injrtenzrre, des souches non-typables d'H. i~flrrenzoe,d'autres especes d'Hnen~ophilrrset certaines Enterobacteries. Cette activitk bactericide a ete mesuree par la technique des plages en ensernencant la souche Rd cornme indicateur et a aussi ete demontree dans des cultures mixtes en bouillon d'une souche productrice et d'une souche indicatrice. La lyse immediate par ce facteur des bacteries sensibles n'est pas evidente. Ce facteur est sensible la trypsine mais resistant h la dCoxyribonuclease, au traiternent par le 2-mercaptoethanol, a la lipase, al'a-amylase et h un chauffage de2O rnindansun bain-marie a 100°C. L'activite ne depend pas d'une augmentation de la concentration des ions Ca2+ ou Mg2+ qui sont necessaires a la propagation des phages HPICl et S2. Ce facteur bactericide n'est pas sediment6 apres 6 h d'ultracentrifugation a 150000 x g . Un traitement par les rayons ultravioletsou la mitomycine C n'entraine pas I'apparition de phages ou de particules d'allure phagique et ne cause pas d'augmentation de I'activite bactericide. Ce facteur bactericide n'est pas une bacteriocine typique "d'allure colicine" de faible poids moleculaire par le fait qu'il n'est pas inductible, a un champ d'activite plus large et ne tue pas selon une cinetique d'un "coup unique." La caracterisation preliminaire indique qu'il s'agit d'une proteine thermostable toxique vis-a-vis d e certaines especes bacteriennes. [Traduit par le journal]

Introduction There have been extensive studies into batterial antagonism between normal flora and pathogens of the gut (7, 11). In addition, batteria] antagonism or interference has been noted in the respiratory tract. For example, streptococci from the respiratory tract produce a low molecular weight factor which is resistant to nuclease, lipase, and protease activity and acts against both Gram-positive and Gram-negative 'Received March 10, 1975.

organisms (19). In turn, this streptococcus is inhibited by certain other flora of the upper respiratory tract (4). H a e m o ~ h i l u sspecies, One of the more common groups inhabiting the upper to respiratory tract (159 181, have been induce interferon production (5) and to inhibit ciliary motion in mammalian cell cultures (6). However, there have been no previous observations of H. influeflzaeproducing factors that might affect other flora. During studies on the bacteriophage of H. injluenzae we observed bactericidal activity among certain H . inguenzae

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strains. This report consists of our preliminary observations of a bactericidal substance produced by H. influenzae 6. Materials and Methods Bacterial Strains The clinical isolates of H. inprrenzae were obtained from the Bacteriology Laboratory, Strong Memorial Hospital, Rochester, New York. All required both hemin and nicotinamide adenine dinucleotide for aerobic growth and did not produce hemolysis on 7% rabbit blood agar. Serotyping was done with Hyland (Hyland Laboratory, Costa Mesa, California) H. inflrrenzae typing sera a-f according to the manufacturer's instructions: 28 H. influenzae strains typed as b while 32 H. it~fie~rzae were non-typable. H. i11/71renzae a, r , d, e, a n d f were obtained from Dr. J. Stuy of The Florida State University, Tallahassee, Florida. H. in7fl1rer1zaestrain C25, a rough mutant originally derived from a type d encapsulated organism, was provided by Dr. S. Goodgal, Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Other bacterial strains were clinical isolates identified in the Bacteriology Laboratory, Strong Memorial Hospital. H. inflrrenzae b (strain 105) and C25 were studied as the bactericidal factor producer and sensitive strains, respectively. Changes in turbidity during growth of bacteria in liquid culture were measured with a Klett-Summerson colorimeter (500-570 nm). Media Brain heart infusion (BHI (Difco) (3.7%)) was supplemented with 2 pg/ml nicotinamide adenine dinucleotide (NAD) (Sigma) and a 1 :3000 final dilution of lysed, defibrinated horse red blood cells (1) or I0 ~ g / m lhemin (Kodak) (16). The solid growth medium contained 2.7% BHI, 0.75% eugonbroth (Baltimore Biological Laboratories (BBL)), 1.2% agar (BBL), and was supplemented with NAD and hemin as above. Dilutions were made in buffer which contained 0.6% NazHPO,, 0.3% KHzPO,, 0.05% NaCI, and 0.1% NH4CI (16). Bactericidal Factor Prodrrction The supplemented BHI (sBHI) was inoculated from an overnight broth culture and shaken in a 37 "C water bath shaker at 110 rpm until the culture reached stationary phase (300 Klett units), The cells were removed by centrifugation at 5000 x g for 15 min at 4 "C; the supernatant was decanted and filtered through a 0.45-pm pore size filter (Millipore). ( N H 4 ) 8 0 4 was added slowly to the filtrate to 50% saturation and stirred for 1 h at 4 "C. The precipitate was collected by centrifugation at 12 000 x g for 30 min at 4 "C, and resuspended in 0.01 M TrisZ-HCI buffer pH 8.0 at 5% of the original volume. Further concentration was achieved with a Diaflo ultrafiltration apparatus (Amicon) with an XM 50 membrane filter. The liquid retained in the membrane cell was filtersterilized and heated in a 100 "C or 65 "C water bath for 20 min. The precipitate was removed from suspension by centrifugation at 5000 x g for 15 min at 4 ' C . The supernatant, which retained the activity, was stored at 4 "C.

Bactericidal Assay Three types of biological assays were used to measure the activity of the bactericidal factor. Cells for bacterial lawns were grown to about lo8 cells/ml (30 Klett units) and stored in 15% glycerol at -70°C. There was no detectable loss of viability of stored cells for at least 2 weeks. The assays were performed as follows. (I) Plate assay-This was used for qualitative screening of bacterial strains. About 5 x lo6 cells/ml were swabbed onto sBHI plates and overstreaked with test strains and the plates were then incubated for 18 h a t 37 "C. Sensitive lawns showed zones of clearing around streaks of producer strains. For qualitative assay of the bacterial factor from broth cultures, 10 p1 of test solution was spotted on lawns of sensitive strains, and the plates incubated as above. (2) Qrrantitatiue assay-Circular wells, 2 x 2 mm, were cut into sBH1 agar plates (20 ml per plate). Lawns were prepared a s above and 10-pl samples of test solutions were added to the wells, and the plates were then incubated as above. Zones ofclearing were measured along two axes by a Fisher-Lilly antibiotic zone reader. All zones were standardized to millimetres per milligram of protein per millilitre. Protein estimations were performed according to Lowry (10). (3) Suruiuor curue.r-The activity of the extract was also assayed by measuring the proportion of bacteria surviving in the presence of the bactericidal factor for up to 30 min. Strain C25 was grown to 108cells/ml a n d diluted to lo4 cellslml into reaction mixtures containing dilutions of the bacterial factor, or preparations from non-producing strains, or of B H I in buffer. These were incubated at 22 "C and samples were taken at appropriate times, diluted to stop further reaction, and plated o n sBHI agar for determination of viability. T o determine the effect of the bactericidal factor o n the cell's integrity, turbidity changes were measured in a Klett colorimeter, and a spectrophotometer at 585 nm. About lo8 cellslml were reacted with the bactericidal factor at a dilution which gave 95% killing of sensitive cells by 15 min a t 22°C. Turbidity changes were monitorcd for 2 h. Mixed Crtlture Grorvth T o ascertain activity in mixed cultures of strains 105 and C25, a streptomycin-resistant mutant of strain C25 (C25 StrR4)was selected that was resistant to 200 ~ g / m l streptomycin. Briefly, a culture of C25 was grown to contain 5 x lo9 cells/ml, the cells pelleted by centrifugation, and resuspended in the salts buffer at 10% the original volume. Aliquots of this suspension were plated on sBHI agar containing 200 pg/ml streptomycin sulfate (Calbiochem). After overnight incubation at 37 "C in 5% C 0 2 , colonies were picked and transferred on the streptomycin-con taining agar. Those selected were similar to the parental C25 strain in rate of growth, lack of streptomycin dependence, phage propagation, and sensitivity to the bactericidal factor. Overnight precultures of C25 StrR4and 105 were diluted so that a culture flask contained each strain in ratio o f 1 :I. A second flask with C25 StrR4and 105 in a ratio of 1 :lo respectively was also prepared. The mixtures of the two strains were monitored by duplicate plate counts o n sBHI agar and sBHI agar with 200 pg/ml streptomycin. Control cultures of single strains were also monitored.

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VENEZIA AND ROBERTSON: BACTERICIDAL SUBSTANCE FROM HAEMOPHILUS

FIG. 1. Qualitative test for inhibition of sensitive H. influenzae strain C25 by H. inflrrenzae encapsulated strains: (A) type a ; (B) type b; (C) typec; (D) type d ; (E) type e ; (F) type f. Theseserotypes were overstreaked onto a lawn of a sensitive strain. Phage or Barleriocin Induction UV and mitomycin C induction of H. influenzae b strains were performed according to Stuy (17) and Gromkova et a/. (9). After treatment, the cells were pelleted by centrifugation at 5000 x g for 15 min. The supernatants were filter-sterilized and spun at 40 000 x g for 90 min, the liquid decanted, and then centrifuged at 150 000 x g for 6 h in a Beckman model LS-50 ultracentrifuge. Any pellet was resuspended in a minimal volume of the salts buffer or 0.01 M Tris-HC1 buffer, pH 8. A drop of this suspension was air-dried on carbon-coated 250-mesh copper grids, negatively stained with 1% phosphotungstic acid, pH 7, and observed with a Zeiss EM4 electron microscope. Both the sediment and supernatant were assayed for bactericidal activity. Selection of Rb Strains Selection of non-typable forms of rough mutants of strain 105 (Rb) was accomplished by the use of specific antibodies t o polyribose phosphate (PRP). P R P is the main capsular component of H. influenzae b. A streptomycin-resistant mutant of strain 105 was selected as above. Then about log cells/ml were reacted with an equal volume of antiserum for 30 min at 37 "C and overnight at 4 "C. Most of the cells agglutinated and precipitated out of suspension. A 0.1-ml volume of the culture was spread on sBHI plates and after 18 h incubation at 37 "C in 5% COz, gray, noniridescent colonies were observed and subcultured. These strains were shown to lack a capsule by their failure to agglutinate with typing serum and by increased phage adsorption over the parental strain

(unpublished data). All maintained the steptomycin resistance of the parental strain. In addition, we obtained a rough mutant of H. influenzae b (Strain Santo, Grace Leidy, New York) from Dr. J. Stuy for study along with our R b strains. Enzymes and Chenlicals T o determine the effect of enzymes and 2-mercaptoethanol, the bactericidal factor was incubated for 30 min at 37°C with each of the following reagents: 1 mg/ml 2 x recrystallized trypsin (Worthington Lab), 0.5% protease (Sigma), 500 bg/ml deoxyribonuclease (Worthington Lab), 10 mg/ml wheat germ lipase (Worthington Lab), 10 bg/ml a-amylase (Worthington Lab), and 0.01 M and 0.1 M 2-mercaptoethanol (Sigma). T h e treated preparations were then measured for bactericidal activity by the plate assay.

Results Range of Inhibitory Activity All 28 H. injfuenzae b strains produced a bactericidal substance which killed all 32 nontypable H. injfuenzae strains. None of the 28 type b strains and Rb strains was susceptible to the factor when tested on solid medium by the plate assay. Of the non-typable H. injfuenzae strains none produced any bactericidal activity except for the Rb mutants, which are non-typable

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TABLE 1 Range of inhibitory activity of the bactericidal factor

Test organism Acinetobacter calcoaccticus Citrobacter species Diplococcus pneumoniae Diphtheroid Esclrevichia coli Flavobacterium species Haetnophilus lraetnolyticus Haetnophilrrs infllret~zae (non-typable) H. itrj711enzaeb H. it~/?rrenzaea H, i~rflrre~rzae c H. itrjfrretrzae d H . itrflrietrzne e H. itrj7~retizae,f' H. itrflrretrzae R b H . paraitrfl~retrzae

Qualitative plate assay, No. resistantlno. tested 111 013 111 111 6/29 111 113 0132 28/28 012 o/ I o/ 1 012 012 515 2/10

strains of H. irlfluenzae b. Other species of Haernopl~iluswere also sensitive; these included H. haemolyticus, H. parallaernolyticus, 8 of 10 strains of H. parair?fluer?zae, and encapsulated H. influeilzae types a, c, d, e, and f (Fig. 1). A variety of Gram-negative bacilli were found to be sensitive, i.e. 23 of 29 E. coli strains, 3 Citrobacter strains, 2 Shigella soilnei strains, and 2 of 4 Salnlonella strains. The substance did not inhibit any of the Gram-positive organisms tested (Table 1). H. influenzae is a facultative anaerobe and when grown under anaerobic conditions (10% H,, and 5% CO, in N,), the bactericidal substance could not be detected. It was exhibited, however. when lawns of a sensitive strain. overstreaked with a producer strain, were incubated anaerobically then set in air at room temperature for several hours. However, all anaerobic bacteria tested, including Propionibacterium acnes, Clostrirliun~perfringens, Peptococcus sp., Peptostreptococcus sp., and Bacteroides fragillis, were resistant t o the bactericidal activity of a producer strain and also an extract of the bactericidal factor Extraction of Bactericidal Factor The factor is extractable from culture filtrates of cells throughout exponential phase and maximally from stationary-phase broth cultures by precipitation with ammonium sulfate at 50% saturation. The bactericidal activity of the factor is readily observable by quantitative assay on

I

Test organism H . parahaemolyricus Klebsiella species Neisseria gonorrhoeae N. meningit idis Pvoteus mivabilis P. morgani P.rettgeri P. uulgaris Pseudonlonas aeruginosa SaImoneIIa types B C C, D Shigella sonnei Sraph.vlococcus arrreus S . epideritiridis Streptococcris (alpha-henlolytic) Streptococcus (beta-hemolytic) Streptococcus (non-hemolytic) Streptococcus (enterococcus)

Qualitative plate assay, No. resistantlno. tested 016 313 2/ 2 111 818 111 111

111 212 2(C,D)/4 o/ 2 ]/I 2/ 2 212 212 111 111

lawns of sensitive strains when the factor is concentrated to 5% of the original volume. T h e action of mitomycin C does not increase the uroduction o r release of the factor as measured by its biological activity. Further concentration is achieved by use of a Diaflo filtration membrane with a particle retention size of about 50 000 Daltons and greater, but not by a filter with 100 000 Daltons pore size. The range of activity of this extract is the same as that produced by H. injuenzae b strains on solid medium. T h e activity is sensitive to proteolytic agents such a s trypsin and protease but resistant to inactivation by treatment with lipase, deoxyribonuclease, 2-mercaptoethanol, a-amylase, and heating in a I00 "C water bath for 20 min. It is stable throughout the pH range from 2 to 1 1 and does not significantly decrease in activity on storage at 4 "C for a t least 2 weeks, at pH 7. Mixed Cultures T o monitor any selective advantage a producer strain of the bactericidal factor may have when coexisting with a sensitive strain, mixed broth cultures of strains 105 and a streptomycinresistant mutant of C25 (C25 StrR4)were grown. In addition t o antibiotic resistance. the two strains can be distinguished by colonial morphology. Strain 105 is an encapsulated type b which exhibits white, iridescent colonies o n translucent sBHI agar, while C25 is a nontypable derivative of a type d organism which

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produces gray, noniridescent colonies. The two strains grew equally well until late exponential phase was reached, after which time strain 105 continued to maintain a normal growth rate while the colony-forming units per millilitre of strain C25 diminished significantly (Figs. 2, 3). This coincided with the maximal production or release of the bactericidal factor from strain 105 (Figs. 4, 5). The slight decrease in activity which occurs during stationary phase is reproducible. Preliminary Characterization The activity of the bactericidal factor and its susceptibility to proteolytic enzymes indicated to us that it may be a bacteriocin or a bacteriophage. However, we were unable to observe phage or phage-like particles after induction by UV light or mitornycin C . The clear area produced either by an overstreak or an extract from culture filtrates could not be transferred by phage 0 1 2 3 4 5 6 7 8 9 1 0 propagating techniques. Increased Ca2+ and TlME MOURS) Mg2+ concentration was not necessary for bactericidal activity but has been reported as FIG. 3. Growth of a mixed culture containing both 105 necessary for HPlCl and S2 phage adsorption and C25 StrR4in a colony-forming ratio of lo:], respec(8,9). Also, phage-like particles were not evident tively. Symbols are the same as in Fig. 2. by electron microscopy in the sediment after centrifugation (40 000 x g for 90 min and 150 000 x g for 6 h) of filtrates of stationaryphase broth cultures. Furthermore, the bacteri-

1

6

0

0

1

2

3 4 5 6 TlME (HOURS)

7

8

9

1

FIG. 2. Growth of a mixed culture containing both H. influenzae b (105) and H. influenzae (C25 StrR4) in a colony-forming ratio of 1 :I ; 105 (0) is a producer of the bactericidal factor and C25 ( A ) is sensitive to it; 105 ( 0 )and C25 (A)show normal growth rates when cultured separately.

FIG. 4. Assay of the production or release of the bactericidal factor during the growth of H. influenzae h (105) in sBHI. Cell-free aliquots were precipitated with ammonium sulfate, concentrated t o 5% of their volume, and tested for biological activity by measurement of zones of inhibition (mm) on a lawn of a sensitive strain, C25. These were standardized to milligrams per millilitre of total protein by Lowry determinations.

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Frc. 5. Quantitative biological assay of inhibition of C25 by the bactericidal factor isolated at various r e H. itlflr~etlzaeh (105). Zones were measured in rnillirnetres on two axis. Wells times from a growing c u l t ~ ~ of proceeding from the upper left across to upper right; then middle left to middle right; and from lower left to lower right represent points of assay during growth of 105 shown in Fig. 4.

cidal activity remained in the supernatant. This suggests that phage or a high molecular weight bacteriocin is not responsible for the activity. Survival curves using a crude extract of the bactericidal factor suggested that "single-hit" kinetics of killing by bacteriocins was unlikely (Figs. 6, 7). In addition, a decrease in optical density of cell suspensions did not occur in the presence of a concentration of the bactericidal factor, which would give 9 5 x killing by 15 min. Since the activity was restricted t o type b strains of H. influenzae, it was thought that the specific capsular component, polyribose phosphate (PRP), might be the bactericidal factor. However, purified P R P (courtesy of Dr. Porter Anderson, Children's Hospital, Boston, Massachusetts) was not found t o have bactericidal activity. In addition, the unencapsulated R b strains retained the capacity t o inhibit sensitive strains.

Discussion We were able to observe bactericidal activity by all type b strains tested. This bactericidal

factor was extracted from culture filtrates of producer strains in late log and stationary phases of growth. It appears to be a non-dialyzable protein that is thermostable, and between 50 000 and 100 000 Daltons in size based upon retention by membrane filters. The range of activity includes representatives of the genus Haemophilus and certain Enterobacteriaceae. None of the Gram-positive and obligately anaerobic bacteria tested were susceptible. Based upon the available data, we believe the factor may prove to be a bacteriocin. In general, there appear t o be bacteriocins of two types (12): those which resemble phage o r phage-like particles and those which are simple protein o r proteins associated with cell wall components. The Haemophilus bactericidal factor fits the criteria of the latter group, i.e. a low molecular weight bacteriocin, since phage-like particles were not observed in sediments after high-speed centrifugations (3). In addition, the activity remained in the supernatants after such treatment. The interpretation of the results is somewhat clouded by the observations that the bactericidal

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VENEZIA A N D ROBERTSON: BACTERICIDAL SUBSTANCE FROM HAEMOPHILUS

0

1.6

MG

3.2 48 6.4 TOTAL P R O T E I N

FIG.7. Proportion of bacteria surviving in buffer when treated with'increasing concentrations of the Hnernophilr,~ bactericidal factor for a period of 5 min at room temperature. Data points taken from Fig. 6.

I

antigen is believed to be polyribitol phosphate since it is present in the polysaccharide capsules of H. influenzae type b and E. coli strains. We do not suggest that this polymer is the active agent 0.1 0 T I M E (MINUTES) or attachment site for the bactericidal factor we have isolated, since polyribitol phosphate is FIG. 6. Proportion of bacteria surviving in buffer present in Gram-positive organisms and antisera when treated with various concentrations of the Ilae~noto it cross-react with H. influenza b (2). Indeed, phil~is bactericidal factor. Bactericidal factor was prethese same Gram-positive organisms were shown pared as described in the text and reaction occurred at room temperature with 7 x lo4 cells. Symbols: 0, to be resistant to the bactericidal activity. How1.6 mg total protein; 0 , 3.2 mg total protein; A, 4.8 mg ever, this may indicate that other surface polytotal protein; A, 6.4 mg total protein. The control, BHI mers are shared by both Haemophilus species plus buffer or preparation from non-producing cells, and sensitive Enterobacteriaceae and these act showed no decrease in percentage of survivors over the as receptor sites. This hypothesis is under total time period. investigation. The possibility that more than one activity on sensitive cells does not result in bactericidal factor with separate ranges of "single-hit" killing and that there is a lack of activity exists has not been eliminated. induction in production of the bactericidal factor As mentioned previously, only H. influenzae b by UV light or mitomycin C treatment which is and Rb strains were producers of the bactericidal typical of the colicins (12, 13). factor and were themselves resistant. The two The main criterion which distinguishes a low resistant H. parainfluenzae strains, the H. haemomolecular weight bacteriocin from classification lyticus, resistant Salnionella strains, and six as an antibiotic is the narrower range of activity resistant E. coli strains did not exhibit bactericidal or preferential effect on closely related strains. activity to either the Haemophilus producer Since this Haemophilus bactericidal factor affects strains or sensitive strains. Epidemiologically, it members of the family Enterobacteriaceae, it would be important to be able t o identify would appear not to meet this restriction. How- H. influenzae b in the absence of its type-specific ever, other bacteriocins such as colicin G, which capsule by some other genetically controlled acts on Pasturella, have been found to have marker. The Rb strains, including those which activity of a broader range (12). The sensitivity were selected by the type-specific antisera, of certain members of the family Enterobac- appear t o have little or no capsule as compared teriaceae might be explained by the fact that with the parent strains and yet still produce the other investigators have found cross-reactive factor and are resistant to it. Although it would surface antigens between H. influenzae b and be difficult to prove complete loss of capsule, E. coli (14). The main cross-reactive surface this is suggestive that capsular loss is independent

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of production of the bactericidal factor. What is perplexing to us is that none of the non-typable strains tested showed bactericidal activity or resistance to the factor. It would seem that either none of the untypable H. influenzae strains tested were derived from type b strains or that both detectable capsule and bactericidal activity are lost or selected against as strains become untypable. Purification and characterization of the bactericidal factor are in progress. Other interesting aspects which are being investigated are the possibility that type b strains have a selective advantage in vivo as a result of the bactericidal activity and the apparent lack of production of the bactericidal factor under anaerobiosis.

Acknowledgments We thank Dr. Sol Goodgal, University of Pennsylvania, Dr. Johan Stuy, the Florida State University, and the Diagnostic Laboratory, Strong Memorial Hospital, Rochester, New York, for their cooperation in obtaining strains used in this study; and Ms. Janice Nagel for her helpful discussion during the preparation of this manuscript. This investigation was supported in part by U.S. Public Health Service Training Grant 5-27576-224. 1. ANDERSON, P., R. B. JOHNSTON,JR., and D. H. SMITH.1972. Human serum activities against H a e mopkilrrsinflrrenzae, type b. J. Clin. Invest. 51: 31-38. 2. ARGAMAN, M., T . L I U , and J. B. ROBBINS.1974. Polyribitol-phosphate: an antigen of four Grampositive bacteria cross-reactive with the capsular polysaccharide of Haemophillrs i n j ~ r e n z a etype b. J. Immunol. 112: 649-655. 3. BRADLEY,D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31: 230-314. 4. CROWE,C. E., W. E. SANDERS, JR.,and S. LONGLEY. 1973. Bacterial interference. 11. Role of the normal throat flora in prevention of colinization by group A Streptococcus. J . Infect. Dis. 128: 527-532. E., and T. C. MERIGAN. 1969. An active 5. DECLERCQ,

interferon inducer obtained from Haemophillrs injluenzae type b. J. Immunol. 103: 899-906. DENNY,F. W. 1974. Effect of a toxin produced by Haemophilus injluenzae on ciliated respiratory epi-

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Bactericidal substance produced by Haemophilus influenzae b.

During bacteriophage studies on Haemophilus influenzer, it was observed that encapsulated type b and unencapsulated Rb strains released a bactericidal...
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