Role of rabbit lysozyme in in vitro serum and plasma serum bactericidal reactions against Bacillus subtilis.

The antibacterial activity of purified rabbit lysozyme was kinetically investigated at concentrations comparable to those in normal rabbit serum and plasma serum. The bactericidal capability, lysozyme content, and electrophoretic composition of "purified beta-lysin," fractionated from normal rabbit serum, were also examined. In contrast to the extensive antibacterial activity of dilute normal rabbit serum observed in vitro, rabbit lysozyme was only weakly bactericidal for Bacillus subtilis. Inhibition of lysozyme enzymatic and bactericidal activities in normal rabbit serum by antilysozyme immunoglobulin G slightly reduced the initial rate of killing. The addition of neutralizing antibody or histamine (another lysozyme inhibitor) to partially purified bactericidal serum fractions had no effect on killing kinetics. Increasing the ionic strength of reaction mixtures containing normal serum or partially purified bactericidal fractions to levels which completely inhibited lysozyme activity resulted in stimulation of their respective killing kinetics. The addition of inhibitors to normal rabbit plasma serum completely eliminated its bactericidal activity. With regard to the killing of B. subtilis by rabbit and human blood fractions, these analyses clearly demonstrated that (i) although lysozyme is not a significant antibacterial component of normal rabbit serum, it represents the principal factor in normal rabbit plasma serum; (ii) different primary bactericidal mechanisms which are not detectable by singlepoint analyses operate in the sera of different species; and (iii) purified beta-lysin isolated from normal rabbit serum by the classical procedure is a heterogenous mixture of components.     

Neutralization of human serum beta-lysin by sodium polyanetholsulfonate and sodium amylosulfate.

Normal fresh and heat-inactivated (56 degrees C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 x 10(4) colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 mug of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed beta-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished beta-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded beta-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl(2) completely abrogated beta-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37 degrees C) completely removed beta-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both beta-lysin and lysozyme activity. Addition of either 63 to 500 mug of sodium polyanetholsulfonate per ml or 63 to 500 mug of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized beta-lysin activity for the entire observation period of 22 h.     

Studies of bactericidal activity to Escherichia coli of porcine serum and colostral immunoglobulins and the role of lysozyme with secretory IgA

Gel filtration and immune inhibition techniques were used to study bactericidal activities of IgG, IgM and IgA against smooth strains of Escherichia coli 0141 and 08 in sow serum and colostrum and post-colostral piglet serum. Bactericidal activity in sow sera was primarily associated with IgM and a low molecular weight IgG component, 7S IgG activity was less frequently observed. In colostral whey fractions and post-colostral piglet sera, in the absence of lysozyme, bactericidal antibody activity was associated with IgM and 7S IgG. In post-colostral serum bactericidal antibody was also attributable to a low molecular weight form of IgG.

IgA in serum from the sow and neonate showed no bactericidal activity, even in the presence of lysozyme, whereas in colostrum secretory 11S IgA had bactericidal activity, but only in the presence of complement and lysozyme.

     

Variable Neutralization of Several Nonspecific Antibacterial Systems in Fresh, Defibrinated Human Blood by Sodium Polyanetholsulfonate and Sodium Amylosulfate

Fresh, defibrinated human blood (80 vol%, i.e., 80% [vol/vol] of a 2-ml final assay volume) from two healthy adult donors killed "delayed serum-sensitive" (DSS) and "promptly serum-sensitive" (PSS) strains of Serratia marcescens, PSS control strain Escherichia coli C, Bacillus subtilis strain ATCC 6633, and Micrococcus lysodeikticus ATCC 4698 in a kinetic manner comparable to that of fresh human serum (80 vol%). However, heat-inactivated (56 degrees C, 30 min), defibrinated human blood revealed markedly reduced or a total lack of beta-lysin activity against the B. subtilis assay strain. Similarly, lysozyme activity of defibrinated blood was diminished somewhat by heat treatment, as determined with the M. lysodeikticus assay strain. Addition of 500 mug of sodium polyanetholsulfonate (SPS) per ml to 80 vol% of fresh, defibrinated human blood completely neutralized blood bactericidal activity against all assay strains of S. marcescens, E. coli C, and B. subtilis; however, SPS at this concentration failed to abolish lysozyme activity for prolonged periods of incubation. Addition of 500 mug of sodium amylosulfate (SAS) per ml to 80 vol% of fresh defibtinated human blood resulted in protection of cell inocula of DSS strains of S. marcescens only; SAS failed to protect cell inocula of the PSS strains of S. marcescens, E. coli C, B. subtilis, and M. lysodeikticus for extended periods of observation. Based on these data, it is recommended that blood culture specimens that are first drawn into specimen containers (such as Vacutainer tubes or the like) at the patient's bedside, and which contain >/=250 mug of SPS per ml, be diluted into suitable broth media with at least >/=250 mug of SPS per ml by the receiving laboratory within 2 to 4 h after procurement of the specimen. This procedure would ensure continued, adequate neutralization of the specimen's inherent beta-lysin, lysozyme, and complement- and antibody-mediated bactericidal activities.     

Agglutination and bactericidal responses of the channel catfish to

Intraperitoneal immunization of channel catfish, , with in Freund's complete adjuvant produced persistent agglutinating antibody titers of hundreds of thousands during a 1-year response. Bactericidal activity of 2 high-titered sera averaged 30% and 20%. Post-immunization bactericidal activity varied little and remained within 10% of pre-immunization bactericidal activity. Fresh channel catfish serum from unimmunized catfish exhibited 100% bactericidal activity against . Lysozyme was present in fresh channel catfish serum at 1.8 μ/ml serum or 34 ng lysozyme/mg protein.     

Bactericidal activity of human lysozyme, muramidase-inactive lysozyme, and cationic polypeptides against Streptococcus sanguis and Streptococcus faecalis: inhibition by chitin oligosaccharides.

The basis of the bactericidal activity of human lysozyme against Streptococcus sanguis was studied. Experiments were designed to evaluate the role of lysozyme muramidase activity in its bactericidal potency. Inactivation of the muramidase activity of lysozyme was achieved by reduction of essential disulfides with dithiothreitol (DTT) or by incubation with the chitin oligosaccharides chitotriose and chitobiose. Muramidase-inactive lysozyme, prepared by reduction with DTT, was equal in bactericidal potency to native lysozyme. Solutions of native chicken egg white lysozyme and human lysozyme exhibited equal bactericidal potency yet differed ca. fourfold with respect to lytic (muramidase) activity. The above results suggested that the bactericidal activity of lysozyme is not dependent upon muramidase activity. Chitotriose and chitobiose were found to inhibit both lytic and bactericidal activities of lysozyme. The bactericidal activity of muramidase-inactive lysozyme (reduction with DTT) was also inhibited by chitotriose and chitobiose. Further investigations demonstrated that chitotriose and chitobiose were also potent inhibitors of the bactericidal activity of the cationic homopolypeptides poly-L-arginine and poly-D-lysine. These latter results suggested that the essential bactericidal property of lysozyme was its extreme cationic nature and that some bacterial endogenous activities, inhibitable by chitotriose and chitobiose, were essential for expression of the bactericidal activity of either native or muramidase-inactive lysozyme or of the cationic homopolypeptides. Experiments with Streptococcus faecalis whole cells, cell walls, and crude autolysin preparations implicated endogenous autolytic muramidases as the bacterial targets of chitotriose and chitobiose. The essentially identical responses of S. sanguis and S. faecalis to chitotriose in bactericidal assays with muramidase-inactive lysozyme and polylysine suggested that muramidase-like enzymes exist in S. sanguis and, furthermore, play an essential role in cationic protein-induced loss of viability of the oral microbe.     

Interrelationship Between Serum Beta-Lysin, Lysozyme, and the Antibody-Complement System in Killing Escherichia coli

The effects of different serum components alone and in conjunction with each other on Escherichia coli B were investigated. In general, the viability, turbidity, and electron microscope results were compatible with the following conclusions. The most efficient killing and destruction of E. coli B occurred when beta-lysin, lysozyme, and the antibody-complement system functioned in cooperation with each other at the serum concentration in isotonic solutions. The addition of sucrose protected the bacteria from the lethal and lytic action of these agents. Elimination of lysozyme from serum had the least effect on bactericidal activity, even though lysozyme treatment caused the cell wall to separate from the cytoplasmic membrane and caused clear areas to appear in the inner granular layer of the cell wall. Beta-lysin removal had an intermediate effect on the serum bactericidal activity. Beta-lysin treatment caused cell walls to collapse, allowed cytoplasmic contents to leak out of the cells, and stopped the separation of cell wall and cytoplasmic membrane, which normally takes place in 0.5 M sucrose solution. Inactivation of the complement eliminated the serum bactericidal activity against E. coli B. After treatment with antibody and complement, the cell walls became thick and indistinct, a portion of the cytoplasmic contents escaped, and patches of the middle layer of the cell wall appeared in freeze-etch preparations. Beta-lysin damaged the cytoplasmic membrane, lysozyme damaged the inner peptidoglycan layer of the cell wall, and the antibody-complement system damaged both the middle lipopolysaccharide layer of the cell wall and the cytoplasmic membrane.     

Agglutination and bactericidal responses of the channel catfish to Salmonella paratyphi

Intraperitoneal immunization of channel catfish, $\text{Ictalurus punctatus}$, with $\text{Salmonella paratyphi}$ in Freund's complete adjuvant produced persistent agglutinating antibody titers of hundreds of thousands during a 1-year response. Bactericidal activity of 2 high-titered sera averaged 30% and 20%. Post-immunization bactericidal activity varied little and remained within 10% of pre-immunization bactericidal activity. Fresh channel catfish serum from unimmunized catfish exhibited 100% bactericidal activity against $\text{S. paratyphi}$. Lysozyme was present in fresh channel catfish serum at 1.8 μ/ml serum or 34 ng lysozyme/mg protein.     

Staphylocidal action of thrombin-induced platelet microbicidal protein is influenced by microenvironment and target cell growth phase.

Thrombin-induced platelet microbicidal protein (tPMP) is a small, cationic peptide released from rabbit platelets following exposure to thrombin in vitro. This peptide exerts potent in vitro microbicidal activity against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. It is known that the microbicidal actions of other cationic antimicrobial peptides (e.g., neutrophil defensins) are influenced by environmental factors and target cell growth phase. However, whether these parameters affect tPMP microbicidal activity has not been studied. Thus, we assessed the in vitro bactericidal activity of tPMP against two tPMP-susceptible strains, Bacillus subtilis ATCC 6633 and S. aureus 502A, in various target cell growth phases or under various microenvironmental conditions. The conditions studied included differing bacterial growth phase (logarithmic versus stationary), temperature (range, 4 to 42 degrees C), pH (range, 4.5 to 8.5), cationicity (range, 0.1 mM to 2 M), anionicity (range, 0.08 to 5 microM), and neutral carbohydrates ranging in molecular weight (MW) from 180 to 37,700 (range, 50 to 500 mM) as well as rabbit platelet-free plasma and serum. tPMP staphylocidal activity was greater against logarithmic- than stationary-phase cells. tPMP bactericidal activity against both B. subtilis and S. aureus was directly correlated with temperature and pH, with microbicidal activity exhibited near the physiological range (37 to 42 degrees C and pH 7.2 to 8.5, respectively). The presence of cations (Na+, K+, Ca2+, and Mg2+) decreased tPMP bactericidal activity in a time- and concentration-dependent manner, with complete inhibition at monovalent or divalent cation concentrations of > or = 250 or > or = 10 mM, respectively. Staphylocidal activity of tPMP was also inhibited by the polyanions polyanetholsulfonic acid and polyaspartic acid, at 0.1 and 0.4 microM, respectively. Coincident exposure with low-MW carbohydrates (glucose, sucrose, and melezitose) did not affect tPMP staphylocidal activity. However, higher-MW carbohydrates (raffinose and dextrans) decreased tPMP activity in a manner directly proportional to their concentration and MW. Solute-mediated inhibition of tPMP bactericidal activity was independent of solute osmolality but directly related to the duration of tPMP-solute coexposure. tPMP enhanced the staphylocidal activities of platelet-free plasma and heat-inactivated serum, while the activity of normal serum was not affected. These collective observations suggest that tPMP retains antimicrobial activities under physiological conditions which are likely to be relevant to host defense in vivo.     

TnphoA-mediated disruption of K54 capsular polysaccharide genes in Escherichia coli confers serum sensitivity.

To assess whether non-K1, group 2 capsular serotypes are important in conferring serum resistance to extraintestinal isolates of Escherichia coli, a K54 blood isolate (CP9) was evaluated as a model pathogen. Transposon mutagenesis (TnphoA) was used to generate isogenic capsule-negative mutants. CP9 was resistant to the bactericidal effects of serum, growing in 80% serum. In contrast, all of the capsule-negative mutants had an increased sensitivity to 80% normal human serum, undergoing a 2- to 3-log kill over 3 h when starting inocula of 10(4) to 10(7) CFU/ml were used. The killing of the capsule-negative strains was mediated through the alternative complement pathway and not by lysozyme or beta-lysins. The protective effect of the K54 capsule against the bactericidal activity of serum was not through inhibition of the complement cascade, nor did it appear to be through a difference in the binding of C3.     

Partial characterization and staphylocidal activity of thrombin-induced platelet microbicidal protein.

Thrombin-induced platelet microbicidal protein (PMP) is considered to play an important role in preventing an important role in preventing streptococcal endocarditis. However, the structural features and functions of PMPs have not been well characterized, and their antibacterial spectra against other common endocarditis pathogens, such as the staphylococci, are not known. Thrombin stimulation of washed rabbit platelets (10(8)/ml) yielded a PMP-rich preparation with a specific activity of approximately 25 U/mg of protein as determined by Bacillus subtilis bioassay. Twenty-eight clinical and laboratory Staphylococcus aureus isolates, exposed to a standardized PMP preparation (100 U/ml for 2 h at 37 degrees C), exhibited a Poisson-distributed heterogeneity to the bactericidal action of PMP, with approximately one-third designated as PMP resistant. Gel filtration chromatography (Sephadex G-50) identified the bioactive moiety within PMP preparations to be in the major protein elution peak; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) presumptively identified PMP as a low-molecular-weight (MW) (8,500) protein present only in such bioactive protein peaks. Both the bioactivity of PMP preparations and the low-MW protein band were removable by specific anionic membranes (e.g., cellulose-acetate/nitrate), as well as by a variety of anionic resins, further corroborating the suspected cationic charge of PMP. In addition, both PMP bioactivity and the low-MW protein band were recoverable by 1.5 M NaCl elution of the anionic membrane filters post-PMP adsorptive removal. Adsorption of bioactive PMP preparations by highly PMP-susceptible B. subtilis (10(8) CFU/ml, 30 min) resulted in a near-complete loss of residual bioactivity; in contrast, adsorption of bioactive PMP preparations with less PMP-susceptible S. aureus strains failed to reduce bioactivity. Significant lysozyme contamination of PMP-rich preparations was ruled out by determination of differences between bioactive PMP preparations and exogenous lysozyme as regards (i) relative heat stabilities; (ii) differential bactericidal activity versus B. subtilis and Micrococcus luteus; and (iii) SDS-PAGE protein profiles. These data show that the bioactive PMP protein moiety is of low MW, is heat stable, is probably cationic (similar to leukocyte-derived defensins), and possesses potent bactericidal activity against a significant percentage of S. aureus isolates.     

Sequential metabolic expressions of the lethal process in human serum-treated Escherichia coli: role of lysozyme.

Several metabolic parameters indicative of Escherichia coli function and integrity were kinetically examined in response to treatment with normal human serum in the presence and absence of functional human lysozyme. Specific inhibition of this enzyme in bacteriolytic and bactericidal reactions was accomplished by using purified rabbit anti-human lysozyme immunoglobulin G. Initiation of the complement-mediated alterations of cytoplasmic membrane integrity, as judged by the leakage of 86Rb from prelabeled cells or the hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by a cryptic strain, was found to be independent of lysozyme action. Furthermore, inhibition of macromolecular synthesis by E. coli in response to serum treatment occurred at the same time regardless of the functional state of lysozyme. Although the rate and extent of bacteriolysis were reduced in the absence of lysozyme, the bactericidal kinetics was unaffected. These results demonstrate that the lethal events associated with the action of antibody and complement on gram-negative bacteria are independent of lysozyme, suggesting an accessory role for this enzyme in immune reactions. A possible temporal sequence of complement-induced effects occurring at the cell surface is presented.     

Impaired bactericidal activity of cord sera against Salmonella strains

Weaker bactericidal activity of cord sera against Salmonella bacilli was shown in comparison with maternal venous sera. No correlation was found between the bactericidal activity and the levels of complement, immunoglobulins and lysozyme in cord sera. Worthy of notice is detected in these researches specificity of bactericidal action of cord sera.     

A kinetic study of the bacteriolytic action of human serum

The killing and lysis of Escherichia coli by human serum have been measured simultaneously at frequent intervals for periods of 30–60 minutes. The kinetic effects of varying the amounts of lysozyme, antibacterial antibody and complement have been studied.

The rate of lysis is largely controlled by the lysozyme concentration but complement is also necessary. Killing is closely related to complement concentration. Antibody is needed in such small amounts that it is rarely a limiting factor.

Inhibition of serum lysozyme by anti-human lysozyme prevents lysis and reduces killing but both are restored to normal by addition of egg-white lysozyme. Both lysis and killing are stopped by bentonite absorption of serum and complete return to normal is not attained by subsequent addition of egg-white lysozyme. In a lysozyme-free system lysis does occur after some delay probably due to the action of complement and antibody alone.

In the presence of adequate complement and antibody the loss of complement (measured haemolytically) in bentonite treated serum is inadequate to account for the fall in bactericidal activity. A new bentonite absorbable factor (BAF) essential for complete serum bactericidal power is postulated.

     

Role of lipopolysaccharide and complement in susceptibility of Haemophilus ducreyi to human serum

The role of lipopolysaccharide (LPS) in the susceptibility of Haemophilus ducreyi to human serum and the mechanism of complement activation by serum-susceptible (Sers) strains were investigated. Serum treated with 2 mM Mg2+ and 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was nonbactericidal, but inulin-treated serum remained bactericidal. Absorption of serum with heat-killed whole cells of an Sers strain removed its bactericidal activity against the absorbing strain and also against other Sers strains. LPS obtained from Sers strains inhibited the bactericidal activity of serum against all Sers strains, whereas LPS from serum-resistant (Serr) strains and an Serr isogenic strain did not. However, high concentrations of LPS from the Serr strain inhibited the bactericidal activity of serum, an indication that part of the structural site involved in serum susceptibility is retained in the LPS of this strain. The LPS of Sers strains exhibited higher anticomplement activity than the LPS of Serr strains. These findings suggest that the classical pathway of complement activation is involved in the serum killing of H. ducreyi and that LPS composition may contribute to their susceptibility to complement-mediated serum bactericidal activity.     

Dynamics of bactericidal properties of human saliva and blood serum during adaptation to arctic conditions

The dynamics of the bactericidal activity of the blood serum and of the lysozyme activity of the blood and saliva was studied in persons living beyond the Arctic circle (Noril'sk). During adaptation to Arctic conditions significant changes take place in the bactericidal activity of the serum and the lysozyme titer of the serum and saliva. Together with changes in the absolute values of these parameters, their circadian rhythms are disturbed. A period of 2 years is not long enough to restore the normal bactericidal activity of the serum or the lysozyme titer.Department of Microbiology, Tomsk Medical Institute. Institute of Clinical and Experimental Medicine, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR D. D. Yablokov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 78, No. 10, pp. 72–74, October, 1974.     

Humoral Factors of Natural Resistance of Bos indicus Cattle Selected for Antibody Titre to Brucella abortus

The concentration of lysozyme, total immunoglobulin and bactericidal activity were measured in sera of Bos indicus cattle, retrospectively screened for specific antibodies to Brucella abortus and classified as being positive reactors or negative reactors. In addition, the effect of complement in the sera was studied to demonstrate complement dependence of antibody-mediated bacterial killing. It was observed that, under the test conditions, serum bactericidal activity and concentration of total immunoglobulin were associated with high specific antibody levels ( P < 0.001). Furthermore, there was a slight decrease in the lytic activity of lysozyme in the sera of animals with high antibody titres.     

Purification and characterization of a phagocytosis-stimulating factor from phagocytosing polymorphonuclear neutrophils: comparison with granule basic proteins.

Phagocytosis-stimulating factor (PSF) was purified by copper chelate chromatography and characterized in comparison with basic proteins in the granule of polymorphonuclear neutrophils. By copper chelate chromatography, PSF was eluted at pH 3.7; whereas cationic protein, lysozyme, and lactoferrin were eluted at pH 5.6, 5.1, and 4.0, respectively. Purified PSF has an approximate molecular weight of 16,000 and an isoelectric point at 8.7, which differ from those of basic proteins, such as cationic protein, lysozyme, and lactoferrin. Anionic substances such as DNA and heparin did not influence the phagocytosis-stimulating activity of PSF, whereas that of the granule basic protein fraction from resting polymorphonuclear neutrophils was abolished. PSF had little bactericidal activity against Escherichia coli and Staphylococcus aureus, whereas the granule basic protein fraction from resting PMNs had strong bactericidal activity against E. coli and weak activity against S. aureus. These results indicate that PSF is a basic protein which is distinguishable from cationic protein, lysozyme, and lactoferrin.     

Determination of serum bactericidal activity with the aid of luminous bacteria

Nonmarine luminous bacteria belonging to the genus Vibrio cholerae were extremely sensitive to the bactericidal activity of human serum. Luminous bacteria incubated in a medium containing serum showed a decrease in their in vivo luminescence that was directly proportional to the decrease in the viable count and was a function of the serum concentration. Both immunoglobulins and the complement system were required to exert the serum bactericidal activity. Serum lacking immunoglobulins or certain complement components, especially C3, did not affect the luminescence. The bactericidal effect of the serum on luminous bacteria was diminished by the presence of lipopolysaccharide or by pretreatment of the serum with different species of killed bacteria. As found in other systems, the bacteriolytic activity of serum was only augmented by lysozyme, but was not lysozyme dependent; although the luminous bacteria were converted into spheroplasts in serum containing 0.5 M sucrose, their in vivo luminescence was almost not affected. This system could easily distinguish between the C classical pathway and the properdin pathway. Ethylene glycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, which inhibits only the classical complement pathway, did not inhibit the decrease in luminescence as did EDTA. Thus, it was possible to distinguish between deficiencies in complement components participating in both pathways and complement components that were involved only in the classical pathway. This system could also be used as a substitute to the hemolytic system in complement fixation tests.     

The influence of protein binding on the antibacterial activity of faropenem against Haemophilus influenzae

The effects of albumin and human serum on the pharmacodynamics of faropenem were studied. The protein binding of faropenem was 91-95%, corresponding to the increase in MICs for Haemophilus influenzae in broth supplemented with albumin. Time-kill experiments in albumin-containing medium and in inactivated human serum 50% v/v showed that much higher drug concentrations were needed to achieve a bactericidal effect than were needed in broth. Active human serum alone exerted a strain-dependent bactericidal effect. It was concluded that it is the free fraction of faropenem in serum that has antibacterial activity against H. influenzae.