Loss Of Bactericidal Activity Research Articles

Optical activity of films based on chitosan of various molecular masses and modifications

The effect of chemical (polysalt → polybase reaction) and physicochemical (heat and vapor processing) modifications of the films of chitosan of various molecular masses and prehistories on the optical activity of the polysaccharide is studied. For both of the chitosan chemical modifications, the following dependence that is nontrivial for high-molecular compounds is established: [α] = f(log $${\bar M_\eta }$$ ); as the degree of polymerization decreases, the modulus of [α] of the films increases. The X-ray diffraction study shows that the differences in the optical activity of the samples with different $${\bar M_\eta }$$ are caused by a restriction in the mobility of the macromolecular sections upon formation of the films of high-molecular chitosan and incompleteness of the relaxation processes of the film systems to the state with energetically favorable conformations. The comparison of humidity and optical and biological activities of the initial and dehydrated chitosan films is performed. The chitosan films in the salt modification feature moderate bactericidal activity, which decreases with an increase in $${\bar M_\eta }$$ of the polymer. The thermal processing of the salt modification reduces the antibacterial action; a conversion to the base form is accompanied by the loss of bactericidal activity.

Superior bactericidal activity of N-bromine compounds compared to their N-chlorine analogues can be reversed under protein load.

To investigate and compare the bactericidal activity (BA) of active bromine and chlorine compounds in the absence and presence of protein load. Quantitative killing tests against Escherichia coli and Staphylococcus aureus were performed both in the absence and in the presence of peptone with pairs of isosteric active chlorine and bromine compounds: hypochlorous and hypobromous acid (HOCl and HOBr), dichloro- and dibromoisocyanuric acid, chlorantine and bromantine (1,3-dibromo- and 1,3 dichloro-5,5-dimethylhydantoine), chloramine T and bromamine T (N-chloro- and N-bromo-4-methylbenzenesulphonamide sodium), and N-chloro- and N-bromotaurine sodium. To classify the bactericidal activities on a quantitative basis, an empirical coefficient named specific bactericidal activity (SBA), founded on the parameters of killing curves, was defined: SBA= mean log reductions/(mean exposure times x concentration) [mmol 1(-1) min (-1)]. In the absence of peptone, tests with washed micro-organisms revealed a throughout higher BA of bromine compounds with only slight differences between single substances. This was in contrast to chlorine compounds, whose killing times differed by a factor of more than four decimal powers. As a consequence, also the isosteric pairs showed according differences. In the presence of peptone, however, bromine compounds showed an increased loss of BA, which partly caused a reversal of efficacy within isosteric pairs. In medical practice, weakly oxidizing active chlorine compounds like chloramines have the highest potential as topical anti-infectives in the presence of proteinaceous material (mucous membranes, open wounds). Active bromine compounds, on the other hand, have their chance at insensitive body regions with low organic matter, for example skin surfaces. The expected protein load is one of the most important parameters for selection of a suited active halogen compound.

Contribution of the R8 substituent to the in vitro antibacterial potency of besifloxacin and comparator ophthalmic fluoroquinolones

IntroductionPrevious work has shown that besifloxacin, an 8-chloro-fluoroquinolone, has more potent activity against gram-positive pathogens than moxifloxacin and gatifloxacin, which carry an 8-methoxy group. This study was conducted to determine the contribution of the R7 and R8 substituent to fluoroquinolone antibacterial activity.Materials and methodsBesifloxacin, moxifloxacin, gatifloxacin, their R8 structural analogs, and ciprofloxacin were tested against representative isolates of various gram-positive and gram-negative species and previously characterized fluoroquinolone-resistant mutants of Staphylococcus aureus. Minimum inhibitory and minimum bactericidal concentrations were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Reserpine was used to determine the effect of efflux pumps on antibacterial activity.ResultsIn general, exchanging the R8 residue in besifloxacin slightly reduced the molecule’s potency, while introducing an 8-chloro group in moxifloxacin increased its potency. A similar change in gatifloxacin had little to no effect. Substituting the R8 residues did not increase the susceptibility to the efflux pump inhibitor reserpine or result in a loss of bactericidal activity. In contrast, the positive control, ciprofloxacin, was shown to be a substrate for reserpine and lost bactericidal activity against some fluoroquinolone-resistant isolates of S. aureus.ConclusionThe data presented here show that, depending on the R7 substituent, replacing an 8-methoxy group with an 8-chloro substituent can improve potency or can have little-to-no effect. These findings highlight the importance of the interplay between the R7 and R8 substituents in determining antibacterial potency.

In vitro efficiency of the piperacillin/tazobactam combination against inhibitor-resistant TEM- and complex mutant TEM-producing clinical strains of Escherichia coli

We investigated the bacteriostatic and bactericidal activities of piperacillin/tazobactam against 16 clinical Escherichia coli producing inhibitor-resistant TEM β-lactamases (IRT; 13/16) and complex mutant TEM enzymes (CMT; 3/16). Bacteriostatic activity was evaluated by three methods (disc diffusion, Vitek2 automated system, MIC determination by a microdilution method) and a time-killing study was used to investigate the bactericidal effect against standard (5 × 10(5) cfu/mL) and high inocula (5 × 10(6) cfu/mL). Piperacillin/tazobactam was bacteriostatic against most of the tested strains (15/16). Using a high inoculum, the piperacillin/tazobactam combination was not bactericidal against the 13 IRT-producing strains and one of the CMT-producing strains (1/3). A loss of bactericidal activity was still observed for seven IRT-producing strains (7/13) with a standard bacterial inoculum (<99.9% killing over 24 h). Despite usual in vitro bacteriostatic activity, the piperacillin/tazobactam combination was not bactericidal against most IRT-producing clinical strains of E. coli, especially for the treatment of a high bacterial inoculum. This possible loss of bactericidal effect should be brought to the attention of physicians and may require high dosing regimens for the treatment of severe infections.

Loss of Bactericidal Activity Promotes Infection

Chronic lung disease, including cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease (COPD), is characterized by high concentrations of interleukin-8 (IL-8) in cells around the airway and accumulation of neutrophils, yet the lungs of these patients are frequently colonized by bacteria, such as Pseudomonas aeruginosa . Hartl et al . show that IL-8 acts through the CXCR1 receptor, not the CXCR2 receptor, to promote the bactericidal activity of neutrophils. By comparing neutrophils from the peripheral blood and airway (bronchial alveolar lavage and sputum) of healthy individuals with those from patients with cystic fibrosis, bronchiectasis, or COPD, Hartl et al . found that the airway neutrophils from the diseased lungs had decreased CXCR1 at the surface and exhibited impaired bactericidal activity. The protease elastase is increased in abundance in airway fluids from patients with cystic fibrosis. Further statistical analysis of various factors associated with cystic fibrosis, including infection with P. aeruginosa , suggested that the concentration of free elastase correlated with the loss of CXCR1 on the airway neutrophils. Addition of airway fluids from individuals with cystic fibrosis to peripheral neutrophils caused loss of CXCR1 and impaired bacterial killing, and these effects were prevented if the elastase activity was inhibited pharmacologically in the airway fluid prior to addition to the isolated neutrophils. Cleavage of CXCR1 by elastase produced two soluble CXCR1 fragments that were also detected in the airway fluids from patients with cystic fibrosis, bronchiectasis, or COPD. Application of purified soluble CXCR1 fragments stimulated bronchial epithelial cells to produce IL-8 through a mechanism requiring the Toll-like receptor TLR2. Cystic fibrosis patients who inhaled α1 antitrypsin to inhibit elastase activity for 4 weeks showed increased CXCR1 at the surface of airway neutrophils, decreased abundance of soluble CXCR1 fragments in the airway fluid, and decreased P. aeruginosa in the sputum. In commentary, Sabroe and Whyte describe how failure of mucosal immunity contributes to disease pathogenesis, especially in cystic fibrosis. D. Hartl, P. Latzin, P. Hordijk, V. Marcos, C. Rudolph, M. Woischnik, S. Krauss-Etschmann, B. Koller, D. Reinhardt, A. A. Roscher, D. Roos, M. Griese, Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease. Nat. Med. 13 , 1423-1430 (2007). [PubMed] I. Sabroe, M. K. B. Whyte, Incapacitating the immune system in cystic fibrosis. Nat. Med. 13 , 1417-1418 (2007). [PubMed]

Description of complete DNA sequence of two plasmids from the nukacin ISK-1 producer, Staphylococcus warneri ISK-1

We report the whole DNA sequence of two plasmids, pPI-1 (30.2 kb) and pPI-2 (2.8 kb). These plasmids are from Staphylococcus warneri ISK-1, which produces a lantibiotic, nukacin ISK-1. Curing of pPI-1 resulted in a loss of bactericidal activity in the culture supernatant and the host’s immunity to nukacin ISK-1, suggesting that the biosynthetic genes of the bacteriocin are encoded by pPI-1. Based on the results of a homology search of each open reading flame, pPI-1 is comprised of the following four distinct regions: (1) the nukacin ISK-1 biosynthesis and immunity gene cluster, (2) the thioredoxin gene cluster, (3) the replication region, and (4) a region of Staphylococcus epidermidis ATCC 12228, highly homologous to pSE-12228-05. Gene organization in the nukacin ISK-1 biosynthesis and immunity gene cluster is different from that in other lacticin-481 type gene clusters. The features of the replication protein encoded in the replicating region are somewhat different from other staphylococcus theta-replicating plasmids. pPI-2 comprised a disinfectant resistant gene, qacC, and the whole DNA sequence showed significant similarity to those of other qacC plasmids such as pSK108, suggesting that pPI-2 belongs to the qacC plasmid group.

Effects of storage on the physicochemical and antibacterial properties of human milk

Storage of human milk for limited periods of time is unavoidable in neonatal units and also in the home where increasing numbers of mothers go back to work soon after delivery. Many mothers, convinced of the importance of prolonged exclusive or complementary breast-feeding, often express and store human milk for use during the period of separation. This study examines the effects of different storage methods on the pH and some antibacterial activities of human milk. Portions of milk and colostrum samples from healthy lactating mothers were stored at 4°C and -20°C for periods ranging from one day to 12 weeks. The stored milk samples were analysed for pH, bactericidal and bacteria sequestration activities against a serum-sensitive Escherichia. coli, and compared with freshly collected samples, with and without EDTA. Milk became progressively more acidic during storage. While the bactericidal activities of refrigerated samples diminished rapidly, up to two-thirds of the original activity level was maintained by freezing for up to three months. The ability of milk fat globule membrane to adhere to suspended bacteria was gradually lost in frozen milk samples, while it was greatly enhanced during the first few days in refrigerated samples, before declining sharply. This study shows that loss of bactericidal activity in refrigerated milk is well compensated for by enhanced bacteria sequestration activity, and allays any fears that might arise concerning the suitability of stored human milk for infant consumption.

Characterization of human bactericidal antibodies to Bordetella pertussis.

The Bordetella pertussis BrkA protein protects against the bactericidal activity of complement and antibody; however, some individuals mount an immune response that overcomes this bacterial defense. To further characterize this process, the bactericidal activities of sera from 13 adults with different modes of exposure to B. pertussis (infected as adults, occupational exposure, immunized with an acellular vaccine, or no identified exposure) against a wild-type strain and a BrkA complement-sensitive mutant were evaluated. All of the sera killed the BrkA mutant, suggesting past exposure to B. pertussis or cross-reactive organisms. Several samples had no or minimal activity against the wild type. All of the sera collected from the infected and occupationally exposed individuals but not all of the sera from vaccinated individuals had bactericidal activity against the wild-type strain, suggesting that some types of exposure can induce an immune response that can overcome the BrkA resistance mechanism. Adsorbing serum with the wild-type strain removed the bactericidal antibodies; however, adsorbing the serum with a lipopolysaccharide (LPS) mutant or an avirulent (bvg mutant) strain did not always result in loss of bactericidal activity, suggesting that antibodies to either LPS or bvg-regulated proteins could be bactericidal. All the samples, including those that lacked bactericidal activity, contained antibodies that recognized the LPS of B. pertussis. Bactericidal activity correlated best with the presence of the immunoglobulin G3 (IgG3) antibodies to LPS, the IgG subtype that is most effective at fixing complement.

Biological activity of serum antibodies to a nonacylated form of lipoprotein D of Haemophilus influenzae.

Protein D, a surface-exposed 42-kDa membrane lipoprotein, is well conserved among both type b and nontypeable Haemophilus influenzae strains, and it is considered a vaccine against H. influenzae infections. Here, we report the large-scale purification of a nonacylated form of protein D (PDm) from the periplasmic space of Escherichia coli overexpressing PDm. Screening of human sera for levels of antibodies to PDm demonstrated that the immunoglobulin G (IgG) antibody level is above background levels in infants less than 6 months of age. Following a drop to background values in the age group 6 months to 1 year, IgG antibody levels start to increase, together with IgA antibody levels, after 1 year of age. The first appearance of serum IgM antibodies is in 6-month- to 1-year-old infants whose IgG antibody levels have dropped to the postnatal background level. Affinity-purified antibodies from humans and from PDm-immunized rats detected epitopes of protein D which are normally exposed on the bacterial surface. Affinity-isolated human anti-PDm antibodies eluted in acidic buffer were not bactericidal against H. influenzae. Loss of bactericidal activity may occur in this buffer, as was demonstrated in pooled human sera with high bactericidal activity after incubation in the same buffer. Hyperimmunization of rats with PDm induced high levels of serum IgG and IgA antibodies against PDm and significant bactericidal activity against homologous and heterologous H. influenzae strains.

Bactericidal activity of the bovine myeloperoxidase system against bacteria associated with mastitis

Myeloperoxidase (MPO) is a lysosomal enzyme found in the primary granules of mammalian neutrophils. Together with MPO, peroxide and halide form a system of defense against bacteria. The present investigation was undertaken to study the bactericidal effects of the bovine-MPO/peroxide/halide system on pathogenic bacteria associated with bovine mastitis. We demonstrated that MPO together with oxidizing agents generated by xanthine oxidasae, hypoxanthine and chloride form a potent antibacterial system against the common udder pathogens Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae and Escherichia coli in a synthetic medium. However, when milk was added to the reaction mixture, the bactericidal properties of this enzyme system were completely inhibited. Loss of bactericidal activity in the milk-containing cultures was unable to be restored by increasing the concentration of MPO. Nor did an increase in concentrations of hypoxanthine and xanthine oxidase, or the replacement of the above-mentioned peroxidase generating system with a high concentration of hydrogen peroxide, significantly elevated the bactericidal activity that was inhibited by milk. The addition of bovine serum albumin to the synthetic medium reduced the bactericidal activity of the MPO/peroxide/chloride system in a dose-dependent manner. Therefore, milk proteins probably form adducts with strong bactericidal agents that are generated by the MPO system and thereby reduce the bacterical potential of this system.

Antibodies to lipooligosaccharide of a Brazilian purpuric fever isolate of Haemophilus influenzae biogroup aegyptius lack bactericidal and protective activity.

The immunological basis for protection against Brazilian purpuric fever (BPF), a fulminant infection of young children associated with bacteremia with Haemophilus influenzae biogroup aegyptius, is unknown. Candidate antigens to which protective antibodies may be directed include cell surface proteins and lipooligosaccharide (LOS). We studied the activity of antisera to LOS purified from a BPF H. influenzae biogroup aegyptius isolate. Anti-LOS antisera contained anti-LOS antibody by enzyme immunoassay and immunoblot and no detectable anti-outer membrane protein antibodies by immunoblot. Anti-LOS antisera had minimal bactericidal activity and were not protective against the homologous strain in an infant rat model of bacteremia. Antiserum to whole bacterial cells had a titer of anti-LOS antibody similar to that of anti-LOS antisera and was bactericidal and protective. Removal of anti-LOS antibodies from anti-whole cell antiserum by affinity chromatography did not result in a loss of bactericidal activity. Serum from a normal adult contained anti-LOS antibodies and had bactericidal activity. However, anti-LOS antibodies purified from this serum did not have detectable bactericidal activity. These studies suggest that anti-LOS antibodies produced in rats are not bactericidal and do not contribute to protection against experimental bacteremia with BPF strains of H. influenzae biogroup aegyptius.

Bactericidal effect of Fe 2+, ceruloplasmin, and phosphate

Fe 2+, when combined with ceruloplasmin or phosphate, was bactericidal to Escherichia coli at pH 5.0, and when Fe 2+, ceruloplasmin, and phosphate were combined, a bactericidal effect was observed under conditions, i.e., short incubation period, in which Fe 2+ plus ceruloplasmin and Fe 2+ plus phosphate were ineffective. Bactericidal activity increased with the ceruloplasmin or phosphate concentration to a maximum and then decreased as their concentration was further increased. Fe 2+ was oxidized in the presence of ceruloplasmin, phosphate, or, in particular, a combination of the two. A bactericidal effect was observed when there was only a partial loss of Fe 2+, with more extensive oxidation resulting in a loss of bactericidal activity. The bactericidal effect of Fe 2+ plus ceruloplasmin and/or phosphate was unaffected by catalase or superoxide dismutase and was not associated with iodination. Fe-EDTA was also bactericidal at an Fe 2+: EDTA molar ratio of 1:0.5, where Fe 2+ was partially oxidized. However, in contrast to Fe 2+ plus ceruloplasmin and/or phosphate, bactericidal activity was inhibited by catalase and was associated with iodination. Combinations of Fe 2+ and Fe 3+ were not bactericidal under the conditions employed. A requirement for Fe 2+ plus either a product of Fe 2+ oxidation or an iron ceruloplasmin and/ or phosphate chelate for bactericidal activity is proposed.

Feasibility Studies on Radiation Sterilization of Cephradine

AbstractA comprehensive study was carried out to examine and evaluate the feasibility of radiation sterilization of cephradine antibiotic in the solid dry state. Absorbed gamma ray doses in the range of 5–25 kGy caused a dose-dependent loss of bactericidal activity. Moreover, radiation caused a dose-dependent degradation of the antibiotic and decrease in cephradine content due to radiation conversion to cephalexin. The former deleterious effect was detected spectrophotometrically and confirmed by high-performance liquid chromatography (HPLC). The latter effect was detected by NMR spectroscopy and by HPLC. The study revealed a noticeable change in the tint of the irradiated powdered drug which is dose-dependent and clearly remarkable at high radiation doses.

High-level penicillin resistance among isolates of enterococci. Implications for treatment of enterococcal infections.

To determine the extent and clinical significance of high-level penicillin-resistant Enterococcus faecium at our institution. Surveillance of clinical enterococcal isolates, in-vitro susceptibility and timed survival studies, and determination of antibiotic efficacy in an experimental model of enterococcal endocarditis. For a 6-month period, 14% of enterococcal isolates (30 of 212) were identified as E. faecium. One third of the isolates were highly resistant to penicillin G (minimum inhibitory concentration [MIC], greater than or equal to 200 micrograms/mL) but did not produce beta-lactamase. The findings from in-vitro survival studies showed that this high-level resistance resulted in the loss of bactericidal activity normally observed when an aminoglycoside antibiotic agent is combined with penicillin. An experimental rat model of endocarditis provided in-vivo data that confirmed our in-vitro observations. After the rats received therapy for 72 hours, penicillin G either alone or in combination with gentamicin did not significantly decrease the numbers of enterococci in vegetations on heart valves compared with untreated controls (P = 0.62 and P = 0.58, respectively). Enterococcus faecium accounts for a notable proportion of clinical enterococcal isolates. Many strains from patients at our institution, as well as from patients at other institutions throughout the country, are highly resistant to penicillin. Because high-level penicillin resistance has important therapeutic implications, periodic surveillance and MIC testing of significant enterococcal isolates, especially E. faecium, are suggested.

Susceptibility of Campylobacter jejuni to strain-specific bactericidal activity in sera of infected patients.

Campylobacter jejuni is a common cause of inflammatory enteritis, which in normal hosts is usually self-limited and resolves without antibiotic therapy. C. jejuni bacteremia is very rare. We examined sera for bactericidal activity that might be important in limiting the extent of C. jejuni infection in man. We studied the ability of nonimmune sera and homologous and heterologous immune sera from infected patients to kill different fresh case isolates of C. jejuni in vitro. The reduction of the log10 concentration of viable C. jejuni (log10 killing) by fresh sera from nonimmune donors was only 0.2. Log10 killing by homologous acute sera varied from 0 to 3.8 (mean, 1.8). Convalescent sera showed remarkable log10 killing of only homologous C. jejuni, with values of 2.7 to 4.4 (mean, 3.7). The bactericidal effects of acute and convalescent sera were abrogated by heat and EDTA chelation, indicating mediation by complement. The role of classical complement pathway activation was supported by chelating sera with magnesium EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid] and by reconstitution experiments with heat-inactivated sera, C2-deficient serum, and purified C2. The requirement of specific antibody for the serum bactericidal effect was indicated by the loss of bactericidal activity when immune sera were absorbed with homologous but not heterologous whole C. jejuni isolates. The presence of specific antibodies was further documented by agglutination of only homologous C. jejuni suspensions by heat-inactivated immune sera. Studies with polymorphonuclear leukocytes suggested that ingestion and killing of two C. jejuni strains were modest and variable in the presence of heat-inactivated homologous serum. In summary, the data document a potent serum bactericidal effect that develops rapidly and specifically during C. jejuni enteritis and may be an important factor in host defense against C. jejuni.

Safety and immunogenicity of a group 29E meningococcal capsular polysaccharide vaccine in adults.

A serogroup 29E Neisseria meningitidis capsular polysaccharide vaccine at a dose of 50 micrograms was injected subcutaneously into 10 adult human volunteers. One vaccinate experienced a mild systemic reaction; one complained of moderate to severe local pain and tenderness. The vaccine induced significant homologous binding and bactericidal antibody by 2 weeks and significant binding antibody against the heterologous serogroup Z capsular polysaccharide by 4 weeks. Although binding antibody rose during the first 4 weeks and then declined slowly over the subsequent 4 months, bactericidal antibody response declined substantially by 4 and 8 weeks for both polysaccharides. The increase in group 29E bactericidal activity was no longer significant at 4 and 8 weeks; loss of bactericidal activity against group Z was significant by 8 weeks. Bactericidal activity again rose between 8 and 26 weeks, becoming significantly increased over prevaccination levels for group 29E and increased, though not significantly (P = 0.085), over prevaccination levels for group Z. As a result of the failure of induced antibody to increase bactericidal activity, only 40% of vaccinates achieved a greater than 2 log2 increase in lytic activity against group 29E, and none achieved this large an increase in lytic activity against group Z.

Inhibition of Neisseria gonorrhoeae by a bacteriocin from Pseudomonas aeruginosa.

Supernatants from broth-grown cultures of Pseudomonas aeruginosa PA 103 exhibited bactericidal activity against Neisseria gonorrhoeae. The concentration of the bactericidal substance increased significantly after induction by mitomycin C. Purification was effected by salt fractionation, chromatography on diethylaminoethyl-cellulose, and sedimentation by centrifugation at 100,000 x g for 90 min. Electron microscopy of this purified preparation revealed structures resembling R-type pyocins in both the contracted and uncontracted state. Pyocins in the contracted state were observed in association with the gonococcal cell surface. No loss of bactericidal activity was observed after treatment with proteolytic enzymes. Standard pyocin typing procedures identified the pyocin pattern as 611 131. The bactericidal activity of this pyocin was examined on various species of Neisseria. Out of 56 strains of N. gonorrhoeae from disseminated and nondisseminated infections, all were susceptible to pyocin 611 131. However, only 3 of 20 strains of N. meningitidis and 5 of 16 strains of N. lactamica were susceptible. The bactericidal activity that pyocin 611 131 has for N. gonorrhoeae and other species of Neisseria is significant because it departs from the expected specificity that heretofore has distinguished bacteriocins from most "classical" antibiotics.

BACTERICIDAL ACTIVITY OF THE BLOOD OF ACTIVELY IMMUNIZED WAX MOTH LARVAE

The blood of normal wax moth larvae is not bactericidal for Pseudomonas aeruginosa. The blood becomes moderately bactericidal when larvae are actively immunized against P. aeruginosa. This activity was measured by a modification of Nagington's technique for the estimation of typhoid antibody. Bactericidal activity appears to be the only measurable type of antibody response against P. aeruginosa. Cell-free blood was as active as whole blood; 0.02 ml of immune serum kills about 1000 organisms. The blood of normal wax moth larvae is bactericidal for Shigella dysenteriae but the blood of insects immunized against either this organism or P. aeruginosa shows no increase in activity against S. dysenteriae. A number of non-specific agents, both protein and non-protein, did not stimulate bactericidal activity in serum after their injection into normal larvae. Immune sera prepared against some strains of P. aeruginosa were not active against other strains. Storage at 37 °C or absorption with zymosan both result in blackening of immune blood and loss of bactericidal activity. Bactericidal activity is evident only during the immune state of the insect, i.e. from about 18 hours until the third day after vaccination; it develops at the same time that inhibition of melanization was observed in the blood from vaccinated larvae.