Challenge Organisms Research Articles

Studies on the evaluation of preservative efficacy V. Effect of concentration of micro-organisms on the antimicrobial activity of phenol

An investigation of the effect of the concentration of Staphylococcus aureus on the antimicrobial activity of phenol has been performed. For this purpose, a kinetic approach to the screening of antimicrobial activity of phenol was successfully applied. The values of the parameters D, E (the time for elimination of any size population of organism) and E ̀ (the time for elimination of 10 2 organisms/ml) were calculated. The results show that the preservative efficacy is not affected by the concentration of challenge organisms up to the point where the system becomes overloaded.

Monoclonal Antibodies for treatment of Gram-Negative Infections

Monoclonal antibody technology has permitted researchers to dissect out the protective antibody response to conserved regions of gram-negative bacillary lipopolysaccharides (endotoxins). Some anticore antibodies can bind to lipid A and have a neutralizing, but not opsonic, activity; these antibodies are usually IgM. IgG antibodies to outer core regions may be weakly opsonic. The outcome of animal protection studies is critically dependent on the choice of challenge organism, dose, timing and rate of antibody administration, and additional factors such as antimicrobial therapy. Protective activity against a wide variety of gram-negative bacillary challenges with the IgM anticore and lipid A reactive antibody, which we have designated E5, is reviewed. Protection in a therapeutic model is demonstrable when the antibody is used in conjunction with appropriate antimicrobial therapy. This antibody is now being assessed in clinical trials. Optimal use of monoclonal antibodies may involve a "cocktail" of antibodies with complementary binding specificities.

Studies on the evaluation of preservative efficacy. IV. The determination of antimicrobial characteristics of some pharmaceutical compounds in aqueous solutions Int. J. Pharm., 46 (1988) 141–147.

An investigation of the antimicrobial activity and its concentration dependence of some pharmaceutical compounds other than preservatives (mercuric oxycyanide (MO), ethyl alcohol (EA), propylene glycol (PG) and sodium metabisulphite (SM)) has been performed. For this purpose the kinetic approach to the screening of antimicrobial activity of preservatives was successfully applied. D-values and concentration exponents (η) were used as parameters in determining microbiocidal activity. Aspergillus niger, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were employed as challenge organisms. All the substances tested exerted antimicrobial activity within the concentration ranges usually applied in pharmaceutical preparations. EA and PG had about the same η-value (3–6), while SM and MO had values of 1–2 and 0.4–3, respectively.

In vivo Studies with Dimethyldithiocarbamate, a Possible New Antimicrobial for Use against Aspergillus fumigatus in Poultry

Dimethyldithiocarbamate (DmDTC), the carbamate analogue, was tested for therapeutic efficacy in a series of in vivo challenge trials using 5- and 10-week-old white leghorn chickens. Challenge organisms were Pasteurella multocida X-73, Escherichia coli O1:K1, and Aspergillus fumigatus. Birds were evaluated for survival rates, lesion scores, and the rate at which the bacteria or mold could be reisolated following challenge. Results showed DmDTC to be ineffective against P. multocida and E. coli at the treatment levels and in the form used in these trials, but DmDTC significantly reduced lesion scores and inhibited the rate of isolation of A. fumigatus compared with untreated infected birds.

Rocky Mountain spotted fever: a disease in need of microbiological concern

Rocky Mountain spotted fever, a life-threatening tick-transmitted infection, is the most prevalent rickettsiosis in the United States. This zoonosis is firmly entrenched in the tick host, which maintains the rickettsiae in nature by transovarian transmission. Although the incidence of disease fluctuates in various regions and nationwide, the problems of a deceptively difficult clinical diagnosis and little microbiologic diagnostic effort persist. Many empiric antibiotic regimens lack antirickettsial activity. There is neither an effective vaccine nor a generally available assay that is diagnostic during the early stages of illness, when treatment is most effective. Microbiology laboratories that offer only the archaic retrospective Weil-Felix serologic tests should review the needs of their patients. Research microbiologists who tackle these challenging organisms have an array of questions to address regarding rickettsial surface composition, structure-function analysis, and pathogenic and immune mechanisms, as well as laboratory diagnosis.

Feasibility of Using Proteins from Salmonella gallinarum vs. 9R Live Vaccine for the Prevention of Fowl Typhoid in Chickens

Proteins from a field strain of Salmonella gallinarum MSG1 were compared with 9R live vaccine strain for their protection against experimental fowl typhoid in chickens. Proteins from S. gallinarum gave better protection than the 9R live vaccine as measured by clearance of challenge organism from internal organs. Proteins given twice with an adjuvant at 200 micrograms/100 g body weight resulted in 95% protection, compared with 60% protection with 9R given orally. The 9R live vaccine produced more hepatic and splenic lesions and, when administered orally as a single dose, was the least protective (60%). In the group vaccinated subcutaneously with a single dose of 9R without an adjuvant, both the challenge strain and the 9R vaccine strain were isolated from the ovaries of some birds. All chickens vaccinated with 9R strain or with proteins developed antibodies detectable by microagglutination test, and in some vaccinated groups as many as 100% of the birds developed antibody levels detected by seroagglutination.

Disinfectant Testing Using a Modified Use-Dilution Method: Collaborative Study

An initial collaborative study of the AOAC use-dilution method (UDM), used for bactericidal disinfectant efficacy testing, demonstrated extreme variability of test results among the 18 laboratories testing identical hospital disinfectants. In an effort to improve the method, 32 changes were made by the UDM Task Force. These changes represented improvements in quality assurance practices and elimination of method variability; however, the basic framework of the method was retained. A second collaborative trial was conducted to determine if the interlaboratory variability of test results could be reduced to an acceptable level using the modified UDM. Twelve of the original 18 laboratories participated in the second study. Each laboratory processed 60 penicylinders (P) for each of the 6 randomly selected, federally registered disinfectants and 3 test organisms (Staphylococcus aureus, Salmonella choleraesuis, Pseudomonas aeruginosa). The number of positive penicylinders (greater than 1 positive P/60 replicates = failure) for the 6 products when P. aeruginosa was used as the challenge organism ranged 1-30, 0-36, 0-15, 0-5, 0-3, and 0-60 for the 3 quaternaries and 3 phenolics, respectively. The results of the variance components analysis for P. aeruginosa and the other 2 organisms showed that the variance components for laboratories were not significantly reduced for any organism in this study. Such interlaboratory variability of results questions the use of the original or the modified UDM for registration purposes.

Efficacy of an immunomodulating drug in immunodeficient and immunocompetent mice

Bacillus subtilis culture filtrate (BSR R) was examined for its immunomodulating ability in the immunodeficient beige mouse and the immunocompetent Han:NMRI mouse strain. Following various application schemes the mice were challenged either subcutaneously with Escherichia coli or intranasally with Streptococcus pneumoniae. The mortality rates and the median of survival time were determined. Following subcutaneous application of BSK R Han:NMRI mice challenged with E. coli showed a decreased in mortality regardless of the application scheme. Using the beige mouse a protective effect was observed only after single applications of relatively low doses. Using Sc. pneumoniae as the challenge organism, a subcutaneous application of BSK R caused a decrease in mortality only if it was given on the day prior to infection. In both infection models the intranasal application of BSK R and/or of saline proved to be beneficial by either significantly decreasing the mortality or considerably increasing the survival time of beige and Han:NMRI mice.

Screening of fungal strains employed in the testing of plastics materials

The problems of selecting tests method and suitable challenge organisms for plastics materials are discussed. Enzyme activities of fungal strains employed in the testing of plastics have been investigated and compared with isolates of these organisms held elsewhere. The ability of organisms to clear a polycaprolactone diol medium has been assessed semiquantitatively and the results discussed in relation to the selection of organisms.

The role of interleukin 1 and 2 in generation of acquired resistance against mouse typhoid infection afforded by dialyzable factor from Salmonella typhimurium.

Dialyzable factor (DF) prepared from a ribosomal fraction of Salmonella typhimurium was tested for its ability to induce interleukin 1 (IL 1) and 2 (IL 2) production, in relation to acquired resistance, after an intraperitoneal injection of DF. IL 1 production in vitro by peritoneal macrophages of DF-treated mice reached the maximum 4 days after injection, at the time when the nonspecific local resistance via macrophages directly activated with DF became apparent (Kita et al, Microbiol. Immunol. 28:807, 1984). Concanavalin A-induced IL 2 production by splenocytes of DF-treated mice reached the maximal level between days 6 and 8, and it could be enhanced even on day 14. Antigen-induced blastogenic responses of splenocytes from DF-treated mice reached the maximal level 14 days after treatment. Although DF did not show the mitogenic activity to normal splenocytes, T cells of DF-treated mice could respond to S. typhimurium. On the contrary, T cells of normal mice could respond to heat-killed cells of S. typhimurium when they were cultured with macrophages which had been directly stimulated in vitro with DF. Furthermore, T cells from DF-treated mice could respond to antigens of different species of bacteria, and especially to Listeria monocytogenes. These results suggest that T cells of DF-treated mice, being at the intermediate stage of activation via monokines including IL 1 which is produced by macrophages stimulated with DF, are able to proliferate immediately after the administration of challenging organisms as a second signal, and also that the specificity of the response may be defined by the challenging organisms.

Protection against experimental salmonellosis in mice and sheep by immunisation with aromatic-dependent Salmonella typhimurium

Mice immunised by the oral or intraperitoneal route with a live aromatic-dependent strain of Salmonella typhimurium exhibited significantly less protection against oral challenge with 50 LD50 of an ovine isolate of S. typhimurium (12313) than when a bovine isolate with the same O antigens and phage-type as strain 12313 was used as the challenge organism. When challenged with 10 LD50, however, protection against both strains was significantly better than that obtained when mice were vaccinated with killed vaccines (heat-killed, acetone-killed or irradiated) even when the antigenic mass of the killed vaccine was increased by up to 500-fold in an attempt to compensate for the expected limited multiplication of the mutant organism. Sheep immunised with the live mutant strain by either the intramuscular or oral route were protected against oral challenge with the virulent ovine isolate of S. typhimurium; unimmunised sheep died of acute enteritis within 7 days, although there was no evidence of systemic invasion by the challenge organism. After challenge, immunised animals ate more food than the unimmunised controls and suffered only transient, mild diarrhoea. Serum antibody titres against O and H antigens measured by direct or antiglobulin tests were significantly higher in sheep immunised by the intramuscular route than in those immunised orally. Sheep in both immunised groups developed skin swellings within 30 min after intradermal inoculation with purified homologous lipopolysaccharide indicating development of immediate-type hypersensitivity, but only those immunised by the intramuscular route showed significant indurated skin swellings characteristic of delayed-type hypersensitivity 48 and 72 h post-inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of pulmonary defense mechanisms by acute exposures to nitrogen dioxide

The effect of acute exposures to NO2 on the antibacterial defenses of the murine lung was assessed following inhalation challenges with Staphylococcus aureus, Proteus mirabilis, and Pasteurella pneumotropica. Animals were challenged with the bacteria and exposed for 4 hr to increasing concentrations of NO2 after which pulmonary bactericidal activity was quantitated. With S. aureus pulmonary antibacterial defenses were suppressed at NO2 levels of 4.0 ppm and greater. Exposure to 10.0 ppm enhanced the intrapulmonary killing of P. mirabilis which correlated with an increase in the phagocytic cell populations lavaged from the lungs; at 20.0 ppm bactericidal activity against P. mirabilis was impaired. Pulmonary antibacterial defenses against P. pneumotropica were impaired at 10.0 ppm which correlated with a decrease in the retrieved phagocytic lung cell population. Reversing the order of treatment (ie., NO2 exposure prior to bacterial challenge) raised the threshold concentration for NO2-induced impairment of intrapulmonary bacterial killing. With S. aureus the effect was not observed at 5.0 ppm but at 10.0 ppm and with P. mirabilis not at 20.0 ppm but at 30.0 ppm intrapulmonary killing was enhanced. Exposures up to 20.0 ppm of NO2 did not effect the physical translocation mechanisms of the lung as quantitated by declines in pulmonary radiotracer activity following aerogenic challenge with 32P-labeled staphylococci. These studies demonstrate that NO2 modulates pulmonary antibacterial defenses and points to the importance of the challenge organism and the exposure protocol in establishing a threshold dose for the adverse effect.

An Ultrastructural Study of HeLa Cell Invasion with Salmonella typhi GIFU 10007

Scanning electron micrograph of HeLa S3 monolayered cells, inoculated with viable bacteria of a Salmonella typhi strain GIFU 10007, revealed that the extended microvilli tangled the bacteria within 10 min after inoculation. The micrographs of HeLa cells, at 1 hr after inoculation, indicate the following: shortening of bacterium-attached microvilli, subsiding of tangled bacteria into microvilli bush, and then attachment of bacterial soma to cell surface making the cell membrane depressed. The transmission electron micrographs, at 1 hr after inoculation, demonstrated the findings of interaction between HeLa cell and S. typhi 10007, similar to those observed on scanning electron micrographs. Hair-like fine structures from the soma of challenge organisms were also observed. They were in contact with HeLa cell microvilli and cell membrane. The bacteria were first partially and then totally surrounded by the HeLa cell plasma membrane. One, two, or several bacteria with intact outer membrane were enclosed in intracytoplasmic membrane-bound vacuoles. Fragmented vacuolar membrane was still visible around the intracellularly accumulated bacteria at 24 hr after inoculation. The viable cells of S. typhi 10007 are regarded as internalizing into HeLa cells by a process of endocytosis and to multiply within the membrane-bound vacuoles.

Invasiveness of Salmonella typhi strains in HeLa S3 monolayer cells.

The internalization and intracellular multiplication, i.e., the invasiveness, of Salmonella typhi strains recently isolated from typhoid fever patients were confirmed in HeLa cell monolayers. When stained with Giemsa solution, intracellular bacteria were 0.6 X 1.2 micron in size and stained purple, whereas extracellular bacteria associated or not with the HeLa cell surface were 1.0 X 3.0 micron and stained deep blue. Strain GIFU 10007 was internalized into 23% of the HeLa cells within 10 min after inoculation. About 90% of the HeLa cells were infected after 24 hr incubation in kanamycin (KM)-containing medium. Intracellular multiplication of the challenge organism was verified by a large number of intracellular bacteria after 24 hr incubation in KM-containing medium by both light-microscopy of the Giemsa stained preparation and viable counts of intracellular bacteria. The viable counts of strain 10007 showed an increase of more than 40-fold within 24 hr after inoculation, whereas in the four other less or non-infective strains, recovery of viable bacteria was poor or nil. Strains which were highly invasive usually failed to show strong adhesion. The contribution of Vi antigen to the internalization of challenge organisms was not proved. Infective strains, when killed by formalin were still adhesive, but were not internalized. The same strains, when killed by boiling, were neither adhesive nor internalized. From these findings it was concluded that the internalization and multiplication of infective S. typhi strains in cultured HeLa cells should be regarded as an invasion rather than phagocytosis by host cells, and such invasiveness could be an indicator to estimate the virulence of S. typhi strains.

Experimental Salmonella arizonae infection of sheep.

At approximately 12 weeks gestation, eight ewes were dosed orally with 3·9 × 109, Salmonella arizonae (61:k:1, 5, 7). No abnormal clinical signs were observed and the challenge organism was recovered from the faeces of all the ewes for up to two weeks. At approximately 16 weeks gestation, the original eight ewes and seven other ewes were dosed with 7·5 × 109, S. arizonae. Abnormal clinical signs were not observed and all the ewes, with the exception of a barren ewe, lambed within five days of the expected date. Faecal excretion of S. arizonae was sporadic, the last isolation occurring 28 weeks after the second challenge. S. arizonae was not isolated from any of the lambs at birth and during the two months suckling period it was isolated from only one of the lambs. Five ewes were served again and S. arizonae was recovered periodically during the nine weeks prior to parturition. At post-mortem examination of the ewes, S. arizonae was isolated from only one of them. In a second experiment, 10 lambs were infected with S. arizonae doses ranging from 2·5 × 104 to 4·3 × 108. One lamb which had low serum immunoglobulins died after challenge with 106 organisms. The other lambs excreted S. arizonae in their faeces for up to six days and it was recovered from the large intestine of one at post-mortem examination.

Cutaneous infection in normal and immunocompromised mice.

Since a model of staphylococcal skin infection adequately reflecting human disease was unavailable, a self-limiting animal infection model specific for virulent Staphylococcus species was developed. A virulent strain of S. aureus, NCTC 9789 (ATCC 27700), was used to develop an infection model in adult, male CF-1 mice treated with 0 to 150 mg of cyclophosphamide (CY) per kg 4 days before challenge. Bacteria were inoculated onto the dorsal side of shaved mice at 0 to 10(6) CFU per mouse. Simultaneously, the skin was gently scraped to remove the superficial layers without drawing blood. The wound was occluded with impermeable film secured with surgical tape. At a CY dose of 50 mg/kg and an inoculum of 10(5) CFU, 89% of the mice (96 of 108) developed large abscesses (approximately 15-mm diameter). Mice which were not immunocompromised developed fewer abscesses (20 of 68). Generally, no abscesses formed when the mice were not wounded (1 of 62), occluded (0 of 89), or inoculated (11 of 50). The abscesses developed 24 to 48 h after challenge and persisted for 2 to 3 weeks. The challenge organism was isolated from the abscesses. The rates of abscess formation of three additional S. aureus strains varied widely in normal and CY-treated mice. Three strains of S. epidermidis, one of Micrococcus varians, and one of S. saprophyticus failed to cause abscesses. Bacterial proliferation studies demonstrated that a strain of S. aureus and a strain of S. epidermidis proliferated to the same levels 48 h after challenge. Immunosuppression and wounding had little effect on the levels of proliferation of S. aureus (P greater than 0.2). Without occlusion, however, S. aureus proliferated to significantly lower levels (P less than 0.005). This model may be be useful for screening topical anti-infective agents or studying the mechanisms of bacterial pathogenesis and host response.

Production of Brucella abortus-specific protein A-reactive antibodies (IgG2) in infected and vaccinated cattle

The IgG2 anti-Brucella antibody response of cattle to Brucella vaccination and infection was measured. Three groups of animals were studied; Group 1 contained 11 non-vaccinated cows, Group 2, 17 cows vaccinated with a low dose of Strain 19 vaccine and Group 3, 17 cows vaccinated with a high dose of Strain 19 vaccine. All animals were challenged at Week 33 with an infectious isolate of B. abortus (Strain 2308). Studies of the IgG2 antibodies response indicated an absolute correlation between anti-Brucella IgG2 levels and infection of the animal. All animals showing reciprocal titers of ⩾3000 (16 of 45 tested) were found to be positive for the challenge organism at slaughter. Animals with reciprocal IgG2 titers ⩽1000 (29 of 45 tested) were found to be negative for the challenge organism at the time of slaughter. The predictive value of IgG2 antibody levels for infection held for animals in all three groups and consequently this suggests that monitoring of specific IgG2 anti-Brucella antibody levels may be of value in detection of Brucella-infected cattle.

Inhibition of macrophage phagocytosis by Bacteroides fragilis in vivo and in vitro.

A number of investigators have provided experimental evidence for synergistic effects in mixed infections with Escherichia coli and Bacteroides fragilis. In vitro studies have suggested that competition for serum opsonins and diminished subsequent phagocytosis by polymorphonuclear leukocytes might explain these effects. In the present study we evaluated the effect of B. fragilis on macrophage phagocytosis. It was shown that peritoneal macrophages from mice injected intravenously 6 to 12 h earlier with 10(8) CFU of encapsulated B. fragilis were markedly suppressed in their phagocytic ability. Injections of laboratory-passaged, less-encapsulated B. fragilis, other bacteria, or latex particles were either not suppressive of macrophage phagocytosis or less effective. When peritoneal macrophages were treated in vitro for 24 h with the same challenge organisms prior to assessing their phagocytic capacity, encapsulated B. fragilis also proved significantly more suppressive than challenges with other organisms or latex particles. We conclude that suppression of macrophage phagocytosis by B. fragilis seems to be an important mechanism contributing to synergistic effects described for mixed aerobic and anaerobic infections.

Increased susceptibility to lethal Candida infections in burned mice preinfected with Pseudomonas aeruginosa or pretreated with proteolytic enzymes

Lethal Candida infections in burn patients are frequently preceded by or occur concomitantly with bacterial infections, which are often due to Pseudomonas aeruginosa. In this study, we developed a burned, mixed-challenge mouse model, which was designed to determine whether and how a recent bacterial infection could influence the development of subsequent candidosis. In this model, burned mice that were preinfected with a sublethal challenge of elastase-producing P. aeruginosa strain WR-5 and then sublethally challenged with Candida albicans exhibited a mortality rate of 60%, while unburned mice challenged in the same way and burned mice that received only one challenge organism exhibited mortality rates of less than 10%. Quantitative microbial counts performed with the kidneys, livers, and eschars of burned mice challenged with both organisms indicated that the deaths were due to Candida infection. Substitution of an elastase-negative P. aeruginosa strain for strain WR-5 in the model resulted in significantly lower mortality rates and lower microbial numbers in the organs. When the Pseudomonas enzyme elastase was substituted for the elastase-positive bacteria in the model, both the mortality rates and the organ counts were comparable to the values found after preinfection with strain WR-5. Another protease, thermolysin, was substituted for the elastase and produced similar mortality results. When the protease inhibitor alpha 2-macroglobulin was given to burned mice infected with the two organisms, it prevented the deaths due to Candida infection. We concluded that this model is one way to study bacterial-fungal infections in burned mice, that recent Pseudomonas infections could predispose burned mice to fatal candidosis, and that the proteolytic activity generated by the bacteria was primarily responsible for the establishment of lethal fungal infections.

Elimination of challenge bacteria from the blood and intestines of ayu Plecoglossus altivelis vaccinated by immersion.

Ayu Plecoglossus alaivelis were immersed for 3 min in a 5% NaCl solution containing 1% lyophilized formalin-killed Vibrio anguillarurm. Two months after vaccination, each of the four experimental groups vaccinated fish were infected by intraperitoneal (i.p) injection of either 103, 104, or 106 cells of V. anguillarum PT479 streptomycin resistant (Smr)-strain or exposed to 106 cells/ml of PT479 Smr strain. The time period of elimination of the infecting organism depended on the number of cells of V. anguillarum given to the fish. The challenge organisms wera virtually eliminated within 6h after i.p. injection of 103 or 104 PT479 Smr cells in the blood and intestinal tracts. On the other hand, organisms increased 12h after injection of 103 or 104 cells/ml in non-vaccinated fish, reaching 106 cells/ml of blood and 105 cells/g in the intestinal tracts by 72h. The challenge organisms were observed in the blood and intestinal tracts of vaccinated fish Within 24h after i.p. injection of 106 cells. Subscquentry, the organisms decreased to 10 cells or less. The number of organisms increased in non-vaccinated fish after i.p. injection of 106 cells. The number of challenge organisms was 10 cells/ml or g, or less, in the blood and the intestinal tracts of vaccinated fish 72h after exposure to 106 cells. The challenge organisms were also ob-served in the blood of non-vaccinated fish for 72h.