Subcutaneous Abscesses In Mice Research Articles

Innate and mild Th17 cutaneous immune responses elicited by subcutaneous infection of immunocompetent mice with Cladosporium cladosporioides

Cladosporium cladosporioides is a dematiaceous hyphomycete that is pathogenic in the superficial and deep tissues of both immunodeficient and immunocompetent humans and animals. Our aim was to evaluate the antifungal immune responses elicited by C. cladosporioides in immunocompetent mice.Hence, we subcutaneously injected suspensions of C. cladosporioides spores into immunocompetent mice to investigate the anti-fungal immune responses in the skin. We collected skin tissue samples for histopathological examination, immunofluorescence staining, and quantitative real-time polymerase chain reaction analysis.We observed subcutaneous abscesses in mice after subcutaneous injection of C. cladosporioides. A large number of inflammatory cells, including dendritic cells, macrophages, and neutrophils, infiltrated the focal abscess, with comparatively few infiltrating inflammatory cells in the epidermal and dermal layers of the skin. We detected the expression of CD54 in the abscesses and the skin. Gene expression of the pattern recognition receptors Dectin-1 and TLR-2 was higher in infected mice than in controls. Gene expression of the cytokines IL-6, IL-1β, and IL-17A also increased after infection, suggesting that the Th17 signaling pathway may be involved in the anti-fungal response. Although the pathogenicity of C. cladosporioides in healthy mice was weak after subcutaneous infection, resulting in few serious pathological phenomena, it appears that innate and Th17 immune responses play important roles in the cutaneous host response to C. cladosporioides. These findings lay a foundation for further study of the pathogenic mechanism and treatment of C. cladosporioides infection.

The role of the capsule of the Streptococcus milleri group in its pathogenicity.

Study of the pathogenicity of encapsulated strains of the Streptococcus milleri group (SMG) was performed by examination of the ability to cause subcutaneous abscesses in mice and by phagocytosis and phagocytic killing of human polymorphonuclear neutrophils (PMNs) against the organisms. All 3 encapsulated isolates from patients with pneumonia or lung abscess induced abscesses in the mice; however, only 2 of 20 unencapsulated isolates from patients with lung abscess or thoracic empyema did so. The 3 encapsulated strains inhibited more phagocytosis and phagocytic killing of PMNs than the unencapsulated strains. In addition, encapsular material separated from Streptococcus constellatus RZYK001 also inhibited phagocytosis and phagocytic killing in proportion to increasing concentrations of the capsular material. These results suggest that capsular material produced by SMG might be a pathogenic factor.

Co-aggregation as a virulent factor of Streptococcus sanguis isolated from infective endocarditis.

The pathogenicity of strains of the Streptococcus sanguis group, isolated from infective endcarditis, was studied by measuring the development of subcutaneous abscesses in mice after infection with S. sanguis and Actinomyces viscosus either singly or as co-aggregated pairs. The pathogenicity of the co-aggregates was also examined in various viable combinations of the two bacterial species. More abscesses were formed by A. viscosus than the S. sanguis group including clinical isolates. Abscess formation by co-aggregates of combinations of each isolate and A. viscosus produced a higher percentage of abscess formation than those caused by infection with a pure suspension of A. viscosus or tested streptococci. Co-aggregated cells were more resistant to phagocytosis and killing by neutrophils in vivo. These results indicated that S. sanguis group streptococci isolated from infective endocarditis are able to co-aggregate and resist phagocytosis. The ability of co-aggregation of S. sanguis may serve as a survival mechanism in a host defense system and may be linked with virulence of this bacteria.

Interaction of sub-inhibitory concentrations of ciprofloxacin and rifampicin against Staphylococcus aureus

The frequency of the emergence of ciprofloxacin-resistant mutants to sub-inhibitory concentrations of ciprofloxacin, rifampicin, and ciprofloxacin plus rifampicin were compared in a subcutaneous abscess model of experimental Staphylococcus aureus infection in mice. The in vitro findings showed that the combination of ciprofloxacin and rifampicin was bacteriostatically additive for all strains tested when the combination was examined by the chequer-board technique and fractional inhibitory concentration indices determined. Animals were infected with ciprofloxacin-sensitive and ciprofloxacin-resistant test strains (6989S and 6989R) and left untreated for nine days, which showed that ciprofloxacin resistance had no effect on the pathogenicity of the organisms. In treated animals, ciprofloxacin plus rifampicin was found to be at least as effective as ciprofloxacin alone. However, in the murine model, the combination therapy produced significantly fewer high-level ciprofloxacin-resistant mutants than ciprofloxacin alone ( p<0.0005). We conclude that a combination of ciprofloxacin and rifampicin was at least as efficient as either drug alone, and the additional presence of rifampicin reduced the emergence of high level ciprofloxacin-resistant sub-populations in the case of S. aureus subcutaneous abscesses in mice and so the combination may prove to be more efficient than ciprofloxacin alone.

Effect of co-aggregation on the pathogenicity of oral bacteria.

The pathogenicity of oral bacteria was studied by measuring the development of subcutaneous abscesses in mice after infection with Actinomyces viscosus and Streptococcus mitis either singly or as co-aggregated pairs. Heat-treated cells were also tested. The pathogenicity of the co-aggregates was examined in various viable and heat-treated combinations of the two bacterial species. More abscesses were formed by A. viscosus than S. mitis at all the bacterial concentrations tested. Also, abscess formation by co-aggregates of the two strains produced a higher percentage of abscess formation than those caused by infection with pure suspensions of A. viscosus or S. mitis. Co-aggregated cells were more resistant to phagocytosis and killing by neutrophils in vitro and in vivo. Furthermore, A. viscosus in co-aggregates were resistant to killing after engulfment by neutrophils. These results suggest that oral bacteria that are able to co-aggregate may resist phagocytosis, and this ability may be linked with pathogenicity.

Differentiation of enterobacteriaceae, pseudomonas aeruginosa, and Bacteroides and Haemophilus species in gram-stained direct smears

The accuracy of examination of the Gram-stained direct smear to classify presumptively Gram-negative rods into three morphotype groups, that is, (a) Enteric bacteria, (b) Pseudomonas, and (c) Bacteroides or Haemophilus, was evaluated. Randomly selected clinical strains (4–9) each of itEscherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Ps. aeruginosa, B. fragilis, and H. influenzae were used to produce peritonitis or subcutaneous abscesses in mice. A Gram-stained direct smear was prepared from exudate collected from each animal. The direct smears were examined to classify bacteria observed into one of the three morphotype groups. The percent accuracy was 82, 56, and 95, respectively, and 76 overall. The assumption was made that classification was based primarily on differences in length and width of the organisms. To test this hypothesis, we prepared scanning electron photomicrographs from each specimen of exudate and measured the lengths and widths of bacteria. Examination of the Gram-stained direct smear was more accurate for classification of enteric bacteria, H. influenzae, or B. fragilis. Electron microscopy was more accurate for classification of Ps. aeruginosa. The higher length-width radio should be helpful in recognizing Ps. aeruginosa in direct smears.

The role of Escherichia coli haemolysin in the pathogenic synergy of colonic bacteria in subcutaneous abscess formation in mice

The growth of nine species of colonic bacteria--Escherichia coli, Enterococcus faecalis, Bacteroides ovatus, Fusobacterium varium, Clostridium perfringens, Klebsiella pneumoniae, Proteus vulgaris, Staphylococcus aureus and Bifidobacterium adolescentis--was examined after concomitant injection to form experimental subcutaneous abscesses in mice. Injection of a mixture of c. 10(5) cfu of each of the first five strains (E. coli, Ent. faecalis, B. ovatis, F. varium and C. perfringens) resulted in abscess formation in all mice tested when the E. coli strain was haemolytic. E. coli and B. ovatus multiplied and reached a maximum population of c. 10(8) cfu/abscess. When non-haemolytic E. coli was used, injection of greater than or equal to 10(7) cfu was required for abscess formation. The inclusion of partially purified E. coli haemolysin (125 HU50) with c. 10(5) cfu of bacteria including non-haemolytic E. coli resulted in abscess formation in most mice tested. These results indicate that E. coli haemolysin is one factor that may potentiate pathogenic synergy among colonic bacteria especially between E. coli and B. ovatus, during abscess formation.

Enhancement of growth of aerobic, anaerobic, and facultative bacteria in mixed infections with anaerobic and facultative gram-positive cocci

The potential for mutual enhancement of growth of seven strains of anaerobic and facultative gram-positive cocci (AFGPC), seven aerobic and facultative organisms, and two Bacteroides spp. commonly isolated with AFGPC in mixed infection was evaluated. Enhancement was studied by measuring the relative increase in the colony-forming units of the two bacterial components inducing subcutaneous abscesses in mice. Of the 56 combinations, AFGPC were enhanced in 6 and inhibited in 1. The aerobic and facultative bacteria were enhanced in 32 of the 42 combinations and depressed in none. The Bacteroides spp. were enhanced in 12 of the 14 combinations and suppressed in none. The organisms uniformly enhanced by AFGPC were Group A streptococci, Pseudomonas aeruginosa, and Bacteroides fragilis (all seven instances), followed by Escherichia coli (six of seven instances), Bacteroides asaccharolyticus and Klebsiella pneumoniae (five instances each), Staphylococcus aureus (four instances), and Group D streptococci (three instances). It is apparent that in mixed infection with AFGPC the rate of growth of Bacteroides spp. and facultative and aerobic bacteria is enhanced much more than the rate of growth of AFGPC.

Enhancement of growth of aerobic and facultative bacteria in mixed infections with Bacteroides species.

The potential for mutual enhancement of growth of the Bacteroides fragilis and B. melaninogenicus groups and the aerobic and facultative organisms commonly isolated with them in mixed infections was evaluated. Enhancement was studied by measuring the relative increase in CFU of the two bacterial components inducing subcutaneous abscesses in mice. Of the 42 combinations between three isolates each of the B. fragilis and B. melaninogenicus groups and seven aerobic or facultative organisms, Bacteroides spp. were enhanced in only 8 and inhibited in 4. The aerobic and facultative bacteria were enhanced in 31 of the 42 combinations and depressed in 2. The organisms uniformly enhanced by all of the Bacteroides spp. were group A streptococci and Escherichia coli (all six instances), followed by Staphylococcus aureus and Klebsiella pneumoniae (five of six instances), Pseudomonas aeruginosa (four instances), group D streptococci (in three instances only by the B. fragilis group), and Haemophilus influenzae (one instance). It is apparent that the growth rate of facultative and aerobic bacteria is enhanced much more in mixed infections with Bacteroides spp. than that of their anaerobic counterparts.

Transfer of tetracycline or clindamycin resistance among strains of Bacteroides fragilis in experimental abscesses.

The ability of strains of Bacteroides fragilis resistant to clindamycin and/or tetracycline to transfer resistance to a susceptible recipient strain in experimental subcutaneous abscesses in mice was investigated. The results indicated that such transfer took place at a frequency similar to that observed in vitro. Transfer of resistance determinants at infected sites may play a role in the epidemiology of disseminated resistance to antimicrobial agents.

Synergistic effect of bacteroides, Clostridium, Fusobacterium, anaerobic cocci, and aerobic bacteria on mortality and induction of subcutaneous abscesses in mice.

The potential for synergy between aerobic, facultative, and anaerobic bacteria was studied by subcutaneous inoculation of mixtures of these organisms into mice and observation of subsequent mortality and abscess formation. The anaerobic bacteria tested included 12 strains of gram-positive cocci and two strains each of Bacteroides species, Clostridium species, and Fusobacterium species. The facultative and aerobic bacteria included one strain each of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Mortality increased significantly when each aerobic organism was inoculated along with either of the Bacteroides species. A similar increase occurred when the anaerobic gram-positive cocci were inoculated along with P. aeruginosa (four of six combinations) or S. aureus (four of six). The rate of abscess induction increased significantly when 10 of the 12 strains of anaerobic gram-positive cocci were injected along with B. fragilis and when nine of these strains were inoculated along with Bacteroides asaccharolyticus . The results demonstrate synergistic potential between Bacteroides species and all aerobic bacteria tested, between Bacteroides species and most anaerobic gram-positive cocci, and between most anaerobic gram-positive cocci and P. aeruginosa or S. aureus.

Comparison of virulence of clones of two Trichomonas vaginalis strains by the subcutaneous mouse assay.

No statistical differences in virulence were found among five clones isolated from each of two Trichomonas vaginalis strains JH31A and Balt 42. The former strain, isolated from a patient showing no cervical epithelial abnormalities, caused relatively small subcutaneous lesions in mice [mean volume for the noncloned strain, 75.45 +/- 4.43 mm3 (n = 70); mean of means for cloned populations, 77.28 +/- 3.14 mm3 (n = 230)]. The latter, Balt 42, isolated from a woman with an in situ carcinoma of the cervix uteri, produced large subcutaneous abscesses in mice [mean volume for the noncloned strain, 202.28 +/- 12.53 mm3 (n = 55); mean of means for cloned populations, 200.48 +/- 13.72 mm3 (n = 264)]. The apparent homogeneity of T. vaginalis strains with regard to virulence reinforces the dependability of the subcutaneous mouse assay.

Infectivity of organisms recovered from polymicrobial abscesses.

The ability to cause subcutaneous abscesses in mice was used to identify the pathogens among the bacteria recovered from 13 clinical abscesses. A total of 35 isolates (30 anaerobes and 5 aerobes), 16 of which were encapsulated, were recovered from these abscesses. Encapsulated organisms included eight Bacteroides spp. (three Bacteroides asaccharolyticus and one strain each of Bacteroides oralis, Bacteroides intermedius, Bacteroides biacutus, Bacteroides vulgatus, and Bacteroides ruminicola subsp. brevis), three anaerobic gram-positive cocci, two Clostridium spp., and two strains of Escherichia coli. Single organisms, or combinations thereof, obtained from these abscesses were inoculated subcutaneously into mice. All but one of the encapsulated organisms were able to cause abscesses by themselves and were recovered from the abscesses when inoculated with other organisms. Seven non-encapsulated organisms which were also recovered mixed with the encapsulated organisms were never able to cause abscesses nor could they be recovered after they were injected alone. Thirteen strains that did not induce abscesses when injected by themselves into mice survived when injected with other organisms which were encapsulated. In three instances, pairs of non-encapsulated organisms belonging to the last group were able to induce an abscess and survive in it when inoculated together. It was shown that the possession of a capsule by a clinical isolate increases the likelihood that it is a major contributor to the infectious process.

Antibiotic levels in infected and sterile subcutaneous abscesses in mice.

The pharmacokinetic properties of 10 antimicrobial agents were examined in sterile and infected encapsulated subcutaneous abscesses in mice. The inoculum for sterile abscesses was autoclaved cecal contents; that for infected abscesses was autoclaved cecal contents combined with Bacteroides fragilis. The antimicrobial agents examined were rosaramicin, clindamycin, chloramphenicol, metronidazole, and six beta-lactam antibiotics. All antimicrobial agents entered abscesses, produced peak levels of biological activity that were somewhat delayed in comparison to serum levels, and were present in negligible levels 8 hr after administration. The highest concentration in abscesses was achieved with rosaramicin and clindamycin, with peak levels of 43%--63% of the peak serum level. Peak levels of other antimicrobial agents in sterile abscesses were 13%--27% of the peak serum level. Levels of biologically active during were significantly lower in infected abscesses than in sterile abscesses for antimicrobial agents that are inactivated by B. fragilis beta-lactamase.