agar and Pneumococcal-Infections

Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspA-based vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1-5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.

Topics: Agar; Animals; Antibodies, Bacterial; Bacterial Proteins; Capsules; Membrane Proteins; Mice; Pneumococcal Infections; Pneumococcal Vaccines; Polysaccharides; Streptococcus pneumoniae

Since the introduction of pneumococcal conjugate vaccine, there have been warnings of an increase in infections caused by non-vaccine type of Streptococcus pneumoniae strains. Among them, nonencapsulated Streptococcus pneumoniae (NESp) has been reported to cause invasive infections, especially in children and the elderly. Due to low virulence, however, basic experimental reports on invasive infections are limited.. We applied a liquid-agar method to establish a mouse model of invasive NESp infection. Mice were intratracheally administered a bacterial suspension including agar. With this technique, we investigated the pathogenicity of NESp and the effect of Pneumococcal surface protein K (PspK), a specific surface protein antigen of NESp. NESp wild-type strain (MNZ11) and NESp pspK-deleted mutant strain (MNZ1131) were used in this study. The survival rate, number of bacteria, cytokine/chemokine levels in the bronchoalveolar lavage fluid, and histology of the lung tissue were evaluated.. Mice that were intratracheally administered MNZ11 developed lethal pneumonia with bacteremia within 48 h. Conversely, MNZ1131 showed predominantly low lethality without significant pro-inflammatory cytokine production. NESp was found to cause severe pneumonia and bacteremia upon reaching the lower respiratory tract, and PspK was a critical factor of NESp for developing invasive infections.. The current study demonstrated the ability of NESp to develop invasive diseases, especially in connection with PspK by use of a mouse pneumonia model.

Topics: Agar; Animals; Bacteremia; Cytokines; Mice; Pneumococcal Infections; Pneumonia, Pneumococcal; Streptococcus pneumoniae; Virulence

To produce two supplemented agar types in order to store pneumococci for several months at room temperature.. Todd-Hewitt/Hemoglobin/Yeast/Charcoal/Agar (TH-HYC) and Todd-Hewitt/Skim-Milk/Yeast/Charcoal/Agar (TH-SYC) were used to prepare two supplemented agar types. Nineteen pneumococci isolated from patients or asymptomatic carriers displaying diverse serotypes and multilocus sequence types (MLST) were subcultured and stored onto supplemented agar types, in four different tests, at room temperature.. At the end of all tests (4-6 months) all noncontaminated subcultures were viable and maintained all phenotypic characteristics. Survival-time curves revealed a slow decrease of viable CFU over time on agar types, but at the end the number of viable CFU was satisfactory (≥2+ of growth). Decreasing of CFU was significantly higher for clinical versus nasopharyngeal isolates. Subcultures contamination rates were 6.25% and 14.58% after 2 and 6 months of storage, respectively.. TH-HYC and TH-SYC agar types allowed the viability of pneumococci with several serotypes, MLST, and genetic profiles, after 6 months of storage at room temperature. We consider that these agar types are a valid alternative to preserve pneumococci over an extended period, especially when methods as cryopreservation or lyophilization are not available, and are useful for transporting strains between laboratories.

Topics: Agar; Bacteriological Techniques; Colony Count, Microbial; Culture Media; Humans; Microbial Viability; Pneumococcal Infections; Preservation, Biological; Streptococcus pneumoniae; Time Factors

Streptococcus pneumoniae grows well and generally exhibits typical morphology on Columbia blood agar, whereas Haemophilus influenzae requires a more complex medium to meet its growth requirements - usually chocolated blood agar - on which S. pneumoniae is less easily recognisable. Therefore, a single medium that produces typical morphology of S. pneumoniae and facilitates the growth of H. influenzae would have considerable potential advantages. It has been claimed that blood agar supplemented with nicotinamide adenine dinucleotide (NAD) is such a medium. However, despite its routine use in several large diagnostic laboratories its performance has never been properly evaluated. In the present study, 1724 sputum samples were examined in four laboratories. The isolation rates of H. influenzae and S. pneumoniae on NAD-supplemented blood agar (SBA) were compared with those on a two-plate combination of plain blood (BA) and chocolated blood agar (CBA). The two-plate combination performed significantly better for both organisms; isolation rates for H. influenzae were increased from 8.16% on SBA to 11.07% on BA plus CBA and for S. pneumoniae from 4.18% to 4.68%. Isolation rates were also compared after incubation for 24 and 48 h. With the two-plate combination, isolation rates for H. influenzae and S. pneumoniae were increased by 0.98% and 0.16%, respectively, and for SBA by 0.57% and 0.32% after 48 h. However, despite this increase, SBA still performed less well than the two-plate combination.

Topics: Agar; Bacteriological Techniques; Cacao; Culture Media; Haemophilus Infections; Haemophilus influenzae; Heme; Humans; NAD; Pneumococcal Infections; Sputum; Streptococcus pneumoniae

A prospective study of the natural history of pneumococcal infection, which involves serial culture studies in healthy infants from 6 weeks of age onward, is in progress in our laboratory. This report describes results of a comparison of several methods for the isolation and identification of Streptococcus pneumoniae from the nasopharynges and throats of these infants. Sheep blood agar, sheep blood agar with gentamicin sulfate (gentamicin agar), and mouse inoculation with 4-h broth cultures were used. Gentamicin agar proved superior to plain sheep blood agar as a solid culture medium, especially in enhancing the recovery of pneumococci from throat cultures. With gentamicin agar, similar carrier rates were found for both culture sites (nasopharynx and throat). In addition, gentamicin agar proved superior to mouse inoculation for the recovery of carrier strains from 131 nasopharyngeal culture samples processed by both methods. Sixty of 131 samples were positive for pneumococci, 25% of which would have been missed had mouse inoculation alone been used. In only three instances did we recover a strain by mouse inoculation that failed to grow on gentamicin agar; conversely, 15 strains were isolated on gentamicin agar but could not be recovered from mice. The latter observation might be explained by the fact that certain carrier strains may be relatively mouse avirulent. The use of blood agar containing gentamicin appears to offer a simple and inexpensive method for the recovery of S. pneumoniae and, in our opinion, provides an ideal method for the identification of pneumococcal carriers as well as for the recovery of these strains from clinical material such as sputum or ear exudates, where other and less fastidious organisms may also be present.

Topics: Agar; Animals; Bacteriological Techniques; Carrier State; Evaluation Studies as Topic; Gentamicins; Humans; Infant; Mice; Nasopharynx; Pharynx; Pneumococcal Infections; Streptococcus pneumoniae

Topics: Agar; Animals; Anti-Bacterial Agents; Culture Media; Drug Resistance, Microbial; Drug Synergism; Enterobacteriaceae; Enterobacteriaceae Infections; Escherichia coli Infections; Folic Acid Antagonists; Haemophilus Infections; Meningococcal Infections; Mice; Microbial Sensitivity Tests; Pneumococcal Infections; Pyrimidines; Streptococcal Infections; Sulfamethoxazole