Evaluation of Sulfamethoxazole- Trimethoprim Blood Agar Plates for Recovery of Group A Streptococci from Throat Cultures

We compared the selective blood agar medium of Gunn et al. (J. Clin. Microbiol. 5:650-655, 1977) which contains sulfamethoxazole plus trimethoprim (SXT-BA) to the conventional blood agar surface plate (SBA) and a modified blood agar pour plate plus broth method for the recovery of group A streptococci from throat swabs. The influence of CO(2) and ambient air incubation of the SXT-BA and SBA plates was also evaluated. A total of 696 throat swabs from symptomatic children were cultured simultaneously by the five methods and observed after overnight incubation; 204 positive cultures were detected overall. Recovery rates of each individual method were: SXT-BA (CO(2)), 90.7%; SXT-BA (air), 87.7%; pour plate plus broth, 83.3%; SBA (CO(2)), 79.4%; and SBA (air) 77%. Approximately one-half of the false-negative cultures in the SXT-BA (CO(2)) and SXT-BA (air) methods had colony counts of >/=10 to 100 colonies per plate. In contrast, for the SBA (CO(2)), SBA (air), and pour plate plus broth methods, approximately 70% of the false-negative cultures had colony counts of >/=10 to 100/plate. False-positive cultures obtained by the SXT-BA (CO(2)) and SXT-BA (air) methods were 11 and 12.7%, respectively-one-half as high as the rates obtained by the remaining methods. Beta-hemolytic streptococci, groups C, F, and G, are inhibited on the SXT-BA plates and were the primary cause of the higher false-positive rates on SBA and pour plate plus broth methods. An additional 3% positive cultures were obtained by incubating SXT-BA (CO(2)) plates up to 48 h before discarding as negative. We recommend either the SXT-BA (CO(2)) or the SXT-BA (air) method with up to 48 h of incubation for routine use in throat cultures.     

SXT and Taxo A Disks for Presumptive Identification of Group A and B Streptococci in Throat Cultures

A bacitracin (0.04 units, BBL)-SXT (trimethoprim, 1.25 mg, plus sulfamethoxazole, 23.75 mg, BBL) susceptibility test was 94% accurate in presumptively identifying streptococci as either group A, B or not group A and B.     

Localization and Characterization of the Hippuricase Activity of Group B Streptococci

Studies are presented on the isolation, localization, and characterization of hippuricase activity of group B streptococci. Washed, intact cells, live or heat killed at 56 C, exhibited hydrolysis of hippuric acid, but cell-free filtrates of the organism did not. Excellent hippuricase activity was recoverable from supernatant fluids of mechanically disrupted cells, and evidence suggests that it exists largely intracellularly. Characteristics of the hippuricase preparation are consistent with the view that the biologically active principle is an enzyme. A quantitative microtiter technique has been developed which is useful in titrating enzymatic activity and antibody neutralization. Sera from rabbits immunized with filtered preparations neutralized hippuricase activity.     

Izalpinin from Fruits of Alpinia Oxyphylla with Antagonistic Activity Against the Rat Bladder Contractility

Background

Alpinia oxyphylla (Zingiberaceae), an herbaceous perennial plant, its capsular fruit is commonly used in traditional Chinese medicine for the treatment of different urinary incontinence symptoms including frequency, urgency and nocturia. These symptoms are similar to the overactive bladder syndrome. In our lab, we found that the 95% ethanol extract of the capsular fruits exhibited significant anti-muscarinic activity. Some constituents in capsular fruits including flavonoids (e.g., izalpinin and tectochrysin), diarylheptanoids (e.g., yakuchinone A and yakuchinone B) and sesquiterpenes (e.g., nootkatone), are regarded as representative chemicals with putative pharmacological activities.

Objective

This study aimed to evaluate the in vitro antagonistic actions of izalpinin on carbachol-induced contraction of the rat detrusor muscle.

Materials and Methods

In vitro inhibition of rat detrusor contractile response to carbachol was used to study the functional activity of izalpinin. The isolated detrusor strips of rats were mounted in organ baths containing oxygenated Krebs'' solution. The cumulative consecutive concentration-response curves to carbachol-evoked contractions in strips of rat bladder were obtained.

Results

Carbachol induced concentration-dependent contractions of isolated rat bladder detrusor strips. The vehicle DMSO had no impact on the contraction response. The contraction effects were concentration-dependently antagonized by izalpinin, with a mean EC50 value of 0.35 µM. The corresponding cumulative agonist concentration-response curves shifted right-ward.

Conclusions

Izalpinin exhibits inhibitory role of muscarinic receptor-related detrusor contractile activity, and it may be a promising lead compound to treat overactive bladder.     

Screening for Antimicrobial Resistance in Normal Bacterial Flora of the Skin Using the Replica Plating Method

 The replica plating method was evaluated for detection of the antimicrobial resistance of normal bacterial flora of the skin and was compared with the results of a ten-colony method. If ≥10% of the colonies from the master plate grew on a plate containing an antibiotic, the sensitivity of replica plating was comparable to that of a ten-colony method for samples containing resistant bacteria. However, this method classified significantly more samples as resistant to all eight antibiotics tested if the detection breakpoint was lowered to ≥1% of the original colonies. Replica plating is an effective and practical tool for screening skin flora for resistance, also in samples with a low proportion of resistant strains.     

Effect of Exposure to the Atmosphere on the Infectivity of Group A Streptococci

During exposure to the atmosphere, type 14 group A streptococci lost infectivity for mice more rapidly than they declined in viability. The loss of infectivity resulted from phenotypic, not genotypic, changes. Comparison of rates of loss of infectivity during exposure of capsulated and noncapsulated streptococci suggested that the principal effect of atmospheric exposure was on the capsule, a known determinant of infectivity. Accelerated drying promoted the loss of infectivity.     

Characterization of sil in Invasive Group A and G Streptococci: Antibodies against Bacterial Pheromone Peptide SilCR Result in Severe Infection

Group G beta-hemolytic streptococcus (GGS) strains cause severe invasive infections, mostly in patients with comorbidities. GGS is known to possess virulence factors similar to those of its more virulent counterpart group A streptococcus (GAS). A streptococcal invasion locus, sil, was identified in GAS. sil encodes a competence-stimulating peptide named SilCR that activates bacterial quorum sensing and has the ability to attenuate virulence in GAS infections. We found that sil is present in most GGS strains (82%) but in only 25% of GAS strains, with a similar gene arrangement. GGS strains that contained sil expressed the SilCR peptide and secreted it into the growth medium. In a modified murine model of GGS soft tissue infection, GGS grown in the presence of SilCR caused a milder disease than GGS grown in the absence of SilCR. To further study the role of the peptide in bacterial virulence attenuation, we vaccinated mice with SilCR to produce specific anti-SilCR antibodies. Vaccinated mice developed a significantly more severe illness than nonvaccinated mice. Our results indicate that the sil locus is much more prevalent among the less virulent GGS strains than among GAS strains. GGS strains express and secrete SilCR, which has a role in attenuation of virulence in a murine model. We show that the SilCR peptide can protect mice from infection caused by GGS. Furthermore, vaccinated mice that produce specific anti-SilCR antibodies develop a significantly more severe infection. To our knowledge, this is a novel report demonstrating that specific antibodies against a bacterial component cause more severe infection by those bacteria.     

Effect of Bactericidal Substance from Staphylococcus aureus on Group A Streptococci II. Structural Alterations

Ultrastructural alterations brought about by treatment of a β-hemolytic streptococcus with a bactericidal substance from Staphylococcus aureus are described and illustrated. The substance causes an early condensation of nucleoid deoxyribonucleic acid (DNA) and a partial loss of ribosomes. These changes are followed by a dissolution of the cell contents resulting in bacterial “ghosts” composed of empty cell wall and capsule. These morphological findings correlate with known biochemical effects of the bactericidal substance on ribonucleic acid degradation and cessation of DNA and protein synthesis.     

Effect of Bactericidal Substance from Staphylococcus aureus on Group A Streptococci I. Biochemical Alterations

A bactericidal substance isolated from phage type 71 staphylococci has been studied relative to its mechanism of action on streptococcal cells. The substance exerts its effect best at 37 C. No cell lysis occurs as evidenced by lack of alteration in optical density of susceptible cell suspensions. The bactericidal substance results in immediate cessation of protein and deoxyribonucleic acid synthesis as well as degradation of newly and previously formed ribonucleic acid (RNA). The action on RNA seems to be independent of protein synthesis.     

M Antigens of Group A Streptococci Isolated by Means of Immunochromatography. Biochemical and Serological Properties

M proteins of Streptococcus pyogenes types 1 and 12 were purified by immunochromatography on immobilized type-specific opsonizing antibodies. The M proteins were characterized serologically and biochemically. They absorb after immobilization opsonizing antibodies and only precipitate with homologous antisera in immunodiffusion. In SDS PAGE they show one main band, corresponding to mol. weights of 5 × 10⁴ (type 1) and 5.4 × 10⁴ (type 12), accompanied by faint lines, which form with the main band a precipitation line of identity in SDS-crossed immunoelectrophoresis. Both M proteins aggregate blood platelets and clot fibrinogen.     

Oxygen-Stable Hemolysins of Group A Streptococci VIII. Leukotoxic and Antiphagocytic Effects of Streptolysins S and O

Streptolysin S exists in a cell-bound form and as an extracellular complex between a nonspecific carrier (serum, serum albumin, ribonucleic acid [RNA], Triton, Tween) and a hemolytic moiety (probably a peptide) synthesized by streptococci. Although all the forms of streptolysin S, at 100 hemolytic units, killed mouse leukocyte monolayers, the time needed to kill 100% of the cells varied with the different streptolysin S preparations. Whereas 30 min was sufficient for the cell-bound hemolysin to kill all of the cells, 60 and 180 min were required when RNA streptolysin S and serum streptolysin S, respectively, were employed. Addition of 10% mouse serum to RNA streptolysin S or to cell-bound hemolysin delayed the killing of the leukocytes. The delayed killing observed with serum and albumin hemolysins is probably due to competition for the hemolytic moiety between the carrier molecules and target sites (phospholipids) upon the leukocyte membrane. Serum streptolysin S must be constantly incubated with the cells for 90 min for 100% of the cells to undergo cytopathic changes upon subsequent incubation for an additional 90 min. Streptolysin S inhibitor (trypan blue) added to the system after 30 or 60 min of incubation resulted in the killing of 50 and 100% of the leukocytes, respectively, when the cells were further incubated for 120 min. It is suggested that 30 min of incubation was not sufficient for the transfer of enough streptolysin S molecules upon the cell surface to allow killing of all of the cells. Sublethal amounts of streptolysin S, streptolysin O, and saponin suppressed phagocytosis of streptococci by mouse peritoneal macrophages. This effect was abolished by inhibitors of streptolysin S (trypan blue) and of streptolysin O and saponin (cholesterol). With sublethal amounts of streptolysin S, no inhibition of the reduction of nitro blue tetrazolium by nonphagocytosing cells was observed, but these amounts of streptolysin S caused a 50% inhibition of the reduction of nitro blue tetrazolium by phagocytosing leukocytes. It is suggested that some metabolic systems, which are normally enhanced during phagocytosis, have been affected by sublethal doses of streptolysin S. The results indicate that the in vivo production of small amounts of streptolysins S and O by group A streptococci may inhibit phagocytosis and may thus contribute to the invasiveness and pathogenicity of this microorganism.     

Opsonic Antibodies to the Surface M Protein of Group A Streptococci in Pooled Normal Immunoglobulins (IVIG): Potential Impact on the Clinical Efficacy of IVIG Therapy for Severe Invasive Group A Streptococcal Infections

The surface M protein of group A streptococci (GAS) is one of the major virulence factors for this pathogen. Antibodies to the M protein can facilitate opsonophagocytosis by phagocytic cells present in human blood. We investigated whether pooled normal immunoglobulin G (IVIG) contains antibodies that can opsonize and enhance the phagocytosis of type M1 strains of GAS and whether the levels of these antibodies vary for different IVIG preparations. We focused on the presence of anti-M1 antibodies because the M1T1 serotype accounts for the majority of recent invasive GAS clinical isolates in our surveillance studies. The level of anti-M1 antibodies in three commercial IVIG preparations was determined by enzyme-linked immunosorbent assay (ELISA), and the opsonic activity of these antibodies was determined by neutrophil-mediated opsonophagocytosis of a representative M1T1 isolate. High levels of opsonic anti-M1 antibodies were found in all IVIG preparations tested, and there was a good correlation between ELISA titers and opsonophagocytic activity. However, there was no significant difference in the levels of opsonic anti-M1 antibodies among the various IVIG preparations or lots tested. Adsorption of IVIG with M1T1 bacteria removed the anti-M1 opsonic activity, while the level of anti-M3 opsonophagocytosis was unchanged. Plasma was obtained from seven patients with streptococcal toxic shock syndrome who received IVIG therapy, and the level of anti-M1 antibodies was assessed before and after IVIG administration. A significant increase in the level of type M1-specific antibodies was found in the plasma of all patients who received IVIG therapy (P < 0.006). The results reveal another potential mechanism by which IVIG can ameliorate severe invasive group A streptococcal infections.     

Peptic Digestion of Streptococcal M Protein II. Extraction of M Antigen from Group A Streptococci With Pepsin

M protein of group A streptococci was extracted by mild peptic digestion. Optimal amounts of type-specific M protein were released after 20 min of digestion with 0.02 mg of pepsin per ml at pH 5.8. Immunological analysis revealed that, unlike conventional HCl extracts, pepsin extracts lacked the surface C carbohydrate antigen and contained less non-type-specific, heat-stable cellular antigens; they also lacked detectable heat-labile T protein. Similar to HCl extracts, however, the pepsin-extracted M protein precipitated homologous-type M antisera and inhibited type-specific opsonization of homologous group A streptococci. Furthermore, the pepsin extract was capable of inducing type-specific opsonic M antibody in rabbits. This method may provide a useful initial step in the purification of M protein by reducing contaminating antigens.     

Individual Physical Activity Behaviour and Group Composition as Determinants of the Effectiveness of a Childhood Obesity Intervention Program

IntroductionUp to now, there is limited clarity on factors that determine the effectiveness of childhood obesity interventions.ObjectiveThis study intends to uncover individual- and program-level predictors of BMI-SDS and fitness to achieve significant, sustainable health improvements.MethodsData of 249 children with obesity or overweight who participated in an outpatient multidisciplinary program were analysed and compared to 54 waitlist controls. Linear regression models were used to examine associations between individual- and group-level variables and BMI-SDS and fitness.ResultsAmong intervention children, BMI-SDS decreased by 0.19 units and physical fitness increased by 11.5%, versus a BMI-SDS decrease of 0.07 and a 1.8% decrease in fitness in the control group. Participants who reported being physically active before the program start achieved greater improvements in BMI-SDS (β = −0.177, p < 0.05) and physical fitness (β = 0.174, p < 0.05) than inactive peers. BMI-SDS decreased significantly more for members of gender-heterogeneous groups (β = 0.194, p < 0.05) with a narrow age range (β = 0.152, p < 0.05).ConclusionsThe program under review is effective in counteracting juvenile obesity. The results give reason to believe that forming mixed-gender groups with a small age range and providing increased support for reportedly inactive children may improve program effectiveness.     

On the Virulence of Group A Streptococci: Evidence for Virulence Factors Other than M-Protein and Capsule

Group A streptococci from parent cultures (PC) of six different serotypes were selected by rotation with human blood (RHB) or by serial passage via intraperitoneal inoculation in mice (MP). M-protein content of PC, RHB and MP streptococci of each serotype was determined in quintuplicate by radial immunodiffusion against type-specific antisera. Hyaluronic acid content was determined in quintuplicate colorimetrically after treatment of streptococci with hyaluronidase. Data were subjected to variance analysis. LD₅₀ for mice of PC, RHB and MP streptococci, inoculated intraperitoneally, was also determined. It was observed that MP streptococci virulent for mice were rich in M-protein and hyaluronic acid. However, RHB streptococci of M-protein and hyaluronic acid content similar to MP streptococci were not virulent for mice. This dichotomy with respect to mouse virulence infers the existence of as yet unidentified streptococcal virulence factors, in addition to M-protein and capsule.     

Identification, Cloning, and Expression of the CAMP factor gene (cfa) of Group A Streptococci

The CAMP reaction is a synergistic lysis of erythrocytes by the interaction of an extracellular protein (CAMP factor) produced by some streptococcal species with the Staphylococcus aureus sphingomyelinase C (beta-toxin). Group A streptococci (GAS [Streptococcus pyogenes]) have been long considered CAMP negative, and this reaction commonly has been used to distinguish GAS from Streptococcus agalactiae. We here provide evidence that GAS possess this gene and produce an extracellular CAMP factor capable of participating in a positive CAMP reaction. The S. pyogenes CAMP factor is specified by a 774-bp open reading frame homologous to the CAMP factor genes from S. agalactiae and Streptococcus uberis. This gene, designated cfa, was isolated on a 1,256-bp fragment and cloned in Escherichia coli. Recombinant clones of E. coli expressing cfa secreted an active CAMP factor. The deduced 28.5-kDa protein encoded by cfa consists of 257 amino acids, with a predicted 28-amino-acid signal peptide. The cfa gene is widely spread among GAS: 82 of 100 clinical GAS isolates produced a positive CAMP reaction. Of the CAMP-negative strains, 17 of the 18 GAS strains contained the cfa gene. Additionally, CAMP activity was detected in streptococci from serogroups C, M, P, R, and U. The cfa gene was cloned and actively expressed in Escherichia coli and gene fusions were made, placing the beta-galactosidase gene (lacZ) under control of the cfa promoter. These cfa promoter-lacZ fusions were introduced into S. pyogenes via a bacteriophage-derived site-specific integration vector where they showed that the cfa gene has a strong promoter that may be subject to as-yet-unidentified regulatory factors. The results presented here, along with previous reports, indicate that the CAMP factor gene is fairly widespread among streptococci, being present at least in groups A, B, C, G, M, P, R, and U.     

Effect of Body Fluids and Macromolecular Substances on the Lysis of Group A Streptococci by Muramidases of Streptomyces albus

The lysis of group A streptococci by muramidases of Streptomyces albus is strongly inhibited by human, rabbit, and calf serum as well as by human synovial fluids and pus. Rabbit antisera to heat-killed streptococci were no more inhibitory to the lysis of the streptococci by the lytic enzyme than normal rabbit serum. The results indicate that muramidases of S. albus will not be useful for the in vivo treatment of chronic granulomatous lesions which had been induced by insoluble cell wall components of streptococci.