Aminoethyl-isothiourea inhibits the increase in plasma endothelin-1 caused by serogroup A streptococci and prolongs survival in rat peritoneal sepsis.

To elucidate the possible roles of nitric oxide (NO), endothelin-1 (ET-1), and reactive oxygen species (ROS) in the pathophysiology of serogroup A streptococcal (GAS) peritoneal sepsis, we investigated the effects of aminoethylisothiourea (AE-ITU), an inducible NO synthase (iNOS) inhibitor, and a ROS scavenger, and the ET-1 receptor antagonist bosentan. In rats, live GAS inocula, 3 x 10(8) and 1 x 10(9) cfu/kg, entailed a 24-h mortality of 10% and 90%, respectively. GAS caused increases in tissue iNOS activity (9 h), in serum nitrite/nitrate (9-24 h), and in intracellular leukocyte ROS levels (3-6 h). These changes were all prevented by the pre-treatment with AE-ITU. A novel finding was that AE-ITU also prevented the GAS-induced marked increase in plasma ET-1 at 6 h. Short-term (7-h) survival was improved by both AE-ITU and by bosentan. The mechanism(s) for the beneficial effects of AE-ITU may possibly be a combined mode of action; iNOS inhibition, ROS scavenging, and inhibition of the increase in plasma ET-1 caused by GAS.

Murine monoclonal antibodies against pneumococcal capsular polysaccharide types 4, 8, 22F and 19A/19F.

Monoclonal antibodies (MAbs) were produced against pneumococcal capsular polysaccharides after subcutaneous immunization of BALB/c mice with a 23-valent vaccine (Pneumovax N, Merck, Sharp & Dohme). Selected antibodies were tested in ELISA against individual polysaccharides from 23 different pneumococcal types and in a dot blot assay with heat-killed whole bacteria adhered to nitrocellulose paper. Three MAbs (isotype IgM) were found to be specific for types 4, 8 and 22F, respectively, whereas one (isotype IgA) reacted both with 19A and 19F. Very mild acid hydrolysis of the capsular polysaccharides resulted in loss of reaction with the antibodies.

Virulence of Streptococcus pneumoniae in mice: a standardized method for preparation and frozen storage of the experimental bacterial inoculum.

Animal models of pneumococcal infection are important to evaluate the protective capacity of new vaccine candidates. We have established a method to prepare and store the experimental inoculum without loss of virulence or number of bacteria. This allows a standardized inoculum from the same culture batch to be used in several experiments. Pneumococci were cultured to mid-logarithmic growth phase in Todd-Hewitt broth with 17% fetal calf serum. The bacterial broth was distributed into smaller volumes and immediately frozen on liquid nitrogen and stored at -70 degrees C. We have tested the virulence of five different pneumococcal serotypes in BALB/c, C57BL/6, and NIHS mice using inocula prepared by this method and stored without loss of virulence for up to 4 years. Serotypes 1, 4, 5 and 8 were highly virulent for the strains of mice tested whereas type 6B showed lower virulence and a peculiar, protracted course of infection. There were no clear differences in virulence between the different strains of mice with the exception of serotype 6B, which showed higher virulence in BALB/c and NIHS mice than in C57BL/6 mice.

Serotyping and bacteriophage typing of group B streptococci.

Three hundred and eighty-two human streptococcal strains of serogroup B, collected from different sources and from different parts of Norway have been serotyped and phagetyped . The results of serotyping show a predominance of serotype Ia; 150 strains belonged to this type. Only 32 strains belonged to serotype III, a serotype well-known as the dominating cause of neonatal septicemia and meningitis (11, 23). The other serotypes were evenly represented. The phagetyping was performed by the procedure and with phages described by Stringer (19). The strains were lysed by phages at a frequency of 70%. A total of 145 strains (38%) was lysed by one or two phages, while 124 strains (32%) were typable by different phage patterns. When combined with serotyping, phagetyping of group B streptococci is an important tool in characterizing these microbes for epidemiological and other purposes.

Aminoethyl-isothiourea, a nitric oxide synthase inhibitor and oxygen radical scavenger, improves survival and counteracts hemodynamic deterioration in a porcine model of streptococcal shock.

OBJECTIVE: To test the effect of a continuous infusion of the nitric oxide (NO) synthase (S) inhibitor aminoethyl-isothiourea (AE-ITU) on survival time, hemodynamics, and oxygen transport in a porcine model of live group A streptococcal (GAS) sepsis. Furthermore, to examine the role of endothelin-1, histamine, and reactive oxygen species (ROS) in streptococcal shock. DESIGN: Prospective, randomized trial. SETTING: Laboratory at a university hospital. SUBJECTS: Twenty-eight pigs with an average weight of 25 kg. INTERVENTIONS: Sixteen animals received a continuous infusion of live Streptococcus pyogenes 1.3 x 10(10) colony forming units/hr: eight received fluids only, and the other eight received an intravenous infusion of AE-ITU 10 mg/kg/hr starting 30 mins before the GAS challenge. Six control pigs received AE-ITU 10 mg/kg/hr iv for 5 hrs. Another six animals received half the dose of GAS over 5 hrs. MEASUREMENTS AND MAIN RESULTS: GAS infusion caused a rapid increase in pulmonary, hepatic, and systemic vascular resistance, followed by hypotension with a 90% lethality at 4 hrs. Treatment with AE-ITU increased 4-hr survival in septic animals from 1/8 to 8/8 and 5-hr survival from 0/8 to 5/8, prevented hypotension, and increased urine output. AE-ITU attenuated the decrease in cardiac output, liver blood flow, and oxygen delivery, and hepatic arterial blood flow as a fraction of cardiac output increased (all p < .05). Plasma nitrate/nitrite levels decreased in all animals. Inducible NOS and endothelial constitutive NOS activities in liver, gut, and lung were not increased during sepsis, nor were they decreased after AE-ITU. Plasma levels of endothelin-1 and methylhistamine increased in all septic animals and were not modified by AE-ITU. AE-ITU prevented the increase in monocyte ROS production caused by GAS. In control animals, AE-ITU caused an increase in mean arterial pressure, liver blood flow, and oxygen delivery. CONCLUSIONS: In this model of porcine GAS-induced septic shock, which was not associated with enhanced NO production, infusion of the NOS inhibitor AE-ITU prolonged survival, prevented hypotension, and improved cardiac contractility, organ perfusion, and tissue oxygenation. These beneficial effects of AE-ITU might be a result of the combined effect of ROS scavenging and modulation of local NO production, thus improving the balance of vasodilator and vasoconstrictor forces and reducing oxidative stress.

Acute serogroup A streptococcal shock: A porcine model.

To elucidate the pathophysiology of acute shock caused by serogroup A streptococci (GAS), GAS were given intravenously to 25 pigs. Short-time infusions of GAS (n=11) caused variable and unpredictable responses. A continuous infusion of 5x108 cfu/kg/h (n=8) caused pulmonary hypertension, arterial hypotension, and reduced cardiac output and liver perfusion, progressing to circulatory shock within 2-4 h. Halving the infusion rate (n=6) induced a more gradual development of shock and doubled the mean survival time from 2.1 to 4.0 h. Mean tumor necrosis factor-alpha levels (+/-SE) increased from 25+/-1 to 40+/-3 pg/mL. Only slight signs of organ dysfunction were observed, which indicates that this is primarily a model of acute septic shock. Light microscopy revealed moderate inflammatory reactions in lung, liver, and gut biopsy samples, although high numbers of viable, M-typeable GAS were recovered from tissues. The present model may be useful to study mechanisms involved in acute septic shock as well as therapeutic interventions.

Systemic pneumococcal disease in Norway 1995-2001: capsular serotypes and antimicrobial resistance.

A total of 4624 pneumococcal isolates from episodes of systemic pneumococcal disease were received at the Norwegian Institute of Public Health during the period 1995-2001. All isolates were serotyped and tested for susceptibility to benzylpenicillin, lincomycin, erythromycin, tetracycline and trimethroprim sulphamethoxazole. The proportion of strains resistant to these antimicrobial agents remained stable at a low level, ranging from 0.1% for benzylpenicillin to 2.5% for erythromycin. The distribution of serotypes was also stable over the 7 years: serotypes 1, 4, 9, 14, 7, 6 and 23 were the most frequent, representing 70.5% of isolates. Overall, 95.8% of the isolates were of serotypes/groups included in the current 23-valent polysaccharide vaccine, 52.2% were of serotypes/groups included in the 7-valent conjugated vaccine and 85.5% were of serotypes/groups included in the 11-valent conjugated vaccine.