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In a rabbit model, we have previously reported evidence for a pathogenic role of streptococcal IgG Fc-binding proteins (IgGFcBP) in poststreptococcal glomerulonephritis (PSGN). These proteins, of the M protein family, were shown to trigger anti-IgG production and enhance renal deposition of IgG and/or immune complexes (ICs), with resulting activation of complement and cytokine cascades. In the present study, type M12/emm12, group A streptococci (GAS) were found often to bind artificial ICs, viz. peroxidase–anti-peroxidase rabbit IgG (PAP) or tetanus toxoid–anti-tetanus human IgG (TAT), rather than monomeric IgG. Animals injected with each of four IC binding clinical isolates (from patients with scarlet fever or PSGN) showed pronounced inflammatory and degenerative glomerular changes, morphologically similar to human PSGN, with membrane thickening and IgG and complement C3 deposition, as well as secretion of IL-6 and TNF-α by mesangial and endothelial cells. In contrast, non-binding strains (two from asymptomatic carriers and one from a PSGN case) failed to trigger any renal changes. Only the IC binding strains induced elevated titres of anti-IgG. Though the streptococcal binding component(s) has not been demonstrated, the selective binding of ICs by type M12/emm12 strains appears important for the well-known, marked nephritogenic potential of this GAS type.

The pathogenesis of acute poststreptococcal glomerulonephritis (APSGN), a major nonsuppurative complication of group A streptococcal (GAS) throat or skin disease, remains unclear. During the years, various theories based on certain streptococcal extracellular factors, as well as immunological mimicry between streptococci and renal tissue, have been forwarded. We earlier reported that many clinical GAS isolates with documented nephritogenic capacity show non-immune binding of monomeric or aggregated IgG. Moreover, in a rabbit model of APSGN we obtained evidence for an important role of streptococcal IgG Fc binding proteins (IgGFcBPs) belonging to the M family surface proteins; thus, hyperimmunization by whole IgGFcBP-positive streptococci was shown to induce renal glomerular changes with deposition of IgG and complement C3, resembling the picture recorded in human APSGN. These typical renal changes were always preceded by the appearance of circulating anti-IgG antibodies. In the present work, using the same rabbit model, each of two purified IgGFcBPs, isolated from type M22 GAS, were found to elicit glomerular degenerative damage comparable to that caused by whole bacteria, as well as formation of anti-IgG. In addition, the induction by whole streptococci (type M1) of experimental APSGN was inhibited by the i.v. administration of purified human or rabbit IgG Fc, but not Fab, fragment, supporting the importance of Fc-mediated mechanisms in causation of glomerulonephritis. We propose that anti-IgG antibody, induced by streptococcal IgGFcBP, facilitated renal accumulation of IgG-containing complexes, which in turn triggered complement deposition and proinflammatory cascades. Further studies on the possible beneficial effect of IgG Fc fragment in APSGN should be of interest.

Recently, we reported that conjugal transfer of plasmid pERL1, determining i.a. erythromycin resistance (Emr), into group A streptococci could trigger the expression of anti-phagocytic activity, adhesiveness, opacity factor and capacity to bind immunoglobulin Fc-parts and beta 2-microglobulin. In the present study, ethidium bromide treatment of Emr transconjugants allowed the selection of "cured", erythromycin sensitive (Ems) mutants. This procedure did not affect the expression of the abovementioned characteristics. However, when plasmid pERL1 was again transferred to two such mutants, the "secondary", Emr transconjugants obtained showed lack of each of these properties. Our experiments thus demonstrated a "switch-on" as well as a "switch-off" effect, exerted by the same plasmid, pERL1, on some major pathogenic properties of group A streptococci.

Anti-type 12 serum incorporated in agarose-polyethylene glycol gel in a concentration of 1.5% (vol/vol) was found to enable a distinct "rocket" precipitate in electro-immuno assay using hot hydrochloric acid extract of type 12 group A streptococci. This precipitate was removed by trypsin treatment of the extract and on addition of anti-M12 typing serum but not of five other typing sera to the extract. The streptococcal component responsible for this precipitate was eluted from a CM-cellulose ion exchange column at pH 6.5. These findings demonstrated that the precipitate was caused by the M12 antigen. Crossed immuno-electrophoresis of hot hydrochloric acid extracts of three different type 12 group A streptococci showed that the electrophoretic mobility of the M12 antigens was similar in the three extracts. A linear correlation was obtained between the concentration of the M12-antigen and the height of the precipitate obtained in the electro-immuno assay using different dilutions of a standard type 12 extract. M12 antigen could thus be quantitated by the electro-immuno assay. In quantitation experiments, uniformly prepared extracts of five randomly selected, freshly-isolated type 12 strains were found to contain from 130 to 1850% of M12 antigen, respectively (expressed in % of the content of the standard type 12 extract).

Eight freshly isolated type M4 strains of group A streptococci were found to bind between 60 and 80% and 2.5 microgram radiolabelled IgA myeloma protein in a standard test system, while a reference type 4 strain bound only 20%. Commercial human IgG or IgG1 myeloma protein did not inhibit the binding of IgA by the reference type 4 strain or one of the freshly isolated type 4 strains, whereas inhibition was obtained by purified polyclonal IgA myeloma protein. Fc fragments of purified IgA1 myeloma protein, obtained by digestion with gonococcal protease, inhibited binding or radiolabelled IgA1, while the Fab fragments had no inhibitory effect.

A medium ("Y" medium) is described, which was more efficient for the isolation of Yersinia enterocolitica from experimentally infected faecal specimens than desoxycholate-citrate. McConkey, lactose-sucrose-urea (LSU) agar, and Yersinia selective medium (Wauter's medium). The "Y" medium consists of casein hydrolysate and peptone servings as carbon and energy sources. A high selectivity is achieved by its contents of sodium oxalate and bile salts. The oxalate suppresses growth of gram-negative rods, including members of the family Enterobacteriaceae and of Pseudomonas spp., while the bile salts inhibit growth of gram-positive bacteria. In the few instances coliform rods grew on the "Y" medium, they could easily be distinguished by their fermentation of lactose, included in the medium, and the fact that colonies of organisms were surrounded by an opaque zone of precipitated bile salts. The most optimal condition for the isolation of Y. enterocolitica from stools was achieved at incubation of the "Y" medium at 29 degrees C for 2 days.

Various polyelectrolytes were investigated regarding their capacity to inhibit the binding of human IgG to Fc-receptors on group A streptococci, type M1. Of cationic substances, protamine and arginine-rich histone inhibited significantly, while lysine-rich histone, concanavalin A, lysozyme, polymyxin B, ribonuclease and tuftsin did not. Of anionic materials, liquoid was inhibitory, in contrast to chondroitin sulphate, dextran sulphate, DNA and heparin. Washing experiments showed that the inhibition was caused by binding of the polyelectrolytes to the streptococci. The finding that heated IgG inhibited the binding of histone to the streptococci also indicated a close relation between the binding sites for these compounds. Diffusion-in-gel experiments with alkaline extract of M1 demonstrated that the substances blocking the IgG Fc-receptor were bound to polyglycerophosphate, suggesting that the inhibition of the IgG uptake was due to interaction with lipoteichoic acid. Leukocyte and platelet extracts could modify the binding of IgG, probably by an enzymatic digestion of the receptors. The arginine-rich histone was also capable of inhibiting the binding of IgG to type M15 group A streptococci and to one group G strain. However, the polyelectrolytes had no effect on the binding of IgG to Staphylococcus aureus or of IgA to type 4 group A streptococci.

Aggregates of various mammalian beta 2-microglobulin (beta 2m) homologues were tested in binding experiments with group A streptococcal strains of different M types. The binding patterns obtained were similar, suggesting that evolutionarily conserved parts of the beta 2m molecule are responsible for the interaction with group A streptococci. An N-terminally abnormal beta 2m showed binding characteristics similar to those of normal human beta 2m, indicating that the amino-terminal does not participate in this interaction. Aggregates of human IgG Fab fragments, kappa chains and lambda chains, were also analyzed. Whereas several of the beta 2m-reactive M types did not interact with any of these aggregates, all strains binding aggregated Fab, kappa or lambda, also bound aggregated beta 2m. Strains of M types 4, 12, 23 and 53 bound all the tested proteins; M type 1 bound all but IgG Fab, whereas M types 46, 49 and 53 showed affinity for beta 2m and lambda chains only. In inhibition experiments, unlabelled aggregated beta 2m in excess completely blocked the uptake of radiolabelled aggregated IgG Fab, kappa and lambda chains. Conversely, Fab, kappa and lambda aggregates inhibited the binding of radiolabelled beta 2m aggregates. Our results indicate that the differences in reactivity recorded between beta 2m and IgG Fab, kappa and lambda chains, all structurally related, are quantitative rather than qualitative. Thus, a common binding structure for these aggregated proteins on group A streptococci appears probable.

The erythromycin-resistance marker of two plasmids, pSM19035 and pERL1, could be transferred by conjugation in matings within group A streptococci and between group H and group A streptococci, with a frequency of 10(-5) to 8 X 10(-7). Two of the recipient strains used, 22v-1 and 22v-2, showed traces of opacity factor (OF) activity. This activity increased markedly upon transfer of both plasmids to the strains, whereas a number of other recipients, viz. 22h-21, 12Teiko-1 and Challis 6-1, remained OF-negative after conjugation. Furthermore, the transconjugants resulting from matings using strains 22v-1 and 22v-2 as recipients expressed anti-phagocytic activity. Attempts were made to type the transconjugants but they did not belong to type M12, M22 (the types from which the parents were derived) M3 or M1. Concomitant with the expression of anti-phagocytic activity, IgG and IgA Fc-receptor activity occurred in the transconjugants of 22v-1 and 22v-2, whereas the donor and recipient cells were without receptors. It was not possible to demonstrate extrachromosomal DNA in transconjugants possessing anti-phagocytic, OF or Fc-receptor activity, although they retained their ability to serve as donors of the Emr marker. It is suggested that the triggering by plasmids of anti-phagocytic activity, OF and IgG and IgA Fc-receptors is due to insertion of plasmid DNA into the chromosome.

The possibility of inducing immunity in the upper respiratory tract against type M50 group A streptococci was studied in mice. The M50 type exhibited an LD100 a of 10(5) colony-forming units (CFU) when administered intranasally (i.n.), and of 10(7) CFU when introduced intraperitoneally (i.p.). I.n. administered vaccines prepared from M types 12, 18, 30, 49, 50 and 55 were equally efficient in preventing lethal infection after i.n. challenge with type M50. Subcutaneous (s.c.) immunization with type 18 had no effect, whereas s.c. immunization with type 50 was effective against i.n. challenge with type 50. Neither with i.n. nor s.c., did vaccination with type 18 - in contrast to with type 50 - protect against i.p. challenge with type 50. The results strongly suggest that local immunity against M50 group A streptococci can be achieved using a non-type-specific mechanism. Once the local barrier had been overcome, protection against systemic infection was type-specific in accordance with classic concepts.

A type 12 group A strain (1800) was passaged serially through mice 25 times. The ability to servive in normal human blood dropped from a growth index of 52 after the first passage to 1 after four passages. After 14 passages the growth index increased again and stabilized above 30. The virulence for mice increased from a LD100 of 10(8) colony forming units (CFU) to 10-100 CFU after 7 passages and then remained constant. The Mqw antigen disappeared after 4 passages as tested by immunodiffusion, electroimmunoassay and indirect bactericidal tests. Three antisera, raised in rabbits against strains originally belonging to types M3, M12 and M46 but devoid of type antigens after mouse passages showed high bactericidal indices against the 1800 strain after 14 or more passages on mice. Anti-type M1 serum was also found bactericidal for the passaged strains. The IgG Fc-receptor activity of the strain isolated after each mouse passage was tested in hemagglutination experiments with human red blood cells coated with "incomplete" anti-Rh and hot hydrochloric acid extracts of the strains. The capacity to agglutinate "Ripley"-coated cells increased gradually during the first 12 passages and subsequently the titres of the extracts stabilized between 1:160 and 1:320. The HUN coat, useful for detection of the G3m (5) maraker gave titraes increasing with the number of passages while the titres for IgG1 coats kept at 1:4 or below. On background of these results, the possible role of the IgG Fc-receptor as a virulence factor is discussed.

The past 2 decades have brought worrying increases in severe Streptococcus pyogenes diseases globally. To investigate and compare the epidemiological patterns of these diseases within Europe, data were collected through a European Union FP-5-funded program (Strep-EURO). Prospective population-based surveillance of severe S. pyogenes infection diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe (Cyprus, the Czech Republic, Denmark, Finland, France, Germany, Greece, Italy, Romania, Sweden, and the United Kingdom) using a standardized case definition. A total of 5,522 cases were identified across the 11 countries during this period. Rates of reported infection varied, reaching 3/100,000 population in the northern European countries. Seasonal patterns of infection showed remarkable congruence between countries. The risk of infection was highest among the elderly, and rates were higher in males than in females in most countries. Skin lesions/wounds were the most common predisposing factor, reported in 25% of cases; 21% had no predisposing factors reported. Skin and soft tissue were the most common foci of infection, with 32% of patients having cellulitis and 8% necrotizing fasciitis. The overall 7-day case fatality rate was 19%; it was 44% among patients who developed streptococcal toxic shock syndrome. The findings from Strep-EURO confirm a high incidence of severe S. pyogenes disease in Europe. Furthermore, these results have identified targets for public health intervention, as well as raising awareness of severe S. pyogenes disease across Europe.

Cysteine protease-inhibiting proteins of the cystatin superfamily can inhibit the replication of certain viruses and bacteria. The inhibitory centre of human cystatin C, the most widely distributed human cystatin, comprises three peptide segments. The present work describes the synthesis and antibacterial activity of 27 new peptidyl derivatives or cyclopeptides based upon the aminoterminal segment Arg 8 -Leu 9 -Val 10 -Gly 11 . Fourteen of the new compounds displayed antibacterial activity against from 1 up to 9 of 17 clinically important bacterial species tested. Antiproteolytic activity of a compound was usually not required for its antibacterial capacity. Peptidyl diazomethanes generally had a very narrow antibacterial spectrum, inhibiting only Streptococcus pyogenes, whereas cyclopeptides and peptidyl derivatives of the general structure X-Arg-Leu-NH-CH(iPr)-CH 2 -NH-Y had a much wider spectrum. The most potent of these substances displayed approximately equal minimal inhibitory and bactericidal concentrations of about 20 μg/ml for both Staphylococcus aureus and S. pyogenes and were devoid of antiproteolytic activity. Several of the new substances could protect mice against lethal intraperitoneal challenge with S. pyogenes. Though their target remains to be disclosed, the group of substances here reported might be promising for the development of antibacterial drugs and the discovery of novel principles of action.

Previous works in the rabbit with experimental acute poststreptococcal glomerulonephritis have demonstrated a pathogenic role of group A streptococcal M family IgG Fc binding proteins (FcBPs). In the present study, we showed that purified human or rabbit IgG Fc fragments, but not rabbit IgG Fab, were effective in blocking the development of glomerular changes in rabbits with initial steps of experimental glomerulonephritis induced by the IgG binding strain M1 (40/58). The findings were in agreement with our earlier data and might suggest new modes of intervention in this non-suppurative streptococcal disease.

Binding of C1q, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of C1q to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between C1q and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound C1q; alkaline and SDS extracts yielded the 52 kDa component as the main C1q-binding substance. Trypsin treatment of the SDS extracts of two GAS strains suggested the C1q-binding component(s) to be of protein nature. C1q-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human C1q and fibrinogen, conjugated to horse radish peroxidase (HRP) as well as rabbit IgG antibodies complexed to HRP (PAP system). The 52 kDa component was non-reactive with fibrinogen or rabbit IgG. However, C1q-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either fibrinogen (type M5) or both fibrinogen and rabbit IgG (type M76); the molecular mass of these components. 55 kDa and 43-40 kDa, respectively, was in agreement with the reported molecular mass of the M5 and FcRA76 proteins. Our findings suggest that C1q may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with C1q as biologically important.

Two amplification tests for the diagnosis of Chlamydia trachomatis infection, namely the ligase chain reaction (LCx) and the strand displacement assay (ProbeTec), were compared using samples from 1183 patients at sexually transmitted disease clinics. The overall prevalence of positive results was 8.0%, with agreement between the two assays of 98.8%. For endocervical, urethral and male urine samples, agreement was 99.3%, 99.4% and 97.7%, respectively. For ten discrepant samples, alternative amplification assays suggested that the LCx and ProbeTec assays gave erroneous results in six and four cases, respectively. Inhibition of amplification was observed with three (0.25%) urine specimens.

We describe the synthesis and antibacterial properties of a novel antimicrobial peptidyl derivative, (2S)-2-(Nalpha-benzyloxycarbonyl-arginyl-leucylamido-1-[(E)-cinnamoylamido]-3-methylbutane, structurally based upon the inhibitory centre of the human cysteine protease inhibitor, cystatin C. The derivative, here called Cystapep 1, displayed antibacterial activity against several clinically important gram-positive bacteria. It displayed minimal inhibitory and bactericidal concentrations of about 16 microg/ml for both Staphylococcus aureus and Streptococcus pyogenes. In radial agar diffusion assays, groups A, B, C and G streptococci as well as staphylococci were generally susceptible to the action of Cystapep 1, whereas pneumococci and enterococci were less susceptible. No activity against gram-negative bacteria was observed. Cystapep 1 also showed high activity against methicillin-resistant S. aureus (MRSA) and multiantibiotic-resistant coagulase-negative staphylococci (CNS), suggesting that its mechanism of action differs from those of most currently used antibiotics.

Our previous studies have indicated that the IgG-binding M-family proteins (IgGBP) of group A streptococci may be involved in eliciting experimental acute poststreptococcal glomerulonephritis (APSGN) in the rabbit. These surface proteins were also found to trigger production of anti-IgG, which might conceivably act to enhance renal deposition of immune complexes (IC). In the present study, a clinical isolate of serotype M22 (strain AL168), an isogenic double mutant deficient for both the IgGBPs Mrp and Emm, as well as mutants deficient in only one of the proteins were tested for capacity to induce glomerulonephritis. Streptococci to be used for injecting rabbits were heat-killed. Surface-bound IgG was removed by 1 M KSCN and cells were then repeatedly washed in PBS before use. Rabbits were injected intravenously with 10(9) cells three times a week for 8 weeks and, following one month of rest, for another 6 weeks. Deposits of IgG and C3 as well as induced chemokines TNF-alpha, IL-1beta and IL-6 were traced in cryostat sections using specific antibodies and appropriate peroxidase-labelled anti-antibodies. In four rabbits immunized with the double mutant strain, no deposits were found, and as examined by TEM, only subtle and transient renal changes were observed. In contrast, the original strain AL168 induced pronounced inflammatory and degenerative glomerular changes in all four rabbits injected, and deposits of TNF-alpha, IL-1beta and IL-6 were found in mesangial and endothelial cells. Similar deposits and glomerular changes were seen in all eight rabbits injected with the mrp-emm+ mutant and in four out of seven animals receiving the mrp+emm- mutant. There was a highly significant correlation between high levels of circulating anti-IgG and development of APSGN. These results confirm an important role of streptococcal IgGBP in triggering experimental APSGN as earlier proposed by our group. (Less)

The case is described of a 10-week-old preterm infant, a twin boy born at 34 weeks of gestational age. The day after a hernia operation he had a rapidly progressive fulminant Neisseria meningitidis serogroup B septicaemia, of which he died despite immediate and adequate treatment. No secondary cases occurred among other infants on the neonatal intensive care unit. Epidemiological investigation revealed that of 185 bacterial throat cultures performed on 17 infants on the ward, 37 close relatives to the infants and 131 medical personnel in contact with the deceased patient, 4 (2.2%) were asymptomatic carriers of N. meningitidis. Serotyping and pulsed-field gel electrophoresis of the genomic DNA of the N. meningitidis isolates revealed that the infant and his father had closely related strains.

Objectives: to investigate the incidence of intraoperative graft contamination, bacterial species and the influence of change of surgeon's gloves on contamination. Design: a prospective randomised study.Materials and methods : forty patients had implantation of synthetic vascular grafts. All patients received intraoperative cloxacillin (2.0 g) or clindamycin (0.6 g) intravenously. The procedures were randomised to two groups: Group 1 – surgeons changed the gloves before the first contact with the vascular prosthesis and Group 2 – operation without glove change. The growth of all bacterial species from graft segments and from the gloves was recorded. The susceptibility to antibiotics was tested.Results : the number of contaminated grafts was similar in the two groups. Growth of bacteria was recorded from 92.5% (37/40) of the graft segments and 33% (51/156) of glove imprints. Of the cultured species, 75% and 47%, respectively, were identified as coagulase-negative staphylococci (CNS). Twenty-eight per cent of CNS were resistant to cloxacillin, 15% to clindamycin, and 10% to cloxacillin and clindamycin. In all, 25% of the CNS strains were resistant to the prophylactic antibiotic used. In 50% of cases, the antibiogram of the CNS strain recovered from gloves agreed with that of the strain harvested from the graft. Conclusions: a high incidence of graft contamination was found which was not reduced by changing gloves. However, changing gloves did seem to reduce the number of bacterial species.

The serum opacity factor (SOF) of Streptococcus pyogenes is a type-specific lipoproteinase of unknown biological significance. We have sequenced the sof gene and characterized the corresponding SOF protein from a strain of type M63. It was found that sof63 is related to sof22 and that, similar to SOF22 [25], SOF63 binds fibronectin. Moreover, we demonstrate opacity factor activity in a Streptococcus dysgalactiae fibronectin-binding protein FnBA that is structurally related to the SOF proteins of S. pyogenes. Sequence analysis of these three SOF proteins showed a unique periodical pattern of conserved and variable regions. The enzymatically active part of SOF63 was localized to the fragment corresponding to the entire set of conserved and variable sequences, while for fibronectin-binding a single repeat in the C terminal part of the protein was sufficient. The results show that streptococcal SOF proteins form a novel family of bifunctional proteins with lipoproteinase and fibronectin-binding activities.

End-point attached, covalently bound heparin has been shown to be effective in preventing activation of the coagulation cascade by biomaterials. Data concerning its possible influence on bacterial attachment and resistance to biomaterial-associated infection are, so far, lacking. In the present work, the in vitro adherence of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli, one isolate of each species, to plain poly(vinyl chloride) (plain PVC) and heparin coated poly(vinyl chloride) (EPA-PVC) segments was compared. Also, the influence of precoating the segments with human normal plasma for 2 h was studied. 35S-Methionine was used to radiolabel bacteria. The segments were exposed to bacterial suspensions of approximately 10(7) colony forming units (CFU) per milliliter at 37 degrees C for 0.5-6 h. Following repeated washing in phosphate buffered saline (PBS), radioactivity associated with the segments was measured. Plain PVC as compared to EPA-PVC bound significantly more cells of all three tested species. Plasma precoating significantly decreased adherence of the tested species to plain PVC but did not affect the binding to EPA-PVC. However, after precoating with human plasma, EPA-PVC compared to plain PVC showed a higher binding of S. aureus which might possibly be due to bridging effects of fibronectin or other plasma proteins, interacting with S. aureus.

Fifty-three patients with bacterial treatment failure after a 10-day course of treatment with phenoxymethyl penicillin (pcV) for group A streptococcal (GAS) pharyngotonsillitis were randomly assigned to continued treatment with pcV, or to treatment with clindamycin instead. The patients were then followed for 1 year with throat cultures and clinical examination every third month and in the event of symptoms of sore throat. In the first 3-month period, 15/22 patients in the pcV group yielded one or more positive cultures for GAS, all of the same T-type as in the original throat culture, as compared to 3/26 in the clindamycin group (p0.001). All three cases in the clindamycin group were due to a new T-type and thus were re-infections. In the pcV group, owing to repeated treatment failure, 12/22 patients were switched to treatment with clindamycin within the 3-month period following the second treatment. During the remainder of the 1-year follow-up period, sporadic cases of GAS-positive throat cultures occurred in both groups, but there was no significant difference in frequency between the two groups. It is concluded that, in patients with GAS pharyngotonsillitis and failure after pcV treatment, a 10-day course of clindamycin can protect the patient from recurrence for at least 3 months and might be an alternative to tonsillectomy.

Penicillin tolerance in group A streptococci has been suggested to cause treatment failures in pharyngotonsillitis. In the present study, group A streptococci from patients with pharyngotonsillitis, who healed (n = 33) or failed (n = 25) on phenoxymethylpenicillin therapy for 10 days, as well as isolates obtained following the first (n = 25) and second (n = 7) failure were tested for penicillin tolerance by a plate-screening method. For most strains, the survival rate after a 6-h exposure of log-phase bacteria (10(4) CFU) to a phenoxymethylpenicillin concentration of 4 times the minimum inhibitory concentration (MIC) was below 0.1%. Five strains from cases of failure, exhibiting survival rates of 0.2-0.5%, were subjected to time killing kinetic tests with phenoxymethylpenicillin at 12 times the MIC. At 6 h each of the strains from failures showed survival rates below 0.03%. One single group A strain, previously selected in our laboratory, showed a survival rate of 0.4-1.2%, which was close to tolerance as defined. Four streptococcal strains, earlier reported as tolerant, showed survival rates of1% but were found to be group G. Penicillin tolerance does not significantly contribute to failures in penicillin therapy of group A streptococcal pharyngotonsillitis, but seems to be a common property of group C and G streptococci.

The pathogenesis of acute poststreptococcal glomerulonephritis (APSGN), a major nonsuppurative complication of group A streptococcal (GAS) throat or skin disease, remains unclear. During the years, various theories based on certain streptococcal extracellular factors, as well as immunological mimicry between streptococci and renal tissue, have been forwarded. We earlier reported that many clinical GAS isolates with documented nephritogenic capacity show non-immune binding of monomeric or aggregated IgG. Moreover, in a rabbit model of APSGN we obtained evidence for an important role of streptococcal IgG Fc binding proteins (IgGFcBPs) belonging to the M family surface proteins; thus, hyperimmunization by whole IgGFcBP-positive streptococci was shown to induce renal glomerular changes with deposition of IgG and complement C3, resembling the picture recorded in human APSGN. These typical renal changes were always preceded by the appearance of circulating anti-IgG antibodies. In the present work, using the same rabbit model, each of two purified IgGFcBPs, isolated from type M22 GAS, were found to elicit glomerular degenerative damage comparable to that caused by whole bacteria, as well as formation of anti-IgG. In addition, the induction by whole streptococci (type M1) of experimental APSGN was inhibited by the i.v. administration of purified human or rabbit IgG Fc, but not Fab, fragment, supporting the importance of Fc-mediated mechanisms in causation of glomerulonephritis. We propose that anti-IgG antibody, induced by streptococcal IgGFcBP, facilitated renal accumulation of IgG-containing complexes, which in turn triggered complement deposition and proinflammatory cascades. Further studies on the possible beneficial effect of IgG Fc fragment in APSGN should be of interest.

An M-like protein from Streptococcus pyogenes type M15 strain EF1949 (EMML15) was cloned in Escherichia coli and sequenced. Recombinant EMML15 protein revealed a unique binding pattern for human IgG subclasses not described previously. Comparative analysis of the EMML15 amino acid sequence with those of other M-like proteins of opacity factor positive (OF+) serotypes and protein H, and IgG receptor from OF- serotype M1, showed that IgG-binding proteins with common binding of IgG3 were closely related and distinct from streptococcal IgG receptors not binding IgG3. Thus, the Ig-binding proteins from S. pyogenes were subdivided into two main categories according to binding pattern, protein structure, and gene location.

During investigation of the interaction of human lactoferrin (HLf) with variou bacteria, it was found that inStreptococcus pyogenes, HLf binding occurred to agar-rather than broth-grown cells irrespective of the nutrients used. Furthermore, binding of HLf to broth-grown, heat-killed bacteria was induced by overnight incubation on agar media or short-time exposure of the cells to water-soluble agar extract. The binding pattern was revealed in most of 92S. pyogenes strains representing various M-or T-types with no apparent type variation. The component thus bridging the attachment of HLf to the streptococcal cell surface was recovered in extracts of agar-grown cells and isolated by affinity chromatography on HLf-sepharose. By gel filtration in the presence of radiolabeled HLf, this component exhibited similar elution position as crude water-soluble agar extract. Chemical analysis identified the active HLf-binding agar component to be a galactose-rich polysaccharide (GRP). Further binding tests showed that the interaction between streptococci and GRP was stable in the presence of high molar NaCl, KSCN, or urea and was unaffected by various serum or matrix proteins or by streptococcal lipoteichoic acid; however, a moderate inhibition by heparin or bovine mucin was observed. Studies on isogenic mutants ofS. pyogenes did not support the involvement of M-protein or the hyaluronate capsule in the binding of GRP. SDS-PAGE and Western blot analyses revealed a GRP-binding protein of approximately 70 kDa in the cell-wall extracts of two strains ofS. pyogenes, types M19 and M55. Finally, the adherence of (broth-grown)3H-thymidine-labeledS. pyogenes, type M19, to the pharyngeal epithelial cell line DT-562 or to normal tonsillar epithelial cells was inhibited by GRP in a dose-related manner. We thus propose that the streptococcal GRP-binding component may represent a novel surface lectin acting as a mucosal adhesin forS. pyogenes, in accordance with previous data indicating that galactosecontaining sugar moieties may serve as ligands for the adherence of streptococci to pharyngeal cells. Our results also indicate that GRP-like components such as mucin or heparin might act to block epithelial adherence ofS. pyogenes at the mucosal level.

The in-vitro activities of clarithromycin, 14-OH clarithromycin, erythromycin, amoxycillin, metronidazole and omeprazole against Helicobacter pylori were determined at pH 7.2 and 5.5. At pH 5.5 the activities of clarithromycin and erythromycin decreased approximately 16 times while 14-OH clarithromycin was less influenced. Chequerboard titration indicated that the combined activity of clarithromycin and the other compounds was additive.

239 patients with streptococcal pharyngotonsillitis completed treatment with phenoxymethyl penicillin 12.5 mg per kg body weight b.i.d. for 10 days. At examination after completing therapy, throat specimens from 53 patients (22%) yielded growth of group A streptococci of the same. T-type as the initial culture (bacterial treatment failure). 20 of these 53 (38%) had symptoms and signs of tonsillitis (clinical and bacterial treatment failure). 48 of the patients with bacterial failure were randomly allocated to phenoxymethyl penicillin or clindamycin in an open design; 22 of them received a second course of phenoxymethyl penicillin for 10 days and 26 were given clindamycin, 6.5 mg per kg body weight b.i.d. (children) or 300 mg t.i.d. (adults) for 10 days. After completing their treatment, 14 of 22 patients (64%) given phenoxymethyl penicillin harboured the same T-type as in the previous two cultures, while group A streptococci were not recovered from any of the 26 patients receiving clindamycin. In patients with clinical failure after phenoxymethyl penicillin treatment, a new course with this drug is not motivated. In that situation clindamycin seems to be an efficient choice.

Group A and B streptococci (Streptococcus pyogenes and Streptococcus agalactiae) are the only known bacterial pathogens expressing IgA Fc-receptors. However, the IgA binding proteins of the two species have been found genetically unrelated. In the present investigation the binding of human IgA among clinical isolates of group A and group B streptococci was studied and the respective IgA-binding epitopes were compared serologically. Surface binding of radiolabelled, monoclonal human IgA1 occurred in 38% of 115 unselected group A streptococcal isolates. Comparing four predominant T-types, IgA-binding was found in 77% and 85%, respectively, of types T4 and T28 strains but only in 5% and 25%, respectively, of T1 and T12 strains. In group B streptococci, 70% of 58 type Ib strains but only 2% of 399 strains of other serotypes bound IgA. Using rabbit immune sera raised to the two streptococcal species it was found that strains exhibiting IgA Fc-receptors often induced antibodies blocking the binding of IgA to bacteria. Furthermore, the blocking shown by an individual serum was restricted to the streptococcal group used for immunization showing that also the IgA-binding eptiopes in group A and B streptococci are conformationally distinct. Though infections with serotypes often binding IgA, compared to other types, are not known to differ, it is assumed that the non-immune binding of IgA might favour mucosal colonization of the organisms.

Sales of antibiotics have increased in Sweden during the past decade. This has been paralleled by an increase in the frequency of beta-lactamase-producing respiratory tract bacteria. To investigate the effects of regional differences in use of antibiotics on beta-lactamase production in respiratory tract bacteria, we collected nasopharyngeal specimens and information about antibiotic use from 1133 children attending day-care centres in four rural municipalities with low use, and one urban municipality with high use of antibiotics, use being assessed from pharmacy sales. The frequency of beta-lactamase production among isolates of Branhamella catarrhalis and Moraxella nonliquefaciens was significantly higher in the urban municipality. This appeared to be a long-term ecological effect of differences in the level of use of antibiotics between the urban and rural populations, rather than an effect of recent antibiotic treatment of individual patients.

Penicillin tolerance in Streptococcus pyogenes has been suggested as a possible cause of therapeutic failure in streptococcal phryngitis treated with penicillin. In 144 patients with acute group A streptococcal tonsillitis treated with phenoxymethyl penicillin 12.5 mg per kg body weight b.i.d. for 10 days the same T-type was recovered after treatment in 21%. The recovery rate was higher for non-tolerant strains, 23%, than for tolerant strains, 10% (p greater than 0.05). Of patients with a non-tolerant strain 17% had both clinical and bacterial treatment failure in comparison with 5% infected with a tolerant strain (p greater than 0.05). Reinfection with a new serotype occurred in altogether 3%. The present data did not indicate that penicillin tolerance in group A streptococci is of significance in acute tonsillitis treated with phenoxymethylpenicillin for 10 days.

In an attempt to compare the epidemiology of severeStreptococcus pyogenesinfection within Europe, prospective data were collected through the Strep-EURO program. Surveillance for severe cases ofS. pyogenesinfection diagnosed during 2003 and 2004 was undertaken in 11 countries across Europe by using a standardized case definition and questionnaire. Patient data as well as bacterial isolates were collected and characterized by T and M/emmtyping, and selected strains were analyzed for the presence of superantigen genes. Data were analyzed to compare the clinical and microbiological patterns of the infections across the participating countries. A total of 4,353 isolates were collected from 5,521 cases with severeS. pyogenesinfections who were identified. A wide diversity of M/emmtypes (n= 104) was found among theS. pyogenesclinical isolates, but the M/emmtype distribution varied broadly between participating countries. The 10 most predominant M/emmtypes were M/emmtype 1 (M/emm1), M/emm28, M/emm3, M/emm89, M/emm87, M/emm12, M/emm4, M/emm83, M/emm81, and M/emm5, in descending order. A correlation was found between some specific disease manifestations, the age of the patients, and theemmtypes. Although streptococcal toxic shock syndrome and necrotizing fasciitis were caused by a large number of types, they were particularly associated with M/emm1 and M/emm3. Theemmtypes included in the 26-valent vaccine under development were generally well represented in the present material; 16 of the vaccine types accounted for 69% of isolates. The Strep-EURO collaborative program has contributed to enhancement of the knowledge of the spread of invasive disease caused byS. pyogeneswithin Europe and encourages future surveillance by the notification of cases and the characterization of strains, which are important for vaccination strategies and other health care issues.

There is epidemiological evidence of an association between cigarette smoking and gastritis. To find out whether the reason for this might be related to the presence of Helicobacter pylori&lt

Previous studies have shown that intraperitoneal bile increases bacterial growth and mortality in Escherichia coli peritonitis in the rat. The purpose of the present study was to determine a) the influence of bile acids (cholic, deoxycholic, or chenodeoxycholic) and bilirubin on survival, bacterial growth, and superoxide release by peritoneal phagocytes in this model, and b) the effect of bile acids on bacterial growth and endotoxin release when incubated with E. coli in vitro. Each of the bile acids aggravated the E. coli peritonitis, with increased bacterial counts in the peritoneal cavity and in blood and increased mortality. Deoxycholic acid was the most deleterious of the bile acids, causing suppression of superoxide release by peritoneal phagocytes, like whole bile. In vitro, bile acids did not seem to affect growth of E. coli, but cholic and deoxycholic acid seemed to enhance the release of endotoxin. It is concluded that the bile acids are responsible for the noxious effect of bile in E. coli peritonitis. It is suggested that the detergent properties of bile acids aggravate the peritonitis by solubilizing the cell membranes of both bacteria and phagocytes.

Serum IgG antibodies of Helicobacter pylori were detected in single-dilution ELISA using glycine extracted material. Among 148 endoscopy patients 59% displayed antibodies; as expected, a higher occurrence (90%) was found in patients with positive gastric culture for H. pylori than in culture negative patients (37%). Among 68 blood donors the frequency of H. pylori antibodies was 28%. In 73 children less than 15 years of age examined for unrelated disorders the occurrence was 4%. By immunoblotting using the same extract, 3 prominent bands, 29K, 54K and 60K and several weak bands were identified. These were formed by 57%, 92%, and 65%, respectively, of the ELISA positive patient sera. Comparing culture positive and negative patients, the 3 bands occurred more often among the culture positive subjects though between 18 and 61% of the sera from culture negative patients gave either of the bands. When comparing the glycine extracts of 4 different H. pylori strains with separate haemagglutinating patterns no differences in the position of the major bands emerged. By absorption experiments no immunological cross-reactivity with components of Escherichia coli, Klebsiella pneumoniae, Campylobacter jejuni or C. fetus was found. Thus, the glycine extract seemed specific for the detection of antibodies to H. pylori.

The incidence and severity of invasive group A streptococcal infection demonstrate great variability over time, which at least, in part, seems to be related to group A streptococcal type distribution among the human population.An enhanced surveillance study of invasive group A streptococcal infection (746 isolates) was performed in Sweden from April 2002 through December 2004. Noninvasive isolates from either the throat or skin (773 isolates) were collected in parallel for comparison. Clinical and epidemiological data were obtained from 88% of patients with invasive disease and were related to isolate characteristics, including T type, emm sequence type, and the presence of 9 superantigen genes, as well as pulsed-field gel electrophoresis pattern comparisons of selected isolates.The annual incidence was 3.0 cases per 100,000 population. Among the patients with invasive disease, 11% developed streptococcal toxic shock syndrome, and 9.5% developed necrotizing fasciitis. The overall case-fatality rate was 14.5%, and 39% of the patients with streptococcal toxic shock syndrome died (P.001). The T3/13/B3264 cluster accounted for 33% of invasive and 25% of noninvasive isolates. Among this most prevalent type cluster, emm types 89 and 81 dominated. Combined results from pulsed-field gel electrophoresis, emm typing, and superantigen gene profiling identified subgroups within specific emm types that are significantly more prone to cause invasive disease than were other isolates of the same type.This study revealed a changing epidemiology of invasive group A streptococcal infection in Sweden, with emergence of new emm types that were previously not described. The results also suggest that some clones may be particularly prone to cause invasive disease.

Human Legionella infections mainly consist of community-acquired and nosocomial pneumonia and rarely affect children. We describe a nosocomial infection with Legionella pneumophila, serogroup 1, subgroup OLDA, in an immunocompromized 2-y-old girl at a paediatric clinic. L. pneumophila identical to that of the patient was found in the hospital's cold-water but not in the hot-water distribution system. Transmission of Legionella to the girl most probably occurred by Legionella-contaminated cold water mixed and heated by water from the hot-water system. Mixing of hot and cold water probably occurred through thermostatic water mixing valves connected to showers regulated by a handle at the shower head. Nosocomial Legionella infection might thus have occurred, although circulating hot water temperatures never dropped below 53 degrees C and cultures for surveillance of Legionella from central parts of the hot-water system have been consistently negative. Legionellae were successfully eliminated from the hospital's cold-water distribution system by hot water flushing at 73 degrees C for 1h.

Acute rheumatic fever (ARF) and acute poststreptococcal glomerulonephritis (APSGN), two important sequelae of streptococcal throat or skin infections, according to current concepts may be elicited by autoimmune mechanisms due to molecular mimicry between group A streptococci (GAS) and human tissue. In the case of APSGN, however, our experimental data have indicated that GAS immunoglobulin-binding surface proteins (IgG BPs) might be of pathogenic significance by triggering anti-IgG production and immune complex formation leading to renal damage. Thus, rabbits injected with IgG-binding, as opposed to non-binding, GAS strains were found to develop renal deposition of IgG and complement factor C3 and inflammatory and degenerative glomerular changes resembling the picture seen in APSGN. In the present study, cardiac tissue material from rabbits injected with GAS was investigated. After 8 or more weeks of intravenous (i.v.) injections, minimal changes were seen in those animals receiving an IgG non-binding GAS strain, type T27, whereas those animals receiving either of two IgG-binding GAS strains, types M1 or M22, developed strong inflammatory and degenerative myocardial changes accompanied by deposition of IgG and C3. Furthermore, on injecting rabbits with defined mutants of a type M22 strain, the development of myocardial tissue damage proved to be dependent on the presence of streptococcal IgG-binding activity. Our results demonstrate that myocardial tissue changes may be induced in the rabbit by i.v. injection of whole heat-killed GAS of at least two M serotypes. Conceivably, induction of immune complexes by bacterial IgG BPs may lead to myocardial deposition of IgG, in turn triggering a series of events, involving the complement system and proinflammatory cytokines, with resulting tissue damage. Though many virulence factors may be involved in the development of ARF and APSGN, and a given GAS strain will never cause both, our results may suggest a new pathogenetic mechanism common to these two major non-suppurative complications.

A new, 1-step, enzyme-linked immunoassay kit for detection of Group A Streptococci (GAS) in throat samples (QuickVue In-Line One-Step Strep A Test; Quidel Corporation, San Diego, CA) was evaluated for use in a study comprising 536 patients in 8 primary healthcare centres. Compared to conventional culture at the clinical microbiology laboratory, the sensitivity achieved was 73.9% and the specificity 86.8%; these figures were not affected to any major extent by broth enrichment of samples before culturing or following PCR testing of the cysteine proteinase gene for independent diagnosis of GAS. It was also found that most samples containing low numbers of GAS were missed by the rapid test. We therefore evaluated the kit in use in our area (TestPack Plus Strep A Test; Abbott Laboratories, Chicago, IL) in a separate study of 615 patients. Somewhat increased sensitivity (82.8%) and specificity (96.1%) were obtained. As current antigen tests depend on subjective judgement of test outcome, improvements in test design or provision of more detailed instructions may be desirable in order to achieve optimal results.

We describe the synthesis and antibacterial properties of a novel antimicrobial peptidyl derivative, (2S)-2-(Nalpha-benzyloxycarbonyl-arginyl-leucylamido-1-[(E)-cinnamoylamido]-3-methylbutane, structurally based upon the inhibitory centre of the human cysteine protease inhibitor, cystatin C. The derivative, here called Cystapep 1, displayed antibacterial activity against several clinically important gram-positive bacteria. It displayed minimal inhibitory and bactericidal concentrations of about 16 microg/ml for both Staphylococcus aureus and Streptococcus pyogenes. In radial agar diffusion assays, groups A, B, C and G streptococci as well as staphylococci were generally susceptible to the action of Cystapep 1, whereas pneumococci and enterococci were less susceptible. No activity against gram-negative bacteria was observed. Cystapep 1 also showed high activity against methicillin-resistant S. aureus (MRSA) and multiantibiotic-resistant coagulase-negative staphylococci (CNS), suggesting that its mechanism of action differs from those of most currently used antibiotics.

For many years group A streptococci of T type 28 (T28) have been common in southern Sweden; however, since 1995 resistance to both macrolide-lincosamide-streptogramin B (MLS) antibiotics and tetracycline was observed among T28 isolates, which prompted the present studies on clonal relatedness of antibiotic-resistant T28 strains. By extended T typing, 95 of 100 examined tetracycline-resistant strains showed the combination T9-T13-T28; of these, 94 belonged to M type 77 (M77) and one belonged to M73. Three strains were T28-M28 and two were T28-M nontypeable. The serological M77 was confirmed by PCR capture enzyme-linked immunosorbent assay, emm amplicon restriction profiling, and emm sequence typing. Fifty strains were examined for superantigen genes: speA was detected in three blood isolates only, whereas all isolates harbored speB , and only two of the strains were negative for speC . Eighty-nine of the 100 strains were also macrolide resistant, of which 59 were inducibly MLS resistant (IR) and 21 were constitutively MLS resistant (CR), 6 were noninducibly resistant (NI), and 3 had novel subphenotypes recently reported by our group. Resistance genes were determined by PCR and hybridization methods. Eighty-four of the 100 strains harbored tetM. ermB was detected in all CR and IR strains, and mefA was found in all NI strains; both ermB and mefA were identified in two strains with novel subphenotypes. Pulsed-field gel electrophoresis showed that these antibiotic-resistant M77 strains belonged to at least five different clones.

Streptococcus pyogenes , a major human pathogen, is still considered susceptible to beta-lactams, but for other relevant antibiotics, highly variable resistance rates have been reported. Since no data were available from Iran, we tested 1,335 throat isolates from two different regions of the country for their antibiotic susceptibilities and, for comparison, a collection of 80 strains isolated from 1989 to 1991. Erythromycin resistance was uncommon (0.6%), whereas an overall high rate of tetracycline resistance was found, increasing between 1989–1991 and 1995–1997 from 23 to 42%. The tetracycline-resistant strains belonged to more than 10 different T types, the majority being types 4, 11, and B3264. By conventional M typing of 406 tetracycline-resistant isolates, more than 20 different M types were found. Approximately 50% of the strains were nontypeable by T agglutination as well as serological M typing; however, by genotyping by a combined PCR–capture-enzyme-linked immunosorbent assay, many of these strains were successfully emm typed. We conclude that the high rate of tetracycline resistance among Iranian S. pyogenes isolates is due to multiclonal dissemination of resistance within the streptococcal population rather than epidemic spread of single clones.

Two cases of campylobacter septicaemia are described. The first, caused by Campylobacter laridis was associated with gastroenteritis and occurred in a healthy individual. In the second case, a catalase negative species, C. mucosalis was isolated from blood in an immunocompromised patient with symptoms of pneumonia. Both campylobacter strains grew faintly under the routine culture conditions used. Improved diagnostic procedures for Campylobacter species may thus be warranted.

Many group A streptococci (GAS) bind the Fc part of IgG. In the present work, the possible influence of growth time and incubation atmosphere on the expression of the IgG binding activity by GAS of various serotypes was studied. Among 13 GAS reference strains, two categories were distinguished on aerobic incubation at 37 degrees C, one expressing similar IgG binding activity at 6 h and 18 h (types M1, M4, M13, M15), and a second one which showed higher binding at 6 h than at 18 h (M9, M14, M22, M25, M48, M49). Only one strain (M36) bound less IgG at 6 h than at 18 h. Seven of the strains (M5, M6, M22, M25, M36, M48, M49) showed higher binding of IgG when grown in a 5% CO2 atmosphere than in air, whereas one strain (M14) showed a reverse pattern and in the remaining five strains, no influence was found. Protease activity was detected in the growth supernatant of most of the strains. For five selected strains, the time of appearance of supernatant protease activity coincided with a decay of surface IgG binding activity. Purified streptococcal cysteine protease was found to reduce or abolish the binding of IgG by each of three studied strains (M1, M13 and M15) and of type M1 or M15 purified IgG binding material. When tested in the stationary phase, a majority of 62 clinical GAS isolates belonging to 6 different M types showed high protease activity but low binding of IgG. We conclude that streptococcal IgG binding is often better expressed on growth in 5% CO2 atmosphere than in air. Furthermore, due to its sensitivity to streptococcal protease, the IgG binding activity is mostly higher during the logarithmic than during the stationary phase of growth.

Type M50 group A streptococci are exceptional for their virulence in mice. However, intranasal (i.n.) vaccination with heat-killed group A streptococci, either of type M50 or M55, or an M12 strain deficient in M-protein, protected mice against i.n. challenge with M50 streptococci (82, 88 and 83% survival, respectively). Significant resistance against M50 streptococci was also noted by i.n. application of heat-killed Lactobacillus fermenti (81% survival) as well as two strains of pneumococci (50 and 79% survival). In contrast, no protective effect was obtained using heat-killed trypsin-treated M55 streptococci. Nor did vaccination with Escherichia coli and Pseudomonas aeruginosa induce protection against type M50. Thus, M protein was not required for immunity against type M50. The results call for a revision of the hitherto accepted view that M proteins are the only candidates for mucosal vaccines against group A streptococci.