Primary peritonitis due to group A Streptococcus in a previously healthy pediatric patient

Primary peritonitis remains a rare disease in otherwise healthy children, with group A Streptococcus (GAS) being a particularly unusual cause. A case involving a 14-year-old girl, who presented with an ‘acute abdomen’ and was taken to the operating room for urgent laparoscopy, is reported. Abdominal and pelvic structures were only minimally inflamed, as was the appendix. Peritoneal fluid and blood cultures both grew pure cultures of GAS. The patient’s course was complicated by streptococcal toxic shock syndrome. She fortunately made a full recovery. The present report highlights the diagnostic and treatment dilemmas associated with GAS primary peritonitis.     

Induction of group A Streptococcus virulence by a human antimicrobial peptide

Group A streptococci (Streptococcus pyogenes or GAS) freshly isolated from individuals with streptococcal sore throat or invasive ("flesh-eating") infection often grow as mucoid colonies on primary culture but lose this colony appearance after laboratory passage. The mucoid phenotype is due to abundant production of the hyaluronic acid capsular polysaccharide, a key virulence determinant associated with severe GAS infections. These observations suggest that signal(s) from the human host trigger increased production of capsule and perhaps other virulence factors during infection. Here we show that subinhibitory concentrations of the human antimicrobial cathelicidin peptide LL-37 stimulate expression of the GAS capsule synthesis operon (hasABC). Up-regulation is mediated by the CsrRS 2-component regulatory system: it requires a functional CsrS sensor protein and can be antagonized by increased extracellular Mg(2+), the other identified environmental signal for CsrS. Up-regulation was also evident for other CsrRS-regulated virulence genes, including the IL-8 protease PrtS/ScpC and the integrin-like/IgG protease Mac/IdeS, findings that suggest a coordinated GAS virulence response elicited by this antimicrobial immune effector peptide. LL-37 signaling through CsrRS led to a marked increase in GAS resistance to opsonophagocytic killing by human leukocytes, an in vitro measure of enhanced GAS virulence, consistent with increased expression of the antiphagocytic capsular polysaccharide and Mac/IdeS. We propose that the human cathelicidin LL-37 has the paradoxical effect of stimulating CsrRS-regulated virulence gene expression, thereby enhancing GAS pathogenicity during infection. The ability of GAS to sense and respond to LL-37 may explain, at least in part, the unique susceptibility of the human species to streptococcal infection.     

Inhibitor of streptokinase gene expression improves survival after group A streptococcus infection in mice

The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.     

Incomplete Kawasaki disease associated with complicated Streptococcus pyogenes pneumonia: A case report

A three-year-old boy presented with community-acquired pneumonia complicated by empyema. Streptococcus pyogenes (group A streptococcus) was identified on culture of the pleural fluid. The patient improved with antibiotic therapy and drainage of the empyema.During his convalescence, the patient developed persistent fever, lethargy and anorexia. His inflammatory markers were elevated, and repeat cultures were negative. Although the patient had none of the classical mucocutaneous features of Kawasaki disease, an echocardiogram was performed, which revealed coronary artery dilation.The patient was diagnosed with incomplete Kawasaki disease and treated with intravenous immunoglobulin and high-dose acetylsalicylic acid. The fever subsided within 48 h.To the authors’ knowledge, the present report is the first report of Kawasaki disease associated with complicated S pyogenes pneumonia. It emphasizes the importance of considering incomplete Kawasaki disease among children with persistent fever, the role of echocardiography in diagnosis, and the potential link between Kawasaki disease and superantigen-producing organisms such as S pyogenes.     

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG

Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-A resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.     

Acute phlegmonous gastritis complicated by delayed perforation

Here,we report on a case of acute phlegmonous gastritis(PG)complicated by delayed perforation.A51-year-old woman presented with severe abdominal pain and septic shock symptoms.A computed tomography scan showed diffuse thickening of the gastric wall and distention with peritoneal fluid.Although we did not find definite evidence of free air on the computed tomography(CT)scan,the patient’s clinical condition suggested diffuse peritonitis requiring surgical intervention.Exploratory laparotomy revealed a thickened gastric wall with suppurative intraperitoneal fluid in which Streptococcus pyogenes grew.There was no evidence of gastric or duodenal perforation.No further operation was performed at that time.The patient was conservatively treated with antibiotics and proton pump inhibitor,and her condition improved.However,she experienced abdominal and flank pain again on postoperative day 10.CT and esophagogastroduodenoscopy showed a large gastric ulcer with perforation.Unfortunately,although the CT showed further improvement in the thickening of the stomach and the mucosal defect,the patient’s condition did not recover until a week later,and an esophagogastroduodenoscopy taken on postoperative day 30 showed suspected gastric submucosal dissection.We performed total gastrectomy as a second operation,and the patient recovered without major complications.A pathological examination revealed a multifocal ulceration and necrosis from the mucosa to the serosa with perforation.     

Discovery and characterization of sialic acid O-acetylation in group B Streptococcus

Group B Streptococcus (GBS) is the leading cause of human neonatal sepsis and meningitis. The GBS capsular polysaccharide is a major virulence factor and the active principle of vaccines in phase II trials. All GBS capsules have a terminal alpha 2-3-linked sialic acid [N-acetylneuraminic acid (Neu5Ac)], which interferes with complement-mediated killing. We show here that some of the Neu5Ac residues of the GBS type III capsule are O-acetylated at carbon position 7, 8, or 9, a major modification evidently missed in previous studies. Data are consistent with initial O-acetylation at position 7, and subsequent migration of the O-acetyl ester at positions 8 and 9. O-acetylation was also present on several other GBS serotypes (Ia, Ib, II, V, and VI). Deletion of the CMP-Neu5Ac synthase gene neuA by precise, in-frame allelic replacement gave intracellular accumulation of O-acetylated Neu5Ac, whereas overexpression markedly decreased O-acetylation. Given the known GBS Neu5Ac biosynthesis pathway, these data indicate that O-acetylation occurs on free Neu5Ac, competing with the CMP-Neu5Ac synthase. O-acetylation often generates immunogenic epitopes on bacterial capsular polysaccharides and can modulate human alternate pathway complement activation. Thus, our discovery has important implications for GBS pathogenicity, immunogenicity, and vaccine design.     

IgG glycan hydrolysis by a bacterial enzyme as a therapy against autoimmune conditions

EndoS from Streptococcus pyogenes efficiently hydrolyzes the functionally important and conserved N-linked glycan of IgG in human blood. Repeated i.v. administration of EndoS in rabbits completely hydrolyzes the glycans of the whole IgG pool, despite the generation of anti-EndoS antibodies. EndoS administration had no apparent effects on the health of the animals. EndoS hydrolysis of the IgG glycan has profound effects on IgG effector functions, such as complement activation and Fc receptor binding, suggesting that the enzyme could be used as an immunomodulatory therapeutic agent against IgG-mediated diseases. We demonstrate here that EndoS indeed has a protective effect in a mouse model of lethal IgG-driven immune (or idiopathic) thrombocytopenic purpura. EndoS pretreatment of pathogenic antibodies inhibits the development of disease, and the enzyme also rescues mice from already established disease when severe thrombocytopenia and s.c. bleeding have developed. These results identify EndoS as a potential therapeutic agent against diseases where pathogenic IgG antibodies are important and further emphasize antibody glycans as possible targets in future therapies against antibody-mediated autoimmune conditions.     

Lactic acid production by Streptococcus thermophilus alters Clostridium difficile infection and in vitro Toxin A production

Antibiotic treatment to treat specific infections has the potential to effectively target the offending microbe as well as other microbes that colonize sites within a host. Antibiotic-associated diarrhea (AAD) is a classic example resulting from disruption of host microbial communities; 20% of patients with AAD are likely to become colonized with Clostridium difficile. Restoration of a “normal” microbial community within the host using probiotic bacteria is one approach to circumvent AAD and C. difficile infection. The goals of this study were to assess the interactions between Streptococcus thermophilus, a potential probiotic organism and C. difficile using both in vitro and in vivo systems. Exposure of C. difficile to filtered supernatants from S. thermophilus showed a dose-dependent, bactericidal effect due to lactic acid. Additional studies show that levels of lactic acid (10 mM) that did not inhibit bacterial growth had the potential to decrease tcdA expression and TcdA release into the extracellular milieu. In vivo, treatment with viable S. thermophilus significantly increased luminal levels of lactate in the cecum compared with UV-irradiated S. thermophilus. In the context of infection with C. difficile, mice treated with viable S. thermophilus exhibited 46% less weight loss compared with untreated controls; moreover, less pathology, diarrhea, and lower detectable toxin levels in cecal contents were evident more often in S. thermophillus treated mice. A significant, inverse correlation (Spearman r = -0.942, p = 0.017) between the levels of luminal lactate and abundance of C. difficile were noted suggesting that lactate produced by S. thermophilus is a factor impacting the progression of C. difficile infection in the murine system.     

Sequence type 1 group B Streptococcus,an emerging cause of invasive disease in adults,evolves by small genetic changes

The molecular mechanisms underlying pathogen emergence in humans is a critical but poorly understood area of microbiologic investigation. Serotype V group B Streptococcus (GBS) was first isolated from humans in 1975, and rates of invasive serotype V GBS disease significantly increased starting in the early 1990s. We found that 210 of 229 serotype V GBS strains (92%) isolated from the bloodstream of nonpregnant adults in the United States and Canada between 1992 and 2013 were multilocus sequence type (ST) 1. Elucidation of the complete genome of a 1992 ST-1 strain revealed that this strain had the highest homology with a GBS strain causing cow mastitis and that the 1992 ST-1 strain differed from serotype V strains isolated in the late 1970s by acquisition of cell surface proteins and antimicrobial resistance determinants. Whole-genome comparison of 202 invasive ST-1 strains detected significant recombination in only eight strains. The remaining 194 strains differed by an average of 97 SNPs. Phylogenetic analysis revealed a temporally dependent mode of genetic diversification consistent with the emergence in the 1990s of ST-1 GBS as major agents of human disease. Thirty-one loci were identified as being under positive selective pressure, and mutations at loci encoding polysaccharide capsule production proteins, regulators of pilus expression, and two-component gene regulatory systems were shown to affect the bacterial phenotype. These data reveal that phenotypic diversity among ST-1 GBS is mainly driven by small genetic changes rather than extensive recombination, thereby extending knowledge into how pathogens adapt to humans.The recent increase in large-scale DNA sequencing feasibility has allowed for significant advances in understanding the population genetics of bacteria that cause disease in humans (1, 2). A major outcome of the rapid expansion of available bacterial genomes has been the appreciation of the marked intraspecies genetic variability present in a wide variety of human bacterial pathogens (3, 4). This genetic variation can have profound impact on host–pathogen interaction by affecting transmissibility, infection severity, and antimicrobial resistance (2, 5, 6). The observed genetic intraspecies variability can arise via many distinct mechanisms including large-scale events such as recombination and bacteriophage-mediated horizontal gene transfer as well as small-scale genetic changes such as short insertions, deletions, and/or single nucleotide changes (2, 3, 5).Group B Streptococcus (GBS) is a common colonizer of humans that emerged in the 1970s as the leading cause of invasive bacterial disease in neonates and infants less than 3 mo of age (7). GBS is divided into 10 serotypes based on the carbohydrate composition of its sialic acid containing capsule, but gene content at the genomic level does not necessarily correlate with capsular serotype (8, 9). A seven-gene multilocus sequence typing (MLST) allows for the classification of the majority of GBS strains isolated from humans into five major clonal complexes (CCs) with a recent study by Da Cunha et al. showing that the major GBS CCs are primarily derived from a limited number of tetracycline-resistant clones, suggesting a key role of tetracycline resistance in GBS strain emergence (10, 11). CC-17 GBS strains have been particularly well studied given their role as the major cause of severe, invasive infant disease (10, 12). In contrast, serotype V strains cause a larger percentage of invasive disease in nonpregnant adults compared with neonates (13–15). Importantly, rates of invasive GBS disease have been increasing during the past 25 y in nonpregnant adults, with a significant part of the rise resulting from serotype V GBS strains (15–17).Despite the clear and increasing impact of serotype V strains, data are limited regarding molecular epidemiology of serotype V GBS causing invasive disease in nonpregnant adults (14, 15, 18). Only a few studies of serotype V strains have investigated the noncapsular genetic makeup of the strains, and those that have done so have included colonizing and invasive GBS strains isolated from infants or have not described the clinical origin of the tested strains (18, 19). Thus, we sought to analyze a large cohort of clinically well-defined, geographically distinct, and temporally disparate GBS isolates by using a whole-genome approach to elucidate the population structure of serotype V GBS causing invasive disease in nonpregnant adults. Data using non–genome-wide level approaches found that many serotype V strains were closely related, suggesting that a particular clone, rather than a genetically diverse array of strains arising from large-scale recombination, might be responsible for the majority of serotype V disease (18). Thus, we specifically sought to test the hypothesis that genetic diversity among invasive serotype V GBS strains is driven by small genetic changes at loci that are critical to GBS host–pathogen interaction.     

An unusual case of Streptococcus anginosus group pyomyositis diagnosed using direct 16S ribosomal DNA sequencing

Bacteria belonging to the Streptococcus anginosus group (Streptococcus intermedius, Streptococcus constellatus and Streptococcus anginosus) are capable of causing serious pyogenic infections, with a tendency for abscess formation. The present article reports a case of S anginosus group pyomyositis in a 47-year-old man. The pathogen was recovered from one of two blood cultures obtained from the patient, but speciation was initially not performed because the organism was considered to be a contaminant (viridans streptococci group). The diagnosis was ultimately confirmed using 16S ribosomal DNA sequencing of purulent fluid obtained from a muscle abscess aspirate. The present case serves to emphasize that finding even a single positive blood culture of an organism belonging to the S anginosus group should prompt careful evaluation of the patient for a pyogenic focus of infection. It also highlights the potential utility of 16S ribosomal DNA amplification and sequencing in direct pathogen detection from aspirated fluid in cases of pyomyositis in which antimicrobial therapy was initiated before specimen collection.     

Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens

The S100A8/S100A9 heterodimer calprotectin (CP) functions in the host response to pathogens through a mechanism termed “nutritional immunity.” CP binds Mn²⁺ and Zn²⁺ with high affinity and starves bacteria of these essential nutrients. Combining biophysical, structural, and microbiological analysis, we identified the molecular basis of Mn²⁺ sequestration. The asymmetry of the CP heterodimer creates a single Mn²⁺-binding site from six histidine residues, which distinguishes CP from all other Mn²⁺-binding proteins. Analysis of CP mutants with altered metal-binding properties revealed that, despite both Mn²⁺ and Zn²⁺ being essential metals, maximal growth inhibition of multiple bacterial pathogens requires Mn²⁺ sequestration. These data establish the importance of Mn²⁺ sequestration in defense against infection, explain the broad-spectrum antimicrobial activity of CP relative to other S100 proteins, and clarify the impact of metal depletion on the innate immune response to infection.     

Streptococcus pneumoniae meningitis in Alberta pre- and postintroduction of the 7-valent pneumococcal conjugate vaccine

OBJECTIVE:

To describe the epidemiology, clinical characteristics, microbiology and outcomes of patients of all ages with Streptococcus pneumoniae meningitis two years pre- and postintroduction of a S pneumoniae 7-valent conjugate vaccine program in Alberta in children <2 years of age.

METHODS:

Between 2000 and 2004, all cases of invasive pneumococcal disease in Alberta were identified. From this cohort, patients with S pneumoniae meningitis were identified by chart review. Clinical data, laboratory data and in-hospital outcomes were collected.

RESULTS:

Of the 1768 cases of invasive pneumococcal disease identified between 2000 and 2004, 110 (6.2%) had S pneumoniae meningitis. The overall incidence was 0.7 per 100,000 persons and remained unchanged over the study period. The rate in children <2 years of age appeared to fall over time, from 10.5 per 100,000 persons in 2000 to five per 100,000 persons in 2004, although there was insufficient evidence of a statistically significant time trend within any age group. Overall, the mean age was 30 years and 47% were male. In-hospital mortality was 20%, ranging from 6% in those ≤2 years of age to 31% for those ≥18 years of age, despite appropriate antimicrobial therapy.

CONCLUSION:

The high mortality rate associated with S pneumoniae meningitis suggests that prevention by vaccination is critical. In children <2 years of age, there was a downward trend in the rate of S pneumoniae meningitis after implementation of the S pneumoniae 7-valent conjugate vaccine program, but rates were still high.     

From the Cover: A Trojan horse mechanism of bacterial pathogenesis against nematodes

Understanding the mechanisms of host–pathogen interaction can provide crucial information for successfully manipulating their relationships. Because of its genetic background and practical advantages over vertebrate model systems, the nematode Caenorhabditis elegans model has become an attractive host for studying microbial pathogenesis. Here we report a “Trojan horse” mechanism of bacterial pathogenesis against nematodes. We show that the bacterium Bacillus nematocida B16 lures nematodes by emitting potent volatile organic compounds that are much more attractive to worms than those from ordinary dietary bacteria. Seventeen B. nematocida-attractant volatile organic compounds are identified, and seven are individually confirmed to lure nematodes. Once the bacteria enter the intestine of nematodes, they secrete two proteases with broad substrate ranges but preferentially target essential intestinal proteins, leading to nematode death. This Trojan horse pattern of bacterium–nematode interaction enriches our understanding of microbial pathogenesis.     

Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance

Epidemic respiratory infections are responsible for extensive morbidity and mortality within both military and civilian populations. We describe a high-throughput method to simultaneously identify and genotype species of bacteria from complex mixtures in respiratory samples. The process uses electrospray ionization mass spectrometry and base composition analysis of PCR amplification products from highly conserved genomic regions to identify and determine the relative quantity of pathogenic bacteria present in the sample. High-resolution genotyping of specific species is achieved by using additional primers targeted to highly variable regions of specific bacterial genomes. This method was used to examine samples taken from military recruits during respiratory disease outbreaks and for follow up surveillance at several military training facilities. Analysis of respiratory samples revealed high concentrations of pathogenic respiratory species, including Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pyogenes. When S. pyogenes was identified in samples from the epidemic site, the identical genotype was found in almost all recruits. This analysis method will provide information fundamental to understanding the polymicrobial nature of explosive epidemics of respiratory disease.