Streptococcus pseudopneumoniae identification by pherotype: a method to assist understanding of a potentially emerging or overlooked pathogen.

The recent identification of Streptococcus pseudopneumoniae (pseudopneumococcus) has complicated classification schemes within members of the "mitis" streptococcal group. Accurate differentiation of this species is necessary for understanding its disease potential and identification in clinical settings. This work described the use of the competence-stimulatory peptide ComC sequence for identification of S. pseudopneumoniae. ComC sequences from clinical sources were determined for 17 strains of S. pseudopneumoniae, Streptococcus pneumoniae, and Streptococcus oralis. An additional 58 ComC sequences from a range of sources were included to understand the diversity and suitability of this protein as a diagnostic marker for species identification. We identified three pherotypes for this species, delineated CSP6.1 (10/14, 79%), CSP6.3 (3/14, 21%), and SK674 (1/14, 7%). Pseudopneumococcal ComC sequences formed a discrete cluster within those of other oral streptococci. This suggests that the comC sequence could be used to identify S. pseudopneumoniae, thus simplifying the study of the pathogenic potential of this organism. To avoid confusion between pneumococcal and pseudopneumococcal pherotypes, we have renamed the competence pherotype CSP6.1, formerly reported as an "atypical" pneumococcus, CSPps1 to reflect its occurrence in S. pseudopneumoniae.

Sequetyping: serotyping Streptococcus pneumoniae by a single PCR sequencing strategy.

The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.

Pneumococcal sepsis and nasopharyngeal carriage.

PURPOSE OF REVIEW: Streptococcus pneumoniae (the pneumococcus) remains an important cause of invasive disease including bacteraemia. This review highlights recent findings related to pneumococcal bacteraemia, virulence factors, and multiple colonization, including strain competition, biofilm formation, and competence. RECENT FINDINGS: Countries with no vaccination programmes see vaccine serotypes still prevalent in disease, whereas the emergence of nonvaccine serotypes in nasopharyngeal carriage and invasive disease is seen in countries with conjugate vaccination in place. Co-colonizing strains are being uncovered with more sensitive methods, and may act synergistically or compete with each other for survival. Several factors such as iron uptake, quorum signalling and the luxS gene, involved in colonization and virulence, are discussed. The role of quorum sensing signalling molecules and formation of biofilms are being explored. SUMMARY: Epidemiological data suggest that the latest serotype-based conjugate vaccines should provide heightened protection, although serotype replacement is now being seen. Much remains to be elucidated about its biology during multiple colonization, when evolution and adaptation to its host take place. The modes of colonization (biofilm, intracellular or surface adherence to the mucosal epithelium), and whether organisms that cause invasive disease have attenuated ability to colonize the nasopharynx remain to be elucidated.

Impact of cotrimoxazole on carriage and antibiotic resistance of Streptococcus pneumoniae and Haemophilus influenzae in HIV-infected children in Zambia.

This is a substudy of a larger randomized controlled trial on HIV-infected Zambian children, which revealed that cotrimoxazole prophylaxis reduced morbidity and mortality despite a background of high cotrimoxazole resistance. The impact of cotrimoxazole on the carriage and antibiotic resistance of Streptococcus pneumoniae and Haemophilus influenzae as major causes of childhood mortality in HIV-infected children was investigated since these are unclear. Representative nasopharyngeal swabs were taken prior to randomization for 181 of 534 children (92 on cotrimoxazole and 89 on placebo). Bacterial identification and antibiotic susceptibility were performed by routine methods. Due to reduced mortality, prophylactic cotrimoxazole increased the median time from randomization to the last specimen from 48 to 56 months (P = 0.001). The carriage of H. influenzae was unaltered by cotrimoxazole. Carriage of S. pneumoniae increased slightly in both arms but was not statistically significant in the placebo arm. In S. pneumoniae switching between carriage and no carriage in consecutive pairs of samples was unaffected by cotrimoxazole (P = 0.18) with a suggestion that the probability of remaining carriage free was lower (P = 0.10). In H. influenzae cotrimoxazole decreased switching from carriage to no carriage (P = 0.02). Cotrimoxazole resistance levels were higher in postbaseline samples in the cotrimoxazole arm than in the placebo arm (S. pneumoniae, P < 0.0001; H. influenzae, P = 0.005). Cotrimoxazole decreased switching from cotrimoxazole resistance to cotrimoxazole sensitivity in S. pneumoniae (P = 0.002) and reduced the chance of H. influenzae remaining cotrimoxazole sensitive (P = 0.05). No associations were observed between the percentage of CD4 (CD4%), the change in CD4% from baseline, child age at date of specimen, child gender, or sampling month with carriage of either pathogen.

Role of bacterial endotoxin in chronic heart failure: the gut of the matter.

Proinflammatory cytokines are now thought to play a key role in the pathophysiology of chronic heart failure, driving both symptomatic presentation and disease progression. We propose that this proinflammatory state, in turn, may be sustained through a chronic release of enterically derived bacterial endotoxin. Human trials have indicated that bacterial decontamination of the gut with concomitant decrease in lipopolysaccharide (LPS) has a positive outcome on heart disease patients. Antiendotoxin antibodies may thus represent therapeutic agents in this setting. Previously, antiendotoxin antibodies were targeted to the inner hydrophobic lipid A moiety of endotoxin in an attempt to neutralize its toxicity. These antibodies failed because they lacked specificity and bound to LPS weakly. In contrast, our studies on antiendotoxin antibodies have revealed that antibodies targeted to the hydrophilic oligosaccharides of the endotoxin have the potential to bind specifically with high affinity. Development of immunotherapeutics that can reduce systemic LPS or other agents, such as bactericidal/permeability-increasing protein that can neutralize LPS and limit inflammation safely, will enable the role of LPS in chronic heart failure to be elucidated and may pave the way to develop a new generation of effective therapeutic agents that may be directed to the treatment of chronic heart failure.

Quorum sensing and biofilms in the pathogen, Streptococcus pneumoniae.

Bacteria are able to colonize and thrive in a variety of different environments as a biofilm, but only within the last half century new insights have been gained in this complex biosystem. Bacterial biofilms play a major role in human health by forming a defensive barrier against antibacterial chemical therapeutics and other potential pathogens, and in infectious disease when the bacteria invade normally sterile compartments. Quorum sensing is the signaling network for cell-to-cell communication and utilized by bacteria to regulate biofilms and other cellular processes. This review will describe recent advances in quorum sensing and biofilms. Initially, it will focus on Streptococcus pneumoniae biofilm regulation and the involvement of the ComABCDE pathway. As part of this review an original analysis of the genotypic and phenotypic variation of the signaling molecule, ComC and its cognate receptor ComD, firstly within the pneumococcal species and then within the genus Streptococcus will be presented. Additionally, a pathway similar to ComABCDE, the BlpABCSRH that regulates bacteriocin and immunity protein production that inhibit the growth of competing bacteria will be described. This review will then examine a third quorum sensing mechanism in the pneumococcus, the LuxS/AI-2, and present a novel gene and protein sequence comparative analysis that indicates its occurrence is more universal across bacterial genera compared with the Com pathway, with more sequence similarities between bacterial genera that are known to colonize the mucosal epithelium.

Is gonorrhea becoming untreatable?

An estimated 498 million new cases of curable sexually transmitted infections occur worldwide annually. Of these, 106 million are gonococcal infections, rendering gonorrhea the second most prevalent bacterial sexually transmitted infection after chlamydia. A decline in susceptibility to extended-spectrum cephalosporins, as well as treatment failures, have been identified worldwide. This, together with the associated epidemiological and socioeconomic burden, is of increasing concern. Currently, the effectiveness of antibiotic resistance control measures is limited. Barriers include the lack of therapeutic options, the difficulties of reducing high-risk sexual behavior and Neisseria gonorrhoeae's propensity to rapidly acquire resistance determinants. While the disease remains treatable for the moment, we need to anticipate and be prepared for the arrival and spread of untreatable gonorrhea by using a multifaceted approach and search for other, perhaps novel control strategies.

Identifying divergent streptococcal strains using pherotype: implications for pathogen emergence and epidemiology.

Since the late 1980s, new and existing species of oral streptococci have been identified by complex and protracted DNA-based methods that are unsuitable for high-throughput testing in clinical laboratories. Developments of simpler DNA-based tests for routine diagnosis have been thwarted by the similarities of their genomes and their high recombination rates. Thus, phenotypic tests to differentiate oral streptococci, such as hemolysis and optochin sensitivity, remain in use. However, these tests are variable and can lead to misidentification, particularly in closely related species such as Streptococcus pneumoniae and the recently identified Streptococcus pseudopneumoniae. This report highlights recent DNA-based developments in differentiating oral streptococci. Reference will be made to the methods 'sequetyping' for identifying and serotyping the pneumococcus, and 'pherotyping' for distinguishing them from pseudopneumococcus strains. These tests are yet to be evaluated in the clinical setting; nevertheless, the identification of the pseudopneumococcus by pherotyping will enable its epidemiology and pathogenesis to be studied.

The adaptive potential during nasopharyngeal colonisation of Streptococcus pneumoniae.

Adaptation to host defences and antimicrobials is critical for Streptococcus pneumoniae (the pneumococcus) during colonisation of the nasopharynx--its only ecological habitat. The pneumococcus is highly transformable with the genome between different strains varying widely in both gene content and gene sequence. Thus, mixed strains colonising together will expand the genetic reservoir--"supragenome" for this highly transformable microorganism, increasing its adaptive potential. The extent of the phenotypic and genotypic diversity of strains co-colonising in the nasopharynx was determined. In contrast to most carriage studies, which characterise single colonies, a systematic analysis of up to 20 colonies per colonisation was undertaken in Tanzanian children for 12 months. The serotype was determined by conventional serology and confirmed by DNA-based methods. The antibiotype for penicillin and co-trimoxazole was determined from the minimum inhibitory concentration determined by E-test. As representative of the genotype of strains the sequence types (STs) was determined by multilocus sequence typing (MLST). Of 61 colonisation events studied, seven (11.5%) had strains expressing multiple serotypes, with a maximum of five serotypes detected. Four colonisation events also had co-colonisation of penicillin and/or co-trimoxazole susceptible and non-susceptible pneumococci. Sequence typing revealed that 58% were unique to our cohort. Simultaneous colonisation of up to six STs with two expressing serotype 6B was seen. Re-isolation of either the same or different strains of serotype 6B was seen. Genetically related single-locus and double-locus variants were identified in our cohort that differed by multiple nucleotides. Multiple colony characterisation revealed phenotypic and genetic evidence of microevolution and a greater diversity of pneumococcal strains colonising together than previously observed, thus increasing the potential to adapt in response to the host environment during colonisation.

Unravelling the ecology of antibiotic resistant bacteria in the nasopharynx.

To study the dynamics and diversity of pneumococcal carriage and antibiotic resistance, a more thorough and systematic approach has been employed compared with routine surveillance of serotype and anti-biotic resistance. Up to ten pneumococcal isolates from pernasal (nose) and oropharyngeal (throat) sites are isolated and characterised. Our carriage studies have revealed a diverse community of pneumococci with multiple strains colonising the nasopharynx of children. In Tanzanian children less than 6 years of age, up to six serotypes and up to six different antibiotic sensitivities (as distinguished by at least a fourfold difference in the minimum inhibitory concentration) have been found. Serotyping by the Quelling reaction is prone to inaccuracy and requires expensive serological reagents. To improve the accuracy and reduce the costs, an alternative capsular typing DNA-based method has been developed. This chapter will describe the methods we have employed with emphasis on the capsular typing method.

Novel PCR-restriction fragment length polymorphism method for determining serotypes or serogroups of Streptococcus pneumoniae isolates.

Serotyping Streptococcus pneumoniae is a technique generally confined to reference laboratories, as purchasing pneumococcal antisera is a huge investment. Many attempts have been made to modify serological agglutination techniques to make them more accessible, and more recently developments in serotyping have focused on molecular techniques. This paper describes a PCR assay which amplifies the entire capsulation locus between dexB and aliA. Amplicons are digested to produce serotype-specific patterns. We have shown, using 81 epidemiologically unrelated strains representing 46 different serotypes, that the patterns correlate with a 90 to 100% similarity range for the same serotype or serogroup. Prospective testing of 73 isolates of unknown serotype confirmed reliable serotype attribution, and serotype profiles are reproducible on repeated testing. Once our database contains all 90 serotypes, this technique should be fully portable, cost-effective, and useful in any laboratory with sufficient molecular experience.

A reiterative method for calculating the early bactericidal activity of antituberculosis drugs.

Studies of early bactericidal activity (EBA) are important in the rapid evaluation of new antituberculosis drugs. Historically, these have concentrated on the log fall in the viable count in sputum during the first 48 hours of therapy. In this paper, we provide a mathematical model that suggests that the viable count in sputum follows an exponential decay curve with the equation V = S + Me(-kt) (where V is the viable count, M the population of bacteria susceptible to the test drug, S the population susceptible only to sterilizing agents, t the day of sputum collection as related to start of therapy, k the rate constant for the bacteria killed each day, and e the Napierian constant). We demonstrate that data from clinical trials fits the exponential decay model. We propose that future EBA studies should be performed by measuring daily quantitative counts for at least 5 days. We also propose that the comparison of the early bactericidal activity of antituberculosis drugs should be evaluated using the time taken to reduce the viable count by 50% (vt(50)). A further reiterative refinement following a rule set based on statistically the best fit to the exponential decay model is described that will allow investigators to identify anomalous results and thus enhance the accuracy in measuring early bactericidal activity.

Characterization of oligopeptides that cross-react with carbohydrate-specific antibodies by real time kinetics, in-solution competition enzyme-linked immunosorbent assay, and immunological analyses.

Phage displaying random cyclic 7-mer, and linear 7-mer and 12-mer peptides at the N terminus of the coat protein, pIII, were panned with the murine monoclonal antibody, 9-2-L379 specific for meningococcal lipo-oligosaccharide. Five cyclic peptides with two sequence motifs, six linear 7-mers, and five linear 12-mers with different sequence motifs were identified. Only phage displaying cyclic peptides were specifically captured by and were antigenic for 9-2-L379. Monoclonal antibody 9-2-L379 exhibited "apparent" binding affinities to the cyclic peptides between 11 and 184 nm, comparable with lipo-oligosaccharide. All cyclic peptides competed with the binding of 9-2-L379 to lipo-oligosaccharide with EC(50) values in the range 10-105 microm, which correlated with their apparent binding affinities. Structural modifications of the cyclic peptides eliminated their ability to bind and compete with monoclonal antibody 9-2-L379. Mice (C3H/HeN) immunized with the cyclic peptide with optimal apparent binding affinity and EC(50) of competition elicited cross-reactive antibodies to meningococcal lipo-oligosaccharide with end point dilution serum antibody titers of 3200. Cyclic peptides were converted to T-cell-dependent immunogens without disrupting these properties by C-terminal biotinylation and complexing with NeutrAvidin. The data indicate that constrained peptides can cross-react with a carbohydrate-specific antibody with greater specificity than linear peptides, and critical to this specificity is their structural conformation.

Impact of azithromycin administration for trachoma control on the carriage of antibiotic-resistant Streptococcus pneumoniae.

Community distribution of azithromycin has an important role to play in trachoma control. Previous studies have suggested that this may increase the prevalence of macrolide-resistant Streptococcus pneumoniae. S. pneumoniae was isolated from children under 7 years of age in Rombo District, northern Tanzania, before and 2 and 6 months after community-wide administration of azithromycin. Overall carriage rates were 11, 12, and 7%, respectively. Only one macrolide-resistant isolate carrying the mef gene was obtained 6 months after azithromycin administration. This contrasted with cotrimoxazole and penicillin resistance, both of which were common (cotrimoxazole resistance, 42, 43, and 47%, and penicillin resistance, 21, 17, and 16% at baseline, 2 months, and 6 months, respectively). There was a significant association between cotrimoxazole and penicillin resistance (P < 0.0001, Fisher's exact). These data suggest that in communities where macrolide resistance is rare, azithromycin distribution for trachoma control is unlikely to increase the prevalence of resistant organisms.