Transposable multiple antibiotic resistance in Streptococcus pneumoniae

Summary A mobile genetic element, designated Tn1545, was detected in the chromosome of Streptococcus pneumoniae BM4200, a clinical isolate multiply resistant to antibiotics. The 25.3 kb element conferred resistance to kanamycin and structurally related aminoglycosides by synthesis of a 3-aminoglycoside phosphotranferase type III (aphA-3), to macrolide-lincosamide-streptogramin B-type antibiotics (ermAM), and to tetracycline (tetM). Tn1545 was self-transferable to a recombination deficient S. faecalis strain where it was able to transpose to various sites, induce insertional mutations and was apparently cleanly excised. The element also conjugated to and transposed to the chromosome of S. faecalis, S. lactis, S. diacetylactis, S. cremoris, S. sanguis, Staphylococcus aureus, and Listeria monocytogenes. The properties of the conjugative transposon Tn1545 could account for the sudden emergence, rapid dissemination, and stabilisation of multiple resistance to antibiotics in S. pneumoniae in the absence of plasmids.     

Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major pathogen causing potentially life-threatening community-acquired diseases in both the developed and developing world. Since 1967, there has been a dramatic increase in the incidence of penicillin-resistant and multiply antibiotic-resistant pneumococci worldwide. Prevention of access of the antibiotic to the target, inactivation of the antibiotic and alteration of the target are mechanisms that S. pneumoniae has developed to resist antibiotics. Recent studies on antibiotic-tolerant pneumococcal mutants permitted development of a novel model for the control of bacterial cell death.     

Properties and identification of antibiotic drug targets

Background  

We analysed 48 non-redundant antibiotic target proteins from all bacteria, 22 antibiotic target proteins from E. coli only and 4243 non-drug targets from E. coli to identify differences in their properties and to predict new potential drug targets.     

Genetic identification of exported proteins in Streptococcus pneumoniae

A strategy was developed to mutate and genetically identify exported proteins in Streptococcus pneumoniae. Vectors were created and used to screen pneumococcal DNA in Escherichia coli and S. pneumoniae for translational gene fusions to alkaline phosphatase (PhoA), Twenty five PhoA⁺ pneumococcal mutants were isolated and the loci from eight of these mutants showed similarity to known exported or membrane-associated proteins. Homologues were found to: (i) protein-dependent peptide permeases, (ii) penicillin-binding proteins, (iii) Cip proteases, (iv) two-component sensor regulators, (v) the phospho-enolpyruvate:carbohydrate phosphotransferase permeases, (vi) membrane-associated dehydrogenases, (vii) P-type (E₁E₂-type) cation transport ATPases, (viii) ABC transporters responsible for the translocation of the RTX class of bacterial toxins. Unexpectedly one PhoA⁺ mutant contained a fusion to a member of the DEAD protein family of ATP-dependent RNA helicases suggesting export of these proteins.     

Capsular serotype and antibiotic resistance of Streptococcus pneumoniae isolates in Malaysia

Background

Streptococcus pneumoniae is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, that has since become a major public health concern. In this study, the serotypes distribution of pneumococcal isolates was investigated to predict the efficacy of the 7-valent pneumococcal conjugate vaccine (PCV7) among the Malaysian populations.

Methodology/Principal Findings

A total of 151 clinical isolates were serotyped using multiplex PCR assays. Out of them, there were 21.2% penicillin-resistant, 29.1% penicillin-intermediate, and 49.7% penicillin-susceptible S.pneumoniae strains. Serotypes detected among the Malaysian isolates were 1, 3, 10A, 11A/11D, 12F/12A, 14, 15A, 15B/15C, 16F, 18C/18B/18A/18F, 19A, 19F, 23F, 35B, 35F/47F, 6A/6B, 7C/7B/40, 7F/7A, 9V/9A, and 34. Serotype 19F and 23F were the two most prevalent serotypes detected. Serotypes are highly associated with invasiveness of isolates (p = 0.001) and penicillin susceptibility (p<0.001). Serotype 19F was observed to have increased resistance against penicillin while serotype 19A has high invasive tendency. Age of patients was an important factor underlying the pneumococcal serotypes (p = 0.03) and clinical sites of infections (p<0.001). High prevalence of pneumococcal isolates were detected among children <5 years old at nasopharyngeal sites while elderly adults ≥60 years old were at increased risk for pneumococcal bacteremia.

Conclusion/Significance

Current study revealed that a number of serotypes, especially those associated with high penicillin resistance, have been formulated in the PCV7. Therefore, the protections expected from the routine use of PCV7 would be encouraging for the Malaysian. However, it is not possible to predict serotypes that might become predominant in the future and hence continued surveillance of circulating serotypes will be needed.     

Streptococcus pneumoniae proteomics: determinants of pathogenesis and vaccine development

Streptococcus pneumoniae is a major pathogen that is responsible for a variety of invasive diseases. The bacteria gain entry initially by establishing a carriage state in the nasopharynx from where they migrate to other sites in the body. The worldwide distribution of the bacteria and the severity of the diseases have led to a significant level of interest in the development of vaccines against the bacteria. Current vaccines, based on the bacterial polysaccharide, have a number of limitations including poor immunogenicity and limited effectiveness against all pneumococcal serotypes. There are many challenges in developing vaccines that will be effective against the diverse range of isolates and serotypes for this highly variable bacterial pathogen. This review considers how proteomic technologies have extended our understanding of the pathogenic mechanisms of nasopharyngeal colonization and disease development as well as the critical areas in developing protein-based vaccines.     

Evaluation of gene-technological and conventional methods in the identification of Streptococcus pneumoniae

To find reliable methods able to identify the "difficult" Streptococcus pneumoniae isolates, an in-house polymerase chain reaction (PCR) for pneumolysin gene (Ply-PCR) and a commercial RNA hybridisation test (AccuProbe) were evaluated. Selected isolates of suspected pneumococci, sent for confirmation of identification and for serotyping, were classified into four groups based on their optochin sensitivity and capsule reaction. All isolates in Group 1, which consisted of 24 typical, optochin-sensitive, encapsulated pneumococcal strains, were positive in the Ply-PCR and AccuProbe tests. In Group 2, which consisted of 25 optochin-sensitive, but unencapsulated pneumococcal strains, all the isolates were positive in the Ply-PCR test, and 23 were positive in the AccuProbe test. In Group 3, which consisted of 15 atypical, optochin-resistant but encapsulated pneumococci, 12 of the isolates were positive in the Ply-PCR and 12 in the AccuProbe test, and 11 of these 12 strains were positive in both tests. In Group 4, which consisted of 36 equivocal optochin-resistant, unencapsulated isolates, 15 strains were positive in the Ply-PCR test and 8 strains in the AccuProbe test. As a conclusion, the Ply-PCR and AccuProbe tests identified similarly typical optochin-sensitive pneumococci, but gave partly controversial results about atypical pneumococci. Thus, they did not reliably help in the identification of suspected pneumococcal isolates lacking the conventional characteristics of pneumococcus.     

Conjugative transfer of multiple antibiotic resistance markers in Streptococcus pneumoniae.

Two antibiotic-resistant isolates of Streptococcus pneumoniae were investigated for conjugative transfer of their drug resistance markers into streptococcal (groups B and D) and pneumococcal (encapsulated and non-encapsulataed) recipients. Of these, 7 wild-type donor pneumococci transferred all their resistance markers (except Pc [penicillin], Su [sulfonamide], and Tp [trimethoprim]) into group D Streptococcus and non-encapsulated S. pneumoniae recipients at a low frequency (10(-5) to 10(-6)). The resistance markers transferred were Tc (tetracycline); Tc and Cm (chloramphenicol); Tc and MLS (macrolides, lincosamides, and streptogramin B); Tc, MLS, Km (kanamycin), and Cm. The transconjugants obtained retransferred their resistance markers into appropriate streptococcal or pneumococcal recipients or both. The resistance markers of streptococcal transconjugants could not be cured by chemical agents. All attempts to detect extra-chromosomal deoxyribonucleic acid from pneumococcal or streptococcal transconjugants were unsuccessful. The molecular weight of a streptococcal conjugative R plasmid (pIP501) was investigated after transfer into the non-encapsulated S. pneumoniae recipient and was found to be similar to that of the wild-type group B Streptococcus host (20 x 10(6)).     

Bacterial proteomics and identification of potential vaccine targets

Currently, the greatest causes of human morbidity and mortality are infectious diseases. Vaccination remains the most effective measure to lessen this burden on the human population. Many vaccines presently in use were developed using techniques first proposed by Louis Pasteur, which involved the use of inactivated, attenuated live forms, or extracts of pathogenic organisms to immunize the host and provide protection from the disease. The advent of the genomic era has recently led to a new generation of rationally designed vaccines developed using a process termed reverse vaccinology. This approach uses genomic data in silico to identify proteins encoded by the pathogen as potential vaccine candidates. Proteomic technologies serve as an important complement to the reverse vaccinology approach to antigen discovery. Proteomic techniques are able to identify proteins that are expressed by the pathogen during infection of a host and the subset of proteins that reside on the surface of the pathogen. These two traits should be considered central factors to vaccine antigen selection as they assure that the host will be able to mount an effective immune response that leads to lasting protection from the pathogen.     

Process intensification for production of Streptococcus pneumoniae whole-cell vaccine

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.     

Novel vaccine strategies with protein antigens of Streptococcus pneumoniae

Infections caused by Streptococcus pneumoniae (pneumococcus) are a major cause of mortality throughout the world. This organism is primarily a commensal in the upper respiratory tract of humans, but can cause pneumonia in high-risk persons and disseminate from the lungs by invasion of the bloodstream. Currently, prevention of pneumococcal infections is by immunization with vaccines which contain capsular polysaccharides from the most common serotypes causing invasive disease. However, there are more than 90 antigenically distinct serotypes and there is concern that serotypes not included in the vaccines may become more prevalent in the face of continued use of polysaccharide vaccines. Also, certain high-risk groups have poor immunological responses to some of the polysaccharides in the vaccine formulations. Protein antigens that are conserved across all capsular serotypes would induce more effective and durable humoral immune responses and could potentially protect against all clinically relevant pneumococcal capsular types. This review provides a summary of work on pneumococcal proteins that are being investigated as components for future generations of improved pneumococcal vaccines.     

Idiotope vaccine against Streptococcus pneumoniae. A precursor study

An analysis of nominal vs idiotope antigen-induced B cell precursors was performed in A/St mice. With the use of the splenic fragment culture system, the quantity and quality of B cell precursors responding to two anti-idiotope carrier antigens (4C11 hemocyanin and F6 hemocyanin) and nominal antigen (phosphorylcholine-hemocyanin) were compared. In addition, the effect of priming with anti-idiotope-carrier antigens on B cell precursors responding to phosphorylcholine-hemocyanin was determined. We found that one anti-idiotope-carrier antigen, 4C11-hemocyanin, and phosphorylcholine-hemocyanin stimulated similar subpopulations of primary B cells. However, the B cell population stimulated by F6-hemocyanin, the other anti-idiotype complex, was distinct. Furthermore, priming with certain idiotope antigens can direct the phosphorylcholine-hemocyanin response into the expression of idiotypes that may be the most effective in protective immunity. Our results provide essential information for the rational design of idiotype vaccines by clarifying the dynamic relationship of the B cell precursor repertoire with the in vivo antibody response in the response to nominal and idiotope antigens.     

New approaches to antibiotic discovery

New antibiotics are urgently required by human medicine as pathogens emerge with developed resistance to almost all antibiotic classes. Pioneering approaches, methodologies and technologies have facilitated a new era in antimicrobial discovery. Innovative culturing techniques such as iChip and co-culturing methods which use ‘helper’ strains to produce bioactive molecules have had notable success. Exploiting antibiotic resistance to identify antibacterial producers performed in tandem with diagnostic PCR based identification approaches has identified novel candidates. Employing powerful metagenomic mining and metabolomic tools has identified the antibiotic’ome, highlighting new antibiotics from underexplored environments and silent gene clusters enabling researchers to mine for scaffolds with both a novel mechanism of action and also few clinically established resistance determinants. Modern biotechnological approaches are delivering but will require support from government initiatives together with changes in regulation to pave the way for valuable, efficacious, highly targeted, pathogen specific antimicrobial therapies.     

Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery

Streptococcus pneumoniae is a useful tool for the discovery of broad-spectrum antibiotics because of its genetic malleability and importance as a pathogen. Recent publications of complete chromosomal DNA sequences for S. pneumoniae facilitate rapid and effective use of genomics-based technology to identify essential genes encoding new targets for antibacterial drugs. These methods include computational comparative genomics, gene disruption studies to determine essentiality or identify essential genes, and gene expression analysis using microarrays and gel-based proteomics. We review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it the best species for the identification and validation of new antibiotic targets.     

Review on chemogenomic approaches towards hepatitis C viral targets

Hepatitis C virus (HCV) is the most prevalent viral pathogen that infects more than 185 million people worldwide. HCV infection leads to chronic liver diseases such as liver cirrhosis and hepatocellular carcinoma. Direct-acting antivirals (DAAs) are the recent combination therapy for HCV infection with reduced side effects than prior therapies. Sustained virological response (SVR) acts as a gold standard marker to monitor the success of antiviral treatment. Older treatment therapies attain 50-55% of SVR compared with DAAs which attain around 90-95%. The current review emphasizes the recent chemogenomic updates that have been unfolded through structure-based drug design of HCV drug target proteins (NS3/4A, NS5A, and NS5B) and ligand-based drug design of DAAs in achieving a stable HCV viral treatment strategies.     

New Streptococcus pneumoniae clones in deceased wild chimpanzees

In wild chimpanzees in the Ta? National Park, C?te d'Ivoire, sudden deaths which were preceded by respiratory problems had been observed since 1999. Two new clones of Streptococcus pneumoniae were identified in deceased apes on the basis of multilocus sequence typing analysis and ply, lytA, and pbp2x sequences. The findings suggest that virulent S. pneumoniae occurs in populations of wild chimpanzees with the potential to cause infections similar to those observed in humans.     

Proteins of Streptococcus pneumoniae: perspectives for development of pneumococcal vaccine

Streptococcus pneumoniae cell wall and cytoplasmic proteins contribute directly to pathogenesis of pneumococcal infection. Protective effect of pneumococcal proteins such as pneumolysin (Ply), muramylamidase (LytA) and pneumococcal surface protein A (PspA). There is discussion in the literature about development of conjugared pneumococcal vaccines, which should include polysaccharides of invasive serotypes of pneumococci as well as protein antigens of this pathogen, for prevention of infections caused by S. pneumoniae. Researches suggest that such hybrid vaccines will be effective, first of all, for children < 2 years of age and elderly > 65 years old because immune response to polysaccharide vaccines either do not form at all or insufficient for prevention of pneumococcal infection.