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.     

Isolation, characterization, and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae.

A comparative hybridization protocol was used to isolate a small segment of DNA present in the Streptococcus pneumoniae type 19F strain SSZ but absent from strain Rx1, a nonencapsulated derivative of the type 2 strain D39. This segment of DNA is a 1,747-bp insertion sequence, designated IS1202, flanked by 23-bp imperfect inverted repeats and containing a single open reading frame sufficient to encode a 54.4-kDa polypeptide. A 27-bp target sequence is duplicated at either end of the element. IS1202 is not related to any of the currently known insertion elements and is the first reported for S. pneumoniae. Although found predominantly in type 19F strains in up to five copies, it has also been shown to be present in the chromosomes of pneumococci belonging to other serotypes. One of the four IS1202 copies in the encapsulated strain SSZ is located 1,009 bp downstream of the dexB gene, and transformation studies reveal that it is also closely linked to the type 19F capsular polysaccharide synthesis (cps) locus.     

CRISPR Interference Can Prevent Natural Transformation and Virulence Acquisition during In Vivo Bacterial Infection

Pathogenic bacterial strains emerge largely due to transfer of virulence and antimicrobial resistance genes between bacteria, a process known as horizontal gene transfer (HGT). Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci of bacteria and archaea encode a sequence-specific defense mechanism against bacteriophages and constitute a programmable barrier to HGT. However, the impact of CRISPRs on the emergence of virulence is unknown. We programmed the human pathogen Streptococcus pneumoniae with CRISPR sequences that target capsule genes, an essential pneumococcal virulence factor, and show that CRISPR interference can prevent transformation of nonencapsulated, avirulent pneumococci into capsulated, virulent strains during infection in mice. Further, at low frequencies bacteria can lose CRISPR function, acquire capsule genes, and mount?a successful infection. These results demonstrate that CRISPR interference can prevent the emergence of virulence in?vivo and that strong selective pressure for virulence or antibiotic resistance can lead to CRISPR loss in bacterial pathogens.     

Two new loci affecting cell division identified as suppressors of an ftsQ-null mutation in Streptomyces coelicolor A3(2)

We have cloned and sequenced the gene encoding the surface protein antigen PAa (antigen I/II family) from Streptococcus cricetus E49 (serotype a) using degenerate PCR. The deduced amino acid sequence of PAa reveals two repeating regions (A region; alanine-rich region, P region; proline-rich region). Two additional tandem repeats were found in the A region and part of the P region was deleted compared to antigen I/II. Homology and phylogenetic analyses reveal that PAa is homologous to Streptococcus sobrinus PAg rather than Streptococcus mutans PAc. Using degenerate PCR a gene homologous to PAc was identified in Streptococcus intermedius, but not found in Streptococcus rattus or Streptococcus anginosus.     

Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin J.

Lactobacillus helveticus 481 produces a 37-kDa bacteriocin called helveticin J. Libraries of chromosomal DNA from L. helveticus were prepared in lambda gt11 and probed for phage-producing fusion proteins that could react with polyclonal helveticin J antibody. Two recombinant phage, HJ1 and HJ4, containing homologous inserts of 350 and 600 bp, respectively, produced proteins that reacted with antibody. These two phage clones specifically hybridized to L. helveticus 481 total genomic DNA but not to DNA from strains that did not produce helveticin J or strains producing unrelated bacteriocins. HJ1 and HJ4 lysogens produced beta-galactosidase fusion proteins that shared similar epitopes with each other and helveticin J. The intact helveticin J gene (hlv) was isolated by screening a library of L. helveticus chromosomal DNA in lambda EMBL3 with the insert DNA from phage HJ4 as a probe. The DNA sequence of a contiguous 3,364-bp region was determined. Two complete open reading frames (ORF), designated ORF2 and ORF3, were identified within the sequenced fragment. The 3' end of another open reading frame, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. ORF2 could encode an 11,808-Da protein. The L. helveticus DNA inserts of the HJ1 and HJ4 clones reside within ORF3, which begins 30 bp downstream from the termination codon of ORF2. ORF3 could encode a 37,511-Da protein. Downstream from ORF3, the 5' end of another ORF (ORF4) was found. A Bg/II fragment containing ORF2 and ORF3 was cloned into pGK12, and the recombinant plasmid, pTRK135, was transformed into Lactobacillus acidophilus via electroporation. Transformants carrying pTRK135 produced a bacteriocin that was heat labile and exhibited an acitivity spectrum that was the same as that of helveticin J.     

Non-typeable pneumococci circulating in Portugal are of cps type NCC2 and have genomic features typical of encapsulated isolates

Background

Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity.

Results

A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements.

Conclusions

NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-863) contains supplementary material, which is available to authorized users.     

Extensive variation in the ddl gene of penicillin-resistant Streptococcus pneumoniae results from a hitchhiking effect driven by the penicillin-binding protein 2b gene

An internal fragment of the ddl gene, encoding the cytoplasmic enzyme D-alanyl-D-alanine ligase, was sequenced from 566 isolates of Streptococcus pneumoniae and single isolates of Streptococcus mitis and Streptococcus oralis. The 52 alleles found among the S. pneumoniae isolates fell into two groups. Group A alleles were very uniform in sequence and were present in both penicillin-susceptible and penicillin-resistant pneumococci. Group B alleles were much more diverse and were found only in penicillin-resistant isolates. The Streptococcus oralis and Streptococcus mitis alleles were less diverged from group A alleles than some of the group B pneumococcal alleles, suggesting that the latter alleles contain interspecies recombinational replacements. The ddl gene was located 783 bp downstream of the penicillin-binding protein 2b gene (pbp2b). Sequencing of the pbp2b-recR-ddl-murF region of three penicillin-resistant pneumococci that had diverged ddl alleles showed that the whole region from pbp2b to ddl (or beyond) was highly diverged (about 8%) compared with the sequences from three penicillin-susceptible isolates. The high levels of diversity in the group B ddl alleles from penicillin-resistant isolates were ascribed to a hitchhiking effect whereby interspecies recombinational exchanges at pbp2b, selected by penicillin usage, often extend into, or through, the ddl gene. The data allow the average size of the interspecies recombinational replacements to be estimated at about 6 kb.     

Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999     

The Structure and Evolution of Subtelomeric Y'' Repeats in Saccharomyces Cerevisiae

The subtelomeric Y' family of repeated DNA sequences in the yeast Saccharomyces cerevisiae is of unknown origin and function. Y's vary in copy number and location among strains. Eight Y's, from two strains, were cloned and sequenced over the same 3.2-kb interval in order to assess the within- and between-strain variation as well as address their origin and function. One entire Y' sequence was reconstructed from two clones presented here and a previously sequenced 833-bp region. It contains two large overlapping open reading frames (ORFs). The putative protein sequences have no strong homologies to any known proteins except for one region that has 27% identity with RNA helicases. RNA homologous to each ORF was detected. Comparison of the sequences revealed that the known long (Y'-L) and short (Y'-S) size classes, which coexist within cells, differ by several insertions and/or deletions within this region. The Y'-Ls from strain Y55 also differ from those of strain YP1 by several short deletions in the same region. Most of these deletions appear to have occurred between short (2-10 bp) direct repeats. The single base pair polymorphisms and the deletions are clustered in the first half of the interval compared. There is 0.30-1.13% divergence among Y'-Ls within a strain and 1.15-1.75% divergence between strains in the interval. This is similar to known unique sequence variation but contrasts with the 8-18% divergence among the adjacent subtelomeric repeats, X. Subsets of Y's exhibit concerted evolution; however, more than one variant appears to be maintained within strains. The observed sequence variation disrupts the first ORF in many Y's while most of the second ORF including the putative helicase region is unaffected. The structure and distribution of the Y' elements are consistent with having originated as a mobile element. However, they now appear to move via recombination. Recombination can account for the homogenization within subsets of Y's but does not account for the maintenance of different variants.     

Physicochemical surface properties of nonencapsulated and encapsulated coagulase-negative staphylococci

Cell surfaces of three nonencapsulated and three encapsulated coagulase-negative staphylococci were characterized by their surface free energies, zeta potentials, and elemental and molecular compositions. Surface free energies were calculated from contact angle measurements with various liquids. All six strains showed a high surface free energy (103 to 126 mJ.m-2), estimated from the concept of polar and dispersion components. However, the hydrogen-donating surface free energy parameter was zero for all nonencapsulated strains. The zeta potential profile measured as a function of pH in phosphate-buffered saline for the nonencapsulated strains was completely different from that of the encapsulated strains. X-ray photoelectron spectroscopy was used to determine the elements (O, C, N, P, and K) in the outer 2 to 5 nm of the freeze-dried cell surface and showed that the hydrophilic character of the staphylococci was related to oxygen (O/C ratio, approximately 0.52)- and phosphorus (P/C ratio, approximately 0.03)-containing groups. Both the elemental and molecular characterizations (done by infrared spectroscopy) pointed to the presence of polysaccharides and polypeptides on the cell surface of the nonencapsulated and encapsulated strains.     

Nonencapsulated or nontypeable Haemophilus influenzae are more likely than their encapsulated or serotypeable counterparts to have mutations in their fucose operon

Population biology of Haemophilus influenzae can be studied by multilocus sequence typing (MLST), and isolates are assigned sequence types (STs) based on nucleotide sequence variations in seven housekeeping genes, including fucK. However, the ST cannot be assigned if one of the housekeeping genes is absent or cannot be detected by the current protocol. Occasionally, strains of H. influenzae have been reported to lack the fucK gene. In this study, we examined the prevalence of this mutation among our collection of H. influenzae isolates. Of the 704 isolates studied, including 282 encapsulated and 422 nonencapsulated isolates, nine were not typeable by MLST owing to failure to detect the fucK gene. All nine fucK-negative isolates were nonencapsulated and belonged to various biotypes. DNA sequencing of the fucose operon region confirmed complete deletion of genes in the operon in seven of the nine isolates, while in the remaining two isolates, some of the genes were found intact or in parts. The significance of these findings is discussed.     

Physicochemical surface properties of nonencapsulated and encapsulated coagulase-negative staphylococci.

Cell surfaces of three nonencapsulated and three encapsulated coagulase-negative staphylococci were characterized by their surface free energies, zeta potentials, and elemental and molecular compositions. Surface free energies were calculated from contact angle measurements with various liquids. All six strains showed a high surface free energy (103 to 126 mJ.m-2), estimated from the concept of polar and dispersion components. However, the hydrogen-donating surface free energy parameter was zero for all nonencapsulated strains. The zeta potential profile measured as a function of pH in phosphate-buffered saline for the nonencapsulated strains was completely different from that of the encapsulated strains. X-ray photoelectron spectroscopy was used to determine the elements (O, C, N, P, and K) in the outer 2 to 5 nm of the freeze-dried cell surface and showed that the hydrophilic character of the staphylococci was related to oxygen (O/C ratio, approximately 0.52)- and phosphorus (P/C ratio, approximately 0.03)-containing groups. Both the elemental and molecular characterizations (done by infrared spectroscopy) pointed to the presence of polysaccharides and polypeptides on the cell surface of the nonencapsulated and encapsulated strains.     

Relationship Between Serotype A Encapsulation and a 40-kDa Lipoprotein in Pasteurella multocida

Eleven serotype A encapsulated and nonencapsulated strains of Pasteurella multocida were examined with regard to lipoprotein content. Relative amounts of an approximately 40-kDa lipoprotein (Plp-40) were found to correlate directly with the degree of encapsulation in that heavily encapsulated strains exhibited the greatest amounts, while nonencapsulated strains possessed little or no Plp-40. Received: 29 September 1997 / Accepted: 31 October 1997     

Panning of a Phage Display Library against a Synthetic Capsule for Peptide Ligands That Bind to the Native Capsule of Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax with the ability to not only produce a tripartite toxin, but also an enveloping capsule comprised primarily of γ-D-glutamic acid residues. The purpose of this study was to isolate peptide ligands capable of binding to the native capsule of B. anthracis from a commercial phage display peptide library using a synthetic form of the capsule consisting of 12 γ-D-glutamic acid residues. Following four rounds of selection, 80 clones were selected randomly and analysed by DNA sequencing. Four clones, each containing a unique consensus sequence, were identified by sequence alignment analysis. Phage particles were prepared and their derived 12-mer peptides were also chemically synthesized and conjugated to BSA. Both the phage particles and free peptide-BSA conjugates were evaluated by ELISA for binding to encapsulated cells of B. anthracis as well as a B. anthracis capsule extract. All the phage particles tested except one were able to bind to both the encapsulated cells and the capsule extract. However, the peptide-BSA conjugates could only bind to the encapsulated cells. One of the peptide-BSA conjugates, with the sequence DSSRIPMQWHPQ (termed G1), was fluorescently labelled and its binding to the encapsulated cells was further confirmed by confocal microscopy. The results demonstrated that the synthetic capsule was effective in isolating phage-displayed peptides with binding affinity for the native capsule of B. anthracis.     

pspK acquisition contributes to the loss of capsule in pneumococci: molecular characterisation of non-encapsulated pneumococci

With the introduction of the pneumococcal conjugate vaccine (PCV), the number of cases of non-vaccine type pneumococci and non-encapsulated Streptococcus pneumoniae (NESp) infection have increased. In order to clarify how pspK-harbouring NESp might have emerged, we characterised NESp and analysed the correlation between transformation and non-encapsulation. A total of 26 NESp strains were used in this study. The genetic backgrounds were compared using multilocus sequence typing (MLST). The ΔpspK::ermB strain, in which pspK was replaced by ermB in NESp, was constructed by homologous recombination. The genomic DNA of the ΔpspK::ermB strain was transformed into two types of encapsulated S. pneumoniae via transformation. The fitness of the parent and non-encapsulated transformants was compared using the growth curve. All NESp had pspK instead of capsular coding regions and were classified into 14 types by MLST, which indicated that NESp had several genetic backgrounds. Transformation of ΔpspK::ermB genomic DNA resulted in 10⁻⁴‒10⁻⁵ non-encapsulated transformants. Non-encapsulated transformants could grow faster than the encapsulated parent strain. The acquisition of pspK region via transformation contributed to the loss of encapsulation with high frequency. The present results suggest that non-encapsulation through pspK acquisition could be a potential mechanism to evade PCV.     

The capsule polysaccharide synthesis locus of streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E.

To identify a chromosomal region of Streptococcus pneumoniae serotype 14 involved in capsule polysaccharide synthesis, two strategies were used: (i) Tn916 mutagenesis, followed by the characterization of four unencapsulated mutants, and (ii) cross-hybridization with a capsule polysaccharide synthesis gene (cps) probe from S. agalactiae, which has a structurally similar capsule. The two approaches detected the same chromosomal region consisting of two adjacent EcoRI fragments. One of these EcoRI fragments was cloned and hybridized with a cosmid library. This resulted in clone cMKO2. A similar cosmid clone was obtained from an unencapsulated Tn916 mutant, Spnl4.H. Sequence analysis of the two cosmid clones revealed that in the Tn916 mutant, a gene, cps14E, which is homologous to other bacterial genes encoding glycosyl transferases, had been inactivated. An open reading frame immediately downstream of cps14E, designated cps14F, shows no significant homology with any known genes or proteins. A functional assay showed that cps14E encodes a glycosyl transferase and that a gene-specific knockout mutant lacks this enzyme activity, whereas inactivation of cps14F does not have this effect.