A case of diphtheria in Sweden, October 2011.

In October 2011, a child who had arrived in Sweden from Somalia presented with atypical tonsillitis, was treated with penicillin and the symptoms resolved. A throat swab was positive for toxigenic Corynebacterium diphtheriae. The child's family were then vaccinated with diphtheria, tetanus and pertussis vaccine and screened for C. diphtheriae. No secondary cases were found. A high level of adherence to childhood vaccination programmes is an effective way to protect populations against diphtheria.

Pattern of accessory regions and invasive disease potential in Streptococcus pneumoniae.

BACKGROUND: The invasive disease potential (IDP) of Streptococcus pneumoniae differs between serotypes, but the reason for this is unknown. METHODS: A total of 47 pneumococcal isolates from 13 serotypes with different IDPs in humans that belonged to 37 multilocus sequence types were compared by whole genome microarrays and mutant analyses. RESULTS: Approximately 34% of the genes were variable, including 95 genes previously shown by signature-tagged mutagenesis (STM) to be required for invasive disease in mice. Many variable genes were localized to 41 accessory regions (ARs), of which 24 contained genes previously identified by STM as required for invasive disease. Only AR6 and AR34 were preferentially found in isolates of serotypes with high IDPs. Neither AR6, which carries a gene previously identified bySTMas required for invasive disease and encodes a 6-phospho-betaglucosidase, nor the putative adhesin expressed by AR34 was required for mouse virulence in TIGR4. CONCLUSIONS: Pneumococci possess a repertoire of ARs that differ between clones and even between isolates of the same clone. The ARs required for invasive disease in humans may be redundant, as no unique pattern distinguished the most invasive clones from others. The ARs that contained genes previously identified by STM as required for virulence in mice were frequently absent from invasive human isolates. Only 1 AR (AR6) was present in almost all isolates from the serotypes with the highest IDP (1, 4, and 7F), whereas it was missing from many others.

RrgA is a pilus-associated adhesin in Streptococcus pneumoniae.

Adherence to host cells is important in microbial colonization of a mucosal surface, and Streptococcus pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits, RrgA, RrgB and RrgC. We sought to determine which subunit(s) confers adherence. Bacteria deficient in RrgA are significantly less adherent than wild-type organisms, while overexpression of RrgA enhances adherence. Recombinant monomeric RrgA binds to respiratory cells, as does RrgC with less affinity, and pre-incubation of epithelial cells with RrgA reduces adherence of wild-type piliated pneumococci. Non-adherent RrgA-negative, RrgB- and RrgC-positive organisms produce pili, suggesting that pilus-mediated adherence is due to expression of RrgA, rather than the pilus backbone itself. In contrast, RrgA-positive strains with disrupted rrgB and rrgC genes exhibit wild-type adherence despite failure to produce pili by Western blot or immunoelectron microscopy. The density of bacteria colonizing the upper respiratory tract of mice inoculated with piliated RrgA-negative pneumococci was significantly less compared with wild-type; in contrast, non-piliated pneumococci expressing non-polymeric RrgA had similar numbers of bacteria in the nasopharynx as piliated wild-type bacteria. These data suggest that RrgA is central in pilus-mediated adherence and disease, even in the absence of polymeric pilus production.

Clonal success of piliated penicillin nonsusceptible pneumococci.

Antibiotic resistance in pneumococci is due to the spread of strains belonging to a limited number of clones. The Spain(9V)-3 clone of sequence type (ST)156 is one of the most successful clones with reduced susceptibility to penicillin [pneumococci nonsusceptible to penicillin (PNSP)]. In Sweden during 2000-2003, a dramatic increase in the number of PNSP isolates was observed. Molecular characterization of these isolates showed that a single clone of sequence type ST156 increased from 40% to 80% of all serotype 14, thus causing the serotype expansion. Additionally, during the same time period, we examined the clonal composition of two serotypes 9V and 19F: all 9V and 20% of 19F isolates belonged to the clonal cluster of ST156, and overall approximately 50% of all PNSP belonged to the ST156 clonal cluster. Moreover, microarray and PCR analysis showed that all ST156 isolates, irrespective of capsular type, carried the rlrA pilus islet. This islet was also found to be present in the penicillin-sensitive ST162 clone, which is believed to be the drug-susceptible ancestor of ST156. Competitive experiments between related ST156 serotype 19F strains confirmed that those containing the rlrA pilus islet were more successful in an animal model of carriage. We conclude that the pilus island is an important biological factor common to ST156 isolates and other successful PNSP clones. In Sweden, a country where the low antibiotic usage does not explain the spread of resistant strains, at least 70% of all PNSP isolates collected during year 2003 carried the pilus islet.

Binding of human C4BP to the hypervariable region of M protein: a molecular mechanism of phagocytosis resistance in Streptococcus pyogenes.

The amino-terminal hypervariable region (HVR) of streptococcal M protein is required for the ability of this virulence factor to confer phagocytosis resistance. The function of the HVR has remained unknown, but the finding that many HVRs with extremely divergent sequences bind the human complement regulator C4b-binding protein (C4BP) has suggested that this ligand may play a role in phagocytosis resistance. We used the M22 system to study the function of bound C4BP and provide several lines of evidence that C4BP indeed contributes to phagocytosis resistance. First, the ability of anti-HVR antibodies to cause opsonization correlated with their ability to inhibit binding of C4BP. Secondly, a short deletion in the HVR eliminated C4BP binding and also reduced the ability of M22 to confer phagocytosis resistance. Thirdly, the addition of an excess of pure C4BP to a phagocytosis system almost completely blocked the effect of opsonizing anti-HVR antibodies. Together, our data indicate that binding of C4BP to the HVR of M22 plays an important role in phagocytosis resistance, but other properties of M22 also contribute. This study provides the first molecular insight into the mechanisms by which the HVR of an M protein confers phagocytosis resistance.

Isolated hypervariable regions derived from streptococcal M proteins specifically bind human C4b-binding protein: implications for antigenic variation.

Antigenic variation in microbial surface proteins represents an apparent paradox, because the variable region must retain an important function, while exhibiting extensive immunological variability. We studied this problem for a group of streptococcal M proteins in which the approximately 50-residue hypervariable regions (HVRs) show essentially no residue identity but nevertheless bind the same ligand, the human complement regulator C4b-binding protein (C4BP). Synthetic peptides derived from different HVRs were found to retain the ability to bind C4BP, implying that the HVR corresponds to a distinct ligand-binding domain that can be studied in isolated form. This finding allowed direct characterization of the ligand-binding properties of isolated HVRs and permitted comparisons between different HVRs in the absence of conserved parts of the M proteins. Affinity chromatography of human serum on immobilized peptides showed that they bound C4BP with high specificity and inhibition experiments indicated that different peptides bound to the same site in C4BP. Different C4BP-binding peptides did not exhibit any immunological cross-reactivity, but structural analysis suggested that they have similar folds. These data show that the HVR of streptococcal M protein can exhibit extreme variability in sequence and immunological properties while retaining a highly specific ligand-binding function.

Decreased amounts of cell wall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sarA mutants due to up-regulation of extracellular proteases.

Data have been presented indicating that Staphylococcus aureus cell surface protein can be degraded by extracellular proteases produced by the same bacterium. We have found that in sarA mutant cells, which produce high amounts of four major extracellular proteases (staphylococcal serine protease [V8 protease] [SspA], cysteine protease [SspB], aureolysin [metalloprotease] [Aur], and staphopain [Scp]), the levels of cell-bound fibronectin-binding proteins (FnBPs) and protein A were very low compared to those of wild-type cells, in spite of unaltered or increased transcription of the corresponding genes. Cultivation of sarA mutant cells in the presence of the global protease inhibitor alpha(2)-macroglobulin resulted in a 16-fold increase in cell-bound FnBPs, indicating that extracellular proteases were responsible for the decreased amounts of FnBPs in sarA mutant cells. The protease inhibitor E64 had no effect on the level of FnBPs, indicating that cysteine proteases were not involved. Inactivation of either ssp or aur in the prototype S. aureus strain 8325-4 resulted in a threefold increase in the amount of cell-bound FnBPs. Inactivation of the same protease genes in a sarA mutant of 8325-4 resulted in a 10- to 20-fold increase in cell-bound protein A. As the serine protease requires aureolysin to be activated, it can thus be concluded that the serine protease is the most important protease in the release of cell-bound FnBPs and protein A.

Regulation of agr-dependent virulence genes in Staphylococcus aureus by RNAIII from coagulase-negative staphylococci.

Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in Staphylococcus aureus are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, including hla (alpha-toxin), saeB (enterotoxin B), tst (toxic shock syndrome toxin 1), and ssp (serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like spa (protein A) and fnb (fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e., Staphylococcus epidermidis, Staphylococcus simulans, and Staphylococcus warneri. RNAIII from these CoNS turned out to be very similar to that of S. aureus and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5' half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficient S. aureus mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of S. aureus RNAIII, suggesting that these regulatory functions are independent. By creating S. epidermidis-S. aureus RNAIII hybrids, we could also show that both the 5' and 3' halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in S. aureus.

Detection of the response regulator AgrA in the cytosolic fraction of Staphylococcus aureus by monoclonal antibodies.

The expression of many virulence genes in Staphylococcus aureus is controlled by a regulatory RNA molecule, RNAIII, which is encoded by the agr locus. Transcription RNAIII requires the activity of the agrA, B, C and D genes, which code for components of a quorum sensing signal transduction system. In this report we describe the overexpression and purification of the response regulator, AgrA. Monoclonal antibodies were produced and used to detect AgrA in the cytosolic fraction of S. aureus cells. Purified AgrA did not bind to the RNAIII promoter region in a DNA mobility shift experiment. This confirms previous results obtained with protein extracts from agr+ and agr- cells.

Transcriptional control of the agr-dependent virulence gene regulator, RNAIII, in Staphylococcus aureus.

Many of the genes coding for extracellular toxins, enzymes and cell-surface proteins in Staphylococcus aureus are regulated by a 510 nt RNA molecule, RNAIII. Expression of the RNAIII gene is positively controlled by the closely linked agr operon, which encodes a multicomponent signal-transduction system, and by an unlinked operon called sar. We have analysed the 120 bp region that separates the RNAIII promoter, P3, from the divergent agr promoter, P2. By transcription analysis, it was shown that P3 can function in trans of the agr operon. A stretch of 53 bp upstream of P3, containing an interrupted repeat of 7 bp, was found to be required for the agr-dependent expression of RNAIII. A single cytoplasmic protein was shown to bind to at least two sites within this regulatory region. The protein, which was absent in extracts from a sarA mutant, was identified as the sarA product by N-terminal amino acid sequencing. A DNA fragment from the P2 region, encompassing an almost identical repeated DNA motif, competed for the same protein. No interaction between the regulatory DNA sequence and any agr-dependent products could be demonstrated. The results of this study suggest that P3 and P2 are controlled by a mechanism involving the binding of the SarA protein to multiple sites within the regulatory regions immediately upstream of each promoter, and the as yet unknown activity of AgrA.

Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII.

The synthesis of virulence factors in Staphylococcus aureus is controlled by a regulatory RNA molecule, RNAIII, encoded by the agr locus. Transcription of genes coding for secreted toxins and enzymes is stimulated, while transcription of cell-surface protein genes is repressed by RNAIII. In the case of staphylococcal alpha-toxin, RNAIII also seems to stimulate translation by an independent mechanism. In this report we show that in a mutant lacking RNAIII the rate of alpha-toxin (hla) production relative to the cellular concentration of hla mRNA was reduced 10-fold as compared with the wild-type strain. A 75% complementarity between the 5' end of RNAIII and the 5' untranslated region of the hla transcript suggests a direct interaction between the RNAs. A complex of RNAIII and hla mRNA was demonstrated in extracts of total RNA from the wild-type strain, and also with in vitro synthesized RNAs. Ribonuclease T1 digestion experiments revealed that the ribosome binding site of the hla transcript is blocked by intramolecular base-pairing. Hybridization with RNAIII prevents this intramolecular base-pairing and makes the hla mRNA accessible for translation initiation. This is, to our knowledge, the first example of an 'antisense RNA' that stimulates translation of the target mRNA.

DNA hybridization for diagnosis of pertussis.

The aim of the present study was to evaluate a mixed phase DNA hybridization assay for detection of Bordetella pertussis and Bordetella parapertussis in nasopharyngeal aspirates from patients with suspected pertussis. Among 179 consecutive patients with own or parental suspicion of pertussis, the diagnosis was confirmed in 103 patients by serology and in 52/103 (50%) cases also by culture. The remaining 76 patients served as nonpertussis controls. Direct hybridization was positive in 38% samples with serology as reference method, a non-significant difference to the 50% sensitivity for culture. Preculture of samples on membranes for 24, 48 and 72 h gave a significantly higher sensitivity only with 72 h preculture, 69% vs 50% (P = 0.007). The 72h preculture gave, however, also a significant decrease of specificity, 87% vs 100% for routine culture (P = 0.001) and is not a more rapid diagnostic method. The result shows that rapid diagnosis by DNA hybridization can be achieved in a large proportion of pertussis cases. The presence of smaller numbers of bacteria in samples only positive after preculture indicate that DNA hybridization could be a highly sensitive diagnostic method with further development of more rapid amplification systems.

A DNA hybridization test for detection of Bordetella in nasopharyngeal specimens.

A cloned Bam H1 fragment of genomic Bordetella pertussis DNA which recognized a frequently repeated sequence in the genome of B. pertussis was used as a probe in a DNA hybridization assay for the detection of Bordetella. Extensive studies on cross-reactivity were carried out in standardized strains and in cultured swab specimens from patients without suspected pertussis. Hybridizations of cultured clinical specimens from 142 patients with suspected pertussis were in complete agreement with the standard identification methods. The recovery rate of B. pertussis from nasopharyngeal swabs was less than 50%. Therefore the possibility to detect low numbers of B. pertussis in solution (nasopharyngeal aspirates) was investigated. The detection limit of direct hybridization by dot-blot technique was 5 x 10(3)-10(4) B. pertussis. Culturing bacteria on membranes placed on agar plates prior to hybridization showed that the detection limit could be lowered to 10(4), 10(2), and 10(1) cfu after 1, 2 and 3 days' culture, respectively. DNA hybridization under these conditions was found to be sufficiently sensitive and specific for further evaluation in clinical specimens for diagnosis of pertussis.

Cloning of a chromosomal locus (exp) which regulates the expression of several exoprotein genes in Staphylococcus aureus.

Insertion of the erythromycin resistance transposon Tn551 into a single site of the Staphylococcus aureus chromosome resulted in decreased production of alpha-toxin, serine and metallo-proteinases and several other extracellular proteins and a simultaneous increase in the production of protein A. The site of insertion, designated exp, was separate from the structural gene for alpha-toxin and protein A. Hybridization analysis showed that the effect of the insertional mutation on the expression of the alpha-toxin and protein A was at the level of transcription. The chromosomal DNA flanking the transposon and the corresponding DNA of the wild-type strain was cloned in Escherichia coli. Northern blot hybridization experiments revealed that the exp locus codes for a major RNA of approximately 3.5 kb. This RNA was not found in the insertional mutant nor in a spontaneous exp mutant. A map of the exp locus constructed by Northern blot and restriction enzyme analysis showed that the insertional mutation was located in the middle of the coding sequence of the 3.5 kb RNA. The insertional mutant was reverted to wild type by inserting a recombinant plasmid containing most of the coding sequence of the 3.5 kb RNA.