European Antibiotic Awareness Day: a five-year perspective of Europe-wide actions to promote prudent use of antibiotics.

Following the European Union (EU) Council Recommendation on prudent use of antimicrobial agents in human medicine in 2001, and the success of national campaigns, i.e. Belgium and France, the European Centre for Disease Prevention and Control (ECDC) decided to establish the European Antibiotic Awareness Day (EAAD) on 18 November as platform to support national campaigns across Europe. This article provides an overview of EAAD tools, materials, and activities developed during the first five years. It shows that EAAD has been successful due to good cooperation between ECDC and national institutions, strong political and stakeholder support and evidence-based development of campaign materials. EAAD has provided a platform for pre-existing national campaigns and encouraged similar campaigns to develop where neither political support had been secured, nor financial support had been available. As a result, participating countries have continuously expressed strong support for ECDC to continue its work on EAAD. This has been endorsed by a steadily increasing number of countries participating and the growing interest of varied professional and stakeholder organisations. We conclude that EAAD should continue to act as catalyst for discussion and as mechanism to raise awareness of the public and prescribers about prudent use of antibiotics.

Carbapenemase-producing Enterobacteriaceae in Europe: a survey among national experts from 39 countries, February 2013.

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a threat to healthcare delivery, although its extent differs substantially from country to country. In February 2013, national experts from 39 European countries were invited to self-assess the current epidemiological situation of CPE in their country. Information about national management of CPE was also reported. The results highlight the urgent need for a coordinated European effort on early diagnosis, active surveillance, and guidance on infection control measures.

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.

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.

Role of the innate immune system in host defence against bacterial infections: focus on the Toll-like receptors.

The innate immunity plays a critical role in host protection against pathogens and it relies amongst others on pattern recognition receptors such as the Toll-like receptors (TLRs) and the nucleotide-binding oligomerization domains proteins (NOD-like receptors, NLRs) to alert the immune system of the presence of invading bacteria. Since their recent discovery less than a decade ago, both TLRs and NLRs have been shown to be crucial in host protection against microbial infections but also in homeostasis of the colonizing microflora. They recognize specific microbial ligands and with the use of distinct adaptor molecules, they activate different signalling pathways that in turns trigger subsequent inflammatory and immune responses that allows a immediate response towards bacterial infections and the initiation of the long-lasting adaptive immunity. In this review, we will focus on the role of the TLRs against bacterial infections in humans in contrast to mice that have been used extensively in experimental models of infections and discuss their role in controlling normal flora or nonpathogenic bacteria. We also highlight how bacteria can evade recognition by TLRs.

A pneumococcal pilus influences virulence and host inflammatory responses.

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality world-wide. The initial event in invasive pneumococcal disease is the attachment of encapsulated pneumococci to epithelial cells in the upper respiratory tract. This work provides evidence that initial bacterial adhesion and subsequent ability to cause invasive disease is enhanced by pili, long organelles able to extend beyond the polysaccharide capsule, previously unknown to exist in pneumococci. These adhesive pili-like appendages are encoded by the pneumococcal rlrA islet, present in some, but not all, clinical isolates. Introduction of the rlrA islet into an encapsulated rlrA-negative isolate allowed pilus expression, enhanced adherence to lung epithelial cells, and provided a competitive advantage upon mixed intranasal challenge of mice. Furthermore, a pilus-expressing rlrA islet-positive clinical isolate was more virulent than a nonpiliated deletion mutant, and it out-competed the mutant in murine models of colonization, pneumonia, and bacteremia. Additionally, piliated pneumococci evoked a higher TNF response during systemic infection, compared with nonpiliated derivatives, suggesting that pneumococcal pili not only contribute to adherence and virulence but also stimulate the host inflammatory response.

Soluble pilin of Neisseria gonorrhoeae interacts with human target cells and tissue.

Pili of Neisseria gonorrhoeae are phase-variable surface structures that mediate adherence to host target cells. Each pilus is composed of thousands of major pilus subunits, pilins, pilus-associated protein PilC, and possibly other components. Piliated and nonpiliated gonococcal clones may secrete a soluble smaller pilin (S-pilin) that is cleaved after amino acid 39 of the mature pilin protein. Here, purified S-pilin was found to migrate as a 61- to 64-kDa double band on nondenaturing gels, suggesting the formation of tetrameric S-pilin proteins with two isomeric forms. In situ studies of binding to formalin-fixed tissue sections demonstrated the binding of S-pilin to human tissue but not to tissue from mouse or rat organs, showing the presence of a human-specific receptor-binding domain within the pilin polypeptide. Pretreatment of the target tissues with proteinase K decreased gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect gonococcal binding. In overlay assays, purified S-pilin bound to a band with a migration pattern and size similar to those of CD46, a cellular pilus receptor. Further, binding of N. gonorrhoeae to target cells and tissues could be blocked by both CD46 antibodies and purified S-pilin. These data argue that S-pilin interacts with a protein domain(s) of the CD46 receptor on human cells.

Attachment of Neisseria gonorrhoeae to the cellular pilus receptor CD46: identification of domains important for bacterial adherence.

Pili of Neisseria gonorrhoeae mediate binding of the bacteria to human host cells. Membrane cofactor protein (MCP or CD46), a human cell-surface protein involved in regulation of complement activation, acts as a cellular pilus receptor. In this work, we examined which domains of CD46 mediate bacterial adherence. The CD46 expression was quantified and characterized in human epithelial cell lines. N. gonorrhoeae showed the highest adherence to ME180 cells, which have BC1 as the dominant phenotype. The BC isoforms of CD46 were expressed in all cell lines tested. The adherence was not enhanced by high expression of other isoforms, showing that the BC domain of CD46 is important in adherence of N. gonorrhoeae to human cells. To characterize the pilus-binding site within the CD46 molecule, a set of CD46-BC1 deletion constructs were transfected into COS-7 cells. Piliated N. gonorrhoeae attached well to CD46-BC1-expressing COS-7 cells. We show that the complement control protein repeat 3 (CCP-3) and the serine-threonine-proline (STP)-rich domain of CD46 are important for efficient adherence to host cells. Further, partial deletion of the cytoplasmic tail of CD46 results in low bacterial binding, indicating that the cytoplasmic tail takes part in the process of establishing a stable interaction between N. gonorrhoeae and host cells.

pUB2380: characterization of a ColD-like resistance plasmid.

A detailed analysis of the mobilizable, ColE1-like resistance plasmid, pUB2380, is reported. The 8.5-kb genome encodes six (possibly seven) major functions: (1) a ColD-like origin of replication, oriV, with associated replication functions, RNAI and RNAII; (2) a set of active mobilization functions highly homologous to that of ColE1, including the origin of transfer, oriT; (3) a ColE1-like multimer resolution site (cer); (4) a kanamycin-resistance determinant, aph, encoding an aminoglycoside-3'-phosphotransferase type 1; (5) an insertion sequence, IS1294; and (6) two genes, probably cotranscribed, of unknown function(s). The GC content of the various parts of the genome indicates that the plasmid is a hybrid structure assembled from DNA from at least three different sources, of which the replication region, the mobilization functions, and the resistance gene are likely to have originated in the enterobacteriaceae.

IS1294, a DNA element that transposes by RC transposition.

IS1294, found on the ColD-like resistance plasmid pUB2380, is IS91-like. It is an active 1.7-kb insertion sequence that lacks terminal inverted repeats, displays insertion-site specificity, and does not generate direct repeats of the target site. The element has one large open reading frame, tnp(1294), encoding a transposase of 351 amino acids, related to members of the REP family of replication proteins used by RC-plasmids of gram-positive bacteria. IS1294 transposes using rolling-circle replication, initiated at one end of the element, oriIS, and terminated at the other, terIS. oriIS and terIS are highly conserved among like IS elements. oriIS resembles the leading strand replication origins of RC-plasmids; terIS resembles a rho-independent transcription terminator. IS1294 mediates not only its own transposition, but also sequences adjacent to terIS. A transposition model for IS1294 and related elements, involving rolling-circle replication and single-strand DNA intermediates, is presented.

Identification of the plasmid-mobilization potential of the strain Klebsiella pneumoniae ozenae KIIIA isolated from a polluted aquatic environment.

The Klebsiella pneumoniae ozenae KIIIA strain was isolated from the River Rhine soon after a serious mercury pollution episode and was selected for mercury resistance as well as for intergeneric DNA mobilization helper potential. This transfer helper capacity was shown to be related to the presence of a Tn3-like transposable element, Tn5403. Because transposon-mediated fusion was found to be involved in the mobilization potential of KIIIA, the visualization and the identification of the conjugative element, responsible for the transfer, were necessary. Our results show that, in addition to the four nonconjugative plasmids visualized in a previous study, K. pneumoniae ozenae KIIIA harbors two other plasmids, pK130 and pK45, of respective sizes of 130 and 45 kb, but none of these plasmids is involved in the mobilization mechanism. The presence of yet another extrachromosomal element pK225, with a size of 225 kb, was established by indirect methods, since yields of pK225 isolated from KIIIA were low and the plasmid was difficult to visualize directly. However, the integration of this plasmid into the chromosome was not detected. The present paper highlights the problem of detecting some plasmids in bacteria which have been isolated from the environment. For these plasmids, indirect approaches, that detect conjugative functions, constitute a feasible alternative for the investigation of the plasmid content of bacteria, if the direct approach fails. An analysis of the different types of transconjugants indicated that the mercury-resistance marker as well as the mobilization potentials, expressed by KIIIA, are linked to pK225. This plasmid could not be assigned to a described Inc group either by DNA hybridization or by PCR amplification.