- Volume 152, Issue 2, 2006

In past years, several useful genetic tools have been developed to study the molecular biology of Streptococcus pneumoniae. In order to extend the existing spectrum of tools, advantage was taken of the toolbox originally developed for the closely related bacterium Lactococcus lactis, which was adapted for the manipulation of S. pneumoniae. The modified tools are as follows. (i) An improved nisin-inducible (over)expression system (NICE). The nisRK genes, encoding a two-component system essential for transcriptional activation in response to nisin, were integrated into the bgaA locus of S. pneumoniae D39. In this strain, D39nisRK, addition of nisin resulted in the overexpression of several genes placed under the control of the nisin-inducible promoter, while no detectable expression was observed in the absence of nisin. (ii) A lacZ reporter system. Using strain D39nisRK, which lacks endogenous β-galactosidase activity, the usefulness of the lacZ reporter vector pORI13 for the generation of chromosomal transcriptional fusions was demonstrated. In addition, the repA gene, necessary for the replication of pORI13, was introduced into the bgaA locus, thereby generating a background for plasmid-based promoter expression studies. (iii) A simplified chemically defined medium, which supports growth of all sequenced S. pneumoniae strains to a level comparable to that in complex medium. (iv) A system for the introduction of unmarked deletions and mutations into the chromosome, which is independent of the genotype of the target strain. Most of these systems were successfully applied in strains R6 and TIGR4 as well. In addition, the tools offer several improvements and advantages compared to existing ones. Thus, the molecular toolbox for S. pneumoniae has been successfully extended.

Sequence types of pneumococci isolated in Scotland between 1996 and 2003 were compared with those of globally prevalent antibiotic-resistant clones. Multilocus sequence typing was performed on 252 invasive pneumococcal isolates referred to the Scottish Meningococcus and Pneumococcus Reference Laboratory. Isolates were not preselected for antimicrobial resistance, patient age or disease caused. Sequence types were compared with globally significant antimicrobial-resistant clones identified by the Pneumococcal Molecular Epidemiology Network (PMEN). Sequence types identical with three of the 26 PMEN clones were present in the Scottish collection; the clones were the Spain9V-3 clone (sequence type 156, seven isolates), the England14-9 clone (sequence type 9, eight isolates) and the Utah35B-24 clone (sequence type 377, one isolate). Many Scottish isolates related to PMEN clones had lower antimicrobial MICs than those described for the corresponding PMEN type strain. A number of single- (SLVs) and double-locus variants (DLVs) were present. Fifteen SLVs related to PMEN sequence types 37, 67, 90, 81, 156, 236 and 377 were detected. The collection contained 10 DLVs related to PMEN sequence types 37, 156, 173 and 338. The majority of SLVs and DLVs were penicillin- or erythromycin-sensitive variants of the resistant PMEN type strains. Capsule switching in isolates related to the PMEN clones was also detected. The highest levels of penicillin resistance were detected in sequence type 320 (serotype 19F), which is not a PMEN clone. These data suggest that PMEN clones are not widely distributed in disease-causing isolates in Scotland.

The authors aimed to get insights into the population structure of non-(sero)typable pneumococci (NTPn), a specific group of natural atypical pneumococci whose identification is often difficult, and which has remained insufficiently studied. A total of 265 presumptive NTPn, isolated between 1997 and 2003 from the nasopharynx of children, were characterized. Strains were confirmed to be pneumococci on the basis of bile solubility, and PCR detection or Southern blotting hybridization of lytA and psaA, genes ubiquitous in this species. Multilocus sequence typing (MLST) was used to exclude two isolates that gave ambiguous results. Non-typability was confirmed by the Quellung reaction using Omniserum. A total of 213 isolates were considered to be true NTPn. The molecular analysis of the true NTPn by PFGE and MLST showed that this population was genetically diverse, although a dominant cluster, accounting for 66 % of the isolates, was identified. Antimicrobial resistance was observed in most genetic backgrounds, and multidrug resistance to penicillin, erythromycin, clindamycin, tetracycline and sulfamethoxazole-trimethoprim was associated with strains belonging to the dominant cluster. Comparison with PFGE fingerprints and sequence types of large collections of serotypable strains showed that the genetic backgrounds of all but two NTPn were different from those found in serotypable strains. In addition, we found that NTPn strains with similar genetic backgrounds to those identified in our study had been isolated from disease sources in other countries. These observations seem to indicate that NTPn have diverse genetic backgrounds and may have evolved as a distinct group of pneumococcal isolates.

A recent study of pneumococcal colonization in 3198 healthy children of 1–19 years of age in The Netherlands showed pneumococcal colonization in 19 % of the children, with a peak incidence of 55 % at the age of 2 years; an age-related serotype distribution was also found. In the present study, the genetic background and resistance profiles of 578 pneumococcal isolates from the latter study were characterized by means of chromosomal genotyping and susceptibility testing. In contrast to the age-related serotype distribution observed previously, the genetic background of the strains was not age related. Few strains were found showing close homology (>95 %) with the international clones Spain9V-3 (ten isolates showed homology), England14-9 (four isolates), Tennessee23F-4 (two isolates), CSR14-10 (one isolate) and Sweden15A-25 (one isolate). In total, 19 % of strains showed resistance to one or more antibiotics. Resistance to cotrimoxazole, tetracycline, erythromycin and penicillin was found in 12·9, 5·6, 5·0 and 2·7 % of isolates, respectively. Multidrug resistance was found in 1·9 % of strains. In conclusion, pneumococcal colonization isolates from healthy Dutch children represent a heterogeneous, mostly antibiotic susceptible, genetic population.

Malassezia furfur is a dimorphic fungus and a member of the normal cutaneous microflora of humans. However, it is also a facultative pathogen, associated with a wide range of skin diseases. One unusual feature of M. furfur is an absolute dependency on externally provided lipids which the fungus hydrolyses by lipolytic activity to release fatty acids necessary for both growth and pathogenicity. In this study, the cloning and characterization of the first gene encoding a secreted lipase of M. furfur possibly associated with this activity are reported. The gene, MfLIP1, shows high sequence similarity to other known extracellular lipases, but is not a member of a lipase gene family in M. furfur. MfLIP1 consists of 1464 bp, encoding a protein with a molecular mass of 54·3 kDa, a conserved lipase motif and an N-terminal signal peptide of 26 aa. By using a genomic library, two other genes were identified flanking MfLIP1, one of them encoding a putative secreted catalase, the other a putative amine oxidase. The cDNA of MfLIP1 was expressed in Pichia pastoris and the biochemical properties of the recombinant lipase were analysed. MfLip1 is most active at 40 °C and the pH optimum was found to be 5·8. The lipase hydrolysed lipids, such as Tweens, frequently used as the source of fatty acids in M. furfur media, and had minor esterase activity. Furthermore, the lipase is inhibited by different bivalent metal ions. This is the first molecular description of a secreted lipase from M. furfur.

The means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to kill K. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-κB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor.

Although Campylobacter jejuni is a leading cause of food-borne illness, little is known about the mechanisms by which this pathogen mediates prolonged environmental survival or host cell virulence. Although these behaviours represent distinct phenotypes, they share a common requirement of an immobilized state. In order to understand the cellular mechanisms that facilitate a surface-associated lifestyle, transcriptional and translational expression profiles were determined for sessile and planktonic C. jejuni. These investigations indicate that the immobilized bacteria undergo a shift in cellular priorities away from metabolic, motility and protein synthesis capabilities towards emphasis on iron uptake, oxidative stress defence and membrane transport. This pattern of expression partially overlaps those reported for Campylobacter during host colonization, as well as for other species of bacteria involved in biofilms, highlighting common adaptive responses to the conserved challenges within each of these phenotypes. The adaptation of Campylobacter to immobilized growth may represent a quasi-differentiated state that functions as a foundation for further specialization towards phenotypes such as biofilm formation or host cell virulence.