- Volume 53, Issue 1, 2004

The fate of GAS and epithelial cells following internalization was determined in this study. HEp-2 cells harbouring intracellular bacteria were treated with antibiotics to kill extracellular adherent bacteria, washed, and the fate of bacteria and epithelial cells was assessed up to 24 h post-infection. In the absence of antibiotics, massive bacterial growth was apparent in the cell medium, accompanied by extensive cell death, suggesting that intracellular bacteria had multiplied and damaged the monolayer. Addition of the internalization inhibitor, cytochalasin D, either pre- or post-internalization prevented bacterial growth and cell injury; post-internalization treatment with chloramphenicol had the same effect. Analysis of three apoptotic markers in HEp-2 cells – chromatin condensation, DNA laddering and translocation of phosphatidylserine onto the cell-surface membrane – indicated that HEp-2 cells underwent apoptosis. Taken together, the data presented here support a model in which intenalized bacteria can induce their own externalization into the medium by a process that requires both an intact host-cell cytoskeleton and de novo synthesis of bacterial proteins. Concomitantly, intracellular and, apparently, extracellular free bacteria induce apoptosis through their cytotoxic activity, and release essential nutrients required for their growth.

We have reported previously that heat-shock protein 60 kDa (hsp60) of Helicobacter pylori is an important antigen in the pathogenesis of gastric mucosa-associated lymphoid tissue (MALT) lymphoma. In order to investigate associations with host immune reactions and hsp60 antigen, CD40 ligand (CD40L) expression and cytokine production were analysed following stimulation with hsp60. To provide a clear antigen-driven immune response, peripheral blood mononuclear cells (PBMC) from patients with low-grade MALT lymphoma and gastritis and those from healthy volunteers were stimulated with recombinant H. pylori hsp60 and H. pylori cell lysate in the presence of cytokines (IL4 and granulocyte-macrophage colony-stimulating factor). mRNA expression was also analysed by a cDNA microarray containing 1100 genes. Expression of CD40L on PBMCs of patients with MALT lymphoma was increased by cytokines or by combination with stimulation with hsp60 antigens. The production of IL4 in PBMC cultures was increased in patients with MALT lymphoma; however, production of IFN-γ was at low levels. DNA microarray analysis indicated increased levels of HLA-DR and integrin mRNAs. In cases of low-grade MALT lymphoma, adaptive immune responses against hsp60 may be enhanced by host factors, such as antigen presentation and T-cell activation, resulting in B-cell proliferation, which can be demonstrated during chronic H. pylori infection.

A unified, bioinformatics-driven, single nucleotide polymorphism (SNP)-based approach to microbial genotyping has been developed. Multilocus sequence typing (MLST) databases consist of known variants of standardized housekeeping genes. Normally, seven fragments are defined; a sequence type (ST) consists of the variants of these fragments that are found in a particular isolate. A computer program that can identify highly informative sets of SNPs in entire MLST databases has been constructed. The SNPs either define a particular user-specified ST or provide a high value for Simpson's index of diversity (D), and may thus be generally applicable to that species. SNP sets that are diagnostic for Neisseria meningitidis ST-11 and ST-42, and high-D SNP sets for N. meningitidis and Staphylococcus aureus, were identified and real-time PCR methods to interrogate these SNPs were demonstrated. High-D SNP sets were also identified in other MLST databases. This widely applicable approach allows rapid genetic fingerprinting of infectious agents.

The antibacterial activity of the nitrogen heterocyclic sponge constituent cribrostatin 6 was examined. Cribrostatin 6 was bacteriostatic for a variety of Gram-positive species and was bactericidal for the majority of clinical isolates of Streptococcus pneumoniae, including penicillin-resistant strains. Minimum bactericidal concentration/MIC ratios were ⩽2 for 75 % of S. pneumoniae clinical isolates. Kill-curve analysis confirmed the bactericidal action of cribrostatin 6. Bactericidal activity was rather slow, beginning at 2, 4 or 8 h, depending on the strain. The frequency of occurrence of bacterial spontaneous mutations to resistance was ⩽10−7. The maximum tolerated dose of cribrostatin 6 in mice was 750–1000 μg kg−1 day−1. Cribrostatin 6 is a promising lead antibiotic for Gram-positive bacteria, particularly S. pneumoniae, a leading cause of infection and mortality worldwide.

Coagulase-negative staphylococci (CNS) are frequently isolated from blood cultures, where they may be only a contaminant or the cause of bacteraemia. Determining whether an isolate of CNS represents a true CNS bacteraemia is difficult, and there is no single criterion with sufficient specificity. The aim of this study was to assess those clinical, microbiological, pathogenic and genotypic features that characterize true CNS bacteraemia. Twenty patients having two or more blood cultures positive for CNS and 20 patients with only one positive blood culture were studied. Significant bacteraemia was defined according to clinical and laboratory criteria. Incubation time for blood cultures to become positive, macroscopic appearance of colonies, species determination, biotype, susceptibility to antimicrobials, PFGE pattern and adherence capacity were all studied. Clinical bacteraemia was present in 16/20 patients with two or more positive blood cultures and in 2/20 patients with only one positive blood culture. A significant difference was seen in the median time to positivity between the 18 clinical bacteraemias and 22 contaminations (23.6 versus 29.2 h; P = 0.04, Wilcoxon). There was also a significant difference between the two groups in the median absorbance of the slime test (1.36 versus 0.58; P = 0.005). All significant bacteraemias with two or more positive blood cultures had the same species identified, the same antimicrobial susceptibility pattern and the same PFGE pattern. In two patients with true bacteraemia with only one positive blood culture, the incubation time for the culture to turn positive was <24 h and the slime production absorbance was >2.5. The most useful parameters for the diagnosis of true CNS bacteraemia for patients with two positive blood cultures were incubation time until positive, species identification, antimicrobial susceptibility pattern, slime production and PFGE pattern. For patients with only one blood culture positive for CNS, the useful parameters for prediction of true bacteraemia were incubation time until positive and slime production, both of which are simple, low-cost tests.

In order to improve our understanding of the colonization of the pulmonary tract of cystic fibrosis (CF) patients by Pseudomonas aeruginosa, 162 isolates from five different ecological origins were studied. The genetic features of each isolate were determined by random amplification of polymorphic DNA (RAPD) and by searching for eight virulence genes (six known virulence genes, algD, lasB, toxA, plcH, plcN and exoS, and two genes encoding putative neuraminidases, nan1 and nan2). Five RAPD groups were identified. Most of the CF isolates were distributed equally in three of these groups (RA, RB and RC). The CF isolates in RB were related to isolates from a wide variety of origins. The CF isolates in RA were related to a population composed of 65 % of the non-CF isolates from pulmonary tract infections. RC was mainly composed of CF isolates that were related to 30 % of isolates from plants. All genes except exoS and nan1 were present in all isolates. The exoS and nan1 virulence factor genes were most prevalent in CF isolates. exoS, which encodes exoenzyme S, was present in 94 % of CF isolates but also in 80 % of non-CF isolates from pulmonary tract infections. nan1, which encodes a putative neuraminidase, was found in 82.5 % of the isolates from group RC, which was composed largely of CF isolates. In conclusion, three major genogroups of P. aeruginosa isolates, each of which exhibits peculiar genetic features, are able to colonize CF patients. This may have different consequences on the outcome of pulmonary disease.

Two cases of severe community-acquired pneumococcal pneumonia (CAP) admitted to our hospital within 24 h are described, both in young males. These two cases illustrate the usefulness of the British Thoracic Society severity criteria and serve to emphasize the importance of early recognition of adverse physiology in critically ill patients. We should not lose sight of the continued impact of pneumonia in this climate of widespread fear about severe acute respiratory syndrome (SARS).