- Volume 53, Issue 8, 2004

Exposure of the pulmonary pathogen Aspergillus fumigatus to amphotericin B alters membrane permeability as indicated by the escape of amino acids and protein from the mycelium. Amphotericin B exposure for periods of 2–4 h also leads to increased release of the immunosuppressive agent gliotoxin into the surrounding culture medium. Examination of the intracellular gliotoxin concentration following exposure to amphotericin B indicated elevated levels within the hyphae as well as in the culture medium – an effect which was also evident upon exposure of A. fumigatus to DMSO. These results indicate that in parallel with the ability of amphotericin B to act as a fungistatic agent it can also induce the synthesis of gliotoxin and facilitate its release by increasing the permeability of the fungal cell membrane. Increased synthesis of gliotoxin may result from the commencement of secondary metabolism in the presence of amphotericin B. The ability of amphotericin B to enhance the synthesis and release of gliotoxin may exacerbate the effects of the toxin and facilitate fungal invasion of pulmonary tissue.

Recognition of bacterial lipopolysaccharide (LPS) is critical in the host defence against Gram-negative infection. While enterobacterial LPS signals via Toll-like receptor 4 (TLR4), it has recently been reported that the LPS of Leptospira interrogans, Legionella pneumophila, Rhizobium species Sin-1 and at least one strain of Porphyromonas gingivalis are capable of signalling via TLR2. Using a TLR transfection assay and measurement of an NF-κB-sensitive promoter region, the results show that the LPS of Bacteroides fragilis NCTC-9343, Chlamydia trachomatis LGV-1 and Pseudomonas aeruginosa PAC-611 also signal via TLR2 and it is pointed out that all TLR2-signalling LPS discovered to date demonstrate relatively weak endotoxicity in some models and structural features distinct from those LPS shown to signal via TLR4.

To study the influence of normal associated microbiota on systemic immunological responses during experimental Chagas’ disease, germ-free and conventional NIH Swiss mice were infected with Y strain of Trypanosoma cruzi. Although no statistical differences in mortality and parasitaemia were found, conventional mice showed IFN-γ, TNF-α and NO production (P < 0.05) by spleen cell cultures and higher blood levels of immunoglobulins of the IgG2a isotype (P < 0.05) when compared to their germ-free counterparts. Moreover, higher levels of IgG1 were also found in conventional animals. On the other hand, no differences in IL10 production were found between germ-free and conventional mice after infection (P < 0.05). Interestingly, spleen cell cultures from non-infected germ-free mice spontaneously produced higher levels of IL10 than cultures from conventional mice. Moreover, cultures from non-infected germ-free mice responded to T. cruzi antigens with IFN-γ production, contrary to cultures from conventional animals. In conclusion, the presence of the normal microbiota skews the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi in mice. However, the increase in production of cytokines that is linked to resistance to this parasite did not alter the outcome of infection significantly, probably due to high virulence of the Y strain.

Methylobacterium species are environmental opportunistic bacteria, and urinary tract infection (UTI) caused by these pathogens has not yet been documented. Four cases of UTI with Methylobacterium bacteraemia in immunocompetent female patients are reported. Their urine cultures, processed according to standard procedures (i.e. incubation at 35 °C in ambient air for 24 h before incubation at room temperature for a further 24 h), were either negative or positive for Escherichia coli. Specially designed experiments indicated that colonies of Methylobacterium species were visualized on blood agar only after incubation at 35 °C for at least 40 h, and growth was completely suppressed when concurrently incubated with much smaller inocula of E. coli. The isolates were variably susceptible to cephalosporins, but 100 % susceptible to aminoglycosides. This study suggests an underdiagnosis of UTI caused by Methylobacterium species when the standard procedure of processing urine cultures is used, and implies that administration of aminoglycosides is important when treatment of UTIs with cephalosporin fails.

Pyrazinamide (PZA) is an unconventional front line tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. This disparity in PZA activity may reflect differences between the in vivo tissue environment and in vitro culture conditions. This study examined the effect of anaerobic conditions, which exist in granulomatous lesions in vivo, on PZA activity in vitro. Low oxygen enhanced the activity of PZA against Mycobacterium tuberculosis, with anaerobic conditions resulting in greater enhancement than microaerobic conditions. ATPase and respiratory chain enzyme inhibitors enhanced PZA activity under normal atmospheric conditions, but not under anaerobic conditions. Furthermore, the inhibitors did not enhance isoniazid or rifampicin activity. Nitrate as an alternative electron acceptor antagonized PZA activity under anaerobic conditions. These findings provide further support for a proposed mechanism of action of PZA in which the active form of PZA (pyrazinoic acid) depletes the membrane energy reserve. They also provide another explanation for the higher sterilizing activity of PZA within in vivo lesions with low oxygen than under in vitro drug susceptibility testing conditions with ambient oxygen.

The progressive increase of invasive disease and reports of resistance among Aspergillus species emphasizes the need for reproducible antifungal susceptibility testing. Inoculum standardization is a crucial step in such procedures. The objective of this study was to develop a fast and precise method of evaluating the cell density of an Aspergillus spore suspension, as an alternative to spectrometric readings or cell-counting with a haemocytometer. Densimat (bioMérieux) is a portable photometer that shows a good correlation with spectrometric readings and can advantageously replace the cumbersome, time-consuming method of cell-counting. Thus, Densimat brings significant improvement to the reproducibility and feasibility of standardization of a fungal inoculum.

The role of Chlamydophila pneumoniae in the development and exacerbation of juvenile idiopathic arthritis (JIA) was investigated. Blood samples were taken from 60 JIA patients during an active disease period and for 4 weeks after. Synovial fluid samples were obtained from 20 of the 60 patients. In addition, 22 patients with familial Mediterranean fever (FMF) during the active period and 35 healthy children were included in the study as control groups. Synovial fluid samples were also obtained from three children with FMF. IgG, IgM and IgA levels against C. pneumoniae in serum samples were studied by immunofluorescence and IgG antibody and PCR studies were performed for C. pneumoniae DNA in synovial fluid samples. Twenty-nine (48.3 %) patients with JIA, 18 (81.8 %) patients with FMF and 22 (62.8 %) healthy children were found to be pre-infected with C. pneumoniae. Pre-infection with C. pneumoniae among FMF patients was found to be significantly higher than among those with JIA. We did not find a significant difference between JIA patients and healthy children. Chronic C. pneumoniae infection was observed only in six JIA patients, one FMF patient and two healthy children. Synovial fluid antibodies were found at higher than 1/512-fold dilution in one JIA patient and four times higher than normal serum in three JIA patients. C. pneumoniae DNA was not detected in any synovial fluid sample from FMF or JIA patients by PCR. In conclusion, C. pneumoniae infection does not have a triggering or a progressive effect on the clinical situation in JIA aetiopathogenesis, as a result of a multifactorial aetiology. New, extensive and serial studies (especially PCR studies of synovial tissue) are needed in order to confirm the indirect results.

Increasing numbers of studies have documented the widespread distribution of human G9 rotaviruses and demonstrated that these strains may represent a fifth epidemiologically important G serotype. Serotype G9 strains were identified in Hungary for the first time in the 1997–1998 rotavirus season. Contrary to numerous surveys that reported several unexpected P–G combinations among recent G9 isolates (e.g. genotypes P[4], P[6] and P[19]), all Hungarian strains characterized to date possess the globally most common P-type, P[8], which was found among the first G9 isolates that were identified during the 1980s in the USA (WI61) and Japan (F45). To study the genetic variability within Hungarian G9 strains, RNA profile analysis and nucleotide sequencing were performed on a subset of samples that were collected between 1998 and 2001. These strains could be classified into four major RNA profiles, of which two were characteristic for epidemiologically major and two for epidemiologically minor G9 strains. Phylogenetic analysis demonstrated substantial sequence differences between the VP7 gene of Hungarian G9 strains and early strains that were isolated in the USA (WI61), Japan (F45) and India (116E) and a few recently identified isolates, e.g. from China (97'SZ37) and the USA (OM67) (<90 % nucleotide sequence similarity). In contrast, the VP7 genes of Hungarian G9 strains were related very closely to the vast majority of G9 strains that were isolated in a variety of countries over the last several years (>96 % nucleotide sequence similarity). With respect to the VP4 gene, Hungarian G9 rotaviruses fell into two of the major genetic lineages of genotype P[8], one corresponding to the epidemic strains (lineage II; P-like) and the other for two unique strains (lineage I; Wa-like), suggesting independent introduction of distinct P[8],G9 strains into Hungary or genetic reassortment between locally circulating P[8] strains and descendants of G9 isolates that were imported into the country at an earlier time. The unexpected heterogeneity found for G9 VP7 genes from several countries suggests that genetic variation among these strains has not yet been fully explored.

The Mycobacterium avium complex (MAC) is associated with various diseases in humans as a zoonosis. The dnaJ gene was partially sequenced in Schaefer's 28 reference strains of MAC, 14 human MAC isolates and 22 veterinary isolates. From substitutions affecting 21–32 nucleotides, all strains could be classified into 14 groups. Most nucleotide substitutions did not alter amino acid sequences. Approximately 8 % genetic diversity was seen in these strains, which divided into two clusters: cluster I (0.8 % genetic diversity), comprising the reference strain serotypes 1–6, 8–11 and 21 and all isolates; and cluster II (7 % genetic diversity), comprising the remaining reference strains. Analysis of the dnaJ gene in MAC may be useful in epidemiological studies.