- Volume 55, Issue 3, 2006

Natural resistance to infection, which does not depend on antibiotics, is a powerful protective mechanism common to all mankind that has been responsible for the survival of our species during countless millennia in the past. The normal functioning of this complex system of phagocytic cells and tissue fluids is entirely dependent on an extremely low level of free ionic iron (10−18 M) in tissue fluids. This low-iron environment is maintained by the unsaturated iron-binding proteins transferrin and lactoferrin, which depend on well-oxygenated tissues, where a relatively high oxidation–reduction potential (Eh) and pH are essential for the binding of ferric iron. Freely available iron is derived from iron overload, free haem compounds, or hypoxia in injured tissue leading to a fall in Eh and pH. This can severely damage or abolish normal bactericidal mechanisms in tissue fluids leading to overwhelming growth of bacteria or fungi. The challenge for clinical medicine is to reduce or eliminate the presence of freely available iron in clinical disease. In injured or hypoxic tissue, treatment with hyperbaric oxygen might prove very useful by increasing tissue oxygenation and restoring normal bactericidal mechanisms in tissue fluids, which would be of huge benefit to the patient.

An increased catalase activity in Candida spp. has been suggested as a mechanism that reduces amphotericin B activity. Furthermore, resistance to antifungal agents like amphotericin B has been reported in some cancer patients undergoing chemotherapy treatment. In this study we analysed the influence of chemotherapy agents on catalase activity in Candida albicans, the major species involved in yeast infections. Eight strains of C. albicans isolated from HIV-positive patients were exposed to cyclophosphamide, cytarabine, dacarbazine and methotrexate antineoplastic drugs at the concentrations used during therapy. Catalase activity was measured and compared to the control group. Very significant differences (P<0·01) were found when C. albicans was exposed to methotrexate (2 μg ml−1=4 μM). For cyclophosphamide (50 μg ml−1), cytarabine (1 μg ml−1) and dacarbazine (8 μg ml−1), no differences were found (P>0·05) between the control and drug-exposed groups. Although more extensive studies are necessary, these data do suggest that the antineoplastic drug methotrexate contributes to the resistance to antifungal drug therapy by varying catalase activity.

Tularaemia caused by inhalation of type A Francisella tularensis bacteria is one of the most aggressive infectious diseases known, but the reasons for the very rapid spread of the organism from the lungs to internal organs and the ensuing mortality are unknown. The present study used the mouse model to examine in detail the host immune response in the lung. After an aerosol challenge with 20 c.f.u. of the type A strain FSC033, all mice developed clinical signs of severe disease, showed weight loss by day 4 of infection and died the next day. Histopathological findings in the lung revealed acute inflammation and intense vasculitis and perivasculitis on day 4. Gene transcriptional changes in the mouse lung samples were examined on days 1, 2 and 4 of infection using a cDNA microarray with 20 600 mouse clones representing 18 500 genes. In total, 424 genes were found to be differentially expressed, some of which were both up- and downregulated at different time points, 192 of which were upregulated and 234 of which were downregulated for at least one time point. A high percentage of selected genes identified by the microarray analysis were confirmed to be differentially regulated by quantitative real-time PCR. Categorization of the differentially expressed genes showed that those preferentially involved in host immune responses were activated extensively on day 4 but hardly or not at all on days 1 and 2. Further analysis revealed that several of the genes upregulated on day 4 are known to depend on gamma interferon or tumour necrosis factor alpha for their regulation. In keeping with this finding, tumour necrosis factor alpha and gamma interferon levels were found to be increased significantly in bronchoalveolar lavage on day 4.

A novel direct detection system has been developed for eight staphylococcal enterotoxin (SE)-encoding genes (sea, seb, sec, sed, see, seg, seh and sei) in milk. Specific detection by real-time PCR was successful for all SE-encoding genes in the reference strains. Furthermore, a novel DNA-preparation method with good reproducibility [coefficients of variation 0·31, 0·99 and 1·21 % at 106, 104 and 102 c.f.u. (ml milk sample)−1, respectively] was developed to overcome PCR inhibition in the milk samples. The combination of this DNA-preparation method and real-time PCR resulted in high sensitivity [between 1·1×102 and 1·0×104 c.f.u. (ml milk sample)−1] and allowed the completion of the entire procedure within 4 h. Results of an evaluation of this method for the detection of SE-encoding genes using known outbreak milk samples produced results showing good correspondence with the reversed passive latex agglutination assay. In addition, this newly developed system can be applied to clinical samples such as faeces and vomit. Consequently, the system should be useful in the routine direct detection of SE-encoding genes in food-borne-poisoning samples.

Pneumonic plague is an aggressive disease that is clinically difficult to treat. Inhibition of attachment using oligosaccharide receptor mimics may provide an alternative to antibiotics. The virulent Yersinia pestis strain GB was demonstrated to attach to the murine monocyte cell line (J774A.1) and a range of human respiratory epithelial cell lines: nasal (RPMI-2650), bronchial (BEAS2-B) and alveolar (A549). Attachment was greatest to the A549 and BEAS2-B cell lines. Pre-treatment of the cell lines with tunicamycin reduced attachment by 55–65 %, indicating the importance of cell-surface carbohydrates in adhesion. The cell lines displayed differences in the oligosaccharides that inhibited attachment. p-Nitrophenol was the best inhibitor for each cell line. Disaccharides such as GalNAcβ1-3Gal and GalNAcβ1-4Gal were also good inhibitors, particularly for the RPMI-2650 cell line. This demonstrates the potential of oligosaccharides as potential anti-adhesion therapeutics.

The identification of mannitol salt positive, coagulase-negative staphylococci (CNS) is often disregarded when Staphylococcus aureus is screened in clinical samples using mannitol salt agar. However, the emergence of CNS as important human pathogens has indicated that reliable methods for the identification of clinically significant CNS are of great importance in understanding the epidemiology of infections caused by them. The identification and molecular characterization of mannitol salt positive CNS from nasal samples of medical personnel and students is reported here. A total of 84 mannitol salt positive staphylococcal isolates were obtained from 240 nasal samples, of which 15 were CNS. The API STAPH system classified the CNS isolates into six species, and one-third of the isolates were identified with confidence levels of <80 %. 16S–23S rRNA intergenic spacer length polymorphism analysis (ITS-PCR) identified only two species (Staphylococcus haemolyticus and Staphylococcus saprophyticus). This identification was confirmed by antibiotyping, species-specific PCR and PFGE. The results from this study indicate that ITS-PCR is a potentially useful and reliable tool, enabling hospital laboratories to obtain rapid, full and accurate identification of CNS at the species level.

The genetic heterogeneity among Campylobacter jejuni and Campylobacter coli isolates obtained from apparently healthy cattle and sheep was investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 348 Campylobacter isolates, consisting of C. jejuni (n=218) and C. coli (n=130), were analysed. All these isolates were successfully typed by RAPD analysis. The total numbers of band patterns defined by RAPD in cattle and sheep were 42 and 45, respectively. Of the 42 distinct types obtained from cattle, 37 types were observed in C. jejuni isolates (n=115), and the remaining 5 were in C. coli isolates (n=30). Of 45 distinct types obtained from sheep, 21 types were observed in C. jejuni isolates (n=103), and 24 were in C. coli isolates (n=100). It was concluded that a high degree of heterogeneity existed among the C. jejuni and C. coli isolates of healthy cattle and sheep.

A coryneform bacterium was isolated from the bronchoalveolar aspirate of a patient with interstitial pulmonary inflammation. Commercial systems identified the isolate as Corynebacterium sp. or Aureobacterium sp./Corynebacterium aquaticum, but 16S rRNA gene analysis unequivocally attributed it to the genus Microbacterium. This represents the first documented case of Microbacterium pulmonary infection.