- Volume 51, Issue 6, 2002

Comparison of DNA from virulent strains of bacterial pathogens with DNA from less virulent or avirulent close relatives allows the identification of those genomic regions that are present only in virulent strains. Such regions are often associated with pathogenicity islands (PIs) and their characterisation can lead to a greater understanding of the pathogenesis of infectious diseases. There is now a large database of bacterial genomic sequences that provides useful reference information with which to compare the genomes of strains that exhibit variations in virulence or host preferences. Subtractive hybridisation (SH) and its sister method, suppression subtractive hybridisation (SSH), are techniques designed to identify those regions present in one genome but absent from another. The application of these techniques has led to the identification of PIs, mobile genetic elements and variations in virulence gene expression in a range of bacterial pathogens.

Sequential blood samples taken from a pregnant woman with haemolytic uraemic syndrome caused by verocytotoxin (VT)-producing Escherichia coli O157 were used to examine the kinetics of serum antibody production to E. coli O157 lipopolysaccharide (LPS), intimin and the conserved region of the translocated intimin receptor (Tir-M). Umbilical cord blood and two samples of blood from the newborn baby were also examined for antibodies to these antigens. In the mother, antibodies of the IgM class, specific for E. coli O157 LPS, were produced in the initial stages of the infection, reaching a peak at 9 days after onset of diarrhoea and subsiding 3 days later. High levels of IgG class antibodies, specific for E. coli O157 LPS, were detected 8 days after the onset of diarrhoea and were present at high titres on day 18. Serum antibodies of the IgA class to E. coli O157 LPS were not detected. Antibodies binding to Tir-M were detected 8 days after the onset of diarrhoea and high titres of these antibodies were still present on day 18. Serum antibodies to intimin were not detected in the mother and no antibodies to any of the antigens tested were detected in either the baby's blood or cord blood. This study describes for the first time the kinetics of serum antibody production during pregnancy, to selected antigens expressed by E. coli O157.

This study examined polymorphisms in the dihydrofolate reductase (DHFR) gene of Pneumocystis carinii isolates from 27 patients with P. carinii pneumonia (PCP) in Japan. Four substitution sites with two synonymous and two non-synonymous changes were found. Two synonymous substitutions at nucleotide positions 540 and 312 were identified in one and 13 patients, respectively. Two amino acid substitutions (Ala67Val, Cys166Tyr) were found in two different patients. No linkage of amino acid substitutions in DHFR to those in dihydropteroate synthase was observed. The two patients whose isolates showed non-synonymous DHFR mutations were not exposed to DHFR inhibitors before they developed PCP and were treated successfully with co-trimoxazole.

Aspirates of pus from acute suppurative sialadenitis were investigated for the presence of aerobic and anaerobic bacteria. A total of 47 specimens, 32 from parotid, 9 from submandibular and 6 from sublingual glands yielded bacterial growth. Fifty five isolates, 25 aerobic and 30 anaerobic, were isolated from parotid infection: anaerobic bacteria only were detected in 13 (41%) specimens, aerobic or facultative bacteria only in 11 (34%) and mixed aerobic and anaerobic bacteria in 8 (25%). Of a total of 17 isolates, 8 aerobic and 9 anaerobic, from submandibular gland infection: anaerobic bacteria only were detected in 3 (33%) specimens, aerobic or facultative bacteria only in 4 (44%) and mixed aerobic and anaerobic bacteria in 2 (22%). Ten isolates, 5 aerobic and 5 anaerobic, were from sublingual gland infection: anaerobic bacteria only were detected in 2 (33%) specimens, aerobic or facultative bacteria only in 2 (33%) and mixed aerobic and anaerobic bacteria in 2 (33%). The predominant aerobes were Staphylococcus aureus and Haemophilus influenzae while the predominant anaerobes were gram-negative bacilli (including pigmented Prevotella and Porphyromonas spp., and Fusobacterium spp.) and Peptostreptococcus spp. The study highlights the polymicrobial nature and importance of anaerobic bacteria in acute suppurative sialadenitis.

Influenza A virus (IAV)-induced impairment of neutrophil function or survival may be a cause of bacterial superinfection of IAV-infected subjects. This study was performed to determine the mechanism through which the combination of IAV and Escherichia coli co-operatively reduces neutrophil survival. Neutrophil binding of annexin-V and caspase-3 activation was significantly increased by either IAV or E. coli, supporting the concept that the micro-organisms accelerate neutrophil apoptosis. The anti-apoptotic agent granulocyte-macrophage colony stimulating factor (GM-CSF) did not improve, but further reduced, survival of neutrophils treated with IAV and E. coli. As addition of E. coli resulted in greater neutrophil uptake of IAV and greater neutrophil respiratory burst responses to IAV, this study tested whether respiratory burst activation by IAV and E. coli contributes to reducing neutrophil survival. The cell-permeant NADPH oxidase inhibitor, diphenylene iodonium, significantly increased survival of neutrophils treated with either E. coli alone or the combination of IAV and E. coli. In contrast, catalase, which is not cell permeant, did not alter survival of E. coli- and IAV-treated neutrophils. Azide enhanced neutrophil hydrogen peroxide responses to IAV and E. coli, and reduced survival of these cells. These results indicate that co-operative induction of intracellular respiratory burst responses by IAV and E. coli mediates the reduced neutrophil survival caused by these pathogens in vitro.

This report describes the application of reference strand-mediated conformational analysis (RSCA), a novel DNA typing technique, for the identification of clinically significant fungal pathogens. RSCA is a heteroduplex-based conformational method which relies on detecting differences in the DNA conformation of heteroduplexes generated in this study by the annealing of different fungal 18S rRNA amplicons to a common fluorescent-labelled reference (FLR). These heteroduplexes are then observed with laser-based instrumentation and computer software to detect differences in the DNA conformation reproducibly. This technique was shown to generate unique and reproducible profiles for the 18S rRNA gene sequences of a number of medically important fungi, distinguishing different Candida species (C. albicans, C. kefyr, C. dubliniensis, C. lusitaniae, C. guilliermondii, C. tropicalis, C. krusei, C. glabrata, C. sake and C. parapsilosis), and in some cases detecting single nucleotide differences between 18S rRNA sequences. The RSCA technique was further evaluated with 50 human clinical isolates of Candida spp., previously identified by culture techniques, and was shown to identify the isolates correctly. This technique displays enormous potential as an alternative to DNA sequence determination and has the potential to become an automated technique that can be implemented in the routine setting.