- Volume 27, Issue 3, 1988

Antibodies to Pseudomonas aeruginosa were investigated in serum from cystic fibrosis (CF) patients by immunoblotting (Western blotting). The results were compared with determinations of precipitating antibodies in serum by crossed immune electrophoresis (CIE). The number of CIE precipitins is a sensitive and specific indication of infection and is used, with sputum bacteriology, to distinguish between colonisation and invasive lung infection. Immunoblotting was considerably more sensitive than CIE for detecting antibodies to P. aeruginosa. Paired serum samples from 64 CF patients, taken before a diagnosis of P. aeruginosa lung infection and immediately afterwards, showed a marked increase in the number of serum antibodies with the onset of infection. The intensity of the reaction, as shown by the density of blotted bands, was also increased. Laser scanning densitometry of immunoblots, and of photographic negatives taken from them, was used to quantify the increases. Differences in the number and intensity of blotted bands were highly significant between the two groups. The reproducibility of the method was good. An immunoblot assay may be a sensitive and useful method for routine diagnosis of early P. aeruginosa lung infection in CF.

There is little information about the local and systemic antibody response to surface antigens of bacteria growing in situ in infected lesions in man. In this study, Pseudomonas aeruginosa was obtained directly from the infected wounds of two patients with burns and studied without subculture. Outer-membrane proteins (OMPs) were investigated and compared with those of cells cultivated in the laboratory, with the aim of selecting defined growth conditions to give surface antigens more closely resembling those found in vivo. Several high-mol. wt (77 000-101 000) proteins were expressed in the outer membranes of the bacteria from the patients and could be phenotypically induced by cultivating the same isolate in iron-depleted conditions in vitro. Other major OMPs (D, E, F, G and H) were also observed in cells taken from the lesions. Immunoblotting demonstrated that proteins D and E were recognised by different classes of immunoglobulins in the sera of both patients as was flagellar antigen present in the outer-membrane preparation of the P. aeruginosa from patient 1. Iron-regulated membrane proteins (IRMPs) were similarly detected, but more strongly by IgM from patient 1. Furthermore, a marked antibody response to IRMPs was noted at the site of infection. Bands of a similar intensity were seen after absorption of the sera with lipopolysaccharide (LPS) purified from the infecting strain. This indicated that the response observed was directed against OMPs (including IRMPs) and not against contaminating LPS.

The relationships between capsulate and non-capsulate Bacteroides fragilis strains and Escherichia coli, and between capsulate and non-capsulate strains of the B. melaninogenicus group and Streptococcus pyogenes, were studied in a subcutaneous abscess model in mice. Selective antimicrobial agents directed against either aerobic or anaerobic bacteria were used alone or in combination to explore the effect of eradication of one component of the mixed infection. Single agent therapy effective against both aerobic and anaerobic flora was also employed. Single therapy of mixed infection directed at the elimination of only one organism (S. pyogenes, E. coli or Bacteroides sp.) caused significant reductions in the numbers of sensitive organisms and also smaller yet significant decreases in the numbers of insensitive organisms. However, the abscesses were not eliminated after such therapy. Combination therapy or use of a single agent (cefoxitin) directed against the aerobic and anaerobic components of the infection was more effective. Non-capsulate Bacteroides spp. became capsulate after passage in mice mixed with either S. pyogenes or E. coli. Therapy directed at the elimination of S. pyogenes and E. coli did not prevent the emergence of capsulate Bacteroides spp. These data demonstrate the synergy between all members of the B. fragilis group and E. coli and between the B. melaninogenicus group and S. pyogenes, and reiterate the need to direct antimicrobial therapy at the eradication of the aerobic and anaerobic components of mixed infections.

To study the hospital epidemiology of Bacteroides fragilis, 343 isolates from infected hospitalised patients (112), infected out-patients (102), and from the faeces of uninfected hospitalised patients (47) and normal subjects in the community (82), were examined by an immunofluorescence technique. In tests with antisera against strains of 20 distinct serotypes of the fragilis group of Bacteroides, 271 (79%) strains were typable. Similarity between strains was estimated by the Jaccard similarity measure and strains were then serogrouped by cluster analysis; 88.1% of hospital strains were typable but only 71.2% of community strains (p < 0.001). Three serogroups were prevalent among both faecal and infection isolates of hospital strains (p < 0.01). However, no particular serogroup was prevalent among community strains and no difference was found in the distribution of serogroups between strains from faeces and those from infected lesions. One serogroup showed a significant increase (p < 0.05) within the period of study. These findings suggest that strains of B. fragilis infecting hospitalised patients may be acquired in hospital and that they may spread to other patients.

Infections caused by coagulase-negative staphylococci (CNS) are a major problem in immunocompromised patients. It has been claimed that extracellular slime production by CNS predicts pathogenicity and inhibits host defences. Luminol-enhanced neutrophil chemiluminescence (CL) and bacterial killing assays were used to assess the effect of slime production on opsonophagocytosis and killing by polymorphonuclear leucocytes in vitro. There was wide variation in CL induction amongst the 43 strains of Staphylococcus epidermidis examined. The presence of slime had no influence either on the requirement or on the efficiency of opsonisation. Slime-producing and non-slime-producing strains showed a stepwise increase in induced CL up to a serum concentration of 10%, and were dependent on complement for efficient phagocytosis. The bacterial killing assays confirmed the CL results. Our data suggest that extracellular staphylococcal slime has no specific anti-opsonic property in vitro. Opsonophagocytosis may still be hampered in vivo by the physical presence of slime.

The production of haemolysin from Treponema hyodysenteriae was increased by an improved culture method and by repeated incubation of spirochaetes suspended in a buffer containing RNA-core. Ion exchange chromatography on DEAE cellulose followed by gel filtration on Sephadex G100 yielded purified haemolysin free from extraneous protein, as judged by silver-stained polyacrylamide gels. The mol. wt of the purified haemolysin, determined by gel filtration was 19 000, a value similar to that of streptolysin S, but much lower than that previously reported.