- Volume 47, Issue 4, 1998

The protective efficacy of immunisation with heat-killed Pseudomonas aeruginosa on murine gut-derived sepsis was evaluated. Mice were immunised intraperitoneally six times with heat-killed bacteria. This induced mean (SEM) serum IgG and IgM antibodies of 1792 (374.7) and 37.3 (8.9) ELISA units, respectively. Specific pathogen-free mice given P. aeruginosa strain D4 orally died of bacteraemia after administration of cyclophosphamide. Immunisation with heat-killed bacteria significantly increased the survival rate compared with that of control mice immunised with bovine serum albumin. Macroscopic observation revealed marked production of liver abscesses in mice immunised with bovine serum albumin but not in those immunised with heat-killed bacteria. Only low titres of antibody against the exoenzymes alkaline protease, elastase and exotoxin A were observed, and no significant difference between antibody titres to boiled and unboiled suspensions of sonicated P. aeruginosa was detected. This suggests that the main protective antibodies might be those specific to the heat stable antigen (lipopolysaccharide). Immunisation with heat-killed bacteria provided complete protection against death from gut-derived P. aeruginosa sepsis.

High resolution molecular subtyping was applied to Campylobacter jejuni Penner heatstable (HS) 11 isolates from human infections and other sources. Strains were genotyped by restriction fragment length polymorphism analysis involving PCR-based flagellin gene (flaA) profiling with HinfI and DdeI, and pulsed-field gel electrophoretic (PFGE) profiling with SmaI and KpnI. Fla-genes of the strains were highly conserved as most (95%) had the same fla-profile. PFGE analysis of SmaI digests was more discriminatory with 15 profile subtypes identified, although 36% of isolates had a common profile. The study showed that strains of C. jejuni HS11, unlike those of HS1 and the HS4 complex, were relatively homogeneous at the genomic level and that high resolution molecular techniques were essential for detailed epidemiological subtyping.

Enteropathogenic Escherichia coli (EPEC) remain an important world-wide cause of diarrhoeal disease and mortality of infants and young children. Research programmes around the world have, in recent times, made enormous strides towards a better understanding of EPEC pathogenesis, yielding unique insights into the molecular intercourse between host and pathogen. Recombinant DNA and cell biology techniques have provided powerful tools, giving the first intriguing glimpses of a wealth of bacterial products mediating complex host:pathogen interactions involving the subversion of normal host signalling processes. Much has been discovered since 1945, when E. coli was first implicated as a cause of diarrhoea. However, many questions remain unanswered and many more remain unasked. Much remains to be discovered, especially in the area of molecular interactions between host and pathogen and how they relate to the manifestation of disease in the patient.

Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) represent an increasing problem in hospitals. Quick and reliable typing methods are required to obtain information about the relatedness of MRSA isolates and to allow faster implementation of appropriate infection control measures. This investigation describes the distribution of MRSA isolates from 11 hospitals in the Düsseldorf region of Germany, and the ability of six different genotypic typing techniques – pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), 16S–23S rDNA spacer amplification, protein A-gene PCR, PCR characterisation of the hypervariable region (HVR) adjacent to mecA, and coagulase gene-PCR – to detect different unrelated types. Of 7814 S. aureus isolates tested, 489 (6.3%) were MRSA, of which 183 were selected for subsequent molecular analyses on the basis of being the first MRSA isolated from colonised or infected patients. Larger hospitals had a higher incidence of MRSA and a greater variability in genotypes than smaller hospitals. All methods confirmed the presence of two main clonal types. The ability of techniques to detect different unrelated types was found to be as follows: PFGE, 28 types; 16S–23S rDNA spacer-amplification, 10 types; RAPD, nine types; protein A-gene PCR, five types; HVR-PCR, five types; and coa gene-PCR, two types. Combination of PFGE and one other PCR-based method (spacer-amplification, RAPD or protein-A gene PCR) provided the best resolution of types and allowed the identification of subtypes. Similar molecular types were identified with international MRSA isolates. Although PCR-based techniques have the advantage of rapid performance and easy handling, their discriminatory capacity is inferior compared to the more labour intensive PFGE.

The protective efficacies of vaccines prepared from Pseudomonas aeruginosa alkaline protease, elastase and exotoxin A toxoids against gut-derived P. aeruginosa sepsis in mice were evaluated. Specific pathogen-free mice given P. aeruginosa strain D4 orally followed by cyclophosphamide (to promote translocation across the gut wall) died of bacteraemia. Mice immunised with one of the three individual toxoid vaccines were not significantly protected when compared to control mice immunised with bovine serum albumin. Combined immunisation with alkaline protease and elastase toxoids likewise showed no significant protective activity. However, combined immunisation with alkaline protease and exotoxin A toxoids significantly increased the survival rate, which reached 60% (compared with a 7.1% survival rate in the control group). These results show that alkaline protease and exotoxin A play important roles as pathogenic factors in gutderived sepsis and that a combination of the two exoenzyme toxoids represents a logical candidate for vaccination against P. aeruginosa sepsis.

A nested polymerase chain reaction (PCR) assay was investigated for detection of Pneumocystis carinii in 96 respiratory tract specimens from 82 children, of whom 28 were immunocompetent but with chronic lung disorders (CLD), eight had AIDS and P. carinii pneumonia (PCP), 16 had AIDS but no respiratory symptoms, and 30 were healthy immunocompetent children. Gomori methenamine silver stain (GMS) and indirect immunofluorescence assay (IFA) were performed in parallel. Of 36 specimens from children with CLD, 12 were P. carinii PCR-positive compared to 10 positive by GMS-IFA. Of eight specimens from children with AIDS and PCP, seven were P. carinii-positive by PCR and six by GMS-IFA, and of 22 specimens from HIV-positive children without respiratory symptoms, two were positive by PCR and none by GMS-IFA. P. carinii DNA was also detected by PCR in blood samples from four children with P. carinii-positive nasopharyngeal aspirates. Specimens from healthy children were negative for P. carinii by both PCR and GMS-IFA. Of the seven children with CLD, who were P. carinii-positive, two had clinical and microbiological improvement with co-trimoxazole treatment, two improved initially but relapsed, and one had P. carinii cysts persistently in follow-up specimens despite co-trimoxazole treatment. These results suggest an association between P. carinii and exacerbations of CLD in childhood, in the absence of HIV infection or other immunodeficiency syndromes.

Cell-bound haemolytic activity was observed in isolates of Mycobacterium avium complex (MAC) from AIDS patients. M. avium type strains showed negligible activity. None of the culture supernates exhibited any haemolytic activity. Zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulphonate (CHAPS) was used to extract haemolysin from ethanol-treated M. avium complex strain 101 (MAC101) cells. Haemolysin was isolated from CHAPS extract (CE) by metal affinity chromatography and identified as a 32-kDa protein by polyclonal antibodies raised against M. tuberculosis haemolysin. Treatment of CE with trypsin resulted in reduction of haemolytic activity, whereas heating at 100°C for 10 min did not affect its activity. A similar 32-kDa haemolysin was extracted from cells of M. avium K128 which was isolated from a monkey infected with simian immunodeficiency virus (SIV). The haemolysin produced by M. avium strains isolated from AIDS patients may be associated with the pathogenesis of M. avium infection.

Recently, a microplate agglutination test (MPAT) was established for the serological diagnosis of legionella pneumonia. To evaluate its usefulness, this study examined antibody titres in 121 serum samples serially obtained from 40 patients with pneumonia, including 17 cases of confirmed legionella pneumonia. Six of the 17 proven cases became serologically positive within 4 weeks of the onset of pneumonia as assayed by MPAT (cut-off value: four-fold rise to ≥128 in paired sera or ≥256 in a single serum specimen), whereas the remaining 11 cases were serologically negative despite serial examination. Four proven cases who were treated with corticosteroids in the acute phase had antibody titres <8 during the first 4 weeks of infection, after which one case showed an elevation in antibody titre for the first time, 13 weeks after the onset of disease. In contrast, all non-proven cases had antibody titres of ≥64, and only one case developed a four-fold or greater rise in titre. These results indicate that MPAT is a useful method for the laboratory diagnosis of suspected legionella pneumonia, although several false-negative cases were observed. This suggests that the previously established MPAT criteria may require modification, possibly to slightly lower values. These data also indicate that serial examination over the first month of infection may be necessary for serodiagnosis of legionella pneumonia, especially in patients treated with corticosteroids.

Clostridium difficile is a human pathogen that produces two types of toxins, A and B, that cause a potentially lethal gastrointestinal syndrome termed pseudomembranous colitis. Virtually nothing is known about the mechanism of regulation of toxin production in this organism, and cis-regulatory regions of neither toxin have yet been identified, thus prompting this investigation. A motif homologous with the Shine-Dalgarno sequence of Escherichia coli occurs upstream from the putative initiation codon of toxin B, making this region also a candidate to contain a promoter. Therefore, a subgenomic DNA library of C. difficile in a plasmid vector was first constructed encompassing the 5′-end of the toxin B gene. A 450-bp DNA fragment was excised from the subgenomic DNA library clone and subcloned into a promoter-probe plasmid vector that contains two divergently oriented, promoterless genes to assay for promoter function. This subcloned DNA fragment directed the expression of alkaline phosphatase, a reporter gene product of the promoterless vector, thus indicating the presence of a functional promoter. To locate the promoter more precisely, a series of nested deletions of the toxin B promoter subclone was constructed with exonuclease III. The promoter that facilitates expression of the toxin B gene in E. coli was localised, based on alkaline phosphatase activity. The transcriptional initiation site of toxin B mRNA in E. coli was mapped by primer extension analysis, suggesting two closely associated tandem start sites directed by two similarly spaced promoters within this localised region.

The effect of in-vitro granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment of neutrophils, monocytes or monocyte-derived macrophages (MDM) on candidacidal activity was tested. Synergy of effector cells with fluconazole (FCZ) for enhanced killing was also investigated. Incubation of neutrophils with GM-CSF 0.67 μg/L plus Candida albicans Sh27 for 24 h significantly increased candidacidal activity (36% versus 74%). Synergy with FCZ for killing was also significantly increased from 93% to 97% when neutrophils were incubated with GM-CSF. Monocytes cultured with GM-CSF and C. albicans for 24 h had significantly increased fungistatic activity compared to controls and synergy with FCZ for killing was significantly enhanced from 40% to 59%. Monocytes cultured for 3 days with GM-CSF had increased fungistatic activity compared to control MDM and showed synergy with FCZ for significantly enhanced killing (46%) compared to control MDM (12%).

As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shock syndrome (TSS). For that reason an easy and quick test system for determining the toxin production pattern of S. aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier. In the present investigation, a new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation. This procedure generated information concerning the presence of seb, sec-1 and tst genes within 4 h in a single test To analyse the sensitivity and the specificity of this procedure, 100 methicillin-resistant S. aureus (MRSA), 50 coagulase-negative staphylococci and 50 other eubacterial isolates were tested initially with sets of single primer pairs followed by a combined multiplex-PCR. Results of this amplification technique were compared to a conventional and widely used method for toxin detection, reversed passive latex agglutination (RPLA). With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and specificity of the sec-1 primer set were 100% and 82%, respectively. With the sec-1 primer set, two isolates were identified as carrying the corresponding toxin gene although the RPLA test did not show any detectable toxin. The multiplex-PCR rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains and could be easily integrated into a multiplex procedure described previously. The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and mecA genes.

The purpose of this study was to prepare specific sera for use in the rapid detection and identification of the intestinal spirochaete Serpulina pilosicoli. In Western blot analysis, with pig antiserum which was raised against whole cells of S. pilosicoli and absorbed with outer envelope protein extracts from S. hyodysenteriae and S. innocens, a prominent protein with Mr of c. 72 kDa was consistently identified in outer envelope preparations of S. pilosicoli strains. Immunogold labelling demonstrated that this was located on the outer surface of intact S. pilosicoli cells. Two monoclonal antibodies (MAbs), designated C12 and M96, were raised against the protein. Although C12 reacted with a protein band of c. 72 kDa, this was also present in preparations from strains of other Serpulina spp. examined. MAb M96 reacted with an 80-kDa protein which was present only in preparations made from strains of S. pilosicoli. This was used in Western blot analysis and in an immunodot-blot assay with outer envelope extracts to specifically identify S. pilosicoli strains isolated from man, pigs, dogs and poultry. An indirect immunofluorescence test with MAb M96 also was used to detect and identify whole S. pilosicoli cells. Therefore, both the cross-absorbed antiserum and MAb M96 are potentially useful reagents for the detection and identification of S. pilosicoli.