- Volume 45, Issue 4, 1996

The general characteristics and genetics of insertion sequence (IS) elements are well-established. For Escherichia coli IS elements, mechanisms of transposition and mutation are known and their recombinogenic role in the bacterial genome has been investigated. Population models relate the distribution of these IS elements to autoregulation of their transposition. IS200, the smallest known element, is confined to the salmonellae and several lineages of E. coli. It exhibits atypical molecular features. The population dynamics of IS200 make it particularly effective marker of chromosomal genotype in many Salmonella serovars. Molecular epidemiological typing with IS200 has been developed for important serovars in groups D1, C1, C2 and B. Findings for S. Enteritidis, S. Panama, S. Infantis, S. Typhimurium, S. Heidelberg, S. Paratyphi B and S. Java are reviewed. Of the 12 IS elements found in mycobacteria, IS6110, found in Mycobacterium tuberculosis and M. bovis, exhibits the greatest potential for molecular epidemiological applications. Although M. tuberculosis is a single serogroup, and its genome is otherwise highly homogeneous, strains are highly polymorphic with respect to copy number and location of IS6110. A standard IS6110 typing method has been established, together with novel PCR-based approaches to IS6110 fingerprinting.

Yersina enterocolitica bone and joint infections are rare. Over a period of 7 months four patients with deep-seated skeletal infections due to Y. enterocolitica were seen at the University Hospital, Nottingham. Sites of infection included the knee (one patient) the hip (one) and the spine (two). None of the patients had major underlying disease or risk factors for developing invasive Y. enterocolitica infection. The organisms were sensitive to the second- and third-generation cephalosporins, gentamicin and fluoroquinolones. A literature search covering the period 1970-1994 revealed 20 other cases of skeletal infections due to Y. enterocolitica; there was no uniformity in the choice of antimicrobial agent for treating these infections. Oral ciprofloxacin was used as the principal antimicrobial agent in the patients described here and therapeutic success was achieved in three of these patients. Ciprofloxacin should be considered as first line therapy for invasive infections due to Y. enterocolitica.

The existence of epitopes common to different strains in the Neisseria meningitidis transferrin (Tf)-binding protein 2 (TBP2), combined with the ability of polyclonal anti-TBP2 antibodies to inhibit Tf binding and block iron uptake in this species, led to this study on the effect of anti-TBP1+2 monoclonal antibodies (MAbs) to determine the presence of epitopes inside the Tf-binding region. All MAbs used reacted exclusively with the homologous strain when tested by dot-blots of outer membrane vesicles, with the reaction being specific for TBP2 after SDS-PAGE and electroblotting. In contrast, ELISA and iron-uptake blocking assays were also positive with heterologous strains belonging to Rokbi’s group II (high mol.wt TBP2). The results confirmed the two group classification proposed by Rokbi and, in contrast to other studies, indicated the existence of epitopes in the Tf-binding region that are common only to strains of Rokbi’s group II. These epitopes may become denatured after drying for dot-blot assays or after SDS-PAGE and electroblotting.

Listeria monocytogenes adhered to and multiplied intracellularly in murine peritoneal macrophages in the absence of opsonins. The infective process in these cells was evaluated by viable bacterial cell colony counts of intracellular organisms and documented by transmission and scanning electron microscopy. Adherence of listeriae to macrophages involved surface interactions of the prokaryotic cell surface and eukaryotic cell membranes. Subsequent phagocytosis was seen to occur through a process in which host cell-derived pseudopodia surrounded and engulfed organisms leaving them within phagosomes in the cytoplasm of infected cells. This process of uptake of L. monocytogenes by macrophages occurred at 4°. Following invasion of the cell, escape of L. monocytogenes from the phagosome into the cytoplasm was initiated as early as 10 min into the infective process. Intracellular multiplication of bacteria continued for 8 h after inoculation at which point loss of adherent macrophages due to cell lysis was evident. The mean generation time of the organism in these cells was 58 min. The cellular and ultrastructural events of L. monocytogenes adherence to and phagocytosis by murine macrophages in the absence of antibody or complement have been defined.

The heparin-binding properties of six different species of coagulase-negative staphylococci were examined by a particle agglutination assay. Heparin (mol.wt 4000-6000), mildly treated with sodium periodate, was covalently coupled to amino-modified latex beads (0.72 μm diameter). The particle agglutination assay was validated by comparing results with the adhesion (percentage binding of adherent cells) of coagulase-negative staphylococcal strains to heparinised microtitration plates. Of 38 different coagulase-negative staphylococcal strains tested, 30 showed agglutination reactivity with heparincoated latex beads. Strains of different coagulase-negative staphylococcal species agglutinated heparin-coated latex beads to various extents (e.g., cells of Staphylococcus haemolyticus strains reacted more strongly than cells of S. epidermidis strains). The agglutination reaction was significantly inhibited by fucoidan, suramin, λ-carrageenan and other sulphated compounds, but not by non-sulphated carbohydrate polymers such as hyaluronic acid. Agglutination of staphylococcal cells with heparin-coated latex beads was completely blocked by a cell-surface extract. These results suggest that structures responsible for heparin binding are exposed on the cell surface.

The expression of a 60-kDa heat shock protein (HSP60) on the cell surface of Helicobacter pylori was analysed by flow cytometry with polyclonal antibody directed to HSP60. All 13 strains of H. pylori examined expressed HSP60 on the cell surface, although the intensity of expression was different among the strains and depended on culture conditions. There was a correlation between the intensity of HSP60 expressed on the cell surface and the rate of adherence to human gastric carcinoma cells (MKN45) by H. pylori, but not with urease activity and production of vacuolating toxin. By flow cytometric analysis with monoclonal antibody (MAb) 3C8 against HSP60, the reactive epitope in the HSP60 of H. pylori was detected on the surface of MKN45 cells. Furthermore, it was shown that gastric epithelial cells were positively stained with MAb 3C8 in one of two biopsy specimens examined. These results suggest that there is a common epitope showing homology between H. pylori HSP60 and human gastric epithelial cells.

Random amplified polymorphic DNA (RAPD) analysis was evaluated for its capacity to distinguish species and strains within species of groups A, C and G streptococci. The 99 strains tested, previously typed by multilocus enzyme electrophoresis (MLEE), included 41 group A streptococci (Streptococcus pyogenes), 25 group G Streptococcus spp. (GGS), seven S. dysgalactiae, 11 S. equisimilis, four S. canis, three S. equi and eight S. zooepidemicus. The combined data obtained with three single primers distinguished 82 types. RAPD analysis provided taxonomic results that were in general agreement with previous species classification based on DNA-DNA homology and MLEE. The intraspecies typing efficiency of the technique was significantly improved by the parallel use of several primers. RAPD analysis had greater discriminatory power than MLEE for GAS and GGS. There was not total agreement between the two techniques as RAPD distinguished strains with identical electrophoretic types, whereas MLEE differentiated strains with identical PCR types. RAPD analysis did not distinguish all GAS strains with different biotypes and its already high discriminatory power was further enhanced by concomitant biotyping.

A method based on long PCR amplification and restriction endonuclease analysis of the virulence regulon was developed for a rapid (2 days), simple differentiation of group A streptococci. The PCR product size varied from 12.3 kb for serotypes M1 (NCTC 8198) and M12 (NCTC 10085) to 7.8 kb for serotype M6 (NCTC 8302). The fragment patterns formed on HaeIII digestion of the products were unique and this allowed the differentiation of each of the M-type strains (M1, M3, M4, M5, M6, M11, M12, M28, M76 and M78) studied. Contemporary M1 isolates all gave the same fragment pattern but differed from the prototype strain (NCTC 8198) in not having a 1.25-kb fragment. Isolates of serotypes M1 and M3 each had similar patterns, an indication of their clonality and global dispersion. In contrast, more than one restriction fragment length polymorphism (RFLP) pattern was detected among clinical isolates of serotypes M5, M6, M12, M4, M(R)28 and M78. Two strains that were M-protein non-typable by serological means were provisionally classified as M6 by comparisons of Hae III long PCR fragment patterns.

Pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of chromosomal DNA was used to confirm the persistence of methicillin-sensitive Staphylococcus aureus isolates in the sputum of 25 cystic fibrosis patients in five French hospitals. Three-to-eight consecutive isolates, with the same esterase electrophoretic type isolated from each patient over a period of 12-28 months, were analysed. Consecutive isolates with indistinguishable PFGE profiles were found in 12 patients (48%) and consecutive isolates with similar PFGE profiles showing minor differences of one-to-four fragments (similarity coefficient ≥84%) were found in 11 patients. Consecutive isolates with different PFGE profiles were obtained from only two patients, but the profiles found in each patient were more closely related to each other than to other profiles. The results were in agreement with esterase electrophoretic typing for 23 patients, and we considered that those patients were infected with a single persistent strain. For any given patient, variations in antibiotypes and phage types of consecutive isolates were not associated with major genotypic variations. PFGE is useful in confirming the persistence of S. aureus strains in cystic fibrosis patients over long periods.