Electrophoretic analysis of the lipopolysaccharides ofBacteroides spp.

Lipopolysaccharide (LPS) extracts of reference strains and isolates ofBacteroides spp. prepared by the proteinase K method were resolved by tricine-sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis and located by silver staining. A considerable diversity of LPS profiles was evident within theBacteroides genus although profiles were essentially species-specific, with some minor interstrain variations apparent among isolates ofB. uniformis, B. ovatus, B. eggerthii andB. thetaiotaomicron. The LPS of most species consisted of a major rough LPS component of 2–5 kDa and a series of higher molecular weight bands which varied with species.B. vulgatus LPS was distinctive in showing an extensive ladder of multiple repeating oligosaccharide units with molecular weights ranging from 4 to >17 kDaB. stercoris LPS included a high molecular weight (>17 kDa) ladder of repeating oligosaccharide units.B. fragilis andB. thetaiotaomicron differed from most other species in producing a short ladder of repeating oligosaccharide units interspersing the rough LPS and a 5.6 kDa (B. fragilis) or 9 kDa (B. thetaiotaomicron) yellow-staining component. The heterogeneity of LPS profiles within theBacteroides genus may reflect the differences in pathogenicity among the species and prove useful for typing.     

Staphylococcus saprophyticus as a urinary pathogen: a six year prospective survey.

Over six years (1978-83, inclusive) weekly laboratory records of organisms causing urinary tract infection in women aged 15-25 not attending hospital were kept prospectively and analysed. The incidence of infection with Staphylococcus saprophyticus defined by age and sex was confirmed. This organism caused an increasing proportion of infections in young women over the six years studied, and these infections showed noticeable seasonality. All but four isolates of S saprophyticus were sensitive to all the commonly used antimicrobial agents that were tested. This might be because the organism is not often present in the body as a commensal and therefore not subject to the selection pressures exerted by such agents. As infection with S saprophyticus has different clinical connotations from infection with other coagulase negative staphylococci it should be differentiated from them in routine laboratory practice.     

Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica

The Bordetella pertussis wlb locus (wlb_pe, formerly bpl ) is required for the biosynthesis of a trisaccharide that, when attached to the B. pertussis lipopolysaccharide (LPS) core (band B), generates band A LPS. The equivalent loci in Bordetella bronchiseptica (wlb_br) and Bordetella parapertussis (wlb_pa) were identified and cloned. The wlb_br and wlb_pa loci differ from wlb_pe in that they lack the insertion sequence that defines the right-hand terminus of wlb_pe. Deletion of 12 kb of DNA containing the whole wlb locus (Δwlb) by allelic exchange in each of the three bordetellae had no effect on band B biosynthesis, whereas band A biosynthesis was prevented in B. pertussis and B. bronchiseptica. In B. bronchiseptica and B. parapertussis, Δwlb mutants also lacked O-antigen. Reintroduction of the wlb_pe or wlb_br loci on a shuttle vector into the three Δwlb mutants restored the wild-type LPS phenotype in the B. pertussis and B. bronchiseptica mutants. In the case of B. parapertussis, which normally does not synthesize an apparent band A structure, introduction of the wlb_pe or wlb_br loci now enabled the generation of band A. This suggests that the attachment point for band A trisaccharide on the LPS core is present in B. parapertussis, and further suggests that the wild-type wlb_pa locus is not fully functional. Introduction of the wlb_pa locus into the Δwlb_pe, Δwlb_br and Δwlb_pa mutants had interesting consequences. The B. bronchiseptica and B. parapertussis recipients were now able to biosynthesize O-antigen, but no band A was generated. In the B. pertussis recipient, a truncated band A was expressed consistent with a mutation in the wlbH gene, but on Western blotting the expression of a small amount of full-length band A was also seen. Evidence that the wlbH_pa protein is not fully functional was provided by the failure of the wlb_pa locus to fully complement a B. pertussis wlbH (ΔwlbH_pe) mutant. This was supported by DNA sequence data showing that a single amino acid, conserved between homologous proteins from a range of bacteria, is altered in the B. parapertussis WlbH protein.     

Isolation of stable aroA mutants of Salmonella typhi Ty2: properties and preliminary characterisation in mice

Summary Derivatives of the Salmonella typhi strain Ty2 carrying stable mutations in the aroA gene were isolated. The mutations were generated by transducing an aroA::Tn10 marker into Ty2 and selecting for derivatives which were tetracyline sensitive and dependent on aromatic compounds for growth. Isolates that did not revert to aromatic compound independence at a detectable frequency were obtained. An S. typhimurium derived aroA specific DNA probe was used to demonstrate the presence of DNA rearrangements in the aroA region of the chromosome of some of the S. typhi aroA mutants. Most of these isolates still expressed Vi antigen. Aromatic compound dependent mutants of S. typhi were less virulent in mice than S. typhi Ty2 following intraperitoneal challenge with bacteria suspended in mucin. Mice immunised with one of these mutants, named WBL85-1, were protected against a potentially lethal challenge of S. typhi Ty2.     

Attenuated Salmonella Typhimurium Lacking the Pathogenicity Island-2 Type 3 Secretion System Grow to High Bacterial Numbers inside Phagocytes in Mice

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.     

Sterilization of tissue-culture medium by filtration on a large laboratory scale

The design and operation of a permanently-piped plant for sterilizing 10–200 l. of tissue-culture media is described. The plant is sterilized in situ by steam injection, the filtration rate is 2–3 l./min., and the turn-around time for volumes of 100 l. is about 3 hr.