- Volume 48, Issue 3, 1999

The relationship between morphological changes and endotoxin release induced in vitro by carbapenems in a clinical isolate of Pseudomonas aeruginosa was examined. The time-course and magnitude of endotoxin release induced varied among imipenem, panipenem, meropenem and biapenem and related to the morphological changes caused by these agents which variously affected cell shape, cell-wall disintegration and cell lysis. The amount of endotoxin released by carbapenem-treated cells correlated with both the cell-wall morphology and bacterial shape immediately before lysis. Meropenem and biapenem caused markedly increased endotoxin release during cell lysis and cell-wall disintegration, whereas imipenem and panipenem caused much less release of endotoxin.

Fatty acid modifying enzyme (FAME) is an extracellular enzyme that inactivates bactericidal fatty acids by esterifying them to cholesterol. Inactivation of these fatty acids may allow Staphylococcus epidermidis to live for long periods of time on the skin. This study describes the identification and partial characterisation of an extracellular activator of FAME production. Addition of FAME-free concentrated culture filtrate (activator) to S. epidermidis cultures (OD600 = 0.05) caused a 3–5-fold increase in FAME activity. Addition of the activator did not increase the amount of exopolysaccharide produced by S. epidermidis. The mol. wt of this activator was <3000 kDa and it was quite resistant to boiling. Treatment of the activator with proteinase K did not destroy its ability to induce FAME expression. Addition of S. aureus activator to S. epidermidis cultures also increased FAME expression. However, when S. epidermidis activator was added to S. aureus cultures no increase or inhibition in FAME production was observed.

Survival of enteric pathogens exposed to various environmental stresses depends upon a number of protective responses, some of which are associated with induction of virulence determinants. Flagella and fimbriae are putative virulence determinants of Salmonella spp. and ELISAs specific for the detection of flagella and SEF21, SEF14 and SEF17 fimbriae were used to assess the effect of temperature and pH upon their elaboration by isolates of Salmonella serotype Enteritidis in planktonic growth and on the surface of two-dimensional gradient agar plates. For three phage type 4 isolates of Enteritidis of comparative clinical provenance, similar phenotypes for the elaboration of these surface antigens were observed. SEF14 fimbriae were elaborated in planktonic growth at 37°C, but not 20°C, at pH 4.77 and above but not at pH 4.04; whereas on agar gradient plates SEF14 fimbriae were elaborated poorly but with best yields at pH 4.04. SEF17 fimbriae were elaborated in planktonic growth at 20°C, but not at 37°C, at pH 6.18 and above but not at pH 5.09 or below; whereas on agar gradient plates SEF17 fimbriae were elaborated well even at pH 4.65. SEF21 fimbriae were expressed very poorly under all conditions tested. Planktonic growth at 37°C induced least flagella whereas growth at 20°C, and particularly surface growth at lower pH values, induced a ‘hyper-flagellate’ phenotype. Single colonies allowed to form on gradient agar plates were shown to generate different colonial morphologies which were dependent on initial pH. These results demonstrate that the physicochemical environment is an important determinant of bacterial response, especially the induction of putative virulence factors.

The aim of this study was to search for putative microbial agents in Crohn's disease (CD) tissues by bacterial broad-range 16S rDNA PCR combined with genus- and species-specific DNA hybridisation analysis. Biopsies taken both surgically and endoscopically from the terminal ileum of 11 CD patients and 11 control patients were investigated. Significant amounts of eubacteria were demonstrated in biopsies taken endoscopically from both affected and unaffected individuals; the biopsies taken at surgery from control patients were negative. Three of five biopsies taken surgically from CD patients harboured Helicobacter spp.-, Mycobacterium paratuberculosis-, Listeria monocytogenes- and Escherichia coli-like 16S rDNA sequences. These findings show the importance of the sampling method chosen when combined with molecular typing of eubacteria in intestinal tissues. The mixed bacterial flora found in the surgical biopsies from CD patients supports the idea that the enteric microflora enters primary lesions where secondary bacterial colonisers may elicit a chronic inflammatory syndrome.

A total of 81 Pasteurella multocida isolates from healthy and diseased dairy and beef cattle originating from various geographical locations was examined by rRNA gene restriction site polymorphism analysis (ribotyping), restriction endonuclease analysis (REA), SDS-PAGE analysis of whole-cell (WCP) and outer-membrane (OMP) proteins, and capsule and somatic serotyping. Bacterial strains were isolated from nose, lung and in one case testicle, of Holstein and cross-bred beef cattle. The isolates represented for the most part serogroup A3 (88%). Ribotyping was performed on DNA digested with HaeII, electrophoresed and then hybridised with 32P-labelled 16S–23S rRNA from Escherichia coli. Six ribotypes (R1–R6) and 10 REA types were found among the 81 isolates with similar discrimination index (DI) of c. 0.60. Protein profiles revealed reproducibility and high levels of polymorphisms among lung isolates. Isolates were compared according to their geographical habitat, their isolation from dairy or from beef cattle and from nasal cavities or lungs. No correlation was apparent between geographical locations and ribotypes. Overall, isolates obtained from dairy cattle were predominantly R1, whereas those obtained from beef cattle were equally distributed between R1 and R2. R1 was more representative of lung isolates. For some strains, particularly the single isolate ribotypes, good correlation was achieved between WCP analysis, REA types and ribotypes. For others, REA to some extent and WCP profiles were able to discriminate among isolates within ribotypes. The data suggest that a combination of ribotyping, REA and WCP analysis is useful for investigating the epidemiology of bovine P. multocida serogroup A.

The diversity of 103 clinical isolates of the Acinetobacter calcoaceticus–Acinetobacter baumannii complex obtained between 1991 and 1997 from 17 Czech hospitals was studied by ribotyping, biotyping, plasmid profiling and antibiotic susceptibility testing. According to the EcoRI ribotypes, all but one of these isolates were identified to the DNA group level: 77 isolates were allocated to DNA group 2 (A. baumannii), 14 to DNA group 3, 10 to DNA group 13 sensu Tjernberg and Ursing and one to DNA group 1 (A. calcoaceticus). In total, 50 different EcoRI ribotypes and 10 biotypes were observed. Plasmids were found in 92% of the isolates and a high variability in plasmid profiles was found in isolates of the same DNA group. The combination of typing profiles allowed two predominant groups (termed A and B) to be distinguished among the A. baumannii isolates (37 and eight isolates, respectively) that shared a specific ribotype and were highly similar in other properties. These two groups comprised both sporadic and outbreak isolates and were found in most localities. Group A and B isolates were markedly more resistant to antibiotics than most of the remaining isolates, thus representing 85% of all multiresistant isolates. The features of groups A and B corresponded to those of two epidemic clones identified recently among hospital strains in north-western Europe.

Bacteriophage typing is currently the recognised methodology for the typing of methicillin-resistant Staphylococcus aureus (MRSA) in the UK. Bacteriophage typing is less discriminatory and does not type all isolates compared with some molecular methods for typing MRSA. Chromosomal genotyping by pulsed-field gel electrophoresis (PFGE) is increasingly recognised as an improved method for typing MRSA, providing increased discrimination and typability. In this study the results of a comparison of bacteriophage typing and PFGE typing and subtyping are presented for a large collection of isolates from the North-West of England. Isolates belonging to the most frequently isolated epidemic methicillin-resistant Staphylococcus aureus (EMRSA) bacteriophage types 15 and 16 were typed by PFGE with further discrimination of common PFGE types possible into a number of subtypes. These results for a large collection of isolates demonstrate the improved typing of MRSA with PFGE. The widespread acceptance of PFGE for typing MRSA isolates has been hampered by the lack of standardised methodologies. Recently, a standardised PFGE strain typing system, known as the GenePath system has become available. The results of an inter-laboratory comparison of PFGE typing for a collection of isolates demonstrated good reproducibility with this system.