- Volume 48, Issue 1, 1999

Adult female Sprague-Dawley rats were challenged intrabronchially with Bordetella pertussis strain 18-323 embedded in fine agarose beads and the time-course of infection and other events was determined. There was a steady decline in the numbers of B. pertussis recovered from the rat lungs, with clearance of the infection in most animals by day 12. Leucocytosis, lung inflammation and an increase in total serum IgE in the rats as a result of the challenge were highest around day 10, which was coincident with the highest incidence of coughing in such animals. IgG and IgA antibodies to the B. pertussis antigens pertussis toxin and filamentous haemagglutinin were not detected until after this period. The coughing rat model of pertussis resembles the human disease in the relationship between the time course of infection and cough production.

Porins, purified from Salmonella typhi strain 0901 provided 90% protection to BALB/c mice against a lethal dose (300 x LD50) of S. typhi Ty2 when given intraperitoneally. To measure the porin-induced cellular immune responses, macrophages and lymphocytes were isolated from spleen and lamina propria (LP) of porin immunised-challenge mice and of infected and control mice; T-cell phenotypes, lymphocyte proliferation and cytokine production were studied. The secretory IgA (sIgA) antibody level in the intestinal fluid was also measured to study mucosal immune response. After immunisation, the splenic lymphocytes exhibited a significant increase in total T-cell count and CD4+/CD8+ ratio, while the LP lymphocytes (LPL) exhibited an increase in CD4+/CD8+ ratio only. They also exhibited a significant increase in porin-specific proliferative response and cytokine levels (IL-1, IL-2, IFN-γ and IL-4). After immunisation, slgA antibody was also found to be increased. These results suggest that porins given intraperitoneally induce cellular and humoral immune responses both at systemic and mucosal levels.

All episodes of bacteraemia during a 15-year period (1981-1995) in the County of Northern Jutland, Denmark, were analysed with regard to antibiotic resistance. A total of 8840 isolates from 7938 episodes of bacteraemia was identified. Over time, no changes in bacterial aetiology were noted. Three isolates of Staphylococcus aureus were methicillin resistant (0.2%) and six were gentamicin resistant (0.4%). Among coagulase-negative staphylococci a 14% increase in resistance to penicillin was observed (95% confidence intervals, CI: 2-26%). Likewise, the frequency of resistance to methicillin, gentamicin and erythromycin increased, the corresponding figures being 38% (CI: 26-50%), 26% (CI: 14-38%) and 32% (CI: 16-50%), respectively, whereas a 14% decrease in resistance to streptomycin was recorded (CI: 4-24%). A 20% (CI: 2-37%) increase of coagulase-negative staphylococci resistant to three or more antibiotics was observed. The frequency of ampicillin resistance increased by 9% among Escherichia coli (CI: 4-13%) and by 10% (CI: 6-14%) in all Enterobacteriaceae. Among Enterobacteriaceae the level of resistance to third-generation cephalosporins, carbapenems, aminoglycosides and fluoroquinolones remained low (<1%). The frequency of resistance to three or more antibiotics remained fairly stable among Enterobacteriaceae, although a slight increase was noted among E. coli (5%; CI: 0-10%) The recommended regimen for empirical antibiotic treatment in this region (a combination of penicillin G or ampicillin and an aminoglycoside) provided an overall coverage of 94% (CI: 94-95%), although a slight decrease was noted at the end of the period. In conclusion, acquired antibiotic resistance was maintained at a low level compared with most other European countries and regions during the 15-year period studied.

Bifidobacteria are dominant in the gut of full-term infants, although colonisation by them is often delayed in preterm neonates. Bifidobacteria are recognised to have beneficial effects on digestive disorders and they might prevent neonatal necrotising enterocolitis (NEC), a gastrointestinal disease that predominantly affects premature infants. They have been shown to protect gnotobiotic quails against NEC-like lesions when the birds were inoculated with faecal flora from preterm infants, decreasing the clostridial population. The present study was designed to investigate whether oligofructose, which stimulates the activity of bifidobacteria, may enhance their protective role. Experiments were done in eight groups of germ-free quails for 28 days. The groups differed as to their bacterial status, diet and environment. Quails were inoculated with one of two flora from premature twins. The first flora included Bifidobacterium pseudo-catenulatum, Escherichia coli and no clostridia. The second flora included clostridial species and was associated with B. infantis-longum. Caecal bacterial population and metabolism changes were investigated with a lactose (6%) diet versus a lactose-oligofructose (3%-3%) diet, either in a gnotobiotic environment or in an ordinary environment permitting post-colonisation by exogenous bacteria. In both environments and with both flora, oligofructose significantly increased the level of bifidobacteria and this was associated with a decrease of E. coli or C. perfringens and C. ramosum. The bacterial changes in the ordinary environment depended on the initial composition of the microflora and the colonisation resistance against exogenous bacteria was more efficient with the flora that included B. pseudo-catenulatum. The changes in caecal pH and short-chain fatty acids were minimal. It was demonstrated that, irrespective of the environmental conditions, the use of oligofructose helped to prevent the overgrowth of bacteria implicated in necrotising enterocolitis in preterm neonates.

Virulence properties of 31 atypical enteropathogenic Escherichia coli (EPEC) strains isolated from cases of diarrhoea were examined. All except two strains adhered to HEp-2 cells in a localised adherence-like (LAL) pattern. With the exception of two strains, all were fluorescent actin staining (FAS) positive. Gentamicin HEp-2 invasion assay studies showed that all strains were invasive. Transmission electron microscopy of infected HEp-2 cells showed the characteristic attaching and effacing lesion and invasion of the cultured cells. Of the nine strains that hybridised with a DNA probe for α-haemolysin, five were haemolytic within 3 h of incubation, while the remaining strains were haemolytic only after incubation for 24 h. Three strains produced enterohaemolysin on blood agar. None of the 31 strains of E. coli induced fluid accumulation in the rabbit intestinal loop assay or displayed cytotoxic effects in HeLa and Vero cells. All the strains belonging to serotypes O26:H11, O26:H- and O119:H2 expressed intimin β, whereas all the strains from serotype O55:H7 expressed intimin γ. The strains belonging to serogroup O111 expressed a non-typable intimin. The participation of intimin in LAL was supported by adhesion inhibition experiments in which antibodies to intimin significantly reduced the level of LAL.

Control and prevention of methicillin-resistant Staphylococcus aureus (MRSA) infections should include early identification of patients at higher risk of MRSA acquisition and analysis of isolates by discriminatory bacterial DNA typing methods. One hundred and three MRSA isolates cultured between Sept. 1994 and Sept. 1995 from 62 patients in two teaching hospitals (hospital 1, in Rio de Janeiro; hospital 2, in Minas Gerais) were tested for antimicrobial resistance and genomic DNA was analysed by pulsed-field gel electrophoresis (PFGE). Ten profiles were identified: A, B, C, I and J in hospital 1 and A, B, D, E, F, G and H in hospital 2. PFGE patterns A and B were isolated at both hospitals. The majority (80%) of isolates had similar PFGE patterns (type A). Subtype A1 was isolated at both hospitals, but was more frequent in hospital 2 (54%), while subtype A2 predominated in hospital 1 (63%). MRSA isolates were resistant to the majority of antimicrobial agents tested. However, susceptibility to vancomycin alone was found in 32% of the isolates at hospital 1, whereas 48% of isolates from hospital 2 were susceptible to both vancomycin and mupirocin, and 34% demonstrated susceptibility to vancomycin, mupirocin and chloramphenicol. Thirty-nine percent of all isolates were mupirocin-resistant, with 90% of these belonging to PFGE pattern A. Four main risk factors were associated with MRSA infection or colonisation which may be useful in the early identification of patients at risk: >7 days hospitalisation (95%), very dependent patients (84%), invasive procedures (79%) and recent antimicrobial therapy (79%). The data demonstrate that PFGE pattern A is disseminated in both hospitals. However, at both hospitals subtypes of pattern A and the other PFGE types were associated with different antibiotic resistance patterns.

Typing methods utilising DNA technology were applied to a collection of Trichophyton mentagrophytes and T. rubrum isolates from skin and nail infections. The methods included restriction enzyme analysis (REA), hybridisation with the DNA probe poly (dG-dT), randomly amplified polymorphic DNA (RAPD) by PCR and restriction analysis of a segment of PCR-amplified rDNA. All these tests successfully differentiated the species, but few intra-species differences were detected. REA demonstrated some isolate variation, but this was limited and difficult to interpret, making it unsuitable as a typing tool. RAPD demonstrated few variations amongst T. mentagrophytes and none in T. rubrum.

Two enzyme immunoassay (EIA) systems were compared for their ability to detect Borrelia burgdorferi sensu lato specific IgG and IgM antibodies and to differentiate between symptomatic (83 patients with neuroborreliosis) and asymptomatic seropositive subjects (80 healthy controls). Antibody concentrations were determined by EIA; the antigens used were either a sonicate of B. burgdorferi or three recombinant borrelial proteins: the 14-kDa flagellin fragment, the outer surface protein C (22 kDa) and the high molecular mass protein p83 (83 kDa). In the sonicate, EIA, IgG or IgM antibodies to B. burgdorferi, or both, were detected in all patients with neuroborreliosis and in all controls. Pre-absorption of sera with Treponema phagedenis sonicate diminished the sensitivity of detection of borrelial specific IgG (IgG or IgM or both) antibodies in patients with neuroborreliosis from 80 to 57% (100 to 82%) and in the controls from 100 to 32% (100 to 37%). While being specific for B. burgdorferi, the recombinant EIAs proved to be significantly more sensitive than the sonicate EIA: IgG or IgM, or both antibodies against any of the recombinant antigens were detected in 92% of patients with neuroborreliosis and in 24% of controls. The increase in sensitivity in patients with neuroborreliosis was mostly due to the higher detection rate of IgM antibodies in the recombinant EIA (77% versus 48% in the sonicate EIA), while IgG antibodies were demonstrated with similar frequencies in both EIA systems (57% versus 60%). It was concluded that the recombinant EIAs are superior to the sonicate EIA with pre-absorption of cross-reactive antibodies in the confirmation of an acute borrelial infection and in the differentiation between symptomatic and asymptomatic infections.

Borrelia burgdorferi, the causative agent of Lyme disease, was first isolated in 1982 and since then has been regularly isolated from ticks and clinical material in both Continental Europe and the USA. However, only three isolations have been reported in Britain. During the summer of 1997, 128 ticks were collected from two sites in the Highlands of Scotland and examined by the polymerase chain reaction (PCR) and culture. Eleven fresh isolates were obtained from culture and passed up to 22 times. Seven of the tick emulsions were also positive by flagellin gene PCR, and a further one was positive by PCR but negative on culture. All 11 isolate cultures were positive by the flagellin gene PCR. Further studies on four of these isolates confirmed their identity by immunofluorescence, but also detected possible differences between them and B. burgdorferi ACA-1 by enzyme profiles and by PCR with OspA gene primers. Culture of these new strains provides antigens that should improve diagnostic serological tests in Britain.