Objective-To determine rates of colonisation with Haemophilus influenzae type b among household contacts
An Australia-wide survey of the prevalence of resistance to antimicrobial agents among Haemophilus influenzae was conducted on clinically significant isolates collected between July 1988 and September 1990. Laboratories from the capital cities of each Australian state and territory participated. Nine hundred and seventy clinical isolates were examined for beta-lactamase production and the MICs of ampicillin, coamoxiclav, chloramphenicol, cefaclor, ceftriaxone, cefotaxime, tetracycline, rifampicin, trimethoprim, sulphamethoxazole and co-trimoxazole were determined using the NCCLS agar dilution method with Haemophilus Test Medium. A smaller number of isolates were tested against penicillin V, penicillin G, ciprofloxacin, piperacillin and erythromycin in addition. The proportion of beta-lactamase producing strains was higher among invasive strains (21.6%) than non-invasive strains (14.2%) and varies considerably between states. The highest prevalence of ampicillin resistance was found in invasive strains from Canberra (40.8%), the lowest in non-invasive strains from Adelaide (5.1%). Paradoxically, in non-invasive strains, although beta-lactamase production was less common, resistance to other antimicrobials was commoner than in invasive strains and also varied between states.
Outer membrane protein subtyping of 187 isolates of Haemophilus influenzae type b (Hib), isolated from children with invasive Hib disease in Victoria, Australia, showed that a single outer membrane protein subtype (1VA) was responsible for 83% of the infections. It was identical to that responsible for the majority of cases of invasive Hib disease in Europe. Outer membrane protein (OMP) subtyping has been used to epidemiologically link cases of Haemophilus influenzae type b (Hib) disease in Europe and the United States. This report describes OMP subtypes from Hib cases in Victoria, Australia, and compares them with those previously reported. All Hib isolates from patients with invasive disease, admitted to the Royal Children's Hospital in Melbourne, Australia, between February 1988 and February 1990, were stored at-70°C. Cases of invasive Hib disease were defined as those in which Hib was isolated from samples of blood, cerebrospinal fluid (CSF), or other normally sterile sites or from the throats or epiglottides of patients with laryngoscopy-confirmed epiglottitis. Isolates were identified as Hib by colonial morphology, Gram stain, requirement for X and V factors, and agglutination with commercially available polyclonal Hib antiserum (Wellcome Diagnostics, Dartford, United Kingdom). OMP preparations were performed by a method similar to that of Barenkamp et al. (2), with modifications. Cells were harvested by centrifugation at 3,000 x g for 20 min washed, and resuspended in 1.5 ml of 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.4). They were frozen (-70°C), thawed, and sonicated on ice. Cellular debris was removed by centrifugation at 3,000 x g for 20 min. Cell envelopes were sedimented at 11,600 x g for 30 min at 4°C in a microcentrifuge. After extraction with 2% sodium lauryl sarcosinate in HEPES buffer, the detergentinsoluble fraction was washed and recovered by microcentrifugation as described above. The pellets were resuspended in 200 ,ul of sample buffer containing 63 mM Tris base, 10% glycerol, 2.3% sodium dodecyl sulfonate (SDS), and 5% beta-mercaptoethanol (pH 6.8). They were then heated at 100°C for 5 min and stored at-70°C until required. OMP preparations were analyzed by using SDS-polyacrylamide gel electrophoresis (PAGE), with a modified Laemmli 11% gel and a 4 to 24% density gradient gel (2). Gels were run with molecular weight standards (Bio-Rad Laboratories, Richmond, Calif.) and known OMP subtypes (provided by
The two most common manifestations of Haemophilus influenzae type b (Hib) infection in Western communities are meningitis and epiglottitis. The role of antibodies against outer membrane proteins (OMP) in the pathogenesis of these diseases was investigated by Western blotting (immunoblotting) with an OMP antigen prepared from a local Hib strain. Acuteand convalescent-phase serum samples from 25 children with epiglottitis and 20 with meningitis and single serum samples from 19 control children in the same age group were tested. Western blots were evaluated quantitatively by use of graphs generated from a densitometer. OMP antibody was detected in all sera from patients and controls. There was no significant difference between the mean antibody level in acute-phase sera from children with meningitis (336 + 143 arbitrary units) and those from children with epiglottitis (286 + 134 arbitrary units). However, the mean OMP antibody level in sera from healthy controls, with no known history of Hib disease, was significantly higher than that in sera from patients with Hib disease within 2 days of admission to the hospital (patients [n = 35], 282 + 144; controls [n = 19], 425 236; P = 0.007). The difference was due mainly to higher levels, in control sera, of antibody against four proteins, one of which is either P1 or a comigrating protein of 49 kDa. The finding of higher levels of OMP antibody in healthy controls suggests a protective role for antibodies directed against one or more OMP. This information could be exploited in future vaccine development.
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