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A prospective nationwide surveillance of invasive Haemophilus influenzae type b disease among adults (greater than or equal to 16 years old) was conducted in Finland during 1985 through 1988. Thirty-one cases were identified (annual incidence, 0.22/100,000). Of these infections, 71% occurred in patients with severe underlying conditions. The overall case fatality rate was 26%. Septicemia (13 patients) and pneumonia (seven patients) were the most common clinical manifestations of H influenzae type b infection; the others were epiglottitis (six patients), meningitis (three patients), and arthritis (two patients). Epiglottitis occurred in significantly younger patients, all of whom were women and four of whom were previously healthy. Subtyping of the H influenzae type b isolates according to the major outer membrane protein subtype, biotype, and lipopolysaccharide serotype showed that patterns that were uncommon (14%) among children were more common (27%) in the adults.

The sugar composition and the electrophoretic mobility in SDS-polyacrylamide gel electrophoresis of the various lipopolysaccharides (LPS) from clinical isolates of Haemophilus influenzae type b (Hib) were determined to correlate epidemiologic data with compositional data. Rabbit sera specific in Ouchterlony immunodiffusion for 10 different LPS (LPS 1-10) reacted with 647 or 690 Hib strains isolated from patients with invasive disease in various continents. Serotype 1 was predominant and was found in 550 isolates (80%). None of the Hib isolates reacted with antisera specific for LPS of two nonencapsulated isolates (LPS 5 and 6). Sugar analysis by gas-liquid chromatography of trimethylsilylated methyl glycosides revealed that the LPS of the 10 serotypes contained glucose, galactose, L-glycero-D-mannoheptose, and glucosamine in various proportions. LPS 1, 2, 8, and 9 contained the highest amounts of glucose and galactose relative to L-glycero-D-mannoheptose, which is considered present in constant amounts in H. influenzae LPS. LPS 1, 2, and 9 were most frequently found in invasive disease isolates.

Haemophilus influenzae meningitis was studied over a 2-year period in children less than 5 years of age in a population-based, prospective study in The Gambia, West Africa. A total of 77 cases were recorded. The incidence was 60 cases/100,000 children less than 5 years of age and 297 cases/100,000 children less than 1 year of age. The peak prevalence was at the age of 5 months; 83% of the children were less than 1 year old and 45% were less than 6 months old. Only 55% of cases recovered completely. The distance to the nearest hospital was an important predisposing factor for a fatal outcome. Two cases were secondary. Many patients were anemic and underweight. The observations show that H. influenzae meningitis in The Gambia has an incidence as high as that in the USA, but that it has 10-fold more devastating outcome. If the infection is to be prevented by vaccination in The Gambia, immunization will have to be given very early in life.

The titer and specificity of antibodies to the infecting Haemophilus influenzae was determined in sera and sputa from 27 patients with chronic obstructive pulmonary disease (COPD) to analyze the specific immune response. COPD patients had significantly higher serum IgG and IgA antibody titers than 13 healthy controls (mean IgG titers 12,302 and 5,623, respectively; mean IgA titers, 2,398 and 912; p less than 0.001). The mean IgM titers were comparable: 501 and 447, respectively. Specific IgA antibodies were also detectable in the sputum of the COPD patients (mean IgA antibody titer, 776). The local antibody production was determined by calculating the relative coefficient of excretion (RCE) to albumin. The mean RCE of 89.1 for IgA indicated statistically significant local production (p less than 0.02), in contrast to a nonsignificant increase for IgG (mean RCE of 3.6). Specific IgM was below the detection level. Immunoblotting experiments showed that the antibodies in sera from COPD patients and controls were directed against most of the outer membrane proteins of H. influenzae, with individual differences between IgG, IgA, and IgM. The IgA and IgG antibodies in serum had a similar specificity as those in sputum. The appearance or persistence of H. influenzae coincided with minor changes in antibody titer and specificity. From these results we conclude that COPD patients are infected with H. influenzae despite the presence of at least as many antibodies in sputum and serum as in controls and that these antibodies are directed against a variety of antigenic determinants of the infecting strain.

The outer membrane protein composition of Haemophilus influenzae grown under a variety of culture conditions including growth in sputum and serum, and intraperitoneally in rats was analyzed. The pattern of the major outer membrane proteins, a, b,c, d, e and P6 remained very similar under all these conditions. Outer membrane proteins expressed during iron limitation were also expressed in bacteria growing in rats, in serum or in sputum. To determine the expression of the major outer membrane proteins and lipopolysaccharide in patient materials (sputum, cerebrospinal fluid, postmortem tissue) monoclonal antibodies specific for the outer membrane proteins a, b,c, d and P6 as well as lipopolysaccharide were used in immunoblotting. They showed the same reaction patterns with bacteria in the patient materials as with the bacteria isolated from these specimens. We conclude that the major outer membrane components expressed under in vitro conditions are also expressed in various clinical materials during infection.

Ninety-five percent of systemic Haemophilus influenzae infections in childhood are caused by serotype b organisms. The high risk for mortality and serious sequelae and the increase in antibiotic resistance of H influenzae type b are strong arguments for vaccination. Incidences and age distribution of disease caused by H influenzae type b vary considerably among countries. Compiled data for meningitis show the highest incidences to be among Alaskan Eskimos, Navajo and White Mountain Indians, and aboriginals in Australia. High incidences coincide with a peak incidence in a younger age group. This differs from Finland, where 95% of the cases of disease caused by H influenzae type b occur in children older than 7 months of age. In general, incidences are low in industrial and high in nonindustrial areas. For example, in Gambia the highest age-specific incidence is at 4 to 5 months after birth (H.A.B., unpublished data, 1988). This implies that a vaccine and regimen similar to that used in Finland would not be as efficacious if used in Gambia because of differences in demographics and incidence. Vaccines that confer protection at 3 to 4 months of age are, therefore, strongly desired. Outer membrane proteins and lipopolysaccharides of H influenzae type b have been suggested as alternative vaccine candidates in conjugation with the capsular polysaccharide because they apparently can contribute to the virulence of H influenzae type b. The occurrence and immunogenicity of various outer membrane proteins and lipopolysaccharides among H influenzae type b in industrial and nonindustrial countries and their significance as epidemiologic markers for the spread of disease, the type of disease, the age of acquisition, and their association with antibiotic resistance will be reviewed in this article.(ABSTRACT TRUNCATED AT 250 WORDS)

15 out of 21 patients admitted to a pulmonary rehabilitation centre were infected with a non-encapsulated strain of Haemophilus influenzae. All isolates showed identical outer membrane protein patterns, harboured a 40 MD plasmid, produced beta-lactamase, and were resistant to amoxycillin, co-trimoxazole, chloramphenicol, and tetracycline. The strain was first isolated from sputum of another 3 patients in the same hospital ward. 2 of them later introduced it into the rehabilitation centre. The strain spread among the other patients over the next 2 months. The absence of a common iatrogenic source of the organism and its slow spread indicate that the most likely means of transmission was person to person.

1. Freeze-fracture electron microscopy and 31P-NMR spectroscopy on native and electrodialyzed lipopolysaccharide from Escherichia coli K12 cells, both above and below the phase transition temperature, are described. 2. Freeze-fracture electron microscopy of native lipopolysaccharide shows ribbon-like structures below (0 and 22 degrees C) and large vesicles above (37 degrees C) the phase transition temperature. Electrodialyzed lipopolysaccharide (sodium salt) occurs in ribbon-like structures at 0, 22 and 37 degrees C if sodium lipopolysaccharide is hydrated in water. If sodium lipopolysaccharide is hydrated in Tris-HCL/NaCl buffer these ribbon-like structures occur only below the phase transition temperature. Above the phase transition temperature stacked sheets are observed. Moreover, in the latter case, the fracture planes contain particles and pits. Upon etching, sodium lipopolysaccharide when hydrated in water appears to form rods and when hydrated in buffer appears to form mainly stacked lamellae both above (37 degrees C) and below (0 degrees C) the phase transition temperature. 3. High resolution 31P-NMR spectra show that the chemical shifts of the phosphorus atoms in native lipopolysaccharide differ from those in electrodialyzed lipopolysaccharide, probably due to conformational and compositional (the disappearance of ions and (poly)electrolytes) changes. The 31P-NMR spectra of native lipopolysaccharide dispersed in Tris-HCL/NaCl buffer are very broad at 20 and at 40 degrees C indicating little motion. At 22 degrees C electrodialyzed lipopolysaccharide also gives a broad spectrum; at 40 degrees C the spectrum is narrower, indicating more motion, and two peaks are visible. After dispersion in H2o and subsequent addition of buffer, the spectrum of electrodialyzed lipopolysaccharide is narrow both at 20 and 40 degrees C, which can be correlated with the rods observed in freeze etching. After treatment with Ca2+, electrodialyzed lipopolysaccharide shows a very broad spectrum at 40 degrees C probably due to immobilization of the lipopolysaccharide. 4. Freeze-fracture electron microscopy and 31P-NMR spectroscopy of liposomes consisting of native lipopolysaccharide and total phospholipids indicate that the phospholipids and the lipopolysaccharide are mainly organized in bilayers. Lipopolysaccharide in such liposomes undergoes more motion than in the absence of phospholipids. Ca2+ does not influence this behaviour.

In a previous paper (A. Verkleij, L. van Alphen, J. Bijvelt, and B. Lugtenberg, Biochim. Biophys. Acta 466:269-282, 1977) we have hypothesized that particles on the outer fracture face of the outer membrane ([Formula: see text]), with corresponding pits on the inner fracture face of the outer membrane ([Formula: see text]), consist of lipopolysaccharide (LPS) aggregates stabilized by divalent cations and that they might contain protein and/or phospholipid. In the present paper the roles of LPS, cations, and proteins in these [Formula: see text] particles are described more extensively, using a strain that lacks the major outer membrane proteins, b, c, and d (b(-) c(-) d(-)), and has a reduction in the number of [Formula: see text] particles of 75%. To study the role of divalent cations in the formation of [Formula: see text] particles, these b(-) c(-) d(-) cells were grown or incubated with Ca(2+), Mg(2+), or putrescine. The presence of Ca(2+) resulted in the appearance of many [Formula: see text] particles and [Formula: see text] pits. Mg(2+) and putrescine were less effective than Ca(2+). Introduction of these particles was not accompanied by alterations in the relative amounts of LPS and cell envelope proteins. Ca(2+) treatment of a heptoseless derivative of a b(-) c(-) d(-) strain did not result in morphological changes. Incubation of Ca(2+)-treated cells with ethylenediaminetetraacetate caused the disappearance of the introduced particles as well as the release of more than 60% of the cellular LPS. These results strongly support the hypothesis that LPS is involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The roles of various outer membrane proteins in the formation of [Formula: see text] particles were studied by comparing the freeze-fracture morphology of b(-) c(-) d(-) cells with that of cells which contain one of the outer membrane proteins b, c, d, and e or the receptor protein for bacteriophage lambda. The results showed that the presence of any of these five proteins in a b(-) c(-) d(-) background resulted in a large increase in the number of [Formula: see text] particles and [Formula: see text] pits, indicating that these proteins are, independent of each other, involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The simplest explanation for the results is that in wild-type cells each particle consists of LPS complexed with some molecules of a single protein species, stabilized by either divalent cations or polyamines. It is hypothesized that the outer membrane of the wild-type cell contains a heterogeneous population of particles, of which 75% consists of protein b-LPS, protein c-LPS, and protein d-LPS particles. A function of these particles as aqueous pores is proposed.

1. At the growth temperature the total phospholipids isolated from Escherichia coli cells give rise to 31P-NMR spectra which indicate the existence of lamellar, isotropic and hexagonal phases. These phases are also detected by freeze fracture electron microscopy. In particular, the isotropic phase may contain lipidic particles (possibly inverted micelles) associated with the lamellar phase. 2. The cytoplasmic membrane isolated from E. coli cells grown at 37 degrees C is mainly lamellar at 25 degrees C, whereas at 37 and 45 degrees C the presence of some almost isotropic phospholipid motion is indicated. The possible significance of the isotropic phase for the functioning of the cytoplasmic membrane is discussed.

Of 275 consecutive Haemophilus influenzae type b (Hib) strains isolated from children with invasive disease in Finland in 1985-86, 74% were of the common European outer membrane protein (OMP) subtype 1 and 22% were of OMP subtype 1c, which is usually rare. Strains of subtype 1c caused proportionately more meningitis and less epiglottis than did strains of subtype 1c. Furthermore, children with disease due to strains of subtype 1c were younger than those with disease due to strains of subtype 1. The significant difference in association between subtype and the diagnosis of epiglottitis or meningitis remained even when the strong influence of age was accounted for. This finding may suggest a true difference in the virulence between these subtypes not previously demonstrated for Haemophilus influenzae type b.

The hypothesis that intramembraneous particles, observed in the outer membrane of Escherichia coli by freeze-fracture electron microscopy, are the morphological representation of aqueous pores, was tested. A mutant which is deficient in five major outer membrane proteins, b, c, d, e and the phage lambda receptor protein, contains a largely decreased number of intramembraneous particles and also shows a greatly decreased rate of uptake of several solutes. In derivatives of this strain which contain only one of these proteins in large amounts a strong decrease of the number of intramembraneous particles is observed, which is accompanied by a complete restoration of the rate of uptake of those solutes which use pores in which the protein in question is involved. The results provide strong evidence for the notion that an individual pore contains only one protein species, a property which has been found earlier for individual particles. The observed correlation between particles and equeous pores strongly supports the hypothesis that the particles are the morphological representation of pores. Implications of this hypothesis for the structure of the particles are discussed.

The components of the cell envelopes of Escherichia coli O1:K1, O7:K1, O18:K1 and O83:K1 strains were separated on SDS-polyacrylamide gels. Longitudinal slices (50 microns thick) of the gel were incubated with typing sera for E. coli O1, O7, O18 and O83, followed by detection of the bound antibodies with 125I-labelled protein A and autoradiography. The antisera reacted with many cell envelope components of strains both with the homologous O-serotype and heterologous O-serotypes. With O-typing sera cross-reactions with heterologous cells and cells boiled for 2 h were found. Up to 40 serotype-specific bands at regular positions with molecular weights between 12000 and 100000 were demonstrated. Since these bands were also observed when purified lipopolysaccharide and unabsorbed homologous O-typing sera were used, it was concluded that these bands represented lipopolysaccharide molecules with increasing molecular weight, all of which contained O-antigen specific immunodeterminants. The band patterns were not influenced by the growth conditions of the cells or the various isolation procedures for the cell envelopes. Comparison of various strains serotyped as O18 revealed strain differences with respect to their lipopolysaccharide band patterns. In the case of O21- and O83-serotyped strains lipopolysaccharide cross-reactions, which were detected by agglutination, were analysed in detail using the gel immunoradioassay method. These cross-reactions appeared to be caused by the presence of common determinants on their lipopolysaccharides and polysaccharide-like material. The cross-reacting antibodies could be removed by cross-absorption. It is concluded that the immunological detection of lipopolysaccharides and other components of E. coli in gels is an important tool in (1) the control of the specificity of typing antisera, (2) the study of the nature of cross-reacting antigens and (3) the study of the nature and uniformity of the various O- and K-serotypes.

The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Me1 resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptor-complex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.

Freeze fracturing electron microscopy of Escherichia coli K12 cells showed that the outer fracture face of the outer membrane is densily occupied with particles. On the inner fracture face of the outer membrane, pits are visible, which are probably complementary to the particles at opposite fracture face. This observation suggests that the particles are micelle-like. In some mutants which lack one or more major outer membrane proteins the density of particles is reduced. The loss of protein d appeared to a prerequisite for this phenomenon. However, mutants which lack all glucose and heptose-bound phosphate in their lipopolysaccharide also have a reduction in particle density whereas, the amount of protein d is normal. Moreover, loss of lipopolysaccharide by EDTA treatment also caused a reduction in the density of particles. From these results it is hypothesized that the particles consist of lipopolysaccharide aggregates stabilized by divalent cations and probably complexed with protein and/or phospholipid.

Inhibition of adherence of bacteria to epithelial cells contributes to a reduction of infections by these bacteria. We have shown that the Anton blood group antigen, the erythrocyte receptor for Haemophilus influenzae (van Alphen et al. 1986, FEMS Microbiol. Lett. 37, 69-71), occurs in saliva, that the occurrence is not related to the secretor state of the donor of the saliva and that saliva containing Anton antigen could not inhibit the adherence of H. influenzae to oropharynx epithelial cells. Anton antigen was detected in saliva samples of 14 donors by immunoblotting with two different anti-Anton sera. The amount of Anton antigen correlated with the ability of H. influenzae to adhere to the epithelial cells of the donor of the saliva: 4.1 +/- 0.1 Anton antigen units for donors with more than 50 H. influenzae per cell and 1.6 +/- 0.5 units for donors with less adhering epithelial cells. No correlation between the amount of Anton antigen in saliva and secretor status of the donor was observed. Adherence of H. influenzae to epithelial cells was not inhibited by saliva of secretors (N = 11) or non-secretors (N = 3). The same saliva did not inhibit the interaction of the bacteria with Anton antigen bearing erythrocytes as measured by haemagglutination inhibition. This indicates that the amount of Anton antigen in saliva is probably too low to interfere with the interaction of H. influenzae with oropharynx epithelial cells and erythrocytes.

1. The outer membrane of a phospholipase A-deficient mutant of Escherichia coli K12, isolated without the use of EDTA and lysozyme, showed the same freeze-fracture morphology as that seen in cells and remained stable for hours as observed by 31P-NMR. 2. 31P-NMR spectroscopy of the isolated outer membranes revealed that the lipopolysaccharide exists in the same physical state as in phospholipid-lipopolysaccharide liposomes and is most probably arranged in a bilayer at 37 degrees C. The outer membrane contains most or all of the phospholipids at 37 degrees C, and all the phospholipids at 20 degrees C, as a bilayer. 3. The 31P-NMR spectroscopy of the outer membranes from a mutant strain lacking the major outer membrane protein b, c and d (60% of the total outer membrane protein) yields virtually the same spectrum as the wild-type outer membranes, although most of the particles and pits which were observed in wild-type outer membranes in freeze-fracture electron microscopy were absent. 4. Whereas treatment of wild-type outer membranes with calcium ions has no effect on the 31P-NMR spectrum, treatment with EDTA results in more motion of the lipopolysaccharide.

Consecutive Hemophilus influenzae type b (Hib) isolates (333 total) from children with invasive disease in Finland in 1985-1986 were analyzed. All belonged to the common genetic clusters described in the USA and Europe. However, detailed typing demonstrated some characteristics unique to Hib strains in Finland. Of the isolates, 86% belonged to one of four distinct patterns according to the combination of outer membrane protein subtype, biotype, and lipopolysaccharide serotype: 1-I-1 (25%), 1-II-9 (8%), and 1c-I-1 (18%). Pattern 1-II-9 has not been previously reported; it was most commonly found in the most densely populated area of Finland and among children cared for outside the home. Multilocus enzyme electrophoresis revealed that 87% of isolates with the pattern 1c-I-1 belonged to the electrophoretic type 21.8, which is seldom recovered from patients with invasive Hib disease in other countries.

1240 throat samples were processed during different seasons in 11 different communities of The Gambia (West Africa). The carriage rate for Haemophilus influenzae type b ranged from 0 to 33%, but often attained 10% or more, higher than that reported from other open communities. The duration of carriage was short (less than 3 months) and H. influenzae b was found in only 10% of the carriers isolated during the previous or the following survey. Children less than 5 years old carried H. influenzae b in their throat significantly more often than children older than 14 years (P less than 0.05). A high carriage rate did not correlate with the wet or dry season. The carriage rate of children in rural areas was similar to that of children in urban areas. Children in day-care centres or nurseries had a surprisingly low carriage rate (2%). The carriage rate of H. influenzae b was compared to the presence of H. influenzae subspecies in a random sample, which revealed that H. influenzae subspecies was found in 90% of the children under 5 years old. Encapsulated strains of H. influenzae were found in 25% of the same sample, two-thirds of which were not type b. All capsule types were represented. No meningitis cases occurred in the survey populations. We conclude that the prevalence of H. influenzae b in open Gambian communities is similar to that in closed communities elsewhere, but that the kinetics are different from those in closed communities, as persistence of infection in Gambian children is short-lived.

Phospholipids in whole cells of wild type Escherichia coli K12 are not degraded by exogenous phospholipases, whereas those of isolated outer membranes are completely degraded. It is concluded that the resistance of phospholipids in whole cells is due to shielding by one or more other outer membrane components. The nature of the shielding component(s) was investigated by testing the sensitivity of whole cells of a number of outer membrane mutants. Mutants lacking both major outer membrane proteins b and d or the heptose-bound glucose of their lipopolysaccharide, are sensitive to exogenous exogenous phospholipases. Moreover, cells of a mutant which lacks protein d can be sensitized by pretreatment of the cells with EDTA. From these results and from data on the chemical composition of the outer membranes, it is concluded that proteins b and d, the heptose-bound glucose of lipopolysaccharide and divalent cations are responsible for the inaccessibility of phospholipids to to exogenous phospholipases.

The percentage of beta-lactamase producing Haemophilus influenzae strains from patients with meningitis in The Netherlands increased from 0% in 1975/1976 to 4.6% in 1985/1986 (n = 1559). Twenty-three isolates resistant to ampicillin, penicillin, chloramphenicol, rifampicin and/or tetracycline were subtyped to determine if one resistant strain was spreading. (Sub)typing was performed by capsular typing, analysis of the major outer membrane protein patterns on sodium dodecylsulfate gels (SDS-PAGE subtypes), lipopolysaccharide serotyping and biotyping. The (sub)types of the resistant strains were similar to those of sensitive strains, thus indicating that antibiotic resistant strains develop at random.