Single Outer Membrane Research Articles

Perspectives and Challenges on the Potential Use of Exosomes in Bioartificial Pancreas Engineering

Exosomes are enclosed within a single outer membrane and exemplify a specific subtype of secreted vesicles. Exosomes transfer signalling molecules, including microRNAs (miRNAs), messenger RNA (mRNA), fatty acids, proteins, and growth factors, making them a promising therapeutic tool. In routine bioartificial pancreas fabrication, cells are immobilized in polymeric hydrogels lacking attachment capability for cells and other biological cues. In this opinion article, we will discuss the potential role that exosomes and their specific biofactors may play to improve and sustain the function of this bioartificial construct. We will particularly discuss the challenges associated with their isolation and characterization. Since stem cells are an attractive source of exosomes, we will present the advantages of using exosomes in place of stem cells in medical devices including the bioartificial pancreas. We will provide literature evidence of active biofactors in exosomes to support their incorporation in the matrix of encapsulated islets. This will include their potential beneficial effect on hypoxic injury to encapsulated islets. In summary, we propose that the biofactors contained in secreted exosomes have significant potential to enhance the performance of islets encapsulated in polymeric material hydrogels with perm-selective properties to provide immunoisolation for islet transplants as an insulin delivery platform in diabetes.

A novel pathway for outer membrane protein biogenesis in Gram-negative bacteria.

SummaryThe understanding of the biogenesis of the outer membrane of Gram‐negative bacteria is of critical importance due to the emergence of bacteria that are becoming resistant to available antibiotics. A problem that is most serious for Gram‐negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram‐negative bacterial outer membrane is therefore critical to developing new antimicrobial agents, as this membrane makes direct contact with the external milieu, and the proteins present within this membrane are the instruments of microbial warfare, playing key roles in microbial pathogenesis, virulence and multidrug resistance. To date, a single outer membrane complex has been identified as essential for the folding and insertion of proteins into the outer membrane, this is the β‐barrel assembly machine (BAM) complex, which in some cases is supplemented by the Translocation and Assembly Module (TAM). In this issue of Molecular Microbiology, Dunstan et al. have identified a novel pathway for the insertion of a subset of integral membrane proteins into the Gram‐negative outer membrane that is independent of the BAM complex and TAM.

High Pressure Freezing/Freeze Substitution Fixation Improves the Ultrastructural Assessment of Wolbachia Endosymbiont – Filarial Nematode Host Interaction

Background Wolbachia α-proteobacteria are essential for growth, reproduction and survival for many filarial nematode parasites of medical and veterinary importance. Endobacteria were discovered in filarial parasites by transmission electron microscopy in the 1970’s using chemically fixed specimens. Despite improvements of fixation and electron microscopy techniques during the last decades, methods to study the Wolbachia/filaria interaction on the ultrastructural level remained unchanged and the mechanisms for exchange of materials and for motility of endobacteria are not known.Methodology/Principal FindingWe used high pressure freezing/freeze substitution to improve fixation of Brugia malayi and its endosymbiont, and this led to improved visualization of different morphological forms of Wolbachia. The three concentric, bilayer membranes that surround the endobacterial cytoplasm were well preserved. Vesicles with identical membrane structures were identified close to the endobacteria, and multiple bacteria were sometimes enclosed within a single outer membrane. Immunogold electron microscopy using a monoclonal antibody directed against Wolbachia surface protein-1 labeled the membranes that enclose Wolbachia and Wolbachia-associated vesicles. High densities of Wolbachia were observed in the lateral chords of L4 larvae, immature, and mature adult worms. Extracellular Wolbachia were sometimes present in the pseudocoelomic cavity near the developing female reproductive organs. Wolbachia-associated actin tails were not observed. Wolbachia motility may be explained by their residence within vacuoles, as they may co-opt the host cell’s secretory pathway to move within and between cells.Conclusions/SignificanceHigh pressure freezing/freeze substitution significantly improved the preservation of filarial tissues for electron microscopy to reveal membranes and sub cellular structures that could be crucial for exchange of materials between Wolbachia and its host.

Chlamydia co‐opts the rod shape‐determining proteins MreB and Pbp2 for cell division

Chlamydiae are obligate intracellular bacterial pathogens that have extensively reduced their genome in adapting to the intracellular environment. The chlamydial genome contains only three annotated cell division genes and lacks ftsZ. How this obligate intracellular pathogen divides is uncharacterized. Chlamydiae contain two high-molecular-weight (HMW) penicillin binding proteins (Pbp) implicated in peptidoglycan synthesis, Pbp2 and Pbp3/FtsI. We show here, using HMW Pbp-specific penicillin derivatives, that both Pbp2 and Pbp3 are essential for chlamydial cell division. Ultrastructural analyses of antibiotic-treated cultures revealed distinct phenotypes: Pbp2 inhibition induced internal cell bodies within a single outer membrane whereas Pbp3 inhibition induced elongated phenotypes with little internal division. Each HMW Pbp interacts with the Chlamydia cell division protein FtsK. Chlamydiae are coccoid yet contain MreB, a rod shape-determining protein linked to Pbp2 in bacilli. Using MreB-specific antibiotics, we show that MreB is essential for chlamydial growth and division. Importantly, co-treatment with MreB-specific and Pbp-specific antibiotics resulted in the MreB-inhibited phenotype, placing MreB upstream of Pbp function in chlamydial cell division. Finally, we showed that MreB also interacts with FtsK. We propose that, in Chlamydia, MreB acts as a central co-ordinator at the division site to substitute for the lack of FtsZ in this bacterium.

Evidence of direct cell-cell fusion in Borrelia by cryogenic electron tomography

Some Borrelia species are the causative agents of tick-borne Lyme disease responsible for different disabilities depending on species and hosts. Borrelia are highly motile bacterial cells, and light microscopy shows that these spirochetes can associate with each other during movement. Using cryo-electron tomography, we observed closely associated Borrelia cells. Some of these showed a single outer membrane surrounding two longitudinally arranged cytoplasmic cylinders. We also observed fusion of two cytoplasmic cylinders and differences in the surface layer density of fused spirochetes. These processes could play a role in the interaction of Borrelia species with the host's immune system.

The Soluble Form of Bax Regulates Mitochondrial Fusion via MFN2 Homotypic Complexes

In mammals, fusion of the mitochondrial outer membrane is controlled by two DRPs, MFN1 and MFN2, that function in place of a single outer membrane DRP, Fzo1 in yeast. We addressed the significance of two mammalian outer membrane fusion DRPs using an invitro mammalian mitochondrial fusion assay. We demonstrate that heterotypic MFN1-MFN2 trans complexes possess greater efficacy in fusion as compared to homotypic MFN1 or MFN2 complexes. In addition, we show that the soluble form of the proapoptotic Bcl2 protein, Bax,positively regulates mitochondrial fusion exclusively through homotypic MFN2 trans complexes. Together, these data demonstrate functional and regulatory distinctions between MFN1 and MFN2 and provide insight into their unique physiological roles.

The Long and the Short of Bacterial Adhesion Regulation

The Long and the Short of Bacterial Adhesion Regulation

Efflux Pump Contribution to Multidrug Resistance in Clinical Isolates of Pseudomonas aeruginosa

To determine if increased expression of efflux pumps, mutations in the genes encoding regulatory proteins for efflux pumps, or the combination is associated with multidrug-resistant (MDR) Pseudomonas aeruginosa isolates. Microbiologic evaluation of prospectively collected Pseudomonas aeruginosa isolates. University teaching hospital. ISOLATES: One hundred eight unique P. aeruginosa isolates-50 non-MDR and 58 MDR isolates-obtained from pulmonary or blood sources from patients admitted to the intensive care unit between January 1, 1999, and December 31, 2004. Isolates were considered MDR if they were resistant to at least three of the following four drugs: ciprofloxacin, tobramycin, ceftazidime, or imipenem. Possible mutations in efflux regulatory genes mexR, nfxB, and mexZ were analyzed by using polymerase chain reaction amplification and DNA sequencing. Determination of the expression of outer membrane proteins OprM and OprJ was performed by using sodium dodecyl sulfate- polyacrylamide gel electrophoresis immunoblotting. Differences in regulatory gene mutations and outer membrane protein expression were compared between non-MDR and MDR isolates. Among the 108 P. aeruginosa isolates, the MDR isolates were more likely to overexpress OprM compared with non-MDR isolates (64% vs 2%, p<0.001). Mutations in mexR and mexZ were present in 64% and 26% of MDR strains, respectively, but were not associated with OprM overexpression or multidrug resistance. Expression of OprJ was not associated with MDR isolates (odds ratio [OR] 3.7, 95% confidence interval [CI] 0.7-18.5, p=0.11). Mutations in nfxB (12% of MDR strains) were also not associated with multidrug resistance (OR 3.5, 95% CI 0.7-17.8, p=0.13). Eight (100%) of 8 isolates with OprJ expression plus OprM overexpression, 12 (92%) of 13 isolates with combined mexR and mexZ mutations, 5 (100%) of 5 isolates with nfxB plus mexZ mutations, and 16 (100%) of 16 isolates with OprM overexpression plus mexZ mutations were MDR isolates. The presence of one regulatory gene mutation or simple expression of a single outer membrane protein was not linked to multidrug resistance. However, OprM overexpression and multiple efflux regulatory gene mutations or efflux protein expression were associated with MDR P. aeruginosa isolates.

Mitofusins and OPA1 Mediate Sequential Steps in Mitochondrial Membrane Fusion

Mitochondrial fusion requires the coordinated fusion of the outer and inner membranes. Three large GTPases--OPA1 and the mitofusins Mfn1 and Mfn2--are essential for the fusion of mammalian mitochondria. OPA1 is mutated in dominant optic atrophy, a neurodegenerative disease of the optic nerve. In yeast, the OPA1 ortholog Mgm1 is required for inner membrane fusion in vitro; nevertheless, yeast lacking Mgm1 show neither outer nor inner membrane fusion in vivo, because of the tight coupling between these two processes. We find that outer membrane fusion can be readily visualized in OPA1-null mouse cells in vivo, but these events do not progress to inner membrane fusion. Similar defects are found in cells lacking prohibitins, which are required for proper OPA1 processing. In contrast, double Mfn-null cells show neither outer nor inner membrane fusion. Mitochondria in OPA1-null cells often contain multiple matrix compartments bounded together by a single outer membrane, consistent with uncoupling of outer versus inner membrane fusion. In addition, unlike mitofusins and yeast Mgm1, OPA1 is not required on adjacent mitochondria to mediate membrane fusion. These results indicate that mammalian mitofusins and OPA1 mediate distinct sequential fusion steps that are readily uncoupled, in contrast to the situation in yeast.

Effect of Energy Depletion and Antimicrobial Peptides on Single Protein Motility in Living Escherichia coli

Using optical tweezers the motility of a single outer membrane protein in E. coli bacteria, the λ-receptor, was studied. The λ-receptor is a porin that transports nutrients (maltodextrins) across the bacterial membrane. By poisoning the cells with arsenate and azide the bacterial metabolism was stopped. The motility of the exact same λ-receptor was measured before and after energy depletion. After energy depletion there was a significant decrease in the spread of positions visited. We have thus established that there is a difference in the movement of membrane proteins in living and dead cells. The active component of the motion of the λ-receptor in living cells has been modeled as an artificial temperature, which estimates the energy necessary for this active motion. The influence of antimicrobial peptides (AMPs) on the outer membrane of bacteria was investigated using the mobility of the λ-receptor as a membrane marker. With the growing resistance to antibiotics AMPs are gaining increased interested. Using the AMPs polymyxin B (PMB), and the non toxic derivate polymyxin B nonapeptide (PMBN), we have investigated the influence of AMPs on the outer bacterial membrane. Cells exposed to PMB showed a decrease in the spread of position visited by the λ-receptor upon poisoning. PMBN is known to increase the permeability of the outer membrane with out killing the cells. Exposure to PMBN did, however, not influence the mobility of the λ-receptor.

Mitochondrial matrix fragmentation as a protection mechanism of yeast Saccharomyces cerevisiae

It was shown that separate fragments of the inner mitochondrial compartment (mitoplasts) can exist under a single non-fragmented outer membrane. Here we asked whether fragmentation of the inner mitochondria could prevent rupturing of the outer membrane and release of pro-apoptotic molecules from the mitochondrial intermembrane space into the cytoplasm during mitochondrial swelling. First, we showed that in Saccharomyces cerevisiae yeast addition of amiodarone causes formation of electrically separate compartments within mitochondrial filaments. Moreover, amiodarone treatment of Deltaysp2 mutant produced a higher proportion of cells with electrically discontinuous mitochondria than in the wild type, which correlated with the survival of cells. We confirmed the existence of separated mitoplasts under a single outer membrane using electron microscopy. Mitochondria with fragmented matrixes were also detected in cells of the stationary phase. Our data suggest that such fragmentation acts as a cellular protective mechanism against stress.

Transport at the nanoscale: temperature dependence of ion conductance

Temperature dependent ion conductance in nanopores is measured in a wide range of electrolyte concentrations and compared with molecular modeling. Single outer membrane protein F (OmpF) channels from E. coli are reconstituted into planar lipid bilayers. In qualitative agreement with the experimental data, applied-field molecular dynamics unraveled atomistic details of the ion transport. Comparing the temperature dependence of the channel conductance with that of the bulk conductivity in the range from 0 to 90 degrees C revealed that at low salt concentrations the transport is mainly driven along the pore surface. Increasing the salt concentration saturates the surface charge transport and induces ion transport in the center of the nanopore. The confinement of the nanopore then favors the formation of ion pairs. Stepping up the temperature reduces the life time of the ion pairs and increases the channel conductance more than expected from the bulk behavior.

Molecular origin of the cation selectivity in OmpF porin: single channel conductances vs. free energy calculation

Ion current through single outer membrane protein F (OmpF) trimers was recorded and compared to molecular dynamics simulation. Unidirectional insertion was revealed from the asymmetry in channel conductance. Single trimer conductance showed particularly high values at low symmetrical salt solution. The conductance values of various alkali metal ion solutions were proportional to the monovalent cation mobility values in the bulk phase, LiCl<NaCl<KCl<RbCl∼CsCl, but the conductance differences were quantitatively larger than conductivity differences in bulk solutions. Selectivity measurements at low concentration showed that OmpF channels favored permeation of alkali metal ions over chloride and suggested size preference for smaller cations. These results suggest that there are specific interactions between the permeating cation and charged residues lining the channel walls. This hypothesis was supported by computational study which predicted that monovalent cations bind to Asp113 at low concentration. Here, free energy calculations revealed that the affinity of the alkali metal ions to its binding site increased with their atomic radii, Li +∼Na +<K +∼Rb +∼Cs +. A detailed inspection of both experimental and computational results suggested that stronger binding at the central constriction of the channel increases the translocation rate of cations under applied voltage by increasing their local concentration relative to the bulk solution.

Construction of recombinant chimeric human lactoferrin/bovine transferrins.

One of the contributing factors to bacterial pathogenesis is the ability to acquire iron (Fe) in the extremely Fe scarce environment of the host. The families Neisseriaceae and Pasteurellaceae, which include a number of human and veterinary pathogens, express outer membrane receptors that specifically bind and remove Fe from the hosts’ Fe binding proteins, transferrin (Tf) and lactoferrin (Lf)5. The bacterial Lf receptor was initially identified by affinity methods with immobilized human Lf (hLf) as the affinity matrix, using high salt and pH conditions to minimize nonspecific interactions11. These studies identified a single Fe-repressible outer membrane protein, lactoferrin binding protein (Lbp), in constrast to the two transferrin binding proteins, transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB), that were isolated with a Tf affinity column11. Lbp shared several properties with TbpA and once the gene encoding Lbp was cloned9 it was apparent that the predicted protein sequence was homologous to TbpA.

Structures and characteristics of novel siderophores from plant deleterious Pseudomonas fluorescens A225 and Pseudomonas putida ATCC 39167.

When Pseudomonas putida ATCC 39167 and plant-deleterious Pseudomonas fluorescens A225 were grown in an iron-deficient culture medium, they each produced two different novel yellow-green fluorescent pseudobactins: P39167-I, II and PA225-I, II. Pseudobactin P39167-I has a molecular formula of C46H65O23N13 and is monoanionic at neutral pH. P39167-II has the molecular formula of C46H63O22N13 and no charge at neutral pH. Pseudobactin PA225-I has a molecular formula of C46H65O24N13 and is monoanionic at neutral pH whereas pseudobactin PA225-II has the molecular formula of C46H63O23N13 and no charge at neutral pH. All four of the pseudobactins contain a dihydroxyquinoline-based chromophore. The amino acid sequence for the octapeptide in case of pseudobactins from P. putida ATCC 39167 is Chr-Ser(1)-Ala(1)-AcOHOrn-Gly-Ala(2)-OHAsp-Ser(2)-Thr. In case of pseudobactins from P. fluorescens A225, the octapeptide has the sequence Chr-Ser(1)-Ala-AcOHOrn-Gly-Ser(2)-OHAsp-Ser(3)-Thr. For all four pseudobactins (P39167-I, II and PA225-I, II), the serine(1) residue of the octapeptide is attached to the carboxylic acid group on the C-11 of the fluorescent quinoline via an amide bond. Additionally, for pseudobactin P39167-II and PA225-II, the hydroxyl group of the serine(1) residue is also attached to the carboxyl group of threonine residue at the carboxy terminus of the peptide via an ester bond, resulting in a cyclic depsipeptide in contrast to the linear peptide chain of P39167-I and PA225-I. For all four pseudobactins, a malamide group is attached to the C-3 of the quinoline derived chromophore. The three bidentate iron(III) chelating groups in all four pseudobactins consist of a 1,2-dihydroxy aromatic group of the fluorescent chromophore, a hydroxy acid group of beta-hydroxy aspartic acid, and a hydroxamate group from the acylated Ndelta-hydroxyornithine. The amino acid constituents of the pseudobactins P39167 I, II are the same as those in pseudobactin A214, whereas those in A225 I, II are the same as in 7SR1, but in both cases the sequences are different. The uptake results indicate a single outer membrane receptor protein for ferric-pseudobactins in both organisms. The receptor proteins in the two species are similar but not identical.

Mitosis in the flagellate Trypanoplasma borreli (Kinetoplastidea: Bodonida)

Summary The mitosis in both uninucleate and giant multinucleate T. borreli culture forms was studied using transmission electron microscopy. The interphase nucleus is of the vesicular type with one prominent nucleolus and dense peripheral chromatin. Nucleosome-like fibres, occasionally with scattered nodules on them of 21–23 nm in diameter, are found in the karyoplasm. The onset of mitosis is signalled by the decondensation of the peripheral chromatin. No individual chromosomes are visible during the whole division. In the course of mitosis the nucleus elongates, the nucleolus becomes fragmented, the nuclear envelope remains intact, and the kinetochore-like plaques and microtubules appear. At metaphase, the spindle microtubules are connected with fibrillar discs that adhere to the inner envelope membrane near nuclear poles. At anaphase, three main bundles of about 40 microtubules each can be found in the spindle. In giant cells the mitotic figures often lie inside a single outer membrane of the envelope, but at different angles to each other. We infer that mitosis in T. borreli involves a combination of a primitive membraneous mechanism of chromosome separation with a more advanced mode of chromosome transmission including spindle microtubules and associated structures. This type of nuclear division can be designated as intranuclear closed orthomitosis without discernible individual chromosomes.

Nosocomial transmission of disease caused by nontypeable strains of haemophilus influenzae

purpose: The authors evaluated a geographic and temporal cluster of lower respiratory tract infections due to unencapsulated (serologically nontypeable) Haemophilus influenzae to determine whether this event represented the transmission of a single clone. methods and materials: H influenzae was recovered from eight patients at a nursing home and from three patients in an adjacent acute care hospital. Serotypes, biotypes, outer membrane protein profiles, and multilocus enzyme genotypes were determined to characterize bacterial isolates. Patient records were retrospectively examined to determine clinical and epidemiologic characteristics. results: During a 10-day period in September 1991, lower respiratory tract infections caused by H influenzae were diagnosed in four patients residing in a single nursing home unit. Oropharyngeal cultures from four of seven asymptomatic roommates of these patients also grew H influenzae. During the month before and after the nursing home cluster of cases, four other individuals in acute care areas of the hospital had positive sputum cultures for H influenzae. Three of these latter specimens were also available for analysis. All H influenzae isolates were unencapsulated and β-lactamase-negative. Eight of the nine isolates from the nursing home patients (two morphologically distinct colony types of H influenzae were isolated from one case) had a single outer membrane protein profile arbitrarily designated as X and a single multilocus enzyme genotype arbitrarily designated as A. In contrast, none of the isolates from the acute care cases had this profile ( P <-0.02; two-tailed Fisher's exact test). The isolates obtained from two of the patients in acute care areas had an outer membrane protein profile arbitrarily designated as Y and a single multilocus enzyme genotype designated as B. These two patients were contemporaneously hospitalized in adjacent intensive care unit cubicles. The remaining isolates displayed an outer membrane protein profile arbitrarily designated as W. All roommates of the four patients in the nursing home were administered oral rifampin 600 mg daily for 4 days. H influenzae was not recovered from follow-up oropharyngeal cultures obtained 1 week after the completion of therapy. No β-lactamase-negative H influenzae were identified in this unit during the subsequent 9 months. conclusion: This study furnishes strong evidence for the nosocomial transmission of a clone of unencapsulated H influenzae in a nursing home unit. Epidemiologic data showed temporal and geographic clustering of respiratory tract infections and colonization by H influenzae. Outer membrane protein profiles and multilocus enzyme genotype analysis indicated that seven of eight patients at the nursing home carried a single clone of unencapsulated H influenzae. Laboratory and epidemiologic data also demonstrated the presence, and possible nosocomial transmission, of a second clone of unencapsulated H influenzae in a physically separate area of the hospital. Finally, although a causal relationship is not proven, the outbreak ended following the administration of rifampin prophylaxis of asymptomatic carriers.

Spirosymplokos deltaeiberi nov. gen., nov. sp.: variable-diameter composite spirochete from microbial mats.

Large (up to 100 micrometers long), loosely coiled, free-living spirochetes with variable diameters (from 0.4 to 3 micrometers in the same cell) were seen at least 40 times between August 1990 and January 1993. These spirochetes were observed in mud water and enrichment media from highly specific habitats in intertidal evaporite flats at three disjunct localities, one in Spain and two in Mexico. All three are sites of commercial saltworks. Associated with Microcoleus chthonoplastes the large spirochetes from Spain display phototaxis and a composite organization. Shorter and smaller-diameter spirochetes are seen inside both healthy and spent periplasm of larger ones. Small spirochetes attached to large ones have been observed live. From two to twelve spirochete protoplasmic cylinders were seen inside a single common outer membrane. A distinctive granulated cytoplasm in which the granules are of similar diameter (20-32 nanometers) to that of the flagella (26 nanometers) was present. Granule diameters were measured in thin section and in negatively-stained whole-mount preparations. Based on their ultrastructure, large size, variable diameter, number of flagella (3 to 6), and phototactic behavior these unique spirochetes are formally named Spirosymplokos deltaeiberi. Under anoxic (or low oxygen) conditions they formed blooms in mixed culture in media selective for spirochetes. Cellobiose was the major carbon source in 80% seawater, the antibiotic rifampicin was added, mat from the original field site was present and tubes were incubated in the light at from 18-31 degrees C. Within 1-2 weeks populations of the large spirochete developed at 25 degrees C but they could not be transferred to fresh medium.

Characterization of an immunoreactive 17.5-kilodalton outer membrane protein of Haemophilus somnus by using a monoclonal antibody.

A single outer membrane protein (OMP) of Haemophilus somnus, with an apparent molecular mass of 17.5 kDa, was identified in the sodium dodecyl sulfate (SDS)-insoluble fraction after extraction with 1% SDS-0.5 M NaCl-0.1% beta-mercaptoethanol. A hybridoma derived from mice immunized with H. somnus OMP fractions produced a monoclonal antibody (MAb), designated 20-3-5, that bound to the 17.5-kDa OMP of H. somnus. The MAb 20-3-5 epitope was present on 45 of 45 strains of H. somnus tested. MAb 20-3-5 cross-reacted with Haemophilus agni, Histophilus ovis, and Haemophilus haemoglobinophilus but not with 13 other species and subspecies of gram-negative bacteria. Immunoelectron-microscopic and antibody absorption studies revealed that the MAb 20-3-5 epitope is exposed on the surface of bacteria. In an immunoblot analysis, convalescent-phase sera obtained from calves with experimental H. somnus pneumonia contained antibodies to the 17.5-kDa OMP of H. somnus. Future studies will be directed toward examining the role of the 17.5-kDa OMP in immunity to H. somnus infections.

Outer membrane protein subtypes of Haemophilus influenzae type b isolates causing invasive disease in Victoria, Australia, from 1988 to 1990.

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.