Growth of Haemophilus influenzae type b in the presence of bovine aortal endothelial cells

In serum-free medium in the presence of bovine aortal endothelial cells (BAOEC), Haemophilus influenzae type b was capable of extensive proliferation compared to that in serum-free medium alone. An unidentified low-molecular-mass (less than 2000 kDa) compound(s) was, in part, responsible for this phenomenon. There were changes in the outer-membrane protein profiles between broth-grown (the original inoculum) and BAOEC-grown organisms, particularly in the 45-70 kDa range. Both broth- and BAOEC-grown bacteria were serum sensitive in vitro but could be converted to a serum-resistant phenotype, resembling that found in vivo, by incubation in a serum filtrate.     

Molecular characterization of three chloramphenicol acetyltransferases isolated from Haemophilus influenzae.

Three plasmid-mediated chloramphenicol acetyltransferases isolated from different Haemophilus influenzae strains were purified and characterized. All three enzymes had properties in common with the gram-negative family of chloramphenicol acetyltransferase. The Haemophilus enzymes and the enteric type II enzyme were sensitive to 5,5'-dithiobis(2-nitrobenzoic acid), gave the same elution patterns from a highly substituted resin containing a bound chloramphenicol base, and had similar reactions to antisera. All four differed from each other in subunit molecular weight, enzyme activity, and partial protein digestion patterns. The data suggest that the three Haemophilus enzymes belong to the less common type II group and are related, but is not identical, to each other and to the enteric type II enzyme.     

Iron-regulated outer-membrane proteins of Escherichia coli strains associated with enteric or extraintestinal diseases of man and animals

The SDS-PAGE patterns of the iron-regulated outer-membrane proteins from 70 strains of Escherichia coli isolated from various human and animal infections were analysed and the nature of the siderophores produced was examined. Iron-regulated 81 kDa and 74 kDa protein bands seen in SDS-PAGE gels were characterized further by immunoblotting using anti-81 kDa and anti-74 kDa (Cir) sera. The results showed considerable differences between the patterns of the iron-regulated outer-membrane proteins exhibited by the different strains. Nevertheless, three distinct and characteristic profiles, based on the most prominent bands expressed, could be identified, although not all strains produced patterns which matched with one of these. These results suggest the possibility of using the pattern of iron-regulated outer-membrane proteins expressed, as well as siderophores produced, as a new set of markers to characterize groups of pathogenic E. coli.     

Influence of iron restriction on growth and the expression of outer membrane proteins by Haemophilus influenzae and H. parainfluenzae

Abstract The growth of both Haemophilus influenzae and H. parainfluenzae was progressively inhibited in media containing increasing concentrations of the iron chelator desferrioxamine. Iron restriction had little effect on the outer-membrane (OM) protein profiles of type b or non-typable H. influenzae although replacement of haemin with protoporphyrin IX resulted in the induction of an M _r 73 000 protein in the type b strains. H. parainfluenzae , however, responded to iron restriction by inducing several new proteins in the range of M _r 70 000 to 86 000.     

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis

Abstract When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M _r of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N . meningitidis strains induce immune responses in vivo.     

Reduced expression of outer-membrane proteins in beta-lactam-resistant mutants of Enterobacter cloacae

Two antibiotic-resistant mutants of Enterobacter cloacae (AZT-R and AMA-R), obtained by selection with aztreonam and carumonam, were studied. Both mutants were resistant to beta-lactam antibiotics. In addition, AMA-R was also resistant to chloramphenicol, trimethoprim and brodimoprim, whereas AZT-R was hypersensitive to these compounds. Cytoplasmic and outer membranes of these bacteria were separated by sucrose density gradient centrifugation. Analysis of the outer membranes using SDS-PAGE showed marked changes in the bands corresponding to the porins (between 35 and 40 kDa). In the two mutants, the 39 kDa band was reduced to approximately 30% of the wild-type and the 36.5 kDa band was absent. Labelling of the outer membranes with the hydrophobic photolabel 3-(trifluoomethyl)- 3-(m-[125I]iodophenyl)diazirine ([125I]TID) enabled the above bands as well as a 28.8 kDa band to be identified as integral membrane proteins, thus supporting the suggestion that they correspond to porins and OmpA protein, respectively. Whereas the changes observed in outer-membrane proteins are assumed to be responsible for resistance to beta-lactam antibiotics, the basis of hypersensitivity of AZT-R to hydrophobic antibiotics remains to be more clearly defined.     

Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae.

Protein expression by Haemophilus influenzae under iron-limiting growth conditions was examined. The five type b strains and four nontypeable strains studied all expressed a new protein of about 40 kDa when deprived of iron during growth. Most strains also expressed a protein of about 31 kDa under the same growth conditions. Both the 40- and 31-kDa proteins were not expressed by cells grown in iron-replete medium. The 40- and 31-kDa proteins were not expressed in iron-deficient medium to which an excess of ferric nitrate had been added, and therefore it was concluded that their expression was iron regulated. These iron-repressed proteins were localized to the periplasmic space. The amino-terminal sequences of both proteins were determined. The N-terminal sequence of the 40-kDa protein had 81% similarity to the N terminus of Fbp, the major iron-binding protein of Neisseria gonorrhoeae and N. meningitidis. The 31-kDa protein sequence showed no homology with any known protein sequence. As no plasmids were found in the strains, it was concluded that these proteins were chromosomally encoded.     

Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.     

Identification and characterization of a conserved outer-membrane protein of Neisseria gonorrhoeae

A previous study in our laboratory identified a surface-exposed peptidoglycan-associated protein of Neisseria gonorrhoeae which had an apparent molecular mass of 44,000 daltons (44kDa) (Hill and Judd, 1989). This paper reports results which confirm that the 44kDa protein is surface-exposed, and that the protein is expressed in, and is structurally invariant among, 14 strains of N. gonorrhoeae. The fact that the 44kDa outer-membrane protein is found in a conserved form in all gonococci examined strongly suggests that it is crucial to the bacterium's survival. Moreover, it appears that this protein is a penicillin-binding protein (PBP3) (Shafer and Judd, 1991). This invariant, surface-exposed, peptidoglycan-associated outer-membrane protein deserves further investigation to elucidate its role in the immunobiology of N. gonorrhoeae, and its possible use as an immunoprophylactic reagent.     

Properties of the porin of Haemophilus influenzae type b in planar lipid bilayer membranes

The major outer membrane protein (40 kDa) of the bacterium Haemophilus influenzae type b is a porin which forms transmembrane permeability channels. It has an exclusion limit for oligosaccharides of about 1.4 kDa. When this protein was added to the aqueous phase which was bathing a planar lipid bilayer, it caused the conductance of the membrane to increase by several orders of magnitude. At low protein concentrations (2-10 pM), the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was very slightly cation-selective: the pores were about 1.6-times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit. A model is proposed which could explain the differences in apparent pore size.     

Serotypes and biotypes and antibiotic susceptibility of Haemophilus influenzae encountered in a clinical laboratory in Taiwan.

Forty-four serologically and biochemically typable Haemophilus influenzae isolates from clinical specimens in Taiwan were subjected to analysis in their relationship with source of isolation and age distribution. It was found that all isolates from blood and cerebrospinal fluid were serotype b, biotype I, and all were in children less than 4 years of age. Serotypes b and e, biotypes I and III were encountered to have the highest incidence of infection caused by H. influenzae in this area. All H. influenzae isolates were further tested for susceptibility to several selected antibiotics. All strains of this organism were susceptible to erythromycin and chloramphenicol. All but two strains were susceptible to tetracycline, whereas more strains were resistant to carbenicillin, gentamycin, keflin, and penicillin. Thirty-four percent strains were found to be resistant to ampicillin and all were beta-lactamase producer. No direct correlation between ampicillin resistance and serotypes or biotypes was recognized.     

Antibiotic resistance in Streptococcus pneumoniae and Haemophilus influenzae. Report of a study group on bacterial resistance.

Twenty laboratories in England and Scotland took part in 1977 in a survey of antibiotic resistance in Streptococcus pneumoniae and Haemophilus influenzae. In Str pneumoniae 59 (6.8%) of the 866 strains studied were resistant to tetracycline and three to chloramphenicol, and one strain showed a decreased susceptibility to penicillin. The prevalence of resistance to tetracycline was lower than that found in a similar study performed in 1975. Nine hundred and fifty-two strains of H influenzae were examined: 15 (1.6%) were resistant to ampicillin (all were beta-lactamase producers) and 26 (2.7%) to tetracycline. Only two strains were resistant to chloramphenicol and two to trimethoprim. Sixty-three H influenzae strains were capsulated. Thirty-four of these were of Pittman type b, and antibiotic resistance, particularly to ampicillin, was more common in these than in other serotypes or non-typable strains. Some variation was seen in the resistance rate of both H influenzae and Str pneumoniae to tetracycline in strains from different centres, but too few were isolated to assess whether this represented a true geographical difference.     

Prevalence and chemosusceptibility of Neisseria meningitidis and Haemophilus influenzae in a population of central Italy

The purpose of this study was to determine the prevalence of nasopharyngeal carriers of Neisseria meningitidis and Haemophilus influenzae, and to evaluate their chemoresistance. N. meningitidis was more frequently isolated in adolescents (10-15 years). All of meningococci were susceptible to ceftriaxone and rifampin, only one isolate showed reduced susceptibility to chloramphenicol and four strains showed reduced penicillin susceptibility. The results show that these drugs are still effective for prophylaxis and treatment in our area. All strains of H. influenzae were susceptible to penicillin, ceftriaxone, chloramphenicol, rifampin, azithromicin and gentamicin. 6 nontypeable strains were resistant to sulfamethoxazole-trimethoprim, 7 strains of type a and c-f, and 3 non-typeable strains showed reduced susceptibility to tetracycline. In contrast with the current trend in the world, in our area the susceptibility of H. influenzae to betalactams was 100%, therefore these antibiotics are still the drugs of choice for treatment of invasive diseases.     

HEp-2 adhesion and the expression of a 94 kDa outer-membrane protein by strains of Escherichia coli belonging to enteropathogenic serogroups

Sixty strains of Escherichia coli belonging to enteropathogenic serogroups (EPEC) were examined for the ability to adhere to HEp-2 cells, the possession of the genes encoding EPEC adherence factor (EAF) and the ability to express an outer-membrane protein (OMP) of 94 kDa thought to be involved in bacterial adhesion to eukaryotic cells. An absolute correlation was found between HEp-2 adhesion and the possession of the genes encoding EAF. An OMP of 94 kDa was observed in the SDS-PAGE profile of most adhesive strains. In some strains this protein was prone to proteolytic degradation. An antiserum raised to a HEp-2 adhesive strain of EPEC did not react with the 94 kDa OMP of all EPEC which were EAF-positive and HEp-2 adhesive, indicating some interstrain antigenic variation of this protein. Although this 94 kDa protein was surface-exposed, specific antibodies binding to the 94 kDa protein in situ in the outer membrane did not interfere with adhesion of EPEC to HEp-2 cells. Therefore, these studies question the value of this protein as a potential vaccine component.     

Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons.

The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.     

Increase in antibiotic resistance in Haemophilus influenzae in the United Kingdom since 1977: report of study group.

A survey of antibiotic resistance in Haemophilus influenzae was carried out in the United Kingdom with 25 laboratories participating. The incidence of resistance in the 1841 strains examined was: tetracycline 3.1%, ampicillin 6.2%, chloramphenicol 1.03%, trimethoprim 1.4%, and sulphamethoxazole 1.5%. Of the 115 strains resistant to ampicillin, 106 produced beta-lactamase. Seventy-nine strains were capsulate, none of which was chloramphenicol resistant, but nine produced beta-lactamase (11.4%). Comparison of these figures of antibiotic resistance with those from a similar survey performed in 1977 showed a significant increase in resistance of H influenzae to ampicillin, chloramphenicol, and trimethoprim.     

Proteome of Haemophilus ducreyi by 2-D SDS-PAGE and mass spectrometry: strain variation, virulence, and carbohydrate expression

We have analyzed the proteome of several strains of Haemophilus ducreyi by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Over 100 spots were analyzed from the soluble and insoluble protein fractions from the prototype strain 35000HP and 122 distinct proteins were identified. Functions of approximately 80% of the 122 proteins were deduced by identification with close homologues of Haemophilus influenzae. Four additional wild type and three mutant strains were also analyzed that vary in their virulence and/or outer-membrane lipooligosaccharide structures. Overall, the 2-DE gel maps of the wild type and mutant strains were similar to strain 35000HP, suggesting little proteome diversity in relation to carbohydrate expression and/or virulence. An exception was the Kenyan strain 33921 which contained significant differences in its proteome 2-DE map and also synthesizes an unusual LOS with a trisaccharide branch structure. This African strain may represent a prototype of a second clonal group of H. ducreyi.     

Novel aerobactin receptor in Klebsiella pneumoniae

Several Klebsiella pneumoniae strains which produced enterochelin but not aerobactin were nevertheless sensitive to cloacin DF13. In contrast, a strain of serotype K1:O1 which produced both siderophores was cloacin-resistant. Loss by mutation of the O1 but not K1 antigen rendered this strain cloacin-sensitive, indicating that the O1 antigen prevented access of cloacin to the cloacin/aerobactin receptor. Unlike the K1:O1 strain, the aerobactin-negative strains failed to hybridize in a colony blot assay with an aerobactin receptor gene probe prepared from pColV-K30. However, antisera raised against the 74 kDa pColV-K30 aerobactin receptor cross-reacted with a 76 kDa outer-membrane protein in each K. pneumoniae strain. In addition to the 76 kDa protein, the K1:O1 strain also produced a strongly cross-reacting 74 kDa protein. To determine whether these aerobactin-negative strains could use aerobactin, mutants unable to synthesize siderophores were isolated. Aerobactin promoted the growth of these mutants in iron-deficient media. The evidence presented suggests that some K. pneumoniae strains produce an aerobactin iron-uptake system without apparent production of aerobactin and which is probably based on a 76 kDa receptor, the gene for which does not hybridize with aerobactin receptor gene encoded on pColV-K30.     

Antibiotic sensitivity of Haemophilus influenzae isolated from patients with inflammatory lung diseases

In 1980-1986 the sensitivity of 2,045 H. influenzae strains isolated from the bronchial contents of patients with inflammatory lung diseases were studied. This study revealed that 60-80% of H. influenzae cultures circulating in Leningrad were sensitive to tetracyclin, chloramphenicol and erythromycin. During the period of observation the tendency towards the decrease of the number of highly sensitive H. influenzae cultures and the increase of the number of strains resistant to all antibiotic preparations was followed. Most of H. influenzae strains isolated in Leningrad were sensitive to penicillin, oleandomycin and ampicillin. In 1983 the appearance of H. influenzae strains, multiresistant to antibiotics, was noted. In 1986 these strains constituted 4.5% of all isolated cultures.     

Characterization of the genetic locus encoding Haemophilus influenzae type b surface fibrils.

Haemophilus influenzae is a common gram-negative pathogen that initiates infection by colonizing the upper respiratory tract epithelium. In previous work, we reported the isolation of a locus involved in expression of short, thin surface fibrils by H. influenzae type b and presented evidence that surface fibrils promote attachment to human epithelial cells. In the present study, we determined that the fibril locus is composed of one long open reading frame, designated hsf, which encodes a protein (Hsf) with a molecular mass of approximately 240 kDa. The derived amino acid sequence of the hsf product demonstrated 81% similarity and 72% identity to a recently identified nontypeable H. influenzae adhesin referred to as Hia. In experiments with a panel of eight cultured cell lines, the Hsf and Hia proteins were found to confer the same binding specificities, suggesting that hsf and hia are alleles of the same locus. Southern analysis and mutagenesis studies reinforced this conclusion. Further investigation revealed that an hsf homolog is ubiquitous among encapsulated H. influenzae strains and is present in a subset of nontypeable Haemophilus strains as well. We speculate that the hsf gene product plays an important role in the process of respiratory tract colonization by H. influenzae.