Plasmid-mediated NAD independence in Haemophilus parainfluenzae.

The location of the genes coding for NAD independence in four unusual clinical isolates of Haemophilus parainfluenzae was determined by transferring these genes to plasmid-free Haemophilus influenzae Rd by transformation and analysing transformants for the presence of plasmids by agarose gel electrophoresis. All NAD-independent transformants were found to carry a single plasmid species. The plasmids, originally harboured by the four H. parainfluenzae isolates recovered from unrelated sources, were of the same size (5.25 kb). Spontaneous reversion to NAD dependence occurred with a low frequency (0.1 to 0.2% of the progeny of a single clone) in both H. parainfluenzae and H. influenzae Rd. The revertants had lost this small plasmid. Mitomycin C exhibited a plasmid 'curing' effect with a frequency of 'curing' of between 1 and 6% of the surviving clones. It was concluded that the genes conferring NAD independence were located on the small 5.25 kb plasmid.     

Chromosomally integrated conjugative plasmids are common in antibiotic-resistant Haemophilus influenzae.

Twenty-three highly antibiotic-resistant strains of Haemophilus influenzae and two of Haemophilus parainfluenzae without detectable large plasmids were examined for conjugative transfer of their resistance to H. influenzae strain Rd or to other strains. Very inefficient transfer was observed for 18 H. influenzae strains and 1 H. parainfluenzae strain. All H. influenzae transcipients carried a large plasmid, and they were in turn efficient donors of their resistances in standard conjugation crosses with isogenic recipients. This was not seen for the H. parainfluenzae transcipients. It is concluded that most of the original antibiotic-resistant cultures carried an integrated conjugative R plasmid which had been excised in a few cells in each population. It was these cells which transferred resistance in the primary crosses.     

Serological relationship of some V-factor dependent Pasteurellaceae (Haemophilus sp.) from guineapigs and rabbits

Culture of guinea pig and rabbit respiratory tracts for bacteria using X- (haemin) and V- (NAD) factor in agar media detected infection by V-factor dependent Pasteurellaceae (Haemophilus sp.) in three colonies of guinea pigs and a group of rabbits. The 12 Haemophilus strains comprised three API NH codes classed as Haemophilus parainfluenzae and two codes classed as Haemophilus aphrophilus/paraphrophilus. Six cell wall lipid profiles were detected, but these were not related to API NH codes. Both bacteriological properties were used to select strains for serological studies but any relationship between bacteriological and serological properties of the Haemophilus strains was not evident. Varying serological relationships occurred between the newly isolated Haemophilus strains, [Pasteurella] pneumotropica NCTC 8284 and Haemophilus strains previously isolated from rats.     

Anomalous but helpful findings from the BBL Crystal ID kit with Haemophilus spp

Fourteen strains of Haemophilus (12 H. influenzae , 1 H. parainfluenzae and 1 H. aphrophilus ) were processed in BBL Crystal ID Enteric/Nonfermenter, API 20E and API 20NE kits, to determine whether the BBL kit misidentifies, as API kits may do, Haemophilus spp. as Pasteurella spp. The 13 H. influenzae and H. parainfluenzae strains produced uninterpretable colour reactions in the Crystal kit, thus signalling that an inappropriate species had been tested. On the other hand, the API kits (especially 20NE) often confidently 'identified' Haemophilus spp. as Pasteurella spp., giving no warning that this was a misidentification.     

Utilization and metabolism of NAD by Haemophilus parainfluenzae

The utilization of exogenous nicotinamide adenine dinucleotide (NAD) by Haemophilus parainfluenzae was studied in suspensions of whole cells using radiolabelled NAD, nicotinamide mononucleotide (NMN), and nicotinamide ribonucleoside (NR). The utilization of these compounds by H. parainfluenzae has the following characteristics. (1) NAD is not taken up intact, but rather is degraded to NMN or NR prior to internalization. (2) Uptake is carrier-mediated and energy-dependent with saturation kinetics. (3) There is specificity for the beta-configuration of the glycopyridine linkage. (4) An intact carboxamide groups is required on the pyridine ring. The intracellular metabolism of NAD was studied in crude cell extracts and in whole cells using carbonyl-14C-labelled NR, NMN, NAD, nicotinamide, and nicotinic acid as substrates in separate experiments. A synthetic pathway from NR through NMN to NAD that requires Mg2+ and ATP was demonstrated. Nicotinamide was found as an end-product of NAD degradation. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide were not found as intermediates. The NAD synthetic pathway in H. parainfluenzae differs from the Preiss-Handler pathway and the pyridine nucleotide cycles described in other bacteria.     

Origin of small beta-lactamase-specifying plasmids in Haemophilus species and Neisseria gonorrhoeae.

Fifty-nine percent of unselected strains of Haemophilus parainfluenzae were found to carry small, phenotypically cryptic plasmid DNA species. Using filter blot hybridization, we found several plasmids which were homologous to the small beta-lactamase-specifying plasmids pJB1 and pFA7, which were originally isolated from Haemophilus ducreyi and Neisseria gonorrhoeae, respectively. Detailed filter hybridization studies combined with electron microscope heteroduplex analysis suggested that three cryptic plasmids are completely homologous to the non-TnA sequences of pJB1. One cryptic plasmid was found to be highly homologous to pJB603, a small beta-lactamase plasmid previously found in two isolates of H. influenzae. A second group of plasmids were found to carry sequences homologous to pJB1 and other sequences homologous to pJB603. These results strongly suggest that small beta-lactamase plasmids found in Haemophilus species and N. gonorrhoeae may have arisen by insertion of the transposable beta-lactamase-specifying element TnA into small, phenotypically cryptic replicons resident in H. parainfluenzae. Attempts to reproduce such a recombination event in the laboratory were not successful.     

Structural characterization of Haemophilus parainfluenzae lipooligosaccharide and elucidation of its role in adherence using an outer core mutant

The opportunistic pathogen Haemophilus parainfluenzae is a gram-negative bacterium found in the oropharynx of humans. Haemophilus parainfluenzae is a member of the Pasteurellaceae family in which it is most closely related to Haemophilus sengis and Actinobacillus. Characterization of surface displayed lipooligosaccharide has identified components that are crucial in adherence. We examined the oligosaccharide structure of lipooligosaccharide from 2 clinical isolates of H. parainfluenzae. Core oligosaccharide was isolated by standard methods from purified lipooligosaccharide. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy, and mass spectrometry revealing the following structures: R-(1-6)-beta-Glc-(1-4)-D,D-alpha-Hep-(1-6)-beta-Glc-(1-4)- substituting a tri-heptose-Kdo inner core of L,D-alpha-Hep-(1-2)-L,D-alpha-Hep-(1-3)-L,D-alpha-Hep-(1-5)-alpha-Kdo at the 4-position of the proximal L,D-alpha-Hep residue to Kdo, and with a PEtn residue at the 6-position of the central L,D-alpha-Hep residue. In strain 4282, the R substituent is beta-galactose and in strain 4201 there is no substituent at the distal glucose. These analyses have revealed that multiple structural aspects of H. parainfluenzae lipooligosaccharide are comparable with nontypeable Haemophilus influenzae lipooligosaccharide. This study also identified a galactan in strain 4201 and a glucan in strain 4282. Haemophilus parainfluenzae was shown to adhere to a bronchial epithelial cell line to the same degree as nontypeable H. influenzae. However, an H. parainfluenzae mutant lacking the outer core of the lipooligosaccharide showed diminished adherence to the epithelial cells, suggesting that H. parainfluenzae lipooligosaccharide plays a role in tissue colonization.     

Preservation of Haemophilus influenzae and Haemophilus parainfluenzae at -70 degrees C.

We have studied the viability of Haemophilus spp. preserved for 5 to 12 months at -70 degrees C. The following media were used: Laboratoire de Santé Publique du Québec (LSPQ) preservation medium, trypticase soy broth with 10 degrees C (vol/vol) glycerol and 40 degrees C (vol/vol) horse serum (TSBG), and Levinthal's broth (LB) medium. Three clinical isolates of both H. influenzae and H. parainfluenzae were used. After 5 months no differences in viability were observed between strains preserved in TSBG and strains preserved in LB, but a significant loss of viability was observed in strains preserved in LSPQ medium. No significant changes in antimicrobial susceptibility were observed after 5-month storage in any medium. After 12 months, TSBG appeared to be the most suitable cryopreservation medium for the six strains tested. We conclude that TSBG represents a good medium for the maintenance of Haemophilus spp. at -70 degrees C for up to 1 year.     

Application of multivariate analyses of enzymic data to classification of members of the Actinobacillus-Haemophilus-Pasteurella group.

Outer membrane vesicles and fragments from Actinobacillus actinomycetemcomitans, Actinobacillus lignieresii, Actinobacillus ureae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Haemophilus parainfluenzae, Pasteurella haemolytica, and Pasteurella multocida were isolated and examined semiquantitatively for 19 enzyme activities by using the API ZYM micromethod. The enzyme contents of vesicles and fragments were compared with the enzyme contents of whole cells of the same organisms. Enzymic data were analyzed by using principal-component analysis and soft independent modeling of class analogy. This technique allowed us to distinguish among the closely related organisms A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus. A. actinomycetemcomitans was divided into two groups of strains. A. lignieresii fell outside or on the border of the A. actinobacillus class. A. ureae, H. influenzae, H. parainfluenzae, P. haemolytica, and P. multocida fell outside the A. actinomycetemcomitans, H. aphrophilus, and H. paraphrophilus classes.     

Constitutive expression of tetracycline resistance mediated by a Tn10-like element in Haemophilus parainfluenzae results from a mutation in the repressor gene.

The Tn10-like constitutively expressed tetracycline resistance determinant from a Haemophilus parainfluenzae strain was cloned in Escherichia coli. Toxicity resulting from expression on multicopy plasmids necessitated its being cloned on a low-copy plasmid vector or in cells containing the Tn10-encoded repressor. Constitutive expression of tetracycline resistance was found to result from the synthesis of a truncated inactive repressor molecule. Instead of the 23-kilodalton repressor found in other Tn10-containing strains, this determinant encoded a 14.5-kilodalton molecule. The DNA sequence of the 700-base-pair region spanning the repressor gene and promoter-operator regions of the Haemophilus determinant was identical to that of the same region of Tn10, except for the absence of a single T X A base pair in the repressor gene. This deletion leads to premature termination of the protein. Antisera to the repressor suggested that the repressor was also absent in a second independently isolated H. parainfluenzae strain bearing a Tn10-like constitutive tetracycline resistance determinant.     

Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences.

Virtually complete 16S rRNA sequences were determined for 54 representative strains of species in the family Pasteurellaceae. Of these strains, 15 were Pasteurella, 16 were Actinobacillus, and 23 were Haemophilus. A phylogenetic tree was constructed based on sequence similarity, using the Neighbor-Joining method. Fifty-three of the strains fell within four large clusters. The first cluster included the type strains of Haemophilus influenzae, H. aegyptius, H. aphrophilus, H. haemolyticus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans. This cluster also contained A. actinomycetemcomitans FDC Y4, ATCC 29522, ATCC 29523, and ATCC 29524 and H. aphrophilus NCTC 7901. The second cluster included the type strains of A. seminis and Pasteurella aerogenes and H. somnus OVCG 43826. The third cluster was composed of the type strains of Pasteurella multocida, P. anatis, P. avium, P. canis, P. dagmatis, P. gallinarum, P. langaa, P. stomatis, P. volantium, H. haemoglobinophilus, H. parasuis, H. paracuniculus, H. paragallinarum, and A. capsulatus. This cluster also contained Pasteurella species A CCUG 18782, Pasteurella species B CCUG 19974, Haemophilus taxon C CAPM 5111, H. parasuis type 5 Nagasaki, P. volantium (H. parainfluenzae) NCTC 4101, and P. trehalosi NCTC 10624. The fourth cluster included the type strains of Actinobacillus lignieresii, A. equuli, A. pleuropneumoniae, A. suis, A. ureae, H. parahaemolyticus, H. parainfluenzae, H. paraphrohaemolyticus, H. ducreyi, and P. haemolytica. This cluster also contained Actinobacillus species strain CCUG 19799 (Bisgaard taxon 11), A. suis ATCC 15557, H. ducreyi ATCC 27722 and HD 35000, Haemophilus minor group strain 202, and H. parainfluenzae ATCC 29242. The type strain of P. pneumotropica branched alone to form a fifth group. The branching of the Pasteurellaceae family tree was quite complex. The four major clusters contained multiple subclusters. The clusters contained both rapidly and slowly evolving strains (indicated by differing numbers of base changes incorporated into the 16S rRNA sequence relative to outgroup organisms). While the results presented a clear picture of the phylogenetic relationships, the complexity of the branching will make division of the family into genera a difficult and somewhat subjective task. We do not suggest any taxonomic changes at this time.     

Frequency and distribution per species, biotypes, resistance to antibiotics and beta-lactamase production of the haemophils isolated from patients with respiratory diseases

A number of 150 samples were prelevated from respiratory tract secretions of 88 patients with respiratory infections and three healthy subjects; 162 haemophilus strains were isolated, identified and studied and the following results were obtained: H. parainfluenzae was isolated from tonsillitis and laryngitis--over 70%, bronchitis--58% and pharyngitis--56.6%; H. influenzae was isolated from pharyngitis--26.4%, bronchitis--16.1% and tonsillitis--13.6% cases; H. parahaemolyticus from bronchitis--19.3%, tonsillitis--13.6% and laryngitis. H. paraphrophilus was isolated (6.8%) from pharyngitis, tonsillitis, sinusitis, bronchitis and pulmonary abscess and H. paraphrohaemolyticus was isolated--4.5% from pharyngitis, synusitis, bronchitis and pulmonary sarcoidosis. Most of the isolates belonged to biotype II H. influenzae and biotypes II, I, III H. parainfluenzae. Haemophils were 100% sensitive to Ofloxacin and resistant to Cro--13.5%, Do--17.9%, C and Caz--22.2%, Aml--24.6%, Rd--40.7%, Amp--41.9% and Te--63.5%; varying according to the haemophilus species. H. influenzae was resistant to Do--14.2%, Caz and C--21.4%, H. parainfluenzae was resistant to Cro--11%, Do--22%, whilst H. parahaemolyticus was resistant to Do--9% and to Aml, Caz and Cro--13.6%. Haemophils isolated from sputum showed a resistance higher by 12-34% and 6-17% than those isolated from other specimens, such as pharyngeal exudate, where the resistance to rifadin was lower by 10%. beta-lactamases were present in 27.7% of the strains: H. parainfluenzae--36%, H. paraphrohaemolyticus--25%, H. influenzae--17.8% and H. parahaemolyticus--15.7%; in strains from sputum--34.2%, pharyngeal exudate--28.8% and from other specimens--6.6%. No correlations were noticed between the biotype and the clinical manifestation or the resistance to the antibiotic, a higher frequency of beta-lactamase production being reported in H. influenzae biotype V and H. parainfluenzae biotypes II and IV.     

Methylase activities from Haemophilus influenzae that protect Haemophilus parainfluenzae transforming deoxyribonucleic acid from inactivation by Haemophilus influenzae endonuclease R.

Specific methylases that have the properties of deoxyribonucleic acid (DNA) modification enzymes have been isolated from Haemophilus influenzae strain Rd. Two activities ((Methylase IIa and methylase III) were found to protect transforming DNA of H. parainfluenzae from the action of H. influenzae restriction enzymes. To determine the specificty of the protection, a procedure based on biological activity was developed for the separation and purification of the restriction endonucleases from H. influenzae strain Rd. Two endonuclease R activities presumably corresponding to Hind II and Hind III (P. H. Roy and H. O. Smith, 1973; H. O. Smith and K. W. Wilcox, 1970) were characterized by differences in their chromatographic properties, ability to attack T7 DNA, and inactivation of the transforming activity of different markers of H. parainfluenzae DNA. One endonuclease R enzyme (Hind II) attacked T7 DNA and was found to inactivate the dalacin resistance marker (smaller than 0.01% activity remaining) with only a slight effect on the streptomycin resistance marker (83% activity remaining). Methylase IIa treatment protected 40% of the dalacin resistance marker of H. parainfluenzae DNA from inactivation by Hind II. The other restriction activity (Hind III) was inert towards T7 DNA and inactivated the streptomycin resistance marker of H. parainfluenzae DNA (smaller than 0.01% activity remaining) without any effect on the dalacin resistance marker. The methylation of H. parainfluenzae DNA accomplished by methylase III protected 60% of the transforming activity of the streptomycin resistance marker of H. parainfluenzae DNA from the action of Hind III.     

Distribution of diaminopropane, putrescine and cadaverine in Haemophilus and Actinobacillus

Cellular levels of diaminopropane, putrescine and cadaverine, and decarboxylase activities to produce these diamines in six species (16 strains) of Haemophilus and four species (5 strains) of Actinobacillus belonging to the family Pasteurellaceae of the gamma subclass of the class Proteobacteria, were determined by high performance liquid chromatography (HPLC). Diaminopropane was ubiquitously distributed within all Haemophilus and Actinobacillus species, and L-2,4-diaminobutyric acid decarboxylase activity was detected in them. Putrescine and ornithine decarboxylase activity were found in H. aphrophilus, H. parainfluenzae and H. influenzae (type a, b, d, e and f except for type c) but not detected in H. aegyptius, H. parahaemolyticus, H. ducreyi and Actinobacillus species. Cadaverine occurred in H. aphrophilus, H. aegyptius, H. influenzae, H. parainfluenzae, A. actinomycetemcomitans, A. equuli and A. lignieresii, whereas their lysine decarboxylase activity was scarcely detected. Cadaverine was not found in H. parahaemolyticus, H. ducreyi and A. suis. The diamine profile serves as a phenotypic marker for the chemotaxonomic classification of the family Pasteurellaceae.     

Relationships among some R plasmids found in Haemophilus influenzae.

Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 X 10(6) daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSF007 isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSF007 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSF007 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UB2832. We conclude either that the strains we studied received R factors of the same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a commom resident plasmid of H. influenzae.     

Uptake of plasmid deoxyribonucleic acid by Haemophilus

The uptake of circular and linear plasmid RSF0885 deoxyribonucleic acids, (DNAs) obtained from Haemophilus parainfluenzae 14, in both homologous and heterologous recipients was studied and compared with that of chromosomal DNA. High concentrations of divalent cations stimulated the uptake of either circular or linear plasmid DNA in H. parainfluenzae 14 competent cells but did not affect the uptake of chromosomal DNA. The biological activity of linear plasmid DNA was similar to that of circular DNA, and the transforming efficiencies for ampicillin resistance of both molecular forms were stimulated by divalent ions. Plasmid DNA was taken up efficiently either with or without the addition of divalent ions but was not biologically active in the heterologous Haemophilus influenzae Rd recipient. Our results suggest that in H. parainfluenzae 14 some of the steps for chromosomal and plasmid DNA uptake are different.     

Bacteriology and Clinical Significance of Hemolytic Haemophilus in the Throat

Hemolytic Haemophilus are rarely isolated in the clinical laboratory, as they do not grow on sheep or human blood-agar alone. On rabbit blood-agar they grow well and are hemolytic, but they grow less well and are not hemolytic on sheep blood-agar with added X and V factors. A survey was made to determine their incidence in pharyngitis. From 28 of 100 sore throats and from 57 of 100 normal throats, only normal bacterial flora were isolated. beta-Streptococci were present in significant numbers in 9 sore and 11 normal throats; staphylococci in 8 sore and 4 normal throats; pneumococci in 20 sore and 11 normal throats; H. influenzae or H. parainfluenzae in 13 sore and no normal throats; hemolytic Haemophilus in 30 sore and 18 normal throats; enteric bacilli in 1 of each; Candida and Neisseria in 2 sore throats each. All of the 33 hemolytic Haemophilus isolates identified to species were H. parahaemolyticus. All were sensitive in vitro to chloramphenicol, erythromycin, and tetracycline; 30 were sensitive to ampicillin and 30 to penicillin, 26 to novobiocin, and 12 to methicillin. H. influenzae, H. parainfluenzae, and H. haemolyticus are indistinguishable by Gram stain morphology, but H. parahaemolyticus is larger than the other three. Hemolytic and nonhemolytic species are indistinguishable by colonial morphology or by nutritional requirements; only hemolysis gives positive differentiation. Nevertheless, only rarely would this be of clinical importance. H. parahaemolyticus apparently may cause pharyngitis, but it is almost always susceptible to penicillin and rarely if ever causes sequelae.     

Method for isolating mutants overproducing nicotinamide adenine dinucleotide and its precursors

A procedure has been developed for isolating mutants which are defective with respect to nicotinamide adenine dinucleotide (NAD) metabolism. It is based on the well known V-factor requirement of Haemophilus parainfluenzae. This procedure was used to isolate a series of mutants from Escherichia coli. The pyridine metabolism of wild-type and mutant E. coli cells falls in one of four distinct classes. Class A includes wild-type E. coli and represents strains that are normal with respect to pyridine metabolism. Class B mutants have altered internal pools of NAD. The intracellular NAD concentration of different class B mutants varies over a 10-fold range. Class C mutants excrete pyridine mononucleotides, and class D mutants excrete NAD. The production of pyridine nucleotides by class C and D mutants exceeds that of wild-type E. coli by a factor of at least ten. The mutant strains generally have normal generation times and achieve normal cell densities in minimal medium.     

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.     

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.