Rapid Discrimination of Haemophilus influenzae,H. parainfluenzae,and H. haemolyticus by Fluorescence In Situ Hybridization (FISH) and Two Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Platforms

Background

Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level.

Methodology

A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction.

Principal Findings

Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system''s database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients.     

Amplification of DNA at a prophage attachment site in Haemophilus influenzae.

The Escherichia coli plasmids pBR322 and pBR327 can be taken up by Haemophilus influenzae but do not replicate in this organism; however, integration of pBR into the H. influenzae chromosome was achieved by ligation to a fragment of the Haemophilus phage S2 that carried a phage attachment site (attP). Once these sequences were integrated, they could serve as sites of recombination and amplification for homologous (pBR or phage) DNA. Amplification appeared to occur in one of two prophage sites (attB) present in the H. influenzae chromosome. The extent of amplification was different in different cells and reflected the ability of these sequences to undergo rearrangement leading to the formation of a DNA ladder. The ladder was obtained by treatment of DNA with restriction enzymes that cut outside of the inserted DNA, i.e., did not cut in the repeat sequence, and represented different numbers of repeat elements. Reversed-field gel electrophoresis was instrumental in resolving amplified structures. Inasmuch as single-cell isolates gave rise to the same ladder structure, it was assumed that amplification was under regulatory control and that it reproduced the same equilibrium of repeat structures. Transformation of E. coli with the amplified H. influenzae DNA resulted in precise excision and replication of the original monomeric plasmids. This excision was independent of the recA and recBC genes.     

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.     

An Application of Outer Membrane Protein P6-Specific Enzyme-Linked Immunosorbent Assay for Detection of Haemophilus influenzae in Middle Ear Fluids and Nasopharyngeal Secretions

An enzyme-linked immunosorbent assay specific to outer membrane protein P6 (P6-ELISA) was applied for detecting Haemophilus influenzae in middle ear fluids (MEFs) from acute otitis media (AOM) patients and in nasopharyngeal secretions (NPSs) from acute rhinosinusitis patients. P6-ELISA had a sensitivity of 83.3% for MEFs and 71.5% for NPSs and a specificity of 85.6% for MEFs and 92.5% for NPSs, respectively. Real-time PCR exhibited significant differences in the number of ompP1 gene copies among samples determined by P6-ELISA to be positive and negative for H. influenzae. However, because the P6-ELISA test has the reactivity in Haemophilus species include two commensals H. haemolyticus and H. parainfluenzae, it is thus a weak method in order to detect only NTHi correctly. Consequently, diagnosis using the P6-ELISA should be based on an overall evaluation, including the results of other related examinations and clinical symptoms to prevent misleading conclusions in clinical setting.     

Haemophilus parainfluenzae has a limited core lipopolysaccharide repertoire with no phase variation

Cell surface lipopolysaccharide (LPS) is a well characterized virulence determinant for the human pathogen Haemophilus influenzae, so an investigation of LPS in the less pathogenic Haemophilus parainfluenzae could yield important insights. Using a panel of 18 commensal H. parainfluenzae isolates we demonstrate that the set of genes for inner core LPS biosynthesis largely resembles that of H. influenzae, with an additional heptosyltransferase I gene similar to waaC from Pasteurella multocida. Inner core LPS structure is therefore likely to be largely conserved across the two Haemophilus species. Outer core LPS biosynthetic genes are much less prevalent in H. parainfluenzae, although homologues of the H. influenzae LPS genes lpsB, non-phase variable lic2A and lgtC, and losA1, losB1 and lic2C are found in certain isolates. Immunoblotting using antibodies directed against selected LPS epitopes was consistent with these data. We found no evidence for tetranucleotide repeat-mediated phase variation in H. parainfluenzae. Phosphocholine, a phase variable H. influenzae LPS epitope that has been implicated in disease, was absent in H. parainfluenzae LPS as were the respective (lic1) biosynthetic genes. The introduction of the lic1 genes into H. parainfluenzae led to the phase variable incorporation of phosphocholine into its LPS. Differences in LPS structure between Haemophilus species could affect interactions at the bacterial-host interface and therefore the pathogenic potential of these bacteria.     

The specific uptake of cloned Haemophilus DNA.

During the process of transformation $\text{Haemophilus}\text{influenzae}$ cells bind its own DNA but little or no foreign DNA. This specificity for recognition of DNA was studied by cloning Haemophilus DNA in $\text{E. coli}$. Haemophilus DNA fragments were cloned using plasmid pBR322 as a vector. The fragment cH7 cloned in pBR322 was found to be homologous to Haemophilus DNA and shown to bind irreversibly to competent Haemophilus cells. The fact that cH7 isolated from $\text{E. coli}$ lacks Haemophilus modification leads to the conclusion that modification does not play a role in the uptake mechanism. Uptake specificity is a function of recognition sequences that reside in DNA itself.     

Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli

Restriction fragments of DNA from bacteriophage S phi-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of S phi-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.     

Delineation of the Species Haemophilus influenzae by Phenotype,Multilocus Sequence Phylogeny,and Detection of Marker Genes

To obtain more information on the much-debated definition of prokaryotic species, we investigated the borders of Haemophilus influenzae by comparative analysis of H. influenzae reference strains with closely related bacteria including strains assigned to Haemophilus haemolyticus, cryptic genospecies biotype IV, and the never formally validated species “Haemophilus intermedius”. Multilocus sequence phylogeny based on six housekeeping genes separated a cluster encompassing the type and the reference strains of H. influenzae from 31 more distantly related strains. Comparison of 16S rRNA gene sequences supported this delineation but was obscured by a conspicuously high number of polymorphic sites in many of the strains that did not belong to the core group of H. influenzae strains. The division was corroborated by the differential presence of genes encoding H. influenzae adhesion and penetration protein, fuculokinase, and Cu,Zn-superoxide dismutase, whereas immunoglobulin A1 protease activity or the presence of the iga gene was of limited discriminatory value. The existence of porphyrin-synthesizing strains (“H. intermedius”) closely related to H. influenzae was confirmed. Several chromosomally encoded hemin biosynthesis genes were identified, and sequence analysis showed these genes to represent an ancestral genotype rather than recent transfers from, e.g., Haemophilus parainfluenzae. Strains previously assigned to H. haemolyticus formed several separate lineages within a distinct but deeply branching cluster, intermingled with strains of “H. intermedius” and cryptic genospecies biotype IV. Although H. influenzae is phenotypically more homogenous than some other Haemophilus species, the genetic diversity and multicluster structure of strains traditionally associated with H. influenzae make it difficult to define the natural borders of that species.Bacterial diversity is usually expected to be organized into phenotypic and genetic clusters recognized as species. The diversity within species is thought to be confined by natural forces, although uncertainty exists as to the nature of the cohesion that constrains this diversity. There is increasing evidence that recombination occurs between distantly related bacteria and that barriers to this process, which could be used to define species naturally, are not always apparent (7, 11). As the bacterial species concept is central to taxonomy and evolutionary biology and has consequences for other disciplines such as the discrimination of pathogens from related bacteria with no pathogenic potential, there is a need to reevaluate to what extent accepted species are indeed coherent units.The gram-negative, nonmotile, facultatively anaerobic bacterium Haemophilus influenzae is a commensal organism of the pharynx frequently involved in localized infections of the respiratory tract, middle ear, and conjunctiva and sometimes in invasive infections such as meningitis or bacteremia. Presumptive identification is based on inability to synthesize NAD (V factor dependence) and porphyrin/hemin (X factor dependence). The latter characteristic is particularly notable as it is shared with few other microorganisms. Further metabolic characteristics of H. influenzae include fermentation of d-xylose, d-ribose, and d-galactose but not sucrose or d-mannose (17). By DNA analysis the X-factor-dependent species H. influenzae, Haemophilus aegyptius, and Haemophilus haemolyticus constitute a closely related group (4, 5, 14, 35), and housekeeping gene nucleotide dissimilarities between these species are exceeded by intraspecific nucleotide dissimilarities within the commensal species Haemophilus parainfluenzae (33).H. aegyptius was conceived as a species distinct from H. influenzae with a particular propensity to cause conjunctivitis (37). However, the existence of H. aegyptius as a separate species is controversial (5), and the designation H. influenzae biogroup aegyptius has been proposed to include traditional H. aegyptius and the closely related clones responsible for the invasive infection known as Brazilian purpuric fever (3), although formally the epithet aegyptius has priority. H. haemolyticus is distinguished from H. influenzae primarily by its hemolytic action on erythrocytes. A recent characterization of a large number of presumptive H. influenzae isolates from the respiratory tract of patients with chronic obstructive airway disease demonstrated that 102 of 258 strains clustered with the type strain of H. haemolyticus by 16S rRNA gene sequence, although many of the isolates were nonhemolytic. In contrast to H. influenzae, the atypical isolates were not associated with exacerbations of the disease (31), emphasizing the need to discriminate between the two species.Genetic methods have identified unnamed taxa of Haemophilus related to H. influenzae. Variant strains isolated from the genitourinary tract were first reported from Canada (1) and have been further studied by Quentin and coworkers (13, 39). These “cryptic genospecies biotype IV” strains (negative for tryptophanase/indole production and positive for urease and ornithine decarboxylase) are distinguishable from typical strains of H. influenzae by multilocus enzyme electrophoresis, 16S rRNA gene sequence, and DNA hybridization. However, only some biotype IV strains belong to the cryptic genospecies, and the lack of phenotypic characters enabling discrimination from H. influenzae has been an obstacle to the formal validation as a species. Another taxon, “Haemophilus intermedius,” was suggested (but never validly published) by Burbach (4). By DNA hybridization and by selected phenotypic traits, these strains take up an intermediate position between H. influenzae and H. parainfluenzae. Two subspecies were described, “Haemophilus intermedius subsp. intermedius,” capable of synthesizing porphyrin from δ-aminolevulinic acid and of fermenting sucrose, and “Haemophilus intermedius subsp. gazogenes,” characterized by the fermentation of mannose and the production of gas from glucose.A number of investigations have addressed the population structure of H. influenzae with assessment of particular virulence traits associated with specific subgroups or clades (9, 26, 32, 41). In the present study we attempted to define the borders of the species by performing phenotypic characterization, 16S rRNA sequence comparison, multilocus housekeeping gene sequence phylogeny, and detection of putative virulence and marker genes on a collection of 42 strains of H. influenzae and representatives of the above-mentioned closely related bacteria.     

High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: evidence for encapsulation and transfer of head-to-tail plasmid concatemers

Transduction of plasmid pBR322 by cytosine-substituted T4 phages has been studied. Three T4 phage mutants which substitute cytosine for all of hydroxymethylcytosine residues in the DNA, were shown to transduce pBR322 at frequencies of 2 × 10^?2 to 4 × 10^?3 transductants per singly infected cell. Also, three T4 phage strains which partially substitute cytosine for hydroxymethylcytosine, transduced pBR322 at frequencies of 2 × 10^?3 to 2 × 10^?4. The transduction frequencies of pBR322 we attained are at least 10-fold higher than those reported by G. G. Wilson, K. Young, and G. J. Edlin (1979, Nature (London)280, 80–82). We found that multiplicity of infection in preparation of the transducing phage is the most important factor affecting the frequency of pBR322 transduction. When a lysate made at a multiplicity of infection ranging from 0.5 to 0.05 was used as the donor phage, transduction frequency of pBR322 was 10- to 40-fold higher than that of high-m.o.i. lysate. The transduction frequency was not affected by either restriction systems or amber suppressors of the recipient cells. However, no pBR322-containing transductant was obtained when either recA or polA mutants were used as the recipients. DNA from T4dC phage containing pBR322-transducing particles was analyzed on agarose gel electrophoresis after cleavage with restriction endonucleases. It was suggested that the pBR322 DNA in the T4dC phage particles exists as head-to-tail concatemers.     

Cloning of cybB, the gene for cytochrome b561 of Escherichia coli K12

Summary A 37 kb fragment of DNA from an F-prime factor, F100-12, which showed a gene dosage effect on b-type cytochromes, was cloned with a cosmid vector, pHC79. Gel filtration of cytochromes and product analysis of the hybrid plasmids indicated that this fragment contained cybB, the structural gene for cytochrome b561. A chromosomal DNA fragment carrying the cybB gene was cloned by the plaque hybridization technique with Charon 4A as a vector. The gene was subcloned into pBR322 and was located in a 1.3 kb DNA fragment. It was concluded that the cybB gene is located on the chromosome of Escherichia coli K12.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - NADH reduced form of nicotinamide adenine dinucleotide     

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.     

Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.     

Sizing of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis.

The four restriction enzymes ApaI (5'-GGGCCC), EagI (5'-CGGCCG), NaeI (5'-GCCGGC), and SmaI (5'-CCCGGG) were found to produce distributions of DNA fragment sizes useful for mapping of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis. ApaI produced 21 fragments (range, 1.6 to 305 kilobases [kb]), EagI yielded 30 fragments (0.6 to 339 kb), NaeI produced 32 fragments (2.3 to 290 kb), and SmaI yielded 16 fragments (6.0 to 377 kb). Summation of the fragment lengths in each digest yielded estimates for the size of the H. influenzae chromosome ranging from 1,834 kb.     

Genetic Transformation in Haemophilus parainfluenzae Clinical Isolates

Haemophilus parainfluenzae isolates recovered from patients with respiratory diseases were studied for their ability to undergo genetic transformation by isogenic DNA. Two chromosomal markers, streptomycin resistance and nalidixic acid resistance, were tested for transformation efficiencies in H. parainfluenzae recipients from three biotypes. Most efficient in transformation was biotype II, followed by biotype I, while biotype III was nontransformable. Lack of transformation was not owing to poor donor activity of DNA, but to inability of the cells to develop competence. Strains that formed clumps in liquid media were nontransformable. Since the transformable biotype II is one of the prevalent biotypes world wide, one can speculate that DNA transformation probably plays a major role in the spread of drug resistance in H. parainfluenzae. Received: 9 December 1997 / Accepted: 26 February 1998     

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R·PstI site, (5′)C-T-G-C-A^↓-G (3′), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3′-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3′-OH termini, thus regenating the PstI cleavage site sequence. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r^?m^?recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage λcI·O). A single phage-resistant clone was found. The recombinant plasmid, pDI10, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.     

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.     

Cloning of two type II methylase genes that recognise asymmetric nucleotide sequences: FokI and HgaI

Summary The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI.The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI.Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.