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.     

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.     

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.     

Multivariate analysis of quantitative chemical and enzymic characterization data in classification of Actinobacillus, Haemophilus and Pasteurella spp

Chemotaxonomic data for strains of Actinobacillus, Haemophilus and Pasteurella spp. were analysed using three multivariate statistical strategies: principal components, partial least squares discriminant, and soft independent modelling of class analogy. The species comprised Actinobacillus actinomycetemcomitans. Haemophilus aphrophilus, H. paraphrophilus, H. influenzae, Pasteurella multocida, P. haemolytica and P. ureae. Strains were characterized by cell sugar and fatty acid composition, lysis kinetics during EDTA and EDTA plus lysozyme treatment, and methylene blue reduction. In total 23 quantitative variables were compiled from chemotaxonomic analyses of 25 strains. A. actinomycetemcomitans and H. aphrophilus formed distinct classes which differed from those of H. paraphrophilus, H. influenzae and Pasteurella spp. All characterization variables, except those describing fatty acid content, contributed significantly to inter-species discrimination.     

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.     

A rapid PCR based method to distinguish between Lactococcus and Enterococcus

Phenotypic characterisation of Lactococcus and Enterococcus species remains unreliable as strains of both genera have been isolated which do not conform to the traditional criteria for separation of these genera. A bank of 131 isolates was phenotypically characterised by three methods: (a) traditional broth tests, (b) API Rapid ID 32 Strep and (c) BBL Crystal ID kits. Differences in genus designation between commercial kits were evident for 12 strains (9%), while 7 strains (5%) remained unidentified by either kit. Published 16S rRNA sequences were aligned and used to design genus-specific primers which, when used in separate PCR reactions, were capable of distinguishing all type strains of Lactococcus and Enterococcus. These primers did not react with known species of Streptococcus, Pediococcus, Lactobacillus, Leuconostoc or Tetragenococcus. Isolates which could not be identified by phenotype were assigned to either genus on the basis of the gene primers.     

Cellular fatty acid composition of Haemophilus species, Pasteurella multocida, Actinobacillus Actinomycetemcomitans and Haemophilus vaginalis (Corynebacterium vaginale)

The fatty acid composition of 35 Haemophilus influenzae strains was found to be grossly similar and characterized by relatively large amounts of 14:0, 3-OH-14:0, 16:1 and 16:0. The three C18 fatty acids 18:2, 18:1 and 18:0 were also present, but in much lower concentrations. This general pattern was also found for most of the other species of Haemophilus examined (H. aegyptius, H. aphrophilus, H. canis, H. gallinarum, H. haemolyticus, and H. parainfluenzae). Small but distinct quantitative discrepancies were detected for H. ducreyi and the haemin-independent species H. paraphrohaemolyticus, H. paraphrophilus and H. suis. Actinobacillus actinomycetemcomitans was found to be indistinguishable from H. influenzae. Pasteurella multocida also exhibited a fatty acid pattern closely related to that of Haemophilus, but could be distinguished by its higher concentration levels of the C18 fatty acids. The fatty acid pattern of H. vaginalis was considerably different from those of the other species examined. This species lacked 3-OH-14:0 and 18:2 and contained small amounts of 14:0 and 16:0, whereas 18:1 and 18:0 were the major constituents.     

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.     

Naturally occurring NAD-independent Haemophilus parainfluenzae

Four, NAD-independent, clinical isolates of Haemophilus parainfluenzae were recovered from a genital ulcer, a purulent skin lesion, a sputum specimen and a throat swab respectively. With the exception of NAD requirement, the strains exhibited the biochemical characteristics of H. parainfluenzae biotype II. The genetic relationship between these isolates and a standard strain of H. parainfluenzae was determined by testing transforming activities of two chromosomal markers, streptomycin resistance and nalidixic acid resistance. The clinical isolates were efficient donors and recipients in transformation. In addition, we demonstrated transfer of the genes conferring NAD independence to typical, NAD-requiring H. parainfluenzae and Haemophilus influenzae strains.     

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.     

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.     

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.     

Binding of human hemoglobin by Haemophilus influenzae

Abstract Binding of biotinylated human hemoglobin to Haemophilus influenzae was detected when organisms were grown in heme-deplete, but not heme-replete, conditions. Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled human hemoglobin or human hemoglobin complexed with human haptoglobin. Binding was only partially inhibited by rat hemoglobin, bovine hemoglobin, human globin, and bovine globin, and not at all by heme, human serum albumin, bovine serum albumin, human transferrin, or myoglobin. Hemoglobin binding was saturable at 16–20 ng of hemoglobin per 10⁹ cfu. Binding of human hemoglobin was detected in serotypes a-f and serologically non-typable strains of H. influenzae , as well as Haemophilus haemolyticus but not Haemophilus parainfluenzae, Haemophilus aphrophilus, Haemophilus parahaemolyticus , or Escherichia coli .     

Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-lactamase producing Haemophili occurrence in clinical materials and carriage in healthy children

Beta-lactamase production was investigated in 126 H. influenzae and 15 H. parainfluenzae strains isolated in various infections. In H. influenzae the rate of beta-lactamase positive strains was 5.6%, in non-encapsulated strains it was higher (9.7%) than in capsule bearing strains (3.1%). Among beta-lactamase producing H. influenzae strains biotype II was predominant, whereas biotype I prevailed in beta-lactamase positive strains of H. parainfluenzae. A study undertaken in 101 children of a day-care nursery revealed 16.8% carriers of beta-lactamase producing Haemophili. Among the isolated strains we found the double number of H. parainfluenzae than H. influenzae strains showing beta-lactamase activity. This result supports the hypothesis of H. parainfluenzae being the reservoir of resistances plasmids in Haemophili.     

Introduction of transposon Tn916 DNA into Haemophilus influenzae and Haemophilus parainfluenzae.

Enterococcus (Streptococcus) faecalis transposon Tn916 was introduced into Haemophilus influenzae Rd and Haemophilus parainfluenzae by transformation and demonstrated to transpose efficiently. Haemophilus transformants resistant to tetracycline were observed at a frequency of approximately 3 x 10(2) to 5 x 10(3)/micrograms of either pAM120 (pGL101::Tn916) or pAM180 (pAM81::Tn916) plasmid DNAs, which are incapable of autonomous replication in this host. Restriction enzyme analysis and Southern blot hybridization revealed that (i) Tn916 integrates into many different sites in the H. influenzae and H. parainfluenzae genomes; (ii) only the 16.4-kilobase-pair Tn916 DNA integrates, and no vector DNA was detected; and (iii) the Tetr phenotype was stable in the absence of selective pressure. Second-generation Tn916 transformants occurred at the high frequency of chromosomal markers and retained their original chromosomal locations. Similar results were obtained with H. influenzae Rd BC200 rec-1 as the recipient strain, which suggests host rec functions are not required in Tn916 integrative transposition. Transposition with Tn916 is an important procedure for mutagenesis of Haemophilus species.     

Enzymatic/biochemical analysis of Actinomyces with commercial test kits with an emphasis on newly described species

In clinical microbiology laboratories, the identification of Actinomyces-like bacteria can be very laborious and problematic. In the present study, we focused on reactivity patterns of 4 commercial test kits, RapID ANA II, RapID 32A, RapID CB Plus, and BBL Crystal ANR ID, that could be used for rapid preliminary identification of Actinomyces isolates belonging to newly described Actinomyces and closely related species. Out of the 54 strains tested, 25 strains (46%) were correctly identified to the genus/group level by BBL Crystal ANR ID system, 16 strains (30%) by RapID 32 A, 11 strains (20%) by RapID CB Plus, and 7 strains (13%) by RapID ANA II. The main problems with these kits were due to occasional weak enzymatic and sugar fermentation reactions. In conclusion, chromogenic substrate sensitivity and specificity need to be enhanced in order to improve the reliability of the test results of these kits, and the present database updated in order to more precisely identify newly described Actinomyces and closely related species.     

EVALUATION OF THREE LATEX AGGLUTINATION TEST KITS FOR RAPID DETECTION OF CAMPYLOBACTER ISOLATES FROM CHICKEN SAMPLES

Three commercially available latex agglutination test (LAT) kits for the rapid identification of Campylobacter isolates were evaluated against 87 isolates of Campylobacter spp. and 46 strains of non-Campylobacter bacteria (26 species). The performance of three LAT kits, MicroScreen® Campylobacter (Mercia Diagnostics, Shalford, UK), BBL Campyslide^TM (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) and MERITEC^TM-Campy (jcl) (Meridian Diagnostics, Cincinnati, OH, USA), were compared. All sensitivities for the three LAT kits were 100%. Specificities of both MicroScreen® Campylobacter and BBL Campyslide^TM were 100%, but specificity of MERITEC^TM-Campy (jcl) was only 89. 1/. The detection limit ranges (log CFU/ml) of MicroScreen®. Campylobacter, BBL Campyslide^TM and MERITEC^TM-Campy (jcl) against five Campylobacter strains were 7.7–9.1, 7.4–8.0 and 8.5–9.4, respectively. BBL Campyslide^TM was more sensitive (10X) than the other two LAT kits (p < 0.01). A one-day procedure was proposed to identify the suspicious colonies on selective agar by performing the LAT kit, catalase, oxidase and morphological observation.     

Action of Haemophilus Endodeoxyribonuclease on Biologically Active Deoxyribonucleic Acid

An enzyme similar to that described by Smith and Wilcox (15) for Haemophilus influenzae which attacks foreign deoxyribonucleic acid (DNA) but not its own has been isolated and purified from H. parainfluenzae. The enzyme degrades foreign DNA to limited sizes and can destroy the transforming activity of H. influenzae and Bacillus subtilis DNA. The enzyme can also destroy the biological activity of H. influenzae phage and prophage DNA. On the other hand, the H. influenzae endodeoxyribonuclease can destroy the transforming activity of H. parainfluenzae DNA but not its own DNA. It also attacks B. subtilis DNA and its transforming activity.     

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.