Identification of clinical Pasteurella isolates by MALDI-TOF -- a comparison with VITEK 2 and conventional microbiological methods.

The aim of this study was to compare the performance of four methods that are widely used in the clinical microbiology laboratory for identification of Pasteurella species. The 4 methods evaluated were VITEK2, VITEK MS (BioMerieux), and Bruker Biotyper MS (Bruker) as well as traditional biochemical tests. Sequencing of the sodA gene was used as the reference method. Sixty-five isolates of Pasteurella spp. from 65 patients were analyzed. One Pasteurella multocida isolate from American Type Culture Collection (Manassas, VA, USA) was used as a reference. Traditional biochemical tests accurately identified 62/66 (94%) isolates. Both Bruker and Vitek matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) identified 59/66 (89%) strains, but VITEK2 could only identify 32/66 (48.5%) isolates correctly. The mean time to identification using biochemical tests was 20 hours; VITEK2 took 6 hours and MALDI-TOF approximately 10 minutes. In conclusion, MALDI-TOF is a quick method, which accurately identified most isolates of Pasteurella to the species level. Thus, MALDI-TOF constitutes a valuable diagnostic tool in the clinical laboratory.

Pasteurella multocida sialic acid aldolase: a promising biocatalyst.

Sialic acid aldolases or N-acetylneuraminate lyases (NanAs) catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D: -mannosamine (ManNAc). A capillary electrophoresis assay was developed to directly characterize the activities of NanAs in both Neu5Ac cleavage and Neu5Ac synthesis directions. The assay was used to obtain the pH profile and the kinetic data of a NanA cloned from Pasteurella multocida P-1059 (PmNanA) and a previously reported recombinant Escherichia coli K12 NanA (EcNanA). Both enzymes are active in a broad pH range of 6.0-9.0 in both reaction directions and have similar kinetic parameters. Substrates specificity studies showed that 5-O-methyl-ManNAc, a ManNAc derivative, can be used efficiently as a substrate by PmNanA, but not efficiently by EcNanA, for the synthesis of 8-O-methyl Neu5Ac. In addition, PmNanA (250 mg l(-1) culture) has a higher expression level (2.5-fold) than EcNanA (94 mg l(-1) culture). The higher expression level and a broader substrate tolerance make PmNanA a better catalyst than EcNanA for the chemoenzymatic synthesis of sialic acids and their derivatives.

Survey of restriction-modification systems and transformation in Mannheimia haemolytica and Pasteurella trehalosi.

A significant obstacle to molecular studies of Mannheimia (Pasteurella) haemolytica, has been its resistance to genetic transformation. The lack of competence of many M. haemolytica strains has been attributed to the presence of restriction modification systems. In this study, representative strains of 12 M. haemolytica serotypes and four Pasteurella trehalosi serotypes were successfully transformed by electroporation using a recombinant vector derived from the native M. haemolytica A1 serotype plasmid pNSF2176. Transformation was achieved despite PCR-based evidence for the presence of genes encoding a type I restriction enzyme, phaI, and a type II restriction enzyme hsdM, in each of the M. haemolytica strains.

Mannheimia haemolytica serotype 1 and Pasteurella trehalosi serotype 10 culture supernatants contain fibrinogen-binding proteins.

Fibrinogen-binding proteins were found in the culture supernatants of Mannheimia haemolytica serotype 1 (ATCC 43270) and Pasteurella trehalosi serotype 10 (ECO-100). Sheep fibrinogen was biotinylated and shown to bind to proteins in the culture supernatants by modified western blot. Fibrinogen-binding proteins in the culture supernatant may be important virulence factors leading to the characteristic fibrinous pneumonia caused by these organisms and may be critical antigenic targets for immune prophylaxis.

Conservation of expression and N-terminal sequences of the Pasteurella haemolytica 31-kilodalton and Pasteurella trehalosi 29-kilodalton periplasmic iron-regulated proteins.

This study examined the conservation of expression of a 31-kDa iron-regulated protein by serotypes of Pasteurella haemolytica and Pasteurella trehalosi associated with pasteurellosis of cattle and sheep. A polyclonal antibody prepared against the purified 31-kDa periplasmic iron-regulated protein from P. haemolytica serotype A1 showed that all P. haemolytica serotypes expressed similar 31-kDa proteins with identical N-terminal sequences, whereas P. trehalosi serotypes expressed immunologically different 29-kDa proteins with a different N-terminal sequence. Antibody to the 31-kDa iron-regulated protein was a useful tool to distinguish similarities and differences of the iron-regulated proteins of P. haemolytica and P. trehalosi.

Fatty acid profiles of "Pasteurella" piscicida: comparison with other fish pathogenic gram-negative bacteria.

The fatty acid methyl ester (FAME) profiles of "Pasteurella" piscicida were determined by gas chromatography and subjected to numerical analysis in comparison with those obtained for Vibrio anguillarum, Aeromonas salmonicida and Pasteurella species of clinical origin. The bacterial species studied shared important characteristics with respect to their FAME content: in all of them the saturated and unsaturated fatty acids of 16 carbon atoms were the predominant fatty acids. However, distinguishing features could be detected for each pathogen. Using either single linkage or complete linkage algorithms, strains were divided into four phena that corresponded to the different species, but showed a high degree of correlation among them. Although single linkage discriminated strains better within each phenum, complete linkage was more useful to establish the relationships among clusters. The results obtained support the idea that "Pasteurella" piscicida is related to members of the genera Vibrio and Aeromonas and indicate the need for exhaustive genetic studies to clarify the taxonomic position of this fish pathogen.

Capsular polysaccharide expressed by Pasteurella piscicida grown in vitro.

Pasteurella piscicida grown in a glucose-rich medium produces a capsule that can be see under light and electron microscopy. The capsular polysaccharide was purified and characterized by chemical and HPLC analysis. The polymer has the composition glucose/mannose/N-acetylgalactosamine/galacturonic acid/acetic acid in the molar ratios of approximately 2.5:1.3:0.5:0.4:2.5. The polysaccharide was immunogenic in rabbits and did not cross-react with antibodies against the O-antigen lipopolysaccharide.

The characterization of ion channels formed by Pasteurella multocida dermonecrotic toxin.

The influence of the dermonecrotic lethal toxin (approximately 120 kDa) produced by Pasteurella multocida serovarian D on planar phospholipid bilayers was studied. It was found that the toxin is able to increase the conductance of the bilayers by formation of low-conductive and cation-selective ion channels [27 pS at 4.0 M KCl, pH 7.5; zero current potential equals to -14.5 +/- 0.5 mV at threefold transmembrane gradient KCl (120 mM/40 mM)]. In biionic conditions the channels displayed weak selectivity between Na, K and Ca ions. The shapes of current-voltage characteristics (which were measured at different pH and salt concentrations) indicate that an energetic barrier for passing ions is situated near the center of the water pore of the ion channels. The effective diameter of the ion channel's water pore was established to be equal to 2.1 +/- 0.3 nm.

Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment.

The stability of Pasteurella piscicida strains in seawater and sediment microcosms at different temperatures (6 and 20 degrees C) was investigated during a 1-month period. Three strains of P. piscicida showed similar survival kinetics. By a standard plate count method they survived in water and sediment for only 6 to 12 days, depending on the strain and type of microcosm. During this starvation period, the metabolic activity of the cells was reduced by more than 80%. Culturable cells of each P. piscicida strain persisted better in sediment than in water, as well as at 20 degrees C compared to 6 degrees C. However, in all the microcosms, the acridine orange direct counts remained at about 10(5) cells per ml during the experimental period, which demonstrated that P. piscicida possesses a capacity to enter a viable but not culturable state. Moreover, dormant cells were always resuscitated by the addition of fresh medium to the microcosms, since we recovered numbers of culturable cells similar to the acridine orange direct counts. These resuscitated cells exhibited the same respiration rate as that seen prior to the start of the experiments. Although the biochemical, physiological, and serological characteristics; lipopolysaccharides; membrane proteins; and plasmid content of P. piscicida strains were unaffected during the starvation conditions, the dormant cells were smaller (dwarf cells) and had increased surface hydrophobicity. The starved cells maintained their infectivity and pathogenic potential for fish, with 50% lethal doses similar to those of the original strains.

Multivariate chemosystematics demonstrate two groups of Actinobacillus actinomycetemcomitans strains.

Chemical analysis by us has indicated that Actinobacillus actinomycetemcomitans is not a homogeneous species. The present study used chemometric methods and a multitude of chemical characters to examine this further. Strains were characterized by cell sugar and fatty acid contents, lysis kinetics during EDTA and EDTA plus lysozyme exposure, methylene blue reduction, and API ZYM enzymatic assessment of whole cells and outer membrane vesicles/fragments. In total, 41 quantitative variables were analyzed from each of 9 strains and treated with principal component analysis and soft independent modeling of class analogy. These methods divided A. actinomycetemcomitans into 2 strain groups. One group contained ATCC 33384, ATCC 29522, FDC 2112 and FDC 2043; the other comprised ATCC 29524, ATCC 29523, FDC 2097, FDC 511 and FDC Y4. With an F-test, the groups (classes) of A. actinomycetemcomitans strains could be distinguished at 95% confidence limits. Both groups were distinct from members of the genera Haemophilus and Pasteurella (Haemophilus aphrophilus, Haemophilus paraphrophilus, Haemophilus influenzae, Pasteurella multocida and Pasteurella haemolytica).

Characterization of the family Pasteurellaceae on the basis of cellular lipids and carbohydrates.

Selected strains representing established and newly described taxa in the family Pasteurellaceae were investigated for their cellular lipid and carbohydrate composition to clarify the taxonomic significance of such features. Methylated cellular fatty acids and acetylated derivatives of the cellular carbohydrates were determined by capillary gas chromatography using a flame ionization detector. In part the carbohydrates were identified by mass spectrometry. Phospholipids were determined by thin layer chromatography, the lipoquinones by high pressure liquid chromatography. The cellular fatty acid patterns proved to be uniform with minor variations, but the separation from the Neisseriaceae and from Moraxella was possible. Also the distribution of the phospholipids was uniform within the family. The lipoquinone contents were useful for the discrimination of groups within the family not necessarily reflecting the degree of genomic relatedness. The analysis of the cellular carbohydrates resulted in a common sugar pattern with all members of the family and characteristic carbohydrate profiles discriminating groups, often to the species level. All of the cytochemical features considered were useful for the characterization of the family Pasteurellaceae.

Purification and characterization of protein H, the major porin of Pasteurella multocida.

Protein H (B. Lugtenberg, R. van Boxtel, D. Evenberg, M. de Jong, P. Storm, and J. Frik, Infect. Immun. 52:175-182, 1986) is the major polypeptide of the outer membrane of Pasteurella multocida, a bacterium pathogenic for humans and animals. We have purified this protein to homogeneity by size exclusion chromatography after selective extraction with surfactants and demonstrated its pore-forming ability after reincorporation into planar lipid bilayers. In these experiments, the current through the pores was a linear function of the applied voltage in the range of -50 to +50 mV. Voltages beyond +/- 50 mV tended to partially close the channels, giving rise to apparent negative resistances. These observations suggest that protein H channels are probably not voltage regulated in vivo. With the patch clamp technique, single-channel conductance fluctuations of 0.33 nS were recorded in 1 M KCl. Electrophoretic and circular dichroism analyses showed that protein H forms homotrimers stable in sodium dodecyl sulfate at room temperature, with a high content of beta-sheet secondary structure. Upon boiling, the trimers were fully dissociated into monomers with an increase of alpha helix and irregular structure, at the expense of beta sheets. The apparent molecular mass of fully denatured monomers ranged between 37 and 41.8 kDa, depending on the electrophoretic system used for analysis. The trimeric arrangement of protein H was confirmed by image analysis of negatively stained, two-dimensional crystal arrays. This morphological study revealed, in agreement with electrophoretical data, a trimeric structure with an overall diameter of 7.7 nm. Each monomer appeared to contain a pore with an average diameter of 1 nm. Quantitative comparisons revealed that the amino acid composition (hydropathy index of -0.40) and the N-terminal sequence (determined over 36 residues) of protein H are similar to those of bacterial general porins, notably porin P2 of Haemophilus influenzae. We conclude from this set of structural and functional data that protein H of P. multocida is a pore-forming protein related to the superfamily of the nonspecific bacterial porins.

Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida.

The pathobiological activities in vivo and in vitro of live cells and extracellular products (ECP) of eleven Pasteurella piscicida strains of different origin were examined. Infectivity trials showed that P. piscicida did not possess strict host specificity since the majority of the isolates were virulent for gilthead seabream, rainbow trout and turbot, with LD50 values ranging between 10(3) and 10(6) live cells. However, none of the strains tested were pathogenic for mice (LD50 > 10(8) cells)). In addition, the ECP were strongly toxic for fish (LD50 ranging from 1.0 to 4.6 micrograms protein per g fish), which clearly demonstrates their important role in the pathogenesis of pasteurellosis. All the ECP samples were cytotoxic for fish and homoiothermic cell lines, possessed notable phospholipase activity and displayed haemolytic activity for sheep, salmon and turbot erythrocytes (but not for trout erythrocytes). However, the production of proteolytic enzymes differed among the P. piscicida strains. Although no strain displayed elastase activity, five isolates (the Japanese and Italian strains) hydrolysed casein and gelatin. All these biological activities in vivo and in vitro were lost after heat treatment (100 degrees C for 10 min). The general enzymic patterns of both live cells and ECP evaluated by the API-ZYM system also revealed some variation among the P. piscicida isolates. Generally, whole cells showed a wider range of enzymic activities than ECP. The results presented here are important for the selection of strains in the development of effective polyvalent pasteurellosis vaccines containing both whole cells and ECP.

Characterization and comparison of antimicrobial susceptibilities and outer membrane protein and plasmid DNA profiles of Pasteurella haemolytica and certain other members of the genus Pasteurella.

The outer membrane protein (OMP), plasmid, and antimicrobial resistance profiles of Pasteurella haemolytica serotypes 1 through 12, a bovine isolate of P multocida, a chicken isolate of P multocida, and an unidentified Pasteurella species of bovine origin were examined. Isolates of P haemolytica serotypes belonging to the same biotype possessed similar OMP profiles. Biotype A isolates contained 2 prominent OMP of 43 kilodaltons (kD) and 29 kD, whereas biotype-T serotypes contained 3 major OMP of 43, 36, and 25 kD. The major OMP profiles of the 2 P multocida isolates and the unidentified Pasteurella species were different from each other and from P haemolytica isolates. Plasmid DNA screening indicated both plasmid-containing and plasmid-free P haemolytica and P multocida isolates. Multiple drug resistance was found in pasteurellae isolates with and without plasmids. However, a relationship between drug resistance and plasmid isolation was found in 3 of 4 haemolytica serotype 1 field isolates, all of which contained a 2.51-megadalton plasmid and had multiple drug resistance for benzylpenicillin, ampicillin, streptomycin, and tetracycline.

Capillary gas chromatography of cellular carbohydrates as a means for the differentiation of fastidious, slow-growing or anaerobic gram-negative bacteria--a review.

The phenotypic differentiation of microaerophilic or anaerobic Gram-negative bacteria of human origin is traditionally based on the analysis of metabolic characteristics. Due to outstanding growth requirements of many kinds of parasitic bacteria, e.g. of the facultatively and strictly anaerobic members of the human periodontal flora, conventional biochemical tests are time-consuming and sometimes lead to doubtful results. Commercial diagnostic test kits often produce misidentifications because of insufficient databases or inappropriate test combinations. In this situation, cytochemical methods can provide an alternative. Especially in the case of Gram-negative bacteria which contain considerable amounts of carbohydrates in their cell wall lipopolysaccharide layers, capillary gas chromatography of peracetylated carbohydrate derivatives prepared from bacterial whole cell hydrolysates proved to be useful to characterize individual species, and sometimes even subspecies. As soon as a pure culture is available, the derivatization of the cellular carbohydrates to their peracetylated aldononitriles and peracetylated O-methyloximes, respectively, needs only about four hours and the gas chromatographic spectra are easy to evaluate.