The Gram-negative bacterium Haemophilus parasuis is the etiologic agent of Glässer’s disease in pigs, and causes significant economic losses to the swine industry. This bacterium has been classified as a member of the family Pasteurellaceae in the genus Haemophilus , but phylogenetic relatedness has not been adequately examined to support this genus classification. Phenotypically, all 38 strains of H. parasuis tested were positive for catalase activity, oxidase activity, V-factor requirement, and acid formation from maltose and d-galactose without gas. All strains were negative for X-factor requirement, formation of indole from tryptophan, urease, l-arabinose, and α-glucosidase activity. To determine whether H. parasuis belongs to one of the current Pasteurellaceae genera 40 H . parasuis genomes, plus those of representative Pasteurellaceae , were subjected to phylogenetic analysis of concatenated, multi-protein alignments. Sequence variation at 16S rRNA and rpoB loci allowed the 15 reference serovars of H. parasuis to be integrated into the whole-genome tree. The phylogenetic analysis showed H. parasuis to be a distinct and tight clade whose sister taxon is the genus Bibersteinia . Within H. parasuis two clades were identified with individual serovars distributed between the two. As a result, H. parasuis was confirmed as a member of the family Pasteurellaceae , but was distinct from other genera in this family. Therefore, we propose the name Glaesserella parasuis, gen. nov., comb. nov. for bacterial strains currently classified as H. parasuis . The reference strain of this species is ATCC 19417 (1374)T, NCTC 4557T, DSM 21448T, CCUG 3712T.

Histophilus somni ( Haemophilus somnus ) is an obligate inhabitant of the mucosal surfaces of bovines and sheep and an opportunistic pathogen responsible for respiratory disease, meningoencephalitis, myocarditis, arthritis, and other systemic infections. The identification of an exopolysaccharide produced by H. somni prompted us to evaluate whether the bacterium was capable of forming a biofilm. After growth in polyvinyl chloride wells a biofilm was formed by all strains examined, although most isolates from systemic sites produced more biofilm than commensal isolates from the prepuce. Biofilms of pneumonia isolate strain 2336 and commensal isolate strain 129Pt were grown in flow cells, followed by analysis by confocal laser scanning microscopy and scanning electron microscopy. Both strains formed biofilms that went through stages of attachment, growth, maturation, and detachment. However, strain 2336 produced a mature biofilm that consisted of thick, homogenous mound-shaped microcolonies encased in an amorphous extracellular matrix with profound water channels. In contrast, strain 129Pt formed a biofilm of cell clusters that were tower-shaped or distinct filamentous structures intertwined with each other by strands of extracellular matrix. The biofilm of strain 2336 had a mass and thickness that was 5- to 10-fold greater than that of strain 129Pt and covered 75 to 82% of the surface area, whereas the biofilm of strain 129Pt covered 35 to 40% of the surface area. Since H. somni is an obligate inhabitant of the bovine and ovine host, the formation of a biofilm may be crucial to its persistence in vivo, and our in vitro evidence suggests that formation of a more robust biofilm may provide a selective advantage for strains that cause systemic disease.

Pasteurella multocidais an important multihost animal and zoonotic pathogen that is capable of causing respiratory and multisystemic diseases, bacteremia, and bite wound infections. The glycosaminoglycan capsule ofP. multocidais an essential virulence factor that protects the bacterium from host defenses. However, chronic infections (such as swine atrophic rhinitis and the carrier state in birds and other animals) may be associated with biofilm formation, which has not been characterized inP. multocida. Biofilm formation by clinical isolates was inversely related to capsule production and was confirmed with capsule-deficient mutants of highly encapsulated strains. Capsule-deficient mutants formed biofilms with a larger biomass that was thicker and smoother than the biofilm of encapsulated strains. Passage of a highly encapsulated, poor-biofilm-forming strain under conditions that favored biofilm formation resulted in the production of less capsular polysaccharide and a more robust biofilm, as did addition of hyaluronidase to the growth medium of all of the strains tested. The matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS), as determined by gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion. However, a putative glycogen synthesis locus was not differentially regulated when the bacteria were grown as a biofilm or planktonically, as determined by quantitative reverse transcriptase PCR. Therefore, the negatively charged capsule may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix from encasing large numbers of bacterial cells. This is the first detailed description of biofilm formation and a glycogen EPS byP. multocida.IMPORTANCEPasteurella multocidais an important pathogen responsible for severe infections in food animals, domestic and wild birds, pet animals, and humans.P. multocidawas first isolated by Louis Pasteur in 1880 and has been studied for over 130 years. However, aspects of its lifecycle have remained unknown. Although formation of a biofilm byP. multocidahas been proposed, this report is the first to characterize biofilm formation byP. multocida. Of particular interest is that the biofilm matrix material contained a newly reported amylose-like glycogen as the exopolysaccharide component and that production of capsular polysaccharide (CPS) was inversely related to biofilm formation. However, even highly mucoid, poor-biofilm-forming strains could form abundant biofilms by loss of CPS or followingin vitropassage under biofilm growth conditions. Therefore, the carrier state or subclinical chronic infections withP. multocidamay result from CPS downregulation with concomitant enhanced biofilm formation.

Francisella tularensis is a zoonotic, Gram-negative coccobacillus that causes tularemia in humans and animals. F. tularensis subspecies tularensis (type A) and F. tularensis subspecies holarctica (type B) are antigenically similar and more virulent than Francisella novicida in humans. The genetic locus that encodes the LPS O antigen was found to be substantially different between the type B live vaccine strain (LVS) and F. novicida. One LVS-specific gene with homology to a galactosyl transferase was selected for allelic replacement using a sacB–chloramphenicol expression suicide plasmid, and recombinants were screened for colony morphology on Congo red agar that matched that of F. novicida. Two mutants (WbtIS187Y and WbtIG191V) were isolated that contained substitutions in conserved motifs in the sugar transamine/perosamine synthetase (WbtI) of the O-antigen locus, and the latter mutant was extensively tested and characterized. WbtIG191V grew at the same rate as the parent strain in Chamberlain's defined medium, completely lacked O antigen, was serum-sensitive but could grow in a mouse macrophage cell line, had increased resistance to sodium deoxycholate, and was highly attenuated in mice. Complementation of WbtIG191V with the wild-type wbtI gene in trans restored normal LPS synthesis, phenotypic properties similar to the parent, and virulence in mice. Immunization with WbtIG191V protected mice against a relatively low-dose intraperitoneal challenge with LVS, but was less protective against a high-dose challenge. These results indicate that complete loss of O antigen alters the surface phenotype and abrogates virulence in F. tularensis, but also compromises the induction of full protective immunity against F. tularensis infection in mice.

Francisella tularensis is a category-A select agent and is responsible for tularemia in humans and animals. The surface components of F. tularensis that contribute to virulence are not well characterized. An electron-dense capsule has been postulated to be present around F. tularensis based primarily on electron microscopy, but this specific antigen has not been isolated or characterized. A capsule-like complex (CLC) was effectively extracted from the cell surface of an F. tularensis live vaccine strain (LVS) lacking O-antigen with 0.5% phenol after 10 passages in defined medium broth and growth on defined medium agar for 5 days at 32°C in 7% CO₂. The large molecular size CLC was extracted by enzyme digestion, ethanol precipitation, and ultracentrifugation, and consisted of glucose, galactose, mannose, and Proteinase K-resistant protein. Quantitative reverse transcriptase PCR showed that expression of genes in a putative polysaccharide locus in the LVS genome (FTL_1432 through FTL_1421) was upregulated when CLC expression was enhanced. Open reading frames FTL_1423 and FLT_1422, which have homology to genes encoding for glycosyl transferases, were deleted by allelic exchange, and the resulting mutant after passage in broth (LVSΔ1423/1422_P10) lacked most or all of the CLC, as determined by electron microscopy, and CLC isolation and analysis. Complementation of LVSΔ1423/1422 and subsequent passage in broth restored CLC expression. LVSΔ1423/1422_P10 was attenuated in BALB/c mice inoculated intranasally (IN) and intraperitoneally with greater than 80 times and 270 times the LVS LD₅₀, respectively. Following immunization, mice challenged IN with over 700 times the LD₅₀ of LVS remained healthy and asymptomatic. Our results indicated that the CLC may be a glycoprotein, FTL_1422 and -FTL_1423 were involved in CLC biosynthesis, the CLC contributed to the virulence of F. tularensis LVS, and a CLC-deficient mutant of LVS can protect mice against challenge with the parent strain.

Haemophilus somnusisolates from cases of thrombotic meningoencephalitis, pneumonia, and other disease sites are capable of undergoing a high rate of phase variation in the oligosaccharide component of their lipooligosaccharides (LOS). In contrast, the LOS of commensal strains isolated from the normal reproductive tract phase vary little or not at all. In addition, the LOS ofH. somnusshares conserved epitopes with LOS fromNeisseria gonorrhoeae,Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates ofH. somnuswith CMP-N-acetylneuraminic acid (CMP-NeuAc) or NeuAc added to the medium resulted in incorporation of NeuAc into the LOS. However, NeuAc was not incorporated into the LOS of commensal isolates and one disease isolate following growth in medium containing CMP-NeuAc or NeuAc. Sialylated LOS was detected by an increase in the molecular size or an increase in the amount of the largest-molecular-size LOS electrophoretic bands, which disappeared following treatment with neuraminidase. Sialylated LOS could also be detected by reactivity withLimax flavusagglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment.H. somnusstrain 2336 LOS was found to contain some sialic acid when grown in medium lacking CMP-NeuAc or NeuAc, although supplementation enhanced NeuAc incorporation. In contrast strain 738, an LOS phase variant of strain 2336, was less extensively sialylated when the growth medium was supplemented with CMP-NeuAc or NeuAc, as determined by electrophoretic profiles and electrospray mass spectrometry. The sialyltransferase ofH. somnusstrain 738 was confirmed to preferentially sialylate the Galβ-(1-3)-GlcNAc component of the lacto-N-tetraose structure by capillary electrophoresis assay. Enhanced sialylation of the strain 2336 LOS inhibited the binding of monoclonal antibodies to LOS by enzyme immunoassay and Western blotting. Furthermore, sialylation of the LOS enhanced the resistance ofH. somnusto the bactericidal action of antiserum to LOS. Sialylation and increased resistance to killing by normal serum also occurred in a deletion mutant that was deficient in the terminal Gal-GlcNAc disaccharide. LOS sialylation may therefore be an important virulence mechanism to protectH. somnusagainst the host immune system.