Thomas J. Inzana

Transferable Antibiotic Resistance Elements in Haemophilus influenzae Share a Common Evolutionary Origin with a Diverse Family of Syntenic Genomic Islands

Transferable antibiotic resistance in Haemophilus influenzae was first detected in the early 1970s. After this, resistance spread rapidly worldwide and was shown to be transferred by a large 40- to 60-kb conjugative element. Bioinformatics analysis of the complete sequence of a typical H. influenzae conjugative resistance element, ICEHin1056, revealed the shared evolutionary origin of this element. ICEHin1056 has homology to 20 contiguous sequences in the National Center for Biotechnology Information database. Systematic comparison of these homologous sequences resulted in identification of a conserved syntenic genomic island consisting of up to 33 core genes in 16 beta- and gamma-Proteobacteria. These diverse genomic islands shared a common evolutionary origin, insert into tRNA genes, and have diverged widely, with G+C contents ranging from 40 to 70% and amino acid homologies as low as 20 to 25% for shared core genes. These core genes are likely to account for the conjugative transfer of the genomic islands and may even encode autonomous replication. Accessory gene clusters were nestled among the core genes and encode the following diverse major attributes: antibiotic, metal, and antiseptic resistance; degradation of chemicals; type IV secretion systems; two-component signaling systems; Vi antigen capsule synthesis; toxin production; and a wide range of metabolic functions. These related genomic islands include the following well-characterized structures: SPI-7, found in Salmonella enterica serovar Typhi; PAP1 or pKLC102, found in Pseudomonas aeruginosa; and the clc element, found in Pseudomonas sp. strain B13. This is the first report of a diverse family of related syntenic genomic islands with a deep evolutionary origin, and our findings challenge the view that genomic islands consist only of independently evolving modules.

Haemophilus somnus Induces Apoptosis in Bovine Endothelial Cells In Vitro

ABSTRACT Haemophilus somnus causes pneumonia, reproductive failure, infectious myocarditis, thrombotic meningoencephalitis, and other diseases in cattle. Although vasculitis is commonly seen as a result of systemic H. somnus infections, the pathogenesis of vascular damage is poorly characterized. In this study, we demonstrated that H. somnus (pathogenic isolates 649, 2336, and 8025 and asymptomatic carrier isolates 127P and 129Pt) induce apoptosis of bovine endothelial cells in a time- and dose-dependent manner, as determined by Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end labeling, DNA fragmentation, and transmission electron microscopy. H. somnus induced endothelial cell apoptosis in as little as 1 h of incubation and did not require extracellular growth of the bacteria. Viable H. somnus organisms induced greater endothelial cell apoptosis than heat-killed organisms. Since viableH. somnus cells release membrane fibrils and blebs, which contain lipooligosaccharide (LOS) and immunoglobulin binding proteins, we examined culture filtrates for their ability to induce endothelial cell apoptosis. Culture filtrates induced similar levels of endothelial cell apoptosis, as did viable H. somnus organisms. Heat inactivation of H. somnus culture filtrates partially reduced the apoptotic effect on endothelial cells, which suggested the presence of both heat-labile and heat-stable factors. We found thatH. somnus LOS, which is heat stable, induced endothelial cell apoptosis in a time- and dose-dependent manner and was inhibited by the addition of polymyxin B. These data demonstrate that H. somnus and its LOS induce endothelial cell apoptosis, which may play a role in producing vasculitis in vivo.

Incorporation of N-Acetylneuraminic Acid into Haemophilus somnus Lipooligosaccharide (LOS): Enhancement of Resistance to Serum and Reduction of LOS Antibody Binding

ABSTRACT Haemophilus somnus isolates 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 of H. somnus shares conserved epitopes with LOS from Neisseria gonorrhoeae, Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates of H. somnus with 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 with Limax flavus agglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment. H. somnus strain 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 of H. somnus strain 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 of H. somnus to 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 protect H. somnus against the host immune system.

Characterization and Comparison of Biofilm Development by Pathogenic and Commensal Isolates of Histophilus somni

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.

Complete Genome Sequence of Haemophilus somnus (Histophilus somni) Strain 129Pt and Comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd

Haemophilus somnus can be either a commensal of bovine mucosal surfaces or an opportunistic pathogen. Pathogenic strains of H. somnus are a significant cause of systemic disease in cattle. We report the genome sequence of H. somnus 129Pt, a nonpathogenic commensal preputial isolate, and the results of a genome-wide comparative analysis of H. somnus 129Pt, Haemophilus influenzae Rd, and Haemophilus ducreyi 35000HP. We found unique genes in H. somnus 129Pt involved in lipooligosaccharide biosynthesis, carbohydrate uptake and metabolism, cation transport, amino acid metabolism, ubiquinone and menaquinone biosynthesis, cell surface adhesion, biosynthesis of cofactors, energy metabolism, and electron transport. There were also many genes in common among the three organisms. Our comparative analyses of H. somnus 129Pt, H. influenzae Rd, and H. ducreyi 35000HP revealed similarities and differences in the numbers and compositions of genes involved in metabolism, host colonization, and persistence. These results lay a foundation for research on the host specificities and niche preferences of these organisms. Future comparisons between H. somnus 129Pt and virulent strains will aid in the development of protective strategies and vaccines to protect cattle against H. somnus disease.

Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

ABSTRACT The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kanr) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kanr gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Δcps1N and strain 4074Δcps1B, respectively. Strain 4074Δcps1N produced no detectable CP, but strain 4074Δcps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Δcps1N to produce 4074Δcps1N(pABcps101), 4074Δcps1N(pJMLcps53), and 4074Δcps1N(pABcps55), respectively. Strain 4074Δcps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Δcps1N(pJMLcps53) and 4074Δcps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Δcps1N(pABcps101) ≥ strain 4074Δcps1N > strain 4074Δcps1B. Strain 4074Δcps1N(pJMLcps53) was less virulent than strain 4074Δcps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

Haemophilus Somnus: Antigen Analysis and Immune Responses

Haemophilus sonrnus causes several disease syndromes in cattle and sheep but is also carried asymptomatically, especially on the genital mucosa. The factors involved in determining whether disease or asymptomatic carriage result are only beginning to be defined. Both host and bacteria contribute to the outcome of the dynamic interaction. To gain some insight into H. sonrnus/host interactions, it is necessary to consider what is known of the disease or asymptomatic conditions seen as well as current information on virulence factors and host immune response. This review constitutes the biased synthesis of the reviewers’ thoughts on available data relative to H. sonrnus infection.

Detection of methicillin-resistant staphylococci by biosensor assay consisting of nanoscale films on optical fiber long-period gratings.

Methicillin-resistance among Staphylococcus species is a major health problem in hospitals, communities, and animals. There is a need for culture-free diagnostic assays that can be carried out rapidly, and maintain a high degree of sensitivity and specificity. To address this need an ionic self-assembled multilayer (ISAM) film was deposited on the surface of a long-period grating (LPG) optical fiber by immersion alternately in poly-allylamine hydrochloride and in poly-1-[p-(3-carboxy-4-hydroxyphenylazo) benzenesulfonamido]-1,2-ethandiyl (PCBS), resulting in terminal carboxyl groups on the LPG-ISAM. The terminal carboxyl groups were covalently conjugated to monoclonal antibodies (MAb) specific to penicillin-binding-protein 2a of methicillin resistant (MR) staphylococci. After exposure of the LPG-ISAM to 10(2) colony forming units (CFU)/ml of MR S. aureus (MRSA) for 50 min., light transmission was reduced by 19.7%. In contrast, after exposure to 10(6) CFU/ml of methicillin-sensitive S. aureus (MSSA) attenuation of light transmission was less than 1.8%. Exposure of the LPG-ISAM to extracts of liver, lungs, or spleen from mice infected with MRSA attenuated light transmission by 11.7-73.5%. In contrast, exposure of the biosensor to extracts from MSSA-infected mice resulted in 5.6% or less attenuation of light transmission. When the sensor was tested with 36 strains of MR staphylococci, 15 strains of methicillin-sensitive staphylococci, 10 strains of heterologous genera (all at 10(4) CFU/ml), or tissue samples from mice infected with MRSA, there was complete agreement between MR and non-MR bacteria determined by antibiotic susceptibility testing and the biosensor assay when the cutoff value for attenuation of light transmission was 6.3%. Thus, the biosensor described has the potential to detect MR staphylococci in clinical samples with a high degree of sensitivity and specificity.

Prevalence and characterization of methicillin-resistant Staphylococcus aureus isolates from healthy university student athletes

BackgroundThe prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has been increasing in the general population, and there is concern that close or physical contact, such as in professional and collegiate sports, may increase spread of MRSA. We sought to determine the prevalence of MRSA colonization of male and female athletes from 9 different sports at a major, Division I University during a 12-week period, and determine the USA and SCCmec type from select isolates.MethodsSwabs for culture of MRSA were obtained from nasal, axillary, and inguinal sites from healthy, asymptomatic student athletes and support staff each week for 12xa0weeks. Select MRSA isolates were typed by pulsed field gel electrophoresis (PFGE), and the genes encoding for MecA, cassette chromosome recombinase (Ccr), and several toxins were determined by multiplex polymerase chain reaction (PCR). Discrepant results were clarified by multi-locus sequence typing (MLST) and spa typing.ResultsThirty-five percent (78/223) of test subjects were positive for MRSA during the study period, resulting in isolation of 139 MRSA isolates. However, 47% (37/78) of MRSA-positive participants carried MRSA in axillary or inguinal sites, but not in the anterior nares. There was significant correlation between MRSA carriage and participation in wrestling (76%, 19/25; adjusted odds ratio 29.7, 95% CI 5.8-151.5) and baseball (44%, 17/39; adjusted odds ratio 4.4, 95% CI 1.1- 17.4), compared with a staff prevalence of 18.1% (4/22), but other factors were not examined. Multiplex PCR analysis indicated that of the 32 isolates examined 26 could be typed, and all of these carried the SCCmec type IV cassette. PFGE typing identified USA types 300, 400, 500, 700, and 800. However, one isolate was not a known USA type, but was identified as a novel ST951 by MLST, and as spa type t216. Of the strains typed from the same individual, there was consistency, but also variation and alternation of the SCCmec and spa types isolated from individual subjects. Various staphylococcal toxin genes were identified in 31 of the 32 isolates analyzed.ConclusionsColonization by MRSA was greater in some student athletes than the average carriage rate for the general population, and only 53% of MRSA carriers were identified by nasal cultures. Carriage of MRSA clones on the same individual and transmission to contacts could vary over time, indicating colonization can be a dynamic process that may be difficult to control.

A variety of novel lipid A structures obtained from Francisella tularensis live vaccine strain

F. tularensis is a Gram-negative coccobacillus that causes tularemia. Its LPS has nominal biological activity. Currently, there is controversy regarding the structure of the lipid A obtained from F. tularensis live vaccine strain (LVS). Therefore, to resolve this controversy, the purification and structural identification of this LPS was crucial. To achieve this, LPS from F. tularensis LVS was acid hydrolyzed to obtain crude lipid A that was methylated and purified by HPLC and the fractions were analyzed by MALDI-TOF MS. The structure of the major lipid A species was composed of a glucosamine disaccharide backbone substituted with four fatty acyl groups and a phosphate (1-position) with a molecular mass of 1505. The major lipid A component contained 18:0[3–O(16:0)] in the distal subunit and two 18:0(3-OH) fatty acyl chains at the 2- or 3-positions of the reducing subunit. Additional variations in the lipid A species include: heterogeneity in fatty acyl groups, a phosphate or a phosphoryl galactosamine at the 1-position, and a hexose at the 4′ or 6′ position, some of which have not been previously described for F. tularensis LVS. This analysis revealed that lipid A from F. tularensis LVS is far more complex than originally believed.