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PubMed1982

Plasmid-coded ampicillin resistance in Haemophilus ducreyi.

Seven of the 96 ampicillin-resistant isolates of Haemophilus ducreyi reported in the preceding article (Bilgeri et al., Antimicrob. Agents Chemother. 22:686-688, 1982) were investigated and found to harbor plasmids of 3.95, 5.2, 5.8, and 6.4 megadaltons. All except the 5.8-megadalton plasmid have been shown to code for beta-lactamase. The 6.4- and 5.2-megadalton plasmids of three isolates were conjugally transferable to a streptomycin-resistant mutant of H. ducreyi at high frequencies, perhaps due to the presence in these strains of a high-molecular-weight plasmid.

Ampicillin↗
PubMed1993

Comparison of the in vitro activities of various parenteral and oral antimicrobial agents against endemic Haemophilus ducreyi.

The in vitro susceptibilities to various antimicrobial agents of 100 strains of beta-lactamase-producing Haemophilus ducreyi recently isolated from patients in New Orleans, La., were determined by an agar dilution method. All strains were highly susceptible to ceftizoxime, ceftriaxone, ceftazidime, azithromycin, erythromycin, ciprofloxacin, and sparfloxacin. beta-Lactam-beta-lactamase inhibitor combinations were less active, but all strains were susceptible to them. Doxycycline exhibited the poorest activity, and the rate of resistance to doxycycline varied depending on the time after inoculation that the MIC was determined.

Anti-Bacterial Agents↗
PubMed1985

Role of lipopolysaccharide and complement in susceptibility of Haemophilus ducreyi to human serum.

The role of lipopolysaccharide (LPS) in the susceptibility of Haemophilus ducreyi to human serum and the mechanism of complement activation by serum-susceptible (Sers) strains were investigated. Serum treated with 2 mM Mg2+ and 20 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was nonbactericidal, but inulin-treated serum remained bactericidal. Absorption of serum with heat-killed whole cells of an Sers strain removed its bactericidal activity against the absorbing strain and also against other Sers strains. LPS obtained from Sers strains inhibited the bactericidal activity of serum against all Sers strains, whereas LPS from serum-resistant (Serr) strains and an Serr isogenic strain did not. However, high concentrations of LPS from the Serr strain inhibited the bactericidal activity of serum, an indication that part of the structural site involved in serum susceptibility is retained in the LPS of this strain. The LPS of Sers strains exhibited higher anticomplement activity than the LPS of Serr strains. These findings suggest that the classical pathway of complement activation is involved in the serum killing of H. ducreyi and that LPS composition may contribute to their susceptibility to complement-mediated serum bactericidal activity.

Animals↗
PubMed1993

Neutralizing antibodies to Haemophilus ducreyi cytotoxin.

Neutralizing antibodies against cytotoxin produced by Haemophilus ducreyi bacteria were studied in rabbits by an assay employing HEp-2 cells and diluted crude cytotoxin preparations from the organism. Antisera to 12 different H. ducreyi strains were prepared by immunization of rabbits with bacterial sonicates combined with Freund's adjuvant. The antibody response during infection with H. ducreyi was studied in two groups of rabbits which were infected with five live strains by either single or multiple intradermal injections. Neutralizing antibodies in hyperimmune sera to sonicates from 12 H. ducreyi strains tested against their homologous cytotoxin preparations were present with titers ranging from 1:80 to 1:640. Similar antibody titers against heterologous cytotoxin preparations were recorded, indicating immunological similarity or identity between cytotoxins from the various H. ducreyi strains. Three strains did not produce cytotoxin, and these strains did not induce toxin-neutralizing antibodies. Hyperimmune sera to other gram-negative bacteria had no detectable neutralizing capacity, indicating species specificity of the H. ducreyi cytotoxin. Cytotoxin-neutralizing antibodies were not detectable in rabbit sera before infection with H. ducreyi. Repeated single injections with live bacteria resulted in development of low levels of neutralizing antibodies. Multiple primary injection of live bacteria of the cytotoxin-producing strain CCUG 7470 resulted in a low immune response to the cytotoxin preparation from the same strain. A booster infection resulted in the development of neutralizing antibodies in all rabbits infected with cytotoxin-producing strains. The antibody titers determined against the homologous cytotoxin preparation were similar to those recorded for two heterologous cytotoxin preparations.

Animals↗
PubMed1996

A hemoglobin-binding outer membrane protein is involved in virulence expression by Haemophilus ducreyi in an animal model.

Haemophilus ducreyi exhibits a requirement for exogenously supplied heme for aerobic growth in vitro. Nine of ten wild-type isolates of H. ducreyi were shown to contain a readily detectable hemoglobin-binding activity. Spontaneous hemoglobin-binding-negative mutants of two of these wild-type isolates lost the ability to express an outer membrane protein with an apparent molecular mass of approximately 100 kDa. Similarly, the single wild-type isolate that lacked the ability to bind hemoglobin also appeared to lack expression of this same 100-kDa protein. A monoclonal antibody (5A9) to this 100-kDa protein was used to identify a recombinant clone which possessed an H. ducreyi chromosomal fragment containing the gene encoding the 100-kDa protein; this protein was designated hemoglobin utilization protein A (HupA). Nucleotide sequence analysis of the hupA gene revealed that the predicted protein, with a calculated molecular mass of 108 kDa, was similar to TonB-dependent outer membrane proteins of other bacteria. Increasing the concentration of heme in the growth medium resulted in decreased expression of the HupA protein. Mutant analysis was used to prove that the HupA protein was essential for the utilization by H. ducreyi of both hemoglobin and hemoglobin-haptoglobin as sources of heme in vitro. In addition, it was found that an isogenic hupA mutant was less virulent than the wild-type parent strain in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi.

Amino Acid Sequence↗
PubMed1998

Involvement of the Haemophilus ducreyi gmhA gene product in lipooligosaccharide expression and virulence.

The lipooligosaccharide (LOS) present in the outer membrane of Haemophilus ducreyi is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that had 87% identity with the protein product of the gmhA (isn) gene of Haemophilus influenzae. In H. influenzae type b, inactivation of the gmhA gene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396-402, 1996). The H. ducreyi gmhA gene was able to complement a gmhA-deficient Escherichia coli strain, a result which confirmed the identity of this gene. When the gmhA gene of H. ducreyi was inactivated by insertion of a cat cartridge, the resultant H. ducreyi gmhA mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type H. ducreyi strain and its isogenic gmhA mutant were used in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi, the gmhA mutant was found to be substantially less virulent than the wild-type parent strain. The H. ducreyi gmhA gene was amplified by PCR from the H. ducreyi chromosome and cloned into the pLS88 vector. When the H. ducreyi gmhA gene was present in trans in gmhA mutant 35000.252, expression of the gmhA gene product restored the virulence of this mutant to wild-type levels. These results indicate that the gmhA gene product of H. ducreyi is essential for the expression of wild-type LOS by this pathogen.

Aldose-Ketose Isomerases↗
PubMed2000

Localization of Haemophilus ducreyi at the pustular stage of disease in the human model of infection.

To localize Haemophilus ducreyi in vivo, human subjects were experimentally infected with H. ducreyi until they developed a painful pustule or for 14 days. Lesions were biopsied, and biopsy samples were fixed in 4% paraformaldehyde, and cryosectioned. Sections were stained with polyclonal anti-H. ducreyi antiserum or H. ducreyi-specific monoclonal antibodies (MAbs) and fluorescently tagged secondary antibodies and examined by confocal microscopy. We identified H. ducreyi in 16 of 18 pustules but did not detect bacteria in the one papule examined. H. ducreyi was observed as individual cells and in clumps or chains. Staining with MAbs 2D8, 5C9, 3B9, 2C7, and 9D12 demonstrated that H. ducreyi expresses the major pilus subunit, FtpA, the 28-kDa outer membrane protein Hlp, the 18-kDa outer membrane protein PAL, and the major outer membrane protein (MOMP) or OmpA2 in vivo. By dual staining with polyclonal anti-H. ducreyi antiserum and MAbs that recognize human skin components, we observed bacteria within the neutrophilic infiltrates of all positively staining pustules and in the dermis of 10 of 16 pustules. We were unable to detect bacteria associated with keratinocytes in the samples examined. The data suggest that H. ducreyi is found primarily in association with neutrophils and in the dermis at the pustular stage of disease in the human model of infection.

Adult↗
PubMed2000

Evaluation of an isogenic major outer membrane protein-deficient mutant in the human model of Haemophilus ducreyi infection.

Haemophilus ducreyi expresses 2 OmpA homologs, designated MOMP and OmpA2, whose genes are arranged in tandem on the chromosome. Northern blot analysis indicated that momp and ompA2 are transcribed independently. Sequences of the momp open reading frame (ORF) lacking the transcriptional start site were amplified by PCR, and an Omega-Km2 cassette was ligated into the ORF. A plasmid containing this construction was electroporated into H. ducreyi 35000HP, and an isogenic MOMP-deficient mutant (35000HP-SMS2) was generated by allele exchange. In Southern blotting, 35000HP-SMS2 contained one copy of the Omega-Km2 cassette in momp. 35000HP and 35000HP-SMS2 had similar outer membrane protein (OMP) and lipooligosaccharide profiles and growth rates except for up-regulation of a putative porin protein in the mutant. Five subjects were inoculated with three doses of live 35000HP-SMS2 on one arm and two doses of live 35000HP and one dose of a heat-killed control on the other arm in a double-blind escalating dose-response trial. Pustules developed at 7 of 10 sites inoculated with 35000HP and at 6 of 15 sites inoculated with 35000HP-SMS2 (P = 0.14). 35000HP and 35000HP-SMS2 were recovered at similar rates from daily surface cultures and semiquantitative cultures. The data suggest that expression of MOMP is not required for pustule formation by H. ducreyi in the human model of infection.

Adult↗
PubMed2001

Transcription of candidate virulence genes of Haemophilus ducreyi during infection of human volunteers.

Haemophilus ducreyi expresses several putative virulence factors in vitro. Isogenic mutant-to-parent comparisons have been performed in a human model of experimental infection to examine whether specific gene products are involved in pathogenesis. Several mutants (momp, ftpA, losB, lst, cdtC, and hhdB) were as virulent as the parent in the human model, suggesting that their gene products did not play a major role in pustule formation. However, we could not exclude the possibility that the gene of interest was not expressed during the initial stages of infection. Biopsies of pustules obtained from volunteers infected with H. ducreyi were subjected to reverse transcription-PCR. Transcripts corresponding to momp, ftpA, losB, lst, cdtB, and hhdA were expressed in vivo. In addition, transcripts for other putative virulence determinants such as ompA2, tdhA, lspA1, and lspA2 were detected in the biopsies. These results indicate that although several candidate virulence determinants are expressed during experimental infection, they do not have a major role in the initial stages of pathogenesis.

Adult↗
PubMed2001

DsrA-deficient mutant of Haemophilus ducreyi is impaired in its ability to infect human volunteers.

Haemophilus ducreyi produces an outer membrane protein called DsrA, which is required for serum resistance. An isogenic dsrA mutant, FX517, was constructed previously in H. ducreyi 35000. Compared to its parent, FX517 cannot survive in normal human serum. When complemented in trans with a plasmid containing dsrA, FX517 is converted to a serum-resistant phenotype (C. Elkins, K. J. Morrow, Jr., and B. Olsen, Infect. Immun. 68:1608-1619, 2000). To test whether dsrA was transcribed in vivo, we successfully amplified transcripts in five biopsies obtained from four experimentally infected human subjects. To test whether DsrA was required for virulence, six volunteers were experimentally infected with 35000 and FX517 and observed for papule and pustule formation. Each subject was inoculated with two doses (70 to 80 CFU) of live 35000 and 1 dose of heat-killed bacteria on one arm and with three doses (ranging from 35 to 800 CFU) of live FX517 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent. However, mutant papule surface areas were significantly smaller than parent papules. The pustule formation rate was 58% (95% confidence interval [CI] of 28 to 85%) at 12 parent sites, and 0% (95% CI of 0 to 15%) at 18 mutant sites (P = 0.0004). Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of DsrA facilitates the ability of H. ducreyi to progress to the pustular stage of disease.

Adult↗
PubMed2001

Expression of cytolethal distending toxin and hemolysin is not required for pustule formation by Haemophilus ducreyi in human volunteers.

Haemophilus ducreyi makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by H. ducreyi in humans.

Adult↗
PubMed2001

Haemophilus ducreyi lipooligosaccharide mutant defective in expression of beta-1,4-glucosyltransferase is virulent in humans.

The lipooligosaccharide (LOS) of Haemophilus ducreyi contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans.

Adult↗
PubMed2004

The LspB protein is involved in the secretion of the LspA1 and LspA2 proteins by Haemophilus ducreyi.

The LspA1 and LspA2 proteins of Haemophilus ducreyi 35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of the Bordetella pertussis filamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. The lspA2 open reading frame is flanked upstream by a gene, lspB, that encodes a predicted protein with homology to the B. pertussis FhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. The H. ducreyi lspB gene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that the lspB gene was transcribed together with the lspA2 gene on a single mRNA transcript. Polyclonal H. ducreyi LspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction of H. ducreyi 35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids from H. ducreyi lspB and lspA1 lspB mutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of the lspB mutant with the wild-type lspB gene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, the lspB mutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that the H. ducreyi LspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.

Amino Acid Sequence↗
PubMed2005

Trafficking pathways and characterization of CD4 and CD8 cells recruited to the skin of humans experimentally infected with Haemophilus ducreyi.

T-cell homing to infected skin is not well studied in humans. We examined sites experimentally infected with Haemophilus ducreyi by immunohistochemistry and flow cytometry for expression of receptors and ligands involved in cutaneous T-cell homing and determined the phenotypes of the T cells that trafficked to skin. Endothelial cells expressed E-selectin in infected but not uninfected skin, while peripheral node addressin (PNAd) was minimally expressed in all samples. CC chemokine ligand 27 (CCL27) was expressed in the epidermis and endothelium of both infected and uninfected skin. Interestingly, CCL21, a chemokine thought to be associated principally with T-cell trafficking in the lymphatic compartment, was highly expressed on the endothelium of infected skin. Few naive cells were present in experimental lesions, emphasizing the combined role of PNAd and CCL21 in trafficking of this subset. Memory cells (CD45RA-) dominated both CD4 and CD8 T-cell populations at the site of infection. Effector memory (CD45RA- CD27-) CD4+ and CD8+ T cells were enriched in lesions. Although the CC chemokine receptor 7-positive (CCR7+) population of both central memory (CD45RA- CD27+) and effector memory cells was not enriched in the skin compared to peripheral blood, CCR7+ cells were not precluded from entering infected skin. Taken together with our previous work (D. Soler, T. L. Humphreys, S. M. Spinola, and J. J. Campbell, Blood 101:1677-1683, 2003), these studies led us to propose a model of memory T-cell trafficking to skin in response to experimental H. ducreyi infection.

Adult↗
PubMed2006

A DltA mutant of Haemophilus ducreyi Is partially attenuated in its ability to cause pustules in human volunteers.

Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.

Adolescent↗
PubMed1980

Isolation and identification of Haemophilus ducreyi in a clinical study.

Seventeen strains of Haemophilus ducreyi were isolated from genital lesions which were negative for syphilis by dark-field examination. Media used for primary isolation at various times during the study were enriched chocolate agar, chocolate agar plus vancomycin (3 microgram/ml), rabbit blood agar plus vancomycin (3 micrograms/ml), fetal bovine serum agar, and fetal bovine serum agar plus vancomycin (3 micrograms/ml). H. ducreyi was isolated on chocolate agar plus vancomycin from 10 of 14 patients found to be positive on one or more media, on rabbit blood agar plus vancomycin from 16 of 17 patients, and on fetal bovine serum agar plus vancomycin from 9 of 11 patients. Sera from six animal species were tested to determine if any would support the growth of H. ducreyi. Horse and rabbit sera supported light growth of some strains. Fetal bovine serum supported good growth of all strains included in the study. Biochemical and physiological tests were done on the 17 isolates, a reference strain of H. ducreyi, and two reference strains of Haemophilus haemoglobinophilus. The results agreed with those reported by Kilian, except that H. ducreyi produced alpha-hemolysis in stabs on rabbit blood agar and was oxidase positive, three strains were urease positive, and CO2 improved the growth of seven strains. All 17 isolates were beta-lactamase positive. The reference strains were beta-lactamase negative.

Agar↗
PubMed1982

Isolation and cultivation of Haemophilus ducreyi.

A useful method for isolating and recognizing Haemophilus ducreyi from chancres and buboes of male patients is presented. A total of 41 clinical isolates of H. ducreyi were recovered from 33 patients over an 8-year period, and the experience with the 15 most recent isolates is presented in detail. Chocolate agar supplemented with 1% Iso VitaleX and 5% sheep blood agar were prepared, using Trypticase soy and Mueller-Hinton Agar bases, and incubation conditions included ambient, capneic, and anaerobic environments. Mueller-Hinton agar was clearly superior over Trypticase soy agar for isolation of H. ducreyi, although there was little difference between 5% sheep blood and supplemented chocolate agar. Growth in ambient air and under anaerobiasis was poor or lacking, whereas growth in 5 to 7% CO2 was good to luxuriant. Heat-inactivated and fresh (unheated)human blood clot tubes also were used for selective isolation. Although the rates of isolation from the two types of clot tube were not significantly different, unheated clot tubes were superior to heated clot tubes because of reduced level of contaminants. Gram stain characteristics taken from blood clot tubes and solid media, cellular and colonial morphology of the bacilli, and lack of oxidase, catalase, and biochemical activity except nitrate reductase were determinant factors. The results of this study demonstrated that successful isolation of H. ducreyi can be achieved with a minimal amount of resources and expertise.

Anaerobiosis↗
PubMed1989

DNA probes for the identification of Haemophilus ducreyi.

Haemophilus ducreyi ATCC 33922, a virulent, well-characterized strain, was used to construct a genomic library in a bacteriophage expression vector. Three DNA fragments were selected for use as probes on the basis of their ability to encode H. ducreyi-specific proteins, as demonstrated by reactivity with rabbit polyclonal antiserum. With DNA-DNA hybridization, the three probes, labeled with 32P, reacted strongly with 16 strains of H. ducreyi obtained from a variety of sources. Thirty-seven other bacterial isolates, representing 33 different species and including organisms likely to be encountered in the urogenital tract, were also tested with the three probes. Twenty-eight of these isolates, including the genital pathogen Neisseria gonorrhoeae, showed no hybridization with the probes. In addition, herpes simplex virus-infected tissue culture cells and Treponema pallidum-infected rabbit testicular fluid were also completely nonreactive. Nine isolates, six belonging to other Haemophilus species and three belonging to Pasteurella species, reacted weakly with the probes when approximately 3.0 x 10(7) to 6.0 x 10(7) CFU was tested. When 10(5) to 10(6) CFU of these organisms was tested, the weak reactions could no longer be seen. Yet this number of H. ducreyi still reacted strongly. In fact, the three probes consistently detected 10(4) CFU of H. ducreyi in pure and mixed cultures and even produced a weak signal when only 10(3) CFU was present. It is clear from our results that use of these probes will greatly facilitate the laboratory diagnosis of this genital pathogen.

Animals↗