First reported case of chancroid in the Czech Republic.

We describe the first case of chancroid seen in the Czech Republic, diagnosed in a 40-year-old heterosexual HIV-positive man. Despite genital localization of the ulcer, the transmission of Haemophilus ducreyi infection in our patient remains unclear, as he denied having sexual intercourse and he did not travel outside the Czech Republic for several months before the ulcer appeared. The correct diagnosis has been revealed by a multiplex nucleic acid amplification test. Physicians in countries in the eastern and central Europe region should be aware that chancroid can occur in their patients.

Single-Dose Azithromycin for the Treatment of Haemophilus ducreyi Skin Ulcers in Papua New Guinea.

BACKGROUND: Haemophilus ducreyi (HD) and Treponema pallidum subspecies pertenue (TP) are major causative agents of cutaneous ulcer (CU) in the tropics. Azithromycin is recommended to treat sexually transmitted HD infections and has good in vitro activity against HD strains from both genital and skin ulcers. We investigated the efficacy of oral single-dose azithromycin on HD-CU. METHODS: We conducted a community-based cohort study in Lihir Island, Papua New Guinea, from October 2014 through May 2016. Consenting patients with skin ulcers >1 cm in diameter were eligible for this study and had collected a lesional swab for polymerase chain reaction (PCR). All participants were treated with single-dose azithromycin (30 mg/kg) and were followed up for assessment of clinical resolution. We retrospectively classified patients according to PCR results into HD, TP, and PCR-negative groups. The primary endpoint was healing rates of HD-CU at 14 days after treatment. RESULTS: We obtained full outcome data from 246 patients; 131 (53.3%) were HD PCR positive, 37 (15.0%) were TP positive, and 78 (31.7%) were negative for all tests. Healing rates were 88.5% (95% confidence interval [CI], .82-.93) in the HD group, 78.4% [95% CI, .63-.89] in the TP group, and 74.4% (95% CI, .64-.83) in the PCR-negative group. If we included the participants with improved ulcers, the healing rates increased to 94.7%, 97.3%, and 89.7% respectively. HD cases classified as not healed all converted to HD-negative PCR. CONCLUSIONS: Based upon clinical resolution and PCR conversion to HD negative, a single oral dose of azithromycin is efficacious for the treatment of HD-CU. These results have implications for the treatment of individual patients and for the use of antibiotics in public health strategies to control CU in the tropics.

Haemophilus ducreyi Cutaneous Ulcer Strains Are Nearly Identical to Class I Genital Ulcer Strains.

BACKGROUND: Although cutaneous ulcers (CU) in the tropics is frequently attributed to Treponema pallidum subspecies pertenue, the causative agent of yaws, Haemophilus ducreyi has emerged as a major cause of CU in yaws-endemic regions of the South Pacific islands and Africa. H. ducreyi is generally susceptible to macrolides, but CU strains persist after mass drug administration of azithromycin for yaws or trachoma. H. ducreyi also causes genital ulcers (GU) and was thought to be exclusively transmitted by microabrasions that occur during sex. In human volunteers, the GU strain 35000HP does not infect intact skin; wounds are required to initiate infection. These data led to several questions: Are CU strains a new variant of H. ducreyi or did they evolve from GU strains? Do CU strains contain additional genes that could allow them to infect intact skin? Are CU strains susceptible to azithromycin? METHODOLOGY/PRINCIPAL FINDINGS: To address these questions, we performed whole-genome sequencing and antibiotic susceptibility testing of 5 CU strains obtained from Samoa and Vanuatu and 9 archived class I and class II GU strains. Except for single nucleotide polymorphisms, the CU strains were genetically almost identical to the class I strain 35000HP and had no additional genetic content. Phylogenetic analysis showed that class I and class II strains formed two separate clusters and CU strains evolved from class I strains. Class I strains diverged from class II strains ~1.95 million years ago (mya) and CU strains diverged from the class I strain 35000HP ~0.18 mya. CU and GU strains evolved under similar selection pressures. Like 35000HP, the CU strains were highly susceptible to antibiotics, including azithromycin. CONCLUSIONS/SIGNIFICANCE: These data suggest that CU strains are derivatives of class I strains that were not recognized until recently. These findings require confirmation by analysis of CU strains from other regions.

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro.

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, ß-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and ß-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

Antimicrobial activity of plant extracts against sexually transmitted pathogens.

Comprehensive management of sexually transmitted infections (STIs) using vaginal or rectal microbicide-based intervention is one of the strategies for prevention of HIV infection. Herbal products have been used for treating STIs traditionally. Herein, we present in vitro activity of 10 plant extracts and their 34 fractions against three sexually transmitted/reproductive tract pathogens - Neisseria gonorrhoeae, Haemophilus ducreyi and Candida albicans. The plant parts were selected; the extracts/fractions were prepared and screened by disc diffusion method. The minimum inhibitory and minimum cidal concentrations were determined. The qualitative phytochemical analysis of selected extracts/fractions showing activity was performed. Of the extracts/fractions tested, three inhibited C. albicans, ten inhibited N. gonorrhoeae and five inhibited H. ducreyi growth. Our study demonstrated that Terminalia paniculata Roth. extracts/fractions inhibited growth of all three organisms. The ethyl acetate fraction of Syzygium cumini Linn. and Bridelia retusa (L.) Spreng. extracts was found to inhibit N. gonorrhoeae at lowest concentrations.

Assessing the antibiotic potential of essential oils against Haemophilus ducreyi.

BACKGROUND: Haemophilus ducreyi is the bacterium responsible for the genital ulcer disease chancroid, a cofactor for the transmission of HIV, and it is resistant to many antibiotics. With the goal of exploring possible alternative treatments, we tested essential oils (EOs) for their efficacy as antimicrobial agents against H. ducreyi. METHODS: We determine the minimum inhibitory concentration (MIC) of Cinnamomum verum (cinnamon), Eugenia caryophyllus (clove) and Thymus satureioides (thyme) oil against 9 strains of H. ducreyi using the agar dilution method. We also determined the minimum lethal concentration for each oil by subculturing from the MIC plates onto fresh agar without essential oil. For both tests, we used a 2-way ANOVA to evaluate whether antibiotic-resistant strains had a different sensitivity to the oils relative to non-resistant strains. RESULTS: All 3 oils demonstrated excellent activity against H. ducreyi, with MICs of 0.05 to 0.52 mg/mL and MLCs of 0.1-0.5 mg/mL. Antibiotic-resistant strains of H. ducreyi were equally susceptible to these 3 essential oils relative to non-resistant strains (p=0.409). CONCLUSION: E. caryophyllus, C. verum and T. satureioides oils are promising alternatives to antibiotic treatment for chancroid.

Resveratrol is cidal to both classes of Haemophilus ducreyi.

Resveratrol, a polyphenolic phytoalexin, is produced by plants in response to infection and has antibacterial activity. Haemophilus ducreyi is a Gram-negative bacterium that is the causative agent of the sexually transmitted disease chancroid. This study employed minimum cidal concentration (MCC) assays to evaluate the potential of resveratrol as a microbicide against H. ducreyi. Five class I and four class II strains of H. ducreyi tested had MCCs ≤500 µg/mL. Resveratrol was also tested against Lactobacillus spp., part of the natural vaginal flora. Representative strains of Lactobacillus were co-cultured with H. ducreyi and 500 µg/mL resveratrol; in all cases, Lactobacillus was recovered in greater numbers than H. ducreyi. These results show that resveratrol is not only bacteriostatic but is bactericidal to H. ducreyi, confirming the compound's potential for use as a topical microbicide to prevent chancroid.

Permeases of the sap transporter are required for cathelicidin resistance and virulence of Haemophilus ducreyi in humans.

BACKGROUND: Haemophilus ducreyi encounters several classes of antimicrobial peptides (APs) in vivo and utilizes the sensitive-to-antimicrobial-peptides (Sap) transporter as one mechanism of AP resistance. A mutant lacking the periplasmic solute-binding component, SapA, was somewhat more sensitive to the cathelicidin LL-37 than the parent strain and was partially attenuated for virulence. The partial attenuation led us to question whether the transporter is fully abrogated in the sapA mutant. METHODS: We generated a nonpolar sapBC mutant, which lacks both inner membrane permeases of the Sap transporter, and tested the mutant for virulence in human volunteers. In vitro, we compared LL-37 resistance phenotypes of the sapBC and sapA mutants. RESULTS: Unlike the sapA mutant, the sapBC mutant was fully attenuated for virulence in human volunteers. In vitro, the sapBC mutant exhibited significantly greater sensitivity than the sapA mutant to killing by LL-37. Similar to the sapA mutant, the sapBC mutant did not affect H. ducreyi's resistance to human defensins. CONCLUSIONS: Compared with the sapA mutant, the sapBC mutant exhibited greater attenuation in vivo, which directly correlated with increased sensitivity to LL-37 in vitro. These results strongly suggest that the SapBC channel retains activity when SapA is removed.

Cervicovaginal fluid and semen block the microbicidal activity of hydrogen peroxide produced by vaginal lactobacilli.

BACKGROUND: H2O2 produced by vaginal lactobacilli is believed to protect against infection, and H2O2-producing lactobacilli inactivate pathogens in vitro in protein-free salt solution. However, cervicovaginal fluid (CVF) and semen have significant H2O2-blocking activity. METHODS: We measured the H2O2 concentration of CVF and the H2O2-blocking activity of CVF and semen using fluorescence and in vitro bacterial-exposure experiments. RESULTS: The mean H2O2 measured in fully aerobic CVF was 23 +/- 5 microM; however, 50 microM H2O2 in salt solution showed no in vitro inactivation of HSV-2, Neisseria gonorrhoeae, Hemophilus ducreyii, or any of six BV-associated bacteria. CVF reduced 1 mM added H2O2 to an undetectable level, while semen reduced 10 mM added H2O2 to undetectable. Moreover, the addition of just 1% CVF supernatant abolished in vitro pathogen-inactivation by H2O2-producing lactobacilli. CONCLUSIONS: Given the H2O2-blocking activity of CVF and semen, it is implausible that H2O2-production by vaginal lactobacilli is a significant mechanism of protection in vivo.

Haemophilus ducreyi SapA contributes to cathelicidin resistance and virulence in humans.

Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides (APs). The organism's genome contains homologs of genes sensitive to antimicrobial peptides (sap operon) in nontypeable Haemophilus influenzae. In this study, we characterized the sap-containing loci of H. ducreyi 35000HP and demonstrated that sapA is expressed in broth cultures and H. ducreyi-infected tissue; sapA is also conserved among both class I and class II H. ducreyi strains. We constructed a nonpolar sapA mutant of H. ducreyi 35000HP, designated 35000HPsapA, and compared the percent survival of wild-type 35000HP and 35000HPsapA exposed to several human APs, including alpha-defensins, beta-defensins, and the cathelicidin LL-37. Unlike an H. influenzae sapA mutant, strain 35000HPsapA was not more susceptible to defensins than strain 35000HP was. However, we observed a significant decrease in the survival of strain 35000HPsapA after exposure to LL-37, which was complemented by introducing sapA in trans. Thus, the Sap transporter plays a role in resistance of H. ducreyi to LL-37. We next compared mutant strain 35000HPsapA with strain 35000HP for their ability to cause disease in human volunteers. Although both strains caused papules to form at similar rates, the pustule formation rate at sites inoculated with 35000HPsapA was significantly lower than that of sites inoculated with 35000HP (33.3% versus 66.7%; P = 0.007). Together, these data establish that SapA acts as a virulence factor and as one mechanism for H. ducreyi to resist killing by antimicrobial peptides. To our knowledge, this is the first demonstration that an antimicrobial peptide resistance mechanism contributes to bacterial virulence in humans.

Killing of Neisseria gonorrhoeae, Streptococcus agalactiae (group B streptococcus), Haemophilus ducreyi, and vaginal Lactobacillus by 3-O-octyl-sn-glycerol.

The microbicide candidate octylglycerol inactivates sexually transmitted bacterial pathogens at concentrations which spare normal vaginal flora (lactobacillus). Standard minimum microbicidal concentration assays and time-kill assays revealed the drug concentrations and times required for inactivation. Octylglycerol concentrations must exceed the binding capacity of any human serum albumin to be effective.

A role for Haemophilus ducreyi Cu,ZnSOD in resistance to heme toxicity.

The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast, under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase. Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity.

Haemophilus ducreyi is resistant to human antimicrobial peptides.

We examined the susceptibility of Haemophilus ducreyi to antimicrobial peptides likely to be encountered in vivo during human infection. H. ducreyi was significantly more resistant than Escherichia coli to the bactericidal effects of all peptides tested. Class I and II H. ducreyi strains exhibited similar levels of resistance to antimicrobial peptides.

The Hd0053 gene of Haemophilus ducreyi encodes an alpha2,3-sialyltransferase.

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted genital ulcer disease. Different lipooligosaccharide (LOS) structures have been identified from H. ducreyi strain 35000, including those sialylated glycoforms. Surface LOS of H. ducreyi is considered an important virulence factor that is involved in ulcer formation, cell adhesion, and invasion of host tissue. Gene Hd0686 of H. ducreyi, designated lst (for lipooligosaccharide sialyltransferase), was identified to encode an alpha2,3-sialyltransferase that is important for the formation of sialylated LOS. Here, we show that Hd0053 of H. ducreyi genomic strain 35000HP, the third member of the glycosyltransferase family 80 (GT80), also encodes an alpha2,3-sialyltransferase that may be important for LOS sialylation.

In vitro and in vivo activity of combination antimicrobial agents on Haemophilus ducreyi.

OBJECTIVES: Development of single dose antibiotic treatments for chancroid has been followed by drug-resistant Haemophilus ducreyi in endemic areas. We examined the activity and interactions of antimicrobial agents and combinations against H. ducreyi. METHODS: We evaluated the in vitro susceptibility of three virulent strains of H. ducreyi to ceftriaxone, azithromycin, rifabutin and streptomycin, and each two-drug combination by the agar dilution method. We then tested each two-antibiotic combination for activity by the chequerboard method. Lastly, we chose the antibiotic combination with the lowest fractional inhibitory concentration index (FICI) and tested combined sub-therapeutic doses, the highest doses which had no effect alone on lesion healing compared with controls, for in vivo interaction in the temperature-dependent rabbit model of H. ducreyi infection. RESULTS: Each H. ducreyi strain was susceptible in vitro to each antibiotic and two-antibiotic combination, and combined ceftriaxone and streptomycin had the lowest FICI at 0.63. In five treated animals versus three untreated controls, combined sub-therapeutic doses of ceftriaxone (0.05 mg/kg) and streptomycin (10 mg/kg) reduced mean (SD) duration of culture positivity from 7.3 (1.1) to 2.6 (1.7) days (P<0.001), time to 50% reduction in lesion size from 9.7 (1.5) to 5.8 (0.8) days (P<0.005), and time to resolution of ulcer from 11.7 (2.3) to 6.6 (1.7) days (P<0.05). CONCLUSIONS: Ceftriaxone and streptomycin have in vivo synergic interaction against H. ducreyi lesions in the temperature-dependent rabbit model of infection. Antibiotic combinations may be evaluated clinically as single-dose therapy for chancroid.

Porphyrin-based compounds exert antibacterial action against the sexually transmitted pathogens Neisseria gonorrhoeae and Haemophilus ducreyi.

A series of porphyrin based compounds without (nMP) or with (MP) metals were found to have potent bactericidal action in vitro against the sexually transmitted pathogens Neisseria gonorrhoeae and Haemophilus ducreyi. nMP and MP did not show bactericidal activity against five species of lactobacilli. An MP containing gallium had the capacity to block a gonococcal infection in a murine vaginal model, indicating that its development as a topical microbicide to block sexually transmitted bacterial infections is warranted. In contrast to other bacterial species, loss of the gonococcal haemoglobin uptake system encoded by hpuB or energy supplied through the TonB-ExbB-ExbD system did not significantly affect levels of MP-susceptibility in gonococci. In contrast, mutations in gonococci that inactivate the mtrCDE-encoded efflux pump were found to enhance gonococcal susceptibility to nMPs and MPs while over-production of this efflux pump decreased levels of gonococcal susceptibility to these compounds.

The activity of p-methoxybenzylisothiocyanate against Neisseria gonorrhoeae, Haemophilus ducreyi, and other microorganisms.

p-Methoxybenzylisothiocyanate was isolated from Lepidium bonariense and found to be responsible for the plants antimicrobial and STD activity. MIC determinations were conducted for p-methoxybenzylisothiocyanate on Haemophilus ducreyi, Neisseria gonorrheae, Candida albicans, Bacillus subtilus, Micrococcus luteus, Staphylococcus aureus, Enterobacter sp., Escherichia coli, Klebsiella pneumoniae, and Psuedomanas aeruginosa. An in vitro cellular toxicity assay showed that at 100 microM (17,9 microg/mL) p-methoxybenzylisothiocyanate is not toxic to living cells.

Genetic analysis of a pyocin-resistant lipooligosaccharide (LOS) mutant of Haemophilus ducreyi: restoration of full-length LOS restores pyocin sensitivity.

DNA sequence and Southern blot analyses were used to determine the genetic defect of a Haemophilus ducreyi pyocin-resistant lipooligosaccharide (LOS) mutant, HD35000R. The region of the HD35000R chromosome containing the suspected mutation was amplified, and sequence analysis detected a 3,189-bp deletion. This deletion resulted in the loss of the entire waaQ gene, another open reading frame that encodes a putative homolog to a hypothetical protein (HI0461) of H. influenzae, the gene encoding an argininosuccinate synthase homolog, and a change in the 3' sequence of the lgtF gene. Southern blot analysis confirmed that no genomic rearrangements had occurred. Isogenic LOS mutants and the respective complemented mutants were evaluated for susceptibility to pyocin C. The mutants expressing truncated LOS were resistant to lysis by pyocin C, and complementation restored sensitivity to the pyocin. We conclude that HD35000R is defective in both glycosyltransferase genes and that pyocin resistance is due to truncation of the full-length LOS molecule.

Episome profiles and mobilizable beta-lactamase plasmid in Haemophilus ducreyi.

Chancroid caused by Haemophilus ducreyi has been described as a significantly predisposing factor of HIV heterosexual transmission in an endemic region of both diseases. The fastidious, H. ducreyi has been reported world wide with various antimicrobial susceptibility patterns. A high tendency of drug resistances has generally been found among isolates derived in Thailand. In this study, the plasmids of H. ducreyi were isolated and analysed from 63 clinically derived organisms. Twenty-nine out of 63 isolates (46%) revealed the same plasmid profiles. Plasmid DNA was further cloned into Escherichia coli and transformants were selected. A 3.6 kb plasmid (pCb) carrying ampicillin resistance was subsequently identified. The pCb conferred resistance to various beta-lactam antibiotics including penicillin G, carbenicillin, piperacillin, cefazolin, cefoperazone, ampicillin-sulbactam, and amoxicillin-clavulanate but not to cefoxitin. Co-resistance to streptomycin, chloramphenicol and tetracycline was not detected. Beta-lactamase gene was located on the major pCb fragment of EcoRI and AatII cutting.