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Purpose. The standard approach to screening sexually transmitted infections (STIs) has often been restricted to urogenital specimens. Most current guidelines, however, also recommend testing extra-genital sites, including rectal locations, because asymptomatic rectal carriage of pathogens has often been reported. The aim of our study was to evaluate self-collected rectal swabs to screen bacterial STIs in HIV-infected patients in Marseille, France. Methodology. Between January 2014 and December 2015, 118 HIV-infected patients (93 males and 25 females) agreed to self-sample anal swabs for detection of bacterial STI. Detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Mycoplasma genitalium and Haemophilus ducreyi was performed using in-house qPCR assay. Results/Key findings. Bacterial STIs were found in 8 % (9/118) of the patients. C. trachomatis was the most commonly detected bacterium (4.2 %) followed by N. gonorrhoeae (2.5 %), M. genitalium (1.7 %) and T. pallidum (0.8 %). All the positive patients were males. The rectal carriage of pathogenic bacteria was fortuitously discovered for seven men (78 %) who did not present rectal signs of STIs and was suspected for two men who presented proctitis (22 %). Conclusion. In conclusion, testing extra-genital sites is crucial for the diagnosis of STIs in men and women presenting or not concomitant urogenital infections in order to detect asymptomatic carriage with the aim of controlling and preventing transmission to their sexual partners.

The authors have previously shown that the periodontal pathogen Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans Y4 contains an operon for a genotoxin known as the cytolethal distending toxin (Cdt). The cdt locus in strain Y4 is flanked by remnants of heterologous plasmid and integrase sequences. In this study, the DNA sequence immediately downstream from the cdt locus on the Y4 chromosome was examined. The extended sequence contained a region that had all the characteristics of a typical bacterial pathogenicity or genomic island. The genomic island (GIY4-1) was approximately 22 kb long, was flanked by a bacteriophage attachment (att) sequence and contained a full-length integrase/resolvase gene (xerD). A total of 22 complete and partial ORFs represented putative DNA replication/DNA binding/conjugation proteins as well as hypothetical proteins. GIY4-1 was most closely related to putative genomic islands in Haemophilus ducreyi 35000HP and Haemophilus influenzae 86-028NP and to a chromosomal region in Haemophilus somnus 129PT. GIY4-1 was not present in HK1651, which was used as the prototype strain for genomic sequencing of A. actinomycetemcomitans. Several sequences in GIY4-1 were homologous to ORFs found on the A. actinomycetemcomitans plasmid pVT745. None of the identified ORFs in GIY4-1 appeared to encode potential virulence genes. However, several unique observations supported the possibility that the cdt locus of A. actinomycetemcomitans Y4 was originally contained within the genomic island.

A physical and rudimentary genetic map of the Haemophilus ducreyi strain 35000 genome was constructed. Pulsed-field gel electrophoresis was used to separate restriction fragments of H. ducreyi DNA digested with Sfil, I-Ceul, or Sfil plus I-Ceul. The sizes of the fragments were determined, and the circular chromosome was estimated to be 1757 kbp. The six I-Ceul fragments and four Sfil fragments were ordered into macrorestriction maps using Southern blot hybridization with random H. ducreyi clones as probes. It was shown that both H. ducreyi and the distantly related Haemophilus influenzae have six rrn operons marked by the locations of the I-Ceul sites. However, the two species displayed distinct I-Ceul restriction patterns. A second H. ducreyi strain, CIP542, displayed an identical I-Ceul pattern to that of H. ducreyi 35000, but Sfil digests of teh two strains were distinct. The orientation of the six rrn operons was determined and thirteen identified H. ducreyi genes positioned on the map of strain 35000.

The cell wall and outer structures of Haemophilus ducreyi bacteria were investigated. The 24-kDa outer protein from two strains was purified with an SDS-PAGE preparative continuous-elution electrophoresis cell. The protein was further characterised by SDS-PAGE and immunoblotting, and the immunological properties were investigated by ELISA. Localisation on the bacterial surface was investigated by immuno-electron-microscopy with a polyclonal antiserum raised against the purified protein. A triple-laminar cell wall typical of gram-negative bacteria, close cellular contact between bacterial cells and outer blebs were seen on thin sections. An additional high mol. wt band of c. 165 kDa was seen when not treated by heating to 100 degrees C. A high density fibrilla-like material was detected on the bacterial cell and in the environment by negative staining and immuno-electron-microscopy with antisera specific for the 24-kDa protein. The surface localisation of the 24-kDa protein was confirmed by an ELISA technique with the specific antiserum and whole bacterial cells as antigen. The presence of antibodies to the 24-kDa protein was demonstrated in antisera to 13 strains of H. ducreyi, indicating antigenic identity or within-species cross-reactivity. Low titres of antibodies to this protein were also detected in 19 antisera raised against different strains of gram-negative bacteria, indicating cross-reactivity with other species. Antibody response to the 24-kDa protein in rabbits immunised subcutaneously with live bacteria resulted in a secondary IgG response. Of 28 sera from patients with culture-verified chancroid, 26 manifested high titres of IgG antibodies to the 24-kDa protein, thus indicating the involvement of this antigen in the disease process in man.

Haemophilus influenzae is a V factor-dependent species. A plasmid conferring V factor independence in Haemophilus parainfluenzae and Haemophilus ducreyi was transferred to plasmid-free H. influenzae Rd by DNA transformation. The growth characteristics of the transformants in a complex and a chemically defined medium were compared, and the ability of several exogenous pyridine nucleotides and precursors to support growth was examined. Although the transformants appeared to be V factor independent in a complex medium, in a chemically defined medium they exhibited both V factor-dependent and nicotinamide-dependent growth. Because of the inability of the plasmid-free H. influenzae Rd to utilize nicotinamide for growth, it was concluded that the genes conferring this function were plasmid linked. Our results indicate that the V factor requirement, as it is presently defined, is not suitable to serve as a definitive taxonomic criterion for species determination in the family Pasteurellaceae.

Summary. An explant adult foreskin cell culture (FS2-3) was compared with human lung carcinoma cell culture (A549) with regard to the ability of Haemophilus ducreyi to produce a cytopathic effect. The survival of H. ducreyi for up to 26 days in FS2-3 cells was far greater than in any previously described in-vitro culture system. H. ducreyi survived for up to 7 days in A549 cells. The H. ducreyi cells grew and formed “fungal-like” microcolonies on the eukaryotic monolayer. Portions of the microcolonies remained attached despite extensive washing. Transmission electronmicroscopy indicated that, at 48 h after infection, the H. ducreyi cells did not penetrate the FS2-3 cells but they were closely associated with them; there was only a 2-5 nm gap between the H. ducreyi cell wall and the FS2-3 membrane. The virulent H. ducreyi strains R018 and 35000 produced a cytopathic effect on FS2-3 cells that did not appear to be due to a soluble toxin. These strains did not produce any CPE on A549 cells. H. influenzae and the avirulent H. ducreyi strain CIP542, inoculated in the same concentration and incubated for the same length of time, did not produce CPE on FS2-3 cells. This study demonstrated that the use of FS2-3 foreskin cell culture provided an in-vitro approach for evaluating the cytopathic effect of virulent H. ducreyi whereby, unlike in other in-vitro systems, viability of the micro-organism could be readily maintained.

Summary. The ability of various haem-and non-haem-iron-containing compounds to support the growth of iron-limited cultures of Haemophilus ducreyi was assessed in a plate bioassay. Only haemin or the haem-containing proteins, bovine haemoglobin, human haemoglobin and bovine catalase, but not equine cytochrome C1 1 1, were capable of serving as the sole exogenous iron source. Complexes of haptoglobin-haemoglobin and haem-serum albumin retained the ability to function as iron substrates. In contrast, no growth was observed with FeCI3, human lactoferrin and human transferrin. Siderophore production was not detected with a universal chemical assay. Outer-membrane-protein profiles derived from iron-starved cultures revealed four iron-regulated polypeptides of 65, 50, 40·5 and 40·5 Kda. These results indicate that haem can supply the requisite iron for growth of H. ducreyi.

Summary Part of a ribosomal ribonucleic acid (rRNA) cistron of Haemophilus ducreyi was enzymically amplified using conserved primers within the rRNA molecules, cloned in a plasmid vector, and sequenced. From the nucleotide sequence, eight oligonucleotides complementary to different regions in the 16S and 23S rRNA molecules were selected, chemically synthesized, and used as hybridization probes. Hybridization experiments with at least 41 H. ducreyi strains and 13 or 14 non-H. ducreyi strains revealed that all eight oligonucleotide probes were highly reliable and completely specific for H. ducreyi strains. Comparisons of 16S rRNA sequences confirm that H. ducreyi is a member of the Pasteurellaceae though not closely related to other species in this family.

Summary The enzymic activity of 29 Haemophilus ducreyi strains on 28 substrates is described. The results are compared with those of seven other authors. There is agreement only about the presence of alkaline phosphatase and arginine aminopeptidase and the lack of glycosidases. Possible reasons for the contradictions in the eight reports are discussed.

The Gram reactions and cell wall topographies of six strains of Haemophilus piscium, including the type strain, and four strains assigned to Haemophilus ducreyi were studied to elucidate the taxonomic position of the two species. All strains were predominantly gram negative when stained by the Gram method but showed differences in staining between individual cells. The six strains of H. piscium and one strain of H. ducreyi exhibited a cell wall of the gram-negative type, whereas the remaining three strains of H. ducreyi exhibited a cell wall structure typical of gram-positive microorganisms. The study thus confirms the classification of H. piscium as a gram-negative bacterium but indicates that the name H. ducreyi has been used for different groups of bacteria. Only one of the strains of H. ducreyi investigated can legitimately be assigned to the genus Haemophilus, and more information is required to settle the taxonomic position of the remaining three strains.

Summary The preliminary characterisation of an unusual gram-negative bacillus isolated from genital ulcers in Swaziland is reported. Like Haemophilus ducreyi, it is an oxidase positive, nitrate-reductase-positive gram-negative rod that forms streptobacillary chains in some circumstances; it was therefore called the “ducreyi-like bacterium” (DLB). Distinguishing features of DLB are production of α-haemolysis on horse-blood agar, stimulation of growth by a microaerophilic atmosphere and by a factor produced by Staphylococcus aureus, a strongly positive porphyrin test, and a remarkable ability to undergo autolysis. DLB has a guanine + cytosine value of c. 50 mole% but it cannot be classified, even at the genus level, until more taxonomic data are obtained.

Three Australian isolates of “Histophilus ovis,” ten strains of “Haemophilus somnus” from North America, Australia, and Europe, and two American strains of “Haemophilus agni” were investigated by the deoxyribonucleic acid (DNA)-DNA hybridization (renaturation) method to determine their genetic interrelationships and their levels of relatedness to recognized members of the family Pasteurellaceae Pohl 1981. Our results confirmed that “Haemophilus somnus,” “Histophilus ovis,” and one of the “Haemophilus agni” strains studied represent one genetically homogeneous species. This species exhibited up to 41% DNA relatedness to Haemophilus haemoglobinophilus, whereas only insignificant levels of relatedness or no measurable DNA binding was observed with the type species of the genera Actinobacillus, Haemophilus, and Pasteurella and with Haemophilus aphrophilus, Haemophilus ducreyi, Haemophilus paragallinarum, Haemophilus parainfluenzae, Haemophilus segnis, Pasteurella avium, Pasteurella ureae, and “Actinobacillus seminis.” On the other hand, one of the “Haemophilus agni” strains studied (Hoerlein strain M650-1343) was included in the species “Actinobacillus seminis” (DNA binding value, 91%). So far, only low levels of genetic relatedness with “Actinobacillus seminis” and currently recognized members of the family Pasteurellaceae have been detected. The problems of the generic affiliation of the “Histophilus ovis” group, Haemophilus haemoglobinophilus, and “Actinobacillus seminis” and the phenotypic differentiation of these organisms are discussed.

Deoxyribonucleic acid relatedness studies (S1 nuclease method) showed that 25 strains of Haemophilus ducreyi from diverse geographic origins form a homogeneous deoxyribonucleic acid hybridization group. A total of 17 type or reference strains of established or proposed Haemophilus species were 0 to 6% related to H. ducreyi at the optimal temperature. These findings do not support the assignment of H. ducreyi to the genus Haemophilus.

Summary Some of the characteristics of 42 clinical isolates of Haemophilus ducreyi are reported. Only six of the 42 strains were able to grow on horse-blood agar. All strains gave a positive oxidase test with tetramethyl-p-phenylenediamine and a negative result with dimethyl-p-phenylenediamine. All of 15 test strains were negative in the porphyrin test. Tests for haemin requirement were inconclusive because of difficulties encountered in obtaining growth on a basal medium.

We found that Haemophilus ducreyi, the etiologic agent of chancroid, contains two isoprenoid quinone structural types, demethylmenaquinone and menaquinone, with six- and seven-unit unsaturated isoprene side chains, respectively. Eleven strains of H. ducreyi and a control strain of Haemophilus influenzae biogroup aegyptius (Haemophilus aegyptius) were grown in a liquid medium with oxygen as the terminal electron acceptor. Quinones were extracted from bacterial membrane preparations and isolated by high-performance liquid chromatography. The chemical structure of both quinone types was determined by using ultraviolet and mass spectroscopy and nuclear magnetic resonance analysis. The presence of both a demethylmenaquinone and a menaquinone in H. ducreyi represents a major physiological and chemotaxonomic difference between H. ducreyi and other Haemophilus species.