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Journal of Clinical Microbiology, January 2006, p. 132-137, Vol. 44, No. 1
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.1.132-137.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of Haemophilus ducreyi Isolates from Different Geographical Locations

J. Mbwana,^1,2 I. Bölin,¹ E. Lyamuya,² F. Mhalu,² and T. Lagergård^1*

Departments of Medical Microbiology and Immunology, Sahlgrenska Academy, University of Göteborg, S-405 30 Göteborg, Sweden,¹ the Department of Medical Microbiology and Immunology, Muhimbili University College of Health Sciences, Dar es Salaam, Tanzania²

Received 27 April 2005/ Returned for modification 5 September 2005/ Accepted 9 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The technique of random amplified polymorphic DNA (RAPD) was adapted and optimized to study Haemophilus ducreyi isolates. A panel of 43 strains isolated from chancroid patients from different countries in Africa, Europe, North America, and Asia were characterized. The strains were also studied with respect to lipooligosaccharide (LOS) migration and immunoblotting patterns and the presence of cytolethal distending toxin genes. The RAPD method with the OPJ20 primer generated nine banding patterns (1 to 9). The majority of the isolates were clustered into two major profiles, 14 and 13 strains into profiles 1 and 2, respectively, and just a few strains revealed patterns 3 and 4. The isolates from Thailand were exceptional in that they showed greater diversity and were represented by six different RAPD patterns, i.e., patterns 3 and 5 to 9. The LOS migration and immunoblotting analyses revealed two different patterns, which indicated long and short forms of LOS; the former was found in 20/23 tested strains. Two strains that expressed the short form of LOS were grouped into RAPD pattern 4. The absence of cdtABC genes was observed in only 4/23 strains, and three of these isolates were assigned to RAPD pattern 4. Our results showed limited genotypic and phenotypic variations among H. ducreyi strains, as supported by the conserved RAPD and LOS profiles shared by the majority of the studied strains. However, the RAPD method identified differences between strains, including those from different geographic areas, which indicate the potential of RAPD as an epidemiological tool for the typing of H. ducreyi isolates in countries where chancroid is endemic.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Haemophilus ducreyi is a fastidious gram-negative coccobacillus that causes chancroid, a sexually transmitted disease that is characterized by genital ulceration and which is accompanied by suppurative regional lymphadenopathy in 50% of infected patients (29). Chancroid is still common in many developing countries and has been associated with increases in the rates of acquisition and transmission of the human immunodeficiency virus through sexual contact (9, 27).

The epidemiology of chancroid is poorly understood due to the lack of typing methods that would permit differentiation among strains of H. ducreyi. The development of typing methods to characterize strains is important in epidemiological studies and could be used to address questions as to the geographical distribution of strains. Strains of H. ducreyi have been characterized phenotypically by outer membrane protein profiling (18), aminopeptidase profiling (30), lectin agglutination (11), indirect immunofluorescence (25), immunotyping (21), and plasmid analysis (22). These methods provide some level of differentiation between strains, although the differences observed have been small and the pattern distributions limited.

The serological analysis of lipopolysaccharide from many gram-negative bacteria has provided the serotyping system used in epidemiological studies. Electrophoretic analysis of the lipooligosaccharide (LOS) of H. ducreyi reveals migration patterns that are similar to those of other mucosal pathogens such as other Haemophilus species and Neisseria species and indicates the lack of the repeating polysaccharide O antigen, which is characteristic of the lipopolysaccharides of most gram-negative enteric bacteria (17). The LOS structures of certain H. ducreyi strains have been chemically characterized. Two conserved glycoforms, with pentasaccharide and with disaccharide lactose branches (hexasaccharide and nonasaccharide, short and long LOS, respectively), with terminating galactose residues that are partially modified with sialic acid, have been identified by analytical and immunological studies (2, 14). Structurally defined epitopes of 10 H. ducreyi LOSs have beenstudied using monoclonal antibodies (MAbs). The MAb MAHD6 recognizes an epitope that is present only in the pentasaccharide branch of LOS, which suggests the possibility of distinguishing between two phenotypic groups of isolates, based on the expression of long and short LOS (1, 2).

Genotyping techniques are now used frequently for epidemiological investigations of infectious disease agents. Ribotyping, which is a molecular technique based on restriction fragment length polymorphism of rRNA genes, has been used to characterize H. ducreyi isolates in different studies from, for example, Kenya and South Africa and from a chancroid outbreak in the United States (8, 20, 23). Molecular methods employing DNA analysis are less affected by variations in growth conditions of H. ducreyi, as happens in phenotypic typing. For example, it has been reported in a Mississippi and Louisiana population study that isolates from the 1950s and 1960s have ribotypes that are comparable to those of isolates obtained in the 1990s (8).

Random amplified polymorphic DNA (RAPD) is a PCR-based method, which was first described in 1990 (34, 36) and has been used extensively to discriminate between strains of different bacterial species, albeit not with H. ducreyi (4, 7, 24, 26, 31). In this method, an arbitrary DNA sequence is used as a single primer that targets unspecified genomic sequences in order to generate a genetic profile, which is an array of amplified DNA fragments that is specific for each strain (33).

An alternative genotyping method, pulsed-field gel electrophoresis (PFGE), which is used in numerous and diverse applications, is considered to be the most discriminatory technique and the "gold standard" in identifying different strains of, for example, Haemophilus influenzae (19).

In the present study, we adapted the RAPD typing method to characterize genotypic variations among H. ducreyi isolates from Africa (Tanzania and Senegal), Southeast Asia (Thailand), Europe, and North America. The differences/similarities in RAPD patterns were confirmed using PFGE. The LOS migration patterns, reactivity with specific monoclonal antibodies, and the presence of H. ducreyi cytolethal distending toxin (HdCDT) genes were used to study the phenotypic variations of those isolates, and these profiles were correlated with the RAPD patterns of the isolates.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. The strains included in this study were 43 clinical isolates from chancroid patients from Tanzania (seven isolates from 2001), Senegal (eight isolates from 1991), Thailand (eight isolates from 1986), Europe (12 isolates collected between 1952 and 1983), and North America (eight isolates collected between 1971 and 1983). The three reference strains 35000 (Canada, 1976), CCUG 7470 (France, 1976), and CCUG 4438 (=CIP 542) (Vietnam, 1954) were also included. The isolates were cultivated on Grand Lux chocolate agar plates (Department of Bacteriology, Sahlgrenska Hospital, Göteborg, Sweden) that contained 5% brain heart infusion agar, 1% horse blood, 1.5% horse serum, 0.06% yeast autolysate, and 0.015% IsoVitaleX.

Identification of H. ducreyi strains. The species specificity of all isolates was confirmed by PCR, developed and evaluated previously (1, 3). The primers used to amplify the 758-bp fragment were the H. ducreyi 16S rRNA-specific sequence 5'-CCCTTTGCAGGTTTGCCGCCCTC-3' and the nonspecific sequence U3 (5'-GTGCCTGCAGCGCGGTAAT-3'), which was derived from the highly conserved U3 region of Escherichia coli 16S rRNA.

RAPD fingerprinting. DNA samples were extracted from the strains using the DNeasy Miniprep kit (QIAGEN), according to the manufacturer's instructions, and the concentration of the extracted DNA was determined using a Smart Spec 3000 spectrophotometer (Bio-Rad Laboratories, Hercules, CA). The extracted DNA was stored at 4°C until required. The primers used in this study consisted of Oligo 10-mer kits E and J from QIAGEN Operon Biotechnologies GmbH (Hilden, Germany).

Initially, tests were carried out to optimize reaction conditions and to identify an appropriate primer. Forty arbitrarily chosen primers were first tested with three well-characterized reference strains of H. ducreyi (strains 35000, 4438, and 7470) from the Culture Collection Centre, University of Göteborg (CCUG). Of the 40 10-mer oligonucleotide primers tested, only two, OPE15 and OPJ20, gave polymorphic DNA patterns for the three strains tested. The most informative patterns, i.e., those that gave the most bands, were obtained with a DNA concentration of 300 ng in a 25-µl reaction mixture, with 20 pmol of primer and 2.5 mM MgCl₂.

PCR was carried out in a final volume of 25 µl of reaction mixture that contained 2.5 mM MgCl₂, 20 pmol primer, 250 mM of each of the deoxynucleotide triphosphates, 1 U Taq polymerase (Promega, Madison, WI), and 300 ng of template DNA in 1x PCR buffer (Promega). The PCR consisted of an initial denaturation step at 94°C for 4 min, followed by 40 cycles of heat denaturation at 94°C for 1 min, primer annealing at 36°C for 1 min, and extension at 72°C for 2 min in a Touch Gene thermocycler (Techne, Cambridge, United Kingdom).

The amplification products were electrophoresed in a 1% agarose gel (Shelton Scientific Inc., Peosta, IA) in Tris-borate buffer. The gels were stained with ethidium bromide and photographed under UV transillumination. A 1-kb DNA ladder (Tamro Lab, Göteborg, Sweden) was used in each gel as the molecular size standard. A negative control, which consisted of the reaction mixture with water instead of DNA, was included in each run. RAPD-PCR bands were interpreted visually. In order to resolve small bands that were not detectable in the 1% agarose gels, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with 4 to 20% gradient Tris-borate gels (Bio-Rad Laboratories) followed by silver staining (Bio-Rad Laboratories). The gels were stained according to the manufacturer's instructions.

The reproducibility of RAPD fingerprinting was confirmed by comparing the fingerprinting patterns obtained from duplicates run on different days using primer OPE15 with DNA from strain CCUG 7470 after several subcultures. The RAPD patterns obtained on different days and from different subcultures were consistent.

PFGE. PFGE was performed as described previously (13), to verify the differences in all nine RAPD patterns obtained with OPJ20 primer and to investigate the similarities between the strains. Briefly, the DNA samples were digested with SmaI and the fragments were electrophoresed in a 1% agarose gel using the Gene Path system (Bio-Rad Laboratories). The following PFGE gel electrophoresis conditions were used: initial switch time of 1 s, final switch time of 23 s, run time of 23 h, angle of 120°, and gradient of 4 V/cm, which was followed by a second round with an initial switch time of 10 s and a final switch time of 17 s, a run time of 4 h, an angle of 120°, and a gradient of 4 V/cm. Good resolution of the banding pattern was obtained using this program. Nine isolates with different patterns and two isolates that had similar RAPD patterns were analyzed. Two reference strains (CCUG 7470 and CCUG 4438) with different RAPD profiles were also included. The PFGE patterns were compared visually and evaluated using the criteria of Tenover et al. (28). The isolates with differences in zero, three or fewer, four to six, or more than six bands were considered identical, related, possibly related, and unrelated, respectively.

Isolation and purification of LOS. We selected 23 isolates from Tanzania, Senegal, and Thailand, which were representative of all nine different RAPD patterns, as well as two reference strains, CCUG 7470 and CCUG 4438. LOS was extracted using the phenol-water extraction procedure described previously (15), with a slight modification. Briefly, organisms grown on Grand Lux medium were harvested in phosphate-buffered saline (PBS) and centrifuged. The pellets were suspended in 20 volumes of distilled water, an equal volume of phenol was then added, and the mixture was heated at 65°C for 30 min, with mixing every 10 min. The mixture was kept at 4°C overnight. The following day, the aqueous layer was removed and saved. An equal volume of distilled water was added, and the mixture was heated again at 65°C for 20 min, with vigorous shaking every 10 minutes. The aqueous layer was removed as before and combined and dialyzed against tap water, followed by dialysis against distilled water overnight at 4°C. The dialysate was centrifuged and lyophilized. Thereafter, the LOS was weighed and dissolved in Tris buffer and treated with DNase, RNase, and protease, followed by overnight dialysis in this buffer. This was followed by another phenol step, as described above. Finally, the dialysate was lyophilized and stored at 4°C until required.

SDS-PAGE and immunoblot analyses. The LOS preparations were analyzed by SDS-PAGE using a 15% polyacrylamide gel. The gel was loaded with 10 µg of purified LOS and electrophoresed at 200 V for 1 h. After electrophoresis, the LOS bands were visualized by silver staining (Bio-Rad Laboratories). The LOS migration pattern of each strain was compared with those of the reference strains.

After electrophoresis, the gel was transferred to a nitrocellulose membrane (Amersham Biosciences, United Kingdom). Blotting was carried out at 100 V for 30 min. The nitrocellulose membrane was blocked with 1% bovine serum albumin in PBS for 1 h and then incubated overnight with the MAb MAHD6. The membrane was washed three times in PBS and incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS for 90 min. The membrane was washed a further three times in PBS, and color development was achieved with 4-chloro-1-naphthol (Bio-Rad) and hydrogen peroxide. The results were compared with those obtained for the reference strains.

Detection of the HdCDT A, B, and C genes. The presence of individual cdtA, cdtB, and cdtC genes in 23 H. ducreyi isolates from Africa (Tanzania and Senegal) and from Thailand was analyzed by PCR described and evaluated previously (3). There was a correlation between the presence of HdCDT genes and toxic activity in H. ducreyi isolates (3).

Top Abstract Introduction Materials and Methods Results Discussion References

 
RAPD patterns of the H. ducreyi isolates. Primers OPE15 and OPJ20 were used to test a panel of 43 H. ducreyi isolates, which included 7 isolates from Tanzania, 8 isolates from Senegal, 8 from Thailand, 12 from Europe, and 8 from North America. The RAPD profiles obtained using the OPE15 primer are presented in Fig. 1. OPE15 gave only three RAPD patterns, and Table 1 shows the distribution of the three RAPD patterns among the 43 isolates tested. The three reference strains included in this study revealed two patterns with the OPE15 primer: CCUG 4438 belonged to pattern 1, and strains 35000 and CCUG 7470 belonged to pattern 2. The majority of the strains were clustered into patterns 1 and 2 (44% and 49%, respectively) (Table 1).

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FIG. 1. Silver-stained polyacrylamide gel showing three RAPD profiles generated by primer OPE15. Lanes: M, size marker; 1, strain 23D; 2, 8224; 3, 7470; 4, 5619; 5, 31592; 6, 067; 7, 5629; 8, 4438; 9, 7311; 10, 3564; 11, 3560; 12, 5621; 13, 3570; 14, 3571; 15, 3581; 16, 3581, 17, 3569.

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TABLE 1. Distribution of different RAPD patterns obtained with primer OPE15

The OPJ20 primer was more discriminatory: nine RAPD profiles were obtained (Fig. 2) and were designated patterns 1 to 9 (Table 2). The three reference strains included in this study revealed a different pattern each, 1, 2, and 3, for strains 35000, CCUG 7470, and CCUG 4438, respectively. The isolates from Tanzania, Senegal, and North America revealed four RAPD profiles, whereas the isolates from Europe produced three banding patterns. The isolates from Thailand were more diverse, showing six RAPD profiles (patterns 3 and 5 to 9), while three isolates had similar RAPD profiles and five had profiles that were different from each other. Overall, 14/43 (33%) and 13/43 (30%) of the tested strains were clustered in RAPD patterns 1 and 2, respectively, which indicates a lack of diversity among the H. ducreyi isolates, with the exception of those from Thailand. When the patterns obtained by OPE15 and OPJ20 primers were compared, further discrimination within OPE15 patterns 1, 2, and 3 was observed. We found that strains displaying pattern 1 with OPE 15 could be divided further into nine patterns, those of pattern 2 were divided into six profiles, and those belonging to pattern 3 showed only one profile. The results indicate that RAPD can be used to distinguish between different H. ducreyi strains, provided that a suitable primer is selected.

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FIG. 2. Silver-stained polyacrylamide gel showing nine RAPD profiles generated by primer OPJ20. Lanes: M, size marker; 1, strain 23D; 2, 8224; 3, 7470; 4, 5619; 5, 31592; 6, 067; 7, 5629; 8, 4438; 9, 7311; 10, 3564; 11, 3560; 12, 5621; 13, 3570; 14, 3571; 15, 3582; 16, 3581; 17, 3569.

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TABLE 2. Distribution of different RAPD patterns obtained with primer OPJ20

To confirm the diversity of the nine RAPD patterns obtained by the OPJ20 primer, which was more discriminatory, each isolate representing a different RAPD profile was analyzed by PFGE. In addition, similarity between two strains with pattern 1 shown by OPJ20 was investigated. The nine isolates with different RAPD profiles also had different PFGE patterns, while the two isolates with similar profiles differed by only one band (not shown). Two reference strains (CCUG 7470 and 4438) that displayed different profiles by the two primers revealed different patterns in PFGE analysis, indicating that they were different.

Characterization of the LOS structures and HdCDT genes of H. ducreyi strains. We selected 23 isolates, representing all nine different RAPD patterns from African countries and from Thailand. The three reference strains represented isolates with long LOS structure, strains 35000 (RAPD type 1) and CCUG 7470 (RAPD type 2), and short LOS, CCUG 4438 (RAPD type 5). The strains were analyzed by SDS-PAGE and immunoblotting. The majority of H. ducreyi strains (20/23) showed high-molecular-weight LOS (Fig. 3) and reacted with MAb MAHD6. Only three strains showed LOS with a low molecular weight. These studies indicate that H. ducreyi strains express two patterns of LOS, which cannot be used to differentiate isolates.

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FIG. 3. LOS migration patterns of H. ducreyi stains investigated by SDS-PAGE and silver staining. Lanes: M, protein standard marker; 1, strain 7470; 2, 5619; 3, 5621; 4, 067; 5, 3560; 6, 3569; 7, 23D; 8, 4438; 9, 5A; 10, 5622; 11, 3563; 12, 3581.

Furthermore, the presence of individual HdCDT genes was investigated by PCR. Of the 23 isolates, four isolates (two from Senegal, one from Tanzania, and one from Thailand) did not carry the HdCDT genes. Three of these isolates displayed the type 4 RAPD profile, and the single Thailand strain manifested the type 9 RAPD pattern. The results are summarized in Tables 3 and 4.

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TABLE 3. Characteristics of the 15 H. ducreyi isolates from Africa and reference strains

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TABLE 4. Characteristics of the eight H. ducreyi isolates from Thailand

Top Abstract Introduction Materials and Methods Results Discussion References

 
The development of typing methods that allow the discrimination of H. ducreyi isolates and that are easy to perform in developing countries would allow us to address questions concerning the geographical distribution and modes of transmission and reinfection with this pathogen, as well as facilitating the identification of strains of differing virulence.

In this study, we attempted to determine the usefulness of RAPD in genotyping and molecular characterization using H. ducreyi isolates selected from different geographical locations: Africa (Tanzania and Senegal), Europe, Thailand, and North America. We also attempted to correlate the virulence determinants of LOS structure and the presence of HdCDT genes with the RAPD profiles. The RAPD fingerprinting technique of using arbitrary oligonucleotides to prime DNA synthesis at a low annealing temperature has been reported to be a powerful typing method for many bacterial species (12) but has not been used previously for H. ducreyi. Unlike traditional PCR analysis, which requires specific knowledge of DNA sequences and the application of target-specific sequences, RAPD does not require any specific knowledge of the DNA sequences of the target organism.

We found that H. ducreyi isolates from different geographic regions could be assigned to nine RAPD profiles when the OPJ20 primer was used and to only three profiles with the OPE15 primer. This indicates some differences among H. ducreyi strains when primer OPJ20 is used; however, the majority of the strains, except those from Thailand, were clustered mainly in two patterns, RAPD types 1 and 2. These results suggest the importance of selecting the right primer when using RAPD, as noted in other studies (12). The clustering of H. ducreyi isolates observed using RAPD provides evidence that the majority of H. ducreyi strains are related at the genetic level, but some local diversity may occur as seen in strains from Thailand, especially when the OPJ20 primer is used. These observations were confirmed by studies using the ribotyping method. In one study of 30 local strains from South Africa, about 13 patterns were found, but the majority of strains were clustered in two patterns for each restriction enzyme used (20). In another study of 44 strains from California and Nairobi, Kenya, a diversity of strains was found among African isolates but not American isolates (23).

The taxonomic position of H. ducreyi as a Haemophilus species has been questioned; H. ducreyi was originally placed in the genus Haemophilus because of the common requirement for hemin (X factor) and a G+C content that was within the acceptable range for Haemophilus spp. However, Casin et al. (6) demonstrated that H. ducreyi was unrelated to true hemophili such as H. influenzae by the DNA hybridization test. Using RAPD we observed that H. ducreyi strains are genetically homogeneous in contrast to the diversity shown by the species of H. influenzae, where distinct genetic polymorphisms were identified using a similar method (17).

PFGE has been reported to have more discriminatory power than the ribotyping and RAPD techniques used for other bacterial species, such as H. influenzae, Streptococcus faecium, Pasteurella haemolytica, and Salmonella enterica serovar Enteritidis (5, 10, 12, 24). However, other investigators have used RAPD and PFGE and obtained comparable findings (32). In the present study H. ducreyi RAPD profiles were confirmed by PFGE analysis. Two strains with similar banding patterns in RAPD differed only in one band, and this could be due to a point mutation according to the interpretation criteria put forward by Tenover et al. (28). This observation indicates that both RAPD and PFGE techniques can be used to characterize the diversity of H. ducreyi strains. However, it should be mentioned that RAPD is more rapid and accessible in resource-poor countries, such as those in Africa, than other molecular methods that require expensive equipment (12, 19, 23).

The phenotypic characteristics are not very useful in the differentiation of H. ducreyi isolates. An earlier study by our group and by others has shown that H. ducreyi isolates express two oligosaccharide structures of LOS, i.e., the hexasaccharide and nonasaccharide (2, 15, 16). The present study of 23 strains confirmed that the LOS structure of H. ducreyi is very homogenous, which is in contrast to the LOS/lipopolysaccharide of other gram-negative, related bacteria. Recently, two classes of H. ducreyi were proposed, based on expression of serum resistance (DsrA) protein and the migration pattern of LOS (35). The low variability of LOS is in accordance with our study. The ability to produce HdCDT is a common feature among H. ducreyi isolates (3). It appears that the RAPD pattern 4 includes these few strains that have short LOS and are HdCDT negative. However, the number of these strains is too low to draw clear conclusions, and this observation needs further evaluation.

In conclusion, the RAPD method can distinguish between different H. ducreyi isolates. The majority of strains isolated from different geographical areas and at different time points are genetically related and are clustered into two main RAPD profiles. The characterization of LOS cannot be used as a classification system due to the high levels of similarity of the LOSs from different strains. The RAPD method can be used as a primary screening method to discriminate between H. ducreyi isolates in epidemiological studies in areas where chancroid is endemic.

 
This work was supported by the Swedish Agency for Research Co-operation in Developing Countries (SIDA/SAREC).

We are grateful to Christina Wellinder of the Department of Bacteriology, Sahlgrenska Hospital, Göteborg, Sweden, for technical support and advice on the PFGE technique and Hinda Ahmed for her valuable ideas. We also thank the clinicians and technicians at both the Mbeya Referral Hospital and the IDC/STD clinic in Dar Es Salaam, Tanzania, for collecting clinical samples and technical support, respectively. We thank Vincent Collins, Department of Rheumatology and Inflammation Research, Göteborg University, for revising the English text of the manuscript.

 
* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology., The Sahlgrenska Academy, Göteborg University, Box 435, S-405 30 Göteborg, Sweden. Phone: 46 31 773 62 20. Fax: 46 31 773 62 10. E-mail: teresa.lagergard{at}microbio.gu.se.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, January 2006, p. 132-137, Vol. 44, No. 1
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.1.132-137.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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