Molecular characterization of Haemophilus ducreyi by ribosomal DNA fingerprinting.

Intraspecies genotypic heterogeneity among Haemophilus ducreyi isolates was examined by using genomic fingerprints with rRNA from Escherichia coli as a probe. DNA from 44 isolates of H. ducreyi was digested by restriction endonuclease HincII or HindIII, separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with 32P-labeled 16S and 23S rRNA. HincII digests yielded four hybridization patterns (ribotypes), whereas HindIII digests yielded eight ribotypes. Four HincII and five HindIII ribotypes were observed among 14 H. ducreyi isolates collected within a period of 1 month in Kenya, where chancroid is endemic. In contrast, one HincII and two HindIII ribotypes were observed among 28 isolates collected during the Orange County, Calif., chancroid epidemic that occurred in 1981 and 1982. The plasmid content, in conjunction with ribotyping, provided additional differentiation among some isolates of H. ducreyi. This study demonstrates that ribotyping of H. ducreyi may be used to study the epidemiology of chancroid.     

Novel plasmid combinations in Haemophilus ducreyi isolates from Thailand.

Thirty isolates of Haemophilus ducreyi collected in Thailand in 1984 were characterized by plasmid content. Three novel plasmids with estimated molecular masses of 1.8, 2.6, and 2.8 MDa were observed in 29 isolates, in addition to the 3.2-, 5.7-, and 7.0-MDa beta-lactamase and 4.4-MDa sulfonamide resistance plasmids. At least three of the seven plasmids were observed in each of the 29 isolates. The number and diversity of plasmids observed in these isolates of H. ducreyi distinguish them from strains previously described.     

Molecular characterization of isolates of Haemophilus paragallinarum from China by ribotyping.

Ribotyping profiles were determined for 12 Page serovar A isolates of Haemophilus paragallinarum from five outbreaks of infectious coryza in Hebei province in the People's Republic of China. Four enzymes were used to generate restriction digests of chromosomal DNA: HaeIII, HindIII, HpaII and SspI. The digests were probed with a digoxigenin-11-dUTP-labelled DNA probe produced by PCR amplification of the 16S rDNA of the type strain of H. paragallinarum. Only one profile was seen among the 12 isolates when using either HindIII or SspI. Three different ribotype profiles were seen with HpaII, while four different profiles were detected with HaeIII. Cluster analysis of the profiles generated by HpaII and HaeIII corresponded with the known epidemiological history of the isolates. These results are the first molecular characterization of isolates of H. paragallinarum from China and confirm the existence of genetic diversity in the H. paragallinarum population in that country.     

Restriction fragment length polymorphisms distinguish Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates from different geographical locations.

Genetic variability among Leptospira borgpetersenii serovar hardjo type hardjo-bovis isolates representing several geographical regions was determined by restriction endonuclease analysis. Five previously unidentified EcoRI digestion patterns and one previously unidentified HhaI digestion pattern were seen with the various isolates. The copy number and genomic distribution of an L. borgpetersenii insertion sequence (IS1533) was determined. Hardjo-bovis isolate 033 (the type strain for hardjo-bovis) contained 40 well dispersed copies of IS1533. IS1533 probes were used to compare hardjo-bovis isolates by DNA blot hybridization analysis. Use of these probes showed the presence of additional genetic heterogeneity among hardjo-bovis isolates, which restriction endonuclease analysis did not show. Pulsed-field gel electrophoretic analysis of DNAs from several isolates suggested that some polymorphisms arose by genomic rearrangements. All hardjo-bovis isolates were categorized into 14 distinct groups on the basis of common hybridization and endonuclease digestion patterns. Most of these groups were isolated from distinct geographical regions, suggesting that several different clonal populations of hardjo-bovis exist.     

Molecular characterization of Mycobacterium tuberculosis isolates from different regions of Bulgaria

Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7_TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes.     

Molecular analysis of the Haemophilus ducreyi groE heat shock operon.

Chancroid is a sexually transmitted genital ulcer disease caused by Haemophilus ducreyi. Previously, we developed diagnostic DNA probes for H. ducreyi (L. M. Parsons, M. Shayegani, A. L. Waring, and L. H. Bopp, J. Clin. Microbiol. 27:1441-1445, 1989). In the present study, DNA sequencing of one of the diagnostic probes revealed two adjacent open reading frames (ORFs). These H. ducreyi ORFs and the encoded proteins show significant homology with the groE genes and GroES and GroEL heat shock proteins from several bacterial pathogens and with conserved eukaryotic 60-kDa heat shock proteins. The first H. ducreyi ORF (groES) is preceded by sequences similar to those of the Escherichia coli consensus heat shock promoters and is 288 nucleotides long and is capable of encoding a protein of 10.3 kDa. The second ORF (groEL) is 1,641 nucleotides long and is capable of encoding a protein of 57.8 kDa. Northern (RNA blot) analysis demonstrated the presence of a high level of groE mRNA in exponential-phase H. ducreyi grown in hemin broth at the organism's optimal growth temperature (33 degrees C), with increased levels seen following heat shock. Heat shock also increased the thermostability of the organisms, since stressed cells were more resistant to the lethal effects of rapid chilling. Electrophoretic analysis and immunoblots demonstrated that the predominant protein produced by exponential-phase H. ducreyi was a heat-inducible, immunoreactive protein of approximately 60 kDa (GroEL). Also, H. ducreyi groE mRNA and GroEL were expressed and inducible by heat in E. coli. This is the first report describing the cloning, sequencing, and expression of H. ducreyi protein-encoding genes.     

Identification and characterization of the N-acetylglucosamine glycosyltransferase gene of Haemophilus ducreyi

Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted ulcerative disease. In the present study, the Neisseria gonorrhoeae lgtA lipooligosaccharide glycosyltransferase gene was used to identify a homologue in the genome of H. ducreyi. The putative H. ducreyi glycosyltransferase gene (designated lgtA) was cloned and insertionally inactivated, and an isogenic mutant was constructed. Structural studies demonstrated that the lipooligosaccharide isolated from the mutant strain lacked N-acetylglucosamine and distal sugars found in the lipooligosaccharide produced by the parental strain. The isogenic mutant was transformed with a recombinant plasmid containing the putative glycosyltransferase gene. This strain produced the lipooligosaccharide glycoforms produced by the parental strain, confirming that the lgtA gene encodes the N-acetylglucosamine glycosyltransferase.     

Enzymic activity of Haemophilus ducreyi

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.     

Virulence factors of Haemophilus ducreyi

We investigated the susceptibility of virulent and avirulent strains of Haemophilus ducreyi to the bactericidal activity of normal human serum and to phagocytosis and killing by human polymorphonuclear leukocytes (PMNL). Strains were defined as virulent if intradermal inoculation into a rabbit produced a typical necrotic lesion. Nonvirulent strains produced no cutaneous lesions in rabbits. Virulent strains were resistant to the complement-mediated lethal action of normal human and rabbit sera, whereas avirulent strains were susceptible (greater than 95% kill, 60 min). Virulent strains were relatively resistant to phagocytosis and killing by human PMNL, in contrast to the avirulent strains. In past studies polymyxin resistance has been correlated with virulence in H. ducreyi. In our studies, polymyxin resistance could not be correlated with virulence, since polymyxin-sensitive mutants obtained from polymyxin-resistant parent strains remained virulent for rabbits and resistant to bactericidal action of normal serum and phagocytosis and killing by human PMNL. Similarly, polymyxin-resistant mutants obtained from polymyxin-sensitive parent strains remained avirulent for rabbits and susceptible to bactericidal action of normal serum and PMNL. The acquisition of polymyxin resistance was accompanied by the loss of a 47,000-molecular-weight protein. The association of serum resistance and resistance to phagocytosis and killing by human PMNL with virulent strains, as defined by the rabbit intradermal test, suggests that these factors may mediate the pathogenicity of H. ducreyi.     

Cellular internalization of cytolethal distending toxin from Haemophilus ducreyi

The chancroid bacterium Haemophilus ducreyi produces a toxin (HdCDT) which is a member of the recently discovered family of cytolethal distending toxins (CDTs). These protein toxins prevent the cyclin-dependent kinase cdc2 from being activated, thus blocking the transition of cells from the G(2) phase into mitosis, with the consequent arrest of intoxicated cells in G(2). It is not known whether these toxins act by signaling from the cell surface or intracellularly only. Here we report that HdCDT has to undergo at least internalization before being able to act. Cellular intoxication was inhibited (i) by removal of clathrin coats via K(+) depletion, (ii) by treatment with drugs that inhibit receptor clustering into coated pits, and (iii) in cells genetically manipulated to fail in clathrin-dependent endocytosis. Intoxication was also completely inhibited in cells treated with bafilomycin A1 or nocodazole and in cells incubated at 18 degrees C, i.e., under conditions known to block the fusion of early endosomes with downstream compartments. Moreover, disruption of the Golgi complex by treatment with brefeldin A or ilimaquinone blocked intoxication. In conclusion, our data indicate that HdCDT enters cells via clathrin-coated pits and has to be transported via the Golgi complex in order to intoxicate cells. This is the first member of the family of CDTs for which cellular internalization and some details of the pathway have been demonstrated.     

Monoclonal antibody characterization of reference isolates of different serogroups of Haemophilus paragallinarum.

Previously, a panel of five monoclonal antibodies (Mabs) was used to study the antigens of strains 0083, 0222 and Modesto of Haemophilus paragallinarum and marked antigenic differences were noted. To establish if these differences were serogroup specific, more reference strains were examined with these Mabs. It was not possible to detect any relationship between the antigens recognized by the Mabs and the serogroup of the reference strain. None of the Mabs produced reacted with the haemagglutinins of the reference strains. The F1 Mab detected an outer membrane protein of 39 kDa, while the V1 Mab detected a lipopolysaccharide of between 13.8 to 14 kDa. Mabs VF1 and VF2 both recognized antigens of 39 kDa of unknown chemical nature and with extremely low frequency of occurrence among strains and isolates. The VF3 Mab detected a lipopolysaccharide with multiple bands at 37 to 39 kDa, which broke down after freezing and thawing to multiple bands of 29 to 32 kDa. These results imply that the haemagglutinins, which are the major typing and protective antigens remain undetected by this panel of Mabs.     

Plasmid analysis of Shigella dysenteriae type 1 isolates obtained from widely scattered geographical locations.

Plasmid profiles and antimicrobial susceptibility patterns of 343 strains of Shigella dysenteriae type 1, obtained from 18 different geographical locations, were analyzed. Three plasmids, with molecular sizes of 140, 6, and 2 megadaltons (MDa), were present in 94, 98, and 96%, respectively, of the 343 strains isolated during either epidemic or nonepidemic periods from 1965 to 1987. In addition to these plasmids, 83% of the strains harbored a 4-MDa plasmid and 25% harbored a 20-MDa plasmid. Various plasmid profiles were observed in which the 140-, 6-, and 2-MDa plasmids occurred commonly, irrespective of the place of isolation and drug resistance pattern of the strains. Certain profiles showed significant association with drug resistance patterns. These findings suggest that three plasmids, of molecular sizes 140, 6, and 2 MDa, are unique to S. dysenteriae type 1 strains and may indicate the global spread of a pathogenic bacterial clone. Additionally, these core plasmids, plus plasmids of various other sizes, could be used to identify emerging subclones which are causing both epidemic and sporadic disease. Thus, plasmid profiles of S. dysenteriae type 1 strains can be used to monitor possible pandemic strains as well as individual epidemic strains.     

Differences in crop bacterial community structure between hoatzins from different geographical locations

The hoatzin is the only known folivorous bird with foregut fermentation, and is distributed in Venezuela in rivers of the central savannas to the eastern Orinoco River. Differences in diet are expected to affect the digestive microbiota and we hypothesized that hoatzins from different habitats might have a different crop microbiota. We thus characterized the microbiota of six birds from the Cojedes and Orinoco Rivers using the G2 PhyloChip and, in parallel, we compared plant availability and foraging behavior of the hoatzins from the two locations. Plant composition differed between the 2 locations, which shared 5 out of 18 plant families and 1 plant genus--Coccoloba--that was highly consumed in both locations. The PhyloChip detected ～1600 phylotypes from 42 phyla. There was a core microbiota with ~50% of the OTUs shared by at least 4 of the 6 individuals, but there were also differences in the crop microbiota of animals from the two regions. There existed a higher relative abundance of Alphaproteobacteria and Actinobacteria in the crops of birds from the Cojedes River and of Clostridia and Deltaproteobacteria in the crops of birds from the Orinoco River. The results showed both a core crop microbiota and also the bacterial taxa responsible for geographical differences among individuals from the two locations with different vegetation, suggesting an effect of both diet and geography in shaping the crop bacterial community of the hoatzin.     

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.     

Characteristics of Haemophilus ducreyi in culture

Growth on different media and the influence of culture conditions were studied on 19 recently isolated strains of Haemophilus ducreyi, none of which had more than four passages on artificial media. The results were compared with 10 laboratory strains, which had an unknown number of passages in vitro. For all strains, growth was best on 30% rabbit blood agar and on Bieling agar. The laboratory strains showed a tendency to grow better on chocolate agar than did the fresh isolates. Of 19 fresh clinical isolates, 12 were CO2 dependent, and 2 needed extra moisture for growth. From the 10 laboratory strains, only one needed CO2 and none needed extra moisture. All 29 strains grew under anaerobic conditions. Of the 19 fresh clinical isolates, 12 grew at 22 degrees C, but only 2 of the 10 laboratory strains grew at this temperature. The laboratory strains grew better than the fresh isolates at 37 degrees C, and the optimal pH for all strains was pH 6.5 to 7.0. All strains showed starch aggregation.     

Molecular genetic characterization of canine and rangiferine Brucella isolates from different regions of Russia

The aim of this study was to carry out a comparative molecular genetic characterization of Brucella isolates obtained from dogs and deer in Russia using current methods of typing. Isolates from dogs (n = 19) and deer (n = 2) were studied by PCR using primers to species and biovar specific Brucella differentiating gene target sequences and multiple locus variable number tandem repeats analysis (MLVA) with 12 pairs of primers to known variable loci of the Brucella genome. As a result of differentiating gene targets PCR identification, isolates from dogs were characterized as B. canis species, and those from deer as B. suis biovar 4. The MLVA molecular typing method of isolates revealed five identical and seven variable loci. A dendrogram constructed on the basis of MLVA-12 data combined all the isolates into a genetically related cluster together with reference strains B. canis RM6/66 and B. suis 1330, Tomsen, 686, 40 biovars 1–4. Seventeen MLVA genotypes have been revealed in isolates with a Hunter and Gaston discriminatory index (HGDI) = 0.9842. Molecular genetic characterization of B. canis and B. suis Russian isolates geographically distant from each other confirm the existing data that there is a close genetic relation between these two Brucella species. In addition, using the MLVA approach, the genetic diversity of these isolates and the association of closely related genotypes with a geographical region of primary isolation have been shown. In order to improve the system of epidemiological control of brucellosis in Russia, it is necessary to perform MLVA typing of isolates obtained from dogs and deer, which pose an epidemiological threat for human health.     

Swine model of Haemophilus ducreyi infection.

Haemophilus ducreyi is a strict human pathogen that causes sexually transmitted genital ulcer disease. We infected domestic swine with H. ducreyi 35000, resulting in the development of cutaneous ulcers histologically resembling human chancroid lesions. Intraepidermal lesions progressed from pustules to ulcers containing polymorphonuclear leukocytes and were accompanied by a dermal inflammatory infiltrate containing T cells and macrophages. H. ducreyi was recovered from lesions up to 17 days after inoculation, and pigs did not develop immunity to reinfection with the challenge strain. Features of the model include inoculation through abrasions in the epidermis, ambient housing temperatures for infected pigs, the ability to deliver multiple different inocula to a single host, and the availability of monoclonal antibodies against porcine immune cells permitting immunohistochemical characterization of the host immune response to H. ducreyi infection.     

Characterization of pili expressed by Haemophilus ducreyi

Twelve strains of Haemophilus ducreyi isolated primarily from chancroid outbreaks in North America were examined for the presence of pili by transmission electron microscopy. We identified piliated cells in 10 of the 12 strains. Pilin extracts were prepared from the mechanically sheared cells of the 12 H. ducreyi strains as well as the stably piliated H. influenzae strain R890 and its non-piliated parent R906. Pili were present in 12 out of 12 H. ducreyi extracts and in the R890 extract but not in the R906 preparation. Pili were purified by cycles of differential pH solubilization and crystallization. In SDS-PAGE, the preparation consisted predominantly of a protein whose apparent relative molecular mass was 24,000 (24 k), and an electron micrograph showed that the preparation contained pili. Three H. ducreyi strains were passed 52 times on agar plates, and extracts prepared from these strains contained pili. There was no evidence of binding of erythrocytes obtained from nine mammalian and avian species to colonies of one of the stably piliated H. ducreyi strains. We conclude that H. ducreyi expressed pili, that the relative molecular mass of the pilin monomer was 24 k, that pilus expression was not readily lost in passage and that H. ducreyi pili may not bind to an erythrocyte receptor.     

Molecular characterization of vancomycin-resistant Enterococcus faecium isolates from Korea

A total of 98 vancomycin-resistant Enterococcus faecium (VREF) isolates from four tertiary-care hospitals in Korea during the period between 1998 and 2004 were analyzed for genotypic characteristics using the multiplex PCR, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and esp gene analysis. Ninety-two isolates of VREF with VanA phenotype and five of six isolates with VanB phenotype possessed the vanA gene. MLST analysis revealed 9 sequence types (STs), which belonged to a single clonal complex (CC78, clonal lineage C1). Five strains showing incongruence between phenotype and genotype (VanB-vanA) did not belong to the same genotypic clone. The esp gene was detected in all VREF strains, showing 12 different esp repeat profiles. Data suggest that an epidemic clonal group of VREF, CC78 with esp gene, is also present in Asia and has differentiated into multiple diverse genotypic clones during the evolutionary process.     

Molecular characterization of Giardia duodenalis isolates from domestic ferrets

Giardia duodenalis is a pathogen that has been found in a variety of mammals, including humans, and consists of host-specific (assemblages C, D, E, F, and G) and zoonotic (assemblages A, B) genetic groups. Ferrets are popular pets, and they, like humans, are hosts to this pathogen. The genetic characteristics of the Giardia population in ferrets are unclear because only one ferret isolate has been genotyped. To develop a more complete picture of the genetic characteristics of the Giardia population in domestic ferrets two additional Giardia isolates, recovered independently from two ferrets suffering from intestinal or hepatic giardiasis, were analyzed genetically. The sequences of both isolates at three loci, small subunit ribosomal RNA (SSUrDNA, 292 bp), ß-giardin (734 bp), and glutamate dehydrogenase (GDH, 713 bp) were identical to each other, but the sequences of triosephosphate isomerase locus (TPI, 512 bp) had five substitutions between isolates. Sequence homology and phylogenetic analyses at four loci identified both isolates as members of the assemblage A. Moreover, the sequences of SSUrDNA, ß-giardin, and GDH from both isolates were identical to those of the previous ferret isolate genotyped as assemblage A within the regions of overlap. The result obtained in the present study indicates that at least two genetically different types in assemblage A exist in domestic ferrets. In addition, there have been no reported human and animal isolates with the same sequence as those from ferret isolates at all four loci examined, suggesting that the present ferret isolates might be host-specific.