An emerging zoonotic clone in the Netherlands provides clues to virulence and zoonotic potential of Streptococcus suis.

Streptococcus suis is a zoonotic swine pathogen and a major public health concern in Asia, where it emerged as an important cause of bacterial meningitis in adults. While associated with food-borne transmission in Asia, zoonotic S. suis infections are mainly occupational hazards elsewhere. To identify genomic differences that can explain zoonotic potential, we compared whole genomes of 98 S. suis isolates from human patients and pigs with invasive disease in the Netherlands, and validated our observations with 18 complete and publicly available sequences. Zoonotic isolates have smaller genomes than non-zoonotic isolates, but contain more virulence factors. We identified a zoonotic S. suis clone that diverged from a non-zoonotic clone by means of gene loss, a capsule switch, and acquisition of a two-component signalling system in the late 19th century, when foreign pig breeds were introduced. Our results indicate that zoonotic potential of S. suis results from gene loss, recombination and horizontal gene transfer events.

Streptococcus gallolyticus meningitis in adults: report of five cases and review of the literature.

We describe the incidence and patient characteristics of Streptococcus gallolyticus meningitis. We identified S. gallolyticus meningitis in a nationwide cohort of patients with community-acquired bacterial meningitis, and performed a systematic review and meta-analysis of all reported adult cases in the literature. Five cases were identified (0.3%) in a cohort of 1561 episodes of bacterial meningitis. In one patient, bowel disease (colon polyps) was identified as a predisposing condition for S. gallolyticus infection, whereas no patients were diagnosed with endocarditis. In a combined analysis of our patients and 37 reported in the literature, we found that the median age was 59 years. Predisposing factors were present in 21 of 42 patients (50%), and mainly consisted of immunosuppressive therapy (seven patients), cancer (four patients), and alcoholism (four patients). Colon disease was identified in 15 of 24 patients (63%) and endocarditis in five of 27 patients (18%). Co-infection with Strongyloides stercoralis was identified in 14 of 34 patients (41%), ten of whom were infected with human immunodeficiency virus or human T-lymphotropic virus. Outcomes were described for 37 patients; eight died (22%) and one (3%) had neurological sequelae. S. gallolyticus is an uncommon cause of bacterial meningitis, with specific predisposing conditions. When it is identified, consultation with a cardiologist and gastroenterologist is warranted to rule out underlying endocarditis or colon disease. Stool examinations for Strongyloides stercoralis should be performed in patients who have travelled to or originate from endemic areas.

Genome sequence of Neisseria meningitidis serogroup B strain H44/76.

Neisseria meningitidis is an obligate human pathogen. While it is a frequent commensal of the upper respiratory tract, in some individuals the bacterium spreads to the bloodstream, causing meningitis and/or sepsis, which are serious conditions with high morbidity and mortality. Here we report the availability of the genome sequence of the widely used serogroup B laboratory strain H44/76.

Chlamydophila psittaci infections in The Netherlands.

Psittacosis, caused by Chlamydophila psittaci, is a well described but sporadically occurring clinical entity, which mainly presents as community-acquired pneumonia. Diagnosis used to be relatively difficult. However, new molecular techniques, such as real-time polymerase chain reaction, increased detection of cases. Furthermore, genotyping of the ompA gene can be used as a tool to trace the possible source of an outbreak or to link a specific bird to a particular patient.

More than 25 years of urogenital Chlamydia trachomatis research in the Netherlands.

This article provides an overview of the Dutch work performed on urogenital Chlamydia trachomatis infections which started over 30 years ago. We will review past PhD research, 50% of which involved C. trachomatis as the main focus of the thesis, as well as research by current PhD fellows investigating (partially) C. trachomatis, and publications from Dutch authors or co-authors and the main discussion forums.

Inclusion proteins of Chlamydiaceae.

Chlamydiaceae are obligate intracellular pathogens with family members among the etiological agents of several human diseases, such as blinding trachoma, sexually transmitted disease (Chlamydia trachomatis) and pneumonia (Chlamydophila pneumoniae, Chlamydophila psittaci). The bacteria replicate intracellularly in a membrane-bound vacuole termed inclusion. The chlamydial inclusion is effectively separated from eukaryotic endocytic pathways. More than two decades ago it was already speculated that Chlamydiae might modify the inclusion membrane through the insertion of chlamydial-derived components. However, because the classical genetic approaches cannot be applied to manipulate these bacteria, it took more than 10 years before definitive proof was obtained that Chlamydiae indeed actively modify the inclusion membrane by the insertion of proteins of chlamydial origin, first observed by Rockey et al. in 1995. This review will focus on the structural and functional aspects of inclusion proteins of Chlamydiaceae, thereby summarizing data obtained by in vitro studies and comparative genomics.

Description of the ICTI consortium: an integrated approach to the study of Chlamydia trachomatis infection.

The use of an integrated approach to the study of Chlamydia trachomatis infection of the female genital tract, presented at the mini-symposium "Chlamydia trachomatis infections" and described in the thesis of Joseph M. Lyons, has resulted in the creation of the ICTI consortium. The ICTI consortium is based on strong interaction and collaboration between basic scientists, clinicians, epidemiologists, and health care policy makers. This translational approach will help to further the valuable insight into the immunopathogenesis of this sexually transmitted infection (STI) and the development of new intervention strategies, including the vaccines and screening programs necessary to effectively diagnose, treat and prevent C. trachomatis infection. A background of the need for this integrated approach is presented and the goals and participants of the consortium are described.

Development of an internally controlled real-time PCR assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system.

A real-time PCR assay with a DNA purification and inhibition control (internal control; IC) was developed to detect Chlamydophila psittaci DNA in human clinical samples. Novel C. psittaci-specific primers targeting the ompA gene were developed. The IC DNA contained the same primer-binding sites and had the same length and nucleotide content as the C. psittaci DNA amplicon, but had a shuffled probe-binding region. The lower limit of detection was 80 target copies/PCR, corresponding to 6,250 copies/mL in a clinical sample. Specificity was tested using reference strains of 30 bacterial species. No amplification was observed from any of these samples. Respiratory samples from eight patients were positive with this PCR. Six of these patients were confirmed as positive for C. psittaci with serological testing. Two patients had increasing antibody titres, but did not fulfil criteria proposed previously for serologically proven Chlamydia spp. infection. The real-time PCR described in this paper is a sensitive, specific and rapid method to detect C. psittaci DNA in human clinical respiratory samples.

Normal IncA expression and fusogenicity of inclusions in Chlamydia trachomatis isolates with the incA I47T mutation.

To investigate the correlation between the incA I47T mutation in Chlamydia trachomatis and the nonfusogenic phenotype, the incA genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the incA I47T mutation is not associated with the nonfusogenic phenotype.

NmeSI restriction-modification system identified by representational difference analysis of a hypervirulent Neisseria meningitidis strain.

Neisseria meningitidis is a gram-negative bacterium that may cause meningitis, sepsis, or both. The increase in the incidence of meningococcal disease in various countries in the past 2 decades is mainly due the genotypically related lineage III meningococci. The chromosomal DNA differences between lineage III strains and non-lineage III strains were identified using representational difference analysis. Thus, a 1.8-kb locus that is specific for lineage III meningococci was identified. The locus contains three open reading frames encoding the NmeSI restriction-modification system. The methyltransferase gene was cloned and expressed in Escherichia coli. Site AGTACT was found to be modified by the enzyme. In conclusion, lineage III strains differ from endemic strains by the presence of a specific restriction-modification system. This restriction-modification system may contribute to the clonal and hypervirulent character of lineage III strains by influencing horizontal gene transfer and transcription.

PCR assessment of Chlamydia trachomatis infection of semen specimens processed for artificial insemination.

In order to ascertain the microbiological quality of stored semen specimens processed for artificial insemination by a donor (AID), we developed a PCR assay targeting the chlamydial plasmid to detect Chlamydia trachomatis in semen. The lower limit of detection of this assay corresponded to 2.5 to 5 elementary bodies per microl of semen. A total of 669 cryopreserved ejaculates from 97 asymptomatic donors were tested for C. trachomatis infection. Twelve ejaculates, originating from four donors, were found to be positive, indicating a 4% prevalence of C. trachomatis infection among the donor population studied. Cross-contamination between the cryopreserved specimens in the storage container was studied by typing using sequence analysis of PCR-amplified omp1 genes of the strains. Two donors were infected with serovar E, one was infected with serovar F, and one was infected with serovar K. For two donors, the duration of C. trachomatis positivity could be assessed. One donor donated C. trachomatis-positive semen for at least 4 successive months, and the other did so for at least 16 months. The occurrence of C. trachomatis infection in cryopreserved donor semen indicates that ejaculates from donors not tested for a C. trachomatis infection just prior to donation should be tested for infection by a direct test such as the PCR described here. Direct testing of semen specimens will detect not only donors with an active infection but also C. trachomatis-infected ejaculates already stored and will thus improve the microbiological quality of AID, since discrepancies in the presence of C. trachomatis in urine and semen specimens have been reported.

Accurate detection of male subclinical genital tract infection via cervical culture and DNA hybridization assay of the female partner.

The accuracy of the PACE2 DNA hybridization assay of the cervix and cervical culture in female partners for the diagnosis of male subclinical genital tract infection were assessed in a male infertility population. A total of 184 men were screened for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis. Seventy-one men were identified with a positive test for one or more of the above mentioned micro-organisms. The overall prevalence of bacterial infection was 39%. Female partners of all men were tested with the PACE2 DNA hybridization assay to detect a C. trachomatis infection. Sensitivity was 100% and specificity was 100%. In 67 female partners (94%) of men who tested positive for U. urealyticum and/or M. hominis, a cervical swab culture was performed. The sensitivity of the cervical swab culture was 100%. In view of the high prevalence of U. urealyticum and M. hominis in the male genital tract and the role these sexually transmitted pathogens may play in infertility, one might question whether all couples should be screened for the presence of these pathogens. Transurethral swab culture after digital prostatic massage is disincentive to men. The cervical culture in their female partner, performed as part of the routine fertility work-up, is a suitable alternative to detect the presence of these micro-organisms in the male genital tract.

Value of detecting leukocytospermia in the diagnosis of genital tract infection in subfertile men.

OBJECTIVES: To evaluate whether detection of leukocytospermia in a routine semen analysis is of diagnostic value in selecting men with an "actual" microbial infection and to assess the association between leukocytospermia and a history of bacterial and viral infections. DESIGN: Prospective clinical study. SETTING: Infertility clinic at the Center for Reproductive Medicine, Academic Medical Center, Amsterdam, the Netherlands. PATIENT(S): One hundred eighty-four men among subfertile couples attending our infertility clinic. INTERVENTION(S): The number of leukocytes was assessed in three semen samples. Serologic tests were performed, as was transurethral culture after digital prostatic massage. MAIN OUTCOME MEASURE(S): Diagnosis of actual bacterial and viral infections in relation to seminal leukocyte concentrations. The association of a history of sexually transmitted diseases with seminal leukocyte concentration. RESULT(S): An actual bacterial infection was present in 39% of men, and 11% of men had an actual viral infection. The area under the receiver operating curve, which was used to determine whether detection of leukocytospermia was of diagnostic value in identifying men with actual bacterial or viral infections, was 0.55 and 0.56 for bacterial and viral infection, respectively. A past infection with N. gonorrhoeae was associated with the presence of leukocytospermia. A past viral infection was not associated with leukocytospermia. CONCLUSION(S): Detection of leukocytospermia appears to be of no diagnostic value for selection of men with actual microbial infections, but leukocytospermia is associated with a history of gonorrhea.

Type III secretion genes identify a putative virulence locus of Chlamydia.

Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence-associated type III secretion loci. The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.

Construction of recombinant neisserial Hsp60 proteins and mapping of antigenic domains.

Here we report the cloning and expression, in Escherichia coli, of PCR-amplified DNA encoding the 63-kDa stress-inducible protein of Neisseria gonorrhoeae strains VP1 and PID2, Neisseria meningitidis 2996 and the commensal Neisseria flavescens. DNA sequence analysis revealed in all cases one open reading frame of 541-544 amino acids corresponding to a protein of approximately 57,000 Da. The various neisserial proteins were > 96% identical at the amino acid level and showed extensive homology with proteins belonging to the Hsp60 heat-shock-protein family. We constructed defined glutathione S-transferase fusion polypeptides of the gonococcal Hsp60 homologue to locate antigenic domains on the recombinant protein. Variation in the immunoreactivity of two monoclonal antibodies recognizing a conserved and a neisseria-unique antigenic Hsp60 determinant, respectively, could thus be deduced to result from single amino acid substitutions. Analysis of the antibody response in patients' sera demonstrated reactivity with the same fusion polypeptides in six out of nine sera, indicating that neisserial Hsp60 is expressed during the natural infection and that distinct domains on the protein are immunodominant in vivo.

Immunogenicity of the meningococcal stress protein MSP63 during natural infection.

Acute- and convalescent-phase sera from 40 patients with meningococcal disease were evaluated for immunoreactivity with the meningococcal member of the hsp60 stress protein family. The IgG response was measured by ELISA, using bacterial cell lysate of the corresponding patients' strain, and purified hsp60 proteins from Neisseria meningitidis (MSP63), Escherichia coli (GroEL) and Mycobacterium bovis BCG (65K) as antigens. Analysis of the antibody responses revealed that 24/35 patients (69%) with elevated anti-meningococcal titres, generated anti-MSP63 antibodies during the time course of infection. Twelve of these patients generated antibodies specific for MSP63, in six patients anti-MSP63 levels exceeded anti-GroEL/65K antibodies. In the remaining six patients, equal levels of anti-MSP63 and anti-GroEL/65K were measured. We conclude that MSP63 is expressed and immunogenic during natural meningococcal infection, and that individual subjects have a restricted response to the antigen, resulting in the recognition of Neisseria-specific hsp60 epitopes and/or cross-reactive hsp60 determinants.

Identification and characterization of a cross-reactive and a unique B-cell epitope on the hsp60 homologue from Neisseria gonorrhoeae.

Two monoclonal antibodies (G6 and 7B), generated against a 63-kDa stress protein (GSP63) from Neisseria gonorrhoeae strain VP1, were used to investigate the antigenic heterogeneity of GSP63 among the Neisseriaceae and its antigenic relationship with the Hsp60 heat-shock protein family. Immunoblotting experiments demonstrated antibody reactivity with all pathogenic Neisseria tested and with some of the commensal strains. One of the antibodies (7B) cross-reacted with the 65-kDa M. bovis BCG heat-shock protein and with 14 out of the 21 similarly sized proteins in other bacterial species. The other antibody (G6) specifically recognized neisserial GSP63 homologues. These results demonstrate that GSP63 is a conserved neisserial antigen bearing both a unique neisserial B-cell epitope and a more widely distributed Hsp60 epitope.

Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae.

Iron limitation, glucose deprivation, and growth under low oxygen supply (environmental stress) increased the expression of several proteins of Neisseria gonorrhoeae, including a 63-kilodalton protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This gonococcal stress protein (GSP63) was detected in the cytosol and copurified with lithium acetate-derived outer membranes. Successful purification of the protein was achieved by sucrose density gradient centrifugation and by chromatography on phenyl-Sepharose. Gel filtration of the purified protein revealed a molecular weight of approximately 450,000, suggesting that in its native state, the protein consists of a multimer of six to eight subunits. Isoelectric focusing indicated a pI of 5.2. Immunoblotting experiments using a polyclonal antiserum raised against the purified protein demonstrated cross-reactivity with a protein of the same electrophoretic mobility as GSP63 in all eight gonococcal isolates tested. N-terminal amino acid sequencing of the protein revealed up to 65% homology with members of the Hsp60 heat shock protein family, suggesting that GSP63 is related to this group of proteins. This relationship was further substantiated by the immunological cross-reactivity of GSP63 with mycobacterial Hsp60 and the ATP-binding activity of the gonococcal stress protein.