Robert J. Suchland

Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates

We previously reported that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogens tryptophan synthase (trpBA) genes. Here, we have applied functional genomics to extend this work and find that the paradigm established for reference serovars also applies to clinical isolates - specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme. Moreover, functional enzyme activity was directly correlated to IFN-gamma resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We speculate that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-gamma-mediated eradication and thus establish persistent infection.

Methodologies and Cell Lines Used for Antimicrobial Susceptibility Testing of Chlamydia spp.

ABSTRACT In vitro susceptibility testing was performed on strains of Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci under various conditions, including the cell line utilized, the time between infection and the addition of an antimicrobial, the concentration of inoculum, and the effect of multiple passage on the minimal chlamydicidal concentrations for the antibiotics doxycycline, azithromycin, erythromycin, ofloxacin, and tetracycline. With macrolides, the MIC varied depending upon the cell line utilized. With all antimicrobials, the MIC was related to the time at which the antimicrobial was added after infection. By an optimized cell culture passage method, all strains of chlamydia tested demonstrated survival after exposure to high levels (>100 times the MIC) of antimicrobials. Furthermore, upon retest, these surviving organisms did not demonstrate increased MICs. Thus, this phenomenon does not reflect selection of antimicrobial-resistant mutants but rather survival of some organisms in high antimicrobial concentrations (heterotypic survival). An additional 44 clinical isolates of C. trachomatis from patients with single-incident infections were tested against those from patients with recurrent or persistent infections, and heterotypic survival was seen in all isolates tested; hence, in vitro resistance did not correlate with the patients apparent clinical outcome.

Isolates of Chlamydia trachomatis that occupy nonfusogenic inclusions lack IncA, a protein localized to the inclusion membrane.

ABSTRACT The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole, termed an inclusion, throughout their developmenal cycle. When an epithelial cell is infected with multipleChlamydia trachomatis elementary bodies, they are internalized by endocytosis into individual phagosomal vacuoles that eventually fuse to form a single inclusion. In the course of large-scale serotyping studies in which fluorescent antibody staining of infected cells was used, a minority of strains that had an alternate inclusion morphology were identified. These variants formed multiple nonfusogenic inclusions in infected cells, with the number of independent inclusions per cell varying directly with the multiplicity of infection. Overall the nonfusogenic phenotype was found in 1.5% (176 of 11,440) of independent isolates. Nonfusing variants were seen in C. trachomatis serovars B, D, D−, E, F, G, H, Ia, J, and K. The nonfusing phenotype persisted through repeated serial passage, and the phenotype was consistent in four mammalian host cell lines. Fluorescence microscopy and immunoblotting with antisera directed at proteins in the C. trachomatis inclusion membrane revealed that one such protein, IncA, was not detected in the inclusion membrane in each tested nonfusogenic strain. The distributions of other chlamydial proteins, including one additional Inc protein, were similar in wild-type and variant strains. TheincA coding and upstream regions were amplified and sequenced from the prototype serovar D and two nonfusing serovar D(s) strains. Three nucleotide changes were discovered in the D(s)incA gene, leading to two amino acid changes within the predicted D(s) IncA sequence. These studies demonstrate a subgroup of variant C. trachomatisisolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an alteredincA sequence that modifies the characteristic hydrophobic domain of the IncA protein.

Genome Sequencing of Recent Clinical Chlamydia trachomatis Strains Identifies Loci Associated with Tissue Tropism and Regions of Apparent Recombination

ABSTRACT The human pathogen Chlamydia trachomatis exists as multiple serovariants that have distinct organotropisms for different tissue sites. Culture and epidemiologic data have demonstrated that serovar G is more prevalent, while serovar E is less prevalent, for rectal isolates from men having sex with men (MSM). The relative prevalence of these serovars is the opposite for isolates from female cervical infections. In contrast, the prevalence of serovar J isolates is approximately the same at the different tissue sites, and these isolates are the only C-class strains that are routinely cultured from MSM populations. These correlations led us to hypothesize that polymorphisms in open reading frame (ORF) sequences correlate with the different tissue tropisms of these serovars. To explore this possibility, we sequenced and compared the genomes of clinical anorectal and cervical isolates belonging to serovars E, G, and J and compared these genomes with each other, as well as with a set of previously sequenced genomes. We then used PCR- and restriction digestion-based genotyping assays performed with a large collection of recent clinical isolates to show that polymorphisms in ORFs CT144, CT154, and CT326 were highly associated with rectal tropism in serovar G isolates and that polymorphisms in CT869 and CT870 were associated with tissue tropism across all serovars tested. The genome sequences collected were also used to identify regions of likely recombination in recent clinical strains. This work demonstrated that whole-genome sequencing along with comparative genomics is an effective approach for discovering variable loci in Chlamydia spp. that are associated with clinical presentation.

Horizontal Transfer of Tetracycline Resistance among Chlamydia spp. In Vitro

ABSTRACT There are no examples of stable tetracycline resistance in clinical strains of Chlamydia trachomatis. However, the swine pathogen Chlamydia suis is commonly tetracycline resistant, both in America and in Europe. In tested U.S. strains, this resistance is mediated by a genomic island carrying a tet(C) allele. In the present study, the ability of C. suis to mobilize tet(C) into other chlamydial species was examined. Differently antibiotic resistant strains of C. suis, C. trachomatis, and Chlamydia muridarum were used in coculture experiments to select for multiply antibiotic resistant progeny. Coinfection of mammalian cells with a naturally occurring tetracycline-resistant strain of C. suis and a C. muridarum or C. trachomatis strain containing selected mutations encoding rifampin (rifampicin) or ofloxacin resistance readily produced doubly resistant recombinant clones that demonstrated the acquisition of tetracycline resistance. The resistance phenotype in the progeny from a C. trachomatis L2/oflR-C. suis R19/tetR cross resulted from integration of a 40-kb fragment into a single ribosomal operon of a recipient, leading to a merodiploid structure containing three rRNA operons. In contrast, a cross between C. suis R19/tetR and C. muridarum MoPn/oflR led to a classical double-crossover event transferring 99 kb of DNA from C. suis R19/tetR into C. muridarum MoPn/oflR. Tetracycline resistance was also transferred to recent clinical strains of C. trachomatis. Successful crosses were not obtained when a rifampin-resistant Chlamydophila caviae strain was used as a recipient for crosses with C. suis or C. trachomatis. These findings provide a platform for further exploration of the biology of horizontal gene transfer in Chlamydia while bringing to light potential public health concerns generated by the possibility of acquisition of tetracycline resistance by human chlamydial pathogens.

Differential Regulation of Inflammatory Cytokine Secretion by Human Dendritic Cells upon Chlamydia trachomatis Infection

ABSTRACT Chlamydia trachomatis is an obligate intracellular gram-negative bacterium responsible for a wide spectrum of diseases in humans. Both genital and ocular C. trachomatis infections are associated with tissue inflammation and pathology. Dendritic cells (DC) play an important role in both innate and adaptive immune responses to microbial pathogens and are a source of inflammatory cytokines. To determine the potential contribution of DC to the inflammatory process, human DC were infected with C. trachomatis serovar E or L2. Both C. trachomatis serovars were found to infect and replicate in DC. Upon infection, DC up-regulated the expression of costimulatory (B7-1) and cell adhesion (ICAM-1) molecules. Furthermore, chlamydial infection induced the secretion of interleukin-1β (IL-1β), IL-6, IL-8, IL-12p70, IL-18, and tumor necrosis factor alpha (TNF-α). The mechanisms involved in Chlamydia-induced IL-1β and IL-18 secretion differed from those of the other cytokines. Chlamydia-induced IL-1β and IL-18 secretion required infection with viable bacteria and was associated with the Chlamydia-induced activation of caspase-1 in infected host cells. In contrast, TNF-α and IL-6 secretion did not require that the Chlamydia be viable, suggesting that there are at least two mechanisms involved in the Chlamydia-induced cytokine secretion in DC. Interestingly, an antibody to Toll-like receptor 4 inhibited Chlamydia-induced IL-1β, IL-6, and TNF-α secretion. The data herein demonstrate that DC can be infected by human C. trachomatis serovars and that chlamydial components regulate the secretion of various cytokines in DC. Collectively, these data suggest that DC play a role in the inflammatory processes caused by chlamydial infections.

The relationship of serovar to clinical manifestations of urogenital Chlamydia trachomatis infection.

Background and Goal Studies assessing the relationship of Chlamydia trachomatis serovars or genotypes to clinical manifestations of urogenital disease have produced contradictory results. Possible reasons for this include small sample sizes insufficient to reliably detect associations and failure to address potential confounding factors. Utilizing a large dataset and multivariate analysis to address confounding factors, we undertook this study of the relationship of chlamydial serovars to specific clinical manifestations of urogenital disease. Study Design This was a cross-sectional study of 480 women and 700 heterosexual men with urogenital chlamydial infection attending a sexually transmitted diseases clinic from 1995 to 1999 and a literature review. Results Women (89%) and men (86%) were infected predominately with serovars D, E, F, Ia, and J. After controlling for age and race, we found that women who reported abdominal pain and/or dyspareunia were more often infected with serovar F (P = 0.048). An association of specific clinical manifestations with serovars was not detected in men. Conclusion We conclude that clinical manifestations of C trachomatis urogenital infection are not strongly influenced by the infecting serovar.

Longitudinal assessment of infecting serovars of Chlamydia trachomatis in Seattle public health clinics: 1988-1996

Background There have been few longitudinal studies of fluctuations in the prevalence of Chlamydia trachomatis serovars in a given community; such studies could improve our understanding of the epidemiology and transmission of C trachomatis. Study Design This was a longitudinal study of 7110 female patients (62%) and 4344 male patients (38%) presenting with first-time infections to health department clinics between 1988 and 1996. Goal The goal was to ascertain trends in the proportion of infecting serovars over the 9-year study period and determine independent relationships between serovar and age, gender, race, and year of infection. Results Serovar E was the most prevalent (32%), followed by F (18%) and D (13%). Being female, African American, and infected with serovar B was associated with young age (P < 0.001, P < 0.001, and P = 0.09, respectively). Class C serovars were found in older patients (P < 0.001). Over the 9-year period, the percentage of infections with serovar types F and G increased (P = 0.007, P = 0.009), those with I and K decreased (P < 0.001, P = 0.008), and those with B, D, D-, E, H, Ia, and J remained stable. The age of those with positive Chlamydia cultures decreased (P < 0.001). Conclusion In this population, while the major serovars appeared stable over 9 years, significant changes in the distribution of minor serovars, especially G, were observed over time.

Epidemiology of anorectal chlamydial and gonococcal infections among men having sex with men in Seattle: Utilizing Serovar and Auxotype strain typing

Background Bacterial sexually transmitted diseases (STDs) among men who have sex with men (MSM) have recently increased in Seattle. Goals Serovar and auxotype typing of strains was used to assess the epidemiology of anorectal chlamydial and gonococcal infections among MSM attending an STD clinic. Study Design The prevalences of anorectal chlamydial infection and gonorrhea among MSM attending an STD clinic during the period of 1994 to 1996 were compared with prevalences during 1997 to 1999. A retrospective case–control study of MSM attending an STD clinic between 1997 and 1999 was performed. Anorectal chlamydial isolates were characterized by serovar and gonococcal isolates were characterized by serovar and auxotype. Infected MSM were mapped by residence and strain type. Results Prevalences of anorectal chlamydial and gonococcal infections increased from 4.0% and 6.3%, respectively, during 1994–1996 to 7.6% and 8.7%, respectively, during 1997–1999 (P = 0.004 and P = 0.013 for chlamydial infection and gonorrhea, respectively). Most chlamydial infections were caused by serovars G (47.9%) and D (29.6%), and most gonococcal infections were caused by auxotype/serovar classes Proto/IB-1 (43.3%), Proto/IB-3 (16.5%), and Proto/IB-2 (10.3%). MSM with anorectal chlamydial infection more often had chlamydial urethritis (P = 0.005) and were not white (P = 0.046), in comparison with controls. MSM with anorectal gonorrhea more often had pharyngeal gonorrhea (P < 0.001), had a history of gonorrhea (P = 0.003), and were younger than age 30 years (P = 0.039), in comparison with controls. Residences of MSM with anorectal gonorrhea were clustered in urban areas, whereas those of MSM with anorectal chlamydial infection were more dispersed. Conclusion Prevalences of anorectal chlamydial infection and gonorrhea among MSM in Seattle have increased dramatically over the past 3 years. Serovar and auxotype analyses indicate these increases are not clonal but are due to the spread of unique distributions of strains that differ from those causing urogenital infections in the same community.

Epidemiology and Clinical Manifestations of Unique Chlamydia trachomatis Isolates That Occupy Nonfusogenic Inclusions

Unique Chlamydia trachomatis strains characterized by multiple nonfusing inclusions were recently described. These strains lack evidence of the protein IncA in the inclusion membrane and have mutations in the incA gene. This study evaluated the epidemiology and clinical manifestations of patients infected with nonfusing mutant strains (case patients) and compared them with patients infected with wild-type fusing strains (control subjects). Both male and female case patients had fewer signs of infection than did control subjects (P=.016 and P=.019, respectively). Female case patients also had fewer symptoms of infection (P=.02). Median inclusion-forming unit (ifu) counts were lower in male and female case patients (P=.045 and P=.135, respectively). Thus, nonfusing strains of C. trachomatis more often produce subclinical infections than do normal fusing strains and have lower median ifu counts. From a prevention perspective, the data underscore the importance of screening programs to detect and treat inapparent C. trachomatis infection.