Horizontal transfer of tetracycline resistance among Chlamydia spp. in vitro.

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/ofl(R)-C. suis R19/tet(R) 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/tet(R) and C. muridarum MoPn/ofl(R) led to a classical double-crossover event transferring 99 kb of DNA from C. suis R19/tet(R) into C. muridarum MoPn/ofl(R). 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.

Methodologies and cell lines used for antimicrobial susceptibility testing of Chlamydia spp.

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 patient's apparent clinical outcome.

Quantitative culture of Chlamydia trachomatis: relationship of inclusion-forming units produced in culture to clinical manifestations and acute inflammation in urogenital disease.

The relationship of Chlamydia trachomatis inclusion-forming units in quantitative culture to clinical manifestations and inflammation in urogenital disease was assessed in 1179 patients attending a sexually transmitted diseases clinic. In women, greater inclusion-forming unit counts were associated with cervical mucopus (3000 vs. 450 ifu), amount and character of cervical discharge, > or =30 polymorphonuclear cells (PMNL) per high-power field (hpf) on Gram stain (2050 vs. 320 ifu), and diagnoses of mucopurulent cervicitis (MPC; 2550 vs. 300 ifu) and pelvic inflammatory disease (PID; 3000 vs. 578 ifu). In men, greater inclusion-forming unit counts were associated with urethral discharge (85 vs. 44 ifu), amount and character of discharge, and > or =10 PMNL/hpf (95 vs. 50 ifu). These associations persisted on multivariate analysis. Thus, chlamydial replication is associated with MPC and PID in women, urethritis in men, and inflammation in both. Since infections with high inclusion counts may be the most transmissible, identification and treatment of patients with these chlamydia-associated syndromes is important in control programs.

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.

Evidence for long-term cervical persistence of Chlamydia trachomatis by omp1 genotyping.

Recurrent Chlamydia trachomatis infections are common among sexually active women. Although recurrences with a new chlamydial serovar indicate reinfection, same-serovar recurrences may be due to persistence. Because persistence has important implications for pathogenesis and patient management, we identified 552 women with >3 recurrences over 2 years. Among these, 130 women (24%) had same-serovar recurrences; 58 (45%) were C class serovars (odds ratio, 2.4; 95% confidence interval, 1.7-3.5; P<.0001). Forty-five isolates from 7 women with 3-10 repeated, same-serovar infections over 2-5 years were studied. As determined by omp1 genotyping, 4 women had identical genotypes at each recurrence; 2 women had 1 or 2 amino acid changes following treatment, and one was persistently infected with a unique genotype, Ja. Many intervening culture-negative samples were positive when tested by ligase chain reaction, which suggests persistence. These data demonstrate that cervical infections with C class serovars can persist for years and may have specific biologic properties that allow for modulation of the major outer membrane protein in response to immune selection.

Quantitative Chlamydia trachomatis cultures: correlation of chlamydial inclusion-forming units with serovar, age, sex, and race.

The number of inclusion-forming units (IFUs) observed in quantitative chlamydial cultures may be a surrogate for infectivity or transmissibility. Therefore, we conducted a cross-sectional study of 11,034 patients with Chlamydia trachomatis infection who presented to the Seattle-King County public health department clinics between 1988 and 1996, to determine relationships between the number of IFUs observed in culture and sex, age, race, and serovar class. Of the 11,034 cases of infection we studied, 6801 (62%) were cervical infections in women, and 4233 (38%) were urethral infections in men. The median count was 450 IFU for women and 72 IFU for men (P<.001). Overall, both men and women infected with B-class serovars had significantly higher IFU counts than did those infected with C-class serovars (P<.001). The median IFU count fell consistently with increasing age for both women (625 IFU for those <16 years old to 185 IFU for those >30 years old; P<.001) and men (210 IFU for those <16 years old to 40.5 IFU for those >30 years old; P<.001). We found, by use of multiple regression analysis, that sex, age, race, and serovar class remained independently related to IFU count, with counts being highest among young white women infected with B-class serovars.

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

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 multiple Chlamydia 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. The incA 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. trachomatis isolates that form nonfusing inclusions; the variant phenotype is associated with the absence of detectable IncA and with an altered incA sequence that modifies the characteristic hydrophobic domain of the IncA protein.

Clinical features of Chlamydia trachomatis rectal infection by serovar among homosexually active men.

BACKGROUND AND OBJECTIVES: Because C. trachomatis serovars correlate with the clinical manifestations of cervical infection, we undertook this study to determine whether clinical, behavioral, and laboratory findings correlate with C. trachomatis serovars isolated from rectal infections. GOAL OF THIS STUDY: To correlate C. trachomatis serovar with signs and symptoms of rectal infection. STUDY DESIGN: A cross-sectional study of 454 men with rectal C. trachomatis infection attending an urban sexually transmitted disease (STD) clinic was undertaken. Isolates were thawed, passaged to high titer, and typed using a panel of monoclonal antibodies. Compared to men infected with B complex isolates (164), men with C complex isolates (55) were less likely to report symptoms (OR: 0.4; 95% CI: 0.1-0.8), or to have erythema, bleeding, or mucopus (OR: 0.3; 95% CI: 0.1-0.8). Among men with inclusion counts of more than 100, those infected with FG group versus B complex isolates were more likely to present with mucopus (OR: 10.5; 95% CI: 1.2-95.5), more than 15 polymorphonuclear leukocytes (OR: 19.2; 95% CI: 1.7-219.8), and proctitis (OR: 4.2; 95% CI: 1.1-16.7). CONCLUSION: Signs and symptoms of rectal infection correlate with the serovar of C. trachomatis isolates.

Chlamydia trachomatis IncA is localized to the inclusion membrane and is recognized by antisera from infected humans and primates.

Chlamydia psittaci produces a collection of proteins, termed IncA, IncB, and IncC, that are localized to the chlamydial inclusion membrane. In this report we demonstrate that IncA is also produced by Chlamydia trachomatis. C. trachomatis IncA is structurally similar to C. psittaci IncA and is also localized to the inclusion membrane. Immunoblot analysis demonstrated that sera from C. trachomatis-infected patients and from experimentally infected monkeys both recognized C. trachomatis IncA.

Infection with Chlamydia trachomatis lymphogranuloma venereum serovar L1 in homosexual men with proctitis: molecular analysis of an unusual case cluster.

Among 767 rectal isolates of Chlamydia trachomatis obtained from men over the period 1981-1991, 7 were found to be a rare lymphogranuloma venereum serovar, L1. These isolates were from rectal specimens taken from five male patients in 1982-1983. Six of the seven isolates were available for DNA sequencing studies. All six of these isolates shared the same DNA sequence in the major outer membrane protein (MOMP) gene variable domains and had different MOMP sequences than did the prototype L1, L2, and L3 strains, suggesting a point source outbreak. All five patients infected with serovar L-1 were homosexual men who had symptomatic proctitis characterized by rectal pain, discharge, tenesmus, abnormalities seen on anoscopy, and leukocytes seen on gram stains of rectal specimens. We conclude that an unrecognized point source outbreak of the rare chlamydial L1 serovar occurred among homosexual men in Seattle in 1982-1983, and that the clinical manifestations of L1 infection may be less severe than those of L2 infections.

Clinical manifestations of genital infection due to Chlamydia trachomatis in women: differences related to serovar.

The relationship between the infecting Chlamydia trachomatis serovar and the clinical manifestations of genital tract infection was evaluated in a study of 155 women attending a sexually transmitted diseases clinic; 99 women had lower genital tract infection and 56 had Chlamydia-associated pelvic inflammatory disease (PID). In the group with lower genital tract infection, women with serovar F differed from those with serovars of class B or C in that they exhibited fewer signs of cervical infection, including easily induced bleeding (P = .04), edema of the zone of cervical ectopy (P = .06), and colposcopic evidence of mucopurulent endocervical discharge (P = .007). Serovar F also produced fewer infections with inclusion counts of > or = 1,000 and fewer rectal infections (P = .04). There was no apparent association of any specific serovar with PID. Thus, in this population, serovar F was associated with fewer objective clinical manifestations of mucopurulent endocervical discharge, and the distribution of chlamydial serovars found in PID reflected that found in lower genital tract infection.

Nucleotide sequence of the variable domains within the major outer membrane protein gene from serovariants of Chlamydia trachomatis.

During studies in which we serotyped large numbers of Chlamydia trachomatis clinical isolates by using monoclonal antibodies, three novel serological variants (D-, D*, and I-) were identified. To determine the molecular basis for the altered monoclonal antibody reactions of these strains and other previously identified variants (Da, Ia, and L2a), we determined the nucleotide sequences of the variable domains in their major outer membrane protein genes. Da, D-, and D* variants differed by a single nucleotide and also an amino acid in the carboxy terminus of variable domain IV (VDIV) from the D serovar. The L2a variant also differed from L2 by a single amino acid but in VDII. Ia variants differed in VDI, III, and IV and I- variants differed in all four VDs from the I serovar. These studies demonstrate the potential for using major outer membrane protein VD sequencing as a highly sensitive typing method and further identify immunologically reactive major outer membrane protein epitopes.

Association of genital infection with specific Chlamydia trachomatis serovars and race.

Black race is an important risk marker for Chlamydia trachomatis genital infection. To define whether C. trachomatis serovars differ by ethnic distribution, a panel of monoclonal antibodies was used to serotype 934 urethral and 581 cervical isolates from patients attending a sexually transmitted diseases clinic over 2 years. The overall serovar distribution in cervical and urethral infections was comparable, with B class serovars predominating. Significantly higher inclusion counts were observed both in younger women and in nonblacks regardless of serovar. Serovar D was less frequent among blacks at the urethral site (P = .001), while serovar Ia was more frequent in blacks at both sites (urethral, P < .001; cervical, P = .02). These associations remained significant after adjusting for age and number of inclusion-forming units by multivariate analysis. Thus, specific serovars may be associated with particular racial groups; either behavioral or biologic factors could explain these findings.

Simplified microtiter cell culture method for rapid immunotyping of Chlamydia trachomatis.

Serotyping of Chlamydia trachomatis strains usually requires three to six serial passages in shell vials to attain sufficient antigen for typing procedures. To circumvent this problem, we developed a rapid low-passage method for serotyping of C. trachomatis clinical isolates. Isolates with an inclusion count of greater than or equal to 500 per well in primary isolation were inoculated directly onto cell culture monolayers in microtiter plates for typing. Primary isolates with a lower initial inclusion count were passed one to two times in shell vials until there were greater than or equal to 20 inclusions per well and were then inoculated onto plates for typing. Inclusions were grown to maturity and reacted with a panel of 17 C. trachomatis-specific monoclonal antibodies in pools. Wells were then reacted with a fluorescein isothiocyanate conjugate and read by FA microscopy, and the reaction patterns were compared with prototype strain reaction patterns to determine the serotype. By the microtiter method, we successfully typed 1,711 consecutive C. trachomatis isolates; 1,215 isolates (71%) were typed with no or with one passage. The first 209 isolates typed by the microtiter method were also typed by the dot-enzyme-linked immunosorbent assay serotyping method; 100% agreement was demonstrated among strains that were typeable by both methods. We conclude that the microtiter method is extremely useful for accurate serotyping of large numbers of isolates and requires greatly reduced technician time.

[Serotype distribution of urogenitally located Chlamydia trachomatis in Rotterdam]. / Serotypeverdeling van urogenitaal gelokaliseerde Chlamydia trachomatis in Rotterdam.

The distribution of serotypes in 208 Chlamydia trachomatis strains of urogenital origin isolated from 185 patients (87 women, 98 men) attending a sexually transmitted disease clinic in Rotterdam, the Netherlands, was studied. Typing by monoclonal antisera using a dot enzyme linked immunosorbent assay (ELISA) showed that the most common serotypes were E (found in 45 strains), F (39), D (34) and K (28). Other serotypes detected were H (21), G, I, I', J (2-12) and B (one strain). Mixed infection with two serotypes was detected in two patients. These results indicate that most genital infections with C. trachomatis result from a small number of serotypes, and that these are similar in the Netherlands and Seattle, U.S.A.

Serovar distribution of urogenital Chlamydia trachomatis strains in The Netherlands.

The distribution of serovars in 208 Chlamydia trachomatis strains of urogenital origin isolated from 185 patients attending a sexually transmitted disease clinic in Rotterdam, The Netherlands, was assessed. Typing by monoclonal antisera using a dot enzyme linked immunosorbent assay (ELISA) showed that the most common serovars were E (found in 45 strains), F (39), D (34), and K (28). Other serovars detected were H (21), G, I, I', J (two to 12), and B (one strain). Mixed infection with two serovars was detected in two patients. These results indicate that most genital infections with C trachomatis result from a small number of serovars, and that those serovars are similar in The Netherlands and Seattle, USA.

Detection of multiple serovars of Chlamydia trachomatis in genital infections.

Eight genital isolates of Chlamydia trachomatis that each contained two different serovars were identified by typing with serovar-specific monoclonal antibodies. Mixed infections were confirmed by using serovar-specific monoclonal antibodies in an indirect immunofluorescence assay. For some isolates, fluorescein/rhodamine double-label indirect immunofluorescence with mixtures of IgM and IgG monoclonal antibodies to Chlamydia provided direct visual confirmation of mixed infection. Two isolates contained serovars D and F, and two E and F; one isolate contained serovars Ba and E, one D and J, one I' and F, and one E and J. In a sample of 352 genital isolates of C. trachomatis consecutively typed by dot ELISA with monoclonal antibodies, seven (2%) demonstrated mixed serovars.