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Partial Characterization of Chlamydia trachomatis Isolates Resistant to Multiple Antibiotics Robert B. Jones, Barbara Van Der Pol, David H. Martin, and Marguerite K. Shepard
From the Departments of Medicine, Microbiology and Immunology, and Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, and the Department of Medicine, Louisiana State University School of Medicine, New Orleans
Genital tract infection with Chlamydia trachomatis is highly prevalent in most areas of the world investigated [1-5]. Recovery rates from sexually active men and women undergoing screening in the USA usually range from 9% [6-8] to 28% [9]. Even higher rates are found in individuals seeking medical care because of other sexually transmitted diseases (e.g., gonorrhea) or because of symptoms such as those associated with urethritis [10]or salpingitis [11]. The major adverse consequences of genital chlamydial infections are epididymitis in men [12] and clinical or subclinical salpingitis in women. The latter often produces tubal infertility [13] or ectopic pregnancy [14, 15] as late sequelae. In addition, perinatal transmission resulting in conjunctivitis of the newborn or infant pneumonia is common [16, 17]. Although relative resistance to erythromycin has been reported for C. trachomatis [18], tetracycline resistance has not [19]. Furthermore, attempts to induce tetracycline resistance in vitro have not been successful [20]. Consequently, control efforts against C. trachomatis rely on identifying individuals who are infected or likely to be infected and treating with tetracycline, a tetracycline congener (e.g., doxycycline), or erythromycin [21]. We report here the recovery from five patients of C. trachomatis isolates resistant to tetracy-
Received 27 February 1990; revised 22 June 1990. Informed consent was obtained from subjects according to protocols approved by the Indiana University-Purdue University Committee for the Protection of Human Subjects. Grant support: AI-20110 (National Institutes of Health). Reprints or correspondence: Dr. Robert B. Jones, Department of Medicine, EM 435, 545 Barnhill Dr., Indianapolis, IN 46202. The Journal of Infectious Diseases 1990;162:1309-1315 © 1990 by The University of Chicago. AU rights reserved. 0022-1899/90/6206-0013$01.00
cline and provide preliminary characterization of these isolates.
Case Reports Cases 1 and 2 have been described previously [13], although the antibiotic susceptibilities of the isolates recovered from them have not. Patient 1. A 19-year-old woman had two episodes in 1 year of acute salpingitis requiring hospitalization. At the second episode a left salpingectomy was done and she and her husband were treated with tetracycline, 500 mg orally four times a day for 7 days. She was then treated with 500 mg of erythromycin orally four times a day for 10 days. Ten months after the salpingectomy, an endometrial culture positive for C. trachomatis (isolate IU888) was obtained; the patient and her husband were treated with 100 mg of doxycycline orally twice daily for 14 days. Six days after completing therapy she underwent a microtuboplasty of her right fallopian tube. C. trachomatis was recovered from biopsies of the endometrium (IU918) and fallopian tube (IU919) and from a swab specimen obtained from the endocervix during surgery (IU917). She and her husband were treated with minocycline, 100 mg orally twice daily for 14 days. They had negative lower-genitaltract cultures 2 months later. Patient 2. A 27-year-old woman had a left salpingooophorectomy done for chronic salpingitis with acute exacerbation and was treated with a cephalosporin. Six months later she was treated with tetracycline for "poor cervical mucus" on a postcoital test. Dose and duration of treatment were not recorded, but the usual regimen in the clinic in which she was treated is 250 mg orally four times daily for 10-14 days. Five months later she underwent microsurgical repair of the right fallopian tube. C. trachomatis was recovered from the
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In vitro susceptibility testing was done on urogenital isolates of Chlamydia trachomatis from five patients, four of whom were suspected treatment failures. At least one isolate from each patient was resistant to tetracycline at concentrations ~8 p.g/ml, although 8 8/NT >8/4 4/2 >8/4 >8/>8 >8/>8 >8/NT >8/NT >8/NT 8/NT
IU13028 (D) IU14747 (D) IU14091 (E) UW-6 (F) LGV-434 (L2) UW-3 (D)
0.25/0.25 0.125/0.125 0.l25/NT 0.125/0.12 0.125/NT 0.25/0.25
New Orleans (MIC) >4 NT NT NT NT >4 NT NT >4 NT >4 0.0625 NT NT 0.0625 0.125 0.25
NOTE. Endo, endometrium; Tube, fallopian tube; Cx, endocervix; Ur, urethra; Bu, bubo; NT, not tested; MCC, minimum chlamydicidal concentration. * Data are expressed in ug/ml.
of the cloned isolates were expanded and tested for susceptibility to tetracycline. Immunotyping. Clinical isolates were immunotyped as previously described using a panel of type-specific monoclonal antibodies and a radioimmunoassay [24, 26].
Results C. trachomatis isolates from each patient contained organisms that were resistant to tetracycline at concentrations of 4 to >8 ~g/ml, while clinical isolates and laboratory strains previously found sensitive to tetracycline (data not shown) remained so (table 1). These observations were made independently in two laboratories using slightly different techniques for the susceptibility testing. Concentrations of tetracycline >8 ~g/ml (Indianapolis) or >4 ~g/ml (New Orleans) were not used in the experiments shown. However, in separate experiments isolate IU888 was resistant to concentrations of tetracycline as high as 64 ~g/ml, while IU824 was resistant to 16 ~g/ml but sensitive to 32 ~g/ml. MCCs were similar to and consistent with MICs for both sensitive and resistant strains (table 1). Although the isolates from these patients formed inclusions in the presence of tetracycline, at higher concentrations of tetracycline there was a ~100-fold decrease in the number of inclusions formed, suggesting that only a small proportion of organisms in the population might be resistant (table 2). To further explore this possibility, one isolate (IU824) was
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and the monolayer scraped with a blunt Pasteur pipette. The suspension from each well was then aspirated and (without pooling) inoculated onto a fresh monolayer. The suspension was overlaid with 0.2 ml antibiotic-free media. After centrifugation and incubation for 72 h the wells were stained for inclusions. The MCC was defined as the lowest concentration at which no inclusions were seen. Susceptibilitytestingin New Orleans. The isolates of C trachomatis described here were initially recovered and tested for antimicrobial sensitivity in Indianapolis. To obtain an independent evaluation, aliquots of isolates from four of the five patients and aliquots from control strains were expanded to a titer of >10 7 ifu/ml and frozen at -70°C. These were then shipped on dry ice to New Orleans where laboratory personnel were blinded as to which isolates had been found resistant to tetracycline. The testing protocol followed in New Orleans was similar to that in Indianapolis. Flat-bottom, 24-well plastic plates were seeded with McCoy cells in Eagle's MEM supplemented with 10% fetal bovine serum and 0.01% extra glucose and incubated overnight to achieve monolayer confluence. Isolates were retitered and each well inoculated with a volume of chlamydial suspension sufficient to produce ~20 inclusions per x400 field. Each monolayer was pretreated with 30 J.d of DEAE-dextran, which was aspirated before chlamydial inoculation. After chlamydiae were added, plates were centrifuged at 1000 g for 1 h at room temperature, the inoculum aspirated, and medium containing an appropriate concentration of tetracycline added to each well. The plates were then incubated for 72 h and monolayers stained for inclusions using the Cultureset identification reagent (Ortho). Each tetracycline concentration was tested in triplicate in twofold dilutions from 0.03125 to 4.0 p.g/ml and the MIC taken as the lowest antibiotic concentration that completely inhibited inclusion formation in all three wells. Growth in tetracycline. Six McCoy cell monolayers in shell vials were inoculated with isolate IU824; MEM containing 8 p.g/mltetracycline was added, and the vials were centrifuged and incubated as above. After 72 h, one coverslip was stained to confirm that organisms were present. The other five monolayers were disrupted and the specimens pooled. This sample was then titered and susceptibility testing done without further passage. Limiting dilutions. Clones of single C trachomatis organisms were obtained by a modification of the limiting dilutions technique of Kahle et al. [24]. A suspension of IU824 in transport medium was titered to determine the number of inclusion-forming units present. This suspension was then diluted to concentrations of 10, 5.0, and 1.0 ifu/ml. Plates containing McCoy cell monolayers were inoculated with 0.1 ml of the diluted suspensions. All tissue culture methods were the same as those previously described. Based on the numbers of viable organisms inoculated, these infected monolayers should have yielded an average of 1.0,0.5, and 0.1 inclusions, respectively. However, small numbers of inclusions are difficult to detect visually in unstained monolayers. Consequently, these monolayers were serially passaged three times to expand the number of inclusions so that they were detectable by light microscopy using an inverted microscope at x400 magnification. The suspension that should have yielded an average of 0.1 inclusions per monolayer yielded 15 positive monolayers and 57 negative ones. The probability that a well contained k inclusions was P = (tlk!) X e», where z is the average number of positive wells [25]. Thus, the probability of this dilution providing 0 inclusions per well was .82, 1 inclusion per well, .17, and >1 inclusion per well, .02. Four
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Table 2. Inclusion-forming units (ifu) of Chlamydia trachomatis detected in the presence of tetracycline. Tetracycline concentration (ug/ml) Isolate IU824 1U888 LGV-434 IU14901
0.0
0.063
0.125
0.25
0.5
1.0
2.0
4.0
8.0
9000 17,500 19,000 7000
2500 3650 46.6 22.7
73.3 21.7 0 0
33.7 16.3 0 0
23.3 4.7 0 0
14 6.7 0 0
11 4.7 0 0
9 2.7 0 0
6.7 1.7 0 0
Data are ifu/well.
NOTE.
• • • LGV-434 . . IU824 . . . IU824 Immediately after tetracyline exposure - -
IU824 After tetracyline exposure and passage
~
IU824 Cloned by limiting dilution
6.0
5.0
.
\
\
-E
4.0 \ \ \
j
3.0
"0
\ \ \
....J
2.0
\
\ \
\ 1.0
\ \ \ 0.017
• • • • • • • • • • • • • • • • • • • • • •
••
• • • •
0.063
0.250.5 1
2
4
8
TETRACYLINE (JJg/ml) Figure 1. Susceptibility of Chlamydia trachomatis isolate IU824 to tetracycline after initial expansion in antibiotic-free medium, cloning by limiting dilutions, growth in 8 ILg/ml tetracycline, and growth in 8 ILg/ml tetracycline followed by nine passages in antibiotic-free medium. Control strain was LGV-434.
resistant to tetracycline at 8 JLglml as indicated by the lack of change in number of inclusion-forming units per milliliter observed with increasing tetracycline concentrations. Inhibition of the entire population of the laboratory strain occurred in a narrow range of tetracycline concentrations (0.032-0.125 J,tg/ml). In contrast, while 1'\.199 % of the organisms present in IU824 before in vitro tetracycline exposure were inhibited by low concentrations of tetracycline, 1'\.11 % survived relatively high concentrations. Four of six attempts to expand these fully resistant organisms by passage in antibiotic-free medium were unsuccessful. With each passage the number of inclusion-forming units per milliliter recovered decreased until, after five or six passages, none were identifiable. However, on two attempts, after decreasing for six passages, the number of inclusionforming units began to increase, and after six more passages the isolates were sufficiently expanded to permit repeat sensitivity testing. At this point the entire population of one group was sensitive to ~0.017 ~ml tetracycline (figure 1) and of the other group to 0.032 J,tglml. The four isolates cloned by limiting dilutions from IU824 had inhibition curves identical to that of the parent. One of these is shown in figure 1. Because only a few inclusion-forming units per well were detectable at higher concentrations of tetracycline, we evaluated the effect of varying the inoculum on our ability to detect resistant isolates (table 3). At inocula >5.0 X lQ3 ifu/well both resistant isolates appeared resistant, but at 3 X 103 ifu/well one (IU888) and at 0.8 x 103 ifu/well the other (lU824) appeared sensitive. The size of the inoculum had only a slight effect on the MIC of the sensitive isolate (lUI4091) tested. In addition, two tetracycline-resistant isolates and two control strains were tested for their susceptibility to other antimicrobial agents potentially useful in treating urogenital tract infections. All four were resistant to ampicillin and ceftriaxone, with MICs >64 JLg/ml. The tetracycline-resistant isolates were resistant to doxycycline, minocycline, and erythromycin, with MICs ~8 J,tglml, and to clindamycin and sulfamethoxazole, with MICs >64 JLglml and >400 JLg/ml, respectively; both tetracycline-sensitive strains were sensitive to these antibiotics. One isolate, IU824, was relatively resistant to rifampin, with a MIC of 0.25 J,tg/ml compared with ~0.031 pg/ml for the control strains. Both tetracycline-resistant iso-
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passaged once in MEM containing 8 JLglml tetracycline and the susceptibility of the surviving progeny compared with that of the parent strain and a sensitive laboratory strain, LGV434 (figure 1). In vitro exposure of IU824 to tetracycline resulted in a population of organisms 100% of which were
Antibiotic-Resistant C. trachomatis
1ID 1990;162 (December)
Table 3. Effect of inoculum size on tetracycline MIC for Chlamydia trachomatis. Isolate IU824
IU888
13.0 7.5 5.5 2.5 0.8 0.13 32.5 19.0 8.5 3.0 0.55 0.23 32.5 15.0 6.0 0.41 0.17 0.03
MIC (ug/rnl)
>8 >8 >8 >8 0.25 0.125
>8 >8 8 0.125 0.125 0.125 0.25 0.125 0.125 0.125 0.125 0.125
NOTE. Inoculum size was determined from inclusion-forming unit (ifu) count in antibiotic-free control wells.
lates were as sensitive as the control strains to ciprofloxacin and ofloxacin, with MICs of ~4 ILg/ml and ~l pg/ml, respectively. The inhibitioncurves observed withminocycline, doxycycline, and erythromycin were similar to those seen with tetracycline; that is, the resistant strains showeda 98 %-99 % decrease in the number of inclusion-forming units per milliliter in wells containing drug concentrations abovethe MIC values for the control strains. However, with ceftriaxone, ampicillin, clindamycin, and sulfamethoxazole, little inhibition of inclusion-formingunit formation was seen for the resistant strains at any concentration of antibiotic tested; that is, all members of the population appeared fully resistant. Discussion Our data are the first to our knowledge to demonstrate resistance to tetracycline in C. trachomatis. Moreover, two tetracycline-resistantisolates from two differentpatients were also fully resistant to doxycycline, clindamycin, and erythromycin, in contrast to the relative resistance to erythromycin reported previously [18]. Tetracycline resistance in C. trachomatis is not surprising given the current practice of treating most documented or suspected chlamydial infections with tetracycline or doxycycline. However, we do not yet know whether this represents a recent change in C. trachomatis antibiotic susceptibility. It is possible that the phenomenon is not new but has not been recognized because of the methods usually used for susceptibility testing. In most studies of C. trachomatis antimicrobial susceptibility, inocula have ranged from a few hundred to rv1000 ifu per tissue culture monolayer [18, 19,
27-30], or rv10- to 100-fold less than were used here. However, because resistance is manifestedby only a small proportion of the organism population, a large inoculum is required to detect it (table 3). In addition, the inclusions that occur in the presenceof highconcentrations of tetracycline are small, havealtered morphology, and are difficultto detect, evenwith immunofluorescent staining. With iodine, the stain most frequently used in the past for susceptibility testing [19], their altered morphology makes them even more difficult to identify. Consequently, this heterotypicresistance could havebeen missed previously in assaysusing substantially lower inocula or iodine staining to detect inclusions or both. Also, most previous studies have used laboratory strains that have undergone multiple passages in tissue culture, and it is possible that under these circumstances the ability to express resistance may be lost. This seemed to happen with IU824 after exposure to high concentrations of tetracycline and regrowth in antibiotic-free medium, although it did not happen during the 31 passages to which IU824 was subjected before susceptibility testing. Resistance manifested by a small proportion of organisms in a population could represent a mixed infection with two strains, one sensitiveand numerically predominant, the other resistant and present in small numbers. Alternatively, there may be heterogenous expression of resistance in which all members of a population are capable of expressing resistance but only a small proportion do so at anyone time. This latter phenomenon clearly occurs in methicillin-resistant staphylococci, although the mechanisms regulating the expression of this resistance are not well understood [31-33]. Limitingdilution experiments were done to resolve these two possibilities. If these isolates were mixed populations in which only 1 of 102-103 organisms were resistant, the clonally derived organisms should all be fullysensitive. The fact that the inhibition curves for the progeny were identical to that of the parent indicates heterogenous expression of resistance. It is of particular interest that fully resistant organisms do not grow well and cannot yet be propagated beyonda fewpassages in tissue culture; they die or, as happened twice, completely lose their resistance. This suggests that the mechanism by which they become resistant does not favorlong-term survival of organisms expressing that trait. A similar observation has been made regarding expression of the tetA gene product in Escherichia coli [34]. Although it confers tetracycline resistance, overproduction inhibitsgrowth. However, the molecularmechanismsunderlyingthe resistance we observed in C. trachomatis are not necessarily the same as those previously seen in gram-negative bacteria. Likewise, the clinical significance of antibiotic resistance in C. trachomatis is unknown. So far, few isolates havebeen tested by the methods described here. Besides those described above, 34 initial isolates from adolescents who experienced relapse or reinfection within 15 months have undergone preliminary testing. Of these, 15 (44%) were resistant to tetracy-
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IU14091
Inoculum (ifu/well x 10- 3)
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References 1. Thompson SE, WashingtonAE. Epidemiology of sexually transmitted Chlamydia trachomatis infections. Epidemiol Rev 1983;5:96-123 2. NayyarKC,Cummings M, WeberJ, Benes S, StolzE, FelmanY,McCormack WM. Prevalenceof genital pathogensamongfemaleprostitutes in New YorkCity and in Rotterdam. SexTransmDis 1986;13:105-107 3. Plummer FA, Laga M, Brunham RC, Piot P, Ronald AR, Bhullar V, Mati JY, Ndinya-Achola JO, Chean M, Nsanze H. Postpartum upper genital infectionsin Nairobi, Kenya: epidemiology, etiology, and risk factors. J Infect Dis 1987;156:92-97 4. ToomeyKE, Rafferty MP, Stamm WE. Unrecognized high prevalence of Chlamydia trachomatis cervical infection in an isolated Alaskan Eskimo population. JAMA 1987;258:53-56 5. Chungue E, Cartel JL, Tourneux M, Mahe A, Perolat P, Flye Sainte Marie F, Roux 1. Chlamydia trachomatis genital infections in Tahiti. Eur J Clin Microbiol Infect Dis 1988;7:635-638
6. Handsfield HH, Jasman LL, Roberts PL, Hanson VW, Kothenbeutel RL, StammWE. Criteria for selective screening for Chlamydia trachomatis infection in women attending family planning clinics. JAMA 1986;255:1730-1734 7. PodgoreIK, Holmes KK, Alexander ER. Asymptomatic urethral infections due to Chlamydia trachomatis in male US military personnel. I Infect Dis 1982;146:828 8. Karam GH, Martin DH, Flotte TR, Bonnarens FO, Ioseph IR, MroczkowskiTF, Iohnson WD. Asymptomatic Chlamydia trachomatis infections among sexually active men. J Infect Dis 1986;154:900-903 9. Blythe MI, Katz BP, Orr DP, Caine VA,Iones RB. Historical and clinical factors associated with Chlamydia trachomatis genitourinary infection in female adolescents. I Pediatr 1988;112:1000-1004 10. NettlemanMD, Jones RB, RobertsSO, Katz BP,WashingtonAB, Dittus RS, Quinn TS. Cost-effectiveness of culturing for Chlamydia trachomatis. A study in a clinic for sexuallytransmitted diseases. Ann Intern Med 1986;105:189-195 11. Sweet RL, Schachter J, Robbie MO. Failure of 13-lactam antibiotics to eradicate Chlamydia trachomatisin the endometrium despite apparent clinical cure of acute salpingitis. lAMA 1983;250:2641-2645 12. BergerRE, AlexanderER, Monda GD,AnsellI, McCormickG, Holmes KK. Chlamydia trachomatisas a cause of acute "idiopathic"epididymitis. N Engl I Med 1978;298:301-304 13. Shepard MK, Iones RB. Recovery of Chlamydia trachomatis from endometrialand fallopiantube biopsiesin womenwith infertilityof tubal origin. Fertil SterH 1989;52:322-338 14. Brunham RC, Binns F, McDowellI, Paraskevas M. Chlamydia trachomatis infection in women with ectopic pregnancy. Obstet Gynecol 1986;67:722-726 15. Hartford SL, SilvaPD, diZerega GS, YonekuraML. Serologic evidence of prior chlamydialinfectionin patients with tubal ectopic pregnancy and contralateral tubal disease. Fertil Steril 1987;47:118-121 16. AlexanderER, Harrison HR. Roleof Chlamydia trachomatisin perinatal infection. Rev Infect Dis 1983;5:713-719 17. SchaeferC, Harrison HR, BoyceWT, LewisM. illnesses in infantsborn to womenwith Chlamydia trachomatis infection: a prospectivestudy. Am I Dis Child 1985;139:127-133 18. Mourad A, SweetRL, SuggN, Schachter1. Relativeresistance to erythromycin in Chlamydia trachomatis. Antimicrob Agents Chemother 1980;18:696-698 19. Ehret 1M, Judson FN. Susceptibilitytesting of Chlamydia trachomatis: from eggs to monoclonal antibodies. Antimicrob Agents Chemother 1988;32:1295-1299 20. Iones RB, Ridgway GL, Boulding S, Hunley KL. In vitro activity of rifamycins alone and in combination with other antibiotics against Chlamydia trachomatis. Rev Infect Dis 1983;5(suppl):S556-S561 21. Centers for Disease Control. 1989 sexually transmitted diseases treatment guidelines. MMWR 1989;38(suppl 8):1-43 22. Iones RB, Katz BP, Van Der Pol B, Caine V, Batteiger BE, Newhall WI V. Effect of blind passage and multiple samplings on recovery of Chlamydia trachomatis fromurogenital specimens. J Clio Microbiol 1986;24:1029-1033 23. Iones RB. Antimicrobialsusceptibility testingfor some atypical microorganisms: mycoplasmas, chlamydiae, and rickettsiae. In: Lorian V, ed. Antibiotics in laboratory medicine. 2nd ed. Baltimore: Williams & Wilkins, 1986:346-358 24. KahleP, Wernet P, Rehbein A, KumbierI, PawelecG. Cloning of functionalhuman T lymphocytesby limitingdilution: impact of killer cells and interleukin-2 sources on cloning efficiencies. Scand I Immunol 1981;14:493-502 25. Batteiger BE, Fraiz J, Newhall WI V, Katz BP, Jones RB. Association of recurrent chlamydial infection with gonorrhea. I Infect Dis 1989;159:661-669 26. NewhallWI V, Terho P, Wilde CE m, BatteigerBE, Iones RB. Serovar
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cline at ~8 #Lglml (unpublished data). However, an appropriate control group was not evaluated and, while these data suggest that the resistant phenotype may be fairly common, further studies are indicated. Furthermore, even when present, heterotypic tetracycline resistance may not always or even often result in treatment failure. In the present study, reinfection is a possible explanation for apparent treatment failure in patients 3 and 5. Patient 3 steadfastly denied exposure between cultures but was infected with two different serovars the two times the organism was isolated and. could be expanded, indicating that she either was reinfected or was infected initially with two different serovars. Patient 5 was potentially reexposed. However, tetracycline resistance likely played a major role in the persistent infection in patient 1, and it may do so in other patients whose therapy with tetracycline or doxycycline inexplicably fails. Bacteriologic cures were achieved in one patient retreated with doxycycline and in the two retreated with minocycline, although the isolates were resistant in vitro to these tetracycline analogs. The poor growth of the tetracycline-resistant organisms observed in vitro may also occur in vivo; thus, a repeat or more prolonged course of therapy with tetracycline may be effective even when tetracycline resistance is present. It is also possible that minocycline is more effectivethan other tetracyclines in this setting because of its better tissue penetration [35]. Antimicrobial susceptibility testing of C. trachomatis isolates is time consuming, technically difficult, and expensive. Moreover, it requires the viable organisms, which are not available when direct antigen tests (direct fluorescent antibody or ELISA) are used to identify infected individuals. Consequently, routine susceptibility testing of C. trachomatis is not practical in most clinical settings. However, our data indicate that resistance should be considered when there is solid documentation of treatment failure with standard therapy. Moreover, the role of this form of resistance in the spread and maintenance of C. trachomatis infections in selected populations needs to be fully assessed.
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27.
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31. Chambers HP. Methicillin-Resistant staphylococci. Clin Microbiol Rev 1988;1:173-186 32. Matthews PR, Stewart PRo Resistance heterogeneity in methicillinresistant Staphylococcus aureus. FEMS Microbiol Lett 1984;22: 161-166 33. Hackbarth CJ, Chambers HE Methicillin-resistant staphylococci: genetics and mechanisms of resistance. Antimicrob Agents Chemother 1989; 33:991-994 34. Moyed HS, Bertrand KP. Mutations in multicopy TnlO tet plasmids that confer resistance to inhibitory effects of tet gene expression. J Bacteriol 1983;155:557-564 35. Kucers A, Bennett NM. The use of antibiotics. 4th ed. Philadelphia: 1. B. Lippincott, 1987:999-1000
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determination of Chlamydia trachomatis isolates by using type-specific monoclonal antibodies. J Clin Microbiol 1986;23:333-338 Alexander ER, Skahen P, Holmes KK. Antibiotic susceptibility of Chlamydia trachomatis in cell culture. In: Hobson D, Holmes KK, eds. Nongonococcalurethritis and related infections. Washington, DC: American Society for Microbiology, 1977:223-226 Blackman HJ, YonedaC, Dawson CR; Schachter 1. Antibiotic susceptibility of Chlamydia trachomatis. Antimicrob Agents Chemother 1977;12:673-677 BowieWR, Lee CK, Alexander ER. Prediction of efficacyof antimicrobial agents in treatment of infections due to Chlamydia trachomatis. J Infect Dis 1978;138:655-659 Johannisson G, Semryd A, Lycke E. Susceptibility of Chlamydia trachomatis to antibiotics in vitro and in vivo. Sex Transm Dis 1979;6:50-57
1315
Partial Characterization of Chlamydia trachomatis Isolates Resistant to Multiple Antibiotics Robert B. Jones, Barbara Van Der Pol, David H. Martin, and Marguerite K. Shepard
From the Departments of Medicine, Microbiology and Immunology, and Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, and the Department of Medicine, Louisiana State University School of Medicine, New Orleans
Genital tract infection with Chlamydia trachomatis is highly prevalent in most areas of the world investigated [1-5]. Recovery rates from sexually active men and women undergoing screening in the USA usually range from 9% [6-8] to 28% [9]. Even higher rates are found in individuals seeking medical care because of other sexually transmitted diseases (e.g., gonorrhea) or because of symptoms such as those associated with urethritis [10]or salpingitis [11]. The major adverse consequences of genital chlamydial infections are epididymitis in men [12] and clinical or subclinical salpingitis in women. The latter often produces tubal infertility [13] or ectopic pregnancy [14, 15] as late sequelae. In addition, perinatal transmission resulting in conjunctivitis of the newborn or infant pneumonia is common [16, 17]. Although relative resistance to erythromycin has been reported for C. trachomatis [18], tetracycline resistance has not [19]. Furthermore, attempts to induce tetracycline resistance in vitro have not been successful [20]. Consequently, control efforts against C. trachomatis rely on identifying individuals who are infected or likely to be infected and treating with tetracycline, a tetracycline congener (e.g., doxycycline), or erythromycin [21]. We report here the recovery from five patients of C. trachomatis isolates resistant to tetracy-
Received 27 February 1990; revised 22 June 1990. Informed consent was obtained from subjects according to protocols approved by the Indiana University-Purdue University Committee for the Protection of Human Subjects. Grant support: AI-20110 (National Institutes of Health). Reprints or correspondence: Dr. Robert B. Jones, Department of Medicine, EM 435, 545 Barnhill Dr., Indianapolis, IN 46202. The Journal of Infectious Diseases 1990;162:1309-1315 © 1990 by The University of Chicago. AU rights reserved. 0022-1899/90/6206-0013$01.00
cline and provide preliminary characterization of these isolates.
Case Reports Cases 1 and 2 have been described previously [13], although the antibiotic susceptibilities of the isolates recovered from them have not. Patient 1. A 19-year-old woman had two episodes in 1 year of acute salpingitis requiring hospitalization. At the second episode a left salpingectomy was done and she and her husband were treated with tetracycline, 500 mg orally four times a day for 7 days. She was then treated with 500 mg of erythromycin orally four times a day for 10 days. Ten months after the salpingectomy, an endometrial culture positive for C. trachomatis (isolate IU888) was obtained; the patient and her husband were treated with 100 mg of doxycycline orally twice daily for 14 days. Six days after completing therapy she underwent a microtuboplasty of her right fallopian tube. C. trachomatis was recovered from biopsies of the endometrium (IU918) and fallopian tube (IU919) and from a swab specimen obtained from the endocervix during surgery (IU917). She and her husband were treated with minocycline, 100 mg orally twice daily for 14 days. They had negative lower-genitaltract cultures 2 months later. Patient 2. A 27-year-old woman had a left salpingooophorectomy done for chronic salpingitis with acute exacerbation and was treated with a cephalosporin. Six months later she was treated with tetracycline for "poor cervical mucus" on a postcoital test. Dose and duration of treatment were not recorded, but the usual regimen in the clinic in which she was treated is 250 mg orally four times daily for 10-14 days. Five months later she underwent microsurgical repair of the right fallopian tube. C. trachomatis was recovered from the
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In vitro susceptibility testing was done on urogenital isolates of Chlamydia trachomatis from five patients, four of whom were suspected treatment failures. At least one isolate from each patient was resistant to tetracycline at concentrations ~8 p.g/ml, although 8 8/NT >8/4 4/2 >8/4 >8/>8 >8/>8 >8/NT >8/NT >8/NT 8/NT
IU13028 (D) IU14747 (D) IU14091 (E) UW-6 (F) LGV-434 (L2) UW-3 (D)
0.25/0.25 0.125/0.125 0.l25/NT 0.125/0.12 0.125/NT 0.25/0.25
New Orleans (MIC) >4 NT NT NT NT >4 NT NT >4 NT >4 0.0625 NT NT 0.0625 0.125 0.25
NOTE. Endo, endometrium; Tube, fallopian tube; Cx, endocervix; Ur, urethra; Bu, bubo; NT, not tested; MCC, minimum chlamydicidal concentration. * Data are expressed in ug/ml.
of the cloned isolates were expanded and tested for susceptibility to tetracycline. Immunotyping. Clinical isolates were immunotyped as previously described using a panel of type-specific monoclonal antibodies and a radioimmunoassay [24, 26].
Results C. trachomatis isolates from each patient contained organisms that were resistant to tetracycline at concentrations of 4 to >8 ~g/ml, while clinical isolates and laboratory strains previously found sensitive to tetracycline (data not shown) remained so (table 1). These observations were made independently in two laboratories using slightly different techniques for the susceptibility testing. Concentrations of tetracycline >8 ~g/ml (Indianapolis) or >4 ~g/ml (New Orleans) were not used in the experiments shown. However, in separate experiments isolate IU888 was resistant to concentrations of tetracycline as high as 64 ~g/ml, while IU824 was resistant to 16 ~g/ml but sensitive to 32 ~g/ml. MCCs were similar to and consistent with MICs for both sensitive and resistant strains (table 1). Although the isolates from these patients formed inclusions in the presence of tetracycline, at higher concentrations of tetracycline there was a ~100-fold decrease in the number of inclusions formed, suggesting that only a small proportion of organisms in the population might be resistant (table 2). To further explore this possibility, one isolate (IU824) was
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and the monolayer scraped with a blunt Pasteur pipette. The suspension from each well was then aspirated and (without pooling) inoculated onto a fresh monolayer. The suspension was overlaid with 0.2 ml antibiotic-free media. After centrifugation and incubation for 72 h the wells were stained for inclusions. The MCC was defined as the lowest concentration at which no inclusions were seen. Susceptibilitytestingin New Orleans. The isolates of C trachomatis described here were initially recovered and tested for antimicrobial sensitivity in Indianapolis. To obtain an independent evaluation, aliquots of isolates from four of the five patients and aliquots from control strains were expanded to a titer of >10 7 ifu/ml and frozen at -70°C. These were then shipped on dry ice to New Orleans where laboratory personnel were blinded as to which isolates had been found resistant to tetracycline. The testing protocol followed in New Orleans was similar to that in Indianapolis. Flat-bottom, 24-well plastic plates were seeded with McCoy cells in Eagle's MEM supplemented with 10% fetal bovine serum and 0.01% extra glucose and incubated overnight to achieve monolayer confluence. Isolates were retitered and each well inoculated with a volume of chlamydial suspension sufficient to produce ~20 inclusions per x400 field. Each monolayer was pretreated with 30 J.d of DEAE-dextran, which was aspirated before chlamydial inoculation. After chlamydiae were added, plates were centrifuged at 1000 g for 1 h at room temperature, the inoculum aspirated, and medium containing an appropriate concentration of tetracycline added to each well. The plates were then incubated for 72 h and monolayers stained for inclusions using the Cultureset identification reagent (Ortho). Each tetracycline concentration was tested in triplicate in twofold dilutions from 0.03125 to 4.0 p.g/ml and the MIC taken as the lowest antibiotic concentration that completely inhibited inclusion formation in all three wells. Growth in tetracycline. Six McCoy cell monolayers in shell vials were inoculated with isolate IU824; MEM containing 8 p.g/mltetracycline was added, and the vials were centrifuged and incubated as above. After 72 h, one coverslip was stained to confirm that organisms were present. The other five monolayers were disrupted and the specimens pooled. This sample was then titered and susceptibility testing done without further passage. Limiting dilutions. Clones of single C trachomatis organisms were obtained by a modification of the limiting dilutions technique of Kahle et al. [24]. A suspension of IU824 in transport medium was titered to determine the number of inclusion-forming units present. This suspension was then diluted to concentrations of 10, 5.0, and 1.0 ifu/ml. Plates containing McCoy cell monolayers were inoculated with 0.1 ml of the diluted suspensions. All tissue culture methods were the same as those previously described. Based on the numbers of viable organisms inoculated, these infected monolayers should have yielded an average of 1.0,0.5, and 0.1 inclusions, respectively. However, small numbers of inclusions are difficult to detect visually in unstained monolayers. Consequently, these monolayers were serially passaged three times to expand the number of inclusions so that they were detectable by light microscopy using an inverted microscope at x400 magnification. The suspension that should have yielded an average of 0.1 inclusions per monolayer yielded 15 positive monolayers and 57 negative ones. The probability that a well contained k inclusions was P = (tlk!) X e», where z is the average number of positive wells [25]. Thus, the probability of this dilution providing 0 inclusions per well was .82, 1 inclusion per well, .17, and >1 inclusion per well, .02. Four
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Table 2. Inclusion-forming units (ifu) of Chlamydia trachomatis detected in the presence of tetracycline. Tetracycline concentration (ug/ml) Isolate IU824 1U888 LGV-434 IU14901
0.0
0.063
0.125
0.25
0.5
1.0
2.0
4.0
8.0
9000 17,500 19,000 7000
2500 3650 46.6 22.7
73.3 21.7 0 0
33.7 16.3 0 0
23.3 4.7 0 0
14 6.7 0 0
11 4.7 0 0
9 2.7 0 0
6.7 1.7 0 0
Data are ifu/well.
NOTE.
• • • LGV-434 . . IU824 . . . IU824 Immediately after tetracyline exposure - -
IU824 After tetracyline exposure and passage
~
IU824 Cloned by limiting dilution
6.0
5.0
.
\
\
-E
4.0 \ \ \
j
3.0
"0
\ \ \
....J
2.0
\
\ \
\ 1.0
\ \ \ 0.017
• • • • • • • • • • • • • • • • • • • • • •
••
• • • •
0.063
0.250.5 1
2
4
8
TETRACYLINE (JJg/ml) Figure 1. Susceptibility of Chlamydia trachomatis isolate IU824 to tetracycline after initial expansion in antibiotic-free medium, cloning by limiting dilutions, growth in 8 ILg/ml tetracycline, and growth in 8 ILg/ml tetracycline followed by nine passages in antibiotic-free medium. Control strain was LGV-434.
resistant to tetracycline at 8 JLglml as indicated by the lack of change in number of inclusion-forming units per milliliter observed with increasing tetracycline concentrations. Inhibition of the entire population of the laboratory strain occurred in a narrow range of tetracycline concentrations (0.032-0.125 J,tg/ml). In contrast, while 1'\.199 % of the organisms present in IU824 before in vitro tetracycline exposure were inhibited by low concentrations of tetracycline, 1'\.11 % survived relatively high concentrations. Four of six attempts to expand these fully resistant organisms by passage in antibiotic-free medium were unsuccessful. With each passage the number of inclusion-forming units per milliliter recovered decreased until, after five or six passages, none were identifiable. However, on two attempts, after decreasing for six passages, the number of inclusionforming units began to increase, and after six more passages the isolates were sufficiently expanded to permit repeat sensitivity testing. At this point the entire population of one group was sensitive to ~0.017 ~ml tetracycline (figure 1) and of the other group to 0.032 J,tglml. The four isolates cloned by limiting dilutions from IU824 had inhibition curves identical to that of the parent. One of these is shown in figure 1. Because only a few inclusion-forming units per well were detectable at higher concentrations of tetracycline, we evaluated the effect of varying the inoculum on our ability to detect resistant isolates (table 3). At inocula >5.0 X lQ3 ifu/well both resistant isolates appeared resistant, but at 3 X 103 ifu/well one (IU888) and at 0.8 x 103 ifu/well the other (lU824) appeared sensitive. The size of the inoculum had only a slight effect on the MIC of the sensitive isolate (lUI4091) tested. In addition, two tetracycline-resistant isolates and two control strains were tested for their susceptibility to other antimicrobial agents potentially useful in treating urogenital tract infections. All four were resistant to ampicillin and ceftriaxone, with MICs >64 JLg/ml. The tetracycline-resistant isolates were resistant to doxycycline, minocycline, and erythromycin, with MICs ~8 J,tglml, and to clindamycin and sulfamethoxazole, with MICs >64 JLglml and >400 JLg/ml, respectively; both tetracycline-sensitive strains were sensitive to these antibiotics. One isolate, IU824, was relatively resistant to rifampin, with a MIC of 0.25 J,tg/ml compared with ~0.031 pg/ml for the control strains. Both tetracycline-resistant iso-
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passaged once in MEM containing 8 JLglml tetracycline and the susceptibility of the surviving progeny compared with that of the parent strain and a sensitive laboratory strain, LGV434 (figure 1). In vitro exposure of IU824 to tetracycline resulted in a population of organisms 100% of which were
Antibiotic-Resistant C. trachomatis
1ID 1990;162 (December)
Table 3. Effect of inoculum size on tetracycline MIC for Chlamydia trachomatis. Isolate IU824
IU888
13.0 7.5 5.5 2.5 0.8 0.13 32.5 19.0 8.5 3.0 0.55 0.23 32.5 15.0 6.0 0.41 0.17 0.03
MIC (ug/rnl)
>8 >8 >8 >8 0.25 0.125
>8 >8 8 0.125 0.125 0.125 0.25 0.125 0.125 0.125 0.125 0.125
NOTE. Inoculum size was determined from inclusion-forming unit (ifu) count in antibiotic-free control wells.
lates were as sensitive as the control strains to ciprofloxacin and ofloxacin, with MICs of ~4 ILg/ml and ~l pg/ml, respectively. The inhibitioncurves observed withminocycline, doxycycline, and erythromycin were similar to those seen with tetracycline; that is, the resistant strains showeda 98 %-99 % decrease in the number of inclusion-forming units per milliliter in wells containing drug concentrations abovethe MIC values for the control strains. However, with ceftriaxone, ampicillin, clindamycin, and sulfamethoxazole, little inhibition of inclusion-formingunit formation was seen for the resistant strains at any concentration of antibiotic tested; that is, all members of the population appeared fully resistant. Discussion Our data are the first to our knowledge to demonstrate resistance to tetracycline in C. trachomatis. Moreover, two tetracycline-resistantisolates from two differentpatients were also fully resistant to doxycycline, clindamycin, and erythromycin, in contrast to the relative resistance to erythromycin reported previously [18]. Tetracycline resistance in C. trachomatis is not surprising given the current practice of treating most documented or suspected chlamydial infections with tetracycline or doxycycline. However, we do not yet know whether this represents a recent change in C. trachomatis antibiotic susceptibility. It is possible that the phenomenon is not new but has not been recognized because of the methods usually used for susceptibility testing. In most studies of C. trachomatis antimicrobial susceptibility, inocula have ranged from a few hundred to rv1000 ifu per tissue culture monolayer [18, 19,
27-30], or rv10- to 100-fold less than were used here. However, because resistance is manifestedby only a small proportion of the organism population, a large inoculum is required to detect it (table 3). In addition, the inclusions that occur in the presenceof highconcentrations of tetracycline are small, havealtered morphology, and are difficultto detect, evenwith immunofluorescent staining. With iodine, the stain most frequently used in the past for susceptibility testing [19], their altered morphology makes them even more difficult to identify. Consequently, this heterotypicresistance could havebeen missed previously in assaysusing substantially lower inocula or iodine staining to detect inclusions or both. Also, most previous studies have used laboratory strains that have undergone multiple passages in tissue culture, and it is possible that under these circumstances the ability to express resistance may be lost. This seemed to happen with IU824 after exposure to high concentrations of tetracycline and regrowth in antibiotic-free medium, although it did not happen during the 31 passages to which IU824 was subjected before susceptibility testing. Resistance manifested by a small proportion of organisms in a population could represent a mixed infection with two strains, one sensitiveand numerically predominant, the other resistant and present in small numbers. Alternatively, there may be heterogenous expression of resistance in which all members of a population are capable of expressing resistance but only a small proportion do so at anyone time. This latter phenomenon clearly occurs in methicillin-resistant staphylococci, although the mechanisms regulating the expression of this resistance are not well understood [31-33]. Limitingdilution experiments were done to resolve these two possibilities. If these isolates were mixed populations in which only 1 of 102-103 organisms were resistant, the clonally derived organisms should all be fullysensitive. The fact that the inhibition curves for the progeny were identical to that of the parent indicates heterogenous expression of resistance. It is of particular interest that fully resistant organisms do not grow well and cannot yet be propagated beyonda fewpassages in tissue culture; they die or, as happened twice, completely lose their resistance. This suggests that the mechanism by which they become resistant does not favorlong-term survival of organisms expressing that trait. A similar observation has been made regarding expression of the tetA gene product in Escherichia coli [34]. Although it confers tetracycline resistance, overproduction inhibitsgrowth. However, the molecularmechanismsunderlyingthe resistance we observed in C. trachomatis are not necessarily the same as those previously seen in gram-negative bacteria. Likewise, the clinical significance of antibiotic resistance in C. trachomatis is unknown. So far, few isolates havebeen tested by the methods described here. Besides those described above, 34 initial isolates from adolescents who experienced relapse or reinfection within 15 months have undergone preliminary testing. Of these, 15 (44%) were resistant to tetracy-
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IU14091
Inoculum (ifu/well x 10- 3)
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References 1. Thompson SE, WashingtonAE. Epidemiology of sexually transmitted Chlamydia trachomatis infections. Epidemiol Rev 1983;5:96-123 2. NayyarKC,Cummings M, WeberJ, Benes S, StolzE, FelmanY,McCormack WM. Prevalenceof genital pathogensamongfemaleprostitutes in New YorkCity and in Rotterdam. SexTransmDis 1986;13:105-107 3. Plummer FA, Laga M, Brunham RC, Piot P, Ronald AR, Bhullar V, Mati JY, Ndinya-Achola JO, Chean M, Nsanze H. Postpartum upper genital infectionsin Nairobi, Kenya: epidemiology, etiology, and risk factors. J Infect Dis 1987;156:92-97 4. ToomeyKE, Rafferty MP, Stamm WE. Unrecognized high prevalence of Chlamydia trachomatis cervical infection in an isolated Alaskan Eskimo population. JAMA 1987;258:53-56 5. Chungue E, Cartel JL, Tourneux M, Mahe A, Perolat P, Flye Sainte Marie F, Roux 1. Chlamydia trachomatis genital infections in Tahiti. Eur J Clin Microbiol Infect Dis 1988;7:635-638
6. Handsfield HH, Jasman LL, Roberts PL, Hanson VW, Kothenbeutel RL, StammWE. Criteria for selective screening for Chlamydia trachomatis infection in women attending family planning clinics. JAMA 1986;255:1730-1734 7. PodgoreIK, Holmes KK, Alexander ER. Asymptomatic urethral infections due to Chlamydia trachomatis in male US military personnel. I Infect Dis 1982;146:828 8. Karam GH, Martin DH, Flotte TR, Bonnarens FO, Ioseph IR, MroczkowskiTF, Iohnson WD. Asymptomatic Chlamydia trachomatis infections among sexually active men. J Infect Dis 1986;154:900-903 9. Blythe MI, Katz BP, Orr DP, Caine VA,Iones RB. Historical and clinical factors associated with Chlamydia trachomatis genitourinary infection in female adolescents. I Pediatr 1988;112:1000-1004 10. NettlemanMD, Jones RB, RobertsSO, Katz BP,WashingtonAB, Dittus RS, Quinn TS. Cost-effectiveness of culturing for Chlamydia trachomatis. A study in a clinic for sexuallytransmitted diseases. Ann Intern Med 1986;105:189-195 11. Sweet RL, Schachter J, Robbie MO. Failure of 13-lactam antibiotics to eradicate Chlamydia trachomatisin the endometrium despite apparent clinical cure of acute salpingitis. lAMA 1983;250:2641-2645 12. BergerRE, AlexanderER, Monda GD,AnsellI, McCormickG, Holmes KK. Chlamydia trachomatisas a cause of acute "idiopathic"epididymitis. N Engl I Med 1978;298:301-304 13. Shepard MK, Iones RB. Recovery of Chlamydia trachomatis from endometrialand fallopiantube biopsiesin womenwith infertilityof tubal origin. Fertil SterH 1989;52:322-338 14. Brunham RC, Binns F, McDowellI, Paraskevas M. Chlamydia trachomatis infection in women with ectopic pregnancy. Obstet Gynecol 1986;67:722-726 15. Hartford SL, SilvaPD, diZerega GS, YonekuraML. Serologic evidence of prior chlamydialinfectionin patients with tubal ectopic pregnancy and contralateral tubal disease. Fertil Steril 1987;47:118-121 16. AlexanderER, Harrison HR. Roleof Chlamydia trachomatisin perinatal infection. Rev Infect Dis 1983;5:713-719 17. SchaeferC, Harrison HR, BoyceWT, LewisM. illnesses in infantsborn to womenwith Chlamydia trachomatis infection: a prospectivestudy. Am I Dis Child 1985;139:127-133 18. Mourad A, SweetRL, SuggN, Schachter1. Relativeresistance to erythromycin in Chlamydia trachomatis. Antimicrob Agents Chemother 1980;18:696-698 19. Ehret 1M, Judson FN. Susceptibilitytesting of Chlamydia trachomatis: from eggs to monoclonal antibodies. Antimicrob Agents Chemother 1988;32:1295-1299 20. Iones RB, Ridgway GL, Boulding S, Hunley KL. In vitro activity of rifamycins alone and in combination with other antibiotics against Chlamydia trachomatis. Rev Infect Dis 1983;5(suppl):S556-S561 21. Centers for Disease Control. 1989 sexually transmitted diseases treatment guidelines. MMWR 1989;38(suppl 8):1-43 22. Iones RB, Katz BP, Van Der Pol B, Caine V, Batteiger BE, Newhall WI V. Effect of blind passage and multiple samplings on recovery of Chlamydia trachomatis fromurogenital specimens. J Clio Microbiol 1986;24:1029-1033 23. Iones RB. Antimicrobialsusceptibility testingfor some atypical microorganisms: mycoplasmas, chlamydiae, and rickettsiae. In: Lorian V, ed. Antibiotics in laboratory medicine. 2nd ed. Baltimore: Williams & Wilkins, 1986:346-358 24. KahleP, Wernet P, Rehbein A, KumbierI, PawelecG. Cloning of functionalhuman T lymphocytesby limitingdilution: impact of killer cells and interleukin-2 sources on cloning efficiencies. Scand I Immunol 1981;14:493-502 25. Batteiger BE, Fraiz J, Newhall WI V, Katz BP, Jones RB. Association of recurrent chlamydial infection with gonorrhea. I Infect Dis 1989;159:661-669 26. NewhallWI V, Terho P, Wilde CE m, BatteigerBE, Iones RB. Serovar
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cline at ~8 #Lglml (unpublished data). However, an appropriate control group was not evaluated and, while these data suggest that the resistant phenotype may be fairly common, further studies are indicated. Furthermore, even when present, heterotypic tetracycline resistance may not always or even often result in treatment failure. In the present study, reinfection is a possible explanation for apparent treatment failure in patients 3 and 5. Patient 3 steadfastly denied exposure between cultures but was infected with two different serovars the two times the organism was isolated and. could be expanded, indicating that she either was reinfected or was infected initially with two different serovars. Patient 5 was potentially reexposed. However, tetracycline resistance likely played a major role in the persistent infection in patient 1, and it may do so in other patients whose therapy with tetracycline or doxycycline inexplicably fails. Bacteriologic cures were achieved in one patient retreated with doxycycline and in the two retreated with minocycline, although the isolates were resistant in vitro to these tetracycline analogs. The poor growth of the tetracycline-resistant organisms observed in vitro may also occur in vivo; thus, a repeat or more prolonged course of therapy with tetracycline may be effective even when tetracycline resistance is present. It is also possible that minocycline is more effectivethan other tetracyclines in this setting because of its better tissue penetration [35]. Antimicrobial susceptibility testing of C. trachomatis isolates is time consuming, technically difficult, and expensive. Moreover, it requires the viable organisms, which are not available when direct antigen tests (direct fluorescent antibody or ELISA) are used to identify infected individuals. Consequently, routine susceptibility testing of C. trachomatis is not practical in most clinical settings. However, our data indicate that resistance should be considered when there is solid documentation of treatment failure with standard therapy. Moreover, the role of this form of resistance in the spread and maintenance of C. trachomatis infections in selected populations needs to be fully assessed.
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determination of Chlamydia trachomatis isolates by using type-specific monoclonal antibodies. J Clin Microbiol 1986;23:333-338 Alexander ER, Skahen P, Holmes KK. Antibiotic susceptibility of Chlamydia trachomatis in cell culture. In: Hobson D, Holmes KK, eds. Nongonococcalurethritis and related infections. Washington, DC: American Society for Microbiology, 1977:223-226 Blackman HJ, YonedaC, Dawson CR; Schachter 1. Antibiotic susceptibility of Chlamydia trachomatis. Antimicrob Agents Chemother 1977;12:673-677 BowieWR, Lee CK, Alexander ER. Prediction of efficacyof antimicrobial agents in treatment of infections due to Chlamydia trachomatis. J Infect Dis 1978;138:655-659 Johannisson G, Semryd A, Lycke E. Susceptibility of Chlamydia trachomatis to antibiotics in vitro and in vivo. Sex Transm Dis 1979;6:50-57
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