pooling, ferning, and nitrazine tests. (Párrafo borrado). (Párrafo borrado). Preterm labour was defined as the presence of uterine contractions occurring at a ...
Occurrence and antimicrobial susceptibility of Ureaplasma parvum (Ureaplasma urealyticum biovar 1) and Ureaplasma urealyticum (Ureaplasma urealyticum biovar 2) from patients with adverse pregnancy outcomes and normal pregnant women Scand J Infect Dis 2001;33(8):604-10 (ISSN: 0036-5548) Martinez MA; Ovalle A; Santa-Cruz A; Barrera B; Vidal R; Aguirre R Universidad de Chile, Facultad de Medicina, Instituto de Ciencias Biomédicas, Santiago.
A recent phylogenetic analysis of Ureaplasma urealyticum resulted in the proposal to divide their 2 biovars into species. We used PCR to compare the distribution of species and the presence of the tet(M) and int-Tn resistance determinants in 63 strains of Ureaplasma spp. isolated from the amniotic fluid of patients with an adverse pregnancy outcome and in 22 strains obtained from the lower genital tract of healthy pregnant women. We also determined the antimicrobial susceptibility of the organisms to erythromycin and tetracycline. U. parvum was the most frequent Ureaplasma species detected in our study. Thus, 50/63 (79.4%) invasive isolates and 17/22 (77.3%) lower genital tract isolates corresponded to U. parvum, whereas 12/63 (19%) invasive isolates and 4/22 (18.2%) non-invasive strains corresponded to U. urealyticum. A mixture of species was found in 2 women. We found no significant differences in the antimicrobial susceptibility of isolates according to species or origin of isolation. Sixty-two strains of Ureaplasma spp. (74.7%) were susceptible to erythromycin, and 21 strains (25.3%) were intermediately susceptible. Sixty-eight isolates (81.9%) were susceptible to tetracycline, 2 strains (2.4%) were intermediate and 13 strains (15.7%) were resistant. DNA sequences related to the tet(M) determinant and the int-Tn gene were found in all tetracycline-resistant isolates. Scand J Infect Dis. 2001;33(8):604-10.
Introduction Ureaplasma urealyticum is frequently isolated, 60 to 80%, from the lower genital tract of pregnant women (1,2). Whereas ureaplasmal colonization at this location, has not proved to be associated with a poor pregnancy outcome (1,2), several studies have demonstrated a strong association between the recovery of the organism from the chorionamnion and histologic chorioamnionitis (1-4). Placenta infection with U.urealyticum, or chorioamnionitis has been further related to preterm labour and preterm premature rupture of membranes (1-4). U.urealyticum was the most common microbial isolated as a sole agent or in polymicrobial infection in preterm premature rupture of membranes (63.6%) and preterm labour with intact membranes (53.3%)(21,36). Ureaplasmas are transmitted from mothers to new-borns either in utero, or during passage trough the infected birth canal (1,3-4). Respiratory tract colonization of premature infants has been consistently associated with pneumonia, bronchopulmonary dysplasia and chronic lung disease (5,6). Despite the high rate of maternal colonization, only a small proportion of pregnancies results in a poor outcome. The reasons are yet unknown. U.urealyticum includes 14 serotypes, which can be grouped into two biovars (7,8). Some authors have shown that particular serotypes, or biovars are invasive and associated with disease (8-11), while other studies have not demonstrated such association (12,13). U.urealyticum is susceptible to antibiotics that inhibit protein synthesis, such as erythromycin and tetracycline’s (14,15). Erythromycin is used for treatment in pregnant women and in neonatal settings, whereas tetracycline is widely used for the treatment of nongonococcal urethritis. Resistance of U.urealyticum to both antibiotics has been reported in several studies (15-18). The mechanism of tetracycline resistance has been described, and is generally due to the presence of DNA sequences homologous to the Streptococcus agalactiae tetracycline determinant tet(M)(16). The tet(M) gene is frequently borne by conjugative transposable elements, such as Tn916 and Tn1545, which exhibit a high nucleotide homology (19-20). The aims of our study were to compare by PCR, the distribution of biovars, and presence of tet(M) and int-Tn sequences in strains of U.urealyticum isolated from the amniotic fluid of obstetric patients and from the lower genital tract of control women, and to test the susceptibility of isolates to erythromycin and tetracycline. Materials and methods. Organisms. We studied 85 strains of U.urealyticum, isolated from obstetric patients and control women, at the San Borja Arriarán Hospital, from January 1992 through January 2000. Sixty three isolates corresponded to invasive strains, randomly chosen from a collection of strains obtained from three separate study protocols (21,22). Group I (n=37) were isolated from the amniotic fluid (AF) of women with preterm premature rupture of membranes (PROM) and a gestational age ranging between 24 and 34 weeks, group II (n=20) were recovered from the AF of women with term PROM (37-42 weeks), and group III (n=6) were obtained from the AF of women with preterm labour and intact membranes and a gestational age ranging between 24 and 34 weeks. PROM was diagnosed with sterile speculum examination and a combination of pooling, ferning, and nitrazine tests. (Párrafo borrado). (Párrafo borrado). Preterm labour was defined as the presence of uterine contractions occurring at a frecuency of 4 in 30 minutes for a period of 1 hour and cervical dilatation equal or greater than 1cm and effacement equal or greater than 80%. Gestational age was determined by the date of last menstrual period, early pelvic examination, or ultrasonographic fetal biometry before 24 weeks. Amniocentesis was offered at admission to all patients and performed under ultrasound guidance. No patients had received antibiotics prior to admission. (Párrafo borrado). The control group (n=22) corresponded to cervico-vaginal isolates
obtained from 31 women, during a routine prenatal visit. Women were eligible for study if they did not had signs of genital infection, had not received antibiotics the past 30 days, and had not poor obstetric history. Ureaplasmas were grown in urea broth and A7 agar, identified by their typical appearance of colonies on A7agar, and by their metabolic activity in urea broth, and stored at –70ºC in broth (23,24). Before testing, stock cultures of each isolate were thawed, and serial 10-fold dilutions were made in urea broth to determine the number of colour changing units (CCU) present. Reference strains. Dr. W.Bredt (University of Freiburg, Germany) kindly provided serotypes 1 to 8 of U.urealyticum. A strain of S. agalactiae tetracycline resistant, and harbouring the tet(M), and int-Tn sequences, was obtained in a previous study (25). PCR. The polymerase chain reaction (PCR) for amplification of the 16S rDNA of U.urealyticum was performed as described by Robertson et al (8), using two sets of primers: U3:P6, which amplifies the parvo biovar, and U8:P6, which amplifies the biovar T960. The size of both amplification products corresponded to 1.300 bp. Primers for the amplification of DNA sequences of the tet(M) and int-Tn genes corresponded to the published sequences by Blanchard et al and Barbeyrac et al respectively (26,27). PCRs were performed as described by the authors and yielded the expected size products, i.e. 397-bp DNA fragment of the tet(M) determinant and 579- bp DNA fragment of the int-Tn gene. For the PCR assays, strains were incubated in urea broth, until an alkaline colour shift occurred, usually 18-36 hours. Subsequently, 200 µl of the culture was centrifuged at 13.000 rpm for 30 min, and the pellet was suspended in 100 µl of lysis buffer (10mM Tris hydrochloride (pH8.5), 50mM KCl, 2mM MgCl2, 1% Triton X-100 (Sigma, St.Louis, USA), and 200 µg per ml of proteinase K (Gibco, BRL). Lysates were incubated at – 60ºC for 1 h, followed by 94ºC for 15 min and quickly cooled on ice. DNA was extracted with 0.5-ml phenol-chloroform-isoamyl alcohol (25:24:1), and chloroform:isoamyl alcohol (24:1), and precipitated with ethanol. The pelleted DNA was suspended in double distilled water and used for PCRs. Reactions were done in a total volume of 50 µl containing 1X PCR buffer, 200 µM of each dNTP (Gibco, BRL), 2µM of each oligonucleotide primer, 1.7 - 4mM MgCl2 (depending on the pair of primers used), 2.5 U of Taq DNA polymerase (Gibco, BRL), and 10 µl of the sample DNA. Amplifications were done in a MJ Research thermal cycler. PCR products were visualised under UV light by ethidium bromide staining after agarose gel electrophoresis. The size of DNA bands was estimated using standard molecular weight markers (Gibco BRL). MICs of antibiotics. The minimal inhibitory concentrations (MIC) of erythromycin and tetracycline were determined in duplicate by the broth microdilution method as described by Waytes et al (15). Stock solutions of erythromycin (Sigma) and tetracycline (Sigma) were prepared the day of assay in sterile deionized water. Erythromycin powder was first dissolved in methanol and then diluted in water. Frozen cultures were diluted in urea broth to approximately 103 to 104 CCU/ml and incubated at 35ºC for 2-h prior use. When erythromycin was tested, broth was adjusted to pH 6.6 (28), otherwise, the final pH was 6.0.Serial twofold dilutions of antibiotics were prepared (final volume 25 µl) in the microtiter plate. A total of 175 µl of the adjusted inoculum was then added to each well. The ureaplasmal dilution used as inoculum for the MIC assay, and uninoculated urea broth, was included respectively as positive and negative controls, in each determination. Staphylococcus aureus ATCC 29213 was used as drug control , and tested in Mueller-Hinton broth. Microplates were sealed, incubated at 35ºC in a moist chamber, and examined for colour changes twice daily for 4 days. The MIC was defined as the lowest antibiotic concentration inhibiting growth for 48 h. The susceptibility breakpoint of antibiotics was defined according to the NCCLS guideline (29).
Results. Table 1 shows the distribution of biovars among the 85 isolates of U.urealyticum tested. Parvo was the most frequent biovar detected in our study, both from obstetric patients as from control women. There were no differences observed in the distribution of biovars according to their clinical status, among isolates obtained from obstetric patients. Thus, 29(78.4%) of isolates recovered from preterm PROM cases, 16(80%) of isolates obtained from PROM at term, and 6(100%) of isolates obtained from patients with preterm labour and intact membranes belonged to the parvo biovar. Figure 1 shows the results of PCR amplification of parvo biovar in some patients. The MICs and ranges of erythromycin and tetracycline against 85 strains of U.urealyticum are summarised in Table 2. Both antibiotics showed good activity against ureaplasmas. The drug concentrations required to inhibit 90% of the strains were erythromycin 1 µg/ml and tetracycline, 2 µg/ml. Sixty three isolates (74.1%) were susceptible to erythromycin, and 22 strains (25.9%) were intermediately susceptible. Sixty-eight strains (80%) were susceptible to tetracycline, 2 (2.4%) showed intermediate susceptibility, and 15 strains (17.6%) were resistant. Using primers for the tet(M) determinant, we identified the tet(M) in 13 out of 15 tetracycline resistant strains (86.7%). The same 13 isolates harboured the int-Tn gene. The tet(M) and the intTn genes were not detected in intermediately susceptible or susceptible isolates. Fig 2 and Fig 3 show some of the results of PCR amplification of the tet(M) and int-Tn determinants. Discussion One of the aims of this study was to compare the distribution of U.urealyticum biovars, among obstetric patients and controls. As described by others, most strains of U.urealyticum isolated from patients with obstetric complications and from normal pregnant women in this study, belonged to the biovar parvo ( 9,11,30). Other authors have also demonstrated a predominance of this biovar, in lower respiratory tract and cerebrospinal infections from neonates (13,31). Despite, the higher prevalence of the parvo strains of U.urealyticum, the invasive properties of this organism are not limited to this biovar. Studies using more exact discrimination procedures, such as serotyping and subtyping, have also not conclusively found particular serotypes or subtypes in association with invasive infections (13,30). It will be important to learn about the host characteristics or host conditions that play a role as a contributing factor affecting the risk of invasiveness of this organism. It is difficult to compare the results of susceptibility to erythromycin reported in the literature, due it great variability. All isolates of U.urealyticum included in our study were inhibited by ≤ 4µg/ml, and no strains were found to be resistant to this drug. Our results are in agreement with those reported by Rylander et al (32), who tested genital isolates, and with those of Matlow et al (31), and Waites et al (15 ), who studied neonatal respiratory isolates. In contrast, several studies have reported higher MICs and erythromycin resistance in this organism (17,18,33). Despite of the lack of a standard procedure for testing the antimicrobial susceptibility of Mycoplasmas, the variables that affect the results, e.g., inoculum size, incubation time and pH of the media are largely known (28). Geographical differences, and different sources of the isolates studied, may explain the discrepancies in the susceptibility of this organism to this antibiotic. The first strain of U.urealyticum with a high level of tetracycline resistance, was isolated in 1974 by Ford et al (34). In women, resistant strains were first reported in 1975, by Spaepen et al, from patients with reproductive failure (35). Roberts and Kenny documented in 1986, the presence and spread of the tet(M) resistance determinant from S.agalactiae to clinical isolates of
U.urealyticum (16). Currently, tetracycline resistant ureaplasmas have been reported in different geographical areas (17). In Chile, high level tetracycline resistant isolates were described in 1985 (36). For 3(6%) of 50 genital isolates, MICs of tetracycline were 64 to ≥ 128 µg/ml. Since then, resistance to tetracycline has increased paulatinely, arising 17.6%. Fortunately, isolates have remained susceptible to erythromycin. The tet(M) determinant is frequently encoded in conjugative transposons that account for its broad distribution. In urogenital strains, e.g. S.agalactiae, Enterococcus faecalis, Gardnerella vaginalis, Mycoplasma hominis, and U.urealyticum, tet(M) is harboured in the transposon Tn916, or Tn1545, prototypes of broad range transposons, which exhibit a high nucleotide similarity (19,20).The left terminus of both transposable elements contains the int-Tn determinant, that mediates the specific DNA cleavage required for it excision and integration (20). We demonstrated int-Tn sequences by PCR, in the thirteen ureaplasmas containing the tetracycline determinant tet(M).We failed to demonstrate tet(M) or int-Tn sequences in two phenotipically resistant isolates. Blanchard et al have demonstrated that clinical isolates consist of mixed populations with only some of the cells carrying the tet(M) determinant (26).Our isolates were stored by a variable time, and subcultured in the absence of tetracycline for determining viability and for the CCU determination. This fact could explain a selection for cells not carrying the resistance determinants.
References 1. Cassell GH, Waites KB, Watson HL, Crouse DT, Harasawa R. Ureaplasma urealyticum intrauterine infection: role in prematurity and disease in newborns. Clin Microbiol Rev 6: 69 – 87, 1993 2. Eschenbach DA. Ureaplasma urealyticum and premature birth. Clin Infect Dis 17 (Suppl 1): S100 – 6, 1993 3. Hillier SL, Martius J, Krohn M, Kiviat V, Holmes KK, Eschenbach DA. A case control study of chorioamnionic infection and histologic chorioamnionitis in prematurity. N Engl J Med 319: 972 – 78, 1989 4. Kundsin RB, Leviton A, Allred EN, Poulin SA. Ureaplasma urealyticum infection of the placenta in pregnancies that ended prematurely. Obstet Gynecol 87: 122 – 27, 1996 5. Crouse DT, Odrezin GT, Cutter GR, Reese JM, Hamrick WB, Waites KB, Cassell GH.Radiographic changes associated with tracheal isolation of Ureaplasma urealyticum from neonates. Clin Infect Dis 17 (Suppl 1):S122-30, 1993 6. Cassell GH, Waites KB, Rudd PT , Crouse DT, Canupp KC, Stagno S, Cutter GR. Association of Ureaplasma urealyticum infection of the lower respiratory tract with chronic lung disease and death in very low birth weight infants. Lancet: 240 – 44, 1988 7. Robertson JA, Stemke WG. Expanded serotyping scheme for Ureaplasma urealyticum strains isolated from humans. J Clin Microbiol 15: 873 – 78, 1982 8. Robertson JA, Vekris A, Bebear C, Stemke GW. Polymerase chain reaction using 16S rRNA gene sequences distinguishes the two biovars of Ureaplasma urealyticum. J Clin Microbiol 31: 824 – 30, 1993 9. Naessens A, Foulon W, Breynaert J, Lauwers S. Serotypes of Ureaplasma urealyticum isolated from normal pregnant women and patients with pregnancy complications. J Clin Microbiol 26: 319 – 22, 1988 10. Quinn PA, Li HCS, Th’ng C, Dunn M, Butany J. Serological response to Ureaplasma urealyticum in the neonate. Clin Infect Dis 17 (Suppl 1):S136 – 43, 1993 11. Abele-Horn M, Wolff C, Dressel P, Pfaff F, Zimmermann A. Association of Ureaplasma urealyticum biovars with clinical outcome for neonates, obstetric patients, and gynaecological patients with pelvic inflammatory disease. J Clin Microbiol 35: 1199 – 1202, 1997 12. Robertson JA, Honore LA, Stemke GW. Serotypes of Ureaplasma urealyticum in spontaneous abortion. Pediatr Infect Dis 5: S270 – 72, 1986 13. Zheng X, Watson HL, Waites KB, Cassell GH. Serotype diversity and antigen variation among invasive isolates of Ureaplasma urealyticum from neonates. Infect Immun 60: 3472 – 74, 1992 14. Mc Cormack WM. Susceptibility of Mycoplasmas to antimicrobial agents: clinical implications. Clin Infect Dis 17 (Suppl 1): S200 – 1, 1993 15. Waites KB, Crouse DT, Cassell GH. Antibiotic susceptibilities and therapeutic options for Ureaplasma urealyticum infections in neonates. Pediatr Infect Dis J 11: 23 – 9, 1992 16. Roberts MC, Kenny GE. Dissemination of the tetM tetracycline resistance determinant to Ureaplasma urealyticum. Antimicrob Agents Chemother 29: 350-52, 1986 17. Taylor-Robinson D, Furr PM. Clinical antibiotic resistance of Ureaplasma urealyticum.. Pediatr Infect Dis 5: S335 – 37, 1986 18. Busolo F, Conventi L. In vitro activity of antibiotics against Ureaplasma urealyticum and Chlamydia trachomatis strains from patients with nongonococcal urethritis. Eur J Clin Microbiol Infect Dis 7: 407- 410, 1988 19. Martin P, Trie-Cuot P, Courvalin P. Nucleotide sequence of the tetM tetracycline resistance determinant of the streptococcal conjugative shuttle transposon Tn1545. Nucleic Acid Research 14: 7047 – 59, 1986
20. Rice LB. Tn916 family conjugative transposons and dissemination of antimicrobial resistance determinants. Antimicrob Agents Chemother 42:1871 – 77, 1998 21. Ovalle A, Gómez R, Martínez MA, Rubio R, Fuentes A, Valderrama O, Lira P, Romero R. Antibiotic therapy in patients with preterm premature rupture of membranes: a prospective, randomized, placebo-controlled study with microbiological assessment of the amniotic cavity and lower genital tract. Prenat Neonat Med 2: 213 – 222, 1997 22. Ovalle A, Gómez R, Martínez MA, Giglio MS, Bianchi R, Díaz L, Sáez J, Poblete JP, Romero R. Antibiotic treatment of patients with term premature rupture of membranes: a randomized clinical trial. Prenat Neonat Med 3: 399 – 406, 1998 23. Shepard MC, Lunceford CL. Differential agar medium (A7) for identification of Ureaplasma urealyticum (human T mycoplasmas) in primary cultures of clinical material. J Clin Microbiol 3: 613 – 25, 1976 24. Taylor-Robinson D, Furr PM. Recovery and identification of human genital tract mycoplasmas. Isr J Med Sci 17: 648 – 53, 1981 25. Lâmmler C, Schwarz S, Wibawan IW, Ott E, Bopp V, Martínez-Tagle MA. Comparison of streptococci of serological group B isolated from healthy carriers and active disease in Chile. J Med Microbiol 42: 161 – 64, 1995 26. Blanchard A, Crabb DM, Dybvig K, Diffy LB, Cassell GH.Rapid detection of tetM in Mycoplasma hominis and Ureaplasma urealyticum by PCR: tetM confers resistance to tetracycline but not necessarily to doxycycline.Fems Microbiology Letters 95: 277 – 82, 1992 27. De Barbeyrac B, Dupon M, Rodriguez P, Renaudin H, Bebear C. A Tn1545-like transposon carries the tet(M) gene in tetracycline resistant strains of Bacteroides ureolyticus as well as Ureaplasma urealyticum but not Neisseria gonorrhoeae. J Antimicrob Chemother 37: 223 – 232, 1996 28. Kenny GE, Cartwright FD. Effect of pH, inoculum size, and incubation time in the susceptibility of Ureaplasma urealyticum to erythromycin. Clin Infect Dis 17(Suppl 1): S215-8, 1993 29. National Committee for Clinical Laboratory Standards.1998. Performance standards for antimicrobial susceptibility testing M7-A4. National Committee for Clinical Laboratory Standards, Villanova, Pa. 30. Knox CL, Timms P. Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women. J Clin Microbiol 1998; 36: 3032 – 39 31. Matlow A, Th’ng C, Kovach D, Quinn P, Dunn M, Wang E. Susceptibilities of neonatal respiratory isolates of Ureaplasma urealyticum to antimicrobial agents. Antimicrob Agents Chemother 1998; 42: 1290 - 92 32. Rylander M, Hallander HO. In vitro comparison of the activity of doxycycline, tetracycline, erythromycin and a new macrolide, CP 62993, against Mycoplasma pneumoniae, Mycoplasma hominis and Ureaplasma urealyticum Scand J Infect Dis, Suppl 1988; 53: 12 – 17 33. Renaudin H, Bebear C. Comparative in vitro activity of azithromycin, clarithromycin, erythromycin and lomefloxacin against Mycoplasma pneumoniae, Mycoplasma hominis and Ureaplasma urealyticum. Eur J Clin Microbiol Infect Dis 1990; 9:838 – 41 34. Ford DK, Smith JR. Nonspecific urethritis associated with a tetracycline resistant Tmycoplasma. Br J Vener Dis 1974;50:373 – 74 35. Spaepen MS, Kundsin RB, Horne HW. Tetracycline resistant T-mycoplasmas (Ureaplasma urealyticum) from patients with a history of reproductive failure. Antimicrob Agents Chemother 1975; 9: 1012 – 18 36. Ovalle A, Martínez MA, Gómez R, Sáez J, Menares I, Aspillaga C y Schwarze JE. Parto prematuro con membranas intactas. Microbiología del líquido amniótico y tracto genital
inferior y su relación con los resultados maternoneonatales. Rev Med Chile 2000 Aceptada publicación
Table 1. Distribution of U. urealyticum biovars among obstetric patients and control women.
Obstetric patients Biovar(s)
Control women
(n=63)
p
(n=22)
Parvo
50(79.4%)
17(77.3%)
NS
T960
12(19.0%)
4(18.2%)
NS
1(1.6%)
1(4.5%)
NS
Parvo + T960
Table 2. Susceptibility of U. urealyticum strains to erythromycin and tetracycline.
MIC (µg/ml)a
Origin of strains Compound (Nº of strains)
Invasive (63)
Control (22)
a
Range
Erythromycin
0.06 - 4
Tetracycline
0.06 - >32
Erythromycin
0.06 - 4
Tetracycline
0.06 - >32
50%
90%
0.5
1
1
2
0.5
1
1
2
50% and 90%, MIC for 50 and 90% of isolates, respectively.
