Genetic Basis of Tetracycline Resistance in Urogenital Bacteria

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, 0066-4804/90/020261-04$02.00/0 Copyright C 1990, American Society for Microbiology

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Genetic Basis of Tetracycline Resistance in Urogenital Bacteria MARILYN C. ROBERTS'* AND SHARON L. HILLIER2 Departments of Pathobiology' and Obstetrics and Gynecology,2 University of Washington, Seattle, Washington 98195 Received 12 June 1989/Accepted 13 November 1989

The distributions of the nucleotide sequences related to the tetracycline resistance determinants Tet K, Tet L, Tet M, and Tet 0 were studied by dot blot hybridization with randomly chosen clinical urogenital tract isolates of viridans group streptococci, Streptococcus agalactiae, Enterococcus faecalis, Gardnerella vaginalis, Lactobacillus spp., Fusobacterium nuckatum, Peptostreptococcus spp., and Veilonella parvula. Among the Peptostreptococcus spp., 79% of the isolates hybridized with one (64%) or more (36%) of the probes for Tet K (27%), Tet L (30%), Tet M (75%) and Tet 0 (13%). Of the viridans group streptococci, 82% of the strains hybridized with one (34%) or more (66%) of the four probes. The distribution of the four determinants in this group was as follows: Tet K, 36%; Tet L, 31%; Tet M, 43%; Tet 0, 61%. Twenty-nine percent of the enterococci and forty-six percent of the group B streptococci hybridized with the probes; however, the Tet K, Tet L, and Tet 0 determinants were found in only a few strains, while the Tet M determinant predominated. A total of 29% of the F. nucleatum isolates, 55% of the G. vaginalis isolates, and 26% of the V. parvula isolates hybridized with the Tet M determinant. In contrast, 43% of the Lactobacillus spp. hybridized with the Tet 0 determinant. The data indicate that tetracycline resistance determinants are common to many of the microorganisms isolated from the urogenital tract.

and Tet 0 determinants. A few strains were also tested for the Tet P determinant (1).

Tetracycline is an antibiotic that has a broad spectrum of activity and low toxicity. As a result, it has been used extensively for treatment of a variety of diseases. Tetracyclines are the second most commonly used antibiotic type worldwide; however, their efficacy has been reduced by the increase in microbial resistance to the drug (5, 10). Currently, there are 13 well-characterized tetracycline resistance determinants representing three different mechanisms of resistance. Five of these determinants, Tet A to Tet F, are found only in gram-negative bacteria (11, 12, 14), while Tet K, Tet L, Tet N, and Tet P have previously been described only for gram-positive species (1, 4, 7, 23). Tet M has been found in gram-positive (3-6, 20), gram-negative (8, 13, 20, 23), and cell-wall-free species (18, 19), whereas Tet 0 has been associated with Streptococcus spp., Enterococcus spp. (23), and Campylobacter spp. (22). A number of species of urogenital bacteria, including anaerobic, aerobic, gram-positive, and gram-negative species, are tetracycline resistant, and many carry the Tet M determinant (8, 9, 13, 18, 19). The aim of the present study was to investigate whether other genera found in the urogenital tract, such as Lactobacillus spp., were tetracycline resistant and whether they carried any of the previously described tetracycline determinants. In addition, we examined other urogenital genera known to carry the Tet M determinant to assess whether they also carried some of the other characterized gram-positive tetracycline determinants. Peptostreptococcus spp. and Fusobacterium nucleatum were included in the study because strains from the oral cavity have been shown to be tetracycline resistant (20) and to carry the Tet M determinant. Thus, it was of interest to determine whether strains isolated from the urogenital tract would also be resistant and carry the Tet M determinant. A total of 371 isolates were tested by using DNA-DNA hybridization and radiolabeled probes for the Tet K, Tet L, Tet M,

*

MATERIALS AND METHODS Bacterial strains. Stock clinical isolates from the urogenital tracts of patients attending the Sexually Transrnitted Disease Clinic and University of Washington Medical Center were chosen without knowledge of the tetracycline susceptibilities of the isolates. However, these isolates came from female patients who had not taken any antibiotics in the 2 weeks before the specimens were collected. The isolates included viridans group Streptococcus spp. (100 isolates), Streptococcus agalactiae (group B) (28 isolates), Enterococcusfaecalis (14 isolates), Gardnerella vaginalis (94 isolates), Lactobacillus spp. (21 isolates), F. nucleatum (24 isolates), Peptostreptococcus spp. (71 isolates), and Veillonella parvula (19 isolates). Media. The anaerobic isolates were grown on prereduced brucella agar (Difco Laboratories, Detroit, Mich.) supplemented with 5% sheep blood under anaerobic conditions at 37°C. G. vaginalis and Lactobacillus spp. were grown on either Columbia bilayer agar plates or GC-medium-base (Difco)-supplemented chocolate plates under 5% CO2 at 37°C. The remaining isolates were grown on 5% sheep blood agar plates or supplemented GC medium base. In some cases, the media were supplemented with 10 ,ug of tetracycline per ml. Plasmid preparation. The purification of plasmid DNA by ultracentrifugation in cesium chloride-ethidium bromide was performed as previously described (17). DNA probes. The plasmid pT181 (7) was used for the Tet K probe to screen the strains. Those that were positive were retested by using an 870-base-pair HincIl-fragment probe isolated directly from the plasmid (23) or taken from the chimeric plasmid pAT102 formed by cloning the 870-basepair fragment into pUC8 (23). Plasmid pVB11.15 was used as the Tet L probe (3, 4). This consisted of a 5-kilobase (kb) vector and a 3.7-kb insert. The purified 860- and 1,000base-pair Hincll fragments from the insert were used to

Corresponding author. 261

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confirm the strains which hybridized with the total pVB11.15 plasmid (3, 4). The 1.8-kb HincII fragment from pUW-JKB1 was used as the Tet M probe (2), and the 1.8-kb HincII fragment from pUOA4 was used as the Tet 0 probe (22). The 1.9- and 2.1-kb EcoRI inserts from the plasmid pJIR39 were used as the Tet P probe (1). Preparation of purified DNA. Individual strains were grown on plates, and purified DNA was prepared. For the streptococci, previously described procedures were used, and the same protocol was used for the Lactobacillus spp. and Peptostreptococcus spp. (4). For the anaerobic organisms and G. vaginalis, the Mobiluncus procedure was used (21). DNA-DNA hybridization. The purified restriction fragments or whole plasmids were labeled with two 32P-labeled

nucleotide triphosphates and two unlabeled nucleotide triphosphates by nick translation as previously described (21). Dot blots or Southern blots were prepared on nitrocellulose and hybridized overnight at 42°C under stringent conditions of 50% (vol/vol) formamide, 0.1% (wt/vol) polyvinylpyrrolidone, 0.1% (wt/vol) albumin, 0.1% (wt/vol) Ficoll, 0.1% (wt/vol) sodium dodecyl sulfate, 0.05 M monobasic sodium phosphate (pH 7.4), 0.005 M EDTA, 0.76 M NaCl, and 100 ,ug of boiled calf thymus DNA per ml. The filters were then washed three times for 10 min each at 520C in 0.1% sodium dodecyl sulfate-0.015 M NaCl-0.0015 M sodium citrate and then three more times for 10 min each at 520C in 0.015 M NaCl-0.0015 M sodium citrate (20). Positive and negative controls were included in each set. With this procedure, none of the probes cross-hybridized, even though the Tet K and the Tet L determinants share 69%o homology (23) and the Tet M and Tet 0 determinants share 76% homology (23). Restriction enzymes. All restriction enzymes were purchased from Pharmacia-PL Biochemicals and used as directed by the instructions of the manufacturer. RISULTS Distribution of tbe tour tetracydine resistance determints in Peptostreptococca* spp. and viridans group streptococci. Previously, we have isolated from a periodontal patient an oral Pept6strept&6occeMs anaerobius strain that was tetracycline resistant and, carried the Tet M determinant in its chromosothe (20). Therefore, we were interested in determining whether peptostreptococci isolated from the urogenital tract also carried the Tet M determinant. Seventy-one stock genital isolates of peptostreptococci, selected without kn"owledge of their susceptibilities to tetracycline, were examiined for the presence of Tet K-, Tet L-, Tet M-, and Tet 0-related nucledtide sequences. The set included P. anaerobius, P. asaccharolyticus, P. magnus, P. micros, P. prevotii, and P. tetrddius. From this group of isolates, 15 strains did not hybridize with any of the four probes, while the remaining strains hybridized to one or more of the probes (Table 1). The distributions of the four probes were 27, 30, 75, and 13% for Tet K, Tet L, Tet M, and Tet 0, respectively. Of the 56 hybridizing strains, 36 (64%) hybridized with a single probe, 16 (29%) hybridized with two probes, 3 (5%) hybridized with three probes, and 1 (2%) hybrndized with all four probes. All six peptostreptococcal species carried the various Tet determinants, indicating that the determinants were not limited to P. anaerobius. To verify the dot blot results, individual isolates were selected for firther study. All were tested for the ability to grow on media supplemented with 10 p,g of tetracycline per

TABLE 1. Distribution of the Tet determinants in viridans group

streptococci and Peptostreptococcus spp. Tet determinant(s)"

K, L, M, 0 K,L,M K, L, 0 K, M, 0 L, M, 0 K, M

K,O L,M L, 0

M,O K L M 0 None

No. of strains hybridizing with probeb

Streptococcus

Peptostreptococcus spp. (n = 71)

7 5 4 5 7 3 8 1 5 9 4 2 6 16 18

1 1

spp. (n = 100)

1 0

1 10 0 5 1

0 2 7 24 3 15

a No strains were found which carried only the Tet K and Tet L determinants, while every other combination of the four probes was found in the streptococcal population. The K-M-0, K-O, K-L, and M-O combinations were not found in the Peptostreptococcus spp. bThe strains were assayed by dot blots. Some of the Peptostreptococcus spp. were reconfirmed by using purified DNA with Southern blot analyses.

tnl. We found that all strains which hybridized with one or more of the Tet probes could grow on the supplemented media. DNA was prepared from the strains. In many cases, small (.3-megadalton) plasmids and a few larger (.20megadalton) plasmids were seen. Unfortunately, most of the plasmids were quite fragile and disappeared with storage.

Southern blots were prepared, and the various radiolabeled probes were used. We found that the strains hybridized with the various Tet probes, confirming the dot blot assay. However, it was very' difficult to determine whether any of the determinants were associated with the plasmids observed in the agarose gels, and further work is in progress to determine their exact locations in the various strains. We also examined four strains which did not hybridize with any of the four Tet determinants. We found that three of the strains could grow on media supplemented with 10 pCg of tetracycline per ml, but on retesting with purified DNA they did not hybridize with any of the four probes. We then checked the remaining 11 strains which did hybridize and found that 9 could grow on media supplemented with 10 pXg of tetracycline per ml. Four of the tetracycline-resistant (Tcr) strains, along with 10 other strains which hybridized to one or more of the Tet probes, were tested with the Tet P determinant, originally described for Clostridium perfringens (1). None of the strains hybridized with this probe. For comparison, we examined 100 randomly isolated urogenital viridans group streptococci by dot blot assays for the presence of Tet K-, Tet L-, Tet M-, and Tet 0-related nucleotide sequences (Table 1). The species included in the set were S. acidominimus, S. constellatus, S. intermedius, S. mitis, S. morbillorium, S. sanguis I and IIj and S. salivarius (15). Only 18 strains did not react with one or more of the probes. Fifteen of these were tested, and eleven could grow. on media supplemented with -10 ,ug of tetracycline per ml. The distributions of the determinants were 36, 31, 43, and 61% for Tet K, Tet L, Tet M, and Tet 0, respectively. Of the hybridizing strains, 7% hybridized with all four probes, while 26, 31, and 34% hybnidized with three, two, or one of the probes, respectively (Table 1).

Tcr IN UROGENITAL BACTERIA

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TABLE 2. Distribution of Tet determinants for the other urogenital bacteria Organism

E. faecalis F. nucleatumb Lactobacillus spp.C G. vaginalis S. agalactiae V. parvula

No. of strains hybridizing with probe(s) for Tet determinant(s): K L Ma 0 K, O M, O

1

1

0

3 7

0

52 8 5

No. (%) of that strains did not hybridize

10 (71) 17 (71) 9

11 (55)d

0

42 (45) 15 (54) 14 (74)

3

1

a The Tet M determinant was the most common Tet deterniinant found in this group of bacteria. b Only seven of the F. nucleatum strains were tested with Tet K, Tet L, or Tet 0, while all were tested with Tet M. c Strains were also tested with the Tet P determinant, and none hybridized. d Six of these strains grew on tetracycline-supplemented media.

Both the viridans group streptococci and the peptostreptococci had all four tetracycline determinants present in the population. However, the Tet M determinant predominated (75%) in the peptostreptococci, while in the viridians group streptococci the Tet 0 determinant was the most common (61%), followed by Tet M (43%). Multiple Tet determinants were also more prevalent among the streptococci than among the peptostreptococci. Distribution of the Tet determinants in other urogenital species. Previously we have shown that oral tetracyclineresistant isolates of F. nucleatum and V. parvula carried the Tet M determinant (20). Therefore, we were interested in determining whether urogenital isolates of these two species also carried the Tet M determinant. We examined 24 F. nucleatum isolates and 19 V. parvula isolates for the Tet M determinant. Both were obtained from the same population as the streptococci and peptostreptococci. The Tet M determinant was found in 29o of the F. nucleatum isolates and 26% of the V. parvula isolates (Table 2). Unfortunately, many of the F. nucleatum isolates died, and we were able to examine only seven of the F. nucleatum isolates for the other three Tet determinants, while all 19 V. parvula isolates were examined with the other three Tet probes. None of the strains hybridized with the other three probes. We have previously shown that G. vaginalis isolated from the urogenital tracts of patients in Seattle and Belgium was tetracycline resistant and carried the Tet M determinant (16). As a result of the work on peptostreptococci, the question of whether G. vaginalis could carry tetracycline determinants other than Tet M arose. To answer this question, we examined 94 randomly isolated strains with the four probes and found that 55% of strains hybridized with the Tet M probe but not with the other three probes. Lactobacillus spp. are part of the normal urogenital flora and are thought to play a role in maintaining a healthy microbial environtnent. Very little is known about the susceptibility of this group of bacteria to tetracycline or other antibiotics. We examined 21 Lactobacillus spp. and found that 15 (71%) were able to grow on media supplemented with 10 pg of tetracycline per ml. Of the Tcr strains, 9 of the 15 (60%) hybridized with the Tet 0 probe. None of the strains hybridized with the Tet K, Tet L, Tet M, or Tet P probe (Table 2). We prepared DNA from nine Tcr strains and one tetracycline-susceptible (Tcs) strain. Southern blots were prepared, and hybridization with the Tet 0 probe was performed. Hybridization was seen with the DNA and was

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consistent with the chromosomal location of the determinant. Both E. faecalis and S. agalactiae isolates have been shown to carry the Tet M determinant as well as the Tet K, Tet L, and Tet 0 determinants (4, 5, 23). We examined 14 E. faecalis isolates and 28 S. agalactiae isolates from the same urogenital population (Table 2). With these strains, 29% of the E. faecalis isolates and 46%o of the S. agalactiae isolates hybridized with one or two of the probes tested, with the Tet M determinant predominating in both species (Table 2). Unlike the viridans group streptococci, the E. faecalis and S. agalactiae isolates were less likely to carry multiple tetracycline resistance genes, with only four of the S. agalactiae isolates carrying two determinants and none of the E. faecalis group doing so. DISCUSSION We examined a large number of randomly isolated strains representing a number of different species commonly associated with the urogenital tract. We found a large number of the strains within the various species which hybridized with one or more of the four tetracycline resistance probes (Tables 1 and 2). The highest level of carriage was found in the viridans group streptococci. In this group, 82% of the strains hybridized with one or more of the probes, and an additional 11 of the 15 nonreactive strains were Tcr. All four tetracycline resistance determinants were found in this group of bacteria. We also found that 54 (66%) of the 82 strains examined hybridized with two or more of the probes, while only 28 (34%) of the strains appeared to carry a single determinant. This is significantly different from the recent report by Zilhao et al. (23), in which of a group of 51 tetracycline-resistant nongroupable streptococci, 43 hybridized with either Tet M or Tet 0, 2 hybridized with Tet M and Tet L, and 6 did not hybridize to any of the four probes. However, the source of these strains is unclear, and therefore it is difficult to determine whether the differences between the two studies are due to the difference between strains isolated in France and Seattle or to the anatomical origins of the strains. This study indicated that the three anaerobic Tet Mcontaining species, F. nucleatum, P. anaerobius, and V. parvula, first isolated in the oral flora of patients with periodontal disease (20), can also be found in the urogenital tracts of patients in Seattle. The percentage of tetracyclineresistant F. nucleatum strains was 29% in the urogenital isolates, compared with 4% in the oral isolates (20). Similarly, hybridizing Peptostreptococcus strains occurred in 79% of the urogenital isolates and 0% (0 of 47) of the oral isolates (20). Whether the anatomical locations of these strains influence the carriage of tetracycline resistance determinants is unclear. We have shown that the Tet M determinant is present not only in P. anaerobius but also in the other five Peptostreptococcus spp. we examined in the study and that only 4% of the strains examined were Tcs. We found that 32 of the Peptostreptococcus spp. carried one or more of the other Tet determinants. The level of carriage of Tet K, Tet L, and Tet 0 in the Peptostreptococcus spp. was not as high as that found in the viridans group streptococci. This is the first time that other Tet determinants have been shown to be associated with the Peptostreptococcus spp., and it suggests that gene exchange between Peptostreptococcus spp. and members of the streptococci may be a frequent event. We demonstrated that 62% of the Lactobacillus spp. could

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grow on tetracycline-supplemented media and that 9 of those strains hybridized with the Tet 0 probe but not with the other probes, including the Tet P probe. We found that Tcr Lactobacillus spp., viridans group streptococci, and Peptostreptococcus spp. did not hybridize with any of the four probes tested, suggesting that other undescribed Tet determinants exist in these genera. This is similar to the finding of Zilhao et al. (23). In that study, 18 tetracycline-resistant enterococci and streptococci did not hybridize with any of the four Tet probes. This study provides the first evidence indicating that the host range of all four Tet determinants is larger than first determined. As a result, we predict that as new urogenital organisms are examined, the four Tet determinants will be found in other species and genera of bacteria.

W. E. DeWitt, M. L. Thomas, S. R. Johnson, and S. A. Morse. 1987. Frequency and distribution in the United States of strains

10. 11. 12. 13.

14.

ACKNOWLEDGMENTS We thank V. Burdett, P. Courvalin, S. Khan, J. I. Rood, and D. E. Taylor for strains carrying the cloned tetracycline determinants and J. Lansciardi for technical assistance. M.R. was supported by Public Health Service grant AI24136 from the National Institutes of Health.

15.

LITERATURE CITED 1. Abraham, J., D. I. Berryman, and J. I. Rood. 1988. Hybridization analysis of the class P tetracycline resistance determinant from the Clostridium perfringens R-plasmid, pCW3. Plasmid 19:113-120. 2. Brown, J. T., and M. C. Roberts. 1987. Cloning and characterization of tetM from a Ureaplasma urealyticum strain. Antimicrob. Agents Chemother. 31:1852-1854. 3. Burdett, V. 1986. Streptococcal tetracycline resistance mediated at the level of protein synthesis. J. Bacteriol. 165:564-569. 4. Burdett, V., J. Inamine, and S. Rajagopalan. 1982. Heterogeneity of tetracycline resistance determinants in Streptococcus. J. Bacteriol. 149:995-1004. 5. Clewell, D. B., and C. Gawron-Burke. 1986. Conjugative transposons and the dissemination of antibiotic resistance in Strep-

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Staphylococcus aureus. Plasmid 10:753-765. 8. Knapp, J. S., S. R. Johnson, J. M. Zenilman, M. C. Roberts, and

S. A. Morse. 1988. High-level tetracycline resistance due to Tet M in strains of Neisseria species, Kingella denitrificans, and Eikenella corrodens. Antimicrob. Agents Chemother. 32:765767. 9. Knapp, J. S., J. M. Zenilman, J. W. Biddle, G. H. Perkins,

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of Neisseria gonorrhoeae with plasmid-mediated, high-level resistance to tetracycline. J. Infect. Dis. 155:819-822. Levy, S. B. 1984. Resistance to tetracyclines, p. 191-204. In L. T. Bryan (ed.), Antimicrobial drug resistance. Academic Press, Inc., New York. Marshall, B., S. Morrissey, P. Flynn, and S. B. Levy. 1987. A new tetracycline-resistance determinant, class E, isolated from Enterobacteriaceae. Gene 50:111-117. Marshall, B., C. Tachibana, and S. B. Levy. 1983. Frequency of tetracycline resistance determinant classes among lactose-fermenting coliforms. Antimicrob. Agents Chemother. 24:835-840. Morse, S. A., S. R. Johnson, J. W. Biddle, and M. C. Roberts. 1986. High-level tetracycline resistance in Neisseria gonorrhoeae is result of acquisition of streptococcal tetM determinant. Antimicrob. Agents Chemother. 30:664-670. Park, B. H., M. Hendricks, M. H. Malamy, F. P. Tally, and S. B. Levy. 1987. Cryptic tetracycline resistance determinant (Class F) from Bacteroides fragilis mediates resistance in Escherichia coli by actively reducing tetracycline accumulation. Antimicrob. Agents Chemother. 31:1739-1743. Rabe, L. K., K. K. Winterscheid, and S. L. Hillier. 1988. Association of viridans group streptococci from pregnant women with bacterial vaginosis and upper genital tract infection. J. Clin. Microbiol. 26:1156-1160. Roberts, M. C., S. L. Hillier, J. Hale, K. K. Holmes, and G. E. Kenny. 1986. Tetracycline resistance and tetM in pathogenic urogenital bacteria. Antimicrob. Agents Chemother. 30.810812. Roberts, M. C., and G. E. Kenniy. 1987. Conjugal transfer of transposon Tn916 from Streptococcus faecalis to Mycoplasma hominis. J. Bacteriol. 169:3836-3839. Roberts, M. C., and G. E. Kenny. 1986. Dissemination of the tetM tetracycline resistance determinant to Ureaplasma urealyticum. Antimicrob. Agents Chemother. 29:350-352. Roberts, M. C., L. A. Koutsky, D. LeBlanc, K. K. Holmes, and G. E. Kenny. 1985. Tetracycline resistant Mycoplasma hominis strains containing streptococcal tetM sequences. Antimicrob. Agents Chemother. 28:141-143. Roberts, M. C., and B. J. Moncla. 1988. Tetracycline resistance and TetM in oral anaerobic bacteria and Neisseria perflava-N. sicca. Antimicrob. Agents Chemother. 32:1271-1273. Spiegel, C. A., and M. Roberts. 1984. Mobiluncus gen. nov., Mobiluncus curtisii subsp. curtisii sp. nov., Mobiluncus curtisli subsp. holmesii subsp. nov., and Mobiluncus mulieris sp. nov., curved rods from the human vagina. Int. J. Syst. Bacteriol. 34:177-184. Taylor, D. E., K. Hiratsuka, H. Ray, and E. K. Manavathu. 1987. Characterization and expression of a cloned tetracycline resistance determinant from Campylobacterjejuni. J. Bacteriol. 169:2984-2989. Zilhao, R., B. Papadopoulou, and P. Courvalin. 1988. Occurrence of the Campylobacter resistance gene tetO in Enterococcus and Streptococcus spp. Antimicrob. Agents Chemother. 32:1793-17%.