Detection of tetQ and ermF antibiotic resistance genes in Prevotella and Porphyromonas isolates from clinical specimens and resident microbiota of humans

Alessandra R. Arzese*, Lara Tomasetig and Giuseppe A. Botta

Institute of Microbiology, Faculty of Medicine, University of Udine, 33100 Udine, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gram-negative anaerobes belonging to the genera Fusobacterium, Prevotella and Porphyromonas were investigated for the presence of tetQ and ermF, which have been shown to be spread by conjugal elements. One hundred isolates from either sites of infection or various body sites in healthy subjects were studied. PCR was used to detect tetQ, and DNA–DNA hybridization studies on EcoRI chromosomal digests were undertaken to detect the presence of tetQ and ermF. Antibiotic sensitivity assays were performed on selected isolates to detect tetracycline, erythromycin and penicillin resistance. Twenty Fusobacterium isolates lacked tetQ, and were tetracycline sensitive. Twenty per cent of Prevotella spp. isolates both from clinical specimens and from healthy subjects were found to possess tetQ. Of 20 Porphyromonas isolates tested, one (Porphyromonas levii) from a case of bacterial vaginosis was shown to possess tetQ in the chromosome. The presence of tetQ was always associated with tetracycline resistance. Four isolates of Prevotella melaninogenica and one isolate of Prevotella were ermF-positive, although expression of erythromycin resistance was not consistently associated with detection of this gene. Antibiotic resistance phenotypes of Prevotella isolates were shown to be related to specific chromosomal restriction patterns by hybridization studies: tetracycline resistance and tetracycline/erythromycin resistance are conferred by Bacteroides tetracycline-resistant ERL elements, whereas the tetracycline/penicillin resistance phenotype could be due to spread of elements identified in Prevotella only. Tetracycline/erythromycin-resistant and tetracycline/erythromycin/penicillin-resistant P. melaninogenica isolates were found in this study. It appeared that the presence of tetQ and ermF in Bacteroides and Prevotella contributed to the persistence of antibiotic resistance isolates within the host and to potential spread to other organisms through conjugal elements.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic studies of human isolates belonging to the Bacteroides fragilis group have shown these anaerobic bacteria to possess conjugal elements that mediate gene transfer. Most of these elements have been found to carry antibiotic resistance factors of the tetQ and ermF classes.13 Studies of these conjugative transposons suggest that they play a significant role in the spread of drug resistance.4,5

In the genus Prevotella, which includes several species of extremely oxygen-sensitive Gram-negative anaerobes, similar mechanisms of conjugal gene exchange have been found,6 although they have been less extensively investigated than those detected in Bacteroides. Chromosomal conjugal elements found in Prevotella intermedia and Prevotella denticola were shown to be capable of transferring tetracycline and penicillin resistance to antibiotic-sensitive recipients belonging to the same genus. The transfer of antibiotic resistance was associated with chromosomal elements that were self-transmissible and entirely transferred during mating.7

Evidence that gene transfer between colonic Bacteroides strains and oral anaerobes occurs in nature comes from various sources. In particular, a tetracycline-resistant P. intermedia strain isolated from the periodontal pocket was shown to possess a tetQ fragment whose sequence was identical to that from a colonic Bacteroides clinical isolate.8 The present study therefore set out to investigate the diffusion of resistance factors of the tetQ and ermF classes in Gram-negative anaerobes belonging to the species most frequently encountered in infectious processes. This study was prompted by the growing body of experimental work suggesting the existence of a broad host spectrum genetic flow mediated by conjugal elements, and the evidence of an upward trend in antibiotic resistance among anaerobic human clinical isolates.9,10 The aim of the study was to explore not only clinical isolates but also isolates from the resident microflora of various body sites in healthy subjects. This approach allowed investigation of anaerobes that predominate in various ecological niches (oral cavity, colon and vagina) as reservoirs for antibiotic resistance genes, as suggested in previous studies.4,11


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

Anaerobic isolates were collected over a 2 year period (1995–1997) from cases of bacteraemia and from mixed infections in various body sites. Isolates were also collected from the resident microbiota of the oral cavity, colon and vagina in healthy volunteers. Isolates were cultured on Columbia agar plates enriched with 5% defibrinated sheep blood (Oxoid, Milan, Italy), haemin (10 mg/L) and vitamin K (1 mg/L) (Sigma Chemicals, St Louis, MO, USA). Plates were incubated in anaerobic jars (Oxoid) at 37°C, for 2–10 days, depending on growth rate. Anaerobic isolates were characterized and identified as recommended by reference protocols.12 Species-level identification of isolates was by biochemical test panels provided by commercial systems (rapid ID32A, bioMérieux, Marcy l'Etoile, France; rapid-ANAII, Oxoid). Analysis of metabolic end-products (short-chain fatty acids) by gas–liquid chromatography was also undertaken when required, according to standard procedures.12

A total of 100 isolates belonging to the genera Fusobacterium, Prevotella and Porphyromonas were selected for inclusion in the study. Isolates were from clinical specimens and from various body sites of healthy individuals (one per subject) (Table IGo). The following reference strains were also included in the assays: Prevotella melaninogenica ATCC 25845, Prevotella nigrescens NCTC 9336, Prevotella intermedia ATCC 25611, Porphyromonas gingivalis NCTC 11834, Porphyromonas asaccharolytica NCTC 9337 and B. fragilis ATCC 25285. Control strains, kindly provided by Professor A. A. Salyers and Dr N. B. Shoemaker (University of Illinois at Urbana-Champaign, USA), included: Bacteroides thetaiotaomicron BT DOT (clinical isolate containing conjugal transposon DOT, carrying tetracycline resistance determinant tetQ and erythromycin resistance determinant ermF) and B. thetaiotaomicron BT5482 (type strain lacking both these antibiotic resistance factors).13


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Table I. Anaerobic strains included in the study
 
PCR assay

The presence of tetracycline resistance determinants of the tetQ class was detected by PCR amplification of a 460 bp fragment (nucleotides 1018–1478 of tetQ). This region has been shown to be highly conserved within tetQ carried by different conjugal elements and plasmids, and has been detected in Bacteroides and Prevotella. The primers used were 5' CAT GGA TCA GCA ATG TTC AAT ATC GG 3' and 5' CCT GGA TCC ACA ATG TAT TCA GAG CGG 3'.8 Chromosomal DNA was extracted using the method of Saito & Miura;14 the procedure was performed using an anaerobic cabinet (MDH, Hampshire, UK), to prevent degradation of nucleic acids extracted from Prevotella and Porphyromonas strains. RNA-free chromosomal DNA was used as template in a PCR procedure and mixed with 200 ng of each primer, 2.0 µM deoxynucleoside triphosphate mixture in 100 µL final volume of Taq DNA polymerase buffer (10 mM Tris–HCl, 50 mM KCl, 1.5 mM MgCl2, pH 8.3), and amplified with 2.0 U of Taq DNA polymerase (Boehringer-Mannheim, Mannheim, Germany). Amplification conditions were set at 95°C for 5 min (initial denaturation step), 94°C for 1 min, 55°C for 1 min and 72°C for 1 min (repeated 25 times); reactions were carried out in a thermal cycles apparatus (Perkin-Elmer Cetus, Norwalk, CT, USA). Aliquots of samples were run on 2% agarose gels, stained with ethidium bromide (50 mg/L) and examined under UV light (Transilluminator, Bio-Rad, Hercules, CA, USA).

DNA–DNA hybridization study

Chromosomal DNA from reference or control strains and from anaerobic isolates was digested with EcoRI (Boehringer-Mannheim). Samples were run on 1.5% agarose gels, and transferred to nitrocellulose membrane (Gene Screen Plus, Dupont NEN, Brussels, Belgium) by Southern blot techniques. Probes were applied to detect the presence of tetQ (2.08 kbp EcoRI fragment from plasmid pFND13-2, containing tetQ from Bacteroides DOT transposon);15 the presence of ermF was checked using an 850 bp EcoRI fragment from plasmid pFD214 (containing ermF from Bacteroides Tn4551 conjugative transposon).16 DNA fragments were fluorescein labelled (Renaissance, Dupont NEN) and used to probe EcoRI-digested DNA under high-stringency conditions (42°C, in 50% formamide hybridization buffer), as prescribed by the manufacturer.

Antibiotic sensitivity assay

Antibiotic sensitivity patterns were determined by the agar dilution method.12 Briefly, 5 mL of c. 5 x 105 cfu/mL bacterial suspensions were spotted on Wilkins–Chalgren agar plates (Oxoid), supplemented with 5% sheep blood, 10 mg/L haemin, 1 mg/L vitamin K and containing each of the following antibiotics at various concentrations: tetracycline (2–16 mg/L), erythromycin (1–8 mg/L) and penicillin G (2–16 mg/L). The breakpoints used for interpretation of the sensitive/resistant phenotype of strains to the antibiotics tested were 8 mg/L for tetracycline, 4 mg/L for erythromycin and 8 mg/L for penicillin G, as recommended by the NCCLS.17 Tetracycline and erythromycin resistance were assayed for Fusobacterium, Prevotella and Porphyromonas isolates; Prevotella and Porphyromonas strains were also assayed for penicillin sensitivity. Plates were incubated in an anaerobic jar at 37°C for 48 h, and bacterial growth was checked visually. The assays were performed in duplicate.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Detection of tetQ and ermF in Fusobacterium isolates

All of the Fusobacterium strains, both from clinical specimens and resident microbiota from various body sites, were negative in the tetQ PCR assay and appeared to be tetracycline susceptible. DNA–DNA hybridization studies with ermF were also performed on the strains. None was found to possess the gene in the chromosome, and the antibiotic phenotype was consistently erythromycin sensitive (Ems).

Detection of tetQ and ermF in Prevotella isolates

Prevotella isolates belonging to various species (14/60 strains examined) were found to possess tetQ. Among 22 P. intermedia strains examined, 5/14 from clinical specimens and 2/8 from oral microbiota were found to be tetQ-positive (Figure 1Go) and tetracycline resistant (Tcr) (8 mg/L), whereas tetQ-negative strains were not resistant. PCR findings were confirmed by hybridization. A single EcoRI chromosomal fragment of either 6 or 8 kb was shown to hybridize with tetQ probe (Figure 2Go). In particular, four P. intermedia strains (indicated as PINT 1–4) were characterized by a tetQ-homologous region of 8 kb. A 6 kb EcoRI chromosomal fragment was detected by the tetQ probe in three P. intermedia strains (PINT 5–7) (Table IIGo). None of the P. intermedia strains possessed ermF and all were erythromycin sensitive. Reference strains NCTC 9336 and ATCC 25611 were found to be tetQ-negative and ermF-negative, and sensitive to tetracycline and erythromycin (TcsEms).



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Figure 1. Detection of tetQ from chromosomal DNA of strains under PCR investigation (expected amplicon size 460 bp). Lane 1, BT 12256 positive control strain; lanes 2–7, P. intermedia; lanes 8–10, P. melaninogenica; lane 11, Prevotella sp.; lane 12, P. levii; lane 13, PCR BT 5482 negative control strain; lane 14, PCR blank; lane 15, empty lane; lane 16, DNA size standard (1 kb DNA ladder, Gibco BRL, Gaithersburg, MD, USA). Molecular weight markers (in bp) defining the size of the expected tetQ amplicon are displayed on the right.

 


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Figure 2. Detection of tetQ from EcoRI chromosomal DNA digests of selected anaerobic isolates and control strains by DNA–DNA hybridization study (fluorescein-labelled tetQ probe; 1.5% agarose gel). Lane 1, DNA size standard (l DNA/HindIII fragments, Gibco BRL); lane 2, BT 5482 negative control strain; lane 3, BT 12256 positive control strain; lane 4, empty lane; lane 5, ATCC 25845; lanes 6–8, P. melaninogenica strains PMEL 5, PMEL 1 and PMEL 4; lane 9, NCTC 9336; lane 10, NCTC 11834; lanes 11–13, P. intermedia PINT 5, PINT 6 and PINT 7; lane 14, P. levii PL 1. Molecular weights (in kb) of the DNA size standard are displayed on the left.

 

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Table II. Antibiotic sensitivity profiles of tetQ-positive and/or ermF-positive Prevotella and Porphyromonas isolates
 
For four of the 22 P. melaninogenica strains tested, a 0.46 kb tetQ amplicon was obtained by PCR (Figure 1Go): three strains (PMEL 1–3) were derived from clinical specimens, and one strain (PMEL 4) from vaginal microbiota. The antibiotic sensitivity pattern to tetracycline was consistent with molecular findings for all of the strains (Table IIGo). A single 8 kb EcoRI chromosomal DNA fragment from strains PMEL 1, PMEL 2 and PMEL 3 was found to hybridize with tetQ (Figure 2Go). A 3.5 kb EcoRI fragment from the same isolates was detected by ermF probe (Figure 3Go). These strains were resistant at the breakpoint concentration of erythromycin (4 mg/L). Strain PMEL 4 appeared to harbour a 6 kb EcoRI DNA fragment homologous to tetQ (Figure 2Go): the strain was resistant to tetracycline and erythromycin (TcrEmr), although ermF was not detected (Figure 3Go). In contrast, a 3.8 kb EcoRI chromosomal fragment was detected in P. melaninogenica PMEL 5 by ermF (Figure 3Go), although the strain was Ems (Table IIGo). Reference strain ATCC 25845 was not shown to harbour tetQ or ermF in the chromosome, consistent with its TcsEms phenotype.



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Figure 3. Detection of ermF in EcoRI-digested chromosomal DNA from selected anaerobic isolates and control strains by DNA–DNA hybridization study (fluorescein-labelled ermF probe; 1.5% agarose gel). Lane 1, BT 12256 positive control strain; lane 2, BT 5482 negative control strain; lane 3, DNA size standard (l DNA/HindIII fragments, Gibco BRL); lanes 4–7, P. melaninogenica PMEL 1, PMEL 2, PMEL 4 and PMEL 5; lanes 8 and 9: P. intermedia PINT 1 and PINT 5; lane 10, Prevotella spp. Molecular weights (in kb) of the DNA size standard are displayed on the right.

 
What about Prevotella strains belonging to other species included in the survey? tetQ was detected in an 8 kb EcoRI P. denticola (oral isolate PDEN 1) chromosomal DNA fragment; no homologous regions were found by probing with ermF. Prevotella corporis (strain PCOR 1) from vaginal microbiota was found to be tetQ-positive and ermF-negative. Hybridization assays with the tetQ probe detected a 6 kb fragment in the EcoRI chromosomal digest (data not shown). A Prevotella strain from the oral cavity, not characterized at species level, appeared to be tetQ- negative and TcsEms (Table IIGo). However, a 4 kb EcoRI chromosomal DNA fragment hybridized with the ermF probe (Figure 3Go). The overall incidence of ermF-positive strains at genus level was 8.3%.

Detection of tetQ and ermF in Porphyromonas isolates

The 20 strains belonging to the genus Porphyromonas included one Porphyromonas levii strain (PL 1) isolated from bacterial vaginosis which was shown to possess tetQ in the chromosome and to be Tcr. An 8 kb fragment from the PL 1 EcoRI chromosomal digest was found to hybridize with the probe (Figure 2Go). None of the Porphyromonas isolates was ermF-positive, consistent with their Ems phenotype.

Penicillin sensitivity assay

Penicillin sensitivity testing was performed for Prevotella and Porphyromonas strains found to possess tetQ and/or ermF. At the breakpoint concentration, strains PINT 1, 2, 3 and 4 were penicillin resistant (Penr), whereas PINT 5, 6 and 7 were penicillin sensitive (Pens); PMEL 1, 2 and 3 were Penr, whereas PMEL 4 was Pens. In addition, strains PDEN 1 and PL 1 were found to be Penr. PCOR 1 and Prevotella strains were Pens (Table IIGo).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In Gram-negative anaerobes, studies on genetic exchange by conjugation have focused on the B. fragilis group.13 It has been shown that Bacteroides conjugative transposons can transfer plasmids from Bacteroides to E. coli. Moreover, at least two of these elements (TcrEmr 12256 and TcrEmr 7853) are capable of transfer between Bacteroides and Prevotella under laboratory conditions.1 From studies of natural isolates, it appears that Bacteroides conjugative transposons have spread widely in nature. In fact, most clinical isolates are resistant to tetracycline, and have most commonly proved to carry Tcr ERL-type transposons.4 Therefore transfer of these elements should occur frequently in situ, especially in an environment where Bacteroides are numerically abundant, such as the human colon. In these species the presence of tetQ could be considered a marker of tetracycline resistance, frequently associated with macrolide resistance mediated by ermF.1,2

Conjugal elements have also been found in Prevotella.6,7 Neither the natural host range nor the incidence of such elements in strains of human origin has yet been explored, although molecular studies indicate that these elements are members of the Tcr ERL family.68 An allele of tetQ found in a Tcr P. intermedia oral strain was found to possess a sequence identical to that of a tetQ allele found in a Bacteroides clinical isolate.8 The antibiotic resistance phenotypes conferred by Bacteroides conjugal transposons to recipient strains included Tcr and TcrEmr. However, the gene transfer mediated by conjugal elements found in Prevotella has been associated with the TcrPenr phenotype.6,7

The aim of the present study was to explore the spread of selected antibiotic resistance factors, to be used as markers of the spread of conjugal elements, in the species of Gram-negative anaerobes most frequently encountered in infectious processes. In order to assess the role exerted by resident microbiota as a potential reservoir of resistance genes, isolates from healthy volunteers were also studied.

For Fusobacterium and Porphyromonas strains assayed in this study, horizontal gene transfer by conjugal elements carrying tetQ and/or ermF may occur at a very low incidence. The detection of one tetQ-positive P. levii strain is of significant interest, since the species has not previously been reported to carry this gene. Moreover, the strain was isolated from a case of bacterial vaginosis. Although from the patient history it was not possible to infer whether antibiotic therapy had been applied, it may be hypothesized that the tetracycline resistance was due to selection caused by antibiotic usage.

Prevotella strains both from clinical specimens and from resident microbiota were found to harbour tetQ, in accordance with previous findings.18,19 The overall incidence at genus level was 23%. A single EcoRI chromosomal fragment was shown to hybridize with tetQ, although two distinct patterns could be discerned in the species investigated. Prevotella strains shown to be Tcr Pens were characterized by a 6 kb tetQ EcoRI fragment, whereas TcrPenr strains, including P. levii and P. denticola strains, were characterized by an 8 kb fragment. Strains PMEL 1, 2 and 3 were identical in terms of the size of the EcoRI chromosomal fragment homologous to ermF, an 8 kb fragment homologous to tetQ, as well as their common antibiotic resistance phenotype (TcrEmrPenr). Thus, patterns of antibiotic resistance detected in Prevotella appeared to be related to specific, and distinct, molecular profiles. Tcr appears to be coded by elements of the Bacteroides Tcr ERL class, whereas the TcrPenr phenotype is presumably due to the spread of a different type of element, as described by Guiney & Bouic.6 However, TcrEmr and TcrEmrPenr phenotypes have not been described in Prevotella. Based on available data, it might be hypothesized either that tetQ and ermF are associated with Tcr ERL-type elements, and penicillin resistance is coded by unrelated factors, or that novel types of conjugal elements have been found in these strains. However, strain PMEL 5 and one Prevotella spp. strain were shown to carry ermF in the chromosome, while being Ems at the breakpoint concentration. In contrast, strain PMEL 4 was resistant to erythromycin in the absence of ermF. In the first case, it might be assumed that ermF had been inserted in a locus not suitable for gene expression. In the second case, a resistance factor other than ermF could be involved. It should also be noted that there is a report of a Bacteroides strain carrying in the chromosome a novel macrolide resistance factor, ermG, which was assumed to be associated with a conjugal transposon.20

In conclusion, the presence of tetQ and ermF in Prevotella strains appears to contribute, together with Bacteroides species, to the persistence of antibiotic resistance isolates within the host and to potential spread to other organisms by conjugal elements. In particular, macrolide and ß-lactam resistance factors need to be further explored at the molecular level, since drugs belonging to such classes are frequently used in treating infections involving Gram-negative anaerobes.


    Notes
 
* Corresponding author. Tel: +39-0432-559299; Fax: +39-0432-545526; E-mail: alessandra.arzese{at}kolbe.drmm.uniud.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Salyers, A. A. & Shoemaker, N. B. (1992). Chromosomal gene transfer elements of the Bacteroides group. European Journal of Clinical Microbiology and Infectious Diseases 11, 1032–8.[ISI][Medline]

2 . Salyers, A. A., Shoemaker, N. B., Stevens, A. M. & Li, L. Y. (1995). Conjugative transposons: an unusual and diverse set of integrated gene transfer elements. Microbiology Reviews 59, 579–90.

3 . Hecht, D. W. & Malamy, M. H. (1989). Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis. Journal of Bacteriology 171, 3603–8.[ISI][Medline]

4 . Salyers, A. A. & Shoemaker, N. B. (1995). Conjugative transposons: the force behind the spread of antibiotic resistance genes among Bacteroides clinical isolates. Anaerobe 1, 143–50.[ISI]

5 . Salyers, A. A. & Shoemaker, N. B. (1996). Resistance gene transfer in anaerobes: new insights, new problems. Clinical Infectious Diseases 23, Suppl. 1, 36–43.[ISI]

6 . Guiney, D. G. & Bouic, K. (1990). Detection of conjugal transfer systems in oral, black-pigmented Bacteroides spp. Journal of Bacteriology 172, 495–7.[ISI][Medline]

7 . Guiney, D. G. & Hasegawa, P. (1992). Transfer of conjugal elements in oral black-pigmented Bacteroides (Prevotella) spp. involves DNA rearrangements. Journal of Bacteriology 174, 4853–5.[Abstract]

8 . Nikolich, M. P., Hong, G., Shoemaker, N. B. & Salyers, A. A. (1994). Evidence that conjugal transfer of a tetracycline resistance gene (tetQ) had occurred very recently in nature between the normal microflora of animals and the normal microflora of humans. Applied Environmental Microbiology 60, 3255–60.[Abstract]

9 . Finegold, S. M. & Wexler, H. M. (1996). Present studies of therapy for anaerobic infections. Clinical Infectious Diseases 23, Suppl. 1, 9–14.[ISI]

10 . Snydman, D. R., McDermott, L., Cuchural, G. J., Jr, Hecht, D. W., Iannini, P. B., Harrell, L. J. et al. (1996). Analysis of trends in antimicrobial resistance patterns among clinical isolates of Bacteroides fragilis group species from 1990 to 1994. Clinical Infectious Diseases 23, Suppl. 1, 54–65.[ISI]

11 . Speer, B. S., Shoemaker, N. B. & Salyers, A. A. (1992). Bacterial resistance to tetracycline: mechanisms, transfer, and clinical significance. Clinical Microbiology Reviews 5, 387–99.[Abstract]

12 . Summanen, P., Baron, E. J., Citron, D. M., Strang, C., Wexler, H. M., Finegold, S. M. et al. (1993). Wadsworth Anaerobic Bacteriology Manual, Star Publishing Company, Belmont, California.

13 . Shoemaker, N. B., Barber, R. D. & Salyers, A. A. (1989). Cloning and characterization of a Bacteroides conjugal tetracycline–erythromycin resistance element by using a shuttle cosmid vector. Journal of Bacteriology 171, 1294–302.[ISI][Medline]

14 . Saito, H. & Miura, K. I. (1963). Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochimica et Biophysica Acta 72, 619–29.[ISI]

15 . Nikolich, M. P., Shoemaker, N. B. & Salyers, A. A. (1992). A Bacteroides tetracycline resistance gene represents a new class of ribosome protection tetracycline resistance. Antimicrobial Agents and Chemotherapy 36, 1005–12.[Abstract]

16 . Halula, M. C., Manning, S. & Macrina, F. L. (1991). Nucleotide sequence of ermFU, a macrolide–lincosamide–streptogramin (MLS) resistance gene encoding an RNA methylase from the conjugal element of Bacteroides fragilis V503. Nucleic Acids Research 19, 3453.[ISI][Medline]

17 . National Committee for Clinical Laboratory Standards. (1993). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Third Edition: Approved Standard M11-A3. NCCLS, Villanova, PA.

18 . Leng, Z., Riley, D. E., Berger, R. E., Krieger, J. N. & Roberts, M. C. (1997). Distribution and mobility of the tetracycline resistance determinant tetQ. Journal of Antimicrobial Chemotherapy 40, 551–9.[Abstract]

19 . Lacroix, J. M. & Walker, C. B. (1996). Detection and prevalence of the tetracycline resistance determinant tetQ in the microbiota associated with adult periodontitis. Oral Microbiology and Immunology 11, 282–8.[ISI][Medline]

20 . Cooper, A. J., Shoemaker, N. B. & Salyers, A. A. (1996). The erythromycin resistance gene from the Bacteroides conjugal transposon, TcrEmr 7853, is nearly identical to ermG from Bacillus sphaericus. Antimicrobial Agents and Chemotherapy 40, 506–8.[Abstract]

Received 19 April 1999; returned 26 July 1999; revised 1 September 1999; accepted 29 November 1999