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Characterisation of viridans group streptococci with different levels of Tet(M)-mediated tetracycline resistance
International Journal of Antimicrobial Agents 24 (2004) 439–443
Characterisation of viridans group streptococci with different levels of Tet(M)-mediated tetracycline resistance Paul Stapletona,∗ , Victoria Adamsa , Rachel Pikeb , Victoria Lucasb , Graham Robertsb , Peter Mullanyb , Robin Rowburya , Michael Wilsonb , Hilary Richardsa a
b
Department of Biology, University College London, Gower Street, London, UK Department of Microbiology, Eastman Dental Institute, University College London, Grays Inn Road, London, UK Received 8 May 2004; accepted 8 June 2004
Abstract Streptococcus oralis 264-3, Streptococcus mitis 254-1 and S. mitis 264-1, isolated from the oral cavities of two children were each found to carry the tet(M) gene but exhibited different degrees of reduced susceptibility to tetracycline (tetracycline MICs of 2, 8 and 64 mg/L, respectively). The aim of this study was to determine the molecular basis for the different levels of tetracycline resistance (TcR ) observed. Escherichia coli HB101 carrying the cloned tet(M) genes exhibited similar levels of tetracycline susceptibility to those observed in the parental streptococcal strains (MICs of 1, 16, and 64 mg/L for tet(M) genes from S. oralis 264-3, S. mitis 254-1 and S. mitis 264-1, respectively). DNA sequencing revealed that S. oralis 264-3 had a tet(M) gene highly homologous to tet(M) carried by Tn916 from Enterococcus faecalis (99.6% identity), while the intermediate- and high-level TcR strains had tet(M) sequences that resembled the tet(M) gene of Tn5251 from Streptococcus pneumoniae (99.3% and 99.4% identity, respectively). No differences were observed in the upstream attenuator structure for each of the strains and differences in reduced tetracycline susceptibilities could be attributed to changes in the deduced amino acid sequences of the Tet(M) proteins. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Viridans group streptococci; Tetracycline resistance; Heterogeneous resistance; tet(M)
1. Introduction Tetracycline resistance in oral bacteria is commonly conferred by the tet(M) gene [1,2] which encodes a ribosomal protection protein (RPP) that confers tetracycline resistance through promoting the release of tetracycline from the 30S ribosomal subunit in a GTP-dependent manner [3–5]. Expression of tet(M) is regulated by transcriptional attenuation [6]. The majority of tet(M) genes are located on conjugative transposons, such as Tn916 and Tn5253, that are responsible for their widespread distribution, not only amongst different bacterial species of the oral cavity, but the larger bacterial com∗ Corresponding author. Present address: Department of Pharmaceutics (Microbiology), School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK. Tel.: +44 20 7753 5848; fax: +44 20 7753 5942. E-mail address: [email protected] (P. Stapleton).
munity as a whole [7]. Recombination between evolutionary divergent tet(M) genes has led to the formation of tet(M) genes with mosaic structures [8]. Consequently tet(M) genes exhibit nucleotide variations at defined segments within these genes [9]. The effect of these mosaic structures on tetracycline resistance has not been formally addressed although it is known that streptococci carrying different tet(M) gene subtypes can confer different levels of tetracycline resistance [10]. Since differences in tetracycline resistance could be attributed to isolates carrying more than one copy of the tet(M) gene, or due to changes in tet(M) promoter sequence, attenuator structure, or due to changes in the structure itself, ideally the influence of different tet(M) subtypes should be examined in the same genetic background. In this study we examined three streptococcal isolates that exhibited; (a) reduced tetracycline susceptibility; (b) intermediate-; (c) and high-level tetracycline resistance and show that the different
0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2004.06.003
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P. Stapleton et al. / International Journal of Antimicrobial Agents 24 (2004) 439–443
levels of reduced tetracycline susceptibilities were due to changes in the deduced amino acid sequences of the Tet(M) proteins.
2. Materials and methods 2.1. Bacterial strains Viridans group streptococci from the saliva and plaque samples of children (age range: 5 to 15 years), were isolated on Mueller–Hinton agar containing HgCl2 (40 M) [11]. Isolates were identified to species level with the API identification system (bioM`erieux UK Limited, Basingstoke, United Kingdom) and by carbohydrate substrate analysis [12]. 2.2. Susceptibility testing MICs of tetracycline, ampicillin and erythromycin (all purchased from Sigma–Aldrich, Poole, United Kingdom) were determined by agar dilution technique on isosensitest agar (Oxoid Ltd., Basingstoke, UK) with 5% horse blood for streptococcal isolates or without blood for Escherichia coli HB101 carrying the tet(M) gene. An inoculum of about 104 organisms per spot was used and the MICs were read after 24 h incubation at 37 ◦ C. 2.3. DNA techniques DNA extraction was performed with a Gram-positive DNA isolation kit (Flowgen, Ashby de la Zouch, United Kingdom). Southern blotting to Nylon N+ membranes (Amersham Pharmacia Biotech) and DNA–DNA hybridisation with digoxygenin-labelled probes was carried out according to the manufacturer’s instructions (Roche Molecular Biochemicals, Lewes, United Kingdom). Digoxygeninlabelled tet(M) probe was prepared from Tn916 DNA by PCR by the incorporation of alkali-stable digoxygenin-labelled dUTP (Roche Molecular Biochemicals) in the amplification mixture. PCR amplification was performed as described by Ausubel et al. [13] with Dynazyme EXT thermostable polymerase (Flowgen) and an annealing temperature of 50 ◦ C. Primers used for amplification of the tet(M) gene and promoter regions (2841 bp) were based on GenBank sequence U09422 for Tn916 from Enterococcus faecalis DS16: tetM-F1 (5 -ACGACGTTCTTCAAGCTCTATCCT-3 ) [positions 11492-11515] and tetM-R1 (5 -CATTTTATCGGCTCTGCGTCTTT-3 ) [positions 14310–14332]. Restriction endonuclease digestion with HindIII and DraI was used to distinguish between tet(M) and the closely related tet(O) and tet(S) genes. The amplified tet(M) genes were sub-cloned into the EcoRV site of pMOSBlue (Amersham Pharmacia Biotech) and introduced into competent E. coli HB101 [F− (gtp-proA)62 recA13 leuB6 supE44 ara-14 galK2 lacY1 (mcrC-mrr) mtl-1 proA2 xyl-5
rpsL20] by a heat-shock procedure [14]. The orientation of the insert in pMOSBlue was confirmed by restriction endonuclease digestion with HindIII and only clones with the same insert orientation were used. Primers used to sequence the tet(M) gene sub-cloned into pMOSBlue were: Univm13 (5 -GTTTTCCCAGTCACGACGTTGTA3 ), and T7P (5 -TAATACGACTCACTATAGGG-3 ) binding to sequences within the vector and; tetM-F3 [5 -TCTGTATGCTTTGTATGCCTATGG-3 ], tetM-F4 [5 -TGTGACGAACTTTACCGAATCTGA-3 ], tetM-F5 [5 -TCCCTCTGCTGCAAACGACTG-3 ], tetM-F6 [5 GTATGGTTGGAATGTGACGGACTG-3 ], and tetM-R3 [5 -GTTGTACCTTTGTCCACGCTTCCT-3 ] binding to sequences within the sub-cloned gene. Independently amplified PCR products were sequenced directly to confirm the nucleotide changes found in the cloned genes. DNA sequencing was performed with the aid of an Applied Biosystems automated fluorescent sequencer (model 373A). The nucleotide sequences of the tet(M) genes for S. oralis 264-3, S. mitis 254-1, and S. mitis 264-1 have the accession numbers, AJ580976, AJ580977, and AJ580978, respectively.
3. Results During a study examining mercury and antibiotic resistance in oral bacteria [11], 22 viridans group streptococci were isolated that exhibited varying degrees of reduced susceptibilities to tetracycline (Table 1). Three isolates exhibited Table 1 Antimicrobial resistance profiles and the presence of the tet(M) gene for oral streptococcal isolates with reduced susceptibility to tetracycline Isolate
tet(M)a
MIC (mg/L) Tetracycline Ampicillin Erythromycin
S. mitis 152-2 S. parasanguis 181-2 S. parasanguis 179-1 S. oralis 264-3 S. oralis 283-4 S. oralis 204-4 S. mitis 254-1 S. mitis 160-1 S. oralis 277-2 S. mitis 166-4 S. mitis 173-1 S. parasanguis 181-1 S. oralis 279-1 S. oralis 262-3 S. oralis 271-2 S. oralis 260-1 S. mitis 264-1 S. oralis 271-1 S. oralis 318-1 S. oralis 254-2 S. oralis 262-1 S. oralis 283-4 a
2 2 2 2 2 4 8 16 16 32 32 32 32 32 32 64 64 64 64 64 64 64
0.12 1 0.5 0.03 0.06 0.12 0.016 0.12 0.12 0.016 0.016 1 <0.008 0.06 0.016 <0.008 1 0.25 0.016 0.12 0.016 0.06
(+) tet(M) present, (−) tet(M) absent.
0.03 0.016 1 <0.008 2 0.016 0.06 0.03 1 0.03 0.016 0.03 <0.008 0.5 0.016 0.03 <0.008 0.03 0.016 1 8 2
− − − + + + + − − + + − − + + + + + + − + +
c
d
a
b
Streptococcal isolate. E. coli HB101. Nucleotide positions are based on the adenosine of the initiation codon (ATG) being assigned position 1. The amino acid and position for the corresponding nucleotide are given in brackets. N/d, not determined.
T (Val631 ) T (Val631 ) A (Met458 ) A (Met458 ) C (Pro451 ) C (Pro451 ) T (Ser450 ) T (Ser450 ) T (Leu327 ) T (Leu327 ) A (Gln317 ) G (Gln317 ) A (Lys286 ) A (Lys286 ) T (Asn252 ) A (Lys252 ) G (Gln209 ) T (His209 ) N/dd N/dd N/dd N/dd tet(M) on Tn916 tet(M) on Tn5251
G (Val164 ) A (Met164 )
T (Val631 ) T (Val631 ) C (Ala631 ) A (Met458 ) G (Val458 ) G (Val458 ) C (Pro451 ) A (Gln451 ) A (Gln451 ) T (Ser450 ) G (Ala450 ) G (Ala450 ) G (Arg327 ) T (Leu327 ) T (Leu327 ) T (His317 ) G (Gln317 ) G (Gln317 ) A (Lys286 ) A (Lys286 ) G (Arg286 ) T (Asn252 ) T (Asn252 ) A (Lys252 ) G (Gln209 ) T (His209 ) G (Gln209 ) A (Met164 ) A (Met164 ) G (Val164 ) 1 16 64 2 8 64
951 857 756 627 Recipientb
441
S. oralis 254-3 S. mitis 254-1 S. mitis 264-1
1372 1352 1348 490 Hosta
980
Nucleotide positions for non-conservative sequence differences amongst tet(M) genesc Tetracycline MIC (mg/L) for:
In common with other reports [2,15] the tet(M) resistance determinant was frequently encountered amongst the viridans group streptococci isolated in this study. The isolates were not uniformly resistant to tetracycline despite detection of the same resistance determinant amongst the majority of the isolates. Such differences could have been attributed to the presence of more than one resistance determinant, or to changes affecting gene expression, or the presence of different Tet(M) variants with different abilities to confer tetracycline resistance. Different tet(M) variants have previously been identified in bacteria isolated from periodontal pockets [9] but whether these variants had different tetracycline susceptibilities was not reported. The tet(M) variants identified in this study, resemble the tet(M) subtypes, M1 (S. oralis 264-3) and M2 (S. mitis 254-1 and S. mitis 264-1), reported by Olsik et al. [9]. However, unlike the M1 subtype, the tet(M) gene from S. oralis 264-3 did not confer tetracycline resistance.
Host strain or genetic element
4. Discussion
Table 2 Tetracycline MICs for streptococcal isolates and E. coli HB101 carrying tet(M) genes coding for Tet(M) proteins with different deduced amino acid sequences
reduced susceptibility to ampicillin (MIC of 1 mg/L), and six isolates were resistant to erythromycin (MIC ≥ 1 mg/L). Fourteen of the twenty-two isolates were found to carry the tet(M) gene by PCR and by DNA–DNA hybridisation with a tet(M) probe (Table 1). Three tet(M)-positive isolates, S. oralis 264-3, S. mitis 254-1 and S. mitis 264-1 with varying degrees of reduced tetracycline susceptibilities (tetracycline MICs of 2, 8 and 64 mg/L, respectively) were chosen to be investigated further. Two of the isolates, S. mitis 2641 and S. oralis 264-3 were isolated from the oral cavity of the same child. The tet(M) gene was amplified from the isolates by PCR with a proof-reading DNA polymerase and the resulting products were sub-cloned into pMOSBlue. E. coli HB101 carrying the sub-cloned tet(M) genes exhibited similar levels of reduced tetracycline susceptibility observed in the parental streptococcal strains (tetracycline MICs of 1, 1, 16, and 64 mg/L for HB101, 264-3, 254-1 and 264-1, respectively) (Table 2). DNA sequencing of the tet(M) genes revealed that S. oralis 264-3 had a tet(M) gene highly homologous to that carried by Tn916 from E. faecalis DS16 (99.6% identity) (Table 2), while isolates, S. mitis 264-1 and S. mitis 254-1 had tet(M) sequences that resembled the tet(M) gene of Tn5251 from Streptococcus pneumoniae (99.3% and 99.4% identity, respectively) (Table 2). No differences were observed in the upstream attenuator structure of the tet(M) gene for each of the isolates, but the intermediate- and high-level tetracyclineresistant strains each had a single nucleotide change in the −35 region of the tet(M) promoter (TTTACA to TTGACA). Tet(M) from isolate S. oralis 264-3 had three deduced amino acid differences compared with Tet(M) from Tn916: V164M, Q307H and L327R. Comparison of the deduced amino acid sequences of the intermediate- and high-level tetracyclineresistant isolates revealed five differences: M164V, H209Q, K252N, K286R and V631A.
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Comparison of the nucleotide sequences revealed that the tet(M) gene from S. oralis 264-3 differed from M1 at two nucleotide positions (951, A to T and 980, T to G) that result in changes to the deduced amino acid sequence of the Tet(M) protein (Q317H and L327R). Consequently, either one of these amino acid substitutions or both of them are likely to be responsible for the reduced ability of the Tet(M) protein to confer tetracycline resistance. The existence of tet(M)-positive isolates which remain susceptible to tetracycline has been previously noted amongst Streptococcus pneumoniae clones [10,16]. MIC testing without prior induction with tetracycline has been proposed as an explanation for the existence of tet(M)-positive tetracyclinesensitive clones [10]. In this study, we have sequenced the region upstream from the tet(M) gene, including the promoter and the attenuator structure which is responsible for inducible tetracycline resistance, and found no nucleotide differences when compared with the upstream tet(M) gene sequence from Tn916. Although the ability to induce tetracycline resistance was not investigated in this study, indirect evidence from the sequence of the upstream region of the tet(M) gene, combined with the lack of ability of the tet(M) gene to confer tetracycline resistance when introduced into E. coli, suggests changes in the deduced amino acid sequence of the Tet(M) is the primary factor responsible for tetracycline susceptibility in this case. The region upstream from the tet(M) gene has previously been reported to be highly conserved even amongst tet(M) genes from diverse hosts [17]. In this study, the regions upstream from the tet(M) gene were also highly conserved, although a single nucleotide change in the −35 sequence of the tet(M) promoter was found in the sequences from S. mitis 254-1 and S. mitis 264-1. Since both isolates had the same nucleotide change, alteration to the −35 sequence of the tet(M) gene was not responsible for the differences in the levels of tetracycline resistance observed. Furthermore, the different levels of tetracycline resistance were reproduced when the corresponding tet(M) genes were introduced into E. coli suggesting that changes in the deduced amino acid sequences of Tet(M) were responsible for the phenotypes observed. Tet(M) belongs to a wider family of ribosomal protection proteins (RPP) that includes the closely related Tet(O) and Tet(S) determinants and more divergent determinants such as Otr(A) from Streptomyces [7]. Ribosomal protection proteins share homology with elongation factors, EF-Tu and EF-G involved in protein synthesis and alignments of amino acid sequences of RPPs and elongation factors have identified putative functional regions in Tet(M) and Tet(O) [18,19]. Alterations to certain conserved amino acids are known to affect the function of the RRP and elongation factors [18]: changes to Asn-128 located in the putative GTP-binding domain of Tet(O) results in a decrease in tetracycline resistance [19]. In this study, S. oralis 264-3 had three deduced alterations in the amino acid sequence of Tet(M) (V164M, Q317H, L327R). Of these positions in the amino acid sequence, only the Leu
residue at position 327 shows some conservation: the Leu327 residue is found in Tet(M), Tet(O), Tet(S), Tet(Q) and EF-G. Since this amino acid residue is located within the putative ribosomal-binding domain of Tet(M), the substitution of Arg for Leu-327 may affect binding of Tet(M) to the ribosome and consequently explain the significantly reduced tetracycline resistance observed in S. oralis 264-3. S. mitis 254-1 and S. mitis 264-1 had five deduced amino acid differences between them. All the amino acid substitutions, with the exception of V631A, are found in other RPPs and it is not possible to deduce which amino acid substitutions confer the level of tetracycline resistance observed. However, it appears that different mosaic gene structures can give rise to moderate (found in S. mitis 254-1) and high-level (found in S. mitis 264-1) tetracycline resistance. The significance of these different tet(M) gene variants remains to be determined. Tetracycline MICs for viridans group streptococci have been reported to exhibit a broad range with tetracycline MICs varying in increments throughout the range [20-22]. From this study we have shown that for isolates carrying the tet(M) gene, changes in the deduced amino acid sequence of Tet(M) is the main reason for this variation. However, further work involving site-directed mutagenesis combined with detailed substrate profiling is required to confirm the contributions made by each of the deduced amino acid changes.
Acknowledgements This work was funded by the Medical Research Council (grants G9810729 and G9810341).
References [1] Olsvik B, Olsen I, Tenover FC. Detection of tet(M) and tet(O) using the polymerase chain reaction in bacteria isolated from patients with periodontal disease. Oral Microbiol Immunol 1995;5:299–303. [2] Villedieu A, Diaz-Torres ML, Hunt N, et al. Prevalence of tetracycline resistance genes in oral bacteria. Antimicrob Agents Chemother 2003;47:878–82. [3] Burdett V. Tet(M)-promoted release of tetracycline from ribosomes is GTP dependent. J Bacteriol 1996;178:3246–51. [4] Connell SR, Trieber CA, Stelz U, Einfeldt E, Taylor DE, Nierhaus KH. The tetracycline resistance protein Tet(O) perturbs the conformation of the ribosomal decoding center. Mol Microbiol 2002;45:1463–72. [5] Connell SR, Trieber CA, Dinos GP, Einfeldt E, Taylor DE, Nierhaus KH. Mechanism of Tet(O)-mediated tetracycline resistance. EMBO J 2003;22:945–53. [6] Su YA, He P, Clewell DB. Characterization of the tet(M) determinant of Tn916: Evidence for regulation by transcription attenuation. Antimicrob Agents Chemother 1992;36:769–78. [7] Chopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 2001;65:232–60. [8] Oggioni MR, Dowson CG, Smith JM, Provvedi R, Pozzi G. The tetracycline resistance gene tet(M) exhibits mosaic structure. Plasmid 1996;35:156–63.
P. Stapleton et al. / International Journal of Antimicrobial Agents 24 (2004) 439–443 [9] Olsvik B, Tenover FC, Olsen I, Rasheed JK. Three subtypes of the tet(M) gene identified in bacterial isolates from periodontal pockets. Oral Microbiol Immunol 1996;5:299–303. [10] Doherty N, Trzcinski K, Pickerill P, Zawadzki P, Dowson CG. Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae. Antimicrob Agents Chemother 2000;44:2979–84. [11] Pike R, Lucas V, Stapleton PM, et al. Prevalence and antibiotic resistance profile of mercury-resistant oral bacteria from children with and without mercury amalgam fillings. J Antimicrob Chemother 2002;5:777–83. [12] Beighton D, Hardie JM, Whiley RA. A scheme for the identification of viridans streptococci. J Med Microbiol 1991;35:367–72. [13] Ausubel FM, Brent R, Kingston RE, et al. Current protocols in molecular biology. New York: John Wiley and Sons; 1989. [14] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989. [15] Lacroix JM, Walker CB. Detection and incidence of the tetracycline resistance determinant tet(M) in the microflora associated with adult periodontitis. J Periodontol 1995;66:102–8. [16] Corso A, Severina EP, Petruk VF, Mauriz YR, Tomas A. Molecular characterisation of penicillin-resistant Streptococcus pneumoniae
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isolates causing respiratory disease in the United States. Microbial Drug Resist 1998;4:325–37. Roberts M. Tetracycline resistance determinants: mechanism of action, regulation of expression, genetic mobility, and distribution. FEMS Microbiol Rev 1996;19:1–24. Taylor DE, Chau A. Tetracycline resistance mediated by ribosomal protection. Antimicrob Agents Chemother 1996;40:1–5. Grewal J, Manavathu EK, Taylor DE. Effect of mutational alteration of Asn-128 in the putative GTP-binding domain of tetracycline resistance determinant Tet(O) from Campylobacter jejuni. Antimicrob Agents Chemother 1993;37:2645–9. Doern GV, Ferraro MJ, Brueggemann AB, Ruoff KL. Emergence of high rates of antimicrobial resistance among viridans group streptococci in the United States. Antimicrob Agents Chemother 1996;40:891–4. Teng L-J, Hsueh P-R, Chen Y-C, Ho S-W, Luh K-T. Antimicrobial susceptibility of viridans group streptococci in Taiwan with an emphasis on the high rates of resistance to penicillin and macrolides in Streptococcus oralis. J Antimicrob Chemother 1998;41:621–7. Wisplinghoff H, Reinert RR, Cornely O, Shifert H. Molecular relationships and antimicrobial susceptibilities of viridans group streptococci isolated from blood of neutropenic cancer patients. J Clin Microbiol 1999;37:1876–80.
Characterisation of viridans group streptococci with different levels of Tet(M)-mediated tetracycline resistance Paul Stapletona,∗ , Victoria Adamsa , Rachel Pikeb , Victoria Lucasb , Graham Robertsb , Peter Mullanyb , Robin Rowburya , Michael Wilsonb , Hilary Richardsa a
b
Department of Biology, University College London, Gower Street, London, UK Department of Microbiology, Eastman Dental Institute, University College London, Grays Inn Road, London, UK Received 8 May 2004; accepted 8 June 2004
Abstract Streptococcus oralis 264-3, Streptococcus mitis 254-1 and S. mitis 264-1, isolated from the oral cavities of two children were each found to carry the tet(M) gene but exhibited different degrees of reduced susceptibility to tetracycline (tetracycline MICs of 2, 8 and 64 mg/L, respectively). The aim of this study was to determine the molecular basis for the different levels of tetracycline resistance (TcR ) observed. Escherichia coli HB101 carrying the cloned tet(M) genes exhibited similar levels of tetracycline susceptibility to those observed in the parental streptococcal strains (MICs of 1, 16, and 64 mg/L for tet(M) genes from S. oralis 264-3, S. mitis 254-1 and S. mitis 264-1, respectively). DNA sequencing revealed that S. oralis 264-3 had a tet(M) gene highly homologous to tet(M) carried by Tn916 from Enterococcus faecalis (99.6% identity), while the intermediate- and high-level TcR strains had tet(M) sequences that resembled the tet(M) gene of Tn5251 from Streptococcus pneumoniae (99.3% and 99.4% identity, respectively). No differences were observed in the upstream attenuator structure for each of the strains and differences in reduced tetracycline susceptibilities could be attributed to changes in the deduced amino acid sequences of the Tet(M) proteins. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Viridans group streptococci; Tetracycline resistance; Heterogeneous resistance; tet(M)
1. Introduction Tetracycline resistance in oral bacteria is commonly conferred by the tet(M) gene [1,2] which encodes a ribosomal protection protein (RPP) that confers tetracycline resistance through promoting the release of tetracycline from the 30S ribosomal subunit in a GTP-dependent manner [3–5]. Expression of tet(M) is regulated by transcriptional attenuation [6]. The majority of tet(M) genes are located on conjugative transposons, such as Tn916 and Tn5253, that are responsible for their widespread distribution, not only amongst different bacterial species of the oral cavity, but the larger bacterial com∗ Corresponding author. Present address: Department of Pharmaceutics (Microbiology), School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK. Tel.: +44 20 7753 5848; fax: +44 20 7753 5942. E-mail address: [email protected] (P. Stapleton).
munity as a whole [7]. Recombination between evolutionary divergent tet(M) genes has led to the formation of tet(M) genes with mosaic structures [8]. Consequently tet(M) genes exhibit nucleotide variations at defined segments within these genes [9]. The effect of these mosaic structures on tetracycline resistance has not been formally addressed although it is known that streptococci carrying different tet(M) gene subtypes can confer different levels of tetracycline resistance [10]. Since differences in tetracycline resistance could be attributed to isolates carrying more than one copy of the tet(M) gene, or due to changes in tet(M) promoter sequence, attenuator structure, or due to changes in the structure itself, ideally the influence of different tet(M) subtypes should be examined in the same genetic background. In this study we examined three streptococcal isolates that exhibited; (a) reduced tetracycline susceptibility; (b) intermediate-; (c) and high-level tetracycline resistance and show that the different
0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2004.06.003
440
P. Stapleton et al. / International Journal of Antimicrobial Agents 24 (2004) 439–443
levels of reduced tetracycline susceptibilities were due to changes in the deduced amino acid sequences of the Tet(M) proteins.
2. Materials and methods 2.1. Bacterial strains Viridans group streptococci from the saliva and plaque samples of children (age range: 5 to 15 years), were isolated on Mueller–Hinton agar containing HgCl2 (40 M) [11]. Isolates were identified to species level with the API identification system (bioM`erieux UK Limited, Basingstoke, United Kingdom) and by carbohydrate substrate analysis [12]. 2.2. Susceptibility testing MICs of tetracycline, ampicillin and erythromycin (all purchased from Sigma–Aldrich, Poole, United Kingdom) were determined by agar dilution technique on isosensitest agar (Oxoid Ltd., Basingstoke, UK) with 5% horse blood for streptococcal isolates or without blood for Escherichia coli HB101 carrying the tet(M) gene. An inoculum of about 104 organisms per spot was used and the MICs were read after 24 h incubation at 37 ◦ C. 2.3. DNA techniques DNA extraction was performed with a Gram-positive DNA isolation kit (Flowgen, Ashby de la Zouch, United Kingdom). Southern blotting to Nylon N+ membranes (Amersham Pharmacia Biotech) and DNA–DNA hybridisation with digoxygenin-labelled probes was carried out according to the manufacturer’s instructions (Roche Molecular Biochemicals, Lewes, United Kingdom). Digoxygeninlabelled tet(M) probe was prepared from Tn916 DNA by PCR by the incorporation of alkali-stable digoxygenin-labelled dUTP (Roche Molecular Biochemicals) in the amplification mixture. PCR amplification was performed as described by Ausubel et al. [13] with Dynazyme EXT thermostable polymerase (Flowgen) and an annealing temperature of 50 ◦ C. Primers used for amplification of the tet(M) gene and promoter regions (2841 bp) were based on GenBank sequence U09422 for Tn916 from Enterococcus faecalis DS16: tetM-F1 (5 -ACGACGTTCTTCAAGCTCTATCCT-3 ) [positions 11492-11515] and tetM-R1 (5 -CATTTTATCGGCTCTGCGTCTTT-3 ) [positions 14310–14332]. Restriction endonuclease digestion with HindIII and DraI was used to distinguish between tet(M) and the closely related tet(O) and tet(S) genes. The amplified tet(M) genes were sub-cloned into the EcoRV site of pMOSBlue (Amersham Pharmacia Biotech) and introduced into competent E. coli HB101 [F− (gtp-proA)62 recA13 leuB6 supE44 ara-14 galK2 lacY1 (mcrC-mrr) mtl-1 proA2 xyl-5
rpsL20] by a heat-shock procedure [14]. The orientation of the insert in pMOSBlue was confirmed by restriction endonuclease digestion with HindIII and only clones with the same insert orientation were used. Primers used to sequence the tet(M) gene sub-cloned into pMOSBlue were: Univm13 (5 -GTTTTCCCAGTCACGACGTTGTA3 ), and T7P (5 -TAATACGACTCACTATAGGG-3 ) binding to sequences within the vector and; tetM-F3 [5 -TCTGTATGCTTTGTATGCCTATGG-3 ], tetM-F4 [5 -TGTGACGAACTTTACCGAATCTGA-3 ], tetM-F5 [5 -TCCCTCTGCTGCAAACGACTG-3 ], tetM-F6 [5 GTATGGTTGGAATGTGACGGACTG-3 ], and tetM-R3 [5 -GTTGTACCTTTGTCCACGCTTCCT-3 ] binding to sequences within the sub-cloned gene. Independently amplified PCR products were sequenced directly to confirm the nucleotide changes found in the cloned genes. DNA sequencing was performed with the aid of an Applied Biosystems automated fluorescent sequencer (model 373A). The nucleotide sequences of the tet(M) genes for S. oralis 264-3, S. mitis 254-1, and S. mitis 264-1 have the accession numbers, AJ580976, AJ580977, and AJ580978, respectively.
3. Results During a study examining mercury and antibiotic resistance in oral bacteria [11], 22 viridans group streptococci were isolated that exhibited varying degrees of reduced susceptibilities to tetracycline (Table 1). Three isolates exhibited Table 1 Antimicrobial resistance profiles and the presence of the tet(M) gene for oral streptococcal isolates with reduced susceptibility to tetracycline Isolate
tet(M)a
MIC (mg/L) Tetracycline Ampicillin Erythromycin
S. mitis 152-2 S. parasanguis 181-2 S. parasanguis 179-1 S. oralis 264-3 S. oralis 283-4 S. oralis 204-4 S. mitis 254-1 S. mitis 160-1 S. oralis 277-2 S. mitis 166-4 S. mitis 173-1 S. parasanguis 181-1 S. oralis 279-1 S. oralis 262-3 S. oralis 271-2 S. oralis 260-1 S. mitis 264-1 S. oralis 271-1 S. oralis 318-1 S. oralis 254-2 S. oralis 262-1 S. oralis 283-4 a
2 2 2 2 2 4 8 16 16 32 32 32 32 32 32 64 64 64 64 64 64 64
0.12 1 0.5 0.03 0.06 0.12 0.016 0.12 0.12 0.016 0.016 1 <0.008 0.06 0.016 <0.008 1 0.25 0.016 0.12 0.016 0.06
(+) tet(M) present, (−) tet(M) absent.
0.03 0.016 1 <0.008 2 0.016 0.06 0.03 1 0.03 0.016 0.03 <0.008 0.5 0.016 0.03 <0.008 0.03 0.016 1 8 2
− − − + + + + − − + + − − + + + + + + − + +
c
d
a
b
Streptococcal isolate. E. coli HB101. Nucleotide positions are based on the adenosine of the initiation codon (ATG) being assigned position 1. The amino acid and position for the corresponding nucleotide are given in brackets. N/d, not determined.
T (Val631 ) T (Val631 ) A (Met458 ) A (Met458 ) C (Pro451 ) C (Pro451 ) T (Ser450 ) T (Ser450 ) T (Leu327 ) T (Leu327 ) A (Gln317 ) G (Gln317 ) A (Lys286 ) A (Lys286 ) T (Asn252 ) A (Lys252 ) G (Gln209 ) T (His209 ) N/dd N/dd N/dd N/dd tet(M) on Tn916 tet(M) on Tn5251
G (Val164 ) A (Met164 )
T (Val631 ) T (Val631 ) C (Ala631 ) A (Met458 ) G (Val458 ) G (Val458 ) C (Pro451 ) A (Gln451 ) A (Gln451 ) T (Ser450 ) G (Ala450 ) G (Ala450 ) G (Arg327 ) T (Leu327 ) T (Leu327 ) T (His317 ) G (Gln317 ) G (Gln317 ) A (Lys286 ) A (Lys286 ) G (Arg286 ) T (Asn252 ) T (Asn252 ) A (Lys252 ) G (Gln209 ) T (His209 ) G (Gln209 ) A (Met164 ) A (Met164 ) G (Val164 ) 1 16 64 2 8 64
951 857 756 627 Recipientb
441
S. oralis 254-3 S. mitis 254-1 S. mitis 264-1
1372 1352 1348 490 Hosta
980
Nucleotide positions for non-conservative sequence differences amongst tet(M) genesc Tetracycline MIC (mg/L) for:
In common with other reports [2,15] the tet(M) resistance determinant was frequently encountered amongst the viridans group streptococci isolated in this study. The isolates were not uniformly resistant to tetracycline despite detection of the same resistance determinant amongst the majority of the isolates. Such differences could have been attributed to the presence of more than one resistance determinant, or to changes affecting gene expression, or the presence of different Tet(M) variants with different abilities to confer tetracycline resistance. Different tet(M) variants have previously been identified in bacteria isolated from periodontal pockets [9] but whether these variants had different tetracycline susceptibilities was not reported. The tet(M) variants identified in this study, resemble the tet(M) subtypes, M1 (S. oralis 264-3) and M2 (S. mitis 254-1 and S. mitis 264-1), reported by Olsik et al. [9]. However, unlike the M1 subtype, the tet(M) gene from S. oralis 264-3 did not confer tetracycline resistance.
Host strain or genetic element
4. Discussion
Table 2 Tetracycline MICs for streptococcal isolates and E. coli HB101 carrying tet(M) genes coding for Tet(M) proteins with different deduced amino acid sequences
reduced susceptibility to ampicillin (MIC of 1 mg/L), and six isolates were resistant to erythromycin (MIC ≥ 1 mg/L). Fourteen of the twenty-two isolates were found to carry the tet(M) gene by PCR and by DNA–DNA hybridisation with a tet(M) probe (Table 1). Three tet(M)-positive isolates, S. oralis 264-3, S. mitis 254-1 and S. mitis 264-1 with varying degrees of reduced tetracycline susceptibilities (tetracycline MICs of 2, 8 and 64 mg/L, respectively) were chosen to be investigated further. Two of the isolates, S. mitis 2641 and S. oralis 264-3 were isolated from the oral cavity of the same child. The tet(M) gene was amplified from the isolates by PCR with a proof-reading DNA polymerase and the resulting products were sub-cloned into pMOSBlue. E. coli HB101 carrying the sub-cloned tet(M) genes exhibited similar levels of reduced tetracycline susceptibility observed in the parental streptococcal strains (tetracycline MICs of 1, 1, 16, and 64 mg/L for HB101, 264-3, 254-1 and 264-1, respectively) (Table 2). DNA sequencing of the tet(M) genes revealed that S. oralis 264-3 had a tet(M) gene highly homologous to that carried by Tn916 from E. faecalis DS16 (99.6% identity) (Table 2), while isolates, S. mitis 264-1 and S. mitis 254-1 had tet(M) sequences that resembled the tet(M) gene of Tn5251 from Streptococcus pneumoniae (99.3% and 99.4% identity, respectively) (Table 2). No differences were observed in the upstream attenuator structure of the tet(M) gene for each of the isolates, but the intermediate- and high-level tetracyclineresistant strains each had a single nucleotide change in the −35 region of the tet(M) promoter (TTTACA to TTGACA). Tet(M) from isolate S. oralis 264-3 had three deduced amino acid differences compared with Tet(M) from Tn916: V164M, Q307H and L327R. Comparison of the deduced amino acid sequences of the intermediate- and high-level tetracyclineresistant isolates revealed five differences: M164V, H209Q, K252N, K286R and V631A.
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Comparison of the nucleotide sequences revealed that the tet(M) gene from S. oralis 264-3 differed from M1 at two nucleotide positions (951, A to T and 980, T to G) that result in changes to the deduced amino acid sequence of the Tet(M) protein (Q317H and L327R). Consequently, either one of these amino acid substitutions or both of them are likely to be responsible for the reduced ability of the Tet(M) protein to confer tetracycline resistance. The existence of tet(M)-positive isolates which remain susceptible to tetracycline has been previously noted amongst Streptococcus pneumoniae clones [10,16]. MIC testing without prior induction with tetracycline has been proposed as an explanation for the existence of tet(M)-positive tetracyclinesensitive clones [10]. In this study, we have sequenced the region upstream from the tet(M) gene, including the promoter and the attenuator structure which is responsible for inducible tetracycline resistance, and found no nucleotide differences when compared with the upstream tet(M) gene sequence from Tn916. Although the ability to induce tetracycline resistance was not investigated in this study, indirect evidence from the sequence of the upstream region of the tet(M) gene, combined with the lack of ability of the tet(M) gene to confer tetracycline resistance when introduced into E. coli, suggests changes in the deduced amino acid sequence of the Tet(M) is the primary factor responsible for tetracycline susceptibility in this case. The region upstream from the tet(M) gene has previously been reported to be highly conserved even amongst tet(M) genes from diverse hosts [17]. In this study, the regions upstream from the tet(M) gene were also highly conserved, although a single nucleotide change in the −35 sequence of the tet(M) promoter was found in the sequences from S. mitis 254-1 and S. mitis 264-1. Since both isolates had the same nucleotide change, alteration to the −35 sequence of the tet(M) gene was not responsible for the differences in the levels of tetracycline resistance observed. Furthermore, the different levels of tetracycline resistance were reproduced when the corresponding tet(M) genes were introduced into E. coli suggesting that changes in the deduced amino acid sequences of Tet(M) were responsible for the phenotypes observed. Tet(M) belongs to a wider family of ribosomal protection proteins (RPP) that includes the closely related Tet(O) and Tet(S) determinants and more divergent determinants such as Otr(A) from Streptomyces [7]. Ribosomal protection proteins share homology with elongation factors, EF-Tu and EF-G involved in protein synthesis and alignments of amino acid sequences of RPPs and elongation factors have identified putative functional regions in Tet(M) and Tet(O) [18,19]. Alterations to certain conserved amino acids are known to affect the function of the RRP and elongation factors [18]: changes to Asn-128 located in the putative GTP-binding domain of Tet(O) results in a decrease in tetracycline resistance [19]. In this study, S. oralis 264-3 had three deduced alterations in the amino acid sequence of Tet(M) (V164M, Q317H, L327R). Of these positions in the amino acid sequence, only the Leu
residue at position 327 shows some conservation: the Leu327 residue is found in Tet(M), Tet(O), Tet(S), Tet(Q) and EF-G. Since this amino acid residue is located within the putative ribosomal-binding domain of Tet(M), the substitution of Arg for Leu-327 may affect binding of Tet(M) to the ribosome and consequently explain the significantly reduced tetracycline resistance observed in S. oralis 264-3. S. mitis 254-1 and S. mitis 264-1 had five deduced amino acid differences between them. All the amino acid substitutions, with the exception of V631A, are found in other RPPs and it is not possible to deduce which amino acid substitutions confer the level of tetracycline resistance observed. However, it appears that different mosaic gene structures can give rise to moderate (found in S. mitis 254-1) and high-level (found in S. mitis 264-1) tetracycline resistance. The significance of these different tet(M) gene variants remains to be determined. Tetracycline MICs for viridans group streptococci have been reported to exhibit a broad range with tetracycline MICs varying in increments throughout the range [20-22]. From this study we have shown that for isolates carrying the tet(M) gene, changes in the deduced amino acid sequence of Tet(M) is the main reason for this variation. However, further work involving site-directed mutagenesis combined with detailed substrate profiling is required to confirm the contributions made by each of the deduced amino acid changes.
Acknowledgements This work was funded by the Medical Research Council (grants G9810729 and G9810341).
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