Genetic Diversity of the tet(M) Gene in Tetracycline-Resistant Clonal Lineages of Streptococcus pneumoniae

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Genetic Diversity of the tet(M) Gene in Tetracycline-Resistant Clonal Lineages of Streptococcus pneumoniae
    10.1128/AAC.44.11.2979-2984.2000. 2000, 44(11):2979. DOI: Antimicrob. Agents Chemother. Zawadzki and Christopher G. DowsonNeil Doherty, Krzysztof Trzcinski, Paul Pickerill, Piotr  Streptococcus pneumoniae  Tetracycline-Resistant Clonal Lineages of (M) Gene in tet  Genetic Diversity of the information and services can be found at: These include:  REFERENCES This article cites 45 articles, 27 of which can be accessed free CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new Information about commercial reprint orders: To subscribe to to another ASM Journal go to:  on J  ul   y 2  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om  on J  ul   y 2  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om    A  NTIMICROBIAL   A  GENTS AND  C HEMOTHERAPY ,0066-4804/00/$04.00  0Nov. 2000, p. 2979–2984 Vol. 44, No. 11Copyright © 2000, American Society for Microbiology. All Rights Reserved. Genetic Diversity of the  tet (M) Gene in Tetracycline-ResistantClonal Lineages of   Streptococcus pneumoniae NEIL DOHERTY, KRZYSZTOF TRZCINSKI,† PAUL PICKERILL,‡ PIOTR ZAWADZKI,§  AND  CHRISTOPHER G. DOWSON*  Department of Biological Sciences, University of Warwick,Coventry CV4 7AL, United Kingdom Received 5 May 2000/Returned for modification 8 July 2000/Accepted 31 July 2000 The aim of the present study was to examine the stability and evolution of   tet (M)-mediated resistance totetracyclines among members of different clonal lineages of   Streptococcus pneumoniae . Thirty-two tetracycline-resistant isolates representing three national (Spanish serotype 14, Spanish serotype 15, and Polish serotype23F) and one international (Spanish serotype 23F) multidrug-resistant epidemic clones were all found to be  tet (M) positive and  tet (O),  tet (K), and  tet (L) negative. These isolates all carried the integrase gene,  int , whichis associated with the Tn 1545 -Tn  916   family of conjugative transposons. High-resolution restriction analysis of   tet (M) products identified six alleles,  tet (M)1 to  tet (M)6:  tet (M)1 to  tet (M)3 and  tet (M)5 in isolates of theSpanish serotype 14 clone,  tet (M)4 in both the Spanish serotype 15 and 23F clones, and  tet (M)6, the mostdivergent allele, in the Polish 23F clone. This indicates that  tet (M) variation can occur at the inter- andintraclone levels in pneumococci. Two alleles of   int  were identified, with  int1  being found in all isolates apartfrom members of the international Spanish 23F clone, which carried  int2 . Susceptibility to tetracycline,doxycycline, and minocycline was evaluated for all isolates with or without preincubation in the presence of subinhibitory concentrations of tetracyclines. Resistance to tetracyclines was found to be inducible in isolatesof all clones; however, the strongest induction was observed in the Spanish serotype 15 and 23F clones carrying  tet (M)4. Tetracycline was found to be the strongest inducer of resistance, and minocycline was found to be the weakest inducer of resistance. The gram-positive pathogen  Streptococcus pneumoniae  (thepneumococcus) is a major cause of pneumonia, otitis media,and meningitis (12). The evolution and broad global distribu-tion of multiple antibiotic resistance determinants in bacteriahave resulted in a situation in which pneumococci are com-monly resistant to penicillin, the broad-spectrum cephalospo-rins, macrolides, lincosamides, co-trimoxazole, chlorampheni-col, and tetracyclines, as well as rifampin (11), sulphonamides(42), and fluoroquinolones (13, 24, 30), making the treatmentof serious pneumococcal disease increasingly difficult (17, 22).The transformable nature of   S. pneumoniae  (which has playedan important role, along with point mutations) in the evolutionof resistance [1, 4, 11, 13, 24, 30] has in no small part also led toa population structure characterized by free genetic exchange,punctuated by clonal expansion of successful variants. The beststudied of these are the Spanish 23F, Spanish 6B, and French/ Spanish 9V14 multidrug-resistant clones that have now spreadintercontinentally (see reference 10 for a recent review).One class of antimicrobial agents found most often in clin-ical use is the tetracyclines, broad-spectrum bacteriostaticdrugs shown to be active against pneumococci (33). In someEuropean (9, 16, 23), Asian (35, 36, 41, 47), and African (31,52), countries lack of susceptibility to tetracyclines is the mostfrequently observed resistance phenotype in pneumococci.This situation has also been seen in Poland (45) and the UnitedKingdom (6). In the mid-1990s tetracyclines were the secondmost commonly prescribed antimicrobial drugs after the pen-icillins in both countries (14, 33). The only known mechanismof tetracycline-resistance in  S. pneumoniae  is the protection of the bacterial 30S ribosome subunit against antibiotic bindingby the TetM (2) or TetO (50) proteins, with the  tet (M) genebeing more common than the  tet (O)gene in pneumococci (7,18, 19, 40, 43). Analysis of the nucleotide sequences of   tet (M)genes from a diverse range of bacteria clearly reveals that tet (M) has evolved by recombination (28); however, it is un-clear what is responsible for driving this recombination.The  tet (M)-mediated resistance to tetracyclines and  erm (B)-mediated resistance to macrolides, lincosamides, and strepto-gramins in pneumococci is due to the acquisition of highlymobile conjugative transposons of the Tn 916 -Tn 1545  type andlarge composite structures like Tn  5253  and Tn  3872  which carrythese and other resistance determinants (8, 21). A core ele-ment in the biology of these transposons is the integrase. Al-lelic variation within the integrase gene,  int , can therefore beused to help track the movement and population biology of these conjugative transposons.To progress from a descriptive analysis of antibiotic con-sumption and the evolution of resistance to a more quanti-tative understanding of the dynamics of resistance and theimplications for changing practices in antimicrobial chemo-therapy, an important factor is an understanding of the stabilityor plasticity of the resistance genotype and phenotype. The aimof this study was to examine the stability and evolution of transposon-associated  tet (M)-encoded tetracycline resistanceamong members of four different multiply drug-resistant clonallineages of   S. pneumoniae . This was undertaken by high-reso-lution restriction analysis (HRRA) of allelic variation within * Corresponding author. Mailing address: Department of BiologicalSciences, University of Warwick, Coventry CV4 7AL, United King-dom. Phone: 44 2476 523534.Fax: 44 2476 523568. E-mail:† Present address: National Institute of Hygiene, Chocimska 24,00-791 Warsaw, Poland.‡ Present address: Zeneca Agrochemicals, Jeallots Hill ResearchStation, Bracknell RG42 6ET, United Kingdom.§ Present address: Procter & Gamble, 65824 Schwalbach/Ts, Ger-many.2979   on J  ul   y 2  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   tet (M) and the transposon integrase gene ( int ). The inducibilityof tetracycline resistance in these isolates was also investigated. MATERIALS AND METHODSBacterial strains.  Thirty-two  S. pneumoniae  isolates were included in thisstudy, and 29 of these have already been described. They represented threedifferent national (Spanish serotype 14, Spanish serotype 15 and Polish serotype23F) or international (Spanish 23F) multidrug-resistant epidemic clones identi-fied in previous studies (see Table 1 for details). All isolates were confirmed tobe  S. pneumoniae  by optochin susceptibility and bile solubility tests in previousstudies and were stored at   80°C, in 15% glycerol. All cultures subsequentlygrown from stored stocks were streaked to single colonies prior to use. Strains were grown overnight at 37°C in 5% CO 2  on brain heart infusion (BectonDickinson Europe, Meylan, France) supplemented with 1.5% Bacto Agar (Bec-ton Dickinson) and 5% (vol/vol) defibrinated sheep’s blood (Oxoid, Basingstoke,United Kingdom) (BHI agar). The capsular types of three isolates for which nopublished data were available were determined with Danish pneumococcal typ-ing antisera (Statens Seruminstitut, Copenhagen, Denmark) by the Quellungreaction. Susceptibility testing.  Antibiotic resistance was determined by the agar dilu-tion and the disk diffusion methods. For medium preparation, plate inoculation,and interpretation of results, the methodology recommended by the NationalCommittee for Clinical Laboratory Standards was applied (25, 26), with theexception that MICs were determined by the agar dilution method rather thanthe broth dilution method. Antibiotic dilutions were prepared in Mueller-Hintonagar (Oxoid) supplemented with 5% defibrinated sheep blood. After inoculationthe plates were incubated aerobically for 18 to 20 h at 37°C and were supple-mented with 5% CO 2 . Susceptibility to the following antimicrobial agents wastested: penicillin, cefotaxime, erythromycin, chloramphenicol, rifampin, ofloxa-cin, doxycycline, and minocycline (all supplied by Sigma-Aldrich, Poole, UnitedKingdom) and tetracycline (NBL Gene Sciences, Cramlington, United King-dom).For all isolates, tetracycline, doxycycline, and minocycline MICs were evalu-ated both with and without induction of resistance. For each strain a few coloniesfrom an overnight growth on BHI agar were used to inoculate three BHI agarplates: unsupplemented medium (lack of induction) and BHI agar supplemented with tetracycline at concentrations of 0.5 and 5 mg/liter, doxycycline at 0.5, 2, and5 mg/liter, and minocycline at 0.5, 1, and 5 mg/liter (for induction of resistanceto tetracyclines). Cells were harvested after not longer than 20 h of growth and were used for MIC evaluation.Resistance to tetracycline, doxycycline, and minocycline was also determinedby the disk diffusion method (26) with disks containing 30   g of each antimicro-bial agent (Oxoid). Macrolide and lincosamide resistance phenotypes were de-termined on the basis of the erythromycin MIC evaluation by the agar dilutionmethod and the double-disk test method (15) with erythromycin (15-  g), linco-mycin (15-  g), and clindamycin (2-  g) disks (all from Oxoid). Identification of tetracycline resistance determinants by PCR.  ChromosomalDNA was purified as described previously (48). PCR were carried out withprimers at a concentration of 4 ng/   l of the final reaction volume. Primersequences were as follows:  tet (M) forward, 5  -AGT TTT AGC TCA TGT TGA TG-3  ;  tet (M) reverse, 5  -TCC GAC TAT TTG GAC GAC GG-3  ;  int  forward,5  -GCG TGA TTG TAT CTC ACT-3  ; and  int  reverse, 5  -GAC GCT CCT GTTGCT TCT-3  . Both primer sets were designed on the basis of the availabledatabase sequences of   tet (M) and  int  (GenBank accession numbers X90939 andL29324, respectively). Cycling parameters were denaturation at 95°C for 1 min,annealing at 55 or 50°C for 1 min [for  tet (M) and  int  primers, respectively], andextension at 72°C for 1.5 or 1 min [for  tet (M) and  int  primers, respectively],followed by a final extension step of 72°C for 10 min. The PCR amplicons were visualized by agarose gel electrophoresis as described by Sambrook et al. (39).Detection of the  tet (O),  tet (K), and  tet (L) determinants was performed by PCRby previously described protocols (44). A tetracycline-susceptible isolate of   S. pneumoniae  (isolate R6) was used as a negative control for each of the  tet - and int -specific PCRs  S. pneumoniae  isolates from a previous study (28) were used aspositive controls for  tet (M) and  int , and  Staphylococcus aureus  isolates (44) wereused for  tet (K) and  tet (L).  Streptococcus mutans  strain DL5 was used as a positivecontrol for  tet (O). Products from positive controls were sequenced by using thePCR primers to confirm the identities of the amplicons. HRRA.  HRRA was undertaken essentially as described previously (45, 49).Specifically, 10   l of each PCR product was digested with the following restric-tion enzymes:  Aci I,  Alu I,  Dde I,  Mse I, and  Rsa I. Restriction fragments wereseparated by polyacrylamide gel electrophoresis in 4 and 8% gels and were visualized by staining with ethidium bromide. The restriction footprint of theenzymes used covered 8.03% of the  tet (M) PCR product on the basis of thepublished sequence of the gene (34). Each restriction pattern given by eachdigest was assigned a pattern number. Alleles were determined on the basis of the combined restriction patterns for all five digests. The percent divergencebetween the alleles described was calculated by using the band matching algo-rithm of Nei and Li (27). The same methodology was applied for HRRA of the int  PCR product.  AP PCR and BOX PCR.  PCR-based typing methods were used to establish thedegrees of similarity among the strains analyzed. Arbitrarily primed (AP) PCR was carried out with AP4 (51) and AP7 (46) primers. Each reaction was carriedout in a final volume of 50   l with 20 mM Tris-HCl (pH 8.4) and 50 mM KCl,5 mM MgCl 2 , 0.2 mM deoxynucleoside triphosphates, 1  g of primer, 5 U of   Taq DNA polymerase (Gibco BRL, Paisley, United Kingdom), and approximately 20ng of DNA. Cycling parameters were 95°C for 2 min, followed by 10 cycles of 30 sat 95°C, 30 s at 35°C, and 1 min at 72°C and then 30 cycles of 30 s at 95°C, 30 sat 55°C, and 1 min at 72°C and a final extension step of 72°C for 4 min. BOXPCR was performed as described above, with the following exceptions: 0.2   g of each primer was used per 50-  l reaction mixture. Primers were designed on thebasis of available published BOX sequences (20) and sequences derived by other workers in our laboratory (S. King, unpublished results): BOX A forward, 5  -CCA CGT CAG CKT CRC CTT RCC GT-3  ; BOX A reverse, 5  -CAA GGCGAM GCT GAC RTK GTT TGA-3  . Cycling parameters were 35 cycles of 30 sat 95°C, 30 s at 50°C, and 4 min at 72°C, with a final extension step of 72°C for10 min. Different AP PCR and BOX PCR types were identified the basis of asingle band difference between the electrophoretic patterns of PCR productsdetected after separation through 1% agarose visualized with ethidium bromide. RESULTS On the basis of serotype, AP PCR and BOX PCR genomicpatterns,  tet (M):: int  allele pattern, and resistance profile, all S. pneumoniae  isolates analyzed in this study fell into one of four clusters of related isolates (Table 1). AP PCR and BOXPCR were found to discriminate between isolates of differentclusters, whereas at least two of the four AP PCR or BOX PCRamplification patterns generated for each isolate were identicalto the patterns for other isolates defined as being clonallyrelated in previous studies (3–5, 11, 29, 49; http://mlst.zoo.ox resistance was determined by the agar dilutionmethod for all 32 isolates studied (MIC range, 8 to 64 mg/ liter). PCR screening revealed that all isolates were  tet (M)positive and  tet (K),  tet (L), and  tet (O) negative. Detection of the 1,862-bp fragment from positions 21 to 1882 of the pub-lished sequence of the  tet (M) gene was indicative of the pres-ence of the  tet (M) determinant (34). HRRA of   tetM   gene PCRamplicons revealed that the  tet (M) loci from all of the strainsanalyzed fell into six distinct restriction types [alleles  tet (M)1 to tet (M)6]. The data generated from the  tet (M) allele assign-ments (data not shown) were used to calculate the degree of genetic diversity among the alleles. The results of this analysisare shown in Table 2. The estimated nucleotide divergencebetween different  tet (M) alleles ranged from 0.44% [ tet (M)1and  tet (M)2] to 8% [ tet (M)2 and  tet (M)6]. HRRA did notreveal any variation in  tet (M) within the Polish serotype 23Fclone, Spanish serotype 15 clone, or Spanish serotype 23Fpandemic clone. The Spanish serotype 14 clone was found todiffer in that the isolates examined carried four alleles,  tet (M)1to  tet (M)3 and  tet (M)5, that were divergent by up to 3.18%.Isolates of the Spanish serotype 15 clone and the Spanishserotype 23F pandemic clone both carried  tet (M)4. All isolates were  int  positive, in that they produce the ex-pected 1,046-bp fragment. After testing with a range of en-zymes, two alleles of   int  that could be discriminated on thebasis of a single  Dde I digest were identified. Isolates of allnational clones possessed a common  int  allele type 1, and allmembers of the Spanish 23F clone possessed the  int 2 allele. According to the geometric mean MICs, the highest level of resistance to the tetracyclines tested was observed among iso-lates of the Polish 23F clone (32 mg/liter for tetracycline, 10.96mg/liter for minocycline, and 7.05 mg/liter for doxycycline).The lowest MICs were observed for isolates of the Spanishserotype 15 clone and were equal to 12.13, 3.03, and 4 mg/literfor the same drugs, respectively. These isolates were all shownto possess  tet (M)4.Induction of resistance to the tetracyclines by subinhibitoryconcentrations of tetracycline was observed in nearly all iso-lates of each of the epidemic clones. The highest induction 2980 DOHERTY ET AL. A  NTIMICROB . A  GENTS  C HEMOTHER .   on J  ul   y 2  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om   ratio (calculated as the increase in the geometric mean MICsevaluated for a particular drug and cluster of isolates with and without induction of resistance) was observed for isolates of Spanish serotype 15 and serotype 23F clones, all of whichcarried  tet (M)4. Induction was not noted for two members of the Polish 23F clone (strains 9 and 40). The subinhibitoryconcentrations of minocycline tested did not induce resistanceto tetracycline or doxycycline. However, minocycline did in-duce resistance to itself in four isolates, two members of theSpanish serotype 14 clone (T13 and GM23; both with the tet (M)3 allele) and two of the Spanish serotype 23F clone (SpIand DN88). Subinhibitory concentrations of doxycycline in-duced resistance in 10 isolates: tetracycline resistance in 4isolates, to minocycline resistance in 7 isolates, and doxycyclineresistance in 3 isolates. According to National Committee for Clinical LaboratoryStandards (26)-recommended breakpoints for the disk diffu-sion method, four isolates of the Spanish serotype 15 clone andone isolate of the Spanish serotype 14 clone fit into the cate-gory of intermediate susceptibility. One member of the sero-type 15 clone (isolate B62) would be categorized as susceptible. All other isolates were identified as resistant to tetracycline bythe disk diffusion method. All isolates were found to be penicillin-nonsusceptible S. pneumoniae . The MICs of erythromycin for all Polish iso-lates were  256 mg/liter, and except for two isolates (isolates233 and 234), all isolates were resistant to the lincosamidestested. A high level of resistance to erythromycin indicates thepresence of the MLS B  phenotype coded by  erm (B). In fourisolates (isolates GM49, C69, 89NB and T9) low-level resis-tance to erythromycin (MIC range, 0.5 to 8 mg/liter) withsusceptibility to lincosamides was observed, indicative of thepresence of a macrolide efflux system (15, 38). All other iso-lates were susceptible to the macrolides and lincosamidestested. No single isolate was susceptible to chloramphenicol,and no single isolate was resistant to ofloxacin. All isolates were identified as multidrug resistant, being resistant to drugsof at least three different groups of antimicrobial agents. DISCUSSION The genes that encode tetracycline resistance in bacteria arenumerous and are often evolutionarily distinct. By far the most widely distributed tetracycline resistance determinant in gram-positive bacteria is  tet (M) (37). In this study isolates of differentepidemic clones of multidrug-resistant pneumococci have beenchecked for the presence of four different tetracycline resis-tance determinants,  tet (K),  tet (L),  tet (M), and  tet (O), and allhave been found to be positive only for  tet (M). The identifi-cation of   tet (O)-positive pneumococci has, however, been re-ported in a limited number of South African (50) and North American (18) isolates.Six   tet (M) alleles were identified by HRRA in  S. pneumoniae isolates belonging to four epidemic clones.  tet (M)6, identifiedin Polish isolates, was the most divergent of all alleles detected,differing from  tet (M)2 by 8% at the nucleotide level. This issimilar to the divergence found previously between progenitoralleles of   tet (M) carried by Tn 1545  and  S. aureus , and recom-bination between these alleles resulted in the mosaic structureof the  tet (M) alleles found in a diverse range of organisms (28).The potential origin of   tet (M)6 is under investigation. Themost common allele was  tet (M)4, identified in all isolates of theSpanish serotype 15 and 23F clones. In this study all of themembers of the Spanish 23F clone carried the same  int2  allele, which was unique among the isolates examined.The susceptibilities of the isolates to selected tetracyclines were analyzed to establish if there are any differences in tet-racycline induction or resistance profiles among  S. pneumoniae isolates of different allelic variants of   tet (M). Interestingly, S. pneumoniae  strains that belong to two different epidemicclones (Spanish serotypes 15 and 23F), both of which carry tet (M)4, shared similar profiles of inducible tetracycline resis-tance. For isolates of both clones, the MICs of minocycline were relatively low, ranging from 2 to 4 mg/liter for strainsexamined without induction of resistance. After induction of resistance with tetracycline, increases in the minocycline MICsto 8 and 16 mg/liter were observed for all these isolates. Of thethree tetracyclines tested, minocycline was the weakest inducerof resistance and tetracycline was the strongest inducer of resistance. Only minocycline did not induce resistance to theother tetracyclines tested. However, the induction of resistanceto minocycline by subinhibitory concentrations of the drugobserved in four isolates indicates that minocycline should notbe underestimated as an inducer of resistance.Susceptibility to tetracycline has apparently been observedin  tet (M)-positive isolates of the largest of national epidemicpenicillin-nonsusceptible  S. pneumoniae  clones identified inthe United States in 1996 and 1997 (7) and Romania (32), andintermediate susceptibility (MICs, 2 to 4 mg/liter) has beenobserved in some Italian isolates of the Spanish 23F clone (19).However, by routine susceptibility testing, resistance to tetra-cycline is evaluated without any induction of resistance, and itis therefore possible that some tetracycline-resistant  S. pneu- moniae  strains may have been misidentified as susceptible. Thispossibility was confirmed in this study, when the disk diffusionmethod was used to evaluate resistance to tetracycline in iso-lates of the Spanish serotype 15 clone, for which tetracyclineMICs were the lowest among the clones examined. The ob-served inducibility of resistance in these and the other iso-lates examined suggests that all  tet (M)-positive  S. pneumoniae strains should be considered resistant to all tetracyclines, andthe lack of susceptibility to one of them should be interpretedas resistance to drugs of the whole group. The importance of inducible resistance in the proper identification of resistance totetracyclines in  tet (M)-positive strains of another gram-positivepathogen,  Staphylococcus aureus,  has already been document-ed (44).It has previously been shown that members of the Tn 916 family of transposons can be found in the host chromosome inmultiple copies (35). From the HRRA experiments performedin this work, it is apparent that there was no superposition of different  tet (M) restriction types, indicating a single allele perisolate (multiple copies of the same allele could be present). Acquisition of different  tet (M) alleles within a clonal lineage, asfound for the Spanish serotype 14 clone, may have been me-diated by loss of the srcinal transposon and acquisition of anew transposon carrying a different allele of   tet (M) or by re-combination following transformation by DNA from a differ-ent strain, again carrying a different allele of   tet (M). TABLE 2. Percent nucleotide divergence between S. pneumoniae tet (M) alleles  Allele% Nucleotide divergence for allele:2 3 4 5 6 1 0.44 1.74 1.34 3.03 7.412 1.25 1.54 2.87 8.003 3.18 2.13 6.934 3.33 5.345 5.282982 DOHERTY ET AL. A  NTIMICROB . A  GENTS  C HEMOTHER .   on J  ul   y 2  6  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   a a c . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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