Evidence for tetracycline resistance determinant tet(M) allele replacement in a Streptococcus pneumoniae population of limited geographical origin

International Journal of Antimicrobial Agents 27 (2006) 159–164

Evidence for tetracycline resistance determinant tet(M) allele replacement in a Streptococcus pneumoniae population of limited geographical origin Katarzyna Dzier˙zanowska-Fangrat a , Katarzyna Semczuk a , Paulina G´orska b , Stefania Giedrys-Kalemba c , Maria Kochman d , Alfred Samet e , Stefan Tyski f , Danuta Dzier˙zanowska a , Krzysztof Trzci´nski b,∗ a

Department of Clinical Microbiology and Immunology, Children’s Memorial Health Institute, Aleja Dzieci Polskich 20, 04-736 Warszawa, Poland b Department of Sera and Vaccines Evaluation, National Institute of Hygiene, Chocimska 24, 00-791 Warszawa, Poland c Department of Microbiology and Immunology, Pomeranian Medical University, Powsta´ nc´ow Wielkopolskich 72, 70-111 Szczecin, Poland d Department of Bacteriology, National Institute of Hygiene, Chocimska 24, 00-791 Warszawa, Poland e Department of Clinical Bacteriology, State Hospital No. 1, D˛ ebinki 7, 80-211 Gda´nsk, Poland f Antibiotics and Microbiology Department, National Institute of Public Health, Chełmska 30/34, 00-725 Warszawa, Poland Received 26 July 2005; accepted 6 October 2005

Abstract A collection of 185 Streptococcus pneumoniae isolates was tested for their susceptibility to antipneumococcal drugs, with a focus on the distribution of tetracycline resistance determinants tet(M) and tet(O). Resistance patterns were compared with established correlates of multidrug resistance, and tetracycline-resistant isolates were tested for clonality and allelic variation within tet(M). Resistance to tetracyclines, penicillins and macrolides were all strongly related to multidrug resistance. Over one-quarter of the strains were tetracycline resistant, all via the tet(M)-mediated mechanism. Restriction fragment length polymorphism analysis revealed a high degree of allelic variation within tet(M) and gave evidence of a clonal and horizontal spread of selected alleles. A tet(M) variant that emerged with the onset of epidemic multidrug-resistant strains was replacing old alleles in the population. © 2005 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: tet(M); Tetracycline; Streptococcus pneumoniae

1. Introduction Lack of susceptibility to tetracyclines was the common resistance phenotype in Streptococcus pneumoniae strains isolated in Poland, with a prevalence varying from 30% up to 50%, whereas an increase in resistance to penicillin and macrolides from less than 3% to 15% has been observed within the last decade [1–4]. The change in the profile of resistance was accompanied by the simultaneous emergence ∗ Corresponding author at: Department of Epidemiology, Harvard School of Public Health, 665 Huntington Avenue, Bldg. 1 Room 903, Boston, MA 02115, USA. Tel.: +1 617 432 3269; fax: +1 617 432 3259. E-mail address: [email protected] (K. Trzci´nski).

of the national epidemic clones Poland23F -16 and Poland6B 20 and the introduction of international pandemic clones Spain23F -1 and Spain9V -3 to the country. Isolates of all these multidrug-resistant (MDR) strains were uniformly resistant to tetracycline [5–7]. The only mechanism of tetracycline resistance identified in pneumococci is synthesis of TetM or TetO proteins with a high affinity for antibiotic molecules. These proteins protect the 30S ribosomal subunit, the target site for the drug in bacterial cells [8]. Tetracycline resistance determinants are present in a wide range of bacteria and are usually localised on mobile genetic elements (plasmids, transposons or integrons) capable of horizontal intraspecies and cross-species transfer [9]. This is also true for tet(M), which codes for resistance in

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the vast majority of tetracycline-non-susceptible pneumococcal strains. In streptococci, tet(M) is usually associated with highly mobile conjugative transposons of the Tn916–Tn1545 family. This family often carries other resistance genes, such as erm(B) coding for resistance to macrolides, lincosamides and streptogramins B [10,11]. Comparison of the tet(M) sequences from various bacterial species reveals a mosaic structure, evolved by recombination [12]. Analysis of tet(M) in isolates of epidemic MDR clones revealed heterogeneity, as well as evidence of clonal distribution of allelic variants of tet(M) and possible horizontal movement of the mobile elements carrying tet(M). It also raised the possibility that S. pneumoniae expressing TetM should be considered resistant to all available tetracyclines, including minocycline, the most active of these against Gram-positive cocci [6]. In our study, a collection of S. pneumoniae isolates of limited geographical origin was tested for the presence and distribution of tet(M) and tet(O) tetracycline resistance genes. Isolates were evaluated for their susceptibility to antipneumococcal drugs. Resistance patterns were compared in order to establish correlates of multidrug resistance and the importance of the TetM and TetO component. A subset of isolates resistant to tetracycline was further tested for clonality and for variation within the tet(M) gene. The rationale for this work was to evaluate the role of resistance to tetracyclines in a pneumococcal population at the time of emergence of multidrug resistance. 2. Materials and methods 2.1. Bacterial strains One hundred and eighty-five S. pneumoniae isolates, collected at various healthcare centres in Poland, were analysed. Sixty pneumococci were isolated between 1995 and 1998, either from carriers (28 isolates) or from the cerebrospinal fluid of patients with pneumococcal meningitis (32 isolates). The remaining isolates were collected during 2000–2001 from clinical specimens at the Children’s Memorial Health Institute in Warszawa (53 isolates), State Hospital No. 1 in Gdansk (44 isolates) and the Pomeranian Medical University in Szczecin (28 isolates). In addition, four isolates of all national and international epidemic clones previously identified in Poland (Poland6B -20, Spain9V -3, Poland23F -16 and Spain23F -1) and a strain of Spain14 -5 clone not observed in Poland were used as reference isolates in this study [5,6]. All isolates were confirmed as S. pneumoniae by optochin susceptibility, bile solubility tests and polymerase chain reaction (PCR)-based detection of the pspA gene. Isolates were stored at −80 ◦ C in brain–heart infusion (BHI; BioMerieux, Marcyl’Etoile, France) supplemented with 10% glycerol [13,14]. All cultures were grown overnight at 37 ◦ C in 5% CO2 on tryptic soy agar (BioMerieux) supplemented with 5% defibrinated sheep blood. Capsular types were determined with a Pneumotest kit or serotype-specific sera (Statens Seruminstitut, Copenhagen, Denmark).

2.2. Susceptibility testing Resistance to antibiotics was determined following the National Committee for Clinical Laboratory Standards guidelines, with the exception that minimum inhibitory concentrations (MICs) were determined by the agar dilution method using Mueller–Hinton agar (Oxoid, Basingstoke, UK) supplemented with 5% defibrinated sheep blood instead of the broth dilution method. Streptococcus pneumoniae ATCC 49619 was used as a control strain [15]. Susceptibility to the following antimicrobial agents was determined: penicillin, erythromycin, clindamycin, rifampicin, doxycycline, minocycline, sulphamethoxazole/trimethoprim (co-trimoxazole) (all supplied by Sigma-Aldrich, Poole, UK) and tetracycline (NBL Gene Sciences, Cramlington, UK). To establish whether different mechanisms of resistance co-existed at an increased frequency in the population under study, resistance patterns were analysed using χ2 and Fisher’s exact tests. 2.3. Identification of resistance determinants by PCR Chromosomal DNA was purified with a Genomic DNA Prep Plus kit (A&A Biotechnology, Gdansk, Poland). Detection of the tet(M) and tet(O) determinants was performed as described elsewhere [6]. The presence of erm(B) was detected by PCR using primers and the PCR conditions described by Sutcliffe et al. [16]. 2.4. High-resolution restriction analysis (HRRA) HRRA of tet(M) was performed as previously described [6]. The PCR products were digested with the following restriction enzymes; AluI, Csp6I, Hin6I, HinfI, MnlI, MspI and TaqI (Fermentas AB, Vilnius, Lithuania). Restriction fragments were separated in 2% agarose gels and visualised by staining with ethidium bromide. The restriction pattern generated by each enzyme for a given PCR product was assigned a unique number. Alleles were determined on the basis of combined restriction patterns for all seven digests. The percentage divergence between the alleles was calculated using the Nei and Li band-matching algorithm [17]. 2.5. Arbitrarily-primed (AP)-PCR and BOX-PCR PCR-based typing methods were used to establish similarities amongst the strains analysed. AP-PCR was carried out with an AP7 primer [18]. The BOX A reverse primer was used in BOX-PCR analysis as previously described [6]. AP-PCR and BOX-PCR types were identified based on single band difference between electrophoretic patterns of PCR products detected after separation through 1% agarose and DNA fragment visualisation with ethidium bromide. 2.6. Macrorestriction analysis of chromosomal DNA DNA isolation and SmaI digestion for pulsed-field gel eletrophoresis (PFGE) were performed using a BioRad kit

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as described by Elliott et al. [19]. DNA digests were separated by PFGE in 1% pulse-field grade agarose gels (Bio-Rad, Marnes-la-Coquette, France) with a switch time of 2–30 s for 20 h at a 120◦ angle with a voltage gradient of 6 V/cm at 8 ◦ C. Macrorestriction patterns generated were analysed using criteria described by Tenover et al. [20] to establish the relatedness between the observed fingerprints.

3. Results Of 185 S. pneumoniae isolates tested for susceptibility to antipneumococcal drugs, 49 (26.5%) were identified as resistant to tetracycline (MIC ≥ 8 mg/L). In all of these 49 isolates, MICs of doxycycline and minocycline were ≥2 mg/L. For tetracycline-susceptible isolates, the MIC of any tetracycline tested was never greater than 0.25 mg/L. Thirty-two (17.3%) S. pneumoniae isolates were identified as not susceptible to penicillin, 21 (11.4%) as resistant to erythromycin and 65 (35.1%) as resistant to co-trimoxazole. There was a correlation between the presence of resistance to tetracycline and to erythromycin (Fisher’s exact test, P < 0.001), with only three erythromycin-resistant isolates susceptible to tetracycline. Analysis of macrolide resistance revealed that 14 of 15 isolates resistant to macrolides via erm(B) mechanisms were also positive for tet(M). There was a correlation between lack of susceptibility to tetracyclines and to penicillin (P = 0.003), with 17 isolates identified as resistant via both mechanisms. The same was true for resistance to macrolides and to penicillin (P < 0.0001). The co-trimoxazole-susceptible and -resistant isolates demonstrated no differences in profiles of resistance to other drugs. Multidrug resistance, defined as lack of susceptibility to at least three different classes of antipneumococcal drugs, has been identified in 13 (7%) isolates, all of which were resistant to tetracycline. An amplified fragment of tet(M) (1682 bp in size) was present in all isolates resistant to tetracycline and also in one susceptible isolate. All isolates were negative for the presence of the tet(O) gene by PCR. Forty-nine tet(M)-positive isolates were typed. A subset of 21 of these 49 was selected for analysis of allelic variations within the tet(M) gene, including strains of unique genotypes and isolates identified as related to the Poland6B -20 clone, the only epidemic strain present in the country that was not analysed in a previous study [6]. Combined demographic, typing and susceptibility data for these isolates are presented in Table 1. The combination of restriction enzymes deployed in HRRA generated fingerprints composed of 27–32 fragments as detected in an agarose gel after electrophoresis. Altogether, 13 different fingerprints were identified and unique Roman numbers were assigned to every tet(M) allele identified by HRRA. Based on the presence of fragments common to different HRRA fingerprints, a correlation coefficient was calculated using the Nei and Li algorithm, and a dendrogram showing similarities among these was constructed (Fig. 1) [17]. Isolates analysed in detail are listed in Table 1 in the

Fig. 1. Dendrogram showing similarities among tet(M) alleles of Streptococcus pneumoniae generated by high resolution restriction analysis.

order established by the dendrogram. The allele assignments for each digest pattern group identified in the study are presented in Table 2. The highest numbers of fragments were generated by an MnlI digest of tet(M) PCR product (six to seven fragments) and distinguished seven different restriction fragment length polymorphism (RFLP) patterns. The lowest number of bands was observed in Hin6I digests (from no restriction site present in allele XI to three fragments generated for allele XII) and generated four different RFLP patterns. The dendrogram (Fig. 1) indicates the presence of two clusters of alleles: II–VI and IX–XI, with all MDR strains present in a cluster II–VI. Four alleles were identified in more than one isolate. Of these, allele III was present in all S. pneumoniae of two epidemic MDR clones, namely Poland6B 20 (three isolates) and Spain23F -1 (single isolate), identified by macrorestriction analysis of genomic DNA and by PCRbased typing methods. Alleles II, VIII and X were present in two, two and five isolates, respectively. All these nine isolates were identified by genotyping as unrelated to each other as well as to the other analysed strains. Alleles I, IV, V, VII, IX and XI were unique for single unrelated isolates. The remaining alleles, VI, XII and XIII, were identified only in strains 1, T9 and 234 of MDR clones Spain9V -3, Spain14 -5 and Poland23F -16, respectively, used as reference isolates in the study.

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Table 1 Antibiotic resistance profiles and typing of tet(M)-containing Streptococcus pneumoniae from Poland Strain origin Strain

ST49B ST20 ST21 2739 1274 5975 6409 6204 30 6447 Sp1 ST28 ST17 1 ST65B 5647 6318 ST3 ST15 ST14 ST59A 10954 1423 4380 T9 234

Resistance profilee

Typing results

Year of isolation

Geographical origina

Clinical origin (or clone name)

SmaI::AP:: BOXb

HRRA pattern of tet(M)c

Capsular typed

MIC (mg/L) TET

DOX

MIN

PEN

1997 1996 1996 2001 2001 2000 2000 2000 1996 2000 1998 1996 1996 1996 1997 2000 2000 1995 1996 1996 1997 2001 2001 2001 1991 1994

Warszawa Ostrowiec Kielce Gda´nsk Gda´nsk Szczecin Szczecin Szczecin Warszawa Szczecin Spain Katowice Gorz´ow Wkp Gda´nsk Warszawa Szczecin Szczecin Ciechanow Kalisz Skierniewice Warszawa Gda´nsk Gda´nsk Gda´nsk Spain Warszawa

Carriage CSF CSF Blood Carriage Sinuses Carriage Carriage Poland6B -20 [5] Carriage Spain23F -1 [6] CSF CSF Spain9V -3 [5] Carriage Carriage Carriage CSF CSF CSF Carriage BAL Carriage Sputum Spain14 -5 [6] Poland23F -16 [5]

J::␭::␭ A::␣::␣ G::␥::␥ C::␹::␹ H3 ::␸::␸ H2 ::␫::␫ H2 ::␩::␩ H1 ::␩::␩ H1 ::␩::␩ I1 ::␬::␬ I2 ::␬::␬ B::␤::␤ D::␦::␦ R::␮::␮ M::␲::␲ N::␯::␯ L::о::␯ E::␧::␧ F::␾::␾ O::␪::␪ P::␳::␳ T::␼::␼ U::␻::␻ K::␶::␶ S::υ::υ Q::␴::␴

I II II III III III III III III III III IV V VI VII VIII VIII IX X X X X X XI XII XIII

NVT 7F* 6A* NVT 6 6 6 6 6B 23 23F 4 19A* 9V 8 11 19 9N* 18C* 15A* NVT 6 23 23 14 23F

16 16 16 16 32 32 32 32 16 16 8 0.12 16 8 32 16 16 16 8 8 32 32 8 16 16 16

8 8 8 8 16 16 16 16 4 16 8 0.12 8 4 16 16 16 4 4 4 16 8 4 16 4 8

8 4 8 2 16 16 16 16 8 8 4 0.5 4 8 16 8 8 8 8 8 16 4 8 16 8 8

0.032 0.023 0.032 0.023 0.094 0.19 0.125 0.125 0.125 1.5 2 0.023 1.5 1.5 0.023 0.023 0.023 0.023 0.023 0.023 0.19 0.023 0.023 0.19 3 8

Antibiogram

ERY ERY, CLI ERY, CLI ERY, CLI ERY, CLI ERY, CLI SXT

ERY, CLI, SXT ERY, CLI, SXT SXT

ERY ERY, SXT

HRRA, high-resolution restriction analysis; MIC, minimum inhibitory concentration; CSF, cerebrospinal fluid; BAL, bronchoalveolar lavage. a Town of isolation for strains collected in Poland; country for isolates Sp1 and T9 of Spain23F -1 and Spain14 -5, respectively. b Different capital letters represent unrelated SmaI patterns determined by criteria proposed by Tenover et al. [20], the same letter (different number) indicates related patterns (no more than three fragments different between fingerprints), with a correlation coefficient value above 90%. Greek letters indicate patterns generated by polymerase chain reaction-based typing methods. c Roman numbers correspond to different alleles of tet(M) identified by HRRA; patterns are organised according to similarities observed between complex AluI, Csp6I, Hin6I, HinfI, MnlI, MspI and TaqI restriction patterns (see Fig. 1). d Polysaccharide capsule types were determined using a Pneumotest latex kit; NVT, non-vaccine type, capsule type not included in the 23-valent pneumococcal polysaccharide vaccine; asterisks apply to serotypes determined with type-specific typing sera (Staten Serum Institut, Copenhagen, Denmark). e Susceptibility testing results show MIC values of tetracycline (TET), doxycycline (DOX) minocycline (MIN) and penicillin (PEN); the last column indicates a lack of susceptibility to the following antibiotics: ERY, erythromycin; CLI, clindamycin; SXT, co-trimoxazole.

Table 2 The Streptococcus pneumoniae tet(M) allele assignments for each digest pattern group Allele

I II III IV V VI VII VIII IX X XI XII XIII

Restriction digest pattern/number of fragments produced AluI

Csp6I

Hin6I

HinfI

MnlI

MspI

TaqI

1 2 2 3 3 2 3 3 4 4 4 3 4

1 1 1 1 1 1 2 2 2 2 3 4 5

1 1 1 1 1 1 1 1 1 1 2 3 4

1 2 2 2 1 1 3 3 3 3 3 4 5

1 1 1 1 2 2 3 4 5 4 4 6 7

1 2 2 3 3 3 2 2 1 1 1 4 5

1 1 2 2 2 1 2 2 1 1 3 4 5

Except for isolates of the known epidemic clones Poland6B -20 and Spain23F -1, the serotypes and serogroups identified were unique among isolates, even when they had the same allele of tet(M), as observed for carriers of allele X. 4. Discussion This work has been done to gain an insight into the changes in resistance to antibiotics in an S. pneumoniae population at the time of emergence and spread of multidrug resistance. We chose resistance to tetracycline as a marker of these changes because of its relatively high prevalence in the region and an expected traceable allelic variation of genetic determinants. The prevalence of resistance to antipneumococcal drugs observed in this study was similar to those described in other reports from Poland prior to and during the time when the isolates were collected. In all of these, lack of susceptibility to co-trimoxazole and tetracycline were the

K. Dzier˙zanowska-Fangrat et al. / International Journal of Antimicrobial Agents 27 (2006) 159–164

most frequently observed resistance phenotypes in S. pneumoniae in the country, followed by lack of susceptibility to penicillin and macrolides [1–4]. Despite the fact that the lack of susceptibility to co-trimoxazole was the most common resistance type, analysis of antibiotic patterns revealed that it was not a significant component of multidrug resistance. There was a strong correlation observed between tet(M)mediated resistance to tetracyclines, erm(B)-mediated resistance to macrolides (and lincosamides) and resistance to penicillin. Two of these, resistance to penicillin and to macrolides, are already known to be strongly related in S. pneumoniae strains [21,22]. The presence of these three mechanisms of resistance observed almost exclusively in isolates of epidemic MDR strains indicates that a vertical (clonal) spread of resistances was responsible for an increase in multidrug resistance in the region. In Poland, tetracyclines were, after penicillins, the second most frequently prescribed antibiotics in the 1990s [23]. Although tetracyclines are not first-choice antimicrobials for the treatment of pneumococcal infections, it is likely that S. pneumoniae, frequently colonising humans, was overexposed to these drugs [24]. Genetic determinants of tetracycline resistance are mediated by transposons, often carrying other resistance genes, particularly erm(B). The pressure from tetracyclines selects not only tet(M) but becomes a significant factor in selecting resistance to other antibiotics, as has previously been documented for some Gram-negative bacteria [25]. Although, with the exception of isolate ST17, multidrug resistance was observed only in isolates of epidemic clones, some components of multidrug resistance were present in non-epidemic isolates (2739 and ST59A), indicating that both are prospective candidates for multidrug resistance. Of these, isolate 2739 carried tet(M) allele III of epidemic Poland6B -20 and Spain23F -1 clones, and of Spain serotype 15 national clone [6]. This indicates enhanced promiscuity of a mobile element(s) spreading horizontally in the investigated population. Allele III might be the most successful of all identified so far. Another allele overrepresented in the analysed population was the newly described allele X, present in five unrelated strains. With one exception of isolate ST59A, all isolates carrying this allele were susceptible to other antibiotics tested. Its presence in unrelated isolates gives evidence for its wide horizontal distribution, suggesting its predominance in the region prior to the introduction of MDR clones. With the exception of ST59A, tetracycline MICs for carriers of allele X were lower than those observed among isolates harbouring allele III. A higher level of resistance to tetracyclines and the co-existence of macrolide resistance may favour allele III in its clonal and possible horizontal spread. Since there is a strong relationship between resistance to macrolides and tetracyclines in MDR strains, and traceable heterogeneity within tet(M), analysis of the polymorphisms within tet(M) can potentially be used as a marker of changes in mobile genetic elements carrying the very conserved erm(B) determinant.

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It is interesting to note that the emergence of epidemic MDR strains with their new tet(M) alleles did not increase the overall prevalence of resistance to tetracyclines in the region, most probably reflecting an equilibrium between antibiotic pressure and the cost of resistance. Isolate ST28 showed a tetracycline-susceptible phenotype but was positive for the presence of the tet(M) gene. Streptococcus pneumoniae carrying tet(M) but susceptible to tetracyclines have previously been described, however the mechanisms and significance of this phenomenon are still unknown [26–28]. Unlike in the previously published study on tet(M) diversity in pneumococci, isolates analysed here were not preselected for clonality or for multidrug resistance [6]. Our results highlight the importance of tet(M)-mediated resistance to tetracyclines as a crucial component of multidrug resistance in S. pneumoniae. In addition, they reveal the plasticity of this genetic determinant evolving with the bacterial population at the time of emergence and spread of multidrug resistance.

Acknowledgments This work was supported by Polish Scientific Research Committee grant 6PO5D 006 20. We thank Noman Siddiqi for help in editing the manuscript for English.

References [1] Hryniewicz W, Trzcinski K, Tyski S, Zareba T, Jeljaszewicz J. Antimicrobial susceptibility patterns of some important pathogens isolated in Poland in 1991–1993. Zentralbl Bakteriol 1994;281:263–9. [2] Jacobs MR, Felmingham D, Appelbaum PC, Gruneberg RN. The Alexander Project 1998–2000: susceptibility of pathogens isolated from community-acquired respiratory tract infection to commonly used antimicrobial agents. J Antimicrob Chemother 2003;52:229–46. [3] Semczuk K, Dzierzanowska-Fangrat K, Lopaciuk U, Gabinska E, Jozwiak P, Dzierzanowska D. Antimicrobial resistance of Streptococcus pneumoniae and Haemophilus influenzae isolated from children with community-acquired respiratory tract infections in Central Poland. Int J Antimicrob Agents 2004;23:39–43. [4] Izdebski R, Sadowy E, Hryniewicz W. Molecular mechanisms of tetracycline resistance and clonal diversity of Streptococcus pneumoniae isolated from respiratory tract infections in Poland. In: 15th European Congress of Clinical Microbiology & Infectious Diseases. Copenhagen, Denmark; 2–5 April 2005. [5] Overweg K, Hermans PW, Trzcinski K, Sluijter M, de Groot R, Hryniewicz W. Multidrug-resistant Streptococcus pneumoniae in Poland: identification of emerging clones. J Clin Microbiol 1999;37:1739–45. [6] 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. [7] Sadowy E, Zhou J, Meats E, Gniadkowski M, Spratt BG, Hryniewicz W. Identification of multidrug-resistant Streptococcus pneumoniae strains isolated in Poland by multilocus sequence typing. Microb Drug Resist 2003;9:81–6.

164

K. Dzier˙zanowska-Fangrat et al. / International Journal of Antimicrobial Agents 27 (2006) 159–164

[8] Connell SR, Tracz DM, Nierhaus KH, Taylor DE. Ribosomal protection proteins and their mechanism of tetracycline resistance. Antimicrob Agents Chemother 2003;47:3675–81. [9] 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. [10] Clewell DB, Flannagan SE, Jaworski DD. Unconstrained bacterial promiscuity: the Tn916–Tn1545 family of conjugative transposons. Trends Microbiol 1995;3:229–36. [11] McDougal LK, Tenover FC, Lee LN, et al. Detection of Tn917-like sequences within a Tn916-like conjugative transposon (Tn3872) in erythromycin-resistant isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother 1998;42: 2312–8. [12] Oggioni MR, Dowson CG, Smith JM, Provvedi R, Pozzi G. The tetracycline resistance gene tet(M) exhibits mosaic structure. Plasmid 1996;35:156–63. [13] Murray PR, Baron EJ, Tenover FC, Yolken RH. Manual of clinical microbiology. 7th ed. Washington, DC: ASM Press; 2005. [14] McDaniel LS, McDaniel DO, Hollingshead SK, Briles DE. Comparison of the PspA sequence from Streptococcus pneumoniae EF5668 to the previously identified PspA sequence from strain Rx1 and ability of PspA from EF5668 to elicit protection against pneumococci of different capsular types. Infect Immun 1998;66:4748– 54. [15] National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. Document M7-A5. Wayne, PA: NCCLS; 2000. [16] Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 1996;40:2562–6. [17] Nei M, Li WH. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 1979;76:5269–73.

[18] van Belkum A, Kluytmans J, van Leeuwen W, et al. Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains. J Clin Microbiol 1995;33:1537–47. [19] Elliott JA, Farmer KD, Facklam RR. Sudden increase in isolation of group B streptococci, serotype V, is not due to emergence of a new pulsed-field gel electrophoresis type. J Clin Microbiol 1998;36:2115–6. [20] Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233–9. [21] McCormick AW, Whitney CG, Farley MM, et al. Geographic diversity and temporal trends of antimicrobial resistance in Streptococcus pneumoniae in the United States. Nat Med 2003;9:424–30. [22] Bruinsma N, Kristinsson KG, Bronzwaer S, et al. Trends of penicillin and erythromycin resistance among invasive Streptococcus pneumoniae in Europe. J Antimicrob Chemother 2004;54:1045–50. [23] Kotula Z, Wasiewicz A. The pharmaceutical market in Poland (1994–1996)—comparison of drug utilisation data. Terapia i Leki 1997;24:149–58. [24] Austrian R. Some aspects of the pneumococcal carrier state. J Antimicrob Chemother 1986;18(Suppl. A):35–45. [25] Levy SB. Evolution and spread of tetracycline resistance determinants. J Antimicrob Chemother 1989;24:1–3. [26] Corso A, Severina EP, Petruk VF, Mauriz YR, Tomasz A. Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates causing respiratory disease in the United States. Microb Drug Resist 1998;4:325–37. [27] Porat N, Leibovitz E, Dagan R, et al. Molecular typing of Streptococcus pneumoniae in northeastern Romania: unique clones of S. pneumoniae isolated from children hospitalized for infections and from healthy and human immunodeficiency virus-infected children in the community. J Infect Dis 2000;181:966–74. [28] Setchanova L, Tomasz A. Molecular characterization of penicillinresistant Streptococcus pneumoniae isolates from Bulgaria. J Clin Microbiol 1999;37:638–48.