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Antimicrobial Agents and Chemotherapy, March 2008, p. 1001-1008, Vol. 52, No. 3
0066-4804/08/$08.00+0     doi:10.1128/AAC.00999-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Diversity of Tn1546 and Its Role in the Dissemination of Vancomycin-Resistant Enterococci in Portugal

Carla Novais,^1,2 Ana R. Freitas,¹ João C. Sousa,² Fernando Baquero,^3,4 Teresa M. Coque,^3,4, and Luísa V. Peixe^1,*

REQUIMTE, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal,¹ Laboratório de Microbiologia, Faculdade Ciências da Saúde, Universidade Fernando Pessoa, Porto, Portugal,² Servicio de Microbiologia, Hospital Universitario Ramón y Cajal-CIBER-ESP, Madrid, Spain,³ Unidad de Resistencia a Antibióticos y Virulencia Bacteriana (RYC-CSIC), Madrid, Spain⁴

Received 31 July 2007/ Returned for modification 29 October 2007/ Accepted 26 December 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We characterized the molecular diversity of vanA vancomycin-resistant enterococci (VRE; 176 isolates/87 pulsed-field gel electrophoresis types) from different sources and cities in Portugal (1996 to 2004): (i) food animals (FA; n = 38 isolates out of 31 samples), hospitalized humans (HH; n = 101/101), healthy human volunteers (HV; n = 7/4), and environmental sources (n = 30/10). Some strains were isolated from different hosts and persistently recovered for years. Twenty-four Tn1546 variants were identified, all located on plasmids (30 to 250 kb). Some Tn1546 variants were associated with specific sources such as FA (3 types), HH (11 types), or HV (1 type), while others were recovered from isolates of different origins (8 types). Polymorphisms in the central vanRSHA region of Tn1546 were scarcely detected, while alterations upstream of vanR and downstream of vanA were frequently identified involving mutations (vanS and vanX), deletions (vanY), insertions (IS1216V, ISEf1, and IS19; sequences with or without homology with others available in GenBank databases), and different genetic rearrangements. Most Tn1546 variants contained IS1216V (14 types) or ISEf1 (6 types). IS1216V was found alone or associated with an IS3-like element at different orientations and positions in Tn1546 from human, animal, and environmental samples. ISEf1 was located within vanX-vanY region at nucleotide 9044 of Tn1546 variants mostly associated with clinical isolates, suggesting a common genetic platform. IS19 was observed within the vanX-vanY region in one Tn1546 variant from poultry. Recent spread of VRE in Portugal reflects a complex epidemiology involving both clonal spread and plasmid dissemination containing a variety of Tn1546 types. Apparent Tn1546 heterogeneity among enterococci from human, animal, and environmental sources might reflect frequent genetic exchange events and evolution of particular widely disseminated genetic elements.

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Since their first description in 1988, vancomycin-resistant enterococci (VRE) have been isolated from hospitalized patients, healthy humans, and a wide variety of mammals, insects, and environmental sources (8, 40). Five types of acquired glycopeptide resistance are currently recognized in enterococci (VanA, VanB, VanD, VanE, and VanG), of which VanA is the most commonly found (8). Some of them may be transferred in vivo and in vitro to other bacterial genera such as Bacillus, Streptococcus, Clostridium, Oeskorvia, and Staphylococcus (8), highlighting the concern about the spread of glycopeptide resistance to more virulent human pathogens.

The best-characterized mechanism of resistance is VanA. The genes forming the VanA operon are part of a 10.8-kb Tn3 derivative designated Tn1546, often located on conjugative plasmids (8, 12, 34, 41, 42). Variants of this transposon containing mutations, deletions, and different insertion sequences have been reported, and a presumptive association between specific Tn1546 types and geographical areas and/or environmental sources has been suggested (4, 15, 19, 32, 38, 44, 45). Nevertheless, the epidemiology of VanA is far from being understood as different situations occur in different locations and settings (23). In Europe, the initial VRE scenario consisted of polyclonal enterococcal population with a high diversity of Tn1546 types in the community and with scarce presence in the nosocomial setting (4, 19, 38). However, this epidemiological pattern seems to have changed in the last few years. Five European countries have reported prevalence rates in hospitals of >20%; therefore, frequent reports of nosocomial VRE outbreaks in most countries and a variable presence of these bacteria in community settings have been described (23). Portugal, currently one of the European nations with the highest VRE prevalence rate in invasive isolates in the nosocomial setting (34% in the last 2005 Annual Report of the European Antibiotic Resistance Surveillance System; http://www.earss.rivm.nl), has a frequent recovery rate in animals, healthy humans, and the environment, as seen in previous surveillance studies from our group, where van genes were screened, between 1996 and 2004, among 1,192 enterococci from different sources and geographical areas in Portugal (28-31, 36). In those studies, we described dissemination of specific vancomycin-resistant Enterococcus faecalis and Enterococcus faecium strains among hospitals of different geographical regions or poultry samples from distinct brands and butcher shops for long periods of time and identified the genetic elements harboring vanA in specific isolates (29, 31, 32).

The goal of the present study was to analyze in detail the molecular diversity of Tn1546 in our large collection of enterococci from different sources in order to better understand the ecology of VRE and the possible reasons explaining the recent fast spread of these microorganisms in Portugal.

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Bacterial strains. Tn1546 was characterized in 176 vanA isolates corresponding to 133 E. faecium, 38 E. faecalis, and 5 Enterococcus sp. isolates recovered from the following samples: (i) swabs or meat samples from the inner carcass of retail poultry of nine different commercial brands sold throughout the country, which were collected from two different butcher shops (34 isolates from 27 samples; n = 34/27); (ii) fecal swine samples from Figueira da Foz (center of Portugal; n = 4/4); (iii) human fecal samples from healthy volunteers living in the center and northern part of the country (n = 7/4); (iv) hospital sewage water from the Porto area (n = 22/6) and from urban sewage water draining directly to the sea in a recreational bathing area (n = 2/2); (v) river water samples from the Porto area (n = 6/2); and (vi) human clinical samples from different patients at three hospitals in the center (Coimbra and Viseu) and north of Portugal (Porto) (n = 101 isolates). All isolates were resistant to vancomycin (MIC range, 32 to 256 µg/ml), and 155 out of 176 (88%) were resistant to teicoplanin. Eighty-seven pulsed-field gel electrophoresis (PFGE) profiles were included and were observed among 173 isolates tested (three isolates were not typed): 49 from clinical isolates (49/101 isolates), 24 from poultry (24/34), 2 from swine (2/4), 2 from the river (2/5), 5 from healthy humans (5/7), and 8 from hospital sewage (8/22). Some of the clinical PFGE types (HB, H70, H71, H76, H78, H88, and H100) were isolated from hospitals of different geographical regions and sometimes persisted for long periods of time (clinical PFGE types HB, H70, H78, and H88 and poultry PFGE types P1, P2, P3, P4, and P22) (29, 31, 32). Transference of vancomycin resistance to E. faecalis JH2-2 or E. faecium GE1 was achieved by conjugation in 90% of the strains included in the present study (28-32).

Characterization of Tn1546 elements. The backbone structure of Tn1546 was determined by the PCR-overlapping scheme described by Woodford et al. (45) using other combinations of primers and PCRs when necessary (Fig. 1). Some Tn1546 types from human samples and sewage have previously been characterized by using this PCR-overlapping assay (29, 30, 32). In this study, we analyzed and compared in detail Tn1546 from different sources. Specific mutations at vanX were searched by analysis of restriction fragment length polymorphism patterns of vanX digested with DdeI (35). Mutations in vanS among strains exhibiting teicoplanin susceptibility were analyzed by sequencing. Characterization of PCR-overlapping fragments of unusual size was performed by sequencing in representative strains harboring different Tn1546 sequences or isolated from different animal, human, or environmental sources. PCR products were purified with the GFX PCR purification kit (Amersham Biosciences) and sequenced on an ABI PRISM 377 automated sequencer (PE Biosystems, Foster City, CA). Nucleotide sequences were compared to those in the GenBank databases by using the BLASTN local alignment search tools.

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FIG. 1. Primers used for Tn1546 PCR characterization and genetic maps of Tn1546 types grouped by the presence of common insertion sequences. Primers used were based on the scheme described by Arthur et al. (1). Several other combinations of primers and PCRs represented in the scheme were used when necessary with 132R, 650F, 4511R, vanS, vanX, and ISEf1 primers (18, 19, 27, 32). Tn1546 prototype A corresponds to the original sequence described by Arthur et al. (1). The positions of genes and open reading frames and the direction of transcription are depicted with open arrows. IS elements are represented by triangles; other sequences are designated by rectangles. The positions of the first nucleotide upstream and downstream from the DNA insertion sites are depicted. Deletions are indicated by dots, and sequences that were not characterized are indicated by dots and small lines. Mutations are indicated above each represented Tn1546 type. a, acetyltransferase (GenBank accession no. AY887084); b, DNA sequence did not match any sequence available in GenBank; c, the positions of IS1216V and the vanZ gene were not determined.

Location of Tn1546. Plasmid location of vanA genes was assessed by the method of Barton hybridizing S1 nuclease-digested genomic DNA with a vanA probe following standard procedures (2, 37). Labeling and detection were carried out with ECL enhanced chemiluminescence kits following the manufacturer's instructions (Amersham Life Sciences, Uppsala, Sweden).

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Characterization of Tn1546 elements. The PCR-overlapping assay revealed the presence of 24 variants of Tn1546 (Table 1 and Fig. 1) (28-30, 32). Some Tn1546 variants were only recovered from samples of specific sources such as poultry (types S, PP-1, and PP-11), hospitalized humans (types PP-2, PP-4, PP-9, PP-10, PP-13, PP-23, PP-24, PP-25, PP-27, PP-28, and PP-29), or healthy humans (type PP-6). Other Tn1546 types were detected among isolates of different origins, such as types A, D, X, PP-3, PP-5, PP-15, PP-16, and PP-20 (Table 1).

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TABLE 1. Tn1546 types, PFGE type, date of isolation, source, city, species, range of MICs for glycopeptides, and conjugation frequency of the VRE characterized

We did not find any polymorphism or size variation in the central regions of Tn1546 (vanRSHA) for any of the enterococci studied, with the exception of types A and PP-20. However, alterations upstream of vanR and downstream of vanA were frequently detected (Fig. 1).

Different insertion sequences were identified among Portuguese Tn1546 variants: IS1216V, IS3, ISEf1, and IS19. IS1216V was found alone or associated with an IS3-like element at different orientations and positions in 14 Tn1546 variants from human, animal, and environmental samples. It was located at nucleotide (nt) 3916 and/or 8839 of Tn1546 in seven variants (types X, PP-13, PP-17, PP-20, PP-23, PP-25, and PP-29). This insertion sequence was associated with deletion of vanY (types PP-13, PP-20, PP-25, and PP-29), with duplication of the vanZ region in the 5' end of the transposon (types D1, PP-17, PP-20, and PP-25), or with duplication of vanX genes in the right side of Tn1546 (PP-23). ISEf1 was located within the vanX-vanY region at nt 9044 of Tn1546 in six variants (types PP-2, PP-4, PP-5, PP-6, PP-9, and PP-24), suggesting a common origin. Two nucleotide changes within the right inverted repeat and transposase regions of ISEf1 were identified in relation to previously detected sequences (changes GA and CT at positions 269144 and 269763, respectively; GenBank accession no. AE016830). The insertion of ISEf1 was associated with the duplication of GACTGAAA sequence at bp 9044 to 9051 of Tn1546 (GenBank accession no. EF064799). IS19, also known as ISEfm1, was also observed within the vanX-vanY region in Tn1546 type S recovered from poultry isolates (Fig. 1).

Other sequences were identified within transposase or vanS regions. Some of them showed homology with known sequences as Tn5405 is widely distributed among enterococcal plasmids (41), with a putative resolvase from streptococcal plasmid pRE25, with IS1062 found in the genome of E. faecalis strain V583, or with a putative N-acetyltransferase found in the genome of E. faecium strain DO (GenBank accession no. SAU73027, X92945, X96976, and EAN11147, respectively). Other regions identified within Tn1546 types did not match any sequence available in the GenBank database (within Tn1546 types D4, X, PP-10, PP-23, and PP-29; Fig. 1).

Mutations were also identified among different Tn1546 types. Two variants of Tn1546 type A (this type is identical to the original Tn1546) from single swine and poultry isolates showed mutations in vanS which were susceptible to teicoplanin (MIC, <4 mg/liter) and were designated as types A2 and A3, respectively. Type A2 contained the changes G4796T, C5803G, and T5808A, previously described by Hashimoto et al. in Tn1546 from Japan (16), and type A3 harbored a silent mutation, C5356G. Some isolates carrying type D from swine and healthy humans contained the nucleotide change G8234T within vanX, which was also described in Tn1546 from animals of different European countries such as the United Kingdom, Denmark, Finland, The Netherlands, and Norway (35).

Location of Tn1546. Hybridization of S1 nuclease-digested genomic DNA with a vanA probe showed that this gene was located on plasmids ranging from approximately 30 to 250 kb.

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VRE spread in Portugal is associated with both epidemic strains and horizontal transfer of Tn1546 (28-30, 32; this study). In contrast with other European studies from Italy and Denmark (3, 14), we found an apparent high diversity of Tn1546 in Portugal. Tn1546 heterogeneity has been previously described (18, 19, 38, 44, 46), but only a few works included a representative number of strains from different origins in a particular area, thus precluding conclusions about local epidemiological dynamics (3, 45). Difficulties also arise from the different methods used for this purpose, which complicates comparison among studies (14, 44, 45).

Tn1546 may be located in conjugative plasmids (14, 20, 25, 43, 47) or in larger composite transposons, as documented for isolates with chromosomally located vanA (15). Horizontal transfer of conjugative plasmids containing Tn1546 seems to have played a relevant role in the recent increase of VRE in Portugal, as vanA was plasmid located in most VRE isolates studied and specific plasmids containing the most prevalent Tn1546 variants were identified in representative distinct clones collected from different cities and years (13). Nevertheless, the role of prevalent and persistent E. faecalis and E. faecium strains in the maintenance of the resistance by acquiring different genetic elements must not be underestimated, highlighting the complex epidemiology associated with successful spread of multidrug-resistant enterococci, as illustrated in Poland and the United States, where both successful plasmids and prevalent clones contributed to endemicity (11, 21, 22). Plasmid persistence has been described in areas with both high and low antibiotic selective pressure, as in the United States versus Norway or Denmark, suggesting that factors other than antibiotic pressure might be important in VRE maintenance and epidemiology (17, 20).

The diversity of Tn1546 observed in the present study was attributed to the occurrence of insertions, deletions, and point mutations. Two insertion sequences, IS1216 (IS6 family) and ISEf1 (IS30 family), were the most frequently detected insertion sequences within Tn1546. IS1216V has been extensively found among European transposons containing vanA from different sources and detected at different positions and orientations, as in the present study (18, 38, 44, 46). We identified known insertions of IS1216V such as that associated with a deletion of vanY, which is believed to affect the transcription of vanZ, thus explaining a decrease in MICs of teicoplanin (24, 44), or that detected within vanS in PP-20 (10, 26). In addition, we observed different rearrangements resulting in the duplication of vanZ (at the 5' end) or vanX (at the 3' end) of Tn1546 variants containing IS1216V, some of them previously identified among English isolates (9). IS1216V is one of the main insertion sequences found in the E. faecalis genome and also among enterococcal plasmids widely disseminated in the community (39) (GenBank accession no. AF408195). The diversity found among Tn1546 variants containing this IS might be explained by genetic exchange between sequences containing IS1216, as IS6 family members are involved in mechanisms associated with cointegrative processes and replicative transposition (6, 33). Interestingly, Tn1546 types containing IS1216 (D, X, PP-1, PP-2, PP-11, PP-13, PP15, PP-16, PP-17, PP-20, PP-23, PP-25, PP-28, and PP-29) were isolated from different sources, which might mirror wide dissemination of vancomycin resistance plasmids which have evolved by different recombinatorial events throughout different hosts. Transposons with the same structure in animals and human enterococci have also been described in the United Kingdom and The Netherlands (35, 44), also suggesting horizontal transfer of widespread conjugative plasmids, as recently demonstrated (25).

ISEf1 was first identified within Tn1546 among clinical Portuguese strains (32), but these Tn1546 variants might be currently dispersed among European hospitals as they have recently been identified in hospital outbreak enterococcal strains from Spain and Germany (43; V. Francia, personal communication). In contrast to IS1216V, ISEf1 was always detected at the same position (within the vanX-vanY region at nt 9044) and Tn1546 variants containing ISEf1 were mostly associated with clinical isolates, which may reflect successful spread of particular plasmids in the clinical setting. Although hospitalized patients seem to constitute the main reservoir of these Tn1546 types, the isolation from a single healthy human without previous contact with the hospital setting, from hospital residual waters, and from river waters indicates that a potential dissemination of genetic elements harboring these transposon types in the Portuguese community cannot be discarded.

IS19 (also called ISEfm1 and belonging to the IS982 family) has been found in clinical enterococcal isolates from different continents and located in a variety of genetic elements, including VanD and VanA clusters (5, 18) (GenBank accession no. AF169185 and AY887084). We first detected this insertion sequence in enterococci from poultry and in a different position from that described in Tn1546 from Korea (within vanX-vanY instead of vanS-vanH). This IS family is spread among gram-positive organisms, and its presence among a variety of enterococcal genetic elements and strains might indicate genetic exchange among gram-positive bacteria sharing the same ecological compartment.

Some studies including isolates from different areas have suggested certain host and geographical specificity of particular Tn1546 variants (4, 38, 44). Diverse insertion sequences have been identified within Tn1546, but this heterogeneity has perhaps been overestimated as the genetic background of transposons has not been analyzed in most studies. Particular prevalent plasmids may contain different Tn1546 variants as a consequence of recombinatorial events or genetic exchange with genetic elements of microorganisms belonging to the same exchange communities in particular areas, as we demonstrated for prevalent Tn1546 combinations (7, 13).

In summary, both clonal dissemination and horizontal transfer of particular genetic elements seem to play a relevant role in the recent wide dissemination of VRE in Portugal. The high diversity of Tn1546 types among enterococci isolated from human, animal, and environmental sources may reflect frequent genetic exchange events within particular widely disseminated genetic elements and microorganisms. Detailed characterization of enterococcal plasmids becomes relevant in order to understand differences in the epidemiology of VRE in different geographical areas and settings.

 
This work was partially supported by grants from Fundação para a Ciência e Tecnologia (POCTI/SAU-ESP/61385/2004 and POCI/AMB/61814/2004), from Ministerio de Educación y Ciencia of Spain, Programa Acciones Integradas Hispano-Portuguesas, (H2004-0092), and from the European Union (LSHE-2007-037410).

 
* Corresponding author. Mailing address: Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 4050 Porto, Portugal. Phone: 351-2-22078946. Fax: 351-2-22003977. E-mail: lpeixe{at}ff.up.pt

Published ahead of print on 7 January 2008.

L.P. and T.M.C. contributed equally to the direction, preparation, and writing of the manuscript.

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Antimicrobial Agents and Chemotherapy, March 2008, p. 1001-1008, Vol. 52, No. 3
0066-4804/08/$08.00+0     doi:10.1128/AAC.00999-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Freitas, A. R., Novais, C., Ruiz-Garbajosa, P., Coque, T. M., Peixe, L. (2009). Clonal expansion within clonal complex 2 and spread of vancomycin-resistant plasmids among different genetic lineages of Enterococcus faecalis from Portugal. J Antimicrob Chemother 63: 1104-1111 [Abstract] [Full Text]  

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