Molecular Analysis of tet(W) Gene-Mediated Tetracycline Resistance in Dominant Intestinal Bifidobacterium Species from Healthy Humans

tet(W) was found responsible for tetracycline resistance (MICs,4 to $≥$ 32 µg ml^–1) in dominant bifidobacterial speciesfrom the gastrointestinal tracts of healthy humans. The genefrom Bifidobacterium longum H66 proved to be identical overa 2.6-kbp region to the recently described tet(W) determinantof Butyrivibrio fibrisolvens.

INTRODUCTION

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Introduction

The commensal intestinal microbiota of humans and animals mayact as a reservoir of antibiotic resistance genes that couldultimately be transferred to pathogens (15, 18); in fact, genetransfer between bacterial species in the gastrointestinal tracts(GIT) of mammals is known to occur (11). Bifidobacteria areamong the dominant populations of the human GIT microbiota,where they are thought to play a pivotal role in maintainingthe microbial balance necessary for intestinal health (19).Bifidobacterial strains are therefore frequently used as probioticsin the prophylaxis and therapy of GIT disorders (12). This practice,however, requires that they be screened for acquired antibioticresistance determinants if the latter are not to be propagatedthrough the food chain (14, 15, 18).

Tetracyclines inhibit protein synthesis by preventing the attachmentof aminoacyl-tRNA to the bacterial ribosome (2). The broad-spectrumantimicrobial properties of these agents, the absence of majoradverse side effects, and their low price have led to theirintensive use not only in the treatment of human and animalinfections but also as prophylactic agents and growth promotersin livestock raising and aquaculture (2). This extensive usehas promoted the appearance of resistance and its spread byhorizontal acquisition (2, 13). Resistance is mediated throughefflux proteins, ribosomal protection proteins, or antibiotic-inactivatingenzymes (for a review, see reference 13).

The tet(W) gene, which encodes a ribosomal protection protein,has recently been described for a wide range of gram-positiveand gram-negative bacteria (1, 2), including bifidobacteria(7, 10, 17). This gene has been associated with a conjugativetransposon (TnB1230) in Butyrivibrio fibrisolvens (9) that hasbeen shown to transfer at high frequencies (up to 5.1 x 10^–3transconjugants per recipient) (16).

In a recent survey, a microbroth assay showed atypical ( $≥$ 16 µgml^–1) MICs of tetracycline for human bifidobacterial strainsisolated from fecal samples of healthy adults without a recenthistory of antibiotic treatment (4). The object of this studywas to investigate the nature and molecular organization ofthe tetracycline determinant encoding this resistance.

MICs of tetracycline for bifidobacteria.

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Mics of tetracycline for...

The MICs of tetracycline for a series of susceptible (14 isolates)and resistant (16 isolates) bifidobacterial species which hadbeen assayed by microdilution (4) were precisely determinedby Etest (AB Biodisk, Solna, Sweden). Assays were performedin LSM test agar (6) (90% IsoSensitest, 10% MRS, 1.5% agar;all from Oxoid, New Hampshire, United Kingdom) supplementedwith 0.3 g of cysteine liter^–1. Plates were incubatedat 37°C for 48 h in an anaerobic chamber (atmosphere, 10%H₂-10% CO₂-80% N₂). The MICs for susceptible isolates rangedfrom 0.25 to 1 µg ml^–1, whereas those for isolatesthought to be resistant ranged from 4 to 32 µg ml^–1.MICs obtained for the same strains in duplicate experimentsnever exceeded 1 order of dilution.

To exclude the analysis of replicates, resistant and susceptibleisolates were typed by rapid amplification of polymorphic DNA(RAPD) analysis by PCR. Amplifications were performed usingprimer OPA18 and PCR conditions described elsewhere (8). Sevendifferent RAPD-PCR profiles identifying distinct strains wereobtained among the resistant isolates, as follows: Bifidobacteriumlongum (11 isolates, 5 strains), Bifidobacterium bifidum (5isolates, 1 strain), and Bifidobacterium animalis subsp. animalis(1 isolate, 1 strain).

Amplification and analysis of tetracycline resistance genes.

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Amplification and Analysis of...

The presence of genes encoding ribosomal protection proteinswas checked among resistant and susceptible strains by PCR withtwo pairs of degenerate primers: DI-DII (3) and Tet1-Tet2 (1).Resistant strains all yielded a single PCR product of the samesize when either of the primer pairs was used. Surprisingly,positive amplification was also obtained with DNA from the susceptiblestrain B. longum M21. Additional PCR assays were then performedwith primers TetWF and Tet2, specific for tet(W) (17), and primersDI and TetMR, specific for tet(M) (5). No DNA was amplifiedfrom either resistant or susceptible isolates with primers fortet(M). In contrast, a product of about 1,250 bp was obtainedfor all 16 resistant isolates when primers for tet(W) were used.Purified amplicons were sequenced and the resulting sequencesanalyzed and compared to others held in public databases.

Analysis of the nucleotide sequences showed them to share >99.8%identity. Comparison of the sequences showed a high degree ofsimilarity at the nucleotide level to several tetracycline resistancegenes. In particular, complete identity was seen with an internalsegment of the tet(W) gene from B. fibrisolvens (1). Based onthis homology, primers TetWSacF (5'-CCCTGGAGCTCATGCTCATGTAC-3')and TetWSacR (5'-CCATCGGAGCTCCATAACTTCTG-3') were designed toamplify the whole gene and its surrounding promoter and terminatorregions. Positive amplification was obtained only when DNA fromthe resistant strains was used as a template, suggesting thatthe susceptible strain may harbor a shortened (nonfunctional)version of the gene.

The amplicon was cloned into pUC19 and introduced into Escherichiacoli, which became resistant to tetracycline (MIC, 64 µgml^–1). The insert was then double-strand sequenced. Atthe nucleotide level, analysis of a 2,589-bp segment showedthat the sequence was identical to that of B. fibrisolvens 1.230(1, 9), except for a single oligonucleotide change in the codingregion leading to a conservative amino acid replacement in itsdeduced Tet(W) protein. Further, the B. longum H66 sequencewas found to share around 98% identity to recently reportedtet(W) genes from bifidobacterial species (GenBank accessionno. AF202986) (7, 17). A phylogenetic tree showing the relationshipsof the deduced Tet(W) proteins and those of other ribosome protectionproteins is depicted in Fig. 1. The presence of resistance geneswith nearly identical nucleotide and protein sequences in unrelatedbacterial and bifidobacterial species suggests that recent horizontaltransfer has occurred (1, 16). Intriguing is the fact that differentMICs are repeatedly reported for identical genes (7, 10, 17).Multiple loci, different expression levels of tet(W), and/orthe influence of the genetic background of the strains may accountfor the phenotypic differences.

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FIG. 1. Phylogenetic tree showing the evolutionary relationships of complete Tet(W) and other ribosome protection proteins from tetracycline-resistant bacteria. GenBank accession numbers for the nucleotide sequences used to derive amino acid sequences are given in parentheses. The rooted neighbor-joining tree was generated using DNAman software (Lynnon Corporation, Vaudreuil-Dorion, Quebec, Canada). Numbers beside nodes indicate bootstrap values when these are greater than 95% (based on 500 trials). Bar, 0.05% difference in amino acid sequence.

Location of the tet(W) gene.

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Location of the tet(w)...

The genetic location of tet(W) in the distinct species was assessedby hybridization using as a probe a 1.2-kbp internal segmentof the gene obtained by PCR and labeled with digoxigenin (RocheApplied Science, Basel, Switzerland). Total and plasmid DNAsdigested with the restriction enzymes EcoRI and HindIII (Roche)were hybridized using high-stringency standard conditions (hybridizationat 68°C and two final washing steps in 0.5x SSC [1x SSCis 0.15 M NaCl plus 0.015 M sodium citrate]-0.1% SDS at 68°Cfor 15 min). Figure 2 shows the results obtained with EcoRI-digestedtotal DNA. A positive hybridization signal was obtained withall tetracycline-resistant strains and, as expected, with thesusceptible strain M21. The hybridization signal appeared ata different position in each of the strains (estimated sizes,2.5 to 16 kbp), except for the related strains B. longum B93and B94 (displaying distinct RAPD-PCR patterns but isolatedfrom the same individual). This suggests that the genetic organizationof the loci might be different in each strain. The tet(W) locusis thought to be located on the bacterial chromosome, sinceonly the resistant strain B. bifidum L71 was shown to harbora plasmid (of around 15 kbp), and its hybridization signalsdid not match the position of EcoRI and HindIII plasmid fragments(data not shown).

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FIG. 2. Hybridization of EcoRI-digested chromosomal DNA from different Bifidobacterium strains using a 1.2-kbp PCR amplicon obtained with the specific primers for tet(W) as a probe. The code numbers of the strains are given above the lane numbers. Tetracycline-susceptible strains are asterisked. Lanes 1, 2, 3, 4, 5, 6, and 7, Bifidobacterium longum strains; lane 8, Bifidobacterium animalis E43; lanes 9 and 10, Bifidobacterium bifidum strains. Lanes M, molecular weight marker (digoxigenin-labeled, EcoRI- and HindIII-digested lambda DNA).

Since the tet(W) gene is encoded on a conjugative transposonin B. fibrisolvens 1.230 (TnB1230) (9, 17), primers based ontransposon sequences were also synthesized and used in PCR experiments.However, no amplification products were obtained with any strain,suggesting that there are no sequences related to this mobileunit in these Bifidobacterium species. Further, preliminaryinverse-PCR experiments indicate that tet(W) is inserted intoa putative open reading frame encoding a permease (GenBank accessionno. AE014295) of an ABC transporter system in B. longum NCC2705(data not shown).

In conclusion, these and other results indicate that dominantBifidobacterium species from the human GIT frequently harboracquired tetracycline resistance encoded by a tet(W) gene. Thefact that genes from Bifidobacterium showed more nucleotidechanges than those from species of different genera stronglysuggests independent transfer events. The spread of this geneamong cattle rumen and human GIT organisms may be occurringvery rapidly by unknown mechanisms that merit further research.However, preliminary conjugation experiments showed that tetracyclineresistance from B. longum H66 and L42 did not transfer to susceptiblestrains (data not shown). Once acquired, genes could eitherbe stably maintained in the absence of antibiotic selectionor selected by continued exposure to antibiotics through dietaryintake (5).

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The nucleotide sequence of the tet(W) gene and its surroundingregions from B. longum H66 has been deposited in GenBank (accessionno. DQ060146).

ACKNOWLEDGMENTS

This work was supported by an EU project within the VI FrameworkProgramme (ACE-ART, ref. CT-2003-506214). M.S.A. was awardeda postdoctoral fellowship from the "Secretaría de Estadode Universidades e Investigación" of the Spanish Ministryof Education and Science (SB2004-0165).

FOOTNOTES

* Corresponding author. Mailing address: Instituto de Productos Lácteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300-Villaviciosa, Asturias, Spain. Phone: 34 985 89 21 31. Fax: 34 985 89 22 33. E-mail: baltasar.mayo{at}ipla.csic.es.

Published ahead of print on 25 August 2006.

REFERENCES

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