J Antimicrob Chemother 2013; 68: 17 – 22doi:10.1093/jac/dks351 Advance Access publication 30 August 2012 TetAB(46), a predicted heterodimeric ABC transporter conferring tetracycline resistance in Streptococcus australis isolated from the oral cavity Philip J. Warburton †, Lena Ciric , Avigdor Lerner , Lorna A. Seville , Adam P. Roberts , Peter Mullany and Elaine Allan * Research Department of Microbial Diseases, UCL Eastman Dental Institute, 256 Gray's Inn Road, University College London, London WC1X 8LD, UK *Corresponding author. Tel: +44-(0)20-3456-1256; Fax: +44-(0)20-3456-1127; E-mail: e.allan@ucl.ac.uk †Present address: Department of Life Sciences, Faculty of Science and Technology, Anglia Ruskin University, Cambridge CB1 1PT, UK.
Received 7 July 2012; returned 25 July 2012; revised 31 July 2012; accepted 2 August 2012 Objectives: To identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis iso-lated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally iso-lated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosispatients and as a cause of invasive disease in an elderly patient.
Methods: Agar containing 2 mg/L tetracycline was used for the isolation of tetracycline-resistant organisms.
A genomic library in Escherichia coli was used to isolate the tetracycline resistance determinant. In-frame dele-tions and chromosomal repair were used to confirm function. Antibiotic susceptibility was determined by agardilution and disc diffusion assay.
Results: The tetracycline resistance determinant from S. australis FRStet12 was isolated from a genomic libraryin E. coli and DNA sequencing showed two open reading frames predicted to encode proteins with similarity tomultidrug resistance-type ABC transporters. Both genes were required for tetracycline resistance (to both thenaturally occurring and semi-synthetic tetracyclines) and they were designated tetAB(46).
Conclusions: This is the first report of a predicted ABC transporter conferring tetracycline resistance in amember of the oral microbiota.
Keywords: antibiotic resistance, oral microflora, oral streptococci major facilitator superfamily (MFS) in which proton-motive forceis used to drive efflux.However, other tetracycline efflux proteins Tetracyclines are broad-spectrum antibiotics that are used not belonging to the MFS group have been reported: (i) Tet(35) of extensively to combat bacterial infections in humans and Vibrio harveyiis a member of the H+ antiporter (NhaC) family animals, and have been used as growth promoters in animals, and confers resistance to tetracycline, oxytetracycline and agriculture and aquacultur– Bacterial resistance to tetracyc- minocycline; and (ii) OtrC of Streptomyces rimosus is predicted line is primarily mediated through acquired genes encoding to encode, on distinct polypeptides, the nucleotide-binding one of three main mechanisms: active efflux, ribosomal protec- domain (NBD) and membrane-spanning domain (MSD) typical tion proteins (RPPs), or enzyme-mediated drug inactivation.
of members of the ABC transporter family. No functional Within the oral cavity, ribosomal protection [e.g. tet(M), tet(Q) information is available for and tet(O)] is the most commonly observed mechanism, In this study, we characterize a novel tetracycline resistance whereas tetracycline-inactivating enzymes and efflux mechan- determinant in Streptococcus australis FRStet12, isolated from isms occur less fr pooled saliva from healthy French subjects as part of a study Tetracycline efflux systems have been reported in both investigating antibiotic resistance in bacteria colonizing adult Gram-negative and Gram-positive The best-studied We show that two proteins, each encoding predicted tetracycline efflux genes [e.g. tet(A) and tet(B)] encode mem- ABC transporter subunits, are both required for tetracycline brane-associated, energy-dependent proteins belonging to the # The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Forcommercial re-use, please contact journals.permissions@oup.com Warburton et al.
Materials and methods pVinto S. australis FRStet12 to provide selection for successfultransformation (ErmR).
Sample collection and culture Saliva samples (5 mL) were collected from 20 healthy adult volunteerswho had not received antibiotic therapy in the previous 3 months, from two centres in France (Faculte´ de Parmacie, Universite´ Paris Sud Genetic competence was induced in S. australis FRStet12 using a modi- and INRA-UEPSD, Domain de Vilvert), as previously descriThe fied version of the method reported by Hudson and A single samples were pooled in a sterile 200 mL Duran bottle and processed colony was inoculated into 10 mL of Todd –Hewitt broth (THB) containing within 48 h of collection. A 10-fold dilution series was prepared from 10% horse serum and incubated at 378C, in air+5% CO 1 mL of the sample in Luria–Bertani (LB) broth and spread onto Iso- 2, for 18 h. A 1/40 dilution of the overnight culture was grown in THB plus 10% horse serum Sensitest agar (Oxoid) supplemented with 5% defibrinated horse blood under the same conditions until the optical density at 600 nm was (E&O Laboratories, Bonnybridge, UK) and 2 mg/L tetracycline. The between 0.1 and 0.2. For co-transformation experiments, 1 mg of either plates were incubated in air enriched with 5% CO2 for up to 72 the DtetA(46) or DtetB(46) mutant fragments plus 250 ng of Growth at concentrations .2 mg/L is defined as resistant by the BSAC pVwere added to 1 mL of culture, mixed and incubated for 3 hin the CO2-enriched atmosphere. pVA838 is a streptococcal shuttleplasmid that carries an erythromycin resistance gene as a selectable Identification of the resistance genes markAliquots of 100 mL were then spread on to brain heart infusion Genomic DNA from S. australis FRStet12 was hybridized to a macroarray (BHI) agar supplemented with 5% defibrinated horse blood plus 10 mg/L containing 23 known tetracycline resistance genes [9 RPP genes—M, O, erythromycin and incubated at 378C, in air+5% CO2, for 24 h. Colonies B(P), Q, S, T, W, 32 and 36; 12 efflux genes—A, B, C, D, E, G, H, J, A(P), were then transferred to BHI agar plates supplemented with 5% defibri- Y, Z and 30; and 2 enzymatic inactivation genes—tet(X) and tet(34)], nated horse blood plus 10 mg/L erythromycin and replica plated on to as previously described.The genomic DNA was also tested for the pres- BHI agar plates supplemented with 5% defibrinated horse blood plus ence of RPP-encoding genes by PCR using a set of universal RPP 10 mg/L erythromycin and 5 mg/L tetracycline. All plates were incubated To clone the resistance genes, S. australis FRStet12 genomic DNA was at 378C, in air+5% CO2, for 24–48 h.
partially digested with HindIII, ligated into HindIII-digested, depho-sphorylated pUC19 and transformed into Escherichia coli JM109 compe-tent cells, according to the supplier's instructions (Promega). Bacteria Construction of plasmids for chromosome repair were spread on to LB agar supplemented with 100 mg/L ampicillin and5 mg/L tetracycline and incubated at 378C in air for up to 36 h. A The wild-type tetA(46) or tetB(46) genes were PCR amplified, ligated into tetracycline-resistant clone, designated P9, was isolated and the insert pVA838 and transformed into the mutant strains, as described above.
The tetA(46) gene was amplified using primers ABC1-8F and ABC1-9Reach contain-ing XbaI restriction sites. The tetB(46) gene was amplified using primers Species identification ABC2-7F and ABC2-8R, each containing SphI restriction sites The amplified products Amplification and sequencing of a manganese-dependent superoxide were digested with their respective restriction endonucleases and dismutase (sodA) gene fragment was carried out according to Poyart ligated into either the XbaI or SphI sites of pVA838, creating the recom- et alusing the primers listed in binant plasmids pABC1 and pABC2, respectively. The plasmids were transformed into E. coli a-select bronze competent cells (Bioline) andthe presence of the wild-type gene confirmed by DNA sequencing. BothE. coli strains were grown overnight in LB broth supplemented with 80 mg/L chloramphenicol, at 378C, aerobically with shaking at 200 rpm.
To sequence the HindIII genomic DNA fragment of S. australis in pUC19 Plasmid DNA was extracted using a HiSpeed Plasmid Midi Kit (Qiagen).
from clone P9 (pP9), a walking strategy was employed using theprimers listed in Antibiotic susceptibility testing The MICs of tetracycline, oxytetracycline, doxycycline, chlortetracyclineand tigecycline were determined according to BSAC guidelines.The recommended medium and inoculum (104 cfu/spot) was also used for In-frame deletions in tetA(46) and tetB(46) were created using ‘splicing determination of the acriflavine and ethidium bromide MICs. Triplicate in- by overlapping extension' (SOEing) PCR.The hybrid fragments were dividual colonies of each strain were inoculated into 10 mL BHI broth and ligated into pGEM-T-Easy (Promega) and verified by DNA sequencing.
incubated at 378C, in air+5% CO2, for 18 h. The cells were diluted and The DtetA(46) fragment was created using two sets of primer pairs: then spotted, using a multipoint inoculator, on to Iso-Sensitest agar supplemented with 5% defibrinated horse blood or Iso-Sensitest agar ABC1-3F shared 24 nucleotides of complementary sequence to facilitate supplemented with 5% defibrinated horse blood plus the appropriate the ligation of the two amplicons antibiotic at concentrations of 0.25–32 mg/L. The plates were incubated The ligated mutant fragment, when recombined at 378C, in air+5% CO2, for up to 48 h.
into the genome, resulted in a 51 bp in-frame deletion (bp 1073– 1123 Agar diffusion assays were carried out according to BSAC guidelines inclusive) in tetA(46). The DtetB(46) fragment was created in the same Discs containing the following amounts of antibiotics were laid on to way, using two sets of primer pairs: ABC2-1F/ABC2-2R and ABC2-3F/ agar: tetracycline (10 mg), ciprofloxacin (1 mg), metronidazole (5 mg), ABC2-4R; similarly, recombination of this mutagenic fragment resulted azithromycin (15 mg), ampicillin (2 mg), methicillin (5 mg), oxacillin in a 51 bp in-frame deletion (bp 1090– 1140 inclusive) in tetB(46). The (1 mg), penicillin (1 mg) and gentamicin (10 mg). Plates were incubated DtetA(46) or DtetB(46) mutagenic fragments were co-transformed with at 378C, in air+5% CO2, for 20 h and the zones of inhibition measured.
TetAB(46) in Streptococcus australis tetB(46) under the current naming standThey are pre-dicted to encode non-identical polypeptides of 574 and 578 S. australis FRStet12 was isolated on agar containing 2 mg/L amino acids, respectively, each containing an NBD and an MSD tetracySequencing of a sodA gene fragment showed characteristic of the ABC transporter superfamily. TetA(46) and that the closest relative was the S. australis type strain CIP TetB(46) are most closely related to YheI (36% amino acid iden- 107167 (DQ132987), with 95.2% identity. A phylogenetic tree tity) and YheH (35% identity) of Bacillus subtilis, respectively, showing the relationship between the sodA gene fragments of which, when overexpressed in E. coli, form a heterodimeric S. australis FRStet12 and other streptococcal species is shown MDR-type ABC transporter. TetA(46) and TetB(46) show sequence in the supplementary data similarity with other heterodimeric ABC transporters with experi- To determine whether tetracycline mentally proven function, including LmrCD of Lactococcus lactis resistance was conferred by a previously described mechanism, and EfrAB of Enterococcus faecalis, as well as related systems genomic DNA from S. australis FRStet12 was hybridized to a in the Gram-negative bacteria, Serratia marcescens and E. coli macroarray containing known tetracycline resistance genes and the genomic DNA was also tested for the presence of To determine the function of each gene, 51 bp in-frame RPP-encoding genes by PCR.The genomic DNA failed to hybrid- deletions were created within the sequences predicted to ize to the macroarray and was negative in the RPP PCR, suggest- encode the Walker A box of the ATP-binding site of each gene ing the presence of a rare or novel tetracycline resistance product using SOEing PCR.The mutant gene fragments were determinant. In our study, of a total of 69 Gram-positive, facul- co-trinto S. australis FRStet12 along with plasmid tatively anaerobic, tetracycline-resistant isolates, S. australis pVmaking use of the erythromycin resistance gene on FRStet12 was one of only two isolates that failed to hybridize the plasmid for selection. This plasmid was chosen for with the arrayThe other isolate that failed to hybridize has co-transformation since our experience showed it to be rapidly not been investigated.
lost from other Streptococcus species in the absence of selection.
To identify the gene(s) conferring resistance in S. australis Of 90 erythromycin-resistant clones examined following trans- FRStet12, a library of HindIII-digested genomic DNA was formation with the tetA(46) mutant fragment and pVA838, five created in pUC19, selecting transformants on agar containing were unable to grow on 5 mg/L tetracycline and sequence ana- 100 mg/L ampicillin and 5 mg/L tetracycline. One tetracycline- lysis confirmed the presence of the DtetA(46) allele in all five resistant transformant was selected for further study and (Figure ). The tetracycline-resistant transformants contained designated E. coli P9. The insert from the plasmid in P9 (pP9) wild-type tetA(46) and were erythromycin resistant, indicating was completely sequenced (accession number HQ652506) and they had only taken up pVA838. Despite repeated subculturing found to contain five putative open reading frames (orfs), two without erythromycin (14 passages in total), pVA838 remained of which encoded predicted proteins with similarity to multidrug in the mutant strains. Therefore, to allow comparison between resistance (MDR)-type ABC transporters (Figure These genes, wild-type and mutant strains, an isogenic strain was created which were both subsequently shown to encode resistance to by transforming the wild-type with pVA838. Of 72 erythromycin- tetracycline (see below), have ≤79% amino acid identity to pre- resistant clones examined following transformation of the viously characterized tet genes and were assigned tetA(46) and wild-type with the tetB(46) mutant fragment and pVA838, 7 DtetA(46) mutant FRStet12 tetA(46) FRStet12 tetB(46) Figure 1. Diagram of the cloned genomic DNA fragment from S. australis FRStet12, indicated by the black line. Five putative orfs are indicated in grey[tetA(46); tetB(46); orf3, encodes a putative metalloprotease; orf4, encodes a putative diacylglycerol kinase; and orf5, encodes a putative GTP-bindingprotein—accession number HQ652506]. The vertical broken black lines indicate the point of the in-frame deletions in tetA(46) and tetB(46) and thesequence of the deletion is given beneath.
Warburton et al.
Table 1. Comparison between S. australis TetA(46)/TetB(46) and ABC Table 2. MICs of tetracyclines for S. australis FRStet12 and the tetAB(46) transporters with experimentally proven function Percentage identical Oxytetracycline Doxycycline Chlortetracycline Tigecycline (similar) residues Organism (reference) S. australis (this study) for the DtetA(46) and DtetB(46) mutants. MIC determination of the other tetracyclines (Table showed that the mutants were 2- to 8-fold more sensitive to oxytetracycline, doxycycline, chlortetracycline and tigecycline, indicating that TetAB(46) is also able to export these molecules. As MDR-type transporters often are capable of extrusion of other toxic compounds,we determined the MIC of acriflavine and ethidium bromide butfound no difference between the wild-type and mutant strains.
GenBank proteins numbers: YheI, NP_388852; YheH, NP_388853; SmdA, In addition, we found no difference in the zone size in disc diffu- BAF79679; SmdB, BAF79680; MdlA, P77265; MdlB, P0AAG5; LmrC, Q9CIP6; sion assays with ciprofloxacin, metronidazole, azithromycin, LmrD, Q9CIP5. For the other sequences, Swiss-Prot entries (in parentheses) ampicillin, methicillin, oxacillin, penicillin or gentamicin, indicat- were used: EfrA (Q82ZX7_ENTFA); and EfrB (Q82ZX8_ENTFA). Percentage ing that TetAB(46) is specific for the transport of tetracyclines.
identity and similarity were obtained by sequence alignment using Clustal W To determine whether similar genes were present in other bacteria, BLAST searches were performed. This analysis revealedorthologues of TetAB(46) in the one draft genome of S. australispresent in the database (NCTC 13166) and in all six of the were unable to grow on 5 mg/L tetracycline and sequencing Streptococcus parasanguinis draft genomes present in the data- confirmed the presence of the DtetB(46) allele (Figure ), while base (ATCC 903, ATCC 15912, F0405, F0449, FW213 and SK236).
those that remained resistant to tetracycline contained wild-type The predicted proteins within these streptococcal genomes share ≥95% amino acid identity with TetA(46) and TetB(46). Since both To determine whether tetracycline resistance could be S. australis NCTC 13166 and S. parasanguinis NCTC 55898 (ATCC restored in the FRStet12DtetA(46) and FRStet12DtetB(46) 15912) are resistant to tetracycline (2 mg/L), it is possible that mutants, a chromosome repair was carried out. The wild-type these tetAB(46) orthologues confer this phenotype.
tetA(46) and tetB(46) genes were cloned into pVA838 to createthe recombinant plasmids pABC1 and pABC2, respectively. Inthree independent experiments, transformation of pABC1 intoFRStet12DtetA(46) resulted in colonies on agar containing 5 mg/L tetracycline, whereas transformation with pVA838 alonedid not. Sequence analysis of two tetracycline-resistant transfor- Most tetracycline resistance genes reported in oral bacteria mants from each of the three independent experiments, using encode RPP,,whereas efflux genes are rarely detected.
primers flanking tetA(46) in the chromosome, revealed that in Here, we report the discovery of a predicted tetracycline efflux each case the wild-type tetA(46) had replaced the mutant determinant, tetAB(46) from S. australis. This species, first iso- allele within the chromosome, demonstrating that wild-type lated from the oral cavities of children in Austris an oppor- tetA(46) is required for tetracycline resistance. The transformants tunist pathogen reported in sputum samples of adult cystic contained empty pVA838 and the mutant DtetA(46) allele was fibrosis patientsand in a case of invasive infection, a not detectable by PCR. Transformation of pABC2 into FRStet12D community-acquired meningitis in an elderly tetB(46) restored the ability of the mutant strain to grow on Most tetracycline efflux proteins belong to the MFS family of 5 mg/L tetracycline, whereas transformation with pVA838 did transporters, which are membrane located and exchange a not. Sequence analysis of these transformants confirmed that proton for a tetracycline–cation complex against a concentration wild-type tetB(46) had replaced the mutant allele within gradient. These have been described in both Gram-positive and the chromosome, demonstrating that wild-type tetB(46) is also Gram-negative ABC transporters conferring resistance essential for tetracycline resistance.
to tetracyclines have also been reported. There is one example The MIC of tetracycline was determined for S. australis currently in the tet nomenclature database: OtrC from S. rimosuswhich consists of an NBD and an MSD encoded by DtetA(46) and DtetB(46) isogenic mutants and their correspond- separate genes. While other ABC transporters capable of exporting ing complemented strains. The MIC for the wild-type and the tetracycline have been reported, e.g. SmdAB in S. marcescens complemented strains was 8 mg/L, compared with ,0.25 mg/L these are capable of exporting a number of other compounds TetAB(46) in Streptococcus australis and have not therefore been given a tetracycline resistance The MFS efflux systems transport only the naturally occurring tet- gene designation.
racyclines, with the exception of TetA(B), which also transports Analysis of S. australis FRStet12 revealed two orfs responsible the semi-synthetic analogue, minocycline.The reduced MICs for tetracycline resistance, which are most closely related to YheI in the tetA(46) and tetB(46) mutants indicate that TetAB(46) is and YheH of B. subtilis. YheI and YheH are non-identical ABC trans- able to transport both tetracycline and its semi-synthetic deriva- porter subunits, each containing an NBD and an MSD, which were tives. We have previously shown that S. australis FRStet12 is sen- shown to interact to form a heterodimeric multidrug ABC trans- sitive to minocycline (MIC of 0.25 mg/L).Since MDR-type ABC porter capable of transporting several structurally dissimilar transporters have been shown to extrude toxic molecules other drugs, such as fluorescently labelled ethidium bromide and dau- than we determined the MICs of acriflavine and nomycinAnother related MDR transporter is LmrCD of L. lactis, ethidium bromide, but found no difference between the wild- also shown to be capable of extrusion of a range of structurally type and mutant strains. Thus, our data suggest that TetAB(46) unrelated drugsExpression of E. faecalis EfrA and EfrB together is specific for tetracyclines.
in E. coli confers resistance to a range of drugs, including acrifla- In conclusion, we have identified a novel tetracycline resist- vine, norfloxacin and doxycycline. Further, energy-dependent ance determinant, tetAB(46), in an oral viridans Streptococcus efflux of acriflavine in E. coli harbouring efrAB was also demon- species. TetAB(46) is related to known MDR-type ABC transpor- strateMDR-type ABC transporters from S. marcescens and ters in both Gram-positive and Gram-negative bacteria, but E. coli are also related to TetAB(46): although there are no func- confers resistance only to tetracyclines. Genes highly related tional data for the E. coli systemSmdAB of S. marcescens has to tetAB(46) are present in the genomes of other tetracycline- been shown to confer multidrug, including tetracycline, resistance resistant oral streptococci, including several strains of S. parasan- on E. coli and to be inhibited by ATPase inhibitors guinis and another strain of S. australis.
Of the tetracycline (tet) and oxytetracycline (otr) resistance genes currently listed in the tetracycline gene nomenclaturedatabase (28 code for active efflux, 12 for ribosomal protection, 3 for enzymatic drug We wish to thank Dr Arthur Hosie and Dr Alex Webb for their advice on inactivation and 1 has an unknown mechanism. The two genes ABC transporter structure.
required for tetracycline resistance in S. australis FRStet12 weredesignated tetA(46) and tetB(46) under the current namingstandards.
In-frame deletions in either tetA(46) or tetB(46) demon- strated that both were required for tetracycline resistance in This study has been carried out with financial support from the Wellcome S. australis. The fact that the genes encode non-identical pro- Trust and the Commission of the European Communities, specifically the teins, each containing a predicted MSD and NBD, suggests that Infectious Diseases research domain of the Health theme of the 7th they may function as a heterodimeric ABC transporter, although Framework Programme, contract 241446, ‘The effects of antibiotic ad- confirmation of this requires demonstration of a physical inter- ministration on the emergence and persistence of antibiotic-resistantbacteria in humans and on the composition of the indigenous microbio- action between the two proteins, experiments that are beyond tas at various body sites' and the 5th Framework Research and Techno- the scope of the present study.
logical Development (RTD) Program ‘Quality of Life and Management of During the course of this work, we made two observations on Living Resources', QLK2-CT-2002-00843, ‘Antimicrobial resistance trans- the molecular biology of S. australis that are worthy of discussion.
fer from and between Gram-positive bacteria of the digestive tract and Firstly, we report the maintenance of pVA838 after multiple pas- consequences for virulence'.
sages in the absence of selection, which is unusual in our experi-ence with this plasmid in other streptococci. The stability of‘empty' pVA838 in the mutant strains may have contributed to the second unexpected result obtained in this work: in ex-periments designed to introduce the wild-type alleles in trans, None to declare.
transformation of both mutants with the wild-type allelescloned in pVA838 resulted in chromosome repair, i.e. doublecrossover recombination occurred, resulting in replacement of Supplementary data the mutant allele with the wild-type allele in the chromosome, and this restored the tetracycline resistance phenotype.
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Source: https://pearl.plymouth.ac.uk/bitstream/handle/10026.1/3627/TetAB46,%20a%20predicted%20heterodimeric%20ABC%20transporter%20conferring%20tetracycline%20resistance%20in%20Streptococcus%20australis%20isolated%20from%20the%20oral%20cavity..pdf?sequence=1


34th ECDD 2006/4.4 khat Assessment of khat (Catha edulis Forsk) Substance Identification A. International Nonproprietary Name (INN): - B. Chemical Abstract Service (CAS) Registry Number: - C. Other Names: qat, q'at, kat, kath, gat, chat, tschat (Ethiopia), miraa (Kenya), murungu; the dried leaves of khat are known as Abyssinian tea or Arabian tea;


LEARNING FROM PRACTICE Dapagliflozin: Clinical practice comparedwith pre-registration trial data ANDREW P MCGOVERN1-3, NINA DUTTA1, NEIL MUNRO1-4, KENNETH WATTERS1,2,4, MICHAEL FEHER1,2,4 Abbreviations and acronyms Background: Dapagliflozin is the first sodium-glucose co-transporter 2 (SGLT2) inhibitor to be approved in Europe