JAC Advance Access originally published online on January 15, 2007
Journal of Antimicrobial Chemotherapy 2007 59(2):191-196; doi:10.1093/jac/dkl498
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Efflux-mediated resistance to florfenicol and/or chloramphenicol in Bordetella bronchiseptica: identification of a novel chloramphenicol exporter
Institut für Tierzucht, Bundesforschungsanstalt für Landwirtschaft (FAL), Höltystr. 10, 31535 Neustadt-Mariensee, Germany
* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de
Received 25 August 2006; returned 14 November 2006; revised 14 November 2006; accepted 14 November 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
OBJECTIVES: Twenty florfenicol- and/or chloramphenicol-resistant Bordetella bronchiseptica isolates of porcine and feline origin were investigated for the presence of floR and cml genes and their location on plasmids.
METHODS: The B. bronchiseptica isolates were investigated for their susceptibility to antimicrobial agents by broth micro- or macrodilution and for their plasmid content. Hybridization experiments and PCR assays were conducted to identify resistance genes. Transformation and conjugation studies were performed to show their transferability. Representatives of both types of genes including their flanking regions were sequenced. Moreover, inhibitor studies with the efflux pump inhibitor Phe-Arg-ß-naphthylamide (PAßN) were performed.
RESULTS: The gene floR was found in the chromosomal DNA of 9 of the 18 florfenicol/chloramphenicol-resistant isolates. Sequence analysis revealed that the deduced FloR protein sequence differed by a single amino acid exchange from FloR of Vibrio cholerae. A chloramphenicol-resistant, but florfenicol-susceptible isolate carried a novel plasmid-borne cml gene, designated cmlB1. The CmlB1 protein revealed only 73.8–76.5% identity to known CmlA proteins. The gene cmlB1 was not part of a gene cassette. The results of inhibitor studies with PAßN suggested that a so-far unidentified efflux system might play a role in phenicol resistance of the remaining florfenicol- and/or chloramphenicol-resistant isolates.
CONCLUSIONS: This is to the best of our knowledge the first report of a floR gene in B. bronchiseptica isolates. The identification of the first member of a new subclass of cml genes, cmlB1 from B. bronchiseptica, extends our knowledge on specific chloramphenicol exporters.
Keywords: cmlB1 gene , floR gene , efflux pump inhibitors , respiratory tract pathogen
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Bordetella bronchiseptica is frequently involved in respiratory tract infections of food-producing animals and companion animals.1 Antimicrobial agents are commonly used to treat these infections in animals. Initial studies of antimicrobial resistance in B. bronchiseptica from pigs revealed a decreased susceptibility to most of the antimicrobial agents currently approved for the treatment of respiratory tract infections, such as tilmicosin and ceftiofur, with MIC90 values of 16 mg/L and 32 mg/L, respectively.2 For other antimicrobial agents, such as florfenicol, a fluorinated chloramphenicol derivative, the corresponding MIC values were distinctly lower.2 Following the European Union's ban of chloramphenicol use in food-producing animals in 1994, florfenicol has been approved for the treatment of respiratory tract infections in cattle (1995) and in pigs (2000). By contrast, chloramphenicol is still approved for use in dogs, cats and other non food-producing animals, and based on its favourable susceptibility situation it is used for the control of a wide variety of infections in these animals.
Although florfenicol-resistant B. bronchiseptica isolates from respiratory tract infections in pigs have been detected in recent years,3,4 the genetic basis for this resistance in B. bronchiseptica had not been elucidated. In the present study, we analysed isolates classified as florfenicol/chloramphenicol-resistant or only chloramphenicol-resistant for the presence of known florfenicol and/or chloramphenicol resistance genes. In addition, an efflux pump inhibitor was used to assess whether efflux may play a role in phenicol resistance of B. bronchiseptica.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Bacterial isolates and susceptibility testing
A total of 496 B. bronchiseptica isolates from animals suffering from respiratory tract infections in Germany, including 349 isolates from pigs collected between 2000 and 2003,2 as well as 105 isolates from pigs, 8 isolates from cats and 34 from dogs, all collected between 2004 and 2006, were investigated for their susceptibility to florfenicol and chloramphenicol. Biochemical species identification was confirmed by genus- and species-specific PCR analysis.5 Macrorestriction analysis with XbaI was performed as described previously.6 MIC determination by broth micro- or macrodilution followed the recommendations of the CLSI as laid down in documents M31-A2 and M31-S1.7,8 The reference strains Escherichia coli ATCC 25922 and/or Staphylococcus aureus ATCC 25923 were included for quality control purposes. To induce phenicol resistance gene expression, the strains were grown overnight either on MH-agar plates containing chloramphenicol (0.5 mg/L) or florfenicol (0.5 mg/L). MIC determinations were performed at least twice on independent occasions.
Detection of phenicol resistance genes
PCR assays for the genes conferring combined resistance to chloramphenicol and florfenicol, namely floR, fexA and cfr, but also for the most common chloramphenicol resistance genes catA1, catA2, and catA3 were performed according to previously described protocols.6,9–11 For the detection of the chloramphenicol resistance gene cmlA, previously described primers were used at an annealing temperature of 60°C.12 In addition, all isolates were investigated for class 1 integrons and their associated catB gene cassettes.6 For the gene floR, additional new primers were designed to amplify the entire floR gene and used with the annealing temperature of 50°C. The forward primer (5'-AGGGTTGATTCGTCATGACCA-3') contained the start codon and the reverse primer (5'-CGGTTAGACGACTGGCGACT-3') the stop codon of the floR gene. To detect circular forms of the floR-carrying transposon TnfloR, the primers floRcirc1 and floRcirc2 were used.13 In addition, PCR assays were conducted for the oqxAB operon14 which has recently been described to mediate the efflux of chloramphenicol in addition to that of olaquindox.
Plasmid profiling, transfer experiments and Southern blot hybridization
Plasmid profiles were prepared by alkaline lysis as described.6 Conjugation into E. coli HK225 was performed.6 Electrotransformation into B. bronchiseptica B543 and into E. coli HB101 was carried out as described previously for Pasteurella15 with the Gene Pulser II electroporation system (Bio-Rad, Munich, Germany). Transfer was confirmed by MIC determination and by plasmid isolation with subsequent restriction analysis and PCR assays. Southern blot hybridization was performed with a PCR-generated floR gene probe16 using either EcoRI- or SacI-digested whole cell DNA or uncut plasmid profiles as target DNA. Probe labelling was achieved with the DIG-High Prime DNA labelling and detection system. Hybridization and signal detection followed the recommendations given by the manufacturer (Roche Diagnostics GmbH, Mannheim, Germany).
Sequencing of resistance gene regions
For sequence analysis, the floR and cmlA PCR amplicons were cloned into the vector pCR-blunt (Invitrogen, Groningen, The Netherlands) as described previously.6 To sequence the flanking regions of floR, chromosomal DNA was first digested with the restriction enzyme AgeI and the fragments re-ligated with T4 DNA ligase. Subsequently, inverse PCR with the floRcirc primers was conducted. The resulting amplicon was cloned into pCR-blunt and sequenced. To sequence the flanking regions of the plasmid-borne cmlA-like gene, the plasmid was digested with EcoRI and KpnI and the fragments cloned into pBluescript II SK+ (Stratagene, Amsterdam, The Netherlands). The clones were confirmed by plasmid profiling, restriction analysis, PCR-directed detection of the cmlA-like gene, and expression of chloramphenicol resistance. Sequences were deposited in the EMBL database under accession nos. AM296480 [GenBank] (floR) and AM296481 [GenBank] (cmlB1).
Inhibition of efflux mechanisms
The efflux pump inhibitor Phe-Arg-ß-naphthylamide (PAßN) was used for inhibition studies.17,18 First, the strain-specific susceptibility to the efflux inhibitor was determined by broth macrodilution. Then, MICs of chloramphenicol and florfenicol, but also nalidixic acid, were determined in parallel in the presence and absence of the inhibitor. An inhibitor concentration of 80 mg/L was used, representing 1/4 of the MIC of PAßN.18
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Susceptibility testing
Of all the isolates analysed, only 18 B. bronchiseptica isolates, 17 from pigs and one from a cat, were classified as florfenicol-resistant by CLSI criteria with MICs of florfenicol of 
8 mg/L.7 All florfenicol-resistant isolates (nos. 1–18) exhibited MICs of chloramphenicol of 16 mg/L (Table 1). In addition, two isolates (nos. 19 and 20) were chloramphenicol-resistant, but not florfenicol-resistant, and had MICs for chloramphenicol of 128 mg/L and 32 mg/L, respectively. The high MIC for chloramphenicol of 128 mg/L for isolate no. 19 suggested the presence of a specific chloramphenicol resistance gene. The remaining isolates (nos. 21–23) were used for control purposes and showed low MIC values of 4 mg/L for chloramphenicol and florfenicol (Table 1).
|
FloR-mediated florfenicol/chloramphenicol resistance
In isolates 3–11 (Table 1), the gene floR was detected by PCR. Hybridization studies confirmed that this gene was located in the chromosomal DNA in all nine cases. Plasmid profiling revealed that the floR-carrying strains were plasmid-free. Since all these B. bronchiseptica isolates shared indistinguishable or closely related XbaI macrorestriction patterns, one of these isolates, no. 5, was chosen for further analysis. Analysis of a 1638 bp region including the floR gene and 66 bp in its upstream and 356 bp in its downstream flanking regions revealed a single base-pair exchange as compared with the corresponding sequence of Vibrio cholerae (accession no. AY822603 [GenBank] ). This base-pair exchange resulted in an amino acid exchange of His-202 in B. bronchiseptica versus Arg-202 in V. cholerae. Compared with FloR proteins so far found in other respiratory tract pathogens, FloR from B. bronchiseptica differed by four amino acid exchanges each from FloR of Pasteurella multocida (Leu-178, His-202, Pro-207 and Phe-228 in B. bronchiseptica versus Arg-178, Arg-202, Ala-207 and Tyr-228 in P. multocida) and from FloR of Pasteurella trehalosi (Ile-32, Met-147, His-202 and Met-225 in B. bronchiseptica versus Met-32, Ile-147, Arg-202 and Ile-225 in P. trehalosi).19,20 Although the determined upstream and downstream flanking regions of floR were identical to the sequence of TnfloR,13 a circular intermediate (which would have confirmed the mobility of floR) could not be detected in any of the nine floR-carrying isolates.
In the remaining nine florfenicol-resistant isolates, the floR gene was not detectable by PCR or by specific hybridization. In addition, none of the other two so-far known florfenicol resistance genes, cfr and fexA, was detectable. PCR assays for genes conferring resistance to chloramphenicol only did not yield amplicons in any of the 18 florfenicol-resistant isolates, thus confirming that no additional chloramphenicol resistance gene is present in these isolates.
CmlB1-mediated chloramphenicol resistance
Solely in isolate no. 19, which had an MIC for chloramphenicol of 128 mg/L, was a cmlA-like gene detected. Plasmid profiling as well as conjugation and transformation experiments revealed its localization on a non-conjugative plasmid of approx. 50 kb. Sequencing of the entire gene and analysis of the deduced amino acid sequence showed that the corresponding gene product differed distinctly from the amino acid sequences of all so-far known CmlA proteins. Based on a multi-sequence alignment with all CmlA amino acid sequences currently deposited in the databases, the 421 amino acid protein from B. bronchiseptica showed identities of only 73.7 to 76.5% to the different CmlA proteins, with least identity to the CmlA4 protein of Salmonella enterica serovar Agona21 and the highest identity to CmlA5 from Acinetobacter baumannii.22 Based on this level of identity, the chloramphenicol exporter from B. bronchiseptica was considered as the first representative of a novel class of CmlA-like proteins but different from the CmlA proteins. Therefore, it was designated CmlB1. A phylogenetic tree (Figure 1) confirmed the evolutionary distance of the CmlB1 protein from the different CmlA variants. In this regard, it should be noted that the CmlA protein sequences as deposited in the databases varied in size between 390 and 437 amino acids with most of the CmlA proteins having a size of 419 amino acids. The cmlA genes coding for proteins of 418 and 419 amino acids have been reported to start with GTG start codon.21 This unusual start codon has also been identified in the novel cmlB1 gene. A closer look at the reading frames for the 390 amino acid proteins (accession nos. AAY43147 [GenBank] , AAY43150 [GenBank] , ABH07981 [GenBank] , ABB71444 [GenBank] , CAD31707 [GenBank] ) strongly suggested that these cmlA genes also have the GTG start codon rather than the proposed ATG start codon and thus code for a protein of 419 amino acids as well. A wrong annotation of the start codon (ATC at positions 43 516–43 518) in the nucleotide sequence of the cmlA5 gene of A. baumannii (CT025832 [GenBank] ) resulted in the uncommon size of 437 amino acids of the respective gene product (CAJ77046 [GenBank] ). Most likely, the cmlA5 gene of A. baumannii also starts with GTG (at positions 43 570–43 572) and codes for a 419 amino acid protein.
|
The analysis of a 2291 bp region encompassing the cmlB1 gene revealed a 582 bp region in the upstream part which differed only by 1 bp from the respective part in the whole genome sequence of B. bronchiseptica strain RB50 (BX640441 [GenBank] ).23 Immediately downstream of the cmlB1 gene, an incomplete reading frame was detected which resembled the N-terminus of a transposase from Marinobacter aquaeolei VT8 (ZP_00818190). Although many cmlA genes are part of gene cassettes located in class 1 integrons,24 no structures resembling the 5'- and 3'-conserved segments of integrons were detectable upstream and downstream of the cmlB1 gene. Moreover, no 59-base element was detectable downstream of the translational termination codon of cmlB1.
In the area immediately upstream of the cmlB1 gene, a putative regulatory region comprising a small reading frame for a 9 amino acid peptide and two pairs of imperfect inverted repeated (IR) sequences of 12 and 10 bp, respectively, were detected. Such an arrangement has also been described for the cmlA1 gene of Tn1696 and is assumed to play a role in the chloramphenicol-inducible expression of the cmlA1 gene by attenuated translation.25 The IR1 sequence in the cmlB1 upstream region was detected immediately after the translational stop codon of the small reading frame, whereas the IR4 sequence comprised the start of the cmlB1 gene. Calculation of the mRNA stabilities suggested that IR1 : IR2 (
G = 90.3 kJ/mol) and IR3 : IR4 (G = 79.4 kJ/mol), but also IR2 : IR3 (G = 74.4 kJ/mol) may be able to form stable mRNA secondary structures.26 In addition, the small reading frame also contained a ribosome stall sequence 5'-AAGAAAGCAGAC-3' which was indistinguishable from that in the small reading frame upstream of the inducibly expressed chloramphenicol resistance gene of the staphylococcal plasmid pC194.27 All these sequence features may support the assumption that cmlB1 expression is also regulated by translational attenuation. MIC determination for the original cmlB1-carrying B. bronchiseptica isolate and its B. bronchiseptica B543 and E. coli HB101 transformants revealed up to 16-fold increase in the MICs of chloramphenicol and up to 8-fold increase in the MICs of florfenicol after pre-incubation in subinhibitory concentrations of chloramphenicol or florfenicol (Table 2).
|
Inhibition of efflux-mediated phenicol resistance
To investigate efflux inhibition, we used three of the nine floR-carrying isolates, all nine florfenicol-resistant but floR-negative isolates, the two chloramphenicol-resistant isolates and, as controls, three isolates with lower MICs for chloramphenicol and florfenicol of 4 mg/L. The MIC values of the antimicrobial agents in the absence and in the presence of the efflux inhibitor PAßN are shown in Table 1. In isolates carrying floR, a 2- to 4-fold decrease in the MICs of both phenicols was seen in the presence of PAßN. In contrast, floR-negative florfenicol-resistant isolates showed a distinctly more pronounced susceptibility to both phenicols in the presence of PAßN, as illustrated by an 8- to 32-fold decrease in the corresponding MICs. A very similar situation was seen with the MICs for the B. bronchiseptica isolates classified as intermediately susceptible to florfenicol (MIC 4 mg/L) (Table 1).
Since PAßN interferes with multi-drug efflux systems of the resistance-nodulation-division (RND) family, it may be possible that one or more such systems, which are widespread among Gram-negative bacteria,28 are also present in B. bronchiseptica and may play a role in phenicol resistance. In other bacteria, such as Salmonella enterica, it has been shown that the MIC of florfenicol dropped distinctly in the presence of the efflux pump inhibitor PAßN.29 Efflux systems of the RND family, like AcrAB-TolC, can also export other antimicrobials such as the quinolone nalidixic acid.28 In good accordance with the results for florfenicol and chloramphenicol, the MICs of nalidixic acid for the B. bronchiseptica isolates also dropped by three to seven dilution steps in the presence of PAßN (Table 1). While isolates 1, 2, 12–18 and 20 showed MICs of nalidixic acid of 64 mg/L and 128 mg/L, the isolates with the phenicol-specific efflux pumps FloR or CmlB1 and the isolates 21–23 used for control purposes had lower MICs for nalidixic acid of 16 mg/L. In the presence of PAßN, an MIC for nalidixic acid of 1–2 mg/L was determined for these B. bronchiseptica isolates, indicating that they may also harbour one or more efflux system(s) not further specified—putatively also of the RND family—exporting phenicols and/or nalidixic acid. Enhanced expression of RND systems in resistant isolates have been described for the AcrAB-TolC tripartite pump from E. coli and S. enterica.28 In S. enterica these pumps conferred lower susceptibility to chloramphenicol, florfenicol and quinolones, but not to ampicillin or streptomycin.30,31 In the genome of the completely sequenced B. bronchiseptica isolate RB50,23 several putative efflux proteins have been identified. One cluster of genes shows homology to genes encoding the RND efflux system common in E. coli: genes encoding an AcrA homologue (CAE34795 [GenBank] ), followed by two genes encoding proteins similar to AcrB (CAE34794 [GenBank] , CAE24793), and followed by a gene encoding a protein similar to TolC (CAE34792 [GenBank] ). Further work is needed to clarify whether these putative efflux proteins from B. bronchiseptica act as a multi-drug transporter and, if so, what is the substrate spectrum of this efflux system.
In conclusion, the results of this study showed that at least two different phenicol-specific efflux pumps of the MF superfamily, encoded by the genes floR and cmlB1, but also a further unspecified efflux system, confer resistance to phenicols in B. bronchiseptica. These data complement recent findings on chloramphenicol resistance genes catB2 and catB3, coding for chloramphenicol-inactivating enzymes, in porcine B. bronchiseptica.6
|
Acknowledgements |
|---|
Kristina Kadlec is supported by a scholarship of the H. Wilhelm Schaumann foundation.
Transparency declarations
None to declare.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
1 Brockmeier SL, Halbur PG, Thacker EL. (2002) Porcine respiratory Disease Complex. In Brogden KA and Guthmiller JM (Eds.). Polymicrobial Diseases(ASM Press, Washington, DC) pp. 231–58.
2
Kadlec K, Kehrenberg C, Wallmann J, et al. (2004) Antimicrobial susceptibility of Bordetella bronchiseptica isolates from porcine respiratory tract infections. Antimicrob Agents Chemother 48:4903–6.
3
Priebe S and Schwarz S. (2003) In vitro activities of florfenicol against bovine and porcine respiratory tract pathogens. Antimicrob Agents Chemother 47:2703–5.
4
Kehrenberg C, Mumme J, Wallmann J, et al. (2004) Monitoring of florfenicol susceptibility among bovine and porcine respiratory tract pathogens collected in Germany during the years 2002 and 2003. J Antimicrob Chemother 54:572–4.
5 Hozbor D, Fouque F, Guiso N. (1999) Detection of Bordetella bronchiseptica by the polymerase chain reaction. Res Microbiol 150:333–41.[Medline]
6
Kadlec K, Kehrenberg C, Schwarz S. (2005) Molecular basis of resistance to trimethoprim, chloramphenicol and sulphonamides in Bordetella bronchiseptica. J Antimicrob Chemother 56:485–90.
7 Clinical and Laboratory Standards Institute. (2002) Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals – Second edition: Approved standard M31-A2(CLSI, Wayne, PA, USA).
8 Clinical and Laboratory Standards Institute. (2002) Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Informational Supplement: M31-S31(CLSI, Wayne, PA, USA).
9 Kehrenberg C, Schwarz S, Jacobsen L, et al. (2005) A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503. Mol Microbiol 57:1064–73.[CrossRef][ISI][Medline]
10
Kehrenberg C and Schwarz S. (2004) fexA, a novel Staphylococcus lentus gene encoding resistance to florfenicol and chloramphenicol. Antimicrob Agents Chemother 48:615–8.
11
Blickwede M and Schwarz S. (2004) Molecular analysis of florfenicol-resistant Escherichia coli isolates from pigs. J Antimicrob Chemother 53:58–64.
12
Keyes K, Hudson C, Maurer JJ, et al. (2000) Detection of florfenicol resistance genes in Escherichia coli isolated from sick chickens. Antimicrob Agents Chemother 44:421–4.
13
Doublet B, Schwarz S, Kehrenberg C, et al. (2005) Florfenicol resistance gene floR is part of a novel transposon. Antimicrob Agents Chemother 49:2106–8.
14
Hansen LH, Johannesen E, Burmolle M, et al. (2004) Plasmid-encoded multidrug efflux pump conferring resistance to olaquindox in Escherichia coli. Antimicrob Agents Chemother 48:3332–7.
15
Kehrenberg C and Schwarz S. (2001) Molecular analysis of tetracycline resistance in Pasteurella aerogenes. Antimicrob Agents Chemother 45:2885–90.
16
Doublet B, Schwarz S, Nussbeck E, et al. (2002) Molecular analysis of chromosomally florfenicol-resistant Escherichia coli isolates from France and Germany. J Antimicrob Chemother 49:49–54.
17 Li XZ and Nikaido H. (2004) Efflux-mediated drug resistance in bacteria. Drugs 64:159–204.[CrossRef][ISI][Medline]
18
Preisler A, Mraheil MA, Heisig P. (2006) Role of novel gyrA mutations in the suppression of the fluoroquinolone resistance genotype of vaccine strain Salmonella Typhimurium vacT (gyrA D87G). J Antimicrob Chemother 57:430–6.
19
Kehrenberg C, Meunier D, Targant H, et al. (2006) Plasmid-mediated florfenicol resistance in Pasteurella trehalosi. J Antimicrob Chemother 58:13–17.
20
Kehrenberg C and Schwarz S. (2005) Plasmid-borne florfenicol resistance in Pasteurella multocida. J Antimicrob Chemother 55:773–5.
21
Michael GB, Cardoso M, Schwarz S. (2005) Class 1 integron-associated gene cassettes in Salmonella enterica subsp. enterica serovar Agona isolated from pig carcasses in Brazil. J Antimicrob Chemother 55:776–9.
22 Fournier PE, Vallenet D, Barbe V, et al. (2006) Comparative genomics of multidrug resistance in Acinetobacter baumannii. PLoS Genet 2:e7.[CrossRef][Medline]
23 Parkhill J, Sebaihia M, Preston A, et al. (2003) Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat Genet 35:32–40.[CrossRef][ISI][Medline]
24 Schwarz S, Kehrenberg C, Doublet B, et al. (2004) Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol Rev 28:519–42.[CrossRef][ISI][Medline]
25 Stokes HW and Hall RM. (1991) Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation. Plasmid 26:10–9.[CrossRef][ISI][Medline]
26 Tinoco I, Borer P, Dengler B., et al. (1973) Improved estimation of secondary structure in ribonucleic acid. Nature New Biol 246:171–2.
27
Horinouchi S and Weisblum B. (1982) Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150:815–25.
28
Poole K. (2005) Efflux-mediated antimicrobial resistance. J Antimicrob Chemother 56:20–51.
29 Baucheron S, Imberechts H, Chaslus-Dancla E, et al. (2002) The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar Typhimurium phage type DT204. Microb Drug Resist 8:281–9.[CrossRef][ISI][Medline]
30
Baucheron S, Tyler S, Boyd D, et al. (2004) AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium DT104. Antimicrob Agents Chemother 48:3729–35.
31 Quinn T, O'Mahony R, Baird AW, et al. (2006) Multi-drug resistance in Salmonella enterica: efflux mechanisms and their relationships with the development of chromosomal resistance gene clusters. Curr Drug Targets 7:849–60.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||