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RESEARCH LETTER

Combined treatment with the antibiotics kanamycin and streptomycin promotes the conjugation ofEscherichia coli Peng-Yi Zhang, Pei-Pei Xu, Zhi-Jie Xia, Jing Wang, Juan Xiong & Yue-Zhong Li

State Key Laboratory of Microbial Technology, school of Life Science, Shandong University, Jinan, China

Correspondence:Yue-Zhong Li, State Key

Laboratory of Microbial Technology, School

of Life Science, Shandong University, Jinan

250100, China. Tel./fax: +86 531 88564288;

e-mail: lilab@sdu.edu.cn

Received 22 July 2013; revised 6 September

2013; accepted 17 September 2013. Final

version published online October 2013.

DOI:10.1111/1574-6968.12282

Editor: Anthony George

Keywords

Escherichia coli; dual-selective antibiotics;

conjugative transfer; promotion.Abstract It is widely accepted that antibiotics provide a critical selective pressure for the horizontal transfer of antibiotic resistance between bacterial species. This study demonstrated that a combination of low doses of kanamycin and streptomycin, which inhibited the growth of recipient and donor cells, respectively, had posi- tive effects on the transmission of the conjugation plasmids pRK2013, pSU2007, and RP4 fromEscherichia coliDH5ato HB101 at their minimum inhibitory concentrations (MICs). Administration of either antibiotic alone as well as other antibiotics in combination or alone did not have this effect. Two- dimensional electrophoresis revealed that 60 proteins were downregulated and

14 proteins were upregulated in the conjugation ofE. coliDH5a(pRK2013)

and HB101 in the presence of kanamycin and streptomycin. Of these proteins,

64 were subsequently identified by mass spectrometry. Two antibiotic-induced

genes encoding oligopeptide-binding protein (OppA) and ribose-binding pro- tein (RbsB) were further confirmed by quantitative real-time PCR. When these genes were deleted, the number of transconjugants decreased in the same fash- ion as when the cells were treated with kanamycin and streptomycin. These results indicate that the process ofE. coliconjugation may be promoted by combination treatment with kanamycin and streptomycin and that two proteins potentially participated in this process.Introduction

Conjugation is one of the major methods by which

genetic materials are transferred between bacterial cells, and it is mediated by self-transmissible and mobilizable plasmids, conjugative transposons, and integrative conju- gative elements. Conjugation is also one of the most important methods of genetic manipulation in the labora- tory. Bacterial conjugation serves as the major mechanism for the horizontal transfer of antibiotic resistance, which increases genetic variability over evolutionary history. During conjugation, donor and recipient cells are in physical contact and establish conjugation channels for DNA transfer (Chenet al., 2005; Dubey & Ben-Yehuda,

2011).

Because of their inhibitory effects, the antibiotics that

are used in classical conjugation procedures are not addedto the mating medium but to the selection medium forthe exclusive growth of transconjugants. However,positive effects of low doses of single DNA-damagingantibiotics have been previously observed during horizon-

tal gene transfers (Hastingset al., 2004). For example, low doses of DNA-damaging antibiotics induced SOS responses in donor cells, facilitating the transfer of inte- grating conjugative elements or plasmids (Beaberet al.,

2004). Low doses of aminoglycosides or fluoroquinolones

induced competence inStreptococcus pneumoniavia the comregulon, thus improving transformation efficiency, whereas no induction was observed when novobiocin, rifampicin, vancomycin, or ampicillin were used (Prudhommeet al., 2006). These reports indicate that low doses of antibiotics that do not kill bacterial cells may facilitate the transfer of resistant genes necessary for

cell survival. However, these reports only described theFEMS Microbiol Lett348(2013) 149-156ª2013 Federation of European Microbiological Societies.

Published by John Wiley & Sons Ltd. All rights reserved

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effect of a single antibiotic on the transfer of resistance among bacteria; the effects of combined antibiotics on the conjugation process have not been widely reported.

A previous study from our laboratory, aimed at

improving the genetic manipulation inSorangium cellulosum,revealed that the minimum inhibitory concentrations (MICs) of dual-selective antibiotics (chloramphenicol and gentamicin) added to the mating medium improved the transmission of the conjugation plasmid, pRK2013 (Xiaet al., 2008). The mating medium included a combination of two antibiotics that inhibited donor and recipient cells, respectively, and were termed dual-selective antibiotics. In this study, we examined the universality of the above phenomenon by performing conjugation experiments betweenE. colistrains and investigating the effects of treatment with dual-selective antibiotics and other combinations of antibiotics on this process. The findings indicate that bacteria may enhance the expression of key survival genes and rapidly generate antibiotic-resistant bacterial strains as a result of environ- mental exposure to subminimal inhibitory concentrations of antibiotics.

Materials and methods

Strains and plasmids

TheE. colistrains, plasmid-containing DH5a(TaKaRa

Biotechnol, China) and streptomycin-resistant HB101 (TaKaRa Biotechnol), were used as the donor and recipient strains for conjugation, respectively. The self-transmissible plasmids used for the conjugative transfer included the kanamycin-resistance plasmids, pRK2013 and pSU2007 (IncP family), and the ampicillin-, kanamycin-, and tetra- cycline-triple-resistance plasmid, RP4 (IncW family). These strains and plasmids are listed in Table 1.

The MICs of kanamycin and ampicillin on HB101 and

streptomycin on DH5a(pRK2013), DH5a(RP4), and

DH5a(pSU2007) were measured using the double-

dilution method according to the previously described methods (Davidet al., 2007).

Conjugation betweenE. colistrains

Classical conjugation was performed according to the pre- viously reported methods (Bradleyet al., 1980; Jaouaet al.,

1992; Koppet al., 2003). The improved protocol for conju-

gation in the presence of antibiotics was as follows:E. coli strains were cultured at 37°C with shaking at 200 r.p.m. until the late log-phase in liquid Luria-Bertani medium (LB medium: 10 g L ?1 tryptone, 5 g L ?1 yeast extract, and

10 g L

?1

NaCl, pH 7.0), which was supplemented with

antibiotics when required. The cells were harvested bycentrifugation and washed and resuspended in LB medium

to a density of 2910 9 cells mL ?1 . Then, 50lL of each plasmid-harboring donor was mixed with 50lL of the recipient (HB101) in a 1.5-mL eppendorf tube and supple- mented with 900lL of liquid LB medium containing anti- biotics to a total volume of 1 mL. After mixing completely, the cells were incubated at 37°C without shaking for 20 h at 2-h or 4-h intervals. The conjugation process was then interrupted by vortexing. The mixture was immediately serially diluted 10-fold and plated on LB medium contain- ing different antibiotics (kanamycin 30lgmL ?1 , strepto- mycin 30lgmL ?1 , ampicillin 100lgmL ?1 , as shown in Table 2) to select and count the donor, recipient, and transconjugant. The concentrations of antibiotics used for conjugation, incubation, and selection were listed in Table 2. The control group was subjected to the same pro- cedure but without antibiotics. All tests were carried out in triplicate. The transconjugant was verified by agarose elec- trophoresis (Supporting Information, Fig. S1). The conju- gation efficiency was expressed as the ratio of the number of transconjugants to the initial number of recipients (1910 8 cells mL ?1 throughout the text; Pooleet al.,

2006).

Two-dimensional electrophoresis analysis

After 12 h of mating incubation between DH5a

(pRK2013) and HB101 in the presence or absence of kanamycin and streptomycin, the mixture was harvested to assay for protein changes. The cells were washed twice using TRIS MgSO 4 buffer (TM buffer) containing 10 mM Tris (pH 8.0) and 5 mM magnesium sulfate, and ultra- sonically disrupted in a lysis buffer containing 8 M urea,

2 M thiourea, 4% (w/v) CHAPS, 10 mM phen-

ylmethanesulfonyl fluoride, 40 mM dithiothreitol, and

2% Pharmalyte (pH 4

-7). The lysate was centrifuged at

12 000gat 4°C for 30 m, and the supernatant was

collected. The protein concentration was determined using the BCA kit (Promega, Madison, WI). Two-dimensional electrophoresis (2-DE) was performed according to the manufacturer's instructions with modifications (Bio-Rad, Hercules, CA). Briefly, an Immobiline DryStrip pH 4 -7 (17 cm) was used for the one-dimension isoelectric focus- ing gel. 2D SDS-polyacrylamide gel electrophoresis was then performed with a 12% acrylamide gel at 10 mA/gel for 1 h and then at 30 mA per gel for 5 h. After electro- phoresis, the gels were fixed in 10% trichloroacetic acid solution for 1 h and stained using Coomassie Brilliant Blue G-250 followed by destaining in a solution contain- ing 40% methanol and 10% acetic acid (Otaniet al.,

2001; Horiuchiet al., 2002). The differences in protein

patterns between the two conjugation conditions were analyzed by

PDQUESTsoftware version 8.0 (Bio-Rad). The

FEMS Microbiol Lett348(2013) 149-156ª2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

150P.-Y. Zhanget al.Downloaded from https://academic.oup.com/femsle/article/348/2/149/731497 by guest on 20 October 2023

identification of protein spots was performed by matrix-assisted laser desorption ionization/tandem time- of-flight mass spectrometry (MALDI-TOF/TOF; ABI, Foster, CA) at Nankai University. The peptide mass fingerprinting and MS/MS ion searching were performed at http://www.matrixscience.com.

Quantitative real-time PCR analysis

Total RNA was extracted from the mixture after 12 h of mating between DH5a(pRK2013) and HB101 in the presence or absence of kanamycin and streptomycin using the Promega SV total RNA purification kit. Any

Table 2.The antibiotics and their concentrations used for conjugation, incubation, and selection, and MICs of streptomycin on DH5a

(pRK2013), DH5a(RP4), and DH5a(pSU2007), kanamycin on HB101, and ampicillin on HB101

Strains MIC Conjugation Incubation Selection

DH5a(pRK2013) Sm8*Km14Sm8 Km30 Km30

DH5a(RP4) Sm20 Km14Sm20 Km30Ap100 Km30Ap100

Ap34Sm20

Km14Ap34Sm20

DH5a(pSU2007) Sm10 Km14Sm10 Km30 Km30

HB101 Km14

Km14Sm8Sm30Sm30

Km14Sm20

Km14Sm10

HB101Ap34

Ap34Sm20Sm30Sm30

HB101 (pRK2013)--Km30Sm30Km30Sm30

HB101 (RP4)--Km30Ap100Sm30Km30Ap100Sm30

HB101 (pSU2007)--Km30Sm30Km30Sm30

*Sm8: 8lgmL ?1 streptomycin in the medium.

Km14: 14lgmL

?1 kanamycin in the medium.

Ap34: 34lgmL

?1 ampicillin in the medium; and so on. Table 1.Strains, plasmids, and primers used in this study Strain Phenotype/Feature/Bases Source or reference

E. coli

DH5asupE44ΔlacU169(Φ80lacZΔM15)hsdR17 recA1 endA1 gyrA96 thi-1 relA1TaKaRa Biotechnol HB101supE44,hsdS20,recA13,ara-14,proA2,lacY1,galK2,rpsL20,xyl-5,mtl-1,leuB6,thi-1, Sm r

TaKaRa Biotechnol

Plasmid

pRK2013 RK2 derivative, Km r ; self-transmissibleFigurski & Helinski (1979) pSU2007 Km r derivative of IncW conjugative plasmid

R388; 2.5-kbEcoRI-SalI fragment exchanged for

1.5-kbEcoRI-SalI fragment carrying Km

r -encoding gene of Tn5Martinez & De la Cruz (1988)

RP4 RK2 derivative, Ap

r ,Km r ,Tc r; self-transmissibleSaunders & Grinsted (1972) pKD46Red expression plasmid, bla PBAD gam bet exo ori pSC101Datsenko & Wanner (2000) pKD4Template plasmid for Red system, bla FRT::kan::FRT ori R6KDatsenko & Wanner (2000) pCP20Flp expression plasmid, bla cat cI857kPR flp ori pSC101Cherepanov & Wackernagel (1995)

Primers

oppA -in-forward 5′-GGAAGTTGGTTCGTTGTAGTCAGCA-3′ oppA -in-reverse 5′-GGCGATTGCTGCCTCTTCATTGTGG-3′ rbsB-in-forward 5′-TTGGCACCTTCACCCGCTTTCTTCG-3′ rbsB-in-reverse 5′-AGGCAACGAAAGGTGAAGTGGTGAG-3′

16S-in-forward 5′-CTCCTACGGGAGGCAGCAG-3′

16S-in-reverse 5′-GTATTACCGCGGCTGCTG-3′

oppA -up 5′-ATGACCAACATCACCAAGAGAAGTTTAGTAGCAGCTGGCGTGTAGGCTGGAGCTGCTTC-3′ oppA -down 5′-TTAGTGCTTCACAATGTACATATTCCGGGTATAGGTATTATGAATATCCTCCTTAGTTC-3′ rbsB-down 5′-CTACTGCTTAACAACCAGTTTCAGATCAACCGGATACTTATGAATATCCTCCTTAGTTC-3′ oppA -antis 5′-GATAAAGTAACCTGACAGCAGAAAG-3′ oppA -sen 5′-GTAATGCGATAAAAATCAGACACCG-3′ rbsB-antis 5′-TGCTTTTCTTTATATTACACGTTCA-3′ rbsB-sen 5′-ATAATGGATTGAATTTGTTAGGTGT-3′ Ap, ampicillin; Km, kanamycin; Sm, streptomycin; Tc, tetracycline. FEMS Microbiol Lett348(2013) 149-156ª2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

Combined antibiotics promoteE. coliconjugation151Downloaded from https://academic.oup.com/femsle/article/348/2/149/731497 by guest on 20 October 2023

contaminating genomic DNA was removed using a DNA- free kit (Ambion, Austin, TX). After purification, the RNA was reverse-transcribed to cDNA for quantitative real-time PCR analysis. The PCRs were performed on the Bio-Rad Sequence Detection System using the followingquotesdbs_dbs44.pdfusesText_44

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