Edited by: Daniela Ceccarelli, European Commission, Belgium
Reviewed by: Susana Brom, National Autonomous University of Mexico, Mexico; Joost Hordijk, National Institute for Public Health and the Environment, Netherlands; J. Tony Pembroke, University of Limerick, Ireland
This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
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Previously, we showed that cefotaxime (CTX) exposure increases conjugative transfer of a
Conjugation is one of the most important mechanisms for intra- and inter-species horizontal gene transfer (HGT), and it plays a significant role to accelerate the dispersal of antibiotic resistance genes (
Extended-spectrum beta-lactamase (ESBL) production is a common resistance mechanism against β-lactam antibiotics in Gram-negative bacteria (
It is generally assumed that antibiotics can enhance HGT, and also a substantial amount of data suggest that sub-inhibitory concentrations of antibiotics may significantly increase the conjugation transfer frequency both
In a previous study, we designed an experimental setup for measurement of conjugation frequency in which we could separate conjugation rate from the power of selection by the antibiotic by pre-growing the donor in the presence of [1/2 minimal inhibitory concentration (MIC)] of antibiotics and performing the conjugation in an antibiotic-free environment for 30 or 60 min (
Twenty-five ESBL-producing
Genetics features of commensal
ESBL 1 | CTX-M-1 | ST101 | 3 | |||
ESBL 2 | CTX-M-1 | ST4373 | IncFII, IncFIB(AP001918) | – | ||
ESBL 3 | CTX-M-1 | ST4580 | IncI1, ColpVC, pO111 | 7 | ||
CTX-M-1 | ST58 | 7 | ||||
ESBL 5 | CTX-M-1 | ST165 | – | |||
ESBL 6 | CTX-M-1 | ST295 | IncFIC (FII), IncFIB(AP001918) | – | ||
ESBL 7 | CTX-M-1 | ST362 | IncFII, |
NT |
||
ESBL 8 | CTX-M-1 | ST345 | 3 | |||
ESBL 9 | CTX-M-1 | ST101 | IncFII(29), |
NT |
||
CTX-M-1 | ST647 | IncFII(pSE11), |
293 |
|||
ESBL 11 | CTX-M-1 | ST48 | IncFII(29), |
294 |
NDm | |
CTX-M-1 | ST1640 | IncFII, |
295 |
|||
ESBL 13 | CTX-M-1 | ST4243 | NT |
|||
ESBL 14 | CTX-M-1 | ST117 | IncFII, |
296 |
||
ESBL 15 | CTX-M-1 | ST752 | IncFIC(FII), |
7 | ||
ESBL 16 | CTX-M-1 | ST88 | IncFII, IncI1, Col(MG828), IncFIB(AP001918) | 7 | ||
CTX-M-1 | ST101 | IncFII, |
3 | |||
ESBL 18 | TEM-52 | ST10 | IncFIA, IncFII, IncFIB(AP001918), |
– | ||
ESBL 19 | TEM-52 | ST10 | IncFIA, IncFII, |
– | ||
ESBL 20 | TEM-52 | ST2607 | IncFIC(FII), IncI1, IncN, IncFIB(AP001918) | – | ||
CTX-M-55 | ST2952 | 16 | ||||
CTX-M-14 | ST1310 | – | ||||
CTX-M-14 | ST88 | – | ||||
ESBL 24 | CTX-M-14 | ST34 | IncI1, IncQ1 |
259 | ND |
|
ESBL 25 | SHV-12 | ST115 | IncFII, IncFIB(AP001918), pO111 | – |
Sequencing reads were quality trimmed using Trimmomatic-v0.32 (
The MIC of CTX and AMP was determined on donors and transconjugants from the first conjugation round (see below) using the broth microdilution method [concentrations ranging from 0 to 2048 mg L–1 (CTX) or 0 to 4096 mg L–1 (AMP) by twofold dilution increases] in poly-styrene microtiter plates using the control strain
The 25 strains containing different ESBL plasmids and MG1655/pTF2 (
Transconjugants from the first conjugation round showing significant antibiotic-induced increase in PT and their respective donors were selected for plasmid profiling. Plasmid DNA was purified according to the method described by
The plasmid replicon types of the ESBL plasmids were confirmed in the transconjugants from the first conjugation round using PCR-Based Replicon Typing (PBRT) 2.0 Kit (Diatheva, Fano, Italy). The total DNA was obtained by boiling lysis method exposing cells to 100°C for 10 min, and clarifying the lysate preparations by centrifugation at 15,000 ×
Plasmid DNA was isolated from the transconjugants from the first conjugation round showing significant antibiotic-induced increase in PT using PureLink HiPure Plasmid DNA Purification Kits (Invitrogen, CA, United States), and the plasmid DNA was subjected to pair-end-read format using the Nextera XT DNA Library Preparation Kit and sequenced in an Illumina MiSeq (Illumina, Inc., San Diego, CA, United States) essentially as previously described (
Single colonies of transconjugants from the first conjugation round were grown in LB broth with shaking (125 rpm) overnight at 37°C. The cultures were diluted 1000-fold and grown with and without antibiotics to OD600 = 0.5. The antibiotic concentration was 1/2 MIC. A FastPrep cell disrupter system (Qbiogene, Illkirch, France) and RNeasy Mini Kit (Qiagen, Sollentuna, Sweden) were used to extract total RNA by mechanical disruption. Quantity of the extracted RNA was determined by A260 measurements and purity by A260/280 ratio measurements using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Hvidovre, Denmark). RNA (1 μg) samples were purified by DNA digestion using TURBOTM DNase kit (2 U/μL) (Ambion, Life Technologies, Nærum, Denmark) to remove contaminating genomic DNA.
RNA was reverse-transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Nærum, Denmark). Reverse-transcribed-quantitative real time polymerase chain reaction (RT-qPCR) was performed using FastStart Essential DNA Green Master (Roche, Hvidovre, Denmark) and a LightCycler 96 (Roche, Hvidovre, Denmark). Expression levels of
Statistical analysis used in this study was performed using GraphPad Prism version 7.03. Data are expressed as the means ± standard deviation (SD) from at least three independent experiments. Comparison of gene expression and conjugation frequencies with and without antibiotics were analyzed using Student’s
With the aim of investigating PT of different plasmid types in various
Conjugation transfer frequency was investigated using the 25 diverse strains and the previously investigated MG1655/pTF2 was included as positive control (
Fold changes of antibiotic (CTX, AMP, or CIP)-induced increased conjugation transfer frequency using ESBL
MG1655/pTF2 (IncI1/CTX-M-1) | 30 | 4.63 × 10–3 ± 1.25 × 10–3 | 2.93 × 10–2 ± 0.23 × 10–2 | 6.3∗∗ | 1.30 × 10–2 ± 0.11 × 10–3 | 2.8∗ | 5.73 × 10–3 ± 0.80 × 10–3 | 1.2 |
60 | 1.11 × 10–2 ± 0.24 × 10–2 | 4.09 × 10–2 ± 0.24 × 10–2 | 3.7∗∗ | 6.11 × 10–2 ± 0.51 × 10–2 | 5.5∗ | 6.97 × 10–2 ± 1.04 × 10–2 | 6.3∗ | |
ESBL 4 (IncI1/CTX-M-1) | 30 | 4.11 × 10–4 ± 0.73 × 10–4 | 1.04 × 10–3 ± 0.39 × 10–3 | 2.5 | 3.63 × 10–3 ± 1.13 × 10–3 | 8.8 | 1.39 × 10–3 ± 0.33 × 10–3 | 4.7 |
60 | 5.71 × 10–4 ± 0.74 × 10–4 | 1.9 × 10–3 ± 0.38 × 10–3 | 3.3∗ | 6.53 × 10–3 ± 0.67 × 10–3 | 11.3∗ | 4.67 × 10–3 ± 0.70 × 10–3 | 8.1∗ | |
ESBL 10 (IncI1/CTX-M-1) | 30 | 1.04 × 10–2 ± 0.12 × 10–2 | 3.62 × 10–2 ± 1.22 × 10–2 | 3.5 | 2.93 × 10–2 ± 0.28 × 10–2 | 2.8∗ | 2.44 × 10–2 ± 0.58 × 10–2 | 2.3 |
60 | 1.17 × 10–2 ± 0.11 × 10–2 | 1.02 × 10–1 ± 0.11 × 10–1 | 8.7∗ | 2.97 × 10–2 ± 0.31 × 10–2 | 2.5∗ | 5.26 × 10–2 ± 1.21 × 10–2 | 4.5 | |
ESBL 12 (IncI1/CTX-M-1) | 30 | 8.46 × 10–5 ± 1.69 × 10–5 | 2.44 × 10–4 ± 0.16 × 10–4 | 2.9∗ | 2.85 × 10–4 ± 0.55 × 10–4 | 3.3 | 1.47 × 10–4 ± 0.19 × 10–4 | 1.7 |
60 | 1.47 × 10–4 ± 0.09 × 10–4 | 1.77 × 10–3 ± 0.38 × 10–3 | 12.0∗ | 6.63 × 10–4 ± 1.24 × 10–4 | 4.5∗ | 5.61 × 10–4 ± 1.42 × 10–4 | 3.8 | |
ESBL 17 (IncI1/CTX-M-1) | 30 | 2.63 × 10–5 ± 0.33 × 10–5 | 5.61 × 10–5 ± 0.72 × 10–5 | 2.1∗ | 3.35 × 10–5 ± 1.41 × 10–5 | 1.2 | 3.20 × 10–5 ± 1.41 × 10–5 | 1.2 |
60 | 4.15 × 10–5 ± 1.07 × 10–5 | 1.09 × 10–4 ± 0.20 × 10–4 | 2.6∗ | 4.20 × 10–5 ± 1.54 × 10–5 | 1.0 | 3.29 × 10–5 ± 0.95 × 10–5 | 0.8 | |
ESBL 21 (IncI1/CTX-M-55) | 30 | 2.73 × 10–3 ± 0.40 × 10–3 | 8.96 × 10–3 ± 1.99 × 10–3 | 3.3 | 8.63 × 10–3 ± 2.03 × 10–3 | 3.2 | 9.34 × 10–3 ± 0.66 × 10–3 | 3.4 |
60 | 5.41 × 10–3 ± 0.18 × 10–2 | 1.62 × 10–2 ± 0.27 × 10–2 | 3.3∗ | 1.45 × 10–2 ± 0.11 × 10–3 | 2.7∗ | 1.30 × 10–2 ± 0.15 × 10–2 | 2.4 | |
ESBL 22 (IncF/CTX-M-14) | 30 | 1.61 × 10–4 ± 0.36 × 10–4 | 5.91 × 10–4 ± 1.18 × 10–4 | 3.7∗ | 9.3 × 10–4 ± 0.95 × 10–4 | 5.8∗ | 9.67 × 10–4 ± 0.94 × 10–4 | 6.0∗ |
60 | 2.72 × 10–4 ± 0.94 × 10–4 | 8.69 × 10–4 ± 2.26 × 10–4 | 3.2 | 1.37 × 10–3 ± 0.14 × 10–3 | 5.0∗ | 1.22 × 10–3 ± 0.19 × 10–3 | 4.5∗ | |
ESBL 23 (IncF/CTX-M-14) | 30 | 2.70 × 10–4 ± 0.17 × 10–4 | 7.35 × 10–4 ± 0.55 × 10–4 | 2.7∗ | 3.11 × 10–4 ± 0.28 × 10–4 | 0.9 | 2.87 × 10–4 ± 0.22 × 10–4 | 1.1 |
60 | 3.98 × 10–4 ± 0.70 × 10–4 | 1.07 × 10–3 ± 0.18 × 10–3 | 2.7∗ | 3.79 × 10–4 ± 0.55 × 10–4 | 1.0 | 4.28 × 10–4 ± 0.35 × 10–4 | 1.1 |
Plasmid profiling and PBRT and sequencing of the transconjugants from the first conjugation round confirmed the presence of the replicons of the ESBL plasmids identified by WGS data analysis and allowed further insights into the IncI1 replicons. PBRT revealed that IncI1α and IncI1γ replicons co-existed in two transconjugants, namely 10AMP and 10CIP (transconjugant originating from ESBL10 treated with AMP or CIP, respectively). The occurrence of both replicons was verified in the donor (ESBL10) and transconjugants by analysis of the WGS data, and was also confirmed by plasmid profiling (data not shown). It is interesting to note that in transconjugant 10CTX, only the IncI1α replicon was transferred. An additional case in which the transconjugants harbored other plasmids besides the ESBL plasmid was represented by 12AMP, which harbored a non-typeable plasmid and a Col156 plasmid besides the IncI1α ESBL encoding plasmid. In all remaining cases, the transconjugants harbored only the ESBL encoding plasmids which were either IncI1α (4CTX, 12CTX, 12AMP, 17CTX, 17AMP, 17CIP, 21CTX, 21AMP, and 21CIP) or IncFII (22CTX, 22AMP, 22CIP, 23CTX, 23AMP, and 23CIP).
Previous data had shown that increased PT was linked to up-regulation of the transfer (
Expression of genes involved in conjugation in the transconjugants from the first round of conjugation. Cell samples grown with no or 1/2 MIC of antibiotics (+CTX, AMP, or CIP) were used for RT-qPCR. Data are presented as fold change relative to control without antibiotics. The combination of strain number with antibiotic name represents the transconjugants from the first round of conjugation obtained after exposure to the specified antibiotic. The expression data were normalized to the reference genes,
To investigate the involvement of the SOS response in antibiotic induced PT, we analyzed the expression of
Expression of SOS-responsive genes in the transconjugants from the first round of conjugation. Cell samples grown with no or 1/2 MIC of antibiotics (+CTX, AMP, or CIP) were used for RT-qPCR. Data are presented as fold change relative to control without antibiotics. Note that the different figures are not drawn to the same scale. The combination of strain number with antibiotic name represents the transconjugants from the first round obtained after exposure to the specified antibiotic. The expression data were normalized to two validated reference genes,
In order to investigate whether the up-regulated
Fold changes of antibiotic-induced increased conjugation transfer frequency using transconjugants from first conjugation round as donors and J53-1 as recipient.
30 | MG1655/pTF2 CTX | 1.6 | MG 1655/pTF2 AMP | 1.6 | MG1655/pTF2 CIP | 1.3 |
60 | 1.6 | 2.2 | 3.1 | |||
30 | 4CTX | 28.9∗∗ | 4 AMP | 6.3∗ | 4CIP | 7.6∗ |
60 | 18.7∗∗ | 3.7 | 3.7∗∗ | |||
30 | 10CTX | 8.6∗ | 10 AMP | 3.3 | 10CIP | 6.8 |
60 | 3.0∗ | 1.3 | 2.6 | |||
30 | 12CTX | 3.1 | 12 AMP | 2.6 | 12CIP | 2.0 |
60 | 2.2∗ | 1.8 | 1.6 | |||
30 | 17CTX | 5.3∗∗ | 17 AMP | 8.2∗ | 17CIP | 3.7∗ |
60 | 16.8∗ | 4.0∗ | 3.4 | |||
30 | 21CTX | 6.3 | 21 AMP | 4.7 | 21CIP | 2.4 |
60 | 2.4∗ | 3.5 | 3.8∗ | |||
30 | 22CTX | 2.4 | 22 AMP | 1.4 | 22CIP | 1.8 |
60 | 2.7∗ | 1.8 | 1.7∗ | |||
30 | 23CTX | 1.3 | 23 AMP | 1.4 | 23CIP | 1.2 |
60 | 1.3 | 1.3 | 2.0 |
The increasing occurrence of antibiotic resistance (AMR) among pathogenic bacteria is considered a major problem for public health in recent decades; and conjugation plays a major role in the spread of AMR (
Extended spectrum beta-lactamases genes, and in particular CTX-M beta-lactamases, have become the most prevalent cephalosporin-resistance determinants in Enterobacteriaceae (
Previously, it had been shown that induction of the SOS-response by antibiotics enhanced transfer of plasmids and ICEs in
Previously published results (
In conclusion, our findings reveal that exposure to different antibiotics can increase conjugative transfer of different naturally occurring β-lactam resistance plasmids in a strain-independent manner. Thus, antibiotic-induced conjugation transfer of ESBL plasmids appears to be a more common phenomenon in
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.
GL, LT, VB, and JO designed the study. GL and KB carried out the experiments. GL, VB, and LT analyzed the results. GL and LT wrote the manuscript. All authors commented on and approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Tony P. Bønnelycke and Dan F. Ryttov are thanked for valuable technical assistance.
The Supplementary Material for this article can be found online at: