Mating assay: Plating below a cell density threshold is required for unbiased estimation of transfer frequency or transfer rate

doi:10.21203/rs.3.rs-4373740/v1

Download PDF

Research Article

Mating assay: Plating below a cell density threshold is required for unbiased estimation of transfer frequency or transfer rate

https://doi.org/10.21203/rs.3.rs-4373740/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 28 Aug, 2024

Read the published version in Microbial Ecology →

You are reading this latest preprint version

Mating assays are common laboratory experiments for measuring the rate, frequency, or efficiency at which a plasmid transfers from a population of donor cells to a population of recipient cells. Selective plating remains a widely used quantification method to enumerate transconjugants at the end of such assays. However, transfer frequencies or rates may be inaccurately estimated because plasmid transfer can occur on transconjugant-selective plates rather than only during the intended mating period. We investigated the influence of cell density on this phenomenon. We conducted mating experiments with IncPα plasmid RP4 at a range of cell densities and mating conditions and compared the results to a model of cell-to-cell distance distribution. Our findings suggest that irrespective of the mating mode (solid vs liquid), the enumeration of transconjugants is significantly biased if the plated cell density exceeds 20 Colony Forming Unit (CFU) /mm² (or 1.2x10⁵ CFU per standard 9 cm Petri dish). Liquid mating assays were more sensitive to this bias because the transfer frequency of RP4 is several orders of magnitude lower in suspension compared to surface mating. Therefore, if selective plating is used, we recommend to plate below this density threshold and that negative controls are performed where donors and recipients are briefly mixed before plating at the same dilutions as for the actual mating assay.

Conjugation

Selective plating

Plate counting

Enumeration

Guideline

Plasmid

Conjugation is a phenomenon that greatly facilitates the exchange of genetic information between microbes within a microbial community[1], [2]. It involves the mobilization of genetic elements such as plasmids[3] and integrative and conjugative elements[4], [5].

Understanding the drivers and barriers of conjugation is essential for predicting or hindering the spread of virulence factors and antibiotic resistance genes[6], [7] and facilitating traits such as biotransformation functions[8], [9]. Therefore, it is useful to employ mating assays, where a population of donors transfer plasmids to a population of recipients, to estimate the transfer frequency or transfer rate, i.e. the efficiency of the plasmid transfer.

Typical mating assays are conducted with pure cultures of donors and recipients[10], [11], [12], [13], [14] and mated either in a liquid suspension[10], [11], [13], [15], [16], [17] or on a solid surface[14], [18], [19], [20], [21], [22] because different classes of plasmids transfer more efficiently in one or the other condition, depending on the type of pilus the plasmid encodes[23]. At the end of the mating period, the mating mixture is often plated on agar plates with appropriate selective agents (e.g. antibiotics) to enumerate transconjugants, donors, and recipients after sufficiently long incubation[10], [12], [24], [25], [26], [27].

However, it is essential to acknowledge that experimental bias can arise from this enumeration technique, as many antibiotics seldom kill or inhibit growth of bacteria immediately[28], [29]. Although antibiotics may halt cell division within minutes, cells can remain metabolically active under physiological stress[30]. This phenomenon can provide opportunities for donor cells to transfer a plasmid to recipients on the transconjugant-selective plates if they have been plated at high density (i.e. in close proximity) and thus artefactually inflate the number of transconjugants. This consequently impedes the accurate estimation of plasmid transfer events under the initial mating conditions. The probability of such overestimation increases with increasing density of parent cells plated on the selective plates[31]. Although the issue of plasmid transfer occurring on transconjugant-selective plates has long been recognized[20], and even mentioned as one of the assumptions to consider in appropriate guidelines[32], an examination of recent literature[10], [11], [12], [15], [16], [25], [26], [27], [33], [34], [35], [36] shows this phenomenon has neither been explicitly reported nor has it been regularly taken into consideration.

In this study, we sought to determine the influence of cell densities on plasmid transfer by disentangling the number of transconjugants produced on transconjugant-selective plates relative to those produced during the mating assay, conducted either in liquid suspension or on a solid surface. Hereby, we offer additional insight into this phenomenon and underscore the significance of cell densities for accurate estimations of transfer frequency and rate.

2.1 Bacterial strains and conjugative plasmids

To determine how plasmid transfer occurring on transconjugant-selective plates is influenced by cell densities, we used model strains Escherichia coli (E. coli) K12 MG1655 as donors and recipients along with the IncPα model conjugative plasmid RP4 (Table S1). To prepare for the conjugation assay, we grew the E. coli MG1655 donor carrying the RP4 plasmid in Luria-Bertani medium with tetracycline at 10 µg/ml and ampicillin at 100 µg/ml. The E. coli MG1655 recipient with chromosomal resistances to nalidixic acid and rifampicin was grown in the same medium without antibiotics.

2.2 Preparation of donor and recipient culture

After overnight growth, 200 µl of donor and recipient culture were transferred to flasks with 10 ml fresh medium supplemented with antibiotics and regrown for 4 h at 37˚C while shaking. The cultures were harvested by centrifugation at 7.441xg for 5 minutes at room temperature and washed twice in phosphate-buffered saline (PBS). After washing, the cultures were resuspended in PBS and diluted to an OD600 of 0.5 to obtain an initial cell density of ~ 1x10⁸ CFU/ml (Table S2).

2.3 Liquid mating

Following strain preparation, the donors and recipient cultures, diluted to the same cell densities, were mixed in a 2 ml microcentrifuge tube at a 1:1 ratio (2 ml total) and incubated at 37˚C for 0, 15, 30, 60, and 120 minutes under static conditions. After mating, the tubes were thoroughly vortexed for 1 min and kept on ice. A serial dilution series of the mating mixtures were plated on transconjugant-selective plates (9 cm Petri dish, 6082 mm²) containing 10 µg/ml tetracycline, 100 µg/ml ampicillin, and 50 µg/ml nalidixic acid. Enumeration of donors and recipients were done by spotting several aliquots of 10 µl of a serial dilution on donor-selective plates containing 10 µg/ml tetracycline and 100 µg/ml ampicillin and on recipient selective plates containing 50 µg/ml nalidixic acid. Plates were incubated overnight at 37˚C and inspected for enumeration (Table S3). Plating was carried out in duplicates or triplicates. In addition, liquid mating was repeated with slightly different conditions, including stationary-phased cultures, 480 minutes of mating, and a mating temperature of 25˚C to investigate if such changes could increase the number of transconjugants.

2.4 Solid mating

For solid mating, sterile 25 mm Whatman® glass microfiber filters Grade GF/C (WHA1822025) were placed onto each chamber of a 1225 Millipore Sampling Manifold (XX2702550) and pre-wetted with PBS followed by placing sterile 25 mm Whatman® Cyclopore® polycarbonate filters (WHA70632502) with working area of 280 mm² on top of the glass microfiber filters. Next, 2 ml of PBS was added to all chambers to flush the filters. Following strain preparation, the cultures were diluted to different cell densities and added to each chamber in a 1:1 ratio (2 ml in total) and filtered. Once filtered, the polycarbonate filters were transferred to nutrient-free PBS agar plates to allow for solid mating and incubated at 37˚C for 0, 5, 10, 15, 30, and 60 min. After mating, the polycarbonate filters were transferred to 2 ml Eppendorf tubes containing 1 ml saline and subsequently vortexed vigorously for 1 min to detach cells. Next, cells were diluted, plated, and enumerated in a similar manner as above (Section 2.3). Identically to the liquid mating, solid mating was repeated with slightly different conditions, including stationary-phased cultures, 480 minutes of mating, and a mating temperature of 25˚C to investigate if such changes could increase the number of transconjugants.

2.5 Estimating cell-to-cell distances using distribution function and simulation

The distribution of cells on a surface can be described as a homogenous Poisson point process. The distance to the closest cell (r) has a distribution function F(r) = \(1-{e}^{-\lambda {r}^{2}}\) as previously described[37]. We used a distance of 1 µm or less between cells as an indication of a successful donor-recipient mating pair. The simulation was done using RStudio version 4.3.2. The rpois() function was used to generate a Poisson-distributed number of points representing donor and recipient cells, which were placed randomly across space by generating random coordinates for the cells. The pairwise distances between cells were calculated using the dist() function.

3.1 Plasmid transfer on transconjugant-selective plates generates artefactual transconjugants when a cell density threshold is exceeded

Plating the mating mixture immediately after mixing donor and recipients in the mating assays confirmed that RP4 transfer could readily occur on transconjugant-selective plates, provided the cell density was high enough (Fig. 1, Table S4, S5, and S6).

When plating a mixture of donors and recipients on the transconjugant-selective plates at a high cell density (≥ 200 CFU/mm² or 1.2x10⁶ CFU per standard 9 cm Petri dish), many transconjugant colonies were observed. In contrast, at low cell density (≤ 20 CFU/mm² or 1.2x10⁵ CFU/plate), very few or no transconjugants were observed. In conclusion, plating a high density of donors and recipients from the mating mixture to enumerate transconjugants will lead to artefactual inflation of the transconjugant numbers.

Next, we further investigated these cell concentrations (Fig. 1) to see if the link between cell density and the formation of transconjugants could be captured with a simple model. Assuming a homogenous Poisson process, the probability for a cell to be at 1 µm or less to its nearest neighbor are 0.0002%, 0.002%, 0.02%, and 0.2% for densities of 2, 20, 200, and 2000 CFU/mm², respectively (Fig. 2a and 2b, Fig. S2 and S3).

We then multiplied the individual probability by the number of cells per plate, to estimate the number of transconjugants generated from RP4 transfer on transconjugant-selective plates. Using the estimated probability of individual cells being within a distance equal to or less than 1 µm for each cell density, the expected transconjugant counts were determined to be 2.4x10^− 2, 2.4, 243.3, and 2.4x10⁴ CFU for total numbers of donors and recipients at 1.2x10⁴, 1.2x10⁵, 1.2x10⁶, and 1.2x10⁷ CFU per standard 9 cm Petri dish, respectively. This is consistent with our observations of CFU counts of transconjugants formed on transconjugant-selective plates (Fig. 1) and thus suggests a high transfer efficiency on the transconjugant-selective plates once donor and recipients are at close range. All in all, the distribution function can be leveraged to decide on a cell density when plating, based on the risk one is willing to take, to have a certain number of artefactual transconjugants on the transconjugant-selective plates.

The decision of the admissible number of transconjugants at each density will depend on the efficiency of plasmid transfer during the mating assay and its duration. Furthermore, this model (possibly modified with data from the relevant plasmid) can even be used to correct datasets where plating was used as enumeration technique for artefactual plasmid transfer, for example from published studies.

3.2 Efficient transfer in solid mating eliminates the biased transfer frequencies and transfer rates of RP4 observed in liquid mating

Knowing that the cell density on the transconjugant-selective plates should not exceed a certain threshold (20 CFU/mm²), we explored different modalities of mating assays to evaluate which one can generate enough transconjugants relative to the density of donors and recipients so they can be detected without significant artefacts from the transconjugant-selective plates.

We first tested liquid mating, but irrespective of the mating time, the mating never produced enough transconjugants that could be detected once diluted to the 2 CFU/mm² density (Fig. S1, Table S4 and S5). These observations indicate that RP4 transfer frequencies and rates in liquid mating are likely overestimated when followed by CFU plate counting. Only in one instance, we could estimate the transfer rate in liquid at 1.01x10^− 16 ml•CFU^− 1•min^− 1.

Next, we tested solid mating on filters placed on top of an agar surface, allowing for more efficient mating because of the short, rigid conjugative pili encoded by the RP4 plasmid (Fig. 3, Table S5).

We observed an increase of transconjugant numbers with mating duration for all mating densities (2x10³, 2x10⁴, and 2x10⁵ CFU/mm² of donors and recipients) when plating 2 CFU/mm² on the transconjugant-selective plates (Fig. 3a) in contrast to liquid mating (Fig. S1). While the estimated transfer rates increased during the first 20 minutes of the mating, they then decreased towards the end of the mating period (30–480 min) (Fig. 3B). This may be because following an extended mating period, energy depletion and the absence of new mating pairs hinder further plasmid transfers. Additionally, we observed that by initiating mating at a high cell density (2x10⁵ CFU/mm²), the close proximity of donors and recipients increases the likelihood of cell-to-cell contact and thereby enhancing the transfer rate compared to mating at lower cell densities (2x10³-2x10⁴ CFU/mm²).

Liquid mating followed by plate counting is a simple and easy method to quantify transfer frequencies and transfer rates. Despite the relative ease of the method, one essential aspect that is seldom accounted for is the risk of bias introduced by the artefactual increase of transconjugant counts due to plasmid transfer on the transconjugant-selective enumeration plates.

Our study confirms that there is a limit to the density of donors and recipients that can be plated to avoid plasmid transfer occurrences on transconjugant-selective plates. For RP4, if the number of cells plated exceeds 20 CFU/mm² (or 1.2x10⁵ CFU per standard 9 cm Petri dish), the estimated transfer frequency/rates will be largely inflated, irrespective of mating method. Hence, we recommend that the cell density is maintained below the threshold (e.g. by making appropriate dilutions) when enumerating transconjugants. Similar finding of a threshold of 10⁵ CFU per Petri dish has been previously reported[20].

We also confirmed that the transfer of RP4 occurs at a very low rate (~ 10^− 16 ml•CFU^− 1•min^− 1) in liquid mating compared to solid mating (~ 10^− 14-10^− 11 ml•CFU^− 1•min^− 1). This raises an important question about all the studies that estimate transfer frequencies or rates of RP4 by a combination of liquid mating and plate count enumeration. Indeed, while controls to check for donors and recipients ability to grow on transconjugant-selective plates by spontaneous mutations are regularly included[34], [38], [39], controls for plasmid transfer on the transconjugant-selective plates are not routinely performed. Therefore, when a new transfer assay is developed, we recommend including a t₀ control where plating is performed immediately after mixing donors and recipients to identify the dilution range that avoids plasmid transfer on the transconjugant-selective plates. Such control is also included, as guideline 2a, in a recent set of guidelines for the estimation and reporting of plasmid conjugation rates[32].

While our study focused on RP4, one of the most used models in plasmid ecology, any plasmid that presents much higher transfer efficiency on solid surfaces than in suspension will be at risk of similar spurious inflation of conjugation rate. Such strong preference for transferring on surfaces has been shown for diverse plasmids including IncP-1β plasmid pB10[40], IncW plasmid R388[40], IncP-9 plasmid NAH7K2[40], and Inc18 plasmid pAMβ1[41] with transfer frequencies being several orders of magnitude higher. For such plasmids, we recommend adopting unbiased enumeration techniques^18,25,26 after mating assay in liquid environments.

Addition of nalidixic acid has been reported to counteract plasmid transfer on transconjugant-selective plates[20]. However, in our work, nalidixic acid at bactericidal concentration of 50 µg/ml[44] proved to insufficiently inhibit the donors at high cell densities and thus failed to prevent plasmid transfer on transconjugant-selective plates. In any case, a survey of numerous studies estimating transfer frequencies[10], [11], [12], [15], [16], [25], [27], [33], [34], [38], [39], [45] indicates that nalidixic acid is rarely used in transconjugant-selective plates (Table S3). Instead, combinations of kanamycin, ampicillin, chloramphenicol, streptomycin, tetracycline, and rifampicin are often applied. Moreover, the synergistic and antagonistic behavior of antibiotics on transconjugant-selective plates in the context of transconjugant enumeration is unexplored. It is commonly emphasized that the interaction between bacteriostatic and bactericidal antibiotics results in an antagonistic effect since bacteriostatic drugs antagonize bactericidal drugs that affect dividing cells by inhibiting cell growth[46], [47]. Notable examples of antagonistic effects are interactions between drugs that inhibit the 30S protein synthesis and cell wall synthesis, 50S protein synthesis and DNA replication, as well as folic acid synthesis and cell wall synthesis. In some cases, drug combinations even cause suppression, where one antibiotic alleviates the effect of another [46]. It is therefore conceivable that the combination of tetracycline (bacteriostatic inhibitor of 30S protein synthesis) and ampicillin (bactericidal inhibitor of cell wall synthesis) used in our study may have resulted in an antagonistic drug interaction and diminished the inhibitory effect on the recipients long enough for them to receive plasmids from the donors. Furthermore, the drug combination of tetracycline and nalidixic acid (bactericidal inhibitor of DNA replication) suggests an antagonistic effect directed toward the donors, with evidence of this occurring in time-kill curves[47].

All in all, mating assays are essential for studying the efficiency of mobilization of genetic elements such as plasmids to allow for additional insight into predicting, hindering, or facilitating plasmid transfer. The main goal with this type of experiment is to accurately estimate the transfer frequencies and rates.

We demonstrated that plating mixtures of donors harboring the RP4 plasmid and recipients above a certain threshold (20 CFU/mm² or 1.2x10⁵ CFU per standard 9 cm Petri dish) artefactually increases transconjugants counts because of plasmid transfer occurring post-mating on transconjugant-selective plates. This affects accurate estimations of conjugal plasmid transfer frequencies or transfer rates.

Because plasmids differ in their preferential mating modes (solid surface vs liquid suspension), they can be differentially affected by this problem. For example, for RP4, transconjugants formed in liquid mating assays were undetected because they were diluted out when plating on transconjugant-selective plates at or below the threshold density.

Therefore, as a final remark, we highlight our recommendation of plating below the cell density threshold and/or performing additional controls where donors and recipients are briefly mixed before plating at the same dilutions as for the actual mating. Alternatively, adopting a robust and unbiased enumeration technique can be useful for the prevention of artefactual increase of transconjugants.

Acknowledgments

The authors would like to thank the partners in the JPI/BIOCIDE research project for insightful discussions and collaboration. The authors are grateful for the financial support provided by the Innovation Fund Denmark (IFD) and the Sino-Danish Center (SDC).

Funding

This study is partly funded by the Innovative Fund Denmark (IFD) under File No. 0236-00022B and the Sino-Danish Center (SDC) funding for PhD scholarships.

Competing interest

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

Conceptualization: All authors; Methodology: Z.H.; Formal analysis and investigation: All authors; Writing - original draft preparation: Z.H.; Writing - review and editing: All authors; Funding acquisition: B.F.S.; Resources: B.F.S. and A.D.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Lederberg, J. and Tatum, E. (1946) ‘Gene Recombination in Escherichia Coli’, Nature, 158(558), p. 45.
Davison, J. (1999) ‘Genetic Exchange between Bacteria in the Environment’, Plasmid, 42(2), pp. 73–91.
Rodríguez-Beltrán, J. et al. (2021) ‘Beyond horizontal gene transfer: the role of plasmids in bacterial evolution’, Nature Reviews Microbiology, 19(6), pp. 347–359. Available at: https://doi.org/10.1038/s41579-020-00497-1.
Wozniak, R.A.F. and Waldor, M.K. (2010) ‘Integrative and conjugative elements: Mosaic mobile genetic elements enabling dynamic lateral gene flow’, Nature Reviews Microbiology, 8(8), pp. 552–563. Available at: https://doi.org/10.1038/nrmicro2382.
Burrus, V. et al. (2002) ‘Conjugative transposons: The tip of the iceberg’, Molecular Microbiology, 46(3), pp. 601–610. Available at: https://doi.org/10.1046/j.1365-2958.2002.03191.x.
Koraimann, G. (2018) ‘Spread and Persistence of Virulence and Antibiotic Resistance Genes: A Ride on the F Plasmid Conjugation Module’, EcoSal Plus, 8(1). Available at: https://doi.org/10.1128/ecosalplus.esp-0003-2018.
Huang, J. et al. (2023) ‘Conjugative transfer of streptococcal prophages harboring antibiotic resistance and virulence genes’, ISME Journal, 17(9), pp. 1467–1481. Available at: https://doi.org/10.1038/s41396-023-01463-4.
Liang, B. et al. (2012) ‘Horizontal transfer of dehalogenase genes involved in the catalysis of chlorinated compounds: Evidence and ecological role’, Critical Reviews in Microbiology, 38(2), pp. 95–110. Available at: https://doi.org/10.3109/1040841X.2011.618114.
Li, J. et al. (2021) ‘Mechanistic insights into the success of xenobiotic degraders resolved from metagenomes of microbial enrichment cultures’, Journal of Hazardous Materials, 418(March), p. 126384. Available at: https://doi.org/10.1016/j.jhazmat.2021.126384.
Zhang, Y. et al. (2017) ‘Subinhibitory Concentrations of Disinfectants Promote the Horizontal Transfer of Multidrug Resistance Genes within and across Genera’, Environmental Science and Technology, 51(1), pp. 570–580. Available at: https://doi.org/10.1021/acs.est.6b03132.
Yu, Z. et al. (2021) ‘Nonnutritive sweeteners can promote the dissemination of antibiotic resistance through conjugative gene transfer’, ISME Journal, 15(7), pp. 2117–2130. Available at: https://doi.org/10.1038/s41396-021-00909-x.
Zhang, S. et al. (2019) ‘Copper nanoparticles and copper ions promote horizontal transfer of plasmid-mediated multi-antibiotic resistance genes across bacterial genera’, Environment International, 129(May), pp. 478–487. Available at: https://doi.org/10.1016/j.envint.2019.05.054.
Schmidt, S.B.I. et al. (2022) ‘Biocides Used as Material Preservatives Modify Rates of de novo Mutation and Horizontal Gene Transfer in Bacteria’, Journal of Hazardous Materials, 437(May), p. 129280. Available at: https://doi.org/10.1016/j.jhazmat.2022.129280.
Møller, T.S.B. et al. (2017) ‘Treatment with cefotaxime affects expression of conjugation associated proteins and conjugation transfer frequency of an IncI1 plasmid in Escherichia coli’, Frontiers in Microbiology, 8(NOV), pp. 1–9. Available at: https://doi.org/10.3389/fmicb.2017.02365.
Wang, Y. et al. (2021) ‘Non-antibiotic pharmaceuticals promote the transmission of multidrug resistance plasmids through intra- and intergenera conjugation’, ISME Journal, 15(9), pp. 2493–2508. Available at: https://doi.org/10.1038/s41396-021-00945-7.
Zhang, Y., Gu, A.Z., Cen, T., Li, X., He, M., et al. (2018) ‘Sub-inhibitory concentrations of heavy metals facilitate the horizontal transfer of plasmid-mediated antibiotic resistance genes in water environment’, Environmental Pollution, 237, pp. 74–82. Available at: https://doi.org/10.1016/j.envpol.2018.01.032.
Lopatkin, A.J. et al. (2016) ‘Antibiotics as a selective driver for conjugation dynamics’, Nature Microbiology, 1(6), pp. 1–8. Available at: https://doi.org/10.1038/nmicrobiol.2016.44.
del Campo, I. et al. (2012) ‘Determination of conjugation rates on solid surfaces’, Plasmid, 67(2), pp. 174–182. Available at: https://doi.org/10.1016/j.plasmid.2012.01.008.
Reniero, R. et al. (1992) ‘High frequency of conjugation in Lactobacillus mediated by an aggregation-promoting factor’, Journal of General Microbiology, 138(4), pp. 763–768. Available at: https://doi.org/10.1099/00221287-138-4-763.
van Elsas, J.D. and Smit, E. (1990) ‘Determination of Plasmid Transfer Frequency in Soil: Consequences of Bacterial Mating on Selective Agar Media’, Current Microbiology, 21, pp. 151–157.
Beaber, J.W., Hochhut, B. and Waldor, M.K. (2004) ‘SOS response promotes horizontal dissemination of antibiotic resistance genes’, Nature, 427(6969), pp. 72–74. Available at: https://doi.org/10.1038/nature02241.
Klümper, U. et al. (2017) ‘Metal stressors consistently modulate bacterial conjugal plasmid uptake potential in a phylogenetically conserved manner’, ISME Journal, 11(1), pp. 152–165. Available at: https://doi.org/10.1038/ismej.2016.98.
Getino, M. and De La Cruz, F. (2019) ‘Natural and artificial strategies to control the conjugative transmission of plasmids’, Microbial Transmission, (6), pp. 33–64. Available at: https://doi.org/10.1128/9781555819743.ch3.
Jutkina, J. et al. (2018) ‘Antibiotics and common antibacterial biocides stimulate horizontal transfer of resistance at low concentrations’, Science of the Total Environment, 616–617, pp. 172–178. Available at: https://doi.org/10.1016/j.scitotenv.2017.10.312.
Cen, T. et al. (2020) ‘Preservatives accelerate the horizontal transfer of plasmid-mediated antimicrobial resistance genes via differential mechanisms’, Environment International, 138(January), p. 105544. Available at: https://doi.org/10.1016/j.envint.2020.105544.
Lu, J. et al. (2018) ‘Triclosan at environmentally relevant concentrations promotes horizontal transfer of multidrug resistance genes within and across bacterial genera’, Environment International, 121(October), pp. 1217–1226. Available at: https://doi.org/10.1016/j.envint.2018.10.040.
Lu, J. et al. (2020) ‘Both silver ions and silver nanoparticles facilitate the horizontal transfer of plasmid-mediated antibiotic resistance genes’, Water Research, 169. Available at: https://doi.org/10.1016/j.watres.2019.115229.
Firsov, A.A. et al. (1997) ‘Parameters of bacterial killing and regrowth kinetics and antimicrobial effect examined in terms of area under the concentration-time curve relationships: Action of ciprofloxacin against Escherichia coli in an in vitro dynamic model’, Antimicrobial Agents and Chemotherapy, 41(6), pp. 1281–1287. Available at: https://doi.org/10.1128/aac.41.6.1281.
Norcia, L.J.L., Silvia, A.M. and Hayashi, S.F. (1999) ‘Against Veterinary Pathogenic Bacteria Including Pasteurella ’, Journal of Antibiotics, 52(1), pp. 52–60.
Imlay, J.A. (2013) ‘The molecular mechanisms and physiological consequences of oxidative stress: Lessons from a model bacterium’, Nature Reviews Microbiology, 11(7), pp. 443–454. Available at: https://doi.org/10.1038/nrmicro3032.
Nakazawa, S. et al. (2017) ‘Different transferability of incompatibility (Inc) P-7 plasmid pCAR1 and IncP-1 plasmid pBP136 in stirring liquid conditions’, PLoS ONE, 12(10), pp. 1–15. Available at: https://doi.org/10.1371/journal.pone.0186248.
Kosterlitz, O. and Huisman, J.S. (2023) ‘Guidelines for the estimation and reporting of plasmid conjugation rates’, Plasmid, 126(April), p. 102685. Available at: https://doi.org/10.1016/j.plasmid.2023.102685.
Qiu, Z. et al. (2015) ‘Effects of nano-TiO2 on antibiotic resistance transfer mediated by RP4 plasmid’, Nanotoxicology, 9(7), pp. 895–904. Available at: https://doi.org/10.3109/17435390.2014.991429.
Qiu, Z. et al. (2012) ‘Nanoalumina promotes the horizontal transfer of multiresistance genes mediated by plasmids across genera’, Proceedings of the National Academy of Sciences of the United States of America, 109(13), pp. 4944–4949. Available at: https://doi.org/10.1073/pnas.1107254109.
Wang, Q., Mao, D. and Luo, Y. (2015) ‘Ionic Liquid Facilitates the Conjugative Transfer of Antibiotic Resistance Genes Mediated by Plasmid RP4’, Environmental Science and Technology, 49(14), pp. 8731–8740. Available at: https://doi.org/10.1021/acs.est.5b01129.
Zhang, Y., Gu, A.Z., Cen, T., Li, X., Li, D., et al. (2018) ‘Petrol and diesel exhaust particles accelerate the horizontal transfer of plasmid-mediated antimicrobial resistance genes’, Environment International, 114(February), pp. 280–287. Available at: https://doi.org/10.1016/j.envint.2018.02.038.
Lagido, C. et al. (2003) ‘A model for bacterial conjugal gene transfer on solid surfaces’, FEMS Microbiology Ecology, 44(1), pp. 67–78. Available at: https://doi.org/10.1016/S0168-6496(02)00453-1.
Wang, Y. et al. (2019) ‘Antiepileptic drug carbamazepine promotes horizontal transfer of plasmid-borne multi-antibiotic resistance genes within and across bacterial genera’, ISME Journal, 13(2), pp. 509–522. Available at: https://doi.org/10.1038/s41396-018-0275-x.
Wang, X. et al. (2018) ‘Bacterial exposure to ZnO nanoparticles facilitates horizontal transfer of antibiotic resistance genes’, NanoImpact, 10(November 2017), pp. 61–67. Available at: https://doi.org/10.1016/j.impact.2017.11.006.
Yanagida, K. et al. (2016) ‘Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among pseudomonas putida at different cell densities’, Bioscience, Biotechnology and Biochemistry, 80(5), pp. 1020–1023. Available at: https://doi.org/10.1080/09168451.2015.1127131.
Lampkowska, J. et al. (2008) ‘A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species’, International Journal of Food Microbiology, 127(1–2), pp. 172–175. Available at: https://doi.org/10.1016/j.ijfoodmicro.2008.06.017.
Kosterlitz, O. et al. (2022) ‘Estimating the transfer rates of bacterial plasmids with an adapted Luria-Delbrück fluctuation analysis’, PLoS Biology, 20(7), pp. 1–23. Available at: https://doi.org/10.1371/journal.pbio.3001732.
Alalam, H. et al. (2020) ‘A High-Throughput Method for Screening for Genes Controlling’, 5(6), pp. 1–14.
Crumplin, G.C. and Smith, J.T. (1975) ‘Nalidixic acid: an antibacterial paradox’, Antimicrob.Agents Chemother., 8(3), pp. 251–261. Available at: https://doi.org/10.1128/AAC.8.3.251.
Yang, B. et al. (2022) ‘Paclitaxel and its derivative facilitate the transmission of plasmid-mediated antibiotic resistance genes through conjugative transfer’, Science of The Total Environment, 810, p. 152245. Available at: https://doi.org/10.1016/j.scitotenv.2021.152245.
Bollenbach, T. (2015) ‘Antimicrobial interactions: Mechanisms and implications for drug discovery and resistance evolution’, Current Opinion in Microbiology, 27, pp. 1–9. Available at: https://doi.org/10.1016/j.mib.2015.05.008.
Ocampo, P.S. et al. (2014) ‘Antagonism between bacteriostatic and bactericidal antibiotics is prevalent’, Antimicrobial Agents and Chemotherapy, 58(8), pp. 4573–4582. Available at: https://doi.org/10.1128/AAC.02463-14.
Seoane, J. et al. (2011) ‘An individual-based approach to explain plasmid invasion in bacterial populations’, FEMS Microbiology Ecology, 75(1), pp. 17–27. Available at: https://doi.org/10.1111/j.1574-6941.2010.00994.x.
Lawley, T.D. et al. (2003) ‘F factor conjugation is a true type IV secretion system’, FEMS Microbiology Letters, 224(1), pp. 1–15. Available at: https://doi.org/10.1016/S0378-1097(03)00430-0.
Jia, Y. et al. (2021) ‘Acetaminophen promotes horizontal transfer of plasmid-borne multiple antibiotic resistance genes’, Science of the Total Environment, 782, p. 146916. Available at: https://doi.org/10.1016/j.scitotenv.2021.146916.
Huang, H. et al. (2022) ‘Nitric Oxide: A Neglected Driver for the Conjugative Transfer of Antibiotic Resistance Genes among Wastewater Microbiota’, Environmental Science and Technology [Preprint]. Available at: https://doi.org/10.1021/acs.est.2c01889.
Ding, P. et al. (2022) ‘Antidepressants promote the spread of antibiotic resistance via horizontally conjugative gene transfer’, Environmental Microbiology, 24(11), pp. 5261–5276. Available at: https://doi.org/10.1111/1462-2920.16165.
Zhang, H. et al. (2021) ‘Glyphosate escalates horizontal transfer of conjugative plasmid harboring antibiotic resistance genes’, Bioengineered, 12(1), pp. 63–69. Available at: https://doi.org/10.1080/21655979.2020.1862995.
Tang, H. et al. (2022) ‘Effects of iron mineral adhesion on bacterial conjugation: Interfering the transmission of antibiotic resistance genes through an interfacial process’, Journal of Hazardous Materials, 435(April), p. 128889. Available at: https://doi.org/10.1016/j.jhazmat.2022.128889.
Li, W. et al. (2022) ‘Environmentally relevant concentrations of mercury facilitate the horizontal transfer of plasmid-mediated antibiotic resistance genes’, Science of the Total Environment, 852(July), p. 158272. Available at: https://doi.org/10.1016/j.scitotenv.2022.158272.
He, K. et al. (2022) ‘Low-concentration of trichloromethane and dichloroacetonitrile promote the plasmid-mediated horizontal transfer of antibiotic resistance genes’, Journal of Hazardous Materials, 425, p. 128030. Available at: https://doi.org/10.1016/j.jhazmat.2021.128030.
Liao, J., Huang, H. and Chen, Y. (2019) ‘CO2 promotes the conjugative transfer of multiresistance genes by facilitating cellular contact and plasmid transfer’, Environment International, 129(April), pp. 333–342. Available at: https://doi.org/10.1016/j.envint.2019.05.060.
Guo, M.T., Yuan, Q. Bin and Yang, J. (2015) ‘Distinguishing effects of ultraviolet exposure and chlorination on the horizontal transfer of antibiotic resistance genes in municipal wastewater’, Environmental Science and Technology, 49(9), pp. 5771–5778. Available at: https://doi.org/10.1021/acs.est.5b00644.

No competing interests reported.

SupplementaryInformation.pdf

Download PDF

Journal Publication

published 28 Aug, 2024

Read the published version in Microbial Ecology →

Editorial decision: Revision requested
06 Jun, 2024
Reviews received at journal
06 Jun, 2024
Reviews received at journal
01 Jun, 2024
Reviewers agreed at journal
09 May, 2024
Reviewers agreed at journal
09 May, 2024
Reviews received at journal
09 May, 2024
Reviewers agreed at journal
08 May, 2024
Reviewers invited by journal
07 May, 2024
Submission checks completed at journal
06 May, 2024
Editor assigned by journal
06 May, 2024
First submitted to journal
05 May, 2024

You are reading this latest preprint version

Mating assay: Plating below a cell density threshold is required for unbiased estimation of transfer frequency or transfer rate

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Methods

2.1 Bacterial strains and conjugative plasmids

2.2 Preparation of donor and recipient culture

2.3 Liquid mating

2.4 Solid mating

2.5 Estimating cell-to-cell distances using distribution function and simulation

3. Results

3.1 Plasmid transfer on transconjugant-selective plates generates artefactual transconjugants when a cell density threshold is exceeded

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1