A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species

International Journal of Food Microbiology 127 (2008) 172–175

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

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A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species Joanna Lampkowska a, Louise Feld b, Aine Monaghan c, Niamh Toomey c, Susanne Schjørring e, Bodil Jacobsen d, Hilko van der Voet g, Sigrid Rita Andersen b, Declan Bolton c, Henk Aarts f, Karen A. Krogfelt e, Andrea Wilcks b, Jacek Bardowski a,⁎ a

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland Department of Microbiology and Risk Assesment, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark c Food Safety Department, Teagase-Ashtown Food Research Centre, Ashtown, Dublin 15, Ireland d Chr. Hansen A/S, 10-12 Boege Allé, DK-2970 Hoerholm, Denmark e Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Artilerivej 5, 2300 S, Copenhagen, Denmark f Rikilt — Institute of Food Safety, Bornsesteeg 45, 6708 Wageningen, The Netherlands g Biometris and Rikilt, Wageningen University and Research Centre, NL-6700 AC Wageningen, The Netherlands b

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Article history: Received 23 July 2007 Received in revised form 11 June 2008 Accepted 13 June 2008 Keywords: Antibiotic resistance Conjugation Lactococcus lactis Plasmids Standardization

A B S T R A C T Optimal conditions and a standardized method for conjugation between two model lactococcal strains, Lactococcus lactis SH4174 (pAMβ1-containing, erythromycin resistant donor) and L. lactis Bu2-60 (plasmidfree, erythromycin sensitive recipient), were developed and tested in a inter-laboratory experiments involving five laboratories from different countries. The ultimate goal of the study was to assess the microbial potential of antibiotic resistance transfer among Lactic Acid Bacteria (LAB). The influence of culture age (various OD values) and ratios of donor and recipient cultures as well as filter, solid and liquid mating techniques, were examined in order to optimize the conjugation protocol. In the result of these studies, we concluded that the donor-to-recipient ratio appear to be important; the most efficient technique for conjugation was filter mating and the optimal conditions for gene transfer were observed when late logarithmic cultures of both donor and recipient were used. Comparison of conjugal transfer frequencies between five partner laboratories showed that results are sufficiently intra-laboratory repeatable and interlaboratory comparable. This is the first study of this kind, in which a standardized protocol of conjugal mating for testing antibiotic resistance dissemination among LAB was established and validated. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Besides being widespread commensal bacteria, lactococci have an important application as fermentation starter cultures for cheese manufacture. Several gene transfer mechanisms have been found in lactococci (Gasson, 1990), including an aggregation mediated highfrequency conjugation system. In this work, the pAMβ1 plasmid (26.5 kb) served as a model for transfer of a mobile conjugative element between two lactococcal strains. The plasmid, which was originally isolated from Enterococcus faecalis, was chosen for the study due to its self-transmissibility and constitutive MLS resistance (Clewell et al., 1974). Moreover, pAMβ1 has already been shown to transfer via conjugation to Bacillus, Clostridium, Staphylococcus, Enterococcus, Lactobacillus and Lactococcus (Cocconcelli et al., 1985; Gasson and Davies, 1980; Hespell and Whitehead, 1991; Morelli et al., ⁎ Corresponding author. Tel.: +48 22 659 70 72, +48 22 592 21 45(Secretariat); fax: +48 22 592 21 90. E-mail address: [email protected] (J. Bardowski). 0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2008.06.017

1988; Sasaki et al., 1988; Shrago et al., 1986; Tannock, 1987; Vescovo et al., 1983). An important factor in risk assessment of antibiotic resistance genes is to test their ability to transfer. This study was initiated to optimize and validate (in the inter-laboratory experiment) conjugation protocol for plasmid transfer between lactococcal species, and assess the comparability of obtained results. 2. Materials and methods 2.1. Bacterial strains and growth conditions Two lactococcal strains were used in these studies — Lactococcus lactis SH4174 (Gasson and Davies, 1980) as a donor, which harboured the pAMß1 plasmid, carrying resistance to erythromycin encoded on erm(B) and the plasmid-free L. lactis subsp. lactis biovar diacetylactis Bu2-60 strain (Neve et al., 1984) resistant to rifampicin and streptomycin as a recipient. Both strains were grown at 30 °C for 24–48 h in GM17 medium (Difco Laboratories, Detroit, MI) (M17

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right-side up on non-selective GM17 agar plates and incubated for 18– 20 h at 30 °C. Control cultures of recipient and donor strains alone were treated in the same manner (1 ml of donor or recipient added to 1 ml of PPS. Incubation filters were treated with 2 ml of PBS and rigorously shaken to wash bacteria from the filters, and afterwards treated as described previously for solid and liquid mating. 2.3. Statistics

Fig. 1. Relationship between transfer frequency TC/rec and time of incubation. Each point represents the average value from five independent experiments. Error bars represent standard deviations.

Statistical analysis for the optimization trials was performed using the Wilcoxon test and p b 0.05 was considered as statistically significant. Implementation of the method was tested in a ring trial between five laboratories. Reported values of transfer frequency obtained in the ring trial experiment are the means of five independent repeats. The log10-transformed values were tested for laboratory differences using analysis of variance and pair wise t tests (without multiplicity correction, because control of the pair wise comparison error rate was considered more important than control of the overall error rate). Variability within and between laboratories was quantified. 3. Results and discussion

supplemented with 1.0% glucose) containing either erythromycin (L. lactis SH4174) or rifampicin and streptomycin (L. lactis Bu2-60). Antibiotics (Sigma) were used at the following concentrations: rifampicin, 50 μg ml− 1; streptomycin, 250 μg ml− 1; erythromycin, 5 or 25 μg ml− 1 when selecting for transconjugants and donor, respectively. 2.2. Mating experiments As a starting point for optimizing the conjugation process, a previously published conjugal mating protocol for lactic acid bacteria (LAB) was used (Gevers et al., 2003) to which various modifications were added as described in details in Results and Discussion. 2.2.1. Liquid and solid mating Overnight cultures of donor and recipient strains were diluted in fresh GM17 broth with appropriate antibiotics, and grown to lateexponential growth phase (ca. 1 × 109 to 2 × 109 CFU/ml), and then mixed in 1:1 proportion in a final volume of 200 µl. The mixture was spread on GM17 agar plates (solid mating) or inoculated into 10 ml of GM17 broth medium (liquid mating) and incubated 18–20 h. In solid mating, at the end of incubation time, the cells were collected in 1 ml of PBS (8.5 g/l NaCl — Merck, Darmstadt, Germany, and 1 g/l neutralized bacteriological peptone — Oxoid, Hampshire, England), while cells from liquid mating were collected by centrifugation and resuspended in 1 ml of PBS. Ten-fold serial dilutions were plated on donor-, recipient- and transconjugants-selective agar plates, which were subsequently incubated at 30 °C for 2 days and obtained colonies were counted. Transconjugants were selected on GM17 agar supplemented with rifampicin, streptomycin and erythromycin. Transfer frequencies were expressed as the number of transconjugants colonies per recipient colony formed after the mating period and the reported values were the average of at least four different trials. To evaluate the frequency of spontaneous mutations, 100 µl (undiluted) of the donor and recipient cultures were plated on transconjugantselective agar plates. 2.2.2. Optimal filter mating protocol The donor and recipient strains were grown as described above to OD600 of 1.2 (corresponding to app. 109 CFU/ml). Subsequently 1 ml of donor and 1 ml of recipient culture were mixed. The mixture was filtered through a sterile 0.45-µm pore size membrane filter (HAWP04700, Millipore, Bedford, MA, USA) which was then placed

3.1. Optimization of the mating protocol To optimize the conjugation protocol described by Gevers (Gevers et al., 2003), several experiments were performed to find: the optimal donor and recipient growth phase, the optimal ratio of donor/ recipient and the most favorable mating approach (filter, solid or liquid mating). First, the relationship between transfer frequency and growth stage of recipient and donor was investigated. Thus, growth kinetics of donor and recipient were monitored by measuring densities of the cultures at OD600 and by plating them on selected media at different time points. Filter mating experiments were conducted with overnight cultures of donor and recipient that were subsequently diluted in fresh GM17 broth and grown separately for 3 to 7 h before mixing. The highest number of transconjugants 1.09 × 10− 2 TC/rec (transconjugants per recipient) was found when carrying out the mating experiments with donor and recipient cells grown for 4.5 h to OD 1.2 which corresponded to late-exponential growth phase (Fig. 1). Lower frequencies were observed when cultures grown for shorter periods or those that have already reached the stationary phase were used. To determine the influence of donor/recipient ratio on the transfer frequency, different ratios of donor and recipient (grown to lateexponential growth phase) ranging from 0.1 to 10 were mixed and

Fig. 2. Relationship between the donor/recipient ratio and transfer frequency (TC/rec). Each point represents the average value from five independent experiments. Error bars represent standard deviations.

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Table 1 Conjugal transfer frequencies obtained by different methods

Table 2 Conjugal transfer frequencies obtained by different laboratory

Method

Filter

Solid

Liquid

Transfer frequencya (transconjugants/donor cfu) Efficiencyb

6.0 × 10− 3 (1.22)

1.35 × 10− 3 (0.44)

2.3 × 10− 7 (0.82)

a b

1

0.225

0.00038

Average value of five different trials, standard deviations are given in brackets. Filter mating arbitrary as 1.

mating experiments were performed (Fig. 2). The donor/recipient ratio significantly influenced the transfer frequency. The lowest frequency (8.7 × 10− 4 TC/rec) was observed when the 0.1 ratio was used, whereas the 1:1 ratio resulted in the highest transfer frequency (8.17 × 10− 3 TC/rec). Other studies examining the relationship between transfer frequency and donor/recipient ratio were contradictory. Transfer of pAMß1 occurred at a higher frequency between lactobacilli when the conjugal experiment was performed by mixing the donor and recipient at a 1:10 ratio while other ratios (10:1, 1:1, 1:2, 1:20 and 1:50) produced no transconjugants (Shrago et al., 1986). The same optimal donor/recipient ratio (1:10) was found when transferring pAMß1 from E. faecalis to Lactobacillus plantarum (Sasaki et al., 1988). (Blaiotta et al. 2000) reported that for better conjugative transposition efficiency between lactococci, a donor/recipient 1:8 ratio should be used, whereas another study surprisingly observed, that the transfer frequency was not affected by variations of these ratios when studying transfer of pAMß1 between L. lactis and Lactobacillus sake (Langella et al., 1996). This divergence probably depends on the kind of donor, recipient and mobile elements used in the various studies. Three different conjugation methods (filter, solid and liquid mating) were tested and method-dependent results were obtained. Although the same donor and recipient strains at late-exponential growth stage and 1:1 donor/recipient ratio were used, different results were obtained depending on the mating system (Table 1). Frequencies of transfer ranged from 2.3×10− 7 TC/rec using liquid mating to 6.0×10− 3 TC/rec when filter mating was used. Solid mating resulted in the transferfrequency of 1.35×10−3 (TC/ rec), which is about 5-fold lower than transfer frequencies obtained by filter mating, however it should be emphasized that the protocol has been optimized using the filter mating approach. Sasaki et al. (1988) also reported that filter mating is the best technique for conjugative transfer of pAMβ1 from E. faecalis to L. plantarum. Although solid mating has also been widely used as a mating technique for transferring of pAMβ1 in many studies (Morelli et al., 1988; Cocconcelli et al., 1985; Langella et al., 1996; Vescovo et al., 1983), it appeared to be less effective as transfer frequencies. Our results illustrate, that transfer frequency is significantly affected by all the factors studied (growth phase, donor/recipient ratio and

Laboratory

1 2 3 4 5

Transfer frequency (TC/rec) Geometric mean

Relative standard deviation

0.02866 0.00433 0.00035 0.00333 0.02209

0.7273 1.0133 0.5252 0.9855 1.3101

conjugation methods) and thus should be harmonized before comparing experiments. 3.2. Inter-laboratory calibration assay After optimization, the conjugal protocol was tested in five partner laboratories by performing filter mating conjugation between L. lactis SH4174 and L. lactis Bu2-60 (Fig. 3, Table 2). The within-laboratory variabilities (quantified by the relative standard deviations) were not statistically significantly different (Bartlett's test, p = 0.09). The variability within laboratories was considered to be of a limited magnitude; thus, sufficiently repeatable results within one laboratory were obtained by using the optimized filter mating protocol. There were significant differences among laboratories as found by the analysis of variance on the log-transformed data (p = 0.004). Pair wise t tests with pooled standard errors showed that Labs 3, 4 and 2 scored significantly lower transfer frequencies than Lab 1, while Lab 1 and 5 scored significantly higher than Lab 3 (significance level of the tests 5%). Considering the laboratory partners as a random factor we estimated 0.386 for the between-laboratory variance component at the 10log scale, and 0.446 for the within-laboratory betweenexperiment variance component. The variance components can be transformed to expected ratios between results of two repeated experiments either at the same laboratory partner (these are expected to be within a factor 77 with 95% confidence), or at different laboratories (expected to be within a factor 380 with 95% confidence). Although the between laboratories variability was relatively high, it should be stressed that the majority of transfer frequency data obtained in partner laboratories were quite coherent, laying in the range of 10− 2–10− 4, and indicating high conjugal transfer frequency. The observed differences between the laboratories could be associated with many factors, e.g.: variations in buffer and media preparations or in the laboratory worker leading the experiments. The elaborated conjugal mating protocol can be considered as a good method to test dissemination of antibiotic resistance. We also postulate that the optimization procedure of conjugation for various bacteria can be performed using the approach presented in this work. Finally, the conjugal protocol presented here can be successfully used for testing the role of LAB in antibiotic resistance dissemination in the food chain. Acknowledgments This work was carried out in the frame of the EU-founded project ‘Assessment and Critical Evaluation of Antibiotic Resistance Transferability in Food Chain’ (ACE-ART; CT-2003-506214) of the 6th Framework Program. We also acknowledge Dr. A. Szczepanska for critical reading and improvement of English quality of the manuscript.

Fig. 3. Transfer efficiency obtained by various partners, mean values obtained by testing the same protocol for five independent experiments. Y axis is presented in logarithmic scale. Lab 1 — National Food Institute, Technical University of Denmark. Lab 2 — Chr. Hansen, Denmark. Lab 3 — Statens Serum Institut, Denmark. Lab 4 — Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland. Lab 5 — Teagasc — The National Food Centre, Ireland.

References Blaiotta, G., Ercolini, D., Simeoli, E., Moschetti, G., Villani, F., 2000. Conditions for conjugative transposon transfer in Lactococcus lactis. Lett. Appl. Microbiol. 31, 343–348. Clewell, D.B., Yagi, Y., Dunny, G.M., Schultz, S.K., 1974. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J. Bacteriol. 117, 283–289.

J. Lampkowska et al. / International Journal of Food Microbiology 127 (2008) 172–175 Cocconcelli, P.S., Morelli, L., Vescovo, M., 1985. Conjugal transfer of antibiotic resistances from Lactobacillus to Streptococcus lactis. Microbiol. Aliment. Nutr. 3, 163–165. Gasson, M., 1990. In vivo genetic systems in lactic acid bacteria. FEMS Microbiol Rev. 87, 43–60. Gasson, M.J., Davies, F.L., 1980. Conjugal transfer of the drug resistance plasmid pAMß1 in the lactic streptococci. FEMS Microbiol. Lett. 7, 51–53. Gevers, D., Hurys, G., Swings, J., 2003. In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram-positive bacteria. FEMS Microbiol. Lett. 225, 125–130. Hespell, R.B., Whitehead, T.R., 1991. Introduction of Tn916 and pAMß1 into Streptococcus bovis JB1 by conjugation. Appl. Environ. Microbiol. 57, 2710–2713. Langella, P., Zagorec, M., Ehrlich, S.D., Morel-Deville, F., 1996. Intergeneric and intrageneric conjugal transfer of plasmids pAMß1, pIL205 and pIP501 in Lactobacillus sake. FEMS Microbiol. Lett. 139, 51–56. Morelli, L., Sarra, P.G., Bottazzi, V., 1988. In vivo transfer of pAMß1 from Lactobacillus reuteri to Enterococcus faecalis. J. Appl. Bacteriol. 65, 371–375.

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Neve, H., Geis, A., Teuber, M., 1984. Conjugal transfer and characterization of bacteriocin plasmids in group N (lactic acid) Streptococci. J. Bacteriol. 157, 833–838. Sasaki, Y., Taketomo, N., Sasaki, T., 1988. Factors affecting transfer frequency of pAMß1 from Streptococcus faecalis to Lactobacillus plantarum. J. Bacteriol. 170, 5939–5942. Shrago, A.W., Chassy, B.M., Dobrogosz, W.J., 1986. Conjugal plasmid transfer (pAMß1) in Lactobacillus plantarum. Appl. Environ. Microbiol. 53, 574–576. Tannock, G.W., 1987. Conjugal transfer of plasmid pAMß1 in Lactobacillus reuteri and between lactobacilli and Enterococcus faecalis. Appl. Environ. Microbiol. 53, 2693–2695. Vescovo, M., Morelli, L., Bottazzi, V., Gasson, M.J., 1983. Conjugal transfer of broad-hostrange plasmid pAMß1 into enteric species of lactic acid bacteria. Appl. Environ. Microbiol. 46, 753–755.