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

Short communication

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
b
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

A R T I C L E I N F O A B S T R A C T

Article history: Optimal conditions and a standardized method for conjugation between two model lactococcal strains,
Received 23 July 2007 Lactococcus lactis SH4174 (pAMβ1-containing, erythromycin resistant donor) and L. lactis Bu2-60 (plasmid-
Received in revised form 11 June 2008 free, erythromycin sensitive recipient), were developed and tested in a inter-laboratory experiments
Accepted 13 June 2008
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
Keywords:
Antibiotic resistance
(various OD values) and ratios of donor and recipient cultures as well as filter, solid and liquid mating
Conjugation techniques, were examined in order to optimize the conjugation protocol. In the result of these studies, we
Lactococcus lactis concluded that the donor-to-recipient ratio appear to be important; the most efficient technique for
Plasmids conjugation was filter mating and the optimal conditions for gene transfer were observed when late
Standardization 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 inter-
laboratory 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 high-
frequency 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, En-
terococcus, 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: jacek@ibb.waw.pl (J. Bardowski).
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) conjuga-
tion 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

0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2008.06.017
 J. Lampkowska et al. / International Journal of Food Microbiology 127 (2008) 172–175
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.
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 late-
exponential 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 supple-
mented 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 transconjugant-
selective 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
173
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
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
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 over-
night 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 (transcon-
jugants 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 late-
exponential 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.
174 J. Lampkowska et al. / International Journal of Food Microbiology 127 (2008) 172–175
Table 1
Conjugal transfer frequencies obtained by different methods
Method Filter Solid Liquid
Transfer frequencya 6.0 × 10− 3 (1.22) 1.35 × 10− 3 (0.44) 2.3 × 10− 7 (0.82)
(transconjugants/donor cfu)
Efficiencyb 1 0.225 0.00038
a
Average value of five different trials, standard deviations are given in brackets.
b
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 depend-
ing 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
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.
Table 2
Conjugal transfer frequencies obtained by different laboratory
Laboratory Transfer frequency (TC/rec)
Geometric mean Relative standard deviation
1 0.02866 0.7273
2 0.00433 1.0133
3 0.00035 0.5252
4 0.00333 0.9855
5 0.02209 1.3101
conjugation methods) and thus should be harmonized before compar-
ing 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 between-
experiment 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 Transfer-
ability 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.
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