This in vitro study investigated the conjugation kinetics between ESBL-producing E. coli donors and various Enterobacteriaceae recipients. In this study we used relatively high concentrations of cefotaxime in the agar. This was due to the results from the initial resistance screening, where 8 µg ctx/mL did not affect the growth of the donors negatively in the broth microdilution assay. Surprisingly, these donors were inhibited by 30 µg CTX discs in the agar diffusion trial. The Clinical Laboratory Standards Institute (CLSI) suggests this disc type for screening for ESBL- producing bacteria and 1 µg ctx/mL for broth microdilution (19). Hence, the donors would have failed to be identified in the recommended agar disc diffusion test but easily be recognized as ESBL-producers in the broth microdilution test.
Of the possible six recipient and 5 donor strains, only six suitable mating pairs were found that could be used in kinetic mating experiments. Successful conjugation was observed in 92 – 100 % of the donor/recipient combinations (51 donors, 1 recipient and 48 donors and 1 recipient) investigated (20, 21) compared to 52.2 % in this present study (5 donors, 6 recipients). This rate is affected by chance and a higher number of recipient strains may have altered the outcome. Also, this study used different bacterial species while only E. coli donors and recipients were used in the study by Franiczek and Krzyzanowska (21). The results of this study suggest that the propensity of ESBL-producing donors for gene transfer differ significantly between strains from the same species. E. coli ESBL10682 and E. coli ESBL10717 transferred their plasmids to 4 of the possible 6 and 5 potential recipients respectively. On the contrary, E. coli ESBL10716 did not transfer its plasmid to any of the recipients provided. This suggests that the bla genes were located on the chromosome or a non-conjugative plasmid (20) in this donor. E. coli ESBL10689 and E. coli ESBL10717 were able to perform conjugation with 2 out of 4 or 3 potential recipients, respectively. A plausible reason for the cases were the recipient did not accept the plasmid is that the recipients may already harbor plasmids with the same replicon (22). Also, the incubation time might have been too short, or the initial concentrations of donor and recipients were too low (23). The latter is rather unlikely, as the initial concentration of 105 cfu/mL is quite high and the long incubation time of 22 hours in media increases cell concentrations even further. Furthermore, the transconjugants could have been under detection limit. Here, the detection limit was 3 cfu transconjugants/mL, which makes this option rather unlikely at the given bacterial concentrations and incubation time. Finally, the recipient may have specific endonucleases which destroy the plasmids after uptake and thereby prevent the formation of transconjugants (24, 25). This may be the case for the Proteus mirabilis recipient, which did not mate with any of the given donors.
When co-cultivated, the recipient strains S. marcescens and Salmonella Typhimurium showed lower growth rates than the donor strains (supplementary data). Hence, the donor/recipient ratio and subsequent conjugation frequencies was shifted towards the donor. This effect should be considered when evaluating conjugation events as conjugation frequencies per recipient cfu would have been higher than conjugation rates per donor cfu. Thus, calculation of conjugation events per donor may be biased and other methods of calculation may give different results (26, 27). However, as the present study was designed to find model strains to study conjugation kinetics in detail, this was not the focus of the research. Hence, the calculation of transconjugants/donor was sufficient to compare the different mating pairs.
Conjugation frequencies differed between various donor and recipient strains in the employed in vitro assay. Genera and strain depending variations in conjugation frequency have been described previously (18, 20). In some studies, it was suggested that conjugation occurs more regularly with donor and recipients from different genera. Donor strains belonging to Enterobacter cloacae, E. sakazakii, E. agglomerans, Serratia marcescens, Citrobacter freundii and E. coli showed conjugation frequencies between 10-7 and 10-1 transconjugants per donor with the recipient E. coli K12C600 (20, 21). Correspondingly, conjugation rates were rather low in the present study, when E. coli strains served as recipients (10-9-10-7 transconjugants/donor). On the contrary, Yamaichi et. al., (9) described higher conjugation frequencies for mating pairs of the same species than interspecies donor/recipient combinations. This corresponds with our findings for the E. coli ESBL10689 donor. Thus, this study cannot confirm a general statement to either direction, but rather suggests strain specific differences. These relatively high conjugation rates reported in the literature compared to the frequencies shown in the present study may depend on different strains used. While some studies used different strains as donors and a consistent E. coli recipient (20, 21), the present study used varying donor and recipient strains. In the mentioned studies, especially Citrobacter freundii, a strain not investigated in the present study, showed high conjugation rates, while S. marcescens donors showed rather low conjugation frequencies, comparable to the results of this study. In another study, Enterobacter spp. donors reached an average of 105 transconjugants/donor when co-cultivated with E. coli recipient strains (28) compared to 10-6 transconjugants/donor when used as a recipient for E. coli donors in the present study. Unfortunately, no information on incubation time or cell concentrations was provided. In this study, the highest conjugation frequency of 1.04 × 10-5 transconjugants/donor occurred when E. coli 10682 was co-incubated for 4 hours with the pathogen Salmonella Typhimurium L1219- R32. This frequency corresponds to results obtained from conjugation trials with Klebsiella spp. donors and Salmonella spp. recipients with 24 hours co-incubation (18).
Also, higher initial concentrations of donors and recipients used in studies such as Franiczek et al. (20) or Franiczek and Krzyzanowska (21) (109 cells/mL compared to 105 cells/mL in this study) can explain the differences in conjugation frequencies. The reason for the relatively low initial bacterial concentrations in this study was that cell numbers were chosen according to realistic amounts present in the gastrointestinal tract (17, 29, 30). Therefore, the detection limit must be considered when evaluating the time for the first observed conjugation event. The impact of initial concentrations of the mating pair on the number of transconjugants after a given time of coincubation and thereby the detection limit of conjugation was previously described in a study by Handel et al. (23).
The time until detection of transconjugants differed significantly between donors with the same recipients. Conjugation kinetics for ESBL-carrying plasmids have previously been studied, but mainly with longer time intervals (15). It was also shown in the present study that both time as well as number of conjugation events differed between different strains. Some strains showed a higher CF early during incubation (Salmonella spp./E. coli ESBL 10682, S. marcescens subsp. marcescens DSM 5570/E. coli ESBL 10682) with declining conjugation frequencies, while other strains increased CF at later time points (E. coli IMT 20751/402/E. coli ESBL 10682, E. coli IMT 20751/402/E. coli ESBL 10689). These results suggest that the most severe differences occur within the first day and therefore short time intervals should be chosen when investigating conjugation kinetics.
The aim of this study was to identify mating pairs fitted for future in vivo studies in poultry. These mating pairs should comprise a donor producing an ESBL type with high prevalence in broilers (3) and perform conjugation at bacterial concentrations commonly observed in the hindgut (17, 30). The most fitted mating pairs were E. coli ESBL10682/E. coli IMT 20751/402, E. coli ESBL10682/Salmonella Typhimurium DSM30122, E. coli ESBL10682/Serratia marcescens subsp. marcescens and E. coli ESBL10689/E. coli IMT 20751/402 due to their formation of transconjugants after a relative short incubation period. This will ensure that the passage time of the ingesta, and thereby the time a donor or recipient resides in the intestinal tract if not established there, will be sufficient for conjugation to be detected. To enhance the chance to detect the conjugation events, high transfer rates are preferred (23). Hence, the mating pair E. coli ESBL10682/Salmonella Typhimurium DSM30122 revealed best fitted. Also, Salmonella Typhimurium is a common pathogen of importance for public health. Thus, the chosen mating pair could be used to address research questions focusing on this topic as well. Conjugation frequencies are commonly obtained from in vitro trials. To understand the impact of the complex system in the intestinal tract ex vivo and in vivo trials should follow these studies.