Cooperative antibiotic resistance facilitates horizontal gene transfer

Abstract

The rise of β-lactam resistance among pathogenic bacteria, due to the horizontal transfer of plasmid-encoded β-lactamases, is a current global health crisis. Importantly, β-lactam hydrolyzation by β-lactamases, not only protects the producing cells but also sensitive neighboring cells cooperatively. Yet, how such cooperative traits affect plasmid transmission and maintenance is currently poorly understood. Here we experimentally show that KPC-2 β-lactamase expression and extracellular activity were higher when encoded on plasmids compared with the chromosome, resulting in the elevated rescue of sensitive non-producers. This facilitated efficient plasmid transfer to the rescued non-producers and expanded the potential plasmid recipient pool and the probability of plasmid transfer to new genotypes. Social conversion of non-producers by conjugation was efficient yet not absolute. Non-cooperative plasmids, not encoding KPC-2, were moderately more competitive than cooperative plasmids when β-lactam antibiotics were absent. However, in the presence of a β-lactam antibiotic, strains with non-cooperative plasmids were efficiently outcompeted. Moreover, plasmid-free non-producers were more competitive than non-producers imposed with the metabolic burden of a plasmid. Our results suggest that cooperative antibiotic resistance especially promotes the fitness of replicons that transfer horizontally such as conjugative plasmids.

Fig. 1. Rescue is enhanced when cooperative antibiotic resistance is expressed from plasmids.

A Total intracellular and extracellular β-lactamase activity of strains P_chr (bla_KPC-2 encoded in chromosome), P_conj (bla_KPC-2 encoded in conjugative plasmid), P_non-conj (bla_KPC-2 encoded in non-conjugative plasmid), and N (non-producer) in broth cultures. The dots and lines refer to the data points (n = 9) and means, respectively. p_adj values were derived from one-way ANOVA followed by a post-hoc Tukey test: *: 0.01 < p_adj < 0.05; **: 0.001 < p_adj < 0.01; ***: p_adj < 0.001. B–D Representative fluorescence microscopy images of colonies illustrating producers (P, green), non-producers (N, red) and transconjugants (T, yellow) after one day of co-cultivation. White scale bars represent 5 mm. E Top: schematic diagram showing the use of flow cytometry to distinguish producers (green box) and non-producers (red box) for the first day co-cultures with imipenem (IMP). Bottom: proportion of the original non-producer populations (N) rescued in co-cultures with the three different KPC-2 producers N[P_chr], N[P_conj] or N[P_non-conj], in the presence of IMP and without antibiotics (AB-free), over six days. Co-cultures were initiated at a 1:1 ratio of producers and non-producers. In P_conj co-cultures, N is the sum of the transconjugants (T) and plasmid-free non-producers (N_rem). Lines show the mean of six replicates (n = 6) and shaded areas are 95% confidence intervals of the mean.

Fig. 2. Efficient social conversion by conjugation of cooperative antibiotic resistance.

A Proportion of the original non-producer populations (N) after one day of co-cultivation with either producer P_conj (bla_KPC-2 encoded in conjugative plasmid) or P_non-conj (bla_KPC-2 encoded in non-conjugative plasmid). P_conj or P_non-conj were mixed with N at different start ratios (1:100, 1:1 and 100:1) and co-cultured in the presence of imipenem (IMP), tetracycline (TET), or without antibiotics (AB-free). Here N refers to the original non-producer populations rescued in co-cultures and thus includes transconjugants (T) in the case of P_conj co-cultures. Dots represent individual data points (n = 6) and lines are the mean. B Representative fluorescence microscopy images of colonies illustrating producers (green), non-producers (red) and transconjugants (yellow) after four days of co-cultivation. White scale bars represent 10 mm. C The number of observed conjugation events was calculated based on stereo microscopy images. Each black arrow represents an individual transfer event. Dots represent individual data points (n = 10) and lines are mean. For (A) and (C), p_adj values were derived from two-sided t-test, *: 0.01 < p_adj < 0.05; **: 0.001 < p_adj < 0.01; ***: p_adj < 0.001.

Fig. 3. A distinction between conjugation efficiency and transconjugant abundance for conjugative plasmids when selecting for private vs. public antibiotic resistance.

A Relative differences (IMP/AB-free) in abundances of non-producer populations (N) when cultivated with the different producers [P_chr], [P_conj] and [P_non-conj] in the presence of imipenem (IMP) and without antibiotics (AB-free), started at different initial producer to non-producer ratios (100:1, 1:1, and 1:100). B Conjugation efficiencies ($\frac{\mathit{T}}{\mathit{T}+{\mathit{N}}_{\mathbf{rem}}\left[{\mathit{P}}_{\mathbf{conj}}\right]}$), calculated for P_conj co-cultures exposed to imipenem (IMP), tetracycline (TET), or without antibiotics (AB-free). C Transconjugant abundances ($\frac{\mathit{T}}{\sqrt{{\mathit{P}}_{\mathbf{conj}}\times \left(\mathit{T}+{\mathit{N}}_{\mathbf{rem}}\left[{\mathit{P}}_{\mathbf{conj}}\right]\right)}}$), calculated for P_conj co-cultures exposed to IMP, TET, or AB-free. For (A), (B), and (C), dots represent individual data points (n = 6), and lines connect the mean for co-cultures started at different initial producer to non-producer ratios and incubated for 24 h. Shaded areas are 95% confidence intervals of the mean. The skull and crossbones symbols indicate that no transconjugants were identified in 100:1 samples exposed to TET.

Fig. 4. Both social conversion by conjugation and chromosomal association ameliorate non-cooperators.

A Global populations exposed to imipenem (IMP) and without IMP (AB-free) on day one, from day one to three, and day one to six. P are KPC-2 producers, either P_chr, P_conj, or P_non-conj as specified. T are transconjugants. N are non-producers. B Admixture model where day six global populations not exposed to antibiotics were added with n (1 to 20) global populations exposed to IMP. The yellow line is the total number of producers in the global population mix and the black line is the non-producers.

Fig. 5. Restricted impact of intrusion by non-cooperative plasmids.

A Proportions of cells with the cooperative plasmid (P_conj), the non-cooperative plasmid (N_{conj n-coop}), and plasmid-free non-producers (N) in co-cultures initiated at a 1:1:1 ratio. Colonies were cultivated with imipenem (IMP) or without antibiotics (AB-free) for six days. B Proportion of subpopulations of rescued cells of the initial non-producer population either with the cooperative plasmid, the non-cooperative plasmid, both plasmids, or without plasmids. For (A) and (B), lines are the mean and shaded areas are 95% confidence intervals of the mean (n = 10). The dashed lines are the initial proportion of each strain (33.3%).