Within- and between-host dynamics of producer and non-producer pathogens

Abstract Abstract For infections to be maintained in a population, pathogens must compete to colonize hosts and transmit between them. We use an experimental approach to investigate within-and-between host dynamics using the pathogen Pseudomonas aeruginosa and the animal host Caenorhabditis elegans. Within-host interactions can involve the production of goods that are beneficial to all pathogens in the local environment but susceptible to exploitation by non-producers. We exposed the nematode host to ‘producer’ and two ‘non-producer’ bacterial strains (specifically for siderophore production and quorum sensing), in single infections and coinfections, to investigate within-host colonization. Subsequently, we introduced infected nematodes to pathogen-naive populations to allow natural transmission between hosts. We find that producer pathogens are consistently better at colonizing hosts and transmitting between them than non-producers during coinfection and single infection. Non-producers were poor at colonizing hosts and between-host transmission, even when coinfecting with producers. Understanding pathogen dynamics across these multiple levels will ultimately help us predict and control the spread of infections, as well as contribute to explanations for the persistence of cooperative genotypes in natural populations.

Supplementary Figure 2: Experimental set up for investigating within-host pathogen loads at the primary (12 hours) and secondary (24 hours) exposure stages.Nematodes are washed between transfers.Table S1: Within-host dynamics.Model output for single exposure and post-hoc test.

A) Likelihood ratio test for quasipoisson glm for non-producer CFU's
Table S2: Within-host dynamics A) Model output for two stage exposure and post hoc test output for non-producer CFU's and for B) producer CFU'S.
Tukey post hoc test for non-producer CFU's B) Likelihood ratio test for quasipoisson glm for producer CFU's Tukey post hoc test for producer CFU's Table S3A: Investigating interactions between pathogen and stage of exposure in within-host dynamics for A) Non-producer 2, B) Non-producer 1, C) Producer and D) Producer with the outlier removed.
Table S3B: Differences between pathogen treatments when investigating the interactions between bacteria and stage of exposure in within-host dynamics for A) Non-producer 2, B) Non-producer 1 and C) Producer.
Table S4: Model output for comparing control CFU's when exposed to food to those exposed to a producer or non-producer treatment.
Table S5: Between host dynamics.Model output for single exposure stage and post-hoc tests.
Table S6: Between host dynamics.Model output for two stage exposure and post-hoc tests for A) non-producers and B) producer.Table S7: Investigating host preference and the shedding of pathogens into the environment.A) Output for choice assay t-test and B) Output for shedding into environment assay and associated post-hoc test.

Supplementary Methods -Time Control Analysis 1 2
With a two-pathogen treatment, within-host pathogen load was measured at the secondary 3 exposure stage (24 hours).To investigate within-host pathogen load at the primary exposure 4 stage (12 hours) we conducted an additional assay with an added stage of CFU collection 5 after the primary exposure.We added a time control of nematode food OP50 to this assay 6 (Figure S2).After 12 hours, CFUs were collected from 4-5 clean nematodes, and the 7 remaining nematodes were washed and transferred to the secondary exposure stage (Figure 8 S2).After 12 hours on the second plate, CFUs were collected from 4-5 clean nematodes.This 9 experiment was replicated three times.10 11 We made comparisons between two treatments for each of the three different pathogens: 1) 12 primary exposure to pathogen (CFUs collected at 12 hours); and 2) nematode on lawn of food 13 (OP50) for 12 hours and then secondary exposure to pathogen (CFUs collected at 24 hours).14 We used t-tests to compare mean CFUs per nematode between treatment 1 and treatment 2 15 for each of the three different pathogens (Figure S4).There was no significant difference 16 between mean CFUs per nematode for treatment 1 and treatment 2 for any of the pathogens 17 (Producer: t = -0.616,p = 0.547; Non-producer A: t = -0.469;p = 0.646; Non-producer B: t = 18 -0.462,p = 0.650).

Figure S4
. Time control analysis.The graph shows CFUs per nematode after 12 hours of exposure for each of the three different pathogens under two treatments: 1) primary exposure to pathogen (CFUs collected at 12 hours); and 2) nematode on lawn of food (OP50) for 12 hours and then secondary exposure to pathogen (CFUs collected at 24 hours).Across the three different pathogens, there were no significant differences between mean CFUs per nematode for treatment 1 and treatment 2. Square points with error bars represent mean +/-1 SE. (Results of t-tests comparing treatments 1 and 2 for each pathogen -Producer: t = -0.616,p = 0.547; Nonproducer A: t = -0.469,p = 0.646; Non-producer B: t =-0.462 ; p = 0.650).
Experiments investigating host preference for picking-up pathogens and host shedding of pathogens in the environment.A) Preference assay.Equal density samples of wild type producer (pale) and non-producer (dark) P. aeruginosa strains are placed equidistant from the center of the plate.40 nematodes are added to the center of the plate and left for 6 hours at 20 o C after which the number of nematodes in the bacteria are recorded to calculate a choice index.B) Host shedding of pathogens in the environment assay.Nematodes are exposed to a producer or non-producer for 24 hours, they are then washed and transferred to a new plate.After 12 hours the nematodes are picked off the plate and the CFUs grown from the plate.