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The Effect of Different Nutritional Resources on Gram-Negative Bacterial Communities Isolated from Marine Environments

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The Effect of Different Nutritional Resources on Gram-Negative Bacterial Communities Isolated from Marine Environments The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments Shannon Lynch Microbial Diversity 2018 Mini Project Final Report Introduction The rise of antibiotic and pesticide resistance in clinical, agricultural, and environmental settings had prompted new ways to combat diseases caused by microbial pathogens across multiple disciplines. In particular, treatment options for many multidrug-resistant gram-negative bacterial human pathogens have become limited and pose an urgent threat to human health (Livermore, 2011). One alternative to antibiotics that has been explored to address the problem is the use of Bdellovibrio as potential biocontrol agents (Kadouri & O’Toole, 2004). Bdellovibrio are small gram-negative predatory bacteria that invade the periplasm of other gram- negative bacteria in a variety of wet, aerobic environments, digesting and killing them from the inside (Sockett, 2009; Stolp, 1973). In the attack phase of their life cycle, Bdellovibrio cells are tiny (< 0.2µm) and highly mobile (Sockett, 2009). Once they invade prey cells, they grow and divide, causing the prey cell to form a structure called a bdelloplasts, which then lyses to release a new generation of Bdellovibrio (Sockett, 2009). With a broad host range that includes plant, animal, and human pathogens (Rendulic et al., 2004), and the capacity to kill many antibiotic resistant pathogens in vitro without adversely affecting animal health (Negus et al., 2017), Bdellovibrio shows promise as a control measure in clinical settings. However, studies appear to focus on the effects of Bdellovibrio in the presence of a single prey under various conditions (Kadouri & O’Toole, 2004, Pantanella et al., 2017). In predation assays against Escherichia coli, Medina & Kadouri (2009) demonstrated that a nutrient rich environment favored the development of axenic host independent biofilm variants, but these variants also switch to predatory growth when nutrients are depleted. To understand how microbial community composition changes in the presence of Bdellovibrio in different nutritive environments, the predatory activities of Bdellovibrio must be explored in a multispecies context to better understand its efficacy as a biocontrol agent in applied settings where more complex communities occur. While enriching for bioluminescent bacteria in our first week of the Microbial Diversity Course, we observed signs of Bdellovibrio on a wet mount collected from plaques on a lawn of many different bacteria colonies growing on saltwater complete (SWC) agar media. Bacteria were recovered from sea water collected from Eel Pond on the MBL Village Campus, presenting a unique opportunity to explore changes in a bacterial predator-prey system recovered from a natural environment, and experimentally assess dynamics when nutritional resources are limited or abundant. Statement of Hypothesis: 1) Abundances of bacteria in nutrient rich media will exhibit a lognormal distribution when growing together in the absence of Bdellovibrio, consistent with community patterns; 2) Relative abundances of bacteria in nutrient poor media may or may not change in the absence of Bdellovibrio, but the overall community load will reduce; 3) In the presence of Bdellovibrio, I expect community composition will either remain unchanged or change in various ways depending on the relative susceptibility of community members to Bdellovibrio and its attack rate in either medium. Methods Microcosm Experiment To assess composition changes of a gram-negative bacteria community in the presence of Bdellovibrio when resources are either abundant or limited, I assembled a synthetic community consisting of five marine bacterial strains in either nutrient rich or nutrient poor liquid media with or without Bdellovibrio sp. and determined their relative abundances at the initial and final stage The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments Shannon Lynch Microbial Diversity 2018 Mini Project Final Report of the experiment (Tinitial = 0 h; Tfinal = 48 h). Selected nutrient rich and nutrient poor media included Sea Water Complete (SWC) and Growth Curve Media (GCM) (MBL 2018, Chapter 2), and prey bacteria were isolated from marine environments (Table 1) using previously described protocols (Hansel & Frances, 2006; MBL 2018, Chapter 2). After colony PCR of pure isolates, identities were confirmed through BLASTn searches of sequence data from the 16S rRNA gene in the National Center for Biotechnology Information (NCBI) database. Table 1. Bacterial strains used in the microcosm experiment. Strain Species Classification L-5 Source 12 Vibrio sp. Vibrionaceae Garbage Beach, Woods Hole, MA 56 Pseudomonas umsongenus Pseudomonadaceae Eel Pond, Woods Hole, MA 122 Vibrio cyclitrophicus Vibrionaceae Eel Pond, Woods Hole, MA 162C2LK2G Tenacibaculum discolor Flavobacteriaceae Woods Hole, MA (Lynn Kee, Microbial Diversity Course 2016) AzwK-3b Roseobacter sp. Rhodobacteraceae Elkhorn Slough, California (Hansel & Francis, 2006; Learman & Hansel, 2014) Synthetic Community Assembly I grew each prey strain from a single colony in GCM at 30 C with shaking and then inoculated 1:1 ratios of overnight cultures into 50-mL flasks containing 20mL of either SWC or GCM. The initial OD600 in each flask was 0.03 and communities were maintained at 30 C with shaking for the duration of the experiment. I used three replicates for each of the following treatments: 1) prey community in SWC with Bdellovibrio; 2) prey community in SWC without Bdellovibrio; 3) prey community in GCM with Bdellovibrio; 4) prey community in GCM without Bdellovibrio. For controls, I used one replicate each of SWC and GCM only, and one replicate each of SWC and GCM with Bdellovibrio only. To generate predator inoculum, I re-suspended five three-week old plaques from spread plates intended for Vibrio enrichments (MBL 2018, Chapter 2) in 2mL SWC and passed the suspension through a 0.2µm filter to remove non-Bdellovibrio bacteria. To the filtrate, I added 50µL of a 100µL concentrated sample from Buzzards Bay (2018.Groups3_4.12.07.2, Table 3) that was processed for virus isolations (MBL 2018, Chapter 18), but instead enriched for Bdellovibrio. I added 150µL of the filtrate-concentrated Bdellovibrio mixture to each Bdellovibrio treatment flask. I used the remaining filtrate mix and part of the remaining concentrated Buzzards Bay sample in a Top Agar Spot Test to confirm the presence of Bdellovibrio and produce additional inoculum (see below). Unfortunately, I did not recover plaques from the assay, indicating that I did not have viable inoculum at my initial time point. As such, I pursued an enrichment of Bdellovibrio to repeat the experiment with sufficient inoculum, assessed community changes in the present experiment without a predator, and further assessed the effect of different nutrient environments on the growth and survival of two of the five bacterial strains in a competition experiment (see below). Relative Abundance Measures To assess changes in relative abundances of bacterial strains for each treatment, I sampled 1mL from each flask at Tinitial and Tfinal, and froze each sample at -20 C for further processing. I later extracted the total genomic DNA from each sample using a Maxwell® RSC Cultured Cells DNA Kit and associated protocols (Promega Corporation), quantified extracted DNA using the The effect of different nutritional resources on gram-negative bacterial communities isolated from marine environments Shannon Lynch Microbial Diversity 2018 Mini Project Final Report Integrated QuantusTM Fluorometer (Promega Corporation), and amplified 7ng/µL of the DNA from each sample using the GoTaq Green Master Mix and 515F-EMP and 926R-EMP primers for 16S rRNA amplicon sequencing. After amplicon sequencing, I processed fastq files for each sample using the dada2 denoise-paired command in the Qiime2 pipeline, aligned sequences using mafft, and assigned taxonomy using the 13_8 99% release of the Greengenes reference OTUs. Relative abundances of OTUs at taxonomic level 5 were visualized on bar charts for each treatment/time point. Interspecific Competition Experiment For this experiment, I assessed the effect of nutritional resources on interspecific competition of two species of gram negative bacteria. First, I conducted in vitro antibiotic assays to determine the appropriate concentration of an antibiotic that would effectively eliminate one competitor. I used results from these assays to inform the selection of two strains for the competition assay and to develop a tool to quantify abundances of taxa on spread plates that were growing together in liquid culture. In Vitro Antibiotic Assays For the first assay, I conducted a disk-diffusion antibiotic sensitivity test to assess the extent to which the five strains in the microcosm experiment were affected by ten different antibiotics (Table 2, Fig. 1). Briefly, I spread plated 100µL of overnight cultures representing each strain and 100 µL of sterile water as a control onto SWC agar plates. I then added 5µL of each antibiotic of an appropriate working concentration (www.adgene.org) to 5mm diameter sterile filter paper disks and immediately placed one disk representing each antibiotic around the perimeter of a corresponding strain plate (Fig.1). Sterile disks without antibiotics were applied to plates as an additional control. After 24 h, I observed that 100µg/mL ampicillin did not inhibit the growth of P. umsongenus
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