Analysis of Microbial Diversity – A Novel Metagenomic Approach to Decipher Beach Microbiome M. Khubbar1, B. Fanelli2, N. Hasan2,3, J. Wojnar1, V. Kalve1, R. Colwell2,3, S. Bhattacharyya1,† 1City of Milwaukee Health Department Laboratory, Milwaukee, WI, 2CosmosID, Inc., Rockville, MD, 3University of Maryland, College Park, MD Introduction: Results: Figure 5: Krona plots of bacterial diversity at class Gammaproteobacteria Table 2: Virulence genes detected in Milwaukee beaches The microbial ecology of recreational beach waters is one of the most underexplored The class includes well-known gram-negative pathogens such as Escherichia coli, Salmonella sp, Yersinia pestis, Vibrio cholerae, and Pseudomonas aeruginosa % Total match: fraction of gene sequence recovered by shotgun sequencing ecosystems. It is frequently disrupted by anthropogenic activities, particularly in summer months. Shotgun metagenomics allows for accurate quantication of bacterial, fungal, parasitic, and viral genomes as well as identication of antibiotic resistance and virulence Sphingomonadaceae 0.8% genes. Analysis of beach metagenomes will help not only to monitor but also to quantify more 7 Figure 2: Bacterial diversity comparison based on dissimilarity between samples Pseudomonas stutzeri group 1% microbial burden and their ecological relationship. Pseudomonas aeruginosa 0.9% Betaproteobacteria_u_g 12% Arenimonas_u_s 2% Beta diversity principal coordiante analysis using Bray-Curtis method Pseudomonas balearica 1% Antimicrobial resistance is a major public health challenge. Detection of genes encoding 4 more 1 more 2 more antimicrobial resistance in complex microbial ecosystems such as beach waters allows for both Acinetobacter sp. 907131 2% 1 more 3 more qualitative and quantitative analyses of community resistome and potential transfer to human Acinetobacter sp. 883425 2% 2% Yersinia intermedia Sphingomonadales commensal bacteria and pathogens. This can improve epidemiological surveillance and Alphaproteobacteria_u_g 41% Betaproteobacteria_u_f outbreak investigation of pathogens with emerging antibiotic resistance. Yersiniaceae Arenimonas Betaproteobacteria Acinetobacter calcoaceticus/baumannii complex 11% Virulence factors help bacteria to invade the host, cause diseases, and evade host defenses. The Alphaproteobacteria Pseudomonas 2% Comamonadaceae virulence genes in beach water may indicate the presence of pathogens, and therefore may Proteobacteria Burkholderiales Milwaukee 1% Burkholderiales_u_f3 more Xanthomonadaceae likely adversely impact the health of children and immunocompromised individuals. Gammaproteobacteria Enterobacteriaceae harbor Enterobacterales Enterobacteriaceae 0.9% Rhodocyclaceae Acinetobacter Enterobacterales Pseudomonadales Whole genome shotgun metagenomics was used to detect and identify microbes, virulence and 3 more 42% Salmonella enterica Gammaproteobacteria resistance genes, and to characterize the microbiomes of three Milwaukee area beaches. This Bacteria 1% Salmonella novel approach not only deciphers biodiversity of microorganisms in recreational water, but 3 more Acinetobacter junii 2% Aeromonadaceae Acinetobacter_u_s 0.8% Bacteroidetes likely will allow a greater understanding of their contributions on ecology, beach and human. Firmicutes Tolumonas_u_s 2% Sinobacteraceae Aeromonas Aeromonas veronii 0.9% Chromatiales Actinobacteria 3 more BacteroidalesFlavobacteriales Aeromonas_u_s 2% Actinobacteria Ectothiorhodospiraceae Chromatiaceae 1% Nevskia_u_s 0.8% Clostridiales Streptomycetaceae Methods: Flavobacteriaceae Aeromonas salmonicida 1% 25% Actinobacteria_u_g 1% Thioalkalivibrio_u_s 0.9% Nitrosococcus_u_s 0.8% Bacteroidetes_u_g 1% Bacteroidales_u_g A total of 18 individual pooled beach water samples were collected during the summer of 2016 0.9% Ilumatobacter Aeromonas popoffii 8% at Milwaukee area beaches–Bradford (7), McKinley (5), and South Shore (6)–based on disparity 0.9% Flavobacterium 0.8% Streptomyces between ndings by Colilert, qPCR, and/or Nowcast predictive model. more 2 Aeromonas sobria 7% 2 more 2 Colilert-18 (IDEXX Lab.) was used to detect fecal coliforms. Lyophilized SmartBeads (BioGx, Inc.) Figure 3: Fungi – Relative abundance shown as 100% stacked bar graph more 1 Bradford Beach were used for E. coli quantication via qPCR. For shotgun metagenomics, extracted DNA was quantied using Qubit 4 Fluorometer (Thermo Sandarakinorhabdus 1% Scientic). Nextera XT library prep protocol was used to generate fragment libraries from DNA 5 more 5 samples, followed by 150bp paired-end sequencing on Illumina HiSeq4000 (generated an Betaproteobacteria_u_g 8% average of 40M read pairs/sample). Yersinia intermedia 2% CosmosID bioinformatics platform was used for multi-kingdom microbiome analyses and Serratia_u_s 1% proling of community resistome and virulome. 3 more Sphingomonadaceae Lysobacter_u_s 1% Betaproteobacteria_u_f 36% Salmonella enterica Sphingomonadales Arenimonas_u_s 6% 1 more 4% Comamonadaceae Enterobacteriaceae Burkholderiales_u_f Figure 6: Antibiotic resistance gene markers detected Betaproteobacteria 0.7% Burkholderiales_u_g 8 more Enterobacterales Burkholderiales 0.7% Rubrivivax 4 more Yersiniaceae Table 1: Actual vs Expected Beach Water Quality Postings Alphaproteobacteria_u_g 42% 3 more Alphaproteobacteria Resistance genes were pooled by type of resistance conferred. 6 more Arenimonas Proteobacteria Rhodocyclaceae Shown are cumulative percentages for all genes by drug class. Date of Colilert qPCR Nowcast Model Actual Expected Pseudomonas Gammaproteobacteria Sample ID collection (MPN/ 100 mL) (CCE/ 100 mL) Prediction posting Reason for posting posting 2% Dechloromonas Pseudomonas aeruginosa 6% Xanthomonadaceae Pseudomonadales Enterobacterales Gammaproteobacteria 2.0 BB061516 6/15/16 Open Open Close Advisory Rainfall 1" Open ✖ 3 more En...ae BB062016 6/20/16 Open Open Open Open Colilert results from 3 days ago Open ✔ 0.8% Salmonella Bacteria 1.8 BB080116 8/1/16 Open Open Open Open Colilert results from previous day Open ✔ 1% Deltaproteobacteria Cellvibrio Cellvibrionales Acinetobacter Chromatiales 4% Cellvibrio_u_s 1.6 BB080316 8/3/16 Advisory NP Open Open Colilert results from previous day Advisory ✖ Pseudomonas_u_s 3% Ae...ae BB082416 8/24/16 Open Open Open Open Colilert results from previous day Open ✔ Si...ae 1.4 BB082716 8/27/16 NP: Weekend Open Colilert results from previous day ? ? Aeromonas Bacteroidetes ChromatiaceaeEctothiorhodospiraceae Pseudomonas balearica 2% 3% Thioalkalivibrio_u_s BB083116 8/31/16 Open Open Close Advisory Open ✖ 1 more 1.2 Actinobacteria 2 more 2% Methylomonas_u_s Rheinheimera Acinetobacter sp. CIP 53.82 0.9% 1.0 MB061516 6/15/16 Advisory Close NP Advisory Rainfall 1" Advisory ✔ Flavobacteriales Actinobacteria 1% Solimonas_u_s Acinetobacter sp. 883425 1% 8% Rheinheimera_u_s MB080116 8/1/16 Open Open NP Advisory Colilert results from 3 days ago Open ✖ 0.8 21% Actinobacteria_u_g MB080316 8/3/16 Open NP NP Open Colilert results from previous day Open ✔ Flavobacteriaceae Acinetobacter junii 2% 0.6 ✖ 1 more MB081716 8/17/16 Open Open NP Advisory Colilert results from previous day Open Propionibacteriales Aeromonas_u_s 1% 1 more 0.9% Chitinophagaceae 0.8% Bacteroidetes_u_g MB082416 8/24/16 Open Open NP Open Colilert results from previous day Open ✔ 1% Ilumatobacter 0.4 Pro...eae Acinetobacter calcoaceticus/baumannii complex 3% Aeromonas popoffii 3% Aeromonas salmonicida 1% Aeromonas sobria 3% 0.2 SB053116 5/31/16 Open Close NP Open Colilert results from previous day Open ✔ 0.7% Flavobacterium ✖ 0.9% Propionibacterium SB062016 6/20/16 Close Open NP Open Colilert results from previous day Close 0.0 SB080316 8/3/16 Advisory NP NP Open Colilert results from previous day Advisory ✖ 6 6 6 6 6 6 6 6 6 6 6 6 16 16 16 16 16 16 51 11 31 71 41 1 0 3 7 7 1 3 more 3 151 201 011 031 241 271 311 1 0 0 1 2 3 2 0 1 2 3 6 6 8 8 8 8 8 6 8 8 8 8 05 06 08 08 08 08 ✔ more 1 0 0 0 0 0 0 0 _0 _0 _0 _0 _0 _ _ _ _ _ _ SB081716 8/17/16 Advisory Open NP Advisory Colilert results from previous day Advisory McKinley Beach B B B B B B B B B B B BB_ BB_ BB_ BB_ BB_ BB_ BB_ M M M M M S S S S S S SB082716 8/27/16 NP: Weekend Open Colilert results from previous day ? ? SB083116 8/31/16 Close Close NP Advisory Close ✖ Aminoglycoside resistance Beta-lactam resistance Fluoroquinolone resistance Macrolide resistance Polymyxin resistance Correct Wrong NP: Sulphonamide resistance Tetracycline resistance Trimethoprim resistance Vancomycin resistance Multidrug resistance factors prediction ✔ prediction ✖ Not performed ? Betaproteobacteria_u_g 21% Sandarakinorhabdus 1% 5 more Pseudomonas alcaligenes 1% Figure 4: Protists – Relative abundance heat map Pseudomonas aeruginosa 3% 3 more Pseudomonas balearica 1% Acinetobacter sp. CIP 53.82 1%Pseudomonas_u_s 2% Conclusion: Acinetobacter calcoaceticus/baumannii complex 2% 1 more Acinetobacter sp. 907131 1% Figure 1: Microbial Diversity in Milwaukee Beaches 2 more • A shotgun metagenomic sequencing approach to explore microbial communities oers critical Acinetobacter sp. 883425 1% 1 more 0.0% 0.5% 50% 1% Pseudomonas sp. CFT9 insight, both on ecological and public health perspectives, and underscores the need for 1% Pseudomonas stutzeri Bradford (BB), McKinley (MB), South Shore (SB) Betaproteobacteria_u_f 2% Arenimonas_u_s Sphingomonadaceae microbiome analysis of waters on the shoreline Species of bacteria (n=645), fungi (n=14), parasites (n=20), viruses (incl. bacteriophages) Acinetobacter baumannii 1% 1% Lysobacter_u_s Sphingomonadales Betaproteobacteria 7% Salmonella enterica