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Home , Acinetobacter junii, Aeromonadaceae, Aeromonas, Aeromonas popoffii, Aeromonas veronii, Alishewanella

Analysis of Microbial Diversity – A Novel Metagenomic Approach to Decipher Beach 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 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 such as coli, sp, pestis, cholerae, and 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 quanti cation of bacterial, fungal, parasitic, and viral genomes as well as identi cation of resistance and virulence 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 group 1%

microbial burden and their ecological relationship. 0.9% Betaproteobacteria_u_g 12%

Arenimonas_u_s 2% Beta diversity principal coordiante analysis using Bray-Curtis method 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 sp. 907131 2% 1 more 3 more qualitative and quantitative analyses of community resistome and potential transfer to human Acinetobacter sp. 883425 2%

2% commensal and pathogens. This can improve epidemiological surveillance and Alphaproteobacteria_u_g 41% Betaproteobacteria_u_f

outbreak investigation of pathogens with emerging antibiotic resistance. Acinetobacter calcoaceticus/baumannii complex 11%

Virulence factors help bacteria to invade the host, cause diseases, and evade host defenses. The Pseudomonas 2% virulence genes in beach water may indicate the presence of pathogens, and therefore may Milwaukee 1% Burkholderiales_u_f3 more

likely adversely impact the health of children and immunocompromised individuals. Gammaproteobacteria

Enterobacteriaceae harbor 0.9% Acinetobacter Enterobacterales Whole genome shotgun metagenomics was used to detect and identify microbes, virulence and 3 more 42% Gammaproteobacteria resistance genes, and to characterize the of three Milwaukee area beaches. This Bacteria 1% Salmonella novel approach not only deciphers biodiversity of microorganisms in recreational water, but 3 more 2% Acinetobacter_u_s 0.8% likely will allow a greater understanding of their contributions on ecology, beach and human. Tolumonas_u_s 2% Sinobacteraceae

Aeromonas veronii 0.9% Chromatiales 3 more BacteroidalesFlavobacteriales Aeromonas_u_s 2% Actinobacteria

1% Nevskia_u_s 0.8% Clostridiales Streptomycetaceae Methods: Flavobacteriaceae 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 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 quanti cation via qPCR.

For shotgun metagenomics, extracted DNA was quanti ed using Qubit 4 Fluorometer (Thermo Sandarakinorhabdus 1%

Scienti c). 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- microbiome analyses and Serratia_u_s 1% pro ling 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% 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 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 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 of bacteria (n=645), fungi (n=14), parasites (n=20), viruses (incl. bacteriophages) 1% 1% Lysobacter_u_s

Sphingomonadales Betaproteobacteria 7% Salmonella enterica (n=21) identi ed. Chao diversity index given for bacteria, fungi, protists, and viruses. Arenimonas • Such advanced molecular surveillance can allow development of baselines that can be integrated

auyuan_1 Acinetobacter junii 3% Enterobacteriaceae Comamonadaceae Pseudomonas TCC_18719 Alphaproteobacteria_u_g 25% 2% Limnohabitans into water quality management decisions to protect plant, animal, and human health following the ain_CCMP1984

ain_MSU_1 1%

Alphaproteobacteria Proteobacteria Enterobacterales Species Alpha Diversity 17 more Burkholderiales Acinetobacter_u_s 1% Xanth...aceae CDC’s One Health concept ain_ A ain_PJ_11 ain_LAMB0001 ain_B_31 anch 5% Cellvibrio_u_s TCC_50818 Cellvibrio ain_QSDP1 ain_G5 ain_LU352 0.9% Pseudomonadales ain_CDC

ain_TNS_C_0014 Acinetobacter ain_CA1 ain_H_H_34 V39c5 Cellvibrionales TCC_30864

ain_V1_4 0.7% Burkholderiales_u_f 120 ain_Xbor erens_st r

ain_SN1 Aeromonas_u_s 1% 1 more ain_LEM423

Aeromonadaceae 0.8% Gammaproteobacteria ain_ A TCC_50062 ain_LEM1108 ain_130c TCC_38817 anch Database anch ain_LEM3045 utum_CCAP_1055_1 ain_LEM452 wi_st r ain_SD_75_1 ain_ L ain_109_892 n ain_SB310 ain_JRB310 itaniensis Aeromonas salmonicida 1% Bacteria oelii 4 more anch 2% Ectothiorhodospiraceae anensis_isolate_ T ain_CCAP_1602_1 medium_st r a_cubensis_st r utum_Mf_1_05b_01 ani_236_B r Fungi ultifiliis_st r Gammaproteobacteria Future directions: ysodes ain_ A ico r 0.8% Aeromonas pureum_st r Protist v mibasis_st r asitica_CBS_223_65 xaurelia_st r ietti_st r xicana_MHOM_GT_2001_U1103

100 ahens_ A anica_st r ubi_st r yi_CCMP1516 antum_JPCM5 Bacteria aziliensis_MHOM_BR_75_M2904 abica_st r oelii_ y Aeromonadaceae

Virus wczarzaki_ A vicollis_MX1

uttalli_P19 8 more allax_st r allax_st r

ius_ m • Improved understanding of the ecology and diversity of microbes in recreational waters elia_st r i f i f a_ r a_pseudonana_CCMP1335 a_oceanica_CCMP1005 ium_sp_CCTCC_M209059 ina_tai w ium_catenoides_st r idium_hominis a_ o

yxa_filosa CyanobacteriaVerrucomicrobia 3 more Aeromonas yt r yt r a_ v Chromatiales um_polycephalum_st r Bacteroidetes • Allows comprehensive performance of current beach monitoring and noti cation ndings based on rega r ychia_lemnae_st r wiella_natans_CCMP2755 ychia_lemnae_2x8_2

ymena_borealis_st r Cyanobacteria_u_c ophthi r

80 oc h icha_t r icha_t r

ostelium_pu r ostelium_fi r ostelium_inte r y Actinobacteria x a h amecium_biaurelia_st r amecium_s e Synechococcales

ysa r ytophtho r Chromatiaceae yptospo r water quality standards, as compared to advanced genomic studies ureococcus_anophagef f a r a r onticula_alba_st r et r

P P h Entamoeba_ n Acanthamoeba_quina Acanthamoeba_palestinensis F P h Stylo n Hammondia_hammondi_st r Hemiselmis_andersenii Symbiodinium_mi n Acanthamoeba_r h Oxyt r Guillardia_theta_CCMP2712 Acanthamoeba_lugdunensis Plasmodium_ y Saprolegnia_410_B r Nannochloropsis_gaditana_CCMP526 Schi z Acanthamoeba_393_B r Pseudoperonospo r Thalassiosi r Reticulo m Stylo n Salpingoeca_rosetta_st r T Dict y Dict y Acanthamoeba_polyphaga P Phaeodactylum_t r Acanthamoeba_mau r Chrome r Nannochloropsis_oceanica_st r Hyphoc h Thecamonas_t r Bigel o Thalassiosi r Leishmania_major_st r Saprolegnia_pa r Asco g Dict y A Acanthamoeba_healyi C r Acanthamoeba_378_B r Leishmania_don o Acanthamoeba_castellanii_str_Neff Leishmania_gerbilii_st r Emiliana_huxl e Leishmania_in f Leishmania_tu r Leishmania_en r Plasmodium_reichen o Leishmania_b r Leishmania_a r Nannochloropsis_gaditana_st r Acanthamoeba_pearcei Leishmania_major_st r Leishmania_tropica_L590 Leishmania_aethiopica_L147 Monosiga_br e Leishmania_m e Sarcocystis_neurona_st r Icht h Capsaspo r

Oxyt r Rheinheimera Actinobacteria _ Cytophagales 21% Rheinheimera_u_s e r si t y I n d BB_061516 • Advance knowledge on microbial burden and predictable ratio of illness caused by pathogenic 60 BB 061516 Sy...ae

2% Methylomonas_u_s BB_062016BB_062016 1% Sinobacteraceae

BB_080116BB_080116 1% Verrucomicrobia_u_g microbes during surveillance 0.8% Cyanobium C h a o D i v _

BB_080316BB 080316 Aeromonas popoffii 23% 3% Bacteroidetes_u_g 40 BB_082416BB_082416 • Surveillance of antibiotic resistance and virulence genes to indicate gene transfer among 25% Actinobacteria_u_g

BB_082716BB_082716 0.9% Flavobacteriales

BB_083116BB_083116 2% Algoriphagus commensal and and their impact on public health MB_061516MB_061516 20 MB_080116MB_080116 more 4 • Correlation to health outcomes based on epidemiological, pharmacy and ED visits/hospitalizations

MB_080316MB_080316 Aeromonas sobria 2% MB_081716MB_081716 South Shore Beach data against genomic surveillance

MB_082416MB_082416 2 more 2 0 SB_053116_ more 6 BB_061516BB_062016B BB_080316BB_082416BB_082716BB_083116MB_061516MB_080116MB_080316MB_081716MB_082416SB_053116SB_062016SB_080316SB_081716SB_082716SB_083116 SB 053116 B _ 0 SB_062016SB_062016 80116 SB_080316SB_080316 SB_081716SB_081716 Acknowledgements: The authors gratefully acknowledge University of Wisconsin-Milwaukee Zilber School of Public Health students’ support for collection of beach samples, Beach posting as per Colilert (E. coli presence): Missed Advisory Missed Closure Expected Open SB_082716SB_082716 References faculty for beach modeling, MHD Disease Control & Environmental Health sta for predictive decisions, Joshua Weiner and Nancy Hills for graphics and technical assistance on SB_083116SB_083116 Hasan, Nur A. et al. “Microbial Community Pro ling of Human Saliva Using Shotgun Metagenomic Sequencing.” Ed. Niyaz Ahmed. PLoS ONE 9.5 (2014): e97699. PMC. Web. 31 May 2018. poster design. The study was supported by MHD operations funding. Ondov, Brian D, Nicholas H Bergman, and Adam M Phillippy. “Interactive Metagenomic Visualization in a Web Browser.” BMC Bioinformatics 12 (2011): 385. PMC. Web. 31 May 2018. Leddy, Menu B. et al. “Characterization of Microbial Signatures From Advanced Treated Wastewater Bio lms.” Journal of the American Water Works Association 109:11 (2017): E503. Web. 31 May 2018. †Contact: [email protected]