June 2015 University of Southern California Sea Grant Proposal

PROJECT TITLE: DEVELOPMENT  OF  DIGITAL  RT-­‐PCR  METHODS  TO  QUANTIFY  HUMAN-­‐ASSOCIATED  BACTERIOPHAGE  IN  STORM  WATER  AND  COASTAL  RECREATIONAL  WATERS   PRINCIPAL INVESTIGATORS: Joshua  A.  Steele,  Microbiologist,  Southern  California  Coastal  Water  Research  Project  Adam  C.  Martiny,  Associate  Professor,  University  of  California  Irvine,  Director  UCI  OCEANS     ASSOCIATE INVESTIGATORS: John  F.  Griffith,  Principal  Scientist,  Southern  California  Coastal  Water  Research  Project   FUNDING REQUESTED: 2016-2017 $59,463 Federal/State $50,406 Match 2017-2018 $59,210 Federal/State $50,568 Match STATEMENT OF THE PROBLEM: Concentrations  of  Fecal  Indicator  Bacteria  (e.g.  Enterococcus,  E.  coli,  fecal  coliforms)  in  recreational  waters  are  monitored  to  protect  swimmers  from  exposure  to  waterborne  pathogens.  Despite  these  efforts,  swimmers  may  still  be  at  risk  from  exposure  to  pathogenic  human  viruses  even  when  levels  of  FIB  meet  standards.  The  problem  is  with  the  indicators:  FIB  methods  are  slow,  non-­‐specific  (i.e.  they  cannot  distinguish  the  source  of  the  contamination),  and  do  not  reliably  predict  the  presence  of  human  viruses  in  recreational  water.  Traditional  viral  indicators,  such  as  bacteriophage,  are  better  indicators  of  viral  contamination,  but  the  methods  are  also  slow  (e.g.  24  hour  incubation  before  a  result)  and  insensitive  (i.e.  low  levels  of  bacteriophage  can  be  missed).    However,  F+RNA  coliphage  (bacteriophage  which  infect  E.  coli  and  related  bacteria)  have  been  shown  to  distinguish  between  human  and  non-­‐human  associated  sources  of  contamination  based  on  their  genotype  and  were  better  predictors  of  gastrointestinal  illness  than  FIB  in  two  recent  epidemiological  studies  at  southern  California  Beaches.    Direct  molecular  assays  of  the  F+RNA  coliphage  using  reverse  transcriptase-­‐quantitative  PCR  (RT-­‐QPCR)  provide  a  rapid  method  to  measure  viral  indicators  and  to  track  sources  of  microbial  contamination  in  environmental  water.  In  their  current  form  (using  reverse-­‐transcriptase  PCR),  these  techniques  are  susceptible  to  inhibition  by  organic  compounds  found  in  environmental  waters  and  have  exhibited  low  sensitivity  to  their  target.  Here,  we  propose  to  adapt  these  assays  to  droplet  digital  reverse-­‐transcriptase  quantitative  PCR  (ddRT-­‐QPCR)  to  increase  their  sensitivity  and  decrease  their  susceptibility  to  inhibition.  The  adapted  molecular  assay  will  support  efforts  by  US  EPA  to  develop  a  rapid  coliphage  assay  

INVESTIGATORY QUESTIONS:

1. Can  the  RT-­‐QPCR  assays  for  F+RNA  coliphage  be  successfully  adapted  to  digital  droplet  PCR  and  used  to  quantify  and  distinguish  the  4  genogroups  in  southern  California  storm  water  and  recreational  coastal  waters?  

2. Can  the  molecular  quantification  F+RNA  coliphage  genogroups  be  used  as  a  viral  indicator  in  storm  water  and  near  shore  coastal  waters,  i.e.  will  the  human-­‐associated  coliphage  correlate  with  pathogenic  viruses  in  contaminated  storm  water  and  coastal  waters?  

3. Can  the  molecular  quantification    of  F+RNA  coliphage  genogroups  be  used  to  distinguish  human-­‐associated  from  non-­‐human  associated  contamination  in  storm  water  and  near  shore  coastal  waters?  

MOTIVATION:  Fecal  indicator  bacteria  (FIB),  such  as  Enterococcus  are  monitored  to  protect  swimmers  and  surfers  from  potentially  harmful  microbial  contamination  in  recreational  waters.  Beaches  must  be  posted  or  closed  when  these  indicators  exceed  the  concentration  set  by  state  and  federal  law.  While  these  bacterial  indicators  are  predictive  of  health  risk  when  there  is  an  acute,  human  source  of  contamination  (i.e.  human  sewage),  they  do  not  consistently  predict  the  presence  of  human  pathogenic  viruses  (Jiang  et  al.  2001;  Noble  &  Fuhrman  2001;  Boehm  et  al.  2003;  Jiang  &  Chu  2004;  McQuaig  et  al.  2012),  or  gastrointestinal  illness  (Colford  et  al.  2007).  Further,  they  are  unable  to  distinguish  between  human-­‐associated  and  non-­‐human  associated  sources  of  contamination,  which  can  alter  the  risk  of  swimming-­‐related  illness  (Soller  2010,  2014).    

 Recognizing  the  inadequacy  of  FIB  as  an  indicator  organism,  the  US  Environmental  Protection  Agency  (EPA)  has  embarked  on  a  path  to  develop  water  quality  standards  for  coliphage  (US  EPA  2015).  Recent  epidemiology  studies  conducted  by  our  organization  in  cooperation  with  EPA  in  southern  California  (Mission  Bay  in  San  Diego,  Avalon  Beach  on  Catalina  Island,  and  at  Surfrider  Beach  in  Malibu)  found  an  association  between  F+  coliphage  and  gastrointestinal  illness  (Colford  et  al.  2007,  Griffith  et  al.  submitted).  In  the  two  studies  in  which  genotyping  was  conducted,  genotypes  III  (at  Avalon)  and  III  (at  Malibu)  were  found  to  be  significant  predictors  of  gastrointestinal  illness  (Griffith  et  al.  submitted).    SCCWRP  has  a  history  of  working  together  with  EPA  on  development  of  rapid  water  quality  measurements.    Our  organization  was  instrumental  in  testing  and  promoting  the  QPCR  method  for  Enterococcus  (EPA  Method  1609,  USEPA  2013)  that  was  included  in  the  2012  Recreational  Water  Quality  Criteria  and  we  are  currently  collaborating  with  EPA  Office  of  Water  on  coliphage.    

 It  is  not  surprising  that  FIB  do  not  correlate  with  human  viruses  at  southern  California  beaches,  as  unless  there  is  a  sewage  spill  or  equipment  failure,  our  beaches  are  not  impacted  by  either  treated  or  untreated  wastewater.  Here,  one  of  the  most  likely  routes  for  

viruses  to  reach  beach  water  is  through  contaminated  groundwater.  Unlike  bacteria,  waterborne  viruses  and  bacteriophage  (i.e.  viruses  that  infect  bacteria)  are  not  as  effectively  filtered  out  as  water  moves  through  sand  or  soil.  Further,  bacteriophage  are  more  abundant  than  human  viruses  (since  their  bacterial  hosts  are  much  more  abundant)  which  makes  them  more  attractive  water  quality  indicators  at  beaches  where  the  source  is  groundwater  contaminated  leaking  infrastructure,  rather  than  acute  inputs,  such  as  storm  water  pulses  or  direct  sewage  spills.        F+RNA  coliphage  (i.e.  single  stranded  RNA  viruses  infecting  E.  coli  and  related  bacteria),  bacteriophage  infecting  human-­‐associated  Bacteroides  bacteria  (e.g.  Bacteroides  GB-­‐124  phage  Ebder  et  al.  2007,  McMinn  et  al.  2012),  and  a  bacteriophage  discovered  from  human  gut  microbiome  metagenomes  (crAssphage,  Stachler  &  Bibby  2014)  have  been  proposed  as  potential  fecal  indicators.    The  EPA  is  in  the  process  of  creating  recreational  water  quality  criteria  using  coliphages  as  fecal  indicators  in  ambient  waters.  Towards  this  end,  the  Office  of  Sceince  and  Technology  has  recently  published  a  review  assessing  the  role  of  coliphages  as  fecal  indicator  viruses  (USEPA  2015).      

 F+RNA  coliphage  have  been  used  as  fecal  indicators  to  protect  drinking  water  supplies  through  cultivation  techniques  for  decades.    However,  the  cultivation  is  slow  (24  hours  before  getting  a  result)  and  does  not  provide  any  indication  of  source  of  the  contamination.  Molecular  techniques  targeting  the  genogroups  of  F+RNA  coliphage  can  distinguish  between  human-­‐associated  (genogroups  II  and  III)  and  non-­‐human  associated  (genogroups  I  and  IV)  sources,  but  until  recently  this  was  performed  as  a  post-­‐cultivation  step  (Hsu  et  al.,  1995;  Beekwilder  et  al.  1996;  Cole et al. 2003; Vinje  et  al.  2004;  Long et al. 2005; Stewart-Pullaro et al. 2006),  requiring  additional  time  to  result.  Recently,  direct  molecular  quantification  (through  reverse  transcription-­‐  quantitative  PCR  i.e.  RT-­‐QPCR)  of  the  F+RNA  coliphage  genogroups  from  environmental  sources  has  been  developed  and  used  as  a  source-­‐tracking  tool  (Orgozaly  &  Gantzer  2006,  Orgozaly  et  al.  2009,  Wolf  et  al.  2008,  2010,  Friedman  et  al.  2009,  2011,  Paar  II  et  al.  2015,  Vergara  et  al.  2015).  This  method  is  rapid,  with  the  potential  of  producing  results  within  4  hours  from  sample  collection,  and  specific,  identifying  genogroups  I-­‐IV.      In  addition  to  the  long  incubation  time  many  bacteriophage  cultivation  techniques  are  relatively  insensitive.  Because  bacteriophage  abundance  is  variable  and  phage  can  go  undetected  by  culture  methods,  requiring  non-­‐quantitative  enrichment  in  order  to  enhance  detection.    Molecular  quantification  of  bacteriophage  directly  from  environmental  waters  avoids  the  lengthy  cultivation  process  and,  in  the  case  of  F+RNA  coliphage,.  However,  the  difficulty  of  effectively  capturing  coliphage  from  environmental  water  and  amplifying  their  signal  in  the  frequent  presence  of  inhibitory  substances  such  as  humic  acids  makes  this  kind  of  measurement  difficult.    

Digital  droplet  PCR  (ddPCR)  has  the  potential  to  overcome  some  of  the  problems  associated  with  PCR  inhibition  and  provides  absolute  quantification  even  at  low  target  concentrations.  Unlike  qPCR,  which  has  been  the  most  popular  method  for  quantifying  molecular  targets  for  many  years,  ddPCR  does  not  depend  on  cycle  threshold  and  comparison  to  a  dilution  series  of  reference  standards  for  quantification.  Rather,  ddPCR  

utilizes  dilution  of  the  target  to  1  copy  per  droplet,  followed  by  amplification,  and  then  counting  and  quantification  using  Poisson  statistics  to  determine  the  number  of  gene  copies.  This  technique  both  eliminates  the  need  for  a  standard  curve  and  combats  inhibition  because  the  cycle  at  which  the  fluorescent  signal  appears  is  no  longer  relevant  for  quantification.  Droplets  with  any  positive  signal  are  counted  as  1’s  and  those  without,  as  0’s  regardless  of  the  cycle  at  which  they  appear.  In  addition,  because  of  the  digital  nature  of  the  data,  multiple  wells  can  be  run  concurrently  from  the  same  sample  and  the  results  combined,  raising  sensitivity  and  eliminating  the  limitations  associated  with  qPCR  where  one  may  only  use  a  small  fraction  of  the  DNA  extracted  from  a  sample  in  each  reaction.    

Current  epidemiology  and  Quantitative  Microbial  Risk  Asessment  (QMRA)  studies  in  San  Diego  and  a  QMRA  studies  in  the  Port  of  Los  Angeles  at  inner  Cabrillo  Beach  will  also  provide  a  direct  link  to  water  quality  measurements  and  human  health  outcomes  and  enable  us  to  test  the  relationship  of  the  F+RNA  coliphage  to  pathogens  and  to  FIB  on  the  same  samples.    Inclusion  of  F+RNA  phage  digital  RT-­‐QPCR  assays  will  enhance  both  projects  and  provide  a  comparative  analysis  of  the  utility  of  the  assay  as  a  predictor  of  illness  in  swimmers  and  as  a  fecal  source  tracking  tool.      

  GOALS AND OBJECTIVES:

A. Overall Goals Our  goals  are  to  further  EPA  efforts  to  develop  rapid,  sensitive  bacteriophage  assays  for  water  quality  monitoring  and  as  source  identification  tools  in  coastal  recreational  watersheds.  Specifically  we  aim  to  1)  to  develop  a  sensitive,  robust  droplet  digital  RT-­‐PCR  assay  to  measure  and  distinguish  human-­‐associated  and  non-­‐human-­‐associated  F+RNA  coliphage  genogroups;  and  2)  apply  this  assay  as  microbial  source  tracking  tool  in  coastal  recreational  waters  and  storm  waters.    

B. 2016-2017 Objectives

As  stated  above  our  overall  aim  is  to  develop  an  assay  targeting  F+RNA  coliphage  as  a  viral  indicator  in  coastal  watersheds  and  stormwater.  Specifically  our  objectives  are  to  1)  Adapt  RT-­‐QPCR  methods  for  F+RNA  coliphage  genogroups  to  droplet  digital  RT-­‐PCR;  2)  Collect  stormwater  and  nearshore  coastal  waters  suspected  of  microbial  contamination  and  capture  viruses;  3)  Apply  the  droplet  digital  RT-­‐PCR  assay  as  a  human  and  non-­‐human  associated  microbial  source  tracker  in  stormwater  and  recreational  coastal  waters  likely  to  be  impacted  by  microbial  contamination.  

METHODS:

 We  will  develop  and  test  sensitive  (potentially  detecting  a  single  gene  copy)  and  robust  (resistant  to  PCR  inhibition)  droplet  digital  RT-­‐PCR  methods  adapted  from  recently  developed  RT-­‐QPCR  assays  applied  to  wastewater  and  environmental  samples  (e.g.  Wolf  et  

al.  2010,,  Paar  III  et  al  2015,  Vergara  et  al.  2015).  These  assays  can  distinguish  multiple  F+RNA  coliphage  genotypes  at  once  by  targeting  shared  coat  protein  and  RNA  replicase  genes.            Duplexing  the  digital  RT-­‐QPCR  assay  will  allow  for  two  targets  to  be  detected  in  one  reaction,  doubling  the  information  from  a  given  sample.    Because  it  was  designed  as  a  multiplex  assay  in  environmental  samples  and  sewage,  we  are  planning  to  adapt  the  set  of  primers  and  probes  from  the  viral  tool  box  of  Wolf  et  al.  (2010)  shown  in  Table  1.    Although  there  are  other  primer  sets  for  F+RNA  coliphage  (e.g.  primer  and  probe  sets  developed  by  Friedman  et  al.  2011  and  recently  employed  in  environmental  samples  by  Paar  III  et  al.  2015,  Vergara  et  al.  2015),  we  chose  the  Wolf  et  al.  primer  set  due  to  the  optimization  of  Wolf  et  al.  for  multiplexing  and  the  potentially  broader  range  for  F+RNA  phage  GIII.      After  conversion  of  the  RNA  in  the  sample  to  complementary  DNA  using  reverse  transcriptase,  the  complementary  DNA  will  be  quantified  via  droplet  digital  PCR.  The  multiplexed  primers  and  probes  can  all  be  amplified  at  the  same  temperatures  dissociation  at  95°C  15s,  annealing/extension  at  59°C  60s  for  45  cycles.    Cultivable  bacteriophage  (e.g.  MS2  for  GI,  GA  for  GII,  Qβ  for  GIII,  or  HB  for  GIV)  will  be  used  as  positive  controls  and  for  optimization  during  the  development  of  the  ddRT-­‐QPCR  assay.    We  expect  that  we  will  maintain  similar  specificity  (i.e.  amplifying  only  the  intended  genogroup)  and  will  increase  the  sensitivity  (i.e.  lower  the  limit  of  detection)  compared  to  the  RT-­‐QPCR  assay.  In  order  to  account  for  potential  inhibitory  compounds  found  in  environmental  samples  (e.g.  humic  acids,  phenolic  compounds),  we  will  conduct  both  spike-­‐dilution  tests,  and  use  an  internal  amplification  control  (following  Friedman  et  al.  2011).  This,  in  addition  to  the  RT  control,  will  enable  us  to  determine  robustness  of  the  ddRT-­‐PCR  assay.  We  have  found  in  previous  studies  that  the  nature  of  the  small-­‐volume  ddPCR  itself  tends  to  be  robust  to  inhibition.      The  reactions  will  be  placed  into  droplets  generated  on  the  BioRad  ddPCR  system,  run  on  BioRad  CFX  thermocyclers  and  read  in  the  BioRad  QX-­‐100  or  QX-­‐200  digital  droplet  reader.  The  reagents  and  equipment  have  been  previously  tested  by  SCCWRP  and  represent  the  state-­‐of  –the-­‐art  for  droplet  digital  PCR  (Cao  et  al.  2015,  Steele  et  al.  2015,  in  prep).      In  addition  to  this  technology,  SCCWRP  is  collaborating  with  Arizona  State  University  and  the  Monterey  Bay  Aquarium  Research  Institute  to  develop  an  automated  sampler  and  field-­‐worthy  droplet  digital  PCR  platform.  The  proposed  assays  would  be  ideal  for  adaptation  and  testing  on  the  field  platform  for  microbial  source-­‐tracking  studies.    The  adaptation  and  testing  will  likely  require  the  first  half  of  the  year  1  to  complete.    Once  we  are  assured  that  the  assay  is  performing  satisfactorily,  we  will  apply  the  assay  to  quantify  F+RNA  coliphage  genotypes  in  fresh  and  archived  samples                    

F+RNA phage

Genogroup Primer Name Forward Primer Reverse Primer Probe

GI FphGI GTCCTGCTCRACTTCCTGT

ATGGAATTSCGGCTACCTACA

CGAGACGCTACCWTGGCTATCGC

GII FphGII ACCTATGTTCCGATTCASAGAG

GGTAGGCAAGTCCATCAAAGT

CACTCGCGATTGTGCTGTCCGATT

GIII FphGIII (MX1)

TTTGAGGCTRTGTTGCGACA

CCGTGGSGTACACTCTTG

CGGYCATCCGTCCTTCAAGTTTGC

GIII FphGIII (Qβ)

CCGTCCGTTGAGGGTATGTT

CGAGGSGTACACGCTTG

CGGYCATCCGTCCTTCAAGTTTGC

GIV FphGIV AAGACWGGTCGGTACAAAGT

ARCTTCACCTCGGGAAKTC

CCGGATGAAGGCACTGTCCTGAATC

 We  will  begin  collecting  samples  from  storm  water,  estuaries,  and  marine  waters  in  the  coastal  zone  in  Southern  California  while  the  adaptation  of  the  RT-­‐QPCR  to  digital  PCR  is  underway.  Thus,  we  plan  to  collect  samples  during  the  first  18  months  of  the  project.  This  will  give  us  both  wet  and  dry  seasons  to  collect  storm  water  and  beach  water.    We  will  target  5  locations  throughout  southern  California  likely  to  suffer  from  aging,  leaky  infrastructure  including  watersheds  in  San  Diego,  Orange  County,  and  Los  Angeles  County.    We  anticipate  collecting  storm  water  samples  from  San  Diego  and  Malibu,  near-­‐shore  beach  samples  from  beaches  in  the  City  and  County  of  San  Diego,  Doheny  State  Beach,  and  Newport  Beach  (in  Orange  County),  and  Inner  Cabrillo  Beach,  and  Malibu  (Los  Angeles  County).      

 Doheny  State  Beach  and  Newport  Beach  are  at  the  end  of  urbanized  watersheds.  Newport  Back  Bay  is  less  urbanized,  but  still  has  a  profound  urban  influence  that  ends  in  a  wetland,  water  treatment  ponds  to  reduce  nutrients,  and  a  small  marina.  These  locations  are  all  near  to  UC  Irvine  and  SCCWRP’s  laboratory  and  will  be  easily  accessible.    An  ongoing  study  of  surfer  wet  weather  epidemiology,  water  quality  and  quantitative  microbial  risk  assessment  in  San  Diego  in  one  large,  urbanized  watershed,  and  one  small,  urbanized  watershed  at  SCCWRP  is  directly  measuring  pathogens,  but  virus  samples  can  be  collected  and  archived  for  F+RNA  coliphage  analysis.  The  Martiny  lab  conducts  regular  sampling  for  phytoplankton,  bacteria,  and  viruses,  in  the  near  shore  ocean  off  of  Newport  Beach,  which  is  influenced  by  Orange  County  and  South  Los  Angeles  County  watersheds.    Samples  of  opportunity  alongside  the  Bight  ’13  wet  and  dry  weather  stormwater  sampling  in  Malibu  

Table 1. Primers and Probes from Wolf et al. 2010 used for multiplex digital RT-QPCR

and  the  upcoming  Inner  Cabrillo  Beach  water  quality  and  QMRA  study  (in  the  heavily  urbanized  port  of  Los  Angeles)  which  SCCWRP  is  beginning  this  year.  Malibu,  a  less  urbanized  but  frequently  contaminated  watershed,  and  Doheny  State  Beach  have  both  been  the  focus  of  earlier  epidemiology  and  microbial  source  tracking  studies  (Griffith  et  al.  submitted).    We  also  have  access  to  archived  virus  samples  from  epidemiology  and  water  quality  studies  collected  during  2008-­‐2015  from  Malibu,  Avalon,  Doheny  State  Beach,  and  San  Diego  which  can  be  used  for  F+RNA  coliphage  assay  application.  Viral  samples    from  Newport  Beach,  Newport  Back  Bay,  and  Doheny  will  also  be  collected  and  tested  on  the  automated  sampler  and  portable  ddPCR  system  being  developed  by  the  Monterey  Bay  Aquarium  Research  Institute,  Arizona  State  University,  and  SCCWRP.    The  adapted  ddRT-­‐PCR  assay  will  be  tested  on  the  new  instrument  to  gauge  the  feasibility  of  automating  viral  indicators  and  RT-­‐PCR  on  the  portable  digital  PCR  instrument.  

 Samples  at  all  sites  will  be  taken  in  duplicate.  For  large  volume  samples  we  will  concentrate  the  viruses  using  a  30  kDa  hollow  fiber  filter  with  a  foam  elution  from  InnovaPrep  (recently  tested  on  environmental  samples  at  SCCWRP,  Fig  1).  For  small  volume  samples  we  will  use  the  alumina  coated  nanoCeram  filters  (Li  et  al.  2010;  Ikner  et  al.  2011)  or  multi-­‐cellulose  ester  type  HA  filters  (after  Katayama  et  al.  2002)  both  of  which  capture  viruses  by  electrostatic  charge.  For  the  type  HA  filters,  water  samples  will  be  amended  with  MgCl2  and  acidified  to  pH  3.5  to  allow  viruses  to  adsorb  to  the  filter  membrane  and  then  be  directly  extracted  (Conn  2012,  Katayama  et  al.  2002).  For  the  nanoCeram  filters,  the  viruses  will  adsorb  to  the  nano-­‐aluminum  coating  and  be  eluted  by  a  solution  of  phosphate  buffered  saline  at  pH  9.3,  with  0.1%  NaPP  and  0.05M  glycine  (Ikner  et  al  2011).    Once  viruses  are  captured  they  will  either  be  preserved  and  stored  in  RNAlater  or  flash  frozen  in  liquid  N2  and  stored  at  -­‐80°C  until  extraction.        These  virus  capture  techniques  performed  well  in  a  recent  comparison  of  virus  recovery  from  stormwater,.  HA  filters  showed  the  highest  recovery  at  a  low  (105  phage  gene  copies  per  L),  and  InnovaPrep  showing  the  most  consistent  recovery  with  large  (20L)  volume  samples.    The  filters  or  the  concentrates  will  be  extracted  using  MoBio  environmental  virus  kit  with  mechanical  lysis  using  glass  beads,  β-­‐mercaptoethanol  and  a  phenol:chloroform:isoamyl  alcohol  extraction.  A  standard  RNA  extraction  control  (e.g.  Mouse  Lung  β-­‐actin  RNA)  will  be  added  to  each  sample  (after  Conn  et  al.  2012).  All  RNA  work  will  use  cleaned  dedicated  lab  space  (e.g.  prep-­‐stations  or  hoods)  and  cleaned,  dedicated  pipets  to  avoid  contamination  with  enzymes  that  would  degrade  the  RNA  in  the  samples.  

     All  data  generated  by  the  project  will  cleaned,  formatted,  and  be  made  publicly  available  through  the  California  Environmental  Data  Exchange  Network,  or  on  SCCWRP’s  website.  In  addition,  the  publications  will  be  open-­‐access  and  data  and  protocols  generated  by  this  project  will  be  housed  on  a  publicly  accessible  website  either  in  an  open  sharing  site  such  as  GitHub.  Water  quality  data  collected  by  SCCWRP  is  routinely  incorporated  into  public  databases  including  the  California  Environmental  Data  Exchange  Network  (CEDEN)  which  combines  sample  location,  date,  and  water  quality  information  and  other  metadata.  

RELATED RESEARCH: While  there  has  been  no  human-­‐associated  bacteriophage  research  funded  recently  by  USC  Sea  Grant,  there  have  been  prior  Sea  Grant  projects  that  examined  the  relationship  of  fecal  indicator  bacteria  (FIB)  and  pathogenic  microorganisms  including  bacteria  and  viruses.  Prior  research  by  Jones  and  Fuhrman  examined  the  fate  and  dispersal  of  pathogens  in  stormwater  at  Southern  California  Beaches,  and  will  inform  this  study.  This  work  on  transport  will  also  provide  a  means  to  extend  the  current  research  beyond  the  sites  that  we  are  able  to  test  and  provide  necessary  context  for  the  proposed  research.    Development  of  

Figure 1. Percent recovery of bacteriophage MS2 (F+RNA Coliphage GI) from three different virus capture filtrations. Data from (Steele et al. 2015, in prep.). The recovery is shown as a percent of the bacteriophage MS2 quantity spiked. The high concentration contained 108 phage gene copies per L and the low concentration contained 105 phage gene copies per L.

Percen

t  Recov

ery  

0

10

20

30

40

50

60

70

80

HA NanoCeram InnovaPrep

High Concentration

Low Concentration

Filter TypeFilter  Type  

molecular  assays  for  viruses  have  been  funded  in  the  past  by  USC  Sea  Grant  in  research  performed  by  SCCWRP  and  USC  including  hepatitis  A  virus  assays  (research  to  J.F.  Griffith)  and  enterovirus  assays  (Noble  et  al.  2003,  Fuhrman  et  al.  2005)  in  coastal  water  and  storm  water.    Research  on  the  correlation  between  human  enteric  viruses  and  FIB  by  Noble  and  Fuhrman  was  crucial  to  understanding  the  limitations  of  FIB  and  exploring  the  behavior  of  viruses  as  the  basis  for  rejecting  the  links  between  FIB  and  pathogens  in    beach  water  (Noble  &  Furhman  2001,  Fuhrman  et  al.  2005).    The  proposed  work  will  add  to  the  physical  oceanography  and  molecular  assays  performed  as  USC  Sea  Grant  projects.    We  note  that  while  there  is  unlikely  a  direct  connection,  the  Sea  Grant  studies  by  Shipe  and  Sanudo-­‐Wilhelmy  looking  at  nutrient  and  metal  inputs  and  their  effect  on  phytoplankton,  along  with  the  harmful  algal  bloom  research  are  related  in  that  the  proposed  research  would  be  able  to  identify  likely  sources  of  nutrient  and  pathogen  contamination;  particularly  at  locations  with  groundwater  seeping  into  the  beach,  contaminated  by  leaky  infrastructure.          Current  research  at  the  Southern  California  Coastal  Water  Research  Project  is  complementary  to  the  proposed  research.    Two  ongoing  studies  are  using  source  identification  and  direct  pathogen  quantification  to  inform  a  Quantitative  Microbial  Risk  Assessment  of  urbanized  coastal  and  harbor  waters.  The  Surfer  Health  Study  in  San  Diego  is  currently  pairing  health  outcomes  during  wet  weather  events  to  water  quality  measurements  using  fecal  indicators,  direct  measurement  of  pathogens  by  ddPCR,  and  the  microbial  source  identification  for  QMRA.    A  separate  QMRA  study  will  begin  this  year  to  measure  the  water  quality,  quantify  pathogens,  and  predict  risk  at  Inner  Cabrillo  Beach  in  Los  Angeles  Harbor.    SCCWRP  also  coordinates  the  Southern  California  Bight  Montioring  Program  acting  as  the  source  of  QPCR  standards  and  the  data  analysis  clearing  house  for  microbial  measurements  using  QPCR.    SCCWRP  also  performs  sampling  at  5  sites  in  watersheds  in  Northern  Los  Angeles  County..  SCCWRP  is  also  collaborating  with  the  Monterey  Bay  Aquarium  Research  Instititue  and  Arizona  State  Unviersity  to  develop  an  automated  environmental  sampler  for  microbial  samples  (including  viruses)  and  a  portable,  field-­‐based  droplet  digital  quantitative  PCR  machine.  This  research  will  enable  SCCWRP  to  add  capabilities  to  this  project  and  make  a  wider  comparison  between  quantitative  molecular  assays  for  microbial  source  tracking.  Combining  the  proposed  F+RNA  phage  digital  PCR  assays  with  the  bight  samples,  and  to  other  archived  samples  where  available,  will  also  allow  for  correlation  to  earlier  cultivation  assays  and  other  pathogen  and  FIB  measurements  

 SCCWRP  recently  coordinated  a  27-­‐laboratory  comparative  study  of  41  microbial  source-­‐tracking  assays  referred  to  collectively  as  the  Source  Identification  Protocol  Project  (SIPP;  Boehm  et  al.  2013)  and  specifically  investigated  virus  and  coliphage  assays  in  13  labs.  Although  pathogenic  viruses  and  F+RNA  coliphage  genotypes  were  analyzed,  the  coliphage  assays  relied  on  cultivation  prior  to  genotyping.  (Harwood  et  al.  2013).  SCCWRP  has  also  been  at  the  forefront  of  developing  rapid  molecular  methods  for  microbial  water  quality  and  has  developed  many  bacterial  and  viral  assays  over  the  past  15  years,  collaborating  with  the  US  EPA  and  leading  academic  laboratories  including  development  of  pathogenic  virus  assays  (e.g.  Gregory  et  al.  2011,  Love  et  al  2014),  pathogenic  bacteria  assays  (e.g.  Lu  et  al.  2012),  and  molecular  indicator  methods  targeting  functional  genes  and  fecal  indicator  bacteria  (e.g.  Johnston  et  al.  2010,  Converse  et  al  2012).    The  microbiology  department  at  

SCCWRP  has  developed  digital  PCR  assays  for  Enterococcus  and  a  human-­‐specific  Bacteroides  marker  (HF183)  and  applied  them  to  water  quality  analysis  in  the  Bight  Monitoring  Program  (Cao  et  al.  2015).    In  addition,  SCCWRP  is  in  the  process  of  adapting  human  adenovirus,  human  norovirus,  murine  norovirus,  and  coliphage  MS2  to  digital  PCR  (Cao  et  al.  in  prep,  Steele  et  al  2015,  in  prep).    The  proposed  research  would  add  another  tool  to  the  cultivation  independent  source  identification  toolbox  and  provide  a  rapid  viral  indicator  that  can  be  used  for  source  identification.      In  collaboration  with  other  groups  at  UCI  and  USC,  the  Martiny  lab  manages  a  time-­‐series  MiCRO  (Microbes  In  the  Coastal  Region  of  Orange  County)  at  Newport  Pier  (Allison  et  al.,  2012).  This  time-­‐series  is  located  in  conjunction  with  a  SCCOOS  automatic  shore  station  and  includes  continuous  measurements  of  salinity,  temperature  and  chlorophyll  as  well  as  weekly  samples  of  dissolved  and  organic  nutrients,  bacterial  and  phytoplankton  counts  using  flow  cytometry,  DNA  measurements  of  bacterial  diversity  and  various  extra  cellular  enzyme  activites.  The  Martiny  lab  also  examines  the  molecular  analyzes  of  bacterial  diversity  in  the  broader  region  of  Orange  County  and  have  a  large  collection  of  DNA  samples  that  can  be  used  in  this  project.  The  Martiny  lab  also  has  ample  experience  with  complex  molecular  analyses  and  harbors  extensive  equipment  for  the  proposed  work.    Coliphage  plaque  assays  have  been  a  part  of  EPA  standard  methods  1601  and  1602  for  recreational  water  quality  for  over  a  decade  (EPA  2001a,b)  and  have  been  found  to  be  useful  indicators  of  viruses  in  environmental  waters  (LeClerc  2000).  The  US  Environmental  Protection  Agency  has  recently  announced  that  it  is  reviewing  F+RNA  coliphage  (including  direct  quantification  via  RT-­‐QPCR)    as  a  fecal  viral  indicator  of  water  quality  and  will  incorporate  coliphage  assays  into  the  new  water  quality  criteria  (EPA  2015).  Earlier  genotyping  work  was  performed  on  cultivated  coliphage  plaques  as  a  secondary  identification  step  (Hsu  et  al.,  1995;  Beekwilder  et  al.,  1996;;  Vinje  et  al.,  2004).  Further  genotyping  work  has  shown  that  genogroups  I  and  IV  are  more  associated  with  non-­‐human  E.coli    sources,  i.e.  animal  fecal  material,  and  genogroups  II  and  III  are  more  often  associated  with  human  E.  coli  sources  i.e.  fecal  material  (Cole et al., 2003; Long et al., 2005; Stewart-Pullaro et al., 2006).    Direct  quantification  of  the  coliphage  genogroups  without  cultivation  has  been  developed  to  get  around  limitations  of  cultivation  methods  and  to  compare  coliphage  indicators  to  pathogenic  virus  measurements  through  methods  such  as  RT-­‐PCR  line  blots  (Love  et  al.  2008)  or  RT-­‐QPCR  (Kirs and Smith, 2007; Ogorzaly and Gantzer, 2006, Orgozaly et al. 2009 Wolf et al., 2008, 2010, Flannery et al. 2013, Paar III et al. 2015, Vergara et al. 2015).    US  EPA  has  also  developed  assays  to  identify  genogroups  I-­‐IV  from  both  cultivated  F+RNA  coliphage  (Friedman  et  al.  2009,  2011)  and  directly  quantifying  them  from  environmental  RT-­‐QPCR  (Paar  III  et  al.  2015)  as  well.  However,  these  molecular  assays  have  rarely  been  applied  to  in  California  coastal  waters  and  only  a  few  have  been  applied  to  California  estuaries  and  watersheds.  The  proposed  work  will  not  only  adapt  the  method  to  a  new  molecular  technology,  but  will  also  be  the  first  to  apply  these  molecular  methods  widely  to  Southern  California  stormwater  and  coastal  waters.      

BUDGET-RELATED INFORMATION:

A. Budget Explanation/Detailed Justification $109,869  is  requested  ($59,463  Federal  and  $50,406  matching)  for  the  first  year  and  $109,778  is  requested  ($59,210  Federal  and  $50,568  matching)  for  the  second  year  to  perform  the  proposed  research.      SEA  GRANT  TRAINEE  One  Sea  Grant  Trainee  is  requested  for  the  2-­‐year  duration  of  the  proposed  research  for  9  months  at  50%  time  (4.5  months)  each  year.  The  Sea  Grant  Trainee  will  be  a  graduate  student  at  UC  Irvine  and  is  expected  to  perform  the  bulk  of  the  lab  work  after  training  by  the  PIs  and  SCCWRP  staff,  The  Sea  Grant  Trainee  will  get  assistance  from  SCCWRP  staff  and  the  Principal  and  Associate  Investigators  in  planning,  sample  collection,  processing,  data  analysis  and  manuscript  preparation.    Southern  California  Coastal  Water  Research  Project  Budget  Justification    SALARIES  AND  WAGES  Salary  support  is  requested  for  Dr.  Joshua  Steele.  As  the  PI,  Dr.  Steele  will  be  responsible  for  project  planning  and  management  of  the  study.  Also,  Dr.  Steele  will  oversee  data  analysis  and  publications  along  with  the  mentoring  of  the  Sea  Grant  Trainee.  Dr.  Steele  will  spend  one  month  annually  on  this  study  at  a  starting  base  salary  of  78,960.  Salary  support  is  also  requested  for  Dr.  John  Griffith  who  will  be  responsible  for  project  planning,  mentoring  the  Sea  Grant  Trainee,  contribution  to  data  analysis  and  manuscript  preparation.  Dr.  Griffith  will  spend  two  weeks  annually  on  the  study  at  a  starting  base  salary  of  $124,848.  Salary  equivalent  to  2.25  months  annually  for  lab  technicians  to  collect  samples  is  also  included.      EMPLOYEE  BENEFITS  Fringe  benefits  for  SCCWRP  employees  are  calculated  at  52.2%  of  the  base  salary  rate.    MATERIAL  AND  SUPPLIES  Materials  and  supplies  are  requested  at  $26,000  for  year  1  ($18000  Federal  and  $8000  match)  and  $20,000  ($15000  Federal  and  $5000  match)  in  year  2.  This  includes  $12,000  in  ddPCR,  QPCR,  and  reverse  transcriptase  reagents  and  supplies,  primers  and  fluorescent  Taqman  probes,  $8000  in  sample  collection  and  filtration  supplies,  $2000  for  coliphage  cultivation  positive  controls,  E.coli  hosts,  and  media,  and  $4000  for  dedicated  pipets  for  RNA-­‐work,  and  lab  expendables  such  as  pipet  tips,  DNAse  and  RNAse-­‐free  water,  lo-­‐bind  tubes  for  nucleic  acid  storage.    

 TRAVEL  Travel  support  is  requested  at  $6000  ($3000  Federal  $3000  match)  in  year  one  and  $4000  ($3000  Federal;  $1000  match  in  year  two)  for  sample  collection  at  potentially  contaminated  beaches  as  outlined  in  the  methods,  this  will  allow  for  mulitple  collection  

trips  to  6  southern  California  beaches  (Malibu,  Avalon,  Inner  Cabrillo  Beach,  Doheny  State  Beach,  San  Diego)  and  transportation  of  the  samples  back  to  the  lab  at  UC  Irvine  or  SCCWRP  on  ice  (if  live)  or  on  dry  ice  (if  captured  on  filters  or  concentrated).  The  average  travel  cost  per  collection  trip  is  $584,  including  gas,  vehicle  use  fees  or  rentals,  ice,  coolers,  dry  ice,  and  food  costs  for  the  sampling  trip.  Annual  travel  support  is  requested  for  the  PI  to  travel  to  a  national  meeting  (e.g.  ASM)  to  present  the  results  from  the  project.  The  projected  costs  for  the  meeting  are  $1500  for  each  meeting  (~$400  flight,  ~$500  registration,  $400  hotel  and  $200  for  meals).    PUBLICATION  COSTS  Costs  for  page  fees,  figure  fees  and  open  access  publications  are  requested  ($0  federal,  $3000  match).      INDIRECT  COSTS  Indirect  costs  are  calculated  using  SCCWRP’s  federally  approved  indirect  rate  of  86.94%  exclusively  on  wages  and  benefits  only.    No  indirect  costs  are  added  to  supplies  or  travel  funds.  

University  of  California  Irvine  Budget  Justification    SALARIES  AND  WAGES  Two  weeks  of   summer  salary   is   requested   for  PI  Adam  Martiny  each  year.  He  will  be  responsible  for  project  planning,  mentoring  the  graduate  student,  contribute  to  the  data  analysis,   and   writing   the   papers.   Actual   salary   rate   was   used.     A   2%   cost   of   living  increase  was  applied   to  each  period  of   this  proposal  as  well  as  an  8%  merit   increase,  where  applicable.    EMPLOYEE  BENEFITS  The  composite  benefit  rate  for  PI  Adam  Martiny  is  12.7%.  The  composite  benefit  rates  are  agreed  upon  by  the  University  of  California  and  the  UC  Office  of  the  President.    TRAVEL  Annual   travel   support   is   requested   for   the   PI   and   graduate   student   to   travel   to   a  national  meeting  (e.g.,  Ocean  Sciences  or  ASM)  to  present  the  results  from  the  project.  The  projected  costs  are  $1500  for  each  meeting  (~$400  flight,  ~$500  registration,  $400  hotel  and  $200  for  meals).    INDIRECT  COSTS  Facilities  and  Administrative  costs  were  estimated   in  accordance  with  UCI’s  approved  indirect   cost   rate   agreement.  The  54.5%   indirect   cost   rate   effective  7/1/11  was  used  based   on   the   nature   of   the   work   proposed.   UCI’s   indirect   cost   rate   agreement   was  approved  by  DHHS,  the  Federal  Cognizant  Audit  Agency  for  UCI  on  4/27/11.  

B. Matching Funds

SCCWRP  will  match  $50,406  in  the  first  year  and    $50,568  in  the  second  year  of  the  proposed  research.    The  match  will  come  from  internal  funding  for  adapting  the  F+RNA  coliphage  digital  RT-­‐QPCR  Assay  ($40,000)  this  will  be  used  for  salary,  benefits,  travel,  sample  collection  and  processing,  and  supplies.    In  addition,  $60,974  in  sample  collection,  processing,  nucleic  acid  extraction,  lab  supplies,  travel,  and  in  kind  salary  will  be  matched  from  three  ongoing  grants  as  follows:  $20,000  from  the  Wet  Weather  Epidemiology  grant  from  the  City  and  County  of  San  Diego  to  SCCWRP  and  it  has  extensive  field  research  in  San  Diego  County.    $25,000  will  be  used  from  the  Inner  Cabrillo  Beach  QMRA  Study  and  $10,974  will  be  used  from  the  Automated  Digital  PCR  study  which  are  California  State  Clean  Beach  Initiative  Grants  and  have  extensive  field  research  and  sampling  components.  Adding  extra  sample  collection  and  processing    in  support  of  this  project  will  help  .  While  not  exclusively  used  for  this  project,  and  thus  not  included  in  matching,  we  note  that  this  project  is  possible  due  to  the  droplet  digital  PCR  machine,  PCR-­‐clean  hoods,  and  biosafety  cabinets  in  the  laboratories  at  SCCWRP.  

ANTICIPATED BENEFITS: This  rapid,  sensitive  detection  of  fecal  indicator  viruses  will  further  efforts  by  EPA  to  develop  coliphage  as  a  water  quality  indicator  for  microbial  monitoring,  used  as  a  source  tracking  tool  by  marine  beach  managers  and  water  quality  regulators,  and  should  also  inform  stormwater  agencies  and  sanitation  agencies  trying  to  prioritize  infrastructure  maintenance  along  heavily  developed  and  populated  beaches.  This  project  will  also  serve  as  a  West  Coast  case  study  for  EPA’s  efforts  to  develop  criteria  for  ambient  water  quality  standards  using  coliphage.  We  note  that  epidemiology  studies  done  at  Doheny  State  Beach,  Avalon,  Malibu,  Ocean  Beach  and  Tourmaline  Surfing  Park  will  also  provide  a  link  to  beachgoer  health  and  a  broader  context  for  these  results  that  will  interest  public  health  agencies.  Visitors  to  the  beaches,  surfers,  and  swimmers  will  also  benefit  from  a  greater  understanding  of  the  beach  water  quality.  Environmental  advocacy  and  citizen  science  groups  such  as  the  Surfrider  Foundation  (see  attached  letter  from    CEO  Chad  Nelsen)  will  also  benefit  from  the  assay  and  source  tracking  results.  The  droplet  digital  RT-­‐PCR  protocols  will  be  available  for  academic  institutions,  private,  and  public  environmental  research  in  water  quality.  SCCWRP  will  also  provide  training  in  the  capture,  extraction,  and  molecular  quantification  of  F+RNA  coliphage  using  ddPCR    to  academic  and  public  environmental  labs.  The  research  will  provide  support  and  training  in  environmental  microbiological  research  and  cutting  edge  molecular  analyses  for  a  graduate  student  (Sea  Grant  Traineeship)  at  UC  Irvine  advised  by  A.  Martiny;  and  will  also  provide  research  support  for  an  early  career  scientist  (J.  Steele).      

COMMUNICATION OF RESULTS:

The  new  OCEANS  Initiative  at  UC  Irvine  lead  by  PI  Martiny  has  a  mission  to  promote  interdisciplinary  research  to  understand  and  improve  California  ocean  health.  It  will  provide  a  platform  to  inform  and  educate  the  public  in  Southern  California  and  communicate  with  the  broader  scientific  community.    SCCWRP  can  communicate  this  information  directly  to  decision  makers  in  California,  and  is  a  leader  in  implementing  new  technologies  and  scientific  methods  for  microbial  beach  water  quality.  This  research  will  be  presented  to  the  SCCWRP  Commission  and  Commission  Technical  Advisory  Group  with  members  from  the  Southern  California  Wastewater  and  Stormwater  Agencies,  the  State  and  Southern  California  Regional  Water  Resources  Agencies,  the  California  Ocean  Protection  Council  and  California  Ocean  Science  Trust,  EPA  Region  IX.  Co-­‐PIs,  Associate  Investigator,  and  the  Sea  Grant  Trainee  will  have  opportunities  to  address  the  State  Water  Resources  Control  Board  Beach  Water  Quality  Working  Group  and  the  Surfrider  Foundation.  The  trainee  and  Co-­‐PIs  will  also  be  encouraged  to  communicate  with  the  EPA  and  stormwater,  sanitation,  and  environmental  agencies  outside  of  California  through  such  venues  as  the  UNC  Water  Microbiology  Conference.     REFERENCES: Allison,  SD,  Chao,  Y,  Farrara,  JD,  Hatosy,  SM  and  AC  Martiny.  Fine-­‐scale  temporal  variation  in  marine  ectoenzymes  of  coastal  southern  California.  Front.  Microbio.  2012.    Beekwilder,  J.,  Nieuwenhuizen,  R.,  Havelaar,  A.H.,  vanDuin,  J.,  1996.  An  oligonucleotide  hybridization  assay  for  the  identification  and  enumeration  of  F-­‐specific  RNA  phages  in  surface  water.  J.  Appl.  Bacteriol.  80  (2),  179–186.    

Boehm,  A.B.,  Fuhrman,  J.A.,  Mrse,  R.D.,  Grant,  S.B.,  2003.  A  tiered  approach  for  the  identification  of  a  human  fecal  pollution  source  at  a  recreational  beach:  Case  study  at  Avalon  Bay,  Catalina  Island,  California.  Environmental  Science  and  Technology  37,  673e680.    

Boehm,  A.B.,  Van  De  Werfhorst,  L.C.,  Griffith,  J.F.,  Holden,  P.A.,  Jay,  J.A.,  Shanks,  O.C.,  Wang,  D.,  Weisberg,  S.B.,  2013.  Performance  of  forty-­‐one  microbial  source  tracking  methods:  A  twenty-­‐seven  lab  evaluation  study.  Water  Research  47:  6812–6828.    

Cao,  Y.,  Raith,  M.R.,  J.F.  Griffith.    2015.    Droplet  digital  PCR  for  simultaneous  quantification  of  general  and  human-­‐associated  fecal  indicators  for  water  quality  assessment.  Water  Research  70:337-­‐349    Cole,  D.,  Long,  S.C.,  Sobsey,  M.D.,  2003.  Evaluation  of  F+  RNA  and  DNA  coliphages  as  source-­‐specific  indicators  of  fecal  contamination  in  surface  waters.  Appl.  Environ.  Microbiol.  69  (11),  6507–6514.    

Colford  Jr.,  J.M.,  Wade,  T.J.,  Schiff,  K.C.,  Wright,  C.C.,  Griffith,  J.G.,  Sandhu,  S.K.,  Burns,  S.,  Hayes,  J.,  Sobsey,  M.,  Lovelace,  G.,  Weisberg,  S.B.,  2007.  Water  quality  indicators  and  the  risk  of  illness  at  non-­‐point  source  beaches  in  Mission  Bay,  California.  Epidemiology  18,  27e35.    

Conn,  K.E.,  Habteselassie,  M.Y.,  Denene  Blackwood,  A.,  Noble,  R.T.,  2012.  Microbial  water  quality  before  and  after  the  repair  of  a  failing  onsite  wastewater  treatment  system  adjacent  to  coastal  waters.  Journal  of  Applied  Microbiology  112  (1),  214–224.    Converse,  R.R.,  Griffith,  J.F.,  Noble,  R.T.,  Haugland,  R.A.,  Schiff,  K.C.,  and  S.B.  Weisberg.    2012.    Correlation  between    quantitative  PCR  and  culture-­‐based  methods  for  measuring  Enterococcus  spp.  over  various  temporal  scales  at    three  California  marine  beaches.    Applied  and  Environmental  Microbiology  78:  1237-­‐1242    Ebdon,  J.;  Muniesa,  M.;  Taylor,  H.  The  application  of  a  recently  isolated  strain  of  Bacteroides  (GB-­‐124)  to  identify  human  sources  of  faecal  pollution  in  a  temperate  river  catchment.  Water  Res.  2007,  41  (16),  3683−3690.    

Flannery  J,  Keaveney  S,  Rajko-­‐Nenow  P,  O'Flaherty  V,  Doré  W  (2013)  Norovirus  and  FRNA  bacteriophage  determined  by  RT-­‐qPCR  and  infectious  FRNA  bacteriophage  in  wastewater  and  oysters.  Water  Res  47:5222–5231    Friedman  SD,  Cooper  EM,  Casanova  L,  Sobsey  MD,  Genthner  FJ  (2009)  A  reverse  transcription-­‐PCR  assay  to  distinguish  the  four  genogroups  of  male-­‐specific  (F+)  RNA  coliphages.  J  Virol  Meth  159:47–52    Friedman  SD,  Cooper  EM,  Calci  KR,  Genthner  FJ  (2011)  Design  and  assessment  of  a  real  time  reverse  transcription-­‐PCR  method  to  genotype  single-­‐stranded  RNA  male-­‐specific  coliphages  (Family  Leviviridae).  J  Virol  Meth  173:196–202    Fuhrman,  J.A.,  Liang,  X.L.,  Noble,  R.T.,  2005.  Rapid  detection  of  enteroviruses  in  small  volumes  of  natural  waters  by  real-­‐time  quantitative  reverse  transcriptase  PCR.  Applied  and  Environmental  Microbiology  71  (8),  4523e4530.    

Griffith,  J.F.,  Weisberg,  S.B.,  Arnold,  B.F.,  Cao,  y.,  Schiff,  K.C.,  and  Colford,  J.M.  submitted  Epidemiologic  evaluation  of  alternate  microbial  water  quality  indicators  at  three  California  Beaches.  Water  Research    Harwood  VJ,  Boehm  AB,  Sassoubre  LM,  Vijayavel  K,  Stewart  JR,  Fong  T-­‐T,  Caprais  M-­‐P,  Converse  RR,  Diston  D,  Ebdon  J,  Fuhrman  JA,  Gourmelon  M,  Gentry-­‐Shields  J,  Griffith  JF,  Kashian  DR,  Noble  RT,  Taylor  H,  Wicki  M  (2013)  Performance  of  viruses  and  bacteriophages  for  fecal  source  determination  in  a  multi-­‐laboratory,  comparative  study.  Water  Res  47:6929–6943    Hsu,  F.C.,  Shieh,  Y.S.C.,  Vanduin,  J.,  Beekwilder,  M.J.,  Sobsey,  M.D.,  1995.  Genotyping  male-­‐  specific  RNA  coliphages  by  hybridization  with  oligonucleotide  probes.  Appl.  Environ.  Microbiol.  61  (11),  3960–3966.    

Ikner,  L.A.,  Soto-­‐Beltran,  M.,  Bright,  K.R.,  (2011).  New  method  using  a  positively  charged  microporous  filter  and  ultrafiltration  for  concentration  of  viruses  from  tap  water.  Applied  and  Environmental  Microbiology  77  (10),  3500–3506.    Jiang  SC,  Chu  W  (2004)  PCR  detection  of  pathogenic  viruses  in  southern  California  urban  rivers.  J  Appl  Microbiol  97:17–28    Jiang,  S.C.,  Noble,  R.,  Chui,  W.P.,  2001.  Human  adenoviruses  and  coliphages  in  urban  runoff  impacted  coastal  waters  of  Southern  California.  Applied  and  Environmental  Microbiology  67,  179-­‐184.      Johnston  C,  Ufnar  JA,  Griffith  JF,  Gooch  JA,  Stewart  JR  (2010)  A  real-­‐time  qPCR  assay  for  the  detection  of  the  nifH  gene  of  Methanobrevibacter  smithii,  a  potential  indicator  of  sewage  pollution.  J  Appl  Microbiol  109:1946–1956    Katayama,  H.H.,  Shimasaki,  A.A.,  Ohgaki,  S.S.,  (2002).  Development  of  a  virus  concentration  method  and  its  application  to  detection  of  enterovirus  and  norwalk  virus  from  coastal  seawater.  Applied  and  Environmental  Microbiology  68  (3),  1033–1039.    Kirs  M,  Smith  DC  (2007)  Multiplex  Quantitative  Real-­‐Time  Reverse  Transcriptase  PCR  for  F+-­‐Specific  RNA  Coliphages:  a  Method  for  Use  in  Microbial  Source  Tracking.  Applied  and  Environmental  Microbiology  73:808–814    Leclerc,  H.,  Edberg,  S.,  Pierzo,  V.,  Delattre,  J.M.,  2000.  Bacteriophages  as  indicators  of  enter-­‐  ic  viruses  and  public  health  risk  in  groundwaters.  J.  Appl.  Microbiol.  88  (1),  5–21.    

Li,  D.,  Shi,  H.-­‐C.,  Jiang,  S.C.,  (2010).  Concentration  of  viruses  from  environmental  waters  using  nanoalumina  fiber  filters.  Journal  of  Microbiological  Methods  81  (1),  33–38.    Long,  S.C.,  El-­‐Khoury,  S.S.,  Oudejans,  S.J.G.,  Sobsey,  M.D.,  Vinje,  J.,  2005.  Assessment  of  sources  and  diversity  of  male-­‐specific  coliphages  for  source  tracking.  Environ.  Eng.  Sci.  22  (3),  367–377.    

Love  DC,  Vinje  J,  Khalil  SM,  Murphy  J,  Lovelace  GL,  Sobsey  MD  (2008)  Evaluation  of  RT-­‐PCR  and  reverse  line  blot  hybridization  for  detection  and  genotyping  F+  RNA  coliphages  from  estuarine  waters  and  molluscan  shellfish.  J  Appl  Microbiol  104:1203–1212    Love  DC,  Rodríguez  RA,  Gibbons  CD,  Griffith  JF,  Yu  Q,  Stewart  JR,  Sobsey  MD  (2014)  Human  viruses  and  viral  indicators  in  marine  water  at  two  recreational  beaches  in  Southern  California,  USA.  Journal  of  Water  and  Health  12:136–16    Lu  J,  Ryu  H,  Domingo  JWS,  Griffith  JF,  Ashbolt  N  (2011)  Molecular  Detection  of  Campylobacter  spp.  in  California  Gull  (Larus  californicus)  Excreta.  Applied  and  Environmental  Microbiology  77:5034–5039    

McMinn  BR,  Korajkic  A,  Ashbolt  NJ  (2014)  Evaluation  of  Bacteroides  fragilis  GB-­‐124  bacteriophages  as  novel  human-­‐associated  faecal  indicators  in  the  United  States.  Lett  Appl  Microbiol  59:115–121    McQuaig,  S.,  Griffith,  J.F.  and  V.J.  Harwood.    2012.    Association  of  fecal  indicator  bacteria  with  human  viruses  and     microbial  source  tracking  markers  at  coastal  beaches  impacted  by  nonpoint  source  pollution.    Applied  and     Environmental  Microbiology  78:6423-­‐6432    Muniesa,  M.;  Lucena,  F.;  Blanch,  A.  R.;  Payan,  A.;  Jofre,  J.  Use  of  abundance  ratios  of  somatic  coliphages  and  bacteriophages  of  Bacteroides  thetaiotaomicron  GA17  for  microbial  source  identification.  Water  Res.  2012,  46  (19),  6410−6418.    

Noble,  R.T.,  Fuhrman,  J.A.,  2001.  Enteroviruses  detected  by  reverse  transcriptase  polymerase  chain  reaction  from  the  coastal  waters  of  Santa  Monica  Bay,  California:  low  correlation  to  bacterial  indicator  levels.  Hydrobiologia  460,  175e184.    

Noble,  R.T.,  Allen,  S.M.,  Blackwood,  A.D.,  Chu,  W.,  Jiang,  S.C.,  Lovelace,  G.L.,  Sobsey,  M.D.,  Stewart,  J.R.,  Wait,  D.A.,  2003.  Use  of  viral  pathogens  and  indicators  to  differentiate  between  human  and  non-­‐human  fecal  contamination  in  a  microbial  source  tracking  comparison  study.  Journal  of  Water  and  Health  1  (4),  195e207.    

Ogorzaly  L,  Gantzer  C  (2006)  Development  of  real-­‐time  RT-­‐PCR  methods  for  specific  detection  of  F-­‐specific  RNA  bacteriophage  genogroups:  Application  to  urban  raw  wastewater.  J  Virol  Meth  138:131–139  

Ogorzaly  L,  Tissier  A,  Bertrand  I,  Maul  A,  Gantzer  C  (2009)  Relationship  between  F-­‐specific  RNA  phage  genogroups,  faecal  pollution  indicators  and  human  adenoviruses  in  river  water.  Water  Res  43:1257–1264  

Paar  J  III,  Doolittle  MM,  Varma  M,  Siefring  S,  Oshima  K,  Haugland  RA  (2015)  Development  and  evaluation  of  a  culture-­‐independent  method  for  source  determination  of  fecal  wastes  in  surface  and  storm  waters  using  reverse  transcriptase-­‐PCR  detection  of  FRNA  coliphage  genogroup  gene  sequences.  Journal  of  Microbiological  Methods  112:28–35  

Soller  JA,  Schoen  ME,  Bartrand  T,  Ravenscroft  JE,  Ashbolt  NJ  (2010)  Estimated  human  health  risks  from  exposure  to  recreational  waters  impacted  by  human  and  non-­‐human  sources  of  faecal  contamination.  Water  Research  44:4674–4691  

Soller  JA,  Schoen  ME,  Varghese  A,  Ichida  AM,  Boehm  AB,  Eftim  S,  Ashbolt  NJ,  Ravenscroft  JE  (2014)  Human  health  Risk  implications  of  multiple  sources  of  faecal  indicator  bacteria  in  a  recreational  waterbody.  Water  Research:1–35  

Stachler  E,  Bibby  K  (2014)  Metagenomic  Evaluation  of  the  Highly  Abundant  Human  Gut  Bacteriophage  CrAssphage  for  Source  Tracking  of  Human  Fecal  Pollution.  Environ  Sci  Technol  Lett  1:405–409  

Steele,  J.A.,  Raith,  M.R.,  Layton,  B.A.,  Blackwood,  A.D.,  Noble,  R.  T.,  Griffith,  J.F.  2015  Comparison  of  Three  Filtration  Methods  to  Capture  Pathogenic  Viruses  and  Bacteria  from  Brackish  Storm  Water.  ASM  General  Meeting  Abstracts    Stewart-­‐Pullaro  J,  Daugomah  JW,  Chestnut  DE,  Graves  DA,  Sobsey  MD,  Scott  GI  (2006)  F  +RNA  coliphage  typing  for  microbial  source  tracking  in  surface  waters.  J  Appl  Microbiol  101:1015–1026    U.S.  Environmental  Protection  Agency,  2001.  Method  1601:  Male-­‐specific  (F+)  and  Somatic  Coliphage  in  Water  by  Two-­‐step  Enrichment  Procedure.  Office  of  Water,  EPA  EPA  821-­‐R-­‐01-­‐030.  Washington,  DC.      U.S.  Environmental  Protection  Agency,  2001.  Method  1602:  Male-­‐specific  (F+)  and  Somatic  Coliphage  in  Water  by  Single  Agar  Layer  (SAL)  Procedure.  Office  of  Water,  EPA  821-­‐R-­‐01-­‐029.  Washington,  DC.      U.S.  Environmental  Protection  Agency.  2013.  Method  1609:  Enterococci  in  Water  by  TaqMan®  Quantitative  Polymerase  Chain  Reaction  (qPCR)  with  Internal  Amplification  Control  (IAC)  Assay.  Office  of  Water,  EPA-­‐820-­‐R-­‐13-­‐005.  Washignton,  DC.    U.S.  Environmental  Protection  Agency,  2015.  Review  Of  Coliphages  As  Possible  Indicators  Of  Fecal  Contamination  For  Ambient  Water  Quality.  Office  of  Water,  EPA  820-­‐R-­‐15-­‐098  Washington,  DC.    Vinje,  J.,  Oudejans,  S.J.G.,  Stewart,  J.R.,  Sobsey,  M.D.,  Long,  S.C.,  2004.  Molecular  detection  and  genotyping  of  male-­‐specific  coliphages  by  reverse  transcription-­‐PCR  and  reverse  line  blot  hybridization.  Appl.  Environ.  Microbiol.  70  (10),  5996–6004.    

Vergara  GGRV,  Goh  SG,  Rezaeinejad  S,  Chang  SY,  Sobsey  MD,  Gin  KYH  (2015)  Evaluation  of  FRNA  coliphages  as  indicators  of  human  enteric  viruses  in  a  tropical  urban  freshwater  catchment.  Water  Res  79:39–47    Wolf  S,  Hewitt  J,  Rivera-­‐Aban  M,  Greening  GE  (2008)  Detection  and  characterization  of  F+  RNA  bacteriophages  in  water  and  shellfish:  Application  of  a  multiplex  real-­‐time  reverse  transcription  PCR.  J  Virol  Meth  149:123–128    Wolf  S,  Hewitt  J,  Greening  GE  (2010)  Viral  Multiplex  Quantitative  PCR  Assays  for  Tracking  Sources  of  Fecal  Contamination.  Applied  and  Environmental  Microbiology  76:1388–1394      

 

Projected Work Schedule Project Title: DEVELOPMENT  OF  DIGITAL  RT-­‐PCR  METHODS  TO  QUANTIFY  HUMAN-­‐ASSOCIATED  BACTERIOPHAGE  IN  STORM  WATER  AND  COASTAL  RECREATIONAL  WATERS  

Activities 2016-2017 F M A M J J A S O N D J

Adapt RT-QPCR assays to ddPCR Begin X X X X X End

Sample Collection and

Processing Begin X X X X X X X X X

F+RNA analysis on fresh and

archived samples

Begin X X X X

Data Analysis Begin X

Progress Reports X X

Presentations X X

Manuscript Preparation

Projected Work Schedule Project Title: DEVELOPMENT  OF  DIGITAL  RT-­‐PCR  METHODS  TO  QUANTIFY  HUMAN-­‐ASSOCIATED  BACTERIOPHAGE  IN  STORM  WATER  AND  COASTAL  RECREATIONAL  WATERS  

Activities 2017-2018 F M A M J J A S O N D J

Adapt RT-QPCR assays to ddPCR

Sample Collection and

Processing X X X

F+RNA analysis on fresh and

archived samples

X X X X X

Data Analysis X X X X X X X X End

Progress Reports X X

Presentations X X X X

Manuscript Preparation Begin X X

OMB Control No. 0648-0362

Expiration Date 1/31/2018

SEA GRANT BUDGET FORM 90-4

GRANTEE: Southern California Coastal Water Research Project GRANT/PROJECT NO.:

DURATION (months 12

12 months 1 Yr.A. SALARIES AND WAGES: man-months

1. Senior PersonnelNo. of People

Amount of Effort Sea Grant Funds Matching Funds

a. (Co) Principal Investigator: 1 1.00 4,935 1,645b. Associates (Faculty or Staff): 1 0.50 1,300 3,902

Sub Total: 2 1.50 6,235 5,547

2. Other Personnela. Professionals:b. Research Associates:c. Res. Asst./Grad Students:d. Prof. School Students:e. Pre-Bachelor Student(s):f. Secretarial-Clerical:g. Technicians: 2 2.25 1,800 7,916h. Other: Sea Grant Trainee 1 4.5

Total Salaries and Wages: 5 8.25 8,035 13,463

B. FRINGE BENEFITS: 52.6% 4,226 7,082Total Personnel (A and B): 12,261 20,545

C. PERMANENT EQUIPMENT: 0 0

D. EXPENDABLE SUPPLIES AND EQUIPMENT: 18,000 8,000

E. TRAVEL:1. Domestic 3,000 1,0002. International

Total Travel: 3,000 1,000

F. PUBLICATION AND DOCUMENTATION COSTS: 3,000

G. OTHER COSTS:1 - UCI subcontractor- Co-PI Adam Martiny 15,542 0234567

Total Other Costs: 15,542 0

TOTAL DIRECT COST (A through G): 48,803 32,545

INDIRECT COST (86.94% on wages/benefits only ): 1 10,660 17,861INDIRECT COST (Off campus % of $ ):

Total Indirect Cost: 10,660 17,861

TOTAL COSTS: 59,463 50,406

PRINCIPAL INVESTIGATOR: Joshua A. Steele, Adam C. Martiny

BRIEF TITLE: Human-associated Coliphage Detection using Digital PCR in C

OMB Control No. 0648-0362

Expiration Date 1/31/2018

SEA GRANT BUDGET FORM 90-4

GRANTEE: Southern California Coastal Water Research Project GRANT/PROJECT NO.:

DURATION (months 12

12 months 1 Yr.A. SALARIES AND WAGES: man-months

1. Senior PersonnelNo. of People

Amount of Effort Sea Grant Funds Matching Funds

a. (Co) Principal Investigator: 1 1.00 5,082 1,695b. Associates (Faculty or Staff): 1 0.50 1,340 5,358

Sub Total: 2 6,422 7,053

2. Other Personnela. Professionals:b. Research Associates:c. Res. Asst./Grad Students:d. Prof. School Students:e. Pre-Bachelor Student(s):f. Secretarial-Clerical:g. Technicians: 2 2.25 2,500 6,642h. Other: Sea Grant Trainee 1 4.5

Total Salaries and Wages: 5 6.8 8,922 13,695

B. FRINGE BENEFITS: 52.6% 4,693 7,204Total Personnel (A and B): 13,614 20,899

C. PERMANENT EQUIPMENT:

D. EXPENDABLE SUPPLIES AND EQUIPMENT: 15,000 5,000

E. TRAVEL:1. Domestic 3,000 3,0002. International

Total Travel: 3,000 3,000

F. PUBLICATION AND DOCUMENTATION COSTS: 3,500

G. OTHER COSTS:1 - UCI subcontractor - Co PI Adam Martiny 15,760 0234567

Total Other Costs: 15,760 0

TOTAL DIRECT COST (A through G): 47,374 32,399

INDIRECT COST (86.94% on wages/benefits only ): 65% 11,836 18,169INDIRECT COST (Off campus of $ ):

Total Indirect Cost: 11,836 18,169

TOTAL COSTS: 59,210 50,568

PRINCIPAL INVESTIGATOR: Joshua A. Steele, Adam C. Martiny

BRIEF TITLE: Human-associated Coliphage Detection using Digital PCR in C

Joshua  A.  SteeleScien&st  (Microbiologist) Phone:  (714)  755-­‐3218Southern  California  Coastal  Water  Research  Project   Fax:          (714)  755-­‐3299Costa  Mesa,  California,  92626 Email:  joshuas@sccwrp.org

PROFESSIONAL  PREPARATION:  Ph.D. Biology,  University  of  Southern  California,  2010B.S. Molecular  Biology,  University  of  California,  San  Diego,  2000

PROFESSSIONAL  EXPERIENCE:2014  –  Present   Scien&st  -­‐  Southern  California  Coastal  Water  Research  Project  2013-­‐2014 Associate  Research  Scien&st  -­‐  California  Ins&tute  of  Technology,  Division  of  Geology  and  

Planetary  Sciences2010-­‐2013   Postdoctoral  Scholar  in  Geomicrobiology,  California  Ins&tute  of  Technology,  Division  of  

Geology  and  Planetary  Sciences2008-­‐2009 NOAA  Sea  Grant  Knauss  Marine  Policy  Fellow:  Legisla&ve  Assistant  for  Rep.  Sam  Farr  (CA-­‐17);  

U.S.  House  of  Representa&ves

SELECTED  PUBLICATIONS:

Steele,  J.A.,  Raith,  M.R.,  Layton,  B.A.,  Blackwood,  A.D.,  Noble,  R.  T.,  Griffith,  J.F.  2015    Comparisonof  Three  Filtra&on  Methods  to  Capture  Pathogenic  Viruses  and  Bacteria  from  Brackish  Storm  Water.  ASM  General  Mee&ng  Abstracts

Marlow  JJ,  Steele  JA,  Case  DH,  Connon  SA,  Levin  LA  and  Orphan  VJ  2014  Microbial  abundance  and  diversity  paeerns  associated  with  sediments  and  carbonates  from  the  methane  seep  environments  of  Hydrate  Ridge,  OR.  Front.  Mar.  Sci.  1:44.  doi:  10.3389/fmars.2014.00044

Marlow,  J.J.,  Steele,  J.  A.,  Ziebis,  W.,  Thurber,  A.  R.,  Levin,  L.  A.,  &  Orphan,  V.  J.  2014  Carbonate-­‐hosted  methanotrophy  represents  an  unrecognized  methane  sink  in  the  deep  sea.  Nature  Communica&ons.,  5,  5094.  doi:10.1038/ncomms6094

Cram,  J.  A.,  Chow,  C.-­‐E.  T.,  Sachdeva,  R.,  Needham,  D.  M.,  Parada,  A.  E.,  Steele,  J.  A.,  &  Fuhrman,  J.  A.  2014.  Seasonal  and  interannual  variability  of  the  marine  bacterioplankton  community  throughout  the  water  column  over  ten  years.  Isme  Journal..  doi:10.1038/ismej.2014.153

Glass,  J.B.,  H.  Yu,  J.A.  Steele,  K.S.  Dawson,  S.  Sun,  K.Chourey,  R.L.  Hekch,  V.J.  Orphan.  2014  Geochemical,  metagenomic  and  metaproteomic  insights  into  trace  metal  u&liza&on  of  methane-­‐oxidizing  microbial  consor&a  in  sulfidic  marine  sediments.  Environmental  Microbiology  16(6),  1592–1611.  doi:10.1111/1462-­‐2920.12314

Hatosy,  S.M.,  J.B.H.  Mar&ny,  R.  Sachdeva,  J.  Steele,  J.A.  Fuhrman,  A.C.  Mar&ny  2013  Beta-­‐diversity  of  marine  bacteria  depends  on  temporal  scale.  Ecology  94  (9),  1898-­‐1904

Chow,  C.E.T.,  R.  Sachdeva,  J.A.  Cram,  J.A.  Steele,  D.M.  Needham,  A.  Patel,  A.E.  Parada,  J.A.  Fuhrman  In  press.  Temporal  variability  and  coherence  of  eupho&c  zone  bacterial  communi&es  over  a  decade  in  the  Southern  California  Bight  ISME  Journal  7,  2259–2273;  doi:10.1038/ismej.2013.122

Tavormina,  P.L.,  W.  Ussler  III,  J.A.  Steele,  S.A.  Connon,  M.G.  Klotz,  V.  J.  Orphan.  2013.  Depth  distribu&on  of  Cu-­‐MMO  variants  through  the  oxygen  minimum  zone  along  the  Costa  Rica  convergent  margin.  Environmental  Microbiology  Reports  5  (3),  414-­‐423

Gilbert,  J.A.,  J.A.  Steele,  J.G.  Caporaso,  L.  Steinbrück,  P.  Somerfield,  J.  Reeder,  B.  Temperton,  S.  Huse,  I.  Joint,  A.C.  McHardy,  R.  Knight,  J.A.  Fuhrman,  D.  Field.  2012  Defining  seasonal  marine  microbial  community  dynamics.  ISME  Journal  6:298-­‐308.

Steele,  J.A.,  P.D.  Countway,  L.  Xia,  P.  D.  Vigil,  J.M.  Beman,  D.Y.  Kim,  C.  T.  Chow,  R.  Sachdeva,  A.C.  Jones,  M.S.  Schwalbach,  J.  M.  Rose,  I.  Hewson,  A.  Patel,  F.  Sun,  D.A.  Caron,  J.A.  Fuhrman.  2011  Marine  bacterial,  archaeal,  and  pro&stan  associa&on  networks  reveal  ecological  linkages  ISME  Journal  5:1414-­‐1425.

Xia,  L.  C.,  J.A.  Steele,  J.  Cram,  Z.G.  Cardon,  S.L.  Simmons,  J.J.  Vallino  ,  J.A.  Fuhrman,  F.  Sun  2011.  Extended  local  similarity  analysis  (eLSA)  of  microbial  community  and  other  &me  series  data  with  replicates.  BMC  Systems  

Biology  5:S15.Beman,  J.M.,  J.A.  Steele,  and  J.A.  Fuhrman.  2011  Co-­‐occurrence  paeerns  for  abundant  marine  archaeal  and  bacterial  

lineages  in  the  deep  chlorophyll  maximum  of  coastal  California.  ISME  Journal  5:1077-­‐1085.

Fuhrman  J.A.,  J.A.  Steele.  2008  Community  Structure  of  Marine  Bacterioplankton:  Paeerns,  Networks,  and  Rela&onships  to  Func&on.  Aqua&c  Microbial  Ecology  53:69-­‐81.

Fuhrman,  J.A.,  J.A.  Steele,  I.  Hewson,  M.  S.  Schwalbach,  M.V.  Brown,  J.  Green,  Brown,  J.H.  2008  A  La&tude  Diversity  Gradient  in  Planktonic  Marine  Bacteria.  Proceedings  of  the  Na&onal  Academy  of  Sciences  105:7774-­‐7778.

Patel,  A.,  R.  Noble,  J.A.  Steele,  M.S.  Schwalbach,  I.  Hewson,  J.A.  Fuhrman  2007  Virus  and  Prokaryote  Enumera&on  from  Planktonic  Marine  Environments  by  Epifluorescence  Microscopy  with  SYBR  Green  I.  Nature  Protocols  2:269-­‐276.

Ruan,  Q.,  D.  Duea,  M.S.  Schwalbach,  J.A.  Steele,  J.A.  Fuhrman,  F.  Sun  2006  Local  Similarity  Analysis  Reveals  Unique  Associa&ons  Among  Marine  Bacterioplankton  Species  and  Environmental  Factors.  Bioinforma&cs  22:  2532-­‐2538.

Fuhrman,  J.  A.,  Hewson,  I.,  Schwalbach,  M.  S.,  Steele,  J.  A.,  Brown,  M.  V.,  &  Naeem,  S.  2006.  Annually  reoccurring  bacterial  communi&es  are  predictable  from  ocean  condi&ons.  Proceedings  of  the  Na&onal  Academy  of  Sciences,  103(35):  13104–13109.

Ruan,  Q.,  Steele,  J.  A.,  Schwalbach,  M.  S.,  Fuhrman,  J.  A.,  &  Sun,  F.  2006.  A  dynamic  programming  algorithm  for  binning  microbial  community  profiles.  Bioinforma&cs,  22(12),  1508–1514.

Hewson,  I.,  D.G.  Capone,  J.  A.  Steele,  J.A.  Fuhrman.  2006  Influence  of  Amazon  and  Orinoco  Offshore  Surface  Water  Plumes  on  Oligotrophic  Bacterioplankton  Diversity  in  the  West  Tropical  Atlan&c.  Aqua&c  Microbial  Ecology  43:11-­‐22.

Hewson,  I,  J.A.  Steele,  D.G.  Capone,  J.A.  Fuhrman.  2006  Remarkable  Heterogeneity  in  Meso-­‐  and  Bathypelagic  Bacterioplankton  Community  Composi&on.  Limnol.  Oceanography  51:1274-­‐1283.

Hewson,  I,  J.A.  Steele,  D.G.  Capone,  J.A.  Fuhrman.  2006  Temporal  and  Spa&al  Scales  of  Oligotrophic  Surface  Water  Bacterioplankton  Assemblage  Varia&on.  Marine  Ecology  Progress  Series  311:67-­‐77.

Steele,  J.A.,  F.  Ozis,  J.A.  Fuhrman,  J.S.  Devinny.  2005.  Structure  of  Microbial  Communi&es  in  Ethanol  Biofilters.  Chemical  Engineering  Journal  113:135-­‐143.

SYNERGISTIC  ACTIVITIES:-­‐Doctoral  Fellow,  NSF-­‐IGERT:  Env.  Studies,  Policy  and  Engineering-­‐  Sustainable  Ci&es  Program,  USC  (2003-­‐2004)-­‐Par&cipant  USC  Sustainable  Ci&es  Internship  in  Hong  Kong  SAR,  China  2004-­‐Sea  Grant  Trainee,  Beach  Water  Quality  Study,  2002

RECENT  COLLABORATORS:

R.  Noble  (UNC),  J.  Stewart  (UNC),  A.  Mar&ny  (UCI),  V.  Orphan  (Caltech),    L.  Levin  (UCSD),  W.  Ziebis  (USC),  J.  Fuhrman  (USC),  J.  Gilbert  (ANL/U.  Chigago),  F.  Sun  (USC)

Assoc. Prof. Adam C. Martiny University of California – Irvine Department of Earth System Science Department of Ecology and Evolutionary Biology 3208 Croul Hall, Irvine, CA 92697 http://ess.uci.edu/researchgrp/amartiny/adam-martiny-lab

Tel: (949) 824 9713 Fax: (949) 824 3874 Home: (949) 5725 636 E-mail: amartiny@uci.edu

A. Professional Preparation: Massachusetts Institute of Technology

Ocean Microbiology, Post Doctoral scholar

2003 - 06

Technical University of Denmark Environmental Microbiology Ph.D. 2003

Technical University of Denmark Chemical Engineering M.S. 2000

B. Scientific Appointments: UCI OCEANS, Director University of California – Irvine Associate Professor, Dept. of Earth System Science & Dept. of Ecology and Evolutionary Biology University of California – Irvine

2015 – current 2012 – current

Visiting Professor University of Copenhagen

2012 – 13

Assistant Professor, Dept. of Earth System Science & Dept. of Ecology and Evolutionary Biology University of California – Irvine

2006 – 12

C. Selected publications (50 total): Galbraith, E and AC Martiny. A simple nutrient-dependence mechanism for

predicting the stoichiometry of marine ecosystems. PNAS. 2015.

Mouginot, C, Zimmerman, AE, Bonachela, JA, Fredricks, H, Allison, SD Van Mooy, BAS and AC Martiny. Resource allocation by the marine cyanobacterium Synechococcus WH8102 in response to different nutrient supply ratios. Limnol. Oceanogr. 2015.

Berlemont , R and AC Martiny. Genomic potential for polysaccharides deconstruction in bacteria. Appl. Environ. Microbiol. 2015.

Johnson, ZI, and AC Martiny. New tools for quantifying phytoplankton community structure. Annu. Rev. Mar. Sci. 2015.

Batmalle, C, Chiang, HI, Zhang, K, Lomas, MW and AC Martiny. Development and bias assessment of a method for targeted metagenomic sequencing of marine Cyanobacteria. Appl. Environ. Microbiol. 2014.

Lomas, MW, Bonachela, JA, Levin, SA and AC Martiny. Impact of ocean phytoplankton diversity on phosphate uptake. PNAS. 2014.

Hatosy, SM, Martiny, JBH, Sachdeva, R, Steele, J, Fuhrman, JA, and AC Martiny. Beta-diversity of marine bacteria depends on temporal scale. Ecology. 2013.

Flombaum, P, Gallegos, JL, Gordillo, RA, Rincón, J, Zabala, LL, Jiao, N, Karl, DM, Li, WKW, Lomas, MW, Veneziano, D, Vera, CS, Vrugt, JA, and AC Martiny. Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus. PNAS. 2013.

Allison, SD, Chao, Y, Farrara, JD, Hatosy, SM and AC Martiny. Fine-scale temporal variation in marine ectoenzymes of coastal southern California. Front. Microbio. 2012.

Rusch, D.B., Martiny, A.C., Dupont, C.L., Halpern, A.L., and J.C. Venter. Characterization of Prochlorococcus clades from iron depleted oceanic regions. PNAS 107:16184-89. 2010.

Martiny, AC, Kathuria, SK, and P Berube. Widespread metabolic potential for nitrite and nitrate assimilation among Prochlorococcus ecotypes. PNAS. 2009.

Kettler, G, Martiny, AC, Huang, K, Zucker, J, Coleman, ML, Rodrigue, S, Chen, F, Lapidus, A, Ferriera, S, Johnson, J, Steglich, C, Richardson, P, Church, GM and SW Chisholm. Patterns and implications of gene gain and loss in the evolution of Prochlorococcus. PLoS Genet. 2007.

Martiny, AC, Jørgensen, TM, Albrechtsen, HJ, Arvin, E, and S Molin. Long-term succession in structure and diversity of a biofilm formed in a model drinking water distribution system. 2003.

D. Synergistic Activities: • Founder and director of a new interdisciplinary academic initiative at UC

Irvine named UCI OCEANS. The goal of this initiative is to elevate the level and visibility of oceans-related research, strengthen connections with a local community that deeply values ocean health, recruit and engage top students at all levels. The initiative will focus on the ocean system and integrate urban-ocean couplings.

• Director of the Flow Cytometry and Cell sorting Facility for research and training at UCI

• Collaboration with Minority Science Programs for mentoring undergraduate students at UC-Irvine

• Collaboration with Orange County K-12 teachers to use art in the science curriculum

• PI for Gateway program for Community College students in Orange County. The program provides research opportunities for underrepresented students.

E. Graduate and Postdoctoral Advisors: Søren Molin, DTU, Hans-Jørgen Albrechtsen, DTU, Erik Arvin, DTU Sallie Chisholm, MIT F. Ph.D. Thesis Advisor (6 total) and Postgraduate-Scholar Sponsor (5 total): Grad Students: Chau Pham, Cecilia Batmalle Georgia Tech, Alyssa Kent UCI, Stephen Hatosy UCI, Allison Moreno UCI, Catherine Garcia UCI. Post docs: Pedro Flombaum CLIMA, Renaud Berlemont, CSULB, Agathe Talarmin KAUST, Nathan Garcia UCI, Junhui Li UCI.

John  F.  GriffithPrincipal  Scien,stDepartment  of  Microbiology Phone:  (714)  755-­‐3228Southern  California  Coastal  Water  Research  Project   Home:  (714)  756-­‐0990Costa  Mesa,  California,  92626 Email:  johng@sccwrp.org

PROFESSIONAL  PREPARATION:  Ph.D. Biology,  University  of  Southern  California,  2006B.S. Biology  and  environmental  Studies,  University  of  Southern  California,  1995

PROFESSSIONAL  EXPERIENCE:2010  -­‐  Present Principal  Scien,st,  Microbiology,  Southern  California  Coastal  Water  Research  Project  2006  -­‐  2010 Supervising  Scien,st,  Southern  California  Coastal  Water  Research  Project2005  -­‐  2006 Senior  Scien,st,  Southern  California  Coastal  Water  Research  Project2001-­‐  2005 Microbiologist,  Southern  California  Coastal  Water  Research  Project

SELECTED    PUBLICATIONS:

Cao,  Y.,  Raith,  M.R.  and  J.F.  Griffith.    2015.    Droplet  digital  PCR  for  simultaneous  quan,fica,on  of   general  and  human-­‐associated  fecal  indicators  for  water  quality  assessment.  Water  Research  70:337-­‐349

Love,  D.C.,  Rodriguez,  R.A.,  Gibbons,  C.D.,  Griffith,  J.F.,  Stewart,  J.R.  and  M.D.Sobsey.    2014.    Human  viruses  and  viral  indicators  in  marine  water  at  two  recrea,onal  beaches  in  southern  California,  USA.  Journal  of  Water  and  Health  12:136-­‐150

Yau,  V.,  Schiff,  K.C.,  Arnold,  B.F.,  Griffith,  J.F.,  Gruber,  J.S.,  Wright,  C.C.,  Wade,  T.J.,  Burns,  S.,  hayes,  J.M.,  McGee,  C.,  Gold,  M.,  Caa,  Y.,  Boehm,  A.B.,  Weisberg,  S.B.  and  J.M.  Colford.    2014.    Effect  of  submarine  groundwater  discharge  on  bacterial  indicators  and  swimmer  health  at  Avalon  Beach,  CA,  USA.    Water  Research  59:23-­‐36

Riedel,  T.E.,  Zimmer-­‐Faust,  A.G.,  Thulsiraj,  V.,  Madi,  T.,  Hanley,  K.T.,  Eben,er,  D.L.,  Byappanahalli,  M.,  Layton,  B.,  Raith,  M.,  Boehm,  A.B.,  Griffith,  J.F.,  Holden,  P.A.,  Shanks,  O.C.,  Weisberg,  S.B.,  and  J.A.  Jay.    2014.    Detec,on  limits  and  cost  comparisons  of  human-­‐  and  gull-­‐associated  conven,onal  and  quan,ta,ve  PCR  assays  in  ar,ficial  waters.    Journal  of  Environmental  Management  136:112-­‐120

Arnold,  B.M.,  Schiff,  K.C.,  Griffith,  J.F.,  Gruber,  J.S.,  Yau,  V.,  Wright,  C.C.,  Wade,  T.J.,  Burns,  S.,  Hayes,  J.M.,  McGee,  C.,  Gold,  M.,  Cao,  Y.,  Weiseberg,  S.B.  and  J.M.  Colford.    2013.    Swimmer  illness  associated  with  marine  water  exposure  and  water  quality  indicators:  Impact  of  widely  used  assump,ons.  Epidemiology  24:845-­‐53.  doi:  10.1097/01.ede.0000434431.06765.4a

Raith,  M.,  Eben,er,  D.,  Cao,  Y.,  Griffith,  J.  and  S.  Weisberg.    2013.    Factors  affec,ng  the  rela,onship  between  quan,ta,ve  polymerase  chain  reac,on  (qPCR)  and  culture-­‐based  enumera,on  of  Enterococcus  in  environmental  waters.  Journal  of  Applied  Microbiology  116:737-­‐746.  DOI:  10.1111/jam.12383

Ryu,  H.,  Henson,  M.,  Elk,  M.,  Toledo-­‐Hernandez,  C.,  Griffith,  J.,  Blackwood,  D.,  Noble,  R.,  Gourmelon,  M.,  Glassmeyer,  S.  and  J.W.  Santo  Domingo.    2013.  Development  of  quan,ta,ve  PCR  assays  targe,ng  the  16S  rRNA  genes  of  Enterococcus  spp.  and  their  applica,on  to  the  iden,fica,on  of  Enterococcus  species  in  environmental  samples.    Applied  and  Environmental  Microbiology  79:196-­‐204

Bourlat,  S.J.,  Borja,  A.,  Gilbert,  J.,  Taylor,  M.I.,  Davies,  N.,  Weisberg,  S.B.,  Griffith,  J.F.,  Lejeri,  T.,  Field,  D.  and  J.  Benzie.    2013.    Genomics  in  marine  monitoring:  New  opportuni,es  for  assessing  marine  health  status.  Marine  Pollu,on  Bulle,n  DOI:10.1016/j.marpolbul.2013.05.042

Cao,  Y.,  Van  De  Werkorst,  L.C.,  Dubinsky,  E.A.,  Badgley,  B.D.,  Sadowsky,  M.J.,  Andersen,  G.L.,  Griffith,  J.F.  and  P.A.  Holden.    2013.    Evalua,on  of  molecular  community  analysis  methods  for  discerning  fecal  sources  and  human  waste.  Water  Research  DOI:10.1016/j.watres.2013.02.06

Harwood,  V.J.,  Boehm,  A.B.,  Sassoubre,  L.M.,  Kannappan,  V.,  Stewart,  J.R.,  Fong,  T-­‐T.,  Caprais,  M-­‐P.,  Converse,  R.R.,  Diston,  D.,  Ebdon,  J.,  Fuhrman,  J.A.,  Gourmelon,  M.,  Gentry-­‐Shields,  J.,  Griffith,  J.F.,  Kashian,  D.R.,  Noble,  R.T.,  Taylor,  H.  and  M.  Wicki.    2013.    Performance  of  viruses  and  bacteriophages  for  fecal  source  determina,on  in  a  Mul,-­‐laboratory,  compara,ve  Study.    Water  Research  DOI:10.1016/j.watres.2013.04.064

Schriewer,  A.,  Goodwin,  K.D.,  Sinigalliano,  C.D.,  Cox,  A.M.,  Wanless,  D.,  Bartkowiak,  J.,  Eben,er,  D.L.,  Hanley,  K.T.,  Ervin,  J.,  Deering,  L.A.,  Shanks,  O,  C.,  Peed,  L.A.,  Meijer,  W.G.,  Griffith,  J.F.,  Santodomingo,  J.,  Jay,  J.A.,  Holden,  P.A.  and    Stefan  Wuertz.    2013.    Performance  evalua,on  of  canine-­‐associated  Bacteroidales  assays  in  a  mul,-­‐laboratory  comparison  study.    Water  Research  DOI:10.1016/j.watres.2013.03.062

Boehm,  A.B.,  Van  De  Werkorst,  L.C.,  Griffith,  J.F.,  Holden,  P.A.,  Jay,  J.A.,  Shanks,  O.C.,  Wang,  D.  and  S.  B.  Weisberg.    2013.    Performance  of  forty-­‐one  microbial  source  tracking  methods:  a  twenty-­‐seven  lab  evalua,on  study.    Water  Research    DOI:10.1016/j.watres.2012.12.046

Cao,  Y.,  Van  De  Werkorst,  L.C.,  Scol,  E.A.,  Raith,  M.R.,  Holden,  P.A.  and  J.F.Griffith.    Bacteroidales  terminal  restric,on  fragment  length  polymorphism  (TRFLP)  for  fecal  source  differen,a,on  in  comparison  to  and  in  combina,on  with  universal  bacteria  TRFLP    Water  Research  DOI:10.1016/j.watres.2013.03.060

Layton,  B.A.,  Cao,  Y.,  Eben,er,  D.L.,  Hanley,  K.,  Ballesté,  E.,  Brandão,  J.,  Byappanahalli,  M.,  Converse,  R.,  Farnleitner,  A.H.,  Gentry-­‐Shields,  J.,  Gidley,  M.L.,  Gourmelon,  M.,  Lee,  C.S.,  Lee,  J.,  Lozach,  S.,  Madi,  T.,  Meijer,  W.G.,  Noble,  R.,  Peed,  L.,  Reischer,  G.H.,  Rodrigues,  R.,  Rose,  J.B.,  Schriewer,  A.,  Sinigalliano,  C.,  Srinivasan,  S.,  Stewart,  J.,Van  De  Werkorst,  L.C.,  Wang,  D.,  Whitman,  R.,  Wuertz,  S.,  Jay,  J.,  Holden,  P.A.,  Boehm,  A.B.,    

                           Shanks,  O.,  and  J.F.  Griffith.    2013      Performance  of  Human  Fecal  Anaerobe-­‐Associated  PCR-­‐Based  Assays  in  a                                Mul,-­‐Laboratory  Method  Evalua,on  Study  Water  Research  DOI:10.1016/j.watres.2013.05.060Converse,  R.R.,  Griffith,  J.F.,  Noble,  R.T.,  Haugland,  R.A.,  Schiff,  K.C.,  and  S.B.  Weisberg.    2012.    Correla,on  between  

quan,ta,ve  PCR  and  culture-­‐based  methods  for  measuring  Enterococcus  spp.  over  various  temporal  scales  at  three  California  marine  beaches.    Applied  and  Environmental  Microbiology  78:  1237-­‐1242

McQuaig,  S.,  Griffith,  J.F.  and  V.J.  Harwood.    2012.    Associa,on  of  fecal  indicator  bacteria  with  human  viruses  and  microbial  source  tracking  markers  at  coastal  beaches  impacted  by  nonpoint  source  pollu,on.    Applied  and  Environmental  Microbiology  78:6423-­‐6432

Dubinsky,  E.A.,  Esmaili,  L.,  Hulls,  J.R.,  Cao,Y.,  Griffith,  J.F.  and  G.  L.  Andersen.  2012.    Applica,on  of  Phylogene,c  Microarray  Analysis  to  Discriminate  Sources  of  Fecal  Pollu,on.    Environmental  Science  and  Technology  46:4340–4347

Colford  Jr,  J.M,,  Schiff,  K.C.,  Griffith,  J.F.,  Yau,  V.,  Arnold,  B.F.,  Wright,  C.C.,  Gruber,  J.S.,  Wade,  T.J.,  S  Burns,  Hayes,  J.,  McGee,  C.,  Gold,  M.,  Cao,  Y.,  Noble,  R.T.,  Haugland,  R.  and  S.B.  Weisberg.    2012.    Using  rapid  indicators  for  Enterococcus  to  assess  the  risk  of  illness  aqer  exposure  to  urban  runoff  contaminated  marine  water.  Water  Research  46:2176-­‐2186

Goodwin,  K.D.,  McNay,  M.,  Cao,  Y.,  Eben,er,  D.,  Madison,  M.  and  J.F.  Griffith.    2012.    A  mul,-­‐beach  study  of  Staphylococcus  aureus,  MRSA,  and  enterococci  in  seawater  and  beach  sand.  Water  Research  46:4195-­‐4207

Shanks,  O.C.,  Sivaganesan,  M.,  Peed,  L.,  Kelty,  C.,  Blackwood,  A.D.,  Greene,  M.R.,  Noble,  R.,  Bushon,  R.,  Stelzer,  E.A.,  Kinzelman,  J.,  Anna'eva,  T.,  Sinigalliano,  C.,  Wanless,  D.,  Griffith,  J.F.,  Cao,  Y.,  Weisberg,  S.,  Harwood,  V.J.,  

Fuhrman,  J.A.,  Griffith,  J.F.,  and  M.S.  Schwalbach.  2000.    Prokaryo,c  and  viral  diversity  in  marine  plankton.    Ecological  Research.    17:183-­‐194

Stol,  L.D.,  T.P.  Hayden,  and  J.F.  Griffith.    1996    Benthic  Foraminifera  at  the  Los  Angeles  County  Whites  Point  Ousall  Revisited.    Journal  of  Foraminiferal  Research.    26  357-­‐368

Griffith,  J.F.  and  S.B.  Weisberg.  2011.  Challenges  in  Implemen,ng  New  Technology  for  Beach  Water  Quality  Monitoring:  Lessons  Learned  form  a  California  Demonstra,on  Project.  Marine  Technology  Society  Journal  45:65-­‐73

SYNERGISTIC  ACTIVITIES:Doctoral  Fellow,  NSF-­‐IGERT:  Env.  Studies,  Policy  and  Engineering-­‐  Sustainable  Ci,es  Program,  USC  (1999-­‐2000)-­‐Sea  Grant  Trainee,  Pathogenic  Viruses  in  the  Coastal  Ocean,  1995  -­‐  2001

RECENT  COLLABORATORS:M   Sadowsky   (U  MN),   R.   Noble   (UNC),   J.   Stewart   (UNC),   A.   Boehm   (Stanford),   J.   Jay   (UCLA),   P.   Holden   (UCSB),   J.  Fuhrman  (USC),  V.  Harwood  (USF),  B.  Arnold  (UCB),  J.  Colford  (UCB)  

THESIS  ADVISORY  &  POSTGRADUATE-­‐SCHOLAR  SPONSOR:

C.Lee  (UCLA),  R.  Converse  (UNC,  Chapel  Hil)

SUMMARY PROPOSAL FORM PROJECT TITLE: DEVELOPMENT  OF  DIGITAL  RT-­‐PCR  METHODS  TO  QUANTIFY  HUMAN-­‐  ASSOCIATED   BACTERIOPHAGE   IN   STORM   WATER   AND   COASTAL   RECREATIONAL  WATERS   OBJECTIVE: Our overall goals are to adapt and further develop a rapid, sensitive water quality monitoring and source-tracking tool that can be used to track the movement of viruses in coastal recreational waters and can be used to track human-associated contamination. Specifically we aim to 1) to develop a sensitive, robust droplet digital RT-PCR assay to measure and distinguish human-associated and non-human-associated F+RNA coliphage genogroups; and 2) apply this assay as microbial source tracking tool in coastal recreational waters and storm waters. METHODOLOGY: We will develop and test sensitive (potentially detecting a single gene copy) and robust (resistant to PCR inhibition) droplet digital RT-QPCR methods adapted from recently developed multiplex Reverse Transcriptase-QPCR assays applied to wastewater and environmental samples. These assays can distinguish multiple F+RNA coliphage genotypes at once by targeting shared coat protein and RNA replicase genes. Using this assay, we will quantify F+RNA coliphage genotypes collected from storm water, estuaries, and marine waters in the coastal zone in Southern California. Specifically we will target beaches likely to suffer from aging, leaky infrastructure and collect and filter 1-5L of seawater to capture the viruses by adsorption onto electronegative mixed cellulose ester filters. We also will draw on a sample archive previously collected by SCCWRP and the Martiny lab at UC Irvine consisting of large (20L) and small (0.5-1L) volume storm water samples from San Diego and Malibu, and near-shore beach samples from Ocean Beach and Tourmaline Surfing Park (San Diego), Doheny State Beach, Newport Beach (Orange County), Avalon, and Malibu (Los Angeles County). RATIONALE: Levels  of  fecal  indicator  bacteria  (FIB)  are  used  to  monitor  the  recreational  water  quality  to  protect  swimmers  from  exposure  to  pathogens  found  in  fecal  material,  but  are  an  imperfect  indicator.  The  main  limitation  is  that  concentrations  of  FIB  have  been  shown  to  be  poorly  correlated  with  the  presence  of  human  enteric  viruses  (e.g.  Human  Norovirus,  Enterovirus  or  Adenovirus)  that  are  responsible  for  the  majority  of  gastrointestinal  illnesses  in  swimmers.  Other  limitations  include  discerning  the  source  of  FIB  (human  or  non-­‐human),  the  dilution  and  degradation  of  FIB  in  the  environment,  and  the  physical  removal  of  bacteria  as  they  are  transported  through  groundwater.    Viruses  and  bacteriophage  (i.e.  viruses  that  infect  bacteria)  are  not  filtered  out  by  sand  or  soil  at  the  same  rate  as  much  larger  bacteria.  Further,  bacteriophage  are  more  abundant  than  human  viruses  (since  their  bacterial  hosts  are  much  more  abundant)  which  makes  them  more  attractive  water  quality  indicators  at  beaches  where  the  source  of  contamination  is  leaking  infrastructure,  rather  than  acute  inputs,  such  as  storm  water  pulses  or  sewage  spills.    F+RNA  coliphage  (i.e.  

viruses  infecting  E.  coli),  bacteriophage  infecting  human-­‐associated  Bacteroides  bacteria  (e.g.  Bacteroides  GB-­‐124  phage),  and  a  bacteriophage  discovered  from  human  gut  microbiome  metagenomes  (crAssphage)  have  been  proposed  as  potential  fecal  indicators.      Traditional  cultivation  techniques  are  slow,  taking  up  to  18-­‐24  hours  to  quantify  the  number  of  phage,  followed  by  molecular  analysis  to  identify  phage  genotypes.    Adding  to  these  difficulties,  bacteriophage  abundance  is  variable  and  can  go  undetected  by  culture  methods,  requiring  non-­‐quantitative  enrichment  cultivation  in  order  to  enhance  detection.    Molecular  quantification  of  bacteriophage  directly  from  environmental  waters  avoids  the  lengthy  cultivation  process  and,  in  the  case  of  F+RNA  coliphage,  measures  genotypes  associated  with  human  (genotypes  II  and  III)  and  non-­‐human  (genotypes  I  and  IV)  fecal  sources.  

DATA SHARING PLAN: All  data  generated  by  the  project  will  cleaned,  formatted,  and  be  made  publicly  available  through  the  California  Environmental  Data  Exchange  Network,  or  on  SCCWRP’s  website.  In  addition,  the  publications  will  be  open-­‐access  and  data  and  protocols  generated  by  this  project  will  be  housed  on  a  publicly  accessible  website  either  in  an  open  sharing  site  such  as  GitHub.    

                                                                                                                  July 7, 2015 Dr. Joshua Steele Department of Microbiology Southern California Coastal Water Research Project 3535 Harbor Blvd. Suite 110 Costa Mesa, CA 92626 Dear Dr. Steele,  I am pleased to hear about your proposal to USC Sea Grant seeking to develop a digital PCR assay for human-associated coliphage in the urban ocean. At the Surfrider Foundation, we are interested in innovative techniques to measure the health of the coastal ocean and in rapid, sensitive methods for monitoring coastal water quality. As you know from our previous collaborations, we are especially interested in the links between coliphage as a measure of viral contamination and gastrointestinal illness in swimmers and surfers. We would find this technique a useful addition to the rapid, molecular water quality methods that we can use to measure coastal health. Sincerely,

Dr. Chad Nelsen CEO

Global Headquarters P.O. Box 6010 San Clemente, CA USA 92674-6010 Phone: (949) 492 8170 Fax: (949) 492 8142 Email: info@surfrider.org www.surfrider.org