1. Development of an Assay to Detect Reportable Bacterial Populations that Support Harmful Algae Blooms on
the Southern California Coast
Alicia Romero1,2
, Eric Izaguirre1,4
, Jason Nefalar1
, Duan Nguyen1
, Leticia Lopez1
, Tiffany Sidwell1,3
, Theodore Peterson1,4
, and Dr. James Harber1
Oxnard College Biotechnology Summer Institute and HSI- STEM, Oxnard CA 93033
Oxnard College Biology Program1
, California Polytechnic State University, San Luis Obispo2
, University of California, Los Angeles3
, California State University, Channel Islands4
AbstractAbstract
Algae ResultsAlgae Results
Algal Association with Bacterial PopulationsAlgal Association with Bacterial Populations
Gram Stains of Bacterial IsolatesGram Stains of Bacterial Isolates
Conclusion and Future ConsiderationsConclusion and Future Considerations
Further InformationFurther Information
Contact information:
Alicia Romero
aromer05@calpoly.edu
Dr. James Harber
Associate Professor, Oxnard College
Oxnard College
4000 S. Rose Ave.
Oxnard, CA 93033
Jim.Harber@gmail.com
Acknowledgments:
Dr. Cynthia Herrera, Oxnard College HSI-STEM Grant Director
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Methods and MaterialsMethods and Materials
Figure 1 (Above): Cepheid SmartCycler PCR results: Pseudo-nitzschia Genus Primer; Highlighted are Samples
collected from Santa Monica 07/05/2012 (top: FAM, bottom: melt curve) Positive signals are confirmed by the
convergence at ~86 degrees in the melt analysis. Negative control confirmed by different melting temperature of
the primer than of the dsDNA in the positive samples.
Figure 2 (Above): Cepheid SmartCycler PCR results: L. Polyedrum primer; Highlighted are Samples
collected from Point Mugu 07/05/2012 (top: FAM, bottom: melt curve) Positive signals correlate with
presence of L. Polyedrum in sample and are confirmed by the primer than of the dsDNA in the positive
samples. Positive signals are confirmed by onvergence of melting temperatures at ~78 degrees. Negative
controls confirmed by different melting temperature of the primer.
Assay for Antibiotic Resistant Aquatic BacteriaAssay for Antibiotic Resistant Aquatic Bacteria
Recent studies indicate that certain algal species share a
symbiotic relationship with bacterial communities in regards to
their exchange of nutrients including NH3. In particular,
estuarine communities are known to have higher populations of
Vibro species because of their nitrogen fixating capabilities that
give them an advantage over other aquatic bacteria in the
nitrogen poor estuarine communities. Samples collected from
the Naval Base beach located at the mouth of an estuary were
found to contain ampicillin resistant Vibrio parahemolyticus. The
Naval Base water samples were filtered, frozen and analyzed
using scanning electron microscopy where a number of diatoms
were visualized indicating that our target HAB diatom species
may also be present with further verification using genetic
detection. A full genome sequence of the most prevalent
bacteria in the ocean, a species of cyanobacteria, revealed that
the organism is missing the genes for photosynthesis. The algae
provides carbon for the cyanobacteria, which is missing the
genes to produce its own, and the bacteria provides nitrogen for
the algae as mentioned above. This further supports the concept
of a symbiotic relationship between algae and ocean- dwelling
bacteria.
SCCOOS
HABs Link
Transgenic Plants &
Antibiotic resistance
markers, 2009
Discovery of
HAB symbiosis
2012
Env. Health News
sewage plants source
of resistance, 2009
Red Tide Surfing Video
Bacterial ResultsBacterial Results
Much of southern California’s coast is home to thriving bacterial communities capable of
supporting growing numbers of detectable harmful algae blooms (HABs) that are responsible
for poisoning of fish, seizures in marine mammals and both Amnesic and Paralytic Shellfish
Poisoning in humans. The symbiosis between the algae and bacterial communities is predicated
on an exchange of nutrients from nitrogen-fixing bacteria, including those of the genus, Vibrio.
Seawater samples collected from several shoreline sampling points along the Southern California
Bight were analyzed via real time PCR and shown to contain relevant isolates of both bacterial
and phytoplankton species, including Vibrio parahemolyticus, the neurotoxin producing
dinoflagellate, Lingulodinium polyedrum, and diatoms of the genus Psuedo-nitzschia. Bacterial isolates
were also proven to be carriers of the beta- lactamase (ampicillin resistance) gene through real
time PCR, suggesting that horizontal gene transfer between aquatic and terrestrial bacterial
communities is likely. Genetic detection of the toxic algae species was accomplished via a
protocol of: i) seawater collection and filtration ii) DNA extraction, purification, and quantitation
iii) real time PCR amplification of indicative genetic sequences using species specific primers.
Bacterial isolates were prepared in largely the same manner, with additional selection and
isolation achieved by plating on MacConkey’s agar, replica plating onto ampicillin tryptic soy
agar, and single colony isolation. This provided the starting material for the DNA extraction and
real time PCR. Amplified 16S rRNA and ampicillin resistance sequences were quantified and
sequenced allowing identification of the bacterial isolates by BLAST submission. Further
investigation of bacterial communities and an expansion of this research may provide a basis for
the utilizing genetic markers of indicator species to detect HABs and their correlative bacterial
communities.
Illustration 4: Frozen filters of seawater samples from Santa Monica and Naval Base Point Mugu used
to visualize diatoms using scanning electron microscopy. Scales of pictures are indicated in white
margins.
a) c)
d) e) f)
h)g)
N Fixing by
Vibro
parahaemolyticus
in estuaries
Figure 3 (Above): Cepheid Smartcycler PCR results (Top- FAM, Bottom- melt curve).
V3F/R 16S Primers used on specimens from Table 1. Positive signals are confirmed by the
convergence at ~89 degrees in the melt analysis.
Table 1 (Left): PCR fragments were amplified using a Nanodrop Spectrophotometer and
submitted for sequencing to Operon. Amplified sequences were submitted into BLAST to
determine the most likely genus and species by the best alignment of submitted sequences
with cataloged sequences. Specimens a, b, and d 16S sequences had no significant similarity
when submitted into BLAST. Their AMP resistance gene was used to identify them.
AMPF/R primers were utilized to span the bla gene from plasmid pUC19. Specimen IDs
indicate where samples were collected. OS- Oxnard Shores Beach; PM- Point Mugu State
Beach; SB- Santa Barbara State Beach; Solimar Beach. All samples were collected in July of
2012.
Illustration 1: Aerial view of the coastal topography of Naval Base Point Mugu.
Illustration 2,3: Left- Point Mugu State Beach. Right- Leo Carillo State Beach.
Illustration 5: Light microscopy visualization of Gram stains of specimens from Table 1.
Illustration 6: Light microscopy visualization of Gram stains of specimens from Table 2. From right to
left: CIH1, NB1, NB2.
Table 2 (Below): BLAST ID of amplified V3F/R
16S and AMPF/R resistance gene sequences.
Specimen IDs indicate where samples were
collected. CIH- Channel Islands Harbor; NB-
Naval Base Point Mugu Family Beach. All samples
were collected in July of 2013.
Figure 4 (Above): Cephied Smartcycler PCR results (Top- FAM, Bottom- melt curve). Highlighted
samples are the results of the 16S reactions for the specimens from Table 2. Positive signals are
confirmed by the convergence at ~90 degrees in the melt analysis.
Ampicillin resistant genes were detected following isolation of bacteria on ampicillin Tryptic Soy Agar from
the seawater samples. PCR primers were developed for an early lineage generic laboratory plasmid’s
ampicillin resistance gene sequence (beta-lactamase bla gene from pUC19). The antibiotic resistance
observed from isolates in this research suggests that marine ecosystems contain a significant genetic identity
with that of known terrestrial antibiotic resistant genetic constructs. The origins and specific gene transfer
mechanisms of this pool of antibiotic resistance genes is a subject for future exploration. The source of
these terrestrial based antibiotic resistance genes could be attributed to storm runoff containing bacteria
from household pets treated with antibiotics, agricultural application of bacteria in transgenic plants, and
livestock treated with antibiotics for the improvement of herd productivity. Literature suggests that public
sewage system effluents contain antibiotic disinfectants and partially metabolized antibiotic compounds
excreted by patients treated for microbial infections. Evidence that healthy humans excrete antibiotic
resistant bacteria propagated by trace amounts of antibiotics in the food industry also contribute to the
source of terrestrial resistance genes. The combined population of antimicrobial agents and antibiotic
resistant bacteria flow into municipal wastewater, then into the sewage treatment plant, where they are
passed into the ocean. Once present in the marine ecosystem, the various bacteria species can rapidly
acquire antibiotic resistance genes by horizontal transfer. An expansion of this research could be aimed
towards investigating specific mechanisms for gene transfer and resistance gene sequence analysis to
determine the origins of the resistance genes.
Specimen BLAST ID
Query
Sequence
a) OS1
Klebsiella
pneumoniae
str. OB4
AMP_F
b) PM1
Klebsiella
variicola str.
XF9
AMP_F
c) PM2
Klebsiella
pneumoniae
str. OB4
V3_16S
d) PM3
Klebsiella
pneumoniae
str. JM45
AMP_F
e) PM4
Pseudomonas
aeruginosa str.
Ps004
V3_16S
f) PM6
Pseudomonas
aeruginosa str.
902
V3_16S
g) PM15
Pseudomonas
aeruginosa str.
902
V3_16S
h) SB
Pseudomonas
sp.
V3_16S
i) Solimar
Bacillus cereus
str. THt1-9
V3_16S
Specimen BLAST ID
CIH1
Vibrio parahaemolyticus
RIMD 2210633
NB1
Vibrio parahaemolyticus
O1:K33 str. CDC_K4557
NB2
Vibrio parahaemolyticus
RIMD 2210633
Results of this research revealed the presence of antibiotic resistant aquatic bacterial
populations and single celled algae species that comprise the bulk of harmful algae blooms
present during the summer months off the coast of Southern California. Genetic detection
of these harmful algal species and bacterial populations was achieved by PCR and provided
the basis for further analysis of the 16S and beta lactamase bla (AMP) genes of the bacterial
isolates. Amplified 16S and/or AMP gene sequences were submitted into BLAST to
identify the isolates. With growing concern for the origins and mechanisms of antibiotic
gene transfer between aquatic and terrestrial bacterial communities, further analysis of the
AMP genes could be used to determine the origin of these resistance genes. Overall, this
research provided a successful protocol for environmental bacterial identification and
genetic detection of harmful algae species. Using techniques demonstrated in this research,
further investigation of the relationship between the bacterial communities and harmful
algae species could lead to the identification of one or more indicator species. Utilization of
genetic detection of these indicator species would provide a simple and comprehensive
method to predict and quantify HAB species. This approach would not only encompass the
issues directly involved with HABs but also the upstream issues associated with these
bacterial communities. Agencies responsible for the safety of the coastal waters and food
supply, such as the Coast Guard, US Navy, EPA, USDA and FDA, could take advantage of
this approach for HAB detection in order to make informed decisions regarding antibiotic
gene usage strategy, human antibiotic use, waste water treatment, food supply monitoring
and the development of reliable ocean monitoring.
Pseudoo-nitzschia from
Centers for Oceans
and Human Health
b)
i)