bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 Bacteriological and histopathological findings in cetaceans
2 that stranded in the Philippines from 2017 to 2018
3
4 Marie Christine M. Obusan1, Jamaica Ann A. Caras1,2, Lara Sabrina L. Lumang1, Erika Joyce S.
5 Calderon1, Ren Mark D. Villanueva1, Cristina C. Salibay3, Maria Auxilia T. Siringan4, Windell L.
6 Rivera5, Joseph S. Masangkay6, and Lemnuel V. Aragones2
7
8 Microbial Ecology of Terrestrial and Aquatic Systems, Institute of Biology, College of Science,
9 University of the Philippines Diliman, Quezon City 1101, Philippines1
10 Marine Mammal Research Stranding Laboratory, Institute of Environmental Science and
11 Meteorology, College of Science, University of the Philippines Diliman, Quezon City 1101,
12 Philippines2
13 College of Science and Computer Studies, De La Salle University-Dasmariñas, City of Dasmariñas
14 Cavite 4115, Philippines3
15 Microbiological Research and Services Laboratory, Natural Sciences Research Institute, University of
16 the Philippines Diliman, Quezon City 1101, Philippines4
17 Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of
18 Science, University of the Philippines Diliman, Quezon City 1101, Philippines5
19 College of Veterinary Medicine, University of the Philippines Los Baños, College, Batong Malake,
20 Los Baños, Laguna 4031, Philippines6
21
22
23 Abstract
24 The relatively high frequency of marine mammal stranding events in the Philippines provide
25 many research opportunities. A select set of stranders (n=21) from 2017 to 2018 were sampled for
26 bacteriology and histopathology. Pertinent tissues and bacteria were collected from eight species (i.e.
1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
27 Feresa attenuata, Kogia breviceps, Globicephala macrorhynchus, Grampus griseus, Lagenodelphis
28 hosei, Peponocephala electra, Stenella attenuata and Stenella longirostris) and were subjected to
29 histopathological examination and antibiotic resistance screening, respectively. Lesions that were
30 observed in tissues of 19 cetaceans include congestion, hemorrhage, edema, hemosiderosis,
31 glomerulopathy, Zenker’s necrosis, atrophy, atelectasis, and parasitic cysts. These lesions may be
32 associated with factors possibly contributing to the death, debility, and stress of the animals during
33 their strandings. On the other hand, the resistance profiles of 24 bacteria (belonging to genera
34 Escherichia, Enterobacter, Klebsiella, Proteus, and Shigella) that were isolated from four cetaceans
35 were determined using 18 antibiotics. All 24 isolates were resistant to at least one antibiotic class, and
36 79.17% were classified as multiple antibiotic resistant (MAR). The MAR index values of isolates
37 ranged from 0.06 to 0.39 with all the isolates resistant to erythromycin (100%; n=24) and susceptible
38 to imipenem, doripenem, ciprofloxacin, chloramphenicol, and gentamicin (100%; n=24). The
39 resistance profiles of these bacteria can be used as basis for selecting antibiotics needed in the medical
40 management of stranded cetaceans that need to be rehabilitated. Overall, the histopathological and
41 bacteriological findings of the study demonstrate the challenges faced by cetacean species in the wild,
42 such as but not limited to, biological pollution through land-sea movement of effluents, fisheries
43 interactions, and anthropogenic activities.
44
45 Keywords: cetaceans, histopathological assessment, lesions, antibiotic resistant bacteria, stranding
46 events
47
48 Introduction
49 The surveillance of wildlife health is part of an early warning system for detecting the
50 emergence or resurgence of disease threats. In the case of cetacean populations in the Philippines,
51 perhaps the most practical way of investigating their health is through their stranding events. A
52 marine mammal is considered stranded when it runs aground, or in a helpless position such as when it
53 is ill, weak, or simply lost [1]. While the event itself deserves attention, as it is not normal for any
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54 marine mammal to strand for no apparent reason, each stranded individual can give information on
55 the abundance, distribution, health, and other ecological characteristics of its free-living counterparts
56 [2], as well as threats faced by its population [3]. It is important that stranding events be responded as
57 quickly as possible, since some stranded animals may quickly die depending on the size of the animal
58 and extent of human intervention [4].
59
60 Biases exist in investigating the factors involved in cetacean strandings; easy-to-detect
61 circumstances such as obvious injuries (especially those intentionally inflicted by humans) are likely
62 to be more reported, whereas the role of diseases or parasites may be underestimated. The capacity to
63 detect the presence of pathogens or parasites of stranded cetaceans depends on resources, such as the
64 presence of a stranding network with the capability to respond to stranding events as well as
65 availability of expertise for conducting necropsy and other protocols for case investigation.
66 Nonetheless, whether or not a pathological condition is the underlying cause of a stranding, stranded
67 animals are good representatives for monitoring wildlife health. Also, while live stranders provide
68 good biological samples for laboratory analyses, a dead or decomposing carcass on the beach is just as
69 useful in providing specimens and other information.
70
71 The available literature on bacteria that were isolated from marine mammals worldwide
72 support the significance of investigating Gram-negative species and their antibiotic resistance or
73 susceptibility. Antibiotic susceptibility patterns have been described for populations and individuals
74 of Atlantic bottlenose dolphins, Pacific bottlenose dolphins, Risso’s dolphins, California sea lions,
75 beluga whale, sea otters and pinnipeds [5-8]. Strains of zoonotic bacteria resistant to multiple
76 antibiotics used for human and animal treatments were isolated from these animals, and some of those
77 bacteria were recognized by the American Biological Safety Association (ABSA) as human
78 pathogens. Associations between increased prevalence of antibiotic resistant bacteria in marine
79 mammals and proximity to human activities were strongly suggested [5,7,9-11]. The antibiotic
80 susceptibility profiles of bacteria isolated from cetaceans found in the Philippines where previously
3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
81 reported, wherein more than half of the bacteria (n=14) had single or multiple resistances to a
82 selection of antibiotics [12].
83
84 On the other hand, histopathological assessments proved to be useful in determining probable
85 causes of death or debility of stranded cetaceans worldwide [13-17]. Tissue lesions help confirm
86 parasitic and bacterial infections, co-morbidities, physical injuries (e.g., brought about by fisheries or
87 human interactions) and bioaccumulation of chemical compounds (e.g., persistent organic pollutants)
88 in cetaceans [16, 18-22]. Histopathological assessment is a practical and informative tool that
89 provides pathological evidence and reinforce the necropsy being done in dead cetaceans as part of the
90 stranding response.
91
92 In this study, swab and tissue samples collected from cetaceans that stranded locally from
93 February 2017-April 2018 were subjected to bacterial isolation (with subsequent antibiotic resistance
94 screening) and histopathological assessment. Data on antibiotic resistant bacteria, parasites, and tissue
95 lesions in cetaceans are valuable in evaluating the factors that may be associated with their local
96 stranding events, observed to have increased in recent years [23-24]. Out of 29 confirmed species in
97 the country, 28 species were reported to have stranded from 2005-2018 [24]. A yearly average of 105
98 cetaceans strandings occurred in the country from 2014 to 2018 [24]; 229 events were recorded by the
99 Philippine Marine Mammal Stranding Network (PMMSN) in collaboration with the Bureau of
100 Fisheries and Aquatic Resources (BFAR) from 2017 (n=121) to 2018 (n=108) involving 118 dead and
101 108 live (n=3 unknown) stranders.
102
103 Materials and methods
104 All biological samples were collected in coordination with PMMSN and the Marine Mammal
105 Research and Stranding Laboratory (MMRSL) of the Institute of Environmental Science and
106 Meteorology (IESM), University of the Philippines, Diliman (UPD). The marine mammal stranding
107 response and tissue collection is a nationwide effort which is part of the Memorandum of Agreement
4 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
108 (MOA) between PMMSN and BFAR. Laboratory work was done at Microbial Ecology of Terrestrial
109 and Aquatic Systems Laboratory (METAS) of the Institute of Biology, UPD.
110
111 Sample collection
112 Cetaceans that stranded in the Philippines from February 2017 to April 2018 (Fig 1) were
113 opportunistically sampled for tissues and swabs by veterinarians who were trained by PMMSN.
114 Tissues were obtained during necropsy. Swabs were collected from routine (i.e., blowhole, anus,
115 genital slit) and non-routine sites (e.g., organ sites and skin lesions suspected for infection) based on
116 animal disposition and physical preservation [1]. Stranded cetaceans were characterized in terms of
117 species, sex, age class, stranding type, stranding site, and stranding season (Table 1).
118
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119
120
121 Fig 1. Sites of cetacean stranding events from February 2017-April 2018
122
123
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124 Table 1. Stranded cetaceans responded for sampling in the Philippines during the year 2017 and 2018.
Strander PMMSN Code Species Region Date Stranding Age Class Condition Sex
Code Type
S01 Lh03R5270217 Fraser's dolphin V 27-Feb-17 Single Adult Alive Male
Lagenodelphis hosei
S02 Sl21R5040317 Spinner dolphin V 04-Mar-17 Single Adult Alive Female
Stenella longirostris
S03 Lh03R11010317 Fraser’s dolphin XI 09-Mar-17 Single Adult Dead Female
Lagenodelphis hosei
S04 Gg04R4A290316 Risso’s dolphin IV-A 29-Mar-17 Single Subadult Alive Unknown
Grampus griseus
S05 Fa02R5020517 Pygmy killer whale V 02-May-17 Mass Adult Alive Unknown
Feresa attenuata
S06 Gg15R5090517 Risso’s dolphin V 09-May-17 Single Adult Alive Unknown
Grampus griseus
S07 Pe04R1300417 melon-headed whale I 30 April 2017 Single Unknown Dead Female
Peponocephala
electra
S08 Kb07R11160517 Pygmy sperm whale XI 16-May-17 Single Adult Alive Male
Kogia breviceps
S09 Gg10R1150617 Risso’s dolphin I 15-Jun-17 Single Neonate Alive Female
Grampus griseus
S10 Sa18R1210617 pantropical spotted I 21-Jun-17 Single Subadult Dead Female
dolphin
Stenella attenuata
S11 Gg02R3230617 Risso’s dolphin III 23-Jun-17 Single Adult Dead Unknown
Grampus griseus
S12 Pe06R12030717 melon-headed whale XII 03-Jul-17 Single Adult Alive Male
Peponocephala
electra
S13 Sa03R4A280717 pantropical spotted IV-A 28-Jul-17 Single Subadult Alive Female
dolphin
Stenella attenuata
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S14 Sl06R11310817 spinner dolphin XI 31-Aug-17 Single Subadult Alive Female
Stenella longirostris
S15 Sl23R1300917 spinner dolphin I 30-Sep-17 Single Subadult Dead Female Stenella longirostris
S16 Kb02R5091117 pygmy sperm whale V 09-Nov-17 Single Adult Alive Female
Kogia breviceps
S17 Lh04R2011217 Fraser’s dolphin II 01-Dec-17 Single Adult Alive Female
Lagenodelphis hosei
S18 Gm11R151217 short-finned pilot I 05-Dec-17 Single Adult Alive Female
whale
Globicephala
macrorhynchus
S19 Sa03R9160118 pantropical spotted
dolphin IX 16-Jan-18 Single Adult Alive Female
Stenella attenuata
S20 Lh01R9170418 Fraser’s dolphin IX 17-Apr-18 Single Adult Dead Female Lagenodelphis hosei
S21 Kb01R9260418 pygmy sperm whale IX 27-Apr-18 Single Adult Alive Female Kogia breviceps 125
126
127
128
129
130
131
132
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133
134
135
136
137
138 Histopathological assessment
139 Tissue samples (< 1 cm3 each) were preserved in 10% neutral buffered formalin, processed by
140 paraffin-embedded technique, sectioned at 5 µm, and subjected to hematoxylin and eosin (H&E)
141 staining. Tissue sectioning and H&E staining technique were performed at a private hospital
142 Providence Hospital, Quezon City where tissue sections were stained with hematoxylin in water,
143 dehydrated using a series of increasing concentrations of alcohol, and applied with eosin as a
144 counterstain. Stained specimens were passed through xylol and toluol before mounting [25]. Using
145 light microscopy technique, stained tissue samples were observed for the following pathologies:
146 inflammation; fibrosis; granuloma lesions; edema; presence of parasite cysts (e.g., nematodes and
147 protozoan parasites); endothelial damage (including endothelial deposits); presence of macrophages;
148 granules; microthrombi formation; and hemorrhage.
149
150 Bacterial isolation and antibiotic resistance screening
151 Swab samples in transport media (e.g., Amies) were stored at 4°C and were sent to the
152 laboratory within 18-24 hours. Swabs were then enriched in Tryptic Soy Broth (TSB) for 18-24 hours
153 at 37 °C. From the enriched media, inocula were streaked on MacConkey Agar (MCA).
154 Morphologically distinct Gram-negative colonies were sub-cultured and purified.
155
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156 Pure bacterial isolates were identified using 16S rRNA gene amplification. Bacterial DNA
157 was extracted from the purified isolates using either the GF-1 Bacterial DNA Extraction Kit (Vivantis
158 Technologies) following manufacturer’s instructions, or the Boil Lysis Method following Ahmed and
159 Dablool (2017) [26]. The universal 16S rRNA bacterial gene was amplified from the DNA of isolates
160 through polymerase chain reaction (PCR). The primers used for targeting the 16S rRNA gene were
161 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1541R (5’-AAGGAGGTGATCCANCCRCA-3’)
162 [27-28]. The PCR reaction mix consisted of: dNTPs, MgCl2, Taq DNA polymerase, DNA template,
163 forward and reverse primers, and nuclease-free water. The thermal cycler conditions were as follows:
164 initial denaturation for 2 min at 95 °C, 30 cycles of denaturation for 30 s at 94 °C, annealing for 30 s
165 at 55-60 °C, extension for 30 s at 72 °C, and final extension for 7 min at 72 °C. Positive controls (E.
166 coli ATCC® 25922) and blanks (DNA-free templates) were included. PCR products were subjected to
167 agarose gel electrophoresis (AGE) to detect target DNA band. PCR products were then sent to a
168 sequencing facility for DNA purification and sequencing. PreGap4 and Gap4 (Staden Package 2.0)
169 were used to obtain the consensus sequences [29]. Sequence homologies were determined using
170 NCBI BLASTn search and further analyses were done using BioEdit [30-31].
171
172 Kirby-Bauer Disk Diffusion Assay was performed to determine the sensitivity of the bacterial
173 isolates to the following antibiotics: carbapenems (imipenem, meropenem, ertapenem, doripenem),
174 penicillins (ampicillin), cephems (cephalothin, ceftriaxone, cefoxitin), fluoroquinolones
175 (moxifloxacin, ciprofloxacin, ofloxacin), aminoglycosides (amikacin, gentamicin), tetracyclines
176 (tetracycline, oxytetracycline), phenicols (chloramphenicol), folate pathway inhibitors (trimethoprim-
177 sulfamethoxazole), and macrolides (erythromycin). These antibiotics were chosen based on (1)
178 inclusion in the priority list of WHO for antibiotic resistance research; (2) known use in agriculture
179 and aquaculture; (3) reported susceptibility profiles of bacteria isolated from marine animals
180 worldwide; (4) use during rehabilitation of stranded marine mammals; and (5) known spectrum
181 activity [5,32-35]. To ensure that only acquired resistances will be observed, antibiotics to which the
182 bacterial isolates have intrinsic resistances were excluded in the assay. The reactions of the isolates to
183 the antibiotics were described as Susceptible (S), Intermediate (I), or Resistant (R) based on Clinical 10 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
184 and Laboratory Standards Institute (CLSI) M31-A2 (2002), M100-S24 (2014), and European
185 Committee on Antimicrobial Susceptibility Testing (EUCAST) v 8.0 (2018). E. coli ATCC® 25922
186 was used as the control [36-38]. Multiple Antibiotic Resistance (MAR) Index values were computed
187 using the formula: (# of resistant antibiotics / total # of antibiotics tested) [39]. MAR indices greater
188 than 0.2 were interpreted to come from sources where antibiotics are often used [32,39-40]. Also,
189 MAR isolates were interpreted as those that are resistant to three or more antibiotic classes [41].
190
191 Results
192 In this study, tissue samples and bacterial isolates were obtained from 21 stranded cetaceans
193 representing eight species (Feresa attenuata, Kogia breviceps, Globicephala macrorhynchus,
194 Grampus griseus, Lagenodelphis hosei, Peponocephala electra, Stenella attenuata, and Stenella
195 longirostris). The stranding events of these cetaceans were responded in different regions of the
196 Philippines, mostly in Region I, Region V, and Region IX, and the rest in Region II, Region III,
197 Region IV-A, Region XI, and Region XII. Majority of the cetaceans (13) were female, while there are
198 only three males. The sex of the remaining five cetaceans are unknown. For the age group, 15 were
199 adults, five were sub-adults, and one was a neonate.
200
201 A total of 75 tissue samples from 19 out of 21 cetaceans were available for histopathological
202 assessment (Table 2). The following lesions were observed in 44 tissue samples: congestion in
203 54.54% (n=24; brain, cardiac muscle, kidney, liver and lungs), hemorrhage in 13.63% (n=6; brain and
204 kidney), edema in 13.63% (n=6; kidney, liver and lungs), hemosiderosis in 6.82% (n=3; brain and
205 kidney), glomerulopathy in 20.45% (n=9; kidney), Zenker’s necrosis in 2.27% (n=1; skeletal muscle),
206 atelectasis in 2.27% (n=1 lung), and parasitic cysts in 31.81%; T. gondii cysts in 11.36% (n=5; kidney
207 and cardiac and skeletal muscles) Sarcocystis sp. cysts in 4.54% (n=2; skeletal muscle), and
208 unidentified cysts in 15.91% (n=7; kidney, skeletal and cardiac muscles) (Fig 2).
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209 Table 2. Histopathological findings in tissues of cetaceans that stranded in the Philippines from February to April 2018.
Strande Species Region Date Remarks Brain Cardiac Kidney Skeletal Liver Lungs Likely r No. / in the muscle muscle Cause of Code Necropsy Stranding Report S01 Lagenodelphis V 27-Feb- unidentifi hosei 17 ed parasites on eyes ; moderate unknown P. NAL NAL NTS NTS congestion cyst delphini cyst in the skeletal muscle S02 Stenella V 04- moderate NAL NAL NAL NTS NTS longirostris Mar-17 congestion S03 Lagenodelphis XI 09- moderate severe Shark hosei Mar-17 moderate congestion; congestion; attack NAL NTS NTS congestion T. gondii hemorrhage; cysts edema S04 Grampus IV-A 29- severe severe NAL NAL NTS NTS griseus Mar-17 congestion congestion; S05 Feresa V 02- moderate to unidentified attenuata May-17 severe cysts; congestion; NTS NAL NTS NTS hemosideros hemorrhage; is hemosiderosis S06 Grampus V 09- skeletal griseus May-17 muscular NAL NAL NTS atrophy; NTS NTS Zenker’s necrosis; S07 Peponocephala I 30- swollen T. gondii electra April- glomerulus; cysts 17 NTS NAL hemosideros NTS NTS nematode is; cyst S08 Kogia XI 16- with breviceps May-17 encysted Sarcocysti NAL NAL NTS NTS NTS parasites s cyst in the
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muscle- blubber section, putative P. delphini; nematodes in the stomach S09 Grampus I 15-Jun- severe severe griseus 17 diffused congesti hepatic severe nematode on; NTS NAL sinusoida congestion cyst focal l pulmona congesti ry edema on S10 Stenella I 21-Jun- moderate Acoustic longirostris 17 congestion; moderate trauma T. gondii hemorrhage; to severe atelectasi NAL NAL cysts membranous congesti s glomerulopa on thy S11 Grampus III 23-Jun- NTS NAL NTS NTS NTS NTS griseus 17 S12 Peponocephala XII 02-Jul- membranous hepatic pulmona electra 17 NAL NAL glomerulopa NAL edema ry edema thy S13 Stenella IV-A 28-Jul- glomerulopa NAL NAL NTS NTS NTS attenuata 17 thy; edema S14 Stenella XI 31- Abundant attenuata Aug-17 parasites in the glomerulopa stomach; thy with T. gondii external moderate lymphocytic cyst fresh NTS NTS NTS congestion aggregation; Sarcocysti wound T. gondii s cyst due to cysts cookie cutter shark bite S15 Stenella 30-Sep- Acoustic I NAL NAL NTS NAL NAL NAL longirostris 17 trauma
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S16 Kogia V 09- rope tied breviceps Nov-17 in hemorrhage; severe peduncle unknown severe NTS NTS NTS congestion area of congestion fluke S17 Lagenodelphis II 01-Dec- glomerulopa unidentifi NTS NTS NTS NTS hosei 17 thy; edema ed cyst S18 Globicephala I 05-Dec- hemorrhage; Moderate unidentifi macrorhynchus 17 NAL Glomerulopa NTS NTS Congestion ed cyst thy S19 NTS severe hemorrhage; NTS NTS NTS congestion severe Stenella 16-Jan- IX congestion; attenuata 18 glomerulopa thy S20 severe severe severe NTS NTS NTS Lagenodelphis 17-Apr- congestion congestion congestion; IX hosei 18 glomerulopa thy S21 NTS moderate hemorrhage; NTS NTS NTS Kogia 27-Apr- IX congestion glomerulopa breviceps 18 thy 210
211 NAL, no apparent lesion; NTS, no tissue sample; Blank entries means no data
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212
a b c
d e f
g h i
Fig 2. Some of the lesions observed in tissues. (a) hemorrhage in S03 kidney; (b) severe congestion in S11 liver; (c) edema in S12 liver; (d) Hemosiderosis characterized by the presence of brown granular pigments in S05 kidney; (e) glomerulopathy in S14 kidney;
(f) Zenker’s necrosis characterized by hyaline degeneration, loss of striations, and muscle fiber waviness in S06 skeletal muscle; (g) atelectasis (collapsed alveoli) in S11 lungs; (h) T. gondii cyst in skeletal muscle of S14; and (i) Sarcocystis cyst in skeletal muscle of
S08.
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213
214 On the other hand, a total of 24 bacteria were isolated from four cetaceans (S12, S16, S17,
215 S18). Based on 16S rRNA gene, these isolates were confirmed to have 98-100% sequence similarities
216 to species belonging to the following genera: Escherichia (n=6), Enterobacter (n=8), Klebsiella
217 (n=5), Proteus (n=4), and Shigella (n=1) (Table 3). These isolates were resistant to at least one
218 antibiotic class tested, and 79.17% were classified as multiple antibiotic resistant (i.e., resistant to at
219 least three antibiotic classes). The MAR index values of the isolates ranged from 0.06 to 0.39. (Fig 3).
220
221 Table 3. Genotypic identification of bacteria isolated from stranded cetaceans.
Nearest Phylogenetic NCBI* Accession Source Cetacean (Code) Swab Site Isolate Code Affiliation (% Sequence Number Similarity)
S12-A Enterobacter cloacae (99%) MH101512.1
S12-B Klebsiella aerogenes (99%) CP024883.1
S12-C Escherichia hermannii (98%) JN644551.1 S12 urine S12-D Enterobacter sp. (99%) KC236445.1
S12-E Enterobacter cloacae (100%) KY492312.1
S12-F Enterobacter cloacae (99%) JN644583.1
S16-G Enterobacter ludwigii (99%) JQ659806.1
S16-H Escherichia hermannii (99%) JN644551.1
S16-I Enterobacter cloacae (99%) KM538690.1 S16 blowhole S16-J Klebsiella pneumoniae (99%) FO203501.1
S16-K Klebsiella pneumoniae (99%) KJ803907.1
S16-L Shigella sp. (99%) KU362661.1
S17-M Klebsiella pneumoniae (99%) CP020847.1 genital Slit S17-N Escherichia coli (99%) AP017620.1
blowhole S17-O Enterobacter cloacae (99%) CP010512.1 S17 S17-P Escherichia coli (99%) JQ661149.1
Klebsiella quasipneumoniae wound S17-Q CP014696.2 (99%)
S18 anus S17-R Proteus mirabilis (99%) CP015347.1
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brainstem S17-S Proteus mirabilis (99%) CP015347.1
cerebellum S17-T Proteus mirabilis (99%) CP004022.1
lungs S18-U Escherichia fergusonii (99%) KJ803900.1
S18-V Enterobacter tabaci (99%) NR_146667.2
blowhole S18-W Proteus mirabilis (99%) CP015347.1
S18-X Escherichia coli (99%) CP027060.1 222
223 *National Center for Biotechnology Information 224 MAR Index
Isolate Code 225
226
227 Fig 3. MAR (multiple antibiotic resistance) index values of Enterobacteriaceae isolated from
228 cetaceans that straded in the Philippines from July to December 2017.
229
230 Discussion
231 Lesions in tissues of stranded cetaceans
232
233 Lesions in tissues of stranded cetaceans were associated with bycatch, trauma, parasitic and
234 bacterial infections, and presence of persistent organic pollutants in stranded cetaceans [13-
235 15,17,20,42-44]. A previous study in the Philippines involved the histopathological assessment of
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236 renal tissues which corroborated the results of molecular and culture methods for a suggested case of
237 leptospirosis in a melon-headed whale (Peponocephala electra) [45]. However, the diagnosis of
238 diseases through histopathological assessment of tissues of stranded marine mammals is often
239 complex and difficult, involving many factors. Also, lesions may be caused by the stranding event
240 itself and may mask pre-existing diseases.
241
242 When a cetacean strands, it is highly likely to have congestion in the liver and other organs
243 due to the pressure from the weight of its body lying on the thorax as well as immotility preventing
244 venous circulation [46]. The present study supports this, as congestion is the most observed type of
245 lesion in hepatic and other organ tissues. Congestion in brain and kidneys of cetaceans has also been
246 associated with acoustic trauma [47]. Acoustic trauma was suggested as the cause of some previously
247 reported cetacean stranding events in the Philippines, possibly due to blast fishing activities near the
248 stranding sites [23,48]. In addition, the stranding event itself can induce trauma and stress myopathy
249 on the animal, causing congestion, hemorrhage, and skeletal and cardiac muscle degeneration such as
250 in the case of Zenker’s necrosis [15,49-50]. These lesions were observed in 86% of tissues.
251
252 Glomerulopathy was observed in 20% of tissues with lesions. Comparably, membranous
253 glomerulonephritis was a common finding among stranded cetaceans in Brazil [17]. This lesion was
254 suggested to be associated with microbial infections or chronic exposure of cetaceans to metals such
255 as cadmium, copper, and zinc, but this remains speculative due to the lack of toxicological analyses
256 [44,51].
257
258 Parasites in cetaceans may predispose these animals to bacterial infections, cardiovascular
259 complications, septicemia and other conditions, which are also frequently reported as probable causes
260 of death during their stranding events [16,52-53]. Until this study, there was no histopathological
261 evidence of coccidian cysts such as T. gondii and Sarcocystis sp. in muscles of locally-stranded
262 cetaceans, with earlier reports on T. gondii detection using serological and molecular methods [45,54].
263 During the past two decades, coccidian infections have been detected in marine mammals that
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264 stranded along the coast of the northeastern Pacific Ocean [55-56]. These infections include
265 encephalitis, myositis, hepatitis and myocarditis [57-58]. A better understanding of the biology,
266 epidemiology, and pathogenesis of tissue-encysting coccidian organisms that parasitize marine
267 mammals is needed to properly assess the risks and burden of protozoal disease in aquatic ecosystems
268 [56-58]. In this study, cetaceans Lagenodelphis hosei (S03), Peponocephala electra (S07), Stenella
269 longirostris (S10), and Stenella attenuata (S14) were found to have T. gondii cysts in their cardiac,
270 kidney, and skeletal tissues while cetaceans Kogia breviceps (S08), and Stenella attenuata (S14) were
271 found to have Sarcocystis spp. in their skeletal muscles. The transmission of these parasites is still
272 poorly understood in marine mammals, although it is known that they are found in striated muscles of
273 intermediate hosts [59-62]. The most likely modes of transmission of these parasites to aquatic
274 animals are via ingestion of water-borne oocysts or sporocysts originating from sewage runoff or
275 through infected prey [56-57,63-65].
276
277 In addition, the presence of Phyllobothrium delphini in the muscles and blubber of two
278 sampled cetaceans was reported in the necropsy reports. This parasite has been documented in many
279 cetacean species, commonly in the subcutaneous blubber with typical concentration in the perigenital
280 region [66]. Siquier and Le Bas (2003) suggested that Fraser’s dolphins (Lagenodelhis losei) could
281 act as intermediate or accidental hosts for P. delphini, and that definitive host infection could occur
282 through predation. There is a need for more evidence to confirm the role of cetaceans in the life cycle
283 of this parasite [67].
284
285 The consumption of muscles with these parasites is one of the major routes of transmission
286 among hosts, including humans. This route of transmission is unlikely to involve cetaceans in the
287 Philippines, as hunting and killing of marine mammals are prohibited under Section 4 of Republic Act
288 9147 (Wildlife Resources Conservation and Protection Act of the Philippines). Still, there were local
289 reports of fishermen butchering cetaceans for food consumption (pers comm., BFAR Region V).
290
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291 Antibiotic resistant bacteria from stranded cetaceans
292 Overall, the bacterial isolates have resistances to carbapenems and third-generation
293 cephalosporins. Enterobacteriaceae resistant to carbapenems and third-generation cephalosporins are
294 considered a research priority for the discovery of new antibiotic agents [35]. As the “last line of
295 defense” against multiple antibiotic resistant bacteria, the detection of carbapenem-resistant strains is
296 a troubling point of concern as carbapenems are fourth- generation antibiotics recommended for
297 critical Gram-negative infections [68]. To the best knowledge of the authors, only Greig et al. (2007)
298 had so far used imipenem and meropenem for antibiotic susceptibility tests on bacteria isolated from
299 cetaceans. Greig et al. reported imipenem-resistant E. coli in bottlenose dolphins, but all of their
300 isolates were still susceptible to meropenem at the time [5].
301
302 All isolates were most resistant to erythromycin. The high frequency of resistance against this
303 antibiotic is said to be due to acquired macrolide–lincosamide–streptogramin B (MLS) resistance
304 genes, which is common among Enterobacteriaceae [69-70]. More than 50% of the isolates were also
305 resistant to cephalothin, ampicillin, and moxifloxacin. It must be noted that the isolated Klebsiella
306 spp., and Escherichia spp., bacterial species often reported as pathogenic to cetaceans, were resistant
307 to erythromycin [71-72]. Similarly, high resistance to erythromycin, cephalothin, and ampicillin of E.
308 coli isolated from bottlenose dolphins in Florida and South Carolina was reported [5-6]. Extra-
309 intestinal pathogenic E. coli isolated from resident killer whales of San Juan Islands, Washington,
310 were found to be resistant to aminoglycosides, sulfonamides, and tetracycline [73]. Resistances
311 against cephalothin and ampicillin were also observed in bacteria isolated from dolphins, whales, and
312 seals in the Northeastern United States Coast [32]. An overall high prevalence (88%) of resistance to
313 at least one antibiotic was found among bacteria isolated from wild bottlenose dolphins in Florida,
314 with highest resistances against erythromycin followed by ampicillin [6]. A previous study on
315 antibiotic susceptibility patterns of bacteria isolated from stranded cetaceans in the Philippines
316 reported the highest resistance (47%) to cefazolin [12].
317
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318 The foregoing findings suggest that the mentioned antibiotics may not be good options for
319 treating bacterial infections caused by Enterobacteriaceae in cetaceans. Based on the results of the
320 study, the use of the following antibiotics must be considered in the medical management of stranded
321 individuals (in order of decreasing potency): imipenem, doripenem, ciprofloxacin, chloramphenicol,
322 gentamicin> meropenem, amikacin, ceftriaxone, trimethoprim-sulfamethoxazole> ertapenem>
323 ofloxacin> tetracycline> cefoxitin> oxytetracycline> moxifloxacin> ampicillin, cephalothin>
324 erythromycin. Susceptibilities to amikacin and gentamicin were also reported among bacteria isolated
325 from marine mammals in Florida, South Carolina, and Northeastern US Coast [5,32].
326
327 The cetacean species sampled in this study generally inhabit deep waters, but their physiology
328 entails a regular need to go up the surface to sequester oxygen from the air for breathing, thus
329 exposing themselves to sewage outflows and other forms of pollution that eventually reach them from
330 the nearby coast [74-76]. The presence of bacteria (and associated antibiotic resistances) in these
331 cetaceans indicate biological pollution and presence of antibiotic resistance in their habitats [77-79].
332 In this study, 33.33% of the isolates from cetaceans had MAR indices greater than 0.2, suggesting that
333 the isolates may have developed resistance from sources that the cetaceans were exposed to, such as
334 bodies of water highly polluted with antibiotics, including domestic, industrial and hospital sewage
335 outflows, water-treatment facilities, and the like [75-76]. As the use of antibiotics stems from
336 anthropogenic activities, this implies the need to regulate and monitor the use and improper disposal
337 of antibiotics to water bodies.
338
339
340 Conclusion
341 Twenty-one cetaceans that stranded in different parts of the Philippines were sampled for
342 histopathological assessment and bacterial isolation and antibiotic resistance screening.
343 Histopathological findings showed congestion as the mostly observed lesion, followed by
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344 glomerulopathy, hemorrhage, and edema. In the absence of conclusive data on the specific causes of
345 the mortality or morbidity of the cetaceans in relation to the stranding event, the observed lesions
346 provide indications of possible involvement of acoustic trauma, stress, and chronic infections caused
347 by microbial infections and exposure to chemical pollutants. On the other hand, bacteriological
348 findings showed more than 50% of the isolated bacteria are multiple antibiotic resistant and that all of
349 them are resistant to erythromycin and susceptible to imipenem, doripenem, ciprofloxacin,
350 chloramphenicol, and gentamicin. While these information can serve as guide in the medical
351 management of stranded cetaceans during rehabilitation, they also indicate the extent of antimicrobial
352 resistance in the marine environment. As sentinels, cetaceans are demonstrating the threats faced by
353 their populations in the wild, and monitoring their health through stranded representatives is a
354 practical approach that can help improve conservation efforts. As local stranding network expands
355 and veterinary and research expertise improve, more robust data from bacteriological and
356 histopathological assessments of cetaceans are expected to be available in the coming years.
357
358 Acknowledgments
359 We thank the Philippine Marine Mammal Stranding Network (PMMSN) and the Bureau of
360 Fisheries and Aquatic Resources (BFAR) for the nationwide cetacean stranding response. Likewise,
361 we thank Honey Leen M. Laggui for the preparation of Fig 1.
362
363 Authors’ contributions
364 MCMO, JAAC, LSLL, EJSC, and RMDV performed the laboratory procedures. LVA led the
365 cetacean stranding response with MCMO, JAAC, and RMDV. MCMO and LVA received funding for
366 the study through project grants BIO-19-1-05 (UP-NSRI) and 171704SOS (UP-OVCRD)
367 respectively. MCMO, LVA CCS, MATS, WLR, and JSM conceptualized the project, collated and
368 analyzed all the data, and interpreted the results. All authors contributed to the writing and approved
369 the final version of the manuscript.
22 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.30.403568; this version posted November 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
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