Whole-genome analysis of extraintestinal pathogenic Escherichia coli (ExPEC) MDR ST73 and ST127 isolated from endangered southern resident killer whales (Orcinus orca).

BACKGROUND: Limited studies have investigated the microbial diversity of wild marine mammals. OBJECTIVES: This study characterized Escherichia coli isolates collected from fresh faecal samples of endangered southern resident killer whales (Orcinus orca) located by detection dogs. METHODS: WGS of each strain was done to determine ST (using MLST), clonotype (C:H), antimicrobial resistance and virulence profile. Conjugation experiments were done to determine the mobility of the tet(B) tetracycline resistance gene. RESULTS: All isolates belonged to extraintestinal pathogenic E. coli (ExPEC) clonal lineages ST73 (8/9) and ST127 (1/9), often associated with human community-acquired urinary tract disease. Clonotyping using fumC and fimH alleles showed divergence in clonal lineages, with ST73 isolates belonging to the C24:H10 clade and the ST127 isolate belonging to C14:H2. The eight ST73 isolates carried multiple acquired antibiotic resistance genes, including aadA1, sul1 and tet(B), encoding aminoglycoside, sulphonamide and tetracycline resistance, respectively. Conjugative transfer of the resistance gene tet(B) was observed for three of the eight isolates. ST127 did not carry any of these acquired resistance genes. Virulence-associated genes identified included those encoding adhesins (iha, papC, sfaS), toxins (sat, vat, pic, hlyA, cnf1), siderophores (iutA, fyuA, iroN, ireA), serum survival/protectins (iss, ompT), capsule (kpsM) and pathogenicity island marker (malX). CONCLUSIONS: Orca whales can carry antibiotic-resistant potentially pathogenic strains of E. coli. Possible sources include contamination of the whale's environment and/or food. It is unknown whether these isolates cause disease in southern resident killer whales, which could contribute to the ongoing decline of this critically endangered population.

Host-derived population genomics data provides insights into bacterial and diatom composition of the killer whale skin.

Recent exploration into the interactions and relationship between hosts and their microbiota has revealed a connection between many aspects of the host's biology, health and associated micro-organisms. Whereas amplicon sequencing has traditionally been used to characterize the microbiome, the increasing number of published population genomics data sets offers an underexploited opportunity to study microbial profiles from the host shotgun sequencing data. Here, we use sequence data originally generated from killer whale Orcinus orca skin biopsies for population genomics, to characterize the skin microbiome and investigate how host social and geographical factors influence the microbial community composition. Having identified 845 microbial taxa from 2.4 million reads that did not map to the killer whale reference genome, we found that both ecotypic and geographical factors influence community composition of killer whale skin microbiomes. Furthermore, we uncovered key taxa that drive the microbiome community composition and showed that they are embedded in unique networks, one of which is tentatively linked to diatom presence and poor skin condition. Community composition differed between Antarctic killer whales with and without diatom coverage, suggesting that the previously reported episodic migrations of Antarctic killer whales to warmer waters associated with skin turnover may control the effects of potentially pathogenic bacteria such as Tenacibaculum dicentrarchi. Our work demonstrates the feasibility of microbiome studies from host shotgun sequencing data and highlights the importance of metagenomics in understanding the relationship between host and microbial ecology.

Captive bottlenose dolphins and killer whales harbor a species-specific skin microbiota that varies among individuals.

Marine animals surfaces host diverse microbial communities, which play major roles for host's health. Most inventories of marine animal surface microbiota have focused on corals and fishes, while cetaceans remain overlooked. The few studies focused on wild cetaceans, making difficult to distinguish intrinsic inter- and/or intraspecific variability in skin microbiota from environmental effects. We used high-throughput sequencing to assess the skin microbiota from 4 body zones of 8 bottlenose dolphins (Tursiops truncatus) and killer whales (Orcinus orca), housed in captivity (Marineland park, France). Overall, cetacean skin microbiota is more diverse than planktonic communities and is dominated by different phylogenetic lineages and functions. In addition, the two cetacean species host different skin microbiotas. Within each species, variability was higher between individuals than between body parts, suggesting a high individuality of cetacean skin microbiota. Overall, the skin microbiota of the assessed cetaceans related more to the humpback whale and fishes' than to microbiotas of terrestrial mammals.

Respiratory Microbiome of Endangered Southern Resident Killer Whales and Microbiota of Surrounding Sea Surface Microlayer in the Eastern North Pacific.

In the Salish Sea, the endangered Southern Resident Killer Whale (SRKW) is a high trophic indicator of ecosystem health. Three major threats have been identified for this population: reduced prey availability, anthropogenic contaminants, and marine vessel disturbances. These perturbations can culminate in significant morbidity and mortality, usually associated with secondary infections that have a predilection to the respiratory system. To characterize the composition of the respiratory microbiota and identify recognized pathogens of SRKW, exhaled breath samples were collected between 2006-2009 and analyzed for bacteria, fungi and viruses using (1) culture-dependent, targeted PCR-based methodologies and (2) taxonomically broad, non-culture dependent PCR-based methodologies. Results were compared with sea surface microlayer (SML) samples to characterize the respective microbial constituents. An array of bacteria and fungi in breath and SML samples were identified, as well as microorganisms that exhibited resistance to multiple antimicrobial agents. The SML microbes and respiratory microbiota carry a pathogenic risk which we propose as an additional, fourth putative stressor (pathogens), which may adversely impact the endangered SRKW population.

Disseminated mycosis in a killer whale (Orcinus orca).

Hematological findings in a female killer whale (Orcinus orca) undergoing rehabilitation after sudden severe anorexia revealed continuing increases in serum lactate dehydrogenase and aspartate aminotransferase activities as well as fibrinogen concentration. Serologic evidence of herpesvirus infection and skin vesicles were detected 2 weeks into the treatment regimen of antibiotics and corticosteroids. The whale showed signs of improvement after treatment with anti-herpesvirus drugs, but sudden severe anorexia reappeared, along with marked elevation of fibrinogen concentration that continued until the death. Postmortem examination revealed multiple light tan foci of necrosis in the skeletal and cardiac muscles, and lung consolidation. Microscopic findings indicated disseminated fungal granulomas in the skeletal and cardiac muscles, as well as myocarditis, mycotic embolic thromboarteritis of cardiac blood vessels, and bronchopneumonia with numerous typical Aspergillus-like fungi. Mucor-like structures in granulomas in the heart and skeletal muscle and Aspergillus-like fungi in the lungs were identified using periodic acid-Schiff, Gomori methenamine silver stain, and immunohistochemistry. The present case involves dual infection with Mucor and Aspergillus species in a killer whale with concurrent herpesvirus.

Salmonella Newport omphaloarteritis in a stranded killer whale (Orcinus orca) neonate.

Salmonella enterica serovar Newport (Salmonella Newport) was isolated from multiple tissues in a neonate killer whale (Orcinus orca) that stranded dead in 2005 along the central coast of California, USA. Necrotizing omphaloarteritis and omphalophlebitis was observed on histologic examination suggesting umbilical infection was the route of entry. Genetic analysis of skin samples indicated that the neonate had an offshore haplotype. Salmonellosis has rarely been identified in free-ranging marine mammals and the significance of Salmonella Newport infection to the health of free-ranging killer whales is currently unknown.

Properties of corynephage attachment site and molecular epidemiology of Corynebacterium ulcerans isolated from humans and animals in Japan.

Sporadic reports of Corynebacterium ulcerans infection in humans and animals have become increasingly common throughout the world. Between 2001 and 2006, five human cases, in addition to isolation of the bacterium from the carcasses of Orcinus orca and Panthera leo, were reported in Japan. While an isolate from P. leo generated only phospholipase D (PLD), the other isolates produced both PLD and diphtheria-like toxin (DLT). Pulsed-field gel electrophoresis analysis showed that isolates from P. leo and humans were genetically homologous. Southern blotting found that a human isolate was lysogenized by two corynephages coding DLT. Sequence analysis of the region of the DLT gene revealed that the integration in C. ulcerans occurred in the same manner as that in C. diphtheriae.

Investigation for presence of Neospora caninum, Toxoplasma gondii and Brucella-species infection in killer whales (Orcinus orca) mass-stranded on the coast of Shiretoko, Hokkaido, Japan.

Twelve killer whale (Orcinus orca) were hemmed in by ice floes, and nine died on the Aidomari coast in the Nemuro Strait in Rausu, Shiretoko, Hokkaido, Japan on 8 February 2005. Tissue samples collected from 8 whales were tested for Neospora caninum, Toxoplasma gondii, and Brucella species DNA by polymerase chain reaction (PCR) assay. Gamma-globulin isolated from blood samples by ammonium sulfate precipitation was tested for antibodies to these pathogens by means of agglutination tests and immunoblotting. None of the 8 tissue samples had antibodies to the pathogens, when subjected to agglutination tests. In immunoblotting, one sample (sample No.5) showed antibody binding to N. caninum antigens. In the PCR assay, none of the samples was positive. Further study is necessary to examine the prevalence of the pathogens in marine mammals inhabiting this area.