Genomic scan of a healthcare-associated NDM-1-producing Citrobacter freundii ST18 isolated from a green sea turtle impacted by plastic pollution.

BACKGROUND: Carbapenemase-producing Citrobacter freundii has been reported as a leading cause of healthcare-associated infections. Particularly, C. freundii belonging to the sequence type (ST) 18 is considered to be an emerging nosocomial clone. OBJECTIVES: To report the genomic background and phylogenomic analysis of a multidrug-resistant NDM-1-producing C. freundii ST18 (strain CF135931) isolated from an endangered green sea turtle affected by plastic pollution in Brazil. METHODS: Genomic DNA was extracted and sequenced using the Illumina NextSeq platform. De novo assembly was performed by CLC Workbench, and in silico analysis accomplished by bioinformatics tools. For phylogenomic analysis, publicly available C. freundii (txid:546) genome assemblies were retrieved from the NCBI database. RESULTS: The genome size was calculated at 5 290 351 bp, comprising 5263 total genes, 4 rRNAs, 77 tRNAs, 11ncRNAs, and 176 pseudogenes. The strain belonged to C. freundii ST18, whereas resistome analysis predicted genes encoding resistance to ß-lactams (blaNDM-1, blaOXA-1, blaCMY-117, and blaTEM-1C), aminoglycosides (aph(3'')-Ib, aadA16, aph(3')-VI, aac(6')-Ib-cr, and aph(6)-Id), quinolones (aac(6')-Ib-cr), macrolides (mph(A) and erm(B)), sulphonamides (sul1 and sul2), tetracyclines (tetA and tetD), and trimethoprim (dfrA27). The phylogenomic analysis revealed that CF135931 strain is closely related to international human-associated ST18 clones producing NDM-1. CONCLUSION: Genomic surveillance efforts are necessary for robust monitoring of the emergence of drug-resistant strains and WHO critical priority pathogens within a One Health framework. In this regard, this draft genome and associated data can improve understanding of dissemination dynamics of nosocomial clones of carbapenemase-producing C. freundii beyond hospital walls. In fact, the emergence of NDM-1-producing C. freundii of global ST18 in wildlife deserves considerable attention.

Case report: Blindness associated with Learedius learedi trematode infection in a green sea turtle, Chelonia mydas, of the northern Red Sea.

Spirorchiid blood flukes are widespread in sea turtles, causing disease and mortality in their populations, with high prevalence in several ocean basins. Besides being leading parasitic causes of sea turtle strandings in several parts of the world, these infectious agents can cause endocarditis, vasculitis, thrombosis, miliary egg granulomas, and aneurysms, which ultimately may compromise the survival of green sea turtles. More severe cases may also result in multifocal granulomatous meningitis or pneumonia, both of which can be fatal. Herein, we report the first case of severe trematode infection, Caused by Learedius learedi, in a green sea turtle in the northern Red Sea; this infection is associated with bilateral blindness. Necropsy revealed multiple granulomas with intralesional trematode eggs in the optic nerve, eyes, spleen, heart, and lungs. The parasite was identified as Learedius learedi through specific primers of the ribosomal genome and COI sequences obtained from GenBank. Altogether, these findings emphasize the importance of recognizing the systemic nature of this particular fluke infection to ultimately protect the lives of these marine animals and ensure the sustainability of these species in the wild.

New Delhi metallo-ß-lactamase-1-producing Citrobacter portucalensis belonging to the novel ST264 causing fatal sepsis in a vulnerable migratory sea turtle.

Olive ridley (Lepidochelys olivacea) turtles migrate across tropical regions of the Atlantic, Pacific, and Indian Oceans. Worryingly, olive ridley populations have been declining substantially and is now considered a threatened species. In this regard, habitat degradation, anthropogenic pollution, and infectious diseases have been the most notorious threats for this species. We isolated a metallo-ß-lactamase (NDM-1)-producing Citrobacter portucalensis from the blood sample of an infected migratory olive ridley turtle found stranded sick in the coast of Brazil. Genomic analysis of C. portucalensis confirmed a novel sequence type (ST), named ST264, and a wide resistome to broad-spectrum antibiotics. The production of NDM-1 by the strain contributed to treatment failure and death of the animal. Phylogenomic relationship with environmental and human strains from African, European and Asian countries confirmed that critical priority clones of C. portucalensis are spreading beyond hospital settings, representing an emerging ecological threat to marine ecosystems.

Systemic oxalosis in a free-ranging green turtle (Chelonia mydas).

A female juvenile green turtle (Chelonia mydas), found alive in Guanabara Bay, Rio de Janeiro, Brazil, was weak, dehydrated and cachectic, with a healed fracture in the caudal portion of the carapace. Despite supportive treatment, the animal died after 9 days. At necropsy the main lesions were pallor of visceral organs, arthritis and deposits of whitish granular material in the wall of large arteries and the trachea. Histopathological analysis revealed mild to severe deposition of crystals, consistent with calcium oxalate, in both kidneys and the spleen, heart, small intestine, pancreas, thymus and salt gland, as well as bacterial meningitis, septic arthritis, spirorchidiasis and a fibropapilloma on the nictitating membrane. The main pathological findings were suggestive of septic shock, mainly due to the bacterial meningitis and septic arthritis, with systemic oxalosis and spirorchidiasis as contributing lesions. Although renal oxalosis has been described in green turtles as an incidental finding, presumably due to ingestion of oxalate-containing plants, this turtle had an unusual systemic deposition of oxalate crystals.

Genomic Analysis of a Highly Virulent NDM-1-Producing Escherichia coli ST162 Infecting a Pygmy Sperm Whale (Kogia breviceps) in South America.

Carbapenemase-producing Enterobacterales are rapidly spreading and adapting to different environments beyond hospital settings. During COVID-19 lockdown, a carbapenem-resistant NDM-1-positive Escherichia coli isolate (BA01 strain) was recovered from a pygmy sperm whale (Kogia breviceps), which was found stranded on the southern coast of Brazil. BA01 strain belonged to the global sequence type (ST) 162 and carried the bla NDM-1, besides other medically important antimicrobial resistance genes. Additionally, genes associated with resistance to heavy metals, biocides, and glyphosate were also detected. Halophilic behavior (tolerance to > 10% NaCl) of BA01 strain was confirmed by tolerance tests of NaCl minimal inhibitory concentration, whereas halotolerance associated genes katE and nhaA, which encodes for catalase and Na+/H+ antiporter cytoplasmic membrane, respectively, were in silico confirmed. Phylogenomics clustered BA01 with poultry- and human-associated ST162 lineages circulating in European and Asian countries. Important virulence genes, including the astA (a gene encoding an enterotoxin associated with human and animal infections) were detected, whereas in vivo experiments using the Galleria mellonella infection model confirmed the virulent behavior of the BA01 strain. WHO critical priority carbapenemase-producing pathogens in coastal water are an emerging threat that deserves the urgent need to assess the role of the aquatic environment in its global epidemiology.

Phylogeographical Landscape of Citrobacter portucalensis Carrying Clinically Relevant Resistomes.

During a surveillance study conducted to assess the occurrence and genomic landscape of critical priority pathogens circulating at the human-animal-environment interface in Brazil, as part of the Grand Challenges Explorations-New Approaches to Characterize the Global Burden of Antimicrobial Resistance program, two multidrug-resistant (MDR) Citrobacter portucalensis carrying blaCTX-M-15 extended-spectrum ß-lactamase (ESBL) genes, isolated from green sea turtles, were characterized. Genomic and phylogeographical analysis of C. portucalensis genomes available in public databases revealed the intercontinental dissemination of clades carrying different arrays of clinically relevant genes conferring resistance to carbapenems, broad-spectrum cephalosporins, cephamycins, aminoglycosides and fluoroquinolones, disinfectants, and heavy metals. Our observations suggest that C. portucalensis could be emerging as critical priority bacteria of both public and One Health importance worldwide. IMPORTANCE The global spread of antibiotic-resistant priority pathogens beyond the hospital setting is a critical issue within a One Health context that integrates the human-animal-environment interfaces. On the other hand, next-generation sequencing technologies along with user-friendly and high-quality bioinformatics tools have improved the identification of bacterial species, and bacterial resistance surveillance. The novel Citrobacter portucalensis species was proposed in 2017 after taxonomic reclassification and definition of the strain A60T isolated in 2008. Here, we presented genomic data showing the occurrence of multidrug-resistant C. portucalensis isolates carrying blaCTX-M-15 ESBL genes in South America. Additionally, we observed the intercontinental dissemination of clades harboring a broad resistome to clinically relevant antibiotics. Therefore, these findings highlight that C. portucalensis is a global MDR bacteria that carries intrinsic blaCMY- and qnrB-type genes and has become a critical priority pathogen due to the acquisition of clinically relevant resistance determinants, such as ESBL and carbapenemase-encoding genes.

Colistin-resistant Enterobacter kobei carrying mcr-9.1 and blaCTX-M-15 infecting a critically endangered franciscana dolphin (Pontoporia blainvillei), Brazil.

The emergence of mobile mcr genes mediating resistance to colistin is a critical public health issue that has hindered the treatment of serious infections caused by multidrug-resistant pathogens in humans and other animals. We report the emergence of the mcr-9.1 gene in a polymyxin-resistant extended-spectrum ß-lactamase (ESBL)-producing Enterobacter kobei infecting a free-living franciscana dolphin (Pontoporia blainvillei), threatened with extinction in South America. Genomic analysis confirmed the presence of genes conferring resistance to clinically relevant ß-lactam [blaCTX-M-15 , blaACT-9 , blaOXA-1 and blaTEM-1B ], aminoglycoside [aac(3)-IIa, aadA1, aph(3'')-Ib and aph(6)-Id], trimethoprim [dfrA14], tetracycline [tetA], quinolone [aac(6')-Ib-cr and qnrB1], fosfomycin [fosA], sulphonamide [sul2] and phenicol [catA1 and catB3] antibiotics. The identification of mcr-9.1 in a CTX-M-15-producing pathogen infecting a critically endangered animal is of serious concern, which should be interpreted as a sign of further spread of critical priority pathogens and their resistance genes in threatened ecosystems.

Comparative Cytogenetics of Four Sea Turtle Species (Cheloniidae): G-Banding Pattern and in situ Localization of Repetitive DNA Units.

Sea turtles are considered flagship species for marine biodiversity conservation and are considered to be at varying risk of extinction globally. Cases of hybridism have been reported in sea turtles, but chromosomal analyses are limited to classical karyotype descriptions and a few molecular cytogenetic studies. In order to compare karyotypes and understand evolutive mechanisms related to chromosome dif-ferentiation in this group, Chelonia mydas, Caretta caretta, Eretmochelys imbricata, and Lepidochelys olivacea were cytogenetically characterized in the present study. When the obtained cytogenetic data were compared with the putative ancestral Cryptodira karyotype, the studied species showed the same diploid number (2n) of 56 chromosomes, with some variations in chromosomal morphology (karyotypic formula) and minor changes in longitudinal band locations. In situ localization using a 18S ribosomal DNA probe indicated a homeologous microchromosome pair bearing a 45S ribosomal DNA locus and size heteromorphism in all 4 species. Interstitial telomeric sites were identified in a microchromosome pair in C. mydas and C. caretta. The data showed that interspecific variations occurred in chromosomal sets among the Cheloniidae species, in addition to other Cryptodira karyotypes. These variations generated lineage-specific karyotypic diversification in sea turtles, which will have considerable implications for hybrid recognition and for the study, the biology, ecology, and evolutionary history of regional and global populations. Furthermore, we demonstrated that some chromosome rearrangements occurred in sea turtle species, which is in conflict with the hypothesis of conserved karyotypes in this group.

Chelonid Alphaherpesvirus 5 DNA in Fibropapillomatosis-Affected Chelonia mydas.

Fibropapillomatosis is a panzootic and chronic disease among Chelonia mydas-usually associated with anthropogenic impacts. This study contributes towards understanding fibropapillomatosis implications for C. mydas populations as a reflector of environmental quality, via prevalence and histological, molecular and blood analyses at a World Heritage site in southern Brazil. Sixty-three juvenile C. mydas (31.3-54.5 cm curved carapace length-CCL) were sampled during two years. Eighteen specimens (~ 29%) had tumours (which were biopsied), while 45 had none. Degenerative changes in the epidermis and Chelonid alphaherpesvirus 5 DNA detection with three variants support a herpesvirus infection. Phylogenetic analysis indicated that variants A and B were similar to a herpesvirus lineage from the Atlantic group, but variant C was similar to a herpesvirus from the eastern Pacific lineage and represents the first published case for marine turtles off Brazil. Significantly lower levels of seven blood parameters, but greater numbers of eosinophils, were observed in tumour-afflicted animals. These observations were attributed to metabolism efficiencies and/or differences in diet associated with temporal-recruitment bias and disease development, and greater non-specific immune stimulation. While most animals had adequate body condition independent of disease, longer-term studies are required to elucidate any protracted population effects.

Genetic background of CTX-M-15-producing Enterobacter hormaechei ST114 and Citrobacter freundii ST265 co-infecting a free-living green turtle (Chelonia mydas).

CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae have become identified in marine ecosystem constituting a serious ecological issue. In this respect, although contamination of coastal waters and seafood, and even colonization of seabirds and fishes have been increasingly reported, molecular data are lacking to elucidate the clinical impact of ESBL producers in infected marine animals. In this study, using a genomic approach, we have analysed the genetic background of CTX-M-15-producing Enterobacter hormaechei (belonging to the international human clone ST114) and Citrobacter freundii (ST265) co-infecting a free-living green turtle (Chelonia mydas) suffering from septic arthritis, which progressed to generalized coelomitis and death. Wide resistome of these pathogens contributed to treatment failure and death of the animal.

Parasitic Ulcerous Caseous Gastroesophagitis Associated with Rameshwarotrema uterocrescens Rao, 1975 (Digenea: Pronocephalidae) in a Juvenile Green Turtle ( Chelonia mydas , Linnaeus 1758 [Testudines: Cheloniidae]): A Case Report.

Here we report a case of ulcerative caseous gastroesophagitis associated with Rameshwarotrema uterocrescens, Rao, 1975 (Digenea: Pronocephalidae), in a juvenile green turtle ( Chelonia mydas ) from southern Brazil. Similar pathologies have been reported only in adult green turtles from Costa Rica. This paper presents the second report of parasitic esophagitis due to R. uterocrescens and the first occurrence in juvenile green turtles along coastal Brazil.

Trace elements distribution in hawksbill turtle (Eretmochelys imbricata) and green turtle (Chelonia mydas) tissues on the northern coast of Bahia, Brazil.

Concentrations of elements (As, Al, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Sb, Se, Sr, V, Zn) were determined in liver, kidneys and bones of Eretmochelys imbricata and Chelonia mydas specimens found stranded along the northern coast of Bahia, Brazil. Results showed that the concentrations of Cd, Cu, Ni and Zn in the liver and kidneys of juvenile C. mydas were the highest found in Brazil. We also observed a significant difference (p<0.05) on the bioaccumulation of trace elements between the two species: Al, Co, Mo, Na and Se in the liver; Al, Cr, Cu, K, Mo, Ni, Pb, Sr and V in the kidneys; and Al, Ba, Ca, Cd, Mn, Ni, Pb, Se, Sr and V in the bones. This study represents the first report on the distribution and concentration of trace elements in E. imbricata in the Brazilian coast.

Avaliação da resposta inflamatória hematológica em cascavéis (Crotalus durissus Linnaeus, 1758) inoculadas com BCG / Assessment of blood inflammatory response in BCG stimulated rattlesnakes (Crotalus durissus Linnaeus, 1758)

A criação de serpentes peçonhentas em cativeiro para produção de soros antipeçonhas possui crescente importância para a saúde pública devido ao aumento do número de notificações de acidentes ofídicos a cada ano no Brasil. Iniciado no século XX, ainda hoje essa atividade apresenta alguns desafios como a instalação de doenças no plantel. O hemograma é um exame de triagem clínica que auxilia no diagnóstico de diversas moléstias que acometem diferentes espécies de animais, no entanto ainda pouco estudado em serpentes. A caracterização das alterações hematológicas em cascavéis inoculadas experimentalmente com BCG pode servir de base na utilização deste exame no auxílio ao diagnóstico de infecções bacterianas na espécie. Dessa forma, foram realizados exames hematológicos em 10 serpentes da espécie Crotalus durissus pertencentes ao plantel da Divisão de Herpetologia do Instituto Vital Brazil. Os animais foram divididos em dois grupos (Grupos 1 e 2), homogêneos entre si em relação ao peso e proporção sexual. Os dois grupos foram inoculados com BCG e submetidos à coleta de sangue antes da inoculação e em três momentos pós-inoculação (3º, 5º, e 7º dias para o Grupo 1 e 11º, 17º e 21º dias para o Grupo 2). O hemograma foi realizado por método semidireto pela utilização de líquido de Natt e Herrick e as lâminas foram coradas pelo Giemsa. Observou-se anemia discreta, com redução dos valores de concentração de hemoglobina corpuscular média e da hemoglobina globular média no Grupo 1 que foi relacionada à doença inflamatória. A trombocitopenia observada no Grupo 2 sugeriu a atuação deste tipo celular em processos inflamatórios. Um único animal do Grupo 1 apresentou granulocitose e alguns animais apresentaram discreta azurofilia. Observaram-se alterações morfológicas nos leucócitos. Os granulócitos apresentaram granulações grosseiras e os azurófilos apresentaram aumento de tamanho e grandes vacúolos. De forma geral, a inoculação de BCG em cascavéis desencadeia ...

The high demand for anti-venom production in response to the increased number of cases of snakebite envenomation highlights the importance of raising and breeding venomous snakes in captivity. Knowledge of types of venoms and anti-venoms is of great interest to public health. The maintenance of venomous serpents in captivity started in the early twentieth century, but still nowadays it is a challenge to manage and prevent diseases in captive fauna. Hematology is commonly used for general health assessment and illness detection. However, data on serpent blood analysis are still scarce. Alterations in hematological parameters were experimentally induced in rattlesnakes by the inoculation of BCG. Based on this, hemograms can be used as a health auxiliary diagnosis method for bacterial diseases in this species. In this study, blood samples were taken from 10 healthy specimens of rattlesnakes (Crotalus durissus) born and bred in captivity in the Herpetological Division of Vital Brazil Institute. Animals were divided into two groups (group 1 and 2) with similar live weight and sex proportion, and were then inoculated subcutaneously with BCG (Bacillus Calmette-Guérin). Blood samples were taken before and after inoculation at three experimental times (days 3, 5 and 7 for group 1 and days 11, 17 and 21 for group 2). Hematological analysis was performed through semi-direct technique, blood samples were diluted in Natt and Herrick solution and smears were stained by Giemsa. Serpents from group 1 developed discrete anemia due to the inflammatory syndrome, and showed significant decrease of MCH and MCHC. Granulocytes were characterized by the presence of rough granules. The azurophils varied in shape and size and showed large amount of cytoplasmic vacuoles. The thrombocytopenia observed in group 2 suggests that these cells participate in the inflammatory process. A single individual from group 1 showed granulocytosis and a few animals showed a discrete ...