Genomic features and comparative analysis of a multidrug-resistant Acinetobacter bereziniae strain infecting an animal: a novel emerging one health pathogen?

Acinetobacter bereziniae has recently gained medical notoriety due to its emergence as a multidrug resistance and healthcare-associated pathogen. In this study, we report the whole-genome characterization of an A. bereziniae strain (A321) recovered from an infected semiaquatic turtle, as well as a comparative analysis of A. bereziniae strains circulating at the human-animal-environment interface. Strain A321 displayed a multidrug resistance profile to medically important antimicrobials, which was supported by a wide resistome. The novel Tn5393m transposon and a qnrB19-bearing ColE1-like plasmid were identified in A321 strain. Novel OXA-229-like ß-lactamases were detected and expression of OXA-931 demonstrated a 2-64-fold increase in the minimum inhibitory concentration for ß-lactam agents. Comparative genomic analysis revealed that most A. bereziniae strains did not carry any antimicrobial resistance genes (ARGs); however, some strains from China, Brazil, and India harbored six or more ARGs. Furthermore, A. bereziniae strains harbored conserved virulence genes. These results add valuable information regarding the spread of ARGs and mobile genetic elements that could be shared not only between A. bereziniae but also by other bacteria of clinical interest. This study also demonstrates that A. bereziniae can spill over from anthropogenic sources into natural environments and subsequently be transmitted to non-human hosts, making this a potential One Health bacteria that require close surveillance.

Gram-negative bacterial diversity and evidence of international clones of multidrug-resistant strains in zoo animals.

Enterobacterales and Pseudomonas aeruginosa have been colonizing or infecting wild hosts and antimicrobial-resistant strains are present in mammals and birds. Furthermore, international high-risk clones of multidrug-resistant Escherichia coli are identified and the implications of multidrug-resistant Gram-negative bacteria in zoo animals are discussed.

Hypermucoviscous/hypervirulent and extensively drug-resistant QnrB2-, QnrS1-, and CTX-M-3-coproducing Klebsiella pneumoniae ST2121 isolated from an infected elephant (Loxodonta africana).

The rapid dissemination of extended-spectrum ß-lactamases (ESBLs)-producing Enterobacterales from different spheres worldwide over recent years has become a serious problem in both human and veterinary medicine. CTX-M-3-type ESBL has only been reported on few occasions, and in Brazil the blaCTX-M-3 gene has been identified only once in clinical strains. In this study, we aimed to molecularly characterize a hypermucoviscous (hm), hypervirulent (hv), and extensively drug-resistant (XDR) Klebsiella pneumoniae strain isolated from a lung tissue culture of an infected elephant. The A246 strain belonged to ST2121 and presented hm phenotype, hypervirulence-associated genes, and carried blaCTX-M-3 and plasmid-mediated quinolone resistance genes (qnrB2 and qnrS1) on an IncFII-IncQ1-IncM1 multireplicon plasmid (pA246-CTX-M-3, ∼ 162 kb). A novel genetic context of blaCTX-M-3, in which a 482-bp ISEcp1 was truncated by an IS26, was also harbored by pA246-CTX-M-3. Furthermore, in vivo experiments revealed that the hm/hv A246 strain killed 100 % of the Galleria mellonella larvae at 72 h post-infection. Our findings evidence the intercontinental dissemination of a rare K. pneumoniae ST2121 and the multidrug resistance IncFII-IncQ1-IncM1 plasmid. Therefore, to the best of our knowledge, this is the first report of an XDR K. pneumoniae coproducing CTX-M-3, QnrB2, and QnrS1 isolated from captive wild animals.

Comparative analyses of coproscopical techniques to diagnose enteroparasites in a group of captive Indian peafowl (Pavo cristatus).

Captive animals commonly have infections by direct life cycle parasites, since they are easily transmitted between individuals. However, diagnosing these infections in the laboratory is challenging due to the wide variety of parasite, their life stages and to the variety of available diagnose techniques, being difficult to choose the best one. The present study sampled a group of captive Indian peafowl (Pavo cristatus) from São Paulo Zoological Park Foundation, São Paulo, Brazil, to test and compare different coproscopical techniques commonly applied in veterinarian clinical analysis laboratories: direct smear, concentrations by sodium chlorite, sucrose, zinc sulphate, faecal sedimentation and formalin-ether followed by modified Ziehl-Neelsen staining. Sensitivity, specificity, predictive values (positive and negative) and Cohen's kappa index were calculated. In total 108 samples were processed and parasites found were: nonsporulated coccidian oocysts (91.7%), Capillarinae eggs (89.8%), unidentified nematode larvae (75%), Ascarididae eggs (63%), unidentified nematode adults (60.2%), unidentified nematode eggs (42.6%), strongylid-like eggs (42.6%), Cryptosporidium spp. (28.7%), flagellated (15.7%) and ciliated (10.2%) protozoans, trematode eggs (0.9%), Acanthocephala eggs (0.9%), Adeleidae oocysts (0.9%) and Cruzia sp. eggs (0.9%). Sensitivity and specificity varied considerably between parasite groups. Cohen's Kappa index reinforces the recommendation of applying more than one technique to diagnose enteroparasites infections.

Comparative analysis of multidrug resistance plasmids and genetic background of CTX-M-producing Escherichia coli recovered from captive wild animals.

Multiple interlinked factors are associated with the global resistome, whereas multidrug-resistant (MDR) pathogens have been related to increased mortality rates in humans and animals. CTX-M-type is the most prevalent extended-spectrum ß-lactamase (ESBL) among Enterobacteriaceae, which raises concern worldwide. Zoological gardens have a high density of animals that live very close to each other and to humans. Therefore, this study aimed to investigate through the whole-genome sequencing (WGS) MDR Escherichia coli lineages obtained from captivity wild animals in a zoo. Genetic background showed a wide resistome for antimicrobials (e.g., blaCTX-M-65, blaCTX-M-8, blaCMY-2, qnrB19), metals (e.g., pcoABCDERS, silABCEP, merACDEPRT), and antibacterial biocides (e.g., sugE, mdfA) among MDR CTX-M-producing E. coli belonging to CC155 and CC156. Mobilome analysis revealed several plasmids, and eight of them were completely characterized, which showed different backbone-encoding genes. Comparative analysis of plasmids blaCTX-M-65/IncHI2-ST3, blaCTX-M-8/IncI1-ST113, and IncQ1 showed a high identity among plasmids obtained from humans and animals worldwide distributed. Besides, several virulence genes, CRISPR, and prophage-related sequences were also detected. The occurrence of MDR E. coli belonging to CCs closely related to humans and food-producing animals and the high similarity among the plasmids from MDR E. coli carrying clinically significant antimicrobial resistance genes may indicate intercontinental dissemination of these lineages and plasmids. Therefore, these findings contribute to the monitoring of antimicrobial resistance and the human-animal-environment interface worldwide. Key Points • Wide resistome for antimicrobials, metals, and antibacterial biocides. • Multidrug resistance plasmids (blaCTX-M-65/IncHI2-ST3, blaCTX-M-8/IncI1-ST113). • Co-occurrence of plasmid-mediated resistance and virulence genes.

Giardia spp., ten years of parasitological data in the biggest zoo of Latin America

Giardia spp. (Diplomonadida: Hexamitidae) is an important and widely studied protozoan parasite with worldwide distribution. Nowadays have six described species, and the most important probably is Giardia duodenalis due to the zoonotical potential that some assemblages have. Many studies analysing samples from wild animals have detected Giardia in captive environment, including the zoonotic type. The aim of this study was to determine the prevalence of Giardia sp. in wild captive animals at São Paulo Zoo, using conventional parasitological techniques (direct smear, passive flotation with saturated sodium chloride solution and simple gravity sedimentation), from 2006 to 2016. In total, 7066 coprological exams were performed during this period with samples from mammals, birds, reptiles and amphibians. The prevalence of Giardia infections was of 1.5% (103/7066). Mammals had the higher prevalence of infections with 2% (77/3872), followed by birds with 1.1% (25/2186) and reptiles with only one positive sample (1/894). All samples from amphibians were negative. Representatives of thirteen families presented positive results for this parasite: Dromaidae, Phasianidae, Ramphastidae, Cervidae, Giraffidae, Canidae, Felidae, Herpestidae, Myrmecophagidae, Callithrichidae, Cebidae, Hylobatidae and Dipsadidae. This study presents the first report of Giardia sp. in Pavo muticus and Brachyteles arachnoides. Infections were prevalent in Cebidae and Ramphastidae species. The findings of this study highlight the importance of identifying which Giardia assemblage are involved in the infections and if they may have a zoonotic potential.

Ticks infesting captive and free-roaming wild animal species at the São Paulo Zoo, São Paulo, Brazil / Carrapatos infestando espécies de animais silvestres cativas e de vida livre no Zoológico de São Paulo, São Paulo, Brasil

Abstract Ticks are ectoparasites of worldwide distribution that affect vertebrates and can transmit pathogens to animals and humans. The Zoological Park Foundation of São Paulo (FPZSP) is located in a Conservation Unit in one of the most important remaining fragments of the Atlantic Rainforest biome in the suburbs of São Paulo, Brazil. The FPZSP houses more than 3,000 wild animals on exhibit, in breeding programs and in environmental education programs, and also attracts migratory birds and free-roaming wildlife. This study focused on identifying the diversity of tick species that infest captive and free-roaming animals at the FPZSP. The collection of ticks kept at the FPZSP contains 523 specimens that were collected from different host species between 1990 and 2017. Ten tick species were found. In addition, Amblyomma aureolatum (Pallas) was found on stray cats living in the Atlantic forest fragment in the FPZSP. This study reveals a low occurrence of parasitism in captive animals and a high diversity of tick species collected from hosts in this Atlantic forest fragment, contributing information about host-parasite relationships and potential vectors of zoonotic diseases, since the vectors of Brazilian spotted fever, A. aureolatum and Amblyomma sculptum Berlese, were found in some hosts.

Resumo Os carrapatos são ectoparasitas da classe Arachnida, conhecidos como importantes vetores de agentes infecciosos para animais e humanos. A Fundação Parque Zoológico de São Paulo (FPZSP) localizada em uma Unidade de Conservação do bioma Mata Atlântica e inserida na cidade de São Paulo, mantém mais de 3.000 animais selvagens cativos, abrigando ainda diversas espécies silvestres nativas da Mata Atlântica. Devido à importância do conhecimento dos carrapatos que parasitam a fauna selvagem, o objetivo deste trabalho foi identificar a coleção de carrapatos da FPZSP e analisar a diversidade desses parasitas em animais cativos e de vida livre. O acervo de carrapatos mantidos pela FPZSP contém 523 exemplares que foram coletados de diferentes espécies hospedeiras, desde 1990 até 2017. Dez espécies de carrapatos foram encontradas. Além dos animais silvestres, foram encontrados Amblyomma aureolatum (Pallas) em gatos domésticos errantes que circulam nas áreas da FPZSP. Este trabalho demonstra a baixa ocorrência de parasitismo em animais cativos e a alta diversidade de carrapatos nos hospedeiros deste fragmento de Mata Atlântica, contribuindo com informações sobre a relação parasita-hospedeiro e potenciais vetores de doenças zoonóticas, já que os vetores da Febre Maculosa Brasileira A. aureolatum e Amblyomma sculptum Berlese, foram encontrados em alguns hospedeiros.

Ticks infesting captive and free-roaming wild animal species at the São Paulo Zoo, São Paulo, Brazil.

Ticks are ectoparasites of worldwide distribution that affect vertebrates and can transmit pathogens to animals and humans. The Zoological Park Foundation of São Paulo (FPZSP) is located in a Conservation Unit in one of the most important remaining fragments of the Atlantic Rainforest biome in the suburbs of São Paulo, Brazil. The FPZSP houses more than 3,000 wild animals on exhibit, in breeding programs and in environmental education programs, and also attracts migratory birds and free-roaming wildlife. This study focused on identifying the diversity of tick species that infest captive and free-roaming animals at the FPZSP. The collection of ticks kept at the FPZSP contains 523 specimens that were collected from different host species between 1990 and 2017. Ten tick species were found. In addition, Amblyomma aureolatum (Pallas) was found on stray cats living in the Atlantic forest fragment in the FPZSP. This study reveals a low occurrence of parasitism in captive animals and a high diversity of tick species collected from hosts in this Atlantic forest fragment, contributing information about host-parasite relationships and potential vectors of zoonotic diseases, since the vectors of Brazilian spotted fever, A. aureolatum and Amblyomma sculptum Berlese, were found in some hosts.

Parasitological surveillance in a rat (Rattus norvegicus) colony in São Paulo Zoo animal house

Rattus norvegicus (Mammalia: Rodentia) is a widespread and synanthropic rodent, broadly used in medical experiments. It can also be used for feeding captive animals in zoos. Parasitological surveys are important to guarantee the health of both the animals and the staff responsible for their management. The aim of this study was to identify intestinal parasites of Rattus norvegicus offered as food to captive animals from São Paulo Zoo, and demonstrate the importance of sanitary hurdling, disease control and biosecurity. The identified protozoan parasites were Eimeria sp., Entamoeba sp., Spironucleus sp., Giardia sp., Tritrichomonas sp., Chilomastix sp., unidentified cysts and non-sporulated coccidians oocysts (Isospora/Eimeria). The following helminths were found: Syphacia muris, Rodentolepis nana and Aspiculuris tetraptera.

Plasmodium (Novyella) nucleophilum from an Egyptian Goose in São Paulo Zoo, Brazil: microscopic confirmation and molecular characterization.

Plasmodium (Novyella) nucleophilum was identified using microscopy and PCR, in an Egyptian Goose (Alopochen aegyptiacus) that died in São Paulo Zoo, Brazil. This parasite is characterized by elongated gametocytes, small meronts with scant cytoplasm, less than eight merozoites and mainly for having all the stages appressed to the nuclei of infected erythrocytes. Additionally, Plasmodium (Haemamoeba) sp. was identified by microscopy in the same blood sample. The latter parasite lacks nucleophilic blood stages and is characterized by large roundish trophozoites, each with a large prominent centrally collated vacuole. This co-infection was not confirmed by PCR amplification of the mitochondrial cytochrome b (cytb) gene and sequencing; only one Plasmodium sp. cytb sequence was detected in the blood sample. Since parasitemia of P. nucleophilum (2.4%) was much higher than that of P. (Haemamoeba) sp. (0.2%), PCR may have favored the amplification of the cytb sequence of the former. Phylogenetic analysis is in agreement with this conclusion because the reported cytb sequence was positioned in the same branch of sequences of several Novyella species. This is the first assignment of the mitochondrial cytb gene sequence to P. nucleophilum. The P. (Haemamoeba) parasite is particularly similar to Plasmodium (Haemamoeba) tejerai, because its advanced trophozoites and young erythrocytic meronts possess a large vacuole with prominent pigment granules arranged around it, the characteristic features of development in this species. For definitive identification of P. (Haemamoeba) species, mature meronts and gametocytes are required; however, these were absent from the thin blood smear. Representative images of the blood stages of P. nucleophilum and P. (Haemamoeba) sp. are provided. Together with microscopy data, the P. nucleophilum cytb sequence will assist in molecular identification (barcoding) of this Plasmodium species in other birds.