A Tropical Composting Operation Unit at São Paulo Zoo as a Soure of Bacterial Proteolytic Enzymes

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the Sao Paulo Zoo Park (SPZPF), Sao Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.

Genomic Surveillance of Yellow Fever Virus Epidemic Waves in São Paulo, Brazil, 2017 – 2018

São Paulo (SP), a densely populated state in southeast Brazil that contains one of the world´s largest urban regions, has experienced its largest yellow fever virus (YFV) outbreak in decades. Surveillance in non-human primates (NHP) is important in order to detect YFV early during an epidemic or epizootic, to quantify the magnitude of the outbreak in NHP, and to evaluate the risk of YFV spillover infection in human populations. To better understand the genetic diversity and spatial distribution of YFV during the current outbreak in southeast Brazil, we generated 46 new virus genomes from YFV positive cases identified in 18 different municipalities in SP, mostly sampled from non-human primates between April 2017 and February 2018. Our data show that most NHP cases in São Paulo state were likely caused by the introduction of a single YFV lineage from Minas Gerais to São Paulo. Phylogenetic and phylogeographic analyses of these data indicate that YFV spread southwards from Minas Gerais into São Paulo state at a typical rate of <1km per day. These results shed light in the sylvatic transmission of yellow fever in highly fragmented forested regions in São Paulo state and highlight the importance of continued operational research and surveillance of zoonotic pathogens in sentinel populations.

Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 – 2018

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species.

Healthcare-associated carbapenem-resistant OXA-72-producing Acinetobacter baumannii of the clonal complex CC79 colonizing migratory and captive aquatic birds in a Brazilian Zoo

Carbapenem resistance in Acinetobacter baumannii is a public health issue globally, mainly due to the production of carbapenem hydrolyzing class D ß-lactamases (CHDLs). In Brazil, OXA-23 and OXA-143 CHDLs have been prevalent in A. baumannii from clinical settings, with some OXA-23 reports in the environmental samples, whereas OXA-72 has begun to be increasingly reported. This study aims to perform the genomic and microbiological characterization of carbapenem-resistant A. baumannii isolates recovered from migratory birds and captive birds inhabiting a lake within a Brazilian Zoo. Four hundred and eighty-one gram-negative bacilli were recovered from choanal and cloacal swabs obtained from 50 migratory birds and 37 captive birds present at the zoo’s lake between July and August of 2012. Among all GNB, nine OXA-72-producing A. baumannii were detected from the microbiota of four migratory and five captive aquatic birds. The OXA-72-producing A. baumannii isolates were submitted to antimicrobial susceptibility test and PFGE, exhibiting a multidrug-resistant profile and clonal relatedness with OXA-72-positive human isolates circulating for eighteen years in a hospital setting. MLST, plasmid analysis and whole-genome sequencing revealed which all carbapenem-resistant A. baumannii from bird and human hosts belonged to clonal complex 79, and harboured a small plasmid (~16.6-kb in size), named pAC1-BRL, which carried blaOXA-72 gene, macrolide resistance genes msrE and mphE, and the toxinantitoxin system AbkAB. To determine the impact of pAC1-BRL acquisition in the the capacity of a microorganism to survive in a competitive environment (in the following called fitness), the laboratory strain A. baumannii ATCC 19606 was used in the fitness experiments and suggested an increase of its relative fitness after the pAC1-BRL acquisition. In summary, the detection of OXA-72-producing A. baumannii strains belonging to CC79 in aquatic birds is a piece of epidemiological evidence demonstrating that dissemination of high-risk bacteria is extending beyond the hospital.

Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatranscriptomics

Composting is a promising source of new organisms and thermostable enzymes that may be helpful in environmental management and industrial processes. Here we present results of metagenomicand metatranscriptomic-based analyses of a large composting operation in the Sao Paulo Zoo Park. This composting exhibits a sustained thermophilic profile (50°C to 75°C), which seems to preclude fungal activity. The main novelty of our study is the combination of time-series sampling with shotgun DNA, 16S rRNA gene amplicon, and metatranscriptome high-throughput sequencing, enabling an unprecedented detailed view of microbial community structure, dynamics, and function in this ecosystem. The time-series data showed that the turning procedure has a strong impact on the compost microbiota, restoring to a certain extent the population profile seen at the beginning of the process; and that lignocellulosic biomass deconstruction occurs synergistically and sequentially, with hemicellulose being degraded preferentially to cellulose and lignin. Moreover, our sequencing data allowed near-complete genome reconstruction of five bacterial species previously found in biomassdegrading environments and of a novel biodegrading bacterial species, likely a new genus in the order Bacillales. The data and analyses provided are a rich source for additional investigations of thermophilic composting microbiology.

Application of Conventional Heating and Microwave Irradiation for the Biocatalytic Reduction of Fluoroacetophenones by Thermophilic Bacteria

This paper describes the bioreduction of four fluoroacetophenones with thermophilic bacteria (Bacillus sp. FPZSP005, B. subtilis FPZSP088, B. licheniformis FPZSP055 and BC-FPZSP051). The reduction reactions were performed under microwave irradiation and the thermophilic bacteria of the Bacillus genus provided moderate conversions to fluoroalcohols with yields in the 26-42% ranges and 5-62% e.e. When the reactions were carried out under conventional heating, they showed high conversions and selectivities (up to > 99% e.e.). This is the first study in the literature on the use of microwave irradiation with thermophilic bacteria applied on biocatalysis.

Interações eco-epidemiológicas entre cães domésticos e a fauna silvestre em agroecossistemas

Cães e seres humanos compartilham os mesmos caminhos por pelo menos 14-100 mil anos e, atualmente, o cão doméstico (Canis lupus familiaris) e a espécie de carnívoro mais abundante e difundida do planeta, está presente em todos os continentes, exceto Antártida. Como consequência, cães representam um desafio global tanto para conservação da biodiversidade como para saúde pública, e esta problemática e ainda mais grave entre cães mantidos soltos e cães ferais em áreas rurais. No entanto, apesar da abrangência e da gravidade do problema, tanto a intensidade dos impactos causados por cães domésticos, como a eficácia de estratégias de manejo, ainda são incertas. Isso ocorre, pois estudos raramente quantificam aspectos básicos da ecologia dos cães. Aspectos que são cruciais tanto para compreensão dos processos de invasão biológica, como para elaboração de modelos epidemiológicos de dispersão de doenças. E nesta lacuna de conhecimento que buscamos nos aprofundar na presente tese de doutorado. Utilizando como modelo a população de cães de um agroecossistema de Cerrado no Brasil Central, realizamos um estudo longitudinal buscando elucidar como e a estrutura e a dinâmica populacional dos cães; de que forma eles utilizam o espaço e que fatores determinam seus caminhos; e como a população e o movimento dos cães podem ser modulados pelas pessoas. Com os estudos aqui apresentados, trouxemos avanços metodológicos, e reflexões de manejo, que contribuirão de forma substancial para aprofundar nossa compreensão de uma problemática comum a todas as partes do planeta. Vimos que, mesmo em uma população de tamanho estável, a estrutura populacional pode ser extremamente dinâmica, e este fluxo de indivíduos e regido essencialmente pelas pessoas. Observamos também que os cães têm um uso considerável da paisagem, ainda que eles permaneçam a maior parte do tempo ao redor de suas casas. Além disso, constatamos que os padrões de uso do espaço variam substancialmente a depender das características do indivíduo, do manejo e do papel social do cão na comunidade. Por fim, propomos estratégias de manejo que possam simultaneamente mitigar os impactos causados por cães e aprimorar o desempenho de suas funções na comunidade.

Wild and Domestic Canids and Their Interactions in the Transmission Cycles of Trypanosoma Cruzi and Leishmania spp. in an Area of the Brazilian Cerrado.

Trypanosoma cruzi and Leishmania spp. are parasites that infect multiple hosts including canids, considered bioaccumulators of parasites. Deforestation in the Cerrado biome has resulted in the exposure of wild canids to anthropized areas, where they may establish ecological and epidemiological relationships with domestic dogs. We evaluated the infection by trypanosomatids in canids from a Cerrado agroecosystem between 2013 and 2017. Samples of wild canids (blood, bone marrow and skin) and dogs (blood) were collected for parasitological, serological and molecular diagnosis. A total of 414 samples from wild (n = 131) and domestic (n = 283) canids were collected, including recaptures. We obtained five positive hemocultures from Lycalopex vetulus (n = 2), Cerdocyon thous (n = 1) and dogs (n = 2), all characterized as T. cruzi TcIII/V (18S rDNA) and TcIII/V/VI (gGAPDH); one positive skin fragment for Leishmania sp. (C. thous), one positive skin culture (Chrysocyon brachyurus) and one positive fresh blood examination from a dog. Infection by T. cruzi and Leishmania spp. was serologically confirmed in 18% and 4% of the canids, respectively. Active transmission was attested by seroconversion events and occurred despite the low rate of positive parasitological assays. Wild and domestic canids infected by both parasites were detected sharing the same areas, pointing to a possible spillover of parasites among them.

First assessment of hoary fox (Lycalopex vetulus) seasonal ovarian cyclicity by non-invasive hormonal monitoring technique.

Reproduction is key to species survival, and reproductive physiology represents a high priority investigative area for conservation biology, as it provides a basic understanding of critical life-history traits, information that is helpful for the establishment of management strategies. Here, we generated knowledge about the reproductive endocrinology of the hoary fox (Lycalopex vetulus), a small canid (2.5–4 kg) endemic to open areas of the Brazilian Cerrado and listed in the Brazilian National List of Endangered species. Specifically, we utilized non-invasive hormone monitoring methods to assess oestrogen and progestagen metabolites from eight female hoary foxes housed in five zoological institutions in the state of Sao Paulo–Brazil. We observed the elevations of oestrogen and progestagen metabolites between July and September in six of the eight females. No significant evidence of ovarian activity was observed during other months. Two females, who shared the same enclosure, did not show a pattern of reproductive cyclicity. Based on these characteristics, we concluded that captive hoary foxes are seasonal monoestric, with the beginning of the oestrus cycle occurring mainly in July followed by 2 months of the luteal phase when conception does not occur. We suggest the dosage of faecal metabolites of estradiol and progesterone could be used to differentiate the reproductive period from a non-reproductive period in Lycalopex vetulus females, providing relevant information about their reproductive biology that may contribute to species conservation and management strategies, such as increased ex situ reproductive success.