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.

A new mutation in mgrb mediating polymyxin resistance in Klebsiella variicola

Polymyxin resistance is a public health concern -present in humans, animals and the environment -caused by chromosomal-encoding or plasmid-encoding mechanisms. Chromosomal alterations in MgrB are frequently detected in Klebsiella spp., but not yet reported and characterised in Klebsiella variicola ( K. variicola ). This study performed microbiological and genomic characterisation of three polymyxin- resistant K. variicola isolates (M14, M15 and M50) recovered from the microbiota of migratory birds in Brazil. The isolates were submitted to SpeI-PFGE, broth microdilution and whole genome sequencing us- ing Illumina MiSeq for analysis of genetic relatedness, sequence typing and detection of antimicrobial- resistance genes. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance only for polymyxins. Sequences of chromosomal two-component systems (PmrAB, PhoPQ, RstAB, CrrAB) and MgrB were evaluated by blastN and blastP against a polymyxin-susceptible K. variicola (A58243), and mutations with biological effect were checked by the PROVEAN tool. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance for polymyxins. In M14 and M15, phoQ deleterious mutations (D90N, I122S and G385S) were identified, while an mgrB variant containing a single deletion (C deletion on position 93) leading to the production of a non-functional protein was detected in M50. mgrB complementation studies showed restoration of polymyxin susceptibility (64 to ­­ ≤ 0.25 mg/L) as a wild-type mgrB was inserted into the mgrB-deficient M50. This study confirmed the role of a non-functional mgrB variant in conferring polymyxin resistance, stressing the role of this regulator in K. variicola and drawing attention to novel polymyxin resistance mechanisms emerging in wildlife.

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.

Impactos antrópicos sobre uma população de Alouatta clamitans (Cabrera, 1940) em um fragmento de Mata Atlântica no Estado de São Paulo: apontamento de medidas mitigatórias

A degradação e fragmentação dos habitats, assim como as consequências relacionadas, possuem grande influência no declínio das espécies, fato pela pressão antrópica a que esses fragmentos estão submetidos. Primatas do gênero Alouatta são animais que apresentam grande adaptabilidade aos recursos, porém, mesmo sobrevivendo em áreas fragmentadas, sofrem os efeitos da urbanização acelerada, gerando assim maior impacto as populações silvestres. Este estudo monitorou entre 2006 e 2014 os impactos a que os espécimes de Alouatta clamitans estavam submetidos dentro do Parque Estadual Fontes do Ipiranga – SP (PEFI), de forma a compilar e analisar as informações visando propor medidas mitigatórias que almejam minimizar os impactos e auxiliar na manutenção de uma população saudável a longo prazo.