Analysis of twelve genomes of the bacterium Kerstersia gyiorum from brown-throated sloths (Bradypus variegatus), the first from a non-human host.

Kerstersia gyiorum is a Gram-negative bacterium found in various animals, including humans, where it has been associated with various infections. Knowledge of the basic biology of K. gyiorum is essential to understand the evolutionary strategies of niche adaptation and how this organism contributes to infectious diseases; however, genomic data about K. gyiorum is very limited, especially from non-human hosts. In this work, we sequenced 12 K. gyiorum genomes isolated from healthy free-living brown-throated sloths (Bradypus variegatus) in the Parque Estadual das Fontes do Ipiranga (São Paulo, Brazil), and compared them with genomes from isolates of human origin, in order to gain insights into genomic diversity, phylogeny, and host specialization of this species. Phylogenetic analysis revealed that these K. gyiorum strains are structured according to host. Despite the fact that sloth isolates were sampled from a single geographic location, the intra-sloth K. gyiorum diversity was divided into three clusters, with differences of more than 1,000 single nucleotide polymorphisms between them, suggesting the circulation of various K. gyiorum lineages in sloths. Genes involved in mobilome and defense mechanisms against mobile genetic elements were the main source of gene content variation between isolates from different hosts. Sloth-specific K. gyiorum genome features include an IncN2 plasmid, a phage sequence, and a CRISPR-Cas system. The broad diversity of defense elements in K. gyiorum (14 systems) may prevent further mobile element flow and explain the low amount of mobile genetic elements in K. gyiorum genomes. Gene content variation may be important for the adaptation of K. gyiorum to different host niches. This study furthers our understanding of diversity, host adaptation, and evolution of K. gyiorum, by presenting and analyzing the first genomes of non-human isolates.

A comparison of rectal and oral cultivable microbiota in wild and captive black lion tamarins (Leontopithecus chrysopygus, Mikan 1823).

The black lion tamarin (Leontopithecus chrysopygus) is an endangered primate species, restricted to the Atlantic Forest fragments of São Paulo state, Brazil, with an estimated wild population of ~1600 individuals. Integrative studies between zoo (ex situ) and wild (in situ) animals are crucial to modern conservation programs. They can demonstrate a substantial impact with the One Health concept, an interdisciplinary research frontier regarding the relations between human, animal, and environmental health. Studies of wild populations of Leontopithecus spp. are scarce and should be encouraged to provide baseline information to develop preventive and curative medicine in zoos and other conservation programs. Studying these animals in the wild can offer important reference parameters for the species. Comparing bacterial communities between in situ and ex situ populations can help us understand both conditions and the dynamics of potentially pathogenic microorganisms. To increase our understanding of resident microorganisms among these groups, we collected oral and rectal samples from captive (zoo) and wild black lion tamarins. We employed a culture method for the identification of aerobic bacteria. Thirty-three specimens were sampled (24 zoo and 8 wild animals) and 18 bacterial genera were identified. We found primarily Gram-positive bacteria in wild animals, whereas in zoo animals, Gram-negative bacteria were dominant. Some of the bacterial species we identified are potentially pathogenic, whereas several others are being reported here for the first time in this host species. Our results reinforce the importance of integrative studies for the future management and conservation of this endangered primate species.

Enzymatic diversity of filamentous fungi isolated from forest soil incremented by sugar cane solid waste.

Fungi are natural degraders of organic matter which can produce enzymes for many industrial and biotechnological applications. In this context, crude enzymatic extracts of fungal isolates were evaluated regarding their hydrolytic and ligninolytic abilities. The fungal strains were isolated from soil samples from Atlantic Rain Forest Park incremented with sugar cane biomass (filter cake), which allowed the selection of efficient lignocellulolytic enzymes. A total of 190 fungi were isolated and evaluated by endocellulase screenings. Thirteen fungi were selected about their hydrolytic and ligninolytic abilities. Among them, three isolates showed xylanolytic activity. Eleven of the isolates were selected by their cellulolytic abilities. Proteolytic enzymes were also detected for three fungi, allowing the classification as metalloprotease and serine protease. The isolates SPZPF3_47 (Mucor sp.), SPZPF1_129 (Byssochlamys nivea) and SPZPF1_141 (Paecilomyces saturatus) were selected for further investigation on their lignin peroxidase abilities. KM, Vmax and kcat apparent for lignin peroxidases were also determined. The strain of Mucor sp. (SPZPF3_47) was highlighted since this fungal genus was not well described about its isolation in the adopted conditions in our study, and showing ligninolytic abilities.

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 using 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.

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.

A Tropical Composting Operation Unit at São Paulo Zoo as a Source 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 São Paulo Zoo Park (SPZPF), São 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.

Decreased AIRE expression and global thymic hypofunction in Down syndrome.

The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity to organ-specific autoimmune disorders, and higher susceptibility to infections, among other features. Considering that AIRE (autoimmune regulator) is located on 21q22.3, we analyzed protein and gene expression in surgically removed thymuses from 14 DS patients with congenital heart defects, who were compared with 42 age-matched controls with heart anomaly as an isolated malformation. Immunohistochemistry revealed 70.48 ± 49.59 AIRE-positive cells/mm(2) in DS versus 154.70 ± 61.16 AIRE-positive cells/mm(2) in controls (p < 0.0001), and quantitative PCR as well as DNA microarray data confirmed those results. The number of FOXP3-positive cells/mm(2) was equivalent in both groups. Thymus transcriptome analysis showed 407 genes significantly hypoexpressed in DS, most of which were related, according to network transcriptional analysis (FunNet), to cell division and to immunity. Immune response-related genes included those involved in 1) Ag processing and presentation (HLA-DQB1, HLA-DRB3, CD1A, CD1B, CD1C, ERAP) and 2) thymic T cell differentiation (IL2RG, RAG2, CD3D, CD3E, PRDX2, CDK6) and selection (SH2D1A, CD74). It is noteworthy that relevant AIRE-partner genes, such as TOP2A, LAMNB1, and NUP93, were found hypoexpressed in DNA microarrays and quantitative real-time PCR analyses. These findings on global thymic hypofunction in DS revealed molecular mechanisms underlying DS immune phenotype and strongly suggest that DS immune abnormalities are present since early development, rather than being a consequence of precocious aging, as widely hypothesized. Thus, DS should be considered as a non-monogenic primary immunodeficiency.

Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp. nov.

A Gram-negative, rod-shaped, non-spore-forming and nitrogen-fixing bacterium, designated ICB 89(T), was isolated from stems of a Brazilian sugar cane variety widely used in organic farming. 16S rRNA gene sequence analysis revealed that strain ICB 89(T) belonged to the genus Stenotrophomonas and was most closely related to Stenotrophomonas maltophilia LMG 958(T), Stenotrophomonas rhizophila LMG 22075(T), Stenotrophomonas nitritireducens L2(T), [Pseudomonas] geniculata ATCC 19374(T), [Pseudomonas] hibiscicola ATCC 19867(T) and [Pseudomonas] beteli ATCC 19861(T). DNA-DNA hybridization together with chemotaxonomic data and biochemical characteristics allowed the differentiation of strain ICB 89(T) from its nearest phylogenetic neighbours. Therefore, strain ICB 89(T) represents a novel species, for which the name Stenotrophomonas pavanii sp. nov. is proposed. The type strain is ICB 89(T) ( = CBMAI 564(T)  = LMG 25348(T)).

Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state / Caracterização molecular de bactérias fixadoras de nitrogênio isoladas de plantas brasileiras no estado de São Paulo

Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.

Quatorze linhagens de bactérias fixadoras de nitrogênio foram isoladas de diferentes espécies de plantas, incluindo cassava, milho e cana-de-açúcar, usando condições seletivas desprovidas de nitrogênio. A capacidade de fixar nitrogênio foi verificada por ensaio de redução de acetileno. Todas as linhagens fixadoras de nitrogênio testadas apresentaram hibridização positiva com sonda de gene nifH derivada de Azospirillum brasilense. As linhagens foram caracterizadas por RAPD, ARDRA e sequenciamento do gene 16S rDNA. As análises de RAPD revelaram 8 genótipos, as 6 linhagens restantes foram agrupadas em 3 grupos de RAPD, sugerindo uma origem clonal. ARDRA e seqüências de 16S rDNA foram alocadas em 13 grupos conhecidos de bactérias fixadoras de nitrogênio, incluindo organismos dos gêneros Azospirillum, Herbaspirillum, Pseudomonas e Enterobacteriaceae. Duas linhagens foram classificadas como Stenotrophomonas ssp. Os resultados da identificação molecular baseados em sequencias de 16S rDNA corroboram com dados obtidos em testes morfológicos e bioquímicos.

Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state.

Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.