The mobilome landscape of biocide-resistance in Brazilian ESKAPE isolates.

The increasing frequency of antibiotic-resistant bacteria is a constant threat to global human health. Therefore, the pathogens of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter spp.) are among the most relevant causes of hospital infections responsible for millions of deaths every year. However, little has been explored about the danger of microorganisms resistant to biocides such as antiseptics and disinfectants. Widely used in domestic, industrial, and hospital environments, these substances reach the environment and can cause selective pressure for resistance genes and induce cross-resistance to antibiotics, further aggravating the problem. Therefore, it is necessary to use innovative and efficient strategies to monitor the spread of genes related to resistance to biocides. Whole genome sequencing and bioinformatics analysis aiming to search for sequences encoding resistance mechanisms are essential to help monitor and combat these pathogens. Thus, this work describes the construction of a bioinformatics tool that integrates different databases to identify gene sequences that may confer some resistance advantage about biocides. Furthermore, the tool analyzed all the genomes of Brazilian ESKAPE isolates deposited at NCBI and found a series of different genes related to resistance to benzalkonium chloride, chlorhexidine, and triclosan, which were the focus of this work. As a result, the presence of resistance genes was identified in different types of biological samples, environments, and hosts. Regarding mobile genetic elements (MGEs), around 52% of isolates containing genes related to resistance to these compounds had their genes identified in plasmids, and 48.7% in prophages. These data show that resistance to biocides can be a silent, underestimated danger spreading across different environments and, therefore, requires greater attention.

A reverse-ecology framework to uncover the potential metabolic interplay among 'Candidatus Liberibacter' species, citrus hosts and psyllid vector.

'Candidatus Liberibacter' species have developed a dependency on essential nutrients and metabolites from the host cell, as a result of substantial genome reduction. Still, it is difficult to state which nutrients they acquire and whether or not they are metabolically reliant. We used a reverse-ecology model to investigate the potential metabolic interactions of 'Ca Liberibacter' species, Citrus, and the psyllid Diaphorina citri in the huanglongbing disease pyramid. Our findings show that hosts (citrus and psyllid) tend to support the nutritional needs of 'Ca. Liberibacter' species, implying that the pathogen's metabolism has become tightly linked to hosts, which may reflect in the parasite lifestyle of this important genus.

Re-classification of Streptomyces venezuelae strains and mining secondary metabolite biosynthetic gene clusters.

Streptomyces species have attracted considerable interest as a reservoir of medically important secondary metabolites, which are even diverse and different between strains. Here, we reassess ten Streptomyces venezuelae strains by presenting the highly resolved classification, using 16S rRNA sequencing, MALDI-TOF MS protein profiling, and whole-genome sequencing. The results revealed that seven of the ten strains were misclassified as S. venezuelae species. Secondary metabolite biosynthetic gene cluster (smBGC) mining and targeted LC-MS/MS based metabolite screening of S. venezuelae and misclassified strains identified in total 59 secondary metabolites production. In addition, a comparison of pyrrolamide-type antibiotic BGCs of four misclassified strains, followed by functional genomics, revealed that athv28 is critical in the synthesis of the anthelvencin precursor, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC). Our findings illustrate the importance of the accurate classification and better utilization of misclassified Streptomyces strains to discover smBGCs and their secondary metabolite products.

Metagenomic strategies identify diverse integron-integrase and antibiotic resistance genes in the Antarctic environment.

The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. Integrons were studied using two independent methods. One involved the construction and analysis of intI gene amplicon libraries. In addition, we sequenced 17 metagenomes of microbial mats and soil by high-throughput sequencing and analyzed these data using the IntegronFinder program. As expected, the metagenomic analysis allowed for the identification of novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions. However, some intI genes are similar to sequences previously identified by amplicon library analysis in soil samples collected from non-Antarctic sites. ARGs were analyzed in the metagenomes using ABRIcate with CARD database and verified if these genes could be classified as GCs by IntegronFinder. We identified 53 ARGs in 15 metagenomes, but only four were classified as GCs, one in MTG12 metagenome (Continental Antarctica), encoding an aminoglycoside-modifying enzyme (AAC(6´)acetyltransferase) and the other three in CS1 metagenome (Maritime Antarctica). One of these genes encodes a class D ß-lactamase (blaOXA-205) and the other two are located in the same contig. One is part of a gene encoding the first 76 amino acids of aminoglycoside adenyltransferase (aadA6), and the other is a qacG2 gene.

Adaption of microbial communities to the hostile environment in the Doce River after the collapse of two iron ore tailing dams.

In November 2015, two iron ore tailing dams collapsed in the city of Mariana, Brazil. The dams' collapse generated a wave of approximately 50 million m3 of a mixture of mining waste and water. It was a major environmental tragedy in Brazilian history, which damaged rivers, and cities 660 km away in the Doce River basin until it reached the ocean coast. Shortly after the incident, several reports informed that the concentration of metals in the water was above acceptable legal limits under Brazilian laws. Here the microbial communities in samples of water, mud, foam, and rhizosphere of Eichhornia from Doce River were analyzed for 16S and 18S rRNA-based amplicon sequencing, along with microbial isolation, chemical and mineralogical analyses. Samples were collected one month and thirteen months after the collapse. Prokaryotic communities from mud shifted drastically over time (33% Bray-Curtis similarity), while water samples were more similar (63% Bray-Curtis similarity) in the same period. After 12 months, mud samples remained with high levels of heavy metals and a reduction in the diversity of microeukaryotes was detected. Amoebozoans increased in mud samples, reaching 49% of microeukaryote abundance, with Discosea and Lobosa groups being the most abundant. The microbial communities' structure in mud samples changed adapting to the new environment condition. The characterization of microbial communities and metal-tolerant organisms from such impacted environments is essential for understanding the ecological consequences of massive anthropogenic impacts and strategies for the restoration of contaminated sites such as the Doce River.

Robust DNA protocols for tropical soils.

Studies in the Amazon are being intensified to evaluate the alterations in the microbial communities of soils and sediments in the face of increasing deforestation and land-use changes in the region. However, since these environments present highly heterogeneous physicochemical properties, including contaminants that hinder nucleic acids isolation and downstream techniques, the development of best molecular practices is crucial. This work aimed to optimize standard protocols for DNA extraction and gene quantification by quantitative real-time PCR (qPCR) based on natural and anthropogenic soils and sediments (primary forest, pasture, Amazonian Dark Earth, and várzea, a seasonally flooded area) of the Eastern Amazon. Our modified extraction protocol increased the fluorometric DNA concentration by 48%, reaching twice the original amount for most of the pasture and várzea samples, and the 260/280 purity ratio by 15% to values between 1.8 to 2.0, considered ideal for DNA. The addition of bovine serum albumin in the qPCR reaction improved the quantification of the 16S rRNA genes of Archaea and Bacteria and its precision among technical replicates, as well as allowed their detection in previously non-amplifiable samples. It is concluded that the changes made in the protocols improved the parameters of the DNA samples and their amplification, thus increasing the reliability of microbial communities' analysis and its ecological interpretations.

Structuring biofilm communities living in pesticide contaminated water.

The wide use of pesticides in agriculture expose microbiota to stressful conditions that require the development of survival strategies. The bacterial response to many pollutants has not been elucidated in detail, as well as the evolutionary processes that occur to build adapted communities. The purpose of this study was to evaluate the bacterial population structure and adaptation strategies in planktonic and biofilm communities in limited environments, as tanks containing water used for washing herbicide containers. This biodiversity, with high percentage of nonculturable microorganisms, was characterized based on habitat and abiotic parameters using molecular and bioinformatics tools. According to water and wastewater standards, the physicochemical conditions of the tank water were inadequate for survival of the identified bacteria, which had to develop survival strategies in this hostile environment. The biodiversity decreased in the transition from planktonic to biofilm samples, indicating a possible association between genetic drift and selection of individuals that survive under stressful conditions, such as heating in water and the presence of chlorine, fluorine and agrochemicals over a six-month period. The abundance of Enterobacter, Acinetobacter and Pseudomonas in biofilms from water tanks was linked to essential processes, deduced from the genes attributed to these taxonomic units, and related to biofilm formation, structure and membrane transport, quorum sensing and xenobiotic degradation. These characteristics were randomly combined and fixed in the biofilm community. Thus, communities of biofilm bacteria obtained under these environmental conditions serve as interesting models for studying herbicide biodegradation kinetics and the prospects of consortia suitable for use in bioremediation in reservoirs containing herbicide-contaminated wastewater, as biofilters containing biofilm communities capable of degrading herbicides.

Genotypic and phenotypic characterization of biofilm production by Staphylococcus aureus strains isolated from bovine intramammary infections in Colombian dairy farms.

The ability of Staphylococcus aureus to form biofilms is an important virulence factor because this has been associated with persistent bovine intramammary infections. Different mechanisms of biofilm formation have been described in S. aureus; however, the process has been found to be mainly driven by the ica and bap genes. The presence of the ica and bap genes, as well as the biofilm formation in vitro were evaluated in 229 S. aureus strains isolated from bovine milk collected from different regions of Department of Antioquia, Colombia. Three different genotypes grouped into three separate clusters were identified from in vitro assays. Genotype 1 (ica positive and bap negative) was the most prevalent (78.17%), followed by genotype 2 (ica and bap positive) (12.66%) and genotype 0 (ica and bap negative) (9.17%). Biofilm formation was observed in 81.26% of the strains from which 100% of genotype 2 isolates showed biofilm formation. The biofilms formed by genotype 2 isolates were also found to have the highest optical density (>2.4). These results showed that most of the S. aureus strains were capable of biofilm formation, suggesting the virulence potential particularly in bap-positive strains.

Recovery and functional validation of hidden soil enzymes in metagenomic libraries.

The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.

Microbe Profile: Saccharomyces eubayanus, the missing link to lager beer yeasts.

Saccharomyces eubayanus was described less than 10 years ago and its discovery settled the long-lasting debate on the origins of the cold-tolerant yeast responsible for lager beer fermentation. The largest share of the genetic diversity of S. eubayanus is located in South America, and strains of this species have not yet been found in Europe. One or more hybridization events between S. eubayanus and S. cerevisiae ale beer strains gave rise to S. pastorianus, the allopolyploid yeasts responsible for lager beer production worldwide. The identification of the missing progenitor of lager yeast opened new avenues for brewing yeast research. It allowed not only the selective breeding of new lager strains, but revealed also a wild yeast with interesting brewing abilities so that a beer solely fermented by S. eubayanus is currently on the market.

MLSTar: automatic multilocus sequence typing of bacterial genomes in R.

Multilocus sequence typing (MLST) is a standard tool in population genetics and bacterial epidemiology that assesses the genetic variation present in a reduced number of housekeeping genes (typically seven) along the genome. This methodology assigns arbitrary integer identifiers to genetic variations at these loci which allows us to efficiently compare bacterial isolates using allele-based methods. Now, the increasing availability of whole-genome sequences for hundreds to thousands of strains from the same bacterial species has allowed us to apply and extend MLST schemes by automatic extraction of allele information from the genomes. The PubMLST database is the most comprehensive resource of described schemes available for a wide variety of species. Here we present MLSTar as the first R package that allows us to (i) connect with the PubMLST database to select a target scheme, (ii) screen a desired set of genomes to assign alleles and sequence types, and (iii) interact with other widely used R packages to analyze and produce graphical representations of the data. We applied MLSTar to analyze more than 2,500 bacterial genomes from different species, showing great accuracy, and comparable performance with previously published command-line tools. MLSTar can be freely downloaded from http://github.com/iferres/MLSTar.