Long-term occurrence of multiple antimicrobial drug resistant Klebsiella pneumoniae isolates harboring virulent potential in a tertiary hospital from Brazil.

Klebsiella pneumoniae strains are globally associated with a plethora of opportunistic and severe human infections and are known to spread genes conferring antimicrobial resistance. Some strains harbor virulence determinants that enable them to cause serious disease in any patient, both in the hospital and in the community. The aim of this study was to determine the frequency of antimicrobial resistance and virulence traits (by gene detection and string test) among 83 K. pneumoniae isolates obtained from patient cultures of a scholar tertiary hospital in the Midwestern Brazil (Brasília, DF). Antimicrobial susceptibility analysis showed that 94% (78/83) of the isolates presented one of the following resistance profiles: resistant (R, 39), multidrug-resistant (MDR, 29), or extensively drug-resistant (XDR, 10). Several MDR and XDR strains harbored multiple virulence genes and displayed hypermucoviscous phenotype. These characteristics were observed among isolates obtained throughout all the sample collection period (2013 - 2017). The K2 serotype gene, a molecular marker of hypervirulence, was detected in three isolates, one of which classified as XDR. Sequence typing revealed the occurrence of isolates belonged to high-risk (ST13) and multiple resistance-spreading clones (ST105). Thus, our findings showed the occurrence of virulent potential isolates that also presented MDR/XDR phenotypes from 2013 to 2015. This study also indicates the probable convergence of virulence and resistance since at least 2013 in Brazil.

Scalable and versatile container-based pipelines for de novo genome assembly and bacterial annotation.

Background: Advancements in DNA sequencing technology have transformed the field of bacterial genomics, allowing for faster and more cost effective chromosome level assemblies compared to a decade ago. However, transforming raw reads into a complete genome model is a significant computational challenge due to the varying quality and quantity of data obtained from different sequencing instruments, as well as intrinsic characteristics of the genome and desired analyses. To address this issue, we have developed a set of container-based pipelines using Nextflow, offering both common workflows for inexperienced users and high levels of customization for experienced ones. Their processing strategies are adaptable based on the sequencing data type, and their modularity enables the incorporation of new components to address the community's evolving needs. Methods: These pipelines consist of three parts: quality control, de novo genome assembly, and bacterial genome annotation. In particular, the genome annotation pipeline provides a comprehensive overview of the genome, including standard gene prediction and functional inference, as well as predictions relevant to clinical applications such as virulence and resistance gene annotation, secondary metabolite detection, prophage and plasmid prediction, and more. Results: The annotation results are presented in reports, genome browsers, and a web-based application that enables users to explore and interact with the genome annotation results. Conclusions: Overall, our user-friendly pipelines offer a seamless integration of computational tools to facilitate routine bacterial genomics research. The effectiveness of these is illustrated by examining the sequencing data of a clinical sample of Klebsiella pneumoniae.

Complete Genome Sequence of Pantoea stewartii RON18713 from Brazil Nut Tree Phyllosphere Reveals Genes Involved in Plant Growth Promotion.

The Amazonian rainforest is a hyper-diverse ecosystem in the number of species and the myriad of intertaxon relationships that are mostly understudied. In order to characterize a dominant and economically important Amazonian species, the Brazil nut tree (Bertholletia excelsa Bonpl.), at the genome level, wegenerated high-coverage long-read sequencing data from the leaves of a single individual. The genome assembly revealed an unexpected discovery: two circular contigs that could be assigned to the chromosome and a plasmid of a Pantoea stewartii strain. Comparative genomics revealed that this strain belongs to the indologenes subspecies and displays high synteny with other strains isolated from diseased leaves of the neotropical palm Bactris gasipaes Kunth. Investigation of pathogenicity-related genes revealed the absence of the entire type III secretion system gene cluster in the plasmid, which was otherwise highly similar to a plasmid from an isolate known to cause disease in Dracaena sanderiana Mast. In contrast, several genes associated with plant-growth promoting traits were detected, including genes involved in indole-3-acetic acid (IAA) production, phosphate solubilization, and biosynthesis of siderophores. In summary, we report the genome of an uncultivated P. stewartii subsp. indologenes strain associated with the Brazil nut tree and potentially a plant growth-promoting bacteria.

Genomic and physiological characterization of Novosphingobium terrae sp. nov., an alphaproteobacterium isolated from Cerrado soil containing a mega-sized chromid.

A novel bacterial strain, designated GeG2T, was isolated from soils of the native Cerrado, a highly biodiverse savanna-like Brazilian biome. 16S rRNA gene analysis of GeG2T revealed high sequence identity (100%) to the alphaproteobacterium Novosphingobium rosa; however, comparisons with N. rosa DSM 7285T showed several distinctive features, prompting a full characterization of the new strain in terms of physiology, morphology, and, ultimately, its genome. GeG2T cells were Gram-stain-negative bacilli, facultatively anaerobic, motile, positive for catalase and oxidase activities, and starch hydrolysis. Strain GeG2T presented planktonic-sessile dimorphism and cell aggregates surrounded by extracellular matrix and nanometric spherical structures were observed, suggesting the production of exopolysaccharides (EPS) and outer membrane vesicles (OMVs). Despite high 16S rDNA identity, strain GeG2T showed 90.38% average nucleotide identity and 42.60% digital DNA-DNA hybridization identity with N. rosa, below species threshold. Whole-genome assembly revealed four circular replicons: a 4.1 Mb chromosome, a 2.7 Mb extrachromosomal megareplicon, and two plasmids (212.7 and 68.6 kb). The megareplicon contains a few core genes and plasmid-type replication/maintenance systems, consistent with its classification as a chromid. Genome annotation shows a vast repertoire of carbohydrate-active enzymes and genes involved in the degradation of aromatic compounds, highlighting the biotechnological potential of the new isolate. Chemotaxonomic features, including polar lipid and fatty acid profiles, as well as physiological, molecular, and whole-genome comparisons showed significant differences between strain GeG2T and N. rosa, indicating that it represents a novel species, for which the name Novosphingobium terrae is proposed. The type strain is GeG2T (= CBMAI 2313T = CBAS 753 T).

Multidrug-Resistant (MDR) Klebsiella variicola Strains Isolated in a Brazilian Hospital Belong to New Clones.

Klebsiella variicola is mainly associated with opportunistic infections and frequently identified as Klebsiella pneumoniae. This misidentification implies a wrong epidemiology result as well as incorrect attribution to K. pneumoniae as the etiology of some severe infections. Recently, huge efforts have been made to study K. variicola, however, the biological aspects of this species are still unclear. Here we characterized five K. variicola strains initially identified as K. pneumoniae, with a Vitek-2 System and 16S rRNA sequencing. One-step multiplex polymerase chain reaction and Whole Genome Sequencing (WGS) identified them as K. variicola. Additionally, WGS analysis showed that all the strains are closely related with K. variicola genomes, forming a clustered group, apart from K. pneumoniae and K. quasipneumoniae. Multilocus sequence typing analysis showed four different sequence types (STs) among the strains and for two of them (Kv97 and Kv104) the same ST was assigned. All strains were multidrug-resistant (MDR) and three showed virulence phenotypes including invasion capacity to epithelial cells, and survival in human blood and serum. These results showed the emergence of new K. variicola clones with pathogenic potential to colonize and cause infection in different tissues. These characteristics associated with MDR strains raise great concern for human health.

A Novel Multidrug Resistant, Non-Tn4401 Genetic Element-Bearing, Strain of Klebsiella pneumoniae Isolated From an Urban Lake With Drinking and Recreational Water Reuse.

Antimicrobial resistance (AMR) is an increasing and urgent issue for human health worldwide, as it leads to the reduction of available antibiotics to treat bacterial infections, in turn increasing hospital stays and lethality. Therefore, the study and genomic surveillance of bacterial carriers of resistance in and outside of clinical settings is of utter importance. A colony of multidrug resistant (MDR) bacteria identified as Klebsiella spp., by 16S rDNA amplicon sequencing, has been isolated from an urban lake in Brazil, during a drug-degrading bacterial prospection. Genomic analyses revealed the bacteria as Klebsiella pneumoniae species. Furthermore, the in silico Multilocus Sequence Typing (MLST) identified the genome as a new sequence type, ST5236. The search for antimicrobial resistance genes (ARGs) detected the presence of genes against beta-lactams, fosfomycin, acriflavine and efflux pumps, as well as genes for heavy metal resistance. Of particular note, an extended-spectrum beta-lactamase gene (blaCTX-M-15) has been detected in close proximity to siphoviridae genes, while a carbapenemase gene (KPC-2) has been found in an extrachromosomal contig, within a novel non-Tn4401 genetic element (NTEKPC). An extrachromosomal contig found in the V3 isolate is identical to a contig of a K. pneumoniae isolate from a nearby hospital, which indicates a putative gene flow from the hospital network into Paranoá lake. The discovery of a MDR isolate in this lake is worrisome, as the region has recently undergone periods of water scarcity causing the lake, which receives treated wastewater effluent, and is already used for recreational purposes, to be used as an environmental buffer for drinking water reuse. Altogether, our results indicate an underrepresentation of environmental K. pneumoniae among available genomes, which may hamper the understanding of the population dynamics of the species in the environment and its consequences in the spread of ARGs and virulence genes.