Brevundimonas brasiliensis sp. nov.: a New Multidrug-Resistant Species Isolated from a Patient in Brazil.

To increase knowledge on Brevundimonas pathogens, we conducted in-depth genomic and phenotypic characterization of a Brevundimonas strain isolated from the cerebrospinal fluid of a patient admitted in a neonatal intensive care unit. The strain was identified as a member of the genus Brevundimonas based on Vitek 2 system results and 16S rRNA gene sequencing and presented a multidrug resistance profile (MDR). Several molecular and biochemical tests were used to characterize and identify the species for in-depth results. The draft genome assembly of the isolate has a total length of 3,261,074 bp and a G+C of 66.86%, similar to other species of the genus. Multilocus sequence analysis, Type (Strain) Genome Server, digital DNA-DNA hybridization, and average nucleotide identity confirmed that the Brevundimonas sp. studied represents a distinct species, for which we propose the name Brevundimonas brasiliensis sp. nov. In silico analysis detected antimicrobial resistance genes (AMRGs) mediating resistance to ß-lactams (penP, blaTEM-16, and blaBKC-1) and aminoglycosides [strA, strB, aac(6')-Ib, and aac(6')-Il]. We also found AMRGs encoding the AcrAB efflux pump that confers resistance to a broad spectrum of antibiotics. Colistin and quinolone resistance can be attributed to mutation in qseC and/or phoP and GyrA/GyrB, respectively. The Brevundimonas brasiliensis sp. nov. genome contained copies of type IV secretion system (T4SS)-type integrative and conjugative elements (ICEs); integrative mobilizable elements (IME); and Tn3-type and IS3, IS6, IS5, and IS1380 families, suggesting an important role in the development and dissemination of antibiotic resistance. The isolate presented a range of virulence-associated genes related to biofilm formation, adhesion, and invasion that can be relevant for its pathogenicity. Our findings provide a wealth of data to hinder the transmission of MDR Brevundimonas and highlight the need for monitoring and identifying new bacterial species in hospital environments. IMPORTANCE Brevundimonas species is considered an opportunistic human pathogen that can cause multiple types of invasive and severe infections in patients with underlying pathologies. Treatment of these pathogens has become a major challenge because many isolates are resistant to most antibiotics used in clinical practice. Furthermore, there are no consistent therapeutic results demonstrating the efficacy of antibacterial agents. Although considered a rare pathogen, recent studies have provided evidence of the emergence of Brevundimonas in clinical settings. Hence, we identified a novel pathogenic bacterium, Brevundimonas brasiliensis sp. nov., that presented a multidrug resistance (MDR) profile and carried diverse genes related to drug resistance, virulence, and mobile genetic elements. Such data can serve as a baseline for understanding the genomic diversity, adaptation, evolution, and pathogenicity of MDR Brevundimonas.

Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil.

Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including ß-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to ß-lactamases (bla IND-13, bla CIA-3, bla TEM-116, bla OXA-209, bla VEB-15), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.

A Systematic Immuno-Informatic Approach to Design a Multiepitope-Based Vaccine Against Emerging Multiple Drug Resistant Serratia marcescens.

Serratia marcescens is now an important opportunistic pathogen that can cause serious infections in hospitalized or immunocompromised patients. Here, we used extensive bioinformatic analyses based on reverse vaccinology and subtractive proteomics-based approach to predict potential vaccine candidates against S. marcescens. We analyzed the complete proteome sequence of 49 isolate of Serratia marcescens and identified 5 that were conserved proteins, non-homologous from human and gut flora, extracellular or exported to the outer membrane, and antigenic. The identified proteins were used to select 5 CTL, 12 HTL, and 12 BCL epitopes antigenic, non-allergenic, conserved, hydrophilic, and non-toxic. In addition, HTL epitopes were able to induce interferon-gamma immune response. The selected peptides were used to design 4 multi-epitope vaccines constructs (SMV1, SMV2, SMV3 and SMV4) with immune-modulating adjuvants, PADRE sequence, and linkers. Peptide cleavage analysis showed that antigen vaccines are processed and presented via of MHC class molecule. Several physiochemical and immunological analyses revealed that all multiepitope vaccines were non-allergenic, stable, hydrophilic, and soluble and induced the immunity with high antigenicity. The secondary structure analysis revealed the designed vaccines contain mainly coil structure and alpha helix structures. 3D analyses showed high-quality structure. Molecular docking analyses revealed SMV4 as the best vaccine construct among the four constructed vaccines, demonstrating high affinity with the immune receptor. Molecular dynamics simulation confirmed the low deformability and stability of the vaccine candidate. Discontinuous epitope residues analyses of SMV4 revealed that they are flexible and can interact with antibodies. In silico immune simulation indicated that the designed SMV4 vaccine triggers an effective immune response. In silico codon optimization and cloning in expression vector indicate that SMV4 vaccine can be efficiently expressed in E. coli system. Overall, we showed that SMV4 multi-epitope vaccine successfully elicited antigen-specific humoral and cellular immune responses and may be a potential vaccine candidate against S. marcescens. Further experimental validations could confirm its exact efficacy, the safety and immunogenicity profile. Our findings bring a valuable addition to the development of new strategies to prevent and control the spread of multidrug-resistant Gram-negative bacteria with high clinical relevance.

NS1 codon usage adaptation to humans in pandemic Zika virus.

BACKGROUND: Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. OBJECTIVES: To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. FINDINGS: The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian epidemic isolate (ZBR) produced more NS1 protein than the MR766 African lineage (ZAF) did, as indicated by proteomic data from infections of neuron progenitor cells-derived neurospheres. Although ZBR replicated more efficiently in these cells, the differences observed in the stoichiometry of ZIKV proteins were not exclusively explained by the differences in viral replication between the lineages. MAIN CONCLUSIONS: Our findings suggest that natural, silent translational selection in the second half of 20th century could have improved the fitness of Asian ZIKV lineage in human and mosquito cells.

Does adaptation to vertebrate codon usage relate to flavivirus emergence potential?

Codon adaptation index (CAI) is a measure of synonymous codon usage biases given a usage reference. Through mutation, selection, and drift, viruses can optimize their replication efficiency and produce more offspring, which could increase the chance of secondary transmission. To evaluate how higher CAI towards the host has been associated with higher viral titers, we explored temporal trends of several historic and extensively sequenced zoonotic flaviviruses and relationships within the genus itself. To showcase evolutionary and epidemiological relationships associated with silent, adaptive synonymous changes of viruses, we used codon usage tables from human housekeeping and antiviral immune genes, as well as tables from arthropod vectors and vertebrate species involved in the flavivirus maintenance cycle. We argue that temporal trends of CAI changes could lead to a better understanding of zoonotic emergences, evolutionary dynamics, and host adaptation. CAI appears to help illustrate historically relevant trends of well-characterized viruses, in different viral species and genetic diversity within a single species. CAI can be a useful tool together with in vivo and in vitro kinetics, phylodynamics, and additional functional genomics studies to better understand species trafficking and viral emergence in a new host.

High Prevalence of Multidrug-Resistant Klebsiella pneumoniae Harboring Several Virulence and ß-Lactamase Encoding Genes in a Brazilian Intensive Care Unit.

Klebsiella pneumoniae is an important opportunistic pathogen that commonly causes nosocomial infections and contributes to substantial morbidity and mortality. We sought to investigate the antibiotic resistance profile, pathogenic potential and the clonal relationships between K. pneumoniae (n = 25) isolated from patients and sources at a tertiary care hospital's intensive care units (ICUs) in the northern region of Brazil. Most of K. pneumoniae isolates (n = 21, 84%) were classified as multidrug resistant (MDR) with high-level resistance to ß-lactams, aminoglycosides, quinolones, tigecycline, and colistin. All the 25 isolates presented extended-spectrum beta-lactamase-producing (ESBL), including carbapenemase producers, and carried the bla KPC (100%), bla TEM (100%), bla SHV variants (n = 24, 96%), bla OXA-1 group (n = 21, 84%) and bla CTX-M-1 group (n = 18, 72%) genes. The K2 serotype was found in 4% (n = 1) of the isolates, and the K1 was not detected. The virulence-associated genes found among the 25 isolates were mrkD (n = 24, 96%), fimH-1 (n = 22, 88%), entB (100%), iutA (n = 10, 40%), ybtS (n = 15, 60%). The genes related with efflux pumps and outer membrane porins found were AcrAB (100%), tolC (n = 24, 96%), mdtK (n = 22, 88%), OmpK35 (n = 15, 60%), and OmpK36 (n = 7, 28%). ERIC-PCR was employed to determine the clonal relationship between the different isolated strains. The obtained ERIC-PCR patterns revealed that the similarity between isolates was above 70%. To determine the sequence types (STs) a multilocus sequence typing (MLST) assay was used. The results indicated the presence of high-risk international clones among the isolates. In our study, the wide variety of MDR K. pneumoniae harboring ß-lactams and virulence genes strongly suggest a necessity for the implementation of effective strategies to prevent and control the spread of antibiotic resistant infections.

NS1 codon usage adaptation to humans in pandemic Zika virus

BACKGROUND Zika virus (ZIKV) was recognised as a zoonotic pathogen in Africa and southeastern Asia. Human infections were infrequently reported until 2007, when the first known epidemic occurred in Micronesia. After 2013, the Asian lineage of ZIKV spread along the Pacific Islands and Americas, causing severe outbreaks with millions of human infections. The recent human infections of ZIKV were also associated with severe complications, such as an increase in cases of Guillain-Barre syndrome and the emergence of congenital Zika syndrome. OBJECTIVES To better understand the recent and rapid expansion of ZIKV, as well as the presentation of novel complications, we compared the genetic differences between the African sylvatic lineage and the Asian epidemic lineage that caused the recent massive outbreaks. FINDINGS The epidemic lineages have significant codon adaptation in NS1 gene to translate these proteins in human and Aedes aegypti mosquito cells compared to the African zoonotic lineage. Accordingly, a Brazilian epidemic isolate (ZBR) produced more NS1 protein than the MR766 African lineage (ZAF) did, as indicated by proteomic data from infections of neuron progenitor cells-derived neurospheres. Although ZBR replicated more efficiently in these cells, the differences observed in the stoichiometry of ZIKV proteins were not exclusively explained by the differences in viral replication between the lineages. MAIN CONCLUSIONS Our findings suggest that natural, silent translational selection in the second half of 20th century could have improved the fitness of Asian ZIKV lineage in human and mosquito cells.

Biological and phylogenetic characteristics of West African lineages of West Nile virus.

The West Nile virus (WNV), isolated in 1937, is an arbovirus (arthropod-borne virus) that infects thousands of people each year. Despite its burden on global health, little is known about the virus' biological and evolutionary dynamics. As several lineages are endemic in West Africa, we obtained the complete polyprotein sequence from three isolates from the early 1990s, each representing a different lineage. We then investigated differences in growth behavior and pathogenicity for four distinct West African lineages in arthropod (Ap61) and primate (Vero) cell lines, and in mice. We found that genetic differences, as well as viral-host interactions, could play a role in the biological properties in different WNV isolates in vitro, such as: (i) genome replication, (ii) protein translation, (iii) particle release, and (iv) virulence. Our findings demonstrate the endemic diversity of West African WNV strains and support future investigations into (i) the nature of WNV emergence, (ii) neurological tropism, and (iii) host adaptation.

Phenotypic and Genotypic Features of a Salmonella Heidelberg Strain Isolated in Broilers in Brazil and Their Possible Association to Antibiotics and Short-Chain Organic Acids Resistance and Susceptibility.

Salmonella enterica serovar Heidelberg is a human pathogen also found in broilers. A strain (UFPR1) has been associated with field reports of resistance to short-chain organic acids (SCOA) in broilers in the South of Brazil, but was susceptible to a Bacillus subtilis-based probiotic added in feed in a related study. This work aimed to (i) report clinical symptoms caused by SH UFPR1 in broilers, (ii) study its susceptibility to some antibiotics in vitro, and (iii) SCOA in vivo; and (iv) relate these phenotypic observations with its genome characteristics. Two in vivo trials used 1-day-old chicks housed for 21 days in 8 sterilized isolated negative pressure rooms with 4 battery cages of 12 birds each. Birds were challenged or not with 107 CFU/bird of SH UFPR1 orally and exposed or not to SCOA in a 2 × 2 factorial design. Zootechnical parameters were unaffected (P > 0.05), no clinical signs were observed, and few cecal and hepatic histologic and immune-related alterations were seen, in birds challenged with SH. Formic and propionic acids added together in drinking water, fumaric and benzoic acid in feed (Trial 1), and coated calcium butyrate in feed (Trial 2) did not reduce the SH isolation frequencies seen in cecum and liver in broilers after SH challenge (P > 0.05). SH UFPR1 was susceptible to amikacin, amoxicillin + clavulanate, ceftiofur, cephalexin, doxycycline and oxytetracycline; and mildly susceptible to ampicillin + sulbactam, cephalothin, ciprofloxacin, enrofloxacin, and gentamycin in an in vitro minimum inhibitory concentration model using Mueller-Hinton agar. The whole genome of SH UFPR1 was sequenced and consisted of a circular chromosome, spanning 4,760,321 bp with 52.18% of GC-content encoding 84 tRNA, 22 rRNA, and 4,427 protein-coding genes. The comparison between SH UFPR1 genome and a multidrug-resistant SL476 strain revealed 11 missing genomic fragments and 5 insertions related to bgt, bgr, and rpoS genes. The deleted genes codify proteins associated with cell cycle regulation, virulence, drug resistance, cellular adhesion, and salt efflux which collectively reveal key aspects of the evolution and adaptation of SH strains such as organic acids resistance and antibiotic sensitivity and provide information relevant to the control of SH in poultry.