Identification and characterisation of SARS-CoV-2 and Human alphaherpesvirus 1 from a productive coinfection in a fatal COVID-19 case.

BACKGROUND: During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection. OBJECTIVES: We aimed to determine and characterise the etiological agent responsible for the poor outcome. METHODS: Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection. FINDINGS: Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR. MAIN CONCLUSIONS: We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.

An Emerging Clone, Klebsiellapneumoniae Carbapenemase 2-Producing K. pneumoniae Sequence Type 16, Associated With High Mortality Rates in a CC258-Endemic Setting.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae has become a global priority, not least in low- and middle-income countries. Here, we report the emergence and clinical impact of a novel Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) sequence type (ST) 16 clone in a clonal complex (CC) 258-endemic setting. METHODS: In a teaching Brazilian hospital, a retrospective cohort of adult KPC-KP bloodstream infection (BSI) cases (January 2014 to December 2016) was established to study the molecular epidemiology and its impact on outcome (30-day all-cause mortality). KPC-KP isolates underwent multilocus sequence typing. Survival analysis between ST/CC groups and risk factors for fatal outcome (logistic regression) were evaluated. Representative isolates underwent whole-genome sequencing and had their virulence tested in a Galleria larvae model. RESULTS: One hundred sixty-five unique KPC-KP BSI cases were identified. CC258 was predominant (66%), followed by ST16 (12%). The overall 30-day mortality rate was 60%; in contrast, 95% of ST16 cases were fatal. Patients' severity scores were high and baseline clinical variables were not statistically different across STs. In multivariate analysis, ST16 (odds ratio [OR], 21.4; 95% confidence interval [CI], 2.3-202.8; P = .008) and septic shock (OR, 11.9; 95% CI, 4.2-34.1; P < .001) were independent risk factors for fatal outcome. The ST16 clone carried up to 14 resistance genes, including blaKPC-2 in an IncFIBpQIL plasmid, KL51 capsule, and yersiniabactin virulence determinants. The ST16 clone was highly pathogenic in the larvae model. CONCLUSIONS: Mortality rates were high in this KPC-KP BSI cohort, where CC258 is endemic. An emerging ST16 clone was associated with high mortality. Our results suggest that even in endemic settings, highly virulent clones can rapidly emerge demanding constant monitoring.

High-Quality Draft Genome Sequence Resources of Eight Xylella fastidiosa Strains Isolated from Citrus, Coffee, Plum, and Hibiscus in South America.

Xylella fastidiosa subsp. pauca, once confined to South America and infecting mainly citrus and coffee plants, has been found to be associated with other hosts and in other geographic regions. We present high-quality draft genome sequences of X. fastidiosa subsp. pauca strains J1a12, B111, U24D, and XRB isolated from citrus plants in Brazil, strain Fb7 isolated from a citrus plant in Argentina and strains 3124, Pr8x, and Hib4 isolated, respectively, from coffee, plum, and hibiscus plants in Brazil. Sequencing was performed using Roche 454-GS FLX, MiSeq-Illumina or Pacific Biosciences platforms. These high-quality genome assemblies will be useful for further studies about the genomic diversity, evolution, and biology of X. fastidiosa.

Analysis of isolates from Bangladesh highlights multiple ways to carry resistance genes in Salmonella Typhi.

BACKGROUND: Typhoid fever, caused by Salmonella Typhi, follows a fecal-oral transmission route and is a major global public health concern, especially in developing countries like Bangladesh. Increasing emergence of antimicrobial resistance (AMR) is a serious issue; the list of treatments for typhoid fever is ever-decreasing. In addition to IncHI1-type plasmids, Salmonella genomic island (SGI) 11 has been reported to carry AMR genes. Although reports suggest a recent reduction in multidrug resistance (MDR) in the Indian subcontinent, the corresponding genomic changes in the background are unknown. RESULTS: Here, we assembled and annotated complete closed chromosomes and plasmids for 73 S. Typhi isolates using short-length Illumina reads. S. Typhi had an open pan-genome, and the core genome was smaller than previously reported. Considering AMR genes, we identified five variants of SGI11, including the previously reported reference sequence. Five plasmids were identified, including the new plasmids pK91 and pK43; pK43and pHCM2 were not related to AMR. The pHCM1, pPRJEB21992 and pK91 plasmids carried AMR genes and, along with the SGI11 variants, were responsible for resistance phenotypes. pK91 also contained qnr genes, conferred high ciprofloxacin resistance and was related to the H58-sublineage Bdq, which shows the same phenotype. The presence of plasmids (pHCM1 and pK91) and SGI11 were linked to two H58-lineages, Ia and Bd. Loss of plasmids and integration of resistance genes in genomic islands could contribute to the fitness advantage of lineage Ia isolates. CONCLUSIONS: Such events may explain why lineage Ia is globally widespread, while the Bd lineage is locally restricted. Further studies are required to understand how these S. Typhi AMR elements spread and generate new variants. Preventive measures such as vaccination programs should also be considered in endemic countries; such initiatives could potentially reduce the spread of AMR.

Genome of Rhizobium leucaenae strains CFN 299(T) and CPAO 29.8: searching for genes related to a successful symbiotic performance under stressful conditions.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici "group" are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian "Cerrados" soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299(T) and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions. RESULTS: The genomes of R. leucaenae strains CFN 299(T) and CPAO 29.8 were estimated at 6.7-6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299(T) is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different. CONCLUSION: A detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899(T) and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.

The genome of Anopheles darlingi, the main neotropical malaria vector.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

Comparative genomics of Bradyrhizobium japonicum CPAC 15 and Bradyrhizobium diazoefficiens CPAC 7: elite model strains for understanding symbiotic performance with soybean.

BACKGROUND: The soybean-Bradyrhizobium symbiosis can be highly efficient in fixing nitrogen, but few genomic sequences of elite inoculant strains are available. Here we contribute with information on the genomes of two commercial strains that are broadly applied to soybean crops in the tropics. B. japonicum CPAC 15 (=SEMIA 5079) is outstanding in its saprophytic capacity and competitiveness, whereas B. diazoefficiens CPAC 7 (=SEMIA 5080) is known for its high efficiency in fixing nitrogen. Both are well adapted to tropical soils. The genomes of CPAC 15 and CPAC 7 were compared to each other and also to those of B. japonicum USDA 6T and B. diazoefficiens USDA 110T. RESULTS: Differences in genome size were found between species, with B. japonicum having larger genomes than B. diazoefficiens. Although most of the four genomes were syntenic, genome rearrangements within and between species were observed, including events in the symbiosis island. In addition to the symbiotic region, several genomic islands were identified. Altogether, these features must confer high genomic plasticity that might explain adaptation and differences in symbiotic performance. It was not possible to attribute known functions to half of the predicted genes. About 10% of the genomes was composed of exclusive genes of each strain, but up to 98% of them were of unknown function or coded for mobile genetic elements. In CPAC 15, more genes were associated with secondary metabolites, nutrient transport, iron-acquisition and IAA metabolism, potentially correlated with higher saprophytic capacity and competitiveness than seen with CPAC 7. In CPAC 7, more genes were related to the metabolism of amino acids and hydrogen uptake, potentially correlated with higher efficiency of nitrogen fixation than seen with CPAC 15. CONCLUSIONS: Several differences and similarities detected between the two elite soybean-inoculant strains and between the two species of Bradyrhizobium provide new insights into adaptation to tropical soils, efficiency of N2 fixation, nodulation and competitiveness.

De novo transcriptome analysis of Hevea brasiliensis tissues by RNA-seq and screening for molecular markers.

BACKGROUND: The rubber tree, Hevea brasiliensis, is a species native to the Brazilian Amazon region and it supplies almost all the world's natural rubber, a strategic raw material for a variety of products. One of the major challenges for developing rubber tree plantations is adapting the plant to biotic and abiotic stress. Transcriptome analysis is one of the main approaches for identifying the complete set of active genes in a cell or tissue for a specific developmental stage or physiological condition. RESULTS: Here, we report on the sequencing, assembling, annotation and screening for molecular markers from a pool of H. brasiliensis tissues. A total of 17,166 contigs were successfully annotated. Then, 2,191 Single Nucleotide Variation (SNV) and 1.397 Simple Sequence Repeat (SSR) loci were discriminated from the sequences. From 306 putative, mainly non-synonymous SNVs located in CDS sequences, 191 were checked for their ability to characterize 23 Hevea genotypes by an allele-specific amplification technology. For 172 (90%), the nucleotide variation at the predicted genomic location was confirmed, thus validating the different steps from sequencing to the in silico detection of the SNVs. CONCLUSIONS: This is the first study of the H. brasiliensis transcriptome, covering a wide range of tissues and organs, leading to the production of the first developed SNP markers. This process could be amplified to a larger set of in silico detected SNVs in expressed genes in order to increase the marker density in available and future genetic maps. The results obtained in this study will contribute to the H. brasiliensis genetic breeding program focused on improving of disease resistance and latex yield.

Comparative analysis of the complete genome of KPC-2-producing Klebsiella pneumoniae Kp13 reveals remarkable genome plasticity and a wide repertoire of virulence and resistance mechanisms.

BACKGROUND: Klebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial and community-acquired infections. A wide repertoire of virulence and antimicrobial resistance genes is present in K. pneumoniae genomes, which can constitute extra challenges in the treatment of infections caused by some strains. K. pneumoniae Kp13 is a multidrug-resistant strain responsible for causing a large nosocomial outbreak in a teaching hospital located in Southern Brazil. Kp13 produces K. pneumoniae carbapenemase (KPC-2) but is unrelated to isolates belonging to ST 258 and ST 11, the main clusters associated with the worldwide dissemination of KPC-producing K. pneumoniae. In this report, we perform a genomic comparison between Kp13 and each of the following three K. pneumoniae genomes: MGH 78578, NTUH-K2044 and 342. RESULTS: We have completely determined the genome of K. pneumoniae Kp13, which comprises one chromosome (5.3 Mbp) and six plasmids (0.43 Mbp). Several virulence and resistance determinants were identified in strain Kp13. Specifically, we detected genes coding for six beta-lactamases (SHV-12, OXA-9, TEM-1, CTX-M-2, SHV-110 and KPC-2), eight adhesin-related gene clusters, including regions coding for types 1 (fim) and 3 (mrk) fimbrial adhesins. The rmtG plasmidial 16S rRNA methyltransferase gene was also detected, as well as efflux pumps belonging to five different families. Mutations upstream the OmpK35 porin-encoding gene were evidenced, possibly affecting its expression. SNPs analysis relative to the compared strains revealed 141 mutations falling within CDSs related to drug resistance which could also influence the Kp13 lifestyle. Finally, the genetic apparatus for synthesis of the yersiniabactin siderophore was identified within a plasticity region. Chromosomal architectural analysis allowed for the detection of 13 regions of difference in Kp13 relative to the compared strains. CONCLUSIONS: Our results indicate that the plasticity occurring at many hierarchical levels (from whole genomic segments to individual nucleotide bases) may play a role on the lifestyle of K. pneumoniae Kp13 and underlie the importance of whole-genome sequencing to study bacterial pathogens. The general chromosomal structure was somewhat conserved among the compared bacteria, and recombination events with consequent gain/loss of genomic segments appears to be driving the evolution of these strains.

Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins.

BACKGROUND: Metarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins. RESULTS: We determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity. CONCLUSIONS: The data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.

Immune response stability to the SARS-CoV-2 mRNA vaccine booster is influenced by differential splicing of HLA genes.

Many molecular mechanisms that lead to the host antibody response to COVID-19 vaccines remain largely unknown. In this study, we used serum antibody detection combined with whole blood RNA-based transcriptome analysis to investigate variability in vaccine response in healthy recipients of a booster (third) dose schedule of the mRNA BNT162b2 vaccine against COVID-19. The cohort was divided into two groups: (1) low-stable individuals, with antibody concentration anti-SARS-CoV IgG S1 below 0.4 percentile at 180 days after boosting vaccination; and (2) high-stable individuals, with antibody values greater than 0.6 percentile of the range in the same period (median 9525 [185-80,000] AU/mL). Differential gene expression, expressed single nucleotide variants and insertions/deletions, differential splicing events, and allelic imbalance were explored to broaden our understanding of the immune response sustenance. Our analysis revealed a differential expression of genes with immunological functions in individuals with low antibody titers, compared to those with higher antibody titers, underscoring the fundamental importance of the innate immune response for boosting immunity. Our findings also provide new insights into the determinants of the immune response variability to the SARS-CoV-2 mRNA vaccine booster, highlighting the significance of differential splicing regulatory mechanisms, mainly concerning HLA alleles, in delineating vaccine immunogenicity.

SARS-CoV-2 Research in Dogs (Canis lupus familiaris) and Felines (Felis silvestris catus) Domiciled in an International Border Region (Paraguay and Brazil).

Introduction: COVID-19 is an infectious disease caused by SARS-CoV-2 that has become a serious threat to public health owing to its rapid spread from aerosols from infected people. Despite being considered a strictly human disease, there are reports in the literature about animals with confirmed presence of the virus. Aim: Owing to the scarcity of scientific literature on the potential for infection of animals and their importance for One Health, the objective of this work was to research SARS-CoV-2 RNA in felines (Felis silvestris catus) and dogs (Canis lupus familiaris) domiciled. Materials and Methods: Oropharyngeal swabs were collected from domestic dogs and cats belonging to patients diagnosed with COVID-19 from August to October 2021 and residents of the northwest and west regions of Paraná, Brazil. Results: Of the 34 samples collected, 14 were from dogs and 20 from cats. Three of these samples tested positive in real-time PCR, and two of them were also positive in the immunochromatographic test. After testing positive in real-time PCR, the samples underwent genetic sequencing using the Illumina COVIDSeq test. Of the 34 samples collected, three (9%), all of them female and from the feline species, tested positive in real-time PCR, with two of these (67%) also testing positive in the immunochromatographic test. Regarding sequencing, it was possible to sequence the three samples aligned with the AY.101 lineage, corresponding to the Delta variant. Conclusion: The occurrence of SARS-CoV-2 infection in dogs and cats is seen as an unintended event with significant implications for public health, including its potential transmission to other animal species. Further research is required to enhance our understanding of how this disease spreads among these animals and its broader impact on One Health initiatives.

Monophyletic origin of magnetotaxis and the first magnetosomes.

Horizontal gene transfer (HGT), the transfer of genetic material other than by descent, is thought to have played significant roles in the evolution and distribution of genes in prokaryotes. These include those responsible for the ability of motile, aquatic magnetotactic bacteria (MTB) to align and swim along magnetic field lines and the biomineralization of magnetosomes that are responsible for this behaviour. There is some genomic evidence that HGT might be responsible for the distribution of magnetosome genes in different phylogenetic groups of bacteria. For example, in the genomes of a number of MTB, magnetosome genes are present as clusters within a larger structure known as the magnetosome genomic island surrounded by mobile elements such as insertion sequences and transposases as well as tRNA genes. Despite this, there is no strong direct proof of HGT between these organisms. Here we show that a phylogenetic tree based on magnetosome protein amino acid sequences from a number of MTB was congruent with the tree based on the organisms' 16S rRNA gene sequences. This shows that evolution and divergence of these proteins and the 16S rRNA gene occurred similarly. This suggests that magnetotaxis originated monophyletically in the Proteobacteria phylum and implies that the common ancestor of all Proteobacteria was magnetotactic.

Comparative genomic analysis of magnetotactic bacteria from the Deltaproteobacteria provides new insights into magnetite and greigite magnetosome genes required for magnetotaxis.

Magnetotactic bacteria (MTB) represent a group of diverse motile prokaryotes that biomineralize magnetosomes, the organelles responsible for magnetotaxis. Magnetosomes consist of intracellular, membrane-bounded, tens-of-nanometre-sized crystals of the magnetic minerals magnetite (Fe3O4) or greigite (Fe3S4) and are usually organized as a chain within the cell acting like a compass needle. Most information regarding the biomineralization processes involved in magnetosome formation comes from studies involving Alphaproteobacteria species which biomineralize cuboctahedral and elongated prismatic crystals of magnetite. Many magnetosome genes, the mam genes, identified in these organisms are conserved in all known MTB. Here we present a comparative genomic analysis of magnetotactic Deltaproteobacteria that synthesize bullet-shaped crystals of magnetite and/or greigite. We show that in addition to mam genes, there is a conserved set of genes, designated mad genes, specific to the magnetotactic Deltaproteobacteria, some also being present in Candidatus Magnetobacterium bavaricum of the Nitrospirae phylum, but absent in the magnetotactic Alphaproteobacteria. Our results suggest that the number of genes associated with magnetotaxis in magnetotactic Deltaproteobacteria is larger than previously thought. We also demonstrate that the minimum set of mam genes necessary for magnetosome formation in Magnetospirillum is also conserved in magnetite-producing, magnetotactic Deltaproteobacteria. Some putative novel functions of mad genes are discussed.

Antimicrobial resistance and genetic background of non-typhoidal Salmonella enterica strains isolated from human infections in São Paulo, Brazil (2000-2019).

Salmonella enterica causes Salmonellosis, an important infection in humans and other animals. The number of multidrug-resistant (MDR) phenotypes associated with Salmonella spp. isolates is increasing worldwide, causing public health concern. Here, we aim to characterize the antimicrobial-resistant phenotype of 789 non-typhoidal S. enterica strains isolated from human infections in the state of São Paulo, Brazil, along 20 years (2000-2019). Among the non-susceptible isolates, 31.55, 14.06, and 13.18% were resistant to aminoglycosides, tetracycline, and ß-lactams, respectively. Moreover, 68 and 11 isolates were considered MDR and Extended Spectrum ß-Lactamase (ESBL) producers, respectively, whereas one isolate was colistin-resistant. We selected four strains to obtain a draft of the Genome Sequence; one S. Infantis (ST32), one S. Enteritidis (ST11), one S. I 4,[5],12:i:- (ST19), and one S. Typhimurium (ST313). Among them, three presented at least one of the following antimicrobial resistance genes (AMR) linked to mobile DNA: blaTEM-1B, dfrA1, tetA, sul1, floR, aac(6')-laa, and qnrE1. This is the first description of the plasmid-mediated quinolone resistance (PMQR) gene qnrE1 in a clinical isolate of S. I 4,[5],12:i:-. The S. Typhimurium is a colistin-resistant isolate, but did not harbor mcr genes, but it presented mutations within the mgrB, pmrB, and pmrC regions that might be linked to the colistin-resistant phenotype. The virulence pattern of the four isolates resembled the virulence pattern of the highly pathogenic S. Typhimurium UK-1 reference strain in assays involving the in vivo Galleria mellonella model. In conclusion, most isolates studied here are susceptible, but a small percentage present an MDR or ESBL-producer and pathogenic phenotype. Sequence analyses revealed plasmid-encoded AMR genes, such as ß-lactam and fluoroquinolone resistance genes, indicating that these characteristics can be potentially disseminated among other bacterial strains.

Seroprevalence, Prevalence, and Genomic Surveillance: Monitoring the Initial Phases of the SARS-CoV-2 Pandemic in Betim, Brazil.

The COVID-19 pandemic has created an unprecedented need for epidemiological monitoring using diverse strategies. We conducted a project combining prevalence, seroprevalence, and genomic surveillance approaches to describe the initial pandemic stages in Betim City, Brazil. We collected 3239 subjects in a population-based age-, sex- and neighborhood-stratified, household, prospective; cross-sectional study divided into three surveys 21 days apart sampling the same geographical area. In the first survey, overall prevalence (participants positive in serological or molecular tests) reached 0.46% (90% CI 0.12-0.80%), followed by 2.69% (90% CI 1.88-3.49%) in the second survey and 6.67% (90% CI 5.42-7.92%) in the third. The underreporting reached 11, 19.6, and 20.4 times in each survey. We observed increased odds to test positive in females compared to males (OR 1.88 95% CI 1.25-2.82), while the single best predictor for positivity was ageusia/anosmia (OR 8.12, 95% CI 4.72-13.98). Thirty-five SARS-CoV-2 genomes were sequenced, of which 18 were classified as lineage B.1.1.28, while 17 were B.1.1.33. Multiple independent viral introductions were observed. Integration of multiple epidemiological strategies was able to adequately describe COVID-19 dispersion in the city. Presented results have helped local government authorities to guide pandemic management.

Cellular Growth Arrest and Efflux Pumps Are Associated With Antibiotic Persisters in Streptococcus pyogenes Induced in Biofilm-Like Environments.

Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections; however, none of them have fully elucidated this phenomenon. In this study, we show that GAS strains have the ability to form antimicrobial persisters when inoculated on abiotic surfaces to form a film of bacterial agglomerates (biofilm-like environment). Our data suggest that efflux pumps were possibly involved in this phenomenon. In fact, gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persisters arising in the presence of penicillin. Phenotypic reversion assay and whole-genome sequencing indicated that this event was due to non-inherited resistance mechanisms. The persister cells showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, the proteomic analysis revealed that susceptible cells express higher levels of ribosome proteins. It is remarkable that previous studies have reported the recovery of S. pyogenes viable cells from tissue biopsies of patients presented with GAS invasive infections and submitted to therapy with antibiotics. The persistence phenomenon described herein brings new insights into the origin of therapeutic failures in S. pyogenes infections. Multifactorial mechanisms involving protein synthesis inhibition, cell growth impairment and efflux pumps seem to play roles in the formation of antimicrobial persisters in S. pyogenes.

Genome and plasmid context of two rmtG-carrying Enterobacter hormaechei isolated from urinary tract infections in Brazil.

OBJECTIVES: Enterobacter hormaechei is an important causative agent of severe infections in critically ill patients. Aminoglycosides are among the main antibiotics for the treatment of E. hormaechei infections, however the development of antimicrobial resistance is an increasing problem. RmtG is a 16S rRNA methyltransferase, a class of enzymes conferring high-level resistance to clinically relevant aminoglycosides. The aim of this study was to characterise the full genetic context of plasmids harbouring the rmtG gene in two aminoglycoside-resistant E. hormaechei isolated in Brazil. METHODS: ThermtG-harbouring plasmids were transferred to an Escherichia coli J53 recipient strain and were fully sequenced using a MiSeq sequencing system. Complete genome assemblies were accomplished using a combination of Newbler v.3.0, SPAdes 3.10.0 and phrap/cross_match programs. Plasmid sequences were annotated using RAST server and were then manually curated using BLAST databases and ISfinder. Easyfig 2.0 was used to map and compare regions of interest containing rmtG in both plasmids. RESULTS: Both isolates carried thermtG gene on an IncA/C plasmid of ˜152kb and ˜235kb, respectively, associated with a Tn3 transposon. The plasmids contain a transfer region as well as genes involved in plasmid stability and resistance to ß-lactams, sulfonamides and quaternary ammonium compounds. One of the plasmids also carried the mrk operon encoding type 3 fimbriae. CONCLUSION: This first detection ofrmtG in E. hormaechei supports the ability for horizontal transfer. The location in complex genetic platforms carried by Tn3 transposons in IncA/C plasmids may facilitate dissemination to other Gram-negative pathogens, further limiting treatment options.

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination.

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations.

Draft Genome Sequence of the Fungus Penicillium brasilianum (Strain LaBioMMi 136), a Plant Endophyte from Melia azedarach.

Penicillium brasilianum (strain LaBioMMi 136) has been reported to be a great producer of secondary metabolites and a source of enzymes of biotechnological interest. Here, we report the draft genome sequence of P. brasilianum (strain LaBioMMi 136), isolated as an endophyte from the plant Melia azedarach (family Meliaceae).