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The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other β-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6')-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies.

Highlights • Epidemiological success of KPC has been linked to plasmids carrying blaKPC genes. • An IncQ1 small plasmid carrying blaKPC-2 was found in pandemic Escherichia coli ST648. • Plasmid analysis revealed blaKPC-2 on an NTEKPC-IId element with the aph(3')-VIa gene. • Plasmid phylogeny confirmed >99% identity with IncQ/blaKPC-2 from Klebsiella pneumoniae. • The emergence and rapid expansion of IncQ1/blaKPC-2 to novel hosts is discussed., Objectives The global success of carbapenem-resistant pathogens has been attributed to large plasmids carrying blaKPC genes circulating among high-risk clones. In this study, we sequenced the genome of a carbapenem-resistant Escherichia coli strain (Ec351) isolated from a human infection. Phylogenomic analysis based on single nucleotide polymorphisms (SNPs) as well as the comparative resistome and plasmidome of globally disseminated blaKPC-2-positive E. coli strains with identical sequence type (ST) were further investigated. Methods Total DNA was sequenced using an Illumina NextSeq 500 platform and was assembled using Unicycler. Genomic data were evaluated through bioinformatics tools available from the Center of Genomic Epidemiology and by in silico analysis. Results Genomic analysis revealed the convergence of a wide resistome and virulome in E. coli ST648, showing a high-level phylogenetic relationship with a KPC-2-positive ST648 cluster identified in the USA and association with international clade 2. Additionally, the emergence of an IncQ1 small plasmid (pEc351) carrying blaKPC-2 (on an NTEKPC-IId element), aph(3')-VIa, and plasmid regulatory and replication genes in the pandemic clone ST648 is reported. Conclusion Identification of a blaKPC-2-positive IncQ1 plasmid in a high-risk E. coli clone represents rapid adaptation and expansion of these small plasmids encoding carbapenemases to novel bacterial hosts with global distribution, which deserves continued monitoring.

This study used whole-genome sequencing to analyze the first case of NDM-1-producing Acinetobacter baumannii belonging to the novel sequence type 1465/CC216 recovered in Brazil. The study identified an unusual plasmid carrying blaNDM-1 gene, in which some genes of the Tn125 transposon were lost. Besides, on the chromosome, the strain reported here presented blaOXA-106 gene, a variant of blaOXA-51 gene, and blaADC-25 with ISAba1 upstream. The isolation of new STs of A. baumannii carrying blaNDM-1 genes elicits our concerns about the possible spread of these genes among clinically relevant bacteria.

BackgroundOngoing research of the mosquito microbiome aims to uncover novel strategies to reduce pathogen transmission. Sequencing costs, especially for metagenomics, are however still significant. A resource that is increasingly used to gain insights into host-associated microbiomes is the large amount of publicly available genomic data based on whole organisms like mosquitoes, which includes sequencing reads of the host-associated microbes and provides the opportunity to gain additional value of these initially host-focused sequencing projects.MethodsTo analyse non-host reads from existing genomic data, we developed a snakemake workflow called MINUUR (MicrobialINsightsUsingUnmappedReads). Within MINUUR, reads derived from the host-associated microbiome were extracted and characterised using taxonomic classifications and metagenome assembly followed by binning and quality assessment. We applied this pipeline to five publicly availableAedes aegyptigenomic datasets, consisting of 62 samples with a broad range of sequencing depths.ResultsWe demonstrate that MINUUR recovers previously identified phyla and genera and is able to extract bacterial metagenome assembled genomes (MAGs) associated to the microbiome. Of these MAGS, 42 are high-quality representatives with >90% completeness and ConclusionsOur metagenomics workflow, MINUUR, was applied to a range ofAedes aegyptigenomic samples to characterise microbiome-associated reads. We confirm the presence of key mosquito-associated symbionts that have previously been identified in other studies and recovered high-quality bacterial MAGs. In addition, MINUUR and its associated documentation are freely available on GitHub (https://github.com/aidanfoo96/MINUUR) and provide researchers with a convenient workflow to investigate microbiome data included in the sequencing data for any applicable host genome of interest.

The emergence and rapid spread of carbapenemase-producing Enterobacterales represents a serious public health concern. Critically, these global priority bacteria have begun to be reported in companion animals, implying a potential risk of cross-transmission between humans and pets. Using long-read (MinION) and short-read (Illumina) sequencing technologies, we have identified and characterized a hypermucoviscous KPC-2-producing Klebsiella pneumoniae strain belonging to the high-risk international clone ST11/CG258, in a dog with urinary tract infection. Strikingly, the blaKPC-2 gene was carried by a 54-kb IncN plasmid assignated to ST15, which shared 99.8 and 96.8% pairwise identity with IncN-pST15 plasmids from human and environmental K. pneumoniae strains, respectively; all come from an area with high endemicity of KPC-2. Our findings suggest that IncN-pST15 plasmids conferring carbapenem resistance can play as important a role as clonal transmission of K. pneumoniae, representing another major challenge for One Health.

A multidrug-resistant CTX-M-15-producing Klebsiella pneumoniae (KpP1 strain) was isolated from a native Amazonian fish (Brachyplatystoma filamentosum) at the Brazilian Amazon. The strain was identified by MALDI-TOF. The genome was extracted, purified and a Nextera DNA Flex library was prepared and sequenced by Illumina platform. The sequenced genome was de novo assembled using Unicycler and in silico prediction accomplished by curated bioinformatics tools. The size of the genome is 5.6 Mb with 5715 genes. Whole-genome sequencing analysis revealed the presence of wide resistome, with genes conferring resistance to clinically relevant antibiotics, heavy metals and disinfectants. The KpP1 strain was assigned to the sequence type ST3827, KL111 (wzi113) and O3b locus. Native freshwater fish sold in wet markets of the Amazonian region could be an important vehicle for transmission of multidrug-resistant bacteria to humans. This study may give genomic insights on the spread of critical-priority WHO pathogens in a One Health context., Highlights • A multidrug-resistant Klebsiella pneumoniae was isolated from a native Amazonian fish. • Genomic analysis revealed that K. pneumoniae belonged to ST3827, KL111 and O3b locus. • A wide resistome against heavy metals, disinfectants, and relevant antibiotics was predicted. • ESBL production was associated with the blaCTX-M-15 gene carried on an IncFII(K) plasmid. • Native freshwater fishes could be a vehicle for transmission of critical-priority pathogens.

The emergence and dissemination of high-risk clones of Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBLs) in animal infections is a critical issue. We report the detection and g...

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 ˜152 kb and ˜235 kb, respectively, associated with a Tn3 transposon. The plasmids contain a transfer region as well as genes involved in plasmid stability and resistance to β-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.

Wolbachia is a genus of obligate bacterial endosymbionts that infect a diverse range of arthropod species as well as filarial nematodes, with its single described species, Wolbachia pipientis , divided into several ‘supergroups’ based on multilocus sequence typing. Wolbachia strains in mosquitoes have been shown to inhibit the transmission of human pathogens, including Plasmodium malaria parasites and arboviruses. Despite their large host range, Wolbachia strains within the major malaria vectors of the Anopheles gambiae and Anopheles funestus complexes appear at low density, established solely on PCR-based methods. Questions have been raised as to whether this represents a true endosymbiotic relationship. However, recent definitive evidence for two distinct, high-density strains of supergroup B Wolbachia within Anopheles demeilloni and Anopheles moucheti has opened exciting possibilities to explore naturally occurring Wolbachia endosymbionts in Anopheles for biocontrol strategies to block Plasmodium transmission. Here, we utilize genomic analyses to demonstrate that both Wolbachia strains have retained all key metabolic and transport pathways despite their smaller genome size, with this reduction potentially attributable to degenerated prophage regions. Even with this reduction, we confirmed the presence of cytoplasmic incompatibility (CI) factor genes within both strains, with wAnD maintaining intact copies of these genes while the cifB gene was interrupted in wAnM, so functional analysis is required to determine whether wAnM can induce CI. Additionally, phylogenetic analysis indicates that these Wolbachia strains may have been introduced into these two Anopheles species via horizontal transmission events, rather than by ancestral acquisition and subsequent loss events in the Anopheles gambiae species complex. These are the first Wolbachia genomes, to our knowledge, that enable us to study the relationship between natural strain Plasmodium malaria parasites and their anopheline hosts.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family Enterobacteriaceae belong to Salmonella , first found in Salmonella enterica Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. Screening of assembly graphs assembled from public read sets identified linear plasmid structures in a further 13 K . pneumoniae species complex genomes. We used these 25 linear plasmid sequences to query all bacterial genome assemblies in the National Center for Biotechnology Information database, and discovered an additional 61 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function; however, each phylogroup carried its own unique toxin–antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug-resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.

Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family

Background: Ongoing research of the mosquito microbiome aims to uncover novel strategies to reduce pathogen transmission. Sequencing costs, especially for metagenomics, are however still significant. A resource that is increasingly used to gain insights into host-associated microbiomes is the large amount of publicly available genomic data based on whole organisms like mosquitoes, which includes sequencing reads of the host-associated microbes and provides the opportunity to gain additional value from these initially host-focused sequencing projects. Methods: To analyse non-host reads from existing genomic data, we developed a snakemake workflow called MINUUR (Microbial INsights Using Unmapped Reads). Within MINUUR, reads derived from the host-associated microbiome were extracted and characterised using taxonomic classifications and metagenome assembly followed by binning and quality assessment. We applied this pipeline to five publicly available Aedes aegypti genomic datasets, consisting of 62 samples with a broad range of sequencing depths. Results: We demonstrate that MINUUR recovers previously identified phyla and genera and is able to extract bacterial metagenome assembled genomes (MAGs) associated to the microbiome. Of these MAGS, 42 are high-quality representatives with >90% completeness and Conclusions: Our metagenomics workflow, MINUUR, was applied to a range of Aedes aegypti genomic samples to characterise microbiome-associated reads. We confirm the presence of key mosquito-associated symbionts that have previously been identified in other studies and recovered high-quality bacterial MAGs. In addition, MINUUR and its associated documentation are freely available on GitHub and provide researchers with a convenient workflow to investigate microbiome data included in the sequencing data for any applicable host genome of interest.

WGS-based surveillance has significantly improved the ability to track global spread and emergence of multidrug-resistant clones of clinically relevant pathogens. In this study, we performed the genomic characterization and comparative analysis of an Acinetobacter baumannii (strain Ac56) belonging to the sequence type ST374, which was isolated for the first time in Brazil, in 1996. Genomic analysis of Ac56 predicted a total of 5373 genes, with 3012 being identical across nine genomes of A. baumannii isolates of ST374 from European, Asian, North and South American countries. GoeBURST analysis grouped ST374 lineages into clonal complex CC3 (international clone IC-III). Resistome analysis of ST374 clone predicted genes associated with resistance to heavy metals and clinically relevant beta-lactams and aminoglycosides antibiotics. In this regard, in two closely related A. baumannii strains, the intrinsic blaADC gene was linked to the insertion sequence ISAba1; including the Ac56 strain, where it has been possibly associated with intermediate susceptibility to meropenem. Other four carbapenem-resistant A. baumannii strains carried the ISAba1/blaOXA-23 gene array, which was associated with the transposon Tn2008 or with Tn2006 in an AbaR4-type resistance island. While most virulence genes were shared for A. baumannii strains of ST374, three isolates from Thailand harbored KL49 capsular loci, previously identified in the hypervirulent A. baumannii LAC-4 strain. Analysis of thirty-four predicted plasmids showed eight major groups, of which GR-6 (LN-1) and GR-2 (LN-2) were prevalent. All strains, including the earliest isolate Ac56 harbored at least one complete prophage, whereas none CRISPR-associated (cas) gene was detected. In summary, genomic data of A. baumannii ST374 reveal a potential of this lineage to become a successful clone.

Hemodialysis patients are at high risk for bloodstream infections associated with highest morbidity and mortality rates. Bacterial species not commonly related to such infections has been hardly identified by traditional methods. Pseudocitrobacter is a novel genus of the order Enterobacterales that is associated with carbapenemase genes and nosocomial infection. In this context, we have investigated nine cases of bloodstream infections by carbapenem-resistant Gram-negative bacilli in patients assisted at a hemodialysis unit in Brazil. The infections were caused by a metallo-β-lactamase (IMP-1)-producing clone (> 90% XbaI-PFGE similarity) of Pseudocitrobacter vendiensis, displaying a multidrug-resistant profile to broad-spectrum cephalosporins, carbapenems, chloramphenicol, and trimethoprim-sulfamethoxazole. S1-PFGE and Southern blot hybridization revealed that bla(IMP-1) was carried by a 200-kb IncC/ST3 plasmid. Patients were successfully treated with amikacin, and strict disinfection procedures and hand washing protocols were reinforced. We report the emergence of P. vendiensis, a recently described species of the genus, in bloodstream infections of patients undergoing hemodialysis. Considering the epidemic potential of carbapenemase-producing Enterobacterales in hospital settings, surveillance of this emerging pathogen is of utmost importance.

Linear plasmids are extrachromosomal DNA that have been found in a small number of bacterial species. To date, the only linear plasmids described in the Enterobacteriaceae family belong to Salmonella, first found in Salmonella Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. We used this collection to search public sequence databases and discovered an additional 74 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function, however each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.Significance as a BioResource to the communityThis study provides the first report of linear plasmids identified within the Klebsiella pneumoniae species complex and the first report in Enterobacteriaceae besides Salmonella.We present the first comparative analysis of linear plasmid sequences in Enterobacteriaceae, however whilst this family is highly clinically significant, the functional and/or evolutionary importance of these plasmids is not yet clear. To facilitate future studies to address these questions, we have publicly deposited (i) the collection of linear plasmid sequence data; (ii) isolates representative of each of the distinct linear plasmid phylogroups.Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary informationWhole genome sequence reads from Klebsiella pneumoniae isolates sequenced in this study have been deposited in NCBI SRA under the accession numbers listed in Table S1.Representative annotated sequences of one linear plasmid per phylogroup have been deposited in FigShare, doi 10.26180/16729126.A copy of all linear plasmid sequences that we assembled from publicly available genome sequence reads are available in FigShare, doi 10.26180/16531365. Read accessions for these are given in Table S1.Eleven representative K. pneumoniae isolates harbouring linear plasmids described in this study have been deposited with the National Collection of Type Cultures (NCTC) and are available for purchase under the NCTC accession numbers listed in Table S1.K. pneumoniae 1194/11 (representative of phylogroup B) has been deposited in the Microorganisms Collection Center, Adolfo Lutz Institute, São Paulo, Brazil. To request strain 1194/11 (IAL 3063, SISGEN ABBF09B), contact: Microorganisms Collection CenterCulture Collection LaboratoryInstituto Adolfo Lutz, Sao Paulo State Department of HealthAddress: Av Dr Arnaldo, 351, 10 floor, room 1020Phone number: +55 11 3068-2884Zip code 01246-000, São Paulo, BrazilE-mail: colecaoial@ial.sp.gov.brAlignments of terminal inverted repeat sequences for each phylogroup can be found in Data S1, available on FigShare, doi 10.26180/16531371.

Klebsiella pneumoniae is an opportunistic pathogen that can cause several infections, mainly in hospitalised or immunocompromised individuals. The spread of K. pneumoniae emerging virulent and multidrug-resistant clones is a worldwide concern and its identification is crucial to control these strains especially in hospitals. This article reports data related to multi-resistant K. pneumoniae strains, isolated from inpatients in the city of Manaus, Brazil, harbouring virulence and antimicrobial resistance genes, including high-risk international clones belonging to clonal group (CG) 258. Twenty-one strains isolated from different patients admitted to four hospitals in the city of Manaus, located in the state of Amazonas, Northern Brazil (Amazon Rainforest region) were evaluated. The majority of strains (61.9 % n = 13) were classified as multidrug-resistant (MDR), and five strains (23.8 %) as extensively drug-resistant (XDR). Several virulence and antimicrobial resistance genes were found among the strains and eight strains (38.1 %) presented the hypermucoviscous phenotype. MLST analysis demonstrated a great diversity of STs among the strains, totaling 12 different STs (ST11, ST23, ST198, ST277, ST307, ST340, ST378, ST462, ST502, ST3991, ST3993 and ST5209). Four of these (ST11, ST23, ST307 and ST340) belong to CG258.

Klebsiella pneumoniae is an opportunistic pathogen that can cause several infections, mainly in hospitalised or immunocompromised individuals. The spread of K. pneumoniae emerging virulent and multidrug-resistant clones is a worldwide concern and its identification is crucial to control these strains especially in hospitals. This article reports data related to multi-resistant K. pneumoniae strains, isolated from inpatients in the city of Manaus, Brazil, harbouring virulence and antimicrobial-resistance genes, including high-risk international clones belonging to clonal group (CG) 258. Twenty-one strains isolated from different patients admitted to four hospitals in the city of Manaus, located in the state of Amazonas, Northern Brazil (Amazon Rainforest region) were evaluated. The majority of strains (61.9% n = 13) were classified as multidrug-resistant (MDR), and five strains (23.8%) as extensively drug-resistant (XDR). Several virulence and antimicrobial-resistance genes were found among the strains and eight strains (38.1%) presented the hyper-mucoviscous phenotype. MLST analysis demonstrated a great diversity of STs among the strains, totaling 12 different STs (ST11, ST23, ST198, ST277, ST307, ST340, ST378, ST462, ST502, ST3991, ST3993 and ST5209). Three of these (ST11, ST23 and ST340) belong to CG258.

The increasing prevalence and dissemination of carbapenemase-producing

Background Carbapenemase-producing Acinetobacter baumannii has been recognized as a critical priority pathogen by the World Health Organization (WHO). Objective We hereby report the identification and the draft genome sequence of a carbapenem-resistant A. baumannii isolated from a patient with community-onset urinary tract infection, in a Chilean Patagonian city. Methods The whole genome was sequenced on an Illumina NextSeq platform and de novo assembled using Unicycler v.0.4. Resistome analysis and epidemiological investigation (based on MLST data and Pasteur scheme) were performed using bioinformatics tools available from Center for Genomic Epidemiology. Results The genome size was calculated at 3,890,824 bp, with a GC content of 39.1%, and comprising 3,864 total genes, 30 tRNAs, 3 rRNAs, 4 ncRNAs, and 109 pseudogenes. Carbapenem-resistant A. baumannii Ab3_Ch strain belonged to the international sequence type ST15 (clonal complex, CC15), and harboured the ISAba-1-blaOXA-219 gene array, along to blaTEM-1B and blaADC-6 β-lactamase genes, and aac(3)-IIa and aph(3’)-VIa aminoglycoside resistance genes. Additionally, efflux pump encoding genes (abaF, abaQ, abeS, adeI, adeK, adeL, adeN, adeR, adeS, and amvA) were identified, and mutations in the quinolone resistance-determining region (QRDR) of gyrA (Ser81Leu) and parC (Ser84Leu) were considered responsible for fluoroquinolone resistance. Conclusion This genome sequence data could be used for comparative genomic studies of critical priority A. baumannii strains, as well as to understand the specific features of hospital-associated A. baumannii lineages of international clonal complexes emerging in community settings.

The dissemination of antimicrobial resistance genes and the bacterium that harbor them have increasingly become a public concern, especially in low- and middle-income countries. The present study used whole-genome sequencing to analyze 10 KPC-2-producing Klebsiella pneumoniae isolates obtained from clinical specimens originated from Brazilian hospitals. The study documents a relevant "snapshot" of the presence of class 1 integrons in 90% of the strains presenting different gene cassettes (dfrA30, dfrA15, dfrA12, dfrA14, aadA1, aadA2, and aac(6')Iq), associated or not with transposons. Two strains presented nonclassical integron (lacking the normal 3'conserved segment). In general, most strains showed a complex resistome, characterizing them as highly resistant. Integrons, a genetically stable and efficient system, confer to bacteria as highly adaptive and low cost evolution potential to bacteria, even more serious when associated with high-risk clones, indicating an urgent need for control and prevention strategies to avoid the spread of resistance determinants in Brazil. Despite this, although the class 1 integron identified in the KPC-2-producing K. pneumoniae clones is important, our findings suggest that other elements probably have a greater impact on the spread of antimicrobial resistance, since many of these important genes were not related to this cassette.

A colistin-resistant Salmonella enterica 4, [5],12:i:- sequence type (ST) 34 harbouring mcr-3.1 was recovered from a patient who travelled to China 2 weeks prior to diarrhoea onset. Genomic analysis revealed the presence of the mcr-3.1 gene located in the globally disseminated IncHI2 plasmid, highlighting the intercontinental dissemination of the colistin-resistant S. enterica 4, [5],12:i:- ST34 pandemic clone.

We report the occurrence and genomic features of multidrug-resistant vancomycin-resistant Enterococcus faecium vanA belonging to a novel sequence type (designated ST1336), carrying a Tn1546-like element, in marine brown mussels (Perna perna) from anthropogenically affected coastal waters of the Atlantic coast of Brazil, highlighting a potential source of dissemination for related ecosystems, with additional consequences for seafood safety and quality.

Assembly of bacterial genomes from long-read data (generated by Oxford Nanopore or Pacific Biosciences platforms) can often be complete: a single contig for each chromosome or plasmid in the genome. However, even complete bacterial genome assemblies constructed solely from long reads still contain a variety of errors, and different assemblies of the same genome often contain different errors. Here, we present Trycycler, a tool which produces a consensus assembly from multiple input assemblies of the same genome. Benchmarking using both simulated and real sequencing reads showed that Trycycler consensus assemblies contained fewer errors than any of those constructed with a single long-read assembler. Post-assembly polishing with Medaka and Pilon further reduced errors and yielded the most accurate genome assemblies in our study. As Trycycler can require human judgement and manual intervention, its output is not deterministic, and different users can produce different Trycycler assemblies from the same input data. However, we demonstrated that multiple users with minimal training converge on similar assemblies that are consistently more accurate than those produced by automated assembly tools. We therefore recommend Trycycler+Medaka+Pilon as an ideal approach for generating high-quality bacterial reference genomes.Data availabilitySupplementary figures, tables and code can be found at: github.com/rrwick/Trycycler-paperReads, assemblies and reference sequences can be found at: bridges.monash.edu/articles/dataset/Trycycler_paper_dataset/14890734

Klebsiella pneumoniae is a leading cause of antimicrobial-resistant (AMR) healthcare-associated infections, neonatal sepsis and community-acquired liver abscess, and is associated with chronic intestinal diseases. Its diversity and complex population structure pose challenges for analysis and interpretation of K. pneumoniae genome data. Here we introduce Kleborate, a tool for analysing genomes of K. pneumoniae and its associated species complex, which consolidates interrogation of key features of proven clinical importance. Kleborate provides a framework to support genomic surveillance and epidemiology in research, clinical and public health settings. To demonstrate its utility we apply Kleborate to analyse publicly available Klebsiella genomes, including clinical isolates from a pan-European study of carbapenemase-producing Klebsiella, highlighting global trends in AMR and virulence as examples of what could be achieved by applying this genomic framework within more systematic genomic surveillance efforts. We also demonstrate the application of Kleborate to detect and type K. pneumoniae from gut metagenomes., Klebsiella pneumoniae is a pathogen of increasing public health concern and antimicrobial resistance is becoming more prevalent. Here, the authors describe a K. pneumoniae genotyping tool, Kleborate, that can be used to identify lineages and detect antimicrobial resistance and virulence loci.

Extended-spectrum β-lactamases (ESBL)-producing Enterobacterales are a leading cause of human and animal infections, being classified as critical priority pathogens by the World Health Organization. Epidemiological studies have shown that spread of ESBL-producing bacteria is not a problem restricted to hospitals, but also represents a growing problem involving environmental and food safety., The global spread of antibiotic-resistant bacteria and their resistance genes is a critical issue that is no longer restricted to hospital settings, but also represents a growing problem involving environmental and food safety. In this study, we have performed a microbiological and genomic investigation of critical priority pathogens resistant to broad-spectrum cephalosporins and showing endophytic lifestyles in fresh vegetables sold in a country with high endemicity of extended-spectrum β-lactamases (ESBLs). We report the isolation of international high-risk clones of CTX-M-15-producing Escherichia coli, belonging to clonal complexes CC38 and CC648, and Klebsiella pneumoniae of complex CC307 from macerated tissue of surface-sterilized leaves of spinach, cabbage, arugula, and lettuce. Regardless of species, all ESBL-positive isolates were able to endophytically colonize common bean (Phaseolus vulgaris) seedlings, showed resistance to acid pH, and had a multidrug-resistant (MDR) profile to clinically relevant antibiotics (i.e., broad-spectrum cephalosporins, aminoglycosides, and fluoroquinolones). Genomic analysis of CTX-M-producing endophytic Enterobacterales revealed a wide resistome (antibiotics, biocides, disinfectants, and pesticides) and virulome, and genes for endophytic fitness and for withstanding acidic conditions. Transferable IncFIB and IncHI2A plasmids carried blaCTX-M-15 genes and, additionally, an IncFIB plasmid (named pKP301cro) also harbored genes encoding resistance to heavy metals. These data support the hypothesis that fresh vegetables marketed for consumption can act as a figurative Trojan horse for the hidden spread of international clones of critical WHO priority pathogens producing ESBLs, and/or their resistance genes, to humans and other animals, which is a critical issue within a food safety and broader public and environmental health perspective. IMPORTANCE Extended-spectrum β-lactamases (ESBL)-producing Enterobacterales are a leading cause of human and animal infections, being classified as critical priority pathogens by the World Health Organization. Epidemiological studies have shown that spread of ESBL-producing bacteria is not a problem restricted to hospitals, but also represents a growing problem involving environmental and food safety. In this regard, CTX-M-type β-lactamases have become the most widely distributed and clinically relevant ESBLs worldwide. Here, we have investigated the occurrence and genomic features of ESBL-producing Enterobacterales in surface-sterilized fresh vegetables. We have uncovered that international high-risk clones of CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae harboring a wide resistome and virulome, carry additional genes for endophytic fitness and resistance to acidic conditions. Furthermore, we have demonstrated that these CTX-M-15-positive isolates are able to endophytically colonize plant tissues. Therefore, we believe that fresh vegetables can act as a figurative Trojan horse for the hidden spread of critical priority pathogens exhibiting endophytic lifestyles.

The emergence and rapid spread of carbapenemase-producing Enterobacterales represents a serious public health concern. Critically, these global priority bacteria have begun to be reported in companion animals, implying a potential risk of cross-transmission between humans and pets. Using long-read (MinION) and short-read (Illumina) sequencing technologies, we have identified and characterized a hypermucoviscous KPC-2-producing Klebsiella pneumoniae strain belonging to the high-risk international clone ST11/CG258, in a dog with urinary tract infection. Strikingly, the bla

During a microbiological and genomic surveillance study to investigate the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli from community-acquired urinary tract infections (UTI) and commercial meat samples, in a Brazilian city with a high occurrence of infections by ESBL-producing bacteria, we have identified the presence of CTX-M (−55, −27, −24, −15, −14 and −2)-producing E. coli belonging to the international clones ST354, ST131, ST117, and ST38. The ST131 was more prevalent in human samples, and worryingly the high-risk ST131-C1-M27 was identified in human infections for the first time. We also detected CTX-M-55-producing E. coli ST117 isolates from meat samples (i.e., chicken and pork) and human infections. Moreover, we have identified the important clone CTX-M-24-positive E. coli ST354 from human samples in Brazil for the first time. In brief, our results suggest a potential of commercialized meat as a reservoir of high-priority E. coli lineages in the community. In contrast, the identification of E. coli ST131-C1-M27 indicates that novel pandemic clones have emerged in Brazil, constituting a public health issue.

Emergent hypervirulent Klebsiella pneumoniae has been responsible for severe diseases, representing a serious threat to public health. We report the whole-genome sequencing of a novel ST3994-K2 clone, a single locus variant of ST86 K2, which is considered a worrying hypervirulent clone that emerged in several parts of the world. The strain K. pneumonia Kpi144 was isolated in 2013 from a blood culture of a 69-year-old male patient admitted to a tertiary hospital in Teresina, state of Piauí, northeastern Brazil. The strain was susceptible to 41 antibiotics tested, presented hypermucoviscous phenotype and a virulent behavior was observed in the Galleria mellonella infection model. Moreover, the virulome showed several virulence genes. To the best of our knowledge, this is the first worldwide report of a novel ST3994-K2 K. pneumoniae clone, an SLV of ST86 K2, which is considered a worrying virulent clone that has emerged in several parts of the world, including South America and Brazil.

The emergence of invasive Haemophilus influenzae infections in vaccinated patient is a public health concern. We have investigated the genomic basis of invasiveness and possible vaccine failure in H. influenzae causing invasive disease in vaccinated and unvaccinated children in Brazil. Three H. influenzae strains isolated from blood cultures of pediatric patients were sequenced. Serotype, MLST, resistome and virulome were predicted using bioinformatic tools, whereas single nucleotide polymorphisms (SNPs) analysis of cap loci and the presence of the putative virulence-enhancing IS1016-bexA partial deletion were predicted in silico. Infections were caused by H. influenzae type a (Hia), type b (Hib) and nontypeable (NTHi), belonging to international high-risk clones of sequence types ST23, ST6 and ST368, respectively, which have been identified in North American, European and Asian countries. Convergence of ampicillin resistance and virulence in Hib-ST6 was supported by blaTEM-1B and deletion in the bexA gene, whereas presence of SNPs in the cap-b locus was associated with antigenic modifications of the capsule structure. Hia-ST23 and NTHi-ST368 strains carried galU, lpsA, opsX, rfaF, iga1, lgtC and lic1/lic2 virulence genes, associated with colonization, adaptation and damage to the lung, or invasiveness. In summary, deletion in the bexA gene and presence of SNPs in the cap locus of Hib could be contributing to invasive disease and possible vaccine failure in pediatric patients, whereas serotype replacement of Hib with type “a” and NTHi strains denotes the ability of non-vaccine serotypes to re-colonize vaccinated patients. Finally, the dissemination of international high-risk clones of H. influenzae emphasizes the importance of monitoring changes in the molecular epidemiology of invasive H. influenzae disease.

Funder: KEH is supported by a Senior Medical Research Fellowship from the Viertel Foundation of Australia RJB through a UKRI MRC New Investigator Research Grant (held by KSB, MR/R020787/1), Shigella sonnei is the most common agent of shigellosis in high-income countries, and causes a significant disease burden in low- and middle-income countries. Antimicrobial resistance is increasingly common in all settings. Whole genome sequencing (WGS) is increasingly utilised for S. sonnei outbreak investigation and surveillance, but comparison of data between studies and labs is challenging. Here, we present a genomic framework and genotyping scheme for S. sonnei to efficiently identify genotype and resistance determinants from WGS data. The scheme is implemented in the software package Mykrobe and tested on thousands of genomes. Applying this approach to analyse >4,000 S. sonnei isolates sequenced in public health labs in three countries identified several common genotypes associated with increased rates of ciprofloxacin resistance and azithromycin resistance, confirming intercontinental spread of highly-resistant S. sonnei clones and demonstrating the genomic framework can facilitate monitoring the spread of resistant clones, including those that have recently emerged, at local and global scales.

Integrative conjugative elements (ICEs) are widespread in many bacterial species, often carrying antibiotic resistance determinants. In the present work, we screened a collection of Proteus mirabilis clinical isolates for the presence of type 1 SXT/R391 ICEs. Among the 76 isolates analyzed, 5 of them carry such elements. The complete sequences of these elements were obtained. One of the isolates carried the CMY-2 beta-lactamase gene in a transposon and is nearly identical to the element ICEPmiJpn1 previously described in Japan, and later shown to be present in other parts of the world, indicating global spread of this element. Nevertheless, the Brazilian isolate carrying ICEPmiJpn1 is not clonally related to the other lineages carrying the same element around the world. The other ICEs identified in this work do not carry known antibiotic resistance markers and are diverse in variable gene content and size, suggesting that these elements may be responsible for the acquisition of other advantageous traits by bacteria. Some sequences carried by these elements in Brazilian strains were not previously found in other SXT/R391 variants.

This study used whole-genome sequencing to analyze the first case of NDM-1-producing

Serratia fonticola is a human pathogen widely found in the environment, with birds being reported as possible natural hosts. During an epidemiological and genomic surveillance study conducted to monitor the occurrence of extended-spectrum b-lactamase (ESBL)-producing Enterobacterales in South American wild birds, we identified an ESBL-positive S. fonticola in a fecal sample collected from a Hudsonian Whimbrel, during its non-breeding range on the Pacific Coast of Chile. Whole genome sequencing analysis and in silico modeling revealed a novel variant of the class A ESBLs FONA family, designated FONA-7, which shows 96.28% amino acid identity with FONA-6; with amino acid substitutions occurring in the signal peptide sequence (Thr22/Ser), and in the mature protein (Ser39/Asn and Thr227/Ile). This finding denotes that migratory birds can be potential vectors for the transboundary spread of ESBL-producing bacteria, creating a further theoretical riskfor the origin of novel plasmid-encoded b-lactamases. Fil: Fuentes Castillo, Danny. Universidade de Sao Paulo; Brasil Fil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Cerdeira, Louise. Universidade de Sao Paulo; Brasil Fil: Cardenas-Arias, Adriana. Universidade de Sao Paulo; Brasil Fil: Moura, Quézia. Universidade de Sao Paulo; Brasil Fil: Oliveira, Flavio A.. Universidade Estadual de Campinas; Brasil Fil: Levy, Carlos E.. Universidade Estadual de Campinas; Brasil Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Catão-Dias, José L.. Universidade de Sao Paulo; Brasil Fil: Lincopan, Nilton. Universidade de Sao Paulo; Brasil

Klebsiella quasipneumoniae is an emerging pathogen in human medicine. We report draft genome sequences of NDM-1- and KPC-2-producing K. quasipneumoniae strains from inpatients in Brazil. K. quasipneumoniae subsp. quasipneumoniae and K. quasipneumoniae subsp. similipneumoniae harbored broad resistomes. These data could contribute to a better understanding of acquired resistance in K. quasipneumoniae.

In this study, we describe the frequency of virulence genes in Klebsiella pneumoniae carbapenemase-2-producing Klebsiella pneumoniae (KPC-KP), including hypervirulent (hv) and hypermucoviscous (hm) strains by whole-genome sequencing. We also evaluate the capacity for biofilm formation by using phenotypic techniques. The occurrence of several virulence genes (fimABCDEFGHIK, mrkABCDFHJ, ecpA, wabG, entB, ugE, irp1, irp2, traT, iutA and ureADE) and a high frequency of hvhmKPC-KP isolates was found. Most hospital-associated lineages of KPC-KP belong to the international clonal group 258 (CG258). Biofilm formation was a constant feature among 90.9 % of KPC-KP strains. This report suggests a close relationship between ST437 and weak biofilm production, given that all weakly biofilm-producing strains belonged to this sequence type. This also supports the dissemination of KPC-KP containing numerous virulence determinants belonging to the biofilm-producing CG258 type in Brazil, including hv and hm strains. These factors allow this pathogen to cause infections, leading to its rapid expansion and persistence in hospital settings.