Emergence of carbapenem-resistant Pseudomonas aeruginosa ST179 producing both IMP-16 and KPC-2: a case study of introduction from Peru to Spain.

We describe four cases of a novel carbapenem-resistant Pseudomonas aeruginosa ST179 clone carrying the blaKPC-2 or blaKPC-35 gene together with blaIMP-16, imported from Peru to Spain and isolated from leukemia patients. All isolates were multidrug-resistant but remained susceptible to fosfomycin, cefiderocol, and colistin. Whole-genome sequencing revealed that blaKPC-2 and blaKPC-35 were located in an IncP6 plasmid, whereas blaIMP-16 was in a chromosomal type 1 integron. This study highlights the global threat of multidrug-resistant P. aeruginosa clones and underscores the importance of monitoring and early detection of emerging resistance mechanisms to guide appropriate treatment strategies. The importation and spread of such clones emphasize the urgent need to implement strict infection control measures to prevent the dissemination of carbapenem-resistant bacteria. IMPORTANCE: This is the first documented case of a Pseudomonas aeruginosa ST179 strain carrying the blaKPC-35 gene, and it represents the first report of a P. aeruginosa co-harboring blaIMP-16 and either blaKPC-2 or blaKPC-35, which wre imported from Peru to Spain, highlighting a threat due to the capacity of spreading carbapenem-resistance via plasmid conjugation.

Clonal dissemination of Acinetobacter radioresistens among Humboldt penguins (Spheniscus humboldti) inhabiting a barren northern Peruvian island.

Acinetobacter spp. are often isolated from natural sources, but knowledge about their presence in wild animals is fragmented and uncomplete. The present study aimed to characterize a series of Acinetobacter radioresistens isolated from Humboldt penguins (Spheniscus humboldti). Fifteen Humboldt penguins from an inhabited northern Peruvian island were sampled. Microorganisms were identified by MALDI-TOF MS. Antibiotic susceptibility to 12 antimicrobial agents was established, and clonal relationships were determined. A representative isolate was selected for whole genome sequencing (WGS). A. radioresistens were isolated from the feces of 12 (80%) Humboldt penguins, being susceptible to all the antimicrobial agents tested, except eight cefotaxime-intermediate isolates. All A. radioresistens were clonally related. WGS showed that the isolate belonged to ST1972, the presence of two chromosomal encoded carbapenemases (blaOXA-23 and a putative subclass B3 metallo-ß-lactamase), and a series of point mutations in antibiotic-resistance related chromosomal genes, which were considered as polymorphisms. In addition, a few virulence factors, including a capsule-encoding operon, superoxide dismutases, catalases, phospholipases and a siderophore receptor were identified. The present results suggest that A. radioresistens may be a common member of the gut microbiota of Humboldt penguins, but further studies in other geographical areas are needed to establish this finding.

Diverse Populations of Staphylococcus pseudintermedius Colonize the Skin of Healthy Dogs.

Staphylococcus pseudintermedius is a commensal bacterium of the canine skin but is also a key opportunistic pathogen that is responsible for most cases of pyoderma in dogs. The current paradigm indicates that infection arises when predisposing factors alter the healthy skin barrier. Despite their importance, the characteristics of the S. pseudintermedius populations colonizing the skin of healthy dogs are yet largely unknown. Here, we retrieved 67 complete circular genomes and 19 associated plasmids from S. pseudintermedius isolated from the skin of 9 healthy dogs via long-reads Nanopore sequencing. Within the S. pseudintermedius populations isolated from healthy skin, multilocus sequence typing (MLST) detected 10 different STs, distributed mainly by the host. 39% of the 18 representative genomes isolated herein were methicillin-resistant S. pseudintermedius (MRSP), and they showed, on average, a higher number of antibiotic resistance genes and prophages than did the methicillin-sensitive (MSSP). In summary, our results revealed that the S. pseudintermedius populations inhabiting the skin of healthy dogs are relatively diverse and heterogeneous in terms of MLST and methicillin resistance. In this study, all of the 67 commensal S. pseudintermedius populations that were isolated from healthy dogs contained antibiotic resistance genes, indicating the extent and severity of the problem of antimicrobial resistance in staphylococci with zoonotic potential. IMPORTANCE Staphylococcus pseudintermedius is a commensal canine bacterium that can become an opportunistic pathogen and is responsible for most cases of canine pyoderma. It can also cause occasional zoonotic infections. Infections caused by antibiotic-resistant Staphylococcus are a global concern. Skin commensal Staphylococcus pseudintermedius is understudied. To provide insight into the commensal strains circulating in healthy dogs, we performed whole-genome sequencing of 67 S. pseudintermedius isolates from different skin sites in 9 healthy dogs. Through the bioinformatic analysis of these genomes, we identified a genomic diversity that is more complete than those afforded by traditional molecular typing strategies. We identified 7 new STs. All of the isolates harbored genes associated with antibiotic resistance, and 39% of the representative genomes were methicillin-resistant. Our data provide critical insights for future skin infection control and antibiotic surveillance within veterinary medicine.

Concordance between Antimicrobial Resistance Phenotype and Genotype of Staphylococcus pseudintermedius from Healthy Dogs.

Staphylococcus pseudintermedius, a common commensal canine bacterium, is the main cause of skin infections in dogs and is a potential zoonotic pathogen. The emergence of methicillin-resistant S. pseudintermedius (MRSP) has compromised the treatment of infections caused by these bacteria. In this study, we compared the phenotypic results obtained by minimum inhibitory concentration (MICs) for 67 S. pseudintermedius isolates from the skin of nine healthy dogs versus the genotypic data obtained with Nanopore sequencing. A total of 17 antibiotic resistance genes (ARGs) were detected among the isolates. A good correlation between phenotype and genotype was observed for some antimicrobial classes, such as ciprofloxacin (fluoroquinolone), macrolides, or tetracycline. However, for oxacillin (beta-lactam) or aminoglycosides the correlation was low. Two antibiotic resistance genes were located on plasmids integrated in the chromosome, and a third one was in a circular plasmid. To our knowledge, this is the first study assessing the correlation between phenotype and genotype regarding antimicrobial resistance of S. pseudintermedius from healthy dogs using Nanopore sequencing technology.

Whole-Genome Sequencing and De Novo Assembly of 67 Staphylococcus pseudintermedius Strains Isolated from the Skin of Healthy Dogs.

We have de novo assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 Mbp size and 99.43% completeness, 2,386 coding sequences, 19 complete rRNAs, 59 tRNAs, and 4 noncoding RNAs. We released 51 single-contig complete genomes and 16 genomes with a circular main contig using Nanopore sequencing.

Novel canine high-quality metagenome-assembled genomes, prophages and host-associated plasmids provided by long-read metagenomics together with Hi-C proximity ligation.

The human gut microbiome has been extensively studied, yet the canine gut microbiome is still largely unknown. The availability of high-quality genomes is essential in the fields of veterinary medicine and nutrition to unravel the biological role of key microbial members in the canine gut environment. Our aim was to evaluate nanopore long-read metagenomics and Hi-C (high-throughput chromosome conformation capture) proximity ligation to provide high-quality metagenome-assembled genomes (HQ MAGs) of the canine gut environment. By combining nanopore long-read metagenomics and Hi-C proximity ligation, we retrieved 27 HQ MAGs and 7 medium-quality MAGs of a faecal sample of a healthy dog. Canine MAGs (CanMAGs) improved genome contiguity of representatives from the animal and human MAG catalogues - short-read MAGs from public datasets - for the species they represented: they were more contiguous with complete ribosomal operons and at least 18 canonical tRNAs. Both canine-specific bacterial species and gut generalists inhabit the dog's gastrointestinal environment. Most of them belonged to Firmicutes, followed by Bacteroidota and Proteobacteria. We also assembled one Actinobacteriota and one Fusobacteriota MAG. CanMAGs harboured antimicrobial-resistance genes (ARGs) and prophages and were linked to plasmids. ARGs conferring resistance to tetracycline were most predominant within CanMAGs, followed by lincosamide and macrolide ones. At the functional level, carbohydrate transport and metabolism was the most variable within the CanMAGs, and mobilome function was abundant in some MAGs. Specifically, we assigned the mobilome functions and the associated mobile genetic elements to the bacterial host. The CanMAGs harboured 50 bacteriophages, providing novel bacterial-host information for eight viral clusters, and Hi-C proximity ligation data linked the six potential plasmids to their bacterial host. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAG discovery and assignment of extra-chromosomal elements to their bacterial host. This will provide essential information for studying the canine gut microbiome in veterinary medicine and animal nutrition.

Clonal Spread and Intra- and Inter-Species Plasmid Dissemination Associated With Klebsiella pneumoniae Carbapenemase-Producing Enterobacterales During a Hospital Outbreak in Barcelona, Spain.

Objectives: The study aimed to characterize the clonal spread of resistant bacteria and dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at a tertiary hospital in Catalonia, Spain. Methods: Isolates were recovered from surveillance rectal swabs and diagnostic samples. Species identification was by matrix-assisted laser desorption ionization-time time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of bla genes was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and long-read sequencing were used to characterize resistance plasmids. Results: From July 2018 to February 2019, 125 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies identified a major clone of Klebsiella pneumoniae belonging to sequence type ST15 and additional isolates of K. pneumoniae, Escherichia coli and Enterobacter sp. from different STs. All isolates but one carried the bla KPC-2 allelic variant. The bla KPC-2 gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn4401 element designated NTEKPC-pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTEKPC-pMC-2-1 module was highly conserved. Conclusion: We report a hospital outbreak caused by the clonal dissemination of KPC-producing ST15 K. pneumoniae but also the intra- and inter-species transmission of the bla KPC-2 gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant high-risk clones.

Whole genome sequencing and de novo assembly of Staphylococcus pseudintermedius: a pangenome approach to unravelling pathogenesis of canine pyoderma.

BACKGROUND: Staphylococcus pseudintermedius is the main aetiological agent of canine pyoderma. Whole genome sequencing is the most comprehensive way of obtaining relevant genomic information about micro-organisms. HYPOTHESIS/OBJECTIVES: Oxford Nanopore technology enables quality sequencing and de novo assembly of the whole genome of S. pseudintermedius. Whole genome analysis of S. pseudintermedius may help to better understand the pathogenesis of canine pyodermas. METHODS AND MATERIALS: Twenty-two strains of S. pseudintermedius isolated from the skin of five healthy dogs and 33 strains isolated from skin of 33 dogs with pyoderma were analysed. DNA was extracted and sequenced using Oxford Nanopore MinION, a new technology that delivers longer reads in a hand-held device. The pangenome was analysed and visualised with Anvi'o 6.1. RESULTS: Nanopore technology allowed the sequencing and de novo assembly of the genomes of 55 S. pseudintermedius strains isolated from healthy dogs and from dogs with pyoderma. The average genome size of S. pseudintermedius was 2.62 Mbp, with 48% being core genome. Pyoderma isolates contained a higher number of antimicrobial resistance genes, yet the total number of virulence factors genes did not change between isolates from healthy dogs and from dogs with pyoderma. Genomes of meticillin-resistant S. pseudintermedius (MRSP) strains were larger than those of meticillin-susceptible (MSSP) strains (2.80 Mbp versus 2.59 Mbp), as a consequence of a greater presence of antimicrobial resistance genes, phages and prophages. CONCLUSIONS AND CLINICAL IMPORTANCE: This technique allows much more precise and easier characterisation of canine S. pseudintermedius populations and may lead to a better understanding of the pathogenesis of canine pyodermas.

Long-read metagenomics retrieves complete single-contig bacterial genomes from canine feces.

BACKGROUND: Long-read sequencing in metagenomics facilitates the assembly of complete genomes out of complex microbial communities. These genomes include essential biologic information such as the ribosomal genes or the mobile genetic elements, which are usually missed with short-reads. We applied long-read metagenomics with Nanopore sequencing to retrieve high-quality metagenome-assembled genomes (HQ MAGs) from a dog fecal sample. RESULTS: We used nanopore long-read metagenomics and frameshift aware correction on a canine fecal sample and retrieved eight single-contig HQ MAGs, which were > 90% complete with < 5% contamination, and contained most ribosomal genes and tRNAs. At the technical level, we demonstrated that a high-molecular-weight DNA extraction improved the metagenomics assembly contiguity, the recovery of the rRNA operons, and the retrieval of longer and circular contigs that are potential HQ MAGs. These HQ MAGs corresponded to Succinivibrio, Sutterella, Prevotellamassilia, Phascolarctobacterium, Catenibacterium, Blautia, and Enterococcus genera. Linking our results to previous gastrointestinal microbiome reports (metagenome or 16S rRNA-based), we found that some bacterial species on the gastrointestinal tract seem to be more canid-specific -Succinivibrio, Prevotellamassilia, Phascolarctobacterium, Blautia_A sp900541345-, whereas others are more broadly distributed among animal and human microbiomes -Sutterella, Catenibacterium, Enterococcus, and Blautia sp003287895. Sutterella HQ MAG is potentially the first reported genome assembly for Sutterella stercoricanis, as assigned by 16S rRNA gene similarity. Moreover, we show that long reads are essential to detect mobilome functions, usually missed in short-read MAGs. CONCLUSIONS: We recovered eight single-contig HQ MAGs from canine feces of a healthy dog with nanopore long-reads. We also retrieved relevant biological insights from these specific bacterial species previously missed in public databases, such as complete ribosomal operons and mobilome functions. The high-molecular-weight DNA extraction improved the assembly's contiguity, whereas the high-accuracy basecalling, the raw read error correction, the assembly polishing, and the frameshift correction reduced the insertion and deletion errors. Both experimental and analytical steps ensured the retrieval of complete bacterial genomes.

Transmission of Similar Mcr-1 Carrying Plasmids among Different Escherichia coli Lineages Isolated from Livestock and the Farmer.

Colistin use has mostly been stopped in human medicine, due to its toxicity. However, nowadays, it still is used as a last-resort antibiotic to treat hospital infections caused by multi-drug resistant Enterobacteriaceae. On the contrary, colistin has been used in veterinary medicine until recently. In this study, 210 fecal samples from pigs (n = 57), calves (n = 152), and the farmer (n = 1) were collected from a farm where E. coli harboring mcr-1-mcr-3 was previously detected. Samples were plated, and mcr-genes presence was confirmed by multiplex-PCR. Hybrid sequencing which determined the presence and location of mcr-1, other antibiotic resistance genes, and virulence factors. Eighteen colistin resistant isolates (13 from calves, four from pigs, and one from the farmer) contained mcr-1 associated with plasmids (IncX4, IncI2, and IncHI2), except for two that yielded mcr-1 in the chromosome. Similar plasmids were distributed in different E. coli lineages. Transmission of mcr-1 to the farmer most likely occurred by horizontal gene transfer from E. coli of calf origin, since plasmids were highly similar (99% coverage, 99.97% identity). Moreover, 33 virulence factors, including stx2 for Shiga toxin E. coli (STEC) were detected, highlighting the role of livestock as a reservoir of pathotypes with zoonotic potential.

Dissemination of NDM-producing Klebsiella pneumoniae and Escherichia coli high-risk clones in Catalan healthcare institutions.

OBJECTIVES: To characterize the clonal spread of carbapenem-resistant Klebsiella pneumoniae and Escherichia coli isolates between different healthcare institutions in Catalonia, Spain. METHODS: Antimicrobial susceptibility was tested by disc diffusion. MICs were determined by gradient diffusion or broth microdilution. Carbapenemase production was confirmed by lateral flow. PCR and Sanger sequencing were used to identify the allelic variants of resistance genes. Clonality studies were performed by PFGE and MLST. Plasmid typing, conjugation assays, S1-PFGE plus Southern blotting and MinION Oxford Nanopore sequencing were used to characterize resistance plasmids. RESULTS: Twenty-nine carbapenem-resistant isolates recovered from three healthcare institutions between January and November 2016 were included: 14 K. pneumoniae isolates from a tertiary hospital in the south of Catalonia (hospital A); 2 K. pneumoniae isolates from a nearby healthcare centre; and 12 K. pneumoniae isolates and 1 E. coli isolate from a tertiary hospital in Barcelona (hospital B). The majority of isolates were resistant to all antimicrobial agents, except colistin, and all were NDM producers. PFGE identified a major K. pneumoniae clone (n = 27) belonging to ST147 and co-producing NDM-1 and CTX-M-15, with a few isolates also harbouring blaOXA-48. Two sporadic isolates of K. pneumoniae ST307 and E. coli ST167 producing NDM-7 were also identified. blaNDM-1 was carried in two related IncR plasmid populations and blaNDM-7 in a conjugative 50 kb IncX3 plasmid. CONCLUSIONS: We report the inter-hospital dissemination of XDR high-risk clones of K. pneumoniae and E. coli associated with the carriage of small, transferable plasmids harbouring blaNDM genes.

Hybrid Assembly from a Pathogenic Methicillin- and Multidrug-Resistant Staphylococcus pseudintermedius Strain Isolated from a Case of Canine Otitis in Spain.

Here we report the genome assembly, using a hybrid approach with Illumina and Nanopore sequencing, of a pathogenic Staphylococcus pseudintermedius strain isolated from a case of canine otitis. Genome assembly confirmed the antimicrobial resistance profile (disk diffusion testing) with specific genes and mutations.

Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon.

Background: Profiling the microbiome of low-biomass samples is challenging for metagenomics since these samples are prone to contain DNA from other sources (e.g. host or environment). The usual approach is sequencing short regions of the 16S rRNA gene, which fails to assign taxonomy to genus and species level. To achieve an increased taxonomic resolution, we aim to develop long-amplicon PCR-based approaches using Nanopore sequencing. We assessed two different genetic markers: the full-length 16S rRNA (~1,500 bp) and the 16S-ITS-23S region from the rrn operon (4,300 bp). Methods: We sequenced a clinical isolate of Staphylococcus pseudintermedius, two mock communities and two pools of low-biomass samples (dog skin). Nanopore sequencing was performed on MinION™ using the 1D PCR barcoding kit. Sequences were pre-processed, and data were analyzed using EPI2ME or Minimap2 with rrn database. Consensus sequences of the 16S-ITS-23S genetic marker were obtained using canu. Results: The full-length 16S rRNA and the 16S-ITS-23S region of the rrn operon were used to retrieve the microbiota composition of the samples at the genus and species level. For the Staphylococcus pseudintermedius isolate, the amplicons were assigned to the correct bacterial species in ~98% of the cases with the16S-ITS-23S genetic marker, and in ~68%, with the 16S rRNA gene when using EPI2ME. Using mock communities, we found that the full-length 16S rRNA gene represented better the abundances of a microbial community; whereas, 16S-ITS-23S obtained better resolution at the species level. Finally, we characterized low-biomass skin microbiota samples and detected species with an environmental origin. Conclusions: Both full-length 16S rRNA and the 16S-ITS-23S of the rrn operon retrieved the microbiota composition of simple and complex microbial communities, even from the low-biomass samples such as dog skin. For an increased resolution at the species level, targeting the 16S-ITS-23S of the rrn operon would be the best choice.