Small Plasmids Research Articles

Characterization of blaKPC-2-Carrying Plasmid pR31-KPC from a Pseudomonas aeruginosa Strain Isolated in China.

This work aimed to characterize a 29-kb blaKPC-2-carrying plasmid, pR31-KPC, from a multidrug resistant strain of Pseudomonas aeruginosa isolated from the sputum of an elderly patient with multiple chronic conditions in China. The backbone of pR31-KPC is closely related to four other blaKPC-2-carrying plasmids, YLH6_p3, p1011-KPC2, p14057A, and pP23-KPC, none of which have been assigned to any of the known incompatibility groups. Two accessory modules, the IS26-blaKPC-2-IS26 unit and IS26-ΔTn6376-IS26 region, separated by a 5.9-kb backbone region, were identified in pR31-KPC, which was also shown to carry the unique resistance marker blaKPC-2. A comparative study of the above five plasmids showed that p1011-KPC2 may be the most complete plasmid of this group to be reported, while pR31-KPC is the smallest plasmid having lost most of its conjugative region. Regions between the iterons and orf207 in the backbone may be hot spots for the acquisition of exogenous resistance entities. The accessory regions of these plasmids have all undergone several biological events when compared with Tn6296. The further transfer of blaKPC-2 in these plasmids may be initiated by either the Tn3 family or IS26-associated transposition or homologous recombination. The data presented here will contribute to a deeper understanding of blaKPC-2 carrying plasmids in Pseudomonas.

Genomic analysis of the nomenclatural type strain of the nematode-associated entomopathogenic bacterium Providencia vermicola

BackgroundEnterobacteria of the genus Providencia are mainly known as opportunistic human pathogens but have been isolated from highly diverse natural environments. The species Providencia vermicola comprises insect pathogenic bacteria carried by entomoparasitic nematodes and is investigated as a possible insect biocontrol agent. The recent publication of several genome sequences from bacteria assigned to this species has given rise to inconsistent preliminary results.ResultsThe genome of the nematode-derived P. vermicola type strain DSM_17385 has been assembled into a 4.2 Mb sequence comprising 5 scaffolds and 13 contigs. A total of 3969 protein-encoding genes were identified. Multilocus sequence typing with different marker sets revealed that none of the previously published presumed P. vermicola genomes represents this taxonomic species. Comparative genomic analysis has confirmed a close phylogenetic relationship of P. vermicola to the P. rettgeri species complex. P. vermicola DSM_17385 carries a type III secretion system (T3SS-1) with probable function in host cell invasion or intracellular survival. Potentially antibiotic resistance-associated genes comprising numerous efflux pumps and point-mutated house-keeping genes, have been identified across the P. vermicola genome. A single small (3.7 kb) plasmid identified, pPVER1, structurally belongs to the qnrD-type family of fluoroquinolone resistance conferring plasmids that is prominent in Providencia and Proteus bacteria, but lacks the qnrD resistance gene.ConclusionsThe sequence reported represents the first well-supported published genome for the taxonomic species P. vermicola to be used as reference in further comparative genomics studies on Providencia bacteria. Due to a striking difference in the type of injectisome encoded by the respective genomes, P. vermicola might operate a fundamentally different mechanism of entomopathogenicity when compared to insect-pathogenic Providencia sneebia or Providencia burhodogranariea. The complete absence of antibiotic resistance gene carrying plasmids or mobile genetic elements as those causing multi drug resistance phenomena in clinical Providencia strains, is consistent with the invertebrate pathogen P. vermicola being in its natural environment efficiently excluded from the propagation routes of multidrug resistance (MDR) carrying genetic elements operating between human pathogens. Susceptibility to MDR plasmid acquisition will likely become a major criterion in the evaluation of P. vermicola for potential applications in biological pest control.

The complete genome of 2,6-dichlorobenzamide (BAM) degrader Aminobacter sp. MSH1 suggests a polyploid chromosome, phylogenetic reassignment, and functions of plasmids

Aminobacter sp. MSH1 (CIP 110285) can use the pesticide dichlobenil and its recalcitrant transformation product, 2,6-dichlorobenzamide (BAM), as sole source of carbon, nitrogen, and energy. The concentration of BAM in groundwater often exceeds the threshold limit for drinking water, requiring additional treatment in drinking water treatment plants or closure of the affected abstraction wells. Biological treatment with MSH1 is considered a potential sustainable alternative to remediate BAM-contamination in drinking water production. We present the complete genome of MSH1, which was determined independently in two institutes at Aarhus University and KU Leuven. Divergences were observed between the two genomes, i.e. one of them lacked four plasmids compared to the other. Besides the circular chromosome and the two previously described plasmids involved in BAM catabolism, pBAM1 and pBAM2, the genome of MSH1 contained two megaplasmids and three smaller plasmids. The MSH1 substrain from KU Leuven showed a reduced genome lacking a megaplasmid and three smaller plasmids and was designated substrain MK1, whereas the Aarhus variant with all plasmids was designated substrain DK1. A plasmid stability experiment indicate that substrain DK1 may have a polyploid chromosome when growing in R2B medium with more chromosomes than plasmids per cell. Finally, strain MSH1 is reassigned as Aminobacter niigataensis MSH1.

Ubiquitous Conjugative Mega-Plasmids of Acinetobacter Species and Their Role in Horizontal Transfer of Multi-Drug Resistance.

Conjugative mega-plasmids play a special role in adaptation since they carry a huge number of accessory genes, often allowing the host to develop in new niches. In addition, due to conjugation they are able to effectively spread themselves and participate in the transfer of small mobilizable plasmids. In this work, we present a detailed characterization of a recently discovered family of multiple-drug resistance mega-plasmids of Acinetobacter species, termed group III-4a. We describe the structure of the plasmid backbone region, identify the rep gene and the origin of plasmid replication, and show that plasmids from this group are able not only to move between different Acinetobacter species but also to efficiently mobilize small plasmids containing different mob genes. Furthermore, we show that the population of natural Acinetobacter strains contains a significant number of mega-plasmids and reveal a clear correlation between the living conditions of Acinetobacter strains and the structure of their mega-plasmids. In particular, comparison of the plasmids from environmental and clinical strains shows that the genes for resistance to heavy metals were eliminated in the latter, with the simultaneous accumulation of antibiotic resistance genes by incorporation of transposons and integrons carrying these genes. The results demonstrate that this group of mega-plasmids plays a key role in the dissemination of multi-drug resistance among Acinetobacter species.

Recovery of small plasmid sequences via Oxford Nanopore sequencing.

Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of ~4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimaeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols.

Complete Genome Sequence of Escherichia coli Sequence Type 1193 Isolate AVS0096, Recovered from River Water in Switzerland.

ABSTRACTEscherichia coli sequence type 1193 (ST1193) is an important cause of multidrug-resistant extraintestinal infections. Here, we report the complete genome sequence of strain AVS0096, isolated from river water in Switzerland in 2020. The genome consists of a chromosome (4.9 Mbp), a multidrug resistance plasmid (101 kb), and two small plasmids.

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

BackgroundSynechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host.ResultsAn optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature.ConclusionsThis study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.

HI1 and I1 Resistance Plasmids from Salmonella enterica Serovar Typhimurium Strain SRC27 Are Epidemic.

Conjugative plasmids are a major contributor to the global spread of antibiotic resistance determinants, but the tracking of their evolutionary history is often neglected. Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SRC27 was isolated from an equine infection in Australia in 1999. SRC27 was known to carry conjugative HI1 and I1 resistance plasmids. In this study, SRC27 was sequenced to determine the relationship between these HI1 and I1 resistance plasmids it was known to carry and HI1 and I1 resistance plasmids circulating worldwide. The resistance genes in the HI1 plasmid, pSRC27-H, are all located in a single complex 34.7 kb resistance region. The backbone sequence and location of the pSRC27-H resistance island were used to identify the most closely related HI1 plasmids among the >90 that have been sequenced since 2011. This defined a sublineage of 20 type 2 HI1 plasmids that have been circulating in Europe, Asia, North America, and Australia since at least 1993. The overall resistance gene content of these HI1 plasmids differs, indicating extensive evolution in situ through the acquisition of additional transposons and deletion or replacement of ancestral regions. The I1 plasmid contains a complete copy of Tn5393a, containing the strAB genes that confer resistance to streptomycin. The precise location of Tn5393a in the backbone also defined a globally disseminated sublineage of I1 plasmids, many of which have also acquired additional resistance determinants. The sequence revealed that SRC27 also carried two additional plasmids, the pSLT-type FIB(S):FII(S) virulence plasmid and a small cryptic theta-replicating Col156 plasmid.

Cas9-Cleavage Sequences in Size-Reduced Plasmids Enhance Nonviral Genome Targeting of CARs in Primary Human T Cells.

T cell genome editing holds great promise to advance a range of immunotherapies but is encumbered by the dependence on difficult-to-produce and expensive viral vectors. Here, small double-stranded plasmid DNA modified to mediate high-efficiency homologous recombination is designed. The resulting chimeric antigen receptor (CAR)-T cells display a similar phenotype, transcriptional profile, and in vivo potency to CAR-T cells generated using adeno-associated viral vector. This method should simplify and accelerate the use of precision engineering to produce edited T cells for research and clinical purposes.

A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation

Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb. We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.

Molecular Characterization of Multidrug-Resistant Yersinia enterocolitica From Foodborne Outbreaks in Sweden.

The foodborne pathogen Yersinia enterocolitica causes gastrointestinal infections worldwide. In the spring of 2019, the Swedish Public Health Agency and Statens Serum Institut in Denmark independently identified an outbreak caused by Yersinia enterocolitica 4/O:3 that after sequence comparison turned out to be a cross-border outbreak. A trace-back investigation suggested shipments of fresh prewashed spinach from Italy as a common source for the outbreak. Here, we determined the genome sequences of five Y. enterocolitica clinical isolates during the Swedish outbreak using a combination of Illumina HiSeq short-read and Nanopore Technologies’ MinION long-read whole-genome sequencing. WGS results showed that all clinical strains have a fully assembled chromosome of approximately 4.6 Mbp in size and a 72-kbp virulence plasmid; one of the strains was carrying an additional 5.7-kbp plasmid, pYE-tet. All strains showed a high pathogen probability score (87.5%) with associated genes for virulence, all of which are closely related to an earlier clinical strain Y11 from Germany. In addition, we identified a chromosomally encoded multidrug-resistance cassette carrying resistance genes against chloramphenicol (catA1), streptomycin (aadA1), sulfonamides (sul1), and a mercury resistance module. This chromosomally encoded Tn2670 transposon has previously been reported associated with IncFII plasmids in Enterobacteriaceae: a Shigella flexneri clinical isolate from Japan in 1950s, a Klebsiella pneumoniae outbreak from Australia in 1997, and Salmonella enterica serovar Typhimurium. Interestingly, we identified an additional 5.7-kbp plasmid with tetB (encoding an ABC transporter), Rep, and its own ORI and ORIt sites, sharing high homology with small tetB-Rep plasmids from Pasteurellaceae. This is the first time that Tn2670 and Pasteurellaceae plasmids have been reported in Y. enterocolitica. Taken together, our study showed that the Swedish Y. enterocolitica outbreak strains acquired multi-antibiotic and metal-resistance genes through horizontal gene transfer, suggesting a potential reservoir of intraspecies dissemination of multidrug-resistance genes among foodborne pathogens. This study also highlights the concern of food-chain contamination of prewashed vegetables as a perpetual hazard against public health.

Phenotypic and molecular characterizations of carbapenem-resistant Acinetobacter baumannii isolates collected within the EURECA study

Multi-drug-resistant Acinetobacter baumannii isolates are key pathogens that contribute to the global burden of antimicrobial resistance. This study aimed to investigate the phenotypic and molecular characteristics of carbapenem-resistant A. baumannii (CRAB) isolates from the EURECA clinical trial. In total, 228 CRAB clinical strains were recovered from 29 sites in 10 European countries participating in the EURECA study between May 2016 and November 2018. All strains were reconfirmed centrally for identification and antimicrobial susceptibility testing, and were then subjected to DNA isolation and whole-genome sequencing (WGS), with analysis performed using BacPipe v.1.2.6. K and O typing was performed using KAPTIVE. Overall, 226 (99.1%) strains were confirmed as CRAB isolates. The minimum inhibitory concentration (MIC90) results of imipenem and meropenem were >16 mg/L. WGS showed that the isolates mainly harboured blaOXA-23 (n=153, 67.7%) or blaOXA-72 (n=70, 30.1%). Four blaOXA-72 isolates from Serbia co-harboured blaNDM-1. An IS5 transposase family element, ISAba31, was found upstream of the blaOXA-72 gene harboured on a small (~10-kb) pSE41030-EUR plasmid. The majority of isolates (n=178, 79.1%) belonged to international clone II. Strains belonging to the same sequence type but isolated in different countries or within the same country could be delineated in different clusters by core-genome multi-locus sequence typing (MLST). Whole-genome/core-genome MLST showed high diversity among the isolates, and the most common sequence type was ST2 (n=153, 67.7%). The EURECA A. baumannii strain collection represents a unique, diverse repository of carbapenem-resistant isolates that adds to the existing knowledge of A. baumannii epidemiology and resistance genes harboured by these strains.

An improved direct metamobilome approach increases the detection of larger-sized circular elements across kingdoms

Mobile genetic elements (MGEs) are instrumental in natural prokaryotic genome editing, permitting genome plasticity and allowing microbes to accumulate genetic diversity. MGEs serve as a vast communal gene pool and include DNA elements such as plasmids and bacteriophages (phages) among others. These mobile DNA elements represent a human health risk as they can introduce new traits, such as antibiotic resistance or virulence, to a bacterial strain. Sequencing libraries targeting environmental circular MGEs, referred to as metamobilomes, may broaden our current understanding of the mechanisms behind the mobility, prevalence and content of these elements. However, metamobilomics is affected by a severe bias towards small circular elements, introduced by multiple displacement amplification (MDA). MDA is typically used to overcome limiting DNA quantities after the removal of non-circular DNA during library preparations. By examining the relationship between sequencing coverage and the size of circular MGEs in paired metamobilome datasets with and without MDA, we show that larger circular elements are lost when using MDA. This study is the first to systematically demonstrate that MDA is detrimental to detecting larger-sized plasmids if small plasmids are present. It is also the first to show that MDA can be omitted when using enzyme-based DNA fragmentation and PCR in library preparation kits such as Nextera XT® from Illumina.

Genomic analysis of an extensively drug-resistant Pseudomonas aeruginosa ST312 harbouring IncU plasmid-mediated blaKPC-2 isolated from ascitic fluid

The Klebsiella pneumoniae carbapenemase (KPC) is disseminated worldwide mostly by plasmids. However, in Pseudomonas aeruginosa chromosomal mutations are more frequently responsible for resistance to carbapenems than the acquisition of mobile elements harbouring carbapenemases genes. Indeed, although uncommon, KPC-2-producing P. aeruginosa has appeared more frequently, including in Brazil. Here we report the first genomic analysis of a plasmid-mediated KPC-2 in an extensively drug-resistant (XDR) P. aeruginosa isolated in Santa Catarina, Brazil. Antimicrobial susceptibility testing was performed according to CLSI 2020 guidelines. The genome was sequenced using an Illumina MiSeq platform and the data were analysed using SPAdes and Prokka. In silico predictions were fulfilled using curated bioinformatics tools. Pseudomonas aeruginosa strain MIMA_PA2.1 (JACGTM000000000) was classified as XDR, belongs to sequence type 312 (ST312) and harbours the blaKPC-2 gene located on a small (7975 bp) IncU plasmid. This plasmid showed 86.3% identity with a non-conjugative plasmid (KC609322) carrying the blaKPC-2 gene from a multidrug-resistant P. aeruginosa (ST1006) from Colombia isolated in 2006. Besides the blaKPC-2 gene, other resistance genes to β-lactams, aminoglycosides, phenicol, fosfomycin and quinolones were detected, the last two also associated with mobile genetic elements other than the IncU plasmid described here. This is the first genomic report of the presence of the blaKPC-2 gene carried by Pseudomonas in Southern Brazil and highlights the adaptability of blaKPC-2 to different mobile elements. This draft genome might be useful for comparative genomic analyses to monitor the spread of plasmid-mediated blaKPC in P. aeruginosa in Latin America.

Molecular typing and antimicrobial resistance profiling of 33 mastitis-related Staphylococcus aureus isolates from cows in the Comarca Lagunera region of Mexico

Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphylococcus aureus. Little is known about the S. aureus lineages causing mastitis in Mexican cattle. The aim of this study was to type S. aureus isolates causing mastitis in cows from the Comarca Lagunera region in Mexico in 2015–2016. Multi-locus variable number tandem repeat fingerprinting (MLVF) of 33 S. aureus isolates obtained from 210 milk samples revealed the MLVF clusters A (n = 1), B (n = 26), C (n = 5) and D (n = 1). Spa-typing showed that clusters A and B represent the spa-type t224, cluster C includes spa-types t3196 and t416, and cluster D represents spa-type t114. The different spa-types were mirrored by the masses of protein A bands as detected by Western blotting. Antimicrobial susceptibility testing showed that one isolate was susceptible to all antimicrobials tested, whereas all other strains were resistant only to benzylpenicillin. These findings show that only four S. aureus lineages, susceptible to most antimicrobials, were responsible for causing mastitis at the time of sampling. Lastly, many isolates carried the same small plasmid, designated pSAM1. The high prevalence of pSAM1 amongst the antimicrobial-susceptible isolates suggests an association with bovine colonization or mastitis rather than antimicrobial resistance.

Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli.

Antimicrobial resistance (AMR) is a major threat to public health worldwide. Currently, AMR typing changes from phenotypic testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g., plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suitable techniques, standardized parameters and approved guidelines for reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E. coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long-read sequencing were assembled individually by Unicycler or Flye or a combination of both (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g., qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed to detect certain duplications and was unsuitable for genome finishing. Overall, the hybrid assembly led to the most comprehensive typing results, especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assemblies currently represents the best approach for reliable AMR typing and risk assessment.

Genomic data reveals the emergence of an IncQ1 small plasmid carrying blaKPC-2 in Escherichia coli of the pandemic sequence type 648

ObjectivesThe 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. MethodsTotal 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. ResultsGenomic 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. ConclusionIdentification 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.

Benzalkonium chloride and heavy metal resistance profiles of Listeria monocytogenes strains isolated from fish, fish products and food-producing factories in Poland

Phenotypic and genotypic resistance to benzalkonium chloride (BC), cadmium and arsenic was tested (by susceptibility assays and molecular methods) in 287 Listeria monocytogenes strains isolated from fish and fish products, and food-producing factories in Poland. Overall, 40% of the isolates were resistant to BC, 56% to cadmium and 41% to arsenic (57% displayed resistance to more than one of the tested compounds). Among BC-resistant isolates, the most commonly detected resistance determinant was the qacH gene (83%). Three distinct types of cadA gene determining resistance to cadmium were detected, with the cadA1 variant predominant (88%), while most arsenic-resistant isolates (86%) harbored the arsA gene associated with a Tn554-like transposon (one strain harbored two copies of arsA in different arsenic resistance cassettes). 53% of all tested isolates contained plasmids (from 4 kb to > 90 kb in size), which were classified into 11 groups (p1–p11) based on their restriction patterns. Interestingly, 12 isolates harbored the small mobilizable pLMST6-like plasmid pLIS3 encoding multidrug efflux pump EmrC. Clustering analysis of PFGE patterns revealed that these isolates represent several diverse bacterial populations, which strongly suggests mobility of the pLMST6-like plasmids among L. monocytogenes strains and their role in dissemination of BC resistance.

Generation of a 100-billion cyclic peptide phage display library having a high skeletal diversity.

Phage display is a powerful technique routinely used for the generation of peptide- or protein-based ligands. The success of phage display selections critically depends on the size and structural diversity of the libraries, but the generation of large libraries remains challenging. In this work, we have succeeded in developing a phage display library comprising around 100 billion different (bi)cyclic peptides and thus more structures than any previously reported cyclic peptide phage display library. Building such a high diversity was achieved by combining a recently reported library cloning technique, based on whole plasmid PCR, with a small plasmid that facilitated bacterial transformation. The library cloned is based on 273 different peptide backbones and thus has a large skeletal diversity. Panning of the peptide repertoire against the important thrombosis target coagulation factor XI enriched high-affinity peptides with long consensus sequences that can only be found if the library diversity is large.

Benchmarking of long-read assemblers for prokaryote whole genome sequencing.

Background:Data sets from long-read sequencing platforms (Oxford Nanopore Technologies and Pacific Biosciences) allow for most prokaryote genomes to be completely assembled - one contig per chromosome or plasmid. However, the high per-read error rate of long-read sequencing necessitates different approaches to assembly than those used for short-read sequencing. Multiple assembly tools (assemblers) exist, which use a variety of algorithms for long-read assembly. Methods:We used 500 simulated read sets and 120 real read sets to assess the performance of eight long-read assemblers (Canu, Flye, Miniasm/Minipolish, NECAT, NextDenovo/NextPolish, Raven, Redbean and Shasta) across a wide variety of genomes and read parameters. Assemblies were assessed on their structural accuracy/completeness, sequence identity, contig circularisation and computational resources used. Results:Canu v2.1 produced reliable assemblies and was good with plasmids, but it performed poorly with circularisation and had the longest runtimes of all assemblers tested. Flye v2.8 was also reliable and made the smallest sequence errors, though it used the most RAM. Miniasm/Minipolish v0.3/v0.1.3 was the most likely to produce clean contig circularisation. NECAT v20200803 was reliable and good at circularisation but tended to make larger sequence errors. NextDenovo/NextPolish v2.3.1/v1.3.1 was reliable with chromosome assembly but bad with plasmid assembly. Raven v1.3.0 was reliable for chromosome assembly, though it did not perform well on small plasmids and had circularisation issues. Redbean v2.5 and Shasta v0.7.0 were computationally efficient but more likely to produce incomplete assemblies. Conclusions:Of the assemblers tested, Flye, Miniasm/Minipolish,NextDenovo/NextPolish and Raven performed best overall. However, no single tool performed well on all metrics, highlighting the need for continued development on long-read assembly algorithms.