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Antimicrobial resistance (AMR) is a major threat to global public health that continues to grow owing to selective pressure caused by the use and overuse of antimicrobial drugs. Resistance spread by plasmids is of special concern, as they can mediate a wide distribution of AMR genes, including those encoding extended-spectrum β-lactamases (ESBLs). The CTX-M family of ESBLs has rapidly spread worldwide, playing a large role in the declining effectiveness of third-generation cephalosporins. This rapid spread across the planet is puzzling given that plasmids carrying AMR genes have been hypothesized to incur a fitness cost to their hosts in the absence of antibiotics. Here, we focus on a WT plasmid that carries the bla CTX-M 55 ESBL gene. We examine its conjugation rates and use head-to-head competitions to assay its associated fitness costs in both laboratory and wild Escherichia coli strains. We found that the wild strains exhibit intermediate conjugation levels, falling between two high-conjugation and two low-conjugation laboratory strains, the latter being older and more ancestral. We also show that the plasmid increases the fitness of both WT and lab strains when grown in lysogeny broth and Davis-Mingioli media without antibiotics, which might stem from metabolic benefits conferred on the host, or from interactions between the host and the rifampicin-resistant mutation we used as a selective marker. Laboratory strains displayed higher conjugation frequencies compared to WT strains. The exception was a low-passage K-12 strain, suggesting that prolonged laboratory cultivation may have compromised bacterial defences against plasmids. Despite low transfer rates among WT E. coli, the plasmid carried low fitness cost in minimal medium but conferred improved fitness in enriched medium, indicating a complex interplay between plasmids, host genetics and environmental conditions. Our findings reveal an intricate relationship between plasmid carriage and bacterial fitness. Moreover, they show that resistance plasmids can confer adaptive advantages to their hosts beyond AMR. Altogether, these results highlight that a closer study of plasmid dynamics is critical for developing a secure understanding of how they evolve and affect bacterial adaptability that is necessary for combating resistance spread.

Antimicrobial resistance (AMR) is a major threat to global public health that continues to grow owing to selective pressure caused by the use and overuse of antimicrobial drugs. Resistance spread by plasmids is of special concern, as they can mediate a wide distribution of AMR genes, including those encoding extended-spectrum β-lactamases (ESBLs). The CTX-M family of ESBLs has rapidly spread worldwide, playing a large role in the declining effectiveness of third-generation cephalosporins. This rapid spread across the planet is puzzling given that plasmids carrying AMR genes have been hypothesized to incur a fitness cost to their hosts in the absence of antibiotics. Here, we focus on a WT plasmid that carries the bla CTX-M 55 ESBL gene. We examine its conjugation rates and use head-to-head competitions to assay its associated fitness costs in both laboratory and wild Escherichia coli strains. We found that the wild strains exhibit intermediate conjugation levels, falling between two high-conjugation and two low-conjugation laboratory strains, the latter being older and more ancestral. We also show that the plasmid increases the fitness of both WT and lab strains when grown in lysogeny broth and Davis-Mingioli media without antibiotics, which might stem from metabolic benefits conferred on the host, or from interactions between the host and the rifampicin-resistant mutation we used as a selective marker. Laboratory strains displayed higher conjugation frequencies compared to WT strains. The exception was a low-passage K-12 strain, suggesting that prolonged laboratory cultivation may have compromised bacterial defences against plasmids. Despite low transfer rates among WT E. coli, the plasmid carried low fitness cost in minimal medium but conferred improved fitness in enriched medium, indicating a complex interplay between plasmids, host genetics and environmental conditions. Our findings reveal an intricate relationship between plasmid carriage and bacterial fitness. Moreover, they show that resistance plasmids can confer adaptive advantages to their hosts beyond AMR. Altogether, these results highlight that a closer study of plasmid dynamics is critical for developing a secure understanding of how they evolve and affect bacterial adaptability that is necessary for combating resistance spread.

Electroporation is a technique to introduce DNA constructs into cells using electric current. Here, we present a protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage. We describe steps for setting up and conducting electroporation. We then detail procedures for collecting, fixing, and mounting embryos and counting expression. This protocol can be used to study the expression of enhancers via reporter assays, manipulating cells using genes or modified genes such as dominant negatives, and genome editing. For complete details on the use and execution of this protocol, please refer to Song, et al.1.

The detection of mobile genetic elements is crucial for exploring the ecology and evolution of microbial communities, and it has diverse implications in biotechnology and public health. geNomad is a computational framework that enables researchers to precisely identifiy and annotate plasmids and viruses in sequencing data on a large scale.

Identifying and characterizing mobile genetic elements in sequencing data is essential for understanding their diversity, ecology, biotechnological applications and impact on public health. Here we introduce geNomad, a classification and annotation framework that combines information from gene content and a deep neural network to identify sequences of plasmids and viruses. geNomad uses a dataset of more than 200,000 marker protein profiles to provide functional gene annotation and taxonomic assignment of viral genomes. Using a conditional random field model, geNomad also detects proviruses integrated into host genomes with high precision. In benchmarks, geNomad achieved high classification performance for diverse plasmids and viruses (Matthews correlation coefficient of 77.8% and 95.3%, respectively), substantially outperforming other tools. Leveraging geNomad's speed and scalability, we processed over 2.7 trillion base pairs of sequencing data, leading to the discovery of millions of viruses and plasmids that are available through the IMG/VR and IMG/PR databases. geNomad is available at https://portal.nersc.gov/genomad .

Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.

UNLABELLED: Cryptic conjugative plasmids lack antibiotic-resistance genes (ARGs). These plasmids can capture ARGs from the vast pool of the environmental metagenome, but the mechanism to recruit ARGs remains to be elucidated. To investigate the recruitment of ARGs by a cryptic plasmid, we sequenced and conducted mating experiments with Escherichia coli SW4848 (collected from a lake) that has a cryptic IncX (IncX4) plasmid and an IncF (IncFII/IncFIIB) plasmid with five genes that confer resistance to aminoglycosides (strA and strB), sulfonamides (sul2), tetracycline [tet(A)], and trimethoprim (dfrA5). In a conjugation experiment, a novel hybrid Tn21/Tn1721 transposon of 22,570 bp (designated Tn7714) carrying the five ARG mobilized spontaneously from the IncF plasmid to the cryptic IncX plasmid. The IncF plasmid was found to be conjugative when it was electroporated into E. coli DH10B (without the IncX plasmid). Two parallel conjugations with the IncF and the new IncX (carrying the novel Tn7714 transposon) plasmids in two separate E. coli DH10B as donors and E. coli J53 as the recipient revealed that the conjugation rate of the new IncX plasmid (with the novel Tn7714 transposon and five ARGs) is more than two orders of magnitude larger than the IncF plasmid. For the first time, this study shows experimental evidence that cryptic environmental plasmids can capture and transfer transposons with ARGs to other bacteria, creating novel multidrug-resistant conjugative plasmids with higher dispersion potential. IMPORTANCE: Cryptic conjugative plasmids are extrachromosomal DNA molecules without antibiotic-resistance genes (ARGs). Environmental bacteria carrying cryptic plasmids with a high conjugation rate threaten public health because they can capture clinically relevant ARGs and rapidly spread them to pathogenic bacteria. However, the mechanism to recruit ARG by cryptic conjugative plasmids in environmental bacteria has not been observed experimentally. Here, we document the first translocation of a transposon with multiple clinically relevant ARGs to a cryptic environmental conjugative plasmid. The new multidrug-resistant conjugative plasmid has a conjugation rate that is two orders of magnitude higher than the original plasmid that carries the ARG (i.e., the new plasmid from the environment can spread ARG more than two orders of magnitude faster). Our work illustrates the importance of studying the mobilization of ARGs in environmental bacteria. It sheds light on how cryptic conjugative plasmids recruit ARGs, a phenomenon at the root of the antibiotic crisis.

Plasmids are extrachromosomal DNA found in microorganisms. They often carry beneficial genes that help bacteria adapt to harsh conditions. Plasmids are also important tools in genetic engineering, gene therapy, and drug production. However, it can be difficult to identify plasmid sequences from chromosomal sequences in genomic and metagenomic data. Here, we have developed a new tool called PlasmidHunter, which uses machine learning to predict plasmid sequences based on gene content profile. PlasmidHunter can achieve high accuracies (up to 97.6%) and high speeds in benchmark tests including both simulated contigs and real metagenomic plasmidome data, outperforming other existing tools.

Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli 'helper' strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA.

Background: Non-tuberculous mycobacteria (NTM) are a diverse group of environmental bacteria that are increasingly associated with human infections and difficult to treat. Plasmids, which might carry resistance and virulence factors, remain largely unexplored in NTM. Methods: We used publicly available complete genome sequence data of 328 NTM isolates belonging to 125 species to study gene content, genomic diversity, and clusters of 196 annotated NTM plasmids. Furthermore, we analyzed 3755 draft genome assemblies from over 200 NTM species and 5415 short-read sequence datasets from six clinically relevant NTM species or complexes including M. abscessus, M. avium complex, M. ulcerans complex and M. kansasii complex, for the presence of these plasmids. Results: Between one and five plasmids were present in approximately one-third of the complete NTM genomes. The annotated plasmids varied widely in length (most between 10 and 400 kbp) and gene content, with many genes having an unknown function. Predicted gene functions primarily involved plasmid replication, segregation, maintenance, and mobility. Only a few plasmids contained predicted genes that are known to confer resistance to antibiotics commonly used to treat NTM infections. Out of 196 annotated plasmid sequences, 116 could be grouped into 31 clusters of closely related sequences, and about one-third were found across multiple NTM species. Among clinically relevant NTM, the presence of NTM plasmids showed significant variation between species, within (sub)species, and even among strains within (sub)lineages, such as dominant circulating clones of Mycobacterium abscessus. Conclusions: Our analysis demonstrates that plasmids are a diverse and heterogeneously distributed feature in NTM bacteria. The frequent occurrence of closely related putative plasmid sequences across different NTM species suggests they may play a significant role in NTM evolution through horizontal gene transfer at least in some groups of NTM. However, further in vitro investigations and access to more complete genomes are necessary to validate our findings, elucidate gene functions, identify novel plasmids, and comprehensively assess the role of plasmids in NTM. [ABSTRACT FROM AUTHOR]

This brief review explores the intricate interplay between bacteriophages and plasmids in the context of antibiotic resistance gene (ARG) dissemination. Originating from studies in the late 1950s, the review traces the evolution of knowledge regarding extrachromosomal factors facilitating horizontal gene transfer and adaptation in bacteria. Analyzing the gene repertoires of plasmids and bacteriophages, the study highlights their contributions to bacterial evolution and adaptation. While plasmids encode essential and accessory genes influencing host characteristics, bacteriophages carry auxiliary metabolic genes (AMGs) that augment host metabolism. The debate on phages carrying ARGs is explored through a critical evaluation of various studies, revealing contrasting findings from researchers. Additionally, the review addresses the interplay between prophages and plasmids, underlining their similarities and divergences. Based on the available literature evidence, we conclude that plasmids generally encode ARGs while bacteriophages typically do not contain ARGs. But extra-chromosomaly present prophages with plasmid characteristics can encode and disseminate ARGs. [ABSTRACT FROM AUTHOR]

Bacterial plasmids and other extrachromosomal DNA elements frequently carry genes with important fitness effects for their hosts. Multicopy plasmids can additionally carry distinct alleles of host-fitness-relevant genes on different plasmid copies, allowing for heterozygosity not possible for loci on haploid chromosomes. Plasmid-mediated heterozygosity may increase the fitness of bacterial cells in circumstances where there is an advantage to having multiple distinct alleles (heterozyogote advantage); however, plasmid-mediated heterozygosity is also subject to constant loss due to random segregation of plasmid copies on cell division. We analyze a multitype branching process model to study the evolution and maintenance of plasmid-mediated heterozygosity under a heterozygote advantage. We focus on an evolutionary rescue scenario in which a novel mutant allele on a plasmid must be maintained together with the wild-type allele to allow population persistance (although our results apply more generally to the maintenance of heterozygosity due to heterozygote advantage). We determine the probability of rescue and derive an analytical expression for the threshold on the fitness of heterozygotes required to overcome segregation and make rescue possible; this threshold decreases with increasing plasmids copy number. We further show that the formation of cointegrates from the fusion of plasmid copies increases the probability of rescue. Overall, our results provide a rigorous quantitative assessment of the conditions under which bacterial populations can adapt to multiple stressors through plasmid-mediated heterozygosity. Many of the results are furthermore applicable to the related problem of the maintenance of incompatible plasmids in the same cell under selection for both. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CRhvKP) poses a significant global health threat due to its enhanced virulence and resistance. This study analyzed 5,036 publicly available K. pneumoniae genomes from China (2005–2023), identifying 1,538 CRhvKP genomes, accounting for 44.6% of carbapenem-resistant isolates and 69.5% of hypervirulent isolates. Predominant carbapenemases included bla KPC (92.1%), with an increasing prevalence of bla NDM and bla OXA-48-like genes. Most isolates (93.6%) carried both aerobactin and yersiniabactin genes. The genetic background showed high diversity, characterized by 36 sequence types (STs) and 22 capsule types, with high-risk endemic STs such as ST11, ST15, and ST23 being predominant. ST23 demonstrated enhanced virulence, whereas ST11 carried more resistance genes but showed minimal presence of iroBCDN genes. A core genome MLST analysis revealed that 89.0% of CRhvKP isolates clustered into 131 clonal groups, indicating widespread dissemination, particularly in eastern China. CR and hv plasmids, primarily IncF, IncH, and IncR types, showed distinct community structures, with CR plasmids demonstrating higher mobility and diversity. Crucially, we identified 40 CR-hv convergent plasmids across five STs, likely resulting from plasmid fusions, which have become increasingly prevalent in eastern China over the last decade. Furthermore, chromosomal integration of hv genes and bla KPC-2 was detected, underscoring the stable inheritance of these traits. Class 1 Integrons were present in 84.5% of CRhvKP strains, most notably in ST11 and least in ST23. These integrons harbored genes that confer resistance to various antibiotics, including bla IMP and bla VIM, with their content varying across different STs. This study highlights the genetic complexity, rapid dissemination, and increasing prevalence of CRhvKP in China, emphasizing the urgent need for enhanced genomic surveillance and targeted interventions to mitigate the threat posed by these multidrug-resistant and hypervirulent strains. [ABSTRACT FROM AUTHOR]

The emergence of multidrug-resistant (MDR) Salmonella is recognized as a significant public health problem worldwide. This study investigated the occurrence of MDR Salmonella serovars in chicken meat from wet markets in Metro Manila, Philippines from February to July 2022. Using whole genome sequencing (WGS) and phenotypic antimicrobial resistance (AMR) testing, the serovar, drug resistance, and virulence profiles of Salmonella isolates were characterized. Out of 253 chicken cut samples, 95 S. enterica isolates representing 15 distinct serovars were recovered. The most common was S. enterica serovar Infantis (51.58%), followed by S. Brancaster (9.47%), S. Anatum (7.37%), S. London (7.37%), S. Uganda (6.32%), and S. Derby (4.21%). Phenotypic AMR testing revealed that 73.68% of the isolates were resistant to at least one drug class, and 45.26% were MDR. A wide array of antimicrobial resistance genes (ARGs) associated with resistance to 12 different drug classes was identified, including three β-lactamase gene variants: bla CTX-M-65, bla TEM-1, and bla TEM-176. Some of these ARGs were located on MDR plasmids, such as those on IncFIB(K)_1_Kpn3, IncFIA(HI1)_1_HI1, and IncX1_1. A total of 131 virulence genes were detected, some of which conferred pESI-like characteristics to S. Infantis. These findings highlight a potential public health risk posed by pathogenic MDR Salmonella in chicken meat and underscore the urgent need for further research and coordinated AMR surveillance in the Philippines, aiming to stimulate national efforts to combat AMR. [ABSTRACT FROM AUTHOR]

This study aimed to characterize antibiotic-resistance plasmids present in microorganisms from sprout samples using exogenous plasmid capture. Fresh mung bean sprouts were predominantly colonized by bacteria from the phyla Proteobacteria and Bacteroidetes. To capture plasmids, a plasmid-free Escherichia (E.) coli CV601 strain, containing a green fluorescent protein gene for selection, was used as the recipient strain in exogenous plasmid capture experiments. Transconjugants were selected on media containing cefotaxime or tetracycline antibiotics. While no cefotaxime-resistant transconjugants were obtained, 40 tetracycline-resistant isolates were obtained and sequenced by Illumina NextSeq short read and Nanopore MinION long read sequencing. Sequences were assembled using Unicycler hybrid assembly. Most of the captured long plasmids carried either the tet (A) or tet (D) resistance gene, belonged to the IncFI or IncFII replicon types, and were predicted as conjugative. While the smaller plasmids contained the tet (A) tetracycline resistance gene as well as additional quinolone (qnr S1), sulfonamide (sul 1) and trimethoprim (dfr A1) resistance genes, the larger plasmids only contained the tet (D) resistance gene. An exception was the largest 192 kbp plasmid isolated, which contained the tet (D), as well as sulfonamide (sul1) and streptomycin (aad A1) resistance genes. The smaller plasmid was isolated from different sprout samples more often and showed a 100% identity in size (71,155 bp), while the 180 kbp plasmids showed some smaller or larger differences (in size between 157,683 to 192,360 bp). This suggested that the plasmids obtained from the similar sprout production batches could be clonally related. Nanopore MinION based 16S metagenomics showed the presence of Enterobacter (En.) cloacae , En. ludwigii , En. kobei , Citrobacter (C.) werkmanii , C. freundii , Klebsiella (K.) oxytoca and K. pneumonia , which have previously been isolated from fresh produce in Germany. These bacteria may harbor antibiotic resistance genes on plasmids that could potentially be transferred to similar genera. This study demonstrated that bacteria present in sprouts may act as the donors of antibiotic resistance plasmids which can transfer resistance to other bacteria on this product via conjugation. [ABSTRACT FROM AUTHOR]

A comparative genomic analysis of feline coronavirus (FCoV) and feline panleukopenia virus (FPLV) was performed. Based on the conserved regions of the two viruses, specific probes and real-time PCR (qPCR) primers were designed, and a duplex TaqMan qPCR-based assay was established for detecting FCoV and FPLV. The results showed high analytical specificity, and no cross-response with other feline viruses was observed. This method is highly versatile and can be used to detect all FCoV strains stored in laboratories and recombinant plasmids constructed according to the sequences of blank FCoV strains in laboratories. The analytical sensitivity of this method in detecting FCoV and FPLV was as low as 50 copies/μL, which is approximately 20-fold greater than that of conventional PCR. The coefficients of variation (CVs) for the intra- and interbatch coefficients of variation were less than 2%. After 75 clinical samples were tested, the percentage of FCoV- and FPLV-positive samples was 5.34% greater than that of conventional PCR methods, a finding robustly supported by sequencing identification. As validated by clinical samples, the method was sensitive, specific, general, and reproducible and holds great potential for the rapid identification and diagnosis of FCoV and FPLV infections, as well as for epidemiological investigations. Key points: • One-step duplex TaqMan real-time PCR detection method can detect FCoV and FPLV in clinical samples simultaneously and steadily. • Almost all the currently known FCoV and FPLV strains can be detected. • This method has high sensitivity, specificity and generality. [ABSTRACT FROM AUTHOR]

The complete genome of Legionella lytica PCM 2298 was sequenced and analyzed to provide insights into its genomic structure, virulence potential, and evolutionary position within the Legionella genus. The genome comprised a 3.2 Mbp chromosome and two plasmids, pLlyPCM2298_1 and pLlyPCM2298_2, contributing to a total genome size of 3.7 Mbp. Functional annotation identified 3,165 coding sequences, including genes associated with known virulence factors such as the major outer membrane protein (MOMP), the macrophage infectivity potentiator (Mip), and a comprehensive set of secretion systems (type II, type IVA, and type IVB Dot/Icm type IV secretion system). Notably, L. lytica contributed 383 unique genes to the Legionella pangenome, with 232 identified effector proteins, of which 35 were plasmid-encoded. The identification of unique genes, particularly those on plasmids, suggests an evolutionary strategy favoring horizontal gene transfer and niche adaptation. The effector repertoire included proteins with domains characteristic of host interaction strategies, such as ankyrin repeats and protein kinases. Comparative analyses showed that while L. lytica shares core virulence traits with other Legionella species, it has distinct features that may contribute to its adaptability and pathogenic potential. These findings underscore the genomic diversity within the genus and contribute to a deeper understanding of Legionella's ecological and clinical significance. A custom web application was developed using the R Shiny library, enabling users to interactively explore the expanded Legionella pangenome through UpSet plots. [ABSTRACT FROM AUTHOR]

Introduction: Despite a scarcity of data, before 2022 Ukraine was already considered a high-prevalence country for carbapenemase-producing Enterobacterales (CPE), and the situation has dramatically worsened during the full-scale war with Russia. The aim of this study was to analyse CPEs isolated in Poland from victims of war in Ukraine. Methods: The study included 65 CPE isolates from March 2022 till February 2023, recovered in 36 Polish medical centres from 57 patients arriving from Ukraine, differing largely by age and reason for hospitalisation. All isolates were sequenced by MiSeq and ten Klebsiella pneumoniae isolates also by MinION. Taxonomy, clonality and resistomes were analysed for all CPEs, whereas phylogeny, serotypes, virulomes and plasmids were characterised for K. pneumoniae, and partially for Escherichia coli ST131, using various bioinformatic tools. Results: Multifactorial diversity of the isolates reflected the patients' clinical-epidemiological heterogeneity. The CPEs represented six species. Klebsiella pneumoniae was the most prevalent with 50 isolates and 15 sequence types (STs), mainly ST395, ST307, ST11, ST147 and ST23, producing NDM (-1/-5), OXA-48 (-48/-1242) or KPC (-2/-3)-like carbapenemases. Each of the STs produced groups of loosely related isolates, clusters of close relatives and/or unique isolates, correlating with K serotypes and carbapenemases. Many of these, especially NDM-1- and/or OXA-48-producing ST395 and ST307, were related to Russian organisms. Others, for example, NDM-1-producing ST11, clustered with those from Poland. Numerous K. pneumoniae isolates had specific virulence genes, including aerobactin iuc, largely due to spread of pNDM-MAR plasmids, showing both resistance and virulence. Two E. coli ST131 isolates belonged to clades B or C1 and produced KPC-3 or NDM-1, respectively. Conclusions: Together with similar studies from Germany and The Netherlands, this work has documented broad dissemination of CPE in Ukraine, driven by a number of specific K. pneumoniae lineages circulating over a large territory of Eastern Europe. [ABSTRACT FROM AUTHOR]

Background Enterobacter hormaechei is an important pathogen in humans and animals, which, in addition to its intrinsic AmpC, can acquire a wide variety of genes conferring resistances to extended-spectrum cephalosporins (ESCs) and carbapenems (CPs). In France, human clinical outbreaks of E. hormaechei resistant to ESC or carbapenem were reported. Objectives To study E. hormaechei isolates from cats and dogs (=59) as well as from horses (n = 55) presenting a non-susceptible phenotype to beta-lactams in order to determine which clones, resistance genes and plasmids are circulating in France. Material and methods E. hormaechei isolates (n  = 114) were short-read sequenced and five isolates were long-read sequenced to better characterize the plasmids carrying ESC- and CP-resistance determinants. Phenotypes were characterized by antibiograms using the disc diffusion method. Results A clear divergence in the molecular epidemiology was observed depending on the host. In cats and dogs, most of the isolates presented an overexpressed ampC gene or the bla CTX-M-15 gene carried by an IncHI2 plasmid, and eight isolates (8/59, 13.6%) presented the bla OXA-48 carbapenemase gene. Thirty-two isolates (32/59, 54.2%) belonged to the human high-risk clones ST78, ST114 and ST171. Contrarily, in horses, ESC resistance was mostly due to the bla SHV-12 and bla CTX-M-15 genes carried by an IncHI2 plasmid, and high-risk clones were rarely identified (5/55, 9.0%). Discussion Potential selection by antibiotic use (which is on an increasing trend in France for cats, dogs and horses), the dissemination capacities of both conjugative IncHI2 plasmids and high-risk clones, and possible transfers of resistant bacteria between humans and animals strongly indicate that E. hormaechei should be closely monitored. [ABSTRACT FROM AUTHOR]

1. Salmonella Gallinarum strains isolated from a southern Brazil fowl typhoid outbreak were subjected to phenotypic and genotypic analyses to identify genetic elements that could improve prevention and control strategies. 2. Whole-genome sequencing revealed the presence of the aac(6')-Iaa gene, conferring aminoglycoside resistance, along with novel chromosomal point mutations, including the first detection of parE p.S451F in Salmonella Gallinarum. 3. Additionally, IncFII(S) plasmid replicons, Salmonella pathogenicity islands and 105 virulence genes associated with cell adhesion, invasion and antimicrobial peptide resistance were identified. 4. These findings shed light on the molecular mechanisms of fowl typhoid and provide crucial insights into emerging antimicrobial resistance and virulence factors. [ABSTRACT FROM AUTHOR]

The emergence of Salmonella enterica serotype Dublin (S. Dublin) presents significant challenges to animal and human health. We analyzed 109 S. Dublin isolates from bovine submissions to the Penn State Animal Diagnostic Laboratory between 2011 and 2022. Using whole genome sequencing, we assessed their phenotypic and genotypic resistance patterns and correlated these traits with case histories and pathology reports. Core-genome analysis identified cgSTs with similar allelic profiles between our isolates and those from the U.S. and Canada, while some cgSTs were unique to our study. Histopathologic findings suggest a predominance of respiratory and gastroenteric/hepatic lesions, aligning with the histopathological case definition for S. Dublin infection. Critically, all isolates were multidrug-resistant, particularly to ampicillin (87%), ceftiofur (89%), chlortetracycline (94%), oxytetracycline (94%), enrofloxacin (17%), florfenicol (94%), sulfadimethoxine (97%), and trimethoprim (20%). Plasmid genomic analysis unveiled distinct plasmid types including virulence, resistance, and hybrid plasmids, carrying unique compositions of virulence genes and antimicrobial resistance. These findings underscore the importance of managing calf movement to control the introduction and dissemination of new cgSTs in Pennsylvania and potentially nationwide. Furthermore, they emphasize the urgent need to mitigate S. Dublin transmission, combat antimicrobial resistance, and enhance surveillance efforts to effectively protect animal and human health. [ABSTRACT FROM AUTHOR]

The antibiotic resistance of pathogenic microorganisms is currently one of most major medical problems, causing a few million deaths every year worldwide due to untreatable bacterial infections. Unfortunately, the prognosis is even worse, as over 8 million deaths associated with antibiotic resistance are expected to occur in 2050 if no new effective antibacterial treatments are discovered. The Hfq protein has been discovered as a bacterial RNA chaperone. However, subsequent studies have indicated that this small protein (composed of 102 amino acid residues in Escherichia coli) has more activities, including binding to DNA and influencing its compaction, interaction with biological membranes, formation of amyloid-like structures, and others. Although Hfq is known to participate in many cellular processes, perhaps surprisingly, only reports from recent years have demonstrated its role in bacterial antibiotic resistance. The aim of this narrative review is to discuss how can Hfq affects antibiotic resistance in bacteria and propose how this knowledge may facilitate developing new therapeutic strategies against pathogenic bacteria. We indicate that the mechanisms by which the Hfq protein modulates the response of bacterial cells to antibiotics are quite different, from the regulation of the expression of genes coding for proteins directly involved in antibiotic transportation or action, through direct effects on membranes, to controlling the replication or transposition of mobile genetic elements bearing antibiotic resistance genes. Therefore, we suggest that Hfq could be considered a potential target for novel antimicrobial compounds. We also discuss difficulties in developing such drugs, but since Hfq appears to be a promising target for drugs that may enhance the efficacy of antibiotics, we propose that works on such potential therapeutics are encouraged. [ABSTRACT FROM AUTHOR]

Epidemiology of shigellosis has drastically changed in recent years due to globalization and sexual risk behaviors. Here, through whole-genome sequencing, we characterized two ESBL-producing Shigella sonnei strains (ShSoBUH1 and ShSoBUH2) carrying a blaCTX−M−15 among men who have sex with men (MSM), who had not recently traveled and presented sexual risk behaviors. Both strains harbored IncB/O/K/Z and IncFII plasmids, which carry aadA1, aadA5, sul1, sul2, dfrA1, dfrA17, mph(A), erm(B), tet(B), qacE and blaCTX−M−15 genes conferring resistance to 2nd and 3rd generation cephalosporins, cotrimoxazole, erythromycin, azithromycin and quinolones. IncFII plasmids containing blaCTX−M−15 from ShSoBUH1 and ShSoBUH2 presented 99,8–99,9% similarity with plasmids from another five CTX-M-15 S. sonnei strains detected in Belgium and Switzerland. A single-nucleotide polymorphism (SNP) analysis determined that the study strains differed by 361 SNPs, belonging to different clusters. To the best of our knowledge, this is the first report describing two extensively drug-resistant (XDR) CTX-M-15 S. sonnei strains in MSM. [ABSTRACT FROM AUTHOR]

The hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses a substantial challenge to the global health care. However, the mechanism behind its evolution and transmission remain elusive. Here, four virulence plasmid types were identified from 310 hv-CRKP isolates collected nationwide during 2017-2018, based on their aerobactin (iuc locus) lineage and IncFIB replicons. Notably, pIUC1-IncFIB(K)37 and pIUC1-IncFIB(Mar), representing two epidemic virulence plasmids in Asia and Europe, respectively, accounted for >90% of the hv-CRKP episodes. Analysis of 494 K. pneumoniae isolates (376 from 2010-2013; 118 from 2017-2018) and 2578 public K. pneumoniae genomes indicated the notable role of IncFIB(Mar) plasmids in the hv-CRKP emergence and spread. Conjugation assays showed the helper IncFIB(Mar) plasmid could efficiently transfer into a hypervirulent strain and uniquely retromobilize with pIUC1-IncFIB(K)37 back into CRKP. Thereafter, the IncFIB(Mar) plasmid either lost rapidly or recombined with pIUC1-IncFIB(K)37, generating the hybrid pIUC1-IncFIB(Mar) plasmid. Our findings elucidated formation, evolution, and dissemination trajectories of the two major hv-CRKP strains in different regions. [ABSTRACT FROM AUTHOR]

The emergence of multidrug-resistant (MDR) Raoultella isolates is linked to the acquisition of antibiotic resistance genes (ARGs) with plasmids playing a pivotal role in this process. While plasmid-mediated transmission of ARGs in Raoultella has been extensively reported, limited attention has been given to genetically dissecting the modular structures of plasmids. This study aims to elucidate the genomic features of novel incompatible plasmids in MDR Raoultella by presenting 13 complete plasmid sequences from four isolates, along with an analysis of 16 related plasmids from GenBank. These 29 plasmids were classified into five distinct groups: IncFII single-replicon plasmids, dual-replicon plasmids containing the IncFII replicon, IncHI plasmids, IncR plasmids, and IncX8 plasmids. A new incompatible group, IncFIIp23141−CTXM, was identified, alongside five newly designated Inc groups based on previously determined sequences, namely IncFIIpKPC2_EC14653, IncFIIpCP020359, IncFIIpCP024509, IncFIIpKOX−137, and IncFIIpKDO1. Furthermore, this research marks the first report of four Inc groups of plasmids within Raoultella, namely IncFIIp23141−CTXM plasmid, IncFIIpKPC2_EC14653 plasmid, IncX8 plasmid, and IncFIIpCP020359: IncFIB-7.1 dual-replicon plasmid. Moreover, novel mobile genetic elements, including two unit transposons (Tn6806 and Tn6891), one IS-based transposition unit (Tn6561), and four insertion sequences (ISRor6, ISRor7, ISRor8, and ISRor9) were discovered. Notably, this is the first report of mcr-9 in clinical Raoultella strains. At least 49 ARGs conferring resistance against 11 different categories of antimicrobials were identified on these 13 plasmids. Overall, this research deepens the understanding of incompatible plasmids in Raoultella, serving as a reference for exploring antibiotic resistance profiles and plasmid diversity in MDR Raoultella. [ABSTRACT FROM AUTHOR]

Aim: Located in the Southwest Indian Ocean area (SIOA), the two French overseas territories (FOTs) of Reunion and Mayotte islands are heavily impacted by antimicrobial resistance. The aim of this study was to investigate all cases of NDM-5 and OXA-181 carbapenemase-producing Escherichia coli (CPEc) in these two FOTs between 2015 and 2020, to better understand the regional spread of these last-line treatment resistant bacteria. Methods: All E. coli isolates not susceptible to ertapenem from various public and private hospitals on Reunion and Mayotte islands were screened for carbapenemase production. Clinical and microbiological data were collected for each case. Genotypic analysis of the isolates was carried out using WGS to determine the clonality relationship between the isolates and the genetic support of the carbapenemase-encoding genes. Results: A total of 92 isolates of NDM-5 (n = 67) and OXA-181 (n = 25) CPEc was collected from Reunion (n = 55) and Mayotte (n = 37) islands. Whole-genome sequencing identified 4 majors STs (ST58, ST167, ST405 and ST410). Genotypic analysis demonstrated numerous intra-ST possible cross transmission events, including strains isolated in both islands. Finally, all isolates (100%) carried the blaNDM-5 or blaOXA-181 genes on plasmids (IncF2, IncX3), most of which were conserved and identified in various STs. Conclusion: We highlighted the dual dissemination of successful plasmids and the worrying circulation of high-risk clones via patients transfer between these two FOTs. It is therefore essential to effectively screen these patients for CPEc carriage on admission and to take these plasmids into account when investigating intra- or inter-hospital CPEc outbreaks. [ABSTRACT FROM AUTHOR]

Background: F plasmids are abundant in E. coli, carrying a variety of genetic cargo involved in fitness, pathogenicity, and antimicrobial resistance. ColV and pUTI89-like plasmids have drawn attention for their potential roles in various forms of extra-intestinal pathogenicity. However, the rates of their carriage and the overall diversity of F plasmids in E. coli bloodstream infections (BSI E. coli) remain unknown. Methods: We performed a t-SNE-based cluster analysis of predicted F plasmids from a collection of 4711 BSI E. coli draft genomes to describe their diversity and abundance. We also screened them for markers of ColV and pUTI89-like plasmids, F plasmid replicon sequence types (RST) and E. coli sequence types (ST) to understand how genetic features were related to plasmid clusters. Results: Predicted F plasmids in BSI E. coli draft genomes were embedded within five major clusters based on a model of complete F plasmid sequences. Nearly half of the clustered sequences belonged to two major clusters, which were associated with ColV and pUTI89-like marker genes, respectively. Genomes from the ColV cluster featured F2:A-:B1 and F24:A-B1 RSTs in association with ST95, ST58 and ST88, whilst the pUTI89-like cluster was mostly F29:A-:B10 linked to ST73, ST69, ST95 and ST131. Plasmids associated with different lineages of ST131 formed additional major clusters, whilst F51:A-:B10 plasmids in ST73 were also common. Conclusions: ColV and pUTI89-like plasmid markers are predominant in BSI E. coli that carry F plasmids. These markers are associated with distinct clusters of plasmids across diverse sequence types of E. coli. We hypothesise that their abundance in BSI E. coli is partially driven by carriage of backbone genes previously shown to contribute to virulence in models of bloodstream infection. Their carriage by pandemic E. coli STs suggests clonal expansion also plays a role in their success in BSI. Ecological pathways via which these plasmids evolve, and spread are likely to be distinct as other studies show ColV is strongly associated with poultry and food animal production, whereas pUTI89-like plasmids appear to be mostly human-restricted. [ABSTRACT FROM AUTHOR]

Conjugation is a complex phenomenon involving multiple plasmid, bacterial, and environmental factors. Here we describe an IncI1 plasmid encoding multidrug antibiotic resistance to aminoglycosides, sulfonamides, and third-generation cephalosporins. This plasmid is widespread geographically among animal, human, and environmental sectors. We present data on the transmissibility of this plasmid from Salmonella enterica ser. Kentucky into 40 strains of S. enterica (10 strains each from serovars Enteritidis, Heidelberg, Infantis, and Typhimurium). Thirty seven out of 40 strains were able to take up the plasmid. Rates of conjugation were variable between strains ranging from 10−8 to 10−4. Overall, serovars Enteritidis and Typhimurium demonstrated the highest rates of conjugation, followed by Heidelberg, and then Infantis. No relationships were observed between the recipient cell surface and rate of conjugation. Recipient cell numbers correlated positively with conjugation rate and strains with high conjugation rates had marginally but significantly higher growth parameters compared to strains that took up the plasmid at lower frequencies. Environmental conditions known to impact cell growth, such as temperature, nutrient availability, and the presence of antibiotics, had a modulating effect on conjugation. Collectively, these results will further understanding of plasmid transmission dynamics in Salmonella, which is a critical first step towards the development of mitigation strategies. [ABSTRACT FROM AUTHOR]

Parasitic elements often spread to hosts through the delivery of their nucleic acids to the recipient. This is particularly true for the primary parasites of bacteria, bacteriophages (phages) and plasmids. Although bacterial immune systems can sense a diverse set of infection signals, such as a protein unique to the invader or the disruption of natural host processes, phage and plasmid nucleic acids represent some of the most common molecules that are recognized as foreign to initiate defense. In this review, we will discuss the various elements of invader nucleic acids that can be distinguished by bacterial host immune systems as "non-self" and how this signal is relayed to activate an immune response. Prokaryotes sense viral and plasmid nucleic acids to start defense pathways against these invaders. This review examines the different mechanisms of DNA and RNA recognition and how these distinguish self from foreign to avoid autoimmunity. We also describe the immune response that follows the detection of invader nucleic acids. [ABSTRACT FROM AUTHOR]

Conjugation plays a major role in dissemination of antimicrobial resistance genes. Following transfer of IncF-like plasmids, recipients become refractory to a second wave of conjugation with the same plasmid via entry (TraS) and surface (TraT) exclusion mechanisms. Here, we show that TraT from the pKpQIL and F plasmids (TraTpKpQIL and TraTF) exhibits plasmid surface exclusion specificity. The cryo-EM structures of TraTpKpQIL and TraTF reveal that they oligomerise into decameric champagne bottle cork-like structures, which are anchored to the outer membrane via a diacylglycerol and palmitic acid modified α-helical barrel domain. Unexpectedly, we identify chromosomal TraT homologues from multiple Gram-negative phyla which form numerous divergent lineages in a phylogenetic tree of TraT sequences. Plasmid-associated TraT sequences are found in multiple distinct lineages, including two separate clades incorporating TraT from Enterobacteriaceae IncF/F-like and Legionellaceae F-like plasmids. These findings suggest that different plasmid backbones have acquired and co-opted TraT on independent occasions. Outer membrane protein TraT protects bacteria from secondary F plasmid conjugation. Here, the authors report cryo-EM structure of decameric cork-like TraT and its evolutionary history as a chromosomal gene that has been incorporated into multiple plasmid families. [ABSTRACT FROM AUTHOR]

Host-pathogen interactions represent a dynamic evolutionary process, wherein both hosts and pathogens continuously develop complex mechanisms to outmaneuver each other. Borrelia burgdorferi, the Lyme disease pathogen, has evolved an intricate antigenic variation mechanism to evade the host immune response, enabling its dissemination, persistence, and pathogenicity. Despite the discovery of this mechanism over two decades ago, the precise processes, genetic elements, and proteins involved in this system remain largely unknown. The vls locus, which is the site of antigenic variation, has been notoriously challenging to manipulate genetically due to its highly conserved structural features, even with significant advancements in molecular biology and genetic engineering for this highly segmented pathogen. Our study highlights the pivotal role of plasmid topology in facilitating in trans gene recombination. We demonstrate that gene conversion can occur in trans when a copy of vlsE gene is present on a linear plasmid, contrary to previous observations suggesting a cis arrangement is required for vlsE recombination. Significantly, employing this in trans gene conversion strategy with a linear plasmid, we have, for the first time, achieved targeted genetic mutation of putative cis-acting elements in the native vlsE gene. This has unveiled a potentially crucial role for the 17 bp direct repeats that flank the central variable cassette region of vlsE. Furthermore, we validated the reliability and reproducibility of our mutational approach by successfully inserting stop codons at two distinct sites within the central variable cassette of vlsE. Thus, this study presents a significant methodological innovation enabling the direct manipulation of the vls locus and lays the groundwork for systematic exploration of specific mutations affecting the mechanism of antigenic variation. As a result, it creates new avenues for research and raises intriguing questions that could guide the development of novel methods to explore host-pathogen interactions of the agent of Lyme disease. Author summary: Antigenic variation is one of the key strategies employed by Borrelia burgdorferi, the causative agent of the Lyme disease, to evade the host's adaptive immune response. Despite being recognized for over 25 years, the precise mechanisms underlying this immune evasion system remain poorly understood. This knowledge gap is mainly because the vls locus, the site of antigenic variation, has many highly conserved features and secondary structures that complicate genetic manipulation efforts. In this study, for the first time, we successfully introduced mutations in various elements of the vlsE gene, a central component of the antigenic variation system. We demonstrate that the plasmid topology significantly influences gene conversion process and identify the 17 bp direct repeat DNA sequences as potentially crucial when a vlsE gene copy is present on a separate linear plasmid. Additionally, we validated our method for introducing mutations, proving its effectiveness by creating stop codons at multiple sites within the vlsE gene. This new approach provides a robust framework for investigating the impact of specific genetic alterations on the bacterium's antigenic variability. Such insights are crucial for a deeper understanding of host-pathogen interactions to combat Lyme disease. [ABSTRACT FROM AUTHOR]

Detecting alterations in plasmid structures is often performed using conventional molecular biology. However, these methods are laborious and time-consuming for studying the conditions inducing these mutations, which prevent real-time access to cell heterogeneity during bioproduction. In this work, we propose combining both flow cytometry and fluorescence-activated cell sorting, integrated with mechanistic modelling to study conditions that lead to plasmid recombination using a limonene-producing microbial system as a case study. A gene encoding GFP was introduced downstream of the key enzymes involved in limonene biosynthesis to enable real-time kinetics monitoring and the identification of cell heterogeneity according to microscopic and flow cytometric analyses. Three different plasmid configurations (one correct and two incorrect) were identified through cell sorting based on subpopulations expressing different levels of GFP at 10 and 50 µM IPTG. Higher limonene production (530 mg/L) and lower subpopulation proportion carrying the incorrect plasmid (12%) were observed for 10 µM IPTG compared to 50 µM IPTG (96 mg/L limonene and more than 70% of cell population carrying the incorrect plasmid, respectively) in 100 mL production culture. We also managed to derive exploratory hypotheses regarding the plasmid recombination region using the model and successfully validated them experimentally. Additionally, the results also showed that limonene production was proportional to GFP fluorescence intensity. This correlation could serve as an alternative to using biosensors for a high-throughput screening process. The developed method enables rapid identification of plasmid recombination at single-cell level and correlates the heterogeneity with bioproduction performance. Key points: • Strategy to study plasmid recombination during bioproduction. • Different plasmid structures can be identified and monitored by flow cytometry. • Mathematical modelling suggests specific alterations in plasmid structures. [ABSTRACT FROM AUTHOR]

Prokaryotes (Bacteria and Archaea) encode a highly diversified arsenal of defence systems that protect them against mobile genetic elements, such as phages and plasmids. In turn, mobile genetic elements encode anti-defence systems that allow them to escape the activity of these defence systems. This has resulted in an evolutionary arms race in which defence systems and anti-defence systems evolve and adapt continuously, driving intriguing innovation and enormous diversification on both sides. Over 150 prokaryotic defence systems have been identified to date. Anti-defence systems are known for only a subset of these, but more are being discovered at a steady rate. Despite an increasing understanding of the highly diverse molecular mechanisms of anti-defence systems, their diverse evolutionary origins, the selective pressures they are subjected to, and their ecological importance and implications often remain obscure. In this review, we describe the diverse strategies that phage and plasmid anti-defence systems employ to escape host defence systems. We explore the evolutionary origins of anti-defence systems and describe different factors that exert selective pressure, affecting their maintenance and diversification. We describe how, in turn, defence systems themselves evolved to act upon anti-defence mechanisms, thereby adding a new layer to the co-evolutionary battle between prokaryotes and their mobile genetic elements. We discuss how the continuous selective pressures found in dynamic microbial communities promote the retention and diversification of these anti-defence systems. Finally, we consider the ecological implications for both hosts and their mobile genetic elements, noting how the balance of defence and anti-defence strategies can shape microbial community composition, influence horizontal gene transfer, and impact ecosystem stability. In this Review, Niault et al. discuss how anti-defence systems allow phages and plasmids to escape prokaryotic defence systems, with a focus on their evolutionary origins, the selective pressures that promote their retention and diversification, and the ecological factors that influence their interactions with defence systems. [ABSTRACT FROM AUTHOR]

Introduction: The conjugative transfer of antibiotic resistance genes (ARGs) mediated by plasmids occurred in different intestinal segments of mice was explored. Methods: The location of ARG donor bacteria and ARGs was investigated by qPCR, flow cytometry, and small animal imaging. The resistant microbiota was analyzed by 16S rRNA gene amplification sequencing. Results: The small intestine was the main site for the location of ARG donor bacteria and ARGs. The intestinal microbiota richness of the small intestine (duodenum and jejunum) and the large intestine (cecum, colon, and rectum) increased, and the ileum microbiota richness decreased under the action of donor bacteria. The differences in the number of bacteria in the small intestine and the large intestine, as well as the relative richness of Firmicutes from the small intestine to the large intestine, decreased. By contrast, the relative abundance of Proteobacteria increased. The intake of resistant plasmids alleviated the impact of antibiotics on intestinal microbiota, particularly increasing the proportion of Proteobacteria and Bacteroides, which were presumed to be susceptible to ARGs. Discussion: The acquisition of ARGs by intestinal microbes is an important reason why infectious diseases are difficult to cure, which brings risks to human health and intestinal microecology. [ABSTRACT FROM AUTHOR]

Background: The emergence of colistin resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant public health concern, as colistin has been the last resort for treating such infections. This study aimed to investigate the prevalence and molecular characteristics of colistin-resistant CRKP isolates in Central South China. Methods: CRKP isolates from twelve hospitals in Central South China were screened for colistin resistance using broth microdilution. The epidemiological characteristics, virulome, resistome, plasmid replicons and two-component systems associated with colistin resistance of colistin-resistant isolates were explored by whole-genome sequencing. The mgrB gene and the relative expression of the pmrC and pmrK genes were analyzed by polymerase chain reaction (PCR) and real-time quantitative PCR, respectively. The bacterial virulence was evaluated through a Galleria mellonella larvae infection model. Results: Of the 429 nonduplicate CRKP isolates, 26 (6.1%) were colistin-resistant and they included eight clonal clusters. Six distinct sequence type (ST)-capsule loci (KL) types were identified: ST11-KL64, ST11-KL47, ST963-KL16, ST307-KL102, ST751-KL64 and ST5254-KL47. 88.5% (23/26) of them were found to carry at least one carbapenemase gene, including blaKPC−2 (65.4%, 17/26) and blaNDM−1 (7.7%, 2/26), as well as coharbouring blaKPC−2 and blaNDM−1 (15.4%, 4/26). Diverse mutations of colistin resistance-related genes were observed, with mgrB inactivation by insertions and the T157P deleterious mutation in pmrB being detected in 57.7% and 42.3% of the colistin-resistant isolates, respectively. In addition, a novel deleterious mutation, R248P, in the crrB gene was found in two ST11 isolates. 88.5% of the 26 isolates presented an increase in pmrK transcription, and 69.2% of them had an overexpression of the pmrC gene. All the 16 ST11-KL64 isolates and one ST751-KL64 isolate (65.4%, 17/26) carried at least two hypervirulence biomarkers and showed high virulence in vivo. Conclusions: This study highlights the presence of different colistin resistance mechanisms in isolates belonging to the same clone and identified multiple clonal transmission clusters in colistin resistant isolates, including the globally high-risk ST11 and ST307 clones, of which a significant proportion exhibited high virulence. Consequently, it is crucial to enforce measures to prevent the ongoing spread of colistin resistance. [ABSTRACT FROM AUTHOR]

Background Tigecycline is an antimicrobial agent with a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. However, mobile tigecycline resistance gene clusters, such as tnfxB-tmexCD-toprJ , have spread globally. The prevalence of tigecycline-resistant Enterobacterales in clinical settings in Japan is unknown. Objectives To investigate the tnfxB-tmexCD-toprJ gene cluster in the genome sequences of Enterobacterales clinical isolates in Japan. Methods We investigated the tnfxB-tmexCD-toprJ cluster from the genome sequences of 5143 Enterobacterales isolates collected from 175 hospitals around Japan between 2019 and 2020 as part of a national genomic surveillance program for antimicrobial-resistant bacteria. Results The tnfxB1-tmexCD1-toprJ1 cluster was detected in two Klebsiella pneumoniae isolates in 2019. One isolate possessed a 299.4 kb IncFIB(K) plasmid, pJBBGAAF19431, and the other possessed a 224.9 kb IncHI1B/IncFIB(K) hybrid plasmid, pJBEAACG19501, co-carrying multiple antimicrobial resistance genes, including extended-spectrum β-lactamase genes, bla OXA-1 and bla CTX-M-27, respectively, along with tnfxB1-tmexCD1-toprJ1. The genetic context of the tnfxB1-tmexCD1-toprJ1 -surrounding structure on pJBBGAAF19431 was similar to that of a K. pneumoniae plasmid pHNAH8I-1 from a chicken in China in 2017, and the cluster was embedded in an apparently intact mobile DNA element: strand-biased circularizing integrative element. The tnfxB1-tmexCD1-toprJ1 on pJBEAACG19501 was embedded in a Tn 3 family transposon related to Tn As1. The plasmid pJBEAACG19501 was highly similar to that of K. pneumoniae , isolated from humans in China in 2021. Conclusions tmexCD-toprJ was present in Japan as of 2019. Even in Japan, where the clinical use of tigecycline is significantly rare, tmexCD-toprJ -harbouring multidrug-resistant Enterobacterales is a public health threat and requires continuous monitoring. [ABSTRACT FROM AUTHOR]

Background 'One Health' initiatives to tackle the rising risk of antimicrobial resistance (AMR) have flourished due to increasing detection of Enterobacterales producing extended-spectrum beta-lactamases (ESBLs) and carbapenemases (CPs). Objectives This study aimed to conduct an in-depth holistic analysis of Escherichia coli (Ec) and Klebsiella pneumoniae (Kp) isolates recovered from landfill-foraging white stork faecal samples and clinical isolates from a nearby hospital. Methods Faecal samples (n  = 211) were collected from storks foraging at two landfills in Spain. Ec/Kp stork isolates were recovered on selective media and whole-genome sequencing (WGS), together with isolates obtained from the nearby hospital. These genomic data were compared with public genomes from different contexts (clinical, environmental, or animal hubs) to understand global transmission dynamics. Results A wide range of bla ESBL/ bla pAmpC (bla CTX-M/ bla SHV-12/ bla DHA) were detected in 71 stork samples (33.6%), while bla CP (bla KPC/ bla NDM/ bla OXA-48/ bla VIM) were identified in 28 (13.3%) samples. Clonal and plasmid transmissions were evidenced inside and between both landfills. Mapping against 10 624 public Ec/Kp genomes and from those of nearby hospital revealed that identical strains (<10 allelic differences with Ec-ST38/ST131 and Kp-ST512 lineages) and epidemic plasmids (full identity/coverage with IncN/ bla KPC-2, IncF/ bla KPC-3, IncX3/ bla NDM-7, IncL/ bla OXA-48) were found from clinical isolates in countries located along the storks' migration routes. Conclusions Storks may be contaminated by bacterial isolates from a likely human origin and become non-human reservoirs of critical genes, which can be dispersed over long distances. Identifying strains/plasmids along the stork's routes that are identical or closely related to those described here opens new perspectives for large-scale research to understand the AMR transmission dynamics. [ABSTRACT FROM AUTHOR]

Hydroxytyrosol, a fine chemical, is widely utilized in food and pharmaceutical industries. In this study, we constructed a pathway to produce hydroxytyrosol by co-expressing tyrosin-phenol lyase (TPL), L-amino acid dehydrogenase (aadL), α-keto acid decarboxylase (KAD), aldehyde reductase (yahK) and glucose dehydrogenase (gdh). We changed combinations between plasmids with different copy numbers and target genes, resulting in 84% increase in hydroxytyrosol production. The yield of hydroxytyrosol was further increased 89.3% by optimizing the temperature and pH. Finally, 55.3 mM hydroxytyrosol was produced within 14 h by fed-batch biotransformation. This study provides a novel approach for hydroxytyrosol production. [ABSTRACT FROM AUTHOR]

Structural maintenance of chromosomes (SMC) complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC-family Wadjet complexes structurally resemble the widespread MukBEF but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves plasmid DNA; however, the molecular mechanism underlying plasmid recognition remains unclear. Here, we use in vitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric loop extruder that simultaneously reels in DNA from both sides of a loop and that this activity requires a dimeric JetABC supercomplex. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44-kb-pair plasmid, stalls, and cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across all kingdoms of life. [Display omitted] • Wadjet is a symmetric DNA loop extruder requiring a dimeric JetABC supercomplex • Wadjet fully extrudes a surface-anchored 44-kbp plasmid, stalls, and then cleaves it • Loop extrusion promotes selective plasmid cleavage by JetABCD for DNA-based immunity • Loop extrusion is conserved among SMC complexes across all domains of life Pradhan et al. use single-molecule imaging to show that the bacterial SMC Wadjet complex is a symmetric loop extruder that reels DNA from both sides of a loop as a dimer. On a plasmid DNA, Wadjet extrudes the full length of the plasmid, stalls, and subsequently cleaves the DNA. These data reveal the role of loop extrusion in the specific plasmid restriction by Wadjet. [ABSTRACT FROM AUTHOR]

Aims This study evaluated the phenotypic and genotypic traits of mcr-1.1 -harboring Escherichia coli isolates from chickens, pigs, humans, and farm environments. The resistome and the mobile genetic elements associated with the spread of mcr-1.1 in Southern Brazil were also characterized. Methods and results The 22 mcr-1.1 -harboring E. coli isolates from different origins were selected for antimicrobial susceptibility testing and whole genome sequencing for characterization of the resistome, plasmids, and sequence types. All isolates presented several resistance genes and harbored the mcr-1.1 gene in a highly similar IncX4 plasmid. Furthermore, the mcr-1.1 gene co-occurred with the mcr-3.12 gene in a multidrug-resistant isolate from the farm environment. Conclusions These findings demonstrate that the mcr-1.1 gene in E. coli isolates from Brazil is spreading mainly by horizontal transfer of the IncX4 plasmid. The co-occurrence of mcr-1.1 and mcr-3.12 highlights pig farming as an important reservoir of colistin resistance. [ABSTRACT FROM AUTHOR]

As an emerging environmental contaminant, antibiotic resistance genes (ARGs) in tap water have attracted great attention. Although studies have provided ARG profiles in tap water, research on their abundance levels, composition characteristics, and potential threat is still insufficient. Here, 9 household tap water samples were collected from the Guangdong-Hong Kong-Macao Greater Bay Area (GBA) in China. Additionally, 75 sets of environmental sample data (9 types) were downloaded from the public database. Metagenomics was then performed to explore the differences in the abundance and composition of ARGs. 221 ARG subtypes consisting of 17 types were detected in tap water. Although the ARG abundance in tap water was not significantly different from that found in drinking water plants and reservoirs, their composition varied. In tap water samples, the three most abundant classes of resistance genes were multidrug, fosfomycin and MLS (macrolide-lincosamide-streptogramin) ARGs, and their corresponding subtypes ompR, fosX and macB were also the most abundant ARG subtypes. Regarding the potential mobility, vanS had the highest abundance on plasmids and viruses, but the absence of key genes rendered resistance to vancomycin ineffective. Generally, the majority of ARGs present in tap water were those that have not been assessed and are currently not listed as high-threat level ARG families based on the World Health Organization Guideline. Although the current potential threat to human health posed by ARGs in tap water is limited, with persistent transfer and accumulation, especially in pathogens, the potential danger to human health posed by ARGs should not be ignored. [ABSTRACT FROM AUTHOR]

This study characterized the genome of three Salmonella enterica serovar Heidelberg (S. Heidelberg) strains with different antimicrobial resistance (AMR) profile that were inoculated as a cocktail into fresh pine wood shavings (PWS). The strains were isolated from feces (SH-AAFC), carcass (SH-ARS), and thigh (SH-FSIS) of broiler chicken. SH-AAFC harbored an antimicrobial resistant gene (ARG) blaCMY-2 on an IncI1 plasmid while SH-FSIS harbored multiple ARGs (floR, cmlA1, tet(A), blaTEM-1B, ant(2″)-Ia, aph(6)-Id, aph(3″)-Ib, and sul2) on an IncC plasmid. SH-ARS was pan-susceptible. The die-off of Salmonella was determined at days 0, 1, 7, 14, and 21. Antibiotic susceptibility tests and whole genome sequencing were performed on 77 isolates. At 21 days post-inoculation, Salmonella abundance decreased by 4.4 Log10 CFU/g with the water activity of PWS being correlated with Salmonella survival. SH-AAFC clonal populations survived longer in PWS than SH-FSIS and SH-ARS populations. SH-AAFC clones persisting in litter carried higher copy number of Col plasmids than their ancestors, while some SH-ARS clones acquired a lysogenic bacteriophage from SH-FSIS populations. These results suggest that mobile genetic determinants such as plasmids (which could carry ARGs) and bacteriophage plays roles in the persistence of S. Heidelberg in the PWS used as broiler litter. Highlights: S. Heidelberg survived up to 21 days in PWS which is often used as broiler bedding. S. Heidelberg abundance and survival was correlated with the water activity of PWS. S. Heidelberg strains that carried higher copy numbers of small Col plasmids were the dominant strains isolated from PWS at later time points. S. Heidelberg strains harboring transmissible plasmid carrying AmpC-like beta-lactamase gene persisted longer in PWS without antibiotic pressures for AMR. [ABSTRACT FROM AUTHOR]

Importance: Urinary tract infections (UTIs) are common and significant health issues in pets. Although extensive international research exists on their prevention and treatment, a notable gap remains in analyzing the characteristics of the causative bacteria. Objective: To investigate the phylogroup, antimicrobial resistance (AMR), and molecular genotype of Escherichia coli isolates from dogs and cats with UTIs in animal clinics in Korea. Methods: Uropathogenic E. coli (UPEC) strains were analyzed for phylogenetic grouping polymerase chain reaction, AMR, transferable resistance plasmids, and multilocus sequence typing. Results: Sixty-seven UPECs were isolated from urine samples of dogs (n = 57) and cats (n = 10). Regarding age, the incidence of UTI was the highest in the 11 to 15 years range (46.3%, 31/67). Regarding sex, it accounted for 58.2% (n = 39) in female dogs and 11.9% (8/67) in female cats. Phylogroup B2 was the most frequent (n = 51, 75.0%) among all strains, followed by D (16.2%), A (7.4%), and B1 (1.5%). Thirty-seven (55.2%) UPECs were multidrugresistant (MDR), and 24 (35.8%) of them belonged to phylogroup B2. Extended-spectrum cephalosporin and carbapenemase genes were detected in 18 (26.9%) UPECs and plasmids carrying these resistance genes were conjugated between strains. Thirty sequence types (STs) were identified among the total strains. Among the UPECs (n = 51) with phylogroup B2, 23 STs were identified, with ST73 being the most frequent (n = 12, 17.9%), followed by ST131 (n = 9, 13.4%). Conclusions and Relevance: Phylogroup B2 strains, particularly ST73 and ST121, were most prevalent in UPECs from Korean companion dogs and cats. For MDR UPECs, appropriate antibiotic selection is essential for the treatment of UTIs. [ABSTRACT FROM AUTHOR]