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Abstract Background The burden of sepsis worsens due to the continuation of emerging pathogens such as multidrug-resistant Pantoea species. Methods A multicenter study was conducted between October 2019 and September 2020 at four hospitals located in central, southern, and northern parts of Ethiopia. A total of 1416 sepsis patients were recruited and blood cultures were performed. At each study site, positive cultures were characterized by their colony characteristics, gram stain, and conventional biochemical tests. All Pantoea species were identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF) and subjected to whole genome sequencing (WGS) using Illumina HiSeq 2500. The phylogeny structure of Pantoea isolates was calculated using IQ-TREE v1.6.12 from single-nucleotide polymorphisms detected by Snippy v.4.6.0 and filtered by Gubbins v.2.3.4. Average nucleotide identity was estimated by using OrthoANI v.0.93.1 on Shovill v.1.1.0 assemblies. Antimicrobial resistance genes and plasmid replicons were detected using ARIBA v.2.14.6. Phylogenetic trees were visualized using iTOLv.6.5.2. Results Multiple Pantoea species include: P. dispersa (n = 19), P. septica (n = 1), and a novel Pantoea spp. (n = 1), were identified among sepsis patients. All P. dispersa isolates and the novel Pantoea species were isolated at Dessie Referral Hospital and displayed phylogenetic clonality, including the ubiquity of an IncM1 plasmid and identical antimicrobial resistance (AMR) gene profiles, encoding bla CTX−M−15, bla TEM−1D, bla SCO−1, and aac(3)-lla. The novel Pantoea spp. isolate harboured bla CTX−M−9 and bla TEM−1D and carried an IncN3 plasmid replicon. The P. septica was isolated at Tikur Anbessa Specialized Hospital in Addis Ababa and carried no detectable acquired AMR genes. Conclusion The emerging Pantoea spp. carrying multiple AMR genes were identified from sepsis patients. Implementation of strong infection prevention strategies and building surveillance capacity with advanced bacteriology laboratories capable of identifying multidrug-resistant emerging pathogens is strongly recommended.

Uropathogenic Escherichia coli (UPEC) is the main cause of urinary tract infections (UTIs) and carries virulence and resistance factors often found in mobilizable genetic elements, such as plasmids or pathogenicity islands (PAIs). UPEC is part of the extraintestinal pathogenic E. coli (ExPEC), but hybrid strains possessing both diarrheagenic E. coli (DEC) and ExPEC traits, termed “hypervirulent”, present a significant health threat. This study assessed the prevalence of UPEC PAIs, ExPEC sequence types (ST), DEC genes, carbapenemase and extended-spectrum β-lactamase (ESBL) phenotypes, resistance genotypes, and plasmids in 40 clinical isolates of UPEC. Results showed that 72.5% of isolates had PAIs, mainly PAI IV536 (53%). ESBL phenotypes were found in 65% of β-lactam-resistant isolates, with 100% of carbapenem-resistant isolates producing carbapenemase. The predominant ESBL gene was blaCTX-M-2 (60%), and the most common resistance gene in fluoroquinolone and aminoglycoside-resistant isolates was aac(6′)Ib (93%). Plasmids were present in 57% of isolates, and 70% belonged to the ST131 clonal group. Molecular markers for DEC pathotypes were detected in 20 isolates, with 60% classified as hybrid pathotypes. These findings indicate significant pathogenic potential and the presence of hybrid pathotypes in E. coli UTI clinical isolates in the Mexican population.

ABSTRACT Antibiotic resistance genes (ARGs) in Staphylococcus aureus can disseminate vertically through successful clones, but also horizontally through the transfer of genes conveyed by mobile genetic elements (MGEs). Even though underexplored, MGE/ARG associations in S. aureus favor the emergence of multidrug-resistant clones, which are challenging therapeutic success in both human and animal health. This study investigated the interplay between the mobilome and the resistome of more than 10,000 S. aureus genomes from human and animal origin. The analysis revealed a remarkable diversity of MGEs and ARGs, with plasmids and transposons being the main carriers of ARGs. Numerous MGE/ARG associations were identified, suggesting that MGEs play a critical role in the dissemination of resistance. A high degree of similarity was observed in MGE/ARG associations between human and animal isolates, highlighting the potential for unrestricted spread of ARGs between hosts. Our results showed that in parallel to clonal expansion, MGEs and their associated ARGs can spread across different strain types sequence types (STs), favoring the evolution of these clones and their adaptation in selective environments. The high variability of MGE/ARG associations within individual STs and their spread across several STs highlight the crucial role of MGEs in shaping the S. aureus resistome. Overall, this study provides valuable insights into the complex interplay between MGEs and ARGs in S. aureus, emphasizing the need to elucidate the mechanisms governing the epidemic success of MGEs, particularly those implicated in ARG transfer.IMPORTANCEThe research presented in this article highlights the importance of understanding the interactions between mobile genetic elements (MGEs) and antibiotic resistance genes (ARGs) carried by Staphylococcus aureus, a versatile bacterium that can be both a harmless commensal and a dangerous pathogen for humans and animals. S. aureus has a great capacity to acquire and disseminate ARGs, enabling efficient adaption to various environmental or clinical conditions. By analyzing a large data set of S. aureus genomes, we highlighted the substantial role of MGEs, particularly plasmids and transposons, in disseminating ARGs within and between S. aureus populations, bypassing host barriers. Given that multidrug-resistant S. aureus strains are classified as a high-priority pathogen by global health organizations, this knowledge is crucial for understanding the complex dynamics of transmission of antibiotic resistance in this species.

IntroductionThe rising prevalence of Extended-Spectrum Beta-Lactamase (ESBL)-producing Klebsiella species (spp.) poses a significant threat to human and animal health and environmental safety. To address this pressing issue, a comprehensive study was undertaken to elucidate the burden and dissemination mechanisms of ESBL-Klebsiella spp. in dairy cattle farms.MethodsFifty-seven Klebsiella species were isolated on CHROMagar™ ESBL plates and confirmed with MADLI-TOF MS and whole genome sequenced from 14 dairy farms.Results and discussionSix families of beta-lactamase (bla) (blaCTX−M, blaSHV, blaTEM, blaOXY, blaOXA, and blaSED) were detected in ESBL-Klebsiella spp. genomes. Most (73%) of isolates had the first three types of beta-lactamase genes, with blaSHV being the most frequent, followed by blaCTX−M. Most (93%) isolates harbored two or more bla genes. The isolates were genotypically MDR, with 26 distinct types of antibiotic resistance genes (ARGs) and point mutations in gyrA, gyrB, and parC genes. The genomes also harbored 22 different plasmid replicon types, including three novel IncFII. The IncFII and Col440I plasmids were the most frequent and were associated with blaCTXM−27 and qnrB19 genes, respectively. Eighteen distinct sequence types (STs), including eight isolates with novel STs of K. pneumoniae, were detected. The most frequently occurring STs were ST353 (n = 8), ST469 (n = 6), and the novel ST7501 (n = 6). Clusters of ESBL-Klebsiella strains with identical STs, plasmids, and ARGs were detected in multiple farms, suggesting possible clonal expansion. The same ESBL variant was linked to identical plasmids in different Klebsiella STs in some farms, suggesting horizontal spread of the resistance gene. The high burden and dual spread mechanism of ESBL genes in Klebsiella species, combined with the emergence of novel sequence types, could swiftly increase the prevalence of ESBL-Klebsiella spp., posing significant risks to human, animal, and environmental health. Immediate action is needed to implement rigorous surveillance and control measures to mitigate this risk.

ABSTRACT Lyme disease, caused by spirochetes in the Borrelia burgdorferi sensu lato clade within the Borrelia genus, is transmitted by Ixodes ticks and is currently the most prevalent and rapidly expanding tick-borne disease in Europe and North America. We report complete genome sequences of 47 isolates that encompass all established species in this clade while highlighting the diversity of the widespread human pathogenic species B. burgdorferi. A similar set of plasmids has been maintained throughout Borrelia divergence, indicating that they are a key adaptive feature of this genus. Phylogenetic reconstruction of all sequenced Borrelia genomes revealed the original divergence of Eurasian and North American lineages and subsequent dispersals that introduced B. garinii, B. bavariensis, B. lusitaniae, B. valaisiana, and B. afzelii from East Asia to Europe and B. burgdorferi and B. finlandensis from North America to Europe. Molecular phylogenies of the universally present core replicons (chromosome and cp26 and lp54 plasmids) are highly consistent, revealing a strong clonal structure. Nonetheless, numerous inconsistencies between the genome and gene phylogenies indicate species dispersal, genetic exchanges, and rapid sequence evolution at plasmid-borne loci, including key host-interacting lipoprotein genes. While localized recombination occurs uniformly on the main chromosome at a rate comparable to mutation, lipoprotein-encoding loci are recombination hotspots on the plasmids, suggesting adaptive maintenance of recombinant alleles at loci directly interacting with the host. We conclude that within- and between-species recombination facilitates adaptive sequence evolution of host-interacting lipoprotein loci and contributes to human virulence despite a genome-wide clonal structure of its natural populations.IMPORTANCELyme disease (also called Lyme borreliosis in Europe), a condition caused by spirochete bacteria of the genus Borrelia, transmitted by hard-bodied Ixodes ticks, is currently the most prevalent and rapidly expanding tick-borne disease in the United States and Europe. Borrelia interspecies and intraspecies genome comparisons of Lyme disease-related bacteria are essential to reconstruct their evolutionary origins, track epidemiological spread, identify molecular mechanisms of human pathogenicity, and design molecular and ecological approaches to disease prevention, diagnosis, and treatment. These Lyme disease-associated bacteria harbor complex genomes that encode many genes that do not have homologs in other organisms and are distributed across multiple linear and circular plasmids. The functional significance of most of the plasmid-borne genes and the multipartite genome organization itself remains unknown. Here we sequenced, assembled, and analyzed whole genomes of 47 Borrelia isolates from around the world, including multiple isolates of the human pathogenic species. Our analysis elucidates the evolutionary origins, historical migration, and sources of genomic variability of these clinically important pathogens. We have developed web-based software tools (BorreliaBase.org) to facilitate dissemination and continued comparative analysis of Borrelia genomes to identify determinants of human pathogenicity.

Plasmids (circular DNA molecules) represent an ingenious strategy for horizontal gene transfer in prokaryotes and eukaryotic cells. Plasmids harbored in bacteria are responsible for the spread of traits such as antibiotic resistance, virulence factors, and the machinery for the horizontal gene transfer e.g., type IV secretion systems. Remarkably, Bacillus thuringiensis (Bt) cryptic plasmids encode and carry genes that, under the host environment, replicate and concomitate with sporulation, producing parasporal crystalline proteins of two major types, crystalline (Cry) and cytolytic (Cyt), the former toxic against different orders of insects such as Lepidopterans, Coleopterans, and Dipterans (Cry proteins, MW 50–130 KDa); Cyt proteins, produced by B. thuringiensis subspecies israelensis (Bti)(MW 27-kDa) are toxic against Dipterans, i.e., mosquitoes and black flies. The X-Ray tridimensional structure for both types of toxins, formed by three domains, mostly of beta sheets antiparallel (Domain II and Domain III) linked through loops of different lengths. Domain I is a bundle of alpha helices. This structure is characterized by five conserved blocks, implying a conservation in the mode of action. Cyt proteins possess two alpha helices and some beta sheets with a structure similar to the antimicrobial peptides. Indeed, the mode of action proposed is mediated by the toxin-lipid interaction that hypothetically could result in transmembrane ionic channel formation. Several pieces of evidence support the action of both toxins in insects and mammals. The question is to what extent these Bt/Bti plasmid-encoded Cry or Cyt genes can be applied as bioinsecticides individually or in combination with Lysinibacillus sphaericus. The feasibility of being considered a promising and safe biological strategy for crop pests and vector-borne neglected infectious diseases is an issue pinpointed in the present review.

Colorectal cancer (CRC) is one of the leading oncogenic disorders, both in terms of incidence and mortality. The etiology of the disease is certainly multifactorial. Various risk factors like alcohol consumption, smoking, CRC family history, inflammatory bowel disease, hormone therapy, aspirin/nonsteroidal anti-inflammatory drugs use, higher body mass index, consumption of red and/or processed meat, insufficient physical activity, and decreased intake of fruit and vegetables have been pointed out; however, there is not enough support evidence for a single particular causative mechanism. Recently, gut bacterial microbiota has been shown to influence significantly the pathogenesis of CRC. However, little attention is paid to the putative impact of plasmids in gut flora.

Abstract Background Carbapenem-resistant Escherichia coli (CREC) has been considered as WHO priority pathogens, causing a great public health concern globally. While CREC from patients has been thoroughly investigated, the prevalence and underlying risks of CREC in healthy populations have been overlooked. Systematic research on the prevalence of CREC in healthy individuals was conducted here. We aimed to characterize CREC collected from healthy populations in China between 2020 and 2022 and to compare the genomes of CREC isolates isolated from healthy individuals and clinical patients. Methods We present a nationwide investigation of CREC isolates among healthy populations in China, employing robust molecular and genomic analyses. Antimicrobial susceptibility testing, whole-genome sequencing, and bioinformatics were utilized to analyze a cohort of CREC isolates (n = 113) obtained from fecal samples of 5 064 healthy individuals. Representative plasmids were extracted for third-generation nanopore sequencing. We previously collected 113 non-duplicate CREC isolates (59 in 2018, 54 in 2020) collected from ICU patients in 15 provinces and municipalities in China, and these clinical isolates were used to compare with the isolates in this study. Furthermore, we employ comparative genomics approaches to elucidate molecular variations and potential correlations between clinical and non-clinical CREC isolates. Results A total of 147 CREC isolates were identified from 5 064 samples collected across 11 provinces in China. These isolates were classified into 64 known sequence types (STs), but no dominant STs were observed. In total, seven carbapenemase genes were detected with bla NDM-5 (n = 116) being the most prevalent one. Genetic environments and plasmid backbones of bla NDM were conserved in CREC isolated from healthy individuals. Furthermore, we compared clinical and healthy human-originated CRECs, revealing noteworthy distinctions in 23 resistance genes, including bla NDM-1, bla NDM-5, and bla KPC (χ 2 test, p

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a prevalent issue in China, with its spread primarily attributed to the presence of the plasmid-borne carbapenemase genes, blaKPC and blaNDM. However, instances of plasmids containing both blaKPC-2 and blaNDM-1have never been reported. Methods: In this study, the genomic and microbiological characteristics of hybrid plasmids containing both blaKPC-2 and blaNDM-1 were identified in Chinese clinical CRKP isolates by Illumina combined with ONT nanopore sequencing technology. Results: The newly identified plasmid was formed via IS26-mediated recombination and has been shown to be transferable to Escherichia coli. It substantially elevates the minimum inhibitory concentration (MIC) of meropenem by 4000-fold in E. coli, surpassing the MIC values observed in E. coli strains that carry either blaKPC-2 and blaNDM-1 alone, as previously demonstrated in our study. Notably, the co-occurrence of the KPC–NDM fusion plasmid and a pLVPK-like virulence plasmid was observed in these organisms. In vivo experiments revealed that the isolates harbouring the pLVPK-like virulence plasmid exhibited a significantly higher lethality rate in Galleria mellonella. Conclusions: The increased antibiotic resistance brought by this novel fusion plasmid and its accompanying virulence factors pose a serious potential threat to human health and deserve our vigilance.

Abstract Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-β-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 108 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 108 CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

The global upsurge in antibiotic resistant bacteria (ARB) is putting immense pressure on healthcare. The spreading of antimicrobial resistance is facilitated by mobile genetic elements, most especially plasmids. The widespread use of antibiotics in clinical and veterinary environments creates selective pressure that drives the evolution of ARB. Plasmids contribute to the propagation of AR in different types of clinical infections. The role plasmids play in this evolution necessitates their utilization in molecular surveillance to detect the emergence of ARB and track the spread of AR plasmids. Recent technologies like replicon typing and whole genome sequencing (WGS) have become the gold standard for molecular epidemiology of plasmids for the detection and control of epidemics in clinical settings. Unfortunately, access to such technologies is limited in low- and middle-income countries (LMICs). The major aim of this review is to examine the specific contributions of plasmids to the upsurge of AR in clinical settings and elucidate the various replicon types that have been attributed to specific antibiotic-resistant infections in healthcare settings. Healthcare in LMICs should be supported to build capacity in WGS and molecular surveillance to effectively prevent and control AR bacterial infections.

ABSTRACT Bacterial relatedness measured using select chromosomal loci forms the basis of public health genomic surveillance. While approximating vertical evolution through this approach has proven exceptionally valuable for understanding pathogen dynamics, it excludes a fundamental dimension of bacterial evolution—horizontal gene transfer. Incorporating the accessory genome is the logical remediation and has recently shown promise in expanding epidemiological resolution for enteric pathogens. Employing k-mer-based Jaccard index analysis, and a novel genome length distance metric, we computed pangenome (i.e., core and accessory) relatedness for the globally important pathogen Salmonella enterica serotype Typhi (Typhi), and graphically express both vertical (homology-by-descent) and horizontal (homology-by-admixture) evolutionary relationships in a reticulate network of over 2,200 U.S. Typhi genomes. This analysis revealed non-random structure in the Typhi pangenome that is driven predominantly by the gain and loss of mobile genetic elements, confirming and expanding upon known epidemiological patterns, revealing novel plasmid dynamics, and identifying avenues for further genomic epidemiological exploration. With an eye to public health application, this work adds important biological context to the rapidly improving ways of analyzing bacterial genetic data and demonstrates the value of the accessory genome to infer pathogen epidemiology and evolution.IMPORTANCEGiven bacterial evolution occurs in both vertical and horizontal dimensions, inclusion of both core and accessory genetic material (i.e., the pangenome) is a logical step toward a more thorough understanding of pathogen dynamics. With an eye to public, and indeed, global health relevance, we couple contemporary tools for genomic analysis with decades of research on mobile genetic elements to demonstrate the value of the pangenome, known and unknown, annotated, and hypothetical, for stratification of Salmonella enterica serovar Typhi (Typhi) populations. We confirm and expand upon what is known about Typhi epidemiology, plasmids, and antimicrobial resistance dynamics, and offer new avenues of exploration to further deduce Typhi ecology and evolution, and ultimately to reduce the incidence of human disease.

ABSTRACT Comprehensive whole-genome sequencing was performed on two multi-drug-resistant Escherichia coli strains isolated from cattle manure from a typical dairy farm in Poland in 2020. The identified strains are resistant to beta-lactams, aminoglycosides, tetracyclines, trimethoprim/sulfamethoxazole, and fluoroquinolones. The complete sequences of the harbored plasmids revealed antibiotic-resistance genes located within many mobile genetic elements (e.g., insertional sequences or transposons) and genes facilitating conjugal transfer or promoting horizontal gene transfer. These plasmids are hitherto undescribed. Similar plasmids have been identified, but not in Poland. The identified plasmids carried resistance genes, including the tetracycline resistance gene tet(A), aph family aminoglycoside resistance genes aph(3″)-lb and aph (6)-ld, beta-lactam resistance genes blaTEM-1 and blaCTX-M-15, sulfonamide resistance gene sul2, fluoroquinolone resistance gene qnrS1, and the trimethoprim resistance gene dfrA14. The characterized resistance plasmids were categorized into the IncY incompatibility group, indicating a high possibility for dissemination among the Enterobacteriaceae. While similar plasmids (99% identity) have been found in environmental and clinical samples, none have been identified in farm animals. These findings are significant within the One Health framework, as they underline the potential for antimicrobial-resistant E. coli from livestock and food sources to be transmitted to humans and vice versa. It highlights the need for careful monitoring and strategies to limit the spread of antibiotic resistance in the One Health approach.IMPORTANCEThis study reveals the identification of new strains of antibiotic-resistant Escherichia coli in cattle manure from a dairy farm in Poland, offering critical insights into the spread of drug resistance. Through whole-genome sequencing, researchers discovered novel plasmids within these bacteria, which carry genes resistant to multiple antibiotics. These findings are particularly alarming, as these plasmids can transfer between different bacterial species, potentially escalating the spread of antibiotic resistance. This research underscores the vital connection between the health of humans, animals, and the environment, emphasizing the concept of One Health. It points to the critical need for global vigilance and strategies to curb the proliferation of antibiotic resistance. By showcasing the presence of these strains and their advanced resistance mechanisms, the study calls for enhanced surveillance and preventive actions in both agricultural practices and healthcare settings to address the imminent challenge of antibiotic-resistant bacteria.

IntroductionAcinetobacter baumannii contributes significantly to the global issue of multidrug-resistant (MDR) nosocomial infections. Often, these strains demonstrate resistance to carbapenems (MDR-CRAB), the first-line treatment for infections instigated by MDR A. baumannii. Our study focused on the antimicrobial susceptibility and genomic sequences related to plasmids from 12 clinical isolates of A. baumannii that carry both the blaOXA-58 and blaNDM-1 carbapenemase genes.MethodsWhole-genome sequencing with long-read technology was employed for the characterization of an A. baumannii plasmid that harbors the blaOXA-58 and blaNDM-1 genes. The location of the blaOXA-58 and blaNDM-1 genes was confirmed through Southern blot hybridization assays. Antimicrobial susceptibility tests were conducted, and molecular characterization was performed using PCR and PFGE.ResultsMultilocus Sequence Typing analysis revealed considerable genetic diversity among blaOXA-58 and blaNDM-1 positive strains in Brazil. It was confirmed that these genes were located on a plasmid larger than 300 kb in isolates from the same hospital, which also carry other antimicrobial resistance genes. Different genetic contexts were observed for the co-occurrence of these carbapenemase-encoding genes in Brazilian strains.DiscussionThe propagation of blaOXA-58 and blaNDM-1 genes on the same plasmid, which also carries other resistance determinants, could potentially lead to the emergence of bacterial strains resistant to multiple classes of antimicrobials. Therefore, the characterization of these strains is of paramount importance for monitoring resistance evolution, curbing their rapid global dissemination, averting outbreaks, and optimizing therapy.

ABSTRACT Bradyrhizobium is known for fixing atmospheric nitrogen in symbiosis with agronomically important crops. This study focused on two groups of strains, each containing eight natural variants of the parental strains, Bradyrhizobium japonicum SEMIA 586 (=CNPSo 17) or Bradyrhizobium diazoefficiens SEMIA 566 (=CNPSo 10). CNPSo 17 and CNPSo 10 were used as commercial inoculants for soybean crops in Brazil at the beginning of the crop expansion in the southern region in the 1960s–1970s. Variants derived from these parental strains were obtained in the late 1980s through a strain selection program aimed at identifying elite strains adapted to a new cropping frontier in the central-western Cerrado region, with a higher capacity of biological nitrogen fixation (BNF) and competitiveness. Here, we aimed to detect genetic variations possibly related to BNF, competitiveness for nodule occupancy, and adaptation to the stressful conditions of the Brazilian Cerrado soils. High-quality genome assemblies were produced for all strains. The core genome phylogeny revealed that strains of each group are closely related, as confirmed by high average nucleotide identity values. However, variants accumulated divergences resulting from horizontal gene transfer, genomic rearrangements, and nucleotide polymorphisms. The B. japonicum group presented a larger pangenome and a higher number of nucleotide polymorphisms than the B. diazoefficiens group, possibly due to its longer adaptation time to the Cerrado soil. Interestingly, five strains of the B. japonicum group carry two plasmids. The genetic variability found in both groups is discussed considering the observed differences in their BNF capacity, competitiveness for nodule occupancy, and environmental adaptation.IMPORTANCEToday, Brazil is a global leader in the study and use of biological nitrogen fixation with soybean crops. As Brazilian soils are naturally void of soybean-compatible bradyrhizobia, strain selection programs were established, starting with foreign isolates. Selection searched for adaptation to the local edaphoclimatic conditions, higher efficiency of nitrogen fixation, and strong competitiveness for nodule occupancy. We analyzed the genomes of two parental strains of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens and eight variant strains derived from each parental strain. We detected two plasmids in five strains and several genetic differences that might be related to adaptation to the stressful conditions of the soils of the Brazilian Cerrado biome. We also detected genetic variations in specific regions that may impact symbiotic nitrogen fixation. Our analysis contributes to new insights into the evolution of Bradyrhizobium, and some of the identified differences may be applied as genetic markers to assist strain selection programs.

ABSTRACT: Objectives: Tigecycline is a last-resort antibiotic used for treatment of infections with carbapenem-resistant Klebsiella pneumoniae. The aim of the study was to understand the genetic mechanism of resistance and the genetic context of resistance genes in two tigecycline-resistant K. pneumoniae strains isolated from sewage in Bergen, Norway. Methods: Complete genome sequencing of the two strains was accomplished using a combination of short-read Illumina MiSeq-based and long-read Oxford Nanopore MinION-based sequencing. Conjugation experiments were performed using filter mating and a green fluorescent protein (GFP)-tagged Escherichia coli strain. Results: The complete genome sequences of strain K6-320.1 and strain K7-325 were assembled into two contigs for each strain, one contig representing the complete circular chromosomes of 5 223 440 bp (K6-320.1) and 5 263 092 bp (K7-325), respectively, and the other representing plasmids with sizes of 276 509 bp (pK6-320.1) and 246 731 bp (pK7-325). Strain K6-320.1 belonged to sequence type (ST)869, whereas strain K7-325 belonged to the pathogenic ST307. Both plasmids belonged to the IncFIB(K)/IncFII(K) group and carried several antibiotic resistance genes (ARGs), including tet(A) and blaCTX-M. Both plasmids (pK6-320.1 and pK7-325) were transferred to a GFP-tagged E. coli strain, leading to the acquisition of resistance against multiple classes of antibiotics. Several heavy-metal resistance genes (HMRGs) encoding resistance against silver (sil), copper (pco), and arsenic (ars) were also present on both plasmids. Conclusions: Our study demonstrates the presence of multidrug-resistant K. pneumoniae strains carrying conjugative plasmids encoding both ARGs and HMRGs that have potential for persistence in the environment and human microbiota.

ABSTRACT: Objectives: To the best of our knowledge, no genomic descriptions of blaVIM-11-harbouring plasmids are available in literature so far. The aim of this study was to describe the genomic features of three blaVIM-11-harbouring plasmids recovered from Pseudomonas aeruginosa isolated in Argentina in different periods. Methods: blaVIM-11-harbouring plasmids from three clinical P. aeruginosa isolates were transferred by transformation into P. aeruginosa PAO-1. Then, genomic DNA of these transformants was extracted and sequenced using NovaSeq 6000 System-Illumina. De novo assemblies were generated using Unicycler program and reads were mapped against a reference genome of P. aeruginosa PAO-1. Plasmids sequences were predicted identifying the reads that did not map the reference sequence of PAO-1. These reads were recovered and assembled de novo. In silico predictions were carried out using bioinformatics tools. Results: One Plasmid (pP6VIM-11) was distributed in 2 contigs, a second plasmid (pPOta2VIM-11) was found in a single contig, and the last one (pP936401VIM-11) was fragmented into 4 contigs. pP6VIM-11 and pPOta2VIM-11 belonged to the IncP-1β group, displaying 64% of coverage and 83.9% of identity among them. pP936401VIM-1 plasmid corresponded to the IncN group. The bioinformatic analysis revealed that blaVIM-11 was located in a class 1 integron, flanked by insertion sequences, exhibiting potential for its dissemination. However, none of the plasmids were conjugative. Conclusion: This study corresponded to the first description and deposit of blaVIM-11-harbouring plasmids in P. aeruginosa, which expands the limited knowledge about their molecular epidemiology.

Abstract Antimicrobial resistance (AMR) in bacteria is a major public health threat and conjugative plasmids play a key role in the dissemination of AMR genes among bacterial pathogens. Interestingly, the association between AMR plasmids and pathogens is not random and certain associations spread successfully at a global scale. The burst of genome sequencing has increased the resolution of epidemiological programs, broadening our understanding of plasmid distribution in bacterial populations. Despite the immense value of these studies, our ability to predict future plasmid-bacteria associations remains limited. Numerous empirical studies have recently reported systematic patterns in genetic interactions that enable predictability, in a phenomenon known as global epistasis. In this perspective, we argue that global epistasis patterns hold the potential to predict interactions between plasmids and bacterial genomes, thereby facilitating the prediction of future successful associations. To assess the validity of this idea, we use previously published data to identify global epistasis patterns in clinically relevant plasmid-bacteria associations. Furthermore, using simple mechanistic models of antibiotic resistance, we illustrate how global epistasis patterns may allow us to generate new hypotheses on the mechanisms associated with successful plasmid-bacteria associations. Collectively, we aim at illustrating the relevance of exploring global epistasis in the context of plasmid biology.

Plasmid shuttle vectors are a common tool used to study yeast physiology. The majority of yeast plasmids have been optimized for Saccharomyces cerevisiae lab strain compatibility, relying on auxotrophic complementation as their selective property. We sought to construct a series of plasmid shuttle vectors to extend functionality beyond strains with auxotrophic requirements, and test compatibility across a diverse panel of yeasts. We constructed 18 plasmids which were successfully maintained by yeasts from several genera. From a panel of 24 yeast strains, these plasmids were maintained by 18 yeasts, spanning 11 species within the genera Lachancea, Metschnikowia, Pichia, Saccharomyces, and Torulaspora. Additionally, an integrated gene expression reporter was assayed for functional compatibility with the 18 strains. Plasmid-derived gene expression was observed for 13 strains, spanning five species within the Saccharomyces genus, in addition to Torulaspora delbrueckii. These results indicate that this plasmid series is broadly useful for advancements and applications within academia, biotechnology, and the food and fermentation industries for research utilizing diverse Saccharomyces and non-Saccharomyces yeasts.

Abstract Background mcr-1-positive Escherichia coli has emerged as a significant threat to human health, veterinary health, and food safety in recent years. After the prohibition of colistin as a feed additive in animal husbandry in China, a noticeable reduction in both colistin resistance and the prevalence of mcr-1 was observed in E. coli from animals and humans. Objectives To assess the prevalence of the colistin resistance gene mcr-1 and characterize its genetic context in E. coli strains derived from fecal and meat samples from food-producing animals in China. Methods A total of 1,353 fecal samples and 836 food samples were collected between 2019 and 2020 in China. E. coli isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their susceptibility to colistin were determined using the broth microdilution method. The colistin-resistant E. coli isolates were screened for the presence of mcr by PCR analysis and sequencing. The minimal inhibitory concentrations (MICs) of 15 antimicrobial agents against the mcr-1-positive strains were further tested using the agar dilution method, conjugation assays were performed, and whole genome sequencing was performed using Illumina HiSeq. Results In total, 1,403 E. coli strains were isolated. Thirteen isolates from chicken meat (n = 7), chickens (n = 3), and pigs (n = 3) were resistant to colistin with MIC values of 4 to 16 mg/L, and carried mcr-1. All mcr-1-positive strains, except for isolate AH20PE105, contained multiple resistance genes and exhibited multidrug-resistant phenotypes. They belonged to 10 sequence types (STs), including a novel ST (ST14521). mcr-1 was located on IncI2 (n = 9), IncX4 (n = 2), and IncHI2 (n = 2) plasmids, which were highly similar to other mcr-1-carrying plasmids sharing the same incompatibility type. Seven mcr-1-carrying plasmids could be successfully conjugally transferred to E. coli C600. Conclusions While the low prevalence of mcr-1 (0.93%) identified in this study may not immediately seem alarming, the very emergence of this gene merits attention given its implications for colistin resistance and public health. Hence, ongoing surveillance of mcr-1 in E. coli remains crucial.

Introduction. Carbapenemase-producing Klebsiella pneumoniae (CP-Kp), which are international high-risk clones, have become a problem of utmost importance. CP-Kps, adapting to the hospital environment, evolve into convergent pathotypes. Such variants combine traits of two genetic lineages: multidrug resistant (MDR) and hypervirulent. The pathotypes, along with MDR K. pneumoniae, pose an exceptional threat to young patients during systemic infection. The objective of this study is the detailed molecular genetic analysis of MDR isolates of K. pneumoniae detected during the monitoring of resistant Gram-negative bacteria at the National Medical Research Center for Children’s Health in 2014–2021. Materials and methods. Whole-genome sequencing with a subsequent bioinformatics analysis of eight MDR isolates from the bloodstream and cerebrospinal fluid. Results. MDR isolates belonged to 4 sublineages (SL): SL307, SL395, SL29 and SL1198. In the genomes of 6 pangrug-resistant (PDR) isolates, genes associated with resistance to all categories of antibiotics recommended for Enterobacteriaceae therapy were identified. Plasmids were present in all genomes. In 6 isolates, plasmids contained heavy metal ion resistance operons in addition to antibiotic resistance genes. Prophages within the plasmids were also involved in the transfer of resistance genes. The ST395 isolate from the cerebrospinal fluid belonged to the convergent pathotype in terms of resistance and virulence. Comparison of genomes within SLs revealed recombination events in the K- and O-locus regions and the Yersiniabactin operon. Conclusion. Thus, in a sample of resistant K. pneumoniae isolated from bloodstream and cerebrospinal fluid, 6 PDR isolates were detected, one of which belongs to the convergent pathotype ST395.

Background. Currently, it is customary to distinguish two pathotypes of Klebsiella pneumoniae — classical and hypervirulent, which have the ability to cause community-acquired infections in healthy people. It has been shown that an increase in virulence is associated with the acquisition of additional genetic material — a plasmid carrying a cluster of aerobactin genes. Aim. To assess the prevalence of the aforementioned virulent plasmids around the globe and in Russia in particular, as well as to identify their key genetic features. Materials and methods. Plasmid sequences were downloaded from PLSDB and BV-BRC databases, annotated with the Abricate and Kleborate programs; cluster analysis was performed using the mge-cluster program, and phylogenetic analysis was performed using the Parsnp program. Results. 296 plasmid sequences isolated from 23 countries from clinical isolates of K.pneumoniae between 2006 and 2021 with a peak in 2019 were analyzed, with more than half of the plasmids coming from China. More than 30 sequence types were identified, among which ST11 and ST23 were predominant. Replicon genes of the IncFIB group were identified in almost all plasmids studied. The pre dominant type of aerobactin in the studied sequences was the first type (iuc1); sequences with iuc3 and iuc5 were also identified. Salmochelin synthesis genes were identified in only 37.1% of sequences; the yersiniabactin cluster was identified in two plasmids from China. 32.1% of plasmids carried resistance genes, of which 7.4% carried extended-spectrum beta-lactase genes and 5% contained carbapenemase genes. Nine clusters of sequences were obtained; almost all plasmids from Russia were assigned to one cluster and were NDM-positive. Together with plasmids from other European countries (Great Britain, Norway, Czech Republic), they formed a separate branch on the phylogenetic tree. Conclusion. Virulent plasmids carrying the aerobactin synthesis gene cluster are distributed globaly, and almost a third of them also carry antibiotic resistance genes.

The bacteria Azospirillum brasilense, used as biofertilizers, have a signifi cant positive eff ect on the growth and development of plants. The genome of the strain A. brasilense Sp7 is represented by a chromosome and numerous plasmids with molecular weight of 90, 115, and over 300 MDa. Genomic rearrangements that cause changes in the “plasmid profi le” can contribute to the formation of subpopulations or phenotypic variants in a bacterial population. There is little data on the role of such rearrangements in the adaptation of A. brasilenseto dynamic environmental conditions. The ability of azospirilla to form biofi lms also has a determined signifi cance for the successful functioning of the plant-microbial association and the resistance of bacteria and plants to various abiotic stresses. The purpose of this work consisted of the analysis of the genomic rearrangements in spontaneous derivatives of A. brasilense Sp7 and the assessment of the resistance of their biofi lms to drying, water stress and oxidative stress. PCR analysis to detect changes in the structure of genomic DNA was performed using primers corresponding to known conservative motifs in repetitive bacterial nucleotide sequences. The relative amount of the biofi lm biomass was assessed by measuring the crystal violet A540 desorbed after staining. The level of relative respiratory activity of cells in biofi lms was determined by the fl uorometric resazurin test. The non-penetrating osmotic agent PEG 6000 was used to create the osmotic/water stress model. It was shown that rearrangements in genomic DNA contribute to the formation of stable phenotypic variants of the Sp7 strain, which form biofi lms in diff erent ways under water stress conditions. A derived strain of A. brasilense Sp7.8, the biofi lm population of which is more resistant to water stress compared to the parent strain was selected.

Objectives: This study reports the genomic characterization of the multidrug resistant Salmonella Newport strain 195_20 recovered from the diarrheic faeces of a foal in Brazil and co-harbouring the mcr-9, blaCMY-2 and qnrB19 antibiotic resistance genes. Methods: Bacterial isolate positive for mobile colistin resistance gene (mcr-9) was submitted to antimicrobial susceptibility testing by disk diffusion and broth microdilution for colistin and polymyxin B. The isolate was submitted to whole genome sequencing by Illumina technology and Nanopore Sequencing. Conjugation assays, plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinIon long reads sequencing. Results: Isolate 195_20 was identified as sequence type ST45, resistant to penicillin and cephalosporins (ampicillin, ceftazidime, ceftriaxone and cefotaxime), aminoglycosides (streptomycin and gentamicin), phenicol (chloramphenicol), quinolones and fluoroquinolones (nalidixic acid, ciprofloxacin, and pefloxacin), folate pathway antagonists (sulfonamides and trimethoprim-sulfamethoxazole), and tetracycline. A transferable IncHI2/IncHI2A plasmid sized ca. 262kb was found to carry the mcr-9 gene in a module consisting of IS903-mcr-9-wbuC-IS26. In addition, an 174kb IncC and a 48kb IncN plasmid were also identified in the 195_20 isolate, carrying blaCMY-2 and qnrB19, respectively. Conclusions: Not surprisingly, isolate 195_20 was susceptible to polymyxins, possibly due to absence of qseBC regulatory operon. Presence of mobile colistin resistance (mcr-9), third-generation cephalosporins (blaCMY-2) and quinolone (qnrB19) resistance determinants in zoonotic pathogens from animals in close contact with humans alerts for the possible route of transmission between these different reservoirs.

ABSTRACTPlasmids are the primary vectors of horizontal transfer of antibiotic resistance genes among bacteria. Previous studies have shown that the spread and maintenance of plasmids among bacterial populations depend on the genetic makeup of both the plasmid and the host bacterium. Antibiotic resistance can also be acquired through mutations in the bacterial chromosome, which not only confer resistance but also result in changes in bacterial physiology and typically a reduction in fitness. However, it is unclear whether chromosomal resistance mutations affect the interaction between plasmids and the host bacteria. To address this question, we introduced 13 clinical plasmids into a susceptible Escherichia coli strain and three different congenic mutants that were resistant to nitrofurantoin (ΔnfsAB), ciprofloxacin (gyrA, S83L), and streptomycin (rpsL, K42N) and determined how the plasmids affected the exponential growth rates of the host in glucose minimal media. We find that though plasmids confer costs on the susceptible strains, those costs are fully mitigated in the three resistant mutants. In several cases, this results in a competitive advantage of the resistant strains over the susceptible strain when both carry the same plasmid and are grown in the absence of antibiotics. Our results suggest that bacteria carrying chromosomal mutations for antibiotic resistance could be a better reservoir for resistance plasmids, thereby driving the evolution of multi-drug resistance.IMPORTANCEPlasmids have led to the rampant spread of antibiotic resistance genes globally. Plasmids often carry antibiotic resistance genes and other genes needed for its maintenance and spread, which typically confer a fitness cost on the host cell observed as a reduced growth rate. Resistance is also acquired via chromosomal mutations, and similar to plasmids they also reduce bacterial fitness. However, we do not know whether resistance mutations affect the bacterial ability to carry plasmids. Here, we introduced 13 multi-resistant clinical plasmids into a susceptible and three different resistant E. coli strains and found that most of these plasmids do confer fitness cost on susceptible cells, but these costs disappear in the resistant strains which often lead to fitness advantage for the resistant strains in the absence of antibiotic selection. Our results imply that already resistant bacteria are a more favorable reservoir for multi-resistant plasmids, promoting the ascendance of multi-resistant bacteria.

The surfaces of hospital kitchen equipment could be a major source of transmission of resistant pathogens to patients. Hence, the objective of this paper was to assess the bacteriological and plasmid profiles of surfaces of tables, sinks, chopping-boards, gas cookers and freezer handles hospital kitchen equipment in Benin City, Nigeria using appropriate standard microbiological techniques. Bacterial plasmids were isolated and separated using the agarose gel electrophoresis. Plasmid curing was performed using acridine orange. The antimicrobial sensitivity pattern showed that the bacterial isolates exhibited varying degree of resistance to the antibiotics. Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were highly resistant to the antibiotics, having multiple antibiotic resistances (MAR) index of 0.6. Micrococcus spp. and Klebsiella pneumoniae were the least resistant to the antibiotics. Plasmid analysis revealed the presence of single and multiple-banded plasmids with sizes ranging from 100-1000bp. After curing, all the isolates (except S. aureus, E. coli and P. aeruginosa) were sensitive to all the antibiotics, indicating a significant reduction in antibiotic resistance after curing. The results revealed that plasmids played a significant role in conferring resistance on the isolates. Regular cleaning and disinfection should be strictly observed in hospital kitchens to prevent outbreaks and spread of resistant pathogens in hospitals.

Introduction: To consider the growing health issues caused by antibiotic resistance from a “one health” perspective, the contribution of meat production needs to be addressed. While antibiotic resistance is naturally present in microbial communities, the treatment of farm animals with antibiotics causes an increase in antibiotic resistance genes (ARG) in the gut microbiome. Pigs are among the most prevalent animals in agriculture; therefore, reducing the prevalence of antibiotic-resistant bacteria in the pig gut microbiome could reduce the spread of antibiotic resistance. Probiotics are often studied as a way to modulate the microbiome and are, therefore, an interesting way to potentially decrease antibiotic resistance.Methods: To assess the efficacy of a probiotic to reduce the prevalence of ARGs in the pig microbiome, six pigs received either treatment with antibiotics (tylvalosin), probiotics (Pediococcus acidilactici MA18/5M; Biopower® PA), or a combination of both. Their faeces and ileal digesta were collected and DNA was extracted for whole genome shotgun sequencing. The reads were compared with taxonomy and ARG databases to identify the taxa and resistance genes in the samples.Results: The results showed that the ARG profiles in the faeces of the antibiotic and combination treatments were similar, and both were different from the profiles of the probiotic treatment (p < 0.05). The effects of the treatments were different in the digesta and faeces. Many macrolide resistance genes were detected in a higher proportion in the microbiome of the pigs treated with antibiotics or the combination of probiotics and antibiotics. Resistance-carrying conjugative plasmids and horizontal transfer genes were also amplified in faeces samples for the antibiotic and combined treatments. There was no effect of treatment on the short chain fatty acid content in the digesta or the faeces.Conclusion: There is no positive effect of adding probiotics to an antibiotic treatment when these treatments are administered simultaneously.

ABSTRACTMembers of Providencia, although typically opportunistic, can cause severe infections in immunocompromised hosts. Recent advances in genome sequencing provide an opportunity for more precise study of this genus. In this study, we first identified and characterized a novel species named Providencia zhijiangensis sp. nov. It has ≤88.23% average nucleotide identity (ANI) and ≤31.8% in silico DNA-DNA hybridization (dDDH) values with all known Providencia species, which fall significantly below the species-defining thresholds. Interestingly, we found that Providencia stuartii and Providencia thailandensis actually fall under the same species, evidenced by an ANI of 98.59% and a dDDH value of 90.4%. By fusing ANI with phylogeny, we have reclassified 545 genomes within this genus into 20 species, including seven unnamed taxa (provisionally titled Taxon 1–7), which can be further subdivided into 23 lineages. Pangenomic analysis identified 1,550 genus-core genes in Providencia, with coenzymes being the predominant category at 10.56%, suggesting significant intermediate metabolism activity. Resistance analysis revealed that most lineages of the genus (82.61%, 19/23) carry a high number of antibiotic-resistance genes (ARGs) and display diverse resistance profiles. Notably, the majority of ARGs are located on plasmids, underscoring the significant role of plasmids in the resistance evolution within this genus. Three species or lineages (P. stuartii, Taxon 3, and Providencia hangzhouensis L12) that possess the highest number of carbapenem-resistance genes suggest their potential influence on clinical treatment. These findings underscore the need for continued surveillance and study of this genus, particularly due to their role in harboring antibiotic-resistance genes.IMPORTANCEThe Providencia genus, known to harbor opportunistic pathogens, has been a subject of interest due to its potential to cause severe infections, particularly in vulnerable individuals. Our research offers groundbreaking insights into this genus, unveiling a novel species, Providencia zhijiangensis sp. nov., and highlighting the need for a re-evaluation of existing classifications. Our comprehensive genomic assessment offers a detailed classification of 545 genomes into distinct species and lineages, revealing the rich biodiversity and intricate species diversity within the genus. The substantial presence of antibiotic-resistance genes in the Providencia genus underscores potential challenges for public health and clinical treatments. Our study highlights the pressing need for increased surveillance and research, enriching our understanding of antibiotic resistance in this realm.

Escherichia coli can harbor a broad repertoire of virulence and antimicrobial resistance (AMR) genes, which can be exchanged across the human gastrointestinal microflora, thus posing a public health risk. In this study, six batches of artisanal soft cheese and a 6-month ripened fermented dried sausage were investigated to assess the occurrence, phylogeny, and genomic traits (AMR, virulence, and mobilome) of E. coli. Thirty and three strains isolated from salami and cheese food chains, respectively, were confirmed as E. coli by whole genome sequencing. The accumulation of single nucleotide polymorphism differences within small clusters of strains encompassing batches or processing stages, combined with high serotype and phylogroup diversity, suggested the occurrence of different contamination phenomena among the facilities. A total of eight isolates harbored plasmid-mediated resistance genes, including one cheese strain that carried an IncQ1 plasmid carrying AMR determinants to macrolides [mph(B)], sulfonamides (sul1, sul2), trimethoprim (dfrA1), and aminoglycosides [aph(3'')-Ib and aph(6)-Id]. A pool of virulence-associated genes in the class of adhesion, colonization, iron uptake, and toxins, putative ColV-positive iron uptake systems sit, iro, or iuc (eight salami and two cheese), plasmid-encoded hemolysin operon hlyABCD (one salami), and potential atypical enteropathogenic E. coli (three salami environment) were reported. Overall, our findings underscore the importance of routine surveillance of E. coli in the artisanal food chain to prevent the dissemination of AMR and virulence.

ABSTRACTMultidrug-resistant Escherichia coli is a leading cause of global mortality. Transfer of plasmids carrying genes encoding beta-lactamases, carbapenamases, and colistin resistance between lineages is driving the rising rates of hard-to-treat nosocomial and community infections. Multidrug resistance (MDR) plasmid acquisition commonly causes transcriptional disruption, and while a number of studies have shown strain-specific fitness and transcriptional effects of an MDR plasmid across diverse bacterial lineages, fewer studies have compared the impacts of different MDR plasmids in a common bacterial host. As such, our ability to predict which MDR plasmids are the most likely to be maintained and spread in bacterial populations is limited. Here, we introduced eight diverse MDR plasmids encoding resistances against a range of clinically important antibiotics into E. coli K-12 MG1655 and measured their fitness costs and transcriptional impacts. The scale of the transcriptional responses varied substantially between plasmids, ranging from >650 to

The presence of methicillin-resistant or -susceptible S. aureus in pig nostrils has been known for a long time, but the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli has hardly been investigated. Here, we collected 25 E. coli recovered from nasal samples of 40 pigs/10 farmers of four farms. Nine ESBL-producing isolates belonging to ST48, ST117, ST847, ST5440, ST14914 and ST10 were retrieved from seven pigs. All blaESBL genes (blaCTX-M-32, blaCTX-M-14, blaCTX-M-1, blaCTX-M-65, and blaSHV-12) were horizontally transferable by conjugation through plasmids belonging to IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1) types. IncI1-plasmids displayed different genetic environments: i) IS26-blaSHV-12-deoR-IS26, ii) wbuC-blaCTX-M-32-ISKpn26 (IS5), and iii) IS930-blaCTX-M-14-IS26. The IncHI2-plasmid contained the genetic environment IS903-blaCTX-M-65-fipA with multiple resistance genes associated either to: a) Tn21-like transposon harbouring genes conferring aminoglycosides/beta-lactams/chloramphenicol/macrolides resistance located on two atypical class 1 integrons with an embedded ΔTn5393; or b) Tn1721-derived transposon displaying an atypical class 1 integron harbouring aadA2-arr3-cmlA5-blaOXA-10-aadA24-dfrA14, preceding the genetic platform IS26-blaTEM-95-tet(A)-lysR-floR-virD2-ISVsa3-IS3075-IS26-qnrS1, as well as the tellurite resistance module. Other plasmids harbouring clinically relevant genes were detected, such as a ColE-type plasmid carrying the mcr-4.5 gene. Chromosomally encoded genes (fosA7) or integrons (intI1-dfrA1-aadA1-qacE-sul1/intI1-IS15-dfrA1-aadA2) were also identified. Finally, an IncY plasmid harbouring a class 2 integron (intI2-dfrA1-sat2-aadA1-qacL-IS406-sul3) was detected but not associated with a blaESBL gene. Our results evidence that pig nostrils might favour the spread of ESBL-E. coli and mcr-mediated colistin-resistance. Therefore, enhanced monitoring should be considered, especially in a sector where close contact between animals in intensive farming increases the risk of spreading antimicrobial resistance.

Abstract Background Avian pathogenic Escherichia coli (APEC) are the causative agents of colibacillosis in chickens, a disease which has significant economic impact on the poultry industry. Large plasmids detected in APEC are known to contribute to strain diversity for pathogenicity and antimicrobial resistance, but there could be other plasmids that are missed in standard analysis. In this study, we determined the impact of sequencing and assembly factors for the detection of plasmids in an E. coli whole genome sequencing project. Results Hybrid assembly (Illumina and Nanopore) combined with plasmid DNA extractions allowed for detection of the greatest number of plasmids in E. coli, as detected by MOB-suite software. In total, 79 plasmids were identified in 19 E. coli isolates. Hybrid assemblies were robust and consistent in quality regardless of sequencing kit used or if long reads were filtered or not. In contrast, long read only assemblies were more variable and influenced by sequencing and assembly parameters. Plasmid DNA extractions allowed for the detection of physically smaller plasmids, but when averaged over 19 isolates did not significantly change the overall number of plasmids detected. Conclusions Hybrid assembly can be reliably used to detect plasmids in E. coli, especially if researchers are focused on large plasmids containing antimicrobial resistance genes and virulence factors. If the goal is comprehensive detection of all plasmids, particularly if smaller sized vectors are desired for biotechnology applications, the addition of plasmid DNA extractions to hybrid assemblies is prudent. Long read sequencing is sufficient to detect many plasmids in E. coli, however, it is more prone to errors when expanded to analyze a large number of isolates.

Cong Zhou,1 Hui Zhang,1 Maosuo Xu,1 Yajuan Liu,1 Baoyu Yuan,2 Yong Lin,1 Fang Shen1 1Department of Clinical Laboratory, Shanghai Fifth People’s Hospital, Fudan University, Shanghai, People’s Republic of China; 2Department of Clinical Laboratory, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaCorrespondence: Fang Shen; Yong Lin, Department of Clinical Laboratory, Shanghai Fifth People’s Hospital, Fudan University, No. 128, Ruili Road, Minhang District, Shanghai, 200240, People’s Republic of China, Tel +86 18021073261 ; +86 18718168695, Email shenfang5th@aliyun.com; linyong7007@163.comBackground: Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused an extremely severe health challenge and public concern. However, the underlying mechanisms of fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we report a hv-CRKP-associated fatal infection and reveal a reduction in virulence due to the acquisition of aminoglycoside resistance.Methods: The bacterial identification, antimicrobial susceptibility, hypermucoviscosity, virulence factors, MLST and serotypes were profiled.The clonal homology and plasmid acquisition among hv-CRKP strains were detected by XbaI and S1-PFGE. The virulence potential of the strains was evaluated using Galleria mellonella larvae infection model, serum resistance assay, capsular polysaccharide quantification, and biofilm formation assay. Genomic variations were identified using whole-genome sequencing (WGS).Results: Four K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from an 86-year-old patient with severe pneumonia. Whole-genome sequencing (WGS) showed that all four hv-CRKP strains belonged to the ST11-KL64 clone. PFGE analysis revealed that the four ST11-KL64 hv-CRKP strains could be grouped into the same PFGE type. Under the pressure of antibiotics, the antimicrobial resistance of the strains increased and the virulence potential decreased. Further sequencing, using the Nanopore platform, was performed on three representative isolates (WYKP586, WYKP589, and WYKP594). Genomic analysis showed that the plasmids of these three strains underwent a large number of breaks and recombination events under antibiotic pressure. We found that as aminoglycoside resistance emerged via acquisition of the rmtB gene, the hypermucoviscosity and virulence of the strains decreased because of internal mutations in the rmpA and rmpA2 genes.Conclusion: This study shows that ST11-KL64 hv-CRKP can further evolve to acquire aminoglycoside resistance accompanied by decreased virulence to adapt to antibiotic pressure in the host.Keywords: hypervirulent carbapenem-resistant Klebsiella pneumoniae, within-host evolution, plasmids, whole-genome sequencing

Abstract Backgrounds Despite recent advances, many cancers are still detected too late for curative treatment. There is, therefore, a need for the development of new diagnostic methods and biomarkers. One approach may arise from the detection of extrachromosomal circular DNA (eccDNA), which is part of cell‐free DNA in human plasma. Aims First, we assessed and compared two methods for the purification of eccDNA from plasma. Second, we tested for an easy diagnostic application of eccDNA liquid biopsy‐based assays. Materials & Methods For the comparison we tested a solid‐phase silica purification method and a phenol/chloroform method with salt precipitation. For the diagnostic application of eccDNA we developed and tested a qPCR primer‐based SNP detection system, for the detection of two well‐established cancer‐causing KRAS mutations (G12V and G12R) on circular DNA. This investigation was supported by purifying, sequencing, and analysing clinical plasma samples for eccDNAs containing KRAS mutant alleles in 0.5 mL plasma from 16 pancreatic ductal adenocarcinoma patients and 19 healthy controls. Results In our method comparison we observed, that following exonuclease treatment a lower eccDNA yield was found for the phenol/chloroform method (15.7%–26.7%) compared with the solid‐phase purification approach (47.8%–65.9%). For the diagnostic application of eccDNA tests, the sensitivity of the tested qPCR assay only reached ~10−3 in a background of 105 wild type (wt) KRAS circular entities, which was not improved by general amplification or primer‐based inhibition of wt KRAS amplification. Furthermore, we did not detect eccDNA containing KRAS in any of the clinical samples. Discussion A potential explanation for our inability to detect any KRAS mutations in the clinical samples may be related to the general low abundance of eccDNA in plasma. Conclusion Taken together our results provide a benchmark for eccDNA purification methods while raising the question of what is required for the optimal fast and sensitive detection of SNP mutations on eccDNA with greater sensitivity than primer‐based qPCR detection.

Abstract Background Paenarthrobacter nicotinovorans ATCC 49919 uses the pyridine-pathway to degrade nicotine and could provide a renewable source of precursors from nicotine-containing waste as well as a model for studying the molecular evolution of catabolic pathways and their spread by horizontal gene transfer via soil bacterial plasmids. Results In the present study, the strain was sequenced using the Illumina NovaSeq 6000 and Oxford Nanopore Technology (ONT) MinION platforms. Following hybrid assembly with Unicycler, the complete genome sequence of the strain was obtained and used as reference for whole-genome-based phylogeny analyses. A total of 64 related genomes were analysed; five Arthrobacter strains showed both digital DNA-DNA hybridization and average nucleotide identity values over the species threshold when compared to P. nicotinovorans ATCC 49919. Five plasmids and two contigs belonging to Arthrobacter and Paenarthrobacter strains were shown to be virtually identical with the pAO1 plasmid of Paenarthrobacter nicotinovorans ATCC 49919. Moreover, a highly syntenic nic-genes cluster was identified on five plasmids, one contig and three chromosomes. The nic-genes cluster contains two major locally collinear blocks that appear to form a putative catabolic transposon. Although the origins of the nic-genes cluster and the putative transposon still elude us, we hypothesise here that the ATCC 49919 strain most probably evolved from Paenarthrobacter sp. YJN-D or a very closely related strain by acquiring the pAO1 megaplasmid and the nicotine degradation pathway. Conclusions The data presented here offers another snapshot into the evolution of plasmids harboured by Arthrobacter and Paenarthrobacter species and their role in the spread of metabolic traits by horizontal gene transfer among related soil bacteria.

ABSTRACT: Objectives: This study aimed to characterize Escherichia coli isolates from cloacal samples of white stork nestlings, with a special focus on extended-spectrum β-lactamases (ESBLs)-producing E. coli isolates and their plasmid content. Methods: Cloacal samples of 88 animals were seeded on MacConkey-agar and chromogenic-ESBL plates to recover E. coli and ESBL-producing E. coli. Antimicrobial susceptibility was screened using the disc diffusion method, and the genotypic characterization was performed by polymerase chain reaction (PCR) and subsequent sequencing. S1 nuclease Pulsed-Field-Gel-Electrophoresis (PFGE), Southern blotting, and conjugation essays were performed on ESBL-producing E. coli, as well as whole-genome sequencing by short- and long-reads. The four blaESBL-carrying plasmids were completely sequenced. Results: A total of 113 non-ESBL-producing E. coli isolates were collected on antibiotic-free MacConkey-agar, of which 27 (23.9%) showed a multidrug-resistance (MDR) phenotype, mainly associated with β-lactam-phenicol-sulfonamide resistance (blaTEM/cmlA/floR/sul1/sul2/sul3). Moreover, four white stork nestlings carried ESBL-producing E. coli (4.5%) with the following characteristics: blaSHV-12/ST38-D, blaSHV-12/ST58-B1, blaCTX-M-1/ST162-B1, and blaCTX-M-32/ST155-B1. Whole-genome sequencing followed by Southern blot hybridizations on S1-PFGE gels in ESBL-positive isolates proved that the blaCTX-M-1 gene and one of the blaSHV-12 genes were carried by IncI1/pST3 plasmids, while the second blaSHV-12 gene and the blaCTX-M-32 gene were located on IncF plasmids. The two blaSHV-12 genes and the two blaCTX-M genes had similar but non-identical close genetic environments, as all four genes were flanked by a variety of insertion sequences. Conclusion: The role played by several genetic platforms in the mobility of ESBL genes allows for interchangeability on a remarkably small scale (gene-plasmid-clones), which may support the spread of ESBL genes.

Enterobacter hormaechei has emerged as a significant pathogen within healthcare settings due to its ability to develop multidrug resistance (MDR) and survive in hospital environments. This study presents a genome-based analysis of carbapenem-resistant Enterobacter hormaechei isolates from two major hospitals in the United Arab Emirates. Eight isolates were subjected to whole-genome sequencing (WGS), revealing extensive resistance profiles including the blaNDM-1, blaOXA-48, and blaVIM-4 genes. Notably, one isolate belonging to ST171 harbored dual carbapenemase genes, while five isolates exhibited colistin resistance without mcr genes. The presence of the type VI secretion system (T6SS), various adhesins, and virulence genes contributes to the virulence and competitive advantage of the pathogen. Additionally, our isolates (87.5%) possessed ampC β-lactamase genes, predominantly blaACT genes. The genomic context of blaNDM-1, surrounded by other resistance genes and mobile genetic elements, highlights the role of horizontal gene transfer (HGT) in the spread of resistance. Our findings highlight the need for rigorous surveillance, strategic antibiotic stewardship, and hospital-based WGS to manage and mitigate the spread of these highly resistant and virulent pathogens. Accurate identification and monitoring of Enterobacter cloacae complex (ECC) species and their resistance mechanisms are crucial for effective infection control and treatment strategies.

Abstract Background Bacteria of the Borrelia burgdorferi sensu lato (s.l.) complex can cause Lyme borreliosis. Different B. burgdorferi s.l. genospecies vary in their host and vector associations and human pathogenicity but the genetic basis for these adaptations is unresolved and requires completed and reliable genomes for comparative analyses. The de novo assembly of a complete Borrelia genome is challenging due to the high levels of complexity, represented by a high number of circular and linear plasmids that are dynamic, showing mosaic structure and sequence homology. Previous work demonstrated that even advanced approaches, such as a combination of short-read and long-read data, might lead to incomplete plasmid reconstruction. Here, using recently developed high-fidelity (HiFi) PacBio sequencing, we explored strategies to obtain gap-free, complete and high quality Borrelia genome assemblies. Optimizing genome assembly, quality control and refinement steps, we critically appraised existing techniques to create a workflow that lead to improved genome reconstruction. Results Despite the latest available technologies, stand-alone sequencing and assembly methods are insufficient for the generation of complete and high quality Borrelia genome assemblies. We developed a workflow pipeline for the de novo genome assembly for Borrelia using several types of sequence data and incorporating multiple assemblers to recover the complete genome including both circular and linear plasmid sequences. Conclusion Our study demonstrates that, with HiFi data and an ensemble reconstruction pipeline with refinement steps, chromosomal and plasmid sequences can be fully resolved, even for complex genomes such as Borrelia. The presented pipeline may be of interest for the assembly of further complex microbial genomes.

Wastewater treatment plants (WWTPs) are considered reservoirs of antibiotic resistance genes (ARGs). Given that plasmid-mediated horizontal gene transfer plays a critical role in disseminating ARGs in the environment, it is important to inspect the transfer potential of transmissible plasmids to have a better understanding of whether these mobile ARGs can be hosted by opportunistic pathogens and should be included in One Health’s considerations. In this study, we used a fluorescent-reporter-gene based exogenous isolation approach to capture extended-spectrum beta-lactamases encoding mobile determinants from sewer microbiome samples that enter an urban water system (UWS) in Denmark. After screening and sequencing, we isolated a ∼73 Kbp IncN plasmid (pDK_DARWIN) that harboured and expressed multiple ARGs. Using a dual fluorescent reporter gene system, we showed that this plasmid can transfer into resident urban water communities. We demonstrated the transfer of pDK_DARWIN to microbiome members of both the sewer (in the upstream UWS compartment) and wastewater treatment (in the downstream UWS compartment) microbiomes. Sequence similarity search across curated plasmid repositories revealed that pDK_DARWIN derives from an IncN backbone harboured by environmental and nosocomial Enterobacterial isolates. Furthermore, we searched for pDK_DARWIN sequence matches in UWS metagenomes from three countries, revealing that this plasmid can be detected in all of them, with a higher relative abundance in hospital sewers compared to residential sewers. Overall, this study demonstrates that this IncN plasmid is prevalent across Europe and an efficient vector capable of disseminating multiple ARGs in the urban water systems.

IntroductionSurveillance is vital for monitoring the increasing risk of antimicrobial resistance (AMR) in bacteria leading to failures in humans and animals to treat infections. In a One Health context, AMR bacteria from livestock and food can transfer through the food chain to humans, and vice versa, which can be characterized in detail through genomics. We investigated the critical aspects of AMR and the dynamics of AMR in poultry in the UK.MethodsIn this study, we performed whole genome sequencing for genomic characterization of 761 extended-spectrum cephalosporinases (ESCs) harboring Escherichia coli isolated from poultry caeca and meat through EU harmonized monitoring of AMR in zoonotic and commensal bacteria from 2016 and 2018 and UK national monitoring in 2020.ResultsThe most common ESC in 2016 and 2018 was blaCTX-M-1; however, 2020 had a greater diversity of ESCs with blaCTX-M-55 dominant in chickens and blaCTX-M-15 more prevalent in turkeys. Co-resistance to sulphonamides, tetracycline, and trimethoprim was widespread, and there were several positive correlations between the sequence types (STs) and ESC genes. We identified certain AMR genotypes and STs that were frequent each year but not as successful in subsequent years, e.g., ST350 harboring blaCTX-M-1, sul2, and tetA-v4.Phylogenetic comparison of isolates prevalent in our panel with global ones from the same STs available in public databases showed that isolates from the UK generally clustered together, suggesting greater within-country than between-country transmission.DiscussionWe conclude that future genomic surveillance of indicator organisms will be invaluable as it will enable detailed comparisons of AMR between and within neighboring countries, potentially identifying the most successful sequence types, plasmids, or emerging threats.

The integration of next-generation sequencing into the identification and characterization of resistant and virulent strains as well as the routine surveillance of foodborne pathogens such as Salmonella enterica have not yet been accomplished in the Philippines. This study investigated the antimicrobial profiles, virulence, and susceptibility of the 105 S. enterica isolates from swine and chicken samples obtained from slaughterhouses and public wet markets in Metropolitan Manila using whole-genome sequence analysis. Four predominant serovars were identified in genotypic serotyping, namely, Infantis (26.7%), Anatum (19.1%), Rissen (18.1%), and London (13.3%). Phenotypic antimicrobial resistance (AMR) profiling revealed that 65% of the isolates were resistant to at least one antibiotic, 37% were multidrug resistant (MDR), and 57% were extended-spectrum β-lactamase producers. Bioinformatic analysis revealed that isolates had resistance genes and plasmids belonging to the Col and Inc plasmid families that confer resistance against tetracycline (64%), sulfonamide (56%), and streptomycin (56%). Further analyses revealed the presence of 155 virulence genes, 42 of which were serovar-specific. The virulence genes primarily code for host immune system modulators, iron acquisition enzyme complexes, host cell invasion proteins, as well as proteins that allow intracellular and intramacrophage survival. This study showed that virulent MDR S. enterica and several phenotypic and genotypic AMR patterns were present in the food chain. It serves as a foundation to understand the current AMR status in the Philippines food chain and to prompt the creation of preventative measures and efficient treatments against foodborne pathogens.

ABSTRACT The ability to better understand the function of proteins expressed by bacteria has typically relied upon the development of genetic mutant strains. This approach has been especially challenging for plasmid-encoded genes, as most of the previously described allelic replacement vectors are inefficient for plasmid genome mutation as they either rely on plasmid-derived counterselection toxins or depend on other strategies such sacB, tetA, and rpsL which have been proven to be less efficient for mutant selection. Integrative vectors lack chromophore indicators, thus requiring laborious screening or excision of the vector’s backbone relies on the introduction of a flippase (FLP)-expressing plasmid. The allelic replacement vector, designated here as pDG1, expresses an X-Gal hydrolyzing enzyme (BgaB) that can be used for blue/white screening allowing identification of colonies that integrated and successfully removed the mutagenesis plasmid without a bias for those still carrying it. pDG1 was further improved by including the rhamnose-inducible Tse2 toxin as a potent counterselection system. The efficacy of pDG1 was validated by deleting portions of the plasmid-encoded VirB4/D4 type IV secretion system and aerobactin-synthesizing operons in Salmonella enterica. The integrative vectors, which contain an ΦC31attP site and genes encoding ΦC31 integrase (int), can seamlessly integrate to target ΦC31attB on Salmonella plasmids or chromosome. These vectors were improved by inserting bgaB and FLP-encoding genes so that, following integration, most of the vector’s backbone encompassing int, bgaB, and FLP genes can be excised by FLP, without the need for another FLP-expressing vector, creating white colonies carrying a stably integrated target gene. As such, we were able to integrate a 9.3-kb DNA fragment to Salmonella chromosome and flipped out most of the integrated vector in one step, leaving the target fragment in the chromosome. IMPORTANCE In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens including in vivo studies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.

Multidrug resistant Acinetobacter baumannii is one of the most important nosocomial pathogens worldwide. During the last decades it has become a major threat for healthcare settings due to the high antibiotic resistance rates among these isolates. Many resistance determinants are coded by conjugative or mobilizable plasmids, facilitating their dissemination. The majority of plasmids harbored by Acinetobacter species are less than 20 Kb, however, high molecular weight elements are the most clinically relevant since they usually contain antibiotic resistance genes. The aim of this work was to describe, classify and determine the genetic content of plasmids harbored by carbapem resistant A. baumannii isolates belonging to predominant clonal lineages circulating in hospitals from Alexandria, Egypt. The isolates were subjected to S1-Pulsed Field Gel Electrophoresis experiments to identify high molecular weight plasmids. To further analyze the plasmid content and the genetic localization of the antibiotic resistance genes, isolates were sequenced by Illumina Miseq and MinION Mk1C and a hybrid assembly was performed using Unicycler v0.5.0. Plasmids were detected with MOBsuite 3.0.3 and Copla.py v.1.0 and mapped using the online software Proksee.ca. Replicase genes were further analyzed through a BLAST against the Acinetobacter Plasmid Typing database. Eleven plasmids ranging in size from 4.9 to 205.6 Kb were characterized and mapped. All isolates contained plasmids, and, in many cases, more than two elements were identified. Antimicrobial resistance genes such as blaOXA-23, blaGES-like, aph(3’)-VI and qacEΔ1 were found in likely conjugative large plasmids; while virulence determinants such as septicolysin or TonB-dependent receptors were identified in plasmids of small size. Some of these resistance determinants were, in turn, located within transposons and class 1 integrons. Among the identified plasmids, the majority encoded proteins belonging to the Rep_3 family, but replicases of the RepPriCT_1 (Aci6) family were also identified. Plasmids are of high interest as antibiotic resistance control tools, since they may be used as genetic markers for antibiotic resistance and virulence, and also as targets for the development of compounds that can inhibit transfer processes.

Since first being identified in 1999, disease outbreaks from Edwardsiella ictaluri remain a significant health challenge for the farmed catfish sector in Vietnam. To better understand the population structure of E. ictaluri circulating in Vietnamese striped catfish (Pangasianodon hypophthalmus) farms, this study aimed to investigate the genetic diversity of 83 E. ictaluri isolates recovered from natural disease outbreaks occurring in the Mekong Delta region between 2001 and 2021. Pulsed-field gel electrophoresis resolved isolates into 15 pulsotypes following restriction digest with speI, with a Simpson’s diversity index of 0.8548. The genetic fingerprints of isolates recovered from recent outbreaks across different provinces were highly shared (100% similarity), suggesting clonal expansion within the production systems situated in the Mekong Delta region. Findings from this study also showed that E. ictaluri populations circulating this region have changed over time, as isolates recovered between 2001 and 2011 were genetically distinct from those recovered after 2017. Furthermore, isolates recovered after 2004 and 2010 lacked the virulence gene traD and a 5.8 Kb plasmid DNA fragment, respectively. Findings from this study illustrate the need for continued epidemiological monitoring of E. ictaluri to ensure prevention and treatment strategies in the Vietnamese striped catfish sector remain robust and effective.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 strains with the T allele in the translocated intimin receptor polymorphism (tir) 255 A > T gene associate with human disease more than strains with an A allele; however, the allele is not thought to be the direct cause of this difference. We sequenced a diverse set of STEC O157:H7 strains (26% A allele, 74% T allele) to identify linked differences that might underlie disease association. The average chromosome and pO157 plasmid size and gene content were significantly greater within the tir 255 A allele strains. Eighteen coding sequences were unique to tir 255 A allele chromosomes, and three were unique to tir 255 T allele chromosomes. There also were non-pO157 plasmids that were unique to each tir 255 allele variant. The overall average number of prophages did not differ between tir 255 allele strains; however, there were different types between the strains. Genomic and mobile element variation linked to the tir 255 polymorphism may account for the increased frequency of the T allele isolates in human disease.

ABSTRACTRecombinant plasmids are essential tools in molecular biotechnology, and reliable plasmid assembly methods have, therefore, become a prerequisite for the successful cloning and transfer of genes. Among the multitude of available plasmid assembly strategies, in vivo homologous recombinational cloning in yeast has emerged as a cost-effective and relatively simple method. Since we use this method routinely in our group for assembling large plasmids with secondary metabolite gene clusters and for direct heterologous production of polyketides in Saccharomyces cerevisiae, we developed an exercise module for undergraduate students where they would get hands-on experience with these molecular practices. The exercises target several molecular techniques, including PCR, restriction enzyme digestion, and yeast recombinational cloning. The students will learn about plasmid assembly and yeast transformation methods by performing these experiments while inherently acquiring new skills valuable for their subsequent laboratory work or projects.

Carbapenem-resistant ST11_KL64 Klebsiella pneumoniae emerged as a significant public health concern in Taiwan, peaking between 2013 and 2015, with the majority of isolates exhibiting OXA-48 as the sole carbapenemase. In this study, we employed whole-genome sequencing to investigate the molecular underpinnings of ST11_KL64 isolates collected from 2013 to 2021. Phylogenomic analysis revealed a notable genetic divergence between the ST11_KL64 strains in Taiwan and those in China, suggesting an independent evolutionary trajectory. Our findings indicated that the ST11_KL64_Taiwan lineage originated from the ST11_KL64 lineage in Brazil, with recombination events leading to the integration of ICEKp11 and a 27-kb fragment at the tRNAASN sites, shaping its unique genomic landscape. To further elucidate this unique sublineage, we examined the plasmid contents. In contrast to ST11_KL64_Brazil strains, which predominantly carried blaKPC-2, ST11_KL64_Taiwan strains exhibited the acquisition of an epidemic blaOXA-48-carrying IncL plasmid. Additionally, ST11_KL64_Taiwan strains consistently harbored a multi-drug resistance IncC plasmid, along with a collection of gene clusters that conferred resistance to heavy metals and the phage shock protein system via various Inc-type plasmids. Although few, there were still rare ST11_KL64_Taiwan strains that have evolved into hypervirulent CRKP through the horizontal acquisition of pLVPK variants. Comprehensive characterization of the high-risk ST11_KL64 lineage in Taiwan not only sheds light on its epidemic success but also provides essential data for ongoing surveillance efforts aimed at tracking the spread and evolution of ST11_KL64 across different geographical regions. Understanding the molecular underpinnings of CRKP evolution is crucial for developing effective strategies to combat its emergence and dissemination.

Providencia as an opportunistic pathogen can cause serious infection, and moreover the emergence of multi-drug-resistant Providencia strains poses a potentially life-threatening risk to public health. However, a comprehensive genomic study to reveal the population structure and dissemination of Providencia is still lacking. In this study, we conducted a genomic epidemiology analysis on the 580 global sequenced Providencia isolates, including 257 ones sequenced in this study (42 ones were fully sequenced). We established a genome sequence-based species classification scheme for Providencia, redefining the conventional 11 Providencia species into seven genocomplexes that were further divided into 18 genospecies, providing an extensively updated reference for Providencia species discrimination based on the largest Providencia genome dataset to date. We then dissected the profile of antimicrobial resistance genes and the prevalence of multi-drug-resistant Providencia strains among these genocomplexes/genospecies, disclosing the presence of diverse and abundant antimicrobial resistance genes and high resistance ratios against multiple classes of drugs in Providencia. We further dissected the genetic basis for the spread of blaNDM-1 in Providencia. blaNDM-1 genes were mainly carried by five incompatible (Inc) groups of plasmids: IncC, IncW, IncpPROV114-NR, IncpCHS4.1-3, and IncpPrY2001, and the last three were newly designated in this study. By tracking the spread of blaNDM-1-carrying plasmids, IncC, IncpPROV114-NR, IncpCHS4.1-3, and IncpPrY2001 plasmids were found to be highly involved in parallel horizontal transfer or vertical clonal expansion of blaNDM-1 among Providencia. Overall, our study provided a comprehensive genomic view of species differentiation, antimicrobial resistance prevalence, and plasmid-mediated blaNDM-1 dissemination in Providencia.

Background and objectives: Fluoroquinolones are a class of broad-spectrum antimicrobials typically used for the treatment of lower urinary tract infections. We aimed to determine the frequency of quinolone resistance genes in Klebsiella pneumoniae isolates from urinary tract infections in Guilan Province, Iran. Methods: The resistance of 114 clinical isolates of K. pneumoniae to common fluoroquinolones and the minimum inhibitory concentration of ciprofloxacin were determined by disk diffusion and broth microdilution methods, respectively. Frequency of five plasmid-mediated quinolone resistance (PMQR) genes including qnrA, qnrB, qnrS, qepA, and aac (6')-Ib-cr was determined by PCR. Results. According to phenotypic assays, 60 isolates (52.6%) were resistant to at least one quinolone compound, 42 isolates (36.8%) were resistant to all tested quinolones, and 28 isolates (24.6%) showed a high level of ciprofloxacin resistance. In addition, aac(6')-Ib-cr was the most common PMQR gene (𝑛 = 44), followed by qnrS (𝑛 = 32), and qnrB (𝑛 = 21). Conclusion: The possible dissemination of PMQR genes poses a serious threat to the management of infections by resistant Klebsiella pneumoniae.