ColE1 Plasmid Research Articles

Whole genome sequencing and antimicrobial resistance among clinical isolates of Shigella sonnei in Addis Ababa, Ethiopia.

Shigellosis is an acute gastroenteritis infection and one of Ethiopia's most common causes of morbidity and mortality, especially in children under five. Antimicrobial resistance (AMR) has spread quickly among Shigella species due to inappropriate antibiotic use, inadequacies of diagnostic facilities, and unhygienic conditions. This study aimed to characterize Shigella sonnei (S. sonnei) using whole genome sequence (WGS) analysis in Addis Ababa, Ethiopia. The raw reads were quality-filtered and trimmed, and a minimum length of 50bp was retained and taxonomically classified using MiniKraken version 1. The whole genome data were aligned with Antibiotic Resistance Gene (ARG) sequences of the Comprehensive Antibiotic Resistance Database (CARD) by Resistance Gene Identifier (RGI). Plasmids were analyzed using the PlasmidFinder tool version 2.1. Additionally, AMR and virulence genes were screened at the Centre for Genomic Epidemiology (CGE) web-based server. All isolates in our investigation contained genes encoding blaEC-8 and blaZEG-1. Here, 60.7% of the isolates were phenotypically sensitive to cefoxitin among the blaEC-8 genes detected in the genotyping analysis, whereas all isolates were completely resistant to amoxicillin and erythromycin phenotypically. The study also identified genes that conferred resistance to trimethoprim (dfrA). Plasmid Col156 and Col (BS512) types were found in all isolates, while IncFII and Col (MG828) plasmids were only identified in one isolate. This study found that many resistant genes were present, confirming the high variety in S. sonnei strains and hence a divergence in phylogenetic relationships. Thus, combining WGS methods for AMR prediction and strain identification into active surveillance may be beneficial for monitoring the spread of AMR in S. sonnei and detecting the potential emergence of novel variations.

Survival of Antimicrobial Resistant Salmonella Heidelberg Inoculated into Microcosms of Fresh Pine Wood Shavings for Broiler Litter.

This study applied genomic characterizations to a cocktail of three Salmonella enterica serovar Heidelberg (S. Heidelberg) strains different antimicrobial resistance (AMR) profile which were inoculated into fresh pine wood shavings (PWS) microcosms. The strains were isolated from feces (SH-AAFC), carcass (SH-ARS) and thigh (SH-FSIS) of broiler chicken. SH-AAFC harbored an antimicrobial resistant gene (ARG) blaCMY-2 on an IncI1 plasmid while SH-FSIS harbored multiple ARGs (floR, cmlA1, tet(A), blaTEM-1B, ant(2'')-Ia, aph(6)-Id, aph(3'')-Ib and sul2) on an IncC plasmid. SH-ARS was pan-susceptible. The die-off of Salmonella was determined at days 0, 1, 7, 14 and 21. Antibiotic susceptibility tests and whole genome sequencing were performed on 77 isolates. At 21 days post-inoculation, Salmonella abundance decreased by 4.4 Log10 CFU/g with the water activity of PWS being correlated with Salmonella survival. SH-AAFC clonal populations survived longer in PWS than SH-FSIS and SH-ARS populations. SH-AAFC clones persisting in litter carried higher copy number of Col plasmids than their ancestors, while some SH-ARS clones acquired a lysogenic bacteriophage from SH-FSIS populations. These results suggests that mobile genetic determinants such as plasmids (which could carry ARGs) and bacteriophage plays roles in the persistence of S. Heidelberg in the PWS used as broiler litter.

Unveiling the microevolution of antimicrobial resistance in selected Pseudomonas aeruginosa isolates from Egyptian healthcare settings: A genomic approach

The incidence of Pseudomonas aeruginosa infections in healthcare environments, particularly in low-and middle-income countries, is on the rise. The purpose of this study was to provide comprehensive genomic insights into thirteen P. aeruginosa isolates obtained from Egyptian healthcare settings. Phenotypic analysis of the antimicrobial resistance profile and biofilm formation were performed using minimum inhibitory concentration and microtiter plate assay, respectively. Whole genome sequencing was employed to identify sequence typing, resistome, virulome, and mobile genetic elements. Our findings indicate that 92.3% of the isolates were classified as extensively drug-resistant, with 53.85% of these demonstrating strong biofilm production capabilities. The predominant clone observed in the study was ST773, followed by ST235, both of which were associated with the O11 serotype. Core genome multi-locus sequence typing comparison of these clones with global isolates suggested their potential global expansion and adaptation. A significant portion of the isolates harbored Col plasmids and various MGEs, all of which were linked to antimicrobial resistance genes. Single nucleotide polymorphisms in different genes were associated with the development of antimicrobial resistance in these isolates. In conclusion, this pilot study underscores the prevalence of extensively drug-resistant P. aeruginosa isolates and emphasizes the role of horizontal gene transfer facilitated by a diverse array of mobile genetic elements within various clones. Furthermore, specific insertion sequences and mutations were found to be associated with antibiotic resistance.

Implementation of the WHO Tricycle protocol for surveillance of extended-spectrum β-lactamase producing Escherichia coli in humans, chickens, and the environment in Madagascar: a prospective genomic epidemiology study

Antimicrobial resistance (AMR) is a major public health threat, affecting not only people but also animals and the environment. The One Health dimension of AMR is well known; however, data are lacking on the circulation of resistance-conferring genes, particularly in low-income countries. In 2017, WHO proposed a protocol called Tricycle, focusing on extended-spectrum β-lactamase (ESBL)-Escherichia coli surveillance in the three sectors (humans, animals, and the environment). We implemented Tricycle in Madagascar to assess ESBL-E coli prevalence and describe intrasector and intersector circulation of ESBL-E coli and plasmids. In this prospective study, we collected blood culture data from hospitalised patients with a suspected bloodstream infection processed from May 1, 2018, to April 30, 2019, and rectal swabs from healthy pregnant women from July 30, 2018, to April 27, 2019, both from three hospitals in Antananarivo, Madagascar; and caeca from farm chickens and surface waters from the Ikopa river, wastewater, and slaughterhouse effluents in the Antananarivo area, Madagascar, from April 9, 2018, to April 30, 2019. All samples were tested for ESBL-E coli. The genomes of all isolates were sequenced using a short-read method on NextSeq 500 and NovaSeq 6000 platforms (Illumina, San Diego, CA, USA) and those carrying plasmid replicons using an additional long-read method on a MinION platform (Oxford Nanopore Technologies, Oxford, UK). We characterised genomes of isolated strains (sequence type, resistance and virulence gene content, and plasmid replicons). We then compared isolates using the variant calling method (single-nucleotide polymorphism). Data from 1056 blood cultures were collected and 289 pregnant women, 246 chickens, and 28 surface waters were sampled. Of the blood cultures, 18 contained E coli, of which seven (39%) were ESBL. ESBL-E coli was present in samples from 86 (30%) of 289 pregnant women, 140 (57%) of 246 chickens, and 28 (100%) of 28 surface water samples. The wet season (November to April) was associated with higher rates of carriage in humans (odds ratio 3·08[1·81-5·27]) and chickens (2·79 [1·65-4·81]). Sequencing of 277 non-duplicated isolates (82 from pregnant women, 118from chickens, and 77 from environmental samples) showed high genetic diversity (90 sequence types identified) with sector-specific genomic features. Single nucleotide polymorphism (SNP) analysis revealed that 169(61%) of 277 isolates grouped into 44 clusters (two or more isolates) of closely related isolates (<40 SNPs), of which 24 clusters contained isolates from two sectors and five contained isolates from all three sectors. ESBL genes were all blaCTX-M variants (215 [78%] of 277 being blaCTX-M-15) and were located on a plasmid in 113 (41%) of 277isolates. These ESBL-carrying plasmids were mainly IncF (63 [55%] of 114; one strain carried two plasmids) and IncY (42 [37%] of 114). The F31/36:A4:B1 (n=13) and F-:A-:B53 (n=8) pMLST subtypes, and the IncY plasmids, which were all highly conserved, were observed in isolates of differing genetic backgrounds from all sectors and were transferable invitro by conjugation. Despite sector-specific population structures, both ESBL-E coli strains and plasmids are circulating among humans, chickens, and the environment in Antananarivo, Madagascar. The Tricycle protocol can be implemented in a low-income country and represents a powerful tool for investigating dissemination of AMR from a One Health perspective. Fondation Mérieux and INSERM, Université Paris Cité.

Genomic insights into antimicrobial resistant Salmonella in internationally traded chicken meat: First baseline findings in the United Arab Emirates

Non-typhoidal Salmonella is among the most prevalent foodborne zoonoses, challenging food safety and One Health worldwide. A dearth of knowledge exists regarding non-typhoidal Salmonella prevalence and genomic features within the United Arab Emirates (UAE), one of the top markets in chicken meat consumption worldwide. In this study, Salmonella was detected in 16 out of 254 (6.30 %, 95 % confidence interval: 3.64 %, 10.03 %) samples of imported frozen chicken carcasses sampled from UAE retails. Among the recovered serotypes, S. Minnesota; ST 548 (10/16) emerged as the most prevalent, trailed by S. Heidelberg; ST 15 (4/16), and then S. Kentucky; ST 198, and ST 152 (2/16). Antimicrobial resistance (AMR) was most prevalent against tetracycline (93.7 %), followed by ampicillin (68.7 %), and extended-spectrum cephalosporins (ceftriaxone and cefoxitin) (56.2 %); with 68.7 % (11/16) of the isolates classified as multidrug resistant (MDR). Whole-genome sequencing (WGS) analysis revealed the existence of 14 AMR genes, among which the blaCMY-2, an AmpC cephalosporinases resistance gene, presented in 10 of the 16 isolates. Among the ten out of the eleven MDR Salmonella isolates, both IncC and Col(pHAD28) plasmid incompatibility types were concurrently featured. The range of virulence genes varied from 149 to 165 genes, with an average of 168 genes per isolate. Except for one isolate, all other isolates possessed type III secretion system (TTSS) related genes known to be encoded by the Salmonella pathogenicity island-1 (SPI-1). This study contributes to our global understanding of Salmonella epidemiology, specifically focusing on the Middle East. The insights gained from this study are significant in shaping import risk analyses aimed at mitigating Salmonella exposure risks through globally traded chicken. The research underscores the value of WGS as a crucial tool for substantiating evidence-based food safety hazard assessment.

Positively charged GFP-chromophore-based fluorescent sensor for DNA

Fluorescence titration of the positively charged green fluorescent protein (GFP) chromophore analogue 1 with E. Coli plasmid DNA [pcDNA3.1(−)] confirms that 1 serves as a fluorescent probe for sensing E. Coli plasmid DNA. The binding constant K between the DNA and 1 is as large as 2.9 × 105. The sensing mechanism is that the intermolecular steric hindrance generated between 1 and E. Coli plasmid DNA by electrostatic attraction restricts the φ-torsion around P-bond and τ-torsion around I-bond of 1, enhancing fluorescence of 1.

The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

Hfq-Antisense RNA I Binding Regulates RNase E-Dependent RNA Stability and ColE1 Plasmid Copy Number.

The mechanisms and consequences of gene regulation by Hfq on trans-encoded small RNAs (sRNAs) have been well studied and documented. Recent employment of Genomic SELEX to search for Hfq-binding motifs has indicated that Hfq might frequently regulate gene expression controlled by cis-antisense RNAs. Here, we use the classic ColE1 plasmid antisense RNA-based regulation model (i.e., RNA I) to study the role of Hfq in controlling antisense regulatory functions. We show that Hfq exhibits a high binding affinity for RNA I and that binding limits RNase E cleavage, thereby stabilizing RNA I and reducing the plasmid copy number. Full-length RNA I displays a binding affinity for Hfq in the sub-micromolar range. In vivo overexpression of Hfq prolongs RNA I stability and reduces the ColE1 plasmid copy number, whereas deletion of hfq reduces RNA I stability and increases the plasmid copy number. RNA I predominantly binds to the proximal face of Hfq and exhibits competitive ability against a chromosome-borne proximal face-bound sRNA (DsrA) for Hfq binding. Through its strong promoter and high gene dosage features, plasmid-encoded antisense RNA I results in high RNA I expression, so it may antagonize the effects of trans-encoded RNAs in controlling target gene expression.

Revolutionizing and identifying novel drug targets in Citrobacter koseri via subtractive proteomics and development of a multi-epitope vaccine using reverse vaccinology and immuno-informatics

Citrobacter koseri is a gram-negative rod that has been linked to infections in people with significant comorbidities and immunocompromised immune systems. It is most commonly known to cause urinary tract infections. Thus, the development of an efficacious C. koseri vaccine is imperative, as the pathogen has acquired resistance to current antibiotics. Subtractive proteomics was employed during this research to identify potential antigenic proteins to design an effective vaccine against C. koseri. The pipeline identified two antigenic proteins as potential vaccine targets: DP-3-O-acyl-N-acetylglucosamine deacetylase and Arabinose 5-phosphate isomerase. B and T cell epitopes from the specific proteins were forecasted employing several immunoinformatic and bioinformatics resources. A vaccine was created using a combination of seven cytotoxic T cell lymphocytes (CTL), five helper T cell lymphocyte (HTL), and seven linear B cell lymphocyte (LBL) epitopes. An adjuvant (β-defensin) was added to the vaccine to enhance immunological responses. The created vaccine was stable for use in humans, highly antigenic, and non-allergenic. The vaccine’s molecular and interactions binding affinity with the human immunological receptor TLR3 were studied using MMGBSA, molecular dynamics (MD) simulations, and molecular docking analyses. E. coli (strain-K12) plasmid vector pET-28a (+) was used to examine the ability of the vaccine to be expressed. The vaccine shows great promise in terms of developing protective immunity against diseases, based on the results of these computer experiments. However, in vitro and animal research are required to validate our findings. Communicated by Ramaswamy H. Sarma

Genomic analysis of Salmonella enterica from Metropolitan Manila abattoirs and markets reveals insights into circulating virulence and antimicrobial resistance genotypes.

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.

Comprehensive Genomic Characterization of Cronobacter sakazakii Isolates from Infant Formula Processing Facilities Using Whole-Genome Sequencing.

Cronobacter sakazakii is an opportunistic pathogen linked to outbreaks in powdered infant formula (PIF), primarily causing meningitis and necrotizing enterocolitis. Whole-genome sequencing (WGS) was used to characterize 18 C. sakazakii strains isolated from PIF (powdered infant formula) manufacturing plants (2011-2015). Sequence Type (ST) 1 was identified as the dominant sequence type, and all isolates carried virulence genes for chemotaxis, flagellar motion, and heat shock proteins. Multiple antibiotic resistance genes were detected, with all isolates exhibiting resistance to Cephalosporins and Tetracycline. A significant correlation existed between genotypic and phenotypic antibiotic resistance. The plasmid Col(pHAD28) was identified in the isolates recovered from the same PIF environment. All isolates harbored at least one intact phage. All the study isolates were compared with a collection of 96 publicly available C. sakazakii genomes to place these isolates within a global context. This comprehensive study, integrating phylogenetic, genomic, and epidemiological data, contributes to a deeper understanding of Cronobacter outbreaks. It provides valuable insights to enhance surveillance, prevention, and control strategies in food processing and public health contexts.

First Report of Aeromonas veronii as an Emerging Bacterial Pathogen of Farmed Nile Tilapia (Oreochromis niloticus) in Brazil.

Brazil is one of the world's leading producers of Nile tilapia, Oreochromis niloticus. However, the industry faces a major challenge in terms of infectious diseases, as at least five new pathogens have been formally described in the last five years. Aeromonas species are Gram-negative anaerobic bacteria that are often described as fish pathogens causing Motile Aeromonas Septicemia (MAS). In late December 2022, an epidemic outbreak was reported in farmed Nile tilapia in the state of São Paulo, Brazil, characterized by clinical signs and gross pathology suggestive of MAS. The objective of this study was to isolate, identify, and characterize in vitro and in vivo the causative agent of this epidemic outbreak. The bacterial isolates were identified as Aeromonas veronii based on the homology of 16S rRNA (99.9%), gyrB (98.9%), and the rpoB gene (99.1%). A. veronii showed susceptibility only to florfenicol, while it was resistant to the other three antimicrobials tested, oxytetracycline, enrofloxacin, and amoxicillin. The lowest florfenicol concentration capable of inhibiting bacterial growth was ≤0.5 µg/mL. The phenotypic resistance of the A. veronii isolate observed for quinolones and tetracycline was genetically confirmed by the presence of the qnrS2 (colE plasmid) and tetA antibiotic-resistant genes, respectively. A. veronii isolate was highly pathogenic in juvenile Nile tilapia tested in vivo, showing a mortality rate ranging from 3 to 100% in the lowest (1.2 × 104) and highest (1.2 × 108) bacterial dose groups, respectively. To our knowledge, this study would constitute the first report of highly pathogenic and multidrug-resistant A. veronii associated with outbreaks and high mortality rates in tilapia farmed in commercial net cages in Brazil.

The rare Salmonella enterica serovar Isangi: genomic characterization of the antimicrobial resistance, virulence potential and epidemiology of Brazilian strains in comparison to global isolates.

Introduction. Salmonella enterica serovar Isangi (S. Isangi) is a rare non-typhoidal serovar, related to invasive nosocomial infections in various countries and to increasing antimicrobial resistance rates.Gap statement. Despite existing reports on S. Isangi, there is a lack of information of specific traits regarding this serovar, which could be improved through genomic analyses.Aim. Our goals were to characterize the antimicrobial resistance, virulence potential and genomic relatedness of 11 S. Isangi strains from Brazil in comparison to 185 genomes of global isolates using whole-genome sequencing (WGS) data.Methodology. Phenotypic resistance was determined by disc-diffusion. The search for resistance genes, plasmids, prophages, Salmonella pathogenicity islands (SPIs) and virulence genes, plus multi-locus sequence typing (MLST) and core-genome MLST (cgMLST) were performed using WGS.Results. Brazilian S. Isangi strains showed phenotypic resistance to nalidixic acid, ciprofloxacin and streptomycin, and harboured antimicrobial resistance [qnrB19, aac(6')-Iaa, mdsAB] and heavy metal tolerance (arsD, golST) genes. Col(pHAD28) and IncFII(S) plasmids, virulence genes related to adherence, macrophage induction, magnesium uptake, regulation and type III secretion systems, 12 SPIs and eight prophages were detected. The 185 additional global genomes analysed harboured resistance genes against 11 classes of antimicrobial compounds, 22 types of plasmids, 32 prophages, 14 SPIs, and additional virulence genes related to serum resistance, stress adaptation and toxins. Sequence type (ST)216 was assigned to genomes from Brazil and other countries, while ST335 was the most frequent ST, especially among South African genomes. cgMLST showed that Brazilian genomes were more closely related to genomes from European and African countries, the USA and Taiwan, while the majority of South African genomes were more closely related among each other.Conclusion. The presence of S. Isangi strains from Brazil and different countries showing a close genomic correlation, antimicrobial resistance profiles to drugs used in human therapy and a large number of virulence determinants reinforced the need for stronger initiatives to monitor rare non-typhoidal Salmonella serovars such as S. Isangi in order to prevent its dissemination among human and non-human sources.

Newly Established Genetic System for Functional Analysis of MetSV.

The linear chromosome of the Methanosarcina spherical virus with 10,567 bp exhibits 22 ORFs with mostly unknown functions. Annotation using common tools and databases predicted functions for a few genes like the type B DNA polymerase (MetSVORF07) or the small (MetSVORF15) and major (MetSVORF16) capsid proteins. For verification of assigned functions of additional ORFs, biochemical or genetic approaches were found to be essential. Consequently, we established a genetic system for MetSV by cloning its genome into the E. coli plasmid pCR-XL-2. Comparisons of candidate plasmids with the MetSV reference based on Nanopore sequencing revealed several mutations of yet unknown provenance with an impact on protein-coding sequences. Linear MetSV inserts were generated by BamHI restriction, purified and transformed in Methanosarcina mazei by an optimized liposome-mediated transformation protocol. Analysis of resulting MetSV virions by TEM imaging and infection experiments demonstrated no significant differences between plasmid-born viruses and native MetSV particles regarding their morphology or lytic behavior. The functionality of the genetic system was tested by the generation of a ΔMetSVORF09 mutant that was still infectious. Our genetic system of MetSV, the first functional system for a virus of methanoarchaea, now allows us to obtain deeper insights into MetSV protein functions and virus-host interactions.

Phylogenomics of Globally Spread Clonal Groups 14 and 15 of Klebsiella pneumoniae.

Klebsiella pneumoniae sequence type 14 (ST14) and ST15 caused outbreaks of CTX-M-15 and/or carbapenemase producers worldwide, but their phylogeny and global dynamics remain unclear. We clarified the evolution of K. pneumoniae clonal group 14 (CG14) and CG15 by analyzing the capsular locus (KL), resistome, virulome, and plasmidome of public genomes (n = 481) and de novo sequences (n = 9) representing main sublineages circulating in Portugal. CG14 and CG15 evolved independently within 6 main subclades defined according to the KL and the accessory genome. The CG14 (n = 65) clade was structured in two large monophyletic subclades, CG14-I (KL2, 86%) and CG14-II (KL16, 14%), whose emergences were dated to 1932 and 1911, respectively. Genes encoding extended-spectrum β-lactamase (ESBL), AmpC, and/or carbapenemases were mostly observed in CG14-I (71% versus 22%). CG15 clade (n = 170) was segregated into subclades CG15-IA (KL19/KL106, 9%), CG15-IB (variable KL types, 6%), CG15-IIA (KL24, 43%) and CG15-IIB (KL112, 37%). Most CG15 genomes carried specific GyrA and ParC mutations and emerged from a common ancestor in 1989. CTX-M-15 was especially prevalent in CG15 (68% CG15 versus 38% CG14) and in CG15-IIB (92%). Plasmidome analysis revealed 27 predominant plasmid groups (PG), including particularly pervasive and recombinant F-type (n = 10), Col (n = 10), and new plasmid types. While blaCTX-M-15 was acquired multiple times by a high diversity of F-type mosaic plasmids, other antibiotic resistance genes (ARGs) were dispersed by IncL (blaOXA-48) or IncC (blaCMY/TEM-24) plasmids. We first demonstrate an independent evolutionary trajectory for CG15 and CG14 and how the acquisition of specific KL, quinolone-resistance determining region (QRDR) mutations (CG15), and ARGs in highly recombinant plasmids could have shaped the expansion and diversification of particular subclades (CG14-I and CG15-IIA/IIB). IMPORTANCE Klebsiella pneumoniae represents a major threat in the burden of antibiotic resistance (ABR). Available studies to explain the origin, the diversity, and the evolution of certain ABR K. pneumoniae populations have mainly been focused on a few clonal groups (CGs) using phylogenetic analysis of the core genome, the accessory genome being overlooked. Here, we provide unique insights into the phylogenetic evolution of CG14 and CG15, two poorly characterized CGs which have contributed to the global dissemination of genes responsible for resistance to first-line antibiotics such as β-lactams. Our results point out an independent evolution of these two CGs and highlight the existence of different subclades structured by the capsular type and the accessory genome. Moreover, the contribution of a turbulent flux of plasmids (especially multireplicon F type and Col) and adaptive traits (antibiotic resistance and metal tolerance genes) to the pangenome reflect the exposure and adaptation of K. pneumoniae under different selective pressures.

ESBL- and Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae among Bivalves from Portuguese Shellfish Production Areas.

Bivalves are filter-feeding organisms and biomarkers of bacterial pollution. Our study aimed to analyze the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Escherichia coli among bivalves. A total of 522 bivalve samples were collected along Portuguese shellfish production areas. Homogenized samples were screened for E. coli contamination on corresponding selective plates, allowing for concomitant growth of Klebsiella pneumoniae. E. coli growth was observed in 39% of the samples. Subsequent selective screening identified nine samples (4.4%) contaminated with ESBL producers, corresponding to E. coli (n = 7) and K. pneumoniae (n = 2), while a single carbapenemase-producing K. pneumoniae (0.5%) was identified. ESBLs were all CTX-M-types commonly identified in human isolates, i.e., CTX-M-32 (n = 4), CTX-M-15 (n = 4), and CTX-M-14 (n = 1). The carbapenemase producer harbored the blaGES-5 gene located on a ColE plasmid. Clonality was evaluated by multilocus sequence typing, identifying E. coli backgrounds as ST10, ST23, ST540, ST617, ST746, SLV206, and SLV2325, commonly identified among environmental and human strains. The K. pneumoniae isolates belonged to ST834, ST15, and DLV644. The occurrence of ESBL- and carbapenemase-producing Enterobacteriaceae in bivalves reveals how the marine environment constitutes a reservoir of critical bacterial pathogens, thus potentially representing a risk to human health.

The Emerging Nosocomial Pathogen Klebsiella michiganensis: Genetic Analysis of a KPC-3 Producing Strain Isolated from Venus Clam.

The recovery and characterization of a multidrug-resistant, KPC-3-producing Klebsiella michiganensis that was obtained from Venus clam samples is reported in this study. A whole-genome sequencing (WGS) analysis using Illumina and Nanopore technologies of the K. michiganensis 23999A2 isolate revealed that the strain belonged to the new sequence type 382 (ST382) and carried seven plasmid replicon sequences, including four IncF type plasmids (FII, FIIY, FIIk, and FIB), one IncHI1 plasmid, and two Col plasmids. The FIB and FIIk plasmids showed high homology to each other and to multireplicon pKpQIL-like plasmids that are found in epidemic KPC-K. pneumoniae clones worldwide. The strain carried multiple β-lactamase genes on the IncF plasmids: blaOXA-9 and blaTEM-1A on FIB, blaKPC-3 inserted in a Tn4401a on FIIK, and blaSHV-12 on FIIY. The IncHI1-ST11 harbored no resistance gene. The curing of the strain caused the loss of all of the bla genes and a rearrangement of the IncF plasmids. Conjugal transfer of the blaOXA-9, blaTEM-1A and blaKPC-3 genes occurred at a frequency of 5 × 10-7, using K. quasipneumoniae as a recipient, and all of the bla genes were transferred through a pKpQIL that originated from the recombination of the FIB and FIIk plasmids of the donor. A comparison with 31 K. michiganensis genomes that are available in the NCBI database showed that the closest phylogenetic relatives of K. michiganensis 23999A2 are an environmental isolate from soil in South Korea and a clinical isolate from human sputum in Japan. Finally, a pan-genome analysis showed a large accessory genome of the strain as well as the great genomic plasticity of the K. michiganensis species. IMPORTANCE Klebsiella michiganensis is an emerging nosocomial pathogen, and, so far, few studies describe isolates of clinical origin in the environment. This study contributes to the understanding of how the dissemination of carbapenem-resistance outside the hospital setting may be related to the circulation of pKpQIL-like plasmids that are derived from epidemic Klebsiella pneumoniae strains. The recovery of a carbapenem-resistant isolate in clams is of great concern, as bivalves could represent vehicles of transmission of pathogens and resistance genes to humans via the food chain. The study demonstrates the plasticity of K. michiganensis genome, which is probably useful to multiple environment adaptation and to the evolution of the species.

Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality.

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells—a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality.

Sequence analysis and plasmid mobilization of a 6.6-kb kanamycin resistance plasmid, pSNC3-Kan, from a Salmonella enterica serotype Newport isolate

Research on the transfer of antibiotic resistance plasmids has been mainly focused on the large multi-drug resistance conjugative plasmids, while the transmission of small mobilizable plasmids remains under-investigated. A series of diverse ColE-like kanamycin resistance plasmids (“KanR plasmids”) from Salmonella enterica were characterized previously. In this study, the 6.6-kb pSNC3-Kan from a Salmonella enterica serotype Newport isolate was investigated. It possessed highly conserved RNA I/II and Tn602 (IS903-aph-IS903) regions to two other KanR plasmids pSe-Kan and pSBardo-Kan, but carried a mobC-mobA/BD operon. The mobilization proteins encoded by the mob operon of pSNC3-Kan showed high sequence identity (~95%) to those of an E. coli plasmid pEC34B, except that MobE was not present; and were much less conserved to those of another KanR plasmid pSN11/00Kan (43% - 86% identity). Four structurally different KanR plasmids were investigated for their ability to be mobilized by the conjugal transfer (tra) genes from F and IncP plasmids. Transfer genes derived from IncP plasmids can efficiently mobilize KanR plasmids possessing the mob operons (mobC-mobA/BD), such as pSNC3-Kan and pSN11/00Kan, in bi-parental mating experiments. On the other hand, F tra genes were able to mobilize pU302S, pSNC3-Kan and pSe-Kan, but not pSN11/00Kan. A plasmid-borne mob operon was not required for mobilization of the oriT(F)-bearing pSe-Kan by the F tra genes. This study underscores the complexity of plasmid interaction and the importance of how small mobilizable plasmids may contribute to the spread of antibiotic resistance genes.

Recurrent bacteremia with a hypermucoviscous Escherichia coli isolated from a patient with perihilar cholangiocarcinoma: insights from a comprehensive genome-based analysis

BackgroundEscherichia coli (E. coli) is a common human pathogen, responsible for a broad spectrum of infections. Sites of infection can vary, but the hepato-biliary system is of particular concern due to the infection-associated formation of gallstones and the spread of pathogens from the bile ducts into the bloodstream.Case presentationThe presented case is striking, as the detected isolate showed a positive string test. This hypermucoviscous phenotype is atypical for E. coli and a particular feature of hypervirulent Klebsiella pneumoniae (K. pneumoniae) variants.ObjectivesTo provide new insights into the genomic background of an E. coli strain with an unusual hypermucoviscous phenotype using hybrid short- and long-read sequencing approaches.ResultsComplete hybrid assemblies of the E. coli genome and plasmids were done and used for genome based typing. Isolate 537–20 was assigned to the multilocus sequence type ST88 and serotype O8:H4. The strain showed a close relationship to avian pathogenic strains. Analysis of the chromosome and plasmids revealed the presence of several virulence factors, such as the Conserved Virulence Plasmidic (CVP) region on plasmid 537-20_1, including several iron acquisition genes (sitABCD, iroABCDEN, iucABCD, hbd) and the iutA gene encoding the receptor of the siderophore aerobactin. The hypermucoviscous phenotype could be caused by encapsulation of putative K. pneumoniae origin.ConclusionsHybrid sequencing enabled detailed genomic characterization of the hypermucoviscous E. coli strain, revealing virulence factors that have their putative origin in K. pneumoniae.