Identical Plasmid Research Articles

Emerging carbapenem-resistant Klebsiella pneumoniae in a tertiary care hospital in Lima, Peru.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant public health threat, particularly in low- and middle-income countries (LMICs) with limited surveillance and treatment options. This study examines the genetic diversity, resistance patterns, and transmission dynamics of 66 CRKP isolates recovered over 5 years (2015-2019) after the first case of CRKP was identified at a tertiary care hospital in Lima, Peru. Our findings reveal a shift from blaKPC-2 to blaNDM-1 as the dominant carbapenemase gene after 2017. Lineage ST45 was the most prevalent and persisted for multiple years, followed by high-risk clones ST11 and ST147. The blaNDM-1 gene was carried almost exclusively by a Tn125-like transposon, similar to the one reported in previous studies from two Peruvian hospitals. Long-read sequencing revealed nearly identical blaNDM-carrying plasmids across the four assessed lineages. A comparative analysis of 1,023 South American CRKP genomes confirmed a unique pattern in Peru, where blaNDM-1 (81.4%) outpaced blaKPC-2, which remained dominant (59.4%) elsewhere. In addition, emerging clones ST45 and ST348 found in Peru were rarely found elsewhere in South America, suggesting potential regional adaptation. In conclusion, our study provides a comprehensive picture of the intra-hospital dynamics of these emerging pathogens and provides a framework for studying their genomic diversity in the understudied South American region.IMPORTANCEThis study provides novel insights into the transmission and genetic diversity of carbapenem-resistant Klebsiella pneumoniae, a bacteria responsible for severe infections, with limited treatment options. By examining isolates recovered over 5 years at a major hospital in Lima, Peru, we demonstrated a shift from one type of resistance gene, blaKPC, to another, blaNDM, which is more challenging to treat. Our findings reveal that specific bacterial lineages carrying the blaNDM gene in a specific plasmid are emerging in Peru, including well-known high-risk strains and others rarely found elsewhere in South America. This pattern highlights an urgent need for targeted surveillance and infection control as these strains pose a significant challenge to healthcare systems. Our study provides crucial data on Klebsiella pneumoniae in Peru, contributing to broader efforts to monitor and control antibiotic-resistant infections in South America and globally.

Genetic Characterization, Transmission Pattern and Health Risk Analysis of Intestinal Colonization ESBL-Producing Escherichia coli in Vegetable Farming Population.

The surging prevalence rates of ESBL-producing Escherichia coli (ESBL-Ec) pose a serious threat to public health. To date, most research on drug-resistant bacteria and genes has focused on livestock and poultry breeding areas, hospital clinical areas, natural water environments, and wastewater treatment plants. However, few studies have been conducted on drug-resistant bacteria in vegetable cultivation. In this study, a total of vegetable farmers (n = 59) from six villages were surveyed. Fecal samples were collected from vegetable farmers; we also collected environmental samples, including river water, well water, soil, river sediment, vegetable surface swabs, and fish intestinal tracts. The ESBL-Ec intestinal colonization rate in vegetable farmers was 76.27%. PFGE results indicated two patterns of ESBL-Ec transmission within the vegetable cultivation area: among vegetable farmers, and among river water, river sediments, and vegetable farmers. Based on the phylogenetic analysis, three transmission patterns of ESBL-Ec outside the vegetable cultivation area were inferred: human-human, human-animal-human, and human-animal-environment. Twelve of the isolates carried closely related or identical IncF plasmids carrying blaCTX-M. Whole genome sequencing (WGS) analysis showed that ST569-B2-O134:H31 and ST38-D-O50:H30 were associated with high disease risk. We assessed the health risks of the farming population and provided a reference basis for public health surveillance and environmental management by monitoring the prevalence and transmission of ESBL-Ec in vegetable areas.

Molecular epidemiology of extended-spectrum beta-lactamase-producing-Klebsiella species in East Tennessee dairy cattle farms.

The 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. Fifty-seven Klebsiella species were isolated on CHROMagar™ ESBL plates and confirmed with MADLI-TOF MS and whole genome sequenced from 14 dairy farms. Six families of beta-lactamase (bla) (bla CTX-M, bla SHV, bla TEM, bla OXY, bla OXA, and bla SED) were detected in ESBL-Klebsiella spp. genomes. Most (73%) of isolates had the first three types of beta-lactamase genes, with bla SHV being the most frequent, followed by bla CTX-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 bla CTXM-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.

Clonal background and routes of plasmid transmission underlie antimicrobial resistance features of bloodstream Klebsiella pneumoniae

Bloodstream infections caused by the opportunistic pathogen Klebsiella pneumoniae are associated with adverse health complications and high mortality rates. Antimicrobial resistance (AMR) limits available treatment options, thus exacerbating its public health and clinical burden. Here, we aim to elucidate the population structure of K. pneumoniae in bloodstream infections from a single medical center and the drivers that facilitate the dissemination of AMR. Analysis of 136 short-read genome sequences complemented with 12 long-read sequences shows the population consisting of 94 sequence types (STs) and 99 clonal groups, including globally distributed multidrug resistant and hypervirulent clones. In vitro antimicrobial susceptibility testing and in silico identification of AMR determinants reveal high concordance (90.44–100%) for aminoglycosides, beta-lactams, carbapenems, cephalosporins, quinolones, and sulfonamides. IncF plasmids mediate the clonal (within the same lineage) and horizontal (between lineages) transmission of the extended-spectrum beta-lactamase gene blaCTX-M-15. Nearly identical plasmids are recovered from isolates over a span of two years indicating long-term persistence. The genetic determinants for hypervirulence are carried on plasmids exhibiting genomic rearrangement, loss, and/or truncation. Our findings highlight the importance of considering both the genetic background of host strains and the routes of plasmid transmission in understanding the spread of AMR in bloodstream infections.

Transmission of global clones of NDM-producing Enterobacterales and interspecies spread of IncX3 plasmid harbouring blaNDM-5 in Tokyo

ObjectiveThe aim of this study is to characterise the molecular characteristics of NDM-producing Enterobacterales, which have been on the increase in recent years in Japan, where IMP-producing bacteria are dominant among carbapenemase-producing Enterobacterales. MethodsWe collected 21 strains of NDM-producing Enterobacterales detected between 2015 and 2022 at five hospitals in Tokyo and performed illumina whole genome sequencing. For the seven selected strains, nanopore long-read sequencing was also performed to characterise the plasmids harbouring blaNDM. ResultsFourteen strains were Escherichia coli and all carried blaNDM-5. Among these strains, eight and three were sequence type (ST) 410 and ST167, respectively, and both groups of strains were spread clonally in different hospitals. Two strains of Klebsiella pneumoniae ST147 carrying blaNDM-1 were detected in a hospital, and these strains had also spread clonally. The remainder included Enterobacter hormaechei, Klebsiella quasipneumoniae, Citrobacter amalonaticus, and Klebsiella michiganensis. Plasmid analysis revealed that an identical IncX3 plasmid harbouring blaNDM-5 was shared among four strains of different bacterial species (E. coli, C. amalonaticus, K. michiganensis, and E. hormaechei) detected at the same hospital. In addition, a Klebsiella quasipneumoniae strain detected at a different hospital also carried an IncX3 plasmid with a similar genetic structure. ConclusionsNosocomial spread of multiple multidrug-resistant global clones and transmission of IncX3 plasmids harbouring blaNDM-5 among multiple species were detected as the major pathways of spread of NDM-producing Enterobacterales in Tokyo. Early detection of carriers and measures to prevent nosocomial spread are important to prevent further spread of NDM-producing organisms.

EpiXact-ONT: Long-read whole genome sequencing for rapid outbreak detection and comprehensive plasmid transmission analysis

Background: Healthcare associated infections (HAIs) are a major contributor to patient morbidity and mortality. HAIs are increasingly important due to the rise of multidrug resistant pathogens which can lead to deadly nosocomial outbreaks. Traditional methods for investigating transmissions are slow, costly and have poor detection resolution. In addition, plasmid transmission which can horizontally transfer critical resistance and virulence genes is not part of routine infection control practice due to lack of comprehensive and cost-effective methods capable of identifying both pathogen and/or plasmid transmission. Here we demonstrate the utility of the Oxford Nanopore Technologies (ONT) platform for whole genome sequencing (WGS) based pathogen and plasmids transmission analysis. Methods: We developed a rapid end-to-end process that includes sample preparation, sequencing optimized for generating long-reads and bioinformatics workflow customized for error-prone ONT data. We use Flye to generate de novo assemblies and a secondary bioinformatics step to identify each circular sequence. Individual circular sequences with an Ori (≥1) are identified. For pathogen clonality analysis we perform a pairwise mapping-based chromosomal sequences comparison eliminating need for an external reference genome. Similarly, individual plasmids are separated and compared pairwise. We annotate both the circularized chromosomal and plasmid sequences for known resistance and virulence genes. Results: We performed ONT (and confirmatory Illumina) sequencing of the genomes of 20 bacterial isolates originating from 5 HAI investigations previously performed at Day Zero Diagnostics using epiXact®, our Illumina-based HAI sequencing and analysis lab service. ONT-based clonality determination had 100% agreement with the Illumina based pipeline. We also found that using the outbreak-specific assembled genomes instead of an external reference increased the SNP-calling resolution in the ONT pipeline. We also identified sets of clonal isolates with both identical plasmids and distinct plasmids; as well as sets of non-clonal isolates with identical plasmids and distinct plasmids. In one subset of 7 multi-species isolates, we identified 2-7 circularized plasmid sequences in each isolate, all harboring known resistance genes. 4 plasmids were found in multiple isolates, with each plasmid appearing in between 2 and 4 distinct isolates. Notably, blaNDM was identified in at least 1 plasmid in each isolate. Conclusion: We demonstrate the utility of ONT for comprehensive HAI investigations, establishing the potential to transform healthcare epidemiology with rapid outbreak determination covering pathogen and plasmid transmission in < 2 4 hours from sample receipt.

Dissemination of clinical Escherichia coli strains harboring mcr-1, blaNDM−7 and siderophore-producing plasmids in a Chinese hospital

BackgroundCarbapenem-resistant E. coli (CREco) pose a significant public health threat due to their multidrug resistance. Colistin is often a last-resort treatment against CREco; however, the emergence of colistin resistance gene mcr-1 complicates treatment options.MethodsTwo E. coli strains (ECO20 and ECO21), recovered from hospitalized patients in distinct wards, exhibited resistance to carbapenems and colistin. Whole-genome sequencing and phenotypic characterization were employed to study resistance patterns, plasmid profiles, transferability of resistance and virulence genes, and siderophore production capabilities. Comparative genome analysis was used to investigate the genetic environment of mcr-1, blaNDM−7, and virulence clusters.ResultsBoth E. coli strains exhibited thr presence of both mcr-1 and blaNDM−7 genes, showing high resistance to multiple antibiotics. Genomic analysis revealed the clonal transmission of these strains, possessing identical plasmid profiles (pMCR, pNDM, and pVir) associated with colistin resistance, carbapenem resistance, and virulence factors. Conjugation experiments confirmed the transferability of these plasmids, indicating their potential to disseminate resistance and virulence traits to other strains. Comparative genomic analyses unveiled the distribution of mcr-1 (IncX4-type) and blaNDM (IncX3-type) plasmids across diverse bacterial species, emphasizing their adaptability and threat. The novelty of pVir indicates its potential role in driving the evolution of highly adaptable and pathogenic strains.ConclusionsOur findings underscore the co-occurrence of mcr-1, blaNDM−7, and siderophore-producing plasmids in E. coli, which poses a significant concern for global health. This research is crucial to unravel the complex mechanisms governing plasmid transfer and recombination and to devise robust strategies to control their spread in healthcare settings.

Genetic and phenotypic characterizations of IncX3 plasmids harboring bla NDM-5 and bla NDM-16b in Japan.

The spread of the gene encoding the NDM-type carbapenemase, bla NDM, poses a serious threat to clinical practice and public health. This gene is usually carried by transferable plasmids, which facilitates its rapid spread among various species of Enterobacterales. Plasmids with the IncX3 replicon play a major role in the spread of bla NDM in Asian countries. In this study, we conducted genomic sequencing and a comparative analysis of the IncX3-type plasmids harboring bla NDM-5 and bla NDM-16b, a single-nucleotide variant of bla NDM-5, isolated in a national antimicrobial-resistant bacterial genomic surveillance in Japan (Japan Antimicrobial Resistant Bacterial Surveillance), which was carried out during 2019-2020. We compared five plasmids harboring bla NDM-5 and one plasmid harboring bla NDM-16b. They were highly identical and differed in several nucleotide substitutions or variations in insertion sequences, although they were carried by different species or genotypes. Notably, bla NDM-16b-carrying pJBBDACG-19-0070 was identical to a plasmid carrying bla NDM-5 except for one nucleotide substitution in the bla NDM gene throughout the 46 kb sequences. Transformants carrying these two plasmids showed comparative resistance to carbapenems, indicating that a substitution on the NDM enzyme (Ala233 to Val) did not affect carbapenem resistance, at least under normal experimental conditions. A database search revealed that several identical plasmids carrying bla NDM-5 were reported from neighboring countries, mainly China, before our surveillance period. Our analysis suggested that the IncX3 plasmid harboring bla NDM-5 is spreading in Japan, and bla NDM-16b emerged from a single nucleotide substitution on the plasmid, with widespread dissemination in Asian countries. IMPORTANCE IncX3 plasmids harboring bla NDM-5 play a major role in the spread of carbapenem resistance in Asia, particularly in China, in clinical and environmental settings. In this study, we present that Enterobacterales isolates carrying IncX3 plasmids harboring bla NDM-5 have been disseminated in Japan, where their identification was previously rare. In addition, bla NDM-16b, a single-nucleotide variant of bla NDM-5, was found to be carried by an identical IncX3 plasmid. A comparative sequence analysis revealed that the bla NDM-16b gene emerged from a single nucleotide substitution on an IncX3 plasmid harboring bla NDM-5. The bla NDM-16b gene did not confer elevated carbapenem resistance compared to bla NDM-5 in our assay using transformants carrying the plasmid harboring either of these genes, although the A233V substitution was reported to confer stability to the enzyme in ion-depleted conditions. Nevertheless, vigilance regarding the emergence of novel variants is required.

A novel virulence plasmid encoding yersiniabactin, salmochelin, and RmpADC from hypervirulent Klebsiella pneumoniae of distinct genetic backgrounds.

Hypervirulent Klebsiella pneumoniae (hvKP) is increasingly reported worldwide as a major clinical and public health threat. The virulence of hvKP is attributed largely to the carriage of virulence plasmids (KpVPs). To date, two dominant types of KpVP have been identified, namely, KpVP-1 and KpVP-2. In this study, we reported two hvKP strains from bloodstream infections that carry highly identical virulence plasmids that exhibited <40% coverage compared with KpVP-1 and KpVP-2. This novel virulence plasmid was designated KpVP-3. The two hvKP have different genetic backgrounds, which belonged to ST29-K54 and ST111-K63, respectively. They were both positive for the string test, highly virulent on the Galleria mellonella infection model, and possess high-level macrophage-killing resistance in vitro. Apart from the intrinsic non-susceptibility to ampicillin, both strains were susceptible to commonly used antibiotics. The virulence plasmid carried virulence genes rmpADC, iroBCDN (iro1), and the ybt locus (ybt4) which was not present on either KpVP-1 or KpVP-2. It did not carry antimicrobial resistance genes but carried an incomplete conjugation machinery containing only the traH and traF genes. The KpVP-3 plasmid was stably maintained in both hvKP strains and could not be eliminated with SDS treatment or by serial passage on stress-free agar plates. KpVP-3 was non-self-transmissible under experimental conditions. Data mining suggested KpVP-3-type plasmids have emerged in different countries including China, Australia, and the USA. The emergence of this novel virulence plasmid might pose a potential threat to public health. Heightened efforts are required to study its dissemination mechanism.

From Farm to Fork: Persistence of Clinically Relevant Multidrug-Resistant and Copper-Tolerant Klebsiella pneumoniae Long after Colistin Withdrawal in Poultry Production.

Concerns about colistin-resistant bacteria in animal food-environmental-human ecosystems prompted the poultry sector to implement colistin restrictions and explore alternative trace metals/copper feed supplementation. The impact of these strategies on the selection and persistence of colistin-resistant Klebsiella pneumoniae in the whole poultry production chain needs clarification. We assessed colistin-resistant and copper-tolerant K. pneumoniae occurrence in chickens raised with inorganic and organic copper formulas from 1-day-old chicks to meat (7 farms from 2019 to 2020), after long-term colistin withdrawal (>2 years). Clonal diversity and K. pneumoniae adaptive features were characterized by cultural, molecular, and whole-genome-sequencing (WGS) approaches. Most chicken flocks (75%) carried K. pneumoniae at early and preslaughter stages, with a significant decrease (P < 0.05) in meat batches (17%) and sporadic water/feed contamination. High rates (>50%) of colistin-resistant/mcr-negative K. pneumoniae were observed among fecal samples, independently of feed. Most samples carried multidrug-resistant (90%) and copper-tolerant (81%; silA and pcoD positive and with a MICCuSO4 of ≥16 mM) isolates. WGS revealed accumulation of colistin resistance-associated mutations and F type multireplicon plasmids carrying antibiotic resistance and metal/copper tolerance genes. The K. pneumoniae population was polyclonal, with various lineages dispersed throughout poultry production. ST15-KL19, ST15-KL146, and ST392-KL27 and IncF plasmids were similar to those from global human clinical isolates, suggesting chicken production as a reservoir/source of clinically relevant K. pneumoniae lineages and genes with potential risk to humans through food and/or environmental exposure. Despite the limited mcr spread due to the long-term colistin ban, this action was ineffective in controlling colistin-resistant/mcr-negative K. pneumoniae, regardless of feed. This study provides crucial insights into the persistence of clinically relevant K. pneumoniae in the poultry production chain and highlights the need for continued surveillance and proactive food safety actions within a One Health perspective. IMPORTANCE The spread of bacteria resistant to last-resort antibiotics such as colistin throughout the food chain is a serious concern for public health. The poultry sector has responded by restricting colistin use and exploring alternative trace metals/copper feed supplements. However, it is unclear how and to which extent these changes impact the selection and persistence of clinically relevant Klebsiella pneumoniae throughout the poultry chain. We found a high occurrence of copper-tolerant and colistin-resistant/mcr-negative K. pneumoniae in chicken flocks, regardless of inorganic and organic copper formulas use and a long-term colistin ban. Despite the high K. pneumoniae isolate diversity, the occurrence of identical lineages and plasmids across samples and/or clinical isolates suggests poultry as a potential source of human K. pneumoniae exposure. This study highlights the need for continued surveillance and proactive farm-to-fork actions to mitigate the risks to public health, relevant for stakeholders involved in the food industry and policymakers tasked with regulating food safety.

Enterobacterales plasmid sharing amongst human bloodstream infections, livestock, wastewater, and waterway niches in Oxfordshire, UK.

Plasmids enable the dissemination of antimicrobial resistance (AMR) in common Enterobacterales pathogens, representing a major public health challenge. However, the extent of plasmid sharing and evolution between Enterobacterales causing human infections and other niches remains unclear, including the emergence of resistance plasmids. Dense, unselected sampling is essential to developing our understanding of plasmid epidemiology and designing appropriate interventions to limit the emergence and dissemination of plasmid-associated AMR. We established a geographically and temporally restricted collection of human bloodstream infection (BSI)-associated, livestock-associated (cattle, pig, poultry, and sheep faeces, farm soils) and wastewater treatment work (WwTW)-associated (influent, effluent, waterways upstream/downstream of effluent outlets) Enterobacterales. Isolates were collected between 2008 and 2020 from sites <60 km apart in Oxfordshire, UK. Pangenome analysis of plasmid clusters revealed shared 'backbones', with phylogenies suggesting an intertwined ecology where well-conserved plasmid backbones carry diverse accessory functions, including AMR genes. Many plasmid 'backbones' were seen across species and niches, raising the possibility that plasmid movement between these followed by rapid accessory gene change could be relatively common. Overall, the signature of identical plasmid sharing is likely to be a highly transient one, implying that plasmid movement might be occurring at greater rates than previously estimated, raising a challenge for future genomic One Health studies.

Genetic and phenotypic analyses of mcr-harboring extended-spectrum β-lactamase-producing Escherichia coli isolates from companion dogs and cats in Japan

The emergence of mcr plasmid-mediated colistin-resistant extended-spectrum β-lactamase (ESBL)-producing Enterobacterales among companion dogs and cats poses a risk of the animals acting as reservoirs for cross-species transmission. However, current knowledge of mcr-harboring ESBL-producing Enterobacterales in companion dogs and cats is still limited; thus, the genetic and phenotypic characteristics of the bacterial isolates and plasmids, in companion dogs and cats, remain to be elucidated. Here, we identified mcr gene-harboring ESBL-producing Escherichia coli isolates during whole-genome sequencing of ESBL-producing E. coli isolates from a dog and a cat in Osaka, Japan. Colistin-resistant MY732 isolate from a dog carried two plasmids: mcr-1.1-harboring IncI2 plasmid and blaCTX-M-14-harboring IncFIB plasmid. Conjugation assays revealed that both plasmids can be co-transferred even though the IncFIB plasmid lacked a conjugal transfer gene cassette. The other isolate MY504 from a cat harbored two bla genes and mcr-9 on the identical IncHI2 plasmid. This isolate was not resistant to colistin, which is likely to be due to deletion of the regulatory two-component QseBC system associated with the mcr-9 expression. To the best of our knowledge, this is the first report of a colistin-resistant ESBL-producing E. coli isolate harboring mcr-1 from a companion dog in Japan. Given that the mcr gene-harboring IncI2 and IncHI2 plasmids in this study shared high homology with plasmids from human or animal-derived Enterobacterales, companion dogs and cats may act as important reservoirs for cross-species transmission of the mcr gene in the community, in Japan.

Antimicrobial Resistance Profiles and Molecular Characteristics of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Healthy Cattle and Pigs in Korea.

Antimicrobial-resistant bacteria isolated from food animals pose a major health threat to the public on this planet. This study aimed to determine the susceptibility profiles of Escherichia coli isolated from cattle and pig fecal samples and investigate the molecular characteristics of extended-spectrum β-lactamase (ESBL)-producing E. coli using gene identification, conjugation, and Southern blot approach. Overall 293 E. coli were recovered from cattle (120 isolates) and pigs (173 isolates) in 7 provinces of Korea during 2017-2018. Ampicillin, chloramphenicol, streptomycin, and sulfisoxazole resistance rates were the highest in pigs' isolates (>60%, p ≤ 0.001) compared to that in cattle (3-39%). Multidrug resistance (MDR) was higher in pig isolates (73%) than in cattle (31%), and the MDR profile usually includes streptomycin, sulfisoxazole, and tetracycline. Resistance to critically important antimicrobials such as ceftiofur, colistin, and ciprofloxacin was higher in weaners than those from finishers in pigs. The qnrS gene was detected in 13% of the pig isolates. Eight isolates from pigs and one isolate from cattle were identified as ESBL-producers and ESBL genes belonged to blaCTX-M-55 (n = 4), blaCTX-M-14 (n = 3), and blaCTX-M-65 (n = 2). Notably, the blaCTX-M-65 and qnrS1 genes were found to be carried together in an identical plasmid (IncHI2) in two isolates from finisher pigs. The blaCTX-M-carrying isolates belonged to phylogenetic groups B1 (n = 4), B2 (n = 2), A (n = 2), and D (n = 1). The blaCTX-M genes and non-β-lactam resistance traits were transferred to the E. coli J53 recipient from seven blaCTX-M-positive strains isolated from pigs. The blaCTX-M genes belonged to the IncI1α, IncFII, and IncHI2 plasmids and are also associated with the ISEcp1, IS26, IS903, and orf477 elements. These findings suggested the possibility of blaCTX-M-carrying E. coli transmission to humans through direct contact with cattle and pigs or contamination of food products.

Detection and Typing of Plasmids in Acinetobacter baumannii Using rep Genes Encoding Replication Initiation Proteins.

Plasmids found in Acinetobacter species contribute to the spread of antibiotic resistance genes. They appear to be largely confined to this genus and cannot be typed with available tools and databases. Here, a method for distinguishing and typing these plasmids was developed using a curated, non-redundant set of 621 complete sequences of plasmids from Acinetobacter baumannii. Plasmids were separated into 3 groups based on the Pfam domains of the encoded replication initiation (Rep) protein and a fourth group that lack an identifiable Rep protein. The rep genes of each Rep-encoding group (n = 13 Rep_1, n = 107 RepPriCT_1, n = 351 Rep_3) were then clustered using a threshold of >95% nucleotide identity to define 80 distinct types. Five Rep_1 subgroups, designated R1_T1 to R1-T5, were identified and a sixth reported recently was added. Each R1 type corresponded to a conserved small plasmid sequence. The RepPriCT_1 plasmids fell into 5 subgroups, designated RP-T1 to RP-T5 and the Rep_3 plasmids comprised 69 distinct types (R3-T1 to R3-T69). Three R1, 2 RP and 32 R3 types are represented by only a single plasmid. Over half of the plasmids belong to the 4 most abundant types: the RP-T1 plasmids (n = 97), which include conjugation genes and are often associated with various acquired antibiotic resistance genes, and R3-T1, R3-T2 and R3-T3 (n = 95, 30 and 45, respectively). To facilitate typing and the identification of plasmids in draft genomes using this framework, we established the Acinetobacter Typing database containing representative nucleotide and protein sequences of the type markers (https://github.com/MehradHamidian/AcinetobacterPlasmidTyping). IMPORTANCE Though they contribute to the dissemination of genes that confer resistance to clinically important carbapenem and aminoglycoside antibiotics used to treat life-threatening Acinetobacter baumannii infections, plasmids found in Acinetobacter species have not been well studied. As these plasmids do not resemble those found in other Gram-negative pathogens, available typing systems are unsuitable. The plasmid typing system developed for A. baumannii plasmids with an identifiable rep gene will facilitate the classification and tracking of sequenced plasmids. It will also enable the detection of plasmid-derived contigs present in draft genomes that are widely ignored currently. Hence, it will assist in the tracking of resistance genes and other genes that affect survival in the environment, as they spread through the population. As identical or similar plasmids have been found in other Acinetobacter species, the typing system will also be broadly applicable in identifying plasmids in other members of the genus.

Solution characterization of the dynamic conjugative entry exclusion protein TraG.

The R100 plasmid and the secretion system it encodes are representative of F-like conjugative type IV secretion systems for the transmission of mobile DNA elements in gram-negative bacteria, serving as a major contributor to the spread of antibiotic resistance in bacterial pathogens. The TraG protein of F-like systems consists of a membrane-bound N-terminal domain and a periplasmic C-terminal domain, denoted TraG*. TraG* is essential in preventing redundant DNA transfer through a process termed entry exclusion. In the donor cell, it interacts with TraN to facilitate mating pair stabilization; however, if a mating pore forms between bacteria with identical plasmids, TraG* interacts with its cognate TraS in the inner membrane of the recipient bacterium to prevent redundant donor-donor conjugation. Structural studies of TraG* from the R100 plasmid have revealed the presence of a dynamic region between the N- and C-terminal domains of TraG. Thermofluor, circular dichroism, collision-induced unfolding-mass spectrometry, and size exclusion chromatography linked to multiangle light scattering and small angle x-ray scattering experiments indicated an N-terminal truncation mutant displayed higher stability and less disordered content relative to full-length TraG*. The 45 N-terminal residues of TraG* are hypothesized to serve as part of a flexible linker between the two independently functioning domains.

Novel insights related to the rise of KPC-producing Enterobacter cloacae complex strains within the nosocomial niche.

According to the World Health Organization, carbapenem-resistant Enterobacteriaceae (CRE) belong to the highest priority group for the development of new antibiotics. Argentina-WHONET data showed that Gram-negative resistance frequencies to imipenem have been increasing since 2010 mostly in two CRE bacteria: Klebsiella pneumoniae and Enterobacter cloacae Complex (ECC). This scenario is mirrored in our hospital. It is known that K. pneumoniae and the ECC coexist in the human body, but little is known about the outcome of these species producing KPC, and colonizing or infecting a patient. We aimed to contribute to the understanding of the rise of the ECC in Argentina, taking as a biological model both a patient colonized with two KPC-producing strains (one Enterobacter hormaechei and one K. pneumoniae) and in vitro competition assays with prevalent KPC-producing ECC (KPC-ECC) versus KPC-producing K. pneumoniae (KPC-Kp) high-risk clones from our institution. A KPC-producing E. hormaechei and later a KPC-Kp strain that colonized a patient shared an identical novel conjugative IncM1 plasmid harboring bla KPC-2. In addition, a total of 19 KPC-ECC and 58 KPC-Kp strains isolated from nosocomial infections revealed that high-risk clones KPC-ECC ST66 and ST78 as well as KPC-Kp ST11 and ST258 were prevalent and selected for competition assays. The competition assays with KCP-ECC ST45, ST66, and ST78 versus KPC-Kp ST11, ST18, and ST258 strains analyzed here showed no statistically significant difference. These assays evidenced that high-risk clones of KPC-ECC and KPC-Kp can coexist in the same hospital environment including the same patient, which explains from an ecological point of view that both species can exchange and share plasmids. These findings offer hints to explain the worldwide rise of KPC-ECC strains based on the ability of some pandemic clones to compete and occupy a certain niche. Taken together, the presence of the same new plasmid and the fitness results that showed that both strains can coexist within the same patient suggest that horizontal genetic transfer of bla KPC-2 within the patient cannot be ruled out. These findings highlight the constant interaction that these two species can keep in the hospital environment, which, in turn, can be related to the spread of KPC.

Detection of an IMI-2 carbapenemase-producing Enterobacter asburiae at a Swedish feed mill.

Occurrence of multidrug resistant Enterobacteriaceae in livestock is of concern as they can spread to humans. A potential introduction route for these bacteria to livestock could be animal feed. We therefore wanted to identify if Escherichia spp., Enterobacter spp., Klebsiella spp., or Raoutella spp. with transferable resistance to extended spectrum cephalosporins, carbapenems or colistin could be detected in the environment at feed mills in Sweden. A second aim was to compare detected isolates to previous described isolates from humans and animals in Sweden to establish relatedness which could indicate a potential transmission between sectors and feed mills as a source for antibiotic resistant bacteria. However, no isolates with transferable resistance to extended-cephalosporins or colistin could be identified, but one isolate belonging to the Enterobacter cloacae complex was shown to be carbapenem-resistant and showing carbapenemase-activity. Based on sequencing by both short-read Illumina and long-read Oxford Nanopore MinIon technologies it was shown that this isolate was an E. asburiae carrying a bla IMI-2 gene on a 216 Kbp plasmid, designated pSB89A/IMI-2, and contained the plasmid replicons IncFII, IncFIB, and a third replicon showing highest similarity to the IncFII(Yp). In addition, the plasmid contained genes for various functions such as plasmid segregation and stability, plasmid transfer and arsenical transport, but no additional antibiotic resistance genes. This isolate and the pSB89A/IMI-2 was compared to three human clinical isolates positive for bla IMI-2 available from the Swedish antibiotic monitoring program Swedres. It was shown that one of the human isolates carried a plasmid similar with regards to gene content to the pSB89A/IMI-2 except for the plasmid transfer system, but that the order of genes was different. The pSB89A/IMI-2 did however share the same transfer system as the bla IMI-2 carrying plasmids from the other two human isolates. The pSB89A/IMI-2 was also compared to previously published plasmids carrying bla IMI-2, but no identical plasmids could be identified. However, most shared part of the plasmid transfer system and DNA replication genes, and the bla IMI-2 gene was located next the transcription regulator imiR. The IS3-family insertion element downstream of imiR in the pSB89A was also related to the IS elements in other bla IMI-carrying plasmids.

Fecal carriage and clonal dissemination of blaNDM-1 carrying Klebsiella pneumoniae sequence type 147 at an intensive care unit in Lao PDR

ObjectivesCarbapenemase-producing Enterobacterales (CPE) are high priority targets of global antimicrobial surveillance. Herein, we determined the colonization rate of CPE on admission to intensive care units in Vientiane, Lao PDR in August-September 2019.MethodsData regarding clinical conditions, infection control, and antibiotic usage were collected during admission. Rectal swab samples (n = 137) collected during admission were inoculated to selective chromogenic agars, followed by confirmatory tests for extended-spectrum beta-lactamases and carbapenemases. All CPE isolates were sequenced on Illumina (HiSeq2500), reads assembled using SPAdes 3.13, and the draft genomes used to query a database (https://www.genomicepidemiology.org) for resistome, plasmid replicons, and sequence types (ST). Optical DNA mapping (ODM) was used to characterize plasmids and to determine location of resistance genes. Minimum spanning tree was generated using the Bacterial Isolate Genome Sequence database (BIGSdb) and annotated using iTOL.ResultFrom 47 Enterobacterales isolated on selective agars, K. pneumoniae (25/47) and E. coli (12/47) were the most prevalent species, followed by K aerogenes (2/47), K. variicola (1/47), and K. oxytoca (1/47). The overall prevalence of ESBLs was 51.0%; E. coli 83.3% (10/12) and Klebsiella spp. 41.3% (12/29). Twenty percent of the K. pneumoniae (5/25) isolates were carbapenem-resistant, and 4/5 contained the blaNDM-1 gene. All blaNDM-1 isolates belonged to ST147 and were indistinguishable with cgMLST. ODM showed that the blaNDM-1 gene was located on identical plasmids in all isolates.ConclusionThe prevalence of ESBL-producing Enterobacterales was high, while carbapenemases were less common. However, the detection of clonal dissemination of blaNDM-1-producing K. pneumoniae isolates in one of the intensive care units calls for vigilance. Stringent infection prevention and antimicrobial stewardship strategies are highly important measures.

Tigecycline-resistant Escherichia coli ST761 carrying tet(X4) in a pig farm, China.

This study aimed to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from a pig farm in Shanghai, China, and to elucidate tet(X4) dissemination mechanism in this swine farm. Forty-nine (80.33%) E. coli strains were isolated from 61 samples from a pig farm and were screened for the presence of tet(X). Among them, six (12.24%) strains were positive for tet(X4) and exhibited resistance to tigecycline (MIC ≥ 16 mg/L). They were further sequenced by Illumina Hiseq. Six tet(X4)-positive strains belonged to ST761 with identical resistance genes, resistance profiles, plasmid replicons, and cgMLST type except that additional ColE10 plasmid was present in isolate SH21PTE35. Isolate SH21PTE31, as a representative ST761 E. coli strain, was further sequenced using Nanopore MinION. The tet(X4) in SH21PTE31 was located on IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP31-1, highly similar to other tet(X4)-carrying IncFIA18/IncFIB(K)/IncX1 plasmids from ST761 E. coli and other E. coli lineages in China. These IncFIA18/IncFIB(K)/IncX1 plasmids shared closely related multidrug resistance regions, and could reorganize, acquire or lose resistance modules mediated by mobile elements such as ISCR2 and IS26. Phylogenetic analysis were performed including all tet(X4)-positive isolates obtained in this pig farm combined with 43 tet(X4)-positive E. coli from pigs, cow, pork, wastewater, and patients with the same ST from NCBI. The 50 tet(X4)-carrying E. coli ST761 isolates from different areas in China shared a close phylogenetic relationship (0-49 SNPs). In conclusion, clonal transmission of tet(X4)-positive E. coli ST761 has occurred in this swine farm. E. coli ST761 has the potential to become a high-risk clone for tet(X4) dissemination in China.

Plasmid-Mediated Quinolone Resistance (PMQR) in Two Clinical Strains of Salmonella enterica Serovar Corvallis.

Non-typhoid serovars of Salmonella enterica are one of the main causes of bacterial food-borne infections worldwide. For the treatment of severe cases of salmonellosis in adults, fluoroquinolones are amongst the drugs of choice. They are categorized by the World Health Organization (WHO) as “critically important with highest priority in human medicine”. In the present study, two clinical S. enterica serovar Corvallis isolates (HUA 5/18 and HUA 6/18) from a Spanish hospital, selected on the basis of fluoroquinolone resistance, were characterized. The MICs of ciprofloxacin, determined by E-test, were 0.5 and 0.75 µg/mL for HUA 5/18 and HUA 6/18, respectively, and both were also resistant to pefloxacin but susceptible to nalidixic acid. Whole genome sequencing (WGS) of the isolates was performed with Illumina platform, and different bioinformatics tools were used for sequence analysis. The two isolates belonged to ST1541, and had the Thr57Ser substitution in the ParC protein which is also found in ciprofloxacin susceptible isolates. However, they harbored identical ColE plasmids of 10 kb carrying the qnrS1 gene. In these plasmids, the gene was flanked by defective versions of IS2-like and ISKra4-like insertion sequences. HUA 5/18 and HUA 6/18 were also phenotypically resistant to streptomycin, sulfonamides and tetracycline, with the responsible genes: strA, strB, sul2 and tet(A) genes, being located on a IncQ1 plasmid. ColE plasmids with the qnrS1 gene are widely spread among multiple serovars of S. enterica from different samples and countries. These mobilizable plasmids are playing an important role in the worldwide spread of qnrS1. Thus, their detection in hospitals is a cause of concern which deserves further attention.