Conjugation Assays Research Articles

Dissemination of bla NDM-5 Driven by Horizontal Transfer of IncFIA Plasmid Between Escherichia coli and Klebsiella pneumoniae Co-Isolated from a Patient's Ascitic Fluid.

Understanding the horizontal transfer of resistance genes, such as bla NDM-5, is pivotal in developing strategies to control the spread of resistance. In this study, we isolated two bacterial strains, Escherichia coli (designated GYB01) and Klebsiella pneumoniae (designated GYB02), from a single patient. The aim of our research is to explore the biological characteristics of these strains and to investigate the interspecies horizontal transfer of bla NDM-5. Strain identification and antimicrobial susceptibility testing were conducted using the Vitek 2 system. Both GYB01 and GYB02 were sequenced with the Illumina HiSeq platform. Bioinformatics analysis tools, including multilocus sequence typing, PlasmidFinder, ResFinder, and others, were utilized to analyze the strains. Additionally, conjugation assays and Galleria mellonella infection assays were employed to assess the strains. The isolates exhibited similar antimicrobial resistance profiles and both harbored the bla NDM-5 gene within the IncFIA plasmids (pGYB01-2, 165.8 kb and pGYB02-2, 211.6 kb, respectively). These plasmids (pGYB01-2 and pGYB02-2) shared over 99% homology, suggesting a common ancestral origin. Conjugation experiments confirmed the transferability of the bla NDM-5 carrying IncFIA plasmids among Enterobacteriaceae. GYB02 possessed an iucACD-iutA gene cluster, exhibited high virulence, and tested positive in the string test. Our findings provide direct evidence of potential in vivo interspecies transfer of a multidrug-resistant plasmid, thus enriching our understanding of the mechanisms driving multidrug resistance (MDR) and aiding in the formulation of containment and treatment strategies.

Characterization of an NDM-1-Producing Citrobacter koseri Isolate from China.

The continuous rise in carbapenemase-producing Enterobacteriaceae infections is a major public health concern. However, there is limited information available on New Delhi metallo-β-lactamase-1 (NDM-1) producing Citrobacter koseri. In this study, we isolated a blaNDM-1-carrying C. koseri from a stool sample of an inpatient. Our aim was to investigate the phenotypic and genomic features of this clinically derived carbapenem-resistant C. koseri isolate and to characterize the transmission pattern of the IncFII/IncN plasmid that carries the blaNDM-1 gene. S1-PFGE, Southern blot and conjugation assay confirmed the presence of blaNDM-1 gene in a conjugative plasmid. C. koseri L2395 and transconjugant L2395-EC600 strains showed similar resistance spectrum. Whole-genome analysis revealed that pL2395_NDM is an IncFII/IncN plasmid with a length of 67,839 bp. Moreover, blaNDM-1 gene was found encoded in the ISKpn19-blaNDM-1-ble-tnpF-dsbD-cutA-ISKpn19 cassette array. Phylogenetic analysis revealed that strain L2395 was close to an IMP-4-bearing C. koseri from Australia. Ongoing surveillance will be essential to control and prevent the spread of carbapenem-resistant Citrobacter spp. in the future.

High qnrS retention of ESBL-producing and mcr-harbouring colistin-resistant Escherichia coli in Vietnamese food products.

Plasmid-mediated antibiotic-resistant bacteria's transmission is fatal and a major threat to public health. This study aimed to clarify the presence of plasmid-mediated quinolone resistance（PMQR）genes in extended-spectrum β-lactamase（ESBL）-producing or/and mcr-harbouring colistin（COL）-resistant Escherichia coli（ESBL-COL-EC）isolates from Vietnamese and Japanese chicken meat. Resistance towards ciprofloxacin（CIP）was examined in 308 ESBL-COL-EC isolates; CIP-resistant ESBL-COL-EC isolates were examined for the PMQR gene. Approximately, 71.1% and 38.1% of ESBL-COL-EC and ESBLproducing E. coli isolates from Vietnamese and Japanese chicken meat were CIP-resistant, respectively. Multiplex PCR led PMQR detection showed that 35.2% of CIP-resistant ESBL-COL-EC isolates from Vietnamese food contained PMQR gene, whereas CIP-resistant ESBL-COL-EC isolates from Japanese chicken meat did not. Conjugation assays showed that the transmission of qnrS gene carried by E. coli to Salmonella. In conclusion, ESBL-COL-EC isolates from Vietnamese food are associated with a high frequency of fluoroquinolone resistance and a high distribution of the qnrS gene.

Characterization of an ST38 carbapenem-resistant and highly virulent Escherichia coli carrying conjugatively transferable ColV virulence-resistance and blaNDM-5-positive resistance plasmids.

To characterize an Escherichia coli strain causing bloodstream infection encoding both high-virulence and carbapenem-resistance phenotypes. Antimicrobial susceptibility testing, WGS and bioinformatics analysis were performed to characterize strain E1. The function of the ColV plasmid was investigated by the Galleria mellonella infection model, serum killing and macrophage killing assays. The fitness effect of the ColV plasmid was tested by growth curve, plasmid stability tests and the in vitro competition assay. The conjugation assay was performed to test the transferability of the ColV and blaNDM-5-carrying plasmids. E. coli E1 from bloodstream infection was MDR and highly virulent in the G. mellonella infection model. It belonged to phylogroup D, ST38 and serotype O7:H8. E1 carried a conjugatively transferable IncI1-type blaNDM-5-positive plasmid, which conferred carbapenem resistance, a conjugative IncFIB/FII-type ColV plasmid encoding an array of virulence-associated genes and antibiotic resistance genes blaTEM-1B, strAB and sul2, and seven other plasmids. Co-transfer of the ColV plasmid and the blaNDM-5-positive plasmid was observed. The ColV virulence-resistance hybrid plasmid contributed to the virulence, resistance to serum killing, and macrophage phagocytosis in E. coli E1. The carriage of this ColV plasmid did not constitute an in vitro fitness burden to strain E1 but caused fitness costs to E. coli strain EC600. The emergence of such a highly virulent and resistant strain with conjugative blaNDM-5-positive and ColV plasmids posed a significant threat to public health. Implementation of control measures is needed to prevent such strains from further disseminating in hospital settings and the community.

Extensively drug-resistant Enterobacter ludwigii co-harbouring MCR-9 and a multicopy of blaIMP-1 in South Korea

In this study, we describe an Enterobacter ludwigii clinical isolate that is resistant to both carbapenems and colistin in South Korea. Antimicrobial susceptibility testing revealed that E. ludwigii CRE2104-31 was non-susceptible to all tested antibiotics except fosfomycin. Whole genome sequencing identified a 323-kbp IncHI2 plasmid, pCRE2104-31a, that was co-harbouring mobile colistin resistance (mcr)-9.1 and blaIMP-1. In comparison with other full plasmids, pCRE2104-31a exhibited the closest similarity to a plasmid from the Klebsiella pneumoniae strain CNR48 from France, with 19.9% query coverage and 99% identity. Notably, we observed five tandem repeats of blaIMP-1 and aac(6′)-Il genes, accompanied by multiple attCs within a class I integron on the Tn402-like transposon. The unit of blaIMP-1-attC-aac(6′)-Il-attC might have accumulated due to multiple convergent events. In addition to mcr-9.1 and blaIMP-1, various other antibiotic resistance-associated genes were identified in the plasmid, as follows: blaTEM-1B, aph(3′)-I, aph(3′)-Ia, aac(6′)-Il, aac(6′)-IIc, aac(6′)-IIa, aph(6)-Id, aph(3′')-Ib, aadA2b, aac(6′)-Ib3, sul, dfrA19, qnrB2, aac(6′)-Ib-cr, ere(A), and qacE. A conjugation assay showed that the mcr-9.1/blaIMP-1-co-bearing plasmid was self-transmissible to E. coli J53. However, colistin and carbapenem resistance could not be transferred to E. coli due to high incompatibility. The convergence of mcr and carbapenemase genes is thought to be host-dependent among Enterobacteriaceae. The emergence of extensively drug-resistant E. ludwigii co-harbouring MCR-9.1 and a multicopy of blaIMP-1 would pose a significant threat within the compatible Enterobacteriaceae.

Circular intermediate-mediated horizontal transfer of the chromosome-encoded cfr(C) gene in multi-drug resistant Campylobacter coli from swine sources.

Campylobacter is a major zoonotic pathogen that causes gastrointestinal and, rarely, immune diseases in humans. The antimicrobial-resistance gene cfr(C) carried by Campylobacter and is a cfr-like gene that targets bacterial 23S rRNA through A2503 methylation. cfr(C) confers cross-resistance to five antimicrobial classes (PhLOPSA), including lincosamide, streptogramin A, and pleuromutilin, which are classified as critically important antimicrobials to human by the World Health Organization. To elucidate the genetic variation and horizontal transfer mechanism of cfr(C), we analyzed the genetic background and horizontal transfer unit of Campylobacter-derived cfr(C) through comparative genomic analysis. We identified nine cfr(C)-positive C. coli strains of 157 strains isolated from swine sources. Three novel cfr(C) gene single nucleotide polymorphism (SNP) sites (19delA, 674C > A, and 890 T > C) were identified from nine cfr(C)-positive strains. Among six identified cfr(C) SNP variant types (SNP-I to -VI), five types of randomly inserted cfr(C)-cassettes on chromosome and one type of plasmid-like element were identified, their gene cassette composition differing depending on the cfr(C) variants. Three of six cfr(C) cassette types contained aminoglycoside-streptothricin resistance cluster "aphA3-sat4-aadE." The cfr(C) gene cassette with pcp gene (GC-1, GC-4, and GC-5) formed a pcp-mediated circular intermediate "pcp-hp-cfr(C)-aphA3," which has not been previously reported. Other two cfr(C) cassette-types with ISChh1 formed circular intermediate "ISChh1-aphA3-cfr(C)-lnu (G)-pnp-ant1-hp-ATPase" and "ISChh1-aphA3-cfr(C)-hp." In conjugation assay, the pcp-mediated circular intermediate was naturally transferred to the plasmid of recipient C. coli wild-type strain from swine source, and comparative genomic analysis revealed that cfr(C) encoded in pcp-mediated circular intermediate was inserted into the plasmid of recipient by homologous recombination with pcp and aphA3. This study revealed that novel multidrug resistance gene cfr(C) carried by C. coli from swine sources can be highly genetically diverse and transferable. Moreover, we suggest that the transferability of chromosomal cfr(C) may contribute to the global spread of multidrug resistance against clinically important antimicrobials.

Tn3-like structures co-harboring of blaCTX-M-65, blaTEM-1 and blaOXA-10 in the plasmids of two Escherichia coli ST1508 strains originating from dairy cattle in China

The purpose of this study was to determine the level of horizontal transmission of the blaCTX-M-65 gene and the role of its associated mobile genetic elements (MGEs) in the bovine-derived Escherichia coli. After PCR identification, two plasmids carrying blaCTX-M-65 were successfully transferred to the recipient E. coli J53 Azr through conjugation assays and subsequently selected for Whole-Genome sequencing (WGS) analysis. The resistance profiles of these two positive strains and their transconjugants were also determined through antimicrobial susceptibility tests. Whole genome data were acquired using both the PacBio sequencing platform and the Illumina data platform. The annotated results were then submitted to the Genbank database for accession number recording. For comparison, the genetic environment of plasmids carrying the resistance gene blaCTX-M-65 was mapped using the Easyfig software. WGS analysis revealed Tn3-like composite transposons bearing blaCTX-M-65, blaTEM-1, and blaOXA-10 in the IncHI2-type plasmids of these two E. coli ST1508 strains. A phylogenetic tree was generated from all 48 assembled E. coli isolates blaCTX-M-65, blaTEM-1, and blaOXA-10 from the NCBI Pathogen Detection database with our two isolates, showing the relationships and the contribution of SNPs to the diversity between genetic samples. This study suggests that the transmissibility of blaCTX-M-65 on Tn3-like composite transposons contributes to an increased risk of its transmission in E. coli derived from dairy cattle.

Molecular characteristic of mcr-1 gene in Escherichia coli from aquatic products in Guangdong, China

Aquatic ecosystems serve as a dissemination pathway and a reservoir of both antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to determine the prevalence of colistin-resistant mcr-like genes in Enterobacteriales in aquatic products, which may be contribute to the transfer of ARGs in water environments. The mcr-1-positive Escherichia coli were recovered from 123 freshwater fish and 34 cultured crocodile cecum samples from 10 farmers' markets in Guangdong, China. Minimum inhibitory concentration (MIC) was determined using the agar dilution method. Genotyping was performed using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Conjugation assay was carried out to investigate the transferability of mcr-1. Genomic information was obtained by whole genome sequencing (WGS) and bioinformatic analysis. Forty-four mcr-1 positive isolates showed co-resistance to tetracycline, trimethoprim/sulfamethoxazole, and gentamicin, while they were all sensitive to tigecycline, meropenem, and amikacin. They were typed into sixteen PFGE clusters. ST10 and ST117 were the most popular sequence types, followed by ST1114. S1-PFGE verified the presence of the mcr-1 gene on plasmids in sizes of ∼60 kb (n = 1) and ∼240 kb (n = 3). Whole genome sequencing-based analysis identified mcr-1 integrated in IncHI2 plasmid (n = 3), IncI2 plasmid (n = 2), and bacterial chromosome in two copies (n = 1). In addition to mcr-1, they carried several other antibiotic resistance genes, such as blaCTX-M-14, fosA3, and aac(6')-Ib-cr. These data suggest that aquatic products are an important antibiotic resistance reservoir and highlight possible risks regarding food safety.

Outer membrane vesicles mediating horizontal transfer of the epidemic bla OXA-232 carbapenemase gene among Enterobacterales

ABSTRACT OXA-232 is one of the most common OXA-48-like carbapenemase derivatives and is widely disseminated in nosocomial settings across countries. The bla OXA-232 gene is located on a 6-kb non-conjugative ColKP3-type plasmid, while the dissemination of bla OXA-232 into different Enterobacterales species and the polyclonal dissemination of OXA-232-producing K. pneumoniae revealed the horizontal transfer of bla OXA-232. However, it’s still unclear how this non-conjugative ColKP3 plasmid could facilitate the mobilization of bla OXA-232. Here, we observed the in vivo intraspecies transfer of bla OXA-232 during a nosocomial outbreak of OXA-232-producing K. pneumoniae. We demonstrated the presence of ColKP3 OXA-232 plasmid in the outer membrane vesicles (OMVs) derived from clinical isolates, and OMVs could facilitate the horizontal transfer of bla OXA-232 among Enterobacterales. In contrast, for the most prevalent carbapenemase genes, including bla KPC-2 and bla NDM-1, though the presence of carbapenemase genes and plasmid backbones in the vesicular lumen was observed, OMVs couldn’t promote effective transformation, probably due to the low copy number of plasmids in clinical isolates and the low number of plasmids loaded into vesicles. Conjugation assay revealed that the epidemic IncX3 NDM-1 and IncFII(pHN7A8)/IncR KPC-2 plasmids were conjugative and could be horizontally transferred via independent conjugation or with the help of a co-existent conjugative plasmid. For the large-size and low-copy number conjugative plasmids carrying carbapenemase genes, OMVs-mediated gene exchange may only serve as an alternative pathway for horizontal transfer. In conclusion, diverse mobilization strategies were employed by plasmids harbouring carbapenemase genes, and plasmids display a proper choice of mobility pathway due to their individual properties.

Gelatin In Situ Zymography to Study Gelatinase Activity in Colon Cancer Cells Treated with Platelet Microparticles (PMPs).

The platelet-derived microparticles (PMPs) have been connected with tumor progression and metastatic dissemination. PMPs infiltrate solid tumors and transfer platelet-derived cargo to cancer cells. The functional roles of PMPs in cancer progression are still poorly understood. PMPs, incorporated by colorectal cancer (CRC) cells, were shown to upregulate the expression and activity of matrix metalloproteases (MMPs). To investigate the impact of PMPs on the invasive potential of CRC, we established the protocol of dequenched gelatin(DG), fluorescein conjugate assay. The procedure confirms the activity of two gelatinases, namely, MMP2 and MMP9, that digest denatured collagen (gelatin). This "step-by-step" protocol, with notes and comments implemented to human CRC lines with different phenotypes and migratory potentials, should be sufficient to obtain representative and elegant results.

Mobilizable plasmids drive the spread of antimicrobial resistance genes and virulence genes in Klebsiella pneumoniae

BackgroundKlebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence genes. In comparison to self-transmissible conjugative plasmids, mobilizable plasmids have received much less attention due to their defects in conjugative elements. However, the contribution of mobilizable plasmids to the horizontal transfer of antimicrobial resistance genes and virulence genes of K. pneumoniae remains unclear. In this study, the transfer, stability, and cargo genes of the mobilizable plasmids of K. pneumoniae were examined via genetic experiments and genomic analysis.MethodsCarbapenem-resistant (CR) plasmid pHSKP2 and multidrug-resistant (MDR) plasmid pHSKP3 of K. pneumoniae HS11286, virulence plasmid pRJF293 of K. pneumoniae RJF293 were employed in conjugation assays to assess the transfer ability of mobilizable plasmids. Mimic mobilizable plasmids and genetically modified plasmids were constructed to confirm the cotransfer models. The plasmid morphology was evaluated through XbaI and S1 nuclease pulsed-field gel electrophoresis and/or complete genome sequencing. Mobilizable plasmid stability in transconjugants was analyzed via serial passage culture. In addition, in silico genome analysis of 3923 plasmids of 1194 completely sequenced K. pneumoniae was performed to investigate the distribution of the conjugative elements, the cargo genes, and the targets of the CRISPR-Cas system. The mobilizable MDR plasmid and virulence plasmid of K. pneumoniae were investigated, which carry oriT but lack other conjugative elements.ResultsOur results showed that mobilizable MDR and virulence plasmids carrying oriT but lacking the relaxase gene were able to cotransfer with a helper conjugative CR plasmid across various Klebsiella and Escherichia coli strains. The transfer and stability of mobilizable plasmids rather than conjugative plasmids were not interfered with by the CRISPR–Cas system of recipient strains. According to the in silico analysis, the mobilizable plasmids carry about twenty percent of acquired antimicrobial resistance genes and more than seventy-five percent of virulence genes in K. pneumoniae.ConclusionsOur work observed that a mobilizable MDR or virulence plasmid that carries oriT but lacks the relaxase genes transferred with the helper CR conjugative plasmid and mobilizable plasmids escaped from CRISPR–Cas defence and remained stable in recipients. These results highlight the threats of mobilizable plasmids as vital vehicles in the dissemination of antibiotic resistance and virulence genes in K. pneumoniae.

Occurrence and characterization of NDM-5-producing Escherichia coli from retail eggs

The New Delhi Metallo-β-lactamase (NDM) producing Enterobacterales has been detected from diverse sources but has rarely been reported in retail eggs. In this study, 144 eggshell and 96 egg content samples were collected in 2022 from Guangdong province and were screened for NDM-producing strains. Four Escherichia coli strains (ST3014, ST10, ST1485, and ST14747) recovered from two (1.39%, 2 of 144) eggshells and two (2.08%, 2 of 96) egg content samples were identified as blaNDM−5-positive strains. Oxford Nanopore MinION sequencing and conjugation assays revealed that the blaNDM−5 gene was carried by IncX3 (n = 1), IncI1 (n = 1), and IncHI2 (n = 2). The IncI1-plasmid-carrying blaNDM−5 displayed high homology with one plasmid pEC6563-NDM5 from the human clinic, while the IncHI2 plasmid harboring blaNDM−5 shared highly similar structures with plasmids of animal origin. To the best of our knowledge, this is the first report on the identification of blaNDM−5-positive bacteria in retail eggs. NDM-producing E. coli could be transmitted to humans by the consumption of eggs or direct contact, which could pose a potential threat to human health.

Interphylum dissemination of NDM-5-positive plasmids in hospital wastewater from Fuzhou, China: a single-centre, culture-independent, plasmid transmission study

The global spread of plasmid-borne carbapenem resistance is an ongoing public health challenge; however, the nature of such horizontal gene transfer events among complex bacterial communities remains poorly understood. We examined the in-situ transfer of the globally dominant New Delhi metallo-β-lactamase (NDM)-5-positive IncX3 plasmid (denoted pX3_NDM-5) in hospital wastewater to simulate a real-world, One Health antimicrobial resistance context. For this transmission study, we tagged pX3_NDM-5 with the green fluorescent protein gene, gfp, using a CRISPR-based method and transferred the plasmid to a donor Escherichia coli strain. Bacteria were extracted from a hospital wastewater treatment plant (Fujian Provincial Maternity and Children's Hospital, Fuzhou, China) as the bacterial recipient community. We mixed this recipient community with the E coli donor strain carrying the gfp-tagged plasmid, both with and without sodium hypochlorite (NaClO) as an environmental stressor, and conducted several culture-based and culture-independent conjugation assays. The conjugation events were observed microscopically and quantified by fluorescence-activated cell sorting. We analysed the taxonomic composition of the sorted transconjugal pool by 16S rRNA gene amplicon sequencing and assessed the stability of the plasmid in the isolated transconjugants and its ability to transfer back to E coli. We show that the plasmid pX3_NDM-5 has a broad host range and can transfer across various bacterial phyla, including between Gram-negative and Gram-positive bacteria. Although environmental stress with NaClO did not affect the overall plasmid transfer frequency, it reduced the breadth of the transconjugant pool. The taxonomic composition of the transconjugal pool was distinct from that of the recipient communities, and environmental stress modulated the permissiveness of some operational taxonomic units towards the acquisition of pX3_NDM-5. Notably, pX3_NDM-5 transconjugants included the Gram-positive pathogen Enterococcus faecalis, and the plasmid could subsequently be reconjugated back to E coli. These findings suggest that E faecalis could act as a natural shuttle vector for the wide dissemination of pX3_NDM-5 plasmids. Our culture-independent conjugation model simulates natural environmental conditions and challenges the established theory that Gram-negative and Gram-positive bacteria rarely exchange clinically important plasmids. The data show that plasmids disseminate more widely across genera and phyla than previously thought. These findings have substantial implications when considering the spread of antimicrobial resistance across One Health sectors. The Laboratory of Lingnan Modern Agriculture Project, the National Natural Science Foundation of China, the Natural Science Foundation of Fujian Province of China, and the Outstanding Young Research Talents Program of Fujian Agriculture and Forestry University.

A critical role of outer membrane vesicles in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae

BackgroundThis study aimed to illustrate the status of carbapenem-resistant Enterobacterales (CRE) infections in a Chinese tertiary hospital and to investigate the role of outer membrane vesicles (OMVs) in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP).MethodsThe data of CRE infections was collected from laboratory records, and the CRE isolates from two distinct periods (2015/07 to 2017/07 and 2020/04 to 2021/04) were enrolled to detect the carbapenemase genes by polymerase chain reaction (PCR). Multilocus sequence typing (MLST) was used to analyze the molecular characterization of CRKP. The conjugation assay was performed to verify the transmission of the antibiotic resistance plasmid. The OMVs of CRKP were isolated with a method combining an electrophoretic technique with a 300 kDa cut-off dialysis bag. The protein components in CRKP OMVs were analyzed by liquid chromatography tandem-mass spectrometry (LC–MS/MS), and the meropenem-hydrolyzing bioactivity of KPC in CRKP OMVs was determined with different treatments in vitro.ResultsA total of 178 CRE isolates, including 100 isolates from 2015/07 to 2017/07 and 78 isolates from 2020/04 to 2021/04, were collected for the detection of carbapenemase genes. We found that the carbapenemase gene blaKPC was the most prevalent, followed by blaNDM. By MLST, we found that sequence type (ST) 11 CRKP (96.1%) was the leading type during 2015/07 to 2017/07 and that the ST15 CRKP increased to 46.2% in the late period of 2020/04 to 2021/04. The diameters of Klebsiella pneumoniae OMVs ranged from 100 to 200 nm, and by proteomics analysis the most proteins from OMVs belonged to the “enzyme” group. The KPC enzyme was found in the OMVs from CRKP, and the OMVs could protect inside KPC from proteinase K digestion. Moreover, the KPC enzymes within OMVs, which could be released after Triton X-100 treatment, could hydrolyze meropenem.ConclusionsCRE has increasingly caused infections in hospitals, and blaKPC-positive CRKP infections have constituted a major proportion of infections in the past decade. The OMVs play a critical role in antibiotic resistance in CRKP.

Molecular characterization of IncFII plasmid carrying blaNDM-5 in a Salmonella enterica serovar Typhimurium ST34 clinical isolate in China.

In this study, an IncFII plasmid pIncFII-NDM5 carrying blaNDM-5 was found in carbapenem-resistant Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium), which has conjugative transferability and carried blaNDM-5, bleMBL, mph(A), and blaTEM-1 four resistance genes that can mediate resistance to multiple antibiotics including cephalosporins, beta-lactamase inhibitor combinations, carbapenems, and macrolides. Phylogenetic analysis showed that 1104-65 and 1104-75 were closely related to other S. enterica serovar Typhimurium in this area. The above-mentioned S. enterica serovar Typhimurium chromosome carries blaCTX-M-55, qnrS1, and tet(A) genes, so the antibiotic resistance of isolates will be further enhanced after obtaining the pIncFII_NDM5-like plasmid. Meanwhile, we discovered a novel genetic structure of blaNDM-5 mediated by the IS26 composite transposon, which will expand our understanding of the emergence and spread of carbapenem-resistance genes. Altogether, the presence of the IncFII plasmid pIncFII-NDM5 further underscores the need for vigilant surveillance and appropriate infection control measures to mitigate the impact of carbapenem-resistant S. enterica serovar Typhimurium in clinical settings.

Nosocomial dissemination of blaIMP-4 among Klebsiella pneumoniae by horizontal gene transfer and clonal spread: the epidemic IncN plasmids and the emerging high-risk IMP-4-producing ST101 clone.

To determine the genomic features of IMP-4-producing Klebsiella pneumoniae isolates recovered from paediatric patients and the transmission dynamics of blaIMP-4. IMP-producing K. pneumoniae isolates were collected from paediatric patients in Shanghai Children's Medical Center from 2013 to 2020. WGS was performed for all isolates, and the complete genomes of three IMP-4-producing isolates were generated. The distribution of blaIMP-4-harbouring plasmids was determined, and a conjugation assay was employed to investigate the horizontal transfer of blaIMP-4-harbouring plasmids. We collected 21 blaIMP-carrying K. pneumoniae isolates, with IMP-4 (16/21, 76.2%) as the predominant subtype, followed by IMP-8 (n = 3) and IMP-26 (n = 2). IMP-4-producing isolates displayed a diverse population structure and all blaIMP-4 genes were located on plasmids, including IncN (n = 9), IncHI5 (n = 5), IncFII(K) (n = 1) and IncFII(pKP91) (n = 1), although only IncN plasmids were conjugative. Clonal transmission of ST101 strains carrying IncHI5 blaIMP-4-harbouring plasmids was observed, and the acquisition of blaIMP-4 by the international high-risk ST101 clone constituted a novel combination of ST101 clone and carbapenemase genes. Plasmid analysis demonstrated that the conjugal transfer of the IncHI5 blaIMP-4-harbouring plasmid might be blocked by the ST101 bacterial host. The horizontal transfer of IncN plasmids and clonal spread of the international high-risk ST101 clone facilitated the nosocomial dissemination of blaIMP-4 among K. pneumoniae. The emerging IMP-4-producing ST101 clone displays diverse combinations of carbapenemase genes, and this clone could be a continually evolving threat and warrants prospective monitoring.

Synthetic augmentation of bilirubin metabolism in human pluripotent stem cell-derived liver organoids

UGT1A1 (UDP glucuronosyltransferase family 1 member A1)is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic aniontransporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype. Bilirubin conjugation assays, chromatin immunoprecipitation, and transcriptome analysis elucidated the role of glucocorticoid antagonism in UGT1A1 activation. This antagonism prevents the binding of transcriptional repressor MECP2 at the expense of NRF2 with associated off-target effects. Therefore, we introduced functional GULO (L-gulonolactone oxidase) in human organoids to augment intracellular ascorbate for NRF2 reactivation. This engineered organoid conjugated more bilirubin and protected against hyperbilirubinemia when transplanted in immunosuppressed Crigler-Najjar syndrome rat model. Collectively, we demonstrate that our organoid system serves as a manipulatable model for interrogating hyperbilirubinemia and potential therapeutic development.

Development of a high-throughput platform to measure plasmid transfer frequency.

Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal gene transfer via conjugation mediated by plasmids. This process implies a direct contact between a donor and a recipient bacterium which acquires the antibiotic resistance genes encoded by the plasmid and, concomitantly, the capacity to transfer the acquired plasmid to a new recipient. Classical assays for the measurement of plasmid transfer frequency (i.e., conjugation frequency) are often characterized by a high variability and, hence, they require many biological and technical replicates to reduce such variability and the accompanying uncertainty. In addition, classical conjugation assays are commonly tedious and time-consuming because they typically involve counting colonies on a large number of plates for the quantification of donors, recipients, and transconjugants (i.e., the bacteria that have received the genetic material by conjugation). Due to the magnitude of the antibiotic resistance problem, it is critical to develop reliable and rapid methods for the quantification of plasmid transfer frequency that allow the simultaneous analysis of many samples. Here, we present the development of a high-throughput, reliable, quick, easy, and cost-effective method to simultaneously accomplish and measure multiple conjugation events in 96-well plates, in which the quantification of donors, recipients, and transconjugants is estimated from the time required to reach a specific threshold value (OD600 value) in the bacterial growth curves. Our method successfully discriminates different plasmid transfer frequencies, yielding results that are equivalent to those obtained by a classical conjugation assay.

Detection and genomic characterization of a multidrug-resistant Salmonella Newport co-harbouring blaCMY-2, qnrB19 and mcr-9 from the diarrheic faeces of a foal

ObjectivesThis 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. MethodsBacterial 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. ResultsIsolate 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. ConclusionsNot 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.

Carbapenem resistance determinants and their transmissibility among clinically isolated Enterobacterales in Lebanon

BackgroundThe occurrence of carbapenem-resistant bacterial infections has increased significantly over the years with Gram-negative bacteria exhibiting the broadest resistance range. In this study we aimed to investigate the genomic characteristics of clinical carbapenem-resistant Enterobacterales (CRE). MethodsSeventeen representative multi-drug resistant (MDR) isolates from a hospital setting showing high level of resistance to carbapenems (ertapenem, meropenem and imipenem) were chosen for further characterization through whole-genome sequencing. Resistance mechanisms and transferability of plasmids carrying carbapenemase-encoding genes were also determined in silico and through conjugative mating assays. ResultsWe detected 18 different β-lactamases, including four carbapenemases (blaNDM-1, blaNDM-5, blaNDM-7, blaOXA-48) on plasmids with different Inc groups. The combined results from PBRT and in silico replicon typing revealed 20 different replicons linked to plasmids ranging in size between 80 and 200 kb. The most prevalent Inc groups were IncFIB(K) and IncM. OXA-48, detected on 76-kb IncM1 conjugable plasmid, was the most common carbapenemase. We also detected other conjugative plasmids with different carbapenemases confirming the role of horizontal gene transfer in the dissemination of antimicrobial resistance genes. ConclusionOur findings verified the continuing spread of carbapenemases in Enterobacterales and revealed the types of mobile elements circulating in a hospital setting and contributing to the spread of resistance determinants. The occurrence and transmission of plasmids carrying carbapenemase-encoding genes call for strengthening active surveillance and prevention efforts to control antimicrobial resistance dissemination in healthcare settings.