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512500.9468Do we still need Illumina sequencing data? Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and the Rapid v14 library prep kit for Gram negative bacteria whole genome assemblies. The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read sequencing data that can bridge repetitive and problematic regions. Oxford Nanopore Technologies (ONT) produce long-read sequencing platforms and they are continually improving their technology to obtain higher quality read data that is approaching the quality obtained from short-read platforms such as Illumina. As these innovations continue, we evaluated how much ONT read coverage produced by the Rapid Barcoding Kit v14 (SQK-RBK114) is necessary to generate high-quality hybrid and long-read-only genome assemblies for a panel of carbapenemase-producing Enterobacterales bacterial isolates. We found that 30× long-read coverage is sufficient if Illumina data are available, and that more (at least 100× long-read coverage is recommended for long-read-only assemblies. Illumina polishing is still improving single nucleotide variants (SNVs) and INDELs in long-read-only assemblies. We also examined if antimicrobial resistance genes could be accurately identified in long-read-only data, and found that Flye assemblies regardless of ONT coverage detected >96% of resistance genes at 100% identity and length. Overall, the Rapid Barcoding Kit v14 and long-read-only assemblies can be an optimal sequencing strategy (i.e., plasmid characterization and AMR detection) but finer-scale analyses (i.e., SNV) still benefit from short-read data.202438354391
149610.9457Plasmid-Mediated Co-Occurrence of mcr-1.1 in Extended-Spectrum β-Lactamase (ESBL)-Producing Escherichia coli Isolated From the Indigenous Seminomadic Community in Malaysia. The growing prevalence of commensal antibiotic resistant Escherichia coli poses a significant concern for the global spread of antibiotic resistance. Stool samples (n = 35) from a seminomadic indigenous community in Malaysia, the Jehai, were screened for multidrug-resistant bacteria, specifically the extended-spectrum β-lactamase (ESBL) producers. Subsequently, whole-genome sequencing was used to provide genomic insights into eight ESBL-producing E. coli that colonised eight individuals. The ESBL E. coli isolates carry resistance genes from various antibiotic classes such as the β-lactams (bla (TEM), bla (CTX-M-15) and bla (CTX-M-55)), quinolones (gyrA, qnrS and qnrS1) and aminoglycosides (aph(3')-Ia, aph(6)-Id and aac(3)-IId). Three concerning convergence of ESBL, colistin and metal resistance determinants, with three plasmids from H-type lineage harbouring bla (CTX-M) and mcr-1.1 genes were identified. Using the Oxford Nanopore Technology (ONT) Native Barcoding Kit (SQK-NBD114.24) in conjunction with the R10.4.1 flow cell, which achieved an average read accuracy (Q > 10) of 99.84%, we further characterised the mcr-1.1-bearing plasmids, ranging in size from 25 to 28 kb, from three strains of E. coli. This report represents the first whole genome analysis of multidrug-resistant bacteria, specifically those resistant to colistin, found within the indigenous population in Malaysia. It strongly indicates that the pertinent issue of colistin resistance in the country is far more significant than previously estimated.202440303148
138720.9452Whole-Genome Characterisation of ESBL-Producing E. coli Isolated from Drinking Water and Dog Faeces from Rural Andean Households in Peru. E. coli that produce extended-spectrum β-lactamases (ESBLs) are major multidrug-resistant bacteria. In Peru, only a few reports have characterised the whole genome of ESBL enterobacteria. We aimed to confirm the identity and antimicrobial resistance (AMR) profile of two ESBL isolates from dog faeces and drinking water of rural Andean households and determine serotype, phylogroup, sequence type (ST)/clonal complex (CC), pathogenicity, virulence genes, ESBL genes, and their plasmids. To confirm the identity and AMR profiles, we used the VITEK(®)2 system. Whole-genome sequencing (WGS) and bioinformatics analysis were performed subsequently. Both isolates were identified as E. coli, with serotypes -:H46 and O9:H10, phylogroups E and A, and ST/CC 5259/- and 227/10, respectively. The isolates were ESBL-producing, carbapenem-resistant, and not harbouring carbapenemase-encoding genes. Isolate 1143 ST5259 harboured the astA gene, encoding the EAST(1) heat-stable toxin. Both genomes carried ESBL genes (bla(EC-15), bla(CTX-M-8), and bla(CTX-M-55)). Nine plasmids were detected, namely IncR, IncFIC(FII), IncI, IncFIB(AP001918), Col(pHAD28), IncFII, IncFII(pHN7A8), IncI1, and IncFIB(AP001918). Finding these potentially pathogenic bacteria is worrisome given their sources and highlights the importance of One-Health research efforts in remote Andean communities.202235625336
174030.9446MDR Escherichia coli carrying CTX-M-24 (IncF[F-:A1:B32]) and KPC-2 (IncX3/IncU) plasmids isolated from community-acquired urinary trainfection in Brazil. Acquired antibiotic resistance in bacteria has become an important worldwide challenge. Currently, several bacteria, including Escherichia coli, have multidrug resistance profiles. Genes such as bla CTX-M-24 and bla KPC-2 (carbapenemase) are widespread. This research letter reports about a genomic surveillance study where multidrug-resistant E. coli containing CTX-M-24(IncF [F-:A1:B32]) and KPC-2(IncX3/IncU) plasmids were obtained from community- acquired urinary tract infection in Brazil.202236228665
150540.9440New insights on mcr-1-harboring plasmids from human clinical Escherichia coli isolates. Mobile colistin resistance (mcr) genes were described recently in Gram-negative bacteria including carbapenem-resistant Enterobacterales. There are ten mcr genes described in different Gram-negative bacteria, however, Escherichia coli harboring mcr-1 gene is by far the most frequent combination. In Argentina, mcr-1 gene was characterized only on plasmids belonging to IncI2 group. The aim of this work was to get new insights of mcr-1-harboring plasmids from E. coli. Eight E. coli isolates from a larger collection of 192 clinical E. coli isolates carrying the mcr-1 gene were sequenced using next generation technologies. Three isolates belonged to ST131 high-risk clone, and five to single ST, ST38, ST46, ST226, ST224, and ST405. Eight diverse mcr-1-harboring plasmids were analyzed: IncI2 (1), IncX4 (3), IncHI2/2A (3) and a hybrid IncFIA/HI1A/HI1B (1) plasmid. Plasmids belonging to the IncI2 (n = 1) and IncX4 (n = 3) groups showed high similarity with previously described plasmids. Two IncHI2/HI2A plasmids, showed high identity between them, while the third, showed several differences including additional resistance genes like tet(A) and floR. One IncFIA/H1A/H1B hybrid plasmid was characterized, highly similar to pSRC27-H, a prototype plasmid lacking mcr genes. mcr-1.5 variant was found in four plasmids with three different Inc groups: IncI2, IncHI2/HI2A and the hybrid FIA/HI1A/HI1B plasmid. mcr-1.5 variant is almost exclusively described in our country and with a high frequency. In addition, six E. coli isolates carried three allelic variants codifying for CTX-M-type extended-spectrum-β-lactamases: blaCTX-M-2 (3), blaCTX-M-65 (2), and blaCTX-M-14 (1). It is the first description of mcr-1 harboring plasmids different to IncI2 group in our country. These results represents new insights about mcr-1 harboring plasmids recovered from E. coli human samples from Argentina, showing different plasmid backbones and resistance gene combinations.202438408071
153550.9436Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases. Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes bla(NDM-1) and bla(OXA-58) in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The bla(NDM-1) gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas bla(OXA-58) was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.202133504662
503260.9435Hijacking a small plasmid to confer high-level resistance to aztreonam-avibactam and ceftazidime-avibactam. Acquired β-lactamase-encoding genes are typically carried by large plasmids in Gram-negative bacteria, which also commonly carry multi-copy small plasmids. This study found that mobile genetic elements carrying antimicrobial resistance genes are capable of hijacking small plasmids. This study focused on aztreonam-avibactam (ATM-AVI) as this combination can be used to effectively counter almost all β-lactamases produced by bacteria, and has been recommended against carbapenem-resistant Enterobacterales. A clinical strain (085003) of carbapenem-resistant Escherichia coli was investigated, and mutants (085003R32 and 085003R512) able to grow under 32/4 and 512/4 mg/L of ATM-AVI were obtained as representatives of low- and high-level resistance, respectively, by induction. Comparative genomics showed that 085003R32 and 085003R512 had a single nucleotide mutation of β-lactamase gene bla(CMY-2), encoding a novel CMY with a Thr319Ile substitution, assigned 'CMY-2R'. Cloning and enzyme kinetics were used to verify that CMY-2R conferred ATM-AVI resistance by compromising binding of AVI and subsequent protection of ATM. Mechanisms for the discrepant resistance between 085003R32 and 085003R512 were investigated. Three tandem copies of bla(CMY-2R) were identified on a self-transmissible IncP1 plasmid of 085003R32 due to IS1294 misrecognizing its end terIS and rolling-circle replication. 085003R512 had only a single copy of bla(CMY-2R) on the IncP1 plasmid, but possessed anther bla(CMY-2R) on an already present 4-kb small plasmid. IS1294-mediated mobilization on to this multi-copy small plasmid increased the copy number of bla(CMY-2R) significantly, rendering higher resistance. This study shows that bacteria can employ multiple approaches to accommodate selection pressures imposed by exposure to varied concentrations of antimicrobial agents.202337769749
138870.9434Snapshot Study of Whole Genome Sequences of Escherichia coli from Healthy Companion Animals, Livestock, Wildlife, Humans and Food in Italy. Animals, humans and food are all interconnected sources of antimicrobial resistance (AMR), allowing extensive and rapid exchange of AMR bacteria and genes. Whole genome sequencing (WGS) was used to characterize 279 Escherichia coli isolates obtained from animals (livestock, companion animals, wildlife), food and humans in Italy. E. coli predominantly belonged to commensal phylogroups B1 (46.6%) and A (29%) using the original Clermont criteria. One hundred and thirty-six sequence types (STs) were observed, including different pandemic (ST69, ST95, ST131) and emerging (ST10, ST23, ST58, ST117, ST405, ST648) extraintestinal pathogenic Escherichia coli (ExPEC) lineages. Eight antimicrobial resistance genes (ARGs) and five chromosomal mutations conferring resistance to highest priority critically important antimicrobials (HP-CIAs) were identified (qnrS1, qnrB19, mcr-1, bla(CTX-M1,15,55), bla(CMY-2), gyrA/parC/parE, ampC and pmrB). Twenty-two class 1 integron arrangements in 34 strains were characterized and 11 ARGs were designated as intI1 related gene cassettes (aadA1, aadA2, aadA5, aad23, ant2_Ia, dfrA1, dfrA7, dfrA14, dfrA12, dfrA17, cmlA1). Notably, most intI1 positive strains belonged to rabbit (38%) and poultry (24%) sources. Three rabbit samples carried the mcr-1 colistin resistance gene in association with IS6 family insertion elements. Poultry meat harbored some of the most prominent ExPEC STs, including ST131, ST69, ST10, ST23, and ST117. Wildlife showed a high average number of virulence-associated genes (VAGs) (mean = 10), mostly associated with an ExPEC pathotype and some predominant ExPEC lineages (ST23, ST117, ST648) were identified.202033172096
139180.9431Faecal carriage of extended-spectrum β-lactamase-producing and AmpC β-lactamase-producing bacteria among Danish army recruits. During May and June 2008, 84 Danish army recruits were tested for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing and AmpC β-lactamase-producing bacteria. Three ESBL-producing (CTX-M-14a) Escherichia coli isolates, two AmpC-producing (CMY-2) E. coli isolates and one AmpC-producing (CMY-34) Citrobacter freundii isolate were detected. Two of the CTX-M-14a E. coli isolates had similar pulsed-field gel electrophoresis and multilocus sequence typing profiles, indicating the same origin or transmission between the two army recruits. The bla(CTX-M-14a) genes were transferable to an E. coli recipient. These commensal bacteria therefore constitute a reservoir of resistance genes that can be transferred to other pathogenic bacteria in the intestine.201120718802
150490.9431Identification and Genomic Analyses of a Multidrug Resistant Avian Pathogenic Escherichia coli Coharboring mcr-1, bla (TEM-176) and bla (CTX-M-14) Genes. The emergence and transmission of the colistin-resistance gene mcr and extended-spectrum β-lactamase (ESBL) encoding genes pose a significant threat to global public health. In recent years, it has been reported that mcr-1 and ESBL genes can coexist in single bacteria strain. The objective of this study was to characterize a multidrug-resistant (MDR) avian pathogenic Escherichia coli (APEC) isolate carrying mcr and ESBL encoding genes in China. A total of 200 APEC isolates were collected for antimicrobial susceptibility testing by Kirby-Bauer (K-B) disk method. The MDR strain EC012 were then further analyzed for minimum inhibitory concentrations, antimicrobials resistance genes (ARGs) detection, conjugation, and whole-genome sequencing (WGS). Among all APEC isolates determined by K-B disk method, strain EC012 was resistant to almost all the antimicrobials, including polymyxin B, cefotaxime, and ceftazidime. Moreover, EC012 harbored ARGs mcr-1, bla (TEM-176), and bla (CTX-M-14). WGS analysis revealed that EC012 belonged to epidemic APEC serotype O1:H16 and multilocus sequence type ST295. EC012 consisted of one chromosome and six plasmids, encoding a broad ARGs. The bla (CTX-M-14), mcr-1 or bla (TEM-176) genes were located on conjugative plasmids pEC012-1 or pEC012-5, respectively. These plasmids were successfully transferred to transconjugants and resulted in the resistance to polymyxin B, cefotaxime, and ceftazidime. This study indicated that APEC was a potential reservoir of colistin-resistance gene mcr-1 and ESBL encoding genes, and highlighted the necessity for enhanced monitoring of ARGs dissemination among bacteria from different origins.202440303132
1528100.9431First Report of Coexistence of bla (SFO-1) and bla (NDM-1) β-Lactamase Genes as Well as Colistin Resistance Gene mcr-9 in a Transferrable Plasmid of a Clinical Isolate of Enterobacter hormaechei. Many antimicrobial resistance genes usually located on transferable plasmids are responsible for multiple antimicrobial resistance among multidrug-resistant (MDR) Gram-negative bacteria. The aim of this study is to characterize a carbapenemase-producing Enterobacter hormaechei 1575 isolate from the blood sample in a tertiary hospital in Wuhan, Hubei Province, China. Antimicrobial susceptibility test showed that 1575 was an MDR isolate. The whole genome sequencing (WGS) and comparative genomics were used to deeply analyze the molecular information of the 1575 and to explore the location and structure of antibiotic resistance genes. The three key resistance genes (bla (SFO-1), bla (NDM-1), and mcr-9) were verified by PCR, and the amplicons were subsequently sequenced. Moreover, the conjugation assay was also performed to determine the transferability of those resistance genes. Plasmid files were determined by the S1 nuclease pulsed-field gel electrophoresis (S1-PFGE). WGS revealed that p1575-1 plasmid was a conjugative plasmid that possessed the rare coexistence of bla (SFO-1), bla (NDM-1), and mcr-9 genes and complete conjugative systems. And p1575-1 belonged to the plasmid incompatibility group IncHI2 and multilocus sequence typing ST102. Meanwhile, the pMLST type of p1575-1 was IncHI2-ST1. Conjugation assay proved that the MDR p1575-1 plasmid could be transferred to other recipients. S1-PFGE confirmed the location of plasmid with molecular weight of 342,447 bp. All these three resistant genes were flanked by various mobile elements, indicating that the bla (SFO-1), bla (NDM-1), and mcr-9 could be transferred not only by the p1575-1 plasmid but also by these mobile elements. Taken together, we report for the first time the coexistence of bla (SFO-1), bla (NDM-1), and mcr-9 on a transferable plasmid in a MDR clinical isolate E. hormaechei, which indicates the possibility of horizontal transfer of antibiotic resistance genes.202134220761
1390110.9430Oxacillinase-484-Producing Enterobacterales, France, 2018-2023. We examined the emergence and characteristics of oxacillinase-484-producing Enterobacterales in France during 2012-2023. Genomic analysis identified 2 predominant sequence types in Escherichia coli: ST410 and ST1722. Plasmid analysis revealed that bla(OXA-484) genes were carried mostly on an IncX3-type plasmid associated with genetic elements including insertion sequences IS3000 and ISKpn19.202439320334
3020120.9430Combining sequencing approaches to fully resolve a carbapenemase-encoding megaplasmid in a Pseudomonas shirazica clinical strain. Horizontal transfer of plasmids plays a pivotal role in dissemination of antibiotic resistance genes and emergence of multidrug-resistant bacteria. Plasmid sequencing is thus paramount for accurate epidemiological tracking in hospitals and routine surveillance. Combining Nanopore and Illumina sequencing allowed full assembly of a carbapenemase-encoding megaplasmid carried by multidrug-resistant clinical isolate FFUP_PS_41. Average nucleotide identity analyses revealed that FFUP_PS_41 belongs to the recently proposed new species Pseudomonas shirazica, related to the P. putida phylogenetic group. FFUP_PS_41 harbours a 498,516-bp megaplasmid (pJBCL41) with limited similarity to publicly-available plasmids. pJBCL41 contains genes predicted to encode replication, conjugation, partitioning and maintenance functions and heavy metal resistance. The |aacA7|blaVIM-2|aacA4| cassette array (resistance to carbapenems and aminoglycosides) is located within a class 1 integron that is a defective Tn402 derivative. This transposon lies within a 50,273-bp region bound by Tn3-family 38-bp inverted repeats and flanked by 5-bp direct repeats (DR) that composes additional transposon fragments, five insertion sequences and a Tn3-Derived Inverted-Repeat Miniature Element. The hybrid Nanopore/Illumina approach allowed full resolution of a carbapenemase-encoding megaplasmid from P. shirazica. Identification of novel megaplasmids sheds new light on the evolutionary effects of gene transfer and the selective forces driving antibiotic resistance.201931381486
5123130.9430Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing. Rapid bacterial identification and antimicrobial resistance gene (ARG) detection are crucial for fast optimization of antibiotic treatment, especially for septic patients where each hour of delayed antibiotic prescription might have lethal consequences. This work investigates whether the Oxford Nanopore Technology's (ONT) Flongle sequencing platform is suitable for real-time sequencing directly from blood cultures to identify bacteria and detect resistance-encoding genes. For the analysis, we used pure bacterial cultures of four clinical isolates of Escherichia coli and Klebsiella pneumoniae and two blood samples spiked with either E. coli or K. pneumoniae that had been cultured overnight. We sequenced both the whole genome and plasmids isolated from these bacteria using two different sequencing kits. Generally, Flongle data allow rapid bacterial ID and resistome detection based on the first 1,000-3,000 generated sequences (10 min to 3 h from the sequencing start), albeit ARG variant identification did not always correspond to ONT MinION and Illumina sequencing-based data. Flongle data are sufficient for 99.9% genome coverage within at most 20,000 (clinical isolates) or 50,000 (positive blood cultures) sequences generated. The SQK-LSK110 Ligation kit resulted in higher genome coverage and more accurate bacterial identification than the SQK-RBK004 Rapid Barcode kit.202235369431
2086140.9430Comparative genomic analyses of β-lactamase (bla(CMY-42))-encoding plasmids isolated from wastewater treatment plants in Canada. Wastewater treatment plants (WWTPs) are useful environments for investigating the occurrence, diversity, and evolution of plasmids encoding clinically relevant antibiotic resistance genes (ARGs). Our objective was to isolate and sequence plasmids encoding meropenem resistance from bacterial hosts within Canadian WWTPs. We used two enrichment culture approaches for primary plasmid isolation, followed by screening for antibiotic resistance, conjugative mobility, and stability in enteric bacteria. Isolated plasmids were sequenced using Illumina MiSeq and Sanger sequencing methods. Bioinformatics analyses resolved a multi-resistance IncF/MOB(F12) plasmid, pFEMG (209 357 bp), harbouring resistance genes to β-lactam (bla(CMY-42), bla(TEM-1β), and bla(NDM-5)), macrolide (mphA-mrx-mphR), tetracycline (tetR-tetB-tetC-tetD), trimethoprim (dfrA12), aminoglycoside (aadA2), and sulfonamide (sul1) antibiotic classes. We also isolated an IncI1/MOB(P12) plasmid pPIMR (172 280 bp) carrying similar β-lactamase and a small multi-drug efflux resistance gene cluster (bla(CMY-42)-blc-sugE) to pFEMG. The co-occurrence of different ARGs within a single 24 552 bp cluster in pFEMG - interspersed with transposons, insertion sequence elements, and a class 1 integron - may be of significant interest to human and veterinary medicine. Additionally, the presence of conjugative and plasmid maintenance genes in the studied plasmids corresponded to observed high conjugative transfer frequencies and stable maintenance. Extensive investigation is required to further understand the fitness trade-offs of plasmids with different types of conjugative transfer and maintenance modules.202134077692
5124150.9430Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing. INTRODUCTION: Genome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time. METHODS: In this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%. RESULTS AND DISCUSSION: For a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.202337256057
1497160.9428Colistin-resistant mcr-1-positive Escherichia coli ST1775-H137 co-harboring bla(CTX-M-2) and bla(CMY-2) recovered from an urban stream. The rapid dissemination of colistin resistance mcr-type genes and extended-spectrum β-lactamase-encoding genes at the human-animal-environment interface has raised concerns worldwide. In this study, we performed a genomic investigation of a multidrug (MDR)- and colistin-resistant Escherichia coli strain recovered from an urban stream strongly affected by pollution and used for recreational purposes in Brazil. E. coli strain EW827 was resistant to clinically significant antimicrobials, including polymyxins, extended-spectrum cephalosporins, and fluoroquinolones. Whole-genome sequencing analysis revealed that EW827 strain belonged to ST1775 and carried the fimH137 allele, clinically relevant antimicrobial resistance genes (e.g., mcr-1.1, bla(CTX-M-2), and bla(CMY-2)), tolerance genes to metals, and biocide resistance genes. Moreover, IncX4 and IncI1-ST12 replicon types were identified carrying mcr-1.1 and bla(CMY-2), respectively. A novel genetic environment of the mcr-1.1 gene, in which a 258-bp ∆IS5-like was inserted in the opposite orientation upstream of the mcr-1.1-pap2 element, was also detected. Additionally, the bla(CTX-M-2) gene was harbored by a Tn21-like element on the chromosome. The occurrence of MDR E. coli co-harboring mcr-1.1, bla(CTX-M-2), and bla(CMY-2) in urban water represents a potential risk to humans, animals, and environmental safety. Therefore, epidemiological studies are required to monitoring multidrug-resistant bacteria and their antimicrobial resistance genes in aquatic ecosystems to determine possible routes and fates of these genes.202134823029
5209170.9428Complete Nucleotide Sequence of pGA45, a 140,698-bp IncFIIY Plasmid Encoding bla IMI-3-Mediated Carbapenem Resistance, from River Sediment. Plasmid pGA45 was isolated from the sediments of Haihe River using Escherichia coli CV601 (gfp-tagged) as recipients and indigenous bacteria from sediment as donors. This plasmid confers reduced susceptibility to imipenem which belongs to carbapenem group. Plasmid pGA45 was fully sequenced on an Illumina HiSeq 2000 sequencing system. The complete sequence of plasmid pGA45 was 140,698 bp in length with an average G + C content of 52.03%. Sequence analysis shows that pGA45 belongs to IncFIIY group and harbors a backbone region which shares high homology and gene synteny to several other IncF plasmids including pNDM1_EC14653, pYDC644, pNDM-Ec1GN574, pRJF866, pKOX_NDM1, and pP10164-NDM. In addition to the backbone region, plasmid pGA45 harbors two notable features including one bla IMI-3-containing region and one type VI secretion system region. The bla IMI-3-containing region is responsible for bacteria carbapenem resistance and the type VI secretion system region is probably involved in bacteria virulence, respectively. Plasmid pGA45 represents the first complete nucleotide sequence of the bla IMI-harboring plasmid from environment sample and the sequencing of this plasmid provided insight into the architecture used for the dissemination of bla IMI carbapenemase genes.201626941718
849180.9428Bacterial Genomics for National Antimicrobial Resistance Surveillance in Cambodia. BACKGROUND: Antimicrobial resistance (AMR) surveillance in low- and middle-income countries (LMICs) often relies on poorly resourced laboratory processes. Centralized sequencing was combined with cloud-based, open-source bioinformatics solutions for national AMR surveillance in Cambodia. METHODS: Blood cultures growing gram-negative bacteria were collected at 6 Cambodian hospitals (January 2021 to October 2022). Isolates were obtained from pure plate growth and shotgun DNA sequencing performed in country. Using public nucleotide and protein databases, reads were aligned for pathogen identification and AMR gene characterization. Multilocus sequence typing was performed on whole-genome assemblies and haplotype clusters compared against published genomes. RESULTS: Genes associated with acquired resistance to fluoroquinolones were identified in 59%, trimethoprim/sulfamethoxazole in 45%, and aminoglycosides in 52% of 715 isolates. Extended-spectrum β-lactamase encoding genes were identified in 34% isolates, most commonly blaCTX-M-15, blaCTX-M-27, and blaCTX-M-55 in Escherichia coli sequence types 131 and 1193. Carbapenemase genes were identified in 12% isolates, most commonly blaOXA-23, blaNDM-1, blaOXA-58, and blaOXA-66 in Acinetobacter species. Phylogenetic analysis revealed clonal strains of Acinetobacter baumannii, representing suspected nosocomial outbreaks, and genetic clusters of quinolone-resistant typhoidal Salmonella and extended-spectrum β-lactamase E. coli cases suggesting community transmission. CONCLUSIONS: With accessible sequencing platforms and bioinformatics solutions, bacterial genomics can supplement AMR surveillance in LMICs.202539163245
1397190.9428Genomic Features of an MDR Escherichia coli ST5506 Harboring an IncHI2/In229/bla(CTX-M-2) Array Isolated from a Migratory Black Skimmer. Migratory birds have contributed to the dissemination of multidrug-resistant (MDR) bacteria across the continents. A CTX-M-2-producing Escherichia coli was isolated from a black skimmer (Rynchops niger) in Southeast Brazil. The whole genome was sequenced using the Illumina NextSeq platform and de novo assembled by CLC. Bioinformatic analyses were carried out using tools from the Center for Genomic Epidemiology. The genome size was estimated at 4.9 Mb, with 4790 coding sequences. A wide resistome was detected, with genes encoding resistance to several clinically significant antimicrobials, heavy metals, and biocides. The bla(CTX-M-2) gene was inserted in an In229 class 1 integron inside a ∆TnAs3 transposon located in an IncHI2/ST2 plasmid. The strain was assigned to ST5506, CH type fumC19/fimH32, serotype O8:K87, and phylogroup B1. Virulence genes associated with survival in acid conditions, increased serum survival, and adherence were also identified. These data highlight the role of migratory seabirds as reservoirs and carriers of antimicrobial resistance determinants and can help to elucidate the antimicrobial resistance dynamics under a One Health perspective.202438251370