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512500.9778Do 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
149610.9772Plasmid-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
138720.9768Whole-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
263230.9764Environmental Spread of Extended Spectrum Beta-Lactamase (ESBL) Producing Escherichia coli and ESBL Genes among Children and Domestic Animals in Ecuador. BACKGROUND: There is a significant gap in our understanding of the sources of multidrug-resistant bacteria and resistance genes in community settings where human-animal interfaces exist. OBJECTIVES: This study characterized the relationship of third-generation cephalosporin-resistant Escherichia coli (3GCR-EC) isolated from animal feces in the environment and child feces based on phenotypic antimicrobial resistance (AMR) and whole genome sequencing (WGS). METHODS: We examined 3GCR-EC isolated from environmental fecal samples of domestic animals and child fecal samples in Ecuador. We analyzed phenotypic and genotypic AMR, as well as clonal relationships (CRs) based on pairwise single-nucleotide polymorphisms (SNPs) analysis of 3GCR-EC core genomes. CRs were defined as isolates with fewer than 100 different SNPs. RESULTS: A total of 264 3GCR-EC isolates from children (n = 21), dogs (n = 20), and chickens (n = 18) living in the same region of Quito, Ecuador, were identified. We detected 16 CRs total, which were found between 7 children and 5 domestic animals (5 CRs) and between 19 domestic animals (11 CRs). We observed that several clonally related 3GCR-EC isolates had acquired different plasmids and AMR genes. Most CRs were observed in different homes (n = 14) at relatively large distances. Isolates from children and domestic animals shared the same blaCTX-M allelic variants, and the most prevalent were blaCTX-M-55 and blaCTX-M-65, which were found in isolates from children, dogs, and chickens. DISCUSSION: This study provides evidence of highly dynamic horizontal transfer of AMR genes and mobile genetic elements (MGEs) in the E. coli community and shows that some 3GCR-EC and (extended-spectrum β-lactamase) ESBL genes may have moved relatively large distances among domestic animals and children in semirural communities near Quito, Ecuador. Child-animal contact and the presence of domestic animal feces in the environment potentially serve as important sources of drug-resistant bacteria and ESBL genes. https://doi.org/10.1289/EHP7729.202133617318
520340.9762Draft genome sequence analysis of a novel MLST (ST5028) and multidrug-resistant Klebsiella quasipneumoniae subsp. similipneumoniae (Kp4) strain 456S1 isolated from a pig farm in China. OBJECTIVES: The avian breeding industry is an important element in exposing bacteria to antibiotics. As one of the major animal welfare and economic problems for the poultry industry, multidrug-resistant Klebsiella spp. have become a substantial source of antibiotic resistance genes. In the present work, we reported the draft genome sequence of a novel multilocus sequence type (MLST) (ST5028) Klebsiella quasipneumoniae subsp. similipneumoniae (Kp4) strain 456S1, which was isolated from a pig farm in China with broad-spectrum antimicrobial activities. METHODS: Classical microbiological methods were applied to isolate and identify the strain, genomic DNA was sequenced using an Illumina HiSeq platform, and the reads were de novo assembled into contigs using CLC Genomics Workbench. The assembled contigs were annotated, and whole-genome sequencing (WGS) analysis was performed. RESULTS: WGS analysis revealed that the genome of strain 456S1 comprised a circular chromosome of 5,419,059 bp (GC content, 57.8%), harbouring 12 important antibiotic resistance genes: aac(6')-ib-cr, aadA16, floR, dfrA27, fosA, tet(D), blaOKP-B-3, oqxA, oqxB, qnrB6, sul1 and arr-3. The Klebsiella quasipneumoniae subsp. similipneumoniae (Kp4) 456S1 was also found to belong to a novel sequence type (ST5028) determined by MLST. CONCLUSION: The genome sequence reported herein will provide useful information for antibiotic resistance and pathogenic mechanisms in Klebsiella quasipneumoniae and will be a reference for comparative analysis with genomic features among different sources of clinically important multidrug-resistant strains, especially among bacteria of animal and human origin.202133516893
149750.9761Colistin-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
523560.9760Draft genome sequences of rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 carrying mobile colistin resistance gene mcr-9 isolated from wastewater in South Africa. OBJECTIVES: Antimicrobial-resistant bacteria of the order Enterobacterales are emerging threats to global public and animal health, leading to morbidity and mortality. The emergence of antimicrobial-resistant, livestock-associated pathogens is a great public health concern. The genera Enterobacter and Lelliottia are ubiquitous, facultatively anaerobic, motile, non-spore-forming, rod-shaped Gram-negative bacteria belonging to the Enterobacteriaceae family and include pathogens of public health importance. Here, we report the first draft genome sequences of a rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 in Africa. METHODS: The bacteria were isolated from environmental wastewater samples. Bacteria were cultured on nutrient agar, and the pure cultures were subjected to whole-genome sequencing. Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were trimmed and subjected to de novo assembly. The assembled contigs were analysed for virulence genes, antimicrobial resistance genes, and extra-chromosomal plasmids, and multilocus sequence typing was performed. To compare the sequenced strains with other, previously sequenced E. kobei and L. nimipressuralis strains, available raw read sequences were downloaded, and all sequence files were treated identically to generate core genome bootstrapped maximum likelihood phylogenetic trees. RESULTS: Whole-genome sequencing analyses identified strain MEZLN61 as L. nimipressuralis and strains MEZEK193 and MEZEK194 as E. kobei. MEZEK193 and MEZEK194 carried genes encoding resistance to fosfomycin (fosA), beta-lactam antibiotics (bla(ACT-9)), and colistin (mcr-9). Additionally, MEZEK193 harboured nine different virulence genes, while MEZEK194 harboured eleven different virulence genes. The phenotypic analysis showed that L. nimipressuralis strain MEZLN61 was susceptible to colistin (2 μg/mL), while E. kobei MEZEK193 (64 μg/mL) and MEZEK194 (32 μg/mL) were resistant to colistin. CONCLUSION: The genome sequences of strains L. nimipressuralis MEZLN6, E. kobei MEZEK193, and E. kobei MEZEK194 will serve as a reference point for molecular epidemiological studies of L. nimipressuralis and E. kobei in Africa. In addition, this study provides an in-depth analysis of the genomic structure and offers important information that helps clarify the pathogenesis and antimicrobial resistance of L. nimipressuralis and E. kobei. The detection of mcr-9, which is associated with very low-level colistin resistance in Enterobacter species, is alarming and may indicate the undetected dissemination of mcr genes in bacteria of the order Enterobacterales. Continuous monitoring and surveillance of the prevalence of mcr genes and their associated phenotypic changes in clinically important pathogens and environmentally associated bacteria is necessary to control and prevent the spread of colistin resistance.202336948496
138870.9759Snapshot 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
84980.9759Bacterial 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
106390.9759Enterobacteriaceae resistant to third-generation cephalosporins and quinolones in fresh culinary herbs imported from Southeast Asia. Since multidrug resistant bacteria are frequently reported from Southeast Asia, our study focused on the occurrence of ESBL-producing Enterobacteriaceae in fresh imported herbs from Thailand, Vietnam and Malaysia. Samples were collected from fresh culinary herbs imported from Southeast Asia in which ESBL-suspected isolates were obtained by selective culturing. Analysis included identification by MALDI-TOF mass spectrometry, susceptibility testing, XbaI-PFGE, microarray, PCR and sequencing of specific ESBL genes, PCR based replicon typing (PBRT) of plasmids and Southern blot hybridization. In addition, the quinolone resistance genotype was characterized by screening for plasmid mediated quinolone resistance (PMQR) genes and mutations in the quinolone resistance determining region (QRDR) of gyrA and parC. The study encompassed fifty samples of ten batches of culinary herbs (5 samples per batch) comprising nine different herb variants. The herbs originated from Thailand (Water morning glory, Acacia and Betel leaf), Vietnam (Parsley, Asian pennywort, Houttuynia leaf and Mint) and Malaysia (Holy basil and Parsley). By selective culturing 21 cefotaxime resistant Enterobacteriaceae were retrieved. Array analysis revealed 18 isolates with ESBL genes and one isolate with solely non-ESBL beta-lactamase genes. Mutations in the ampC promoter region were determined in two isolates with PCR and sequencing. The isolates were identified as Klebsiella pneumoniae (n=9), Escherichia coli (n=6), Enterobacter cloacae complex (n=5) and Enterobacter spp. (n=1). All isolates tested were multidrug resistant. Variants of CTX-M enzymes were predominantly found followed by SHV enzymes. PMQR genes (including aac(6')-1b-cr, qnrB and qnrS) were also frequently detected. In almost all cases ESBL and quinolone resistance genes were located on the same plasmid. Imported fresh culinary herbs from Southeast Asia are a potential source for contamination of food with multidrug resistant bacteria. Because these herbs are consumed without appropriate heating, transfer to human bacteria cannot be excluded.201424607424
1214100.9759Plasmid-mediated quinolone resistance genes in fecal bacteria from rooks commonly wintering throughout Europe. This study concerned the occurrence of fecal bacteria with plasmid-mediated quinolone resistance (PMQR) genes in rooks (Corvus frugilegus, medium-sized corvid birds) wintering in continental Europe during winter 2010/2011. Samples of fresh rook feces were taken by cotton swabs at nine roosting places in eight European countries. Samples were transported to one laboratory and placed in buffered peptone water (BPW). The samples from BPW were enriched and subcultivated onto MacConkey agar (MCA) supplemented with ciprofloxacin (0.06 mg/L) to isolate fluoroquinolone-resistant bacteria. DNA was isolated from smears of bacterial colonies growing on MCA and tested by PCR for PMQR genes aac(6')-Ib, qepA, qnrA, qnrB, qnrC, qnrD, qnrS, and oqxAB. All the PCR products were further analyzed by sequencing. Ciprofloxacin-resistant bacteria were isolated from 37% (392 positive/1,073 examined) of samples. Frequencies of samples with ciprofloxacin-resistant isolates ranged significantly from 3% to 92% in different countries. The qnrS1 gene was found in 154 samples and qnrS2 in 2 samples. The gene aac(6')-Ib-cr was found in 16 samples. Thirteen samples were positive for qnrB genes in variants qnrB6 (one sample), qnrB18 (one), qnrB19 (one), qnrB29 (one), and qnrB49 (new variant) (one). Both the qnrD and oqxAB genes were detected in six samples. The genes qnrA, qnrC, and qepA were not found. Wintering omnivorous rooks in Europe were commonly colonized by bacteria supposedly Enterobacteriaceae with PMQR genes. Rooks may disseminate these epidemiologically important bacteria over long distances and pose a risk for environmental contamination.201222731858
1508110.9758First Detection and Genomic Insight into mcr-1 Encoding Plasmid-Mediated Colistin-Resistance Gene in Escherichia coli ST101 Isolated from the Migratory Bird Species Hirundo rustica in Thailand. Background: This study aimed to investigate the occurrence of mcr-1 encoding plasmid-mediated colistin-resistance gene in Escherichia coli isolated from migratory birds in Thailand. Materials and Methods: A total of 178 cloacal swabs from migratory birds was sampled and isolated from 2016 to 2017 in Nan, Trang, and Bangkok, Thailand. The multiplex polymerase chain reaction was used to screen the resistance genes. After screening, a disk diffusion assay and the minimum inhibitory concentration were investigated. The draft genome sequence of isolate 2A85589 was obtained using an Illumina HiSeq X-Ten platform. The genome was assembled using SPAdes 3.0.0. Antimicrobial resistance genes were identified using ResFinder 3.1. Results: We reported E. coli ST101 of isolate 2A85589, an mcr-1-carrying resistance gene isolated from the migratory bird species Hirundo rustica in Thailand. The draft genome of 2A85589 was 4,621,016 bp in size. IncHI1A plasmid was identified using PlasmidFinder with high coverage. In silico analysis detected the presence of eight putative acquired resistance genes, namely blaTEM-1B, mcr-1, mef(A), mef(B), QnrS1, sul3, tet(A), and tet(B), which conferred resistance to β-lactam, colistin, macrolide, quinolone, sulfonamide, and tetracycline. Conclusion: This study underlines the potential risk of the environmental contamination of mcr-1-carrying E. coli isolated from the migratory bird. The long range migration of birds can result in dissemination of mcr-1-carrying bacteria globally. Therefore, plasmid-mediated colistin is an urgent need to be addressed in both human and veterinary medicine for disease control and prevention.201931334682
1190120.9757Co-occurrence of mcr-1, mcr-3, mcr-7 and clinically relevant antimicrobial resistance genes in environmental and fecal samples. Multidrug-resistant bacteria harboring different antimicrobial resistance genes (ARGs) have been detected worldwide. The association of plasmid-mediated colistin resistance genes (mcr-like) and other ARGs in bacteria isolated from animals is a huge concern worldwide. Therefore, this study aimed to investigate the presence of mcr-like genes and clinically relevant ARGs as well as plasmids in samples from a zoo. Fecal and environmental (soil and water) samples were collected from a zoo and the DNA of cultivable aerobic bacteria was extracted. ARGs were screened by PCR and the plasmids were detected using the PCR-based replicon typing method. A total of 74 amplicons from 27 ARGs [mcr-1, mcr-3, mcr-7.1, bla(CTX-M-Gp1), bla(CTX-M-Gp2), bla(CTX-M-Gp9), bla(VEB), bla(PER), bla(CMY), tetA, tetB, tetC, aadA, aac(6')-Ib, aph(3')-Ia, ant(2'')-Ia, qnrA, qnrB, qnrS, oqxA, oqxB, sul1, sul2, sul3, cmlA, mefAE, ermB] and 21 amplicons from eight plasmid families (IncY, ColE-like, IncF(repB), IncFIA, IncFIB, IncHI1, IncFIC, IncP) were detected. These findings reinforce that the zoo acts as a reservoir of clinically relevant ARGs, including mcr-like, and call attention to the monitoring studies in the zoo. Therefore, to the best of our knowledge, this is the first report of the world of mcr-1, mcr-3 and mcr-7.1 in environmental samples from the zoo.202032382766
5616130.9757Whole genome sequencing analysis on antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia. IMPORTANCE: The emergence and rapid increase in the incidence of multidrug-resistant (MDR) bacteria in pig farms has become a serious concern and reduced the choice of effective antibiotics. OBJECTIVE: This study analyzed the phylogenetics and diversity of antibiotic resistance genes (ARGs) and molecularly identified the source of ARGs in antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia. METHODS: Forty-four antibiotic-resistant E. coli isolates from fecal samples from 44 pig farms in Banten Province, Indonesia, were used as samples. The samples were categorized into 14 clusters. Sequencing was performed using the Oxford Nanopore Technologies MinION platform, with barcoding before sequencing with Nanopore Rapid sequencing gDNA-barcoding (SQK-RBK110.96) according to manufacturing procedures. ARG detection was conducted using ResFinder, and the plasmid replicon was determined using PlasmidFinder. RESULTS: Three phylogenetic leaves of E. coli were identified in the pig farming cluster in Banten Province. The E. coli isolates exhibited potential resistance to nine classes of antibiotics. Fifty-one ARGs were identified across all isolates, with each cluster carrying a minimum of 10 ARGs. The ant(3'')-Ia and qnrS1 genes were present in all isolates. ARGs in the E. coli pig farming cluster originated mainly from plasmids, accounting for an average of 89.4%. CONCLUSIONS AND RELEVANCE: The elevated potential for MDR events, coupled with the dominance of ARGs originating from plasmids, increases the risk of ARG spread among bacterial populations in animals, humans, and the environment.202438834513
1397140.9757Genomic 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
2634150.9756Characterization of Escherichia coli and Other Enterobacterales Resistant to Extended-Spectrum Cephalosporins Isolated from Dairy Manure in Ontario, Canada. Extended-spectrum cephalosporins (ESCs) resistance genes, such as bla(CTX-M), bla(CMY), and bla(SHV), have been found regularly in bacteria from livestock. However, information on their distribution in dairy cattle in Canada and on the associated genome sequences of ESC-resistant Enterobacterales is sparse. In this study, the diversity and distribution of ESC-resistant Escherichia coli throughout manure treatments in six farms in Southern Ontario were assessed over a one-year period, and their ESC-resistance plasmids were characterized. The manure samples were enriched using selective media. The resulting isolates were screened via polymerase chain reaction for bla(CTX-M), bla(CMY), and bla(SHV). No E. coli carrying bla(SHV) were detected. Escherichia coli (n = 248) carrying bla(CTX-M) or bla(CMY) underwent whole-genome sequencing using an Illumina MiSeq/NextSeq. These isolates were typed using multilocus sequence typing (MLST) and their resistance gene profiles. A subset of E. coli (n = 28) were sequenced using Oxford Nanopore Technologies. Plasmids were assembled using Unicycler and characterized via the resistance genes pattern, replicon type, plasmid MLST, phylogenetic analysis, and Mauve alignments. The recovery of ESC-resistant Enterobacterales (18 species, 8 genera) was drastically reduced in manure outputs. However, multiple treatment stages were needed to attain a significant reduction. 62 sequence types were identified, with ST10, ST46, ST58, ST155, ST190, ST398, ST685, and ST8761 being detected throughout the treatment pipeline. These STs overlapped with those found on multiple farms. The ESC-resistance determinants included CTX-M-1, -14, -15, -17, -24, -32, -55, and CMY-2. The plasmids carrying bla(CTX-M) were more diverse than were the plasmids carrying bla(CMY). Known "epidemic plasmids" were detected for both bla(CTX-M) and bla(CMY). IMPORTANCE The increase in antimicrobial resistance is of concern for human and animal health, especially when resistance is conferred to extended-spectrum cephalosporins, which are used to treat serious infections in both human and veterinary medicine. Bacteria carrying extended-spectrum cephalosporin resistance genes, including bla(CTX-M) and bla(CMY), are frequently found in dairy manure. Manure treatment influences the loads and diversity of bacteria, including those carrying antimicrobial resistance genes, such as Enterobacterales and Escherichia coli. Any bacteria that survive the treatment process are subsequently applied to the environment. Enterobacterales carrying bla(CTX-M) or bla(CMY) can contaminate soil and crops consumed by humans and animals, thereby increasing the potential for antimicrobial resistance genes to integrate into the human gut microflora through horizontal gene transfer. This furthers the dissemination of resistance. Therefore, it is imperative to understand the effects manure treatments have on ESC-resistance in environmentally applied manure.202336695602
1743160.9755International clones of extended-spectrum β-lactamase (CTX-M)-producing Escherichia coli in peri-urban wild animals, Brazil. CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Escherichia coli clones have been increasingly reported worldwide. In this regard, although discussions of transmission routes of these bacteria are in evidence, molecular data are lacking to elucidate the epidemiological impacts of ESBL producers in wild animals. In this study, we have screened 90 wild animals living in a surrounding area of São Paulo, the largest metropolitan city in South America, to monitor the presence of multidrug-resistant (MDR) Gram-negative bacteria. Using a genomic approach, we have analysed eight ceftriaxone-resistant E. coli. Resistome analyses revealed that all E. coli strains carried bla(CTX-M) -type genes, prevalent in human infections, besides other clinically relevant resistance genes to aminoglycosides, β-lactams, phenicols, tetracyclines, sulphonamides, trimethoprim, fosfomycin and quinolones. Additionally, E. coli strains belonged to international sequence types (STs) ST38, ST58, ST212, ST744, ST1158 and ST1251, and carried several virulence-associated genes. Our findings suggest spread and adaptation of international clones of CTX-M-producing E. coli beyond urban settings, including wildlife from shared environments.202032239649
5124170.9755Oxford 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
1707180.9755Beta-Lactam Antibiotic Resistance Genes in the Microbiome of the Public Transport System of Quito, Ecuador. Multidrug-resistant bacteria present resistance mechanisms against β-lactam antibiotics, such as Extended-Spectrum Beta-lactamases (ESBL) and Metallo-β-lactamases enzymes (MBLs) which are operon encoded in Gram-negative species. Likewise, Gram-positive bacteria have evolved other mechanisms through mec genes, which encode modified penicillin-binding proteins (PBP2). This study aimed to determine the presence and spread of β-lactam antibiotic resistance genes and the microbiome circulating in Quito's Public Transport (QTP). A total of 29 station turnstiles were swabbed to extract the surface environmental DNA. PCRs were performed to detect the presence of 13 antibiotic resistance genes and to identify and to amplify 16S rDNA for barcoding, followed by clone analysis, Sanger sequencing, and BLAST search. ESBL genes bla(TEM-1) and bla(CTX-M-1) and MBL genes bla(OXA-181) and mecA were detected along QPT stations, blaTEM being the most widely spread. Two subvariants were found for bla(TEM-1), bla(CTX-M-1), and bla(OXA-181). Almost half of the circulating bacteria found at QPT stations were common human microbiota species, including those classified by the WHO as pathogens of critical and high-priority surveillance. β-lactam antibiotic resistance genes are prevalent throughout QPT. This is the first report of bla(OXA-181) in environmental samples in Ecuador. Moreover, we detected a new putative variant of this gene. Some commensal coagulase-negative bacteria may have a role as mecA resistance reservoirs.202336767267
1386190.9755ESBL/pAmpC-producing Enterobacterales in common leopard geckos (Eublepharis macularius) and central bearded dragons (Pogona vitticeps) from Portugal. Common leopard geckos (Eublepharis macularius) and central bearded dragon (Pogona vitticeps) are widely kept as pets but can harbor pathogenic bacteria, including antimicrobial-resistant (AMR) bacteria. This study aimed to research the frequency of β-lactamase-producing Enterobacterales in these two reptile species. A total of 132 samples were collected from the oral and cloacal cavities of healthy common leopard geckos and central bearded dragons in the Lisbon area, Portugal. Antimicrobial resistance was assessed for third-generation cephalosporin (3GC)-resistant Enterobacterales. The results revealed that 3GC-resistant Enterobacterales were observed in 17.9% (n = 14/78) of the reptiles. The most commonly identified species were: Citrobacter freundii and Klebsiella aerogenes. Furthermore, some isolates produced extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases (AmpC) encoding genes such as bla (CMY-2), bla (CTX-M-15,) and bla (TEM-1). These findings emphasize the potential role of these reptiles in the spread of AMR bacteria, particularly in urban settings where human- animal interactions are frequent. Given the zoonotic risks, this study emphasizes the importance of continued surveillance and responsible antimicrobial use in both veterinary and human medicine to mitigate the spread of AMR bacteria.202540370835