CReSIL: accurate identification of extrachromosomal circular DNA from long-read sequences.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is found in many eukaryotic species and cell types, including cancer, where eccDNAs with oncogenes drive tumorigenesis. Most studies of eccDNA employ short-read sequencing for their identification. However, short-read sequencing cannot resolve the complexity of genomic repeats, which can lead to missing eccDNA products. Long-read sequencing technologies provide an alternative to constructing complete eccDNA maps. We present a software suite, Construction-based Rolling-circle-amplification for eccDNA Sequence Identification and Location (CReSIL), to identify and characterize eccDNA from long-read sequences. CReSIL's performance in identifying eccDNA, with a minimum F1 score of 0.98, is superior to the other bioinformatic tools based on simulated data. CReSIL provides many useful features for genomic annotation, which can be used to infer eccDNA function and Circos visualization for eccDNA architecture investigation. We demonstrated CReSIL's capability in several long-read sequencing datasets, including datasets enriched for eccDNA and whole genome datasets from cells containing large eccDNA products. In conclusion, the CReSIL suite software is a versatile tool for investigating complex and simple eccDNA in eukaryotic cells.

Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

OBJECTIVES: In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. METHODS: WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. RESULTS: mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. CONCLUSIONS: The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

Genome-associations of extended-spectrum ß-lactamase producing (ESBL) or AmpC producing E. coli in small and medium pig farms from Khon Kaen province, Thailand.

Thailand is undergoing rapid intensification of livestock production where small subsistence farms and medium sized commercial farms coexist. In medium farms, antimicrobials are prescribed by a veterinarian, whereas in small farms antimicrobial use remains largely unsupervised. The impact of these differences as well as other farming practices on the emergence and composition of antimicrobial resistance genes (ARGs) remains largely unknown. We analyzed 363 genomes of extended-spectrum ß-lactamase producing (ESBL) and/or AmpC producing Escherichia coli recovered from humans and pigs at small and medium farms from the Khon Kaen province, Thailand. We tested for genome-wide associations to identify links between ARGs, host, and farm size. Pig isolates from small farms were associated with mcr and qnr genes conferring resistance to colistin and fluoroquinolones, respectively. In contrast, pig isolates from medium farms were associated with ARGs conferring resistance to drugs commonly used on medium farms (i.e., streptomycin). ESBL plasmids from small farms co-carried ARGs conferring resistance to critically important antimicrobials more frequently compared to plasmid from medium farms. Frequent ARG combinations included blaCTX-M-55 + qnrS1 (29.8% vs 17.5% in small and medium farms, respectively), blaCTX-M-55 + qnrS1 + mcr-3.19 (5% vs 0%), blaCTX-M-14 + qnrS1 (9.3% vs 6.2%), and blaCTX-M-14 + qnrS1 + mcr-1.1 (3.1% vs 0%). The co-location on plasmids of ARGs conferring resistance to critically important antimicrobials as defined by the World Health Organization is concerning, and actions to curb their spread are urgently needed. Legislation on limiting antimicrobial sales and initiatives to better inform farmers and veterinarians on appropriate antimicrobial usage and farm biosecurity could help reduce antimicrobial use on farms.

Characterization of Klebsiella pneumoniae complex isolates from pigs and humans in farms in Thailand: population genomic structure, antibiotic resistance and virulence genes.

OBJECTIVES: To define characteristics of Klebsiella pneumoniae complex (hereafter KP) isolates from healthy pigs, farm workers and their household members in Thailand. METHODS: A total of 839 individual rectal swabs from pigs on 164 farms and 271 faecal samples of humans working on pig farms and persons living in the same household in Khon Kaen, Thailand were screened for gut colonization by KP. Genomic sequences were investigated for antibiotic resistance and virulence genes. Phylogenetic analyses were performed in addition to comparison with isolates from previous studies from Thailand. RESULTS: KP was detected in approximately 50% of pig and human samples. In total, 253 KP isolates were obtained: 39% from pigs, 34% from farmers and 26% from individuals living on the same farm but without animal contact. MLST revealed high genetic diversity with 196 different STs distributed over four phylogroups (Kp1 to Kp4). Low prevalence of ESBL-KP (7.5%) and colistin-resistant KP (3.2%) was observed among pigs and humans. Remarkably, four convergent MDR and hypervirulent strains were observed: one from pigs (ST290) and three from humans [ST35, ST3415 (strain 90CP1), ST17 (strain 90CM2)]. Sharing of KP clones among pigs and humans was identified for some STs including ST4788, ST661, ST3541 and ST29. CONCLUSIONS: The study indicated a low prevalence of ESBL and mcr genes among KP isolated from pigs and healthy humans in Thailand and suggested the possibility of zoonotic transmission for a subset of circulating KP clones.

Deciphering Heteroresistance to Colistin in a Klebsiella pneumoniae Isolate from Marseille, France.

Here, we report the description of a colistin-heteroresistant Klebsiella pneumoniae isolate fortuitously isolated from the stool sample of a patient with suspicion of tuberculosis in a public hospital of Marseille, France. In the colistin-resistant subpopulation, a mutation in the mgrB gene leading to a premature stop codon was found, and the hypermucoviscous phenotype was lost. Susceptibility to other antibiotics remained unchanged. To our knowledge, this is the first identification of such a colistin-heteroresistant Klebsiella pneumoniae isolate in France.

Risk factors for acquisition of CTX-M genes in pilgrims during Hajj 2013 and 2014.

Background: Mass gatherings, especially the Hajj pilgrimage, provide favourable conditions for bacterial transmission among participants, which might contribute to the international spread of ESBL-producing Enterobacteriaceae (ESBL-E). Objectives: We conducted an analysis aimed at investigating risk factors for CTX-M gene (blaCTX-M) rectal carriage in 2013 and 2014 Hajj pilgrims. Methods: A total of 218 pilgrims provided paired pre- and post-Hajj rectal samples (2013, 129 pilgrims; 2014, 89 pilgrims). CTX-M genes in rectal samples were identified by PCR and confirmed by sequencing. Pilgrims' characteristics, including possible factors relating to ESBL acquisition, were collected and analysed using XLSTAT version 2016.05.34687 (Addinsoft). For the univariate analysis, the frequencies of nominal data were compared using Pearson's χ2 test and Fisher's exact test, and the means of quantitative data were compared using Student's t-test. A difference was considered significant when P < 0.05. For multivariate analysis, a logistic regression was carried out, considering all the significant data in univariate analysis. Results: The acquisition rates of CTX-M genes among pilgrims during the 2013 and 2014 Hajj were similar, at 31.0% and 34.83%, respectively. Being of Moroccan origin, having chronic conditions, shortness of breath or diarrhoea, and using ß-lactams were associated with higher CTX-M gene acquisition, while being of Algerian origin and using macrolides were associated with lower CTX-M acquisition in univariate analysis. Shortness of breath and diarrhoea remained associated with increased CTX-M gene acquisition and consumption of macrolides with lower CTX-M gene acquisition in multivariate analysis. Conclusions: The possible gut colonization by CTX-M-type ESBL bacteria should be taken into account when prescribing empirical antibiotic treatments for infections that occur in returning Hajj pilgrims.

LBJMR medium: a new polyvalent culture medium for isolating and selecting vancomycin and colistin-resistant bacteria.

BACKGROUND: Multi-drug resistant bacteria are a phenomenon which is on the increase around the world, particularly with the emergence of colistin-resistant Enterobacteriaceae and vancomycin-resistant enterococci strains. The recent discovery of a plasmid-mediated colistin resistance with the description of the transferable mcr-1 gene raised concerns about the need for an efficient detection method for these pathogens, to isolate infected patients as early as possible. The LBJMR medium was developed to screen for all polymyxin-resistant Gram-negative bacteria, including mcr-1 positive isolates, and vancomycin-resistant Gram-positive bacteria. RESULTS: The LBJMR medium was developed by adding colistin sulfate salt at a low concentration (4 µg/mL) and vancomycin (50 µg/mL), with glucose (7.5 g/L) as a fermentative substrate, to a Purple Agar Base (31 g/L). A total of 143 bacterial strains were used to evaluate this universal culture medium, and the sensitivity and specificity of detection were 100% for the growth of resistant strains. 68 stool samples were cultured on LBJMR, and both colistin-resistant Gram-negative and vancomycin-resistant Gram-positive strains were specifically detected. CONCLUSIONS: The LBJMR medium is a multipurpose selective medium which makes it possible to identify bacteria of interest from clinical samples and to isolate contaminated patients in hospital settings. This is a simple medium that could be easily used for screening in clinical microbiology laboratories.

Whole-Genome Sequence of a blaOXA-48-Harboring Raoultella ornithinolytica Clinical Isolate from Lebanon.

We analyzed the whole-genome sequence of ablaOXA-48-harboringRaoultella ornithinolyticaclinical isolate from a patient in Lebanon. The size of theRaoultella ornithinolyticaCMUL058 genome was 5,622,862 bp, with a G+C content of 55.7%. We deciphered all the molecular mechanisms of antibiotic resistance, and we compared our genome to other availableR. ornithinolyticagenomes in GenBank. The resistome consisted of 9 antibiotic resistance genes, including a plasmidicblaOXA-48gene whose genetic organization is also described.

Acquisition of a High Diversity of Bacteria during the Hajj Pilgrimage, Including Acinetobacter baumannii with blaOXA-72 and Escherichia coli with blaNDM-5 Carbapenemase Genes.

Pilgrims returning from the Hajj (pilgrimage to Mecca) can be carriers of multidrug-resistant bacteria (MDR). Pharyngeal and rectal swab samples were collected from 98 pilgrims before and after they traveled to the Hajj in 2014 to investigate the acquisition of MDR bacteria. The bacterial diversity in pharyngeal swab samples was assessed by culture with selective media. There was a significantly higher diversity of bacteria in samples collected after the return from the Hajj than in those collected before (P = 0.0008). Surprisingly, Acinetobacter baumannii strains were isolated from 16 pharyngeal swab samples (1 sample taken during the Hajj and 15 samples taken upon return) and 26 post-Hajj rectal swab samples, while none were isolated from samples taken before the Hajj. Testing of all samples by real-time PCR targeting blaOXA-51 gave positive results for only 1% of samples taken during the Hajj, 21/90 (23.3%) pharyngeal swab samples taken post-Hajj, and 35/90 (38.9%) rectal swab samples taken post-Hajj. One strain of A. baumannii isolated from the pharynx was resistant to imipenem and harbored a blaOXA-72 carbapenemase gene. Multilocus sequence typing analysis of 43 A. baumannii isolates revealed a huge diversity of 35 sequence types (STs), among which 18 were novel STs reported for the first time in this study. Moreover, we also found one Escherichia coli isolate, collected from a rectal swab sample from a pilgrim taken after the Hajj, which harbored blaNDM-5, blaCTX-M-15, blaTEM-1, and aadA2 (ST2659 and ST181). In conclusion, pilgrims are at a potential risk of acquiring and transmitting MDR Acinetobacter spp. and carbapenemase-producing Gram-negative bacteria during the Hajj season.

Acquisition of Extended-Spectrum ß-Lactamases by Escherichia coli and Klebsiella pneumoniae in Gut Microbiota of Pilgrims during the Hajj Pilgrimage of 2013.

We reported the acquisition of extended-spectrum-ß-lactamase (ESBL)-producing bacteria in rectal samples of 129 pilgrims during the 2013 Hajj (pilgrimage to Makkah). When returning from the Hajj, there was a significant increase in the number of pilgrims carrying E. coli resistant to ceftriaxone (P = 0.008). The CTX-M gene was detected in rectal samples, with the detection rate increasing from 10.08% to 32.56% of samples after the Hajj (P < 0.001).

Diversity, functional classification and genotyping of SHV ß-lactamases in Klebsiella pneumoniae.

Interpreting the phenotypes of bla SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla SHV alleles or additional plasmid-borne bla SHV alleles that have extended-spectrum ß-lactamase (ESBL) activity and/or ß-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the ß-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI's Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla SHV-1) is the ancestral chromosomal variant, (ii) ESBL- and BLI-resistant variants have evolved multiple times through parallel substitution mutations, (iii) ESBL variants are mostly mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla SHV. We used matched genome-phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K. pneumoniae isolates carrying one or more bla SHV alleles but no other acquired ß-lactamases to assess genotype-phenotype relationships for bla SHV. This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae), whereby known and novel bla SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve the understanding of resistance mechanisms.

Highly conserved composite transposon harbouring aerobactin iuc3 in Klebsiella pneumoniae from pigs.

Klebsiella pneumoniae is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent K. pneumoniae, which are K. pneumoniae with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This Klebsiella pathotype is capable of causing liver abscesses in immunocompetent persons in the community. K. pneumoniae has not been extensively studied in non-human niches. In the present study, K. pneumoniae isolated from caecal samples (n=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on K. pneumoniae from healthy pigs in Thailand. Lastly, an in-depth plasmid study on K. pneumoniae with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize K. pneumoniae isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (n=299) were positive for K. pneumoniae. Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were iuc3. In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIBK/FIIK plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS3-family IS407-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by K. pneumoniae from several countries and sources, such as human clinical samples. The high occurrence of K. pneumoniae harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for K. pneumoniae with iuc3. Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.

Comparative genomics of two Shewanella xiamenensis strains isolated from a pilgrim before and during travels to the Hajj.

BACKGROUND: Shewanella xiamenensis has been reported in water environment and in patients and can act as the originator of oxacillinase in gram-negative bacteria. In order to assess genome plasticity and its functional properties related diarrhea symptoms in pilgrim, comparisons of draft genome sequences of the two isolates were conducted with other closely related genomes. RESULTS: We isolated S. xiamenensis 111B and 111D strains from a pilgrim before travels to the Hajj and during travels with diarrhea symptom, respectively. Whole-genome sequencing showed that draft genome size of 111B strain was 5,008,191 bp, containing 49 kb of a putative plasmid. The genome size of 111D was 4,964,295 bp containing 225 kb of a putative plasmid that shared the backbone sequences with the hospital wastewater strain T17. Comparatively, two Hajj strains are identical at 97.3% identity and 98.7% coverage. They are closely related to river water strain, AS58 by SNPs analysis. Notably, a novel blaOXA-48 allele blaOXA-547 was identified in 111D, sharing 99.5% identity with blaOXA-546 and blaOXA-894. Multiple copies of virulence specific genes, such as capsular polysaccharide biosynthesis, O-antigen and lasB (vibriolysin related gene) have been identified specifically in 111D, but absent in 111B strain. CONCLUSIONS: The whole genome sequences of S. xiamenensis strain 111B and 111D, including comparative genomic analysis, highlight here the potential for virulence factors that might be related to the cause of diarrhea in humans and also indicate the possible acquisition of pathogenic bacteria, including antibiotic resistance genes or plasmids during the Hajj.

Exploring the Antibiotic Resistance Burden in Livestock, Livestock Handlers and Their Non-Livestock Handling Contacts: A One Health Perspective.

Antibiotics are freqeuently used in the livestock sector in low- and middle-income countries for treatment, prophylaxis, and growth promotion. However, there is limited information into the zoonotic prevalence and dissemination patterns of antimicrobial resistance (AMR) within these environments. In this study we used pig farming in Thailand as a model to explore AMR; 156 pig farms were included, comprising of small-sized (<50 sows) and medium-sized (≥100 sows) farms, where bacterial isolates were selectively cultured from animal rectal and human fecal samples. Bacterial isolates were subjected to antimicrobial susceptibility testing (AST), and whole-genome sequencing. Our results indicate extensive zoonotic sharing of antibiotic resistance genes (ARGs) by horizontal gene transfer. Resistance to multiple antibiotics was observed with higher prevalence in medium-scale farms. Zoonotic transmission of colistin resistance in small-scale farms had a dissemination gradient from pigs to handlers to non-livestock contacts. We highly recommend reducing the antimicrobial use in animals' feeds and medications, especially the last resort drug colistin.

Corrigendum: bla CTX-M-1 /IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

[This corrects the article DOI: 10.3389/fmicb.2020.00333.].