Laboratory Stock Variants of the Archetype Silver Resistance Plasmid pMG101 Demonstrate Plasmid Fusion, Loss of Transmissibility, and Transposition of Tn7/pco/sil Into the Host Chromosome

Salmonella Typhimurium carrying the multidrug resistance (MDR) plasmid pMG101 was isolated from three burns patients in Boston United States in 1973. pMG101 was transferrable into other Salmonella spp. and Escherichia coli hosts and carried what was a novel and unusual combination of AMR genes and silver resistance. Previously published short-read DNA sequence of pMG101 showed that it was a 183.5Kb IncHI plasmid, where a Tn7-mediated transposition of pco/sil resistance genes into the chromosome of the E. coli K-12 J53 host strain had occurred. We noticed differences in streptomycin resistance and plasmid size between two stocks of E. coli K-12 J53 pMG101 we possessed, which had been obtained from two different laboratories (pMG101-A and pMG101-B). Long-read sequencing (PacBio) of the two strains unexpectedly revealed plasmid and chromosomal rearrangements in both. pMG101-A is a non-transmissible 383Kb closed-circular plasmid consisting of an IncHI2 plasmid sequence fused to an IncFI/FIIA plasmid. pMG101-B is a mobile closed-circular 154 Kb IncFI/FIIA plasmid. Sequence identity of pMG101-B with the fused IncFI/IncFIIA region of pMG101-A was >99%. Assembled host sequence reads of pMG101-B showed Tn7-mediated transposition of pco/sil into the E. coli J53 chromosome between yhiM and yhiN. Long read sequence data in combination with laboratory experiments have demonstrated large scale changes in pMG101. Loss of conjugation function and movement of resistance genes into the chromosome suggest that even under long-term laboratory storage, mobile genetic elements such as transposons and insertion sequences can drive the evolution of plasmids and host. This study emphasises the importance of utilising long read sequencing technologies of plasmids and host strains at the earliest opportunity.

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The Small Noncoding DsrA RNA Is an Acid Resistance Regulator in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.186.18.6179-6185.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6179-6185 ◽

Cited By ~ 65

Author(s):

Richard A. Lease ◽

Dorie Smith ◽

Kathleen McDonough ◽

Marlene Belfort

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Resistance Genes ◽

Acid Resistance ◽

Dna Arrays ◽

E Coli ◽

Specific Mrnas ◽

Steady State Levels ◽

Control Translation ◽

K 12

ABSTRACT DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (σs), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.

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The large-scale blast score ratio (LS-BSR) pipeline: a method to rapidly compare genetic content between bacterial genomes

10.7287/peerj.preprints.220v1 ◽

2014 ◽

Author(s):

Jason W Sahl ◽

Greg Caporaso ◽

David A Rasko ◽

Paul S Keim

Keyword(s):

Large Scale ◽

Sequence Data ◽

Parallel Implementation ◽

Genetic Relationships ◽

Clinical Diagnostics ◽

Whole Genome Sequence ◽

Bacterial Isolates ◽

Bacterial Genomes ◽

E Coli ◽

Blast Score

Background. As whole genome sequence data from bacterial isolates becomes cheaper to generate, computational methods are needed to correlate sequence data with biological observations. Here we present the large-scale BLAST score ratio (LS-BSR) pipeline, which rapidly compares the genetic content of hundreds to thousands of bacterial genomes, and returns a matrix that describes the relatedness of all coding sequences (CDSs) in all genomes surveyed. This matrix can be easily parsed in order to identify genetic relationships between bacterial genomes. Although pipelines have been published that group peptides by sequence similarity, no other software performs the large-scale, flexible, full-genome comparative analyses carried out by LS-BSR. Results. To demonstrate the utility of the method, the LS-BSR pipeline was tested on 96 Escherichia coli and Shigella genomes; the pipeline ran in 163 minutes using 16 processors, which is a greater than 7-fold speedup compared to using a single processor. The BSR values for each CDS, which indicate a relative level of relatedness, were then mapped to each genome on an independent core genome single nucleotide polymorphism (SNP) based phylogeny. Comparisons were then used to identify clade specific CDS markers and validate the LS-BSR pipeline based on molecular markers that delineate between classical E. coli pathogenic variant (pathovar) designations. Scalability tests demonstrated that the LS-BSR pipeline can process 1,000 E. coli genomes in ~60h using 16 processors. Conclusions. LS-BSR is an open-source, parallel implementation of the BSR algorithm, enabling rapid comparison of the genetic content of large numbers of genomes. The results of the pipeline can be used to identify specific markers between user-defined phylogenetic groups, and to identify the loss and/or acquisition of genetic information between bacterial isolates. Taxa-specific genetic markers can then be translated into clinical diagnostics, or can be used to identify broadly conserved putative therapeutic candidates.

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Genomic dynamics of species and mobile genetic elements in a prolonged blaIMP-4-associated carbapenemase outbreak in an Australian hospital

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz526 ◽

2020 ◽

Vol 75 (4) ◽

pp. 873-882 ◽

Cited By ~ 3

Author(s):

A Kizny Gordon ◽

H T T Phan ◽

S I Lipworth ◽

E Cheong ◽

T Gottlieb ◽

...

Keyword(s):

Resistance Genes ◽

Genetic Relatedness ◽

Sequence Data ◽

Environmental Isolates ◽

Class 1 Integron ◽

Oxford Nanopore ◽

Class 1 ◽

Long Read ◽

Cleaning Methods ◽

Burns Unit

Abstract Background Hospital outbreaks of carbapenemase-producing organisms, such as blaIMP-4-containing organisms, are an increasing threat to patient safety. Objectives To investigate the genomic dynamics of a 10 year (2006–15) outbreak of blaIMP-4-containing organisms in a burns unit in a hospital in Sydney, Australia. Methods All carbapenem-non-susceptible or MDR clinical isolates (2006–15) and a random selection of equivalent or ESBL-producing environmental isolates (2012–15) were sequenced [short-read (Illumina), long-read (Oxford Nanopore Technology)]. Sequence data were used to assess genetic relatedness of isolates (Mash; mapping and recombination-adjusted phylogenies), perform in silico typing (MLST, resistance genes and plasmid replicons) and reconstruct a subset of blaIMP plasmids for comparative plasmid genomics. Results A total of 46/58 clinical and 67/96 environmental isolates contained blaIMP-4. All blaIMP-4-positive organisms contained five or more other resistance genes. Enterobacter cloacae was the predominant organism, with 12 other species mainly found in either the environment or patients, some persisting despite several cleaning methods. On phylogenetic analysis there were three genetic clusters of E. cloacae containing both clinical and environmental isolates, and an additional four clusters restricted to either reservoir. blaIMP-4 was mostly found as part of a cassette array (blaIMP-4-qacG2-aacA4-catB3) in a class 1 integron within a previously described IncM2 plasmid (pEl1573), with almost complete conservation of this cassette across the species over the 10 years. Several other plasmids were also implicated, including an IncF plasmid backbone not previously widely described in association with blaIMP-4. Conclusions Genetic backgrounds disseminating blaIMP-4 can persist, diversify and evolve amongst both human and environmental reservoirs during a prolonged outbreak despite intensive prevention efforts.

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Molecular and Phenotypic Characteristics ofEscherichia coliIsolates from Farmed Minks in Zhucheng, China

BioMed Research International ◽

10.1155/2019/3917841 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Jianhua Qiu ◽

Zhiyu Jiang ◽

Zijing Ju ◽

Xiaonan Zhao ◽

Jie Yang ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Large Scale ◽

Clonal Complex ◽

Sensitivity Testing ◽

Class 1 Integron ◽

Pcr Screening ◽

E Coli ◽

Antimicrobial Resistance Genes ◽

Class 1

In this study, the prevalence, phenotypes, and clonal relationships ofEscherichia coli(E. coli) strains isolated from minks were investigated. In July 2017, a total of 62 fresh faecal swab samples were randomly collected from one large-scale mink farm in Zhucheng, Shandong Province, China. In all the samples, 50E. colistrains were isolated and then assigned to serotyping, antimicrobial susceptibility test, detection of antimicrobial resistance genes and the Class 1 integrons, and multilocus sequence typing (MLST). Four pathogenic serotypes were identified among all the isolates, while the most common serotype was enterohemorrhagicE. coliO104:H4 (6.0 %). Antimicrobial sensitivity testing revealed that most isolates were susceptible to cefoxitin (96.0 %) and amikacin (82.0 %), while most isolates were resistant to ampicillin (92.0 %) and tetracycline (90.0 %). An analysis of the nucleotide sequences revealed that 7 isolates (14.0%) carried 4 types of Class 1 integron cassette, includingdfrA27+aadA2+qnrA(57.1%),dfrA17+aadA5(14.3%),dfrA12+aadA2(14.3%), anddfrA1+aadA1(14.3%). PCR screening showed that 14 antibiotic resistance genes were presented in 50 isolates, while the most prevalent resistance gene wasqnrS, which was detected in 60.0 % of isolates, followed bysul2(40.0%) andoqxA(38.0%). MLST analysis showed that 32 sequence types (STs) were identified, while ST46 was the predominant genotype among all isolates. Clonal complex 3 (CC3) was dominant. Compared with 340 humanE. coliSTs reported in China, the ST10 clonal complex, known as the largest human clonal complex, was also found in the 50 minkE. coliisolates. Meanwhile, mink-derived strain ST206 formed a new clonal complex, CC206, which was different from human ST strains. Our results showed that farmed minks could be reservoirs of antimicrobial-resistantE. coliwith Class 1 integron cassettes and resistance genes, which were likely to pose a threat to public health. Therefore, continuous inspections and monitoring ofE. coliin minks are essential for detecting and controlling emergingE. coliwith different serovars as well as antibiotic resistance.

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Interaction of a colicinogenic factor with a resistance factor and with the fertility factor F inEscherichia coliK-12

Genetics Research ◽

10.1017/s0016672300012143 ◽

1971 ◽

Vol 17 (2) ◽

pp. 151-159 ◽

Cited By ~ 5

Author(s):

Pearl Cooper

Keyword(s):

Resistance Genes ◽

Recombinant Plasmid ◽

Resistance Factor ◽

E Coli ◽

Fertility Factor ◽

Plasmid Incompatibility ◽

R Factor ◽

Transmissible Plasmid ◽

K 12 ◽

R Factors

SUMMARYThe resistance (R)-factor R538–1drdof three F-like R-factors tested can cause the transfer of the non-transmissible ColV determinant fromE. coliV. The col factor, once transferred toE. coliK-12 strains, was shown to be related to the fertility factor, F, on the basis of entry exclusion and plasmid incompatibility. The col factor was found to recombine with R538–1drd, yielding a transmissible plasmid comprised of the col determinant and the sex factor of the R-factor, but not the resistance genes. The recombinant plasmid was found to be incompatible with both R538–1drdand F, and excluded the entry of R538–1drdbut not F.

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Non-human primates in the Gambia harbour human-associated pathogenic Escherichia coli strains

Access Microbiology ◽

10.1099/acmi.ac2020.po0781 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Ebenezer Foster-Nyarko ◽

Nabil-Fareed Alikhan ◽

Anuradha Ravi ◽

Gaëtan Thilliez ◽

Nicholas Thomson ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

The Gambia ◽

Public Health Importance ◽

Software Environment ◽

E Coli ◽

Antimicrobial Resistance Genes ◽

Long Read ◽

Sequence Types

Increasing contact between humans and non-human primates provides an opportunity for the transfer of potential pathogens or antimicrobial resistance between different host species. We have investigated genetic diversity and antimicrobial resistance in Escherichia coli isolates from a range of non-human primates dispersed across the Gambia: patas monkey (n=1), western colobus monkey (n=6), green monkey (n=14) and guinea baboon (n=22). From 43 stools, we recovered 99 isolates. We performed Illumina whole-genome shotgun sequencing on all isolates and nanopore long-read sequencing on isolates with antimicrobial resistance genes. We inferred the evolution of E. coli in this population using the EnteroBase software environment. We identified 43 sequence types (ten of them novel), spanning five of the eight known phylogroups of E. coli. Many of the observed sequence types and phylotypes from non-human primates have been associated with human extra-intestinal infection and carry virulence characteristics associated with disease in humans, particularly ST73, ST217 and ST681. However, we found a low prevalence of antimicrobial resistance genes in isolates from non-human primates. Hierarchical clustering showed that ST442 and ST349 from non-human primates are closely related to isolates from human infections, suggesting recent exchange of bacteria between humans and monkeys. Our results are of public health importance, considering the increasing contact between humans and wild primates.

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LINKS: Scaffolding genome assemblies with kilobase-long nanopore reads

10.1101/016519 ◽

2015 ◽

Cited By ~ 4

Author(s):

Rene L Warren ◽

Benjamin P Vandervalk ◽

Steven JM Jones ◽

Inanc Birol

Keyword(s):

Genome Assembly ◽

Error Rates ◽

Great Promise ◽

Genomic Information ◽

E Coli ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read ◽

K 12 ◽

Genome Assemblies

Owing to the complexity of the assembly problem, we do not yet have complete genome sequences. The difficulty in assembling reads into finished genomes is exacerbated by sequence repeats and the inability of short reads to capture sufficient genomic information to resolve those problematic regions. Established and emerging long read technologies show great promise in this regard, but their current associated higher error rates typically require computational base correction and/or additional bioinformatics pre-processing before they could be of value. We present LINKS, the Long Interval Nucleotide K-mer Scaffolder algorithm, a solution that makes use of the information in error-rich long reads, without the need for read alignment or base correction. We show how the contiguity of an ABySS E. coli K-12 genome assembly could be increased over five-fold by the use of beta-released Oxford Nanopore Ltd. (ONT) long reads and how LINKS leverages long-range information in S. cerevisiae W303 ONT reads to yield an assembly with less than half the errors of competing applications. Re-scaffolding the colossal white spruce assembly draft (PG29, 20 Gbp) and how LINKS scales to larger genomes is also presented. We expect LINKS to have broad utility in harnessing the potential of long reads in connecting high-quality sequences of small and large genome assembly drafts. Availability: http://www.bcgsc.ca/bioinfo/software/links

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MinION Analysis and Reference Consortium: Phase 1 data release and analysis

F1000Research ◽

10.12688/f1000research.7201.1 ◽

2015 ◽

Vol 4 ◽

pp. 1075 ◽

Cited By ~ 183

Author(s):

Camilla L.C. Ip ◽

Matthew Loose ◽

John R. Tyson ◽

Mariateresa de Cesare ◽

Bonnie L. Brown ◽

...

Keyword(s):

Large Scale ◽

Phase 1 ◽

Public Access ◽

Quality Of Data ◽

E Coli ◽

Oxford Nanopore ◽

Access Programme ◽

K 12 ◽

Consistency Rate

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.

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Transfer of Class 1 Integron-Mediated Antibiotic Resistance Genes from Shiga Toxin-Producing Escherichia coli to a Susceptible E. coli K-12 Strain in Storm Water and Bovine Feces

Applied and Environmental Microbiology ◽

10.1128/aem.00517-08 ◽

2008 ◽

Vol 74 (16) ◽

pp. 5063-5067 ◽

Cited By ~ 20

Author(s):

Supakana Nagachinta ◽

Jinru Chen

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Shiga Toxin ◽

Antibiotic Resistance Genes ◽

Storm Water ◽

Class 1 Integron ◽

E Coli ◽

Class 1 ◽

K 12 ◽

Bovine Feces

ABSTRACT Transfer of class 1 integron-mediated antibiotic resistance genes has been demonstrated under laboratory conditions. However, there is no information concerning the transfer of these genes in an agricultural environment. The present study sought to determine if integron-mediated streptomycin and sulfisoxazole resistance genes could be transferred from Shiga toxin-producing Escherichia coli (STEC) strains 6-20 (O157:H7) and 7-63 (O111:H8) to the susceptible strain E. coli K-12 MG1655 in bovine feces (pH 5.5, 6.0, or 6.5) and storm water (pH 5, 6, 7, or 8) at 4, 15, and 28°C, which are average seasonal temperatures for winter, spring-fall, and summer, respectively, in the Griffin, GA, area. The results indicated that at 28°C, the integron-mediated antibiotic resistance genes were transferred from both of the STEC donors in bovine feces. Higher conjugation efficiencies were, however, observed in the conjugation experiments involving STEC strain 6-20. In storm water, the resistance genes were transferred only from STEC strain 6-20. Greater numbers of transconjugants were recovered in the conjugation experiments performed with pH 6.5 bovine feces and with pH 7 storm water. Antibiotic susceptibility tests confirmed the transfer of integron-mediated streptomycin resistance and sulfisoxazole resistance, as well as the transfer of non-integron-mediated oxytetracycline resistance and tetracycline resistance in the transconjugant cells. These results suggest that the antibiotic resistance genes in STEC could serve as a source of antibiotic resistance genes disseminated via conjugation to susceptible cells of other E. coli strains in an agricultural environment.

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Adaptation of Sucrose Metabolism in the Escherichia coli Wild-Type Strain EC3132†

Journal of Bacteriology ◽

10.1128/jb.184.19.5307-5316.2002 ◽

2002 ◽

Vol 184 (19) ◽

pp. 5307-5316 ◽

Cited By ~ 54

Author(s):

Knut Jahreis ◽

Lars Bentler ◽

Jürgen Bockmann ◽

Stephan Hans ◽

Astrid Meyer ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Hot Spot ◽

Chromosomal Rearrangements ◽

Enteric Bacteria ◽

Transcriptional Level ◽

Wild Type ◽

New Host ◽

E Coli ◽

K 12

ABSTRACT Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.

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