scholarly journals Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

2014 ◽  
Vol 80 (12) ◽  
pp. 3597-3603 ◽  
Author(s):  
Vicki Adams ◽  
Radhika Bantwal ◽  
Lauren Stevenson ◽  
Jackie K. Cheung ◽  
Milena M. Awad ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Genes ◽  
Dna Fragments ◽  
Site Specific ◽  
Content Type ◽  
E Coli ◽  
Marker Recycling ◽  
A Site

ABSTRACTTnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451and Tn4453inClostridium perfringensandClostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids inEscherichia coliand from marked chromosomalC. perfringensmutants. This methodology enabled the construction of aC. perfringensplc virRdouble mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic toE. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes inE. colifor subsequent expression in the heterologous hostC. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

scholarly journals Predicting Antibiotic Resistance in Gram-Negative Bacilli from Resistance Genes

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
G. Terrance Walker ◽  
Julia Quan ◽  
Stephen G. Higgins ◽  
Nikhil Toraskar ◽  
Weizhong Chang ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Unknown Origin ◽  
Bacterial Isolates ◽  
Antibiotic Resistant ◽  
Predictive Values ◽  
Content Type ◽  
Phenotypic Resistance ◽  
E Coli

ABSTRACT We developed a rapid high-throughput PCR test and evaluated highly antibiotic-resistant clinical isolates of Escherichia coli (n = 2,919), Klebsiella pneumoniae (n = 1,974), Proteus mirabilis (n = 1,150), and Pseudomonas aeruginosa (n = 1,484) for several antibiotic resistance genes for comparison with phenotypic resistance across penicillins, cephalosporins, carbapenems, aminoglycosides, trimethoprim-sulfamethoxazole, fluoroquinolones, and macrolides. The isolates originated from hospitals in North America (34%), Europe (23%), Asia (13%), South America (12%), Africa (7%), or Oceania (1%) or were of unknown origin (9%). We developed statistical methods to predict phenotypic resistance from resistance genes for 49 antibiotic-organism combinations, including gentamicin, tobramycin, ciprofloxacin, levofloxacin, trimethoprim-sulfamethoxazole, ertapenem, imipenem, cefazolin, cefepime, cefotaxime, ceftazidime, ceftriaxone, ampicillin, and aztreonam. Average positive predictive values for genotypic prediction of phenotypic resistance were 91% for E. coli, 93% for K. pneumoniae, 87% for P. mirabilis, and 92% for P. aeruginosa across the various antibiotics for this highly resistant cohort of bacterial isolates.

scholarly journals F Plasmids Are the Major Carriers of Antibiotic Resistance Genes in Human-Associated Commensal Escherichia coli

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Craig Stephens ◽  
Tyler Arismendi ◽  
Megan Wright ◽  
Austin Hartman ◽  
Andres Gonzalez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Transposable Elements ◽  
Dna Sequencing ◽  
Resistance Genes ◽  
Bacterial Pathogens ◽  
Acquired Resistance ◽  
Antibiotic Resistance Genes ◽  
Content Type ◽  
E Coli

ABSTRACT The evolution and propagation of antibiotic resistance by bacterial pathogens are significant threats to global public health. Contemporary DNA sequencing tools were applied here to gain insight into carriage of antibiotic resistance genes in Escherichia coli, a ubiquitous commensal bacterium in the gut microbiome in humans and many animals, and a common pathogen. Draft genome sequences generated for a collection of 101 E. coli strains isolated from healthy undergraduate students showed that horizontally acquired antibiotic resistance genes accounted for most resistance phenotypes, the primary exception being resistance to quinolones due to chromosomal mutations. A subset of 29 diverse isolates carrying acquired resistance genes and 21 control isolates lacking such genes were further subjected to long-read DNA sequencing to enable complete or nearly complete genome assembly. Acquired resistance genes primarily resided on F plasmids (101/153 [67%]), with smaller numbers on chromosomes (30/153 [20%]), IncI complex plasmids (15/153 [10%]), and small mobilizable plasmids (5/153 [3%]). Nearly all resistance genes were found in the context of known transposable elements. Very few structurally conserved plasmids with antibiotic resistance genes were identified, with the exception of an ∼90-kb F plasmid in sequence type 1193 (ST1193) isolates that appears to serve as a platform for resistance genes and may have virulence-related functions as well. Carriage of antibiotic resistance genes on transposable elements and mobile plasmids in commensal E. coli renders the resistome highly dynamic. IMPORTANCE Rising antibiotic resistance in human-associated bacterial pathogens is a serious threat to our ability to treat many infectious diseases. It is critical to understand how acquired resistance genes move in and through bacteria associated with humans, particularly for species such as Escherichia coli that are very common in the human gut but can also be dangerous pathogens. This work combined two distinct DNA sequencing approaches to allow us to explore the genomes of E. coli from college students to show that the antibiotic resistance genes these bacteria have acquired are usually carried on a specific type of plasmid that is naturally transferrable to other E. coli, and likely to other related bacteria.

scholarly journals bla OXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands

2021 ◽  
Vol 7 (5) ◽  
Author(s):  
Antoni P. A. Hendrickx ◽  
Fabian Landman ◽  
Angela de Haan ◽  
Sandra Witteveen ◽  
Marga G. van Santen-Verheuvel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
The Netherlands ◽  
Resistance Genes ◽  
Type Species ◽  
Antibiotic Resistance Genes ◽  
Gene Content ◽  
Content Type ◽  
E Coli ◽  
Link Type

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by bla OXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the bla OXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete bla OXA-48-like plasmids for E. coli and K. pneumoniae , respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the bla OXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded bla OXA-48-like alleles. Chromosomally localized bla OXA-48 and bla OXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The bla OXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli . Lastly, K. pneumoniae isolates carrying bla OXA-48 or bla OXA-232 were mostly resistant for meropenem, whereas E. coli bla OXA-48, bla OXA-181 and chromosomal bla OXA-48 or bla OXA-244 isolates were mostly sensitive. In conclusion, the overall bla OXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of bla OXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

scholarly journals Phylum barrier and Escherichia coli intra-species phylogeny drive the acquisition of antibiotic-resistance genes

2021 ◽  
Vol 7 (8) ◽  
Author(s):  
Marie Petitjean ◽  
Bénédicte Condamine ◽  
Charles Burdet ◽  
Erick Denamur ◽  
Etienne Ruppé
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Type Species ◽  
Antibiotic Resistance Genes ◽  
Resistance Pattern ◽  
Content Type ◽  
E Coli ◽  
Link Type ◽  
Specific Distribution

Escherichia coli is a ubiquitous bacterium that has been widely exposed to antibiotics over the last 70 years. It has adapted by acquiring different antibiotic-resistance genes (ARGs), the census of which we aim to characterize here. To do so, we analysed 70 301 E. coli genomes obtained from the EnteroBase database and detected 1 027 651 ARGs using the AMRFinder, Mustard and ResfinderFG ARG databases. We observed a strong phylogroup and clonal lineage specific distribution of some ARGs, supporting the argument for epistasis between ARGs and the strain genetic background. However, each phylogroup had ARGs conferring a similar antibiotic class resistance pattern, indicating phenotypic adaptive convergence. The G+C content or the type of ARG was not associated with the frequency of the ARG in the database. In addition, we identified ARGs from anaerobic, non- Proteobacteria bacteria in four genomes of E. coli , supporting the hypothesis that the transfer between anaerobic bacteria and E. coli can spontaneously occur but remains exceptional. In conclusion, we showed that phylum barrier and intra-species phylogenetic history are major drivers of the acquisition of a resistome in E. coli .

scholarly journals The Transferable Resistome of Produce

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Khald Blau ◽  
Antje Bettermann ◽  
Sven Jechalke ◽  
Eva Fornefeld ◽  
Yann Vanrobaeys ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Blot Hybridization ◽  
Antibiotic Resistance Genes ◽  
Human Pathogens ◽  
Content Type ◽  
E Coli

ABSTRACTProduce is increasingly recognized as a reservoir of human pathogens and transferable antibiotic resistance genes. This study aimed to explore methods to characterize the transferable resistome of bacteria associated with produce. Mixed salad, arugula, and cilantro purchased from supermarkets in Germany were analyzed by means of cultivation- and DNA-based methods. Before and after a nonselective enrichment step, tetracycline (TET)-resistantEscherichia coliwere isolated and plasmids conferring TET resistance were captured by exogenous plasmid isolation. TET-resistantE. coliisolates, transconjugants, and total community DNA (TC-DNA) from the microbial fraction detached from leaves or after enrichment were analyzed for the presence of resistance genes, class 1 integrons, and various plasmids by real-time PCR and PCR-Southern blot hybridization. Real-time PCR primers were developed for IncI and IncF plasmids. TET-resistantE. coliisolated from arugula and cilantro carried IncF, IncI1, IncN, IncHI1, IncU, and IncX1 plasmids. Three isolates from cilantro were positive for IncN plasmids andblaCTX-M-1. From mixed salad and cilantro, IncF, IncI1, and IncP-1β plasmids were captured exogenously. Importantly, whereas direct detection of IncI and IncF plasmids in TC-DNA failed, these plasmids became detectable in DNA extracted from enrichment cultures. This confirms that cultivation-independent DNA-based methods are not always sufficiently sensitive to detect the transferable resistome in the rare microbiome. In summary, this study showed that an impressive diversity of self-transmissible multiple resistance plasmids was detected in bacteria associated with produce that is consumed raw, and exogenous capturing intoE. colisuggests that they could transfer to gut bacteria as well.IMPORTANCEProduce is one of the most popular food commodities. Unfortunately, leafy greens can be a reservoir of transferable antibiotic resistance genes. We found that IncF and IncI plasmids were the most prevalent plasmid types inE. coliisolates from produce. This study highlights the importance of the rare microbiome associated with produce as a source of antibiotic resistance genes that might escape cultivation-independent detection, yet may be transferred to human pathogens or commensals.

scholarly journals Discovery ofmcr-1-Mediated Colistin Resistance in a Highly VirulentEscherichia coliLineage

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Brian M. Forde ◽  
Hosam M. Zowawi ◽  
Patrick N. A. Harris ◽  
Leah Roberts ◽  
Emad Ibrahim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Neonatal Meningitis ◽  
Colistin Resistance ◽  
Content Type ◽  
E Coli ◽  
Virulence Potential ◽  
High Virulence

ABSTRACTResistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene (mcr-1) represents a new threat to global human health. Here we present the complete genome sequence of anmcr-1-positive multidrug-resistantEscherichia colistrain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum β-lactamaseblaCTX-M-1) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated withE. colibacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulentE. colilineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult.IMPORTANCEEscherichia coliST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of anE. coliST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-bornemcr-1gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage.

scholarly journals Draft Genome Sequences of Cefepime-Resistant and -Susceptible Escherichia coli Strains and Imipenem-Resistant and -Susceptible Pseudomonas aeruginosa Strains

2021 ◽  
Vol 10 (48) ◽  
Author(s):  
Kendra Batchelder ◽  
Liz Ward ◽  
Elsa Collins ◽  
Caitlin Miles ◽  
Stefania Palm ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Draft Genome ◽  
Antibiotic Resistance Genes ◽  
Genome Sequences ◽  
Content Type ◽  
E Coli

Draft genome sequences of Escherichia coli and Pseudomonas aeruginosa strains collected from clinical infections were used to determine the prevalence of newly emerging antibiotic resistance genes in Maine. Comparisons between cefepime-resistant and -susceptible E. coli strains and imipenem-resistant and -susceptible P. aeruginosa strains are being conducted.

scholarly journals Comparative Genome Analysis of an Extensively Drug-Resistant Isolate of Avian Sequence Type 167 Escherichia coli Strain Sanji with Novel In Silico Serotype O89b:H9

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Xiancheng Zeng ◽  
Xuelin Chi ◽  
Brian T. Ho ◽  
Damee Moon ◽  
Christine Lambert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
In Silico ◽  
Antibiotic Resistance Genes ◽  
Secretion System ◽  
Sequence Type ◽  
Content Type ◽  
E Coli ◽  
Multiple Paths

ABSTRACT Extensive drug resistance (XDR) is an escalating global problem. Escherichia coli strain Sanji was isolated from an outbreak of pheasant colibacillosis in Fujian province, China, in 2011. This strain has XDR properties, exhibiting sensitivity to carbapenems but no other classes of known antibiotics. Whole-genome sequencing revealed a total of 32 known antibiotic resistance genes, many associated with insertion sequence 26 (IS26) elements. These were found on the Sanji chromosome and 2 of its 6 plasmids, pSJ_255 and pSJ_82. The Sanji chromosome also harbors a type 2 secretion system (T2SS), a type 3 secretion system (T3SS), a type 6 secretion system (T6SS), and several putative prophages. Sanji and other ST167 strains have a previously uncharacterized O-antigen (O89b) that is most closely related to serotype O89 as determined on the basis of analysis of the wzm-wzt genes and in silico serotyping. This O89b-antigen gene cluster was also found in the genomes of a few other pathogenic sequence type 617 (ST617) and ST10 complex strains. A time-scaled phylogeny inferred from comparative single nucleotide variant analysis indicated that development of these O89b-containing lineages emerged about 30 years ago. Comparative sequence analysis revealed that the core genome of Sanji is nearly identical to that of several recently sequenced strains of pathogenic XDR E. coli belonging to the ST167 group. Comparison of the mobile elements among the different ST167 genomes revealed that each genome carries a distinct set of multidrug resistance genes on different types of plasmids, indicating that there are multiple paths toward the emergence of XDR in E. coli. IMPORTANCE E. coli strain Sanji is the first sequenced and analyzed genome of the recently emerged pathogenic XDR strains with sequence type ST167 and novel in silico serotype O89b:H9. Comparison of the genomes of Sanji with other ST167 strains revealed distinct sets of different plasmids, mobile IS elements, and antibiotic resistance genes in each genome, indicating that there exist multiple paths toward achieving XDR. The emergence of these pathogenic ST167 E. coli strains with diverse XDR capabilities highlights the difficulty of preventing or mitigating the development of XDR properties in bacteria and points to the importance of better understanding of the shared underlying virulence mechanisms and physiology of pathogenic bacteria.

scholarly journals Limited and Strain-Specific Transcriptional and Growth Responses to Acquisition of a Multidrug Resistance Plasmid in Genetically Diverse Escherichia coli Lineages

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Steven Dunn ◽  
Laura Carrilero ◽  
Michael Brockhurst ◽  
Alan McNally
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Multidrug Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Growth Responses ◽  
Fitness Costs ◽  
Transcriptional Responses ◽  
Content Type ◽  
E Coli

ABSTRACT Multidrug-resistant (MDR) Escherichia coli strains are a major global threat to human health, wherein multidrug resistance is primarily spread by MDR plasmid acquisition. MDR plasmids are not widely distributed across the entire E. coli species, but instead are concentrated in a small number of clones. Here, we test if diverse E. coli strains vary in their ability to acquire and maintain MDR plasmids and if this relates to their transcriptional response following plasmid acquisition. We used strains from across the diversity of E. coli strains, including the common MDR lineage sequence type 131 (ST131) and the IncF plasmid pLL35, carrying multiple antibiotic resistance genes. Strains varied in their ability to acquire pLL35 by conjugation, but all were able to stably maintain the plasmid. The effects of pLL35 acquisition on cefotaxime resistance and growth also varied among strains, with growth responses ranging from a small decrease to a small increase in growth of the plasmid carrier relative to the parental strain. Transcriptional responses to pLL35 acquisition were limited in scale and highly strain specific. We observed transcriptional responses at the operon or regulon level—possibly due to stress responses or interactions with resident mobile genetic elements (MGEs). Subtle transcriptional responses consistent across all strains were observed affecting functions, such as anaerobic metabolism, previously shown to be under negative frequency-dependent selection in MDR E. coli. Overall, there was no correlation between the magnitudes of the transcriptional and growth responses across strains. Together, these data suggest that fitness costs arising from transcriptional disruption are unlikely to act as a barrier to dissemination of this MDR plasmid in E. coli. IMPORTANCE Plasmids play a key role in bacterial evolution by transferring adaptive functions between lineages that often enable invasion of new niches, including driving the spread of antibiotic resistance genes. Fitness costs of plasmid acquisition arising from the disruption of cellular processes could limit the spread of multidrug resistance plasmids. However, the impacts of plasmid acquisition are typically measured in lab-adapted strains rather than natural isolates, which act as reservoirs for the maintenance and transmission of plasmids to clinically relevant strains. Using a clinical multidrug resistance plasmid and a diverse collection of E. coli strains isolated from clinical infections and natural environments, we show that plasmid acquisition had only limited and highly strain-specific effects on bacterial growth and transcription under laboratory conditions. These findings suggest that fitness costs arising from transcriptional disruption are unlikely to act as a barrier to transmission of this plasmid in natural populations of E. coli.

scholarly journals Draft Genome Sequence of the Shrimp Pathogen Vibrio parahaemolyticus ST17.P5-S1, Isolated in Peninsular Malaysia

2018 ◽  
Vol 7 (11) ◽  
Author(s):  
Sridevi Devadas ◽  
Subha Bhassu ◽  
Tze Chiew Christie Soo ◽  
Fatimah M. Yusoff ◽  
Mohamed Shariff
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Vibrio Parahaemolyticus ◽  
East Coast ◽  
Draft Genome ◽  
Antibiotic Resistance Genes ◽  
Peninsular Malaysia ◽  
Penaeus Vannamei ◽  
Content Type ◽  
Acute Hepatopancreatic Necrosis Disease

We sequenced the genome of Vibrio parahaemolyticus strain ST17.P5-S1, isolated from Penaeus vannamei cultured in the east coast of Peninsular Malaysia. The strain contains several antibiotic resistance genes and a plasmid encoding the Photorhabdus insect-related (Pir) toxin-like genes, pirAvp and pirBvp, associated with acute hepatopancreatic necrosis disease (AHPND).

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