10 research outputs found
Identification of a Type IV-A CRISPR-Cas System Located Exclusively on IncHI1B/IncFIB Plasmids in Enterobacteriaceae
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are diverse immune systems found in many prokaryotic genomes that target invading foreign DNA such as bacteriophages and plasmids. There are multiple types of CRISPR with arguably the most enigmatic being Type IV. During an investigation of CRISPR carriage in clinical, multi-drug resistant, Klebsiella pneumoniae, a Type IV-A3 CRISPR-Cas system was detected on plasmids from two K. pneumoniae isolates from Egypt (isolated in 2002-2003) and a single K. pneumoniae isolate from the UK (isolated in 2017). Sequence analysis of all other genomes available in GenBank revealed that this CRISPR-Cas system was present on 28 other plasmids from various Enterobacteriaceae hosts and was never found on a bacterial chromosome. This system is exclusively located on IncHI1B/ IncFIB plasmids and is associated with multiple putative transposable elements. Expression of the cas loci was confirmed in the available clinical isolates by RT-PCR. In all cases, the CRISPR-Cas system has a single CRISPR array (CRISPR1) upstream of the cas loci which has several, conserved, spacers which, amongst things, match regions within conjugal transfer genes of IncFIIK/ IncFIB(K) plasmids. Our results reveal a Type IV-A3 CRISPR-Cas system exclusively located on IncHI1B/ IncFIB plasmids in Enterobacteriaceae that is likely to be able to target IncFIIK/ IncFIB(K) plasmids presumably facilitating intracellular, inter-plasmid competition
御用留記(天保十三壬寅年十一月一日より三十日迄、益子信彭)
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated cas genes are sequence-specific DNA nuclease systems found in bacteria and archaea. CRISPR/Cas systems use RNA transcripts of previously acquired DNA (spacers) to target invading genetic elements with the same sequence, including plasmids. In this research we studied the relationship between CRISPR/Cas systems and multidrug resistance in Escherichia coli . The presence of Type I-E and Type I-F CRISPR systems was investigated among 82 antimicrobial-susceptible and 96 MDR clinical E. coli isolates by PCR and DNA sequencing. Phylogrouping and MLST were performed to determine relatedness of isolates. RT-PCR was performed to ascertain the expression of associated cas genes. Type I-F CRISPR was associated with the B2 phylogroup and was significantly overrepresented in the susceptible group (22.0%) compared with the MDR group (2.1%). The majority of CRISPR I-F-containing isolates had spacer sequences that matched IncF and IncI plasmids. RT-PCR demonstrated that Type I-F cas genes were expressed and therefore potentially functional. The CRISPR I-F system is more likely to be found in antimicrobial-susceptible E. coli . Given that the Type I-F system is expressed in WT isolates, we suggest that this difference could be due to the CRISPR system potentially interfering with the acquisition of antimicrobial resistance plasmids, maintaining susceptibility in these isolates
Development of teixobactin analogues containing hydrophobic, non-proteogenic amino acids that are highly potent against multidrug-resistant bacteria and biofilms.
Teixobactin is a cyclic undecadepsipeptide that has shown excellent potency against multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). In this article, we present the design, synthesis, and antibacterial evaluations of 16 different teixobactin analogues. These simplified analogues contain commercially available hydrophobic, non-proteogenic amino acid residues instead of synthetically challenging expensive L-allo-enduracididine amino acid residue at position 10 together with different combinations of arginines at positions 3, 4 and 9. The new teixobactin analogues showed potent antibacterial activity against a broad panel of Gram-positive bacteria, including MRSA and VRE strains. Our work also presents the first demonstration of the potent antibiofilm activity of teixobactin analogoues against Staphylococcus species associated with serious chronic infections. Our results suggest that the use of hydrophobic, non-proteogenic amino acids at position 10 in combination with arginine at positions 3, 4 and 9 holds the key to synthesising a new generation of highly potent teixobactin analogues to tackle resistant bacterial infections and biofilms
Development of teixobactin analogues containing hydrophobic, non-proteogenic amino acids that are highly potent against multidrug-resistant bacteria and biofilms
Teixobactin is a cyclic undecadepsipeptide that has shown excellent potency against multidrug-resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). In this article, we present the design, synthesis, and antibacterial evaluations of 16 different teixobactin analogues. These simplified analogues contain commercially available hydrophobic, non-proteogenic amino acid residues instead of synthetically challenging expensive L-allo-enduracididine amino acid residue at position 10 together with different combinations of arginines at positions 3, 4 and 9. The new teixobactin analogues showed potent antibacterial activity against a broad panel of Gram-positive bacteria, including MRSA and VRE strains. Our work also presents the first demonstration of the potent antibiofilm activity of teixobactin analogoues against Staphylococcus species associated with serious chronic infections. Our results suggest that the use of hydrophobic, non-proteogenic amino acids at position 10 in combination with arginine at positions 3, 4 and 9 holds the key to synthesising a new generation of highly potent teixobactin analogues to tackle resistant bacterial infections and biofilms
Detection of new SHV-12, SHV-5 and SHV-2a variants of extended spectrum beta-lactamase in Klebsiella pneumoniae in Egypt.
BACKGROUND: Klebsiella pneumoniae outbreaks possessing extended-spectrum β-lactamase- (ESBL) mediated resistance to third-generation cephalosporins have increased significantly in hospital and community settings worldwide. The study objective was to characterize prevalent genetic determinants of TEM, SHV and CTX-M types ESBL activity in K. pneumoniae isolates from Egypt. METHODS: Sixty five ESBL-producing K. pneumoniae strains, isolated from nosocomial and community-acquired infections from 10 Egyptian University hospitals (2000-2003), were confirmed with double disc-synergy method and E-test. blaTEM, blaSHV and blaCTX-m genes were identified by PCR and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was conducted for genotyping. RESULTS: All isolates displayed ceftazidime and cefotaxime resistance. blaTEM and blaSHV genes were detected in 98% of the isolates' genomes, while 11% carried blaCTX-m. DNA sequencing revealed plasmid-borne SHV-12,-5,-2a (17%), CTX-m-15 (11%), and TEM-1 (10%) prevalence. Among SHV-12 (n=8), one isolate displayed 100% blaSHV-12 amino acid identity, while others had various point mutations: T17G (Leu to Arg, position 6 of the enzyme: n=2); A8T and A10G (Tyr and Ile to Phe and Val, positions 3 and 4, respectively: n=4), and; A703G (Lys to Glu 235: n=1). SHV-5 and SHV-2a variants were identified in three isolates: T17G (n=1); A703G and G705A (Ser and Lys to Gly and Glu: n=1); multiple mutations at A8T, A10G, T17G, A703G and G705A (n=1). Remarkably, 57% of community-acquired isolates carried CTX-m-15. PFGE demonstrated four distinct genetic clusters, grouping strains of different genetic backgrounds. CONCLUSIONS: This is the first study demonstrating the occurrence of SHV-12, SHV-5 and SHV-2a variants in Egypt, indicating the spread of class A ESBL in K. pneumoniae through different mechanisms
Molecular Characterisation of MDR Klebsiella pneumoniae and Escherichia coli isolates from Egypt containing Transferable Resistance to β-Lactam Antibiotics
The significant increase in antimicrobial resistance (AMR) over the past few years is a serious global public health concern, particularly in the developing countries, where infections with resistant organisms are associated with poor clinical reporting and higher cost burdens. The objectives of this research were to characterise multidrug-resistant (MDR) Klebsiella pneumoniae and Escherichia coli isolates from Egypt and evaluate the bacterial fitness associated with the acquisition of transferable, plasmid-based, resistance to β-Lactam antibiotics. A total of 169 MDR Enterobacteriaceae isolates from nosocomial and community-acquired infections were collected from 10 different university teaching hospitals on two surveillance studies and were divided into three groups (group 1: 65 K. pneumoniae from 2000-2003, and group 2: 63 K. pneumoniae from 2009-2011), and (group 3: 41 E. coli from 2009-2011). Phenotypic tests were performed according to CLSI 2013. Genotypic characterisation of blaCTX-M-15, blaOXA-48, and blaVIM distribution were performed by PCR and sequence analysis. The distribution of class 1 and class 2 integrons, and repeat-spacer CRISPR-Cas systems were analysed. Selected resistance plasmids transferability, stability and fitness were evaluated. An increase in multidrug resistance and class 1 integrons over time in the K. pneumoniae isolates was observed. A high prevalence of multidrug resistance, and class 1 and class 2 integrons were observed among the E. coli isolates. Types I-E* and IV-B CRISPR-Cas were detected among the isolates, showing hypervariability, and an inverse correlation with AMR prevalence. The transfer of certain β-Lactam resistance plasmids revealed that positive epistasis plays an important role in stabilising resistance within the analysed transconjugants. The new insights reveal the potential for the stable accumulation of β-lactam resistance and other antibiotic resistances over time in bacterial cells and the lower tendency for losing those resistances even in the absence of antibiotic pressure
Isolation of an antimicrobial resistant, biofilm forming, Klebsiella grimontii isolate from a re-usable water bottle.
A re-usable water bottle was swabbed as part of the citizen science project Swab and Send and a Klebsiella grimontii isolate was recovered on chromogenic agar and designated SS141. Whole genome sequencing of SS141 showed it has the potential to be a human pathogen as it contains the biosynthetic gene cluster for the potent cytotoxin, kleboxymycin, and genes for other virulence factors. The genome also contains the antibiotic resistance genes blaOXY-6-4 and a variant of fosA which is likely to explain the observed resistance to ampicillin, amoxicillin and fosfomycin. We have also shown that SS141 forms biofilms on both polystyrene and polypropylene surfaces, providing a reasonable explanation for its ability to colonise a re-usable water bottle. With the increasing use of re-usable water bottles as an alternative to disposables, and a strong forecast for growth in this industry over the next decade, this study highlights the need for cleanliness comparable to other re-usable culinary items
Mobile colistin resistance gene mcr-1 detected on an IncI1 plasmid in Escherichia coli from meat
Objectives: Mobile colistin resistance (mcr) genes encoded on conjugative plasmids, although described only relatively recently, have been reported globally both in humans and livestock. The genes are often associated with the insertion sequence ISApl1 that can transpose the genes to novel genetic locations. Since its first report, multiple variants of mcr have been discovered in a variety of genetic locations in Escherichia coli, in plasmids and integrated into the chromosome. Methods: Using hybrid assembly of short-read and long-read whole-genome sequencing data, the presence ofmcr-1 was confirmed on an IncI1 plasmid in E. coli. In vitro conjugation assays were performed to determine the potential to transfer between strains. Genetic comparison with previously reported IncI1 plasmids was performed. Results: The genomic sequence identified thatmcr-1 is present on a complete IncI1 plasmid. Comparison with previously reported extended-spectrum β-lactamase (ESBL)-encoding plasmids from E. coli in the Netherlands from the same time period indicated a distinct lineage for this plasmid. Conclusions: The observation ofmcr-1 on an IncI1 plasmid confirms that the genetic region of this gene is actively transposed between genetic locations. This active transposition has consequences for the study of the epidemiology of mcr in populations
Mobile colistin resistance gene mcr-1 detected on an Incl1 plasmid in Escherichia coli from meat
Objectives
Mobile colistin resistance genes (mcr) encoded on conjugative plasmids, although described only relatively recently, have been reported globally in both humans and livestock. The genes are often associated with ISApl1 which can transpose the genes to novel genetic locations. Since the first report, multiple variants of mcr have been discovered on a variety of genetic locations in Escherichia coli, in plasmids and integrated into the chromosome.
Methods
Using hybrid assembly of short-read and long-read WGS data, the presence of mcr-1 was confirmed on an IncI1 plasmid in E. coli. In vitro conjugation assays were performed to determine the potential to transfer between strains. Genetic comparison to previously reported IncI1 plasmids was performed.
Results
The genomic sequence has identified that mcr-1 is present on a complete IncI1 plasmid. Comparison to previously reported ESBL-encoding plasmids from E. coli in the Netherlands from the same time period indicated a distinct lineage for this plasmid.
Conclusion
The observation of mcr-1¬ on an IncI1 plasmid confirms that the genetic region of this gene is actively transposed between genetic locations. This active transposition has consequences for the study of the epidemiology of mcr in populations