Studies on the role of IS1216E in the formation and dissemination of poxtA-carrying plasmids in an Enterococcus faecium clade A1 isolate

2020 ◽  
Vol 75 (11) ◽  
pp. 3126-3130
Author(s):  
Xinxin Shan ◽  
Xin-Sheng Li ◽  
Nannan Wang ◽  
Stefan Schwarz ◽  
Su-Mei Zhang ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Blot Hybridization ◽  
Tetracycline Resistance ◽  
Southern Blot Hybridization ◽  
Conjugative Plasmid ◽  
Fusion Event ◽  
Mobilizable Plasmid ◽  
Antimicrobial Resistance Genes ◽  
Positive Isolate

Abstract Objectives To analyse the role of IS1216E in the dissemination of the phenicol-oxazolidinone-tetracycline resistance gene poxtA in an Enterococcus faecium clade A1 isolate. Methods MICs were determined by broth microdilution. The poxtA-positive isolate was typed by MLST. The two plasmids were characterized by PCR, conjugation, S1-PFGE, Southern blot hybridization and WGS analysis. The presence of translocatable units (TUs) was examined by PCR and sequencing. Results Isolate E1077 contains the 217661 bp conjugative plasmid pE1077-217 and the 23710 bp mobilizable plasmid pE1077-23. pE1077-217 harbours erm(B), aac(A)-aph(D), aadE, spw, lsa(E), lnu(B), aphA3 and dfrG, whereas pE1077-23 carries a Tn6657-like transposon containing poxtA and fexB. pE1077-23 was apparently formed by an IS1216E-mediated composite transposon–plasmid fusion event, involving a replicative transposition process. Conjugation experiments showed that pE1077-23 is mobilizable by pE1077-217. Moreover, a novel 31742 bp plasmid, pT-E1077-31, was found in a transconjugant. WGS analysis indicated that pT-E1077-31 was formed by the integration of a Tn6657-derived, IS1216E-based translocatable unit, which carried fexB and poxtA, into a copy of pE1077-23. Conclusions This study showed the presence of two cointegrate formation events in the formation and spread of a poxtA/fexB-carrying plasmid in E. faecium. One was the integration of a transposon into a plasmid while the other was the integration of a TU into a different site of the same type of plasmid-borne transposon from which it originated. In both events, IS1216E played a major role, suggesting that IS1216E-mediated transposition and translocation processes aid the dissemination and persistence of important antimicrobial resistance genes, such as poxtA, among enterococci.

scholarly journals Detection of a Novel, and Likely Ancestral, Tn916-Like Element from a Human Saliva Metagenomic Library

Genes ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 548
Author(s):  
Liam J. Reynolds ◽  
Muna F. Anjum ◽  
Adam P. Roberts
Keyword(s):  
Resistance Genes ◽  
Metagenomic Library ◽  
Tetracycline Resistance ◽  
Human Saliva ◽  
Fusion Event ◽  
Tetracycline Resistance Genes ◽  
Antimicrobial Resistance Genes ◽  
Genes Encoding ◽  
Gene Fusion Event ◽  
Metagenomic Approach

Tn916 is a conjugative transposon (CTn) and the first reported and most well characterised of the Tn916/Tn1545 family of CTns. Tn916-like elements have a characteristic modular structure and different members of this family have been identified based on similarities and variations in these modules. In addition to carrying genes encoding proteins required for their conjugation, Tn916-like elements also carry accessory, antimicrobial resistance genes; most commonly the tetracycline resistance gene, tet(M). Our study aimed to identify and characterise tetracycline resistance genes from the human saliva metagenome using a functional metagenomic approach. We identified a tetracycline-resistant clone, TT31, the sequencing of which revealed it to encode both tet(M) and tet(L). Comparison of the TT31 sequence with the accessory, regulation, and recombination modules of other Tn916-like elements indicated that a partial Tn916-like element encoding a truncated orf9 was cloned in TT31. Analysis indicated that a previous insertion within the truncated orf9 created the full length orf9 found in most Tn916-like transposons; demonstrating that orf9 is, in fact, the result of a gene fusion event. Thus, we hypothesise that the Tn916-like element cloned in TT31 likely represents an ancestral Tn916.

Characterization of a vancomycin-resistant Enterococcus faecium isolate and a vancomycin-susceptible E. faecium isolate from the same blood culture

2020 ◽  
Author(s):  
Kyriaki Xanthopoulou ◽  
Julia Wille ◽  
Janine Zweigner ◽  
Kai Lucaßen ◽  
Thorsten Wille ◽  
...  
Keyword(s):  
Blood Culture ◽  
Enterococcus Faecium ◽  
Core Genome ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Vana Gene ◽  
Plasmid Analysis ◽  
Vancomycin Resistant ◽  
Faecium Isolate

Abstract Objectives To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture. Methods The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization. Results Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical. Conclusions Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates.

scholarly journals Dissemination of Blandm-5 in Escherichia Coli via the Incx3 Plasmid From Different Regions in China

2021 ◽  
Author(s):  
Xiaofeng Hu ◽  
Lang Yang ◽  
Nian Dong ◽  
Yanfeng Lin ◽  
Ling Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Blot Hybridization ◽  
Carbapenem Resistance ◽  
Southern Blot Hybridization ◽  
Control Measures ◽  
Rrna Gene ◽  
Limited Information ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Antimicrobial Resistance Genes

Abstract Background: Recently, the spread of NDM-5-producing Escherichia coli has become a severe challenge in clinical therapy, which necessitates reliable detection and surveillance methods. However, limited information is available regarding the prevalence and dissemination of the blaNDM-5 gene in Escherichia coli in China. Therefore, we investigated the dissemination of the blaNDM-5 gene in carbapenem-resistant Escherichia coli isolates from different regions in China.Methods: A total of 1,180 carbapenem-resistant enterobacteriaceae strains were obtained from patients admitted to the 20 sentinel hospitals in eight cities. Strains positive for blaNDM-5 were detected using the Vitek 2 compact system, 16S rRNA gene sequencing, PCR, the S1-pulsed-field gel electrophoresis assay, and Southern blot hybridization. The horizontal-transfer capability of the blaNDM gene was assessed by filter mating with a standard E. coli J53 azide-resistant strain as the recipient. Genotyping, susceptibility testing, and whole genome sequencing were performed. Results: Seven strains of blaNDM-5-positive E.coli was detected in 1180 clinical strains from different regions in China. The blaNDM-5-carrying strains showed resistance to multiple tested antibiotics and belonged to two widespread sequence types, ST167 and ST405. Antimicrobial resistance genes including blaCTX-M, blaOXA, blaCMY, and two novel blaTEM variants (blaTEM-230 and blaTEM-231) were also identified. Southern blotting located the blaNDM-5 gene on 46-kb IncX3 plasmids in all isolates, which showed only two single nucleotide differences between EJN003 and the other strains. Conclusions: This study further confirms the increasing occurrence of blaNDM-5-carrying IncX3 plasmids and the dissemination of carbapenem resistance in E. coli isolates via the plasmid from different parts in China, which warrants stringent surveillance and control measures.

scholarly journals A Conjugative Plasmid Carrying the efe Gene for the Ethylene-Forming Enzyme Isolated from Pseudomonas syringae pv. glycinea

1998 ◽  
Vol 88 (11) ◽  
pp. 1205-1209 ◽  
Author(s):  
K. Watanabe ◽  
K. Nagahama ◽  
M. Sato
Keyword(s):  
Southern Blot ◽  
Pseudomonas Syringae ◽  
Direct Evidence ◽  
Blot Hybridization ◽  
Molecular Size ◽  
Southern Blot Hybridization ◽  
Conjugative Plasmid ◽  
Gene Encoding

Previous work suggested that the efe gene encoding the ethylene-forming enzyme was present in the plasmids of three pathovars of Pseudomonas syringae including glycinea, phaseolicola (kudzu strains), and cannabina. However, no direct evidence to support this assumption had been presented. In the current study, we isolated the conjugative plasmid harboring the efe gene (ethylene plasmid) designated pETH2 from P. syringae pv. glycinea MAFF301683. pETH2 was detected by Southern blot hybridization using the efe probe, marked with the transposon mini-Tn5-Km1, and transferred into P. syringae Ni27n, which does not produce ethylene. The transconjugant Ni27n (pETH2) produced ethylene at a level similar to pv. glycinea MAFF301683. In addition, the plasmid designated pCOR2, which encodes coronatine biosynthesis genes, was detected in the same strain. Although the molecular size of the plasmid pCOR2 was not easily distinguishable from pETH2, pCOR2 transferred independently into Ni27n and the transconjugants produced coronatine. These findings suggested that the efe gene has been horizontally dispersed among pathovars of P. syringae by plasmid-mediated conjugation in nature.

Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome

1982 ◽  
Vol 152 (1) ◽  
pp. 215-222
Author(s):  
J A Tobian ◽  
F L Macrina
Keyword(s):  
Streptococcus Mutans ◽  
Blot Hybridization ◽  
Tetracycline Resistance ◽  
Southern Blot Hybridization ◽  
Vector System ◽  
Oral Streptococci ◽  
Helper Plasmid ◽  
Cloning System ◽  
Streptococcus Sanguis ◽  
High Level

A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.

scholarly journals TcpA, an FtsK/SpoIIIE Homolog, Is Essential for Transfer of the Conjugative Plasmid pCW3 in Clostridium perfringens

2007 ◽  
Vol 189 (21) ◽  
pp. 7782-7790 ◽  
Author(s):  
Jennifer A. Parsons ◽  
Trudi L. Bannam ◽  
Rodney J. Devenish ◽  
Julian I. Rood
Keyword(s):  
Clostridium Perfringens ◽  
Essential Role ◽  
Tetracycline Resistance ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Binding Motifs ◽  
Coupling Protein ◽  
Conjugation Process ◽  
Dna Translocase

ABSTRACT The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogen Clostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer in C. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that the tcpA gene was essential for the conjugative transfer of pCW3 and that the tcpB gene was not required for transfer. Furthermore, complementation of a pCW3ΔtcpA mutant with divergent tcpA homologs provided experimental evidence that all of the known conjugative plasmids from C. perfringens use a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process in C. perfringens.

scholarly journals Dissemination ofblaOXA-23in Acinetobacter spp. in China: Main Roles of Conjugative Plasmid pAZJ221 and Transposon Tn2009

2015 ◽  
Vol 59 (4) ◽  
pp. 1998-2005 ◽  
Author(s):  
Li-Lin Liu ◽  
Shu-Juan Ji ◽  
Zhi Ruan ◽  
Ying Fu ◽  
Yi-Qi Fu ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Carbapenem Resistance ◽  
Southern Blot Hybridization ◽  
Conjugative Plasmid ◽  
S1 Nuclease ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Copy Numbers ◽  
Ca 50 ◽  
Genomic Locations

ABSTRACTProduction of the OXA-23 carbapenemase is the most common reason for the increasing carbapenem resistance inAcinetobacterspp. This study was conducted to reveal the genetic basis ofblaOXA-23dissemination inAcinetobacterspp. in China. A total of 63 carbapenem-resistant OXA-23-producingAcinetobactersp. isolates, representing different backgrounds, were selected from 28 hospitals in 18 provinces for this study. Generally, two patterns of plasmids carryingblaOXA-23were detected according to S1-nuclease pulsed-field gel electrophoresis and Southern blot hybridization. A ca. 78-kb plasmid, designated pAZJ221, was found in 23Acinetobacter baumanniiand threeAcinetobacter nosocomialisisolates, while a novel ca. 50-kb plasmid was carried by only two otherA. baumanniiisolates. Three of these isolates had an additional copy ofblaOXA-23on the chromosome. Transformation of the two plasmids succeeded, but only pAZJ221 was conjugative. Plasmid pAZJ221 was sequenced completely and found to carry no previously known resistance genes exceptblaOXA-23. TheblaOXA-23gene of the remaining 35 isolates was chromosome borne. TheblaOXA-23genetic environments were correlated with Tn2009in 57 isolates, Tn2008in 5 isolates, and Tn2006in 1 isolate. The MIC values for the carbapenems with these isolates were not significantly associated with the genomic locations or the copy numbers ofblaOXA-23. Overall, these observations suggest that the plasmid pAZJ221 and Tn2009have effectively contributed to the wide dissemination ofblaOXA-23inAcinetobacterspp. in China and that horizontal gene transfer may play an important role in dissemination of theblaOXA-23gene.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 967-978 ◽  
Author(s):  
C. Viana-Niero ◽  
P. E. de Haas ◽  
D. van Soolingen ◽  
S. C. Leão
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phospholipase C ◽  
Genetic Polymorphisms ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Clinical Isolates ◽  
Significant Similarity ◽  
Genomic Deletions ◽  
Genes Encoding ◽  
Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

scholarly journals Diversity and Antimicrobial Resistance in the Streptococcus bovis/Streptococcus equinus Complex (SBSEC) Isolated from Korean Domestic Ruminants

2021 ◽  
Vol 9 (1) ◽  
pp. 98
Author(s):  
Seon Young Park ◽  
Mingyung Lee ◽  
Se Ra Lim ◽  
Hyemin Kwon ◽  
Ye Seul Lee ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Phylogenetic Analyses ◽  
Tetracycline Resistance ◽  
Current Status ◽  
Streptococcus Bovis ◽  
Limited Information ◽  
Phenotypic Resistance ◽  
Domestic Ruminants ◽  
Antimicrobial Resistance Genes ◽  
Acid Producing Bacteria

S. bovis/S. equinus complex (SBSEC) includes lactic acid-producing bacteria considered as the causative agent associated with acute rumen lactic acidosis in intensive ruminants. Considering the limited information on the detailed characteristics and diversity of SBSEC in Korea and the emergence of antimicrobial resistance (AMR), we investigated the diversity of SBSEC from domestic ruminants and verified the presence of antimicrobial resistance genes (ARGs) against several antimicrobials with their phenotypic resistance. Among 51 SBSEC isolates collected, two SBSEC members (S. equinus and S. lutetiensis) were identified; sodA-based phylogenetic analyses and comparisons of overall genome relatedness revealed potential plasticity and diversity. The AMR rates of these SBSEC against erythromycin, clindamycin, and tetracycline were relatively lower than those of other SBSEC isolates of a clinical origin. An investigation of the ARGs against those antimicrobials indicated that tetracycline resistance of SBSECs generally correlated with the presence of tet(M)-possessing Tn916-like transposon. However, no correlation between the presence of ARGs and phenotypic resistance to erythromycin and clindamycin was observed. Although a limited number of animals and their SBSEC isolates were examined, this study provides insights into the potential intraspecies biodiversity of ruminant-origin SBSEC and the current status on antimicrobial resistance of the bacteria in the Korean livestock industry.

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