scholarly journals A Conjugative MDR pMG1-Like Plasmid Carrying the lsa(E) Gene of Enterococcus faecium With Potential Transmission to Staphylococcus aureus

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiao-Mei Yan ◽  
Jing Wang ◽  
Xiao-Xia Tao ◽  
Hong-Bing Jia ◽  
Fan-Liang Meng ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Complete Sequence ◽  
Reading Frame ◽  
E Gene ◽  
Tertiary Teaching ◽  
Tertiary Teaching Hospital ◽  
Next Generation Sequencing Ngs ◽  
Mating Tests

lsa(E) is a pleuromutilin, lincosamide, and streptogramin A (PLSA phenotype) resistance gene that was first described in S. aureus and was thought to have been transferred from Enterococcus sp. This study aimed to elucidate the prevalence of the lsa(E) gene among E. faecium isolates at a tertiary teaching hospital and to evaluate the transferability of the lsa(E) gene from E. faecium to S. aureus in vitro. A total of 96 E. faecium strains isolated from one hospital in Beijing in 2013 were analysed for quinupristin-dalfopristin (QDA) resistance genes, and multilocus sequence typing (MLST) was performed. The transferability of QDA resistance between ten E. faecium strains and four S. aureus strains was determined by filter mating. Genome sequencing of the transconjugant was performed. A total of 46 E. faecium isolates (46/96, 47.92%) tested positive for lsa(E), while two isolates (2/96, 2.08%) tested positive for lsa(A). Thirty-six lsa(E)-positive strains (36/46, 78.3%) belonged to ST78. Among 40 mating tests, lsa(E) was successfully transferred through one conjugation at a frequency of 1.125 × 10–7 transconjugants per donor. The QDA resistance of the transconjugant N7435-R3645 was expressed at a higher level (MIC = 16 mg/L) than that of the parent S. aureus strain (MIC = 0.38 mg/L). Next-generation sequencing (NGS) analysis of the transconjugant N7435-R3645 showed that the complete sequence of the lsa(E)-carrying plasmid pN7435-R3645 had a size of 92,396 bp and a G + C content of 33% (accession no. MT022086). The genetic map of pN7435-R3645 had high nucleotide similarity and shared the main open reading frame (ORF) features with two plasmids: E. faecium pMG1 (AB206333.1) and E. faecium LS170308 (CP025078.1). The rep gene of pN7435-R3645 showed 100% identity with that of pMG1, although it did not belong to the rep1-19 family but instead a unique rep family. Multiple antibiotic resistance genes, including lsa(E), aadE and lnu(B), erm(B), ant6-Ia, and lnu(B), were present on the plasmid. In conclusion, an lsa(E)-carrying plasmid that can be transferred by conjugation from E. faecium to S. aureus in vitro was identified. This multidrug resistance (MDR) pMG1-like plasmid may act as a vector in the dissemination of antimicrobial resistance among species.

scholarly journals Removal of Antibiotic Resistance Gene-Carrying Plasmids from Lactobacillus reuteri ATCC 55730 and Characterization of the Resulting Daughter Strain, L. reuteri DSM 17938

2008 ◽  
Vol 74 (19) ◽  
pp. 6032-6040 ◽  
Author(s):  
Anna Rosander ◽  
Eamonn Connolly ◽  
Stefan Roos
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Lactobacillus Reuteri ◽  
Antibiotic Resistance Gene ◽  
Genetically Modify ◽  
Antibiotic Resistance Genes ◽  
Probiotic Strain ◽  
Probiotic Properties ◽  
Food Borne

ABSTRACT The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of β-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known β-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The β-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.

scholarly journals Characterization of antibiotic resistance genes in the species of the rumen microbiota

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yasmin Neves Vieira Sabino ◽  
Mateus Ferreira Santana ◽  
Linda Boniface Oyama ◽  
Fernanda Godoy Santos ◽  
Ana Júlia Silva Moreira ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Animal Health ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Ruminal Bacteria ◽  
Genes Encoding

AbstractInfections caused by multidrug resistant bacteria represent a therapeutic challenge both in clinical settings and in livestock production, but the prevalence of antibiotic resistance genes among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico and confirm representative phenotypes in vitro. We find a high abundance of genes encoding tetracycline resistance and evidence that the tet(W) gene is under positive selective pressure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes confirm the expression of the most abundant antibiotic resistance genes. Our data provide insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and reveal the potential role of mobile genetic elements in shaping the resistome of the rumen microbiome, with implications for human and animal health.

scholarly journals Intra- and Interspecies Conjugal Transfer of Tn916-Like Elements from Lactococcus lactis In Vitro and In Vivo

2009 ◽  
Vol 75 (19) ◽  
pp. 6352-6360 ◽  
Author(s):  
Joanna Boguslawska ◽  
Joanna Zycka-Krzesinska ◽  
Andrea Wilcks ◽  
Jacek Bardowski
Keyword(s):  
Antibiotic Resistance ◽  
Lactococcus Lactis ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Raw Milk ◽  
M Gene ◽  
Transfer Resistance

ABSTRACT Tetracycline-resistant Lactococcus lactis strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed L. lactis isolates were able to transfer tetracycline resistance determinants in vitro to L. lactis Bu2-60, at frequencies ranging from 10−5 to 10−7 transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to Enterococcus faecalis JH2-2, whereas no transfer to Bacillus subtilis YBE01, Pseudomonas putida KT2442, Agrobacterium tumefaciens UBAPF2, or Escherichia coli JE2571 was observed. Rats were initially inoculated with the recipient E. faecalis strain JH2-2, and after a week, the L. lactis IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the tet(M) gene showed that this gene was identical to tet(M) localized on the conjugative transposon Tn916. Primer-specific PCR confirmed that the Tn916 transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn916-like antibiotic resistance transposon from L. lactis to E. faecalis. These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria.

scholarly journals Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

2019 ◽  
Author(s):  
Marinelle Rodrigues ◽  
Sara W. McBride ◽  
Karthik Hullahalli ◽  
Kelli L. Palmer ◽  
Breck A. Duerkop
Keyword(s):  
Antibiotic Resistance ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Bacterial Infections ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Conjugative Plasmid ◽  
Antibiotic Resistant ◽  
Resistance Determinants

AbstractThe innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiontEnterococcus faecalisis associated with HAIs and some strains are MDR. Therefore, novel strategies to controlE. faecalispopulations are needed. We previously characterized anE. faecalisType II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers toE. faecalisfor the selective removal of antibiotic resistance genes. Usingin vitrocompetition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistantE. faecalisby several orders of magnitude. Finally, we show thatE. faecalisdonor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinantsin vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.ImportanceCRISPR-Cas nucleic acid targeting systems hold promise for the amelioration of multidrug-resistant enterococci, yet the utility of such tools in the context of the intestinal environment where enterococci reside is understudied. We describe the development of a CRISPR-Cas antimicrobial, deployed on a conjugative plasmid, for the targeted removal of antibiotic resistance genes from intestinalEnterococcus faecalis. We demonstrate that CRISPR-Cas targeting reduces antibiotic resistance ofE. faecalisby several orders of magnitude in the intestine. Although barriers exist that influence the penetrance of the conjugative CRISPR-Cas antimicrobial among target recipientE. faecaliscells, the removal of antibiotic resistance genes inE. faecalisupon uptake of the CRISPR-Cas antimicrobial is absolute. In addition, cells that obtain the CRISPR-Cas antimicrobial are immunized against the acquisition of new antibiotic resistance genes. This study suggests a potential path toward plasmid based CRISPR-Cas therapies in the intestine.

scholarly journals Molecular Characterization of blaIMP–4-Carrying Enterobacterales in Henan Province of China

2021 ◽  
Vol 12 ◽  
Author(s):  
Wentian Liu ◽  
Huiyue Dong ◽  
Tingting Yan ◽  
Xuchun Liu ◽  
Jing Cheng ◽  
...  
Keyword(s):  
Plasmid Stability ◽  
Multidrug Resistant ◽  
Complete Sequence ◽  
Molecular Characteristics ◽  
Carbapenem Resistant ◽  
Predominant Type ◽  
Tertiary Teaching ◽  
Query Coverage ◽  
Tertiary Teaching Hospital ◽  
Positive Isolate

Carbapenem-resistant Enterobacterales (CRE) pose a serious threat to clinical management and public health. We investigated the molecular characteristics of 12 IMP-4 metallo-β-lactamase-producing strains, namely, 5 Enterobacter cloacae, 3 Escherichia coli, 2 Klebsiella pneumoniae, and 2 Citrobacter freundii. These strains were collected from a tertiary teaching hospital in Zhengzhou from 2013 to 2015. The minimum inhibitory concentration (MIC) results showed that each blaIMP–4-positive isolate was multidrug-resistant (MDR) but susceptible to colistin. All of the E. coli belonged to ST167, two C. freundii isolates belonged to ST396, and diverse ST types were identified in E. cloacae and K. pneumoniae. S1-PFGE, Southern blotting, and PCR-based replicon typing assays showed that the blaIMP–4-carrying plasmids ranged from ∼52 to ∼360 kb and belonged to FII, FIB, HI2/HI2A, and N types. N plasmids were the predominant type (8/12, 66.7%). Plasmid stability testing indicated that the blaIMP–4-carrying N-type plasmid is more stable than the other types of plasmids. Conjugative assays revealed that three of the blaIMP–4-carrying N plasmids were transferrable. Complete sequence analysis of a representative N type (pIMP-ECL14–57) revealed that it was nearly identical to pIMP-FJ1503 (KU051710) (99% nucleotide identity and query coverage), an N-type blaIMP–4-carrying epidemic plasmid in a C. freundii strain. PCR mapping indicated that a transposon-like structure [IS6100-mobC-intron (K1.pn.I3)-blaIMP–4-IntI1-IS26] was highly conserved in all of the N plasmids. IS26 involved recombination events that resulted in variable structures of this transposon-like module in FII and FIB plasmids. The blaIMP–4 gene was captured by a sul1-type integron In1589 on HI2/HI2A plasmid pIMP-ECL-13–46.

scholarly journals Enterococcus faecalisCRISPR-Cas is a robust barrier to conjugative antibiotic resistance dissemination in the murine intestine

2018 ◽  
Author(s):  
Valerie J. Price ◽  
Sara W. McBride ◽  
Karthik Hullahalli ◽  
Anushila Chatterjee ◽  
Breck A. Duerkop ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmids ◽  
Resistance Plasmids ◽  
The Impact ◽  
Mammalian Intestine

AbstractCRISPR-Cas systems are barriers to horizontal gene transfer (HGT) in bacteria. Little is known about CRISPR-Cas interactions with conjugative plasmids, and studies investigating CRISPR-Cas/plasmid interactions inin vivomodels relevant to infectious disease are lacking. These are significant gaps in knowledge because conjugative plasmids disseminate antibiotic resistance genes among pathogensin vivo, and it is essential to identify strategies to reduce the spread of these elements. We use enterococci as models to understand the interactions of CRISPR-Cas with conjugative plasmids.Enterococcus faecalisis a native colonizer of the mammalian intestine and harbors pheromone-responsive plasmids (PRPs). PRPs mediate inter- and intraspecies transfer of antibiotic resistance genes. We assessedE. faecalisCRISPR-Cas anti-PRP activity in the mouse intestine and under varyingin vitroconditions. We observed striking differences in CRISPR-Cas efficiencyin vitroversusin vivo. With few exceptions, CRISPR-Cas blocked intestinal PRP dissemination, whilein vitro, the PRP frequently escaped CRISPR-Cas defense. Our results further the understanding of CRISPR-Cas biology by demonstrating that standardin vitroexperiments do not adequately model thein vivoanti-plasmid activity of CRISPR-Cas. Additionally, our work identifies several variables that impact the apparentin vitroanti-plasmid activity of CRISPR-Cas, including planktonic versus biofilm settings, different donor/recipient ratios, production of a plasmid-encoded bacteriocin, and the time point at which matings are sampled. Our results are clinically significant because they demonstrate that barriers to HGT encoded by normal human microbiota can have significant impacts onin vivoantibiotic resistance dissemination.ImportanceCRISPR-Cas is a type of immune system encoded by bacteria that is hypothesized to be a natural impediment to the spread of antibiotic resistance genes. In this study, we directly assessed the impact of CRISPR-Cas on antibiotic resistance dissemination in the mammalian intestine and under varyingin vitroconditions. We observed a robust effect of CRISPR-Cas onin vivobut notin vitrodissemination of antibiotic resistance plasmids in the native mammalian intestinal colonizerEnterococcus faecalis. We conclude that standard laboratory experiments currently do not appropriately model thein vivoconditions where antibiotic resistance dissemination occurs betweenE. faecalisstrains. Moreover, our results demonstrate that CRISPR-Cas encoded by native members of the mammalian intestinal microbiota can block the spread of antibiotic resistance plasmids.

scholarly journals Phenotypic and Genotypic Characterization of Virulence Factors and Susceptibility to Antibiotics in Salmonella Infantis Strains Isolated from Chicken Meat: First Findings in Chile

Animals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 1049
Author(s):  
Lisette Lapierre ◽  
Javiera Cornejo ◽  
Sebastián Zavala ◽  
Nicolás Galarce ◽  
Fernando Sánchez ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Antibiotic Resistance Genes ◽  
Virulence Gene ◽  
Chicken Meat ◽  
Poultry Production ◽  
Production Chain ◽  
Salmonella Serotype

Salmonella Infantis is a zoonotic pathogen that causes gastroenteritis in humans and animals, with poultry being its main reservoir. In Chile, there are no data to characterize S. Infantis strains in poultry production. In this study, 87 S. Infantis strains were isolated from chicken meat for sale in supermarkets in Santiago, Chile, and characterized according to their virulence genes, biofilm formation abilities, antibiotic susceptibility, and resistance genes. Through polymerase chain reaction or PCR, the strains were analyzed to detect the presence of 11 virulence genes, 12 antibiotic resistance genes, and integrase genes. Moreover, disc diffusion susceptibility to 18 antimicrobials and the ability to form biofilm in vitro were evaluated. Results demonstrated six different virulence gene profiles. Ninety-four percent of the strains were multi-resistant to antibiotics with weak biofilm formation abilities, 63.2% of the strains were broad spectrum β- lactam resistant, and the bla CTX-M-65 gene was amplified in 13 strains. Only 3.4% of the strains were fluoroquinolone resistant, and the qnrB gene was amplified in two strains. Colistin resistance was exhibited in 28.7% of the strains, but mrc genes were not amplified in any strain under study. The isolated S. Infantis strains are pathogenic and antibiotic multi-resistant, and thus, this Salmonella serotype should be under surveillance in the poultry food production chain with the aim of protecting public health.

In vitro expression of a mouse tissue specific glutathione-peroxidase-like protein lacking the selenocysteine can protect stably transfected mammalian cells against oxidative damage

1996 ◽  
Vol 74 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Patrick Vernet ◽  
Nicole Rigaudiére ◽  
Norbert Ghyselinck ◽  
Jean Pierre Dufaure ◽  
Joël R. Drevet
Keyword(s):  
Glutathione Peroxidase ◽  
Mammalian Cells ◽  
Expression System ◽  
Complete Sequence ◽  
Protein Domain ◽  
Mouse Tissue ◽  
Reading Frame ◽  
Mammalian Expression ◽  
Mouse Epididymis

The complete sequence of the mouse epididymal protein (MEP24) was cloned. It contains a 663 bp open-reading frame that, after conceptual translation, shows extensive identity with proteins belonging to the glutathione peroxidase (GPX) family. However, a major difference between GPX5 (MEP24) and other known GPXs concerns a protein domain known to be critical for GPX function. To find out what could be the physiological function of such a protein in the mouse epididymis, we have used a mammalian expression system to overexpress the GPX5 protein. Cells constitutively expressing the GPX5 protein were generated and assayed for their ability to metabolize regular substrates of GPX enzymes. Data presented here show that the GPX5-expressing cells can metabolize hydrogen peroxide in a manner that is consistent with a peroxidase activity. However, the substrate preference of the GPX5-expressing cells and their apparent insensitivity to a regular inhibitor of GPX enzymes suggest that the GPX5 protein belongs to a particular class of GPX proteins. Involvement of this protein in the physiology of the mouse epididymis is discussed.Key words: glutathione peroxidase (GPX), mouse epididymis, MEP24, GPX5 cDNA, selenocysteine, GPX5 polyclonal antibody, spermatozoa, CHO-KI cells.

scholarly journals In vitro evalution of antibiotic resistance of Lactobacillus bulgaricus strains isolated from traditional dairy products

2019 ◽  
Vol 37 (No. 1) ◽  
pp. 36-43
Author(s):  
Huiling Guo ◽  
Bilige Menghe
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Fermented Milk ◽  
Lactobacillus Bulgaricus ◽  
Dilution Method ◽  
Resistance Transfer ◽  
Traditional Dairy Products

The antimicrobial susceptibility of 20 Lb. bulgaricus isolates from traditional fermented milk-originated was assessed and then determined the ability to transfer antibiotic resistance genes to other bacteria. The minimum inhibitory concentration of each strain was determined using a standardized dilution method. All the tested strains were found to be susceptible to gentamicin, erythromycin, clindamycin, neomycin, tetracycline, linezolid, chloramphenicol, rifampicin, and quinupristin/dalfopristin, while their susceptibilities to kanamycin, ciprofloxacin, streptomycin, trimethoprim, ampicillin, and vancomycin varied. Polymerase chain reaction (PCR) was used to check whether specific antibiotic resistance genes were present in these Lb. bulgaricus. We detected the rpoB, erm(B), aadA, bla, cat and vanX. Finally, a filter mating assay was applied to investigate the transferability of these resistance markers; and we observe no antibiotic resistance transfer between bacteria. This work demonstrates a low risk of lateral transfer of the antibiotic resistance gene of Lb. bulgaricus.

scholarly journals Screening and Quantification of the Expression of Antibiotic Resistance Genes in Acinetobacter baumannii with a Microarray

2009 ◽  
Vol 54 (1) ◽  
pp. 333-340 ◽  
Author(s):  
Sébastien Coyne ◽  
Ghislaine Guigon ◽  
Patrice Courvalin ◽  
Bruno Périchon
Keyword(s):  
Antibiotic Resistance ◽  
Acinetobacter Baumannii ◽  
Resistance Genes ◽  
Efflux Pump ◽  
Acquired Resistance ◽  
Antibiotic Resistance Genes ◽  
Efflux Pumps ◽  
Single Step ◽  
Resistance Determinants

ABSTRACT An oligonucleotide-based DNA microarray was developed to evaluate expression of genes for efflux pumps in Acinetobacter baumannii and to detect acquired antibiotic resistance determinants. The microarray contained probes for 205 genes, including those for 47 efflux systems, 55 resistance determinants, and 35 housekeeping genes. The microarray was validated by comparative analysis of mutants overexpressing or deficient in the pumps relative to the parental strain. The performance of the microarray was also evaluated using in vitro single-step mutants obtained on various antibiotics. Overexpression, confirmed by quantitative reverse transcriptase PCR, of RND efflux pumps AdeABC, due to a G30D substitution in AdeS in a multidrug-resistant (MDR) strain obtained on gentamicin, and AdeIJK, in two mutants obtained on cefotaxime or tetracycline, was detected. A new efflux pump, AdeFGH, was found to be overexpressed in a mutant obtained on chloramphenicol. Study of MDR clinical isolates, including the AYE strain, whose entire sequence has been determined, indicated overexpression of AdeABC and of the chromosomally encoded cephalosporinase as well as the presence of several acquired resistance genes. The overexpressed and acquired determinants detected by the microarray could account for nearly the entire MDR phenotype of the isolates. The microarray is potentially useful for detection of resistance in A. baumannii and should allow detection of new efflux systems associated with antibiotic resistance.

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