scholarly journals Functional Characterization of IS1999, an IS4 Family Element Involved in Mobilization and Expression of β-Lactam Resistance Genes

2006 ◽  
Vol 188 (18) ◽  
pp. 6506-6514 ◽  
Author(s):  
Daniel Aubert ◽  
Thierry Naas ◽  
Claire Héritier ◽  
Laurent Poirel ◽  
Patrice Nordmann
Keyword(s):  
Resistance Genes ◽  
Consensus Sequence ◽  
Functional Characterization ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmid ◽  
Transposition Frequency ◽  
Transposase Gene ◽  
Reverse Transcription Pcr ◽  
Mutant Derivative ◽  
Rapid Amplification

ABSTRACT IS1999 and a point mutant derivative, IS1999.2, have been described inserted upstream of emerging antibiotic resistance genes bla VEB-1 and bla OXA-48. 5′ Rapid amplification of cDNA ends experiments revealed that expression of these β-lactamase genes was driven by the outward-directed promoter, Pout, located in the IS1999 elements. These findings led us to study IS1999-mediated gene mobilization. Thus, the transposition properties of IS1999 and of IS1999-based composite transposons, made of two copies of IS1999 in different orientations, were investigated. IS1999 or IS1999-based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS1999.2 was 10-fold lower than that of IS1999, and transposition frequencies of the putative natural transposon, Tn1999, were below detection limits of our transposition assay. This reduced transposition frequency of IS1999.2-based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses.

scholarly journals Complete Nucleotide Sequence of the pCTX-M3 Plasmid and Its Involvement in Spread of the Extended-Spectrum β-Lactamase Gene blaCTX-M-3

2007 ◽  
Vol 51 (11) ◽  
pp. 3789-3795 ◽  
Author(s):  
M. Gołębiewski ◽  
I. Kern-Zdanowicz ◽  
M. Zienkiewicz ◽  
M. Adamczyk ◽  
J. Żyliǹska ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmid ◽  
Bacterial Strains ◽  
Extended Spectrum ◽  
Gene Coding ◽  
Kluyvera Ascorbata ◽  
Almost All ◽  
Lactamase Gene

ABSTRACT Here we report the nucleotide sequence of pCTX-M3, a highly conjugative plasmid that is responsible for the extensive spread of the gene coding for the CTX-M-3 extended-spectrum β-lactamase in clinical populations of the family Enterobacteriaceae in Poland. The plasmid belongs to the IncL/M incompatibility group, is 89,468 bp in size, and carries 103 putative genes. Besides bla CTX-M-3, it also bears the bla TEM-1, aacC2, and armA genes, as well as integronic aadA2, dfrA12, and sul1, which altogether confer resistance to the majority of β-lactams and aminoglycosides and to trimethoprim-sulfamethoxazole. The conjugal transfer genes are organized in two blocks, tra and trb, separated by a spacer sequence where almost all antibiotic resistance genes and multiple mobile genetic elements are located. Only bla CTX-M-3, accompanied by an ISEcp1 element, is placed separately, in a DNA fragment previously identified as a fragment of the Kluyvera ascorbata chromosome. On the basis of sequence analysis, we speculate that pCTX-M3 might have arisen from plasmid pEL60 from plant pathogen Erwinia amylovora by acquiring mobile elements with resistance genes. This suggests that plasmids of environmental bacterial strains could be the source of those plasmids now observed in bacteria pathogenic for humans.

scholarly journals Degradation of antibiotic resistance genes and mobile gene elements in dairy manure anerobic digestion

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0254836
Author(s):  
Yi Wang ◽  
Pramod K. Pandey ◽  
Sundaram Kuppu ◽  
Richard Pereira ◽  
Sharif Aly ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Anaerobic Digestion ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Dairy Manure ◽  
Health Concern ◽  
Animal Origin ◽  
Rrna Gene ◽  
Transposase Gene ◽  
Integrase Gene

Antibiotic resistance genes (ARGs) are emerging contaminants causing serious global health concern. Interventions to address this concern include improving our understanding of methods for treating waste material of human and animal origin that are known to harbor ARGs. Anaerobic digestion is a commonly used process for treating dairy manure, and although effective in reducing ARGs, its mechanism of action is not clear. In this study, we used three ARGs to conducted a longitudinal bench scale anaerobic digestion experiment with various temperatures (28, 36, 44, and 52°C) in triplicate using fresh dairy manure for 30 days to evaluate the reduction of gene abundance. Three ARGs and two mobile genetic elements (MGEs) were studied: sulfonamide resistance gene (sulII), tetracycline resistance genes (tetW), macrolide-lincosamide-streptogramin B (MLSB) superfamily resistance genes (ermF), class 1 integrase gene (intI1), and transposase gene (tnpA). Genes were quantified by real-time quantitative PCR. Results show that the thermophilic anaerobic digestion (52°C) significantly reduced (p < 0.05) the absolute abundance of sulII (95%), intI1 (95%), tnpA (77%) and 16S rRNA gene (76%) after 30 days of digestion. A modified Collins–Selleck model was used to fit the decay curve, and results suggest that the gene reduction during the startup phase of anaerobic digestion (first 5 days) was faster than the later stage, and reductions in the first five days were more than 50% for most genes.

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 Azithromycin and Ciprofloxacin can Promote Antibiotic Resistance in Biosolids and Biosolids-amended Soils

2021 ◽  
Author(s):  
Harmanpreet Sidhu ◽  
Hee-Sung Bae ◽  
Andrew Ogram ◽  
George O’Connor ◽  
Fahong Yu
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Sewage Treatment ◽  
Antibiotic Resistance Genes ◽  
Environmental Parameters ◽  
Mobile Elements ◽  
Health Concern ◽  
Amended Soils ◽  
Reverse Transcription Pcr ◽  
Amended Soil

Spread of biosolids-borne antibiotic resistance is a growing public and environmental health concern. Herein we conducted incubation experiments involving biosolids, derived from sewage treatment plants, and biosolids-amended soil. Quantitative reverse transcription PCR (RT-qPCR) was employed to assess responses of select antibiotic resistance genes (ARGs) and mobile elements to environmentally relevant concentrations of two biosolids-borne antibiotics, azithromycin (AZ) and ciprofloxacin (CIP). Additionally, we examined sequence distribution of gyrA (encoding DNA gyrase; site of action of CIP) to assess potential shifts in genotype. Increasing antibiotic concentrations generally increased the transcriptional activities of qnrS (encoding CIP resistance) and ermB and mefE (encoding AZ resistance). The transcriptional activity of intl1 , a marker of Class 1 integrons, was unaffected by CIP or AZ concentrations, but biosolids amendment increased intl1 activity in the soil by 4 to 5 times which persisted throughout incubation. While the dominant gyrA sequences found herein were unrelated to known CIP-resistant genotypes, the increasing CIP concentrations significantly decreased the diversity of genes encoding gyrA , suggesting changes in microbial community structures. This study suggests that biosolids harbor transcriptionally active ARGs and mobile elements that could survive and spread in biosolids-amended soils. However, more research is warranted to investigate these trends under field conditions. IMPORTANCE Although previous studies have indicated that biosolids may be important spreaders of antibiotics and antibiotic resistance genes (ARGs) in environments, the potential activities of ARGs or their responses to environmental parameters have been understudied. This study highlights that certain biosolids-borne antibiotics can induce transcriptional activities of ARGs and mobile genetic elements in biosolids and biosolids-amended soil, even when present at environmentally relevant concentrations. Furthermore, these antibiotics can alter the structure of microbial population expressing ARGs. Our findings indicate the bioavailability of the antibiotics in biosolids and provide evidence that biosolids can promote the activities and dissemination of ARGs and mobile genes in biosolids and soils that receive contaminated biosolids; thus, underscoring the importance of investigating anthropogenically-induced antibiotic resistance in the environment under real-world scenarios.

The effect of synthetic microbial spatial self-organization on the fate of antibiotic resistance genes

2020 ◽  
Author(s):  
Yinyin Ma ◽  
David Johnson
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Pseudomonas Stutzeri ◽  
Trophic Interaction ◽  
Conjugative Plasmid ◽  
Self Organization ◽  
Antibiotic Selection ◽  
Feeding Group ◽  
Lower Magnitude

&lt;p&gt;Biofilms are considered as hotspots for the transfer of antibiotic resistance genes (ARGs), but very few studies have investigated the fate of ARGs (e.g. proliferation or elimination) in situ given different microbial spatial self-organization (SSO). SSO refers to a pervasive process during biofilm formation when microbes arrange themselves non-randomly across surfaces. So far the causes of SSO have been uncovered in a sense, however, the consequences of SSO were largely overlooked. Here, I hypothesize that the magnitude of inter-species intermixing, as one fundamental character of SSO, will determine the fate of ARG-carrying conjugative plasmid in both absence and presence of antibiotic selection. I evaluated this by performing range expansion experiments on agar plates to develop an artificial biofilm using a synthetic microbial community consisting of two isogenic Pseudomonas Stutzeri &lt;em&gt;A1501&lt;/em&gt; who are facultative denitrifiers in anaerobic condition. By knocking out different functional genes responsible for different steps of denitrification I am able to modify the metabolic interactions between these two strains from competing (without trophic interaction) to cross-feeding (with trophic interaction), which will further result in different magnitude of inter-species intermixing. Competing group has lower magnitude due to demixing of two, while cross-feeding group has higher magnitude due to mixing. I observed that in the absence of antibiotic selection plasmid experienced faster pace of elimination in competing group than cross-feeding group, whereas in the presence of antibiotic selection plasmid proliferated more efficiently in cross-feeding group than competing group. These results suggest that SSO is a determining factor of the fate of ARGs in biofilms, which provides a novel perspective of better understanding ARGs-related pressing problems facing our society.&lt;/p&gt;

scholarly journals First Report of Coexistence of blaSFO–1 and blaNDM–1 β-Lactamase Genes as Well as Colistin Resistance Gene mcr-9 in a Transferrable Plasmid of a Clinical Isolate of Enterobacter hormaechei

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenxiu Ai ◽  
Ying Zhou ◽  
Bingjie Wang ◽  
Qing Zhan ◽  
Longhua Hu ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Clinical Isolate ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Mobile Elements ◽  
Conjugative Plasmid ◽  
S1 Nuclease ◽  
Antimicrobial Resistance Genes ◽  
Enterobacter Hormaechei

Many antimicrobial resistance genes usually located on transferable plasmids are responsible for multiple antimicrobial resistance among multidrug-resistant (MDR) Gram-negative bacteria. The aim of this study is to characterize a carbapenemase-producing Enterobacter hormaechei 1575 isolate from the blood sample in a tertiary hospital in Wuhan, Hubei Province, China. Antimicrobial susceptibility test showed that 1575 was an MDR isolate. The whole genome sequencing (WGS) and comparative genomics were used to deeply analyze the molecular information of the 1575 and to explore the location and structure of antibiotic resistance genes. The three key resistance genes (blaSFO–1, blaNDM–1, and mcr-9) were verified by PCR, and the amplicons were subsequently sequenced. Moreover, the conjugation assay was also performed to determine the transferability of those resistance genes. Plasmid files were determined by the S1 nuclease pulsed-field gel electrophoresis (S1-PFGE). WGS revealed that p1575-1 plasmid was a conjugative plasmid that possessed the rare coexistence of blaSFO–1, blaNDM–1, and mcr-9 genes and complete conjugative systems. And p1575-1 belonged to the plasmid incompatibility group IncHI2 and multilocus sequence typing ST102. Meanwhile, the pMLST type of p1575-1 was IncHI2-ST1. Conjugation assay proved that the MDR p1575-1 plasmid could be transferred to other recipients. S1-PFGE confirmed the location of plasmid with molecular weight of 342,447 bp. All these three resistant genes were flanked by various mobile elements, indicating that the blaSFO–1, blaNDM–1, and mcr-9 could be transferred not only by the p1575-1 plasmid but also by these mobile elements. Taken together, we report for the first time the coexistence of blaSFO–1, blaNDM–1, and mcr-9 on a transferable plasmid in a MDR clinical isolate E. hormaechei, which indicates the possibility of horizontal transfer of antibiotic resistance genes.

Integron-Mediated Antibiotic Resistance in Shiga Toxin–Producing Escherichia coli

2009 ◽  
Vol 72 (1) ◽  
pp. 21-27 ◽  
Author(s):  
SUPAKANA NAGACHINTA ◽  
JINRU CHEN
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Shiga Toxin ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmid ◽  
E Coli ◽  
Class 1 Integrons ◽  
Class 1 ◽  
K 12

This study was undertaken to characterize the integrons present in a group of Shiga toxin–producing Escherichia coli (STEC) isolates and the ability of these integrons to transfer antibiotic resistance genes from STEC to E. coli K-12 MG1655. A total of 177 STEC isolates were analyzed for antibiotic susceptibility and the presence of integrons. Class 1 integrons were detected in 14 STEC isolates, and a class 2 integron was identified in 1 STEC isolate. The STEC isolates positive for class 1 integrons were resistant to streptomycin (MICs &gt; 128 μg/ml) and sulfisoxazole (MICs &gt; 1,024 μg/ml), and the isolate positive for the class 2 integron was resistant to streptomycin (MIC of 128 μg/ml), trimethoprim (MIC &gt; 256 μg/ml), and streptothricin (MIC &gt; 32 μg/ml). Results of restriction digestion and nucleotide sequencing revealed that the cassette regions of the class 1 integrons had a uniform size of 1.1 kb and contained a nucleotide sequence identical to that of aadA1. The class 2 integron cassette region was 2.0 kb and carried nucleotide sequences homologous to those of aadA1, sat1, and dfrA1. Results of the conjugation experiments revealed that horizontal transfers of conjugative plasmids are responsible for the dissemination of class 1 integron–mediated antibiotic resistance genes from STEC to E. coli K-12 MG1655. Antibiotic resistance traits not mediated by integrons, such as resistance to tetracycline and oxytetracycline, were cotransferred with the integron-mediated antibiotic resistance genes. The study suggested a possible role of integron and conjugative plasmid in dissemination of genes conferring resistance to antibiotics from pathogenic to generic E. coli cells.

scholarly journals pCTX-M3—Structure, Function, and Evolution of a Multi-Resistance Conjugative Plasmid of a Broad Recipient Range

2021 ◽  
Vol 22 (9) ◽  
pp. 4606
Author(s):  
Izabela Kern-Zdanowicz
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Mobile Genetic Elements ◽  
Conjugative Plasmid ◽  
Conjugation System ◽  
Incompatibility Group ◽  
Genetic Elements ◽  
The Family ◽  
Stable Maintenance

pCTX-M3 is the archetypic member of the IncM incompatibility group of conjugative plasmids (recently referred to as IncM2). It is responsible for the worldwide dissemination of numerous antibiotic resistance genes, including those coding for extended-spectrum β-lactamases and conferring resistance to aminoglycosides. The IncM plasmids acquired during evolution diverse mobile genetic elements found in one or two multiple resistance regions, MRR(s), grouping antibiotic resistance genes as well as mobile genetic elements or their remnants. The IncM plasmids can be found in bacteria inhabiting various environments. The information on the structure and biology of pCTX-M3 is integrated in this review. It focuses on the functional modules of pCTX-M3 responsible for its replication, stable maintenance, and conjugative transfer, indicating that the host range of the pCTX-M3 replicon is limited to representatives of the family Enterobacteriaceae (Enterobacterales ord. nov.), while the range of recipients of its conjugation system is wide, comprising Alpha-, Beta-, and Gammaproteobacteria, and also Firmicutes.

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