scholarly journals Movement of IS26-Associated Antibiotic Resistance Genes Occurs via a Translocatable Unit That Includes a Single IS26 and Preferentially Inserts Adjacent to Another IS26

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Christopher J. Harmer ◽  
Robert A. Moran ◽  
Ruth M. Hall
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Class I ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Unusual Structure ◽  
Gram Negative

ABSTRACTThe insertion sequence IS26plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS26. A model presented for a second mode of IS26movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS26such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26at each end and between each unique segment. This model requires that IS26recognizes another IS26as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS26. This reaction was conservative, with no additional IS26or target site duplication generated, and orientation specific as the IS26s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS26movement when a target IS26was not present. Intact transposase genes in both IS26s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS26target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency.IMPORTANCEResistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram-negative bacteria, IS26is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS26copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS26-flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS26movement do not resemble those of the IS and class I transposons studied to date. IS26uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called “translocatable units.” The IS26mechanism also explains the properties of IS257(IS431) and IS1216, which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.

scholarly journals IS26-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes

mSphere ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Christopher J. Harmer ◽  
Ruth M. Hall
Keyword(s):  
Antibiotic Resistance ◽  
Homologous Recombination ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Class I ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Movement Mechanism

ABSTRACT In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds transposons and brings additional transposons into resistance regions. The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. Recently, Tnp26 was shown to catalyze an additional intermolecular, conservative reaction between two preexisting copies of IS26 in different plasmids. Here, we have investigated the relative contributions of homologous recombination and Tnp26-catalyzed reactions to the generation of a transposon from a TU. Circular TUs containing the aphA1a kanamycin and neomycin resistance gene or the tet(D) tetracycline resistance determinant were generated in vitro and transformed into Escherichia coli recA cells carrying R388::IS26. The TU incorporated next to the IS26 in R388::IS26 forms a transposon with the insertion sequence (IS) in direct orientation. Introduction of a second TU produced regions containing both the aphA1a gene and the tet(D) determinant in either order but with only three copies of IS26. The integration reaction, which required a preexisting IS26, was precise and conservative and was 50-fold more efficient when both IS26 copies could produce an active Tnp26. When both ISs were inactivated by a frameshift in tnp26, TU incorporation was not detected in E. coli recA cells, but it did occur in E. coli recA + cells. However, the Tnp-catalyzed reaction was 100-fold more efficient than RecA-dependent homologous recombination. The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded transposons in the resistance regions found in Gram-negative bacteria. IMPORTANCE In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds transposons and brings additional transposons into resistance regions.

scholarly journals Analytical Performance of Multiplexed Screening Test for 10 Antibiotic Resistance Genes from Perianal Swab Samples

2016 ◽  
Vol 62 (2) ◽  
pp. 353-359 ◽  
Author(s):  
G Terrance Walker ◽  
Tony J Rockweiler ◽  
Rossio K Kersey ◽  
Kelly L Frye ◽  
Susan R Mitchner ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Test Performance ◽  
Health Care Systems ◽  
Bacterial Species ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Resistant Bacteria ◽  
Gene Test

Abstract BACKGROUND Multiantibiotic-resistant bacteria pose a threat to patients and place an economic burden on health care systems. Carbapenem-resistant bacilli and extended-spectrum β-lactamase (ESBL) producers drive the need to screen infected and colonized patients for patient management and infection control. METHODS We describe a multiplex microfluidic PCR test for perianal swab samples (Acuitas® MDRO Gene Test, OpGen) that detects the vancomycin-resistance gene vanA plus hundreds of gene subtypes from the carbapenemase and ESBL families Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integron-mediated metallo-β-lactamase (VIM), imipenemase metallo-β-lactamase (IMP), OXA-23, OXA-48, OXA-51, CTX-M-1, and CTX-M-2, regardless of the bacterial species harboring the antibiotic resistance. RESULTS Analytical test sensitivity per perianal swab is 11–250 CFU of bacteria harboring the antibiotic resistance genes. Test throughput is 182 samples per test run (1820 antibiotic resistance gene family results). We demonstrate reproducible test performance and 100% gene specificity for 265 clinical bacterial organisms harboring a variety of antibiotic resistance genes. CONCLUSIONS The Acuitas MDRO Gene Test is a sensitive, specific, and high-throughput test to screen colonized patients and diagnose infections for several antibiotic resistance genes directly from perianal swab samples, regardless of the bacterial species harboring the resistance genes.

scholarly journals High levels of antibiotic resistance gene expression among birds living in a wastewater treatment plant

2018 ◽  
Author(s):  
Vanessa R. Marcelino ◽  
Michelle Wille ◽  
Aeron C. Hurt ◽  
Daniel González-Acuña ◽  
Marcel Klaassen ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Wastewater Treatment ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Wastewater Treatment Plant ◽  
Antibiotic Resistance Gene ◽  
Treatment Plant ◽  
Antibiotic Resistance Genes ◽  
Resistant Bacteria ◽  
Chloramphenicol Resistance

AbstractAntibiotic resistance is rendering common bacterial infections untreatable. Wildlife can incorporate and disperse antibiotic resistant bacteria in the environment, such as water systems, which in turn serve as reservoirs of resistance genes for human pathogens. We used bulk RNA-sequencing (meta-transcriptomics) to assess the diversity and expression levels of functionally active resistance genes in the microbiome of birds with aquatic behavior. We sampled birds across a range of habitats, from penguins in Antarctica to ducks in a wastewater treatment plant in Australia. This revealed 81 antibiotic resistance genes in birds from all localities, including β-lactam, tetracycline and chloramphenicol resistance in Antarctica, and genes typically associated with multidrug resistance plasmids in areas with high human impact. Notably, birds feeding at a wastewater treatment plant carried the greatest resistance gene burden, suggesting that human waste, even if it undergoes treatment, contributes to the spread of antibiotic resistance genes to the wild. Differences in resistance gene burden also reflected the birds’ ecology, taxonomic group and microbial functioning. Ducks, which feed by dabbling, carried a higher abundance and diversity of resistance genes than turnstones, avocets and penguins, that usually prey on more pristine waters. In sum, this study helps to reveal the complex factors explaining the distribution of resistance genes and their exchange routes between humans and wildlife.

scholarly journals Metagenomics Analysis Reveals the Microbial Communities, Antimicrobial Resistance Gene Diversity and Potential Pathogen Transmission Risk of Two Different Landfills in China

Diversity ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 230
Author(s):  
Shan Wan ◽  
Min Xia ◽  
Jie Tao ◽  
Yanjun Pang ◽  
Fugen Yu ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Microbial Communities ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Gene Diversity ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Pathogen Transmission ◽  
Transmission Risk ◽  
Antimicrobial Resistance Gene

In this study, we used a metagenomic approach to analyze microbial communities, antibiotic resistance gene diversity, and human pathogenic bacterium composition in two typical landfills in China. Results showed that the phyla Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in the two landfills, and archaea and fungi were also detected. The genera Methanoculleus, Lysobacter, and Pseudomonas were predominantly present in all samples. sul2, sul1, tetX, and adeF were the four most abundant antibiotic resistance genes. Sixty-nine bacterial pathogens were identified from the two landfills, with Klebsiella pneumoniae, Bordetella pertussis, Pseudomonas aeruginosa, and Bacillus cereus as the major pathogenic microorganisms, indicating the existence of potential environmental risk in landfills. In addition, KEGG pathway analysis indicated the presence of antibiotic resistance genes typically associated with human antibiotic resistance bacterial strains. These results provide insights into the risk of pathogens in landfills, which is important for controlling the potential secondary transmission of pathogens and reducing workers’ health risk during landfill excavation.

scholarly journals Persistence of Transferable Extended-Spectrum-β-Lactamase Resistance in the Absence of Antibiotic Pressure

2012 ◽  
Vol 56 (9) ◽  
pp. 4703-4706 ◽  
Author(s):  
Jennifer L. Cottell ◽  
Mark A. Webber ◽  
Laura J. V. Piddock
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Fitness Cost ◽  
Resistant Bacteria ◽  
Bacterial Host ◽  
Antibiotic Resistant ◽  
M 14 ◽  
Host Strains

ABSTRACTThe treatment of infections caused by antibiotic-resistant bacteria is one of the great challenges faced by clinicians in the 21st century. Antibiotic resistance genes are often transferred between bacteria by mobile genetic vectors called plasmids. It is commonly believed that removal of antibiotic pressure will reduce the numbers of antibiotic-resistant bacteria due to the perception that carriage of resistance imposes a fitness cost on the bacterium. This study investigated the ability of the plasmid pCT, a globally distributed plasmid that carries an extended-spectrum-β-lactamase (ESBL) resistance gene (blaCTX-M-14), to persist and disseminate in the absence of antibiotic pressure. We investigated key attributes in plasmid success, including conjugation frequencies, bacterial-host growth rates, ability to cause infection, and impact on the fitness of host strains. We also determined the contribution of theblaCTX-M-14gene itself to the biology of the plasmid and host bacterium. Carriage of pCT was found to impose no detectable fitness cost on various bacterial hosts. An absence of antibiotic pressure and inactivation of the antibiotic resistance gene also had no effect on plasmid persistence, conjugation frequency, or bacterial-host biology. In conclusion, plasmids such as pCT have evolved to impose little impact on host strains. Therefore, the persistence of antibiotic resistance genes and their vectors is to be expected in the absence of antibiotic selective pressure regardless of antibiotic stewardship. Other means to reduce plasmid stability are needed to prevent the persistence of these vectors and the antibiotic resistance genes they carry.

scholarly journals Molecular Analysis of Antibiotic Resistance Gene Clusters in Vibrio cholerae O139 and O1 SXT Constins

2001 ◽  
Vol 45 (11) ◽  
pp. 2991-3000 ◽  
Author(s):  
Bianca Hochhut ◽  
Yasmin Lotfi ◽  
Didier Mazel ◽  
Shah M. Faruque ◽  
Roger Woodgate ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Dna Sequences ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Gene Clusters ◽  
Vibrio Cholerae O139 ◽  
El Tor

ABSTRACT Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXTMO10, these genes are clustered within a composite transposon-like structure found near the element's 5′ end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXTET, does not contain the same resistance genes as SXTMO10. In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.

scholarly journals IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Christopher J. Harmer ◽  
Ruth M. Hall
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
High Frequency ◽  
Antibiotic Resistance Genes ◽  
Kanamycin Resistance ◽  
Gram Negative Bacteria ◽  
Critical Determinant ◽  
Gram Negative ◽  
Precise Excision ◽  
Replicative Transposition

ABSTRACTWe recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26from a compound transposon bounded by IS26. In arecAmutant strain, Tn4352, a kanamycin resistance transposon carrying theaphA1agene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26transposase Tnp26. However, the Tnp26 of only one IS26in Tn4352B was required, specifically the IS26downstream of theaphA1agene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352.These G residues are located immediately adjacent to the two G residues at the left end of the IS26that is upstream of theaphA1agene. Transcription oftnp26was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end.IMPORTANCEResistance to antibiotics limits treatment options. In Gram-negative bacteria, IS26plays a major role in the acquisition and dissemination of antibiotic resistance. IS257(IS431) and IS1216, which belong to the same insertion sequence (IS) family, mobilize resistance genes in staphylococci and enterococci, respectively. Many different resistance genes are found in compound transposons bounded by IS26, and multiply and extensively antibiotic-resistant Gram-negative bacteria often include regions containing several antibiotic resistance genes and multiple copies of IS26. We recently showed that in addition to replicative transposition, IS26can use a conservative movement mechanism in which an incoming IS26targets a preexisting one, and this reaction can create these regions. This mechanism differs from that of all the ISs examined in detail thus far. Here, we have continued to extend understanding of the reactions carried out by IS26by examining whether the reverse precise excision reaction is also catalyzed by the IS26transposase.

scholarly journals Longitudinal assessment of antibiotic resistance gene profiles from the infant gut microbiome

2019 ◽  
Author(s):  
Evelyn Loo ◽  
Amanda Zain ◽  
Gaik Chin Yap ◽  
Rikky W Purbojati ◽  
Daniela I Drautz-Moses ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Cohort Study ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Longitudinal Cohort Study ◽  
First Year ◽  
Beta Lactam ◽  
First Year Of Life

Abstract Background: The rapid spread of multidrug- resistant pathogenic bacteria is a worldwide public health concern. Given the high carriage rate of extended spectrum beta-lactamase (ESBL)- producing Enterobacteriaceae in Asia, we aimed to evaluate community prevalence and dynamics by studying the longitudinal changes in antibiotic resistance gene (ARG) profiles and prevalence of ESBL-producing E coli and K. pneumoniae in the intestinal microbiome of infants participating in the Growing Up in Singapore Towards Healthy Outcomes (GUSTO) study, a longitudinal cohort study of pregnant women and their infants. Methods: We analysed the antibiotic resistance genes profile in the first year of life among 75 infants who had stool samples collected at multiple timepoints using metagenomics. Results: The mean number of ARGs per infant increased with age. The most common ARGs identified confer resistance to aminoglycoside, beta-lactam, macrolide and tetracycline antibiotics; all infants harboured these antibiotic resistance genes at some point in the first year of life. Few ARGs persisted throughout the first year of life. Beta-lactam resistant Escherichia coli and Klebsiella pneumoniae were detected in 4 (5.3%) and 32 (42.7%) of subjects respectively. Conclusion: In this longitudinal cohort study of healthy infants living in a region with high endemic antibacterial resistance, we demonstrate that majority of the infants harboured a number of antibiotic resistance genes in their gut and showed that the infant gut resistome is diverse and dynamic over the first year of life.

scholarly journals What is the role of the environment in the emergence of novel antibiotic resistance genes? – A modelling approach

2021 ◽  
Author(s):  
Johan Bengtsson-Palme ◽  
Viktor Jonsson ◽  
Stefanie Heß
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Quantitative Data ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Resistant Bacteria ◽  
Minor Role ◽  
Model Framework ◽  
Animal Pathogens

AbstractIt is generally accepted that intervention strategies to curb antibiotic resistance cannot solely focus on human and veterinary medicine but must also consider environmental settings. While the environment clearly has a role in the transmission of resistant bacteria, it is less clear what role it plays in the emergence of novel types of resistance. It has been suggested that the environment constitutes an enormous recruitment ground for resistance genes to pathogens, but the extent to which this actually happens is unknown. In this study, we built a model framework for resistance emergence and used the available quantitative data on the relevant processes to identify the steps which are limiting the appearance of antibiotic resistance determinants in human or animal pathogens. We also assessed the effect of uncertainty in the available data on the model results. We found that in a majority of scenarios, the environment would only play a minor role in the emergence of novel resistance genes. However, the uncertainty around this role is enormous, highlighting an urgent need of more quantitative data to understand the role of the environment in antibiotic resistance development. Specifically, more data is most needed on the fitness costs of antibiotic resistance gene (ARG) carriage, the degree of dispersal of resistant bacteria from the environment to humans, but also the rates of mobilization and horizontal transfer of ARGs. Quantitative data on these processes is instrumental to determine which processes that should be targeted for interventions to curb development and transmission of resistance.

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