scholarly journals Methods for the targeted sequencing and analysis of integrons and their gene cassettes from complex microbial communities

2021 ◽  
Author(s):  
Timothy M. Ghaly ◽  
Anahit Penesyan ◽  
Alexander Pritchard ◽  
Qin Qi ◽  
Vaheesan Rajabal ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Antibiotic Resistance Genes ◽  
Pcr Amplification ◽  
Gene Cassette ◽  
Human Pathogens ◽  
Diverse Range ◽  
Gene Cassettes ◽  
Wide Range ◽  
Class 1 ◽  
Range Of Functions

AbstractIntegrons are bacterial genetic elements that can integrate mobile gene cassettes. They are mostly known for spreading antibiotic resistance cassettes among human pathogens. However, beyond clinical settings, gene cassettes encode an extraordinarily diverse range of functions important for bacterial adaptation. The recovery and sequencing of cassettes has promising applications, including: surveillance of clinically important genes, particularly antibiotic resistance determinants; investigating the functional diversity of integron-carrying bacteria; and novel enzyme discovery. Although gene cassettes can be directly recovered using PCR, there are no standardised methods for their amplification and, importantly, for validating sequences as genuine integron gene cassettes. Here, we present reproducible methods for the PCR amplification, sequence processing, and validation of gene cassette amplicons from complex communities. We describe two different PCR assays that either amplify cassettes together with integron integrases, or gene cassettes together within cassette arrays. We compare the use of Nanopore and Illumina sequencing, and present bioinformatic pipelines that filter sequences to ensure that they represent amplicons from genuine integrons. Using a diverse set of environmental DNAs, we show that our approach can consistently recover thousands of unique cassettes per sample and up to hundreds of different integron integrases. Recovered cassettes confer a wide range of functions, including antibiotic resistance, with as many as 300 resistance cassettes found in a single sample. In particular, we show that class 1 integrons appear to be collecting and concentrating antibiotic resistance genes out of the broader diversity of cassette functions. The methods described here can be applied to any environmental or clinical microbiome sample.

scholarly journals Distribution and Content of Class 1 Integrons in Different Vibrio cholerae O-Serotype Strains Isolated in Thailand

2000 ◽  
Vol 44 (5) ◽  
pp. 1315-1321 ◽  
Author(s):  
Anders Dalsgaard ◽  
Anita Forslund ◽  
Oralak Serichantalergs ◽  
Dorthe Sandvang
Keyword(s):  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Gene Cassette ◽  
Gene Cassettes ◽  
Class 1 Integrons ◽  
Class 1

ABSTRACT In this study, 176 clinical and environmental Vibrio cholerae strains of different O serotypes isolated in Thailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the aadB,aadA2, blaP1, dfrA1, anddfrA15 gene cassettes, which encode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; β-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the antibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons. However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and 1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982 contained the aadB gene cassette. Surprisingly, several strains harbored two integrons containing different cassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among differentV. cholerae O serotypes of mainly clinical origin in Thailand.

scholarly journals The Evolution of Class 1 Integrons and the Rise of Antibiotic Resistance

2008 ◽  
Vol 190 (14) ◽  
pp. 5095-5100 ◽  
Author(s):  
Michael Gillings ◽  
Yan Boucher ◽  
Maurizio Labbate ◽  
Andrew Holmes ◽  
Samyuktha Krishnan ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Lateral Transfer ◽  
Natural Environments ◽  
Class 1 Integrons ◽  
Wide Range ◽  
Human Food Chain ◽  
Class 1 ◽  
Phylogenetic Signature

ABSTRACT Class 1 integrons are central players in the worldwide problem of antibiotic resistance, because they can capture and express diverse resistance genes. In addition, they are often embedded in promiscuous plasmids and transposons, facilitating their lateral transfer into a wide range of pathogens. Understanding the origin of these elements is important for the practical control of antibiotic resistance and for exploring how lateral gene transfer can seriously impact on, and be impacted by, human activities. We now show that class 1 integrons can be found on the chromosomes of nonpathogenic soil and freshwater Betaproteobacteria. Here they exhibit structural and sequence diversity, an absence of antibiotic resistance genes, and a phylogenetic signature of lateral transfer. Some examples are almost identical to the core of the class 1 integrons now found in pathogens, leading us to conclude that environmental Betaproteobacteria were the original source of these genetic elements. Because these elements appear to be readily mobilized, their lateral transfer into human commensals and pathogens was inevitable, especially given that Betaproteobacteria carrying class 1 integrons are common in natural environments that intersect with the human food chain. The strong selection pressure imposed by the human use of antimicrobial compounds then ensured their fixation and global spread into new species.

scholarly journals Incidence, Distribution, and Spread of Tetracycline Resistance Determinants and Integron-Associated Antibiotic Resistance Genes among Motile Aeromonads from a Fish Farming Environment

2001 ◽  
Vol 67 (12) ◽  
pp. 5675-5682 ◽  
Author(s):  
Anja S. Schmidt ◽  
Morten S. Bruun ◽  
Inger Dalsgaard ◽  
Jens L. Larsen
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Class 1 Integron ◽  
Gene Cassettes ◽  
Class 1 Integrons ◽  
Resistance Determinants ◽  
Class 1

ABSTRACT A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908–4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had “empty” integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n = 6), or E (n = 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly tetApositive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids andtetA among the OTC-resistant aeromonads, tetEand the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.

Comparison of integron-linked antibiotic resistance genes in strains of Salmonella spp. isolated from swine in Chile in 2005 and 2008

2010 ◽  
Vol 56 (6) ◽  
pp. 515-521 ◽  
Author(s):  
Lisette Lapierre ◽  
Betty San Martín ◽  
Carolina Araya-Jordán ◽  
Consuelo Borie
Keyword(s):  
Antibiotic Resistance ◽  
Clavulanic Acid ◽  
Resistance Genes ◽  
Geographical Area ◽  
Antibiotic Resistance Genes ◽  
Gene Cassette ◽  
Salmonella Spp ◽  
Single Class ◽  
Class 1 Integrons ◽  
Class 1

Salmonella spp. isolates obtained from healthy swine in 2008 were analyzed for antibiotic resistance phenotypes and genotypes. The resistance profiles of the 2008 isolates were compared with those of a Salmonella collection isolated from the same geographical area in 2005. The 2008 isolates consisted of strains that were 97% oxytetracycline resistant, 33.3% amoxicillin resistant, 31.8% amoxicillin- plus clavulanic acid resistant, 27.5% trimethoprim–sulfamethoxazole resistant, 17.3% streptomycin resistant, and 7.2% enrofloxacin–ciprofloxacin resistant. The presence of integrons and resistance genes and their topological association in resistant strains was assessed by PCR. The prevalence of class 1 integrons was the highest, at 46.2%, while class 2 integrons were present in 17.9% of the isolates. In strains that harboured class 1 integrons, we identified 3 different gene cassette arrangements; a single class 2 integron arrangement of dfrA1–sat1–aadA1 was found. Comparison of these results with data obtained from the 2005 isolates showed that Salmonella strains resistant to amoxicillin and amoxicillin plus clavulanic acid had clearly emerged over the span of 3 years, along with an increase in the prevalence of class 1 integrons and the acquisition of new gene cassette arrangements. These findings highlight the need for continual monitoring of regional isolates to establish more efficient vigilance programs that can address variations in resistance over short periods of time within the same geographical area.

scholarly journals Class 1 Integrons Potentially Predating the Association with Tn402-Like Transposition Genes Are Present in a Sediment Microbial Community

2006 ◽  
Vol 188 (16) ◽  
pp. 5722-5730 ◽  
Author(s):  
H. W. Stokes ◽  
Camilla L. Nesbø ◽  
Marita Holley ◽  
Martin I. Bahl ◽  
Michael R. Gillings ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Bacterial Species ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Global Scale ◽  
Human Pathogens ◽  
Class 1 Integrons ◽  
Recombination System ◽  
Class 1

ABSTRACT Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the β-proteobacterial class. These data suggest that the dispersal of this class may have begun before the “antibiotic era.”

scholarly journals Antimicrobial Susceptibility Profiles among Pseudomonas aeruginosa Isolated from Professional SCUBA Divers with Otitis Externa, Swimming Pools and the Ocean at a Diving Operation in South Africa

Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 91
Author(s):  
Kevin Maclean ◽  
Fernande Olpa J Pankendem Njamo ◽  
Mahloro Hope Serepa-Dlamini ◽  
Kulsum Kondiah ◽  
Ezekiel Green
Keyword(s):  
South Africa ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Otitis Externa ◽  
Antibiotic Resistance Genes ◽  
Pcr Amplification ◽  
Multi Drug Resistance ◽  
Scuba Divers ◽  
Class 1 ◽  
Synergy Test

SCUBA divers are predisposed to otitis externa caused by Pseudomonas aeruginosa, which is becoming increasingly multi-drug resistant (MDR). The present work assessed the antibiotic resistance profiles of P. aeruginosa obtained from SCUBA divers and their environment in Sodwana Bay, South Africa. Bacterial isolates from a total of 137 random water and ear swab samples were identified using biochemical and molecular methods. P. aeruginosa strains were further evaluated for antibiotic susceptibility using the Kirby–Bauer assay. Double disk synergy test (DDST) to confirm metallo-β-lactamase (MBL) production and PCR amplification of specific antibiotic resistance genes was performed. All (100%) 22 P. aeruginosa isolates recovered were resistant to 6 of the β-lactams tested including imipenem but exhibited susceptibility to trimethoprim–sulfamethoxazole. MBL production was observed in 77% of isolates while the most prevalent extended-spectrum β-lactamase (ESBL) genes present included blaAmpC (86.9%) followed by blaTEM (82.6%). Sulfonamide resistance was largely encoded by sul1 (63.6%) and sul2 (77.3%) genes with a high abundance of class 1 integrons (77.3%) of which 18.2% carried both Intl1 and Intl2. P. aeruginosa found in Sodwana Bay exhibits multi-drug resistance (MDRce) to several pharmaceutically important drugs with the potential to transfer antibiotic resistance to other bacteria if the judicious use of antibiotics for their treatment is not practiced.

scholarly journals Impact of Wastewater Treatment on the Prevalence of Integrons and the Genetic Diversity of Integron Gene Cassettes

2018 ◽  
Vol 84 (9) ◽  
Author(s):  
Xin-Li An ◽  
Qing-Lin Chen ◽  
Dong Zhu ◽  
Yong-Guan Zhu ◽  
Michael R. Gillings ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Wastewater Treatment ◽  
Resistance Genes ◽  
High Throughput Sequencing ◽  
Wastewater Treatment Plants ◽  
Gene Cassette ◽  
Class 1 Integron ◽  
Gene Cassettes ◽  
Class 1 ◽  
The Impact

ABSTRACT The integron platform allows the acquisition, expression, and dissemination of antibiotic resistance genes within gene cassettes. Wastewater treatment plants (WWTPs) contain abundant resistance genes; however, knowledge about the impacts of wastewater treatment on integrons and their gene cassettes is limited. In this study, by using clone library analysis and high-throughput sequencing, we investigated the abundance of class 1, 2, and 3 integrons and their corresponding gene cassettes in three urban WWTPs. Our results showed that class 1 integrons were most abundant in WWTPs and that wastewater treatment significantly reduced the abundance of all integrons. The WWTP influents harbored the highest diversity of class 1 integron gene cassettes, whereas class 3 integron gene cassettes exhibited highest diversity in activated sludge. Most of the gene cassette arrays detected in class 1 integrons were novel. Aminoglycoside, beta-lactam, and trimethoprim resistance genes were highly prevalent in class 1 integron gene cassettes, while class 3 integrons mainly carried beta-lactam resistance gene cassettes. A core class 1 integron resistance gene cassette pool persisted during wastewater treatment, implying that these resistance genes could have high potential to spread into environments through WWTPs. These data provide new insights into the impact of wastewater treatment on integron pools and highlight the need for surveillance of resistance genes within both class 1 and 3 integrons. IMPORTANCE Wastewater treatment plants represent a significant sink and transport medium for antibiotic resistance bacteria and genes spreading into environments. Integrons are important genetic elements involved in the evolution of antibiotic resistance. To better understand the impact of wastewater treatment on integrons and their gene cassette contexts, we conducted clone library construction and high-throughput sequencing to analyze gene cassette contexts for class 1 and class 3 integrons during the wastewater treatment process. This study comprehensively profiled the distribution of integrons and their gene cassettes (especially class 3 integrons) in influents, activated sludge, and effluents of conventional municipal wastewater treatment plants. We further demonstrated that while wastewater treatment significantly reduced the abundance of integrons and the diversity of associated gene cassettes, a large fraction of integrons persisted in wastewater effluents and were consequentially discharged into downstream natural environments.

scholarly journals Molecular Characterization of Class 3 Integrons from Delftia spp.

2007 ◽  
Vol 189 (17) ◽  
pp. 6276-6283 ◽  
Author(s):  
Hai Xu ◽  
Julian Davies ◽  
Vivian Miao
Keyword(s):  
Antibiotic Resistance ◽  
Antibiotic Resistance Genes ◽  
Gene Cassette ◽  
Integrase Gene ◽  
Delftia Acidovorans ◽  
Sequence Identity ◽  
Chromosomal Genes ◽  
Class 1 ◽  
Delftia Tsuruhatensis

ABSTRACT Two environmental strains, Delftia acidovorans C17 and Delftia tsuruhatensis A90, were found to carry class 3 integrons, which have seldom been reported and then only from pathogens in which they are associated with antibiotic resistance genes. The Delftia integrons comprised a highly conserved class 3 integrase gene, upstream and oppositely oriented from a set of three or four gene cassettes that encoded unidentified functions. The A90 integron had one more gene cassette than the C17 integron, but the two were otherwise the same; furthermore, they were located within regions of sequence identity in both strains and linked to chromosomal genes. A screen of other Delftia and related strains did not reveal the presence of additional class 3 integrons. The observations suggest that these integrons were horizontally transferred to Delftia as part of a larger region and reside as chromosomal elements that probably predate transposon dissemination, as has been proposed for certain class 1 integrons.

Quintuplex PCR to Detect Antibiotic Resistance Genes in Streptococcus pneumoniae

2016 ◽  
Vol 1 (2) ◽  
pp. 22 ◽  
Author(s):  
Navindra Kumari Palanisamy ◽  
Parasakthi Navaratnam ◽  
Shamala Devi Sekaran
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Resistance Genes ◽  
Bacterial Pathogen ◽  
Antibiotic Resistance Genes ◽  
Pcr Amplification ◽  
Penicillin Resistance ◽  
Penicillin Binding Proteins ◽  
Efflux Mechanism ◽  
Mic Value

Introduction: Streptococcus pneumoniae is an important bacterial pathogen, causing respiratory infection. Penicillin resistance in S. pneumoniae is associated with alterations in the penicillin binding proteins, while resistance to macrolides is conferred either by the modification of the ribosomal target site or efflux mechanism. This study aimed to characterize S. pneumoniae and its antibiotic resistance genes using 2 sets of multiplex PCRs. Methods: A quintuplex and triplex PCR was used to characterize the pbp1A, ermB, gyrA, ply, and the mefE genes. Fifty-eight penicillin sensitive strains (PSSP), 36 penicillin intermediate strains (PISP) and 26 penicillin resistance strains (PRSP) were used. Results: Alteration in pbp1A was only observed in PISP and PRSP strains, while PCR amplification of the ermB or mefE was observed only in strains with reduced susceptibility to erythromycin. The assay was found to be sensitive as simulated blood cultures showed the lowest level of detection to be 10cfu. Conclusions: As predicted, the assay was able to differentiate penicillin susceptible from the non-susceptible strains based on the detection of the pbp1A gene, which correlated with the MIC value of the strains.

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