Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community

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 beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."     

Molecular characterization of class 3 integrons from Delftia spp

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.     

The evolution of class 1 integrons and the rise of antibiotic resistance

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.     

Class I integrons containing a dhfrI trimethoprim resistance gene cassette in aquatic Acinetobacter spp

The presence of antibiotic resistance gene cassettes in class I integrons was investigated in 24 sulfamethoxazole-resistant and -sensitive Acinetobacter isolates derived from two Danish freshwater trout farms. Integrons were detected in five isolates from one of the fish farms, and their inserts were characterised by DNA sequencing. Each isolate contained a dhfrI gene cassette encoding resistance to trimethoprim and an open reading frame orfC of unknown function identical to the content of an integron previously found in a clinical enterobacterial isolate. Among the five isolates, at least two different strains were differentiated based on phenotypic tests and randomly amplified polymorphic DNA analysis. To our knowledge, this is the first report and characterisation of an integron in environmental bacteria.     

Class 1 and class 2 integrons in multidrug-resistant gram-negative bacteria isolated from the Salmon River, British Columbia

Using an enrichment protocol, we isolated 16 gram-negative, multidrug-resistant strains of known or opportunistic bacterial pathogens from the Salmon River in south-central British Columbia from 2005 to 2009, and investigated the genetic basis of their resistance to a variety of antibiotics. Of the 16 strains, 13 carried class 1 integrons and three carried class 2 integrons. Genes found in cassettes associated with the integrons included those for dihydrofolate reductases (dfrA1, dfrA12, dfrA17, and dfrB7), aminoglycoside adenyltransferases (aadA1, aadA2, aadA5, and aadB), streptothricin acetyltransferase (sat), and hypothetical proteins (orfF and orfC). A new gene cassette of unknown function, orf1, was discovered between dfrA1 and aadA5 in Escherichia sp. Other genes for resistance to tetracycline, chloramphenicol, streptomycin, and kanamycin (tetA, tetB, tetD; catA; strA-strB; and aphA1-Iab, respectively) were outside the integrons. Several of these resistance determinants were transferable by conjugation. The detection of organisms and resistance determinants normally associated with clinical settings attest to their widespread dispersal and suggest that regular monitoring of their presence in aquatic habitats should become a part of the overall effort to understand the epidemiology of antibiotic resistance genes in bacteria.     

Class 1 integrons in Pseudomonas aeruginosa isolates from clinical settings in Amazon region, Brazil

A hundred and six Pseudomonas aeruginosa isolates from clinical cases were screened using PCR for the presence of integrons and associated resistance gene cassettes. Forty-four isolates harboured class 1 integrons (41.5%), of which 29 isolates (66%) also carried gene cassettes. The aacA gene was most frequently found within class 1 integrons (69%), followed by blaOXA family genes (52%). From class 1 integron-positive strains, we detected a total of 15 isolates (34%) carrying no gene cassettes. Restriction fragment-length polymorphism analysis of the integrons variable region revealed some identical structures, as well as distinct profiles indicating heterogeneity among these cassette regions. Multiresistance was observed in 71% of isolates, nevertheless no strong correlation was observed between integron presence and multiresistance. This is the first report showing class 1 integron prevalence and gene cassette content in P. aeruginosa isolates from clinical settings in the Brazilian Amazon.     

Molecular differentiation of common promoters in Salmonella class 1 integrons

The integron is a mobile gene element which harbors antibiotic-resistance gene cassettes capable of site-specific integration. Among the four known types of integrons, the class 1 integron has been associated with multidrug-resistance in pathogenic bacteria. These gene cassettes have been the focus of a series of studies. The gene cassettes share a common promoter, and their expression levels are affected not only by their proximity to the promoter, but also by the strength (weak, hybrid and strong) of the common promoter, P1, as well as the presence of the additional promoter, P2. In this study, we developed molecular methods for the differentiation of promoter structures using PCR, restriction enzyme analysis, and polyacrylamide gel electrophoresis, and have applied them to the characterization of class 1 integrons in 33 non-typhoidal Salmonella serotypes in Korea. Class 1 integrons were detected in four serotypes: S. Derby (SD), S. Istanbul (SI), S. Paratyphi B (SPB), and S. Livingstone (SL), and the amplicon sizes were 1.0 Kb (SD, SI and SPB) and 2.0 Kb (SL). All of the 1.0 kb amplicons harbored gene cassettes (aadA1 or aadA2), but the 2.0 kb amplicon harbored three (dhfrXII-orfF-aadA2) gene cassettes, which conferred streptomycin/spectinomycin (aadA) and trimethoprim (dhfr) resistances. Our promoter structure study revealed three types of promoters; strong P1 (SD), weak P1 (SPB and SL), and weak P1+P2 (SI). In conclusion, the class 1 integrons were detected in Korean NTS, and their promoter structures were found to be variable. Therefore, our methods may prove helpful in terms of our understanding of molecular diversity, as well as the transmission of class 1 integrons and phenotype-genotype relationships in antibiotic-resistance.     

Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China

Aims: To characterize the molecular diversity of class 1 integrons and antibiotic resistance (AR) genes of Enterobacteriaceae strains recovered from aquatic habitats in Jinan, Shandong Province, China. Methods and Results: Six hundred and thirty‐eight antimicrobial‐resistant Enterobacteriaceae isolated from wastewater were examined for class 1 integron. Of these, 293 were positive for the class 1 integrase gene intI1; among these, 34 gene cassettes and 29 AR genes were detected. Twenty‐nine distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP). Seven strains harboring novel gene cassette arrays were subjected to further study, in which antimicrobial susceptibility profiles were determined, and the presence of other AR genes outside of the integrons was assayed. Several of the resistance determinants were found to be transferable by conjugation or transformation. Conclusions: This study established the assessment of class 1 integron and antimicrobial resistance gene patterns among environmental Enterobacteriaceae. Also, a restriction enzyme EcoRII was employed to develop a rapid and simple method for characterizing gene cassette arrays by RFLP analysis, which facilitated further study of novel gene cassette arrays. Significance and Impact of Study: These data not only illustrated the diversity of class 1 integron gene cassettes but also provided direct evidence that integrons mobilized gene cassettes, generating new linkages of resistance genes, and they could be integrated in gene transfer units such as conjugative plasmids to contribute to the dissemination of AR genes by horizontal gene transfer (HGT) in aquatic environments.     

Distribution of class 1 integrons among enteropathogenic Escherichia coli

The aim of this study was to investigate the incidence of and resistance gene content of class 1 integrons among enteropathogenic Escherichia coli (EPEC) and non-EPEC and to investigate intraspecies genetic diversity of EPEC strains isolated from children with diarrhea in Iran. Twenty-eight EPEC and 16 non-EPEC strains isolated from children with diarrhea were tested for the presence of a class 1 integron associated integrase gene (int1). Sequence analysis was performed to identify the resistance gene content of integrons. Genetic diversity and cluster analysis of EPEC isolates were also investigated using enterobacterial repetitive intergenic concensus-polymerase chain reaction (ERIC-PCR) fingerprinting. Twenty-three (82%) EPEC isolates and 11 (68.7%) non-EPEC isolates harbored the int1 gene specific to the conserved integrase region of class 1 integrons. Sequence analysis revealed the dominance of dfrA and aadA gene cassettes among the isolates of both groups. ERIC-PCR fingerprinting of EPEC isolates revealed a high diversity among these isolates. The widespread distribution of 2 resistance gene families (dfrA and aadA) among both groups of EPEC and non-EPEC isolates indicates the significance of integrons in antibiotic resistance transfer among these bacteria. Furthermore, clonal diversity of EPEC isolates harbouring a class 1 integron also suggests the circulation of these mobile elements among a diverse population of EPEC in this country.     

产超广谱β-内酰胺酶细菌中I类整合子的研究进展

产超广谱β-内酰胺酶(Extended-spectrum beta-lactamase, ESBLs)细菌的多重耐药性是临床用药的一大难题, 近年研究发现其耐药性的产生与整合子密切相关, 其中临床最常见、研究最深入的是I类整合子。整合子是一种可移动基因元件, 在整合酶的作用下捕捉外源基因盒并使之表达, 是具有基因整合和切除功能的天然克隆和表达系统。研究表明I类整合子可连续捕捉和整合多种耐药基因, 以质粒或转座子为载体在细菌之间传播耐药性, 使ESBLs细菌多重耐药趋势十分严峻。本文就I类整合子的结构特征、I类整合子对耐药基因盒的整合作用及其与ESBLs细菌耐药性的关系等方面进行综述。     

Gene cassettes and cassette arrays in mobile resistance integrons

Gene cassettes are small mobile elements, consisting of little more than a single gene and recombination site, which are captured by larger elements called integrons. Several cassettes may be inserted into the same integron forming a tandem array. The discovery of integrons in the chromosome of many species has led to the identification of thousands of gene cassettes, mostly of unknown function, while integrons associated with transposons and plasmids carry mainly antibiotic resistance genes and constitute an important means of spreading resistance. An updated compilation of gene cassettes found in sequences of such 'mobile resistance integrons' in GenBank was facilitated by a specially developed automated annotation system. At least 130 different (<98% identical) cassettes that carry known or predicted antibiotic resistance genes were identified, along with many cassettes of unknown function. We list exemplar GenBank accession numbers for each and address some nomenclature issues. Various modifications to cassettes, some of which may be useful in tracking cassette epidemiology, are also described. Despite potential biases in the GenBank dataset, preliminary analysis of cassette distribution suggests interesting differences between cassettes and may provide useful information to direct more systematic studies.     

Class 1 integron in staphylococci

As a major concern in public health, methicillin-resistant staphylococci (MRS) still remains one of the most prevalent pathogens that cause nosocomial infections throughout the world and has been recently labeled as a “super bug” in antibiotic resistance. Thus, surveillance and investigation on antibiotic resistance mechanisms involved in clinical MRS strains may raise urgent necessity and utmost significance. As a novel antibiotic resistance mechanism, class 1 integron has been identified as a primary source of antimicrobial resistance genes in Gram-negative organisms. However, most available studies on integrons had been limited within Gram-negative microbes, little is known for clinical Gram-positive bacteria. Based on series studies of systematic integrons investigation in hundreds of staphylococci strains during 2001–2006, this review concentrated on the latest development of class 1 integron in MRS isolates, including summary of prevalence and occurrence of class 1 integron, analysis of correlation between integron and antibiotic resistance, further demonstration of the role integrons play as antibiotic determinants, as well as origin and evolution of integron-associated gene cassettes during this study period.     

Recovery of diverse genes for class 1 integron-integrases from environmental DNA samples

Class 1 integrons are an important vector for the spread of antibiotic resistance. The core of this genetic element is highly conserved in all class 1 integrons recovered from clinical contexts. Recently, bacteria containing more divergent class 1 integrons have been isolated from environmental samples, suggesting undiscovered diversity in these elements. We performed a culture-independent survey of the class 1 integron-integrase gene (intI1) from environmental DNA, assessing sequence variation using capillary electrophoresis single-strand conformation polymorphism. This analysis allowed informed selection of environments for further investigation based on the diversity of intI1 targets that were present. IntI1 was common in environmental samples and exhibited previously unsuspected sequence diversity. The method allowed discrimination between clinical and environmental variants of intI1.     

Class 1 and class 2 integrons and plasmid-mediated antibiotic resistance in coliforms isolated from ten rivers in northern Turkey

We aimed to determine the molecular mechanisms of antibiotic resistance in coliforms isolated from ten rivers in northern region of Turkey. A total of 183 isolates were tested for antimicrobial susceptibility by disk diffusion and agar dilution methods. Resistance to ampicillin, streptomycin, trimethoprim, tetracycline, and chloramphenicol was detected in 58%, 51.9%, 24%, 28.4%, and 12.5%, respectively. Twelve (6.5%) phylogenetically distant organisms were detected to harbor self-transmissible plasmids ranging 52 to >147 kb in sizes. Resistances to ampicillin, tetracycline, trimethoprim, streptomycin, and nalidixic acid were commonly transferable traits. Transferable nalidixic acid-resistant strains harbored qnrS gene, which was the first report of plasmid-mediated quinolone resistance in bacteria of environmental origin in Turkey. Fourteen and five coliforms harbored class 1 and class 2 integrons, respectively, and some of them were located on transferable plasmids. Sequence analyses of variable regions of the class 1 and 2 integrons harbored various gene cassettes, dfrA1, dfr2d, dfrA7, dfrA16, dfrA17, aadA1, aadA5, bla _oxA-30, and sat1. A gene cassette array, dfrA16 has been demonstrated for the first time in a Citrobacter koseri isolate. Class 1 and class 2-bearing strains were clustered in different groups by BOX-PCR fingerprinting. Rivers in the northern Turkey may act as receptacle for the multi-drug resistant enterobacteria and can serve as reservoirs of the antimicrobial resistance determinants in the environment. The actual risk to public health is the transfer of resistance genes from the environmental bacteria to human pathogens. This study was presented in part at the 2^nd World Conference on Magic Bullets, held October 3–5, 2008 in Nurnberg, Germany.     

Diversity of gene cassette promoters in class 1 integrons from wastewater environments

The diversity of gene cassette promoters in class 1 integrons was investigated in 47 strains isolated from wastewaters. The weak PcW and PcH1 variants predominated, suggesting that, similar to clinical environments, high rates of gene cassette recombination, rather than high expression of gene cassettes, have been preferentially selected in wastewaters.     

Epidemiology and molecular mechanism of integron-mediated antibiotic resistance in <Emphasis Type="Italic">Shigella</Emphasis>

Integrons are gene capture and expression systems that are characterized by the presence of an integrase gene. This encodes an integrase, a recombined site, and a promoter. They are able to capture gene cassettes from the environment and incorporate them using site-specific recombination. The role of integrons and gene cassettes in the dissemination of multidrug resistance in Gram-negative bacteria is significant. In Shigella species, antimicrobial resistance is often associated with the presence of class 1 and class 2 integrons that contain resistance gene cassettes. Multiple and complex expression regulation mechanisms involving mobile genetic elements in integrons have been developed in the evolution of Shigella strains. Knowledge of the epidemiology and molecular mechanisms of antimicrobial resistance in this important pathogen is essential for the implementation of intervention strategies. This review was conducted to introduce the structures and functions of integrons in Shigella species and mechanisms that control integron-mediated events linked to antibiotic resistance.     

Integron-associated gene cassettes in Halifax Harbour: assessment of a mobile gene pool in marine sediments

The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of ∼3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling ( Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins.     

Characterization of integrons and antimicrobial resistance genes in clinical isolates of Gram-negative bacteria from Palestinian hospitals

Sixty Gram-negative bacterial isolates were collected from Palestinian hospitals in 2006. Thirty-two (53.3%) isolates showed multidrug resistance phenotypes. PCR and DNA sequencing were used to characterize integrons and antimicrobial resistance genes. PCR screening showed that 19 (31.7%) and five (8.3%) isolates were positive for class 1 and class 2 integrons, respectively. DNA-sequencing results for the captured antimicrobial resistance gene cassettes within class 1 integrons identified the following genes: dihydrofolate reductases, dfrA1 , dfrA5 , dfrA7 , dfrA12, dfrA17 and dfrA25 ; aminoglycoside adenyltransferases, aadA1, aadA2 , aadA5, aadA12 and aadB ; aminoglycoside acetyltransferase, aac(6')-Ib ; and chloramphenicol resistance gene, cmlA1 . ESBL were identified in 25 (41.7%) isolates. The identified ESBL were bla _CTX-M-15, bla _CTX-M-56, bla _OXA-1, bla _SHV-1, bla _SHV-12, bla _SHV-32 and bla _TEM-1 genes. Moreover, we characterized the plasmid-mediated quinolone resistance genes, aac(6')-Ib-cr and qnrB2 , which were detected in seven (11.7%) and two (3.3%) isolates, respectively. In this study various types of antibiotic resistance genes have been identified in Gram-negative bacteria from Palestinian hospitals, many of which are reported in the Middle East area for the first time.     

Bacterial superintegrons, a source of new genes with adaptive functions

Data on the structural organization of the platform of integrons, gene cassettes, and integrons with integrated cassettes of genes encoding drug resistance are briefly summarized. Data obtained during recent years about superintegrons or chromosomal integrons, characteristics of their organization, the presence of genes with known adaptive and unidentified functions in them, as well as data on the differences between superintegrons and previously described multiple resistance integrons, are considered in more detail. Studies that provide evidence for translocations of gene cassettes from stationary chromosomal integrons into integrons associated with mobile elements resulting in gene flows in natural bacterial populations, which favors their survival and adaptation to changing environment, are also reviewed.