Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment

The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, >1 × 10¹⁹ bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year.     

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.     

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。     

A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria

The arenas and detailed mechanisms for transfer of antibiotic resistance genes between environmental bacteria and pathogens are largely unclear. Selection pressures from antibiotics in situations where environmental bacteria and human pathogens meet are expected to increase the risks for such gene transfer events. We hypothesize that waste-water treatment plants (WWTPs) serving antibiotic manufacturing industries may provide such spawning grounds, given the high bacterial densities present there together with exceptionally strong and persistent selection pressures from the antibiotic-contaminated waste. Previous analyses of effluent from an Indian industrial WWTP that processes waste from bulk drug production revealed the presence of a range of drugs, including broad spectrum antibiotics at extremely high concentrations (mg/L range). In this study, we have characterized the antibiotic resistance profiles of 93 bacterial strains sampled at different stages of the treatment process from the WWTP against 39 antibiotics belonging to 12 different classes. A large majority (86%) of the strains were resistant to 20 or more antibiotics. Although there were no classically-recognized human pathogens among the 93 isolated strains, opportunistic pathogens such as Ochrobactrum intermedium, Providencia rettgeri, vancomycin resistant Enterococci (VRE), Aerococcus sp. and Citrobacter freundii were found to be highly resistant. One of the O. intermedium strains (ER1) was resistant to 36 antibiotics, while P. rettgeri (OSR3) was resistant to 35 antibiotics. Class 1 and 2 integrons were detected in 74/93 (80%) strains each, and 88/93 (95%) strains harbored at least one type of integron. The qPCR analysis of community DNA also showed an unprecedented high prevalence of integrons, suggesting that the bacteria living under such high selective pressure have an appreciable potential for genetic exchange of resistance genes via mobile gene cassettes. The present study provides insight into the mechanisms behind and the extent of multi-drug resistance among bacteria living under an extreme antibiotic selection pressure.     

New sequence types and multidrug resistance among pathogenic Escherichia coli isolates from coastal marine sediments

The spread of antibiotic-resistant microorganisms is widely recognized, but data about their sources, presence, and significance in marine environments are still limited. We examined 109 Escherichia coli strains from coastal marine sediments carrying virulence genes for antibiotic susceptibility, specific resistance genes, prevalence of class 1 and 2 integrons, and sequence type. Antibiotic resistance was found in 35% of strains, and multiple resistances were found in 14%; the resistances detected most frequently were against tetracycline (28%), ampicillin (16.5%), trimethoprim-sulfamethoxazole (13%), and streptomycin (7%). The highest prevalence of resistant strains was in phylogenetic group A, whereas phylogroup B2 exhibited a significantly lower frequency than all the other groups. Sixty percent of multiresistant strains harbored class 1 or 2 integrase genes, and about 50% carried resistance genes (particularly dfrA and aadA) linked to a class 1 integron. Multilocus sequence typing of 14 selected strains identified eight different types characteristic of extraintestinal pathogens and three new allelic combinations. Our data suggest that coastal marine sediment may be a suitable environment for the survival of pathogenic and antimicrobial-resistant E. coli strains capable of contributing to resistance spread via integrons among benthic bacteria, and they highlight a role for these strains in the emergence of new virulent genotypes.     

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.     

Bacterial resistance evolution by recruitment of super-integron gene cassettes

The capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram-negative clinical isolates. The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution. Recently, ancestral chromosomal versions, the super-integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species. SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes. However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli. We introduced a naturally occurring MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae, a species known to harbour a SI. We show that MRIs can randomly recruit genes directly from the cache of SI cassettes. By applying a selective constraint for the development of antibiotic resistance, we demonstrate bacterial resistance evolution through the recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase gene from the V. cholerae SI and its precise insertion into the MRI. The resulting resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency. These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron-mediated recombination events.     

Quantification of class 1 integron abundance in natural environments using real-time quantitative PCR

Integrons are bacterial genetic elements capable of capturing and expressing potentially adaptive genetic material. Class 1 integrons constitute the most intensely studied group of these elements to date, mainly due to their well-established role in the acquisition and dissemination of antibiotic resistance genes in clinical environments. However, virtually nothing is known about the distribution or abundance of class 1 integrons outside of the clinical context. Here we develop a SYBR Green-based real-time quantitative PCR assay capable of quantifying the abundance of class 1 integrons in environmental samples. It was shown that the abundance of the intI1 gene in creek sediment correlates with ecological condition, implying that class 1 integrons provide selective advantages relevant to environmental pressures other than the use of antibiotics. By comparing the quantities of intI1 and 16S rRNA gene in each sample, it was demonstrated that approximately 2.7% of cells potentially harbour a class 1 integron. These findings suggest that class 1 integrons are widespread in natural environments removed from clinical settings and occur in a broader range of host organisms than had previously been assumed on the basis of culture-dependent estimates.     

Marine Sediment Bacteria Harbor Antibiotic Resistance Genes Highly Similar to Those Found in Human Pathogens

The ocean is a natural habitat for antibiotic-producing bacteria, and marine aquaculture introduces antibiotics into the ocean to treat infections and improve aquaculture production. Studies have shown that the ocean is an important reservoir of antibiotic resistance genes. However, there is a lack of understanding and knowledge about the clinical importance of the ocean resistome. We investigated the relationship between the ocean bacterial resistome and pathogenic resistome. We applied high-throughput sequencing and metagenomic analyses to explore the resistance genes in bacterial plasmids from marine sediments. Numerous putative resistance determinants were detected among the resistance genes in the sediment bacteria. We also found that several contigs shared high identity with transposons or plasmids from human pathogens, indicating that the sediment bacteria recently contributed or acquired resistance genes from pathogens. Marine sediment bacteria could play an important role in the global exchange of antibiotic resistance.     

Insects Represent a Link between Food Animal Farms and the Urban Environment for Antibiotic Resistance Traits

Antibiotic-resistant bacterial infections result in higher patient mortality rates, prolonged hospitalizations, and increased health care costs. Extensive use of antibiotics as growth promoters in the animal industry represents great pressure for evolution and selection of antibiotic-resistant bacteria on farms. Despite growing evidence showing that antibiotic use and bacterial resistance in food animals correlate with resistance in human pathogens, the proof for direct transmission of antibiotic resistance is difficult to provide. In this review, we make a case that insects commonly associated with food animals likely represent a direct and important link between animal farms and urban communities for antibiotic resistance traits. Houseflies and cockroaches have been shown to carry multidrug-resistant clonal lineages of bacteria identical to those found in animal manure. Furthermore, several studies have demonstrated proliferation of bacteria and horizontal transfer of resistance genes in the insect digestive tract as well as transmission of resistant bacteria by insects to new substrates. We propose that insect management should be an integral part of pre- and postharvest food safety strategies to minimize spread of zoonotic pathogens and antibiotic resistance traits from animal farms. Furthermore, the insect link between the agricultural and urban environment presents an additional argument for adopting prudent use of antibiotics in the food animal industry.     

Evolution and ecology of antibiotic resistance genes

A new perspective on the topic of antibiotic resistance is beginning to emerge based on a broader evolutionary and ecological understanding rather than from the traditional boundaries of clinical research of antibiotic-resistant bacterial pathogens. Phylogenetic insights into the evolution and diversity of several antibiotic resistance genes suggest that at least some of these genes have a long evolutionary history of diversification that began well before the 'antibiotic era'. Besides, there is no indication that lateral gene transfer from antibiotic-producing bacteria has played any significant role in shaping the pool of antibiotic resistance genes in clinically relevant and commensal bacteria. Most likely, the primary antibiotic resistance gene pool originated and diversified within the environmental bacterial communities, from which the genes were mobilized and penetrated into taxonomically and ecologically distant bacterial populations, including pathogens. Dissemination and penetration of antibiotic resistance genes from antibiotic producers were less significant and essentially limited to other high G+C bacteria. Besides direct selection by antibiotics, there is a number of other factors that may contribute to dissemination and maintenance of antibiotic resistance genes in bacterial populations.     

Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months

Manuring of arable soils may stimulate the spread of resistance genes by introduction of resistant populations and antibiotics. We investigated effects of pig manure and sulfadiazine (SDZ) on bacterial communities in soil microcosms. A silt loam and a loamy sand were mixed with manure containing SDZ (10 or 100 mg per kilogram of soil), and compared with untreated soil and manured soil without SDZ over a 2-month period. In both soils, manure and SDZ positively affected the quotients of total and SDZ-resistant culturable bacteria [most probable number (MPN)], and transfer frequencies of plasmids conferring SDZ resistance in filter matings of soil bacteria and an Escherichia coli recipient. Detection of sulfonamide resistance genes sul1, sul2 and sul3 in community DNA by polymerase chain reaction (PCR) and hybridization revealed a high prevalence of sul1 in manure and manured soils, while sul2 was mainly found in the loamy sand treated with manure and high SDZ amounts, and sul3 was not detected. By PCR quantification of sul1 and bacterial rrn genes, a transient effect of manure alone and a long-term effect of SDZ plus manure on absolute and relative sul1 abundance in soil was shown. The dynamics in soil of class 1 integrons, which are typically associated with sul1, was analysed by amplification of the gene cassette region. Integrons introduced by manure established in both soils. Soil type and SDZ affected the composition of integrons. The synergistic effects of manure and SDZ were still detectable after 2 months. The results suggest that manure from treated pigs enhances spread of antibiotic resistances in soil bacterial communities.     

Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements

The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.     

Integrons: Past,Present, and Future

SUMMARY

Integrons are versatile gene acquisition systems commonly found in bacterial genomes. They are ancient elements that are a hot spot for genomic complexity, generating phenotypic diversity and shaping adaptive responses. In recent times, they have had a major role in the acquisition, expression, and dissemination of antibiotic resistance genes. Assessing the ongoing threats posed by integrons requires an understanding of their origins and evolutionary history. This review examines the functions and activities of integrons before the antibiotic era. It shows how antibiotic use selected particular integrons from among the environmental pool of these elements, such that integrons carrying resistance genes are now present in the majority of Gram-negative pathogens. Finally, it examines the potential consequences of widespread pollution with the novel integrons that have been assembled via the agency of human antibiotic use and speculates on the potential uses of integrons as platforms for biotechnology.     

Identification and characterization of class 1 integron resistance gene cassettes among Salmonella strains isolated from healthy humans in China

Twenty-three strains of Salmonella spp. isolated from healthy humans in Guangdong, China, were examined for their susceptibility to ten common antibiotics and the presence of antibiotic resistance integrons. All the strains were resistant to at least one antibiotic, and 4 strains were positive for the intI1 gene. Polymerase chain reaction using in-F and in-B primers showed the existence of amplicons of 1,009 bp in two, 1,664 bp in one, and 1,009 bp and 1,664 bp in one of the intI1 -positive isolates, respectively. Sequence analysis revealed that the 1,009-bp amplicon harbored gene cassette aadA2, conferring resistance to spectinomycin, and the 1,664-bp amplicon harbored genes aadA5 and dfr17, conferring resistance to spectinomycin, streptomycin and trimethoprim. Meanwhile the experiments of plasmid conjugation and Southern hybridization with intI1 as the DNA probe indicated that all the integrons found in these strains were chromosomal. Because the strains carrying class 1 integrons were isolated from healthy humans, it suggests the need for all-round surveillance of the antibiotic resistance of pathogens.     

Abundance and Dynamics of Antibiotic Resistance Genes and Integrons in Lake Sediment Microcosms

Antibiotic resistance in bacteria causing disease is an ever growing threat to the world. Recently, environmental bacteria have become established as important both as sources of antibiotic resistance genes and in disseminating resistance genes. Low levels of antibiotics and other pharmaceuticals are regularly released into water environments via wastewater, and the concern is that such environmental contamination may serve to create hotspots for antibiotic resistance gene selection and dissemination. In this study, microcosms were created from water and sediments gathered from a lake in Sweden only lightly affected by human activities. The microcosms were exposed to a mixture of antibiotics of varying environmentally relevant concentrations (i.e., concentrations commonly encountered in wastewaters) in order to investigate the effect of low levels of antibiotics on antibiotic resistance gene abundances and dynamics in a previously uncontaminated environment. Antibiotic concentrations were measured using liquid chromatography-tandem mass spectrometry. Abundances of seven antibiotic resistance genes and the class 1 integron integrase gene, intI1, were quantified using real-time PCR. Resistance genes sulI and ermB were quantified in the microcosm sediments with mean abundances 5 and 15 gene copies/10⁶ 16S rRNA gene copies, respectively. Class 1 integrons were determined in the sediments with a mean concentration of 3.8×10⁴ copies/10⁶ 16S rRNA gene copies. The antibiotic treatment had no observable effect on antibiotic resistance gene or integron abundances.     

Genetics of antimicrobial resistance

Antimicrobial resistant strains of bacteria are an increasing threat to animal and human health. Resistance mechanisms to circumvent the toxic action of antimicrobials have been identified and described for all known antimicrobials currently available for clinical use in human and veterinary medicine. Acquired bacterial antibiotic resistance can result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. The most common resistance mechanisms employed by bacteria include enzymatic degradation or alteration of the antimicrobial, mutation in the antimicrobial target site, decreased cell wall permeability to antimicrobials, and active efflux of the antimicrobial across the cell membrane. The spread of mobile genetic elements such as plasmids, transposons, and integrons has greatly contributed to the rapid dissemination of antimicrobial resistance among several bacterial genera of human and veterinary importance. Antimicrobial resistance genes have been shown to accumulate on mobile elements, leading to a situation where multidrug resistance phenotypes can be transferred to a susceptible recipient via a single genetic event. The increasing prevalence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. The versatility with which bacteria adapt to their environment and exchange DNA between different genera highlights the need to implement effective antimicrobial stewardship and infection control programs in both human and veterinary medicine.     

Genetics of Antimicrobial Resistance

Antimicrobial resistant strains of bacteria are an increasing threat to animal and human health. Resistance mechanisms to circumvent the toxic action of antimicrobials have been identified and described for all known antimicrobials currently available for clinical use in human and veterinary medicine. Acquired bacterial antibiotic resistance can result from the mutation of normal cellular genes, the acquisition of foreign resistance genes, or a combination of these two mechanisms. The most common resistance mechanisms employed by bacteria include enzymatic degradation or alteration of the antimicrobial, mutation in the antimicrobial target site, decreased cell wall permeability to antimicrobials, and active efflux of the antimicrobial across the cell membrane. The spread of mobile genetic elements such as plasmids, transposons, and integrons has greatly contributed to the rapid dissemination of antimicrobial resistance among several bacterial genera of human and veterinary importance. Antimicrobial resistance genes have been shown to accumulate on mobile elements, leading to a situation where multidrug resistance phenotypes can be transferred to a susceptible recipient via a single genetic event. The increasing prevalence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. The versatility with which bacteria adapt to their environment and exchange DNA between different genera highlights the need to implement effective antimicrobial stewardship and infection control programs in both human and veterinary medicine.     

Activation of class 1 integron integrase is promoted in the intestinal environment

Class 1 integrons are widespread genetic elements playing a major role in the dissemination of antibiotic resistance. They allow bacteria to capture, express and exchange antibiotic resistance genes embedded within gene cassettes. Acquisition of gene cassettes is catalysed by the class 1 integron integrase, a site-specific recombinase playing a key role in the integron system. In in vitro planktonic culture, expression of intI1 is controlled by the SOS response, a regulatory network which mediates the repair of DNA damage caused by a wide range of bacterial stress, including antibiotics. However, in vitro experimental conditions are far from the real lifestyle of bacteria in natural environments such as the intestinal tract which is known to be a reservoir of integrons. In this study, we developed an in vivo model of intestinal colonization in gnotobiotic mice and used a recombination assay and quantitative real-time PCR, to investigate the induction of the SOS response and expression and activity of the class 1 integron integrase, IntI1. We found that the basal activity of IntI1 was higher in vivo than in vitro. In addition, we demonstrated that administration of a subinhibitory concentration of ciprofloxacin rapidly induced both the SOS response and intI1 expression that was correlated with an increase of the activity of IntI1. Our findings show that the gut is an environment in which the class 1 integron integrase is induced and active, and they highlight the potential role of integrons in the acquisition and/or expression of resistance genes in the gut, particularly during antibiotic therapy.     

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.