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.     

Prevalence of antimicrobial resistance in bacteria isolated from central nervous system specimens as reported by U.S. hospital laboratories from 2000 to 2002

Background

Class 1 integrons contain genetic elements for site-specific recombination, capture and mobilization of resistance genes. Studies investigating the prevalence, distribution and types of integron located resistance genes are important for surveillance of antimicrobial resistance and to understand resistance development at the molecular level.

Methods

We determined the prevalence and genetic content of class 1 integrons in Enterobacteriaceae (strain collection 1, n = 192) and E. coli (strain collection 2, n = 53) from bloodstream infections in patients from six Norwegian hospitals by molecular techniques. Class 1 integrons were also characterized in 54 randomly selected multiresistant E. coli isolates from gastrointestinal human infections (strain collection 3).

Results

Class 1 integrons were present in 10.9% of the Enterobacteriaceae blood culture isolates of collection 1, all but one (S. Typhi) being E. coli. Data indicated variations in class 1 integron prevalence between hospitals. Class 1 integrons were present in 37% and 34% of the resistant blood culture isolates (collection 1 and 2, respectively) and in 42% of the resistant gastrointestinal E. coli. We detected a total of 10 distinct integron cassette PCR amplicons that varied in size between 0.15 kb and 2.2 kb and contained between zero and three resistance genes. Cassettes encoding resistance to trimethoprim and aminoglycosides were most common. We identified and characterized a novel plasmid-located integron with a cassette-bound novel gene (linF) located downstream of an aadA2 gene cassette. The linF gene encoded a putative 273 aa lincosamide nucleotidyltransferase resistance protein and conferred resistance to lincomycin and clindamycin. The deduced LinF amino acid sequence displayed approximately 35% identity to the Enterococcus faecium and Enterococcus faecalis nucleotidyl transferases encoded by linB and linB'

Conclusions

The present study demonstrated an overall low and stable prevalence of class 1 integron gene cassettes in clinical Enterobacteriaceae and E. coli isolates in Norway. Characterization of the novel lincosamide resistance gene extends the growing list of class 1 integron gene cassettes that confer resistance to an increasing number of antibiotics.     

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.     

Identification and molecular characterization of class 1 integrons in multiresistant Escherichia coli isolates from poultry litter

This study describes the prevalence of arrays of class 1 integron cassettes and Qnr determinants (A, B, and S) in 19 fluoroquinolone-resistant Escherichia coli isolates from chicken litter. qnrS and qnrA were the predominant genes in these fluoroquinolone-resistant isolates, and an uncommon array of aacA4-catB3-dfrA1 gene cassettes from a class1 integron was found. Additionally, aadA1 and dfrA1 gene cassettes, encoding resistance to streptomycin and trimethoprim, constituted the most common genes identified and was located on megaplasmids as well on the chromosome. Antibiotic resistance, pulsed-field gel electrophoresis (PFGE), and plasmid data suggest a genetically diverse origin of poultry E. coli isolates.     

临床分离的变形杆菌中整合子分型研究

目的了解临床分离变形杆菌中1、2类整合子的流行现状及其耐药性。方法PCR扩增146株变形杆菌中1、2类整合酶基因,M-H肉汤稀释法检测146株变形杆菌的药敏情况。结果1类整合子检出率为36.9%(54/146),2类整合子检出率为38.4%(56/146),同时携带1、2类整合子的检出率为18.5%(27/146)。结论1、2类整合子在变形杆菌感染临床分离株中所占比例已相当高,1、2类整合子与变形杆菌的多重耐药具有相关性。     

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.     

鲍曼不动杆菌整合子分型与耐药性的初步研究

目的了解临床分离的43株鲍曼不动杆菌中Ⅰ类、Ⅱ类、Ⅲ类整合子的流行病学现状及其耐药性。方法用多重PCR方法扩增30株鲍曼不动杆菌Ⅰ类、Ⅱ类、Ⅲ类整合酶基因,用K—B法检测鲍曼不动杆菌的耐药情况。结果Ⅰ类整合子检出率为79．1％（34／43）,未检出Ⅱ、Ⅲ类整合子。结论Ⅰ类整合子阳性的菌株的耐药率较高,显著高于Ⅰ类整合子阴性的菌株,多重PCR是筛查革兰阴性杆菌整合子的有效方法。     

上呼吸道分离鲍曼不动杆菌1 型整合子的分离与鉴定

【目的】研究I型整合子的结构特征,探讨其与细菌多重耐药之间的相关性。【方法】收集2008年至2009年广州呼吸疾病研究所上呼吸道分离的187株鲍曼不动杆菌,应用K-B纸片扩散法检测耐药性,采用聚合酶链式反应进行I型整合子整合酶基因的检测;扩增整合子的可变区,应用DNA测序技术分析I型整合子基因结构。【结果】I型整合子的阳性率达53.4%。共七种1型整合子基因盒被鉴定,其中首次发现报道一种新的整合子(GenBank:HQ322622)。可变区主要编码氨基糖苷类药物的耐药基因。20种抗菌素耐药的结果均表明携带Ⅰ型整合子的鲍曼不动杆菌耐药率较不携带I型整合子的鲍曼不动杆菌的耐药率明显增高。整合子与鲍曼不动杆菌的多重耐药表型具有密切相关性。【结论】I类整合子相关耐药基因在本院临床分离鲍曼不动杆菌中分布较广泛。整合子在鲍曼不动杆菌耐药性的形成和播散中具有重要作用。     

Unnoticed spread of class 1 integrons in gram-positive clinical strains isolated in Guangzhou, China

A total of 46 gram-positive bacteria isolated from clinical specimens collected in China were subjected to PCR analysis with the intI1-specific primers, and the intI1-positive strains were further analyzed for their resistance gene cassette. All isolates possessed the class 1 integron in their genomes and the array of gene cassettes was dhfrXII-orfF-aadA2, which is very similar to other organisms except in one isolate carrying an additional copy of the class 1 integron containing the aadA2 gene cassette. Altogether, the results indicate that the class 1 integron is widespread in gram-positive clinical strains isolated in Guangzhou, China.     

Integron,Plasmid and Host Strain Characteristics of Escherichia coli from Humans and Food Included in the Norwegian Antimicrobial Resistance Monitoring Programs

Antimicrobial resistant Escherichia coli (n=331) isolates from humans with bloodstream infections were investigated for the presence of class 1 and class 2 integrons. The integron cassettes arrays were characterized and the findings were compared with data from similar investigations on resistant E. coli from meat and meat products (n=241) produced during the same time period. All isolates were obtained from the Norwegian monitoring programs for antimicrobial resistance in human pathogens and in the veterinary sector. Methods used included PCR, sequencing, conjugation experiments, plasmid replicon typing and subtyping, pulsed-field-gel-electrophoresis and serotyping. Integrons of class 1 and 2 occurred significantly more frequently among human isolates; 45.4% (95% CI: 39.9-50.9) than among isolates from meat; 18% (95% CI: 13.2 -23.3), (p<0.01, Chi-square test). Identical cassette arrays including dfrA1-aadA1, aadA1, dfrA12-orfF-aadA2, oxa-30-aadA1 (class 1 integrons) and dfrA1-sat1-aadA1 (class 2 integrons) were detected from both humans and meat. However, the most prevalent cassette array in human isolates, dfrA17-aadA5, did not occur in isolates from meat, suggesting a possible linkage between this class 1 integron and a subpopulation of E. coli adapted to a human host. The drfA1-aadA1 and aadA1 class 1 integrons were found frequently in both human and meat isolates. These isolates were subjected to further studies to investigate similarities with regard to transferability, plasmid and host strain characteristics. We detected incF plasmids with pMLST profile F24:A-:B1 carrying drfA1-aadA1 integrons in isolates from pork and in a more distantly related E. coli strain from a human with septicaemia. Furthermore, we showed that most of the class 1 integrons with aadA1 were located on incF plasmids with pMLST profile F51:A-:B10 in human isolates. The plasmid was present in unrelated as well as closely related host strains, demonstrating that dissemination of this integron also could be attributed to clonal spread. In conclusion, among the systematically collected isolates from two different sources, some significant differences concerning integron prevalence and integron variants were observed. However, closely related plasmids as vehicles for specific class 1 integrons in isolates from meat and from a human with bloodstream infection were found. The occurrence of similar multi-resistance plasmids in bacteria from a food source and from a human clinical sample highlights the possible role of meat as a source of resistance elements for pathogenic bacteria.     

Validated predictive modelling of the environmental resistome

Multi-drug-resistant bacteria pose a significant threat to public health. The role of the environment in the overall rise in antibiotic-resistant infections and risk to humans is largely unknown. This study aimed to evaluate drivers of antibiotic-resistance levels across the River Thames catchment, model key biotic, spatial and chemical variables and produce predictive models for future risk assessment. Sediment samples from 13 sites across the River Thames basin were taken at four time points across 2011 and 2012. Samples were analysed for class 1 integron prevalence and enumeration of third-generation cephalosporin-resistant bacteria. Class 1 integron prevalence was validated as a molecular marker of antibiotic resistance; levels of resistance showed significant geospatial and temporal variation. The main explanatory variables of resistance levels at each sample site were the number, proximity, size and type of surrounding wastewater-treatment plants. Model 1 revealed treatment plants accounted for 49.5% of the variance in resistance levels. Other contributing factors were extent of different surrounding land cover types (for example, Neutral Grassland), temporal patterns and prior rainfall; when modelling all variables the resulting model (Model 2) could explain 82.9% of variations in resistance levels in the whole catchment. Chemical analyses correlated with key indicators of treatment plant effluent and a model (Model 3) was generated based on water quality parameters (contaminant and macro- and micro-nutrient levels). Model 2 was beta tested on independent sites and explained over 78% of the variation in integron prevalence showing a significant predictive ability. We believe all models in this study are highly useful tools for informing and prioritising mitigation strategies to reduce the environmental resistome.     

Detection of Pseudomonas aeruginosa carried a new array of gene cassettes within class 1 integron isolated from a teaching hospital in Nanjing, China

We report here novel array of gene cassettes found in single variable region of class 1 integron disseminated in Pseudomonas aeruginosa isolated from a teaching hospital in Nanjing, Jiangsu Province, China. 29 of 47 (61%) P. aeruginosa strains were confirmed haboured class 1 integron, and all the positive strains have the same variable region confirmed by PCR and RFLP methods. The variable region contained an unreported order of four gene cassettes aac(6′)-II-aadA13-cmlA8-oxa-10. Of those, cmlA8 gene was a variant of cmlA5 encoding non-enzymatic protein which putatively confer resistance to chloramphenicol. Susceptibility testing revealed multidrug-resistant mechanisms were involved in the class 1 integron positive clinical isolates. And the class 1 integron located on an about 15 kb transferable plasmid was certified by conjugation experiment and plasmid DNA analysis. The macro restriction profile indicated those clinical strains were clonally related. These authors contributed equally to this work.     

A plasmid-encoded class 1 integron carrying sat, a putative phosphoserine phosphatase gene and aadA2 from enterotoxigenic Escherichia coli O159 isolated in Japan

A class 1 integron was detected in a single multidrug-resistant strain of enterotoxigenice Escherichia coli (ETEC) O159 after examination of 23 clinical E. coli isolates. This isolate was resistant to streptomycin, kanamycin, gentamicin, chloramphenicol and ampicillin. Sequencing of the class 1 integron identified three-gene cassettes. The first is the streptothricin acetyltransferase gene, sat, which confers resistance to streptothricin. The second is an ORF whose product is a putative phosphoserine phosphatase (PSP), and the last is an aminoglycoside adenyltransferase gene, aadA2, which confers resistance to streptomycin and spectinomycin. The putative PSP gene product was found to be 39%, 38%, 28%, and 27% identical to PSP gene products of Vibrio vulnificus CMCP6, V. vulnificus YJ016, Pseudomonas syringae, and P. aeruginosa, respectively. Southern-blot hybridization showed that this integron is located on a 90 kb plasmid. This is the first report identifying a putative PSP gene in an integron.     

A comparison of antibiotic resistance integrons in cattle from separate beef meat production systems at slaughter

Aims: To compare antibiotic resistance integrons in cattle from three separate grass‐fed, grain‐fed and certified organic cattle production systems at slaughter. Methods and Results: In this study 198 samples from three separate cattle production systems were tested by PCR for the presence of class 1 and class 2 integrons. Integron‐containing bacteria were readily isolated from pen faeces and hide samples regardless of production system. Lower numbers of integron‐containing bacteria were isolated from the remaining sample types. Ninety‐one class 1 and 34 class 2 integron‐containing bacteria were isolated. Characterization of the integrons demonstrated a high degree of similarity across the three production systems with aadA1 and aadA2 routinely present. Integrons harbouring the cassette array cmlA5–bla_OXA‐10–aadA1 and the putative insertion sequence IS1066 were isolated from organic and grass‐fed cattle and have not been described previously. Conclusions: Integrons carrying antibiotic resistance genes were common in cattle from differing production systems at slaughter and the likelihood of presence appears unrelated to the production system. Significance and Impact of the Study: Similar integron arrays are present in different cattle production systems suggesting that their presence may be independent of production practices. This is the first report of two novel integron structures present in Aeromonas.     

Preclinical class 1 integron with a complete Tn402-like transposition module

The presence of integrons was assessed in gut bacteria isolated from wild-caught prawns. A pseudomonad was recovered that contained a Tn402-like class 1 integron with a complete transposition module and two gene cassettes. One cassette was identical to a previously described cassette from a chromosomal class 3 integron in Delftia tsuruhatensis.     

An integron of class 1 is present on the plasmid pCG4 from Gram-positive bacterium Corynebacterium glutamicum

The streptomycin/spectinomycin resistance determinant of the 29-kb plasmid pCG4 from Corynebacterium glutamicum was found to be a part of a typical class 1 integron. The sequence analysis revealed that the integron (designated InCg) identified in this Gram-positive bacterium is almost identical to the integron InC present on the plasmid pSA1700 from the Gram-negative bacterium Pseudomonas aeruginosa. Differences in only two base pairs were found in the 3.8-kb sequence. One base substitution (G→C) is present in the streptomycin/spectinomycin resistance determinant which is thus identical to the aadA2a gene from the integron In6 of the broad-host-range plasmid pSa. The other one (C→G) is present in the extended −10 region of the integron promoter involved in expression of the antibiotic resistance gene. It was shown that this novel version of the integron promoter displays five times higher activity in both C. glutamicum and Escherichia coli than the original one.     

Immobilisation, remineralisation and residual effects in subsequent crops of dairy cattle slurry nitrogen compared to mineral fertiliser nitrogen

About 50–60% of dairy cattle slurry nitrogen is ammonium N. Part of the ammonium N in cattle slurry is immobilised due to microbial decomposition of organic matter in the slurry after application to soil. The immobilisation and the remineralisation influence the fertiliser value of slurry N and the amount of organic N that is retained in soil. The immobilisation and the remineralisation of 15 N-labelled dairy cattle slurry NH₄-N were studied through three growing seasons after spring application under temperate conditions. Effects of slurry distribution (mixing, layer incorporation, injection, surface-banding) and extra litter straw in the slurry on the plant utilisation of labelled NH₄-N from slurry were studied and compared to the utilisation of ¹⁵N-labelled mineral fertiliser. The initial immobilisation of slurry N was influenced by the slurry distribution in soil. More N was immobilised when the slurry was mixed with soil. Surface-banding of slurry resulted in significant volatilisation losses and less residual ¹⁵N in soil. Much more N was immobilised after slurry incorporation than after mineral fertiliser application. After 2.5 years the recovery of labelled N in soil (0–25 cm) was 46% for slurry mixed with soil, 42% for injected slurry, 22% for surface-banded slurry and 24% for mineral fertiliser N. The total N uptake in a ryegrass cover crop was 5–10 kg N/ha higher in the autumn after spring-application of cattle slurry (100–120 kg NH₄-N/ha) compared to the mineral fertiliser N reference, but the immobilised slurry N (labelled N) only contributed little to the extra N uptake in the autumn. Even in the second autumn after slurry application there was an extra N uptake in the cover crop (0–10 kg N/ha). The residual effect of the cattle slurry on spring barley N uptake was insignificant in the year after slurry application (equivalent to 3% of total slurry N). Eighteen months after application, 13% of the residual ¹⁵N in soil was found in microbial biomass whether it derived from slurry or mineral fertiliser, but the remineralisation rate (% crop removal of residual ¹⁵N) was higher for fertiliser- than for slurry-derived N, except after surface-banding. Extra litter straw in the slurry had a negligible influence on the residual N effects in the year after application. It is concluded that a significant part of the organic N retained in soil after cattle slurry application is derived from immobilised ammonium N, but already a few months after application immobilised N is stabilised and only slowly released. The immobilised N has negligible influence on the residual N effect of cattle slurry in the first years after slurry application, and mainly contributes to the long-term accumulation of organic N in soil together with part of the organic slurry N. Under humid temperate conditions the residual N effects of the manure can only be optimally utilised when soil is also covered by plants in the autumn, because a significant part of the residual N is released in the autumn, and there is a higher risk of N leaching losses on soils that receive cattle slurry regularly compared to soils receiving only mineral N fertilisers.     

Changing Trends in the Prevalence of Shigella Species: Emergence of Multi-Drug Resistant Shigella sonnei Biotype g in Bangladesh

Shigellosis, caused by Shigella species, is a major public health problem in Bangladesh. To determine the prevalence and distribution of different Shigella species, we analyzed 10,827 Shigella isolates from patients between 2001 and 2011. S. flexneri was the predominant species isolated throughout the period. However, the prevalence of S. flexneri decreased from 65.7% in 2001 to 47% in 2011, whereas the prevalence of S. sonnei increased from 7.2% in 2001 to 25% in 2011. S. boydii and S. dysenteriae accounted for 17.3% and 7.7% of the isolates respectively throughout the period. Of 200 randomly selected S. sonnei isolates for extensive characterization, biotype g strains were predominant (95%) followed by biotype a (5%). Resistance to commonly used antibiotics including trimethoprim-sulfamethoxazole, nalidixic acid, ciprofloxacin, mecillinam and ampicillin was 89.5%, 86.5%, 17%, 10.5%, and 9.5%, respectively. All isolates were susceptible to ceftriaxone, cefotaxime, ceftazidime and imipenem. Ninety-eight percent of the strains had integrons belonging to class 1, 2 or both. The class 1 integron contained only dfrA5 gene, whereas among class 2 integron, 16% contained dhfrAI-sat1-aadA1-orfX gene cassettes and 84% harbored dhfrA1-sat2 gene cassettes. Plasmids of ∼5, ∼1.8 and ∼1.4 MDa in size were found in 92% of the strains, whereas only 33% of the strains carried the 120 MDa plasmid. PFGE analysis showed that strains having different integron patterns belonged to different clusters. These results show a changing trend in the prevalence of Shigella species with the emergence of multidrug resistant S. sonnei. Although S. flexneri continues to be the predominant species albeit with reduced prevalence, S. sonnei has emerged as the second most prevalent species replacing the earlier dominance by S. boydii and S. dysenteriae in Bangladesh.     

ESBLs大肠埃希菌和肺炎克雷伯杆菌整合子分布及其与ESBLs基因关系研究

目的研究整合子在院内感染ESBLs大肠埃希菌和肺炎克雷伯杆菌中的分布及其与细菌耐药的相关性,探讨Ⅰ类整合子在ESBLs基因水平转移中的作用及其与ESBLs基因型的关系。方法运用K-B法,纸片扩散法,PCR,接合传递试验、套式PCR、质粒谱分析及DNA测序研究携带耐药基因的Ⅰ类整合子与耐药播散的关系。结果产ESBLs和非产ESBLs菌株中Ⅰ类整合酶扩增阳性例数分别是70例（占66.7%）和22例（占22.4%）,2组整合子阳性率差异有统计学意义（P〈0.01）。整合子阳性组中ESBLs、多重耐药菌均明显高于阴性组。产ESBLs菌株中blaSHV、blaCTX和blaTEM基因与整合子经质粒共同转移的频率分别是79.3%、58.5%和45.1%。结论整合子在产ESBLs大肠埃希菌和肺炎克雷伯杆菌中的分布明显高于非产ESBLs菌株,主要为Ⅰ类整合子,并参与产ESBLs菌株多重耐药性的形成,其中blaSHV基因型与整合子经质粒共同转移的频率高于其他两类基因,提示SHV型酶在浙南地区具有更强的扩散优势。