Impact of Wastewater Treatment on the Prevalence of Integrons and the Genetic Diversity of Integron Gene Cassettes.
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.
Project description:Twenty Acinetobacter baumannii strains resistant to various antibiotics were analyzed for integron content and sequences of the amplification products. Sixteen clinical isolates had a class 1 integron, 2 contained an additional class 1 or class 2 integron, but no class 3 integron was detected. Thirteen strains had integrons with a single cassette: aac(3)-Ia (9 strains), ant(2")-Ia (2 strains), or aac(6')-Ib (2 strains); 1 had aac(6')-Ib and oxa20 cassettes and an unknown gene; and 1 had an integron containing ant(2")-Ia and an oxa3 cassette truncated by IS6100. The remaining strains harbored class 1 integrons with gene cassettes previously found in Enterobacteriaceae. One integron had a hybrid structure composed of intI2 and the 3' conserved segment of class 1 integrons. These data indicate that integrons play a major role in multidrug resistance in Acinetobacter.
Project description:In this study, antimicrobial-resistance patterns were analyzed in Escherichia coli isolates from raw (RW) and treated wastewater (TW) of two wastewater treatment plants (WWTPs), their marine outfalls (MOut), and mouth of the Vistula River (VR). Susceptibility of E. coli was tested against different classes of antibiotics. Isolates resistant to at least one antimicrobial agent were PCR tested for the presence of integrons. Ampicillin-resistant E. coli were the most frequent, followed by amoxicillin/clavulanate (up to 32 %), trimethoprim/sulfamethoxazole (up to 20 %), and fluoroquinolone (up to 15 %)-resistant isolates. Presence of class 1 and 2 integrons was detected among tested E. coli isolates with rate of 32.06 % (n = 84) and 3.05 % (n = 8), respectively. The presence of integrons was associated with increased frequency of resistance to fluoroquinolones, trimethoprim/sulfamethoxazole, amoxicillin/clavulanate, piperacillin/tazobactam, and presence of multidrug-resistance phenotype. Variable regions were detected in 48 class 1 and 5 class 2 integron-positive isolates. Nine different gene cassette arrays were confirmed among sequenced variable regions, with predominance of dfrA1-aadA1, dfrA17-aadA5, and aadA1 arrays. These findings illustrate the importance of WWTPs in spreading of resistance genes in the environment and the need for inclusion of at least monitoring efforts in the regular WWTP processes.
Project description:There is increasing evidence that human activity, and especially the resulting effluent, has a major role in the dissemination of bacterial antibiotic-resistance determinants in the environment. Hospitals are the major antibiotic consumers and thus facilitate the spread of antibiotic resistance. Questions are increasingly being raised about the management of hospital effluents, but their involvement in antibiotic-resistance dissemination has never been assessed. Integrons are a paradigm of genetic transfer between the environmental resistome and both commensal and pathogenic bacteria. In order to assess the impact of hospital activities on antibiotic-resistance dissemination in the environment, we monitored integrons and their gene cassettes in hospital effluents, and their release in the environment. We found that bacterial communities present in a hospital effluent contained a high proportion of integrons. In terms of both their gene cassette diversity and gene cassette arrays, the urban effluent and municipal wastewater treatment plant (WWTP) influent were most similar, whereas the hospital effluent and recirculation sludge exhibited very specific patterns. We found that anthropogenic activities led to the release of abundant integrons and antibiotic-resistance gene cassettes, but we observed no specific impact of hospital activities on the receiving environment. Furthermore, although the WWTP did not reduce the normalized integron copy number, it reduced the diversity of gene cassette arrays contained in the raw wastewater, underlining the effect of the biological treatment on the anthropogenic integron pool arriving at the WWTP.
Project description:Integrons are genetic units characterized by the ability to capture and incorporate gene cassettes, thus can contribute to the emergence and transfer of antibiotic resistance. The objectives of this study were: (1) to investigate the presence and distribution of class I and class II integrons and the characteristics of the gene cassettes they carry in Enterobacteriaceae isolated from nosocomial infections at Zagzig University Hospital in Egypt, (2) to determine their impact on resistance, and (3) to identify risk factors for the existence of integrons. Relevant samples and full clinical history were collected from 118 inpatients. Samples were processed; isolated microbes were identified and tested for antibiotic susceptibilities. Integrons were detected by polymerase chain reaction (PCR) and were characterized into class I or II by restriction fragment length polymorphism (RFLP). Integron-positive isolates were subjected to another PCR to detect gene cassette, followed by gene cassette sequencing. Risk factors were analyzed by logistic regression analysis. Seventy-six Enterobacteriaceae isolates were recognized, 41 of them (53.9%) were integron-positive; 39 strains carried class I and 2 strains carried class II integrons. Integrons had gene cassettes encoding different combinations and types of resistance determinants. Interestingly, blaOXA129 gene was found and ereA gene was carried on class I integrons. The same determinants were carried within isolates of the same species as well as isolates of different species. The presence of integrons was significantly associated with multidrug resistance (MDR). No risk factors were associated for integron carriage. We conclude that integrons carrying gene cassettes encoding antibiotic resistance are significantly present among Enterobacteriaceae causing nosocomial infection in our hospital. Risk factors for acquisition remain to be identified.
Project description: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.
Project description:<h4>Objectives</h4>The rapid emergence of drug-resistant Shigella sonnei is a serious public health problem. This study aimed to characterise the antimicrobial resistance patterns, molecular subtypes, and integron types and resistance gene cassettes in S. sonnei from Jiangsu Province, China.<h4>Methods</h4>In total, 340 S. sonnei were collected in 2002-2011 throughout Jiangsu Province. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), PCR amplification of integrons, restriction fragment length polymorphism (RFLP) and DNA sequencing of cassette regions were performed.<h4>Results</h4>Resistance rates to ampicillin (67.7%), nalidixic acid (75.2%), tetracycline (73.7%) and trimethoprim/sulfamethoxazole (68.7%) remained high. Strains from Centre and South Jiangsu showed higher resistance and multiresistance rates compared with the North. PFGE analysis indicated that large-scale clonal transmission among different cities occurred several times during 10 years. Among all strains, 55.9% (190/340) harboured class 1 integrons, 80.3% (273/340) harboured class 2 integrons and 49.4% (168/340) harboured an atypical class 1 integron. Resistance rates to nine antimicrobials in the class 1 integron-positive group were significantly higher than in the negative group (P<0.05). Seven different gene cassettes were detected in class 1 integrons. The most prevalent type was aacA4-cmlA1 (114/286). Class 2 integrons carried the gene cassette array dfrA1-sat1-aadA1, and the atypical class 1 integron carried bla<sub>OXA-30</sub>-aadA1.<h4>Conclusions</h4>The increasing antimicrobial resistance and significant clonal transmission of S. sonnei circulating in Jiangsu were closely related to the high prevalence of integrons and gene cassettes. Long-term cross-regional monitoring of antimicrobial resistance is urgently required for S. sonnei.
Project description:Klebsiella pneumoniae FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An Escherichia coli transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum beta-lactamase was encoded by bla(GES-1), previously described in class 1 integrons from K. pneumoniae ORI-1 and Pseudomonas aeruginosa Pa695. Further sequence analysis demonstrated that the bla(GES-1) gene cassette was located on a new class 3 integron. The integron was 2863 bp long and consisted of an intI3 integrase gene, an attI3 recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of Serratia marcescens AK9373. The bla(GES-1) gene cassette was inserted at the attI3 site. The second gene cassette was the result of a fusion event between bla(OXA-10)-type and aac(6')-Ib gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the bla(GES-1) gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.
Project description:BACKGROUND: The aims of this study were to understand the molecular epidemiology of integron-associated gene cassettes in Acinetobacter baumannii across four hospitals in northern Taiwan and to clarify the relationship between the presence of integrons and antibiotic-resistant phenotypes. METHODS: Sixty-five A. baumannii isolates, collected from the patients of four regional hospitals in northern Taiwan in 2009, were tested for the presence of integrons and their associated gene cassettes. The susceptibility difference between integron-positive and integron-negative A. baumannii strains was analyzed. Antibiotic-resistant phenotypes among A. baumannii with different types of gene cassette array combinations were also compared. RESULTS: Around 72% of the A. baumannii isolates carried class 1 integrase genes. Despite this, only three gene cassette arrays were found in the integrons. Integron-positive strains were significantly more resistant to all the tested antibiotics than the integrase-negative strains. All the four types of A. baumannii with different gene cassette array combinations were multidrug-resistant in nature. Gene cassette array aacA4-catB8-aadA1 existed in all the integron-positive A. baumannii isolates. Repetitive-sequence-based PCR (rep-PCR) results revealed the prevalence of one major cluster of imipenem-resistant A. baumannii strains (84%) in the four regional hospitals. CONCLUSIONS: The presence of integrons with associated antimicrobial resistance gene cassettes can be used as a representative marker of multidrug resistance in A. baumannii. Some prevalent gene cassette arrays may exist among epidemiologically unrelated A. baumannii strains.
Project description:<h4>Background</h4>Antimicrobial resistance among enteric bacteria in Africa is increasingly mediated by integrons on horizontally acquired genetic elements. There have been recent reports of such elements in invasive pathogens across Africa, but very little is known about the faecal reservoir of integron-borne genes.<h4>Methods and findings</h4>We screened 1098 faecal Escherichia coli isolates from 134 mother-child pairs for integron cassettes by PCR using primers that anneal to the 5' and 3' conserved ends of the cassette regions and for plasmid replicons. Genetic relatedness of isolates was determined by flagellin and multi-locus sequence typing. Integron cassettes were amplified in 410 (37.5%) isolates and were significantly associated with resistance to trimethoprim and multiple resistance. Ten cassette combinations were found in class 1 and two in class 2 integrons. The most common class 1 cassette configurations were single aadA1 (23.4%), dfrA7 (18.3%) and dfrA5 (14.4%). Class 2 cassette configurations were all either dfrA1-satI-aadA1 (n = 31, 7.6%) or dfrA1-satI (n = 13, 3.2%). A dfr cassette was detected in 294 (31.1%) of trimethoprim resistant strains and an aadA cassette in 242 (23%) of streptomycin resistant strains. Strains bearing integrons carried a wide range of plasmid replicons of which FIB/Y (n = 169; 41.2%) was the most frequently detected. Nine isolates from five different individuals carried the dfrA17-aadA5-bearing ST69 clonal group A (CGA). The same integron cassette combination was identified from multiple distinct isolates within the same host and between four mother-child pairs.<h4>Conclusions</h4>Integrons are important determinants of resistance in faecal E. coli. Plasmids in integron-containing strains may contribute to dispersing resistance genes. There is a need for improved surveillance for resistance and its mechanisms of dissemination and persistence and mobility of resistance genes in the community and clinical settings.
Project description:Integrons were sought in Acinetobacter isolates from hospitals in the United Kingdom by integrase gene PCR. Isolates were compared by pulsed-field gel electrophoresis, and most belonged to a small number of outbreak strains or clones of A. baumannii, which are highly successful in the United Kingdom. Class 1 integrons were found in all of the outbreak isolates but in none of the sporadic isolates. No class 2 integrons were found. Three integrons were identified among the main outbreak strains and clones. While a particular integron was usually associated with a strain or clone, some members carried a different integron. Some integrons were associated with more than one strain. The cassette arrays of two of the integrons were very similar, both containing gene aacC1, which confers resistance to gentamicin, two open reading frames coding for unknown products (orfX, orfX'), and gene aadA1a, which confers resistance to spectinomycin and streptomycin. The larger of these integrons had two copies of the first (orfX) of the gene cassettes coding for unknown products. The third integron, with a cassette array containing gene aacA4, which codes for amikacin, netilmicin, and tobramycin resistance; a chloramphenicol acetyltransferase, catB8; and gene aadA1, conferring resistance to spectinomycin and streptomycin, was associated with an OXA-23 carbapenemase-producing clone, which has spread rapidly in hospitals in the United Kingdom during 2003 and 2004. These integron cassette arrays have been found in other outbreak strains of A. baumannii from other countries. We conclude that integrons are useful markers for epidemic strains of A. baumannii and that integron typing provides valuable information for epidemiological studies.