scholarly journals EVALUATION OF THE IMPACT OF PLASMID CURING ON ANTIBIOTIC RESISTANCE ON SOME CLINICAL ISOLATES OF ESCHERICHIA COLI

2020 ◽  
Vol 4 (3) ◽  
pp. 323-327
Author(s):  
Mamunu Abdulkadir SULAIMAN ◽  
H.S Muhammad ◽  
Aliyu Muhammad Sani ◽  
Aminu Ibrahim ◽  
Ibrahim Muhammad Hussain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Clinical Isolates ◽  
Diffusion Method ◽  
Plasmid Curing ◽  
Sensitivity Testing ◽  
E Coli ◽  
Risk Of Death ◽  
Mar Index ◽  
The Impact

Multidrug resistance (MDR) exhibited by some strains of Escherichia coli may be due to acquiring mobile genetic element (R-plasmid) by the bacteria, or intrinsically induced by inappropriate use of antibiotics by the hosts.  Infection by such strains may result to prolonged illness and greater risk of death. The study evaluated the impact of curing on antibiotic resistance on selected clinical isolates of E. coli. Twenty clinical isolates of E. coli from our previous studies were re-characterized using conventional microbiological techniques. Antibiotic sensitivity testing was determined by disk diffusion method, MDR selected based on resistance to ≥ 2 classes of antibiotics. Multiple antibiotic resistance (MAR) index was determined as ratio of the number of antibiotic resisted to the total number of antibiotics tested and considered significant if ≥. 0.2. The isolates that showed significant MAR index were subjected to plasmid curing using acridine orange, thereafter, profiled for plasmid and the cured ones were re-tested against the antibiotics they initially resisted. Out of the 20 isolates, 19 (95%) were confirmed as E. coli, all (100%) of which were MDRs, which was highest against augmentin (78.9%) followed by amoxacillin (52.6%). However, after the plasmid curing only 6 (31.6%) out of the 19 isolates cured retained significant MAR index and the level of the significance had reduced drastically in 16 (84.2%) isolates. Conclusively, curing assay can completely eliminate R-plasmid acquired resistance. More studied on plasmid curing agents for possible augmentation of the agents into antibiotics may see the rise of successful antibiotic era again.

scholarly journals Detection of biofilm among uropathogenic Escherichia coli and its correlation with antibiotic resistance pattern

2019 ◽  
Vol 11 (01) ◽  
pp. 017-022 ◽  
Author(s):  
Rashmi M. Karigoudar ◽  
Mahesh H. Karigoudar ◽  
Sanjay M. Wavare ◽  
Smita S. Mangalgi
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Biofilm Formation ◽  
Agar Plate ◽  
Diffusion Method ◽  
Resistance Pattern ◽  
Susceptibility Pattern ◽  
Sensitivity Testing ◽  
E Coli ◽  
Tract Infections

Abstract BACKGROUND: Escherichia coli accounts for 70%–95% of urinary tract infections (UTIs). UTI is a serious health problem with respect to antibiotic resistance and biofilms formation being the prime cause for the antibiotic resistance. Biofilm can restrict the diffusion of substances and binding of antimicrobials. In this context, the present study is aimed to perform in vitro detection of biofilm formation among E. coli strains isolated from urine and to correlate their susceptibility pattern with biofilm formation. MATERIALS AND METHODS: A total of 100 E. coli strains isolated from patients suffering from UTI were included in the study. The identification of E. coli was performed by colony morphology, Gram staining, and standard biochemical tests. The detection of biofilm was carried out by Congo Red Agar (CRA) method, tube method (TM), and tissue culture plate (TCP) method. Antimicrobial sensitivity testing was performed by Kirby–Bauer disc diffusion method on Muller–Hinton agar plate. RESULTS: Of the 100 E. coli strains, 49 (49%) and 51 (51%) were from catheterized and noncatheterized patients, respectively. Biofilm production was positive by CRA, TM, and TCP method were 49 (49%), 55 (55%), and 69 (69%), respectively. Biofilm producers showed maximum resistance to co-trimoxazole (73.9%), gentamicin (94.2%), and imipenem (11.6%) when compared to nonbiofilm producers. Significant association was seen between resistance to antibiotic and biofilm formation with a P = 0.01 (<0.05). CONCLUSION: A greater understanding of biofilm detection in E. coli will help in the development of newer and more effective treatment. The detection of biofilm formation and antibiotic susceptibility pattern helps in choosing the correct antibiotic therapy.

scholarly journals Escherichia coli as Possible Agents of Spread of Multidrug Resistance in Port Harcourt, Rivers State.

2019 ◽  
Vol 4 (1) ◽  
pp. 16-21
Author(s):  
Kome Otokunefor ◽  
Victor Ogechi Osogho ◽  
Chijindu Precious Nwankwo
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Multidrug Resistance ◽  
Clinical Isolates ◽  
Human Populations ◽  
Plasmid Curing ◽  
Port Harcourt ◽  
Genes Encoding ◽  
Mar Index ◽  
High Level

AbstractMultidrug resistance (MDR) continues to be a growing global issue. The problem of MDR is fuelled in part by the spread of the genes encoding resistance horizontally which is linked particularly to conjugation involving plasmids. Studies have demonstrated the presence of plasmids in drug resistant isolates, few have shown a link between these plasmids and drug resistance via plasmid curing especially in our locale. This study set out to explore this link inEscherichia coliisolates from Port Harcourt, Nigeria. Plasmid curing was done on a selection of clinical and non-clinical bacteria using acridine orange and antibiotic susceptibility testing carried out on both cured and uncured variants. Data generated was analysed to ascertain the multiple antibiotic resistance (MAR) index and MDR of each isolate. Data was then compared to ascertain effects of plasmid curing on antibiotic resistance of the isolates. Results revealed a decrease in resistance to 7 of 8 antibiotics following plasmid curing. The highest change was noted in ceftazidime (40%), followed by ofloxacin (26.7%). Plasmid curing caused a shift in MAR index values of isolates from higher to lower indices. At MAR index values of ≤0.25 occurrence increased from 5% to 36.7% while at MAR index values ≥0.75, occurrence reduced from 29.9% to 10.0%. A reduction in the degree of MDR was noted (from 55% to 36.7%). Strikingly, the reduction in MDR level of non-clinical isolates was 30% as opposed to 3.4% in the clinical isolates. This study shows a link between plasmids and antibiotic resistance. For the non-clinical isolates, the high-level link between MDR and plasmid carriage could indicate a higher use of antimicrobials in non-clinical rather than clinical settings. Additionally, it could be an indicator for a higher risk of the transfer of MDR determinants from non-clinical sources to human populations in our locale.

scholarly journals Transmission of Antibiotic-Resistant Escherichia coli from Chicken Litter to Agricultural Soil

2022 ◽  
Vol 9 ◽  
Author(s):  
Dorcas Oladayo Fatoba ◽  
Daniel Gyamfi Amoako ◽  
Akebe Luther King Abia ◽  
Sabiha Yusuf Essack
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Soil Samples ◽  
Diffusion Method ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Before And After ◽  
Chicken Litter ◽  
The Impact ◽  
Amended Soil

A growing concern regarding the use of animal manure as fertilizer is the contamination of soil, plants, and the environment with a variety of antibiotic-resistant and pathogenic bacteria. This study quantified and characterized the antibiotic resistance profiles of Escherichia coli in soil before and after chicken litter application to determine the impact of manure on the soil resistome. Litter and soil samples were collected from a sugarcane field before and after litter application. E. coli was isolated and quantified using the Colilert®-18/Quanti-tray® 2000 and 10 randomly selected isolates from the positive wells of each Quanti-tray were putatively identified on eosin methylene blue agar. Real-time PCR was used to confirm the isolates by targeting the uidA gene. Antibiotic susceptibility test against 18 antibiotics was conducted using the disk diffusion method, and the multiple antibiotic resistance index was calculated. Soil amendment with chicken litter significantly increased the number of antibiotic-resistant E. coli in the soil. Among the 126 E. coli isolates purified from all the samples, 76% showed resistance to at least one antibiotic, of which 54.2% were multidrug-resistant (MDR). The highest percentage resistance was to tetracycline (78.1%), with the least percentage resistance (3.1%) to imipenem, tigecycline, and gentamicin. The isolates also showed resistance to chloramphenicol (63.5%), ampicillin (58.3%), trimethoprim-sulfamethoxazole (39.6%), cefotaxime (30.2%), ceftriaxone (26.0%), cephalexin (20.8%), cefepime (11.5%), amoxicillin-clavulanic acid (11.5%), cefoxitin (10.4%), Nalidixic acid (9.4%), amikacin (6.3%), and ciprofloxacin (4.2%). Of the 54.2% (52/96) MDR, the highest number was isolated from the litter-amended soil (61.5%) and the least isolates from soil samples collected before litter application (1.9%). The relatively higher mean MAR index of the litter-amended soil (0.14), compared to the soil before the amendment (0.04), suggests soil pollution with antibiotic-resistant E. coli from sources of high antibiotic use. E. coli could only be detected in the soil up to 42 days following manure application, making it a suitable short-term indicator of antibiotic resistance contamination. Notwithstanding its relatively short detectability/survival, the application of chicken litter appeared to transfer antibiotic-resistant E. coli to the soil, enhancing the soil resistome and highlighting the consequences of such agricultural practices on public health.

scholarly journals High Levels of Antibiotic Resistance in Isolates From Diseased Livestock

2021 ◽  
Vol 8 ◽  
Author(s):  
Nurul Asyiqin Haulisah ◽  
Latiffah Hassan ◽  
Siti Khairani Bejo ◽  
Saleh Mohammed Jajere ◽  
Nur Indah Ahmad
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Sensitivity ◽  
Clinical Isolates ◽  
Situational Analysis ◽  
Sensitivity Testing ◽  
Resistance Level ◽  
Diagnostic Laboratory ◽  
E Coli ◽  
Livestock Health ◽  
High Level

Overuse of antimicrobials in livestock health and production beyond therapeutic needs has been highlighted in recent years as one of the major risk factors for the acceleration of antimicrobial resistance (AMR) of bacteria in both humans and animals. While there is an abundance of reports on AMR in clinical isolates from humans, information regarding the patterns of resistance in clinical isolates from animals is scarce. Hence, a situational analysis of AMR based on clinical isolates from a veterinary diagnostic laboratory was performed to examine the extent and patterns of resistance demonstrated by isolates from diseased food animals. Between 2015 and 2017, 241 cases of diseased livestock were received. Clinical specimens from ruminants (cattle, goats and sheep), and non-ruminants (pigs and chicken) were received for culture and sensitivity testing. A total of 701 isolates were recovered from these specimens. From ruminants, Escherichia coli (n = 77, 19.3%) predominated, followed by Staphylococcus aureus (n = 73, 18.3%). Antibiotic sensitivity testing (AST) revealed that E. coli resistance was highest for penicillin, streptomycin, and neomycin (77–93%). In addition, S. aureus was highly resistant to neomycin, followed by streptomycin and ampicillin (68–82%). More than 67% of E. coli isolates were multi-drug resistant (MDR) and only 2.6% were susceptible to all the tested antibiotics. Similarly, 65.6% of S. aureus isolates were MDR and only 5.5% were susceptible to all tested antibiotics. From non-ruminants, a total of 301 isolates were recovered. Escherichia coli (n = 108, 35.9%) and Staphylococcus spp. (n = 27, 9%) were the most frequent isolates obtained. For E. coli, the highest resistance was against amoxicillin, erythromycin, tetracycline, and neomycin (95–100%). Staphylococcus spp. had a high level of resistance to streptomycin, trimethoprim/sulfamethoxazole, tetracycline and gentamicin (80–100%). The MDR levels of E. coli and Staphylococcus spp. isolates from non-ruminants were 72.2 and 74.1%, respectively. Significantly higher resistance level were observed among isolates from non-ruminants compared to ruminants for tetracycline, amoxicillin, enrofloxacin, and trimethoprim/sulfamethoxazole.

scholarly journals Antibiotic Resistance of Escherichia Coli Isolated from Lake Nainital, Uttarakhand State, India

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Neha Giri ◽  
Anchal Lodhi ◽  
Devendra Singh Bisht ◽  
Suvarna Bhoj ◽  
Deepak Kumar Arya
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Multiple Drug Resistance ◽  
Health Concern ◽  
Penicillin G ◽  
Resistant Bacteria ◽  
Sample Collection ◽  
E Coli ◽  
Mar Index ◽  
Collection Sites

Researchers have encountered new challenges with the discovery of multiple drug resistance in microbes. Currently, multidrug resistant bacteria are considered a major public health concern and an emerging global epidemic. Presence of Escherichia coli in water is used as a faecal pollution measure. In this study E. coli isolates were collected from 20 sample collection sites at Lake Nainital. 20 E. coli isolates, 1 from each sample collection sites, were examined for their antibiotic response patterns against a panel of widely used 15 antibiotics. The result of this study showed 100% resistance to Penicillin G followed by Erythromycin (80%). All isolates (100%) were found susceptible for Gentamycin. The susceptibilities for Chloramphenicol and Co-trimoxazaole were found next to Gentamycin as 90 and 85% respectively. Multiple antibiotic resistance (MAR) index was also determined. 0.73 MAR index was observed as highest in 1 isolate. 13 out of 20 isolates had more than 0.2 MAR indices. The result reveals the origin of E. coli isolates from an area of high antibiotics use.

scholarly journals A Preliminary Study: Antibiotic Resistance Patterns of Escherichia coli and Enterococcus Species from Wildlife Species Subjected to Supplementary Feeding on Various South African Farms

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 396 ◽  
Author(s):  
Michaela Sannettha van den Honert ◽  
Pieter Andries Gouws ◽  
Louwrens Christiaan Hoffman
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Supplementary Feeding ◽  
Health Concern ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Wildlife Species ◽  
Human Contact ◽  
Resistance Patterns

Studies have shown that antibiotic resistance among wild animals is becoming a public health concern, owing to increased contact and co-habitation with domestic animals that, in turn, results in increased human contact, indirectly and directly. This type of farming practice intensifies the likelihood of antibiotic resistant traits in microorganisms transferring between ecosystems which are linked via various transfer vectors, such as rivers and birds. This study aimed to determine whether the practice of wildlife supplementary feeding could have an influence on the antibiotic resistance of the bacteria harboured by the supplementary fed wildlife, and thus play a potential role in the dissemination of antibiotic resistance throughout nature. Escherichia coli and Enterococcus were isolated from the faeces of various wildlife species from seven different farms across South Africa. The Kirby-Bauer disk diffusion method was used according to the Clinical and Laboratory Standards Institute 2018 guidelines. The E. coli (F: 57%; N = 75% susceptible) and Enterococcus (F: 67%; N = 78% susceptible) isolates from the supplementary fed (F) wildlife were in general, found to be more frequently resistant to the selection of antibiotics than from those which were not supplementary fed (N), particularly towards tetracycline (E. coli F: 56%; N: 71%/Enterococcus F: 53%; N: 89% susceptible), ampicillin (F: 82%; N = 95% susceptible) and sulphafurazole (F: 68%; N = 98% susceptible). Interestingly, high resistance towards streptomycin was observed in the bacteria from both the supplementary fed (7% susceptible) and non-supplementary fed (6% susceptible) wildlife isolates. No resistance was found towards chloramphenicol and ceftazidime.

scholarly journals Retrospective Analysis on Antimicrobial Resistance Trends and Prevalence of β-lactamases in Escherichia coli and ESKAPE Pathogens Isolated from Arabian Patients during 2000–2020

2020 ◽  
Vol 8 (10) ◽  
pp. 1626
Author(s):  
Mahfouz Nasser ◽  
Snehal Palwe ◽  
Ram Naresh Bhargava ◽  
Marc G. J. Feuilloley ◽  
Arun S. Kharat
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Antimicrobial Resistance ◽  
Enterococcus Faecium ◽  
Arab Countries ◽  
Clinical Isolates ◽  
Arab Region ◽  
E Coli ◽  
Extended Spectrum ◽  
Eskape Pathogens

The production of diverse and extended spectrum β-lactamases among Escherichia coli and ESKAPE pathogens is a growing threat to clinicians and public health. We aim to provide a comprehensive analysis of evolving trends of antimicrobial resistance and β-lactamases among E. coli and ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acine to bacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) in the Arabian region. A systematic review was conducted in Medline PubMed on papers published between January 2000 and February 2020 on countries in the Arab region showing different antibiotic resistance among E. coli and ESKAPE pathogens. A total of n = 119,144 clinical isolates were evaluated for antimicrobial resistance in 19 Arab countries. Among these clinical isolates, 74,039 belonged to E. coli and ESKAPE pathogen. Distribution of antibiotic resistance among E. coli and ESKAPE pathogens indicated that E. coli (n = 32,038) was the predominant pathogen followed by K. pneumoniae (n = 17,128), P. aeruginosa (n = 11,074), methicillin-resistant S. aureus (MRSA, n = 4370), A. baumannii (n = 3485) and Enterobacter spp. (n = 1574). There were no reports demonstrating Enterococcus faecium producing β-lactamase. Analyses revealed 19 out of 22 countries reported occurrence of ESBL (Extended-Spectrum β-Lactamase) producing E. coli and ESKAPE pathogens. The present study showed significantly increased resistance rates to various antimicrobial agents over the last 20 years; for instance, cephalosporin resistance increased from 37 to 89.5%, fluoroquinolones from 46.8 to 70.3%, aminoglycosides from 40.2 to 64.4%, mono-bactams from 30.6 to 73.6% and carbapenems from 30.5 to 64.4%. An average of 36.9% of the total isolates were reported to have ESBL phenotype during 2000 to 2020. Molecular analyses showed that among ESBLs and Class A and Class D β-lactamases, blaCTX-M and blaOXA have higher prevalence rates of 57% and 52.7%, respectively. Among Class B β-lactamases, few incidences of blaVIM 27.7% and blaNDM 26.3% were encountered in the Arab region. Conclusion: This review highlights a significant increase in resistance to various classes of antibiotics, including cephalosporins, β-lactam and β-lactamase inhibitor combinations, carbapenems, aminoglycosides and quinolones among E. coli and ESKAPE pathogens in the Arab region.

scholarly journals Virulence Characteristics and Antibiotic Resistance Profiles of Shiga Toxin-Producing Escherichia coli Isolates from Diverse Sources

Antibiotics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 587
Author(s):  
Momna Rubab ◽  
Deog-Hwan Oh
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Shiga Toxin ◽  
Multidrug Resistant ◽  
Control Measures ◽  
Diffusion Method ◽  
Shiga Toxins ◽  
E Coli ◽  
And Control ◽  
High Degree

Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen that causes several gastrointestinal ailments in humans across the world. STEC’s ability to cause ailment is attributed to the presence of a broad range of known and putative virulence factors (VFs) including those that encode Shiga toxins. A total of 51 E. coli strains belonging to serogroups O26, O45, O103, O104, O113, O121, O145, and O157 were tested for the presence of nine VFs via PCR and for their susceptibility to 17 frequently used antibiotics using the disc diffusion method. The isolates belonged to eight different serotypes, including eight O serogroups and 12 H types. The frequency of the presence of key VFs were stx1 (76.47%), stx2 (86.27%), eae (100%), ehxA (98.03%), nleA (100%), ureC (94.11%), iha (96.07%), subA (9.80%), and saa (94.11%) in the E. coli strains. All E. coli strains carried seven or more distinct VFs and, among these, four isolates harbored all tested VFs. In addition, all E. coli strains had a high degree of antibiotic resistance and were multidrug resistant (MDR). These results show a high incidence frequency of VFs and heterogeneity of VFs and MDR profiles of E. coli strains. Moreover, half of the E. coli isolates (74.5%) were resistant to > 9 classes of antibiotics (more than 50% of the tested antibiotics). Thus, our findings highlight the importance of appropriate epidemiological and microbiological surveillance and control measures to prevent STEC disease in humans worldwide.

scholarly journals Constitutive soxR Mutations Contribute to Multiple-Antibiotic Resistance in Clinical Escherichia coli Isolates

2005 ◽  
Vol 49 (7) ◽  
pp. 2746-2752 ◽  
Author(s):  
Anastasia Koutsolioutsou ◽  
Samuel Peña-Llopis ◽  
Bruce Demple
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Dna Sequences ◽  
Single Point ◽  
Constitutive Expression ◽  
Clinical Isolates ◽  
Multiple Antibiotic Resistance ◽  
E Coli ◽  
First Time

ABSTRACT The soxRS regulon of Escherichia coli and Salmonella enterica is induced by redox-cycling compounds or nitric oxide and provides resistance to superoxide-generating agents, macrophage-generated nitric oxide, antibiotics, and organic solvents. We have previously shown that constitutive expression of soxRS can contribute to quinolone resistance in clinically relevant S. enterica. In this work, we have carried out an analysis of the mechanism of constitutive soxS expression and its role in antibiotic resistance in E. coli clinical isolates. We show that constitutive soxS expression in three out of six strains was caused by single point mutations in the soxR gene. The mutant SoxR proteins contributed to the multiple-antibiotic resistance phenotypes of the clinical strains and were sufficient to confer multiple-antibiotic resistance in a fresh genetic background. In the other three clinical isolates, we observed, for the first time, that elevated soxS expression was not due to mutations in soxR. The mechanism of such increased soxS expression remains unclear. The same E. coli clinical isolates harbored polymorphic soxR and soxS DNA sequences, also seen for the first time.

scholarly journals Antibiotic resistance and virulence genes of extraintestinal pathogenic Escherichia coli from tropical estuary, south India

2015 ◽  
Vol 9 (05) ◽  
pp. 496-504 ◽  
Author(s):  
Divya Sukumaran ◽  
Abdulla A Mohamed Hatha
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Multidrug Resistance ◽  
Virulence Factor ◽  
Virulence Genes ◽  
Antimicrobial Agents ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Virulence Factor Genes

Introduction: Escherichia coli strains can cause a variety of intestinal and extraintestinal diseases. Extraintestinal pathogenic E. coli (ExPEC) strains have the ability to cause severe extraintestinal infections. Multidrug resistance among ExPEC could complicate human infections. Methodology: Escherichia coli strains were isolated during the period of January 2010 to December 2012 from five different stations set at Cochin estuary. Susceptibility testing was determined by the disk-diffusion method using nine different antimicrobial agents. A total of 155 strains of Escherichia coli were screened for the presence of virulence factor genes including papAH, papC, sfa/focDE, iutA,and kpsMT II associated with ExPEC. Results: Among the 155 E. coli isolates, 26 (16.77%), carried two or more virulence genes typical of ExPEC. Furthermore, 19.23% of the ExPEC isolates with multidrug resistance were identified to belong to phylogenetic groups B2 and D. Statistically significant association of iutA gene in ExPEC was found with papC (p < 0.001) and kpsMT II (p < 0.001) genes. ExPEC isolates were mainly resistant to ampicillin (23.07%), tetracycline (19.23%), co-trimoxazole (15.38%), and cefotaxime (15.38%). The adhesion genes papAH and sfa/focDE were positively associated with resistance to gentamicin, chloramphenicol, and cefotaxime (p < 0.05). Conclusions: Co-occurrence of virulence factor genes with antibiotic resistance among ExPEC poses considerable threat to those who use this aquatic system for a living and for recreation.

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