Browsing by Author "Reid, R."
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Item Metadata only Adaptability to Various Growth Conditions of Biofilm Associated Extended-Spectrum-Beta-Lactamases Producing Bacteria(OMICS International, 2018-05-22) Baho, S.; Reid, R.; Samarasinghe, S.Extended-Spectrum β-Lactamase (ESBL) producing bacteria are becoming increasingly prevalent in biofilmassociated infections. Bacteria form biofilms that allow their survival in hostile environments. The amount of formed biofilm is affected by external environmental factors. This study investigates the effect of specific parameters (media type, incubation condition, and growth stage) on the amount of produced biofilm on antibiotic resistant bacterial strains, Escherichia coli (CTX-M-15, TEM-3, and IMP-type) and Klebsiella pneumoniae (OXA-48, SHV-18, NDM-1, and KPC-3). The amount of biofilm formed was measured at different time points (6, 12, 24 and 48 h) of incubations under static and shaking conditions, using three different types of media (nutrient broth, LB broth, and AB broth). Statistical tests showed that there was a significant difference in biofilm level (p<0.01) for 64 out of 80 tests (80%) when grown under different types of media. Growing under different incubation conditions also showed a statistical difference in biofilm level (p<0.05) for 76 out of 120 tests (63%). Stage of growth of the same species also showed statistical difference, 20 out of 24 tests (83%) for E. coli and 24 out of 24 tests (100%) for K. pneumoniae. These findings suggested that biofilm formation is highly affected by incubation conditions, strains’ stage of growth, and media type demonstrating that these conditions may play a role in adaptability of the ESBLs on different environmental conditions and their increased prevalence in biofilm associated infections.Item Metadata only Detection of Extended-Spectrum Beta-Lactamases E. coli in Animal Faeces Collected in Urban Parks in Leicester, UK(2017-06-02) Adeyemi, J.; Reid, R.; Baho, S.; Hoosen, H.; del Aguila, C.; Fenoy, S.; Pena, M. A.; Izquierdo, F.; Magnet, A.; Sgamma, Tiziana; Ollero, M. D.; Hurtado, C.; Pena-Fernandez, A.Background: The presence and distribution of antibiotic resistance bacteria in the environment could constitute an emerging public health concern. Different studies have described these bacteria in a range of animals and their possible role in the contamination of the environment, however very little studies have determined these bacteria in urban ecosystems. Recovery and remediation of affected environments with these biological hazards, and the establishment of protection interventions, constitute a challenge that requires a collaborative international response to protect the public, especially in urban ecosystems. A preliminary study carried out by our research group have detected Extended-Spectrum β-Lactamases (ESBL) genes for Gram-negative bacteria in animal faecal samples collected in different urban parks in the city centre of Leicester (United Kingdom). Methods: This study investigated the presence of ESBL-producing genes (blaCTX-M, blaSHV, blaTEM and blaOXA) within Escherichia coli (E. coli) due to its implications for human health. 30 faecal samples were collected in the same parks between August and September of 2016. A veterinarian identified the animal species as follow: 22 avian (18 waterfowls, 4 pigeons), 4 dogs, 3 cats and 1 fox. After appropriate treatment of the samples, CTX-M-1-type producing E. coli was detected by molecular analysis in 5 of the analysed samples (17%); all of them from waterfowls. Results: The results described here, although preliminary, might indicate that waterfowls might be carriers of ESBL E. coli producers in Leicester. Waterfowls may have a possible role in the spread of CTX-M-1 producing E. coli in urban ecosystems although more research is required prior to implementing intervention programs in the monitored environment. Conclusions: Possible control measures may be cleaning frequently urban parks, roads and pavement from animal faeces, especially avian faeces; or banning exposure practices such as feeding these animals, activity that is very popular in the monitored city.Item Metadata only The development and evaluation of a multiplex real-time PCR assay for the detection of ESBL genes in urinary tract infections(Open Access Pub, 2018-08-09) Reid, R.; Samarasinghe, S.Background Overuse of beta-lactam antibiotics has lead to selection for extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, a major cause of antibiotic resistant urinary tract infections (UTIs). Standard detection methods are time-consuming, with disputed accuracy. This study describes a novel real-time PCR method to detect CTX-M, SHV, OXA and TEM. Methods 179 Enterobacteriaceae isolates from UTIs were collected from the Leicester Royal Infirmary, UK. A multiplex Plexor®-based real-time PCR assay detected ESBLs using their specific amplicon melting temperature, during each cycle, removing the need for a melt-curve analysis. Validation was achieved by end-point PCR and disk diffusion. Results The method was able to produce rapid and accurate results, achieving a sensitivity and specificity of 94.9% and 72% respectively, and the assay can differentiate between the different ESBL genes, with ease. Conclusions With further investigation, a Plexor®-based assay could form the basis of a high-throughput kit that health services could use to detect ESBLs or other antibiotic resistance genes.Item Open Access The Distribution of ESBL-Producing Enterobacteriaceae: Leicestershire UK Compared to Worldwide(American Journal of Biomedical Science & Research, 2019-05-21) Reid, R.; Samarasinghe, S.; Varnakulasingam, AtheenaItem Open Access Exploring the presence of ESBL-producing bacteria in different aquatic ecosystems in England.(Elsevier, 2018-09-12) Anjum, U.; Reid, R.; Hoosen, H.; Lobo-Bedmar, M. C.; Pena-Fernandez, A.Recent evidence has shown an increasing presence and distribution of extended-spectrum beta-lactamases (ESBLs) in the environment, which could represent a threat to public health. However, their presence in open water systems in England remains unknown despite the significant role of water in disseminating contamination and as a reservoir for biological hazards. Therefore, three sets of 30 water samples each were collected from different open water environments in Leicestershire (UK) including Leicester city in three periods of time (summer, autumn and winter) in 2017. Water samples were collected in the same locations each season using a portable water pump connected to a foam filter module according to the 1623 method (US Environmental Protection Agency). The environments monitored were: the course of the River Soar and the Grand Union Canal (a canalised section of the River Soar) throughout the city including the River Biam; lakes highly frequented for fishing or leisure (e.g. John Merricks' Lake, Kings Lear’s Lake; Bennion Pools Fishing Lake); and a marina. Water samples were concentrated using the IDEXX® Filta Max system according to manufacturer's instructions and 1623 method. The DNA was extracted from each concentrated water sample with a Fast DNA® Kit. Genotypic prevalence of ESBL-producing bacteria was investigated by means of multiplex PCR. The assay was performed to detect the ESBLs: blaTEM, blaSHV, blaOXA and blaCTX-M using a CTX-M-15 producing strain of Escherichia coli as a positive control. All 90 samples assessed for ESBLs were negative. However these results should be considered inconclusive, as different factors such as dilution in the surface water could have affected them, as we have previously detected the presence of ESBLs (specifically CTX-M-15) in animal faecal samples (6 waterfowls, 3 dogs and 2 foxes) collected in different parks in Leicester city in 2016. Further studies will be needed to determine the presence and distribution of ESBLs in the open water systems in England as the World Health Organisation has highlighted ESBLs as a priority to be controlled to response to the bacterial resistance phenomenon. Information about the presence and distribution of human-pathogenic bacteria harbouring ESBL enzymes is needed to develop national intervention strategies to protect the public.Item Open Access Exploring the presence of human pathogenic free-living amoebas in different water ecosystems in Leicester, UK.(2018-04-24) Anjum, U.; Hoosen, H.; Magnet, A.; Izquierdo, F.; Fenoy, S.; Reid, R.; Pena-Fernandez, A.Background: The presence and distribution of Acanthamoeba spp., Balamuthia mandrillaris and Naegleria fowleri (human pathogenic free-living amoebas, FLA) in different environmental compartments and geographical locations in Europe remains unknown. These FLA can be a public health threat as their cysts are highly resistant to harsh environmental conditions. The aim of this pilot study was to determine the presence of FLA in different water ecosystems close or in Leicester city (Leicestershire, UK) as information on the presence these emerging parasites in the UK is limited in the literature. Materials/methods: A total of 30 water samples were collected from different open water environments in Leicester during winter 2016/17 including: the River Soar and the Grand Union Canal (a canalised section of the River Soar), different lakes highly frequented for fishing or leisure (e.g. John Merricks' Lake, Kings Lears Lake; Bennion Pools Fishing Lake), and a marina near River Soar. The River Soar is rich in wildlife including water birds, fish and plant populations attracting large numbers of users. Water samples were obtained following protocol 1623 described by US EPA and concentrated using IDEXX® Filta Max system following manufacturer's instructions. DNA extraction from concentrated water was performed from each water sample with Fast DNA® Kit. A triplex real-time TaqMan PCR assay was performed to detect FLA; positive controls for the three amoebae were used. Results: All 30 samples assessed for FLA were negative. However these results should be considered as inconclusive as, although rare, several studies have reported the presence of Acanthamoeba spp. in the UK domestic water supplies which may indicate the presence of these human pathogens in other water systems including the environment. Moreover, the incidence of Acanthamoeba keratitis has increased in recent years in England. Conclusions: Further studies will be needed to determine the presence and distribution of FLA in the open water systems monitored to protect the public as recent evidence indicates an increase in infections due to these emerging human pathogens globally. This information is crucial to develop novel strategies to protect humans and increase the awareness of these protozoan parasites in aquatic environments in the UK.Item Metadata only Genotypic Identification of ESBL Producing Urinary Tract Infections In Leicestershire Area, UK(2017-06-02) Reid, R.; Baho, S.; Samarasinghe, S.Background: Uropathogenic E. coli is one of the highest producers of extended-spectrum β-lactamases (ESBLs), a major public health concern. Many studies have described the epidemiology and prevalence of ESBL-producing E.coli. (1) Plasmids can transfer between different species of bacteria, carrying resistance genes with them, facilitating the spread of resistance. This study aimed to investigate the prevalence of ESBL genes and plasmid types in Leicestershire, UK. Methods: 380 uropathogenic E. coli ESBL-producing isolates were collected from the Leicester Royal Infirmary in Leicestershire, UK. Isolates were confirmed to be ESBL producers using the MAST ESBL detection kit. This study investigated the presence of ESBL-producing genes (blaCTX-M, blaSHV, blaTEM and blaOXA) by multiplex PCR.A second multiplex PCR assay identified blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9 and blaCTX-M-25. To investigate the relationship between plasmid type and ESBLs, a multiplex PCR-based replicon typing assay was designed to detect IncFIA, IncI1, IncL/M, IncN and IncFII. Results: Prevalence of ESBL genes was as follows: blaCTX-M (37%) blaOXA (5%), blaTEM and blaSHV (2.4%). Multiple resistance genes were detected in 10.2% of isolates. In the second multiplex assay, the order of prevalence of CTX-M genes was as follows: blaCTX-M-1 (56%), blaCTX-M-9 (11%), blaCTX-M-25 (6%), blaCTX-M-8 (0.5%) and blaCTX-M-2 (0.2%). Multiple resistance genes were detected in 3 isolates. blaCTX-M was found to be associated with all the replicon genes other than incL/M. Conclusions: To our knowledge, this is the first study to analyse the prevalence of uropathogenic ESBLs in Leicestershire. Our findings are in line with other authors findings in Europe. Bacteria use plasmids to transfer genes, including those that cause antibiotic resistance. It was found that CTX-M producing E.coli are associated with multiple plasmids, which can be linked to its rapid spread across the world. Epidemiology and prevalence studies help to inform policy about antibiotic stewardship, with the aim to reduce resistance levels in the future.Item Open Access Genotypic Identification of Extended-Spectrum β-Lactamase (ESBL)Producing Enterobacteriaceae from Urinary Tract Infections in the Leicestershire Area, United Kingdom: A One Health Prospective(Journal of Infectious Diseases and Diagnosis, 2018-10-17) Reid, R.; Al-Bayati, Majid; Samarasinghe, S.Objectives: Urinary Tract Infections (UTIs) are one of the most common infections diagnosed in the United Kingdom (UK). The prevalence of Extended-Spectrum-Β-Lactamase (ESBL) producing UTIs has dramatically risen, limiting treatment options. The emergence and spread of ESBLs is thought to be through the horizontal transmission of antibiotic resistance plasmids IncL/M, IncF, IncN and IncI1. These conjugative plasmids have been described as important vectors and directly linked to major outbreaks of antibiotic resistance. This study aimed to investigate the prevalence of ESBLs in Leicestershire, UK and their relationship with antibiotic resistance plasmids. Methods: 236 ESBL producing uropathogenic Enterobacteriaceae isolates were obtained from the Leicester Royal Infirmary (Leicestershire, UK). ESBL production was confirmed phenotypically via the MAST ID double disc synergy test. ESBL-producing genes (CTX-M, SHV, TEM and OXA) were identified by multiplex PCR. The CTX-M family was then further characterised into (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25) by multiplex PCR. The relationship between ESBL-producing genes and plasmid type was then investigated by multiplex PCRbased replicon typing to detect IncFIA, IncI1, IncL/M, IncN and IncFII. Results: ESBL genes were identified as follows: CTX-M (71.6%), OXA (7.6%), TEM (3.8%) and SHV (3.8%). Multiple resistance genes were detected in 16% of isolates. CTX-M genes were identified as follows: CTX-M-1 (84.1%), CTX-M-9 (12.5%), CTX-M-25 (1.7%), CTX-M-8 (1.1%) and CTX-M-2 (0.6%). Replicon typing results were as follows: IncL/M (29.2%), IncN (14.4%), IncI1 (5.1%), IncFII (27.5%) and IncFIA (23.3%). A combination of IncL/M, IncFII and IncFIA was the most common at 9.8%. A positive correlation between CTX-M and all plasmids except IncI1 was found. Conclusion: CTX-M harbouring Enterobacteriaceae are associated with multiple plasmids, which can be linked to its rapid spread across the world. Prevalence studies help to inform policy about antibiotic stewardship and resistance evolution, aiming to reduce resistance levels in the future.Item Metadata only Phenotypic and genotypic identification of urinary tract infections caused by E. coli in the Leicestershire area.(2017-07-01) Reid, R.; Samarasinghe, S.Backgrounds Uropathogenic E. coli is one of the highest producers of ESBLs (Extended-spectrum β-lactamases), a major public health concern. Objectives This study aims to investigate the prevalence of ESBLs and plasmid types in Leicestershire. Methods 380 urinary E. coli ESBL producing isolates were collected from the Leicester Royal Infirmary. Phenotypic analysis involved the MAST ESBL detection kit. Genotypic prevalence was investigated by using a multiplex PCR assay. Primers for blaCTX-M, blaSHV, blaOXA and blaTEM were designed. A second multiplex PCR assay was designed to identify blaCTX-M-1, blaCTX-M-2, blaCTX-M-8, blaCTX-M-9 and blaCTX-M-25. To investigate the relationship between plasmid type and ESBLs, a multiplex PCR-based replicon typing assay was designed[RR1] to detect IncFIA, IncI1, IncL/M, IncN and IncFII. Conclusions Phenotypic analysis showed blaCTX-M genes confer higher resistance to CTX, than CAZ and CPD. All isolates that showed resistance to CAZ and CPD, were also resistant to CTX. Prominence for ESBL genes was blaCTX-M (37%)> blaOXA (5%), blaTEM and blaSHV (2%). Associations between the ESBL groups were detected. In the second multiplex assay, the most prominent were blaCTX-M-1 (56%) and blaCTX-M-9 (11%), with associations between the blaCTX-M groups detected. blaCTX-M was found to be associated with all replicons other than L/M. This is the first study to analyse the prevalence of uropathogenic ESBLs in Leicestershire. blaCTX-M is the most prominent ESBL in Leicestershire. blaCTX-M can be associated with other ESBLs. blaCTX-M-1 is the most common subgroup. Multiple associated plasmids can increase the spread of resistance, causing the epidemic we see today.Item Open Access Rapid Multiplex Real Time PCR Assay for the Identification of ESBL Genes in Urinary Tract Infections Without the Need for Melting Curve Analysis(American Society for Microbiology- ASM Microbe, 2018-03-20) Reid, R.; Samarasinghe, S.Background: Extended-spectrum β-lactamases (ESBLs) are a major public health concern and have therefore become one of the most studied topics within the field of antibiotic resistance. Current standard detection methods for ESBLs are time-consuming and their accuracy has been called into question. This study aimed to develop a rapid, accurate method to detect ESBLs (blaTEM, blaSHV, blaOXA, and blaCTX-M) using real-time PCR, without the need for a melting curve analysis. Materials/methods:The Plexor® qPCR system was chosen for it high multiplex ability. The system requires a florescent reporter, adjacent to an iso-dC label, to be attached to one of the primers in each pair. Florescence is then quenched by incorporation of a Dabcyl-iso-dGTP label contained in the mastermix, when bound to another DNA strand. Therefore, as product increases, florescence decreases. Monitoring this process each cycle allows us to calculate the Tm of each product, similar to what occurs during the high-resolution melting curve analysis, shown in fig 1. 90 patient isolates from urinary tract infections were obtained from the Leicester Royal Infirmary and tested using the new assay. DNA was extracted by means of boiling colonies for ten minutes at 95⁰C. Results were confirmed by multiplex end-point PCR. Fig 1. (A) Initial denaturation - the primers are unbound, and florescence is high. (B) Annealing/extension - florescence decreases as primers bind and incorporate the Dabcyl-iso-dGTP label, quenching the reporter. (C) Denaturation - as temperature rises between the annealing step and the denaturation step, primers dissociate from the accompanying DNA strand, and the reporter is no longer quenched. Results: The assay correctly identified 97.7% isolates tested, with a sensitivity and specificity of 98.7% and 83.3% respectively. The positive and negative likelihood ratio was 5.92 and 0.02 respectively. Conclusions: The aim was to develop a rapid real-time PCR method to detect ESBL genes. It was found that the method could produce accurate results, without the need for high skill in real-time PCR analysis. The ability to rapidly and accurately detect ESBL genes is an important step in improving antimicrobial stewardship and reducing morbidity and mortality as a result of ESBL-producing pathogenic infections.