Beta-lactam resistant urinary tract infections: prevalence, the development of rapid diagnostics and novel treatments
Background The 21st century has seen antibiotic resistance rise to be a major public health concern. The O’Neill report in 2016 reiterated the importance of antibiotic resistance and proposed many actions, including strengthening surveillance systems, the development of rapid diagnostics, and investment in new classes of antibiotics and alternatives. Urinary tract infections (UTIs) are one of the most common infections diagnosed in the United Kingdom with uropathogenic Escherichia coli (UPEC) the most common cause. It is thought that the main reason why UPEC is so successful at causing UTIs, is its expression of a wide range of virulence factors including adhesins, capsules, toxins and iron-acquisition systems. Overuse of beta-lactam antibiotics to treat UTIs has led to selection for extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, a major cause of antibiotic resistant urinary tract infections (UTIs). The prevalence of ESBL producing UTIs has dramatically risen, limiting treatment options. The most common ESBL types seen today are CTX-M, TEM, OXA and SHV. The emergence and spread of these types 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 directly linked to major outbreaks of antibiotic resistance. Standard detection methods for ESBL-producing UTIs are time-consuming, with disputed accuracy. Patients are often treated empirically with broad-spectrum antibiotics. Reducing the time spent on broad-spectrum antibiotics by prescribing a more appropriate treatment, increases the favorability of the outcome of the patient and shortens the stay in hospital. Aims This study had three aims: to investigate the prevalence of ESBLs and the relationship between plasmids and ESBLs in Leicestershire; to develop a rapid, accurate method to detect ESBLs (TEM, SHV, OXA, and CTX-M) using real-time PCR and to investigate the effect of cranberry (Cysticlean®) on the expression of 10 virulence genes. Methods A total of 236 uropathogenic E. coli ESBL-producing isolates were collected from the Leicester Royal Infirmary. This study identified ESBL genes (CTX-M, SHV, TEM and OXA) and multiple CTX-M gene subtypes by multiplex PCR. A multiplex PCR-based replicon typing assay identified IncFIA, IncI1, IncL/M, IncN and IncFII. A real-time PCR assay was designed using amplicon melting analysis and the Plexor system to detect the ESBL family. A CTX-M-producing E.coli isolate was treated with the cranberry extract Cysticlean®. Taqman qRT-PCR was used to detect the relative expression of the virulence genes SAT, USP, ChuA, SoxS, KPSM, TraT, RecA, IdfB and HcaT and the antibiotic resistance gene CTX-M. Results ESBL genes were identified as follows: CTX-M (71.6%), OXA (7.6%), TEM (3.8%) and SHV (3.8%) with multiple genes detected in 10.2% of isolates. CTX-M-1 (84.1%) was the most frequently detected CTX-M subtype. 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. The qualitative real-time PCR assay correctly identified 97.7% isolates tested, with a sensitivity and specificity of 98.7% and 83.3% respectively. The positive predictive value was 97.5% and the negative predictive value was 90.9%. Results from the qRT-PCR assay showed that Cysticlean® was able to significantly reduce the expression of all the genes investigated. Conclusions This is the first study to analyse the prevalence of uropathogenic ESBLs in Leicestershire. 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. The ability to reduce the expression of critical virulence factors, could lead to the development of alternatives to antibiotics.
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