Isolation and characterisation of imipenem-resistant bacteria from natural environments and clinical settings

• Main Author: Alkhaleefah, Fahd
• Other Authors: Osborn, A. Mark
• Published: University of Hull 2015
• Subjects: 616.9; Biological sciences
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.666464

The development and spread of bacterial resistance to antimicrobials is now recognised as a key threat to public health and society. A small number of antimicrobials, including imipenem and vancomycin, are now considered to be the drugs of ‘last resort’ for treating antibiotic resistant bacteria. This study investigates and characterises antibiotic (imipenem) resistant bacteria in environmental and clinical samples from the U.K. Imipenem resistant (ImR) bacteria were isolated and characterised from river water samples from East Yorkshire and soil samples from Lincolnshire. ImR clinical isolates from different hospitals (York, Sheffield and Hull) were also characterised. Phenotypic resistance to imipenem was observed in 11.2% (75/670 CFU ml⁻¹), 13.3% (145.35 x 10⁵/ 109.1 x 10⁶ CFU g⁻¹) and 38.5% (42/109) of water, soil and clinical bacterial isolates, respectively. The minimum inhibitory concentrations (MICs) of the clinical isolates were generally higher (> 32 mg L⁻¹ in 71.4% of isolates) than those of the environmental isolates, which were around 4 mg L⁻¹ in 63.4% of water isolates and in 42.7% of soil isolates. β-lactamase activity studies showed that the most common β-lactamases among the environmental isolates were class B metallo β-lactamases (MBLs) (84.2%), while class A Klebsiella pneumoniae carbapenemases (KPCs) (40.5%) were the most common β-lactamases observed in the clinical isolates. Higher frequencies of multi-drug resistant (MDR) patterns were detected among the environmental isolates than among the clinical strains. Sequencing of 16S rRNA genes identified 30 (17 species), 96 (27 species), and 42 (11 species) ImR bacteria in water, soil and clinical samples, respectively. The most abundant genera identified were Caulobacter (36.7%), Stenotrophomonas (44.8%) and Stenotrophomonas (40.5%) from water, soil and clinical environments, respectively. PCR products were generated from ImR clinical isolates and some of the environmental isolates using primers targeting β-lactamase genes. Sequence analysis of these products from clinical isolates showed that they were specific and related to β-lactamase genes. However, the products from environmental isolates were not related to known genes characterised from antibiotic resistant clinically important bacteria. This suggests that there is a potentially large and divergent gene pool encoding for imipenem ressitance within natural environments, and that river water and agricultural soil are important as reservoirs of novel antibiotic resistance. Genome sequencing was used to characterise 8 MDR Stenotrophomonas spp. isolates from water, soil and clinical samples. This analysis showed the detection of β-lactamases genes (between 8 and 15 genes per isolate) including class A (L2), B (L1) and C (AmpC), fluoroquinolones resistance genes (between 4-8 genes per isolate), and genes encoding MDR efflux pumps (between 23-32 genes per isolate). Antibiotic resistance genes for other antimicrobials were also observed in small numbers; these represented aminoglycoside, sulphonamide and tetracycline resistance. Genes encoding resistance to heavy metal resistance (between 13-27 genes per isolate) were also observed. Overall, this research has demonstrated the widespread presence of imipenem resistant bacteria in environmental and clinical settings, carrying multiple resistances to other antibiotics. In particular, imipenem resistant Stenotrophomonas spp. were present in all of the environments studied and these bacteria were found to harbour multiple and diverse antibiotic resistance genes, that differed between isolates from environmental and clinical origins.