CARBAPENEM AND COLISTIN RESISTANCE DETECTION: A CHALLENGE FOR THE DIAGNOSTIC MICROBIOLOGY LABORATORY.
Topic/Discipline: Medical Microbiology
Byrne, K.1, Drudy, D.1, Keating, D.2, Coyle, E.2, Donoghue, O.2.
1Technological University Dublin
2Microbiology, St. Vincent’s University Hospital
Biography: I graduated in 2018 with a BSc in Biomedical Science (TUD). My undergraduate research project involved the evaluation of molecular assays for the detection of carbapenem resistant Enterobacterales in screening specimens. This was followed by the evaluation of a rapid method for the detection of colistin resistance in Enterobacterales. I am currently working as a medical scientist in the Microbiology department of the Mater Misericordiae University Hospital.
Background: The global dissemination of carbapenemase producing Enterobacterales (CPE) represents a significant public health issue. Rapid and accurate detection of CPE colonisation through screening is essential to enable effective infection control strategies. Carbapenem resistance detection remains a challenge for the diagnostic microbiology laboratory (1). The EntericBio realtime® CPE assay and the GeneXpert® Carba-R assay are molecular assays designed for the in vitro diagnostic testing and qualitative detection of Enterobacterale produced, carbapenemase genes: KPC, OXA-48, NDM, VIM and IMP . Colistin resistant Enterobacterales are being increasingly reported worldwide. Colistin susceptibility testing also represents a challenge for the diagnostic microbiology laboratory. The Rapid Polymyxin NP kit detects colistin resistance in Enterobacterales (2).
The aim of this study was to evaluate the EntericBio realtime® CPE assay for direct use, the GeneXpert® Carba-R assay and the Rapid Polymyxin NP kit for use with enterobacterial isolates in a diagnostic
Methods: A total of 277 rectal swabs were tested directly using the EntericBio realtime® CPE assay. A total of ten enterobacterial isolates were processed on the GeneXpert Carba-R assay and twelve enterobacterial isolates using the Rapid Polymyxin NP kit. Results: The EntericBio realtime® CPE assay demonstrated a sensitivity, specificity, positive predicted value (PPV) and negative predicted value (NPV) of 100%, 98.95%, 95.1% and 100% respectively. The GeneXpert® Carba-R assay and the Rapid Polymyxin NP kit exhibited sensitivities of 100% and 66.7% and specificities of 80% respectively.
Conclusion: The EntericBio realtime® CPE assay and the GeneXpert® Carba-R assay are rapid and accurate molecular assays for the detection of CPE that facilitate the improvement of infection control strategies. The Rapid Polymyxin NP kit is a rapid and easy to use method for the detection of colistin resistance. However, further study is required to determine its utility in a diagnostic microbiology laboratory.
(1) Humphries, R. M. and McKinnell, J. A. Continuing Challenge for the Clinical Laboratory for Detection of Carbapenem-Resistant Enterobacteriaceae. J Clin Microbiol. 2015; 53(12):3712-3714.
(2) Poirel, L., Jayol, A. and Nordmann, P. Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes. Clinical Microbiology Reviews. 2017;30(2): 558-570
RESISTANCE REVERSAL IN MULTI-DRUG RESISTANT KLEBSIELLA PNEUMONIAE: EXPLORING THE SOXS DEPENDENT REGULON
Topic: Medical Microbiology
Centre for Food Safety University College Dublin,
Wyckham Way, Dundrum, Dublin
Dever, K, Srikumar, S, Anes, J, Fanning, S, Corcoran, D.
Biography: I completed my undergraduate degree in the Galway Mayo Institute of Technology. I have since been lucky enough to work in the University College Dublin Centre for Food Safety, where I completed my thesis, as a research technician in molecular microbiology. In September of 2018 I was offered a permanent position as a medical scientist in the microbiology laboratory of St. Vincent’s University Hospital Dublin, where I continue to work today.
The overuse and misuse of antibiotics has led to the emergence and dissemination of multi-drug resistance in Gram-negative pathogens, jeopardising the advances of modern medicine. The SoxRS regulon plays a key role in bacterial defence against oxidative stress (1). The aim of this project was to determine the role of the transcription factor, SoxS, as a regulator of antibiotic resistance under oxidative stress conditions (2). This was achieved by employing the multi-drug resistant (MDR) wildtype Klebsiella pneumoniae MGH 78578, the deletion mutant Klebsiella
pneumoniae MGH 78578 δsoxS (with soxS deleted from the bacterial chromosome), and the complementation Klebsiella pneumoniae MGH
78578 δsoxS pBADsoxS isolate (where soxS was over-expressed from the plasmid pBAD using an arabinose inducible promoter).
The project involved the interpretation of RNA (ribonucleic-acid)- sequencing data obtained from a broader study, conducted on MDR Klebsiella pneumoniae placed under oxidative stress conditions using paraquat, at the University College Dublin-Centre for Food Safety (UCDCFS). In order to determine if genotypic observations from this data translated into distinct phenotypes, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were performed using six antibiotics namely, colistin, gentamicin, kanamycin, cefotaxime, tetracycline and rifampicin. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was employed for the confirmation of soxS over-expression in the complemented Klebsiella pneumoniae MGH 78578 δsoxS pBADsoxS strain. Finally, the project incorporated the objective to determine the 5’transcription-start-site (5’TSS) of soxS using random amplification of cDNA (complementarydeoxyribonucleic-acid) ends.
The RNA-sequencing data outlined that under oxidative stress conditions certain antibiotic resistance genes were down-regulated in the absence of soxS. This was reflected in the phenotypic MIC and MBC assays which showed antibiotic resistance reversal in the Klebsiella pneumoniae MGH 78578 δsoxS isolate for the antibiotics cefotaxime and tetracycline.
These results highlighted that regulatory molecules, such as soxS, may provide novel targets to mitigate multi-drug resistance in the future.
(1) Seo SW, Kim D, Szubin RO, Palsson B. Genome-wide reconstruction of OxyR and SoxRS transcriptional regulatory networks under oxidative stress in Escherichia coli K-12 MG1655. Cell Reports. 2015;12(8): 1289-1299.
(2) Kohanski MA, Dwyer DJ, Collins JJ. How antibiotics kill bacteria: from targets to networks. Nature Reviews Microbiology. 2010;8(6): 423-435.
READY-TO-EAT DAIRY FOODS: A POTENTIALLY IMPORTANT VECTOR OF ANTIBIOTIC-RESISTANT BACTERIA
Discipline: Medical Microbiology.
Institution: Department of Biological Sciences, Cork Institute of
Authors: Diggin, S., Sleator, RD., Culligan, EP.
Biography: Biomedical Science student from Listowel, Co. Kerry. Currently undertaking clinical laboratory placement at University Hospital Kerry.
A perceived boost in nutritional value and flavour has seen the market for minimally processed, ready-to-eat (RTE) foods grow considerably in recent years. However, mounting food safety concerns have accompanied this demand, with RTE produce being increasingly recognised as a potential vehicle to transfer not only foodborne pathogens, but also antibiotic-resistant bacteria and their associated resistance genes to the consumer (1). This study examined the microbiological quality of RTE, unpasteurised milk and cheeses and investigated the antibiotic-resistance profiles of the bacteria present. Enumeration of the levels of bacteria was determined in CFU/mL and revealed that three out of the four samples tested contained unacceptably high quantities of bacteria (2), indicating poor standards of food hygiene and safety. Five species of bacteria were isolated from the dairy products; Hafnia spp. (cheese), Klebsiella oxytoca, Kluyvera cryocrescens, Escherichia coli and Hafnia paralvei (milk). All isolates were resistant to a minimum of three antibiotics (ampicillin, cephradine and fusidic acid), when subjected to antimicrobial susceptibility test using the agar disc diffusion method. The antimicrobial resistance profile of Hafnia spp. was particularly alarming; displaying resistance
to 8 out of 11 antibiotics tested and PCR revealed positive carriage of the ampC beta-lactamase gene. Moreover, bacterial isolates were shown to be capable of surviving conditions that simulated the human gastrointestinal tract and were resistant to a number of disinfectants, following stress survival testing. These findings suggest that individuals consuming unpasteurised dairy may be routinely inoculated with resistant bacteria and further reinforces the knowledge that raw milk and cheeses may represent a significant environment for the evolution of antibiotic-resistant bacteria and their spread to humans (3). The potential health implications of consuming RTE foods, therefore, need to be carefully re-evaluated. Limiting the spread of resistant bacteria throughout the food chain remains a crucial strategy to combat the global antibiotic resistance crisis.
(1) Hudson, JA., Frewer, LJ., Jones, G., Brereton, PA., Whittingham, MJ., Stewart, G. The agri-food chain and antimicrobial resistance: A review. Trends Food Sci. Technol. 2017; 69:131–147. https://doi.org/10.1016/j.tifs.2017.09.007
(2) European Commission. Regulation (EC) No. 92/46/EEC of 16 June 1992 laying down the health rules for the production and placing on the market of raw milk, heattreated milk and milk-based products. Official Journal of the European Union. 1992; 268, (14):1–32
(3) Verraes, C., Van Boxstael, S., Van Meervenne, E., Van Coillie, E., Butaye, P., Catry, B., de Schaetzen, MA., Van Huffel, X., Imberechts, H., Dierick, K., Daube, G., Saegerman, C., De Block, J., Dewulf, J., Herman, L. Antimicrobial Resistance in the Food Chain: A Review. Int. J. Environ. Res. Public. Health. 2013; 10: 2643–2669. https://doi.org/10.3390/ijerph10072643
EQUIVALENCE STUDY OF THE LYOPHILISED ENTERICBIO® NOROVIRUS ASSAY (EBVP2) TO THE ORIGINAL CE-IVD (EUROPEAN CONFORMITY – IN VITRO DIAGNOSTICS) MARKED ENTERICBIO® NOROVIRUS ASSAY (EBNOV) ON THE MARKET
Topic – Medical Microbiology
Institution – Serosep Ltd., Co. Limerick
Authors – Maunsell, A., O’Leary, J., Corcoran, D.”
Introduction: Norovirus is a major cause of inflammatory gastroenteritis. It peaks during the winter months and early spring giving it the name ‘Winter Vomiting Disease’. This study was carried out to assess the equivalence of the lyophilised EntericBio® Norovirus assay (EBVP2) to the CE-IVD marked EntericBio® Norovirus assay (EBNoV). These assays detect
Norovirus genogroup I and Norovirus genogroup II directly from faecal sample. The equivalence of the assays was assessed under analytical sensitivity, clinical sensitivity and clinical specificity.
Methods: The analytical sensitivity of the EBVP2 assay was examined by testing RNA solution diluted to the concentrations relevant to the limit of detection of the EBNoV assay – 100 genome copies for Norovirus genogroup I and 50 genome copies for Norovirus genogroup II. 24 replicates of each genogroup were tested. The clinical sensitivity was assessed by testing 30 faecal samples using both assays that had previously tested positive for Norovirus. The clinical specificity was assessed by testing 80 samples using both assays that had previously tested negative.
Results: The EBVP2 assay had an analytical sensitivity of 100% at the limit of detection (LOD) previously established for the EBNoV. Both assays had a clinical sensitivity and specificity of 100%. However, there was a difference in Cp values of positive samples in the assays. Reducing the time between heat treatment and testing improved this difference.
Conclusions: These results show that the assays are equivalent but poses questions about the stability of RNA in heated stool preparation solution.