Genomic Surveillance for Antimicrobial Resistance inMannheimia haemolyticaUsing Nanopore Single Molecule Sequencing Technology

Abstract

35Disruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are 36 transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible 37 strain of Mannheimia haemolytica, isolated from pneumonic bovine lung samples, were 38 respectively sequenced at 146x and 111x coverage with Oxford Nanopore Technologies 39 MinION. De novo assembly produced a complete genome for the non-resistant strain and a 40 nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid 41 Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance 42 Database) and ResFinder databases identified genes conferring resistance to different classes of 43 antibiotics including beta lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, 44 sulfonamides and macrolides. Antibiotic resistance phenotypes of the M. haemolytica strains 45 were confirmed with minimum inhibitory concentration (MIC) assays. The sequencing capacity 46 of highly portable MinION devices was verified by sub-sampling sequencing reads; potential for 47 antimicrobial resistance determined by identification of resistance genes in the draft assemblies 48 with as little as 5,437 MinION reads corresponded to all classes of MIC assays. The resulting 49 quality assemblies and AMR gene annotation highlight efficiency of ultra long-read, whole-50 genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.51 52 53 54 55 56Emergence of antimicrobial resistance (AMR) among the most important bacterial pathogens is 57 recognized as a major public health concern. Not only has AMR emerged in hospital 58 environments, it is often identified in community settings, in livestock feedlots and in 59 aquaculture and crop production, suggesting an ever-increasing range of reservoirs of antibiotic-60 resistant bacteria (1-4). Bacterial response to the antibiotic "attack" is the prime example of 61 genetic adaptation through the interplay of immense genetic plasticity, ranging from mutational 62 adaptations and acquisition of genetic material to alteration of gene expression with fitness 63 consequence of the pathogen (5). As a result, understanding the genetic basis of resistance is of 64 paramount importance to design strategies to curtail the emergence and spread of AMR, as well 65 as to devise innovative therapeutic approaches against multidrug-resistant organisms (6). 66With an increased population of pet and farm animals there is a significant risk of 67 humans acquiring drug resistant bacteria from animal sources. One of the major deficiencies in 68 veterinary medicine is the lack of validated data to determine minimum inhibitory concentration 69 (MIC) breakpoints for drug-microbe-host combinations, based on which scientifically sound 70 interpretations can be made regarding whether a pathogen is susceptible or resistant to a specific 71 drug. This is especially true for anaerobic bacteria. The large diversity of domestic and exotic 72 animal sp...