Table_7_Bacterial Genome Wide Association Studies (bGWAS) and Transcriptomics Identifies Cryptic Antimicrobial Resistance Mechanisms in Acinetobacter baumannii.xlsx

Antimicrobial resistance (AMR) in the nosocomial pathogen, Acinetobacter baumannii, is becoming a serious public health threat. While some mechanisms of AMR have been reported, understanding novel mechanisms of resistance is critical for identifying emerging resistance. One of the first steps in identifying novel AMR mechanisms is performing genotype/phenotype association studies; however, performing these studies is complicated by the plastic nature of the A. baumannii pan-genome. In this study, we compared the antibiograms of 12 antimicrobials associated with multiple drug families for 84 A. baumannii isolates, many isolated in Arizona, USA. in silico screening of these genomes for known AMR mechanisms failed to identify clear correlations for most drugs. We then performed a bacterial genome wide association study (bGWAS) looking for associations between all possible 21-mers; this approach generally failed to identify mechanisms that explained the resistance phenotype. In order to decrease the genomic noise associated with population stratification, we compared four phylogenetically-related pairs of isolates with differing susceptibility profiles. RNA-Sequencing (RNA-Seq) was performed on paired isolates and differentially-expressed genes were identified. In these isolate pairs, five different potential mechanisms were identified, highlighting the difficulty of broad AMR surveillance in this species. To verify and validate differential expression, amplicon sequencing was performed. These results suggest that a diagnostic platform based on gene expression rather than genomics alone may be beneficial in certain surveillance efforts. The implementation of such advanced diagnostics coupled with increased AMR surveillance will potentially improve A. baumannii infection treatment and patient outcomes.

鲍曼不动杆菌（Acinetobacter baumannii）作为院内病原菌，其抗菌药物耐药性（Antimicrobial resistance, AMR）正逐渐成为严重的公共卫生威胁。尽管已有部分AMR相关机制被报道，但解析新型耐药机制对于识别新发耐药性至关重要。鉴定新型AMR机制的首要步骤之一为开展基因型/表型关联研究；然而，鲍曼不动杆菌泛基因组（pan-genome）的可塑性使得此类研究的开展极为复杂。本研究针对84株鲍曼不动杆菌分离株（其中多株分离自美国亚利桑那州），比对了覆盖多个药物家族的12种抗菌药物的抗菌谱。针对已知AMR机制的计算机模拟（in silico）基因组筛查未能明确多数药物的耐药关联。随后，我们开展了细菌全基因组关联研究（bacterial genome wide association study, bGWAS），以寻找所有可能的21-mers之间的关联；该方法通常未能识别可解释耐药表型的机制。为降低群体分层带来的基因组噪声，我们比对了4对系统发育相关且药敏谱存在差异的分离株。对这些配对分离株开展RNA测序（RNA-Sequencing, RNA-Seq），并鉴定出差异表达基因。在这些分离株对中，共识别出5种潜在耐药机制，凸显了该物种广谱AMR监测的难度。为验证差异表达结果，我们开展了扩增子测序（amplicon sequencing）。上述结果提示，基于基因表达而非单纯基因组学的诊断平台，或可在部分监测工作中发挥积极作用。将此类先进诊断技术与强化的AMR监测相结合，有望改善鲍曼不动杆菌感染的治疗效果与患者预后。