Gargis A S, McLaughlin H P, Conley A B, Lascols C, Michel P A, Gee J E, Marston C K, Kolton C B, Rodriguez-R L M, Hoffmaster A R, Weigel L M, Sue D
Division of Preparedness and Emerging Infections, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
IHRC-Georgia Tech Applied Bioinformatics Laboratory, Atlanta, Georgia, USA.
mSystems. 2018 Dec 11;3(6). doi: 10.1128/mSystems.00154-18. eCollection 2018 Nov-Dec.
Penicillin (PEN) is a low-cost option for anthrax treatment, but naturally occurring resistance has been reported. β-Lactamase expression (, ) in Bacillus anthracis is regulated by a sigma factor (SigP) and its cognate anti-sigma factor (RsiP). Mutations leading to truncation of RsiP were previously described as a basis for PEN resistance. Here, we analyze whole-genome sequencing (WGS) data and compare the chromosomal regions from 374 B. anthracis strains to determine the frequency of mutations, identify mutations associated with PEN resistance, and evaluate the usefulness of WGS for predicting PEN resistance. Few (3.5%) strains contained at least 1 of 11 different mutations in , , or Nine of these mutations have not been previously associated with PEN resistance. Four strains showed PEN resistance (PEN-R) by conventional broth microdilution, including 1 strain with a novel frameshift in . One strain that carries the same frameshift mutation as that found previously in a PEN-R strain showed a PEN-susceptible (PEN-S) phenotype and exhibited decreased and transcription. An unexpectedly small colony size, a reduced growth rate, and undetectable β-lactamase activity levels (culture supernatant and cell lysate) were observed in this PEN-S strain. Sequence analysis revealed mutations in genes associated with growth defects that may contribute to this phenotype. While B. anthracis mutations cannot be exclusively used to predict resistance, four of the five strains with mutations were PEN-R. Therefore, the B. anthracis region is a useful locus for WGS-based PEN resistance prediction, but phenotypic testing remains essential. Determination of antimicrobial susceptibility of B. anthracis is essential for the appropriate distribution of antimicrobial agents for postexposure prophylaxis (PEP) and treatment of anthrax. Analysis of WGS data allows for the rapid detection of mutations in antimicrobial resistance (AMR) genes in an isolate, but the presence of a mutation in an AMR gene does not always accurately predict resistance. As mutations in the anti-sigma factor RsiP have been previously associated with high-level penicillin resistance in a limited number of strains, we investigated WGS assemblies from 374 strains to determine the frequency of mutations and performed functional antimicrobial susceptibility testing. Of the five strains that contained mutations in , only four were PEN-R by functional antimicrobial susceptibility testing. We conclude that while sequence analysis of this region is useful for AMR prediction in B. anthracis, genetic analysis should not be used exclusively and phenotypic susceptibility testing remains essential.
青霉素（PEN）是治疗炭疽的一种低成本选择，但已有自然产生耐药性的报道。炭疽芽孢杆菌中的β-内酰胺酶表达受一个σ因子（SigP）及其同源抗σ因子（RsiP）调控。导致RsiP截短的突变先前被描述为PEN耐药的基础。在此，我们分析全基因组测序（WGS）数据，并比较374株炭疽芽孢杆菌菌株的染色体区域，以确定突变频率，识别与PEN耐药相关的突变，并评估WGS在预测PEN耐药方面的实用性。少数（3.5%）菌株在rsiP、sigP或bla基因中至少含有11种不同突变中的1种。其中9种突变以前未与PEN耐药相关。4株菌株通过传统肉汤微量稀释法显示出PEN耐药（PEN-R），包括1株rsiP基因有新的移码突变的菌株。1株携带与先前在1株PEN-R菌株中发现的相同移码突变的菌株表现出PEN敏感（PEN-S）表型，且rsiP和bla转录减少。在这株PEN-S菌株中观察到意外小的菌落大小、降低的生长速率以及无法检测到的β-内酰胺酶活性水平（培养上清液和细胞裂解物）。序列分析揭示了与生长缺陷相关的基因中的突变，这些突变可能导致了这种表型。虽然炭疽芽孢杆菌rsiP突变不能单独用于预测耐药性，但5株有rsiP突变的菌株中有4株是PEN-R。因此，炭疽芽孢杆菌rsiP区域是基于WGS预测PEN耐药性的有用位点，但表型检测仍然至关重要。确定炭疽芽孢杆菌的抗菌药敏性对于暴露后预防（PEP）和炭疽治疗中抗菌药物的合理分配至关重要。对WGS数据的分析允许快速检测分离株中抗菌耐药（AMR）基因的突变，但AMR基因中的突变并不总是能准确预测耐药性。由于抗σ因子RsiP中的突变先前已在少数菌株中与高水平青霉素耐药相关，我们研究了374株菌株的WGS组装结果以确定突变频率，并进行了功能性抗菌药敏试验。在5株有rsiP突变的菌株中，通过功能性抗菌药敏试验只有4株是PEN-R。我们得出结论，虽然该区域的序列分析对炭疽芽孢杆菌的AMR预测有用，但不应仅使用基因分析，表型药敏试验仍然至关重要。
