Biswas Chayanika, Marcelino Vanessa R, Van Hal Sebastiaan, Halliday Catriona, Martinez Elena, Wang Qinning, Kidd Sarah, Kennedy Karina, Marriott Deborah, Morrissey C Orla, Arthur Ian, Weeks Kerry, Slavin Monica A, Sorrell Tania C, Sintchenko Vitali, Meyer Wieland, Chen Sharon C-A
Centre for Infectious Diseases and Microbiology-Public Health, Westmead Hospital, Sydney, NSW, Australia.
Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia.
Front Microbiol. 2018 Dec 3;9:2946. doi: 10.3389/fmicb.2018.02946. eCollection 2018.
is a pathogen with reduced susceptibility to azoles and echinocandins. Analysis by traditional multilocus sequence typing (MLST) has recognized an increasing number of sequence types (STs), which vary with geography. Little is known about STs of in Australia. Here, we utilized whole genome sequencing (WGS) to study the genetic diversity of 51 Australian isolates and sought associations between STs over two time periods (2002-2004, 2010-2017), and with susceptibility to fluconazole by principal component analysis (PCA). Antifungal susceptibility was determined using Sensititre YeastOne Y010 methodology and WGS performed on the NextSeq 500 platform (Illumina) with MLST STs inferred by WGS data. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-fluorocytosine resistance were analyzed. Of 51 isolates, WGS identified 18 distinct STs including four novel STs (ST123, ST124, ST126, and ST127). Four STs accounted for 49% of isolates (ST3, 15.7%; ST83, 13.7%; ST7, 9.8%; ST26, 9.8%). Split-tree network analysis resolved isolates to terminal branches; many of these comprised multiple isolates from disparate geographic settings but four branches contained Australian isolates only. ST3 isolates were common in Europe, United States and now Australia, whilst ST8 and ST19, relatively frequent in the United States, were rare/absent amongst our isolates. There was no association between ST distribution (genomic similarity) and the two time periods or with fluconazole susceptibility. WGS identified mutations in the (S629P) and (S663P) genes in three, and one, echinocandin-resistant isolate(s), respectively. Both mutations confer phenotypic drug resistance. Twenty-five percent (13/51) of isolates were fluconazole-resistant (MIC ≥ 64 μg/ml) of which 9 (18%) had non wild-type MICs to voriconazole and posaconazole. Multiple SNPs were present in genes linked to azole resistance such as and , as well as several in ; however, SNPs occurred in both azole-susceptible and azole-resistant isolates. Although no particular SNP in these genes was definitively associated with resistance, azole-resistant/non-wild type isolates had a propensity to harbor SNPs resulting in amino acid substitutions in Pdr1 beyond the first 250 amino acid positions. The presence of SNPs may be markers of STs. Our study shows the value of WGS for high-resolution sequence typing of , discovery of novel STs and potential to monitor trends in genetic diversity. WGS assessment for echinocandin resistance augments phenotypic susceptibility testing.
是一种对唑类和棘白菌素敏感性降低的病原体。通过传统多位点序列分型（MLST）分析已识别出越来越多的序列类型（STs），这些序列类型因地理位置而异。在澳大利亚，关于的STs知之甚少。在这里，我们利用全基因组测序（WGS）研究了51株澳大利亚分离株的遗传多样性，并通过主成分分析（PCA）寻找两个时间段（2002 - 2004年，2010 - 2017年）内STs之间的关联以及与氟康唑敏感性之间的关联。使用Sensititre YeastOne Y010方法测定抗真菌药敏性，并在NextSeq 500平台（Illumina）上进行WGS，通过WGS数据推断MLST STs。分析了与棘白菌素、唑类和5 - 氟胞嘧啶耐药相关基因中的单核苷酸多态性（SNPs）。在51株分离株中，WGS鉴定出18种不同的STs，包括4种新的STs（ST123、ST124、ST126和ST127）。四种STs占分离株的49%（ST3，15.7%；ST83，13.7%；ST7，9.8%；ST26，9.8%）。分裂树网络分析将分离株解析到末端分支；其中许多分支包含来自不同地理区域的多个分离株，但有四个分支仅包含澳大利亚分离株。ST3分离株在欧洲、美国以及现在的澳大利亚都很常见，而在美国相对频繁的ST8和ST19在我们的分离株中很少见/不存在。ST分布（基因组相似性）与两个时间段或与氟康唑敏感性之间没有关联。WGS分别在3株和1株棘白菌素耐药分离株中鉴定出基因（S629P）和基因（S663P）中的突变。这两种突变均赋予表型耐药性。25%（13/51）的分离株对氟康唑耐药（MIC≥64μg/ml），其中9株（18%）对伏立康唑和泊沙康唑具有非野生型MIC。与唑类耐药相关的基因如和中存在多个SNPs，以及中也有几个；然而，SNPs在唑类敏感和唑类耐药分离株中均有出现。尽管这些基因中没有特定的SNP与耐药性明确相关，但唑类耐药/非野生型分离株倾向于在Pdr超过前250个氨基酸位置的区域含有导致氨基酸替换的SNP。SNP的存在可能是STs的标志物。我们的研究显示了WGS在进行高分辨率序列分型、发现新的STs以及监测遗传多样性趋势方面的价值。对棘白菌素耐药性的WGS评估增强了表型药敏试验。
