广西部分家禽养殖场曲霉菌（Aspergillus sp.）遗传多态性及对伊曲康唑敏感性分析
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摘要
曲霉属的真菌是主要存在于土壤、水以及腐败的营养物的一类霉菌。它们产生大量的孢子,这些孢子通过空气能很容易的扩散到环境中,由于这类孢子的普遍存在,人和动物不断的暴露于曲霉菌孢子中。烟曲霉和黄曲霉被认为是引起人和各类动物真菌性疾病的主要病原,相比哺乳动物而言,鸟类更易感。家禽养殖场的环境条件非常适合许多真菌,包括曲霉菌的生长于繁殖。本研究的主要日的是评估广西家禽养殖场分离的主要曲霉菌的遗传多样性及对抗真菌药物的敏感性。
研究第一部分主要对南宁的3个养殖场以及桂林的一个(包含一个孵化场)种鸡场真菌污染的情况进行调查。调查主要采集家禽咽部样品及空气样本,采样时间一般持续几周,同时对孵化场进行了三个孵化周期的监测。经表型鉴定辅助分子生物学方法鉴定,最终共收集到188份烟曲霉和159份黄曲霉。
第二部分建立或应用多位点数目可变串联重复序列(VNTR)多态性对黄曲霉和烟曲霉进行遗传多样性分析。本研究筛选了8个VNTR位点对黄曲霉遗传多样性进行分析,并设计双重反应。利用筛选到的8个多态性位点对91份黄曲霉分离株,其中包括6个参考株进行分型分析,结果共产生78种基因型,其中71种基因型仅出现一次,辛普森多样性指数为0.993。用10个VNTR多态性位点对188个广西烟曲霉分离株进行遗传多样性分析,结果共产生142种基因型。可使用图形算法最小生成树(MST)对黄曲霉或烟曲霉进行聚类分析。这种指纹分型方法能用于很多没有精密设备的国家进行黄曲霉分子流行病学研究,此外,本研究的结果能通过网站进行共享。黄曲霉遗传多样性聚类分析结果与烟曲霉一样与地理分布存在一定的相关性。
第三部分是对来自中国和法国的177个分离株进行抗真菌药物的敏感性分析。绝大部分中国和法国分离株对伊曲康唑敏感,其中中国分离株最小抑菌浓度在0.38～0.75微克/毫升,法国分离株的最小抑菌浓度在0.19～1微克/毫升。来自曾经用化学药物进行预防的养殖场的分离株的最小抑菌浓度并不比从未使用抗真菌药物的高。药敏试验显示4个分离株对伊曲康唑耐受：2株来自中国,2株来自法国。Cyp51A基因测序分析显示有11株分离株发生基因变异(3个耐药株和8个药敏株),共检测到20个点突变,其中11个点突变不引起氨基酸改变,9个点突变导致氨基酸序列的改变。氨基酸突变种有6种突变是已经报导的,但A116R,E130D和Q131H三个位点的突变是新发现的。对抗唑类药物的抗药率和分离耐药株频率的可能趋势应继续进行监控。农业上对于唑类药物的应用,以及环境样品的监控也同样重要。
Fungi of the genus Aspergillus are moulds, which occur most frequently in soil, water and decaying vegetation. They sporulate abundantly and the spores are easily dispersed into the environment by air. As a result of this ubiquitous presence, animals and people are constantly exposed to Aspergillus spores. Aspergillus fumigatus and A. flavus are recognized as predominant causes of fungal diseases in humans and wide range of animals. Birds are much more sensitive that mammals and in avian farms, environmental conditions are favorable to the development of many fungal species, including Aspergillus spp.The objective of the present study was to assess the genetic diversity and antifungal susceptibility of Aspergillus isolates from avian farms in Guangxi, China.
The first part of the work related the dynamic of fungal contamination in3avian farms near the city of Nanning and one farm (including a hatchery) near the city of Guilin. Pharyngeal swabs and air samples were collected during several weeks and3cycles of hatching were monitored. The average contamination level with Aspergillus spp. and Mucorales was significantly different according to the farms. The survey allowed to collect a total number of188A. fumigatus and159A. flavus isolates.
The second part of the work is about the genetic diversity of A. fumigatus and A. flavus. For that purpose, the Multiple Locus Variable-number tandem-repeat (VNTR) Analysis was specifically developed and used. For A. flavus,8VNTR markers were selected and a multiplex reaction was designed. A total number of91A. flavus isolates, including6reference strains were typed with the panel of8VNTRs. This analysis yielded78different genotypes, which corresponds to a combined loci index of0.993. Among all genotypes,71were only found once. The analysis of188A. fumigatus isolates using10VNTR markers led to the resolution of142distinct genotypes. Clusters of A. flavus or A. fumigatus isolates could be defined by using the graphing algorithm Minimum Spanning Tree. The typing method could be used for molecular epidemiological studies of A. flavus in many countries without the need for sophisticated equipment. Furthermore, data obtained by the present method could be easily shared in a web database. The clustering analysis was in accordance with a geographical structuring of A. flavus isolates similar to that detected in A. fumigatus.
The third part of the work is about the antifungal susceptibility of177A. fumigatus isolates collected in avian farms in China and France. Most of the isolates from China were susceptible to itraconazole with a Minimum Inhibitory Concentration (MIC) comprised between0.38and0.75ug/mL Most of the isolates from birds and avian farms in France were susceptible to itraconazole with a MIC comprised between0.19and1μg/mL. MIC values of isolates collected in farms with antifungal chemoprophylaxis were not higher than those of isolates collected from birds that never received antifungal drugs before the sampling. Susceptibility testings demonstrated that4isolates should be considered as resistant to itraconazole:2isolates from avian farms in Guangxi, China and2isolates from avian farms in France. A modification of the Cyp51A sequence was identified in11isolates (3azole-resistant and8azole-susceptible isolates). Twenty-one nucleotidic mutations were detected.Twelve of these mutations is silent and9yielded to amino acid substitutions. Six of these substitutions had already been described whereas mutations A116R, E130D and Q131H were original. Continued surveillance of azole resistance should be maintained to determine the resistance rates and any possible trend of increase in the isolation frequency of resistant strains. Based on the association with agricultural azole use, environmental sampling remains significant as well.
研究第一部分主要对南宁的3个养殖场以及桂林的一个(包含一个孵化场)种鸡场真菌污染的情况进行调查。调查主要采集家禽咽部样品及空气样本,采样时间一般持续几周,同时对孵化场进行了三个孵化周期的监测。经表型鉴定辅助分子生物学方法鉴定,最终共收集到188份烟曲霉和159份黄曲霉。
第二部分建立或应用多位点数目可变串联重复序列(VNTR)多态性对黄曲霉和烟曲霉进行遗传多样性分析。本研究筛选了8个VNTR位点对黄曲霉遗传多样性进行分析,并设计双重反应。利用筛选到的8个多态性位点对91份黄曲霉分离株,其中包括6个参考株进行分型分析,结果共产生78种基因型,其中71种基因型仅出现一次,辛普森多样性指数为0.993。用10个VNTR多态性位点对188个广西烟曲霉分离株进行遗传多样性分析,结果共产生142种基因型。可使用图形算法最小生成树(MST)对黄曲霉或烟曲霉进行聚类分析。这种指纹分型方法能用于很多没有精密设备的国家进行黄曲霉分子流行病学研究,此外,本研究的结果能通过网站进行共享。黄曲霉遗传多样性聚类分析结果与烟曲霉一样与地理分布存在一定的相关性。
第三部分是对来自中国和法国的177个分离株进行抗真菌药物的敏感性分析。绝大部分中国和法国分离株对伊曲康唑敏感,其中中国分离株最小抑菌浓度在0.38～0.75微克/毫升,法国分离株的最小抑菌浓度在0.19～1微克/毫升。来自曾经用化学药物进行预防的养殖场的分离株的最小抑菌浓度并不比从未使用抗真菌药物的高。药敏试验显示4个分离株对伊曲康唑耐受：2株来自中国,2株来自法国。Cyp51A基因测序分析显示有11株分离株发生基因变异(3个耐药株和8个药敏株),共检测到20个点突变,其中11个点突变不引起氨基酸改变,9个点突变导致氨基酸序列的改变。氨基酸突变种有6种突变是已经报导的,但A116R,E130D和Q131H三个位点的突变是新发现的。对抗唑类药物的抗药率和分离耐药株频率的可能趋势应继续进行监控。农业上对于唑类药物的应用,以及环境样品的监控也同样重要。
Fungi of the genus Aspergillus are moulds, which occur most frequently in soil, water and decaying vegetation. They sporulate abundantly and the spores are easily dispersed into the environment by air. As a result of this ubiquitous presence, animals and people are constantly exposed to Aspergillus spores. Aspergillus fumigatus and A. flavus are recognized as predominant causes of fungal diseases in humans and wide range of animals. Birds are much more sensitive that mammals and in avian farms, environmental conditions are favorable to the development of many fungal species, including Aspergillus spp.The objective of the present study was to assess the genetic diversity and antifungal susceptibility of Aspergillus isolates from avian farms in Guangxi, China.
The first part of the work related the dynamic of fungal contamination in3avian farms near the city of Nanning and one farm (including a hatchery) near the city of Guilin. Pharyngeal swabs and air samples were collected during several weeks and3cycles of hatching were monitored. The average contamination level with Aspergillus spp. and Mucorales was significantly different according to the farms. The survey allowed to collect a total number of188A. fumigatus and159A. flavus isolates.
The second part of the work is about the genetic diversity of A. fumigatus and A. flavus. For that purpose, the Multiple Locus Variable-number tandem-repeat (VNTR) Analysis was specifically developed and used. For A. flavus,8VNTR markers were selected and a multiplex reaction was designed. A total number of91A. flavus isolates, including6reference strains were typed with the panel of8VNTRs. This analysis yielded78different genotypes, which corresponds to a combined loci index of0.993. Among all genotypes,71were only found once. The analysis of188A. fumigatus isolates using10VNTR markers led to the resolution of142distinct genotypes. Clusters of A. flavus or A. fumigatus isolates could be defined by using the graphing algorithm Minimum Spanning Tree. The typing method could be used for molecular epidemiological studies of A. flavus in many countries without the need for sophisticated equipment. Furthermore, data obtained by the present method could be easily shared in a web database. The clustering analysis was in accordance with a geographical structuring of A. flavus isolates similar to that detected in A. fumigatus.
The third part of the work is about the antifungal susceptibility of177A. fumigatus isolates collected in avian farms in China and France. Most of the isolates from China were susceptible to itraconazole with a Minimum Inhibitory Concentration (MIC) comprised between0.38and0.75ug/mL Most of the isolates from birds and avian farms in France were susceptible to itraconazole with a MIC comprised between0.19and1μg/mL. MIC values of isolates collected in farms with antifungal chemoprophylaxis were not higher than those of isolates collected from birds that never received antifungal drugs before the sampling. Susceptibility testings demonstrated that4isolates should be considered as resistant to itraconazole:2isolates from avian farms in Guangxi, China and2isolates from avian farms in France. A modification of the Cyp51A sequence was identified in11isolates (3azole-resistant and8azole-susceptible isolates). Twenty-one nucleotidic mutations were detected.Twelve of these mutations is silent and9yielded to amino acid substitutions. Six of these substitutions had already been described whereas mutations A116R, E130D and Q131H were original. Continued surveillance of azole resistance should be maintained to determine the resistance rates and any possible trend of increase in the isolation frequency of resistant strains. Based on the association with agricultural azole use, environmental sampling remains significant as well.
引文
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