抗甲氧西林金黃色葡萄球菌 夫西地酸抗藥基因fusC之減少趨勢

Abstract

夫西地酸 (fusidic acid) 為固醇類抗生素，在臨床上主要用於治療金黃色葡萄球菌引起的皮膚感染和全身系統性感染。目前已知金黃色葡萄球菌可藉由使藥物作用標的基因突變或產生保護藥物作用標的之蛋白質來產生夫西地酸之抗藥性。近年來抗夫西地酸金黃色葡萄球菌日趨增多，根據臺大醫院統計資料顯示，具有夫西地酸抗藥之抗甲氧西林金黃色葡萄球菌 (methicillin-resistant Staphylococcus aureus, MRSA) 的比例逐年上升。為了持續觀察MRSA對夫西地酸抗藥情況，本實驗收集2011至2016年自台大醫院細菌室分離之185株夫西地酸抗藥之MRSA，偵測其夫西地酸抗藥基因。結果顯示，在185株夫西地酸抗藥之MRSA中，有34株(17.8%)帶有fusC基因，並無偵測到帶有fusB、fusD和fusF基因之菌株。而在隨機挑選未知機制的菌株中，以FusA產生L461K突變為主。然而，根據收集台大醫院2008至2010菌株之研究指出，約有59%夫西地酸抗藥之MRSA菌株帶有fusC基因。因此，進一步分析菌株分子流行病學以了解fusC基因比例的下降是否因菌株族群發生改變所造成。根據spa typing、multilocus sequencing及SCCmec分型之結果，發現在2011年，帶有fusC基因之夫西地酸抗藥MRSA分子分型仍與過去相同以ST239-SCCmec type III-t037為主，但在之後比例逐漸下降，至2012年便以ST45-SCCmec type VT-t1081為主，顯示菌株族群有所變化。進一步探討此34株帶有fusC基因之MRSA，其攜帶fusC基因之結構是否與先前研究結果以SCCfusC為主。結果顯示，此34株fusC基因夫西地酸抗藥之MRSA均帶有SCCfusC結構。為了瞭解造成SCCfusC轉移到不同菌株族群之原因，由於type I restriction modification (R-M)系統被報導與細菌調控外來基因插入細菌基因內之相關性，且HsdS相似度愈高不同菌株基因交換頻率愈高，因此比較不同菌株族群type I R-M系統的HsdR、HsdM、HsdS。根據胺基酸序列比對結果顯示，ST239-SCCmec type III-t037與ST45-SCCmec type VT-t1081的HsdR和HsdM氨基酸序列相似度大於99%，但兩者的HsdS氨基酸序列有巨大的差異。此結果表示，造成SCCfusC首先從ST239菌株傳遞至ST45菌株的原因可能與type I R-M系統關聯性較低。 根據以上結果，本實驗發現夫西地酸抗藥之MRSA中帶有fusC基因比例較往年下降，並且主要菌株族群由ST239-SCCmec type III-t037改變為ST45-SCCmec type VT-t1081，然而其攜帶fusC基因之結構卻仍為SCCfusC，推測此結構已不再只存在單一MRSA族群，有傳播至不同族群之情況，但其傳播之原因仍需進一步研究。

Fusidic acid is a steroid antibiotic which has been used as antimicrobial agent to treat skin infection and systemic infections caused by Staphylococcus aureus. However, fusidic acid-resistant S. aureus has been reported worldwide. The fusidic acid resistance rate in National Taiwan University Hospital (NTUH) was approximately 3 to 6%, but the ratio is climbing up especially in methicillin-resistant S. aureus (MRSA) (from 10% to 20%) recently. In this study, a total of 185 fusidic acid-resistant MRSA isolates from 2011 to 2016 was collected. The MICs of fusidic acid ranged from 2 to ≥128 μg/ml. Detection of resistance determinants by PCR indicated that 34 carried fusC (34/185, 18%) but none contained fusB, fusD and fusF. The prevalence of fusC in this study was much lower than previous results (59% MRSA carried fusC). We randomly selected unknown resistant mechanism isolates to test fusA mutation, and then the results revealed that the L461K was the major mutation typ. To understand whether the genetic background of fusidic acid-resistant MRSA had changed, we examined SCCmec types, spa types, multilocus sequence types and mapped SCCfusC element. The molecular typing indicated the prevalent fusidic acid resistant lineage belonged to ST239-SCCmec type III-t037 in 2011, but ST45-SCCmec type VT-t1081 became the major fusidic acid resistant lineage after 2012. The SCCfusC PCR mapping also showed that the SCCfusC structure harbored in 34 fusC-carrying isolates was identical to that reported previously. To understand what lead the SCCfusC transferred to other lineage, we examined type I restriction modification (R-M) system. Type I R-M system has been reported that it is correlated with genes uptake and the exchange of DNA is in high frequency in the isolates with same hsdS. We compared the hsdR, hsdM and hsdS of type I R-M system, fusC isolates and non-fusC isolates showed >99% similarity in the amino acids sequence of HsdR and HsdM. However, the amino acids sequence of HsdS between fusC isolates and non-fusC isolates showed high variety. This indicated that type I R-M system was low relationship with the transfer of SCCfusC from ST239 isolates to ST45 isolates. In conclusion, the prevalence of fusC in MRSA had been recently decreased and the dominant lineage of isolates was changed from ST239-SCCmec type III-t037 to ST45-SCCmec type VT -t1081 among fusC-carrying MRSA. Moreover, SCCfusC is still the main structure that responsible for carrying the fusC gene, so it can be suggested that SCCfusC was transferred to other lineages, but the reason why SCCfusC was transferred to other lineages still need to be investigated.