金黃葡萄球菌及表皮葡萄球菌對於夫西地酸抗藥基因分析

Abstract

夫西地酸 (Fusidic acid)為類固醇類的抗生素，臨床上主要用於治療葡萄球菌所引起的皮膚感染或較為嚴重的全身性症狀。夫西地酸藉由與EF-G-GTP/GDP- ribosome結合，使EF-G/GDP無法離開ribosome而抑制細菌蛋白質的合成，達到藥物作用效果。細菌可經由藥物作用標的基因產生突變 (fusA or rplF point mutation)或表現可以保護藥物作用標的之蛋白質 (FusB-protein family)產生抗藥性。抗藥菌株的發生會限制夫西地酸在臨床上的使用，為了維持夫西地酸能在臨床上有效的應用，進一步了解其抗藥基因的盛行率，抗藥菌株間菌種關係，及分析抗藥基因結構有其不可或缺的重要性。 本研究分析由台大醫院收集的夫西地酸抗藥性葡萄球菌臨床菌株，包含71株金黃葡萄球菌(45株甲氧西林[methicillin]抗藥性金黃葡萄球菌及26株甲氧西林敏感性金黃葡萄球菌)，36株表皮葡萄球菌，10株溶血葡萄球菌25株腐生葡萄球菌，其夫西地酸抗藥基因(fusA點突變、fusB、fusC與fusD)的盛行率及抗藥程度的關聯性，發現在菌種間有明顯的差異。在甲氧西林抗藥性金黃葡萄球菌中，主要由於fusA點突變導致對於夫西地酸高程度抗藥性(22/38 with MIC ≥128 μg/ml，58%)，而大部分的甲氧西林敏感性金黃葡萄球菌(15/26，58%)，表皮葡萄球菌(34/36，94%)及溶血葡萄球菌(8/10，80%)則因帶有fusB抗藥基因引起抗藥(MIC範圍為4 μg/ml至32 μg/ml)。在25株腐生葡萄球菌中，有17株因為帶有fusD的抗藥基因造成夫西地酸抗藥(MIC範圍為2 μg/ml to 16 μg/ml)。fusB、fusC、fusD 抗藥基因導致對於夫西地酸較低程度的抗藥。 於金黃葡萄球菌中，41株具有fusA點突變之金黃葡萄球菌(包含38株甲氧西林抗藥性金黃葡萄球菌及3株甲氧西林敏感性金黃葡萄球菌)，共發現22個不同位點的突變造成21個胺基酸改變，其中R76C，E444K，E444V，C473S，P478S，M651I為第一次在夫西地酸抗藥之金黃葡萄球菌中被發現，並進一步利用定點突變(site-directed mutagenesis)探討E444K，C473S，R76C所扮演的角色。分析甲氧西林敏感性金黃葡萄球菌中fusB上下游的基因片段結構，其結構為partial IS257-aj1-LP- fusB-aj2-aj3-IS257-partial blaZ，此結構與pUB101中相關序列相同。進一步研究菌株間的種系關係，發現在甲氧西林抗藥性金黃葡萄球菌中有兩個主要的基因型分佈，spa type t037-SCCmec type III (t037-III，28/45，62%)及t002-II (13/45，29%)。根據PFGE樹狀圖結果，以80%相似度作為依據，45株甲氧西林抗藥性金黃葡萄球菌可以分為12個不同的pulsotypes，而在26株甲氧西林敏感性金黃葡萄球菌中則可分為15個pulsotypes。 此外，為了瞭解夫西地酸抗藥性表皮葡萄球菌其fusB的基因片段結構，分析34株帶有fusB抗藥基因之表皮葡萄球菌。定序分析aj1-LP-fusB片段發現三種不同的aj1-LP-fusB類型，其帶有相同的LP及fusB但不同的aj1基因序列，第一型為完整的aj1基因，第二型為自93至421核苷酸缺失之aj1基因，第三型為僅剩最後37核苷酸aj1基因。相較帶有第三型aj1-LP-fusB片段之菌株 (MICs，4至16 µg/ml)，帶有第一型及第二型的aj1-LP-fusB片段之菌株可表現相對上較高程度抗藥 (MICs，8至32 µg/ml)。選擇四株帶有不同型的aj1-LP-fusB片段之菌株進一步完成定序，結果顯示在表皮葡萄球菌中，fusB位於噬菌體相關的致病性島嶼 (pathogenicity island)上，但因此基因片段僅具有抗藥基因而無其他毒力因子，分別將此命名為SeRIfusB-2793，SeRIfusB-704，SeRIfusB-7778，SeRIfusB-5907 (S. epidermidis resistance island)。其中，三個抗藥島嶼 (SeRIfusB-2793, SeRIfusB-704, SeRIfusB-5907)嵌入genomic DNA上groEL下游 (相當於金黃葡萄球菌全基因序列44分鐘之位置)，而SeRIfusB-7778則嵌入genomic DNA上rpsR基因下游 (相當於金黃葡萄球菌全基因序列8分鐘之位置)，此四個抗藥島嶼皆嵌入integrase所辨識之att位置。在34帶有fusB菌株中，28株(82%)為嵌入groEL基因下游之抗藥島嶼 (resistance islands)，兩株為嵌入rpsR基因下游之抗藥島嶼，但仍有四株菌株無法確認其嵌入位置及抗藥片段是否為抗藥島嶼。利用PFGE分型發現帶有fusB基因的表皮葡萄球菌菌株間的異質性，此細菌造成的感染應非單一菌株的傳播。在表皮葡萄球菌中，fusB抗藥基因與噬菌體相關的抗藥島嶼有高度關聯性。

Fusidic acid is a steroid antibiotic used as topical agent for skin infection and systemic treatment against staphylococcal infection. Fusidic acid interacting with the elongation factor G (EF-G) blocks bacterial protein synthesis by preventing the release of EF-G/GDP complex from ribosome. Resistance to fusidic acid results from alternation of drug target site and protection of drug target site by FusB-family protein. The emergence of fusidic acid resistance is a problem that could restrict the clinical usage. To maintain the usefulness of fusidic acid, it is important to clarify the prevalence of the resistance mechanisms, the phylogenetic lineages between the resistant isolates, and genetic organizations of resistance determinants. A total of 71 fusidic acid-resistant Staphylococcus aureus (45 methicillin-resistant and 26 methicillin-susceptible), 36 fusidic acid-resistant Staphylococcus epidermidis, 10 fusidic acid-resistant Staphylococcus haemolyticus, and 25 fusidic acid-resistant Staphylococcus saprophyticus isolates were examined for the presence of resistance determinants. The distribution of fusA point mutations, fusB, and fusC was significantly different between staphylococci. Among 45 fusidic acid-resistant MRSA, 38 (84%) had fusA mutations conferring high-level resistance to fusidic acid (22/38 with MIC ≥128 μg/ml), none had fusB, and 7 (16%) had fusC. Most methicillin-susceptible S. aureus (MSSA, 15/26, 58%), S. epidermidis (34/36, 94%) and S. haemolyticus (8/10, 80%) carry fusB determinant (MIC ranges from 4μg/ml to 32 μg/ml). Among 25 S. saprophyticus, 17 (68%) isolates carry fusD determinant (MIC ranges from 2 μg/ml to 16 μg/ml). Low-level resistance to fusidic acid (MICs, ≤32 μg/ml) was found in most fusB- or fusC- or fusD- positive isolates. For 41 isolates (38 MRSA and 3 MSSA) with fusA mutations, a total of 21 amino-acid substitutions in EF-G (fusA gene) were detected. Of which, EF-G, R76C, E444K, E444V, C473S, P478S, and M651I, were identified for the first time. Furthermore, we analyzed the importance of E444K, C473S, and R76C by site-directed mutagenesis. Nucleotide sequencing of fusB and flanking regions in a MSSA isolate revealed the structure of partial IS257-aj1-LP-fusB-aj2-aj3-IS257-partial blaZ, which is identical to the corresponding region in pUB101, and the rest of fusB-carrying MSSA isolates also show similar structures. On the basis of spa and SCCmec typing, two major genotypes, spa type t037-SCCmec type III (t037-III, 28/45, 62%) and t002-II (13/45, 29%), were predominant among 45 MRSA. By pulsed-field gel electrophoresis analysis, 45 MRSA isolates were divided into 12 clusters, while 26 MSSA isolates were divided into 15 clusters. To understand the high prevalence of fusB in fusidic acid-resistant S. epidermidis, analysis of resistance elements in 34 isolates was performed. First, sequence analysis of aj1-LP-fusB region indicated that at least three types were present. Type I contained full-length aj1, type II contained a partial aj1 truncated from 93 to 421 nt, and type III contained a more truncated aj1 that retained only the last 37 nt. Isolates with type I or type II aj1 displayed slightly higher-level of resistance to fusidic acid (MICs, 8 to 32 µg/ml) compared with those with type III aj1 (MICs, 4 to 16 µg/ml). Subsequent sequencing of the flanking regions of fusB from four selected isolates carrying different types of aj1-LP-fusB region revealed that the fusB were all located on phage-related resistance islands (RIs), referred to as SeRIfusB-2793, SeRIfusB-704, SeRIfusB-5907, and SeRIfusB-7778, respectively. Among them, three islands (SeRIfusB-2793, SeRIfusB-704, SeRIfusB-5907) were located downstream of groEL (corresponding to 44’ position based on S. aureus whole genomic sequences), and one (SeRIfusB-7778) was located downstream of the rpsR (corresponding to 8’ position). All of the RIs were inserted into integrase-recognized att sites. Among 34 isolates, the insertion sites of fusB RIs were mostly (28/34, 82%) located downstream of groEL, two were located downstream of rpsR; but four remained unidentified. The pulsotypes distribution indicated that fusB-containing S. epidermidis isolates were heterogeneous. In conclusion, the fusB resistance determinant in S. epidermidis was highly associated with phage-related RIs.