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Characterization of antibiotic resistance genes among staphylococci isolated from ready-to-eat food
ready-to-eat food,Staphylococcus aureus,coagulase-negative staphylococci,antibiotic resistance genes,
|Publication Year :||2019|
|Abstract:||本研究為針對台灣流通率最高之即食食品種類如畜、禽、蛋、奶 (製品)、即食生鮮蔬果、冰品、即食米類製品及低水活性食品共採樣1128件，探討其葡萄球菌汙染情形及抗藥特性。共檢出111株金黃色葡萄球菌(S. aureus)及709株、共23種菌種之凝固酶陰性葡萄球菌(Coagulase-negative staphylococci, CoNS)。不同即食食品之S. aureus檢出率互異，其中以即食生鮮蔬果最高(20.5%)，低水活性最低(0.7%)；若不論菌種類別，仍以即食生鮮蔬果遭受葡萄球菌污染最為顯著(62.2%)，亦常發現單一檢體遭受多株(至多10株)或多種(至多6種)葡萄球菌汙染，且各類即食食品皆有其獨特之菌相分布。相較於其他即食食品(0-7.1%)，即食生鮮蔬果之抗藥性S. aureus檢出率最高(14.2%)，其中有4株為攜帶第四型SCCmec (3株)及第五型(1株)之耐甲氧西林金黃色葡萄球菌(methicillin-resoistant S. aureus, MRSA)(3.6%)，其中3株型別為ST59，1株為ST5；在41株MR-CoNS (5.8%)中，以帶有mec complex class B、ccrA2B2 complex的S. epidermidis檢出率最高 (n=16)，而在各類CoNS菌種，以S. cohnii之耐甲氧西林發生率最高(2/5)，但攜帶的mec complex及ccr complex組合未曾在S. aureus發現。除S. saprophyticus及S. cohnii攜帶已知內源性(intrinsic)抗藥基因fusD及fusF而對fusidic acid具低度抗性，S. xylosus、S. kloosii及S. sciuri亦普遍對於fusidic acid展現低度抗性，進一步發現S. xylosus、S. sciuri及S. kloosii具有fusB family新穎基因、位於染色體鄰近區域皆未發現移動性遺傳因子(mobile genetic element)，推測這些新穎基因不具移動性。在即食食品分離之葡萄球菌發現的其他抗藥性及其相對應之抗藥基因包括Penicillin (blaZ)、Tetracycline (tetK、tetL、tetM)、Erythromycin (ermA、ermB、ermC、msrA/B、ermT)、Gentamicin (aacA-aphD)、Fusidic acid (fusB、fusC)及Trimethoprim/sulfamethoxazole (dfrA、dfrG)，在即食食品之葡萄球菌菌相之主要組成菌種，S. epidermidis及S. warneri對於多種藥物皆展現抗性且攜帶多元抗藥基因；據此進一步探討各類即食食品之風險性，顯示即食生鮮蔬果因遭受攜帶抗藥基因(包含耐甲氧西林抗藥基因mecA)之葡萄球菌汙染比例較高，推測在傳遞抗藥性上之風險性應較其他即食食品高。有鑑於S. cohnii在臨床案例展現高致病力，且MR-S. cohnii所攜帶之SCCmec非S. aureus之常見型別，本研究以長讀定序(long-read sequencing)解析2014-2015年自碗糕(菌株RC35)及冰品(菌株I-104及I-112)分離之MR-S. cohni之SCCmec結構，發現3種SCCmec複合結構：菌株RC35攜帶SCCmec-SCC串聯結構，該SCCmec結構與過去日本泌尿道感染患者所分離之S. saprophyticus TSU33所攜帶之SCCmecTSU33相同，SCC之前大半部與過去中國在傷口檢體分離之S. cohnii所攜帶之SCCWC28相同，尾部則為一連串基因組成，其中包含負責生合成staphyloxanthin之crtOPQMN operon；菌株I-104攜帶SCCI-104up-SCCmecTSU33-SCCRC35串聯結構，其中SCCI-104up之ccrA1B3 complex至其上游8 kb位置之區間與過去泰國自狗檢體分離之S. pseudointermedius AI16菌株之SCCAI16部分區塊結構相近；菌株I-112攜帶SCCmecI-112-SCCI-112串聯結構，其中SCCmecI-112由SCCmecTSU33及SCCI-104-up之3端所組成，SCCI-112則有ccrA1B7 complex以及表現致病力、抗藥性及對抗環境壓力之一連串基因群；另發現菌株RC35、I-104及I-112之共同點除了SCCmec具序列高度保守 之class A mec complex，在串聯結構尾端皆具有crtOPQMN operon，該結構亦出現於臨床檢體、健康人手部分離之S. cohnii菌株。帶有crtOPQMN operon之S. cohnii菌落外觀明顯偏黃，萃取其色素，在波長465 nm下，帶有crtPOQMN operon之菌株吸光值明顯高於未帶crtPOQMN operon之菌株，且經1.5%過氧化氫模擬氧化壓力下，S. cohnii RC35菌株存活率明顯高於未帶crtOPQMN operon的ATCC49330菌株，顯示crtOPQMN operon有啟動且提供S. cohnii抵抗氧化壓力。本研究為首次探討食品分離S. cohnii之SCCmec特性、首次發現SCCmecTUS33-SCCWC28之串聯結構，及首次發現crtOPQMN operon存在於S. saprophyticus group部分菌株，推測crtOPQMN operon有機會藉由SCC或其相關結構進行傳遞，並賦予細菌抵抗宿主免疫能力之優勢，有助於S. cohnii之致病力。|
This study investigated antibiotic resistance of staphylococci isolated from 1128 samples of high-circulating RTE foods in Taiwan. A total of 111 Staphylococcus aureus and 709 coagulase-negative staphylococci (CoNS) comprising 23 species were isolated. The prevalence of S. aureus differed in various category of RTE foods, highest in fresh-cut fruits/vegetables (20.5%) and lowest in low-water activity (LWA) foods (0.7%). The overall staphylococcal contamination was highest in fresh-cut fruits/vegetables (62.2%), in which multiple isolates (up to 10) or species (up to 6) in single sample were frequently found. Distinct distribution of species contributed to unique feature in each category. Prevalence of antibiotic-resistant S. aureus was higher in fresh-cut fruits/vegetables samples (14.2% in 127) compared to other food categories (0-7.1%). A total of 4 MRSA carrying SCCmec type IV or VT were identified (3.6% in 111), in which 3 belonged to sequence type ST59 and one was ST5. Of 41 methicillin-resistant CoNS (5.8% in 709) isolates, S. epidermidis with SCCmec containing mec complex class B and ccrA2B2 complex was most frequent. Among CoNS species, prevalence of methicillin resistance was highest in S. cohnii (2/5), but the combination of mec complex and ccr complex in SCCmec did not appear in S. aureus. In addition to S. saprophyticus and S. cohnii carrying known intrinsic genes (fusD and fusF) conferring low-level resistance to fusidic acid, low-level fusidic acid resistance also commonly found in S. xylosus, S. kloosii and S. sciuri. Novel fusB-family determinants were identified in S. xylosus, S. sciuri and S. kloosii. Owing to mobile genetic elements were not found in the neighboring region of these novel fusB-family determinants, suggesting these novel fusB-family determinants could be immobile. Other kinds of antibiotic resistance and the corresponding determinants were also found in RTE food-isolated staphylococci, including penicillin (blaZ)、tetracycline (tetK、tetL、tetM)、erythromycin (ermA、ermB、ermC、msrA/B、ermT)、gentamicin (aacA-aphD)、fusidic acid (fusB、fusC) and trimethoprim/sulfamethoxazole (dfrA、dfrG). Among CoNS species contributing main composition of staphylococcal community on RTE food, S. epidermidis and S. warneri exhibited higher rate of antibiotic resistance and carried various resistance gene compared to other species. Compared to other categories, higher contamination rate of staphylococci carrying resistance genes (including mecA) was found in fresh-cut fruits/vegetables, suggesting fresh-cut fruits/vegetables had higher potential in transmitting antibiotic resistance. Owing to S. cohnii displaying highly virulent in clinical and frequently carrying SCCmec which could not found in S. aureus, long-read sequencing was applied to discover SCCmec structures in S. cohnii isolated from rice cake (strain RC35) and ice desserts (strain I-104 and I-112), and three novel SCCmec composites were found. A composite SCCmec-SCC structure in strain RC35 was composed of an entire SCCmec TUS33 previously found in S. saprophyticus strain TSU33 isolated from human urinary tract infection, the SCCWC28 in S. cohnii WC28 isolated from wound specimens, and a series of ORFs including staphyloxanthin crtOPQMN operon common in some staphylococcal species. The structure in strain I-104 included an SCCI-104-up carrying ccrA1B3 complex, both which and is upstream 8 kb region displayed high identity to part of SCCAI16 found in S. pseudintermedius isolated from dog, and a composite SCCmec-SCC structure found in strain RC35. The strain I-112 harbored an SCCmec (an SCCmecTSU33 and the 3’ portion of SCCI-104-up), followed by a large SCC containing ccrA1B7 complex, the genes for virulence and resistance to antibiotics and environmental stimuli, and the tail region shared by SCCRC35. The region surrounding the class A mec complex was conserved in the above strains. Moreover, the tail regions of SCCmec-SCC composites in 3 MR-S. cohnii contained crtOPQMN operon, which was also found in S. cohnii isolated from clinical settings and healthy people’s hands and MR-S. saprophyticus isolates from ready-to-eat foods. The colony appearance of S. cohnii containing crtOPQMN operon displayed significant yellow. Absorbance at 465 nm was significantly higher in pigment extracted from S. cohnii carrying crtOPQMN operon than that from S. cohnii without crtOPQMN operon. Compared to S. cohnii ATCC49330 without crtOPQMN operon, S. cohnii RC35 displayed notably more resistant under treatment of 1.5% hydrogen peroxide, suggesting crtPOQMN operon could be functional. This is the first description of novel SCCmec composites in food-isolated S. cohnii, in which the copresence of SCCmecTUS33 and SCCWC28 and novel SCC structure was found. This study first identified crtOPQMN operon on SCC structure, suggesting crtOPQMN operon could transmit among some staphylococci and provide the ability to defense host innate immune system.
|Appears in Collections:||醫學檢驗暨生物技術學系|
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