劉教,劉暢,陳進(jìn),王勉之,熊文廣,曾振靈
多重耐藥大腸桿菌中前噬菌體的分布特征及誘導(dǎo)分離
劉教,劉暢,陳進(jìn),王勉之,熊文廣,曾振靈
華南農(nóng)業(yè)大學(xué)/廣東省獸藥研制與安全評價重點實驗室/國家獸醫(yī)微生物耐藥性風(fēng)險評估實驗室,廣州 510642
【】通過調(diào)查前噬菌體在多重耐藥大腸桿菌中的分布特征、誘導(dǎo)分離以及前噬菌體中耐藥基因與毒力基因的流行狀況,為研究前噬菌體介導(dǎo)耐藥基因在細(xì)菌的傳播提供科學(xué)依據(jù)。挑選前期保存的2018—2019年廣東省分離的131株禽源多重耐藥大腸桿菌進(jìn)行核酸提取及全基因組測序,將二代測序的結(jié)果組裝拼接成全基因組序列,上傳至噬菌體PHASTER網(wǎng)絡(luò)數(shù)據(jù)庫與數(shù)據(jù)庫中已有的噬菌體基因組序列進(jìn)行比對分析。利用CGE數(shù)據(jù)庫比對耐藥基因與毒力基因,從而獲得在前噬菌體上耐藥基因與毒力基因的分布情況。溫和性噬菌體由絲裂霉素C誘導(dǎo)并使用雙層平板法分離純化。131株大腸桿菌藥物敏感性試驗的結(jié)果顯示,氨芐西林、四環(huán)素、氟苯尼考、復(fù)方新諾明的耐藥率均高達(dá)90%以上,其次是頭孢類抗生素以及慶大霉素、環(huán)丙沙星、美羅培南和黏菌素均在50%左右,替加環(huán)素的耐藥率達(dá)到了0.2%,所有菌株都呈現(xiàn)出多重耐藥的現(xiàn)象,均為多重耐藥大腸桿菌。131株多重耐藥大腸桿菌中共檢出736個前噬菌體片段,其中包含329個完整型前噬菌體,其與40個已知數(shù)據(jù)庫噬菌體物種以不同百分比匹配上;可疑型噬菌體有66個,其與20個已知數(shù)據(jù)庫噬菌體物種以不同百分比匹配上;不完整型噬菌體有341個,其與52個已知數(shù)據(jù)庫噬菌體物種以不同百分比匹配上,完整型前噬菌體的基因序列顯示出與已知的噬菌體物種的序列相似性最高,平均為58.53%;131株大腸桿菌中平均前噬菌體數(shù)量為5.6個,平均總含量為152.4 kb。前噬菌體基因組占其宿主基因組的比例分布在0.58%—5.87%,以3.0%為主。前噬菌體基因組長度范圍在2.8—107.9 kb,其中13.0 kb的前噬菌體出現(xiàn)的頻次最高,占所有前噬菌體的9.1%。CGE比對結(jié)果表明,131株多重耐藥大腸桿菌的基因組共在18株前噬菌體序列檢測到耐藥基因(A)、(G)和,其中(A)、(G)和檢出數(shù)分別為16、1和1。71株多重耐藥大腸桿菌前噬菌體中攜帶有6種不同的毒力基因,其中存在部分菌株攜帶2種或者3種毒力基因,有62株前噬菌體攜帶端粒酶RNA基因,16株前噬菌體攜帶血清存活率增加基因,外膜蛋白酶、黏附素基因、和ABC轉(zhuǎn)運蛋白基因分別在2、2、1和1株前噬菌體中檢出。(A)和分別是前噬菌體中最常見的耐藥基因和毒力基因。溫和性噬菌體誘導(dǎo)試驗結(jié)果顯示,前噬菌體的誘導(dǎo)成功率為84.0%,但出現(xiàn)噬菌斑的概率仍比較低。前噬菌體在多重耐藥大腸桿菌中分布廣泛且攜帶有多種耐藥基因和毒力基因,溫和性噬菌體誘導(dǎo)成功率高,具有攜帶耐藥基因及毒力基因水平傳播的風(fēng)險,需要加強和持續(xù)監(jiān)測。
前噬菌體;大腸桿菌;誘導(dǎo);分布特征;耐藥基因
【研究意義】噬菌體是感染細(xì)菌、真菌、藻類、放線菌或螺旋體等微生物的病毒的總稱,因部分能引起宿主菌的裂解,故稱為噬菌體。噬菌體可分為溶原性噬菌體和裂解性噬菌體[1]。溶原性噬菌體,也稱為溫和性噬菌體,由細(xì)菌中的前噬菌體誘導(dǎo)產(chǎn)生,主要是通過將自身基因組整合入細(xì)菌基因組的方式進(jìn)行傳代。這種帶有噬菌體基因組的細(xì)菌稱為溶原性細(xì)菌,而整合入細(xì)菌基因組的噬菌體稱為前噬菌體[2-3]。溫和性噬菌體對其細(xì)菌宿主有重要的生態(tài)和進(jìn)化影響,在面對復(fù)雜的環(huán)境中能夠與細(xì)菌進(jìn)行共進(jìn)化,同時,溫和性噬菌體會攜帶一定的耐藥基因,可導(dǎo)致細(xì)菌耐藥性的傳播[4-6]。研究前噬菌體在多重耐藥大腸桿菌的分布特性對于細(xì)菌耐藥性的防控具有重要意義,為深入研究噬菌體通過轉(zhuǎn)導(dǎo)方式介導(dǎo)耐藥基因水平基因轉(zhuǎn)移提供基礎(chǔ)科學(xué)依據(jù)?!厩叭搜芯窟M(jìn)展】耐藥基因水平轉(zhuǎn)移(horizontal gene transfer,HGT)是細(xì)菌獲得耐藥基因的重要方式之一,且細(xì)菌獲得外源性基因的主要方式有接合轉(zhuǎn)移、轉(zhuǎn)化及轉(zhuǎn)導(dǎo)[7-10]。前噬菌體在細(xì)菌的基因組上廣泛存在,例如在177株鮑曼不動桿菌中鑒定出1 156個前噬菌體,并攜帶耐藥基因包括 OXA-23和NDM-1,前噬菌體是細(xì)菌間耐藥基因水平轉(zhuǎn)移的重要載體[11-16]。然而,噬菌體介導(dǎo)耐藥基因的水平轉(zhuǎn)移起先被認(rèn)為發(fā)生的概率較低,對耐藥基因的傳播能力比較低,沒有引起足夠的重視,后逐漸有文章報道其介導(dǎo)的耐藥基因水平轉(zhuǎn)移能力不可忽視[17-25],并且已在不同種屬細(xì)菌中證實,例如四環(huán)素和鏈霉素抗性葡萄球菌[26-27],四環(huán)素和氯霉素抗性沙門氏菌鼠傷寒沙門氏菌DT104[5],四環(huán)素和慶大霉素抗性腸球菌[28]和耐紅霉素的艱難梭菌[16]。【本研究切入點】目前國內(nèi)外有關(guān)噬菌體的研究大多針對于噬菌體通過轉(zhuǎn)導(dǎo)方式介導(dǎo)耐藥基因水平轉(zhuǎn)移的機制及噬菌體治療和展示方向,對于多重耐藥大腸桿菌中前噬菌體的流行狀況等相關(guān)的報道還比較少。而前噬菌體上耐藥基因和毒力基因的存在對噬菌體和細(xì)菌的共同進(jìn)化機制有重大影響[29-30]?!緮M解決關(guān)鍵問題】本文以多重耐藥大腸桿菌中前噬菌體為研究對象,調(diào)查前噬菌體在多重耐藥大腸桿菌中的分布狀況,以及前噬菌體中耐藥基因與毒力基因在的流行情況,同時利用絲裂霉素C誘導(dǎo)分離溫和性噬菌體,為研究前噬菌體介導(dǎo)耐藥基因在細(xì)菌的傳播提供科學(xué)依據(jù)。
LB瓊脂培養(yǎng)基(Luria-Bertani Agar),LB肉湯(Luria-Bertani Broth)均購于山東海博生物有限公司;麥康凱瓊脂培養(yǎng)基、水解酪蛋白瓊脂(Mueller-Hinton Agar)購自廣東環(huán)凱微生物科技有限公司;Agar購于西格瑪奧德里奇(上海)貿(mào)易有限公司。
絲裂霉素C購自美國MCE公司;細(xì)菌全基因組試劑盒購于廣州碧堯德生物科技有限公司;氨芐西林、慶大霉素、替加環(huán)素、環(huán)丙沙星、美羅培南、黏菌素等12種抗生素均購自生工生物工程(上海)股份有限公司。
將甘油肉湯保存的廣東省2018—2019年禽源大腸桿菌菌株(來自廣東省獸藥研制與安全評價重點實驗室)劃線于麥康凱瓊脂培養(yǎng)基上,37℃恒溫培養(yǎng)箱培養(yǎng)18—24h。
使用瓊脂稀釋法測定細(xì)菌的最小抑菌濃度,選用12種針對革蘭氏陰性菌的抗生素進(jìn)行藥物敏感性的測定。根據(jù)美國臨床實驗室標(biāo)準(zhǔn)化委員會(CLSI)規(guī)定的細(xì)菌藥敏試驗標(biāo)準(zhǔn),以MIC值位于其規(guī)定的敏感、中介、耐藥的折點值范圍作為判斷結(jié)果。
純化已復(fù)活的菌株,涂于LB瓊脂培養(yǎng)基上,過夜培養(yǎng),備用。按照全基因組測序試劑盒說明書進(jìn)行提取,將菌液轉(zhuǎn)移至1.5 mL離心管中,離心棄上清,加入適量Buffer STE, Lysozyme和RNase Solution,渦旋混勻,37℃水浴40 min,加250 μL Buffer DL和10 μL Proteinase K至細(xì)胞重懸液中。70℃消化10 min,加250 μL無水乙醇,渦旋后轉(zhuǎn)移至吸附柱,10 000×離心1 min,棄去濾液加500 μL Buffer GW1, 10 000×離心1 min,棄濾液加入650 μL Buffer GW2,10 000×離心1 min,倒棄流出液,10 000×空離2 min,柱子裝入新1.5 mL離心管,加入50 μL預(yù)熱至70℃ddH2O,放置3 min,離心棄柱子,-20℃暫放,將樣品送至北京諾禾致源生物有限公司進(jìn)行測序。
使用CLC genomics workbench version 10.0.1進(jìn)行組裝,通過在線網(wǎng)站(https://cge.cbs.dtu.dk/)進(jìn)行多基因座序列分型(Multi-locus sequence typing,MLST)。同時,使用PHASTER工具(http://phaster.ca/)進(jìn)行細(xì)菌基因組中前噬菌體的鑒定[31],通過上傳基因組與已知的NCBI病毒基因組數(shù)據(jù)庫,轉(zhuǎn)運RNA和噬菌體在細(xì)菌基因組上的附著位點進(jìn)行匹配,然后對檢測出來的前噬菌體進(jìn)行評分,依據(jù)分?jǐn)?shù)(總分150)分為完整型噬菌體(Intact,>90)可疑型噬菌體(Questionable,70—90之間)和不完整型噬菌體(Incomplete,<70)?;谇懊骅b定的前噬菌體,計算其基因組長度,分析前噬菌體在細(xì)菌基因組上的分布特性,并且計算前噬菌體在細(xì)菌基因組中的占比情況。同時,將提取到的前噬菌體序列與已知數(shù)據(jù)庫噬菌體物種進(jìn)行比對,對它們的相似度進(jìn)行比較分析。
將提取的前噬菌體序列上傳至基因組流行病學(xué)中心主頁網(wǎng)站(https://cge.cbs.dtu.dk//services)進(jìn)行比對,檢測前噬菌體序列上的耐藥基因和毒力基因,并對檢測到的耐藥基因和毒力基因的分布進(jìn)行分析。
復(fù)活前面所用菌株,劃線于麥康凱瓊脂平板上,于37℃恒溫培養(yǎng)箱過夜培養(yǎng),挑取單菌落接種到已高壓的2 mL LB肉湯試管中,37℃,180 r/min搖床培養(yǎng)4 h,用移液槍抽取50 μL菌液至新的含5 mL LB肉湯試管中,搖床培養(yǎng)至OD600為0.3左右,加入絲裂霉素C,使其終濃度為1 μg·mL-1,并設(shè)置對照組,繼續(xù)同樣條件搖床培養(yǎng)12 h,測定其OD600值,若OD值下降明顯,則認(rèn)為其誘導(dǎo)成功。
劃線培養(yǎng)大腸桿菌工程菌C600,接種于5 mL LB肉湯試管中,180 r/min,37℃搖床培養(yǎng),備用;倒薄層的LB瓊脂板備用;將5 mL半固體與100 μL的C600菌液混合加入薄層LB瓊脂板中,搖晃混勻,待瓊脂凝固后,取誘導(dǎo)液滴至平板中,37℃恒溫培養(yǎng)箱培養(yǎng)過夜,次日,觀察是否有噬菌斑的產(chǎn)生。若產(chǎn)生噬菌斑,則將誘導(dǎo)液稀釋并與菌液1﹕1混合鋪雙層平板,過夜培養(yǎng),若有單個噬菌斑,則用槍頭扣取噬菌斑,充分渦旋,使噬菌體釋放,6 000 r/min,4℃離心5 min,用注射器吸取上清液,過0.22 μm的濾膜,以除去細(xì)菌。用雙層平板法重復(fù)三次完成噬菌斑的純化[32-33]。
藥敏試驗結(jié)果表明,131株大腸桿菌對試驗所用的12種抗生素(氨芐西林、頭孢噻肟、頭孢他啶、美羅培南、慶大霉素、阿米卡星、四環(huán)素、替加環(huán)素、氟苯尼考、黏菌素、環(huán)丙沙星、復(fù)方新諾明)的耐藥率分別是96.06%、78.74%、57.48%、44.88%、62.20%、15.75%、93.70%、0.2%、96.06%、53.54%、85.04%和93.70%,呈現(xiàn)出多重耐藥現(xiàn)象(圖1)。
131株多重耐藥大腸桿菌中三類前噬菌體(完整型,可疑型和不完整型)基因組分析結(jié)果(圖2)顯示,前噬菌體占其宿主基因組的比例為0.58%—5.87%,大部分在3%左右,平均每株多重耐藥大腸桿菌中前噬菌體數(shù)量為5.6個,其平均總含量為152.4 kb。統(tǒng)計736個前噬菌體的不同基因組長度的概率分布結(jié)果發(fā)現(xiàn)前噬菌體基因組大小顯示出一個清晰的峰值,峰值噬菌體大小為13 kb,占所有736株前噬菌體的9.1%,前噬菌體最大長度可達(dá)到107.9 kb,占比為0.14%,最小的基因長度為0.28 kb,占總前噬菌體的0.27%(圖3)。
AMP,氨芐西林;CTX,頭孢噻肟;CAZ,頭孢他啶;MEM,美羅培南;GEN,慶大霉素;AMI,阿米卡星;TET,四環(huán)素;TIG,替加環(huán)素;FLR,氟苯尼考;CL,黏菌素;CIP,環(huán)丙沙星;SXT,復(fù)方新諾明
131株多重耐藥大腸桿菌的基因組中累計檢測到736個潛在的前噬菌體序列片段,其中包含了329個完整型前噬菌體(Intact prophage),66個可疑型前噬菌體(Questionable prophage)和341個不完整型前噬菌體(Incomplete prophage);其分別與40、22和52個已知數(shù)據(jù)庫噬菌體物種以8.33%—95%不同百分比匹配上。
由圖4可知,完整型前噬菌體的基因序列顯示出與已知的噬菌體物種的序列相似性最高,平均為58.53%,剩下的兩種前噬菌體(可疑型噬菌體和不完整噬菌體)與已知的噬菌體序列相似度比較低,其平均相似度分別是46.58%和37.83%,其原因可能是完整的前噬菌體的序列更長,或者序列中匹配源ORF數(shù)量多導(dǎo)致與不完整噬菌體序列相比有更高的物種序列相似度。另外,不同的ST型都與特定的已知噬菌體匹配,其匹配的與已知的噬菌體相似率也極其相似。mEp460樣噬菌體、Fels_2樣噬菌體和SfⅡ樣噬菌體在完整前噬菌體序列上分布最廣且相似率最高(圖5)。mEp460樣噬菌體在ST162、ST167、ST48、ST5229和ST6725上均有分布;SfⅤ樣噬菌體主要是分布在ST10中;Fels_2樣噬菌體主要分布在ST162、ST48、ST746中;SfⅡ樣噬菌體則主要是分布在ST10、ST746、ST23中;僅在ST10中檢測到P1樣噬菌體、HK75樣噬菌體,僅在ST7085中檢測到Psp3樣噬菌體(圖5)。
圖2 前噬菌體占其宿主基因組的長度貢獻(xiàn)百分比
圖3 前噬菌體不同基因組長度概率分布圖
耐藥基因與毒力基因檢出結(jié)果(圖6)表明,131株多重耐藥大腸桿菌的基因組共在18株前噬菌體序列檢測到耐藥基因(A)、(G)和,其中(A)、(G)和檢出數(shù)分別為16、1和1,其中16個耐藥基因在完整型前噬菌體上分布,2個在不完整型前噬菌體上分布,可疑型前噬菌體上未找到耐藥基因。(A)基因可作為多藥耐藥的轉(zhuǎn)運體,攜帶(A)基因的菌株對氯霉素、四環(huán)素和紅霉素等抗生素均有耐藥性[34]。(G)屬于林可霉素類耐藥基因在不完整前噬菌體上發(fā)現(xiàn),該前噬菌體與PHAGE__SJ46相似度最高;(黏菌素耐藥基因)位于ST10大腸桿菌上的完整前噬菌體上,檢測到該前噬菌體與PHAGE__P1最為相似。此外,ST10與ST48型多重耐藥大腸桿菌中前噬菌體攜帶耐藥基因概率較高。結(jié)果表明在大腸桿菌的前噬菌體上攜帶耐藥基因的概率比較低,且具有特異性。
圖4 前噬菌體與已知數(shù)據(jù)庫中噬菌體基因組的相似百分比
圖5 前噬菌體上耐藥基因分布圖
如圖6所示,毒力基因的檢測發(fā)現(xiàn)在71株前噬菌體攜帶有6種不同的毒力基因,其中有些菌株攜帶2種或者3種毒力基因。完整型前噬菌體上分布有17個毒力基因,可疑型前噬菌體上分布6個毒力基因,不完整型前噬菌體上則分布有61個毒力基因。62株前噬菌體攜帶端粒酶RNA基因,攜帶的多重耐藥大腸桿菌中ST167型占比較大,但總體來說這些前噬菌體對應(yīng)的多重耐藥大腸桿菌ST型分布無明顯特征,這些前噬菌體大多為不完整型前噬菌體,這些前噬菌體均與數(shù)據(jù)庫中PHAGE__ vB_AbaM_ME3相似;16株前噬菌體攜帶血清存活率增加基因帶的前噬菌體大多為完整型前噬菌體,這些前噬菌體與PHAGE__BP_ 4795相似度高且基本都分布于ST167型多重耐藥大腸桿菌中;外膜蛋白酶、黏附素基因、和ABC轉(zhuǎn)運蛋白基因分別在2、2、1和1株前噬菌體中檢出,可通過加工或者降解多種宿主蛋白質(zhì)使宿主發(fā)病,分布于ST1771型和ST5946型多重耐藥大腸桿菌中可疑型前噬菌體上與前噬菌體PHAGE__DE3相似;黏附素是生物定植的基礎(chǔ),同時也是細(xì)菌致病感染的首要條件,分布于ST29型多重耐藥大腸桿菌中不完整型前噬菌體上,與噬菌體PHAGE__vB_AbaM_ME3相似;分布于ST46型多重耐藥大腸桿菌中可疑型前噬菌體上,與噬菌體PHAGE__vB_AbaM_ME3相似;分布于ST46型多重耐藥大腸桿菌中可疑型前噬菌體上并與噬菌體PHAGE__vB_AbaM_ME3呈現(xiàn)較高相似度。
圖6 毒力基因在前噬菌體上分布圖
通過絲裂霉素C誘導(dǎo)大腸桿菌,結(jié)果顯示與對照組比較得到OD600值下降明顯(OD600值下降0.3以上認(rèn)為下降明顯)的有110株,占比84.0%;通過雙層平板法點板得到噬菌斑的有38株,占比29.0%;分離純化得到27株,占比20.6%;有11株在純化過程中未能出現(xiàn)噬菌斑。
目前,已有文獻(xiàn)報道噬菌體在細(xì)菌間水平基因的轉(zhuǎn)移中發(fā)揮著重要作用[35-36],使用宏基因組學(xué)分析的方法對環(huán)境水流、土壤、人和動物腸道基因組中噬菌體分布的豐度和多樣性以及宏噬菌體基因組中ARGs的特性和對其傳播的影響進(jìn)行研究,發(fā)現(xiàn)噬菌體對于耐藥基因的獲得和傳播有重要作用[37-39],但是對于細(xì)菌基因組中前噬菌體和類前噬菌體的分布、多樣性、豐度及其攜帶耐藥基因的特性和對其傳播的作用研究非常少。因此本研究對131株大腸桿菌基因組中前噬菌體序列進(jìn)行了鑒定,針對大腸桿菌中前噬菌體的分布流行特征及其攜帶ARGs的狀況進(jìn)行分析闡述。
本研究中,131株大腸桿菌前噬菌體有736個,前噬菌體基因組占其宿主菌的基因組的比例在0.58—5.87之間,在宿主基因組中檢測到的前噬菌體與已知噬菌體物種數(shù)據(jù)庫中71種噬菌體以不同的相似百分比匹配上,結(jié)果表明前噬菌體在大腸桿菌中分布廣泛且具有多樣性和高豐度,這與Schmieger和Casjens等[40-41]研究結(jié)果相同。該131株大腸桿菌攜帶大量的ARGs,但檢測到的前噬菌體序列上攜帶的ARGs僅有3種,且概率極小,本研究中的噬菌體基因組上攜帶耐藥基因的數(shù)量明顯低于相關(guān)報道中的環(huán)境微生物樣品中的宏噬菌體中攜帶的耐藥基因的數(shù)量。前噬菌體攜帶ARGs的概率很低,這可能與細(xì)菌的繁殖傳代或者與噬菌體共進(jìn)化過程有關(guān),在不同環(huán)境一些特殊的條件下攜帶耐藥基因的細(xì)菌與前噬菌體相互作用的概率大大提高,前噬菌體上攜帶ARGs的能力提高,導(dǎo)致在噬菌體上檢出相關(guān)的耐藥基因,如先前報道的前噬菌體上攜帶黏菌素耐藥基因3.1[42],以及23和NDM-1[43]。然而,目前關(guān)于前噬菌體介導(dǎo)耐藥基因傳播的影響因素目前還不明確,相關(guān)的研究有待今后更多的試驗來探究。噬菌體編碼的毒力因子可以通過細(xì)菌分泌到細(xì)胞外發(fā)揮作用或者在溶原期通過細(xì)胞裂解擴散到細(xì)胞外起作用[44], 本研究中前噬菌體上攜帶的毒力基因主要是C和,131株大腸桿菌中有71株大腸桿菌上的前噬菌體攜帶毒力基因,表明有增強細(xì)菌毒力的風(fēng)險。
對于檢測到的736個前噬菌體序列經(jīng)過與NCBI數(shù)據(jù)庫中病毒基因組的比較發(fā)現(xiàn),前噬菌體評分(完整型前噬菌體>可疑型前噬菌體>不完整型前噬菌體)越高,其前噬菌體序列與病毒數(shù)據(jù)庫中基因組序列相似度就越高,但是相似度并沒有達(dá)到100%完全相似,其原因可能是在細(xì)菌和噬菌體共同繁殖進(jìn)化中,宿主細(xì)菌與噬菌體相互適應(yīng)各自調(diào)整的結(jié)果[45];也可能是由于數(shù)據(jù)庫中完整的噬菌體基因組序列不足,信息不完整所致。對于溫和性噬菌體的誘導(dǎo)和分離,本研究在溫和性噬菌體的分離中采用的是大腸桿菌工程菌C600作為宿主菌[46],可以發(fā)現(xiàn)OD600下降明顯但是分離出單個噬菌斑的概率不高,分析其原因可能是由于誘導(dǎo)過程的不確定性,細(xì)菌誘導(dǎo)后點板出斑再次純化噬菌斑消失原因可能是細(xì)菌發(fā)生溶原轉(zhuǎn)化,即在噬菌體誘導(dǎo)后細(xì)菌再次溶原化[47],可能需要在特定的宿主菌才能出現(xiàn)噬菌斑[48]。
131株大腸桿菌中前噬菌體達(dá)736個(包括完整和不完整前噬菌體),平均前噬菌體數(shù)量為5.6個/株,平均總含量為152.4 kb,占宿主基因組比例大部分在3%。前噬菌體序列中鑒定出3種耐藥基因和6種毒力基因,其中包含重要黏菌素耐藥基因。前噬菌體在大腸桿菌的基因組中的分布具有多樣性且豐度較高,相同ST型下的前噬菌體序列相似度較高。溫和性噬菌體的誘導(dǎo)率很高,達(dá)到了84.0%。
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Distribution Characteristics of Prophage in Multidrug Resistantas well as Its Induction and Isolation
LIU Jiao, LIU Chang, CHEN Jin, WANG MianZhi, XIONG WenGuang, ZENG ZhenLing
South China Agriculture University/Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation/National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, Guangzhou 510642
【】This study investigated the distribution characteristics of prophage in multi-drug resistant, induction and isolation, as well as the prevalence of drug resistance and virulence genes in prophage, so as to provide a scientific basis for the study of prophage-mediated resistance genes in the spread of bacteria. 【】 131 multi-drug resistantisolating from poultry origin in Guangdong Province from 2018 to 2019 were selected in the laboratory for nucleic acid extraction and whole-genome sequencing. The results of second-generation sequencing were assembled and spliced into a whole-genome sequence and uploaded to the phage. The PHASTER network database was compared and analyzed with the existing phage genome sequences in the database. Drug resistance genes and virulence genes were compared on the CGE database, and then the distribution of drug resistance genes and virulence genes on the prophage were obtained. The mild phage was induced by mitomycin C, separated and purified by using the double-layer plate method. 【】 The results of the drug sensitivity test of 131 strains ofshowed that the drug resistance rates of ampicillin, tetracycline, florfenicol and compound trimethoprim were all more than 90%, followed by cephalosporin antibiotics, gentamicin, ciprofloxacin, meropenem and colistin with all around 50%, and the resistance rate of tigecycline reached 0.2%. All strains showed multi-drug resistance, and they were all multi-drug resistant. A total of 736 prophage fragments were detected in 131 strains of multi-drug resistant, including 329 complete prophage, 66 suspicious phages and 341 incomplete phage, which matched with 40, 20 and 52 known database phage species in different percentages, respectively. The gene sequence of the complete prophage showed that it matched the known phage species better, and the sequence similarity was the highest, with an average of 58.53%. The average number of prophages in 131 strains ofwas 5.6, and the average total content was 152.4 kb. Prophage genome accounted for 0.58% to 5.87% of its host genome, with 3.0% being the dominant. The length of the prophage genome ranged from 2.8 to 107.9 kb, and the 13.0 kb prophage had the highest frequency, accounting for 9.1% of all prophages. CGE comparison results showed that the genomes of 131 strains of multi-drug-resistantdetected resistance genes(A),(G) and(A),(G) and mcr-1 were 16, 1, and 1, respectively. 71 strains of multi-drug resistantprophage carried 6 different virulence genes, and some strains carried 2 or 3 virulence genes. There were 62 prophages carrying the telomerase RNA gene, 16 prophages carrying the serum survival increasing gene, and the outer membrane protease, among which the adhesin gene, thegene and the ABC transporter genewere at 2, 2, 1, and 1, respectively. Mcr-a gene were detected in prophage of 1 strain multi-drug resistant. The(A) gene andgene were the most common resistance genes and virulence genes in prophage, respectively. The results of mild phage induction experiments showed that the success rate of prophage induction was 84.0%, but the probability of plaque appearance was still relatively low. 【】Prophages were widely distributed in multi-drug resistantand carried a variety of resistance genes and virulence genes. Mild phages had a high induction rate, and have the risk of horizontal transmission of resistance genes and virulence genes, and need to be strengthened and sustained monitor.
prophage;; induction; distribution characteristics; resistance genes
10.3864/j.issn.0578-1752.2022.07.017
2021-02-06;
2021-09-30
國家自然科學(xué)基金面上項目(31872524)、廣東省普通高校省級重大科研項目(2017KZDXM006)
劉教,E-mail:1766612575@qq.com。通信作者曾振靈,E-mail:zlzeng@scau.edu.cn
(責(zé)任編輯 林鑒非)