葛玉梅，胡慶豐，朱永澤，周永列，呂火烊

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龜頭包皮炎患者淋病奈瑟菌分離與鑒定及其耐藥機(jī)制研究

葛玉梅，胡慶豐，朱永澤，周永列，呂火烊

目的 分離鑒定龜頭化膿性包皮炎病原菌，檢測(cè)分離菌株耐藥性并了解其耐藥機(jī)制。方法 采取尿道分泌物及龜頭膿包液標(biāo)本，革蘭染色鏡檢并采用Mycoplasma IST 2試劑盒檢測(cè)支原體。上述標(biāo)本接種哥倫比亞血平板、淋病奈瑟菌選擇平板、酵母菌鑒定平板進(jìn)行細(xì)菌分離培養(yǎng)，獲得的菌落用VITEK 2-compact全自動(dòng)細(xì)菌檢測(cè)分析系統(tǒng)進(jìn)行鑒定，另采用PCR檢測(cè)上述標(biāo)本及菌落的淋病奈瑟菌16S rRNA基因。采用K-B法檢測(cè)分離菌株對(duì)5種常用抗生素的敏感性，采用β-內(nèi)酰胺酶和超廣譜β-內(nèi)酰胺酶確證試驗(yàn)了解該菌株產(chǎn)酶情況，PCR檢測(cè)該菌株耐藥相關(guān)tetM、TEM、mefA和ermF基因。 結(jié)果 各標(biāo)本支原體檢測(cè)結(jié)果均為陰性。尿道分泌物標(biāo)本分離培養(yǎng)結(jié)果均為陰性，龜頭膿包液血平板和選擇平板培養(yǎng)結(jié)果為陽(yáng)性。VITEK 2-compact系統(tǒng)和16S rRNA-PCR檢測(cè)結(jié)果顯示分離菌株為淋病奈瑟菌。該菌株產(chǎn)β內(nèi)酰胺酶且對(duì)青霉素G、環(huán)丙沙星、四環(huán)素耐藥，其基因組攜帶tetM、TEM、mefA和ermF基因。結(jié)論 淋病奈瑟菌可引起龜頭化膿性包皮炎，該淋病奈瑟菌菌株多重耐藥并與其攜帶的耐藥基因密切相關(guān)。

龜頭包皮炎；淋病奈瑟菌；分離鑒定；耐藥性；耐藥基因

淋病奈瑟菌(Neisseriagonorrhoeae)俗稱淋球菌，是人類(lèi)常見(jiàn)性傳播疾病淋病(gonorrhea)的病原體[1]。淋病是《中華人民共和國(guó)傳染病防治法》中重點(diǎn)防治的乙類(lèi)傳染病，近年來(lái)其發(fā)病率一直占據(jù)我國(guó)性傳播疾病第2位[2]。早已肯定，淋病奈瑟菌引起人泌尿生殖道急性或慢性化膿性感染，新生兒經(jīng)產(chǎn)道感染后引起淋球菌性結(jié)膜炎[3]。然而，我們從1例龜頭包皮炎患者龜頭膿包液中分離出淋病奈瑟菌，該菌株同時(shí)攜帶四環(huán)素耐藥相關(guān)tetM基因、β-內(nèi)酰胺類(lèi)抗生素耐藥相關(guān)TEM基因、紅霉素耐藥相關(guān)外排基因mefA和甲基化酶基因ermF基因，表現(xiàn)為對(duì)多種抗生素很強(qiáng)的耐藥性。

1 材料和方法

1.1 菌株來(lái)源及分離培養(yǎng) 龜頭包皮化膿性感染患者男性，33歲，無(wú)尿道刺激癥狀，2016年2月27日就診杭州某三甲醫(yī)院泌尿外科。無(wú)菌棉棒采取尿道分泌物及龜頭包皮膿包液標(biāo)本，革蘭染色鏡檢并采用Mycoplasma IST 2試劑盒(梅里埃診斷產(chǎn)品有限公司)檢測(cè)支原體，然后將標(biāo)本分別涂布淋病奈瑟菌巧克力選擇平板、哥倫比亞血平板、酵母菌鑒定平板，35 ℃、5% CO2培養(yǎng)24 h，觀察其生長(zhǎng)情況及菌落形態(tài)。

1.2 菌株鑒定 挑取菌落革蘭染色鏡檢后用VITEK 2-compact型法國(guó)梅里埃VITEK 2-compact全自動(dòng)細(xì)菌檢測(cè)分析系統(tǒng)及其配套的API-NH細(xì)菌鑒定卡進(jìn)行鑒定[4]。此外，采用細(xì)菌基因組DNA提取試劑盒(Axygen)提取尿道口分泌物或龜頭膿包液、淋病奈瑟菌分離株DNA，紫外分光光度法檢測(cè)其純度和濃度。采用淋病奈瑟菌16S rRNA基因通用引物[5]、淋球菌核酸熒光PCR試劑盒(上海復(fù)星長(zhǎng)征醫(yī)學(xué)科學(xué)有限公司)檢測(cè)上述DNA標(biāo)本中淋病奈瑟菌16S rRNA基因片段。引物由上海Invitrogen公司合成，上游引物序列：5′-GCT ACG CAT ACC CGC GTT GC-3′， 下游引物序列：5′-CGA AGA CCT TCG AGC AGA CA-3′。PCR參數(shù)：94 ℃ 5 min；94 ℃ 30 s、55 ℃ 30 s、72 ℃ 1 min，35個(gè)循環(huán)；72 ℃ 10 min。采用溴乙錠預(yù)染色1.5%瓊脂糖凝膠電泳及Bio-Rad成像系統(tǒng)觀察260 bp目的擴(kuò)增片段。

1.3 藥物敏感試驗(yàn) 參照國(guó)內(nèi)臨床通用的美國(guó)臨床實(shí)驗(yàn)室標(biāo)準(zhǔn)化協(xié)會(huì)(CLSI)介紹的方法及判斷標(biāo)準(zhǔn)[5]，采用K-B法檢測(cè)分離菌株對(duì)6種常見(jiàn)抗生素的敏感性。實(shí)驗(yàn)中采用淋病奈瑟菌ATCC49981株為質(zhì)控菌株。

1.4 β-內(nèi)酰胺酶和超廣譜β-內(nèi)酰胺酶確證試驗(yàn) 分別采用顯色頭孢菌素法和雙紙片法進(jìn)行β-內(nèi)酰胺酶和超廣譜β-內(nèi)酰胺酶(ESBLs)確證試驗(yàn)[6-7]。淋病奈瑟菌分離株涂布于紙片頭孢硝噻吩紙片(BioMérieux)上，即刻觀察紙片顏色變化，若出現(xiàn)紅色表明該菌產(chǎn)β-內(nèi)酰胺酶。淋病奈瑟菌分離株涂布于巧克力瓊脂(Oxoid)平板上，然后貼上頭孢他啶、頭孢他啶/克拉維酸、頭孢噻肟、頭孢噻肟/克拉維酸藥物紙片(Oxoid)，37 ℃孵育24 h后觀察結(jié)果。若頭孢他啶/克拉維酸或頭孢噻肟/克拉維酸紙片抑菌環(huán)直徑較頭孢他啶或頭孢噻肟紙片抑菌環(huán)直徑≥5 cm，表明該菌株產(chǎn)ESBLs。實(shí)驗(yàn)中以不產(chǎn)ESBLs大腸埃希菌ATCC25922株為陰性對(duì)照、產(chǎn)ESBLs肺炎克雷伯菌ATCC700603株為陽(yáng)性對(duì)照。

1.5 耐藥基因檢測(cè) 按上法提取淋病奈瑟菌分離株DNA，紫外分光光度法檢測(cè)其純度和濃度[4]。采用文獻(xiàn)報(bào)道的引物以及Ex-Taq PCR試劑盒(TaKaRa)檢測(cè)上述DNA標(biāo)本中TEM、tetM、ermF、mefA耐藥基因片段，反應(yīng)參數(shù)同上[8-10]。引物并由上海Invitrogen公司合成，其序列(見(jiàn)表1)。

表1 淋病奈瑟菌耐藥基因PCR引物
Tab.1 Primers for amplification of drug resistance genes ofN.gonorrhoeae

耐藥基因drugresistancegenes引物序列(5'-3')primersequences產(chǎn)物長(zhǎng)度/bplengthtetMF:GTGGACGAACTTTAC-CGAA501R:GCTTTGTATCTCCAA-GAACACTEM內(nèi)顯F:AGGAAGAGTATGAT-TCAACA535R:CTCGTCGTTTGGTAT-GGCmefAF:ACTATCATTAATCAC-TAGTGC346R:TTCTTCTGGTACTAA-AAGTGGermFF:GGATACGGTTTAGA-TATTGGG295R:TTGAAGGACAATGG-AACCTCC

F and R: upstream and downstream primers respectively

2 結(jié) 果

2.1 臨床標(biāo)本檢查結(jié)果 龜頭膿包液中可見(jiàn)革蘭陰性雙球菌，部分雙球菌位于多形核白細(xì)胞內(nèi)(圖1A)，尿道分泌物未見(jiàn)細(xì)菌，但兩種標(biāo)本支原體檢測(cè)結(jié)果均為陰性。尿道分泌物各種平板培養(yǎng)結(jié)果均為陰性，龜頭膿包液酵母菌鑒定平板培養(yǎng)結(jié)果陰性，但哥倫比亞血平板和淋病奈瑟菌選擇平板培養(yǎng)結(jié)果陽(yáng)性，其菌落細(xì)小、凸起、光滑、圓形、灰白色，革蘭染色鏡檢為革蘭陰性雙球菌(圖1B)。淋病奈瑟菌16S rRNA基因PCR結(jié)果顯示，僅龜頭膿包液以及淋病奈瑟菌選擇平板上菌落檢出淋病奈瑟菌16S rRNA基因片段(圖2)。

圖1 龜頭膿包液(A)及選擇平板上菌落(B)革蘭染色鏡檢結(jié)果(100×)Fig.1 Microscopic examination results of glans pustule (A) and colonies (B) on selective plate after Gram staining (100×)

M: DNA marker(TaKaRa); 1-3: PCR results for detecting urethral secretions,balanus pustule liquids and selective plates of N. gonorrhoeae 16S rRNA genes圖2 不同標(biāo)本淋病奈瑟菌16S核糖體rRNA基因PCR檢測(cè)結(jié)果Fig.2 PCR results for detecting different samples of N. gonorrhoeae 16S rRNA gene

2.2 分離菌株生化反應(yīng)鑒定結(jié)果 分離菌株經(jīng)VITEK 2-compact全自動(dòng)細(xì)菌檢測(cè)分析系統(tǒng)及其API-NH細(xì)菌鑒定卡鑒定為淋病奈瑟菌，鑒定值=99%，各生化反應(yīng)中營(yíng)養(yǎng)瓊脂35 ℃培養(yǎng)、硝酸鹽還原試驗(yàn)NO3(red)、NO2→N2以及麥芽糖、乳糖、蔗糖、果糖發(fā)酵試驗(yàn)結(jié)果均為陰性，但葡萄糖發(fā)酵試驗(yàn)結(jié)果陽(yáng)性。

2.3 藥敏試驗(yàn)結(jié)果 淋病奈瑟菌分離株對(duì)青霉素G、環(huán)丙沙星、四環(huán)素、紅霉素耐藥，但對(duì)頭孢西丁、頭孢噻肟敏感(表2)。

表2 淋病奈瑟菌分離株藥敏試驗(yàn)結(jié)果
Fig.2 Results of drug sensitive test of theN.gonorrhoeaeisolate

抗生素antibioticsKB值KBvalues折點(diǎn)范圍breakpoints結(jié)果判定results青霉素G(penicillinG)626-47耐藥drug-resistant環(huán)丙沙星(ciprofloxacin)627-41耐藥drug-resistant四環(huán)素(tetracycline)2430-38耐藥drug-resistant頭孢噻肟(cefotaxime)45>=31敏感drug-sensitive頭孢西丁(cefoxitin)3323-28敏感drug-sensitive

2.4 β-內(nèi)酰胺酶及ESBLs確證試驗(yàn)結(jié)果 淋病奈瑟菌分離株涂布的頭孢硝噻吩紙片由黃色轉(zhuǎn)變?yōu)榧t色，表明該菌株產(chǎn)β-內(nèi)酰胺酶。頭孢他啶對(duì)淋病奈瑟菌分離株抑菌環(huán)直徑18 mm、頭孢他啶/克拉維酸17 mm、頭孢噻肟32 mm、頭孢噻肟/克拉維酸35 mm。由于頭孢他啶/克拉維酸或頭孢噻肟/克拉維酸抑菌環(huán)直徑與頭孢他啶或頭孢噻肟抑菌環(huán)直徑之差小于5 mm，表明該菌株不產(chǎn)ESBLs。

2.5tetM、TEM、mefA、ermF基因PCR檢測(cè)結(jié)果 淋病奈瑟菌分離株四環(huán)素耐藥相關(guān)tetM基因、β-內(nèi)酰胺酶TEM基因、紅霉素耐藥相關(guān)mefA和ermF基因PCR結(jié)果均為陽(yáng)性(圖3)。

M: DNA marker(TaKaRa); 1: blank control; 2-5: PCR results of tetM,TEM,mefA and ermF genes of the N. gonorrhoeae isolate圖3 淋病奈瑟菌分離株tetM、TEM、mefA、ermF基因PCR檢測(cè)結(jié)果Fig.3 PCR results of tetM,TEM,mefA and ermF genes of the N. gonorrhoeae isolate

3 討 論

淋病是全球流行的人類(lèi)性傳播疾病，臨床上主要表現(xiàn)為泌尿道化膿性炎癥，該菌雖可上行感染引起男性附睪炎、前列腺炎或引起女性陰道炎、子宮炎[11-13]，但未有引起龜頭包皮炎的報(bào)道。因此，臨床上一般采集尿道分泌物檢測(cè)淋病奈瑟菌。本文中患者既無(wú)泌尿道化膿性炎癥的癥狀與體征，其尿道分泌物中也未分離出淋病奈瑟菌或檢出該菌16S rDNA基因片段，但在龜頭包皮膿包液中不僅分離出淋病奈瑟菌，也檢出了該菌16S rDNA基因，表明該患者為淋球菌龜頭包皮炎。

近年來(lái)淋病奈瑟菌臨床分離株對(duì)抗生素、尤其是對(duì)環(huán)丙沙星、青霉素、四環(huán)素等臨床一線抗菌藥物的耐藥性日趨增強(qiáng)[14]。了解淋病奈瑟菌臨床菌株的耐藥譜、耐藥基因型及其相互關(guān)系，不僅可指導(dǎo)臨床合理用藥，同時(shí)也有助于進(jìn)一步揭示耐藥表型與基因型關(guān)系并為開(kāi)發(fā)新的抗菌藥物提供依據(jù)。

青霉素類(lèi)抗生素能與本質(zhì)為細(xì)菌細(xì)胞壁肽聚糖合成相關(guān)內(nèi)肽酶和羧肽酶的青霉素結(jié)合蛋白(penicilin-binding proteins，PBPs)結(jié)合，導(dǎo)致酶分子變構(gòu)失活，細(xì)菌因細(xì)胞壁缺陷而裂解[2]。TEM基因產(chǎn)物β-內(nèi)酰胺酶可水解青霉素類(lèi)抗生素，故攜帶TEM基因細(xì)菌表現(xiàn)為對(duì)青霉素耐藥[15]。環(huán)丙沙星是大環(huán)內(nèi)酯類(lèi)抗生素。mef基因產(chǎn)物為細(xì)菌排出環(huán)丙沙星等大環(huán)內(nèi)酯類(lèi)抗生素的外排蛋白，erm基因產(chǎn)物為rRNA甲基化酶，該酶可使大環(huán)內(nèi)酯類(lèi)抗生素作用靶位核糖體50S亞基發(fā)生改變，降低大環(huán)內(nèi)酯類(lèi)藥物結(jié)合能力而導(dǎo)致耐藥[16]。四環(huán)素類(lèi)抗生素主要通過(guò)與細(xì)菌核糖體30S亞基結(jié)合，抑制氨基酰-tRNA與核糖體結(jié)合而阻斷細(xì)菌蛋白合成[17]。tetM基因產(chǎn)物為核糖體保護(hù)蛋白，可對(duì)抗四環(huán)素類(lèi)抗生素阻斷氨基酰-tRNA與核糖體結(jié)合的作用，從而使細(xì)菌產(chǎn)生對(duì)四環(huán)素類(lèi)抗生素的耐藥性[18]。我們的藥物敏感試驗(yàn)結(jié)果顯示，淋病奈瑟菌分離株對(duì)青霉素G、環(huán)丙沙星、四環(huán)素耐藥，但對(duì)頭孢噻肟和頭孢西丁敏感；β-內(nèi)酰胺酶和ESBLs確證試驗(yàn)結(jié)果顯示，淋病奈瑟菌分離株產(chǎn)β-內(nèi)酰胺酶，但不產(chǎn)ESBLs。我們的PCR檢測(cè)結(jié)果顯示，淋病奈瑟菌分離株具有TEM、tetM、mef和erm基因，與該菌株耐藥譜及其表型相符。

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Isolation and identification ofNeisseriagonorrhoeaestrain from a balanoposthitis patient and drug resistance mechanism of the isolate

GE Yu-mei,HU Qing-feng,ZHU Yong-ze,ZHOU Yong-lie,LYU Huo-yang

(ZhejiangProvincialPeople’sHospital,Hangzhou310014,China)

We isolated and identified the bacterial pathogen in a pyogenic balanoposthitis patient and investigated the drug resistance and its mechanism of the isolate. Urethral secretions and balanus pustule liquids were collected for microscopic examination after Gram-staining and detection of mycoplasma usingMycoplasmaIST 2 kit. The two samples were inoculated on Columbia blood plate,N.gonorrhoeaeselective plate and chromID Candida plate for isolation. The obtained colonies were identified by VITEK 2-compact automatic bacterial detection and analysis system. Moreover,PCR was performed to detect 16S rRNA gene ofN.gonorrhoeaein the samples and colonies. KB method was applied for detecting susceptibility of five common antibiotics against the isolate. The β-lactamase and extended spectrum β-lactamase confirmatory tests were used to investigate the enzyme production of the isolate as well as drug resistance-associated tetM,TEM,mefA and ermF genes in the isolate were detected by PCR. Results showed that all the clinic samples showed negative for mycoplasma. All the isolating cultivation results of urethral secretions were negative while the balanus pustule liquids provided positive isolating cultivation in the blood and selective plates. The VITEK 2-compact system and 16S rRNA-PCR revealed that the isolated strain belongs toN.gonorrhoeae. The isolate can produce β-lactamases and resist to penicillin G,ciprofloxacin and tetracycline. The tetM,TEM,mefA and ermF genes could be found in the isolate’s genome. The patient’s balanoposthitis is caused by infection ofN.gonorrhoeae. The multidrug resistance ofNeisseriagonorrhoeaeisolate is closely associated with its carried resistant genes.

balanoposthitis;Neisseriagonorrhoeae; isolation and identification; drug resistance; resistance gene

Lyu Huo-yang,Email: geyumei1990@hotmail.com

10.3969/j.issn.1002-2694.2017.05.009

浙江省醫(yī)藥衛(wèi)生科技計(jì)劃項(xiàng)目(2017KY004)資助

呂火烊，geyumei1990@hotmail.com

浙江省人民醫(yī)院檢驗(yàn)中心，杭州 310014

R378.1

A

1002-2694(2017)05-0432-04

2016-11-14 編輯：劉岱偉

Supported by the Medical Scientific Research Foundation of Zhejiang Province (No. 2017KY004)