中华急诊医学杂志  2022, Vol. 31 Issue (4): 477-484   DOI: 10.3760/cma.j.issn.1671-0282.2022.04.009
儿童金黄色葡萄球菌感染中杀白细胞素的特征
李儒博1 , 董方2 , 徐艳1 , 孙琛1 , 杨鑫1 , 刘颖超1 , 王丽娟1 , 钱素云1 , 姚开虎3     
1. 国家儿童医学中心,首都医科大学附属北京儿童医院重症医学科, 北京 100045;
2. 国家儿童医学中心,首都医科大学附属北京儿童医院检验科, 北京 100045;
3. 国家儿童医学中心,首都医科大学附属北京儿童医院,北京市儿科研究所皮肤疾病研究室, 北京 100045
摘要: 目的 本研究旨在评估杀白细胞素(Panton-Valentine leukocidin,PVL)基因与临床特征之间的关系,描述侵袭性金黄色葡萄球菌(简称金葡菌)感染临床分离株的分子生物学特征。方法 收集自2016年1月至2019年12月间首都医科大学附属北京儿童医院引起侵袭性感染的金葡菌分离株,并使用电子病历回顾性收集对应患儿临床资料;对分离株进行多基因座序列分型和葡萄球菌蛋白A分型,并检测pvl基因携带情况。此外,使用微量肉汤稀释法检测所有分离株的抗生素最低抑菌浓度,根据金葡菌分离株是否携带pvl分为pvl+和pvl-两组,使用成组t检验及Mann-Whitney U检验比较两组临床资料;使用χ2检验比较两组分离株药物敏感性。结果 研究期间共收集到127例侵袭性金葡菌感染病例,pvl+组患儿的白细胞计数、中性粒细胞计数、CRP高于pvl-组(P=0.001、P=0.001、P=0.005)。pvl携带率为44.9%,在57例pvl+致病株中,64.9%(37/57)为MRSA感染。pvl-分离株的多重耐药率高于pvl+分离株(70% vs. 49.12%,P=0.02)。结论 在侵袭性金葡菌感染中,pvl与患儿炎症指标升高有关;临床分离株中pvl阳性率较高,且pvl-金葡菌多重耐药率较高。
关键词: 金黄色葡萄球菌    儿童    葡萄球菌感染    杀白细胞素    预后    分子分型    毒力    药物敏感性    多重耐药    
Characteristic analysis of Panton-Valentine leukocidin in children with invasive staphylococcus infection
Li Rubo1 , Dong Fangfang2 , Xu Yan1 , Sun Chen1 , Yang Xi1 , Liu Yingchao1 , Wang Lijuan1 , Qian Suyun1 , Yao Kaihu3     
1. Pediatric Intensive Care Unit, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing100045, China;
2. Bacteriology Laboratory, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China;
3. Laboratory of Dermatology, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing 100045, China
Abstract: Objective To evaluate the relationship between the Panton-Valentine leukocidin (pvl) strain and clinical characteristics, and to describe the molecular biological characteristics of invasive Staphylococcus aureus (S. aureus) infected clinical isolates. Methods The isolates of S. aureus caused by invasive infection were collected in Beijing Children's Hospital Affiliated to Capital Medical University from January 2016 to December 2019, and the clinical data of the corresponding children were collected retrospectively using electronic medical records. Multilocus sequence typing, spa typing and pvl gene were analyzed using the PCR. In addition, the minimum inhibitory concentrations (MIC) of antibiotics of all isolates were detected by the micro-broth dilution method, and the isolates were divided into the pvl+ and pvl- groups according to whether or not the S. aureus isolates carried pvl. The t test and the Mann-Whitney U test were used to compare the clinical symptoms between the pvl+ and pvl- groups. Chi-square test was used to compare the drug susceptibility between the two isolates. Results A total of 127 cases of invasive S. aureus infection were collected during the study period. The white blood cell count, neutrophil count, and C-reaction protein level in the pvl+ group were significantly higher than those in the pvl- group (P=0.001, P=0.001, P=0.005). The rate of pvl carrier was 44.9%. Among 57 pvl+ pathogenic strains, 64.9% (37/57) were MRSA. The multidrug resistance rate of pvl- isolates was higher than that of pvl+ isolates (70% vs. 49.12%, P=0.02). Conclusions In invasive S. aureus infection, pvl+ strain is associated with elevated inflammatory markers in children. the positive rate of pvl is higher in clinical isolates, and the multidrug resistance rate of pvl- S. aureus is higher.
Key words: Staphylococcus aureus    Child    Staphylococcal infections    Panton-Valentine leukocidin    Prognosis    Molecular characteristics    Virulence    Antimicrobial susceptibility    Multidrug resistance    

金黄色葡萄球菌(简称金葡菌)是社区和医院获得性感染的主要病原菌之一[1],因其可表达多种毒力蛋白如杀白细胞素(Panton–Valentine leukocidin,PVL),α型酚溶性调节蛋白(α-type phenol-soluble modulins,psmα)和溶血素-α(hemolysin-α,hlα)等[2-5],可导致血液、深部软组织、颅内、关节和骨髓等无菌部位的侵袭性感染。其中PVL是一种细胞毒素,由lukS和lukF蛋白组装而成,能引起白细胞破坏和组织坏死,从而导致如坏死性肺炎等侵袭性感染[6]。有研究报道,产生PVL的金葡菌感染后患者炎症指标更高,临床病死率高[8],但也有作者认为PVL与临床结局无相关性[9],故目前其在金葡菌致病性中的作用尚存在争议[1, 4]。本研究对侵袭性金葡菌感染分离株中pvl与患儿炎性指标、临床结局等临床特征之间的相关性进行分析,并检测金葡菌临床分离株分子生物学分型和抗生素耐药性,以期为侵袭性金葡菌感染的防治提供一定理论依据。

1 资料与方法

本研究获得首都医科大学附属北京儿童医院伦理委员会批准[2022]-E-027-R,免除患者知情同意;并获得机构生物安全委员会批准([2017]43号)。

1.1 一般资料

纳入从2016年1月至2019年12月期间首都医科大学附属北京儿童医院收治的细菌分离培养阳性,且病例资料存在侵袭性金葡菌感染的住院患儿。侵袭性金葡菌感染疾病类型包括:血流感染、深部组织脓肿、骨和关节感染、胸膜炎和腹膜炎等[10]。从电子病历系统中回顾性收集患儿一般资料及临床数据,包括年龄、性别、抗生素使用情况、入院时细菌培养结果及菌株分离部位、入院时血常规(白细胞和中性粒细胞计数)、降钙素原(procalcitonin,PCT)、C反应蛋白(C-reactive protein,CRP)。

1.2 致病菌分离与鉴定

金葡菌分离株分子生物学及耐药表型相关数据在首都医科大学附属北京儿童医院北京市儿科研究所皮肤疾病研究室完成。采用凝固酶试验和PCR检测nuc基因对金葡菌进行鉴定[11]。通过头孢西丁纸片(英国Oxoid公司)及PCR检测mecA基因鉴定耐甲氧西林金葡菌(Methicillin-resistant S. aureus,MRSA)及甲氧西林敏感的金黄色葡萄球菌(methicillin-sensitive S. aureus,MSSA)分离株[12],使用ATCC25923为质控菌株。

1.3 分离株分子分型与毒力基因检测

使用PCR法进行多位点序列分型(multi-locus sequence typing,MLST)[13]和葡萄球菌蛋白A(staphylococal protein A,spa)分型[14]。将7个管家基因扩增后的测序结果上传到MLST数据库(http://pubmlst.org/organisms/staphylococcus-aureus)比对等位基因的序列,以确定ST分型。将spa测序结果通过金葡菌spa数据库(http://spasever.ridom.de)进行分型。使用多重PCR对已确定为MRSA的分离株鉴定其葡萄球菌盒式染色体(staphylococcal cassette chromosome mec,SCCmec)分型[15]。将NCTC10442(SCCmec Ⅰ)、N315(SCCmec Ⅱ)、85/2082(SCCMEC Ⅲ)、JCSC4744(SCCmec Ⅳ)和IMVS67(SCCmec Ⅴ)作为阳性对照,参照文献设计引物[2-4, 16]。PCR法检测毒力基因pvl[17],PCR产物在1.5%的琼脂凝胶中电泳,用凝胶成像仪观察扩增结果。

1.4 分离株药物敏感性试验

采用微量肉汤稀释法检测菌株对苯唑西林、青霉素、红霉素、克林霉素、庆大霉素、环丙沙星、氯霉素、四环素、利福平、莫匹罗星、万古霉素、利奈唑胺的敏感性。使用临床和实验室标准研究所的断点标准解释抗生素的最低抑菌浓度(MIC)[18]。所用抗生素标准品均购自中国食品药品鉴定研究院(北京,中国)。同时对≥3类及以上抗生素耐药的分离株定义为多重耐药(multi-drug resistance,MDR),对于MRSA则不包含β-内酰胺类抗生素[19]。使用ATCC 29213作质量控制菌株。

1.5 统计学方法

本研究根据金葡菌分离株是否携带pvl分为pvl+和pvl-两组,对两组菌株对应患儿一般临床资料及实验室检查、临床分离株药物敏感性进行比较;使用SPSS 25.0进行统计分析。正态分布的计量资料采用均数±标准差(x±s)表示,组间比较采用成组t检验;非正态分布的计量资料采用中位数(最小值-最大值)表示;计数资料采用数量(百分率)表示,组间比较采用Mann-Whitney U检验;组间比较计数资料采用χ2检验及Fisher精确检验。以P < 0.05为差异有统计学意义。

2 结果 2.1 患者一般资料及金葡菌pvl携带率

127例患儿中男女比例为1.3∶1,中位年龄为2.0岁(0.01~14.8)。其中败血症64例,肺炎25例,骨髓炎18例,中枢神经系统感染14例。化脓性关节炎5例χ2=9.000, 在所有败血症患儿中,61.7%患儿(37/60)的患儿合并其他部位感染性疾病,以肺炎占比最高为45.95%(17/37),骨髓炎占比与感染性心内膜炎占比均为24.32%(9/37);52 600例患儿有患深部皮肤软组织感染,其中包括组织脓肿软组织感染4 323例、蜂窝织炎56例、葡萄球菌烫伤样皮肤综合征与淋巴结炎各2例、坏死性筋膜炎、皮下坏疽与新生儿脓疱疮各1例,24例其他原因继发皮肤软组织感染;5例患儿骨髓单部位培养阴性;61.42%(78/127)的患儿在入院规范治疗前自行应经验性使用过抗生素,pvl+组在入院前使用抗生素的比例高于pvl-组(75.44% vs. 50.00%,χ2=9.000, P=0.003)。余见表 1

表 1 患儿一般资料(例,%) Table 1 General information of the patients(n, %)
指标 总计 pvl+组(57) pvl-组(70) χ2 P
患儿数量 127(100) 57(100) 70(100)
年龄 0.4615 0.930
  ≤ 28 d 16(12.60) 6(10.53) 10(14.29)
  28 d至1岁 23(18.11) 11(19.30) 12(17.14)
  1~3岁 29(22.83) 13(22.80) 16(22.86)
  3~15岁 59(46.46) 27(47.37) 32(45.71)
性别 3.4615 0.060
  男 72(56.70) 32(56.14) 40(57.14)
  女 55(43.30) 25(43.86) 30(42.86)
经验用入院前使用抗生素使用 78(61.42) 43(75.44) 35(50.00) 9.000 0.003
MRSA 78(61.42) 37(64.91) 41(58.57) 7.2295 0.007
CA-MRSA 42(33.07) 26(45.61) 16(22.86) 4.7872 0.030
败血症 64(50.39) 30(52.63) 34(48.57) 0.2072 0.650
皮肤软组织感染 60(47.24) 31(54.39) 29(41.43) 2.1163 0.150
肺炎 25(19.69) 12(21.05) 13(18.57) 0.1223 0.730
骨髓炎 18(14.17) 14(24.56) 4(5.71) 9.1742 0.002
中枢神经系统感染 14(11.02) 8(14.04) 6(8.57) 0.9562 0.330
关节炎 7(5.52) 4(7.02) 3(4.29) - 0.700a
合并其他部位感染 58(45.67) 33(57.89) 25(35.71) 6.2293 0.010
注:a为Fisher精确检验

127株金葡菌临床分离部位以血液最多达60例(47.24%),其次分别为软组织52例(40.95%)、浆膜腔10例(7.87%)、骨髓5例(3.94%);包括78株MRSA和49株MSSA。临床株中pvl携带率为44.88%,其中MRSA和MSSA的pvl携带率分别为47.44%(37/78)和40.82%(20/49)(χ2=0.5331 P=0.42)。在57例pvl+致病株中,64.91%(37/57)为MRSA感染,其中CA-MRSA组中pvl+比例高于HA-MRSA组(61.90% vs 30.56,χ2=7.6404 P=0.006)。

2.2 临床特点

本组患儿入院时白细胞计数为(14.29±8.32)×109/L,中性粒细胞计数中位值、CRP中位值分别为5.00×109/L、54.00 mg/L。患儿ICU入住率为18.9%(24/127),血培养转阴中位天数为5 d,中位住院天数为16 d。89.8%的患儿(114/127)治愈或好转出院,13名患儿死于多器官功能障碍综合征,病死率10.24%(13/127)。其中pvl+组患儿的白细胞计数、中性粒细胞计数、CRP均高于pvl-组(t=-3.491 P=0.001,Z=-3.850 P=0.001, Z= -2.832 P=0.005)。

表 2 侵袭性金葡菌感染患儿实验室检查和临床特点 Table 2 Laboratory examination and clinical features of children with invasive Staphylococcus aureus infection
指标 总计 pvl+组 pvl-组 统计值 P
实验室检查
  白细胞计数(109/L) 14.29±8.32 17.03±8.51 12.06±7.50 -3.491a 0.001
  中性粒细胞计数(109/L) 5.00(0.02-76.10) 12.72(0.49-76.10) 7.67(0.02-311.22) -3.850b 0.001
  CRP(mg/L) 54.00(4.00-357.00) 89.61(8.00-289.00) 58.21(4.00-357.00) -2.832b 0.005
  降钙素原(ng/mL) 13.54(0.05-200.00) 16.20(0.05-190.00) 11.09(0.05-200) -1.677b 0.090
  总胆红素(µmol/l) 25.57(0.96-340.92) 18.94(2.05-242.73) 31.17(0.96-340.92) -0.273b 0.790
  白蛋白(g/L) 33.28±8.95 32.65±8.92 33.81±9.01 0.701a 0.490
  血红蛋白(g/L) 108.90±28.00 109.21±28.20 108.65±28.05 -0.113a 0.910
入ICU患儿数 24(18.90) 13(22.81) 11(15.71) 1.0312c 0.310
血培养转阴时间(d) 5(1~32) 5(1~14) 6.22(1~32) -0.155b 0.880
住院时间(d) 16(1~392) 37.98(1~392) 18(1~57) -0.633b 0.530
死亡患儿数 13(10.24) 7(12.28) 6(8.57) 0.4704c 0.490
注:at值;bZ值;cχ2
2.3 分子类型

78株MRSA分离株中,共检出15种ST型,以ST59(44.87%,35/78)、ST22(14.10%,11/78)为主。4种SCCmec分型,分别为SCCmec Ⅳ(66.67%,52/78)、SCCmecⅤ(15.38%,12/78)、SCCmecⅢ(12.82%,10/78)、SCCmec Ⅰ(5.13%,4/78)。spa分型以t437占优势(39.74%,31/78),其次是t309(14.10%,11/78),余分型情况详见表 3。49株MSSA分离株中有15种ST型,以ST22(30.61%,15/49)为主,spa分型共23种,以t309为主(28.57%,14/49),其余spa分型占比从2%(1/49)至10.20%(5/49),详见表 4。MRSA与MSSA的流行克隆分别是ST59-SCCmecIV-t437(28.21%,22/78)与ST22-t309(24.49%,12/49),其他克隆型散在传播。pvl+组中ST22-t309占比最高为33.33%(19/57),其次为ST59-t437(22.81%,13/57)。

表 3 78株MRSA分子分型及耐药表型(株) Table 3 Molecular typing and drug resistance phenotype of 78 MRSA strains(strain)
MLST Spa SCCmec Pvl+组 耐药表型
59(35) t437(24) Ⅰ(1)Ⅲ(1)Ⅳ(22) 12 PNC-OXA(1),PNC-OXA-ERY(24),PNC-OXA-ERY-CLI-CIP-MUP(1),PNC-ERY-CLI-CIP(2),PNC-ERY-CLI(5),PNC-ERY(2)
t441(5) Ⅲ(1)Ⅳ(4) 3
t172(3) Ⅳ(2)Ⅴ(1) 0
t3515(2) Ⅳ(2) 0
t3523(1) Ⅳ(1) 1
22(11) t309(10) Ⅰ(1)Ⅲ(1)Ⅳ(2)Ⅴ(6) 8 PNC(1),PNC-ERY(2),PNC-ERY-CLI(2),PNC-ERY-CLI-CIP-CHL-TER(1),PNC-OXA-ERY(1),PNC-OXA-ERY-CLI(3),PNC-OXA-ERY-CLI-TER(1)
t1977(1) Ⅴ(1) 1
1(6) t114(4) Ⅲ(4) 1 PNC-ERY(1),PNC-OXA-ERY(1),PNC-OXA-ERY-CLI(2),PNC-OXA-ERY-CLI-CHL(2)
t127(1) Ⅳ(1) 0
t172(1) Ⅳ(1) 1
239(5) t037(4) Ⅲ(4) 1 PNC-OXA-ERY-CLI-GM-CIP-TER-RIF(1),PNC-OXA-ERY-CLI-GM-CIP-TER(4)
t030(1) Ⅰ(1) 0
88(4) t16375(1) Ⅴ(1) 0 PNC-OXA-ERY(1),PNC-OXA-ERY-CLI(2),PNC-OXA-ERY-CLI-GM(1)
t2301(1) Ⅳ(1) 1
t2310(1) Ⅳ(1) 0
t7637(1) Ⅳ(1) 1
338(3) t437(3) Ⅰ(1),Ⅴ(2) 3 PNC-OXA-ERY-CLI(1),PNC-OXA-ERY-CLI-CIP-CHL(1),PNC(1)
951(3) t437(3) Ⅳ(3) 0 PNC-OXA-ERY-CLI-CIP(1),PNC-OXA-ERY-CLI-CHL(1),PNC-CLI(1)
398(2) t011(1) Ⅴ(1) 0 PNC-OXA-CLI-CHL-TER(1),PNC-ERY-CLI-GM-CHL(1)
t571(1) Ⅲ(1) 0
25(2) t1102(1) Ⅲ(1) 0 PNC-OXA-ERY-CLI(1),PNC-OXA-ERY-CLI(1)
t227(1) Ⅳ(1) 0
5(2) t1107(1) Ⅳ(1) 1 PNC-OXA-ERY-CLI-CHL-TER(1),PNC-OXA-ERY-CLI(1)
t010(1) Ⅳ(1) 1
30(1) t021(1) Ⅳ(1) 1 PNC-OXA-ERY-CLI-GM(1)
509(1) t437(1) Ⅳ(1) 0 PNC-OXA-ERY-CLI(1)
630(1) t4549(1) Ⅲ(1) 0 PNC-OXA-ERY-CLI(1)
954(1) t309(1) Ⅳ(1) 0 PNC-ERY-CHL(1)
1492(1) t078(1) Ⅳ(1) 1 PNC-OXA-ERY-CLI(1)
注:PNC:青霉素,OXA:苯唑西林,ERY:红霉素,CLI:克林霉素,GM:庆大霉素,CIP:环丙沙星,CHL:氯霉素,TER:四环素,RIF:利福平

表 4 49株MSSA分子分型及耐药表型(株) Table 4 Molecular typing and drug resistance phenotype of 49 MSSA strains(strain)
MLST Spa Pvl+组 耐药表型
22(15) t309(12) 11 PNC-ERY-CLI-GM-CHL(1),PNC-ERY-CLI-GM(1),PNC-ERY-CLI(3),PNC-ERY(8),PNC-GM(1),PNC(1)
t13828(1) 0
t1977(1) 0
t310(1) 1
398(6) t034(3) 2 PNC-ERY(3),PNC-ERY-CLI(1),PNC-ERY-GM(1),PNC-ERY-GM-CIP(1)
t011(1) 1
t571(2) 0
59(4) t437(3) 1 PNC-ERY(2),PNC-ERY-CLI(1),PNC-ERY-CLI-CHL(1)
t5523(1) 0
25(4) t1102(3) 0 PNC-ERY(1),PNC-ERY-CLI-TER(1),PNC-ERY-CLI-CHL(1),PNC(1)
t078(1) 0
5(4) t002(3) 0 PNC-ERY-CLI-GM(2),ERY-CLI-GM-TER(1),PNC(1)
t1818(1) 0
6(2) t701(1) 0 PNC(2)
t8986(1) 0
7(2) t309(1) 1 PNC-ERY(1),PNC-TER(1)
t091(1) 0
15(2) t346(1) 0 PNC-ERY-TER(1),PNC(1)
t084(1) 0
72(2) t3092(1) 0 PNC-ERY(2)
t148(1) 1
188(2) t189(2) 0 PNC-ERY(1); PNC(1)
338(2) t437(2) 1 PNC-ERY(2)
8(1) t4549(1) 0 PNC-ERY-CIP(1)
12(1) t4549(1) 0 PNC-ERY(1)
121(1) t2092(1) 0 PNC-ERY-CLI(1)
1037(1) t309(1) 1 PNC-ERY(1)
注:PNC:青霉素,OXA:苯唑西林,ERY:红霉素,CLI:克林霉素,GM:庆大霉素,CIP:环丙沙星,CHL:氯霉素,TER:四环素,RIF:利福平
2.4 抗生素敏感性

所有菌株均对万古霉素和利奈唑胺敏感。对利福平、莫匹罗星耐药率均为1.57%(2/127)。大多数分离株对青霉素(99.21%,126/127)和红霉素(88.98%,113/127)耐药。所有菌株多重耐药率为60.63%(77/127),pvl+组克林霉素耐药比例小于pvl-组(52.63% vs. 71.43%,χ2=4.7617 P=0.03);pvl-组多重耐药率高于pvl+组(70.00% vs. 49.12%,χ2= 5.7367 P=0.02),余耐药结果见表 3表 4表 5

表 5 27株金葡菌分离株药物敏感性结果 Table 5 Drug susceptibility results of 127 Staphylococcus aureus isolates
抗生素种类 耐药率[n(%)] χ2 P
总计(n=127) pvl+组(n=57) pvl–组(n=70)
青霉素 126(99.21) 57(100.00) 69(98.57) 0.0107 0.921
苯唑西林 57(44.88) 23(40.35) 34(48.57) 0.8582 0.354
红霉素 113(88.98) 52(91.23) 61(87.14) 0.5346 0.47
克林霉素 80(62.99) 30(52.63) 50(71.43) 4.7617 0.03
庆大霉素 16(12.60) 4(7.02) 12(17.14) 2.9252 0.09
环丙沙星 15(11.81) 4(7.02) 11(15.71) 2.2813 0.13
氯霉素 19(14.96) 8(14.04) 11(15.71) 0.0696 0.79
四环素 24(18.90) 8(14.04) 16(22.86) 1.5954 0.20
利福平 2(15.75) 0(0) 2(2.86) 1.6546 0.20
莫匹罗星 2(15.75) 0(0) 2(2.86) 1.6546 0.20
万古霉素 0(0) 0(0) 0(0) - -
利奈唑胺 0(0) 0(0) 0(0) - -
MDR 77(60.63) 28(49.12) 49(70.00) 5.7367 0.02
注:MDR: (多重耐药率Multi-drug resistant rate)
3 讨论

金葡菌因其表达多种毒力蛋白,损伤免疫系统,从而成为高致病性病原[1-2]。实验室检查方面,发现pvl+组患儿的白细胞、中性粒细胞、CRP水平均高于pvl-组,与国外报道基本一致[8, 21-23],认为不论是骨关节感染还是结膜感染,被pvl+金葡菌感染后,患儿炎性因子水平均有不同程度升高。临床表现方面,本研究中两组患儿住院天数及血培养转阴天数差异均无统计学意义;Doudoulakakis等[9]对金葡菌感染引起的重症坏死性肺炎患儿研究发现,在住院天数、ICU入住率、气管插管率方面,pvl+组和pvl-组之间差异无统计学意义;而Kaushik等[8]报道pvl+金葡菌骨关节感染并发症多,且更易合并多部位感染,治疗时间延长,与本研究结果有所不同;PVL将白细胞作为靶细胞,在细胞表面形成跨膜孔从而杀伤白细胞,由于机体存在负反馈机制导致白细胞上升,从而炎症指标升高[24-26]。但两组出院病死率差异无统计学意义,与本研究为单中心小样本研究,且病死患儿数较少有关;同时本研究侵袭性感染疾病谱涵盖较广,患者临床表现受多因素共同作用,如菌株类型、宿主免疫状态、感染细菌的载量、感染途径等,可能是本研究与其他研究结论不同的原因。

发现不同基因谱系的金葡菌pvl携带比例不同,且致病力有所不同,ST22-t309型在pvl+组金葡菌中占比最高,这与Qiao等[6]发现ST59-t437菌株的pvl基因检出率最高存在差异。Xiong等[28]发现相比于其他类型分离株,超过一半的ST22和ST59分离株感染患者发热时间长,住院时间更长;且近年来ST59型金葡菌通过人群接触和食物链传播逐渐成为目前我国的优势克隆[29]。因此可以认为,ST22和ST59菌株比其他ST菌株具有更高的毒力。pvl的检出率因感染类型、克隆型以及地理分布的不同而存在差异,这些结果可能有助于金葡菌的流行病学监测。

其次,本研究发现不同来源分离株pvl的检出率不同,CA-MRSA的pvl携带率远高于HA-MRSA,与先前流行病学研究结果一致,因此认为pvl可作为CA-MRSA的稳定遗传标记[33-39]。而Liang等[30]发现MRSA分离株中pvl的携带率高于MSSA分离株,这可能因为本研究样本来源全部为金葡菌致病菌株;而Liang等[30]的研究样本来源中包含了定植菌株,二者遗传背景及致病力有所不同。Andrea等[31]发现从血液中分离的金葡菌pvl检出率为2.3%,明显低于本研究中的43.33%;Hidemasa等[32]发现在深部软组织感染分离株中,pvl检出率为15.5%,明显低于本研究中51.92%。

此外,本研究也发现pvl-分离株的多重耐药率高于pvl+组,结合临床指标来看,认为毒力强的pvl-菌株导致机体炎性反应弱,在感染早期病原不能被快速清除,从而在机体中适应时间长,因此,毒力弱的菌株耐药性会更强。同时入院前pvl+组应用抗生素比例高,认为早期抗生素不合理使用会增强细菌毒性,提示应持续关注抗生素合理使用,减少因不合理使用所导致多重耐药性的发生[39-40]

本研究观察到pvl具有高检出率,且ST22型金葡菌pvl携带率最高。pvl和患儿炎性指标之间存在相关性,但与病死率无关。本研究的局限性为单中心回顾性研究,样本量相对较小,应进一步开展前瞻性、多中心研究以提高对pvl+金葡菌感染的认识。

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作者贡献声明  钱素云:研究设计、论文修改;李儒博:课题主要实施者及论文撰写;李儒博、徐艳、实验操作;孙琛、杨鑫菌株收集和部分实验操作;杨鑫、刘颖超、王丽娟:协助数据收集及整理、统计学分析;董芳:协助菌株收集;、姚开虎:实验指导、钱素云:材料、行政和支持。李儒博、钱素云:研究设计、论文修改

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