中国医科大学学报  2018, Vol. 47 Issue (6): 537-541, 547

文章信息

岳冬梅, 佟雅洁, 杨凡
YUE Dongmei, TONG Yajie, YANG Fan
儿科患者血培养常见病原菌分布及耐药性特点分析
Analysis of Common Pathogenic Bacteria Distribution and Antibiotic Resistance of Bacteria in Blood Culture among Patients in the Pediatric Department
中国医科大学学报, 2018, 47(6): 537-541, 547
Journal of China Medical University, 2018, 47(6): 537-541, 547

文章历史

收稿日期:2017-12-11
网络出版时间:2018-05-21 10:39
儿科患者血培养常见病原菌分布及耐药性特点分析
岳冬梅 , 佟雅洁 , 杨凡     
中国医科大学附属盛京医院儿科, 沈阳 110004
摘要目的 比较儿科重症监护病房(PICU)与普通病房及门诊(非PICU)患儿血培养阳性的病原菌分布及耐药性特点,为临床合理用药提供参考依据。方法 回顾性分析,2016年我院PICU与非PICU血培养阳性的病原学资料,利用SPSS 19.0统计软件进行数据处理。结果 共分离出病原菌914株,其中PICU 101株(11.05%),非PICU 813株(88.95%)。革兰阳性菌551株(60.28%),以表皮葡萄球菌和人葡萄球菌多见;革兰阴性菌331株(36.21%),以大肠埃希菌和肺炎克雷伯菌多见。PICU与非PICU比较,除肺炎克雷伯菌外,其他病原菌无统计学差异(P > 0.05)。2组中表皮葡萄球菌对左氧氟沙星、环丙沙星、庆大霉素、利福平的耐药率存在统计学差异(P < 0.05);大肠埃希菌对头孢噻肟、头孢他啶、左氧氟沙星、美洛培南、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦以及亚胺培南的耐药率存在统计学差异(P < 0.05);肺炎克雷伯菌对头孢呋辛酯、头孢唑林、头孢曲松、头孢呋辛、头孢孟多、四环素、庆大霉素的耐药率存在统计学差异(P < 0.05)。结论 2组血培养阳性标本中分离出的革兰阳性菌均以表皮葡萄球菌和人葡萄球菌多见,革兰阴性菌以大肠埃希菌和肺炎克雷伯菌多见;2组耐药情况均较严峻,但对一部分抗菌药物耐药性存在差异,应引起高度重视。
关键词儿科    重症监护病房    普通病房    门诊    血培养    病原菌    耐药性    
Analysis of Common Pathogenic Bacteria Distribution and Antibiotic Resistance of Bacteria in Blood Culture among Patients in the Pediatric Department
Department of Pediatrics, Shengjing Hospital, China Medical University, Shenyang 110004, China
Abstract: Objective To conduct a comparative analysis of the distribution and antibiotic resistance characteristics of pathogenic bacteria among patients with positive blood cultures in the pediatric intensive care unit (PICU), and those in the general ward and outpatient department (non-PICU). Methods A retrospective analysis was performed to collect and compare the pathogenic data of patients with positive blood cultures in the department of pediatrics in the PICU and non-PICU in our hospital in 2016. Results A total of 914 strains of pathogenic bacteria was isolated, including 101 strains from the PICU (11.05%) and 813 strains from the non-PICU (88.95%). Among these strains, 551 were Gram-positive bacteria (60.28%), most of which were Staphylococcus epidermidis(S. epidermidis) and Human staphylococcus, along with 331 strains of Gram-negative bacteria (36.21%), which were mostly Escherichia coli(E. coli) and Klebsiella pneumoniae(K. pneumoniae). The drug resistance rates of S. epidermidis to levofloxacin, ciprofloxacin, gentamicin, and rifampin (P < 0.05);E. coli to cefotaxime, ceftazidime, levofloxacin, meropenem, piperacillin/tazobactam, cefoperazone/sulbactam, and imipenem (P < 0.05);and K. pneumoniae to cefuroxime, cefazolin, ceftriaxone, cefuroxime, cefamandole, tetracycline, and gentamicin (P < 0.05) were statistically significant. Conclusion Gram-positive bacteria isolated from the pediatric positive blood culture specimens were mostly S. epidermidis and Human Staphylococcus, and the Gram-negative bacteria were mainly E. coli and K. pneumoniae, both in the PICU and non-PICU. The pathogenic bacteria showed severe drug resistance, and a statistically significant difference in drug resistance to some commonly used antibiotics was found between the 2 groups.

儿童机体抵抗力弱,免疫系统发育尚不成熟,皮肤表面正常菌群极易进入血液引起感染[1-2],严重者会出现菌血症或败血症,病情危急,严重影响预后。然而细菌感染临床表现不典型,缺乏特异性,临床上通过血培养检出病原菌明确诊断,同时根据药敏试验结果选用抗菌药物来提高患儿治愈率[3]。儿科重症监护病房(pediatrics intensive care unit,PICU)患儿病情重,体质弱,插管等介入治疗手段造成的医源性感染较普通病房及门诊(非PICU)多见,感染风险亦高[4-5]。本研究回顾性分析2016年我院PICU与非PICU患儿血培养的病原菌分布及耐药性特点,旨在为感染患儿合理使用药物提供参考依据。

1 材料与方法 1.1 一般资料

收集2016年1月至12月期间我院住院及门诊就诊的血培养阳性患儿的临床资料,共914例,其中PICU患儿101例,非PICU患儿813例。

1.2 病原菌检测及耐药菌鉴定

对怀疑血液感染的患儿在使用抗菌药物前或者患儿发热时,局部严格消毒后采集静脉血液标本(新生儿采集1.5 mL,儿童采集5~10 mL)并分别注入需氧和厌氧血培养瓶中,混合均匀后送我院检验科。根据《全国临床检验操作规程》的要求对标本进行培养,采用法国梅里埃公司Vitek2 Compact全自动微生物鉴定分析系统对病原菌进行分离与鉴定,采用美国西门子公司Walkaway药敏分析仪对药物敏感性进行分析,结果判断依据2016年美国临床和实验室标准协会标准执行。同一患者相同细菌标本类型计为1株。

1.3 统计学分析

采用SPSS 19.0软件对数据进行统计分析处理,计数资料以百分比或率表示,组间比较采用χ2检验。P < 0. 05为差异有统计学意义。

2 结果 2.1 PICU与非PICU病原菌分布比较

结果显示,PICU共送检了2 171份血培养标本,分离出病原菌101株,阳性率4.65%;非PICU共送检了11 320份血培养标本,分离出病原菌813株,阳性率7.18%。PICU与非PICU中革兰阳性菌均以表皮葡萄球菌和人葡萄球菌多见;革兰阴性菌均以大肠埃希菌和肺炎克雷伯菌多见。肺炎链球菌、肺炎克雷伯菌、流感嗜血杆菌、摩式摩根菌、阴沟肠杆菌以及白假丝酵母菌分布上2组比较具有统计学差异(均P < 0.05)。见表 1

表 1 PICU与非PICU血培养病原菌分布[n(%)] Tab.1 Distribution of pathogens in the PICU and non-PICU with blood culture [n (%)]
Pathogenic bacteria PICU Non-PICU P
Gram-positive bacteria 61(60.40) 490(60.27) 0.981
  Staphylococcus epidermidis 23(22.77) 196(24.11) 0.767
  Human staphylococcus 14(13.86) 75(9.23) 0.138
  Streptococcus pneumoniae 6(5.95) 9(1.11) 0.001
  Staphylococcus aureus 3(2.97) 22(2.71) 0.878
  Enterococcus faecium 3(2.97) 15(1.85) 0.443
  Staphylococcus cephalus 3(2.97) 60(7.38) 0.099
  Staphylococcus haemolyticus 0(0.00) 27(3.32) 0.063
  Enterococcus faecalis 2(1.98) 20(2.46) 0.767
  Staphylococcus agalactiae 3(2.97) 12(1.48) 0.265
  Staphylococcus vaughn 1(0.99) 11(1.35) 0.763
  Others 3(2.97) 43(5.29) 0.315
Gram-negative bacteria 35(34.65) 296(36.40) 0.729
  Escherichia coli 14(13.86) 75(9.22) 0.138
  Klebsiella pneumoniae 9(8.91) 164(20.17) 0.006
  Haemophilus influenzae 2(1.98) 2(0.25) 0.013
  Citrobacter freundii 0(0.00) 10(1.23) 0.262
  Morganella morganii 2(1.98) 0(0.00) < 0.001
  Enterobacter cloacae 2(1.98) 1(0.12) 0.002
  Pseudomonas aeruginosa 0(0.00) 8(0.98) 0.317
  Others 6(5.94) 36(4.43) 0.494
Fungi 4(3.96) 27(3.32) 0.738
  Candida membranaceus 0(0.00) 19(2.34) 0.121
  Candida albicans 2(1.98) 2(0.25) 0.013
  Candida parapsilosis 1(0.99) 1(0.12) 0.079
  Candida tropicalis 1(0.99) 1(0.12) 0.079
Others 0(0.00) 4(0.49) 0.480

2.2 PICU与非PICU表皮葡萄球菌、人葡萄球菌药敏试验结果比较

结果显示,血培养阳性标本中分离出的2种主要革兰阳性菌分别为表皮葡萄球菌和人葡萄球菌。PICU与非PICU比较,表皮葡萄球菌对左氧氟沙星、环丙沙星、庆大霉素、利福平耐药率差异存在统计学意义(均P < 0.05);人葡萄球菌耐药率差异均无统计学意义(均P > 0.05)。见表 2

表 2 PICU与非PICU主要革兰阳性菌对常用抗菌药物耐药率(%) Tab.2 Drug resistance rates of the main Gram-positive bacteria to commonly used antimicrobials in the PICU and non-PICU (%)
Antimicrobial Staphylococcus epidermidis Human staphylococcus
PICU Non-PICU P PICU Non-PICU P
Penicillin G 100.00 94.30 0.240 92.86 93.24 0.958
Erythromycin 95.65 86.22 0.200 100.00 93.33 0.320
Oxacillin 91.30 78.76 0.154 78.57 69.33 0.485
Levofloxacin 66.67 31.40 0.003 23.08 13.43 0.373
Clindamycin 60.87 58.67 0.840 64.28 75.00 0.407
Compound sulfamethoxazole 60.87 52.04 0.422 35.71 41.10 0.707
Ciprofloxacin 58.82 33.33 0.039 22.22 16.07 0.648
Gentamicin 34.78 11.86 0.003 0.00 2.70 0.534
Rifampicin 21.74 7.73 0.028 0.00 4.17 0.437
Moxifloxacin 11.11 5.85 0.384 23.08 13.64 0.386
Tetracycline 5.88 10.71 0.534 22.22 32.14 0.549
Vancomycin 0.00 0.00 - 0.00 0.00 -
Linezolid 0.00 0.00 - 0.00 0.00 -
Tegocycline 0.00 0.00 - 0.00 0.00 -
Quinupristin/dafoe leptin 0.00 0.87 0.746 0.00 2.13 0.677
Darpoo mycin 0.00 0.00 - 0.00 0.00 -
Furazolidin 0.00 0.00 - - 6.25 -
Teicoplanin 0.00 0.00 - 0.00 0.00
Ceftaroline fosamil 0.00 0.00 - 0.00 0.00

2.3 PICU与非PICU大肠埃希菌、肺炎克雷伯菌药敏试验结果比较

结果显示,分离出的主要革兰阴性菌分别为大肠埃希菌和肺炎克雷伯菌。PICU与非PICU比较,大肠埃希菌对头孢噻肟、头孢他啶、左氧氟沙星、美洛培南、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦以及亚胺培南的耐药率差异存在统计学意义(均P < 0.05);肺炎克雷伯菌对头孢呋辛酯、头孢唑林、头孢曲松、头孢呋辛、头孢孟多、四环素、庆大霉素的耐药率差异存在统计学意义(均P < 0.05)。见表 3

表 3 PICU与非PICU主要革兰阴性菌对常用抗菌药物的耐药率(%) Tab.3 Drug resistance rates of the main Gram-negative bacteria to commonly used antimicrobials in the PICU and non-PICU (%)
Antimicrobial Escherichia coli Klebsiella pneumoniae
PICU Non-PICU P PICU Non-PICU P
Cefuroxime axetil 100.00 80.76 0.404 66.67 96.36 0.024
Ampicillin 92.86 91.89 0.903 100.00 100.00 -
Cefazolin 90.91 83.56 0.530 62.50 96.30 0.001
Cefatriaxone 88.89 74.24 0.334 71.43 92.54 0.048
Piperacillin 80.00 100.00 0.164 50.00 100.00 0.819
Cefuroxime 55.56 76.79 0.179 60.00 90.16 0.035
Ceftizoxime 55.56 50.91 0.796 40.00 68.64 0.181
Ampicillin/sulbactam 54.55 77.08 0.129 66.67 87.85 0.137
Cefimaddo 50.00 76.09 0.128 40.00 96.12 0.000
Aztreonam 42.86 22.67 0.113 33.33 48.78 0.366
Compound sulfamethoxazole 42.86 58.67 0.274 44.44 65.85 0.191
Tetracycline 41.67 63.08 0.165 33.33 87.20 0.003
Gentamicin 28.57 53.33 0.089 44.44 14.63 0.018
Cefepime 21.43 46.67 0.080 55.56 56.10 0.975
Cefotaxime 20.00 77.78 0.036 50.00 100.00 0.819
Cefimazole 16.67 30.77 0.489 50.00 17.24 0.239
Amoxicillin/clavulanic acid 12.50 43.24 0.104 40.00 71.67 0.140
Ciprofloxacin 9.09 35.42 0.087 16.67 8.41 0.488
Ceftazidime 7.14 40.00 0.018 55.56 58.54 0.860
Levofloxacin 7.14 40.00 0.018 0.00 3.05 0.595
Amikacin 0.00 2.67 0.537 0.00 0.61 0.814
Ertapenem 0.00 17.54 0.117 0.00 17.02 0.233
Furazolidin 0.00 0.00 - 0.00 3.23 0.796
Oxo cephalosporin 0.00 0.00 - 0.00 0.00 -
Chloramphenicol 0.00 7.94 0.312 33.33 42.98 0.641
Meropenem 0.00 32.31 0.021 33.33 29.37 0.835
Piperacillin/tazobactam 0.00 34.67 0.009 22.22 39.02 0.312
Tegocycline 0.00 0.00 - 0.00 0.00 -
Cefoperazone/sulbactam 0.00 40.00 0.004 20.00 49.18 0.201
Ceftaetan 0.00 30.77 0.113 25.00 23.08 0.929
Cefoxitin 0.00 37.50 0.070 0.00 36.21 0.139
Tobramycin 0.00 33.33 0.094 25.00 7.69 0.219
Imipenem 0.00 33.33 0.019 22.22 29.27 0.650

3 讨论

儿童自身免疫功能与成人不同,很容易发生血流感染,血流感染患儿病情进展迅速,易引起全身性感染[6-7]。有研究[8-9]显示,严重感染患儿常起病急骤,病情危急,预后不良,及时有效治疗可显著改善预后。了解本地区患儿血流感染常见病原菌及耐药性特点,是重症感染初期患儿缺乏病原学证据时经验性治疗的关键。本研究结果显示,儿科患者血培养以革兰阳性菌(60.28%)为主,表皮葡萄球菌和人葡萄球菌最多见,与国内外相关报道一致[10-13],表皮葡萄球菌和人葡萄球菌均属于凝固酶阴性葡萄球菌(coagulase-negative staphylococci,CoNS),这类病原菌属于人体皮肤黏膜定植菌[14-15],儿童由于皮肤黏膜发育尚未完全成熟,定植菌容易入血引起血流感染。革兰阴性菌占36.21%,以大肠埃希菌和肺炎克雷伯菌多见,真菌(3.39%)较少见。PICU与非PICU病原菌分布结果显示,肺炎克雷伯菌、肺炎链球菌、流感嗜血杆菌、摩式摩根菌、阴沟肠杆菌以及白假丝酵母菌分布两者具有统计学差异(均P < 0.05),但除肺炎克雷伯菌外,其余病原菌检出的菌株数较少,可能存在一定的误差。

药敏试验结果显示,革兰阳性菌中,PICU中分离的表皮葡萄球菌对青霉素G全部耐药,非PICU中分离的表皮葡萄球菌对青霉素G的耐药率高达90%以上;PICU、非PICU分离出的表皮葡萄球菌对红霉素和苯唑西林的耐药率分别为90%以上、70%~90%,两者差异无统计学意义(P > 0.05),说明我院患儿感染的表皮葡萄球菌已对青霉素G、红霉素和苯唑西林高度耐药,不适合早期经验性抗感染治疗。PICU、非PICU分离出的表皮葡萄球菌对喹诺酮类抗生素(左氧氟沙星、环丙沙星、莫西沙星)、氨基糖苷类抗生素(庆大霉素)、磺胺类(复方新诺明)、克林霉素和利福平的耐药率分别为11.11%~66.67%、5.85%~58.67%,因此,这些可根据临床实际情况作为经验性治疗药物。PICU与非PICU比较,表皮葡萄球菌对左氧氟沙星、环丙沙星、庆大霉素、利福平耐药率存在统计学差异(均P < 0.05)。此外,2组表皮葡萄球菌对糖肽类抗生素(万古霉素、替考拉宁)、人工合成的噁唑烷酮类抗菌药(利奈唑胺)、硝基呋喃类合成抗菌药(呋喃妥因)以及甘氨酰环素类抗生素(替加环素)均敏感,可考虑作为早期经验性治疗药物,但应避免反复应用。PICU中分离的人葡萄球菌对红霉素全部耐药,对青霉素G的耐药率在90%以上,非PICU中分离的人葡萄球菌对青霉素G和红霉素的耐药率均达90%以上,均不适用于经验性抗感染治疗;2组人葡萄球菌对苯唑西林、克林霉素、喹诺酮类抗生素(左氧氟沙星、环丙沙星、莫西沙星)、磺胺类药物(复方新诺明)以及四环素的耐药率在20%~80%之间,可根据实际药敏情况选择用药;2组分离的人葡萄球菌对庆大霉素以及利福平均敏感,但因其药物的不良反应在儿童中应用受到限制;如果应用需要注意不能长期使用,以免产生耐药菌株[16]

革兰阴性菌中,PICU分离的大肠埃希菌对第二代头孢菌素类抗生素(头孢呋辛酯)耐药,对广谱半合成青霉素类(氨苄西林、哌拉西林)以及第一、三代头孢菌素类抗生素(头孢唑林、头孢曲松)耐药率均达80%以上;对阿米卡星、美洛培南、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦、亚胺培南均敏感,应注意避免耐药菌株的产生。上述药物均不适用于早期经验性抗感染治疗。2组在对头孢噻肟、头孢他啶、左氧氟沙星、美洛培南、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦以及亚胺培南的耐药率存在统计学差异(P < 0.05)。PICU分离的肺炎克雷伯菌对广谱半合成青霉素(氨苄西林)耐药,对头孢菌素类抗菌药物,对阿米卡星、厄他培南、头孢西丁、左氧氟沙星均敏感。非PICU分离的肺炎克雷伯菌对广谱半合成青霉素(氨苄西林、哌拉西林)以及第三代头孢菌素类抗生素(头孢噻肟)耐药。2组在对头孢呋辛酯、头孢唑林、头孢曲松、头孢呋辛、头孢孟多、四环素、庆大霉素的耐药率存在统计学差异(P < 0.05)。

综上所述,儿科患者血培养分离的病原菌分布广泛,耐药性严峻。其中主要以CoNS、大肠埃希菌和肺炎克雷伯菌多见。肺炎链球菌、肺炎克雷伯菌、流感嗜血杆菌、摩式摩根菌、阴沟肠杆菌以及白假丝酵母菌分布上PICU与非PICU比较有统计学差异。2组病原菌对部分抗菌药物耐药性存在差异。临床上应加强抗菌药物使用的监管,在有效控制感染基础上延缓耐药菌株的产生。

参考文献
[1]
GILMAN KH, JONATHAN HK, NG TK, et al. Performance evaluation of the verigene Gram-positive and Gram-negative blood culture test for direct identification of bacteria and their resistance determinants from positive blood cultures in Hong Kong[J]. PLoS One, 2015, 10(10): e0139728. DOI:10.1371/journal.pone.0139728
[2]
DONG H, CAO H, ZHENG H. Pathogenic bacteria distributions and drug resistance analysis in 96 cases of neonatal sepsis[J]. BMC Pediatr, 2017, 17(1): 44. DOI:10.1186/s12887-017-0789-9
[3]
PETTERSEN VK, MOSEVOLL KA, LINDEMANN PC, et al. Coordination of metabolism and virulence factors expression of extraintestinal pathogenic escherichia coli purified from blood cultures of patients with sepsis[J]. Mol Cell Proteomics, 2016, 15(9): 2890-2907. DOI:10.1074/mcp.M116.060582
[4]
AMANATI A, KARIMI A, FAHIMZAD A, et al. Incidence of ventilator-associated pneumonia in criticallyⅢchildren undergoing mechanical ventilation in pediatric intensive care unit[J]. Children, 2017, 4(7): 56. DOI:10.3390/children4070056
[5]
LIMA A, MUNIZ VM, ZANDONADE E, et al. Performance of the pediatric index of mortality 2 in a pediatric intensive care unit[J]. Rev Bras Ter Intensiva, 2014, 26(1): 44-50. DOI:10.1007/s00134-012-2580-6
[6]
EYADHY AA, TEMSAH MH, ALHABOOB AN, et al. Asthma changes at a pediatric intensive care unit after 10 years:observational study[J]. Ann Thorac Med, 2015, 10(4): 243-248. DOI:10.4103/1817-1737.165302
[7]
YAO JF, LI N, JIANG J. Clinical characteristics of bloodstream infections in pediatric acute leukemia:a single-center experience with 231 patients[J]. Chin Med J (Engl), 2017, 130(17): 2076-2081. DOI:10.4103/0366-6999.213411
[8]
NEGUSSIE A, MULUGETA G, BEDRU A, et al. Bacteriological profile and antimicrobial susceptibility pattern of blood culture isolates among septicemia suspected children in selected hospitals Addis Ababa, Ethiopia[J]. Int J Biol Med Res, 2015, 6(1): 4709-4717. DOI:10.1155/2014/153747
[9]
LABI AK, OBENG-NKRUMAH N, BJERRUM S, et al. Neonatal bloodstream infections in a Ghanaian Tertiary Hospital:are the current antibiotic recommendations adequate?[J]. BMC Infect Dis, 2016, 16: 598. DOI:10.1186/s12879-016-1913-4
[10]
WEISS SL, FITZGERALD JC, PAPPACHAN J, et al. Global epidemiology of pediatric severe sepsis:the sepsis prevalence, outcomes, and therapies study[J]. Am J Respir Crit Care Med, 2015, 191(10): 1147-1157. DOI:10.1164/rccm.201412-2323OC
[11]
周维, 向慧, 刘国生. 儿童血培养结果阳性标本的病原菌分布情况及耐药性分析[J]. 湖北民族学院学报(医学版), 2017, 34(3): 36-38. DOI:10.13501/j.cnki.42-1590/r.2017.03.012
[12]
吕春兰, 刘开琴, 陈莹莹, 等. 儿童患者血培养分离菌的分布与耐药性[J]. 中国感染与化疗杂志, 2016, 16(5): 644-647. DOI:10.16718/j.1009-7708.2016.05.021
[13]
HEYER NJ, DERZON JH, WINGES L, et al. Effectiveness of practices to reduce blood sample hemolysis in EDs:a laboratory medicine best practices systematic review and meta-analysis[J]. Clin Biochem, 2012, 45: 999-1011. DOI:10.1016/j.clinbiochem.2012.08.002
[14]
ALTINDIS M, KOROGLU M, DEMIRAY T, et al. A multicenter evaluation of blood culture practices, contamination rates, and the distribution of causative bacteria[J]. Jundishapur J Microbiol, 2016, 9(1): e29766. DOI:10.5812/jjm.29766
[15]
SENG R, KITTI T, THUMMEEPAK R, et al. Biofilm formation of methicillin-resistant coagulase negative staphylococci (MR-CoNS) isolated from community and hospital environments[J]. PLoS One, 2017, 12(8): e0184172. DOI:10.1371/journal.pone.0184172
[16]
BETHELL C, GOMBOJAV N, SOLLOWAY M, et al. Adverse childhood experiences, resilience and mindfulness-based approaches:common denominator issues for children with emotional, mental, or behavioral problems[J]. Child Adolesc Psychiatr Clin N Am, 2016, 25(2): 139-156. DOI:10.1016/j.chc.2015.12.001