Trends in antimicrobial resistance of Acinetobacter baumannii clinical isolates from hospitalised patients in Greece and treatment implications

This study analysed the proportions of Acinetobacter baumannii isolates resistant to various antibiotics that were recovered from patients hospitalised in Greek hospitals between 1996 and 2006. The microbiological data were derived from the ongoing WHONET Greek System for the Surveillance of Antibiotic Resistance. There were increases in the proportions of A. baumannii isolates resistant to imipenem from patients hospitalised in intensive care units, medical wards and surgical wards during the study period from 0% to 91%, 8% to 71%, and 5% to 71%, respectively.     

Clonal relationship and the association of the ST218 strain harboring blaOXA-72 gene to mortality in carbapenem-resistant Acinetobacter baumannii bacteremia

Background/purpose

In 2017, the World Health Organization categorized carbapenem-resistant Acinetobacter baumannii (CRAB) as a priority 1, critical antibiotic-resistant bacteria. This study analyzed the clinical outcomes and investigated the molecular epidemiology of CRAB bacteremia in a medical center in Northern Taiwan.

Analysis on drug-resistance and molecular epidemiology of Acinetobacter baumannii isolated from the clinical samples in two Chinese hospitals

In the present study, the drug-resistance genes encodingβ-lactamases, aminoglycoside modifying enzymes, DNA topoisomerases and integron as well as their molecular epidemiology were investigated by means of analyzing the drug-resistance and molecular epidemiology of Acinebacter bau mannii isolated from the clinical samples in two hospitals in Qiangzhou and Huzhou city of Jiangsu and Zhejiang province from July 2000 to March 2005. The minimal inhibitory concentrations (MICs) of these 307 isolates were detected by automatic microbiological system, and 35 strains against 5-fluoro-quinolones were performed by agar dilution assay. Meanwhile, the resistant genes in 80 isolates were amplified by PCR with identification by DNA sequencer. It was found that most of the 307 isolates of A . baumannii were resistant to multiple antibiotics tested, in which the resistance rates of the isolates against piperacillin, piperacillin/tazobactam, amoxacillin/clavulanic acid, cefotaxime, ceftazidime, cefepime, gentamicin, amikacin, ciprofloxacin, chloramphenicol and sulfamethoxazole/trimethoprim were all above 35% , but those of imipenem and meropenem were quite low, ranged only 2.6% and 3.3%. In addition, it was also demonstrated that the positive rates of TEM and SHVβ-lactamase genes accounted for 93.8% and 22.5% respectively, and those of the aminoglycoside-modifying enzyme genes including aacC1, aacC2, aacC3, aacC4, aacC4A, aphA6, ant(2")-I and ant(3") I were 58.8%, 8.8%, 7.5%, 28.8%, 45.0%, 2.5%, 28.8% and 65.0% respectively. The mutations in the quinolone-resistant determining region (QRDR) of gyrA and parC genes indicated that substitution in Ser-83 residue of GyrA protein was most frequently occurred among strains with MIC for ciprofloxacin of more than 4μg/ml, whereas a double mutation at Ser-83 residue of gyrA and Ser-80 of parC was found in strains with MIC of ciprofloxacin of more than 8μg/ml. As to the positive rates of class 1 integron (Int I -1) and qacE△1-sul-1, it was found to be 60.0% and 77.5% respectively, and the rates of resistant genes of strains isolated in these two hospitals varied considerably. The results obtained in the present study indicate the presence of the multiple resistant genes in strains of A. baumannii , and great measures should be taken to control the spread of the resistant strains carrying the resistant genes.     

成都地区肥胖患者GNB3基因825C/T多态性研究

目的 研究G蛋白β3亚单位(G-protein β3 subunit,GNB3)基因825C/T多态性是否与中国人肥胖有关联,为探讨肥胖的分子遗传基础提供依据.方法 应用聚合酶链反应-限制性片段长度多态性分析法,对成都地区270名非肥胖者及129例肥胖患者GNB3基因825C/T多态性位点进行分析,采用酶法和单向免疫扩散法对血脂和载脂蛋白水平进行测定.结果 GNB3基因825C/T位点C、T等位基因的频率在肥胖组为0.531、0.469,在非肥胖组为0.528、0.472,两绀间等传基因的频率差异无统计学意义(P＞0.05).中国人825C/T位点T等位基因频率(0.471,合并组)较德国白人的0.319显著增高(P＜0.01),而低于非洲黑人的0.788(P＜0.01),与日本人的0.487相近(P＞0.05).825C/T位点在非肥胖组TT基因型携带者血清甘油三酯水平高于CT基因型者(P＜0.05);在肥胖组CC基因型携带者血清高密度脂蛋白胆固醇水平较CT基因型者降低(P＜0.05).进一步按件别分层后,这种差异仪在相应各组的男性、女性亚组存在;此外,非肥胖男性TT型者、女性CC型者血清高密度脂蛋白胆固醇、载脂蛋白A Ⅰ水平分别低于和高于相应业组CT型者(P＜0.05),肥胖男性亚组TT型者血清载脂蛋白A Ⅰ水平高于CC型者(P＜0.05).结论 GNB3基因825C/T多态性与中国成都地区汉族人肥胖无关联,但与血清甘油三脂、高密度脂蛋白胆同醇和载脂蛋白A Ⅰ水平含量有关,且具有性别差异存在.     

牙龈卟啉单胞菌prtH基因分布与多态性研究

目的　分析牙龈卟啉单胞菌 (P .gingivalis)prtH基因的遗传多态性 ,从而了解细菌变异与其对牙周致病作用的相关性。方法　以PCR技术从牙周炎患者口腔中分离的P .gingivalis进行prtH基因扩增 ;采用斑点杂交法 ,以生物素标记prtH基因为探针 ,与以引物B进行PCR扩增为阳性的P .gingivalis基因组DNA杂交 ;对PCR产物进行AluⅠ限制性酶切和DNA测序分析。结果　以prtH基因A、B引物对 14 0株临床分离株进行PCR扩增 ,分别在 76例 (引物A)和 117例 (引物B)出现阳性扩增产物 ,阳性率分别为 5 4 .2 %、84 %。对 34例引物B扩增阳性的P .gingivalisDNA进行点杂交检测 ,阳性吻合率为 82 .4 %。与GenBank中菌株ATCC332 77(U15 2 82 )和W83(L2 74 83)的prtH基因序列相比 ,5株P .gingivalisPCR扩增产物的测序存在着缺失和点突变。结论　在慢性牙周炎患者临床P .gingivalis分离菌株中存在着prtH基因的遗传多态性 ,本实验建立了具有高度敏感性和特异性的PCR检测方法     

Facial asymmetry and clinical manifestations in patients with novel insertion of the TCOF1 gene

Su P-H, Liu Y-F, Yu J-S, Chen J-Y, Chen S-J, Lai Y-J. Facial asymmetry and clinical manifestations in patients with novel insertion of the TCOF1 gene. This study explored the role of TCOF1 insertion mutations in Taiwanese patients with craniofacial anomalies. Twelve patients with single or multiple, asymmetrical congenital craniofacial anomalies were enrolled. Genomic DNA was prepared from leukocytes; the coding regions of TCOF1 were analyzed by polymerase chain reaction and direct sequencing. Clinical manifestations were correlated to the TCOF1 mutation. Six of 12 patients diagnosed with hemifacialmicrosomia exhibited a novel insertion mutation 4127 ins G (frameshift) in exon 24 in the TCOF1 gene. All six patients were diagnosed with anomalies on the left side. In addition, four of these six patients had hearing impairment; three had other major anomalies; and two had developmental delay. The insertion caused a frameshift, an early truncation, the loss of two putative nuclear localization signals (residues 1404-1420 and 1424-1440), and the loss of coiled coil domain (1406-1426) in treacle protein. These findings support the existence of two regulators of growth of the mandibular condyles.     

Evaluation of differential gene expression in susceptible and resistant clinical isolates of Klebsiella pneumoniae by DNA microarray analysis

DNA microarray technology was used to evaluate differential gene expression in a susceptible Klebsiella pneumoniae isolate and a resistant clinical derivative. Nineteen genes were up-regulated in the resistant isolate when compared with the susceptible isolate. An ABC transporter-related gene, ycjV, was strongly over-expressed, suggesting the existence of a novel active efflux mechanism. Approximately half of the up-regulated genes coded for ribosomal proteins, or proteins involved in tRNA metabolism. Among 33 downregulated genes, almost one-third were related to nitrogen metabolism. A possible role of fitness in the development of antimicrobial resistance is suggested.     

牙龈卟啉单胞菌临床菌株fimA基因变异性和表达频率及其重组表达产物的致炎作用

目的了解牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）归以基因变异性，构建归以基因原核表达系统，鉴定其表达产物（rFimA）的致炎作用，检测FimA在咯临床菌株中表达频率，了解FimA与慢性牙周炎的关系。方法采用高保真PCR从6株咯临床菌株中扩增fimA基因并测定其核苷酸序列。构建fimA基因原核表达系统，制备rFimA兔抗血清。采用Western blot鉴定rFimA抗原性和免疫反应性。建立ELISA检测38株段临床菌株FimA表达情况和97例慢性牙周炎患者212份龈下菌斑标本中的FimA。检测rFimA诱导人脐静脉内皮EVC-304细胞分泌n1、IL-8、TNF-α和细胞问黏附分子-1（ICAM-1）的作用。结果6株Pg临床菌株fimA基因核苷酸序列完全相同。所构建的原核表达系统rFimA产量高达细菌总蛋白的50％左右。rFimA可与Pg全菌抗血清发生结合反应，免疫家兔可获得高效价抗体。94．7％（36／3S）菌株和91．5％（194／212）龈下菌斑标本ELISA结果阳性。1和10μg的rFimA作用EVC-304细胞48h，其分泌的IL-1α水平升高（P〈0．05）；但作用12h即可出现高水平的ICAM-1（P〈0．05），未发现有诱导IL-8和TNF-α的作用（P〉0．05）。结论 fimA基因序列保守，在Pg临床菌株中有很高的表达频率。rFimA有良好的抗原性和免疫反应性，可作为Pg血清学检测试剂盒和段疫苗的候选抗原。rFimA有较强的诱导细胞分泌ICAM-1的作用。     

Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii

Purpose: The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla_NDM-1 gene using TaqMan probes among clinical isolates. Materials and Methods: Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. Results: TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla_NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12–17) cycles (mean Cp value 14), indicating number of bla_NDM-1 gene copies of 106–108. The wider range of Cp values (15–34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla_NDM-1 gene copies of 103–108. Conclusions: The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla_NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla_NDM-1 gene copies per specimen of DNA.     

丙型肝炎病毒核心蛋白基因重组质粒的构建及其在真核细胞中的表达

目的：构建包含丙型肝炎病毒（ＨＣＶ）核心蛋白（Ｃ）基因片段的重组真核表达载体，并在肝细胞癌细胞株７７２１细胞中表达。方法：将从ｐＢＲＴＭＨＣＶ１－３０１１质粒切下的ＨＣＶＣ基因片段插入ｐｃＤＮＡ３质粒的ＣＭＶ启动子下游，构建真核表达质粒ｐｃＤＮＡＨＣＶ－Ｃ，然后，采用脂质体转染技术，转染７７２１细胞进行瞬时表达，转染细胞裂解煮沸后，通过ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ检测表达的核心抗原。结果：用限制性内切酶酶切后，片段大小与计算值相符。Ｗｅｓｔｅｒｎｂｌｏｔ证实，表达抗原的Ｍｒ约为２２０００。结论：ＨＣＶＣ基因能够插入ｐｃＤＮＡ３真核表达载体，并使其在真核细胞中表达，为进一步ＨＣＶ基因疫苗的研制和探讨抗ＨＣＶ感染打下了基础。     

Amino acid substitution mutations and mRNA expression levels of the pbp5 gene in clinical Enterococcus faecium isolates conferring high level ampicillin resistance

In this study, clinical ampicillin‐resistant Enterococcus faecium isolates with minimum inhibitory concentrations (MICs) for ampicillin in the ranges from 128 to ?512 μg/mL (n = 17) and two ampicillin‐susceptible isolates (MIC 1 μg/mL) were investigated. No β‐lactamase production was detected in these isolates. Alterations in the C‐terminal part of pbp5 and levels of pbp5 mRNA expression were investigated by sequencing and quantitative real‐time qRT‐PCR, respectively. Sequencing analysis revealed five different pbp5 alleles (A to E) having differences in 18 amino acid positions spanning from residue 426 to 642. Allele A (V‐462 → A, H‐470 → Q, M‐485 → A, N‐496 → K, A‐499 → T, E‐525 → D, N‐546 → T, A‐558 → T, G‐582 → S, E‐629 → V, K‐632 → Q, and P‐642 → L) was the most frequent allele. The presence of just two susceptible isolates in allele E suggests a possible correlation between amino acid patterns and MIC, even if there is no discernible correlation with specific single amino acid differences. Also, these were the only isolates that showed much lower expression of class B penicillin‐binding protein 5 (PBP5) compared to isolates with MIC of 128 or greater. Thus, ampicillin MICs were correlated with PBP5 expression.     

cDNA microarray analysis of differential gene expression and regulation in clinically drug-resistant isolates of Candida albicans from bone marrow transplanted patients

Fungi have emerged as the fourth most common pathogens isolated in nosocomial bloodstream infections, and Candida albicans is the most common human fungal pathogen. Only a few antibiotics are effective in the treatment of fungal infections. In addition, the repetition and lengthy duration of fluconazole therapy has led to an increased incidence of azole resistance and treatment failure associated with C. albicans. To investigate the mechanism of drug resistance and explore new targets to treat clinically resistant fungal pathogens, we examined the large-scale gene expression profile of two sets of matched fluconazole-susceptible and -resistant bloodstream C. albicans isolates from bone marrow transplanted (BMT) patients for the first time by microarray analysis. More than 198 differentially expressed genes were identified and they were confirmed and validated by RT-PCR independently. Not surprisingly, the resistant phenotype is associated with increased expression of CDR mRNA, as well as some common genes involved in drug resistance such as CaIFU5, CaRTA2 and CaIFD6. Meanwhile, some special functional groups of genes, including ATP binding cassette (ABC) transporter genes (IPF7530, CaYOR1, CaPXA1), oxidative stress response genes (CaALD5, CaGRP1, CaSOD2, IPF10565), copper transport and iron mobilization-related genes (CaCRD1/2, CaCTR1/2, CaCCC2, CaFET3) were found to be differentially expressed in the resistant isolates. Furthermore, among these differentially expressed genes, some co-regulated with CaCDR1, CaCDR2 and CaIFU5, such as CaPDR16 and CaIFD6, have a DRE-like element and may interact with TAC1 in the promoter region. These findings may shed light on mechanisms of azole resistance in C. albicans and clinical antifungal therapy.     

腹泻儿童粪便分离的大肠杆菌中eaeA基因的检测及携带eaeA菌株的特性研究

用碱性磷酸酶标记的ｅａｅＡ基因寡核苷酸探针，对３７个血清群的２２１株从肯尼亚５岁以下腹泻患儿粪便中分离的大肠杆菌进行检测，并对ｅａｅＡ基因阳性菌株进一步做了ｂｆｐＡ和ｓｌｔ基因检测，ＨＥｐ－２细胞粘附及荧光肌纤蛋白染色（ＦＡＳ）试验。结果表明，ｅａｅＡ基因的携带率为１９．５％，ＥＰＥＣ、ＥＨＥＣ和其它血清群中ｅａｅＡ基因的携带率分别为３１．６％、６６．７％和８．９％，根据ｂｆｐＡ和ｓｌｔ基因检测及ＨＥｐ－２细胞粘附和ＦＡＳ试验的结果，可将携带ｅａｅＡ基因的大肠杆菌分成５种类型。提示ｅａｅＡ基因的分布不仅限于ＥＰＥＣ和ＥＨＥＣ，而且携带ｅａｅＡ基因菌株含有多种毒力决定因子。     

问号钩端螺旋体rLTB/rCTB-LipL32/1免疫保护作用及野生株LipL32基因表达和患者抗体检测

目的构建LTB—LipL32／1和CTB—LipL32／1融合基因原核表达系统并鉴定其表达产物的免疫和佐剂活性及保护作用，了解问号钩端螺旋体(简称钩体)野生株LipL32基因携带、表达及钩体病人血清LipL32基因产物抗体产生频率。方法采用常规分子生物学技术构建LTB—LipL32／1和CTB—LipL32／1融合基因及其原核表达系统。采用SDS-PAGE检测目的重组蛋白rLTB-LipL32／1及rCTB-LipL32／1表达情况。分别采用Western blot和GM1-ELISA检测rLTB-LipL32／1及rCTB—LipL32／1的免疫反应性和佐剂活性。采用PCR和MAT分别检测97株问号钩体野生株LipL32基因及其表达情况。采用ELISA检测228例钩体病人血清LipL32基因产物的抗体。采用豚鼠保护试验检测重组蛋白的免疫保护作用。结果与报道的相关序列比较，LTB—LipL32／1和CTB-LipL32／1融合基因核苷酸序列相似性分别为99．12％～99．71％和98．54％～99．42％，氨基酸序列相似性分别为97．58％-99．63％和96．77％～99．63％。rLTB-LipL32／1和rCTB—LipL32／1表达产量均约为细菌总蛋白的10％，并主要以包涵体形式存在。rLTB—LipL32／1和rCTB-LipL32／1均分别能与LipL32／1兔抗血清和牛GMI结合。97．9％(95／97)问号钩体野生株含有LipL32基因，95．9％(93／97)问号钩体野生株与rLipL32／1和rLipL32／2兔抗血清的MAT结果阳性。97．4％(222／228)和94．7％(216／228)的病人血清rLipL32／1和rLipL32／2抗体阳性。rLTB-LipL32／1、rCTB-LipL32／1和rLipL32／1豚鼠保护率分别为75．0％、75．0％～87．5％、50．O％～62．5％。结论成功构建了LTB—LipL32／1和CTB—LipL32／1融合基因及其原核表达系统。rLTB-LipL32／1和rCTB-LipL32／1融合蛋白有良好的抗原性和佐剂活性及一定的免疫保护作用，具有成为问号钩体属特异性疫苗的良好前景。LipL32／1是不同问号钩体血清群中广泛存在、序列保守、高频率表达的基因。     

循环癌细胞p53表达在大肠癌患者检测中的临床意义

目的探讨从大肠癌患者外周血检测循环癌细胞胡表达的临床意义。方法用流式细胞术，对术前128例大肠癌患者外周血循环癌细胞胡异常表达进行检测，实验数据用SPSS统计软件处理。结果大肠癌外周血循环癌细胞胡异常表达与患者淋巴结转移显著相关（P〈0．01）；p53异常表达与患者肿瘤分化程度密切并呈正相关（r=0．8476，P〈0．05）；p53异常表达与淋巴结转移在左右结肠癌病例中差异具有统计学意义（P〈0．05）。结论外周血中可检出大肠肿瘤细胞生物学标志物胡突变表达，这可为大肠痛预测复发及远端微转移提供了一个有效的手段。     

Cytogenetic analyses of chromosomal rearrangements in Mus minutoides/musculoides from North-West Zambia through mapping of the telomeric sequence (TTAGGG)n and banding techniques

Three specimens of M. minutoides/musculoides from Zambia were cytogenetically studied through G- and C-banding, DAPI staining and fluorescence in-situ hybridization (FISH) with a (TTAGGG)_n telomeric sequence. Biarmed chromosomes were identified according to the current nomenclature as follows: Rb(2.7), Rb(3.12), Rb(4.5), Rb(6.8), Rb(9.16), and the sex chromosomes Rb(1.X), Rb(1.Y) and Rb(1.X_d), originated from the deleted X chromosome. One female showed the diploid number 2n=24; in the two other individuals, the Rb(9.16) occurred in a heteromorphic condition, and, accordingly, the diploid number was 2n=25. FISH showed the sites of telomeric sequences at telomeres of all the chromosomes, and in an interstitial position at the centromeres of all Robertsonian metacentrics, except one – the Rb(6.8), though the patterns of hybridization varied between chromosomes. Sex chromosome pairs, in the male and females, showed a similar C-banding pattern, but revealed clear differences after FISH. Traces of telomeric sequences were found dispersed in the whole-heterochromatic arm of the Rb(1.X_d). No visible bond between C-positive heterochromatin and telomeric sequences were detected in the other either bi- or uniarmed chromosomes, indicating that they may actually represent retained telomeres in the Robertsonian metacentrics. This revised version was published online in July 2006 with corrections to the Cover Date.     

CreERT2 expression from within the c‐Kit gene locus allows efficient inducible gene targeting in and ablation of mast cells

Mast cells are abundantly situated at contact sites between the body and its environment, such as the skin and, especially during certain immune responses, at mucosal surfaces. They mediate allergic reactions and degrade toxins as well as venoms. However, their roles during innate and adaptive immune responses remain controversial and it is likely that major functions remain to be discovered. Recent developments in mast cell‐specific conditional gene targeting in the mouse promise to enhance our understanding of these fascinating cells. To complete the genetic toolbox to study mast cell development, homeostasis and function, it is imperative to inducibly manipulate their gene expression. Here, we report the generation of a novel knock‐in mouse line expressing a tamoxifen‐inducible version of the Cre recombinase from within the endogenous c‐Kit locus. We demonstrate highly efficient and specific inducible expression of a fluorescent reporter protein in mast cells both in vivo and in vitro. Furthermore, induction of diphtheria toxin A expression allowed selective and efficient ablation of mast cells at various anatomical locations, while other hematopoietic cells remain unaffected. This novel mouse strain will hence be very valuable to study mast cell homeostasis and how specific genes influence their functions in physiology and pathology.     

MAGE—1基因在人食管癌中的表达及基因克隆

目的 研究MAGE-1基因在人食管癌中的表达,分析MAGE-1抗原能否作为食管癌的治疗性候选疫苗。方法 采用RT-PCR方法检测了MAGE-1基因在30例食管癌组织和相应癌旁正常组织中的表达;并从食管癌组织中扩增了MAGE-1cDNA全长序列,经酶切后与pET-30a( )质粒载体连接,经初步酶切鉴定后,进行DNA序列分析。获得克隆基因的核苷酸序列。结果 MAGE-1基因在食管癌组织中的表达率为43%（13/30）,在30例相应癌旁正常组织中未见表达;成功地克隆了MAGE-1cDNA全长序列。结论 由于MAGE-1基因在食管癌中高频率表达以及MACE-1抗原具有HLA-I类分子限制性CTL识别表位,因此,MAGE-1抗原可以作为食管癌免疫治疗的候选疫苗。