脆弱类杆菌基因探针的制备及初步应用研究

本文制备了~(32)P、地高辛配基标记的两种脆弱类杆菌染色体DNA探针,检测厌氧菌和需氧菌共94株、临床标本45份,和培养生化鉴定比较:该探针对临床上常见的类杆菌属细菌均能检出,与其他细菌无交叉反应;其中非同位素——地高辛配基标记探针的灵敏度与同位素~(32)P标记探针相近;和培养生化鉴定比较:地高辛配基标记探针的敏感性为89.47%,特异性为92.31%。     

DNA微阵列芯片法与常规生化法在分枝杆菌菌种鉴定方面的比较

目的通过DNA微阵列芯片法进行分枝杆菌菌种鉴定与常规生化法进行比较,分析其特点,提高分枝杆菌菌种鉴定水平,更好的为临床服务。方法选择我院2010年1月至2013年3月从临床标本中分离培养后所得的结核分枝杆菌复合群12株（其中含1株牛型结核分枝杆菌）,非结核分枝杆菌3l株,分别用DNA微阵列芯片法和常规生化法进行鉴定。结果DNA微阵列芯片法进行分枝杆菌菌型鉴定与普通生化培养法鉴定结果符合率为100％,对常规生化法未定型的两株非结核分枝杆菌也分别定型为1株土分枝杆菌,1株耻垢分枝杆菌。结论DNA微阵列芯片法与常规生化法相比在分枝杆菌菌型鉴定中具有更快速、准确的特点,是分枝杆菌菌型鉴定的有利工具。     

两种念珠菌DNA探针的研制及应用

目的：制备两种念珠菌DNA探针并应用于临床诊断。方法：对聚合酶链反应扩增的白色念珠菌标准株的E03基因的125bp片段，用半抗原地高辛标记；合成仪合成的E03基因中25bp特异寡核苷酸，用生物素标记。检测两种探针显色敏感度，并与23株临床分离的白色念珠菌、热带念珠菌，近平滑念珠菌、克柔念珠菌、星形念珠菌及大肠埃希菌等细菌进行斑点杂交试验。结果：Dig-125bpDNA探针显色敏感度为0.01pg，仅与白色念珠菌杂交，且对临床分离株检测的结果与传统厚膜孢子鉴定法相符。Bio-25bpDNA探针显色敏感度为20pg，与白色念珠菌、近平滑念珠菌及星形念珠菌杂交。结论：Bio-25bp DNA探针可用于念珠菌初步鉴定，而Dig-125bp DNA探针可用于白色念珠菌的鉴定。     

用间接免疫荧光法快速诊断拟杆菌感染

脆弱拟杆菌群(Bacteroides fragilis group)包括一群经常从临床感染标本中分离得到的厌氧杆菌。脆弱拟杆菌(Bacteroides fragilis)是其中最重要并且数量最多的一株,约占菌群的70%以上。脆弱拟杆菌具有其他亚群所没有的致病特性,荚膜多糖是其唯一的毒力因素。用常规培养法作拟杆菌感染的诊断常常是缓慢而不可靠的。改良的细菌学技术方法,诸如用预还原培养基,以及提供适宜的厌氧环境等,虽能提高拟杆菌的检出率,但这样也会使原认为是无菌或仅含单一菌株的标本分离出几种细菌来,从而贻误诊断、     

应用膜芯片技术快速鉴定分枝杆菌菌种

目的：利用膜芯片技术建立一种快速、简便的分枝杆菌分子菌种鉴定方法。方法：以DNA直接测序法为对照，通过PCR．SSCP和膜芯片技术分析11种分枝杆菌标准菌株、9种非分枝杆菌和199株分枝杆菌临床分离株的菌种。结果：应用膜芯片技术分析11种分枝杆菌标准菌株和7种非分枝杆菌菌株，特异性100％。199株分枝杆菌临床分离株中，经16S rRNA PCR-SSCP初步菌种鉴定，30株为结核分枝杆菌复合群，应用膜芯片分析，显示与分枝杆菌属探针分枝杆菌和结核分枝杆菌复合群探针a杂交阳性，两种鉴定方法结果一致：169株PCR．SSCP初步鉴定为非结核分枝杆菌的分离株，经芯片分析，58株为龟分枝杆菌，46株为胞内分枝杆菌，33株为堪萨斯、瘰疬、胃和猿猴分枝杆菌复合群，6株为偶然分枝杆菌，15株为戈登分枝杆菌，3株为乌分枝杆菌，2株为海和溃疡分枝杆菌复合群，另6株只与探针分枝杆菌杂交，经测序显示2株为胞内分枝杆菌，但其基因序列与标准菌株不完全相同，1株为土分枝杆菌，1株为迪氏分枝杆菌，1株为草分枝杆菌，1株为新金色分枝杆菌，芯片上无鉴定该菌种的探针。结论：应用膜芯片技术可简便、快速、灵敏、特异地将大多数分枝杆菌鉴定到种，指导临床合理治疗。     

DNA探针杂交技术与涂片镜检法检测结核杆菌对比分析

目的：评价DNA探针杂交技术检测TB的临床应用价值。方法：应用DNA探针杂交法和涂片镜检法检测330份痰液标本。结果：检测治疗前、后结核病患者标本,DNA探针杂交法阳性检出率分别为56.7%、24.2%,明显高于涂片法检出率（P〈0.01）;两种方法检测90份非结核病患者痰液标本符合率均为100%。结论：DNA探针杂交技术适用于结核病诊断、药物疗效判断等方面。     

DNA芯片检测肝组织及血清中乙型肝炎病毒DNA的临床研究

目的：研究DNA芯片检测乙型肝炎和肝硬化患者肝组织及血清中乙型肝炎病毒（HBV）DNA的应用价值。方法：用点样仪将聚合酶链反应（PCR）扩增的HBV DNA探针制成基因芯片。对15例慢性乙型肝炎病人的血清和肝活检组织，99例乙型肝炎后肝硬化肝组织，分别用基因芯片、原位分子杂交、免疫组织化学和雅培试剂检测HBV DNA、乙型肝炎核心抗原(HBcAg)、乙型肝炎表面抗原（HBsAg)和乙型肝炎e抗原（HBeAg)。结果：用基因芯片对15份HBsAg,HBeAg阳性的乙型肝炎患者血清进行检测，HBV DNA均阳性，阳性率符合率为100％；15份肝活检组织标本，免疫组化法检测HBcAg阳性15例，原位分子杂交法和基因芯片检测HBV DNA均阳性14例，阳性率93％。99份肝炎后肝硬化患者肝组织标本，HBcAg阳性67份，HBV DNA阳性53份，基因芯片检测HBV DNA阳性46份，阳性率分别为69％、88％。32例HBcAg、HBV DNA阳性的肝组织中，基因芯片检测HBV DNA均为阴性。结论：肝炎基因诊断芯片可检测肝组织及血清中HBV DNA，诊断准确率高，假阳性率低。     

API法与16S rRNA法鉴定棒状杆菌比较

目的解决API法无法鉴定的棒状杆菌的鉴定问题，比较API法与16S rRNA法鉴定棒状杆菌的不同。方法86株棒状杆菌临床分离株用法国生物梅里埃API Coryne鉴定系统鉴定，经API法鉴定结果无编码的和可信度小于80％的以及两家医院结果不一致的共9株，自行设计引物用16S rRNA鉴定法完成。结果经API法鉴定结果无编码的和可信度小于80％的以及两家医院结果不一致的共9株棒状杆菌用16S rRNA法得以准确鉴定。结论难以用表型鉴定的棒状杆菌用16S rRNA法得以准确鉴定，表型鉴定法的准确性一定程度上受方法学限制。     

双探针实时荧光定量PCR检测对拉米呋啶耐药的乙型肝炎患者HBV变异

目的研究双探针实时荧光定量PCR方法检测对拉米夫定耐药的乙肝患者血清中HBV病毒变异的可行性。方法用双探针MGB实时荧光定量PCR法检测拉米夫定治疗后的乙型肝炎患者血清,确定HBV YMDD变异类型,与测序法进行比对。结果68例标本中本法测出27例YMDD野生株,20例Y IDD变异株,11例YVDD变异株,与直接测序结果一致。另有10例标本测得为混合株,经克隆后测序证实,混合株中存在优势株。直接测序只能检测出优势株。结论双探针MGB实时荧光定量PCR法可以快速、准确地测出HBV DNA混合变异毒株。     

免疫色谱法抗-MPB64单克隆抗体检测结核分枝杆菌菌群方法学评价

目的：用免疫色谱法抗-MPB64单克隆抗体快速检测和诊断结核分枝杆菌结核菌群的方法学评价。方法：共收集20株临床标本分离菌株、11株参考菌株和1株结核分枝杆菌标准菌株，应用免疫色谱法检测在培养基上生长的细菌，并和传统鉴定方法、实时荧光探针定量PCR法(FQ—PCR)作比较研究。结果：用免疫色谱法检测1株标准菌株为阳性，检测11株参考菌株发现用该法能完全区分结核和非结核分枝杆菌。对20株临床分离的标本用免疫色谱检出11株结核菌菌群，检出率为55％；用传统鉴定方法检出10株，其中未能检出的一株为混合菌感染；用FQ-PCR法检出10株，其中未能检出的一株为牛结核菌。免疫色谱法能检测到的最低菌浓度为10^5CFU／ml。免疫色谱法、FQ—PCR法和传统鉴定方法的平均耗时分别为15分钟，1～2天和30天。结论：免疫色谱法是一种简便、快速、准确、敏感和特异性鉴别结核和非结核分枝杆菌的方法，适合在临床推广使用。     

Differences in susceptibilities of species of the Bacteroides fragilis group to several beta-lactam antibiotics: indole production as an indicator of resistance.

Clinical isolates of the members of the Bacteroides fragilis group differ markedly in their susceptibilities to a variety of beta-lactam antibiotics, including cefoperazone, moxalactam, cefotaxime, cefoxitin, cefamandole, cephalothin, cefazolin, and carbenicillin, as determined by dilution techniques. The minimum concentrations required to inhibit at least 50% of the strains tested (MIC50) for the entire B. fragilis group were lowest with moxalactam and cefoxitin, 4 and 8 micrograms/ml, respectively, whereas the MIC90S of cefoperazone, cefotaxime, moxalactam, cefoxitin, and carbenicillin were equivalent (64 micrograms/ml); the MIC90S of cefamandole, cephalothin, and cefazolin were higher. Indole-positive members of the group (B. ovatus, B. thetaiotaomicron, and B. uniformis) were significantly more resistant to every antibiotic tested than were indole-negative members (B. fragilis, B. distasonis, and B. vulgatus). In a 6-month survey of clinical laboratory data, indole-positive strains comprised 40% of the B. fragilis group isolates and 22% of all Bacteroides isolates; B. fragilis was the most common species isolated (23%). The increased use of second-generation and the introduction of third-generation cephalosporins may dictate that clinical microbiology laboratories routinely identify members of the B. fragilis group as to species or, alternatively, test for indole production in addition to performing more extensive susceptibility testing.     

Ertapenem (MK-0826), a new carbapenem: comparative in vitro activity against clinically significant anaerobes

Ertapenem, a new long acting beta-lactam with broad-spectrum antimicrobial coverage, was tested in vitro to compare its activity against 556 clinical isolates of anaerobes to other established agents using a broth microdilution method as recommended by the NCCLS. Against all anaerobes ertapenem inhibited 99.1% of isolates at 4 microg/mL, had a mode MIC of 0.12 microg/mL, and showed activity comparable to imipenem, meropenem, trovafloxacin, piperacillin-tazobactam, and metronidazole. Five of the B. fragilis group (4 B. fragilis, 1 B. vulgatus) isolates tested showed reduced susceptibility (>or=8 microg/mL; <1%) to ertapenem while all isolates of Prevotella, Porphyromonas, Fusobacterium, and Peptostreptococcus were susceptible. Only piperacillin-tazobactam had susceptible MIC's for all test isolates followed by metronidazole and the carbapenems exhibiting low resistance rates (<1%).     

Detection of a specific bacterial antigen in urine of rats with Bacteroides fragilis infection

Human intra-abdominal infections frequently yield Bacteroides fragilis and require specific antimicrobial and surgical therapy. Noninvasive immunologic assessment of this organism might allow more optimum therapy. Therefore we raised antisera in goats to Bacteroides fragilis ATCC 23745 and allowed it to react with a solid-phase capsular polysaccharide-protein antigen extracted from the same organism. Preliminary work disclosed that 10 ng/ml antigen could be detected in competition assays in both saline and dialyzed rat urine. Results were manifest by diminution of bound antiglobulin alkaline phosphatase conjugate in an antigen-mediated antibody-inhibition enzyme-linked immunosorbent assay. Rats were then infected intra-abdominally with (1) B. fragilis ATCC 23745; (2) one of eight recent clinical isolates of B. fragilis; or (3) one of nine isolates representative of Enterobacteriaceae. Seventy-two rat urine samples obtained prior to infection disclosed essentially no assay inhibition: 98.3% +/- 10.3 (1 S.D.). Mean values of reagent antibody activity after incubation with urine aliquots from 24 hr samples collected between 24 and 72 hr were (1) strain 23745 (n = 35) 70.9% +/- 2.6 (S.E.); (2) eight isolates of B. fragilis (n = 49) 86.8% +/- 1.9; (3) nine isolates of Enterobacteriaceae (n = 47) 100.9% +/- 1.0; and (4) shams (n = 29) 95.5% +/- 1.55. Ascribing values less than or equal to 77.7% (2 S.D.) as positive, seven of the eight clinical B. fragilis isolates causing infection were detected in at least one 24 hr urine sample (sensitivity = 87% by organism); 12 of 17 infected rats were correctly identified as positive by at least one urine (sensitivity = 70.6% by rat). Specificity, as assessed in the Enterobacteriaceae group, was 89% (by organism) and 94.5% (by rat). Collectively, these results suggest the presence of a potentially specific, soluble antigen excreted in the urine of rats with B. fragilis infection.     

Antimicrobial susceptibilities of anaerobic bacteria: recent clinical isolates

Minimal inhibitory concentrations of clindamycin, minocycline, metronidazole, penicillin, and carbenicillin were determined by agar dilution against 150 recent clinical isolates of anaerobic bacteria. Ninety-nine percent of Bacteroides fragilis and all B. melaninogenicus, Clostridium perfringens, and Fusobacterium were inhibited by clindamycin at 3.1 mug/ml. Only 58% of other clostridial species were inhibited by this concentration of clindamycin. Minocycline at 3.1 mug/ml inhibited 72% of C. perfringens, 81% of other Clostridium species, and 66, 75, and 100% of B. fragilis, B. melaninogenicus, and Fusobacterium, respectively. Metronidazole at 12.5 mug/ml inhibited all bacteria tested. B. fragilis was resistant to both penicillin and carbenicillin at 6.2 mug/ml. Concentrations of 25 mug/ml for penicillin and 100 mug/ml for carbenicillin were needed to inhibit more than 90% of B. fragilis. Organisms other than B. fragilis were moderately or extremely susceptible to the penicillins.     

Genotyping of Bacteroides fragilis isolates from stool specimens by arbitrarily-primed-PCR

In order to determine genetic relatedness of Bacteroides fragilis isolates from different clinical sources, arbitrarily primed polymerase chain reaction (PCR) (AP-PCR) was used to compare 17 strains isolated from patients with inflammatory bowel disease (IBD) and 20 strains isolated from foals with diarrhea. Three reference ATCC strains were also analyzed. Eighteen unique types were identified with a 22-mer arbitrary primer (ERIC-2) among the 20 patient isolates. Types 1 (enterotoxigenic) and 9 (nonenterotoxigenic), were each found in the stools of two patients. All other isolates showed a distinct and unique DNA banding pattern indicating a high degree of genotypic variability. Eleven types were identified among the foal isolates. Type 20, a nonenterotoxigenic type, was present in 30% of the foals. No correlation was found between the human and horse isolates. No clear relationship between a disease state (diarrhea or IBD) and specific types was observed. AP-PCR will be useful as a rapid method to determine genetic relatedness and in future epidemiologic studies of diarrheal diseases due to B. fragilis.     

First national survey of antibiotic susceptibility of the Bacteroides fragilis group: emerging resistance to carbapenems in Argentina

The antibiotic susceptibility rates of 363 clinical Bacteroides fragilis group isolates collected from 17 centers in Argentina during the period from 2006 to 2009 were as follows: piperacillin-tazobactam, 99%; ampicillin-sulbactam, 92%; cefoxitin, 72%; tigecycline, 100%; moxifloxacin, 91%; and clindamycin, 52%. No metronidazole resistance was detected in these isolates during this time period. Resistance to imipenem, doripenem, and ertapenem was observed in 1.1%, 1.6%, and 2.3% of B. fragilis group strains, respectively. B. fragilis species showed a resistance profile of 1.5% to imipenem, 1.9% to doripenem, and 2.4% to ertapenem. This is the first report of carbapenem resistance in Argentina. The cfiA gene was present in 8 out of 23 isolates, all of them belonging to the B. fragilis species and displaying reduced susceptibility or resistance to carbapenems (MICs ≥ 4 μg/ml). Three out of eight cfiA-positive isolates were fully resistant to carbapenems, while 5 out of 8 isolates showed low-level resistance (MICs, 4 to 8 μg/ml). The inhibition by EDTA was a good predictor of the presence of metallo-β-lactamases in the fully resistant B. fragilis strains, but discrepant results were observed for low-level resistant isolates. B. fragilis was more susceptible to antimicrobial agents than other Bacteroides species. Bacteroides vulgatus species was the most resistant to ampicillin-sulbactam and piperacillin-tazobactam, and B. thetaiotaomicron/ovatus strains showed the highest level of resistance to carbapenems, with an unknown resistance mechanism. B. vulgatus and the uncommon non-Bacteroides fragilis species were the most resistant to moxifloxacin, showing an overall resistance rate of 15.1%.     

Comparison of the activities of penicillin G and new beta-lactam antibiotics against clinical isolates of Bacteroides species.

MICs were determined for 218 clinical isolates of Bacteroides by a broth microdilution method. Imipenem was the most active antibiotic tested. Azlocillin, mezlocillin, and cefoxitin had comparable activities, with resistance among members of the B. fragilis group and B. capillosus. Ceftizoxime was the most active cephalosporin tested. Members of the B. fragilis group showed high levels of resistance to cefotetan and ceftazidime. Resistance to penicillin G varied from 0 to 14%.     

Non-radioactive DNA probe for the rapid identification of Mycobacterium avium complex from clinical specimens.

We evaluated an alkaline phosphatase labelled oligonucleotide probe (SNAP(TM), Syngene Co., Molecular Biosystems, Inc., San Diego CA) for the direct culture identification of Mycobacterium avium complex (MAC) isolated from clinical specimens. Mycobacterial species identified by conventional biochemical methods were retested with this DNA probe using the Centri-Dot(TM) format. The probe accurately identified all 69 pigmented M. avium complex and 15 non-pigmented isolates of M. avium complex. There were no false-positives with 45 non-MAC mycobacteria isolates (10 species) and 16 non-mycobacteria organisms (10 species). The sensitivity and specificity of the SNAP(TM) culture identification for M. avium complex were 100%. The alkaline phosphatase labelled DNA probe is stable for at least 9 months. The procedure can be completed within 2 h and is easily adapted in the clinical laboratory. For the strains encountered in our laboratory, we conclude that the SNAP(TM) hybridization is a rapid, specific, and reliable method for culture identification of M. avium complex.     

Bacteremia due to Bacteroides fragilis group: distribution of species,beta-lactamase production,and antimicrobial susceptibility patterns

A retrospective analysis of susceptibility data on 542 blood isolates of the Bacteroides fragilis group tested from 1987 to 1999 by the same NCCLS-recommended broth microdilution method throughout is presented. Metronidazole, beta-lactam-beta-lactamase inhibitor combinations, carbapenems, and trovafloxacin were the most active agents (susceptibility of >or=93%). Among the cephalosporin-cephamycins, the order of activity was cefoxitin > ceftizoxime > cefotetan = cefotaxime = cefmetazole > ceftriaxone. All isolates were resistant to penicillin G, and 22% were resistant to clindamycin. The susceptibility rates to piperacillin-tazobactam, imipenem, and meropenem were affected least among isolates resistant to cefoxitin or clindamycin. Except for piperacillin-tazobactam, imipenem, and meropenem, the B. fragilis species was more susceptible than were the non-B. fragilis species. These data underscore the importance of susceptibility testing of the B. fragilis group and can serve as a guide in the choice of empirical antimicrobial therapy.