Evaluation of a biochemical test scheme for identifying clinical isolates of Enterococcus faecalis and Enterococcus faecium

AIMS: To evaluate the full test scheme of Facklam and Sahm (1995) for the identification of clinical enterococcal isolates to genus and species level. METHODS AND RESULTS: Fifty-nine clinical isolates, previously provisionally classed as enterococci on the basis of just four biochemical tests of Facklam and Sahm and one other test, were subjected to genus and species identification using the full identification scheme of Facklam and Sahm; 98% of these strains were confirmed to be enterococci and of these, 69% were identified as Enterococcus faecalis and 31% as Enterococcus faecium. Six tests in the scheme (out of 24) gave anomalous or unreliable results for some strains, and two gave unexpected results for the majority of strains presumptively identified as Ent. faecium. CONCLUSIONS: Nine (out of 12) genus tests and nine (out of 12) species tests from the Facklam and Sahm scheme were reliable. Testing for the presence of the Lancefield antigen D was also useful. The majority of presumptive Ent. faecium strains gave different results for the sorbitol and raffinose tests from that expected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the level of reliability for each of the tests in a current enterococcal identification scheme for differentiating clinical isolates, and showed that two tests gave consistently different test results from those expected for Ent. faecium.     

铜绿假单胞菌的MALDI-TOF-MS检测方法的建立

目的 建立利用基质辅助激光解吸电离飞行时间质谱仪( MALDI-TOF-MS)对铜绿假单胞菌的快速检测方法.方法 通过MALDI-TOF-MS法对铜绿假单胞菌进行检测分析,并与生化鉴定方法相比较.结果 MALDI-TOF-MS对铜绿假单胞菌的检测后得到肽指纹图片及相关质谱数据,建立MALDI-TOF-MS对铜绿假单胞菌的快速检测方法.结论 MALDI-TOF-MS方法检测铜绿假单胞菌准确快速、操作简单等特点,可发展成为食品检验铜绿假单胞菌的重要(辅助)工具.     

Evaluation of a molecular identification scheme based on 23S rRNA gene polymorphisms for differentiating canine and feline gastric Helicobacter spp

A scheme for the rapid identification of Helicobacter spp. using restriction fragment length polymorphism digestion profiles of PCR amplified 23S rRNA genes is described. The efficacy of this scheme for speciation of the closely related gastric species H. felis, H. bizzozeronii and H. salomonis was evaluated. It was difficult to distinguish between some RFLP profiles obtained and often, more than one profile was seen with each species examined. Some evidence was found that the 23S rRNA gene copies of these species may not be identical. Moreover, the identification scheme was ineffective in discriminating these species from each other, although they could be differentiated, as a group, from other Helicobacter spp. The results indicate that this scheme should be carefully evaluated with a number of isolates if it is to be applied to additional, highly related Helicobacter spp.     

Multiplex PCR for rapid differentiation of three species in the "Clostridium clostridioforme group"

Clostridium clostridioforme is a relatively antimicrobial resistant, phenotypically heterogeneous anaerobe that has been involved in a variety of infections. 16S rDNA sequencing analysis revealed three principal species in what has been called Clostridium clostridioforme - Clostridium bolteae, C. clostridioforme, and Clostridium hathewayi. Based on the 16S rDNA sequence information we obtained, we developed a cost-effective, timesaving one-step multiplex PCR assay for rapid and accurate differentiation of these three species. The established multiplex PCR identification scheme was applied to the identification of 88 clinical isolates that had previously been identified phenotypically as C. clostridioforme. The identification obtained from multiplex PCR assays showed 100% agreement with 16S rDNA sequencing identification. This scheme will permit more accurate assessment of the role of these three Clostridium species in infection and of the degree of antimicrobial resistance in each of the species.     

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.     

A proposed serotyping system for Flavobacterium psychrophilum

AIMS: To develop a common serological system for rapid and routine identification of the fish pathogen Flavobacterium psychrophilum. METHODS: Thirty-four isolates of Fl. psychrophilum from different fish species and different geographical areas were typed using a slide agglutination test and an enzyme-linked immunosorbent assay (ELISA). RESULTS: Seven host-dependent serovars (1: salmon; 2: trout; 3: trout; 4: eel; 5: carp; 6: tench; 7: ayu) were found. Serovar 2 was divided into two antigenic subgroups (2a and 2b). The results achieved by both slide agglutination and ELISA methods were totally consistent with each other. Although both techniques proved to be simple to carry out and useful, only the ELISA allowed identification of Fl. psychrophilum serovars using unabsorbed antiserum and whole-cells as antigens. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proposes a harmonized scheme for serological identification of Fl. psychrophilum to be used for diagnostic and seroepidemiological studies of the diseases it causes.     

拉曼光谱技术在病原菌感染诊断中的应用进展

快速准确诊断感染性疾病病原体是遏制超级细菌传播和抗生素滥用的重要防线。目前,临床病原菌感染诊断十分依赖于培养手段,导致检测周期长达数日,不但影响了患者的及时诊治,还间接导致抗生素的滥用。拉曼光谱技术是一种无损、高灵敏的分子指纹图谱检测技术,近年来在生物学领域得到广泛应用,其具有的免培养、快速、高特异性、低成本等优点为病原菌感染的诊断提供了新方案。本文阐述了拉曼光谱技术的原理和特点,综述了其在病原菌鉴定和抗菌药物敏感性试验方面的应用进展。     

Taxonomic identification of gram-negative nonfermenting microorganisms in environmental research

The scheme for the identification of Gram-negative nonfermenting microorganisms is proposed. The scheme comprises the most important key signs, such as the cytochrome oxidase reaction determined by the method of Gaby and Hadley, the oxidation/fermentation test, maltose oxidation and motility, as well as additional key signs, among them gelatinase activity, the oxidation of 10% lactose, nitratase activity with the liberation of free nitrogen, the utilization of the sources of carbon and energy (glucose and sodium acetate) in limited media containing ammonium salts and nitrates as the sources of nitrogen. Additional tests for the identification of nonfermenting microorganisms similar in their main key signs are also recommended.     

3D identification of trabecular bone fracture zone using an automatic image registration scheme: A validation study

Accurate identification of the local fracture zone is an important step towards the failure assessment of trabecular bone. In previous in-vitro studies, local fracture zones were visually identified in micro-CT images by experienced observers. This is a time-consuming and observer-dependent approach and it prevents any large-scale analysis of local trabecular fracture regions. The scope of this study is the application and validation of a new registration scheme for the automatic identification of trabecular bone fracture zones. Six human trabecular specimens were extracted from different anatomical sites. Five specimens were mechanically tested and scanned using micro-CT. For each specimen pre- and post-failure micro-CT datasets were obtained. The sixth specimen was scanned twice without any mechanical compression and was used to test the accuracy of the proposed scheme. The registration scheme was applied to the acquired datasets for the automatic identification of the fracture zone. The proposed scheme comprises of a three-dimensional (3D) automatic registration method to define the differences between the two datasets, and the application of a criterion for defining slices of the pre-failure dataset as "broken" or "unbroken". Identifications of the fracture zones were qualitatively validated against visual identification of observers. Furthermore, "full 3D" fracture zone identification, based on the presented scheme, was proposed. The proposed scheme proved to be more accurate and significantly faster than the currently used visual process.     

酒酒球菌的快速特异性PCR鉴定

以Oenococcus oeni苹果酸-乳酸酶基因(mleA)为目标基因,设计了1对特异性引物PmleaL/PmleaR进行酒酒球菌的快速鉴定研究。结果表明,直接以O.oeni的菌落为模板,通过引物对PmleaL/PmleaR的PCR扩增,可得到mleA基因的特异性条带;用此特异性引物进行供试乳酸菌的PCR鉴定,所有O.oeni菌系均得到特异性条带,而供试的其它种类乳酸菌未扩增出目标带。PmleaL/PmleaR可用于O.oeni的快速PCR鉴定。     

Rapid detection of influenza A virus in clinical samples using an ion channel switch biosensor

This report describes the fabrication and successful use of the ion channel switch biosensor (ICSB) for rapid point-of-care detection of influenza A in different types of respiratory specimens. Virus culture -- regarded as the "gold standard" -- and an immunochromatographic rapid point-of-care test for influenza A virus were compared with the biosensor. The ICSB rapid test provided an objective readout within 10 min of specimen inoculation into the ICSB chamber wells, without the need for chemical or other pretreatments. Construction of the ICSB with specific antibodies also enables rapid detection and identification of appropriate influenza A subtypes.     

Rapid Method of Determining Coagulase Activity During Staphylococcal Bacteriophage Typing

The plate test, a modification of the slide test described by Cadness-Graves was developed for the rapid identification of coagulase-positive staphylococci in conjunction with bacteriophage typing. An evaluation of 1,145 cultures by three coagulase-determination methods, the slide, tube, and plate tests, indicates that the plate test is as accurate as the slide tests, and the plate test agrees 97.7% with the tube test.     

FT-IR microspectroscopy: a promising method for the rapid identification of Listeria species

This work presents a pilot study to investigate the potential of fourier transform infrared (FT-IR) microspectroscopy for rapid identification of Listeria at the species level. Using this technique, FT-IR spectra were acquired from 30 strains from five Listeria species. The FT-IR spectra were analysed using stepwise canonical discriminant analysis and partial least-squares regression in a stepwise identification scheme. The results showed that 93% of all the samples were assigned to the correct species, and that 80% of the Listeria monocytogenes strains were correctly identified. In comparison, 100% of the samples, including the L. monocytogenes samples, were correctly identified using spectra acquired by FT-IR macrospectroscopy. The results show that FT-IR microspectroscopy has potential as a rapid screening method for Listeria, which is especially valuable for the food industry.     

Estimation of diagnostic value of the microtest systems for biochemical identification of vibrios groups and their typing according to Heiberg's method

Diagnostic value determination of microtest systems for biochemical identification of vibrios and their groups identification according to Heiberg's was made with the use of 260 collection strains of V. cholerae O1 and non-O1, as well as microorganisms belonging to other genera and families. The newly developed microtest systems were found to have a number of advantages over the traditional test tube method being more economic, ensuring rapid identification, standard and reproducible results.     

Diversity and prevalence of Arcobacter spp. in broiler chickens

Ninety-nine strains of Arcobacter spp., isolated from 10 chicken carcasses purchased from a supermarket and 15 chicken carcasses collected from a poultry abattoir, were speciated using a variety of phenotypic identification methods. All were tested using API Campy test strips and the 16-test (Preston) identification scheme developed for campylobacters. Fifty strains were selected for examination using a more comprehensive probabilistic identification scheme, and the identity of representative strains confirmed by protein profiling using SDS-PAGE. All 25 carcasses yielded Arcobacter butzleri . Three supermarket and 10 abattoir carcasses also carried A. cryaerophilus , and two abattoir carcasses carried A. skirrowii . The API Campy scheme proved unsatisfactory for identifying these strains: only 20 of 99 strains were accurately identified, all of which were A. cryaerophilus , the only Arcobacter sp. included in the database. Moreover, 76 of 99 strains were misidentified. The 16-test scheme identified all the arcobacter strains as A. cryaerophilus, since neither A. butzleri nor A. skirrowii had been described when the scheme was developed. The computer-assisted probabilistic scheme succeeded in identifying all but one strain, the identity of which was clarified by the use of SDS-PAGE. To our knowledge this is the first time that arcobacters other than A. butzleri have been reported in poultry meat or any other food of animal origin. Their high prevalence in poultry products may be of significance to public health.     

Use of Flow Cytometry for Rapid,Quantitative Detection of Poliovirus-Infected Cells via TAT Peptide-Delivered Molecular Beacons

Rapid and efficient detection of viral infection is crucial for the prevention of disease spread during an outbreak and for timely clinical management. In this paper, the utility of Tat peptide-modified molecular beacons (MBs) as a rapid diagnostic tool for the detection of virus-infected cells was demonstrated. The rapid intracellular delivery mediated by the Tat peptide enabled the detection of infected cells within 30 s, reaching saturation in signal in 30 min. This rapid detection scheme was coupled with flow cytometry (FC), resulting in an automated, high-throughput method for the identification of virus-infected cells. Because of the 2-order-of-magnitude difference in fluorescence intensity between infected and uninfected cells, as few as 1% infected cells could be detected. Because of its speed and sensitivity, this approach may be adapted for the practical diagnosis of multiple viral infections.     

食源性相关腹泻肠炎沙门菌感染抗菌药物敏感性

目的了解食源性相关腹泻非伤寒沙门菌感染的菌种分布及其对抗菌药物敏感性,为控制该类细菌的感染及传播提供技术支持。方法对2015-2017年本系统2家社区卫生服务中心就诊的食源性相关腹泻患者粪便(或肛拭子)标本采用直接分离与增菌分离相结合的方法常规培养分离获得42株沙门菌;采用血清凝集法进行快速血清分型,并进行自动化生化鉴定及抗菌药物敏感性试验;通过现况调查进行流行病学危险因素分析。结果自动化生化鉴定结果能够覆盖常规血清学快速鉴定结果,同属沙门菌群;42株沙门菌以肠炎沙门菌、鼠伤寒沙门菌和斯坦利沙门菌为主,其中肠炎沙门菌占全部菌株的23.81%,鼠伤寒沙门菌占19.06%,斯坦利沙门菌占14.29%;其中肠炎沙门菌可对氟喹诺酮类、三四代头孢菌素类与碳青霉烯类等敏感(敏感率可达97.00%以上),而对氨基糖苷类可产生双向耐药。通过现况调查,发现患者有腹痛、腹泻等胃肠道症状,可伴有发热,所有患者48h内有可疑食物暴露史,无水源性案例,均为散发。结论菌种鉴定应以常规快速血清学凝集结果为准,自动化生化鉴定仅供参考,并将鉴定菌种与药敏报告相关联,可根据药敏结果合理选用敏感抗菌药物;应对社区居民开展针对性的健康宣教。     

QuickMod: A tool for open modification spectrum library searches

MS2 library spectra are rich in reproducible information about peptide fragmentation patterns compared to theoretical spectra modeled by a sequence search tool. So far, spectrum library searches are mostly applied to detect peptides as they are present in the library. However, they also allow finding modified variants of the library peptides if the search is done with a large precursor mass window and an adapted Spectrum-Spectrum Match (SSM) scoring algorithm. We perform a thorough evaluation on the use of library spectra as opposed to theoretical peptide spectra for the identification of PTMs, analyzing spectra of a well-annotated modification-rich test data set compiled from public data repositories. These initial studies motivate the development of our modification tolerant spectrum library search tool QuickMod, designed to identify modified variants of the peptides listed in the spectrum library without any prior input from the user estimating the modifications present in the sample. We built the search algorithm of QuickMod after carefully testing different SSM similarity scores. The final spectrum scoring scheme uses a support vector machine (SVM) on a selection of scoring features to classify correct and incorrect SSM. After identification of a list of modified peptides at a given False Discovery Rate (FDR), the modifications need to be positioned on the peptide sequence. We present a rapid modification site assignment algorithm and evaluate its positioning accuracy. Finally, we demonstrate that QuickMod performs favorably in terms of speed and identification rate when compared to other software solutions for PTM analysis.     

Confirmatory identification of Neisseria gonorrhoeae by slide coagglutination

Neisseria gonorrhoeae was identified by the Phadebact gonococcus test, a rapid slide coagglutination technique, and the results obtained were compared with those obtained by conventional methods (Gram stain morphology, oxidase reaction, and carbohydrate utilization tests) for the confirmatory identification of gonococci. Of 308 clinical isolates examined, the coagglutination procedure correctly identified 97.8% of the isolates tested as N. gonorrhoeae and 93.9% of other bacteria as not N. gonorrhoeae. The coagglutination procedure also identified 29 laboratory strains correctly as not N. gonorrhoeae. The slide coagglutination test is easy to perform and offers a valuable alternative to other techniques for the confirmatory identification of N. gonorrhoeae.     

Fast isolation of recombinant baculovirus by antibody screening.

The use of a rapid and efficient scheme for the detection and purification of recombinant baculoviruses is described. The method is based on the detection of foreign proteins in cellular lysates of baculovirus-infected insect cells by antibody screening. The recombinant virus is purified by repeated serial dilutions. The method allows the identification and purification of recombinant viruses within two to three weeks.