High-throughput identification of clinically important bacterial pathogens using DNA microarray

Rapid and accurate detection of pathogenic bacteria is important for the treatment of patients with suitable antibiotics. Here we report the development of a diagnostic DNA microarray for the high-throughput identification of 39 pathogenic bacteria selected based on their high prevalence rate and/or difficulty of cultivation. The 23S ribosomal DNA and 16S–23S rDNA intergenic spacer region were used as target DNAs for pathogen detection. Universal- and species-specific probes were designed based on the unique and common sites within the target DNA sequences. New target DNA sequences were determined for the detection of 19 bacterial pathogens. The usefulness of the designed probes was validated using 39 reference bacteria and also with 515 clinical isolates from various clinical samples including blood, stool, pus, sputum, urine and cerebrospinal fluid. The DNA microarray developed in this study allowed efficient detection of bacterial pathogens with the specificities of 100%. The sensitivities were 100% as well except for the two pathogens, Enterobacter cloacae (75%) and Enterococcus faecium (85%). These results suggest that the DNA microarray-based assay developed in this study outperforms current diagnostic systems with respect to sensitivity, specificity, and high-throughput detection, and thus should be useful in pathogen diagnosis in the clinical setting.     

血源性病原菌早期诊断技术研究进展

医院住院患者常常发生血液病原菌感染，死亡率高，早期快速鉴定病原体给予适当的抗微生物疗法是降低死亡率的关键。 血培养是诊断血液病原菌感染的金标准，然而血培养不仅耗时且阳性率低。 分子生物学方法可大幅度缩减诊断时间，提高诊断的灵敏度与特异性，为临床合理用药与准确治疗提供可靠的依据，进而提高患者的生存率。 本研究就血源性病原菌诊断的分子生物学方法（主要包括核酸杂交技术、核酸扩增技术、荧光实时定量PCR技术、DNA微阵列与基质辅助激光解析电离飞行时间质谱等技术）等进行综述。     

Detection and identification of intestinal pathogens in clinical specimens using DNA microarrays

The detection and identification of intestinal pathogens is critical for clinical patient diagnosis and antimicrobial therapy. No currently available assays with DNA microarrays can simultaneously detect and identify multiple intestinal pathogens, because there is no appropriate method for choosing target probes. To solve the problem we have experimented for facilitating screening of specific probes and developed a rapid (<3h) and reliable assay for simultaneous detection of intestinal pathogens using two universal PCR primers to amplify two variable regions of bacterial 16S and 23S ribosomal DNA (rDNA) genes, and then applied to DNA microarrays, hybridization between probes and amplicons occurred. Through this idea for screening of probes the assay was successful in discriminating 15 genera or species of intestinal pathogens. The limit of detection was approximately 10(3)CFU/mL for one species of pathogen and 10(5)CFU/mL for six species pathogens existing simultaneously in stool. When this assay was applied directly to identify 99 clinical specimens, 80(80.8%) were correctly analyzed, including four with mixed pathogens; 8(8.08%) received negative results due to no corresponding probes in this array and 11(11.11%) belonging to our targets were misidentified due to low-level pathogens and other factors. This approach is also convenient to obtain specific and proper probes while establishing assays for the applications in other aspects using DNA microarrays. In addition, the more species may be added to this system easily and endlessly by screening of candidate target probes in order to increase the power of simultaneous detection.     

Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens

Escherichia coli O157:H7, Salmonella enterica, Listeria monocytogenes and Campylobacter jejuni are considered important pathogens causing the most food-related human illnesses worldwide. Current methods for pathogen detection have limitations in the effectiveness of identifying multiple foodborne pathogens. In this study, a pathogen detection microarray was developed using various 70-mer oligonucleotides specifically targeting the above pathogens. To reduce the cost of detection, each microarray chip was designed and fabricated to accommodate 12 identical arrays which could be used for screening up to 12 different samples. To achieve high detection sensitivity and specificity, target-specific DNA amplification instead of whole genome random amplification was used prior to microarray analysis. Combined with 14-plex PCR amplification of target sequences, the microarray unambiguously distinguished all 4 pathogens with a detection sensitivity of 1 × 10^?4 ng (approximately 20 copies) of each genomic DNA. Applied the assay to 39 fresh meat samples, 16 samples were found to be contaminated by either 1 or 2 of these pathogens. The co-occurrences of Salmonella and E. coli O157:H7, Salmonella and L. monocytogenes in the same meat samples were also observed. Overall, the microarray combined with multiplex PCR method was able to effectively screen single or multiple pathogens in food samples and to provide important genotypic information related to pathogen virulence.     

Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the detection and identification of organisms of clinical and epidemiologic importance. Staphylococcus aureus, one of the most frequent causes of human infections, was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. One hundred clinical isolates of S. aureus, verified by biochemical reactions and latex agglutination and 90 negative control clinical isolates were screened in the assay. Moreover, fifty blood broth samples from blood culture bottles showing Gram-positive cocci in clusters on direct Gram's stain and 25 showing Gram-negative bacilli were screened. The probes, constructed from the nuc gene, correctly detected all S. aureus genomes present without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from other types of clinical specimens.     

Sequence-specific identification of 18 pathogenic microorganisms using microarray technology

We have developed a Multi-Pathogen Identification (MPID) microarray for high confidence identification of eighteen pathogenic prokaryotes, eukaryotes and viruses. Analysis of amplified products from pathogen genomic DNA using microarray hybridization allows for highly specific and sensitive detection, and allows the discrimination between true amplification products and false positive amplification products that might be derived from primers annealing to non-target sequences. Species-specific primer sets were used to amplify multiple diagnostic regions unique to each individual pathogen. Amplified products were washed over the surface of the microarray, and labelled with phycoerythrin-streptavidin for fluorescence detection. A series of overlapping 20-mer oligonucleotide probes hybridize to the entire diagnostic region, while parallel hybridizations on the same surface allow simultaneous screening for all organisms. Comparison to probes that differ by a single mismatch at the central position reduced the contribution of non-specific hybridization. Samples containing individual pathogens were analyzed in separate experiments and the corresponding species-specific diagnostic regions were identified by fluorescence among their highly redundant probe sets. On average, 91% of the 53 660 pathogen probes on the MPID microarray performed as predicted. The limit of detection was found to be as little as 10 fg of B. anthracis DNA in samples that were amplified with six diagnostic primer-pairs. In contrast, PCR products were not observed at this concentration when identical samples were prepared and visualized by agarose gel electrophoresis.     

Development of a base stacking hybridization-based microarray method for rapid identification of clinical isolates

A base stacking hybridization-based microarray method was developed for rapid identification of clinical isolates within 2 h. The oligonucleotide probe sequences for species or genus-level identification were targeted against ribosomal RNA. Isolates were lysed and directly hybridized to the microarray-bound capture probes without conventional DNA or RNA isolation and prior polymerase chain reaction amplification. Five bacterial species encountered frequently in the clinical setting, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae, and one genus Enterococcus, could be discriminated by the microarray-based assay. Identification by this method matched biochemical identification for 150 of 152 clinical strains. This base-stacking hybridization microarray offers a simple, fast (相似文献   

Multiplex PCR pathogen detection in two severely burned patients with suspected septicemia

The case studies reported in this article strive to illustrate the clinical value of multiplex polymerase chain reaction (PCR)-based pathogen detection in patients with burn sepsis. Adult (age ≥18 years) burn patients (≥20% TBSA) presenting with signs and symptoms of burn sepsis were enrolled into a prospective, observational trial. Patients received PCR testing in parallel to routine blood cultures. The authors report two cases in which PCR was used to rapidly detect pathogens in whole blood from burn patients with suspected septicemia. PCR identified Escherichia coli in 5.8 hours in case 1. Blood and sputum cultures required 17 hours for Gram stain results. Empiric ceftriaxone therapy was initiated. Blood cultures required an additional 18 hours to identify the same pathogen detected by PCR. Ceftriaxone was replaced with ciprofloxacin for improved coverage. Follow-up antimicrobial susceptibility results revealed intermediate ciprofloxacin resistance. Meropenam therapy was initiated. In case 2, PCR detected Pseudomonas aeruginosa in 5.45 hours while blood cultures remained negative. Respiratory cultures became positive for P. aeruginosa 2 days later. Serial PCR samples continued detecting P. aeruginosa despite negative blood cultures and appropriate antimicrobial therapy. The patient later became hypotensive and coagulopathic and expired. For both patient cases, PCR identified high-risk pathogens faster than culture methods. In the second patient case, PCR identified the presence of pathogen DNA despite negative cultures before the onset of septic shock and presumptive disseminated intravascular coagulation. These results warrant further investigation to determine the clinical significance of pathogen DNA in burn sepsis.     

DNA probes for antimicrobial susceptibility testing

As DNA probes are used more frequently in the clinical laboratory for the detection and identification of pathogens in clinical samples, a means of determining the antimicrobial susceptibility profile of those pathogens will be required. DNA probes directed to specific resistance determinants offer a solution to this problem. Methods of determining the susceptibility of viruses to antiviral agents can also be accomplished with DNA probes. This article explores the advantages and disadvantages of using hybridization methods for susceptibility testing of organisms contained in clinical samples.     

DNA microarray-based detection of nosocomial pathogenic Pseudomonas aeruginosa and Acinetobacter baumannii

Infection by nosocomial pathogenic bacteria is increasingly becoming a major threat to the patients in the hospital. We have developed a diagnostic DNA microarray for the detection of two important nosocomial pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii. The diagnostic DNA microarray contains the species-specific probes of 15mer oligonucleotides designed based on the sequences of 23S ribosomal DNA. The performance of DNA microarray in diagnosing P. aeruginosa and A. baumannii was evaluated using reference bacteria as well as clinical specimens such as blood, stool, pus, sputum, urine and cerebrospinal fluid. Using this DNA microarray, A. baumannii could be successfully detected in 11 out of 13 clinical specimens, thus giving the sensitivity of 84.6% with the specificity of 100% and the positive predictive value of 100%. P. aeruginosa could also be detected in 25 out of 26 clinical specimens, showing the sensitivity of 96.2%, the specificity of 100%, and the positive predictive value of 100%. These results suggest that two nosocomial pathogens, P. aeruginosa and A. baumannii, can be efficiently diagnosed by using the DNA microarray developed in this study.     

脓毒症常见病原菌多重实时PCR检测方法的建立

目的 建立可同时检测脓毒症患者血液中肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌、凝固酶阴性葡萄球菌、金黄色葡萄球菌和白假丝酵母菌的多重实时PCR方法。方法 根据各病原菌基因组保守序列设计引物和探针,建立多重实时PCR检测方法。对建立的多重实时PCR检测方法进行特异性、敏感性和灵敏度分析,并使用该法对112份脓毒症血培养阳性标本进行验证。结果 成功建立脓毒症常见病原菌的多重实时PCR检测方法。此方法特异性、敏感性良好,灵敏度达10-5 ng/l,与标准检测方法（血培养加生化鉴定）相比,缩短了20 h。112份临床血培养阳性标本中,目标病原菌83例,多重实时PCR检出80例阳性;非目标病原菌29例,多重实时PCR检测均为阴性。检测结果显示,该方法特异性达100%,敏感性达96.39%。结论 该多重实时PCR检测方法具有快速、高效、灵敏、特异的优点,可用于临床重症监护室患者血液常见感染病原菌的诊断。     

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。     

Rapid diagnosis of acute pyogenic meningitis by a combined PCR dot-blot assay

A multiplex PCR was employed to amplify unique conserved sequences of DNA from the pathogens Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae from cerebrospinal fluid samples of patients suffering from acute pyogenic meningitis. The accurate identification of the PCR amplified product was achieved by hybridizing dot-blots of the PCR products to probes which were specific, biotinylated internal sequences of the amplified target DNA. Detection of the hybrids was done in a colour reaction using streptavidin-alkaline phosphatase conjugate and BCIP/NBT substrates. The entire protocol took only 7 h for the correct identification of the pathogen present in clinical samples of cerebrospinal fluid. The sensitivity and specificity were >95%.     

Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system

Sepsis is a leading cause of death. Rapid and accurate identification of pathogens and antimicrobial resistance directly from blood culture could improve patient outcomes. The FilmArray® (FA; Idaho Technology, Salt Lake City, UT, USA) Blood Culture (BC) panel can identify >25 pathogens and 4 antibiotic resistance genes from positive blood cultures in 1 h. We compared a development version of the panel to conventional culture and susceptibility testing on 102 archived blood cultures from adults and children with bacteremia. Of 109 pathogens identified by culture, 95% were identified by FA. Among 111 prospectively collected blood cultures, the FA identified 84 (91%) of 92 pathogens covered by the panel. Among 25 Staphylococcus aureus and 21 Enterococcus species detected, FA identified all culture-proven methicillin-resistant S. aureus and vancomycin-resistant enterococci. The FA BC panel is an accurate method for the rapid identification of pathogens and resistance genes from blood culture.     

Design of oligonucleotide arrays to detect point mutations: molecular typing of antibiotic resistant strains of Neisseria gonorrhoeae and hantavirus infected deer mice

Microarrays are promising tools for use in molecular diagnostics due to their ability to perform a multitude of tests simultaneously. In the case of genotyping many such tests will require discrimination of sequence at the single nucleotide level. A number of challenges exist including binding of optimal quantities of probe to the chip surface, the use of uniform hybridization conditions across the chip and the generation of labeled target. We investigated two model systems to test out the efficacy and ease with which probes can be designed for this purpose. In the first of these we designed primers to identify five mutations found in two genes from N. gonohorroeae, gyrA and parC that have been implicated in ciprofloxacin resistance. In the second system we used a similar strategy to identify four mutations in AT rich mitochondrial DNA from deer mice. These mutations are associated with deer mice subspecies that originate from different geographical regions of Canada and harbor different hantavirus strains. In every case we were able to design probes that could discriminate mutations in the target sequences under uniform hybridization conditions, even when targets were fairly long in length, up to 400 bp. Our results suggest that microarray analysis of point mutations might be very useful for automated identification and characterization of pathogens and their hosts.     

新生儿血培养的病原菌分布及耐药分析

目的 了解本院新生儿血培养标本病原菌的分布及耐药情况.方法 采用BACT/ALERT 3D全自动血培养仪对新生儿血液标本进行血培养,所有菌株采用法国梅里埃公司的ATB Expression系统进行鉴定及药敏试验.结果 42例病原菌中以革兰阳性球菌为主,占全部的83.3%.表皮葡萄球菌在革兰阳性球菌占首位,革兰阳性球菌均对万古霉素均敏感,对苯唑西林耐药率高;革兰阴性杆菌对碳青霉烯类抗菌药物均敏感.结论 新生儿血培养结果为临床医生治疗危重患儿提供了实验依据,对患儿的治疗、抢救有积极的意义.     

Aortic vascular graft infection caused by Cardiobacterium valvarum: A case report

A 53-year-old man with a past medical history of total arch replacement surgery and severe aortic regurgitation presented with a 1-month history of persistent general malaise, anorexia, body weight loss and night sweats. His recent history included gingival hyperplasia for 6 years, gingivitis after tooth extraction 3 years before, prolonged inflammatory status for 4 months, fundal hemorrhage and leg tenderness for 2 months. A pathogen was detected from blood culture, but conventional microbiological examination failed to identify the pathogen. The organism was eventually identified as Cardiobacterium valvarum by 16S rRNA analysis, and the patient was diagnosed with infective endocarditis and prosthetic vascular graft infection. The patient received intravenous antibiotic therapy using a combination of ceftriaxone and levofloxacin for 5 weeks and was discharged with a good clinical course.C. valvarum is a rare human pathogen in clinical settings. Only 10 cases have been reported to date worldwide, and therefore, the clinical characteristics of C. valvarum infection are not fully known. This is a first well-described case of C. valvarum infection in Japan, and further, a first report of aortic prosthetic vascular graft infection worldwide. Identification of C. valvarum is usually difficult due to its phenotypic characteristics, and molecular approaches would be required for both clinicians and microbiologists to facilitate more reliable diagnosis and uncover its clinical picture more clearly.     

Helicobacter cinaedi kidney cyst infection and bacteremia in a patient with autosomal dominant polycystic kidney disease

A 48-year-old man with autosomal dominant polycystic kidney disease (ADPKD) was admitted to our hospital with a 5-day history of lower right back pain, high-grade fever, and arthralgia. He was diagnosed with right kidney cyst infection and bacteremia due to Helicobacter cinaedi (H. cinaedi) based on these symptoms, highly elevated CRP (32.25 mg/dL), abdominal magnetic resonance imaging findings, and the identification of H. cinaedi from blood cultures using PCR and sequence analysis of the 16S ribosomal DNA gene. Intravenous cefotaxime 0.5 g twice daily followed by meropenem 0.5 g twice daily and ciprofloxacin 200 mg twice daily were partially effective; oral doxycycline added at 200 mg/day finally eradicated the infection. Total duration of antimicrobial therapy was 9 weeks. H. cinaedi infections typically present as bacteremia with or without cellulitis in immunocompromised patients such as those with AIDS or malignant disease. To our knowledge, this is the first report describing an ADPKD patient with H. cinaedi cyst infection. Although H. cinaedi infections are increasingly recognized, even in immunocompetent subjects, numerous cases may still be overlooked given that this bacterium is slow-growing, and is difficult to culture, be Gram-stained, and identify on phenotypic tests. Consideration of this bacterium as a possible pathogen and sufficient duration of incubation with molecular testing are necessary in treating ADPKD patients with cyst infection.     

153株血培养病原菌的分布及耐药性分析

目的了解该院2014-2015年血培养中病原菌构成与耐药情况。方法对病房送检的1 260例血培养标本进行细菌培养,阳性标本进行分离鉴定及耐药情况分析。结果 1 260例血培养标本中分离出细菌153株,阳性率12.1%。病原菌中,革兰阴性杆菌以大肠埃希菌和肺炎克雷伯菌为主,革兰阳性球菌以凝固酶阴性葡萄球菌和金黄色葡萄球菌为主;肠杆菌科对亚胺培南、厄他培南的敏感度为100.0%,革兰阳性球菌对万古霉素、利奈唑胺和替加环素的敏感度为100.0%。结论血培养标本病原菌的耐药情况严重,及时监测病原菌变化及耐药趋势,指导临床合理用药非常重要。