Cellular microbiology: can we learn cell physiology from microorganisms?

Cellularmicrobiology is a new discipline that is emerging at the interfacebetween cell biology and microbiology. The application of moleculartechniques to the study of bacterial pathogenesis has made possiblediscoveries that are changing the way scientists view thebacterium-host interaction. Today, research on the molecular basis ofthe pathogenesis of infective diarrheal diseases of necessity transcends established boundaries between cell biology, bacteriology, intestinal pathophysiology, and immunology. The use of microbial pathogens to address questions in cell physiology is just now yieldingpromising applications and striking results.     

Genetic Engineering and Nitrogen Fixation

Molecular cloning is based on isolation of a DNA sequence of interest to obtain multiple copies of it in vitro. Application of this technique has become an increasingly important tool in clinical microbiology due to its simplicity, cost effectiveness, rapidity, and reliability. This review entails the recent advances in molecular cloning and its application in the clinical microbiology in the context of polymicrobial infections, recombinant antigens, recombinant vaccines, diagnostic probes, antimicrobial peptides, and recombinant cytokines. Culture-based methods in polymicrobial infection have many limitation, which has been overcome by cloning techniques and provide gold standard technique. Recombinant antigens produced by cloning technique are now being used for screening of HIV, HCV, HBV, CMV, Treponema pallidum, and other clinical infectious agents. Recombinant vaccines for hepatitis B, cholera, influenza A, and other diseases also use recombinant antigens which have replaced the use of live vaccines and thus reduce the risk for adverse effects. Gene probes developed by gene cloning have many applications including in early diagnosis of hereditary diseases, forensic investigations, and routine diagnosis. Industrial application of this technology produces new antibiotics in the form of antimicrobial peptides and recombinant cytokines that can be used as therapeutic agents.     

Clinical applications of molecular biology for infectious diseases

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.     

What does the future hold for clinical microbiology?

In the past decade, clinical microbiology laboratories have undergone important changes with the introduction of molecular biology techniques and laboratory automation. In the future, there will be a need for more rapid diagnoses, increased standardization of testing and greater adaptability to cope with new threats from infectious microorganisms, such as agents of bioterrorism and emerging pathogens. The combination of the new tools that are now being developed in research laboratories, the general reorganization of clinical laboratories and improved communication between physicians and clinical microbiologists should lead to profound changes in the way that clinical microbiologists work.     

CRISPR-Cas9应用于病毒性传染病防控的研究进展

病毒性传染病是威胁人类健康的重要因素,迫切需要新的治疗方法来降低由急性病毒感染如鼻病毒和登革热病毒以及慢性病毒感染如人类免疫缺陷病毒1和乙型肝炎病毒引起的发病率和死亡率.随着分子生物学技术的发展,靶向序列特异性的基因编辑技术成为传染病治疗的有力工具.其中规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)-CRISPR相关蛋白9(CRISPR associated protein 9,Cas9)凭借其高效、简便、高特异性等特点被广泛应用于细胞系和动物模型中的传染病治疗,从而成为有前景的新型传染病治疗模式.目前,利用病毒和非病毒载体将Cas9以DNA、m RNA或蛋白质的形式递送到细胞中的可行性研究和评估CRISPR-Cas9体内适用性的临床试验已经在进行中.本篇综述中,我们将对CRISPR-Cas9的原理,其应用于传染病治疗的最新研究进展以及该技术面临的挑战和可预测性的解决方法等加以概述,并进一步展望其未来的发展方向.     

Controlling the maturation of pathogen-containing vacuoles: a matter of life and death

Once considered to be contained, infectious diseases of bacterial origin are now making a comeback. A lack of innovative therapies and the appearance of drug-resistant pathogens are becoming increasingly serious problems. A better understanding of pathogen-host interactions at the cellular and molecular levels is necessary to define new targets in our fight against microorganisms. In the past few years, the merging of cell biology and microbiology has started to yield critical and often surprising new information on the interactions that occur between various pathogens and their mammalian host cells. Here we focus on the intracellular routing of vacuoles containing microorganisms, as well as on the bacterial effectors and their host-cell targets that control vacuole maturation. We also describe new approaches for isolating microorganism-containing vacuoles and analysing their molecular composition, which will help researchers to define the molecules and mechanisms governing vacuole biogenesis.     

Applications of sequencing technology in clinical microbial infection

Infectious diseases are a type of disease caused by pathogenic microorganisms. Although the discovery of antibiotics changed the treatment of infectious diseases and reduced the mortality of bacterial infections, resistant bacterial strains have emerged. Anti‐infective therapy based on aetiological evidence is the gold standard for clinical treatment, but the time lag and low positive culture rate of traditional methods of pathogen diagnosis leads to relative difficulty in obtaining the evidence of pathogens. Compared with traditional methods of pathogenic diagnosis, next‐generation and third‐generation sequencing technologies have many advantages in the detection of pathogenic microorganisms. In this review, we mainly introduce recent progress in research on pathogenic diagnostic technology and the applications of sequencing technology in the diagnosis of pathogenic microorganisms. This review provides new insights into the application of sequencing technology in the clinical diagnosis of microorganisms.     

Introduction: microbiology and immunology: lessons learned from Salmonella.

Salmonella enterica, a Gram-negative bacterium, causes significant morbidity and mortality worldwide, and is an excellent model to study bacterial pathogenesis and cellular immune responses. With the development of powerful new technologies, there has been a fusion of research on immunology, molecular biology and cellular microbiology of S. enterica infections. This multidisciplinary research will enhance our understanding of the basic mechanisms of bacterial infections and immunity; it also provides new approaches towards therapeutic and control measures.     

Infectious animal disease and its control

The control of infectious diseases in the main food-producing animals is considered and the main factors involved in the epizootiology of disease are presented. The properties of infectious agents and their natural history together with factors that influence the spread and development of disease are summarized. The factors in intensive animal husbandry that affect the occurrence of infectious disease and its control are considered. These include population density, population movement, management, hygiene and genetic constitution of the host. They encourage the appearance of new diseases, changes in the character of established diseases and the development of pathogenicity in infectious agents that were previously of no importance. Intensive animal husbandry has also increased the importance of multifactorial disease, which includes diseases that require more than one infectious agent or one or more infectious agents plus other factors for their cause. The methods of control of infectious disease currently available are described and the success and difficulties of their control on a global, national and local (farm or enterprise) basis are considered. Examples of diseases of global importance where national and world programmes of control and eradication have been of varying success are described. Examples of diseases that are enzootic throughout the world and the procedures used for their control are also described. The technological opportunities for the improvement of the control of infectious disease in the future are discussed. It is considered that developments in molecular biology and immunology will provide improvements in diagnostic tools and will revolutionize the development of animal resistance to disease and the production and use of vaccines.     

提高临床医学生微生物检验 见习教学质量的探索与实践

临床微生物室见习是临床医学专业学生了解微生物检验的重要窗口,提高其见习教学质量,使其认识到微生物检验在感染性疾病诊治中的作用。除了系统介绍临床微生物检验的工作内容及流程外,我们更注重临床医生与微生物检验的密切联系,强调临床医生对微生物检验前质量控制的重要性。案例教学法联合问题导向教学法,将临床感染病例与微生物检验紧密结合。举例讲解临床医生该如何正确解读微生物检验报告结果,从而合理选择抗菌药物进行有效的抗感染治疗。最后提醒临床医学生注意生物安全及医院感染的发生。通过我们的见习教学,临床医学生表示获益颇丰,意识到微生物检验对感染性疾病诊治的重要作用。     

Molecular methods used in clinical laboratory: prospects and pitfalls

The role of molecular detection, identification and typing or fingerprinting of microorganisms has shifted gradually from the academic world to the routine diagnostic laboratory. Molecular methods have been used increasingly over the past decade to improve the sensitivity, specificity and turn-around time in the clinical laboratory. Molecular methods have also been used to identify new and nonculturable agents. Many high-throughput molecular tests are now available commercially, which impacts on the infrastructure in many of the diagnostic laboratories. In this paper, we take an overall look at the use of molecular methods (prospects vs. pitfalls) based on our clinical and public health experience, particularly as they related to Borrelia burgdorferi, a vector-borne pathogen, Treponema pallidum, a re-emerging sexually transmitted global pathogen, and West Nile virus, a newly recognized virus in North America.     

Enterococcus: review of its physiology, pathogenesis, diseases and the challenges it poses for clinical microbiology

The genus Enterococcus is composed of 38 species, the most important of which are Enterococcus faecalis and Enterococcus faecium—both human intestinal colonizers. Hospitals within the United States and around the world commonly isolate these bacteria because they are a cause of bacteremia, urinary tract infections (UTIs), endocarditis, wound infections, meningitis, intraabdominal and pelvic infections, and nosocomial and iatrogenic infections. Given the ubiquity of enterococci within the human population, it is important for laboratories to be able to distinguish these agents within hospitalized patients from other bacterial genera and also differentiate different species within the Enterococcus genus as well as different strains within each species. Unfortunately, the enterococci are emerging as serious pathogens in both the developed world, where surveillance needs to be improved and speciation procedures are inadequate or cumbersome, and in developing nations, which lack the trained hospital personnel or funding to sufficiently identify enterococci to the genus or species level. This review explores the Enterococcus genus and highlights some of the concerns for national and international clinical microbiology laboratories.     

Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.     

Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection

Identification of etiology remains a significant challenge in the diagnosis of infectious diseases, particularly in resource-poor settings. Viral, bacterial, and fungal pathogens, as well as parasites, play a role for many syndromes, and optimizing a single diagnostic system to detect a range of pathogens is challenging. The TaqMan Array Card (TAC) is a multiple-pathogen detection method that has previously been identified as a valuable technique for determining etiology of infections and holds promise for expanded use in clinical microbiology laboratories and surveillance studies. We selected TAC for use in the Aetiology of Neonatal Infection in South Asia (ANISA) study for identifying etiologies of severe disease in neonates in Bangladesh, India, and Pakistan. Here we report optimization of TAC to improve pathogen detection and overcome technical challenges associated with use of this technology in a large-scale surveillance study. Specifically, we increased the number of assay replicates, implemented a more robust RT-qPCR enzyme formulation, and adopted a more efficient method for extraction of total nucleic acid from blood specimens. We also report the development and analytical validation of ten new assays for use in the ANISA study. Based on these data, we revised the study-specific TACs for detection of 22 pathogens in NP/OP swabs and 12 pathogens in blood specimens as well as two control reactions (internal positive control and human nucleic acid control) for each specimen type. The cumulative improvements realized through these optimization studies will benefit ANISA and perhaps other studies utilizing multiple-pathogen detection approaches. These lessons may also contribute to the expansion of TAC technology to the clinical setting.     

Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.     

全基因组测序在病原菌分型与溯源中的应用研究进展

细菌分子分型已成为监测细菌感染性疾病的暴发流行与明确病原菌传播途径的重要工具.随着全基因组测序技术的日益兴起,公共数据库中已产生大量的细菌基因组数据,迫切需要研究人员充分认识和理解该技术,并掌握多种生物信息学工具挖掘并解读测序数据.本文系统概述了全基因组测序技术与生物信息学工具在病原菌分型与溯源中的应用,并对全基因组测...     

Recent advances in detection and control of infectious hematopoietic necrosis virus in aquaculture

Infectious hematopoietic necrosis (IHN) is one of the most important viral diseases of salmon and trout reared in culture. The disease remains untreatable with avoidance being the only control measure. Much has been learned about the chemical, physical, and serological characteristics of the rhabdovirus causing IHN, but critical gaps exist in our understanding of the biology of the virus in nature. The tools of molecular biology have provided improved methods for detection of pathogens and new strategies for control of viral diseases. This paper reviews several recent improvements in methods for detecting infectious hematopoietic necrosis virus including the application of enzyme-linked immunosorbent assays, development of monoclonal antibodies and DNA probes, and use of the polymerase chain reaction. New strategies for control of IHN through the use of better water treatment, more resistant fish, antiviral drugs or chemicals, and new generation vaccines are discussed.     

Whole-genome sequencing targets drug-resistant bacterial infections

During the past two decades, the technological progress of whole-genome sequencing (WGS) had changed the fields of Environmental Microbiology and Biotechnology, and, currently, is changing the underlying principles, approaches, and fundamentals of Public Health, Epidemiology, Health Economics, and national productivity. Today’s WGS technologies are able to compete with conventional techniques in cost, speed, accuracy, and resolution for day-to-day control of infectious diseases and outbreaks in clinical laboratories and in long-term epidemiological investigations. WGS gives rise to an exciting future direction for personalized Genomic Epidemiology. One of the most vital and growing public health problems is the emerging and re-emerging of multidrug-resistant (MDR) bacterial infections in the communities and healthcare settings, reinforced by a decline in antimicrobial drug discovery. In recent years, retrospective analysis provided by WGS has had a great impact on the identification and tracking of MDR microorganisms in hospitals and communities. The obtained genomic data are also important for developing novel easy-to-use diagnostic assays for clinics, as well as for antibiotic and therapeutic development at both the personal and population levels. At present, this technology has been successfully applied as an addendum to the real-time diagnostic methods currently used in clinical laboratories. However, the significance of WGS for public health may increase if: (a) unified and user-friendly bioinformatics toolsets for easy data interpretation and management are established, and (b) standards for data validation and verification are developed. Herein, we review the current and future impact of this technology on diagnosis, prevention, treatment, and control of MDR infectious bacteria in clinics and on the global scale.     

Causation and disease: effect of technology on postulates of causation.

This paper reviews the technical developments in microbiology that led to the discovery of new infectious agents and the effect of these discoveries on establishing proof of causation. In bacteriology, these advances included the light microscope, bacterial stains, bacterial cultures, and the methods used to isolate clones. In virology, they involved the use of filters to separate viruses from bacteria, the electron microscope, the use of laboratory animals, embryonated eggs, tissue cultures to identify or grow the agent, and the recent development of molecular techniques to detect the presence of antigen in tissues. In immunology, they were based on the discovery of antibodies and of the immune response.     

Development and application of synthetic peptides as vaccines

Vaccination against bacterial and viral diseases has been one of the major achievements in medicine and immunology since the beginning of this century. Extensive vaccination programs have been able to control or, in the case of smallpox, virtually wipe out some of the most dangerous infectious diseases e.g. poliomyelitis, measles, whooping cough, diphtheria and tetanus. However, as this success has been limited mainly to the developed, affluent countries, infectious diseases still remain the worlds largest health problem. Furthermore, vaccines against human parasites are non-existent. Recent advances in immunology and molecular biology including recombinant DNA technology have provided the basis for new approaches to vaccine development.