Causative Agents Of Infectious Diseases Research Articles

Inhibition of the multidrug efflux pump LmrS from Staphylococcus aureus by cumin spice Cuminum cyminum.

Staphylococcus aureusis a serious causative agent of infectious disease. Multidrug-resistant strains like methicillin-resistantS. aureus compromise treatment efficacy, causing significant morbidity and mortality. Active efflux represents a major antimicrobial resistance mechanism. The proton-driven multidrug efflux pump, LmrS, actively exports structurally distinct antimicrobials. To circumvent resistance and restore clinical efficacy of antibiotics, efflux pump inhibitors are necessary, and natural edible spices like cumin are potential candidates. The mode of cumin antibacterial action and underlying mechanisms behind drug resistance inhibition, however, are unclear. We tested the hypothesis that cumin inhibits LmrS drug transport. We found that cumin inhibited bacterial growth and LmrS ethidium transport in a dosage-dependent manner. We demonstrate that cumin is antibacterial toward a multidrug-resistant host and that resistance modulation involves multidrug efflux inhibition.

Исследование токсичности гетероциклических соединений с высокой антимикробной активностью

Проведена комплексная оценка токсичности новых гетероциклических соединений ряда фенилпентендиона, халконов, полифункционально замещенных эфиров, енаминов и семикарбазонов, обладающих высокой антимикробной активностью, на биотест-объектах и белых лабораторных мышах. Определены препараты с низкими показателями острой токсичности из группы фенилпентендиона (2,4-дихлор-1,3,5-трифенил-2-пентен-1,5-дион), халкона (3-(4-(диметиламино)бензилиден)пентан-2,4-дион), полифункционально-замещенных эфиров (2,2’-ди-(3-(3-метокси-4-гидроксифенил)-2,4-диацетил-5-гидрокси-5-метилциклоген-1-ил)диэтиловый эфир) и енаминов (этил-2-метил-4-(1-пиперидил)-6-(3-нитрофенил)циклогекса-1,3-диенкарбоксилат – диенамин), которые могут рассматриваться как кандидаты в эффективные химиотерапевтические средства для борьбы с возбудителями инфекционных заболеваний.

A new epoch in medical microbiology

The development of molecular biology and proteomics technologies has led to revolutionary changes in the methods of identifying causative agents of infectious diseases. The introduction of the newest diagnostic approaches allows us to hope that medical microbiology will reach a qualitatively new level in which studies, as opposed to Koch’s classical bacteriology, do not require culturing microorganisms in growth media. The goal of the authors of this article is to analyze the possibilities, advantages, and drawbacks of immunochemical, molecular–genetic, and mass spectrometry–based methods of identifying microorganisms and, proceeding from this, to outline the main trends in the development of medical microbiological science.

Structural and Functional Diversity of Nairovirus-Encoded Nucleoproteins.

The nairoviruses include assorted tick-borne bunyaviruses that are emerging as causative agents of infectious diseases among humans and animals. As negative-sense single-stranded RNA (-ssRNA) viruses, nairoviruses encode nucleoprotein (NP) that encapsidates the genomic RNA and further forms ribonucleoprotein (RNP) complex with viral RNA-dependent RNA polymerase (RdRp). We previously revealed that the monomeric NP encoded by Crimean-Congo hemorrhagic fever virus (CCHFV) presents a racket-shaped structure and shows unusual DNA-specific endonuclease activity. To examine the structural and biological variation of nairovirus-encoded NPs, here, we systematically solved the crystal structures of NPs encoded by various nairoviruses, including Hazara virus (HAZV), Kupe virus (KUPV), and Erve virus (ERVEV). Combined with biochemical analysis, our results generate a clearer picture to aid in the understanding of the functional diversity of nairovirus-encoded NPs and the formation of nairovirus RNPs. Nairoviruses comprise several tick-borne bunyaviruses that are emerging as causative agents of infectious diseases among humans and animals; however, little is known of the nairovirus genome assembly and transcription mechanisms. Based on the previous study of CCHFV NP reported by different research groups, we systematically investigate here the structural and functional diversity among three different nairoviruses. This work provides important information on nairovirus nucleoprotein function and the formation of RNPs.

The application of pulsed field gel electrophoresis for molecular typing of causative agents of especially dangerous infections

The macro-restriction analysis of the microorganism DNA with the use of gel electrophoresis in pulsed field (PFGE typing, pulse electrophoresis) is applied in molecular biology to study the clonal structure and typing of causative agents of infectious diseases. Determining the degree of the relationship and definition of epidemiological interrelations of studied isolates, as well as studying the evolutionary history of pathogens, is performed by comparing DNA restriction patterns. This review presents an analysis of the use of the pulse electrophoresis in molecular-epidemiological research and the study of phylogeny of especially dangerous infections, cholera, and plague. The possibility of genetic heterogeneity of the Vibrio cholerae and Yersinia pestis populations is demonstrated; territorial and epidemiological characteristics of the spread of different isolate pulso-types, problems and prospects of the PFGE typing method in the system of epidemiological surveillance of cholera and plague in Russia are discussed.

A Case of Liver Abscess with Desulfovibrio desulfuricans Bacteremia.

Desulfovibrio spp. are gram-negative, sulfate-reducing, and anaerobic bacteria found in the digestive tract of humans. Because Desulfovibrio spp. are infrequent causative agents of infectious diseases and are difficult to isolate and to identify from clinical specimens, the appropriate antibiotic therapy to infection with Desulfovibrio spp. has not been determined. We report the first case of liver abscess with bacteremia due to Desulfovibrio desulfuricans to show the clinical presentation and treatment. The patient was successfully treated with intravenous piperacillin-tazobactam and oral amoxicillin-clavulanic acid.

<b>Applications of Molecular Diagnostic Techniques for Infectious Diseases</b>

Diagnosis is concerned with identifying the cause of a disease or precise and consistent outcomes that are results of direct or indirect actions, reactions and interactions between the cause of a disease and the host. That outcome, if accurate, would help the clinician in disease management, or the epidemiologist in identifying trends of diseases or the administrator in policy and decision making. Traditionally, infectious disease diagnosis involves identifying the causative agents of infectious diseases through the direct examination, culture and often immunological tests on clinical specimens. The traditional diagnostic techniques have varied sensitivities and specificities which influence their choice and applicability in a particular setting for the diagnosis of infectious diseases. However, the limitations of many traditional techniques particularly low specificity and long turnaround time often necessitate initiation of treatment before results are made available. Molecular diagnostic techniques involve a variety of techniques that explore the use of nucleic acid molecules for the identification of a particular pathogenic organism. These techniques include nucleic acid-based typing system, nucleic acid analysis without amplification, polymerase chain reaction (PCR) and other nucleic acid amplification techniques. Applications of molecular detection methods for infectious diseases have resolved many of the problems of the traditional diagnostic techniques, due to their exquisite sensitivity and specificity that allow the accurate and timely detection of very small numbers of organisms. This paper examines the principles and applications of molecular biology techniques in the identification of the causative agents of infectious diseases either in a routine setting or as research tools.Keywords: Infectious diseases, Molecular diagnosis, Polymerase Chain Reaction

INsPECT, an open-source and versatile software for automated quantification of (Leishmania) intracellular parasites.

Intracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries. Leishmania sp., Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals, including humans. Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs, analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains. Nevertheless, laborious manual microscopic counting of intracellular parasites is still the most commonly used approach. Here, we present INsPECT (Intracellular ParasitE CounTer), an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining. It offers the possibility to choose between different types of analyses (fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra- from extracellular parasites), and software running modes (automatic or custom). A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI (4′,6-diamidino-2-phenylindole) confirms a good correspondence between digital results and the “gold standard” microscopic counting method with Giemsa. Interestingly, this software is versatile enough to accurately detect intracellular T. gondii parasites on images acquired with High Content Screening (HCS) systems. In conclusion, INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites.

The Impenetrable Veil – from Yellow Fever to Ebola to SARS. Charles H. Calisher, 2013

The Impenetrable Veil – from Yellow Fever to Ebola to SARS. Charles H. Calisher, 2013

К вопросу о причинности в медицине

Представлениям монокаузализма (однопричинности) в развитии инфекционных болезней противопоставлены размышления, согласно которым понятие «причина» включает в себя возбудитель инфекционной болезни, генетические условия, необходимые для развития болезни, и взаимодействие с тканями организма.

MePIC, Metagenomic Pathogen Identification for Clinical Specimens

Next-generation DNA sequencing technologies have led to a new method of identifying the causative agents of infectious diseases. The analysis comprises three steps. First, DNA/RNA is extracted and extensively sequenced from a specimen that includes the pathogen, human tissue and commensal microorganisms. Second, the sequenced reads are matched with a database of known sequences, and the organisms from which the individual reads were derived are inferred. Last, the percentages of the organisms' genomic sequences in the specimen (i.e., the metagenome) are estimated, and the pathogen is identified. The first and last steps have become easy due to the development of benchtop sequencers and metagenomic software. To facilitate the middle step, which requires computational resources and skill, we developed a cloud-computing pipeline, MePIC: "Metagenomic Pathogen Identification for Clinical specimens." In the pipeline, unnecessary bases are trimmed off the reads, and human reads are removed. For the remaining reads, similar sequences are searched in the database of known nucleotide sequences. The search is drastically sped up by using a cloud-computing system. The webpage interface can be used easily by clinicians and epidemiologists. We believe that the use of the MePIC pipeline will promote metagenomic pathogen identification and improve the understanding of infectious diseases.

Generation and Analysis of Large-Scale Data-Driven Mycobacterium tuberculosis Functional Networks for Drug Target Identification

Technological developments in large-scale biological experiments, coupled with bioinformatics tools, have opened the doors to computational approaches for the global analysis of whole genomes. This has provided the opportunity to look at genes within their context in the cell. The integration of vast amounts of data generated by these technologies provides a strategy for identifying potential drug targets within microbial pathogens, the causative agents of infectious diseases. As proteins are druggable targets, functional interaction networks between proteins are used to identify proteins essential to the survival, growth, and virulence of these microbial pathogens. Here we have integrated functional genomics data to generate functional interaction networks between Mycobacterium tuberculosis proteins and carried out computational analyses to dissect the functional interaction network produced for identifying drug targets using network topological properties. This study has provided the opportunity to expand the range of potential drug targets and to move towards optimal target-based strategies.

Multiparametric determination of genes and their point mutations for identification of beta-lactamases

More than half of all currently used antibiotics belong to the beta-lactam group, but their clinical effectiveness is severely limited by antibiotic resistance of microorganisms that are the causative agents of infectious diseases. Several mechanisms for the resistance of Enterobacteriaceae have been established, but the main one is the enzymatic hydrolysis of the antibiotic by specific enzymes called beta-lactamases. Beta-lactamases represent a large group of genetically and functionally different enzymes of which extended-spectrum beta-lactamases (ESBLs) pose the greatest threat. Due to the plasmid localization of the encoded genes, the distribution of these enzymes among the pathogens increases every year. Among ESBLs the most widespread and clinically relevant are class A ESBLs of TEM, SHV, and CTX-M types. TEM and SHV type ESBLs are derived from penicillinases TEM-1, TEM-2, and SHV-1 and are characterized by several single amino acid substitutions. The extended spectrum of substrate specificity for CTX-M beta-lactamases is also associated with the emergence of single mutations in the coding genes. The present review describes various molecular-biological methods used to identify determinants of antibiotic resistance. Particular attention is given to the method of hybridization analysis on microarrays, which allows simultaneous multiparametric determination of many genes and point mutations in them. A separate chapter deals with the use of hybridization analysis on microarrays for genotyping of the major clinically significant ESBLs. Specificity of mutation detection by means of hybridization analysis with different detection techniques is compared.

Molecular taxonomy and identification of pathogenic fungi based on DNA sequence analysis

Although approximately 80,000 fungi are known, less than 1% are associated with human infection. However, their taxonomy has long been insufficient. During the last decade, DNA sequence analysis was introduced to the taxonomy of pathogenic fungi. Taxonomic advances in the field of medical mycology are helping to identify the causative agents of infectious diseases accurately, facilitating diagnosis and treatment. For example, Malassezia furfur was long considered the major microflora in atopic dermatitis, yet recent studies have indicated that this is not the case, as M. furfur is taxonomically heterogeneous and consists of five species. DNA sequence analysis resolved its taxonomic heterogeneity. Similar examples can be seen in "Candida albicans and C. dubliniensis" and "Trichosporon cutaneum and T. asahii". DNA sequence analysis also enables accurate identification of fungi. At present, almost all pathogenic fungi can be identified by determining the D1/D2 26S rDNA and ITS region of rRNA gene. This paper describes the practical taxonomy and identification of pathogenic fungi based on DNA sequence analysis.

In vivo-expressed genes of Pasteurella multocida.

Pasteurella multocida is the causative agent of infectious diseases of economic importance such as fowl cholera, bovine hemorrhagic septicemia, and porcine atrophic rhinitis. However, knowledge of the molecular mechanisms and determinants that P. multocida requires for virulence and pathogenicity is still limited. To address this issue, we developed a genetic expression system, based on the in vivo expression technology approach first described by Mahan et al. (Science 259:686--688, 1993), to identify in vivo-expressed genes of P. multocida. Numerous genes, such as those encoding outer membrane lipoproteins, metabolic and biosynthetic enzymes, and a number of hypothetical proteins, were identified. These may prove to be useful targets for attenuating mutation and/or warrant further investigation for their roles in immunity and/or pathogenesis.

The roots of microbiology and the influence of Ferdinand Cohn on microbiology of the 19th century.

The beginning of modern microbiology can be traced back to the 1870s, and it was based on the development of new concepts that originated during the two preceding centuries on the role of microorganisms, new experimental methods, and discoveries in chemistry, physics, and evolutionary cell biology. The crucial progress was the isolation and growth on solid media of clone cultures arising from single cells and the demonstration that these pure cultures have specific, inheritable characteristics and metabolic capacities. The doctrine of the spontaneous generation of microorganisms, which stimulated research for a century, lost its role as an important concept. Microorganisms were discovered to be causative agents of infectious diseases and of specific metabolic processes. Microscopy techniques advanced studies on microorganisms. The discovery of sexuality and development in microorganisms and Darwin's theory of evolution contributed to the founding of microbiology as a science. Ferdinand Cohn (1828-1898), a pioneer in the developmental biology of lower plants, considerably promoted the taxonomy and physiology of bacteria, discovered the heat-resistant endospores of bacilli, and was active in applied microbiology.

Adherence and invasion-two pathogenicity factors in bacterial and fungal pathogens

Pathogenicity factors such as adhesins, toxins, capsules, and other microbial gene products are involved as causative agents for infectious diseases. Therefore, the pathogenicity of organisms is increasingly studied on a molecular level. In bacteriology, unspecific adherence mechanisms and receptor-specific adhesins have to be distinguished. An adhesin-mediated invasion of pathogenic organisms in eukaryotic host cells could be relevant for pathogenesis. In mycology, various specific adhesins are involved in colonization of the host. Aspartyl proteases and phospholipases are relevant for adherence and invasion of host structures by pathogenic yeasts. Resistance factors have a central function in the distribution of infectious organisms. Gene-transfer, point mutations and efflux mechanisms are involved in the development of antibiotic drug resistance. Antifungal drug resistance does occur predominantly in Candida albicans against azole drugs. As underlying mechanisms point mutations in the ERG11 gene, encoding for the target enzyme of azoles, as well as energy-dependent efflux mechanisms were identified. Whether these mycotic factors are specific virulence factors or "fitness-factors" for a better survival of these organisms in the host, and if a possible alternating effect exists between resistance and virulence mechanisms is currently under investigation.

Water-insoluble camptothecin analogues as potential antiviral drugs.

In addition to being causative agents of infectious diseases in animals and humans, DNA viruses have served as models for the study of eukaryotic molecular mechanisms including replication and transcription. Studies of DNA virus functions utilizing cell-free systems and virus-infected cells in culture, in the presence of the anticancer drug camptothecin (CPT), have demonstrated that CPT is a potent inhibitor of replication, transcription and packaging of double-stranded DNA-containing adenoviruses, papovaviruses and herpesviruses, and the single- stranded DNA-containing autonomous parvoviruses. CPT inhibits viral functions by inhibiting topoisomer- ase I, a host cell enzyme required for initiation and completion of the viral functions. These findings indicate that CPT analogues could be developed for use as potent drugs against DNA viruses.

Subviral pathogens of plants: viroids and viroidlike satellite RNAs

Contrary to earlier beliefs, viruses are not the smallest causative agents of infectious diseases. Single-stranded RNAs as small as 246 nucleotides exist in certain higher plants and cause more than a dozen crop diseases. These RNAs have been termed viroids. Despite their extremely limited information content, viroids replicate autonomously in susceptible cells--that is, they do not require helper functions from simultaneously replicating conventional viruses. Viroids are covalently closed circular molecules with a characteristic rodlike secondary structure in which short helical regions are interrupted by internal and bulge loops. Viroids are not translated; they are replicated by a host enzyme (or enzymes) (probably RNA polymerase II) via oligomeric RNA intermediates by a rolling circle mechanism. Viroidlike satellite RNAs resemble viroids in size and molecular structure, but are found within the capsids of specific helper viruses on which they depend for their own replication. These RNAs are of great interest to molecular biology for at least two reasons: 1) they are the smallest and simplest replicating molecules known, and 2) they may represent living fossils of precellular evolution in a hypothetical RNA world.