PCR and sequencing based methods for detection and typing of pathogenic microorganisms

The potential use of bacteria and viruses as biological terror weapons makes certain highly pathogenic microorganisms a worldwide public health threat. In an outbreak investigation involving a possible deliberate spread of a biological warfare agent there is a need for a fast and reliable diagnostic method. Ideally, the same method should be usable for both bacteria and viruses. To distinguish between natural and deliberate spread, information on the prevalence/incidence of the organism/disease, ecology and natural mechanisms for spread is needed. In addition, knowledge of national and international subtypes reflecting both micro- and macro-evolution of the organisms is required for the analysis. The field of molecular epidemiology has evolved during the last decades with the introduction of several different methods to type and characterize bacteria. There is now a plethora of different molecular techniques available to identify and discriminate between different strains of bacteria. In this thesis, rapid protocols based on pyrosequencing technology were developed and used for discrimination of Bacillus anthracis from closely related bacillus species. The technique was also used for identification and subtyping of Francisella species, including the human pathogen Francisella tularensis and its subspecies. Also a rapid generic protocol for detection and genotyping of infectious agents including bacteria, parasites, and viruses was developed. Tularemia epidemics occur in limited geographical areas, and at variable intervals. The reasons for these irregularities are still unknown. There may be ecological niches in the affected areas, which harbor the bacteria between epidemics, or the bacterium may be reintroduced into an ecological system that permits its amplification. The establishment of a relevant typing system indentifying individual strains is of great importance .One of the most promising methods, MLVA was evaluated as a tool for practical use in epidemiological investigations of tularemia in Sweden. PFGE analysis was used as reference method. Typing data were combined with geomapping (GIS), in order to predict sources of infection and determine possible reservoirs for the different MLVA types. The results indicate that MLVA has the capacity to be used as a standalone typing method in outbreak investigations. It was possible to use this method directly on clinical specimens without isolating the bacterium by cultivation. In most cases, a certain MLVA type was not correlated to a strict geographic location, indicating that subtyping might be of limited use in surveillance and outbreak investigations of tularemia in endemic countries

Issues and Trends in Collection Development for East Asia Legal Materials

The authors delineate the general policy and guidelines for developing foreign and transnational law collections in U.S. law libraries, and they analyze factors that shape East Asian collections, such as law libraries’ preservation and digitization efforts and their related cost-efficiency, and the availability and quality of English translations. The authors then discuss the main sources for Korean, Japanese, and Chinese law

In vitro evaluation of porcupine bezoar extracts as anticancer agent on A549 -A preliminary study

Porcupine bezoar (PB) was reported to possess medicinal properties in old medical manuscript. However, its potential as anticancer agent on human lung cancer cells (A549) is not yet studied. In present study, porcupine bezoar was tested to observe its ability in inhibiting cell growth of cancer cell (A549) and its cytotoxicity on Normal Human Gingival Fibroblast Cell (HGF-1). A549 cells morphology was observed after treated with bezoars for 72 hours. The ability of bezoars to induce DNA damage and apoptosis was analyzed by staining cells with Hoechst 33428(nucleus) and Rhodamine Phalloidin (f-actin). The A549 IC50 is 13.6±1.58μg/ml A549 growths was inhibited in dose-dependent pattern, but no inhibition found on normal HGF-1 cells. Treated A549 morphology shows sign of apoptosis such as DNA fragmentation, cytoplasm shrunk and vacuolation. The finding in this study suggests PB extracts able to inhibit cell growth, induce DNA damage and apoptosis, further analysis need to be done to verify the mechanism

Identification and quantification of quercetin, a major constituent of Artocarpus altilis by targeting related genes of apoptosis and cell cycle: in vitro cytotoxic activity against human lung carcinoma cell lines

Nine phenolic compounds were identified and quantified in Artocarpus altilia fruit. One of the main compounds was quercetin, which is the major class of flavonoids has been identified and quantified in pulp part of A. altilis fruit of methanol extract. The aim of this study was to evaluate in vitro cytotoxic assay. Inhibitory concentration 50% concentration was determined using trypan blue exclusion assay. Apoptosis induction and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell cycle-related regulatory genes were assessed by RT-qPCR study of the methanol extract of pulp part on human lung carcinoma (A549) cell line. A significant increase of cells at G2/M phases was detected (P<0.05). Furthermore, the pulp of the fruit downregulated the expression of antiapoptosis gene BCL-2 and upregulated the expression of pro-apoptosis gene BAX. CASPASE3 was also activated by the fruit, which started a CASPASE-3-depended mitochondrial pathway to induce apoptosis. As the results, the pulp was the most active in terms of all tests, due to high amount of quercetin in pulp part, 78% of total flavonoids. Taken together, these findings suggested that A. altilis induces apoptosis in a mitochondrial-dependent pathway by releasing and upregulating CYTOCHROME C expression and regulates the expression of downstream apoptotic components, including BCL-2 and BAX

Pyrosequencing Bacillus anthracis

Pyrosequencing technology was used to rapidly and specifically identify Bacillus anthracis

Identification of Highly Pathogenic Microorganisms by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Results of an Interlaboratory Ring Trial

In the case of a release of highly pathogenic bacteria (HPB), there is an urgent need for rapid, accurate, and reliable diagnostics. MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive technique that is becoming increasingly important in microbiological diagnostics to complement classical microbiology, PCR, and genotyping of HPB. In the present study, the results of a joint exercise with 11 partner institutions from nine European countries are presented. In this exercise, 10 distinct microbial samples, among them five HPB, Bacillus anthracis, Brucella canis, Burkholderia mallei, Burkholderia pseudomallei, and Yersinia pestis, were characterized under blinded conditions. Microbial strains were inactivated by high-dose gamma irradiation before shipment. Preparatory investigations ensured that this type of inactivation induced only subtle spectral changes with negligible influence on the quality of the diagnosis. Furthermore, pilot tests on nonpathogenic strains were systematically conducted to ensure the suitability of sample preparation and to optimize and standardize the workflow for microbial identification. The analysis of the microbial mass spectra was carried out by the individual laboratories on the basis of spectral libraries available on site. All mass spectra were also tested against an in-house HPB library at the Robert Koch Institute (RKI). The averaged identification accuracy was 77% in the first case and improved to >93% when the spectral diagnoses were obtained on the basis of the RKI library. The compilation of complete and comprehensive databases with spectra from a broad strain collection is therefore considered of paramount importance for accurate microbial identification

The prevalence of rodent-borne zoonotic pathogens in the South Gobi desert region of Mongolia.

peer reviewedThe alpine ecosystems and communities of central Asia are currently undergoing large-scale ecological and socio-ecological changes likely to affect wildlife-livestock-human disease interactions and zoonosis transmission risk. However, relatively little is known about the prevalence of pathogens in this region. Between 2012 and 2015 we screened 142 rodents in Mongolia's Gobi desert for exposure to important zoonotic and livestock pathogens. Rodent seroprevalence to Leptospira spp. was >1/3 of tested animals, Toxoplasma gondii and Coxiella burnetii approximately 1/8 animals, and the hantaviruses being between 1/20 (Puumala-like hantavirus) and <1/100 (Seoul-like hantavirus). Gerbils trapped inside local dwellings were one of the species seropositive to Puumala-like hantavirus, suggesting a potential zoonotic transmission pathway. Seventeen genera of zoonotic bacteria were also detected in the faeces and ticks collected from these rodents, with one tick testing positive to Yersinia. Our study helps provide baseline patterns of disease prevalence needed to infer potential transmission between source and target populations in this region, and to help shift the focus of epidemiological research towards understanding disease transmission among species and proactive disease mitigation strategies within a broader One Health framework

Determination toxic effects of Hystrix Brachyura Bezoar extracts using cancer cell lines and embryo zebrafish (Danio rerio) models and identification of active principles through GC-MS analysis

Ethnopharmacological relevance: Porcupine bezoar (PB) is used as folk medicine for various medical conditions including cancer treatment in Malaysia. However, its toxicity profile has never been thoroughly ascertained to confirm its safe nature as an efficacious traditional medicine in the treatment of cancer as well as other ailments. Aim of the study: This study was aimed to reveal three different PBs’ aqueous extracts(viz. PB-A, PB-B, PB-C) chemical constituent’s profile using GC-MS analysis, anticancer property on A375, HeLa and MCF7 cancer cells, toxicity profile on zebrafish embryo morphology, EC50, LC50 and teratogenicity index. Materials and methods: PBs’ extracts characterization was performed through GC-MS analysis, in vitro anticancer effect was carried out on A375, HeLa and MCF7 cancer cell lines and finally and toxicity properties on three different PBs aqueous extracts (viz. PB-A, PB-B, PB-C) were determined using zebrafish embryo model. Results: The GC-MS analysis revealed 10 similar compounds in all PBs’ extracts. Dilauryl thiodipropionate was found to be a major compound in all PBs’ extracts followed by tetradecanoic acid. An in vitro anticancer study revealed PB extracts exerted median inhibition concentration (IC50) <50 μg/mL, on cancer cells viz. A375, HeLa and MCF7 with no significant toxicity on normal cells viz. NHDF cells. In vivo toxicity of PBs extracts found affecting tail detachment, hatching, craniofacial, brain morphology, soft tissues, edema, spinal, somites, notochord and cardiovascular system (brachycardia, disruption of blood circulation) deformities. The LC50 and EC50 demonstrated PB extracts effect as dose and time dependent with median concentration <150.0 μg/mL. Additionally, teratogenicity index (TI) viz. >1.0 revealed teratogenic property for PB extracts. Conclusions: The findings revealed that all three PBs aqueous extracts possessed anticancer activity and exhibited significant toxicological effects on zebrafish embryos with high teratogenicity index. Hence, its use as an anticancer agent requires further investigation and medical attentions to determine its safe dose

Adaptation of Brucella melitensis Antimicrobial Susceptibility Testing to the ISO 20776 Standard and Validation of the Method

This article belongs to the Special Issue Emerging Themes in Brucella and Brucellosis.Brucellosis, mainly caused by Brucella (B.) melitensis, is associated with a risk of chronification and relapses. Antimicrobial susceptibility testing (AST) standards for B. melitensis are not available, and the agent is not yet listed in the EUCAST breakpoint tables. CLSI recommendations for B. melitensis exist, but they do not fulfill the requirements of the ISO 20776 standard regarding the culture medium and the incubation conditions. Under the third EU Health Programme, laboratories specializing in the diagnostics of highly pathogenic bacteria in their respective countries formed a working group within a Joint Action aiming to develop a suitable method for the AST of B. melitensis. Under the supervision of EUCAST representatives, this working group adapted the CLSI M45 document to the ISO 20776 standard after testing and validation. These adaptations included the comparison of various culture media, culture conditions and AST methods. A Standard Operation Procedure was derived and an interlaboratory validation was performed in order to evaluate the method. The results showed pros and cons for both of the two methods but also indicate that it is not necessary to abandon Mueller–Hinton without additives for the AST of B. melitensis.This research was funded by the EU Health Programme 2014–2020, through the Consumers, Health, Agriculture and Food Executive Agency (CHAFEA, European Commission), the Joint Action EMERGE (CHAFEA n° 677 066) and the Joint Action SHARP (848096-SHARP JA).info:eu-repo/semantics/publishedVersio