Unpacking the Pathogen Box—An Open Source Tool for Fighting Neglected Tropical Disease

The Pathogen Box is a 400-strong collection of drug-like compounds, selected for their potential against several of the world's most important neglected tropical diseases, including trypanosomiasis, leishmaniasis, cryptosporidiosis, toxoplasmosis, filariasis, schistosomiasis, dengue virus and trichuriasis, in addition to malaria and tuberculosis. This library represents an ensemble of numerous successful drug discovery programmes from around the globe, aimed at providing a powerful resource to stimulate open source drug discovery for diseases threatening the most vulnerable communities in the world. This review seeks to provide an in-depth analysis of the literature pertaining to the compounds in the Pathogen Box, including structure–activity relationship highlights, mechanisms of action, related compounds with reported activity against different diseases, and, where appropriate, discussion on the known and putative targets of compounds, thereby providing context and increasing the accessibility of the Pathogen Box to the drug discovery community.     

Transport of selected bacterial pathogens in agricultural soil and quartz sand

The protection of groundwater supplies from microbial contamination necessitates a solid understanding of the key factors controlling the migration and retention of pathogenic organisms through the subsurface environment. The transport behavior of five waterborne pathogens was examined using laboratory-scale columns packed with clean quartz at two solution ionic strengths (10 mM and 30 mM). Escherichia coli O157:H7 and Yersinia enterocolitica were selected as representative Gram-negative pathogens, Enterococcus faecalis was selected as a representative Gram-positive organism, and two cyanobacteria (Microcystis aeruginosa and Anabaena flos-aquae) were also studied. The five organisms exhibit differing attachment efficiencies to the quartz sand. The surface (zeta) potential of the microorganisms was characterized over a broad range of pH values (2-8) at two ionic strengths (10 mM and 30 mM). These measurements are used to evaluate the observed attachment behavior within the context of the DLVO theory of colloidal stability. To better understand the possible link between bacterial transport in model quartz sand systems and natural soil matrices, additional experiments were conducted with two of the selected organisms using columns packed with loamy sand obtained from an agricultural field. This investigation highlights the need for further characterization of waterborne pathogen surface properties and transport behavior over a broader range of environmentally relevant conditions.     

A Rapid and Simple DNA Extraction Procedure to Detect Salmonella spp. and Listeria monocytogenes from Fresh Produce Using Real-time PCR

DNA isolation procedures significantly influence the outcome of PCR-based detection of human pathogens. Unlike clinical samples, DNA isolation from food samples, particularly from fresh and fresh-cut produce has remained a formidable task and has hampered the sensitivity and accuracy of molecular methods. We utilized a commercially available filter-based DNA isolation method (FTA) in conjunction with real-time PCR-based detection of Salmonella spp. and Listeria monocytogenes. The protocol uses filter paper discs impregnated with a patented chemical formulation that lyses cells, immobilizes DNA, and protects it from degradation. Use of the FTA method in conjunction with real-time PCR for the detection of Salmonella spp. and L. monocytogenes was compared with two commercially available DNA isolation procedures that are commonly used for high throughput real-time PCR pathogen detection systems. Both pathogens were successfully detected from artificially inoculated fresh and fresh-cut produce such as alfalfa sprouts, cilantro, green onion, broccoli, prepacked mixed salad, and spinach at low cell numbers (four to seven colony forming units per 25 g initial inoculum level before enrichment). The FTA protocol had distinct advantages of simplicity, biosafety, and compatibility for high throughput screening. This DNA preparation protocol was rapid, sensitive, required minimal handling, and reduced interference from produce-associated inhibitors of real-time PCR. Mention of brand names does not constitute an endorsement by the U.S. Department of Agriculture above others of a similar nature not mentioned.     

Evaluation of low-copy genetic targets for waterborne bacterial pathogen detection via qPCR

Recent developments in water quality research have highlighted difficulties in accurately predicting the incidence of pathogens within freshwater based on the viability, culturability and metabolic activity of indicator organisms. QPCR-driven assays are candidates to replace standard culture-based methods, however, protocols suitable for routine use have yet to be sufficiently validated. The objective of this study was to evaluate five oligonucleotide primers sets (ETIR, SINV, exoT, VS1 and ipaH2) for their potential applicability in qPCR assays to detect contamination from five waterborne bacterial pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, Pseudomonas aeruginosa, and Shigella flexneri). An enrichment-free qPCR protocol was also tested using S. Typhimurium-seeded source water, combining membrane filtration and mechanical, chemical and enzymatic lysis techniques to recover the bacterial cells. All five primer sets were found to have high specificity and sensitivity for the tested organisms. Four of the primers were able to detect pathogen loads as low as 10 cells/mL while 200 cells/mL of C. jejuni were detectable in pure culture. Although sensitivity decreased in an artificially contaminated environmental matrix, it was still possible to detect as few as 10 S. Typhimurium cells without enrichment. The primers and protocols evaluated in this study have demonstrated potential for further validation for possible application alongside traditional indicator techniques.     

Detection of E. coli O157:H7 in raw ground beef by Pathatrix™ immunomagnetic-separation, real-time PCR and cultural methods

Detection of Escherichia coli O157:H7 by conventional cultural methods can be difficult in food matrices with a high background flora such as raw ground beef. Raw ground beef samples, artificially contaminated separately with five strains of E. coli O157:H7 at low (~ 0.2 cfu/g) and high (~ 2 cfu/g) levels, were enriched by two enrichment protocols; buffered peptone water (BPW) at 37 °C for 5 h and 24 h and modified buffered peptone water with pyruvate (mBPWp) for 5 h at 37 °C followed by adding selective agents and incubating at 42 °C to 24 h. Detection of added E. coli O157:H7 by real-time PCR (RTiPCR) and recovery on isolation agars was performed before and after PATHATRIX™ immunomagnetic separation (IMS). RTiPCR detection and cultural recovery of inoculated E. coli O157:H7 after 5 h enrichment were poor at 0.21-0.24 cfu/g. The addition of IMS after 5 h enrichment did not improve RTiPCR detection but markedly improved recovery by culturing. By extending enrichment to 24 h, RTiPCR detection improved to 76% for either enrichment protocol without IMS. When 24 h enrichment was followed by IMS, RTiPCR detection was also further improved. Cultural recovery after 24 h enrichment was 56% and 84% without IMS and 100% and 92% after IMS for BPW and mBPWp respectively. Extended enrichment to 24 h followed by IMS was found to be sensitive and reliable for detection and cultural recovery from raw ground beef using either enrichment method.     

2002年陕西省食品中食源性致病菌监测

为了解陕西省食品中致病菌的污染状况，2002年对陕西省食品中食源性致病菌进行了监测，在5个监测点采集6类468份食品样本，按常规方法分离鉴定沙门氏菌、E．coliO157：H7、单增李斯特氏菌。共分离到沙门氏菌44株，E．coliO157：H75株，单增李斯特氏菌35株。以生肉类食品的污染率为高(38．5％)。分离的E．coliO157：H7菌株中有1株具有stx2、hly、eae毒力基因。调查结果提示食源性致病菌在食品中的污染状况比较严重，应引起有关部门的重视。     

河南疑似烟草PVX的病原种类鉴定及田间发生规律

采用双抗体夹心酶联免疫吸附检测技术和多点田间试验调查的方法,研究了河南疑似烟草PVX的病原种类及田间发生规律。结果表明,河南疑似烟草PVX的病原种类为马铃薯Y病毒;移栽后10～20d发生较轻且发展缓慢,20～60d发生较重且发展迅速,60d以后病情趋于稳定;中烟100和云烟87品种的抗性无显著差异,且均显著低于NC89。     

Performance assessment of electrospun nanofibers for filter applications

The aim of this study was to evaluate the use of nanofiber microfiltration membranes, spun by an innovative electrospinning technique, in water filtration applications. As such, this study bridges between developments in electrospinning techniques for the production of flat sheet membranes and the application of these membranes in water filtration. Three different applications were examined. First, the use of the membrane (functionalized or non-functionalized) for the removal of pathogens was investigated. Second the electrospun flat sheet membranes were applied in a lab scale submerged membrane bioreactor (MBR). Third, the electrospun membranes were applied as stand-alone filter for water treatment. Next to these applications, physical properties such as clean water permeability (CWP) and strength were also examined. The test showed that the electrospun membranes can be used for water filtration applications, but that further research is necessary towards irreversible fouling properties and level of functionalisation.     

Assessment of biofertilizer quality and health implications of anaerobic digestion effluent of cow dung and chicken droppings

Anaerobic digestate have been identified as a rich source of essential plant nutrients. Nevertheless, its safety measured by the concentration of pathogen present is of great concern to end users. This research explored the efficiency of the mesophilic biodigestion process in the stabilization and sanitization of cow dung and chicken droppings. Six (6) kg each of cow dung and chicken droppings were collected fresh and free from impurities, pre-fermented, mixed with water in the ratio 1:1 w/v to form slurry, fed into the respective reactors and digested for 30 days at an average ambient temperature of 30 ± 2 °C. The pH of the medium fluctuated between 6.5 and 8.0. The analysis of the feedstock and effluent of the digesters showed that a total solids reduction of 75.3% and 60.1% were recorded for cow dung and chicken droppings while the reduction in total coliforms was 95% and 70% respectively for the dung and droppings. Microbial analysis of the biofertilizer produced reveals both aerobic and anaerobic organisms which include species of Pseudomonas, Klebsiella, Clostridium, Bacillus, Bacteroides, Salmonella, Penicillum and Aspergillus. Escherichia coli and Shigella spp. were removed while species of Salmonella and Klebsiella were still present in the digestate. Notwithstanding these results, the digestate still requires further treatment for it to be suitable for application on unrestricted crops either as fertilizer; otherwise a health problem would be created as attempt is made to improve soil fertility.