Detection of mutagens in water-distribution systems after disinfection

This research examined the quality of water-before and after distribution-of four drinking-water production plants located in Northern Italy, two of which collected water from local aquifers and two from the River Po. A battery of genotoxicity assays for monitoring drinking-water was performed to assess the quality of the water produced by the treatment plants under study. Three different sampling stations were selected at each plant, one right at the outlet of the treatment plant and two along with the distribution pipelines. Raw river water was also sampled and analysed as a control. The water samples (500 l) were concentrated on silica C18 cartridges and the extracts were tested in in vitro mutagenicity assays (Salmonella/microsome assay with strains TA 98 and TA 100; SOS Chromotest with Escherichia coli strain PQ37); gene conversion, point mutation and mitochondrial DNA mutability assays with the diploid Saccharomyces cerevisiae strain D7 and a toxicity test using the bioluminescent bacterium Vibrio fischeri (Microtox). The Microtox test and the mitochondrial DNA mutability assay showed the greatest sensitivity towards toxic or mutagenic substances in the water extracts considered. The results show that this battery of short-term tests is applicable in the routine monitoring of drinking-water quality before and after distribution.     

Detection of bacterial pathogens in environmental samples using DNA microarrays

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.     

Detection of foodborne pathogens using DNA probes and a dipstick format

The detection of foodborne microorganisms has traditionally been done using microbiologically based methods. Such “gold standard” methods are generally reliable but have the disadvantages of being labor intensive, subjective, and time consuming. Over the last several years, the development of DNA probe-based methods has simplified the methods used to detect organisms such asSalmonella, Listeria, andE. coli by targeting the unique DNA or RNA sequences of these organisms using DNA probes and nonradioactive detection.     

Bioassay and dose measurement in UV disinfection.

A bioassay method was developed to measure the average intensity within a UV disinfection reactor. The survival of spores of Bacillus subtilis was determined as a function of UV dose to prepare a standard curve. Spores were added to unknown systems, and the survival rate was used to determine the average intensity. A modification was used for flow-through reactors by which spores were injected as a spike and collected at a known time after injection. A point source summation method for calculating intensity was verified by bioassay measurements in a simple cylinder. This calculation method was also applied to multiple-lamp reactors.     

Quantitative detection and biological propagation of scrapie seeding activity in vitro facilitate use of prions as model pathogens for disinfection

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤10(1)- to ≥10(5.5)-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants.     

Detection of pathogens in water: from phylochips to qPCR to pyrosequencing

Waterborne pathogens pose a significant threat to human health and a proper assessment of microbial water quality is important for decision making regarding water infrastructure and treatment investments and eventually to provide early warning of disease, particularly given increasing global disasters associated with severe public health risks. Microbial water quality monitoring has undergone tremendous transition in recent years, with novel molecular tools beginning to offer rapid, high-throughput, sensitive and specific detection of a wide spectrum of microbial pathogens that challenge traditional culture-based techniques. High-density microarrays, quantitative real-time PCR (qPCR) and pyrosequencing which are considered to be breakthrough technologies borne out of the 'molecular revolution' are at present emerging rapidly as tools of pathogen detection and discovery. Future challenges lie in integrating these molecular tools with concentration techniques and bioinformatics platforms for unbiased guide of pathogen surveillance in water and developing standardized protocols.     

Detection of PCR amplicons from bacterial pathogens using microsphere agglutination

For rapid and inexpensive detection of polymerase chain reaction (PCR) amplicons, a novel microsphere agglutination assay has been developed. PCR is carried out using biotinylated forward and reverse primers, and the amplified DNA fragments are able to agglutinate streptavidin-coated microspheres (5.7 microm in diameter). Purification of PCR amplicons is unnecessary when initial primer concentrations are 250 nM. Agglutination can be identified visually within 2 min without any additional equipment or reagents. Using listeriolysin (lisA)-specific biotinylated primers, we have successfully detected and identified Listeria monocytogenes lisA+ cells among Salmonella typhimurium, Staphylococcus aureus, Campylobacter jejuni and Escherichia coli O157:H7 cells. The simplicity of this protocol considerably reduces the time and cost of diagnostic PCR experiments. This procedure is potentially useful for various studies and field applications.     

Detection and identification of globally distributed mycobacterial fish pathogens in some ornamental fish in India

Mycobacteriosis is a progressive disease of a wide range of wild and captive, marine and freshwater fish species. Conventional detection of fish Mycobacteria is based on histopathology, culture, and biochemical characteristics. The present study analyzed the occurrence of Mycobacteria in clinically ill ornamental fish of different species, from different places of India. In first group, 60 fish were examined for presence of granulomatous inflammation and acid-fast bacteria. Thirty-eight (63.34 %) fish were positive for granulomatous inflammations. Presences of acid-fast bacteria were detected in 27 (45 %) fish having granulomatous inflammation and in two (3.33 %) fish without granulomatous inflammation. In total, AFB were found in 29 (48.34 %) of the 60 fish examined. In second group, 20 fish having granulomatous inflammation, 12 (60 %) samples were positive using Ziehl–Neelsen (Z-N) staining and 11 (55 %) of them were culture positive. Eight (40 %) samples were Z-N negative but two (10 %) of them were culture positive. In total, 13 (65 %) of the 20 examined fish were culture positive. On the basis of biochemical tests and 16S rRNA sequencing, 13 isolates were identified: five as Mycobacterium fortuitum, five as Mycobacterium gordonae, and three as Mycobacterium chelonae. In comparison of two decontamination methods, 2 % HCl treatment was better than 4 % NaOH treatment. Mycobacteria recovery from decontaminated samples was significantly high on Lowenstein–Jensen medium compared to Middlebrook 7H11 agar and Stonebrink (SB) media. The disease is transmissible from fish to fish and also from fish to human, so the significance of Mycobacteria in ornamental fish should not be overlooked.     

Residual disinfection of some preparations and substances.

The results of our experiments over many years point to the possibility of practical application of disinfectants of residual effect. In our opinion, further investigations should be carried out in the following two directions: detection of new means and preparations showing uniform properties and development of an optimum method of their application. We believe that residual disinfection will play a role in the prophylaxis of hospitalism.     

Enzymatic removal and disinfection of bacterial biofilms.

Model biofilms of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas fluorescens, and Pseudomonas aeruginosa were made on steel and polypropylene substrata. Plaque-resembling biofilms of Streptococcus mutans, Actinomyces viscosus, and Fusobacterium nucleatum were made on saliva-coated hydroxyapatite. The activity of enzymes against bacterial cells in biofilm was measured by fluorescence microscopy and an indirect conductance test in which evolution of carbon dioxide was measured. Glucose oxidase combined with lactoperoxidase was bactericidal against biofilm bacteria but did not remove the biofilm from the substrata. A complex mixture of polysaccharide-hydrolyzing enzymes was able to remove bacterial biofilm from steel and polypropylene substrata but did not have a significant bactericidal activity. Combining oxidoreductases with polysaccharide-hydrolyzing enzymes resulted in bactericidal activity as well as removal of the biofilm.     

Detection of tularemia pathogens in patients with the use of an immunofluorescence reaction

For the first time three cases of the detection of Francisella tularensis, made by means of the direct immunofluorescence test in the fluid obtained from punctured buboes or in purulent matter taken from patients with the ulcerous bubonic form of tularemia, are presented. The simplicity of the test and its capacity of yielding rapid results make it possible to recommend this test, together with other diagnostic methods, for the clinical diagnosis of tularemia.     

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

Detection of black-foot and Petri disease pathogens in soils of grapevine nurseries and vineyards using bait plants

Background and aims

Little information is currently available regarding the number of species of black-foot and Petri disease pathogens present in soil and their capacity to infect grapevine roots and reach the xylem vessels.

Methods

Seedlings of grapevine rootstock 41-B, and cvs. Bobal and Palomino were planted both in pots containing soil samples collected from commercial vineyards and in nursery fields. Roots and xylem vessels were later analyzed for fungal isolation.

Results

Black-foot pathogens: Ilyonectria alcacerensis, I. macrodidyma, I. novozelandica and I. torresensis were frequently isolated from roots of seedlings grown in all soils evaluated, whereas Petri disease pathogens: Cadophora luteo-olivacea, Phaeoacremonium aleophilum, Pm. parasiticum and Phaeomoniella chlamydospora were only isolated from xylem vessels of seedlings grown in nursery soils, with a low incidence. Ilyonectria alcacerensis, I. novozelandica and I. torresensis were isolated for the first time from grapevines in Spain, and Pm. parasiticum and Ca. luteo-olivacea were detected for the first time in nursery soils.

Conclusions

Our results confirm nursery and vineyard soils as an important inoculum source for black-foot pathogens and demonstrate the presence of several Petri disease pathogens in nursery soils.     

Detection of Fusarium wilt pathogens of Psidium guajava L. in soil using culture independent PCR (ciPCR)

Traditional culturing methods take a long time for identification of pathogenic isolates. A protocol has been developed for the detection of Fusarium from soil samples in the early stage of infection. Seventeen soil samples from different locations were collected before the onset of rains to find out the presence of Fusarium spp. population present in the soil of guava orchards and to correlate its presence with incidence of wilt. A PCR based method was developed for the molecular characterization of Fusarium using Fusarium spp. specific primer. DNA extracted by this method was free from protein and other contaminations and the yield was sufficient for PCR amplification. The primer developed in this study was amplifying ∼230 bp in all infected samples while not in healthy soil. The specificity and sensitivity of primer were tested on several Fusarium spp. and found that this primer was amplifying 10⁻⁶ dilution of the fungal DNA. The present study facilitates the rapid detection of Fusarium spp. from infected soil samples of guava collected from different agroclimatic regions in India. A rapid detection method for pathogens and a diagnostic assay for disease would facilitate an early detection of pathogen and lead to more effective control strategies.     

Physiological responses of bacteria in biofilms to disinfection.

In situ enumeration methods using fluorescent probes and a radioisotope labelling technique were applied to evaluate physiological changes of Klebsiella pneumoniae within biofilms after disinfection treatment. Chlorine (0.25 mg of free chlorine per liter [pH 7.2]) and monochloramine (1 mg/liter [pH 9.0]) were employed as disinfectants in the study. Two fluorgenic compounds, 5-cyano-2,3-ditolyl tetrazolium chloride and rhodamine 123, and tritiated uridine incorporation were chosen for assessment of physiological activities. Results obtained by these methods were compared with those from the plate count and direct viable count methods. 5-Cyano-2,3-ditolyl tetrazolium chloride is an indicator of bacterial respiratory activity, rhodamine 123 is incorporated into bacteria in response to transmembrane potential, and the incorporation of uridine represents the global RNA turnover rate. The results acquired by these methods following disinfection exposure showed a range of responses and suggested different physiological reactions in biofilms exposed to chlorine and monochloramine. The direct viable count response and respiratory activity were affected more by disinfection than were the transmembrane potential and RNA turnover rate on the basis of comparable efficiency as evaluated by plate count enumeration. Information revealed by these approaches can provide different physiological insights that may be used in evaluating the efficacy of biofilm disinfection.     

Gaseous disinfection of Cryptosporidium parvum oocysts.

Purified oocysts of Cryptosporidium parvum suspended in approximately 400 microliters of phosphate-buffered saline or deionized water in microcentrifuge tubes were exposed at 21 to 23 degrees C for 24 h to a saturated atmosphere of ammonia, carbon monoxide, ethylene oxide, formaldehyde, or methyl bromide gas. Controls were exposed to air. Oocysts in each tube were then rinsed and resuspended in fresh, deionized water, and 1 million oocysts exposed to each gas were orally administered to each of three to six neonatal BALB/c mice in replicate groups. Histologic sections of ileum, cecum, and colon tissues taken from each mouse 72 h after oral administration of oocysts were examined microscopically to determine if infection had been established. All 15 mice given oocysts exposed to carbon monoxide had numerous developmental stages of cryptosporidium in all three intestinal segments. Of 10 mice given oocysts exposed to formaldehyde, 6 had a few developmental stages of cryptosporidium in the ileum. No mice given oocysts exposed to ammonia, ethylene oxide, or methyl bromide were found to be infected. These findings indicate the efficacy of these low-molecular-weight gases (ammonia, ethylene oxide, and methyl bromide) as potential disinfectants for C. parvum oocysts where soil, rooms, buildings, tools, or instruments might be contaminated.