Inactivation of poliovirus 1 and F-specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers

Several models (animal caliciviruses, poliovirus 1 [PV1], and F-specific RNA bacteriophages) are usually used to predict inactivation of nonculturable viruses. For the same UV fluence, viral inactivation observed in the literature varies from 0 to 5 logs according to the models and the methods (infectivity versus molecular biology). The lack of knowledge concerning the mechanisms of inactivation due to UV prevents us from selecting the best model. In this context, determining if viral genome degradation may explain the loss of infectivity under UV radiation becomes essential. Thus, four virus models (PV1 and three F-specific RNA phages: MS2, GA, and Qbeta) were exposed to UV radiation from 0 to 150 mJ.cm-2. PV1 is the least-resistant virus, while MS2 and GA phages are the most resistant, with phage Qbeta having an intermediate sensitivity; respectively, 6-log, 2.3-log, 2.5-log, and 4-log decreases for 50 mJ.cm-2. In parallel, analysis of RNA degradation demonstrated that this phenomenon depends on the fragment size for PV1 as well as for MS2. Long fragments (above 2,000 bases) for PV1 and MS2 fell rapidly to the background level (>1.3-log decrease) for 20 mJ.cm-2 and 60 mJ.cm-2, respectively. Nevertheless, the size of the viral RNA is not the only factor affecting UV-induced RNA degradation, since viral RNA was more rapidly degraded in PV1 than in the MS2 phage with a similar size. Finally, extrapolation of inactivation and UV-induced RNA degradation kinetics highlights that genome degradation could fully explain UV-induced viral inactivation.     

The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems

Three strains of Listeria monocytogenes (NCTC 11994, a poultry isolate and the Scott A strain) were exposed to a range of pressures (300, 350, 375, 400 and 450 MPa) in 10 mmol l⁻¹ phosphate-buffered saline (PBS) at pH 7·0 for up to 30 min at ambient temperature. Generally, increasing the magnitude and duration of compression resulted in increasing levels of inactivation, although the inactivation kinetics varied depending on the strain and pressure applied. The three strains also exhibited a wide variation in their resistance to high pressure. The resistance of the three strains to high pressure (375 MPa) was also assessed in a series of model food systems containing one of each of the three main food constituents: protein (1, 2, 5 and 8% w/v bovine serum albumin in PBS), carbohydrate (1, 2, 5 and 10% w/v glucose in PBS) and lipid (olive oil (30% v/v) in PBS emulsion). Overall, increasing the concentrations of bovine serum albumin (BSA) and glucose in the suspending medium resulted in decreasing levels of inactivation of all three strains; however, the minimum concentration of BSA and glucose required to increase survival to a level greater than that observed in PBS alone varied depending on the strain and on the duration of the treatment. The survival of all three strains was greater in the olive oil/PBS emulsion than in PBS alone at all treatment times.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada     

Inactivation of Salmonella Typhimurium and Listeria monocytogenes using high-pressure treatments: destruction or sublethal stress?

AIMS: To investigate potential resuscitation of Listeria monocytogenes and Salmonella Typhimurium after high hydrostatic pressure treatments. METHODS AND RESULTS: Pressure treatments were applied at room temperature for 10 min on bacterial suspensions in buffers at pH 7 and 5.6. Total bacterial inactivation (8 log(10) CFU ml(-1) of bacterial reduction) obtained by conventional plating was achieved regarding both micro-organisms. Treatments at 400 MPa in pH 5.6 and 600 MPa in pH 7 for L. monocytogenes and at 350 MPa in pH 5.6 and 400 MPa in pH 7 for S. Typhimurium were required respectively. A 'direct viable count' method detected some viable cells in the apparently totally inactivated population. Resuscitation was observed for the two micro-organisms during storage (at 4 and 20 degrees C) after almost all treatments. In the S. Typhimurium population, 600 MPa, 10 min, was considered as the treatment achieving total destruction because no resuscitation was observed under these storage conditions. CONCLUSIONS: We suggest a delay before performing counts in treated samples in order to avoid the under-evaluation of surviving cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The resuscitation of pathogen bacteria after physical treatments like high hydrostatic pressure has to be considered from the food safety point of view. Further studies should be performed in food products to study this resuscitation phenomenon.     

Synthesis of Indicator Strains and Density of Ribonucleic Acid-Containing Coliphages in Sewage

Escherichia coli strains freshly isolated from natural sources are inefficient indicators of coliphages present in sewage. Four E. coli strains recently isolated from clinical specimens were mutagenized to obtain lac(-) mutants. Such mutants were infected with an F'lac(+) sex factor of E. coli K-12. Pairs of isogenic lac(-) and lac(-)/F'lac(+) strains were used as indicators of coliphages present in sewage, and it was found that such strains can be effectively used for a direct and almost selective enumeration of F-specific coliphage contents of sewage samples. Serological tests were applied to a number of F-specific phages isolated. All the isolates that were tested fell into two distinguishable antigenic classes: members of one class being related to ribonucleic acid (RNA) phage MS2 and those of the other being related to another RNA phage, namely, Qbeta. MS2-related phages have been found to be more widely distributed than the Qbeta related phages. Most habitats sampled were found to yield only one or the other kind of phage. Single-stranded deoxyribonucleic acid-containing F-specific phages were not detectable by the methods employed by us.     

不同悬浮介质对激光衍射法测得的变形指数—渗透压曲线的影响

本文用PBS和PVP两种介质作为激光衍射仪的悬浮介质,系统地观测了悬浮介质物化性能不同对红细胞变形指数(DI)—渗透压曲线的影响,发现:1.在不同悬浮介质中的变形指数—渗透压曲线有显著差别,在高渗区这种差别更明显.2.同一红细胞试样,在相同高渗压下,在不同的悬浮介质中进行交叉实验表明、PBS悬浮介质能使得红细胞变形性得到恢复,而PVP悬浮介质却使得红细胞变形性显著降低.3.在高渗情况下,首次观察到红细胞变形性随时间变化,一开始DI增大,约3小时后趋于稳定,无论PBS还是PVP其趋势皆相似.     

Refined molecular weights for phage, viral and ribosomal RNA.

The RNAs of the Escherichia coli bacteriophages MS2 and Qbeta as well as E. coli 16S ribosomal RNA were examined under identical conditions by electron microscopy using the protein-free benzyldimethylalkylammonium chloride (BAC) spreading technique. From the contour length ratios of the RNAs and the known number of nucleotides for MS2, the chain lengths for Qbeta RNA and 16S RNA were found to be 4790 +/- 150 and 1645 +/- 55 nucleotides. Correcting for the base composition of Qbeta RNA the molecular weight of the Na salt of this RNA is (1.64 +/- 0.06) . 10(6) daltons. Since published values on the relative lengths of Qbeta RNA and several other homogeneous RNAs (E. coli 23S rRNA, E. Coli bacteriophage R17 and f2 RNAs, Pseudomonas aeruginosa phage PP7 RNA and Newcastle disease virus RNA) are available, we are able to calculate the approximate number of nucleotides for these useful standards.     

Bacteriophages and indicator bacteria in human and animal faeces

In an attempt to explain the presence of F-specific (RNA) bacteriophages in waste-water, faecal material from humans and a variety of animals was examined. The phages were detected in appreciable numbers only in faeces from pigs, broiler chickens, sheep and calves but not from dogs, cows, horses and humans. Parallel examinations for somatic coliphages, thermotolerant coliforms, faecal streptococci and spores of sulphite-reducing clostridia revealed the consistent presence of these organisms in all types of samples, albeit in variable numbers. The number of F-specific bacteriophages was related to the total number of somatic coliphages, but phage counts were unrelated to bacterial counts. F-specific RNA phages were grouped by serotyping and all animal isolates were found to belong to either group I (MS2 subtype) or IV (four different subtypes). Among the group IV isolates, most belonged to well-known subtypes SP (24 isolates) or FI (18 isolates) but five isolates were related to phage ID2 and one isolate was a new subtype. In contrast with animal isolates, 19 isolates from hospital wastewater belonged to serogroups II or III.     

Bacteriophages and indicator bacteria in human and animal faeces

In an attempt to explain the presence of F-specific (RNA) bacteriophages in waste-water, faecal material from humans and a variety of animals was examined. The phages were detected in appreciable numbers only in faeces from pigs, broiler chickens, sheep and calves but not from dogs, cows, horses and humans. Parallel examinations for somatic coliphages, thermotolerant coliforms, faecal streptococci and spores of sulphite-reducing clostridia revealed the consistent presence of these organisms in all types of samples, albeit in variable numbers. The number of F-specific bacteriophages was related to the total number of somatic coliphages, but phage counts were unrelated to bacterial counts. F-specific RNA phages were grouped by serotyping and all animal isolates were found to belong to either group I (MS2 subtype) or IV (four different subtypes). Among the group IV isolates, most belonged to well-known subtypes SP (24 isolates) or FI (18 isolates) but five isolates were related to phage ID2 and one isolate was a new subtype. In contrast with animal isolates, 19 isolates from hospital wastewater belonged to serogroups II or III.     

Removal and inactivation of indicator bacteriophages in fresh waters

AIMS: The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis in fresh waters. METHODS AND RESULTS: Removal was studied in two areas of a river. The results showed different removal of each group of microbes. Faecal coliform bacteria were removed faster than any other, whereas SRC and bacteriophages infecting Bact. fragilis were the most persistent. Inactivation was measured by 'in situ' experiments, which showed significant differences in survival of the different groups of bacterial and bacteriophage indicators. The SRC and bacteriophages were more resistant than faecal coliforms and enterococci, with the exception of F-specific RNA bacteriophages in the summer. Inactivation experiments with pure cultures of bacteriophages confirmed that phage B40-8 of Bact. fragilis was the most resistant. CONCLUSIONS: Bacteria and bacteriophages show different resistance to natural inactivation. The use of phages allows information to be obtained in addition to that provided by bacterial indicators. Somatic coliphages and phages infecting Bact. fragilis might supply that indicator function. SIGNIFICANCE AND IMPACT OF THE STUDY: Confirmation was obtained that bacteriophages provided additional information to that provided by bacterial indicators to monitor the natural inactivation of viruses and/or pathogens.     

Fate of viruses in artificial wetlands.

Little is known about the ability of wetlands to remove disease-causing viruses from municipal wastewater. In this study we examined the survival of several indicators of viral pollution (indigenous F-specific bacteriophages, seeded MS2 bacteriophage, and seeded human poliovirus type 1) applied in primary municipal wastewater to artificial wetland ecosystems. Only about 1% of the indigenous F-specific RNA bacteriophages survived flow through the vegetated wetland beds at a 5-cm-day-1 hydraulic application rate during the period from June through December 1985. The total number of indigenous F-specific bacteriophages (F-specific RNA and F-specific DNA phages) was also reduced by about 99% by wetland treatment, with the mean inflow concentration over the period of an entire year reduced from 3,129 to 33 PFU ml-1 in the outflow of a vegetated bed and to 174 PFU ml-1 in the outflow of an unvegetated bed. Such superior treatment by the vegetated bed demonstrates the significant role of higher aquatic plants in the removal process. Seeded MS2 bacteriophage and seeded poliovirus were removed more efficiently than were the indigenous bacteriophages, with less than 0.2% of MS2 and 0.1% of the poliovirus surviving flow at the same hydraulic application rate. The decay rate (k) of MS2 in a stagnant wetlands (k = 0.012 to 0.028 h-1) was lower than that for flowing systems (k = 0.44 to 0.052 h-1), reflecting the enhanced capacity for filtration or adsorption of viruses by the root-substrate complex (and associated biofilm).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modelling the effect of high pressure on the inactivation kinetics of a pressure-resistant strain of Pediococcus damnosus in phosphate buffer and gilt-head seabream (Sparus aurata)

AIMS: The aim of this research was to: (i) determine the inactivation pattern of a pressure-resistant strain of Pediococcus damnosus by high hydrostatic pressure in phosphate buffer (pH 6.7) and gilt-head seabream using the linear, biphasic and Weibull models; and (ii) validate the applicability of the Weibull model to predict survival curves at other experimental pressure levels. METHODS AND RESULTS: A pressure-resistant strain of P. damnosus was exposed to a range of pressures (500, 550, 600 and 650 MPa) in phosphate buffer (pH 6.7) and gilt-head seabream for up to 8 min at ambient temperature (23 degrees C). Inactivation kinetics were described by the linear, biphasic and Weibull models. Increasing the magnitude of the pressure applied resulted in increasing levels of inactivation. Pronounced tailing effect was observed at pressures over 600 MPa. The Weibull and biphasic models consistently produced better fit than the linear model as inferred by the values of the root mean squared error, coefficient of determination (R2) and accuracy factor (A(f)). The scale factor (b) of the Weibull model was linearly correlated with pressure (P) treatment in the whole pressure range. Substituting the b parameter in the initial Weibull function and calculating the shape factor (n) by linear interpolation, high pressure (P) was directly incorporated into the model providing reasonable predictions of the survival curves at 570 and 630 MPa. Comparison between the survival curves in phosphate buffer and gilt-head seabream showed a clear protective effect of the food matrix on the resistance of the micro-organism, especially at 500 and 550 MPa. CONCLUSIONS: The Weibull and biphasic models were more flexible to describe the survival curves of P. damnosus in the experimental pressure range, taking also into account the tailing effect that could not be included in the linear model. The Weibull model could also give reasonable predictions of the survival curves at other experimental pressures in both pressure menstrua. As the food matrix has a protective effect in microbial inactivation, the development of accurate mathematical models should be done directly on real food to avoid under- or over-processing times. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of accurate models to describe the survival curves of micro-organisms under high hydrostatic pressure treatment would be very important to the food industry for process optimisation, food safety and extension of the applicability of high pressure processing.     

Fate of viruses in artificial wetlands

Little is known about the ability of wetlands to remove disease-causing viruses from municipal wastewater. In this study we examined the survival of several indicators of viral pollution (indigenous F-specific bacteriophages, seeded MS2 bacteriophage, and seeded human poliovirus type 1) applied in primary municipal wastewater to artificial wetland ecosystems. Only about 1% of the indigenous F-specific RNA bacteriophages survived flow through the vegetated wetland beds at a 5-cm-day-1 hydraulic application rate during the period from June through December 1985. The total number of indigenous F-specific bacteriophages (F-specific RNA and F-specific DNA phages) was also reduced by about 99% by wetland treatment, with the mean inflow concentration over the period of an entire year reduced from 3,129 to 33 PFU ml-1 in the outflow of a vegetated bed and to 174 PFU ml-1 in the outflow of an unvegetated bed. Such superior treatment by the vegetated bed demonstrates the significant role of higher aquatic plants in the removal process. Seeded MS2 bacteriophage and seeded poliovirus were removed more efficiently than were the indigenous bacteriophages, with less than 0.2% of MS2 and 0.1% of the poliovirus surviving flow at the same hydraulic application rate. The decay rate (k) of MS2 in a stagnant wetlands (k = 0.012 to 0.028 h-1) was lower than that for flowing systems (k = 0.44 to 0.052 h-1), reflecting the enhanced capacity for filtration or adsorption of viruses by the root-substrate complex (and associated biofilm).(ABSTRACT TRUNCATED AT 250 WORDS)     

Biomechanical properties of articular cartilage after high hydrostatic pressure treatment]

AIM: Reconstruction of bone defects due to malignant tumors can be realized by several methods. Up to now, two methods, irradiation and autoclaving, are available for extracorporeally devitalizing resected tumor-bearing osteochondral segments. Previous investigations have shown that human normal and tumor cells in culture were irreversibly impaired when subjected to extracorporeal high hydrostatic pressure (HHP) of 350 MPa. The aim of this study was to examine the biomechanical and immunohistochemical properties of cartilage after exposure to HHP MATERIALS AND METHODS: Osteochondral segments of bovine femoral condyles were exposed to pressure of 300 and 600 MPa (n=20 each). Biomechanical and biological properties of untreated and treated segments were evaluated by repetitive ball indention testing and immunohistochemical labelling aggrecan, link protein and collagen II. The contralateral segments served as untreated control. RESULTS: No significant alterations concerning stiffness and relaxation of osteochondral segments even after 600 MPa were observed. Immunohistochemically, staining was positive in all cases and no differences in the labeling pattern of proteoglycanes were observed between untreated and HHP-treated specimens. CONCLUSION: These findings give hope that HHP eventually will be used as a new gentle way of treating resected cartilage and bone without alteration of biomechanical properties to inactivate tumor cells in order to allow autologous reimplantation.     

Integrated analysis of established and novel microbial and chemical methods for microbial source tracking

Several microbes and chemicals have been considered as potential tracers to identify fecal sources in the environment. However, to date, no one approach has been shown to accurately identify the origins of fecal pollution in aquatic environments. In this multilaboratory study, different microbial and chemical indicators were analyzed in order to distinguish human fecal sources from nonhuman fecal sources using wastewaters and slurries from diverse geographical areas within Europe. Twenty-six parameters, which were later combined to form derived variables for statistical analyses, were obtained by performing methods that were achievable in all the participant laboratories: enumeration of fecal coliform bacteria, enterococci, clostridia, somatic coliphages, F-specific RNA phages, bacteriophages infecting Bacteroides fragilis RYC2056 and Bacteroides thetaiotaomicron GA17, and total and sorbitol-fermenting bifidobacteria; genotyping of F-specific RNA phages; biochemical phenotyping of fecal coliform bacteria and enterococci using miniaturized tests; specific detection of Bifidobacterium adolescentis and Bifidobacterium dentium; and measurement of four fecal sterols. A number of potentially useful source indicators were detected (bacteriophages infecting B. thetaiotaomicron, certain genotypes of F-specific bacteriophages, sorbitol-fermenting bifidobacteria, 24-ethylcoprostanol, and epycoprostanol), although no one source identifier alone provided 100% correct classification of the fecal source. Subsequently, 38 variables (both single and derived) were defined from the measured microbial and chemical parameters in order to find the best subset of variables to develop predictive models using the lowest possible number of measured parameters. To this end, several statistical or machine learning methods were evaluated and provided two successful predictive models based on just two variables, giving 100% correct classification: the ratio of the densities of somatic coliphages and phages infecting Bacteroides thetaiotaomicron to the density of somatic coliphages and the ratio of the densities of fecal coliform bacteria and phages infecting Bacteroides thetaiotaomicron to the density of fecal coliform bacteria. Other models with high rates of correct classification were developed, but in these cases, higher numbers of variables were required.     

Occurrence and numbers of bacteriophages and bacterial indicators in faeces of yellow-legged seagull (Larus cachinnans)

Faeces from feral populations of yellow-legged seagulls from the northern coastal area of Catalonia (North-eastern Spain) contained variable amounts of faecal coliforms, faecal streptococci, somatic coliphages, F-specific bacteriophages and Bacteroides fragilis bacteriophages. Occurrence and numbers of bacterial indicators and bacteriophages in the faeces of yellow-legged seagulls are in the ranges described in the faeces of different animals. The ratios between numbers of bacterial indicators and numbers of bacteriophages are much higher in faeces of seagulls than in treated or raw sewage contributed by out-falls of the same area.     

Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins

Overby, L. R. (University of Illinois, Urbana), G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman. Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins. J. Bacteriol. 92:739-745. 1966.-The ribonucleic acid (RNA) molecules and coat proteins of two RNA coliphages, MS-2 and Qbeta, have been characterized. MS-2 RNA shows an S(20,w) of 25.8 and a molecular weight by light scattering of 10(6). The corresponding parameters for Qbeta-RNA were 28.9 and 0.9 x 10(6). A difference in base composition was reflected in the adenine-uracil ratio, which was 0.95 for MS-2 and 0.75 for Qbeta. The two RNA preparations are readily separated by chromatography on columns of methylated albumin. Both gave identical bouyant densities in cesium sulfate of 1.64 g/ml. The coat protein subunits were of similar molecular weights: 15,500 (Qbeta) and 14,000 (MS-2). They differed, however, in that the Qbeta-protein lacked tryptophan and histidine, whereas the MS-2 protein lacked only histidine.     

Transmission of F'lac plasmid from Escherichia coli K-12 to bacteria of the genus Erwinia]

The F'lac plasmid was transferred by conjugation from Escherichia coli K-12 W1655 to 21 lac- strains of Erwinia spp. (5.2 . 10(-6) to 6.8 . 10(-2) lac+ exconjugants per donor cell). Erw. herbicola and Erw. chrysanthemi were the better recipients than others. The degree of the stability of lac+ genes in Erwinia exconjugants depends on the strains. Stable exconjugants of Erwinia, which harbored F'lac plasmid, were able to utilize lactose, to transfer lac genes by conjugation to Erwinia spp. and E. coli, and were sensitive to the F-specific phages f1, f2, Qbeta. The F'lac plasmid was eliminated from the exconjugants by the treatment with acridine orange, which indicates that this genetic element is not integrated into the Erwinia chromosome.     

A physiological role for tRNA nucleotidyltransferase during bacteriophage infection

Bacteriophage infection of E. coli cells deficient in the enzyme tRNA nucleotidyltransferase (cca mutants) resulted in greatly decreased production of viable progeny phage compared to wild type cells. This decrease amounted to as much as 90% in the case of T-even bacteriophages, and 50-65% for T-odd bacteriophages. However, infection by the RNA phages, Qbeta and f2, was unaffected by the cca mutation. Examination of T4 infection of cca hosts indicated that phage development proceeded normally, that near-normal numbers of progeny particles were formed, but that most of these particles were non-viable. Possible functions for E. coli tRNA nucleotidyltransferase during bacteriophage infection are discussed.     

Structural basis of RNA binding discrimination between bacteriophages Qbeta and MS2

Sequence-specific interactions between RNA stem-loops and coat protein (CP) subunits play vital roles in the life cycles of the RNA bacteriophages, e.g., by allowing translational repression of their replicase cistrons and tagging their own RNA genomes for encapsidation. The CPs of bacteriophages Qbeta and MS2 each discriminate in favor of their cognate translational operators, even in the presence of closely related operators from other phages in vivo. Discrete mutations within the MS2 CP have been shown to relax this discrimination in vitro. We have determined the structures of eight complexes between such mutants and both MS2 and Qbeta stem-loops with X-ray crystallography. In conjunction with previously determined in vivo repression data, the structures enable us to propose the molecular basis for the discrimination mechanism.