Determination of enteroviruses, hepatitis A virus, bacteriophages and Escherichia coli in Adriatic Sea mussels

The aim of the present study was to evaluate the incidence of enteric viruses in mussels and to verify the possibility of using phages as indirect indicators of mussel viral contamination. Mussels (36 samples) collected from three different areas of the Adriatic Sea were analysed to determine the following parameters: Escherichia coli, somatic coliphage (T6 phage), F-Plus (MS2 phage), B40-8 (phage of Bacteroides fragilis), enteroviruses and hepatitis A virus. Most of the results of the bacteriological analysis (most probable number (MPN) ml-1) were in accordance with the bacteriological limits established by European law, with the exception of seven samples. The bacteriophage analyses were always negative for F-Plus and B40-8, with the exception of a few samples, whereas the somatic coliphages were generally between 0 and 20 MPN g-1, with the exception of two samples (110 MPN g-1). The virological analysis showed five samples positive for the presence of enteroviruses and 13 for the presence of hepatitis A virus (in three samples both viruses were present). Most of these samples presented acceptable bacteriological parameters and the bacteriophages were absent or their value was generally very low. The results show that the detection of E. coli and phages does not seem to be a good indicator of viral contamination.     

Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium

Based on results from eight experiments, no overall difference was found between roll tube and three- and five-tube most-probable-number (MPN) methods for estimating total numbers of ruminal bacteria. However, standard errors for the replicate means within an experiment were higher with the MPN procedures. Visual growth and pH were the criteria used for scoring the MPN tubes. Total numbers were significantly higher in MPN medium containing 40% ruminal fluid, as compared with a complete medium without ruminal fluid. By using a broth medium containing ball-milled cellulose and soluble carbohydrates as energy sources, it was possible to estimate both total and cellulolytic ruminal bacterial numbers in the same MPN series. Disappearance of cellulose and decrease in pH were used to determine growth. Values did not differ from those obtained in separate MPN assays. By using this method, diurnal changes in total and cellulolytic bacterial numbers were estimated in sheep fed forage or a concentrate-type diet.     

Bacteriophage development in an immobilized lactic acid bacteria system

Summary Bacteriophages were added to milk fermented byStreptococcus raffinolactis cells immobilized in calcium alginate. Beads containing the immobilized streptococci were used for five consecutive fermentations; pH, free cell and bacteriophage counts were estimated. Free cells increased from 5×10⁶ to 3×10⁸ per mL of milk, over the successive fermentations. Addition of bacteriophages reduced the free cell count by almost 1000 after 3 fermentations, but a gradual increase occurred subsequently. Bacteriophages were inoculated at 100 per mL and gradually attained 5×10⁹ per mL in the system. Rinsing of the system did not have a substantial influence on free cell or phage counts. Presence of bacteriophage reduced slightly the acidification rate in the system.Bacteriophage numeration by two layer agar method gave better results than by most probable number (MPN). MPN counts were greatly influenced byS. raffinolactis inoculation level.Contribution # 099     

Most-probable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium.

Based on results from eight experiments, no overall difference was found between roll tube and three- and five-tube most-probable-number (MPN) methods for estimating total numbers of ruminal bacteria. However, standard errors for the replicate means within an experiment were higher with the MPN procedures. Visual growth and pH were the criteria used for scoring the MPN tubes. Total numbers were significantly higher in MPN medium containing 40% ruminal fluid, as compared with a complete medium without ruminal fluid. By using a broth medium containing ball-milled cellulose and soluble carbohydrates as energy sources, it was possible to estimate both total and cellulolytic ruminal bacterial numbers in the same MPN series. Disappearance of cellulose and decrease in pH were used to determine growth. Values did not differ from those obtained in separate MPN assays. By using this method, diurnal changes in total and cellulolytic bacterial numbers were estimated in sheep fed forage or a concentrate-type diet.     

Most-probable-number technique for the enumeration of aromatic degraders in natural environments

A most-probable-number (MPN) method is described for the enumeration of heterotrophic populations capable of utilizing chlorinated and nonchlorinated benzoates and phenols as sole carbon sources. A correlation coefficient of 0.91 was obtained between the numbers determined by the MPN technique and the standard plate count. The MPN method gave realistic cell counts when population densities were low, and the presence of oligocarbophiles did not give spurious results.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Aims:  To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method.
Methods and Results:  The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0·1 MPN per litre, with a 95% confidence level of 0·01–0·7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0·9 MPN per litre for pond outflow and from not detectable to 8·3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10⁴ MPN per hour.
Conclusion:  This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife.
Significance and Impact of the Study:  This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.     

A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.     

Loss of Lysis Inhibition in Filamentous Escherichia coli Infected with Wild-Type Bacteriophage T4

The plaque enlargement of wild-type T4 bacteriophage observed when assayed in the presence of low concentrations of mitomycin C or after exposure to very low doses of ultraviolet light was studied by using solid as well as liquid culture media. It was found that the filamentous cell formed by the treatment with the agents is responsible for the phenomenon. The filamentous cell was also shown to be characterized not only by the loss of capacity of lysis inhibition but also by a shortening of the latent period. No difference in cellular rigidity could be seen between the filamentous cell and normal cell as far as the analysis from the outside of the cell was concerned, whereas the former cell was shown to be more readily susceptible to phage-induced lysozyme from the inside of the cell. A possible change in the membrane of the filamentous cell and a possible mechanism for lysis inhibition are discussed.     

An indirect fluorescent antibody staining technique for determining population levels ofThiobacillus ferrooxidans in acid mine drainage waters

Thiobacillus ferrooxidans is believed to be responsible for the oxidation of ferrous ion at low pH, the rate-limiting step in the oxidation of pyrite ores and subsequent formation of acid mine drainage (AMD). It has been suggested that efforts to control this environmental problem include procedures that would inhibit this bacterium. At present, a most probable number (MPN) procedure requiring a minimum of 10 days is used to enumerate this microorganism in natural waters. If control of AMD through inhibition ofT. ferrooxidans is to be feasible, it will be necessary to develop a more rapid method to determine population levels to facilitate application of control measures.An indirect fluorescent antibody (FA) staining technique was developed for this purpose which provided reliable estimates within a few hours. Artificial samples containing approximated numbers ofT. ferrooxidans were analyzed using the FA and MPN procedures, and the FA technique more closely approximated expected numbers of cells. The MPN method was excessively conservative, detecting only 3% to 21% of the cells enumerated by the FA procedure.     

Microplate MPN-enumeration of monocyclic- and dicyclic-aromatic hydrocarbon degraders via substrate phase-partitioning

The high aqueous solubility of monoaromatic and some diaromatic oil components may hinder classical growth-based MPN enumeration of bacterial mono- and di-aromatics degraders because these aromatics are toxic in high concentrations. We developed a microplate MPN method for the enumeration of toluene-, xylene-, naphthalene-, biphenyl- and benzothiophene-degraders on the basis of phase-partitioning of substrate between a biologically inert organic phase and an aqueous mineral salt medium. This way, it was possible to maintain non-toxic, aqueous concentrations in the microplate wells. Depletion of aqueous aromatics by growth of the degraders was prevented by the continuous solubilization of aromatics from the silicone phase. The method was validated by MPN enumerating degrader cultures both with phase-partitioned aromatics and with tryptic soy broth as carbon sources. The applicability of the method was demonstrated by MPN-enumerating mono- and di-aromatic degraders in soils of varying hydrocarbon pre-exposure. An erratum to this article can be found at     

Enumeration of Campylobacter in New Zealand recreational and drinking waters

AIMS: To use a published polymerase chain reaction (PCR) method for the detection and identification of thermotolerant Campylobacter species (Camp. jejuni, Camp. coli and Camp. lari) in tandem with a Most Probable Number (MPN) technique to enumerate these species in water samples. METHODS AND RESULTS: An initial study of 42 river water samples compared the use of conventional culture and PCR methods for the detection of Campylobacter in MPN enrichment tubes. It was found that all samples positive by culture were also positive by PCR. Thirty-seven percent more MPN tubes were positive by PCR compared with culture. The MPN/PCR technique was subsequently applied to 96 additional samples collected from rivers, drinking, roof and shallow ground water. Campylobacter was especially prevalent in river water (60% positive) and shallow ground water (75% positive) samples. Drinking water (29.2% positive) and roof water (37.5% positive) also contained Campylobacter, but the numbers detected were very low (maximum 0.3 and 0.56 MPN 100 ml-1, respectively). CONCLUSION: River waters contained Campylobacter at higher levels than any other water type and in a high percentage of the samples. Although Campylobacter was present in treated drinking water, the levels detected were low. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that water may act as a significant transmission route for human campylobacteriosis.     

A rapid luminescent-phage based MPN method for the enumeration of Salmonella typhimurium in environmental samples

Materials such as soils, waters, sewage sludges and foods can contain low numbers of salmonellas. A most-probable-number (MPN) method that utilized a bioluminescent-bacteriophage is described that allowed the specific determination of as few as one Salmonella typhimurium cell/100 ml of material within 24 h. The method was developed with soil, lake water and sewage sludge inoculated with Salm. typhimurium and had an efficiency of 100% when tested against a traditional MPN method. The protocol is rapid, sensitive, inexpensive, has a low operator time compared to the traditional MPN method, allows for the repair of injured cells and is amenable to automation.     

Most-Probable-Number Rapid Viability PCR method to detect viable spores of Bacillus anthracis in swab samples

A comparison of Most-Probable-Number Rapid Viability (MPN RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs from a multi-center validation study was performed. The purpose of the study was to compare environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were typically within 1-log of the values from a plate count method for all three levels of spores tested (3.1 × 10⁴, 400, and 40 spores sampled from surfaces with swabs) even in the presence of debris. The MPN method tended to overestimate the expected result, especially at lower spore levels. Blind negative samples were correctly identified using both methods showing a lack of cross contamination. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols, enhancing its utility for characterization and clearance following a biothreat agent release.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Salmonella in surface waters

Aims: To better understand and manage the fate and transport of Salmonella in agricultural watersheds, we developed a culture‐based, five tube–four dilution most probable number (MPN) method for enumerating dilute densities of Salmonella in environmental waters. Methods and Results: The MPN method was a combination of a filtration technique for large sample volumes of environmental water, standard selective media for Salmonella and a TaqMan confirmation step. This method has determined the density of Salmonella in 20‐l samples of pond inflow and outflow streams as low as 0·1 MPN l^?1 and a low 95% confidence level 0·015 MPN l^?1. Salmonella densities ranged from not detectable to 0·55 MPN l^?1 for pond inflow samples and from not detectable to 3·4 MPN l^?1 for pond outflow samples. Salmonella densities of pond inflow samples were associated with densities of Escherichia coli and faecal enterococci that indicated stream contamination with faeces and with nondetectable pond outflow densities of the faecal indicator bacteria. The MPN methodology was extended to flux determinations by integrating with volumetric measurements of pond inflow (mean flux of 2·5 l s^?1) and outflow (mean flux of 5·6 l s^?1). Fluxes of Salmonella ranged from 100 to greater than 10⁴ MPN h^?1. Conclusions: This is a culture‐based method that can detect small numbers of Salmonella in environmental waters of watersheds containing animal husbandry and wildlife. Significance and Impact of the Study: Applying this method to environmental waters will improve our understanding of the transport and fate of Salmonella in agricultural watersheds, and can be the basis of valuable collections of environmental Salmonella.     

Use of Tetrahymena thermophila to study the role of protozoa in inactivation of viruses in water

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4 degrees C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.     

Discovery of new Mycoplasma pneumoniae antigens by use of a whole-genome lambda display library

Mycoplasma pneumoniae is the leading cause of atypical pneumonia in children and young adults. Bacterial colonization can occur in both the upper and the lower respiratory tracts and take place both endemically and epidemically worldwide. Characteristically, the infection is chronic in onset and recovery and both humoral and cell-mediated mechanisms are involved in the response to bacterial colonization. To identify bacterial proteins recognized by host antibody responses, a whole-genome M. pneumoniae library was created and displayed on lambda bacteriophage. The challenge of such a library with sera from individuals hospitalized for mycoplasmal pneumonia allowed the identification of a panel of recombinant bacteriophages carrying B-cell epitopes. Among the already known M. pneumoniae B-cell antigens, our results confirmed the immunogenicity of P1 and P30 adhesins. Also, the data presented in this study localized, within their sequences, the immunodominant epitopes recognized by human immunoglobulins. Furthermore, library screening allowed the identification of four novel immunogenic polypeptides, respectively, encoded by fragments of the MPN152, MPN426, MPN456 and MPN-500 open reading frames, highlighting and further confirming the potential of lambda display technology in antigen and epitope discovery.     

Separation of T-even Bacteriophage Deoxyribonucleic Acid from Host Deoxyribonucleic Acid by Hydroxyapatite Chromatography

A simple and rapid method is described for separation of T-even bacteriophage deoxyribonucleic acid (DNA) from host (Escherichia coli) DNA by hydroxyapatite column chromatography with a shallow gradient of phosphate buffer at neutral pH. By this method, bacteriophage T2, T4, and T6 DNA (but not T5, T7, or lambda DNA) could be separated from host E. coli DNA. It was found that glucosylation of the T-even phage DNA is an important factor in separation.     

Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria.

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10(1) and 10(5) cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.