Selective bacteriophages reduce the emergence of resistant bacteria in bacteriophage-antibiotic combination therapy.

Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny.

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some ß-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.

Coliphages as Indicators for the Microbial Quality of Treated Wastewater Effluents.

Wastewater effluents are a reliable water source for non-potable water reuse including unrestricted crop irrigation in arid regions suffering from water scarcity. This study was performed to develop and optimize a procedure to concentrate coliphages from 100 L of treated effluent. Moreover, the reduction of coliphages by filtration and disinfection by either chlorine or UV was compared with that of fecal coliform (FC). The adsorption efficiency of MS2 and Qß coliphages by the NanoCeram filter was similar and reached 99.8%. Elution efficiency of MS2 coliphage from the NanoCeram filters by a solution of 1% NaPP and 0.05 M glycine, pH 9.5, was 74  ±  9.5%. The highest reconcentration efficiency of MS2 and Qß coliphages was obtained with polyethylene glycol (PEG) precipitation and reached 76  ±  28% and 90  ±  11%, respectively. In comparison, the reconcentration efficiency of organic flocculation was 0% and 1.3% for Qß and MS2 coliphages, respectively. The overall recovery efficiency of MS2 coliphages from 100 L tertiary effluent was 57  ±  1.5%. Poor reduction was observed for coliphages compared to FC by filtration and chlorine disinfection although; the reduction of FC, as measured by cultivation, was satisfactory and within the guidelines for unrestricted irrigation. High correlation between the reduction of FC and coliphages was recorded for tertiary effluent disinfected by UV irradiation. Monitoring the microbial quality of tertiary effluent using qPCR for the enumeration of FC was found unsuitable, because DNA levels were unaffected by the treatment processes. The results of this study demonstrated that monitoring the microbial quality of tertiary effluent by FC may not reflect the health risks encountered by the application of these effluents and the addition of coliphages to the monitoring programs may allow for accurate assessment of the health risks introduced by the application of tertiary effluent.

Risk-based water quality thresholds for coliphages in surface waters: effect of temperature and contamination aging.

Coliphages, viruses that infect Escherichia coli, have been used for decades to assess surface water quality yet there is no guideline for interpreting their concentrations. The present study uses a quantitative microbial risk assessment (QMRA) framework to derive risk-based surface water quality thresholds for somatic and F+ or male-specific coliphages. The risk-based threshold is the concentration at which the risk of gastro-intestinal illness is simulated to be 32/1000. The framework specifically investigates a simplified hazard scenario where recreational swimmers come into contact with water contaminated with untreated sewage containing coliphages and enteric pathogens. The framework considers exposure to sewage of diverse ages and thus accounts for the decay of coliphages and pathogens over time. As decay rate constants depend on temperature, the model considers the effect of temperature on the risk-based threshold. When exposure to fresh, unaged sewage contamination occurs, the risk-based water quality threshold for somatic and F+ coliphages is 60 PFU per 100 mL and 30 PFU per 100 mL, respectively, and temperature independent. The risk-based threshold decreases as the contamination ages because, on average, coliphages decay more quickly than norovirus, the pathogen that contributes the most to risk. The decrease in the risk-based threshold with contaminant age is equal to the difference in the first order decay rate constants of coliphages and norovirus. Since coliphage decay rate constants are larger at 25 °C than at 15 °C, and norovirus decay rate constants are a weak function of temperature, risk-based thresholds decrease more quickly with age at 25 °C than at 15 °C. For the common case where the age of contamination is unknown, the risk-based threshold for both coliphages is between ∼1 PFU per 100 mL and ∼10 PFU per 100 mL, depending on model assumptions. Future work can apply this QMRA framework for identifying risk-based thresholds for coliphages from different hazards (treated wastewater or animal feces) or from mixtures of contamination of different ages and sources.

Cinnamon Oil Inhibits Shiga Toxin Type 2 Phage Induction and Shiga Toxin Type 2 Production in Escherichia coli O157:H7.

This study evaluated the inhibitory effect of cinnamon oil against Escherichia coli O157:H7 Shiga toxin (Stx) production and further explored the underlying mechanisms. The MIC and minimum bactericidal concentration (MBC) of cinnamon oil against E. coli O157:H7 were 0.025% and 0.05% (vol/vol), respectively. Cinnamon oil significantly reduced Stx2 production and the stx2 mRNA expression that is associated with diminished Vero cell cytotoxicity. Consistently, induction of the Stx-converting phage where the stx2 gene is located, along with the total number of phages, decreased proportionally to cinnamon oil concentration. In line with decreased Stx2 phage induction, cinnamon oil at 0.75× and 1.0× MIC eliminated RecA, a key mediator of SOS response, polynucleotide phosphorylase (PNPase), and poly(A) polymerase (PAP I), which positively regulate Stx-converting phages, contributing to reduced Stx-converting phage induction and Stx production. Furthermore, cinnamon oil at 0.75× and 1.0× MIC strongly inhibited the qseBC and luxS expression associated with decreased AI-2 production, a universal quorum sensing signaling molecule. However, the expression of oxidative stress response genes oxyR, soxR, and rpoS was increased in response to cinnamon oil at 0.25× or 0.5× MIC, which may contribute to stunted bacterial growth and reduced Stx2 phage induction and Stx2 production due to the inhibitory effect of OxyR on prophage activation. Collectively, cinnamon oil inhibits Stx2 production and Stx2 phage induction in E. coli O157:H7 in multiple ways. IMPORTANCE: This study reports the inhibitory effect of cinnamon oil on Shiga toxin 2 phage induction and Shiga toxin 2 production. Subinhibitory concentrations (concentrations below the MIC) of cinnamon oil reduced Stx2 production, stx2 mRNA expression, and cytotoxicity on Vero cells. Subinhibitory concentrations of cinnamon oil also dramatically reduced both the Stx2 phage and total phage induction in E. coli O157:H7, which may be due to the suppression of RNA polyadenylation enzyme PNPase at 0.25× to 1.0× MIC and the downregulation of bacterial SOS response key regulator RecA and RNA polyadenylation enzyme PAP I at 0.75× or 1.0× MIC. Cinnamon oil at higher levels (0.75× and 1.0× MIC) eliminated quorum sensing and oxidative stress. Therefore, cinnamon oil has potential applications as a therapeutic to control E. coli O157:H7 infection through inhibition of bacterial growth and virulence factors.

Bactericidal and virucidal mechanisms in the alkaline disinfection of compost using calcium lime and ash.

In the present study, the bactericidal and virucidal mechanisms in the alkaline disinfection of compost with calcium lime and ash were investigated. Two indicator microorganisms, Escherichia coli and MS2 coliphage, were used as surrogates for enteric pathogens. The alkaline-treated compost with calcium oxide (CaO) or ash resulted primarily in damage to the outer membrane and enzyme activities of E. coli. The alkaline treatment of compost also led to the infectivity loss of the coliphage because of the partial capsid damage and RNA exteriorization due to a raised pH, which is proportional to the amount of alkaline agents added. These results indicate that the alkaline treatment of compost using calcium oxide and ash is effective and can contribute to the safe usage of compost from a mixing type dry toilet.

The effect of chlorine and combined chlorine/UV treatment on coliphages in drinking water disinfection.

Chlorine disinfection is a globally used method to ensure the safety of drinking water. However, it has not always been successful against viruses and, therefore, it is important to find new methods to disinfect water. Seventeen different coliphages were isolated from the treated municipal wastewater. These coliphages and MS2 were treated with different dosages of chlorine in drinking water, and a combined chlorine/ultraviolet irradiation treatment for the chlorine-resistant coliphages. Chlorine disinfection with 0.3-0.5 mg/L total chlorine (free Cl-dosage 0.12-0.21 mg/L) for 10 min achieved 2.5-5.7 Log10-reductions for 11 sensitive coliphages. The six most resistant coliphages showed no reduction with these chlorine concentrations. MS2 was intermediate in chlorine resistance, and thus it is not a good indicator for viruses in chlorine disinfection. In the combined treatment total chlorine of 0.05-0.25 mg/L (free Cl-dosage 0.02-0.08 mg/L) and ultraviolet irradiation (14-22 mWs/cm(2)) were more effective than chlorine alone, and 3-5 Log10-reductions were achieved for the chlorine-resistant strains. The chlorination efficiency could be increased by higher dosages and longer contact times, but this could increase the formation of disinfection by-products. Therefore, the combination treatment is a recommended disinfection method.

Inactivation of Escherichia coli O157 Bacteriophages by Using a Mixture of Ferrous Sulfate and Tea Extract.

Bacteriophages (phages) have been used for biocontrol of Escherichia coli O157 and other pathogenic bacteria in many different matrices and foods, but few studies have included inactivation of residual phages in culture medium before plating and enumeration of surviving host bacteria for the assessment of phage efficacy. This oversight may lead to overestimation of phage efficacy. The ability of virucidal solution containing a mixture of ferrous sulfate [iron(II) sulfate, FeSO4] and tea extract [Fe(II)T] to inactivate residual T5-like, T1-like, T4-like, and rV5-like phages was assessed using E. coli O157 as the host. At concentrations of ≥10 mM FeSO4, all phages were not detected after 20 min in a broth culture model. Compared with the virucidal solution-free samples (1 to 96% recovery), Fe(II)T (10 mM FeSO4 plus 15% tea extract) recovered a greater (P < 0.01) number of E. coli O157 from phage-treated broth culture (97 to 100% recovery) and beef samples (52 to 100% recovery). Moreover, with the addition of Fe(II)T, the number of bacteria surviving after exposure to T5-like or T4-like phages was greater (P < 0.01) than that after exposure to T1-like or rV5-like phages. Consequently, use of a virucide for phage inactivation is recommended to improve the accuracy of evaluations of phage efficacy for biocontrol of E. coli O157.

Ammonia as an in situ sanitizer: inactivation kinetics and mechanisms of the ssRNA virus MS2 by NH3.

Sanitizing human and animal waste (e.g., urine, fecal sludge, or grey water) is a critical step in reducing the spread of disease and ensuring microbially safe reuse of waste materials. Viruses are particularly persistent pathogens and can be transmitted through inadequately sanitized waste. However, adequate storage or digestion of waste can strongly reduce the number of viruses due to increases in pH and uncharged aqueous ammonia (NH3), a known biocide. In this study we investigated the kinetics and mechanisms of inactivation of the single-stranded RNA virus MS2 under temperature, pH and NH3 conditions representative of waste storage. MS2 inactivation was mainly controlled by the activity of NH3 over a pH range of 7.0­9.5 and temperatures lower than 40 °C. Other bases (e.g., hydroxide, carbonate) additionally contributed to the observed reduction of infective MS2. The loss in MS2 infectivity could be rationalized by a loss in genome integrity, which was attributed to genome cleavage via alkaline transesterification. The contribution of each base to genome transesterification, and hence inactivation, could be related to the base pKa by means of a Bronsted relationship. The Bronsted relationship in conjunction with the activity of bases in solution enabled an accurate prediction of MS2 inactivation rates.

SDS-PAGE and IR spectroscopy to evaluate modifications in the viral protein profile induced by a cationic porphyrinic photosensitizer.

Reactive oxygen species can be responsible for microbial photodynamic inactivation due to its toxic effects, which include severe damage to proteins, lipids and nucleic acids. In this study, the photo-oxidative modifications of the proteins of a non-enveloped T4-like bacteriophage, induced by the cationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide were evaluated. Two methods were used: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared spectroscopy. SDS-PAGE analysis showed that the phage protein profile was considerably altered after photodynamic treatment. Seven protein bands putatively corresponding to capsid and tail tube proteins were attenuated and two other were enhanced. Infrared spectroscopy confirmed the time-dependent alteration on the phage protein profile detected by SDS-PAGE, indicative of a response to oxidative damage. Infrared analysis showed to be a promising and rapid screening approach for the analysis of the modifications induced on viral proteins by photosensitization. In fact, one single infrared spectrum can highlight the changes induced to all viral molecular structures, overcoming the delays and complex protocols of the conventional methods, in a much simple and cost effective way.

Influence of environmental variables in the efficiency of phage therapy in aquaculture.

Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 10(5) CFU ml(-1) and a phage concentration of ≈ 10(6-8) plaque forming unit ml(-1) were used. Phage survival was not significantly affected by the natural variability of pH (6.5-7.4), temperature (10-25 °C), salinity (0-30 g NaCl l(-1) ) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in non-marine systems through the addition of salt, whenever this option is feasible and does not affect the survival of aquatic species being cultured.

Efficiency of peracetic acid in inactivating bacteria, viruses, and spores in water determined with ATP bioluminescence, quantitative PCR, and culture-based methods.

The disinfection efficiency of peracetic acid (PAA) was investigated on three microbial types using three different methods (filtration-based ATP (adenosine-triphosphate) bioluminescence, quantitative polymerase chain reaction (qPCR), culture-based method). Fecal indicator bacteria (Enterococcus faecium), virus indicator (male-specific (F(+)) coliphages (coliphages)), and protozoa disinfection surrogate (Bacillus subtilis spores (spores)) were tested. The mode of action for spore disinfection was visualized using scanning electron microscopy. The results indicated that PAA concentrations of 5 ppm (contact time: 5 min), 50 ppm (10 min), and 3,000 ppm (5 min) were needed to achieve 3-log reduction of E. faecium, coliphages, and spores, respectively. Scanning electron microscopy observation showed that PAA targets the external layers of spores. The lower reduction rates of tested microbes measured with qPCR suggest that qPCR may overestimate the surviving microbes. Collectively, PAA showed broad disinfection efficiency (susceptibility: E. faecium > coliphages > spores). For E. faecium and spores, ATP bioluminescence was substantially faster (∼5 min) than culture-based method (>24 h) and qPCR (2-3 h). This study suggests PAA as an effective alternative to inactivate broad types of microbial contaminants in water. Together with the use of rapid detection methods, this approach can be useful for urgent situations when timely response is needed for ensuring water quality.

Persistence of infectious Shiga toxin-encoding bacteriophages after disinfection treatments.

In Shiga toxin-producing Escherichia coli (STEC), induction of Shiga toxin-encoding bacteriophages (Stx phages) causes the release of free phages that can later be found in the environment. The ability of Stx phages to survive different inactivation conditions determines their prevalence in the environment, the risk of stx transduction, and the generation of new STEC strains. We evaluated the infectivity and genomes of two Stx phages (Φ534 and Φ557) under different conditions. Infectious Stx phages were stable at 4, 22, and 37°C and at pH 7 and 9 after 1 month of storage but were completely inactivated at pH 3. Infective Stx phages decreased moderately when treated with UV (2.2-log10 reduction for an estimated UV dose of 178.2 mJ/cm(2)) or after treatment at 60 and 68°C for 60 min (2.2- and 2.5-log10 reductions, respectively) and were highly inactivated (3 log10) by 10 ppm of chlorine in 1 min. Assays in a mesocosm showed lower inactivation of all microorganisms in winter than in summer. The number of Stx phage genomes did not decrease significantly in most cases, and STEC inactivation was higher than phage inactivation under all conditions. Moreover, Stx phages retained the ability to lysogenize E. coli after some of the treatments.

Microbial reduction in wastewater treatment using Fe(3+) and Al(3+) coagulants and PAA disinfectant.

Wastewater is an important source of pathogenic enteric microorganisms in surface water and a major contaminating agent of drinking water. Although primary and secondary wastewater treatments reduce the numbers of microorganisms in wastewater, significant numbers of microbes can still be present in the effluent. The aim of this study was to test the feasibility of tertiary treatment for municipal wastewater treatment plants (WWTPs) using PIX (FeCl3) or PAX (AlCl3) coagulants and peracetic acid (PAA) the disinfectant to reduce microbial load in effluent. Our study showed that both PIX and PAX efficiently reduced microbial numbers. PAA disinfection greatly reduced the numbers of culturable indicator microorganisms (Escherichia coli, intestinal enterococci, F-specific RNA coliphages and somatic DNA coliphages). In addition, pathogenic microorganisms, thermotolerant Campylobacter, Salmonella and norovirus GI, were successfully reduced using the tertiary treatments. In contrast, clostridia, Legionella, rotavirus, norovirus GII and adenovirus showed better resistance against PAA compared to the other microorganisms. However, interpretation of polymerase chain reaction (PCR) analysis results will need further studies to clarify the infectivity of the pathogenic microbes. In conclusion, PIX and PAX flocculants followed by PAA disinfectant can be used as a tertiary treatment for municipal WWTP effluents to reduce the numbers of indicator and pathogenic microorganisms.

Inhibition of development of Shiga toxin-converting bacteriophages by either treatment with citrate or amino acid starvation.

OBJECTIVES: Shiga toxin-producing Escherichia coli (STEC) are pathogenic strains, whose virulence depends on induction of Shiga toxin-converting prophages and their subsequent lytic development. We explored which factors or conditions could inhibit development of these phages, potentially decreasing virulence of STEC. MATERIALS AND METHODS: Lytic development of Shiga toxin-converting bacteriophages was monitored after mitomycin C-provoked prophage induction under various conditions. Phage DNA replication efficiency was assessed by measurement of DNA amount in cells using quantitative polymerase chain reaction. RESULTS: We demonstrated that the use of citrate delayed Shiga toxin-converting phage development after prophage induction. This effect was independent on efficiency of prophage induction and phage DNA replication. However, an excess of glucose reversed the effect of citrate. Amino acid starvation prevented the phage development in bacteria both able and unable to induce the stringent response. CONCLUSIONS: Lytic development of Shiga toxin-converting bacteriophages can be inhibited by either the presence of citrate or amino acid starvation. We suggest that the inhibition caused by the latter condition may be due to a block in prophage induction or phage DNA replication or both. APPLICATIONS: Our findings may facilitate development of procedures for treatment of STEC-infected patients.

[Study of the mechanisms of interaction between intestinal bacteriophages and polyaluminum coagulants].

The authors have investigated the adsorption-elution of intestinal bacteriophages T2 and MS2 as a model of enteric viruses, by using the coagulants aluminum oxychloride (AOX78) and aluminum sulfate (AS). The investigation has ascertained the effective removal of the phages from water with AOX78 (98.7-100%) and the low efficiency of elution of coliphages from the particles of AOX hydrolysis products (0.1-3.7% of the baseline count of bacteriophages), which may suggest that AOX has antiviral activity. The above phenomena produced by AS are less evident.

Bacteriophages carrying Shiga toxin genes: genomic variations, detection and potential treatment of pathogenic bacteria.

Although most Escherichia coli strains occur in the mammalian intestine as commensals, some of them, including enterohemorrhagic E. coli (EHEC), are capable of causing disease in humans. The most notorious virulence factors of EHEC are Shiga toxins, encoded by genes located on genomes of lambdoid prophages. Production and release of these toxins is strongly stimulated after the induction of these prophages. Many antibiotics used to treat bacterial infections stimulate induction of Shiga toxin-converting prophages, enhancing severity of the disease symptoms. Hence, treatment with antibiotics is not recommended if infection with EHEC is confirmed or even suspected. In this light, rapid detection of EHEC is crucial, and understanding the mechanisms of prophage induction and phage development in the human intestine is important to facilitate development of procedures preventing or alleviating Shiga toxin-caused diseases.

Greywater disinfection with the environmentally friendly Hydrogen Peroxide Plus (HPP).

Hydrogen Peroxide Plus (HPP) is a newly developed, stabilized-H(2)O(2)-based compound. This study was aimed at determining the disinfection efficiency of HPP in greywater (GW), assessing HPP dose requirements and evaluating the feasibility of its use in small-scale GW-treatment systems. Fecal coliforms were the most sensitive to treatment, followed by somatic coliphages and F+ bacteriophages. The calculated HPP dose required to reduce fecal coliform counts by 99% was 125mg H(2)O(2)L(-1), with a contact time of 35min. The use of HPP was found feasible and comparable to the use of chlorine for small systems with a flow rate of 5m(3)d(-1). HPP is suggested as an alternative for GW disinfection in small communities and private houses.

Comparative study on the efficiency of peracetic acid and chlorine dioxide at low doses in the disinfection of urban wastewaters.

A comparison was made between the efficiency of low doses of peracetic acid (PAA: 1.5 mg/l) and chlorine dioxide (ClO(2): 1.5 and 2.0 mg/l) in the disinfection of secondary effluents of a wastewater treatment plant. Peracetic acid was seen to be more active than chlorine dioxide and less influenced by the organic content of the waste. Both PAA and ClO(2) (2.0 mg/l) lead to a higher reduction in total and faecal coliforms and E. coli than in phages (somatic coliphages and F-specific RNA bacteriophages) and enterococci. Detection of faecal coliforms and E. coli should therefore be accompanied by a search for these more resistant microorganisms when assessing the conformity of wastewater for irrigation use, or for discharge into surface waters. Coliphages are also considered suitable indicators of the presence of enteric viruses. Although the application of low doses of both disinfectants offers advantages in terms of costs and produces not significant quantities of byproducts, it is not sufficient to obtain wastewater suitable for irrigation according to the Italian norms (E. coli < 10/100 ml in 80 % of samples and <100/100 ml in the remaining samples). Around 65 % of the samples, however, presented concentrations of E. coli lower than the limit of 5,000/100 ml established by Italian norms for discharge into surface waters.

Simple method for plating Escherichia coli bacteriophages forming very small plaques or no plaques under standard conditions.

The use of low concentrations (optimally 2.5 to 3.5 microg/ml, depending on top agar thickness) of ampicillin in the bottom agar of the plate allows for formation of highly visible plaques of bacteriophages which otherwise form extremely small plaques or no plaques on Escherichia coli lawns. Using this method, we were able to obtain plaques of newly isolated bacteriophages, propagated after induction of prophages present in six E. coli O157:H(-) strains which did not form plaques when standard plating procedures were employed.