Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186.

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.

Pathogenic Escherichia coli-Specific Bacteriophages and Polyvalent Bacteriophages in Piglet Guts with Increasing Coliphage Numbers after Weaning.

Postweaning diarrhea in pigs is mainly caused by pathogenic Escherichia coli and is a major source of revenue loss to the livestock industry. Bacteriophages dominate the gut virome and have the potential to regulate bacterial communities and thus influence the intestinal physiology. To determine the biological characterization of intestinal coliphages, we isolated and identified the fecal coliphages of healthy preweaned and postweaned piglets from the Nanjing and Chuzhou pig farms. First, ahead of coliphage isolation, 87 E. coli strains were isolated from healthy or diarrheal fecal samples from three pig farms, of which 8 were pathogenic strains, including enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC). Of the E. coli strains, 87.3% possessed drug resistance to three antibiotics. Using these 87 E. coli strains as indicator hosts, we isolated 45 coliphages and found a higher abundance in the postweaning stage than in the preweaning stage (24 versus 17 in the Nanjing and 13 versus 4 in the Chuzhou farm). Furthermore, each farm had a single most-prevalent coliphage strain. Pathogenic E. coli-specific bacteriophages were commonly detected (9/10 samples in the Nanjing farm and 7/10 in the Chuzhou farm) in guts of sampled piglets, and most had significant bacteriostatic effects (P < 0.05) on pathogenic E. coli strains. Three polyvalent bacteriophages (N24, N30, and C5) were identified. The N30 and C5 strains showed a genetic identity of 89.67%, with mild differences in infection characteristics. Our findings suggest that pathogenic E. coli-specific bacteriophages as well as polyvalent bacteriophages are commonly present in piglet guts and that weaning is an important event that affects coliphage numbers. IMPORTANCE Previous studies based on metagenomic sequencing reported that gut bacteriophages profoundly influence gut physiology but did not provide information regarding the host range and biological significance. Here, we screened coliphages from the guts of preweaned and postweaned piglets against indicator hosts, which allowed us to identify the pathogenic E. coli-specific bacteriophages and polyvalent bacteriophages in pig farms and quantify their abundance. Our approach complements sequencing methods and provides new insights into the biological characterizations of bacteriophage in the gut along with the ecological effects of intestinal bacteriophages.

Bacteriophages as enteric viral indicators in bivalve mollusc management.

Human enteric viruses, such as norovirus and hepatitis A virus, are spread by a variety of routes including faecal-oral transmission. Contaminated bivalve shellfish are regularly implicated in foodborne viral disease outbreaks internationally. Traditionally indicator bacteria, the coliforms and Escherichia coli, have been used to detect faecal pollution in growing waters and shellfish. However, studies have established that they are inadequate as indicators of the risk of human enteric viruses. Bacteriophages have been identified as potential indicators or surrogates for human enteric viruses due to their similarities in morphology, behaviour in water environments and resistance to disinfectant treatments. The somatic coliphages, male-specific RNA coliphages (FRNA coliphages) and the bacteriophages of Bacteroides are the groups recognised as most suitable for water and shellfish testing. In this review, we discuss the rationale and supporting evidence for the application of bacteriophages as surrogates for human enteric viruses in shellfish under a variety of conditions. There is some evidence to support the validity of using bacteriophage levels to indicate viral risk in shellfish in highly contaminated sites and following adverse sewage events.

Bacteriophage LM33_P1, a fast-acting weapon against the pandemic ST131-O25b:H4 Escherichia coli clonal complex.

OBJECTIVES: Amongst the highly diverse Escherichia coli population, the ST131-O25b:H4 clonal complex is particularly worrisome as it is associated with a high level of antibiotic resistance. The lack of new antibiotics, the worldwide continuous increase of infections caused by MDR bacteria and the need for narrow-spectrum antimicrobial agents have revived interest in phage therapy. In this article, we describe a virulent bacteriophage, LM33_P1, which specifically infects O25b strains, and provide data related to its therapeutic potential. METHODS: A large panel of E. coli strains (n = 283) was used to assess both the specificity and the activity of bacteriophage LM33_P1. Immunology, biochemistry and genetics-based methods confirmed this specificity. Virology methods and sequencing were used to characterize this bacteriophage in vitro, while three relevant mouse models were employed to show its in vivo efficacy. RESULTS: Bacteriophage LM33_P1 exclusively infects O25b E. coli strains with a 70% coverage on sequence types associated with high antibiotic resistance (ST131 and ST69). This specificity is due to an interaction with the LPS mediated by an original tail fibre. LM33_P1 also has exceptional intrinsic properties with a high adsorption constant and produces over 300 virions per cell in <10 min. Using animal pneumonia, septicaemia and urinary tract infection models, we showed the in vivo efficacy of LM33_P1 to reduce the bacterial load in several organs. CONCLUSIONS: Bacteriophage LM33_P1 represents the first weapon that specifically and quickly kills O25b E. coli strains. Therapeutic approaches derived from this bacteriophage could be developed to stop or slow down the spread of the ST131-O25b:H4 drug-resistant clonal complex in humans.

Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages.

In Escherichia coli, the major poly(A) polymerase (PAP I) is encoded by the pcnB gene. In this report, a significant impairment of lysogenization by Shiga toxin-converting (Stx) bacteriophages (Φ24B, 933W, P22, P27 and P32) is demonstrated in host cells with a mutant pcnB gene. Moreover, lytic development of these phages after both infection and prophage induction was significantly less efficient in the pcnB mutant than in the WT host. The increase in DNA accumulation of the Stx phages was lower under conditions of defective RNA polyadenylation. Although shortly after prophage induction, the levels of mRNAs of most phage-borne early genes were higher in the pcnB mutant, at subsequent phases of the lytic development, a drastically decreased abundance of certain mRNAs, including those derived from the N, O and Q genes, was observed in PAP I-deficient cells. All of these effects observed in the pcnB cells were significantly more strongly pronounced in the Stx phages than in bacteriophage λ. Abundance of mRNA derived from the pcnB gene was drastically increased shortly (20 min) after prophage induction by mitomycin C and decreased after the next 20 min, while no such changes were observed in non-lysogenic cells treated with this antibiotic. This prophage induction-dependent transient increase in pcnB transcript may explain the polyadenylation-driven regulation of phage gene expression.

Phage-encoded colanic acid-degrading enzyme permits lytic phage infection of a capsule-forming resistant mutant Escherichia coli strain.

In this study, we isolated a bacteriophage T7-resistant mutant strain of Escherichia coli (named S3) and then proceeded to characterize it. The mutant bacterial colonies appeared to be mucoid. Microarray analysis revealed that genes related to colanic acid production were upregulated in the mutant. Increases in colanic acid production by the mutant bacteria were observed when l-fucose was measured biochemically, and protective capsule formation was observed under an electron microscope. We found a point mutation in the lon gene promoter in S3, the mutant bacterium. Overproduction of colanic acid was observed in some phage-resistant mutant bacteria after infection with other bacteriophages, T4 and lambda. Colanic acid overproduction was also observed in clinical isolates of E. coli upon phage infection. The overproduction of colanic acid resulted in the inhibition of bacteriophage adsorption to the host. Biofilm formation initially decreased shortly after infection but eventually increased after 48 h of incubation due to the emergence of the mutant bacteria. Bacteriophage PBECO4 was shown to infect the colanic acid-overproducing mutant strains of E. coli. We confirmed that the gene product of open reading frame 547 (ORF547) of PBECO4 harbored colanic acid-degrading enzymatic (CAE) activity. Treatment of the T7-resistant bacteria with both T7 and PBECO4 or its purified enzyme (CAE) led to successful T7 infection. Biofilm formation decreased with the mixed infection, too. This procedure, using a phage cocktail different from those exploiting solely receptor differences, represents a novel strategy for overcoming phage resistance in mutant bacteria.

Meta-Analysis of the Reduction of Norovirus and Male-Specific Coliphage Concentrations in Wastewater Treatment Plants.

Human norovirus (NoV) is the leading cause of foodborne illness in the United States and Canada. Wastewater treatment plant (WWTP) effluents impacting bivalve mollusk-growing areas are potential sources of NoV contamination. We have developed a meta-analysis that evaluates WWTP influent concentrations and log10 reductions of NoV genotype I (NoV GI; in numbers of genome copies per liter [gc/liter]), NoV genotype II (NoV GII; in gc/liter), and male-specific coliphage (MSC; in number of PFU per liter), a proposed viral surrogate for NoV. The meta-analysis included relevant data (2,943 measurements) reported in the scientific literature through September 2013 and previously unpublished surveillance data from the United States and Canada. Model results indicated that the mean WWTP influent concentration of NoV GII (3.9 log10 gc/liter; 95% credible interval [CI], 3.5, 4.3 log10 gc/liter) is larger than the value for NoV GI (1.5 log10 gc/liter; 95% CI, 0.4, 2.4 log10 gc/liter), with large variations occurring from one WWTP to another. For WWTPs with mechanical systems and chlorine disinfection, mean log10 reductions were -2.4 log10 gc/liter (95% CI, -3.9, -1.1 log10 gc/liter) for NoV GI, -2.7 log10 gc/liter (95% CI, -3.6, -1.9 log10 gc/liter) for NoV GII, and -2.9 log10 PFU per liter (95% CI, -3.4, -2.4 log10 PFU per liter) for MSCs. Comparable values for WWTPs with lagoon systems and chlorine disinfection were -1.4 log10 gc/liter (95% CI, -3.3, 0.5 log10 gc/liter) for NoV GI, -1.7 log10 gc/liter (95% CI, -3.1, -0.3 log10 gc/liter) for NoV GII, and -3.6 log10 PFU per liter (95% CI, -4.8, -2.4 PFU per liter) for MSCs. Within WWTPs, correlations exist between mean NoV GI and NoV GII influent concentrations and between the mean log10 reduction in NoV GII and the mean log10 reduction in MSCs.

Modeling the infection dynamics of bacteriophages in enteric Escherichia coli: estimating the contribution of transduction to antimicrobial gene spread.

Animal-associated bacterial communities are infected by bacteriophages, although the dynamics of these infections are poorly understood. Transduction by bacteriophages may contribute to transfer of antimicrobial resistance genes, but the relative importance of transduction among other gene transfer mechanisms is unknown. We therefore developed a candidate deterministic mathematical model of the infection dynamics of enteric coliphages in commensal Escherichia coli in the large intestine of cattle. We assumed the phages were associated with the intestine and were predominantly temperate. Model simulations demonstrated how, given the bacterial ecology and infection dynamics, most (>90%) commensal enteric E. coli bacteria may become lysogens of enteric coliphages during intestinal transit. Using the model and the most liberal assumptions about transduction efficiency and resistance gene frequency, we approximated the upper numerical limits ("worst-case scenario") of gene transfer through specialized and generalized transduction in E. coli by enteric coliphages when the transduced genetic segment is picked at random. The estimates were consistent with a relatively small contribution of transduction to lateral gene spread; for example, generalized transduction delivered the chromosomal resistance gene to up to 8 E. coli bacteria/hour within the population of 1.47 × 10(8) E. coli bacteria/liter luminal contents. In comparison, the plasmidic blaCMY-2 gene carried by ~2% of enteric E. coli was transferred by conjugation at a rate at least 1.4 × 10(3) times greater than our generalized transduction estimate. The estimated numbers of transductants varied nonlinearly depending on the ecology of bacteria available for phages to infect, that is, on the assumed rates of turnover and replication of enteric E. coli.

Novel approach of using a cocktail of designed bacteriophages against gut pathogenic E. coli for bacterial load biocontrol.

BACKGROUND: This study was conducted to explore new approaches of animal biocontrol via biological control feed. METHOD: White rats were subjected to 140 highly lytic designed phages specific against E. coli. Phages were fed via drinking water, oral injection, and vegetable capsules. Phage feeding was applied by 24 h feeding with 11 d monitoring and 20 d phage feeding and monitoring. Group of rats received external pathogenic E. coli and another group did not, namely groups A and B. RESULTS: Phage feeding for 20 d via vegetable capsules yielded the highest reduction of fecal E. coli, 3.02 and 4.62 log, in rats group A and B respectively. Second best, feeding for 20 d via drinking water with alkali yielded 2.78 and 4.08 log in rats groups A and B respectively. The peak reduction in E. coli output was 5-10 d after phage feeding. Phage control declined after 10th day of feeding. CONCLUSIONS: The use of cocktail of designed phages succeeded in suppressing flora or external E. coli. The phage feed biocontrol is efficient in controlling E. coli at the pre-harvest period, precisely at the 6th-8th day of phage feeding when the lowest E. coli output found.

Features of membrane receptors in bacterial multiplication process and necessary conditions for phage infection of bacteria.

According to the obtained experimental results, the thermal shock (from 37 to 53 °C) not only stops the multiplication process of Escherichia coli bacteria, but also causes bacterial titer to decrease gradually. After this period lasting up to 1 hour, the bacterial cells continue to grow. A similar type of response was observed when bacteria were subjected to acid shock. Increasing acidity of media leads to decrease of bacterial growth process, and finally, their titer curve sharply falls over time. Also, interesting results were obtained about necessary conditions for infecting the bacteria by phages. Particularly, DNA injection from phages into bacterial cells requires most of corresponding bacterial membrane receptors to be occupied by phages. We suppose that this occurs due to autocrine phenomenon when the signaling molecules block the DNA ejection from phage particles. This effect lasts until a certain number of phage particles are attached to the membrane. After that, DNA injection from phage head into the cytoplasm takes place and the process of bacterial infection begins. The real number of phages in a stock is by several orders higher than the number of plaque-forming units in a given stock, which is determined by a classical double-layer agar method.

T4-like phage Bp7, a potential antimicrobial agent for controlling drug-resistant Escherichia coli in chickens.

Chicken-pathogenic Escherichia coli is severely endangering the poultry industry in China and worldwide, and antibiotic therapy is facing an increasing problem of antibiotic resistance. Bacteriophages can kill bacteria with no known activity in human or animal cells, making them an attractive alternative to antibiotics. In this study, we present the characteristics of a novel virulent bacteriophage, Bp7, specifically infecting pathogenic multidrug-resistant E. coli. Phage Bp7 was isolated from chicken feces. Bp7 belongs to the family Myoviridae, possessing an elongated icosahedral head and contractile sheathed tail. It has a 168-kb double-stranded DNA genome. For larger yields, its optimal multiplicity of infection (MOI) to infect E. coli was about 0.001. The latent period was 10 to 15 min, and the burst size was 90 PFU/infected cell. It was stable both at pH 5.0 to 10.0 and at 40°C or 50°C for at least 1 h. Bp7 could infect 46% of pathogenic clinical E. coli strains. Bp7 harbored 791 open reading frames (ORFs) and 263 possible genes. Among the 263 genes, 199 possessed amino acid sequence identities with ORFs of phage T4, 62 had identities with other T4-like phages, and only one lacked any database match. The genome of Bp7 manifested obvious division and rearrangement compared to phages T4, JS98, and IME08. Bp7 is a new member of the "T4-like" genus, family Myoviridae. Its wide host range, strong cell-killing activity, and high stability to pH make it an alternative to antimicrobials for controlling drug-resistant E. coli in chickens.

Phage inhibition of Escherichia coli in ultrahigh-temperature-treated and raw milk.

Escherichia coli can contaminate raw milk during the milking process or via environmental contamination in milk-processing facilities. Three bacteriophages, designated EC6, EC9, and EC11, were investigated for their ability to inhibit the growth of three strains of E. coli in ultrahigh-temperature (UHT) treated and raw bovine milk. A cocktail of the three phages completely inhibited E. coli ATCC 25922 and E. coli O127:H6 in UHT milk at 25 °C and under refrigeration temperatures (5-9 °C). The phage cocktail produced similar results in raw milk; however, E. coli ATCC 25922 and O127:H6 in raw milk controls also declined to below the level of detection at both temperatures. This observation indicated that competition by the raw milk microbiota might have contributed to the decline in viable E. coli cells. A cocktail containing EC6 and EC9 completely inhibited E. coli O5:H-, an enterohemorrhagic strain, in UHT milk at both temperatures. In raw milk, the phage cocktail initially inhibited growth of E. coli O5:H- but regrowth occurred following incubation for 9 h at 25 °C and 144 h at 5-9 °C. In contrast to the other E. coli strains, O5:H- was not inhibited in the raw milk controls. This study demonstrates that bacteriophages are effective biocontrol agents against E. coli host strains in UHT and raw bovine milk at various storage temperatures.

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

In vivo growth rates are poorly correlated with phage therapy success in a mouse infection model.

Two classes of phages yield profoundly different levels of recovery in mice experimentally infected with an Escherichia coli O18:K1:H7 strain. Phages requiring the K1 capsule for infection (K1-dep) rescue virtually all infected mice, whereas phages not requiring the capsule (K1-ind) rescue modest numbers (∼30%). To rescue infected mice, K1-ind phages require at least a 10(6)-fold-higher inoculum than K1-dep phages. Yet their in vivo growth dynamics are only modestly inferior to those of K1-dep phages, and competition between the two phage types in the same mouse reveals only a slight growth advantage for the K1-dep phage. The in vivo growth rate seems unlikely to be the primary determinant of phage therapy success. An alternative explanation is that the success of K1-dep phages is due substantially to their proteomic composition. They encode an enzyme that degrades the K1 capsule, which has been shown in other work to be sufficient to cure infection in the complete absence of phages.

A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12.

We have developed a direct and efficient strategy, based on a three-step method, to select bacterial cell-envelope mutants resistant to bacteriophage infection. Escherichia coli K-12 strain W3110 underwent classical transposon mutagenesis followed by replica plating and selection for mutants resistant to infection by coliphage mEp213. To verify that phage resistance was due to mutations in the cell envelope, we transformed host cells with the viral genome using electroporation and selected those in which virions were subsequently detected in the supernatant. Among the nine mutants resistant to coliphage infection that we selected, six were in the fhuA gene, two were mutated in the waaC gene, and one was mutated in the gmhD gene. The latter two gene products are involved in the synthesis of lipopolysaccharide (LPS). The efficiency of plating and adsorption of phage mEp213 was affected in these mutants. We verified that LPS is required for the efficient infection of phage λ as well. We propose that this mutation-and-selection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.

Quantitative comparison of the RNA bacteriophage Qß infection cycle in rich and minimal media.

As bacteriophages are dependent on the host for multiplication, their infection cycle is expected to be influenced by the host's physiological state. To elucidate how and which steps of the bacteriophage infection cycle are influenced by changes in the physiological state of the host, we quantitatively compared the infection cycle of lytic RNA bacteriophage Qß in Escherichia coli cultured in rich and minimal media. The adsorption rate constants in both media were almost the same. A difference of 15 min in the latent period and an approximately twofold increase in the rate of phage release were observed, although approximately 10(5) molecules of coat proteins, equivalent to approximately 600-1000 phage particles, accumulated in an infected cell prior to burst. Addition of Mg(2+) to minimal medium markedly affected the Qß infection cycle, and these results suggest that Mg(2+) is required for the stages of the infectious cycle after adsorption.

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.

Isolation of generalized transducing bacteriophages for uropathogenic strains of Escherichia coli.

The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.

Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities.

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.

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.