Changes in Escherichia coli to enteric protozoa ratios in rivers: Implications for risk-based assessment of drinking water treatment requirements.

Minimum treatment requirements are set in response to established or anticipated levels of enteric pathogens in the source water of drinking water treatment plants (DWTPs). For surface water, contamination can be determined directly by monitoring reference pathogens or indirectly by measuring fecal indicators such as Escherichia coli (E. coli). In the latter case, a quantitative interpretation of E. coli for estimating reference pathogen concentrations could be used to define treatment requirements. This study presents the statistical analysis of paired E. coli and reference protozoa (Cryptosporidium, Giardia) data collected monthly for two years in source water from 27 DWTPs supplied by rivers in Canada. E. coli/Cryptosporidium and E. coli/Giardia ratios in source water were modeled as the ratio of two correlated lognormal variables. To evaluate the potential of E. coli for defining protozoa treatment requirements, risk-based critical mean protozoa concentrations in source water were determined with a reverse quantitative microbial risk assessment (QMRA) model. Model assumptions were selected to be consistent with the World Health Organization (WHO) Guidelines for drinking-water quality. The sensitivity of mean E. coli concentration trigger levels to identify these critical concentrations in source water was then evaluated. Results showed no proportionalities between the log of mean E. coli concentrations and the log of mean protozoa concentrations. E. coli/protozoa ratios at DWTPs supplied by small rivers in agricultural and forested areas were typically 1.0 to 2.0-log lower than at DWTPs supplied by large rivers in urban areas. The seasonal variations analysis revealed that these differences were related to low mean E. coli concentrations during winter in small rivers. To achieve the WHO target of 10-6 disability-adjusted life year (DALY) per person per year, a minimum reduction of 4.0-log of Cryptosporidium would be required for 20 DWTPs, and a minimum reduction of 4.0-log of Giardia would be needed for all DWTPs. A mean E. coli trigger level of 50 CFU 100 mL-1 would be a sensitive threshold to identify critical mean concentrations for Cryptosporidium but not for Giardia. Treatment requirements higher than 3.0-log would be needed at DWTPs with mean E. coli concentrations as low as 30 CFU 100 mL-1 for Cryptosporidium and 3 CFU 100 mL-1 for Giardia. Therefore, an E. coli trigger level would have limited value for defining health-based treatment requirements for protozoa at DWTPs supplied by small rivers in rural areas.

Impact of Hydrometeorological Events for the Selection of Parametric Models for Protozoan Pathogens in Drinking-Water Sources.

Temporal variations in concentrations of pathogenic microorganisms in surface waters are well known to be influenced by hydrometeorological events. Reasonable methods for accounting for microbial peaks in the quantification of drinking water treatment requirements need to be addressed. Here, we applied a novel method for data collection and model validation to explicitly account for weather events (rainfall, snowmelt) when concentrations of pathogens are estimated in source water. Online in situ ß-d-glucuronidase activity measurements were used to trigger sequential grab sampling of source water to quantify Cryptosporidium and Giardia concentrations during rainfall and snowmelt events at an urban and an agricultural drinking water treatment plant in Quebec, Canada. We then evaluate if mixed Poisson distributions fitted to monthly sampling data ( n = 30 samples) could accurately predict daily mean concentrations during these events. We found that using the gamma distribution underestimated high Cryptosporidium and Giardia concentrations measured with routine or event-based monitoring. However, the log-normal distribution accurately predicted these high concentrations. The selection of a log-normal distribution in preference to a gamma distribution increased the annual mean concentration by less than 0.1-log but increased the upper bound of the 95% credibility interval on the annual mean by about 0.5-log. Therefore, considering parametric uncertainty in an exposure assessment is essential to account for microbial peaks in risk assessment.

Importance of Distributional Forms for the Assessment of Protozoan Pathogens Concentrations in Drinking-Water Sources.

The identification of appropriately conservative statistical distributions is needed to predict microbial peak events in drinking water sources explicitly. In this study, Poisson and mixed Poisson distributions with different upper tail behaviors were used for modeling source water Cryptosporidium and Giardia data from 30 drinking water treatment plants. Small differences (<0.5-log) were found between the "best" estimates of the mean Cryptosporidium and Giardia concentrations with the Poisson-gamma and Poisson-log-normal models. However, the upper bound of the 95% credibility interval on the mean Cryptosporidium concentrations of the Poisson-log-normal model was considerably higher (>0.5-log) than that of the Poisson-gamma model at four sites. The improper choice of a model may, therefore, mislead the assessment of treatment requirements and health risks associated with the water supply. Discrimination between models using the marginal deviance information criterion (mDIC) was unachievable because differences in upper tail behaviors were not well characterized with available data sets ( n<30 ). Therefore, the gamma and the log-normal distributions fit the data equally well but may predict different risk estimates when they are used as an input distribution in an exposure assessment. The collection of event-based monitoring data and the modeling of larger routine monitoring data sets are recommended to identify appropriately conservative distributions to predict microbial peak events.

Legionella pneumophila levels and sequence-type distribution in hospital hot water samples from faucets to connecting pipes.

Recent studies have reported increased levels of Legionella pneumophila (Lp) at points of use compared to levels in primary and secondary components of hot water systems, suggesting possible selection by environmental conditions. In this study, concentrations of Lp in a hospital hot water system were evaluated by profile sampling, collecting successive water samples to determine the prevalence at the faucet (distal) and upstream piping before and after a system intervention to increase temperature. Lp strain diversity was compared between different points of use and different areas of the hot water system (i.e., tap, intermediate piping and main upflow piping). In total, 47 isolates were recovered from 32 positive hot water samples collected from designated taps, showers and recirculation loops; these isolates were subsequently analyzed by sequence-based typing (SBT). Lp levels were comparable between first draw (500 mL) and flushed (2 and 5 min) samples, whereas a decrease was observed in the amount of culturable cells (1 log). Two sequence types (STs) were identified throughout the system. ST378 (sg4/10) was present in 91% of samples, while ST154-like (sg1) was present in 41%; both STs were simultaneously recovered in 34% of samples. Isolated STs displayed comparable tolerance to copper (0.8-5 mg/L) and temperature (55 °C, 1 h) exposure. The ability to replicate within THP1 cells and Acanthamoeba castellanii was similar between the two STs and a comparative environmental outbreak strain. The low Lp diversity and the detection of both Lp sequence types in repeated subsequent samples collected from positive faucets in a hospital wing suggest a minimal impact of the distal conditions on strain selection for the sampled points, as well as a possible adaptation to stressors present in the system, leading to the predominance of a few strains.

Energy Conservation and the Promotion of Legionella pneumophila Growth: The Probable Role of Heat Exchangers in a Nosocomial Outbreak.

OBJECTIVE To determine the source of a Legionella pneumophila serogroup 5 nosocomial outbreak and the role of the heat exchanger installed on the hot water system within the previous year. SETTING A 400-bed tertiary care university hospital in Sherbrooke, Canada. METHODS Hot water samples were collected and cultured for L. pneumophila from 25 taps (baths and sinks) within wing A and 9 taps in wing B. Biofilm (5) and 2 L water samples (3) were collected within the heat exchangers for L. pneumophila culture and detection of protists. Sequence-based typing was performed on strain DNA extracts and pulsed-field gel electrophoresis patterns were analyzed. RESULTS Following 2 cases of hospital-acquired legionellosis, the hot water system investigation revealed a large proportion of L. pneumophila serogroup 5 positive taps (22/25 in wing A and 5/9 in wing B). High positivity was also detected in the heat exchanger of wing A in water samples (3/3) and swabs from the heat exchanger (4/5). The outbreak genotyping investigation identified the hot water system as the source of infections. Genotyping results revealed that all isolated environmental strains harbored the same related pulsed-field gel electrophoresis pattern and sequence-based type. CONCLUSIONS Two cases of hospital-acquired legionellosis occurred in the year following the installation of a heat exchanger to preheat hospital hot water. No cases were reported previously, although the same L. pneumophila strain was isolated from the hot water system in 1995. The heat exchanger promoted L. pneumophila growth and may have contributed to confirmed clinical cases. Infect. Control Hosp. Epidemiol. 2016;1475-1480.

Adaptation in bacterial CRISPR-Cas immunity can be driven by defective phages.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated cas genes serve as a prokaryotic 'adaptive' immune system, protecting against foreign DNA elements such as bacteriophages. CRISPR-Cas systems function by incorporating short DNA 'spacers', homologous to invading DNA sequences, into a CRISPR array (adaptation). The array is then transcribed and matured into RNA molecules (maturation) that target homologous DNA for cleavage (interference). It is unclear how these three stages could occur quickly enough in a naive phage-infected cell to interfere with phage replication before this cell would be irrevocably damaged by the infection. Here we demonstrate that cells can acquire spacers from defective phages at a rate directly proportional to the quantity of replication-deficient phages to which the cells are exposed. This process is reminiscent of immunization in humans by vaccination with inactivated viruses.

Inactivation of dairy bacteriophages by commercial sanitizers and disinfectants.

Many commercial sanitizers and disinfectants have been used over the years to control microbial contamination but their efficacy on phages is often unknown. Here, 23 commercial chemical products, including 21 food-grade sanitizers were tested against virulent dairy phages. These food-grade chemicals included oxidizing agents, halogenated agents, alcohols, quaternary ammonium compounds, anionic acids, iodine-based acids, and an amphoteric chemical. Phage P008 was first exposed to each sanitizer for 2 and 15min at room temperature and at two different concentrations, namely the lowest and highest no-rinse sanitizing concentrations. Organic matter (whey or milk) was also added to the testing solutions. At the end of the exposure period, the test solution was neutralized and the number of infectious phages was determined by plaque assays. The five most efficient sanitizers against phage P008 (<4 log of inactivation) were then tested against virulent lactococcal phages P008, CB13, AF6, P1532 of the 936 group, P001 (c2), Q54, and 1358 as well as Lactobacillus plantarum phage B1 and Streptococcus thermophilus phage 2972 using the same protocol. The oxidizing agents and the quaternary ammonium compounds were the most efficient against all phages although phages CB13 and P1532 were less sensitive to these chemicals than the other phages. This study may help in the selection of appropriate chemicals for controlling phage contamination in industrial factories and research laboratories.

CRISPR-Cas and restriction-modification systems are compatible and increase phage resistance.

Bacteria have developed a set of barriers to protect themselves against invaders such as phage and plasmid nucleic acids. Different prokaryotic defence systems exist and at least two of them directly target the incoming DNA: restriction-modification (R-M) and CRISPR-Cas systems. On their own, they are imperfect barriers to invasion by foreign DNA. Here, we show that R-M and CRISPR-Cas systems are compatible and act together to increase the overall phage resistance of a bacterial cell by cleaving their respective target sites. Furthermore, we show that the specific methylation of phage DNA does not impair CRISPR-Cas acquisition or interference activities. Taken altogether, both mechanisms can be leveraged to decrease phage contaminations in processes relying on bacterial growth and/or fermentation.

The double-edged sword of CRISPR-Cas systems.

A recent paper gives the details on how specific small RNAs can program a protein to cleave an undesired piece of DNA and to provide immunity to a microbial cell.

Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system.

Streptococcus thermophilus, similar to other Bacteria and Archaea, has developed defense mechanisms to protect cells against invasion by foreign nucleic acids, such as virus infections and plasmid transformations. One defense system recently described in these organisms is the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats loci coupled to CRISPR-associated genes). Two S. thermophilus CRISPR-Cas systems, CRISPR1-Cas and CRISPR3-Cas, have been shown to actively block phage infection. The CRISPR1-Cas system interferes by cleaving foreign dsDNA entering the cell in a length-specific and orientation-dependant manner. Here, we show that the S. thermophilus CRISPR3-Cas system acts by cleaving phage dsDNA genomes at the same specific position inside the targeted protospacer as observed with the CRISPR1-Cas system. Only one cleavage site was observed in all tested strains. Moreover, we observed that the CRISPR1-Cas and CRISPR3-Cas systems are compatible and, when both systems are present within the same cell, provide increased resistance against phage infection by both cleaving the invading dsDNA. We also determined that overall phage resistance efficiency is correlated to the total number of newly acquired spacers in both CRISPR loci.

Involvement of the major capsid protein and two early-expressed phage genes in the activity of the lactococcal abortive infection mechanism AbiT.

The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB) Lactococcus lactis to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives: e14 (bIL170 [e14(bIL170)]), orf41 (P008 [orf41(P008)]), and orf6 (p2 [orf6(p2)] and P008 [orf6(P008)]). The genes e14(bIL170) and orf41(P008) are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function. orf6 is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in L. lactis of ORF6(p2) and ORF5(p2), a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.

A reverse transcriptase-related protein mediates phage resistance and polymerizes untemplated DNA in vitro.

Reverse transcriptases (RTs) are RNA-dependent DNA polymerases that usually function in the replication of selfish DNAs such as retrotransposons and retroviruses. Here, we have biochemically characterized a RT-related protein, AbiK, which is required for abortive phage infection in the Gram-positive bacterium Lactococcus lactis. In vitro, AbiK does not exhibit the properties expected for an RT, but polymerizes long DNAs of 'random' sequence, analogous to a terminal transferase. Moreover, the polymerized DNAs appear to be covalently attached to the AbiK protein, presumably because an amino acid serves as a primer. Mutagenesis experiments indicate that the polymerase activity resides in the RT motifs and is essential for phage resistance in vivo. These results establish a novel biochemical property and a non-replicative biological role for a polymerase.

The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.

Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.

P087, a lactococcal phage with a morphogenesis module similar to an Enterococcus faecalis prophage.

The virulent lactococcal phage P087 was isolated from a dairy environment in 1978. This phage was then recognized as the reference member for one of the ten phage groups currently known to infect Lactococcus lactis strains. The double-stranded DNA genome of this Siphoviridae phage is composed of 60,074 bp and is circularly permuted. Five tRNA and 88 orfs were found within an uncommon genome architecture. Eleven structural proteins were also identified through SDS-PAGE and LC-MS/MS analyses. Of note, 11 translated orfs from the structural module of phage P087 have identities to gene products found in a prophage located in the genome of Enterococcus faecalis V583. The alignment of both genomic sequences suggests that DNA exchanges could occur between these two phages which are infecting low G+C bacteria found in similar ecological niches.

Bacteriophages of lactobacillus.

In this review, we are listing Lactobacillus phages that have been reported in peer-reviewed articles published since 1960. Putative phages that are defective or have not been shown to be infectious, such as phage-like particles, are not discussed. Our literature searches led to the identification of 231 Lactobacillus phages, 186 of which have been observed by electron microscopy, with 109 belonging to the Siphoviridae family, 76 to the Myoviridae family, and 1 to the Podoviridae family. Model phages infecting Lb delbrueckii, casei, rhamnosus, plantarum, and gasseri are highlighted, as well as prophages of Lactobacillus hosts. To date, nine complete Lactobacillus phage genomes are available for comparisons and evolution studies. Features such as phage receptors and endolysins are also reviewed, as well as phage-derived genetic tools. Lactobacillus phage research has progressed significantly over the past decade but a thorough understanding of their biology is still lacking. Because of the risks they represent and the knowledge gaps that need to be filled, the outlook for research on Lactobacillus phages is bright.

Crystal structure of ORF12 from Lactococcus lactis phage p2 identifies a tape measure protein chaperone.

We report here the characterization of the nonstructural protein ORF12 of the virulent lactococcal phage p2, which belongs to the Siphoviridae family. ORF12 was produced as a soluble protein, which forms large oligomers (6- to 15-mers) in solution. Using anti-ORF12 antibodies, we have confirmed that ORF12 is not found in the virion structure but is detected in the second half of the lytic cycle, indicating that it is a late-expressed protein. The structure of ORF12, solved by single anomalous diffraction and refined at 2.9-A resolution, revealed a previously unknown fold as well as the presence of a hydrophobic patch at its surface. Furthermore, crystal packing of ORF12 formed long spirals in which a hydrophobic, continuous crevice was identified. This crevice exhibited a repeated motif of aromatic residues, which coincided with the same repeated motif usually found in tape measure protein (TMP), predicted to form helices. A model of a complex between ORF12 and a repeated motif of the TMP of phage p2 (ORF14) was generated, in which the TMP helix fitted exquisitely in the crevice and the aromatic patches of ORF12. We suggest, therefore, that ORF12 might act as a chaperone for TMP hydrophobic repeats, maintaining TMP in solution during the tail assembly of the lactococcal siphophage p2.

The XerC recombinase of Proteus mirabilis: characterization and interaction with other tyrosine recombinases.

XerC and XerD are two site-specific recombinases, which act on different sites to maintain replicons in a monomeric state. This system, which was first discovered and studied in Escherichia coli, is present in several species including Proteus mirabilis, where the XerD recombinase was previously characterized by our laboratory. In this paper, we report the presence of the xerC gene in P. mirabilis. Using in vitro reactions, we show that the two P. mirabilis recombinases display binding and cleavage activity on the E. coli dif site and the ColE1 cer site, together or in collaboration with E. coli recombinases. In vivo, P. mirabilis XerC and XerD are able to resolve and monomerize a plasmid containing two cer sites, increasing its stability. However, P. mirabilis XerC, in combination with E. coli XerD, is unable to perform these functions.