The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by β-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified. In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively. The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

Summary: Replication from the origin of Escherichia coli has traditionally been visualized as two replisomes moving away from each other, each containing a leading and a lagging strand polymerase. Fluorescence microscopy studies of tagged polymerases or forks have, however, indicated that the polymerases may be confined to a single location (or a few locations in cells with overlapping replication cycles). Here, we have analysed the exact replication patterns of cells growing with four different growth and replication rates, and compared these with the distributions of SeqA foci. The SeqA foci represent replication forks because the SeqA protein binds to the newly formed hemimethylated DNA immediately following the forks. The results show that pairs of forks originating from the same origin stay coupled for most of the cell cycle and thus support the replication factory model. They also suggest that the factories consisting of four polymerases are, at the time immediately after initiation, organized into higher order structures consisting of eight or 12 polymerases. The organization into replication factories was lost when replication forks experienced a limitation in the supply of nucleotides or when the thymidylate synthetase gene was mutated. These results support the idea that the nucleotide synthesis apparatus co‐localizes with the replisomes forming a ‘hyperstructure’ and further suggest that the integrity of the replication factories and hyperstructures is dependent on nucleotide metabolism.

The application of bacteriophages as antibacterial agents has many benefits in the “post-antibiotic age”. To increase the number of successfully targeted bacterial strains, phage cocktails, instead of a single phage, are commonly formulated. Nevertheless, there is currently no consensus pipeline for phage cocktail development. Thus, although large cocktails increase the spectrum of activity, they could produce side effects such as the mobilization of virulence or antibiotic resistance genes. On the other hand, coinfection (simultaneous infection of one host cell by several phages) might reduce the potential for bacteria to evolve phage resistance, but some antagonistic interactions amongst phages might be detrimental for the outcome of phage cocktail application. With this in mind, we introduce here a new method, which considers the host range and each individual phage-host interaction, to design the phage mixtures that best suppress the target bacteria while minimizing the number of phages to restrict manufacturing costs. Additionally, putative phage-phage interactions in cocktails and phage-bacteria networks are compared as the understanding of the complex interactions amongst bacteriophages could be critical in the development of realistic phage therapy models in the future.

In Escherichia coli , three mechanisms have been proposed to maintain proper regulation of replication so that initiation occurs once, and only once, per cell cycle. First, newly formed origins are inactivated by sequestration; second, the initiator, DnaA, is inactivated by the Hda protein at active replication forks; and third, the level of free DnaA protein is reduced by replication of the datA site. The datA site titrates unusually large amounts of DnaA and it has been reported that reinitiation, and thus asynchrony of replication, occurs in cells lacking this site. Here, we show that reinitiation in Δ datA cells does not occur during exponential growth and that an apparent asynchrony phenotype results from the occurrence of rifampin-resistant initiations. This shows that the datA site is not required to prevent reinitiation and limit initiation of replication to once per generation. The datA site may, however, play a role in timing of initiation relative to cell growth. Inactivation of active ATP-DnaA by the Hda protein and the sliding clamp of the polymerase was found to be required to prevent reinitiation and asynchrony of replication.

In recent years, the spread of antibiotic-resistant bacteria and efforts to preserve food microbiota have induced renewed interest in phage therapy. Phage cocktails, instead of a single phage, are commonly used as antibacterial agents since the hosts are unlikely to become resistant to several phages simultaneously. While the spectrum of activity might increase with cocktail complexity, excessive phages could produce side effects, such as the horizontal transfer of genes that augment the fitness of host strains, dysbiosis or high manufacturing costs. Therefore, cocktail formulation represents a compromise between achieving substantial reduction in the bacterial loads and restricting its complexity. Despite the abovementioned points, the observed bacterial load reduction does not increase significantly with the size of phage cocktails, indicating the requirement for a systematic approach to their design. In this work, the information provided by host range matrices was analyzed after building phage-bacteria infection networks (PBINs). To this end, we conducted a meta-analysis of 35 host range matrices, including recently published studies and new datasets comprising Escherichia coli strains isolated during ripening of artisanal raw milk cheese and virulent coliphages from ewes’ feces. The nestedness temperature, which reflects the host range hierarchy of the phages, was determined from bipartite host range matrices using heuristic (Nestedness Temperature Calculator) and genetic (BinMatNest) algorithms. The latter optimizes matrix packing, leading to lower temperatures, i.e., it simplifies the identification of the phages with the broadest host range. The structure of infection networks suggests that generalist phages (and not specialist phages) tend to succeed in infecting less susceptible bacteria. A new metric (Φ), which considers some properties of the host range matrices (fill, temperature, and number of bacteria), is proposed as an estimator of phage cocktail size. To identify the best candidates, agglomerative hierarchical clustering using Ward’s method was implemented. Finally, a cocktail was formulated for the biocontrol of cheese-isolated E. coli, reducing bacterial counts by five orders of magnitude.