The microcosm mediates the persistence of shiga toxin-producing Escherichia coli in freshwater ecosystems.

Water is a major route for infection of humans by exotoxin-producing bacteria, including Shiga toxin-producing Escherichia coli (STEC). While STEC has the potential to be present in nearly every type of water source, its distribution is sporadic, and an understanding of factors that govern its emergence and persistence within water is lacking. In this study, we examined the influence of microbe content on STEC persistence in freshwater. We found that depletion of microbes in the water leads to a considerable increase in the persistence of STEC, an effect that can be mitigated by adding grazing protists to the water. STEC strains appear to be more resistant to the impact of grazing protists than E. coli strains that lack the Shiga toxin (stx) gene. Our results demonstrate that the microcosm can dramatically influence the persistence of STEC in aquatic ecosystems and that the overall impact by microbes on STEC strains is fundamentally different from that of non-STEC strains of bacteria. Overall, these results provide insight into why STEC and possibly other exotoxin-producing bacterial pathogens display such variability in abundance, distribution, and persistence in aquatic ecosystems.

Contamination level and location of recreational freshwater influence the ability to predict Escherichia coli concentration by qPCR targeting Bacteroides.

Fecal bacteria are common microbial contaminants in freshwater with the potential to cause human illness. Detection of these microbes have traditionally relied on microbial plating to enumerate colonies of fecal indicator bacteria (FIB) such as Escherichia coli (E. coli), which can take 24 h or longer to complete. Quantitative PCR (qPCR) is a rapid and sensitive method for detection of FIB in recreational water that could compliment or potentially substitute for microbial plating. In this study, we have isolated DNA from the beach water on the shoreline at three different locations of Lake Erie and subjected these samples to qPCR to examine the relative abundance of Bacteroides. These values were compared to colony forming units (CFU) of E. coli. The resultant linear regressions between these different measurements of microbe concentration were used to determine the efficacy of qPCR targeting Bacteroides at predicting E. coli concentrations that are relevant for decision making by recreational water managers. Our findings indicate that the ability of Bacteroides to serve as an early predictive tool for E. coli CFU concentration depends on sample location and level of bacterial contamination, but can be used in some cases to supplement recreational water quality measurement and consequential management.

Athletes Drive Distinctive Trends of COVID-19 Infection in a College Campus Environment.

The COVID-19 pandemic forced most institutions of higher education to offer instruction and activities offsite, impacting millions of people. As universities consider resuming normal operations on campus, evidence-based guidance is needed to enhance safety protocols to reduce the spread of infectious disease in their campus environments. During the 2020/2021 academic year, Gannon University in Erie, PA, USA, was able to maintain most of its operations on campus. Part of Gannon's disease mitigation strategy involved the development of a novel in-house, real-time RT-PCR-based surveillance program, which tested 23,227 samples to monitor the presence of COVID-19 on campus. Temporal trends of COVID-19 infection at Gannon were distinct from statewide data. A significant portion of this variance involved student athletes and associated staff, which identified as a higher incidence risk group compared with non-athletes. Rapid identification of athlete driven outbreaks allowed for swift action to limit the spread of COVID-19 among teammates and to the rest of the campus community. This allowed for successful completion of instruction and a modified season for all sports at Gannon. Our findings provide insights that could prove useful to the thousands of institutions seeking to resume a more traditional presence on campus.

Correlation of shiga toxin gene frequency with commonly used microbial indicators of recreational water quality.

Shiga toxin (Stx) genes produce proteins that are pathogenic to humans, leading to severe gastrointestinal illness. This work focuses on examining the abundance and distribution of stx genes in relation to common microbial indicators in beach water and streams in the vicinity of Presque Isle State Park in Erie, PA. By use of quantitative PCR, the relative abundance levels of stx DNA in over 700 samples in the sampling area were determined. The results demonstrate that the abundance and distribution of stx genes are variable and do not correlate with the abundance of Escherichia coli bacteria, enterococci, or viral particles. These results suggest that microbial indicators of water quality are not adequate in predicting the occurrence of organisms that harbor stx genes and highlight the need for standardized pathogen-specific detection protocols for waters utilized for recreational swimming.

Molecular Mechanisms Governing "Hair-Trigger" Induction of Shiga Toxin-Encoding Prophages.

Shiga toxin (Stx)-encoding E. coli (STEC) strains are responsible for sporadic outbreaks of food poisoning dating to 1982, when the first STEC strain, E. coli O157:H7, was isolated. Regardless of STEC serotype, the primary symptoms of STEC infections are caused by Stx that is synthesized from genes resident on lambdoid prophage present in STEC. Despite similar etiology, the severity of STEC-mediated disease varies by outbreak. However, it is unclear what modulates the severity of STEC-mediated disease. Stx production and release is controlled by lytic growth of the Stx-encoding bacteriophage, which in turn, is controlled by the phage repressor. Here, we confirm our earlier suggestion that the higher spontaneous induction frequency of Stx-encoding prophage is a consequence, in part, of lower intracellular repressor levels in STEC strains versus non-STEC strains. We also show that this lowered intracellular repressor concentration is a consequence of the utilization of alternative binding/regulatory strategies by the phage repressor. We suggest that a higher spontaneous induction frequency would lead to increased virulence.

Monovalent cations regulate DNA sequence recognition by 434 repressor.

The bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout. In indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein's DNA-binding site modulate the affinity of DNA for protein. The conformation and flexibility of a DNA sequence can be influenced by the interaction of the DNA bases or backbone with solution components. We examined the effect of changing the cation-type present in solution on the stability and structure of 434 repressor complexes with wild-type and mutant OR1 and OR3, binding sites that differ in their contacted and non-contacted base sequences. We find that the affinity of repressor for OR1, but not for OR3, depends remarkably on the type and concentration of monovalent cation. Moreover, the formation of a stable, specific repressor-OR1 complex requires the presence of monovalent cations; however, repressor-OR3 complex formation has no such requirement. Changing monovalent cation type alters the ability of repressor to protect OR1, but not OR3, from *OH radical cleavage. Altering the relative length of the poly(dA) x poly(dT) tract in the non-contacted regions of the OR1 and OR3 can reverse the cation sensitivity of repressor's affinities for these two sites. Taken together these findings show that cation-dependent alterations in DNA structure underlies indirect readout of DNA sequence by 434 repressor and perhaps other proteins.

Shiga toxin: expression, distribution, and its role in the environment.

In this review, we highlight recent work that has increased our understanding of the production and distribution of Shiga toxin in the environment. Specifically, we review studies that offer an expanded view of environmental reservoirs for Shiga toxin producing microbes in terrestrial and aquatic ecosystems. We then relate the abundance of Shiga toxin in the environment to work that demonstrates that the genetic mechanisms underlying the production of Shiga toxin genes are modified and embellished beyond the classical microbial gene regulatory paradigms in a manner that apparently "fine tunes" the trigger to modulate the amount of toxin produced. Last, we highlight several recent studies examining microbe/protist interactions that postulate an answer to the outstanding question of why microbes might harbor and express Shiga toxin genes in the environment.

Detection of stx and stx genes in Pennsylvanian white-tailed deer.

Shiga toxin-producing E. coli carrying the stx(1) and/or stx(2) genes can cause multi-symptomatic illness in humans. A variety of terrestrial and aquatic environmental reservoirs of stx have been described. Culture based detection of microbes in deer species have found a low percentage of samples that have tested positive for Stx-producing microbes, suggesting that while deer may contain these microbes, their overall abundance in deer is low. In this study, quantitative PCR (qPCR) was utilized to test for the presence of stx genes in white-tailed deer fecal matter in western Pennsylvania. In this culture independent screening, nearly half of the samples tested positive for the stx(2) gene, with a bias towards samples that were concentrated with stx(2). This study, while limited in scope, suggests that deer may be a greater reservoir for stx than was previously thought.

The role of the minor groove substituents in indirect readout of DNA sequence by 434 repressor.

The sequence of non-contacted bases at the center of the 434 repressor binding site affects the strength of the repressor-DNA complex by influencing the structure and flexibility of DNA (Koudelka, G. B., and Carlson, P. (1992) Nature 355, 89-91). We synthesized 434 repressor binding sites that differ in their central sequence base composition to test the importance of minor groove substituents and/or the number of base pair hydrogen bonds between these base pairs on DNA structure and strength of the repressor-DNA complex. We show here that the number of base pair H-bonds between the central bases apparently has no role in determining the relative affinity of a DNA site for repressor. Instead we find that the affinity of DNA for repressor depends on the absence or presence the N2-NH(2) group on the purine bases at the binding site center. The N2-NH(2) group on bases at the center of the 434 binding site appears to destabilize 434 repressor-DNA complexes by decreasing the intimacy of the specific repressor-DNA contacts, while increasing the reliance on protein contacts to the DNA phosphate backbone. Thus, the presence of an N2-NH(2) group on the purines at the center of a binding site globally alters the precise conformation of the protein-DNA interface.