Yersinia pestis Δail Mutants Are Not Susceptible to Human Complement Bactericidal Activity in the Flea.

Ail confers serum resistance in humans and is a critical virulence factor of Y. pestis, the causative agent of plague. Here, the contribution of Ail for Y. pestis survival in the flea vector was examined. Rat or human but not mouse sera were bactericidal against a Y. pestis Δail mutant at 28°C in vitro. Complement components deposited rapidly on the Y. pestis surface as measured by immunofluorescent microscopy. Ail reduced the amount of active C3b on the Y. pestis surface. Human sera retained bactericidal activity against a Y. pestis Δail mutant in the presence of mouse sera. However, in the flea vector, the serum protective properties of Ail were not required. Flea colonization studies using murine sera and Y. pestis KIM6+ wild type, a Δail mutant, and the Δail/ail+ control showed no differences in bacterial prevalence or numbers during the early stage of flea colonization. Similarly, flea studies with human blood showed Ail was not required for serum resistance. Finally, a variant of Ail (AilF100V E108_S109insS) from a human serum-sensitive Y. pestis subsp. microtus bv. Caucasica 1146 conferred resistance to human complement when expressed in the Y. pestis KIM6+ Δail mutant. This indicated that Ail activity was somehow blocked, most likely by lipooligosaccharide, in this serum sensitive strain. IMPORTANCE This work contributes to our understanding of how highly virulent Y. pestis evolved from its innocuous enteric predecessor. Among identified virulence factors is the attachment invasion locus protein, Ail, that is required to protect Y. pestis from serum complement in all mammals tested except mice. Murine sera is not bactericidal. In this study, we asked, is bactericidal sera from humans active in Y. pestis colonized fleas? We found it was not. The importance of this observation is that it identifies a protective niche for the growth of serum sensitive and nonsensitive Y. pestis strains.

Contributions of Yersinia pestis outer membrane protein Ail to plague pathogenesis.

PURPOSE OF REVIEW: Pathogenic Yersinia have been a productive model system for studying bacterial pathogenesis. Hallmark contributions of Yersinia research to medical microbiology are legion and include: (i) the first identification of the role of plasmids in virulence, (ii) the important mechanism of iron acquisition from the host, (iii) the first identification of bacterial surface proteins required for host cell invasion, (iv) the archetypical type III secretion system, and (v) elucidation of the role of genomic reduction in the evolutionary trajectory from a fairly innocuous pathogen to a highly virulent species. RECENT FINDINGS: The outer membrane (OM) protein Ail (attachment invasion locus) was identified over 30 years ago as an invasin-like protein. Recent work on Ail continues to provide insights into Gram-negative pathogenesis. This review is a synopsis of the role of Ail in invasion, serum resistance, OM stability, thermosensing, and vaccine development. SUMMARY: Ail is shown to be an essential virulence factor with multiple roles in pathogenesis. The recent adaptation of Yersinia pestis to high virulence, which included genomic reduction to eliminate redundant protein functions, is a model to understand the emergence of new bacterial pathogens.

Escherichia coli 0157:H7 virulence factors and the ruminant reservoir.

PURPOSE OF REVIEW: This review updates recent findings about Escherichia coli O157:H7 virulence factors and its bovine reservoir. This Shiga toxin (Stx)-producing E. coli belongs to the Enterohemorrhagic E. coli (EHEC) pathotype causing hemorrhagic colitis. Its low infectious dose makes it an efficient, severe, foodborne pathogen. Although EHEC remains in the intestine, Stx can translocate systemically and is cytotoxic to microvascular endothelial cells, especially in the kidney and brain. Disease can progress to life-threatening hemolytic uremic syndrome (HUS) with hemolytic anemia, acute kidney failure, and thrombocytopenia. Young children, the immunocompromised, and the elderly are at the highest risk for HUS. Healthy ruminants are the major reservoir of EHEC and cattle are the primary source of human exposure. RECENT FINDINGS: Advances in understanding E. coli O157:H7 pathogenesis include molecular mechanisms of virulence, bacterial adherence, type three secretion effectors, intestinal microbiome, inflammation, and reservoir maintenance. SUMMARY: Many aspects of E. coli O157:H7 disease remain unclear and include the role of the human and bovine intestinal microbiomes in infection. Therapeutic strategies involve controlling inflammatory responses and/or intestinal barrier function. Finally, elimination/reduction of E. coli O157:H7 in cattle using CRISPR-engineered conjugative bacterial plasmids and/or on-farm management likely hold solutions to reduce infections and increase food safety/security.

Deletion of Yersinia pestis ail Causes Temperature-Sensitive Pleiotropic Effects, Including Cell Lysis, That Are Suppressed by Carbon Source, Cations, or Loss of Phospholipase A Activity.

Maintenance of phospholipid (PL) and lipopoly- or lipooligosaccharide (LPS or LOS) asymmetry in the outer membrane (OM) of Gram-negative bacteria is essential but poorly understood. The Yersinia pestis OM Ail protein was required to maintain lipid homeostasis and cell integrity at elevated temperature (37°C). Loss of this protein had pleiotropic effects. A Y. pestis Δail mutant and KIM6+ wild type were systematically compared for (i) growth requirements at 37°C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernatants, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild-type phenotype. The Δail mutant grew normally at 28°C but lysed at 37°C when it entered stationary phase, as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immunofluorescence microscopy showed that the Δail mutant did not assemble Caf1 capsule. Expression of heat shock promoter rpoE or rpoH fused to a lux operon reporter were not induced when the Δail mutant was shifted from 28°C to 37°C (P < 0.001 and P < 0.01, respectively). Mutant lysis was suppressed by addition of 11 mM glucose, 22 or 44 mM glycerol, 2.5 mM Ca2+, or 2.5 mM Mg2+ to the growth medium or by a mutation in the phospholipase A gene (pldA::miniTn5, ΔpldA, or PldAS164A). A model accounting for the temperature-sensitive lysis of the Δail mutant and the Ail-dependent stabilization of the OM tetraacylated LOS at 37°C is presented. IMPORTANCE The Gram-negative pathogen Yersinia pestis transitions between a flea vector (ambient temperature) and a mammalian host (37°C). In response to 37°C, Y. pestis modifies its outer membrane (OM) by reducing the fatty acid content in lipid A, changing the outer leaflet from being predominantly hexaacylated to being predominantly tetraacylated. It also increases the Ail concentration, so it becomes the most prominent OM protein. Both measures are needed for Y. pestis to evade the host innate immune response. Deletion of ail destabilizes the OM at 37°C, causing the cells to lyse. These results show that a protein is essential for maintaining lipid asymmetry and lipid homeostasis in the bacterial OM.

Repeated Oral Vaccination of Cattle with Shiga Toxin-Negative Escherichia coli O157:H7 Reduces Carriage of Wild-Type E. coli O157:H7 after Challenge.

Subcutaneous vaccination of cattle for enterohemorrhagic Escherichia coli O157:H7 reduces the magnitude and duration of fecal shedding, but the often-required, repeated cattle restraint can increase costs, deterring adoption by producers. In contrast, live oral vaccines may be repeatedly administered in feed, without animal restraint. We investigated whether oral immunization with live stx-negative LEE+E. coli O157:H7 reduced rectoanal junction (RAJ) colonization by wild-type (WT) E. coli O157:H7 strains after challenge. Two groups of cattle were orally dosed twice weekly for 6 weeks with 3 × 109 CFU of a pool of three stx-negative LEE+E. coli O157:H7 strains (vaccine group) or three stx-negative LEE- non-O157:H7 E. coli strains (control group). Three weeks following the final oral dose, animals in both groups were orally challenged with a cocktail of four stx+ LEE+E. coli O157:H7 WT strains. Subsequently, WT strains at the RAJ were enumerated weekly for 4 weeks. Serum antibodies against type III secretion protein (TTSP), the translocated intimin receptor (Tir), and EspA were determined by enzyme-linked immunosorbent assay (ELISA) at day 0 (preimmunization), day 61 (postimmunization, prechallenge), and day 89 (postchallenge). Vaccine group cattle had lower numbers of WT strains at the RAJ than control group cattle on postchallenge days 3 and 7 (P ≤ 0.05). Also, vaccine group cattle shed WT strains for a shorter duration than control group cattle. All cattle seroconverted to TTSP, Tir, and EspA, either following immunization (vaccine group) or following challenge (control group). Increased antibody titers against Tir and TTSP postimmunization were associated with decreased numbers of WT E. coli O157:H7 organisms at the RAJ.IMPORTANCE The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

Building a biomedical pipeline: the impact of the Idaho IDeA INBRE summer research experience at a primarily undergraduate institution.

Idaho Institutional Development Award (IDeA) Network for Biomedical Research Excellence (INBRE) aims to build biomedical research capacity and enhance the scientific and technology knowledge of the Idaho workforce. A key INBRE Program at The College of Idaho, a primarily undergraduate institution of 1,100 students, is a 10-wk summer fellows research experience. This report documents outcomes from 2005 to present, including demographic trends, faculty and student research productivity, self-reported gains, educational attainment, and career outcomes. Of 103 participants, 83.7% were from Idaho, 26.7% from rural areas, and 23.9% first-generation college students. Faculty and student research productivity (conference presentations and peer-reviewed publications) increased threefold. We found that 91.4% of fellows entered a scientific- or healthcare-related career and that 70.7% completed or are currently enrolled in postgraduate training (51.7% doctoral and 19.0% master's level). Anonymous surveys were uniformly positive, with gains in self-confidence and independent laboratory work. Open-ended responses indicated students valued mentoring efforts and improved awareness of scientific opportunities and competitive preparation for postgraduate training. Lastly, we observed that student research involvement increased college-wide during the award period. These data suggest that the summer fellows program is successfully meeting National Institutes of Health IDeA goals and serving as a pipeline to future health research careers and a scientifically trained Idaho workforce.

Rapid Evolution of Citrate Utilization by Escherichia coli by Direct Selection Requires citT and dctA.

The isolation of aerobic citrate-utilizing Escherichia coli (Cit(+)) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class of E. coli Cit(+) mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-type E. coli strain B and with K-12 and three K-12 derivatives: an E. coli ΔrpoS::kan mutant (impaired for stationary-phase survival), an E. coli ΔcitT::kan mutant (deleted for the anaerobic citrate/succinate antiporter), and an E. coli ΔdctA::kan mutant (deleted for the aerobic succinate transporter). E. coli underwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independent E. coli Cit(+) mutants were isolated from all E. coli derivatives except the E. coli ΔcitT::kan mutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry. E. coli Cit(+) mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of four E. coli Cit(+) mutants revealed the required sequence of mutational events leading to a refined Cit(+) mutant. These events showed amplified citT and dctA loci followed by DNA rearrangements consistent with promoter capture events for citT. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE.IMPORTANCE E. coli cannot use citrate aerobically. Long-term evolution experiments (LTEE) performed by Blount et al. (Z. D. Blount, J. E. Barrick, C. J. Davidson, and R. E. Lenski, Nature 489:513-518, 2012, http://dx.doi.org/10.1038/nature11514 ) found a single aerobic, citrate-utilizing E. coli strain after 33,000 generations (15 years). This was interpreted as a speciation event. Here we show why it probably was not a speciation event. Using similar media, 46 independent citrate-utilizing mutants were isolated in as few as 12 to 100 generations. Genomic DNA sequencing revealed an amplification of the citT and dctA loci and DNA rearrangements to capture a promoter to express CitT, aerobically. These are members of the same class of mutations identified by the LTEE. We conclude that the rarity of the LTEE mutant was an artifact of the experimental conditions and not a unique evolutionary event. No new genetic information (novel gene function) evolved.

Standardized Escherichia coli O157:H7 Exposure Studies in Cattle Provide Evidence that Bovine Factors Do Not Drive Increased Summertime Colonization.

The increased summertime prevalence of cattle carriage of enterohemorrhagic Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) is associated with the increased summertime incidence of human infection. The mechanism driving the seasonality of STEC O157 carriage among cattle is unknown. We conducted experimental challenge trials to distinguish whether factors extrinsic or intrinsic to cattle underlie the seasonality of STEC O157 colonization. Holstein steers (n = 20) exposed to ambient environmental conditions were challenged with a standardized pool of STEC O157 strains four times at 6-month intervals. The densities and durations of rectoanal junction mucosa (RAJ) colonization with STEC O157 were compared by season (winter versus summer), dose (10(9) CFU versus 10(7) CFU), and route of challenge (oral versus rectal). Following summer challenges, the RAJ STEC O157 colonization density was significantly lower (P = 0.016) and the duration was shorter (P = 0.052) than for winter challenges, a seasonal pattern opposite to that observed naturally. Colonization was unaffected by the challenge route, indicating that passage through the gastrointestinal microbiome did not significantly affect the infectious dose to the RAJ. A 2-log reduction of the challenge doses in the second-year trials was accompanied by similarly reduced RAJ colonization in both seasons (P < 0.001). These results refute the hypothesis that cattle are predisposed to STEC O157 colonization during the summer months, either due to intrinsic factors or indirectly due to gastrointestinal tract microbiome effects. Instead, the data support the hypothesis that the increased summertime STEC O157 colonization results from increased seasonal oral exposure to this pathogen.

The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a ß-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006). The E. coli O157 IP (O157-IP) comprised 91 proteins, and included those identified previously using proteomics-based expression library screening, and also proteins comprising DMEM and bovine rumen fluid proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured HEp-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine rectoanal junction squamous epithelial cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to rectoanal junction squamous epithelial cells, and additionally implicate a possible role for the outer membrane protein A regulator, TdcA, in the expression of such adhesins. Our observations have implications for the development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract.

SdiA aids enterohemorrhagic Escherichia coli carriage by cattle fed a forage or grain diet.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis and life-threatening complications. The main reservoirs for EHEC are healthy ruminants. We reported that SdiA senses acyl homoserine lactones (AHLs) in the bovine rumen to activate expression of the glutamate acid resistance (gad) genes priming EHEC's acid resistance before they pass into the acidic abomasum. Conversely, SdiA represses expression of the locus of enterocyte effacement (LEE) genes, whose expression is not required for bacterial survival in the rumen but is necessary for efficient colonization at the rectoanal junction (RAJ) mucosa. Our previous studies show that SdiA-dependent regulation was necessary for efficient EHEC colonization of cattle fed a grain diet. Here, we compared the SdiA role in EHEC colonization of cattle fed a forage hay diet. We detected AHLs in the rumen of cattle fed a hay diet, and these AHLs activated gad gene expression in an SdiA-dependent manner. The rumen fluid and fecal samples from hay-fed cattle were near neutrality, while the same digesta samples from grain-fed animals were acidic. Cattle fed either grain or hay and challenged with EHEC orally carried the bacteria similarly. EHEC was cleared from the rumen within days and from the RAJ mucosa after approximately one month. In competition trials, where animals were challenged with both wild-type and SdiA deletion mutant bacteria, diet did not affect the outcome that the wild-type strain was better able to persist and colonize. However, the wild-type strain had a greater advantage over the SdiA deletion mutant at the RAJ mucosa among cattle fed the grain diet.

The acyl-homoserine lactone synthase YenI from Yersinia enterocolitica modulates virulence gene expression in enterohemorrhagic Escherichia coli O157:H7.

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes the rectoanal junction (RAJ) in cattle, its natural reservoir. Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses acyl-homoserine lactones (AHLs) produced by the microbiota in the rumen to activate the gad acid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine if the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express the Yersinia enterocolitica AHL synthase gene yenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. The yenI(+) EHEC produces oxo-C6-homoserine lactone (oxo-C6-HSL) and had a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formation in vitro compared to the wild type (WT). The yenI(+) EHEC also activated expression of the gad genes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT or yenI(+) EHEC. Although the yenI(+) EHEC colonized the RAJ with efficiency equal to that of the WT, there was a trend for the cattle to shed the WT strain longer than the yenI(+) EHEC.

Physiological levels of glucose induce membrane vesicle secretion and affect the lipid and protein composition of Yersinia pestis cell surfaces.

Yersinia pestis grown with physiologic glucose increased cell autoaggregation and deposition of extracellular material, including membrane vesicles. Membranes were characterized, and glucose had significant effects on protein, lipid, and carbohydrate profiles. These effects were independent of temperature and the biofilm-related locus pgm and were not observed in Yersinia pseudotuberculosis.

Chemical sensing in mammalian host-bacterial commensal associations.

The mammalian gastrointestinal (GI) tract is colonized by a complex consortium of bacterial species. Bacteria engage in chemical signaling to coordinate population-wide behavior. However, it is unclear if chemical sensing plays a role in establishing mammalian host-bacterial commensal relationships. Enterohemorrhagic Escherichia coli (EHEC) is a deadly human pathogen but is a member of the GI flora in cattle, its main reservoir. EHEC harbors SdiA, a regulator that senses acyl-homoserine lactones (AHLs) produced by other bacteria. Here, we show that SdiA is necessary for EHEC colonization of cattle and that AHLs are prominent within the bovine rumen but absent in other areas of the GI tract. We also assessed the rumen metagenome of heifers, and we show that it is dominated by Clostridia and/or Bacilli but also harbors Bacteroidetes. Of note, some members of the Bacteroidetes phyla have been previously reported to produce AHLs. SdiA-AHL chemical signaling aids EHEC in gauging these GI environments, and promotes adaptation to a commensal lifestyle. We show that chemical sensing in the mammalian GI tract determines the niche specificity for colonization by a commensal bacterium of its natural animal reservoir. Chemical sensing may be a general mechanism used by commensal bacteria to sense and adapt to their mammalian hosts. Additionally, because EHEC is largely prevalent in cattle herds, interference with SdiA-mediated cattle colonization is an exciting alternative to diminish contamination of meat products and cross-contamination of produce crops because of cattle shedding of this human pathogen.

Adherence of non-O157 Shiga toxin-producing Escherichia coli to bovine recto-anal junction squamous epithelial cells appears to be mediated by mechanisms distinct from those used by O157.

This study presents evidence that the pattern (diffuse or aggregative) of adherence of clinically relevant non-O157 Shiga toxin-producing Escherichia coli (STEC) to bovine recto-anal junction squamous epithelial cells is similar to that of E. coli O157, although the mechanisms of adherence appear to be distinct. Our results further suggest that novel adhesins, and not Intimin, are likely involved in non-O157 STEC adherence to bovine recto-anal junction squamous epithelial cells. These findings have important implications for the development of efficacious modalities for blocking adherence of non-O157 STEC to bovine gastrointestinal epithelial cells.

Comparison of Yersinia enterocolitica DNA Methylation at Ambient and Host Temperatures.

Pathogenic bacteria recognize environmental cues to vary gene expression for host adaptation. Moving from ambient to host temperature, Yersinia enterocolitica responds by immediately repressing flagella synthesis and inducing the virulence plasmid (pYV)-encoded type III secretion system. In contrast, shifting from host to ambient temperature requires 2.5 generations to restore motility, suggesting a link to the cell cycle. We hypothesized that differential DNA methylation contributes to temperature-regulated gene expression. We tested this hypothesis by comparing single-molecule real-time (SMRT) sequencing of Y. enterocolitica DNA from cells growing exponentially at 22 °C and 37 °C. The inter-pulse duration ratio rather than the traditional QV scoring was the kinetic metric to compare DNA from cells grown at each temperature. All 565 YenI restriction sites were fully methylated at both temperatures. Among the 27,118 DNA adenine methylase (Dam) sites, 42 had differential methylation patterns, while 17 remained unmethylated regardless of the temperature. A subset of the differentially methylated Dam sites localized to promoter regions of predicted regulatory genes including LysR-type and PadR-like transcriptional regulators and a cyclic-di-GMP phosphodiesterase. The unmethylated Dam sites localized with a bias to the replication terminus, suggesting they were protected from Dam methylase. No cytosine methylation was detected at Dcm sites.

Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance.

Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.

Genomic surveillance identifies potential risk factors for SARS-CoV-2 transmission at a mid-sized university in a small rural town.

Understanding transmission dynamics of SARS-CoV-2 in institutions of higher education (IHEs) is important because these settings have potential for rapid viral spread. Here, we used genomic surveillance to retrospectively investigate transmission dynamics throughout the 2020-2021 academic year for the University of Idaho ("University"), a mid-sized IHE in a small rural town. We generated genome assemblies for 1168 SARS-CoV-2 samples collected during the academic year, representing 46.8% of positive samples collected from the University population and 49.8% of positive samples collected from the surrounding community ("Community") at the local hospital during this time. Transmission dynamics differed for the University when compared to the Community, with more infection waves that lasted shorter lengths of time, potentially resulting from high-transmission congregate settings along with mitigation efforts implemented by the University to combat outbreaks. We found evidence for low transmission rates between the University and Community, with approximately 8% of transmissions into the Community originating from the University, and approximately 6% of transmissions into the University originating from the Community. Potential transmission risk factors identified for the University included congregate settings such as sorority and fraternity events and residences, holiday travel, and high caseloads in the surrounding community. Knowledge of these risk factors can help the University and other IHEs develop effective mitigation measures for SARS-CoV-2 and similar pathogens.

Differential virulence of clinical and bovine-biased enterohemorrhagic Escherichia coli O157:H7 genotypes in piglet and Dutch belted rabbit models.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) is an important cause of food and waterborne illness in the developed countries. Cattle are a reservoir host of EHEC O157 and a major source of human exposure through contaminated meat products. Shiga toxins (Stxs) are an important pathogenicity trait of EHEC O157. The insertion sites of the Stx-encoding bacteriophages differentiate EHEC O157 isolates into genogroups commonly isolated from cattle but rarely from sick humans (bovine-biased genotypes [BBG]) and those commonly isolated from both cattle and human patients (clinical genotypes [CG]). Since BBG and CG share the cardinal virulence factors of EHEC O157 and are carried by cattle at similar prevalences, the infrequent occurrence of BBG among human disease isolates suggests that they may be less virulent than CG. We compared the virulence potentials of human and bovine isolates of CG and BBG in newborn conventional pig and weaned Dutch Belted rabbit models. CG-challenged piglets experienced severe disease accompanied by early and high mortality compared to BBG-challenged piglets. Similarly, CG-challenged rabbits were likely to develop lesions in kidney and intestine compared with the BBG-challenged rabbits. The CG strains used in this study carried stx2 and produced significantly higher amounts of Stx, whereas the BBG strains carried the stx2c gene variant only. These results suggest that BBG are less virulent than CG and that this difference in virulence potential is associated with the Stx2 subtype(s) carried and/or the amount of Stx produced.

Role of a new intimin/invasin-like protein in Yersinia pestis virulence.

A comprehensive TnphoA mutant library was constructed in Yersinia pestis KIM6 to identify surface proteins involved in Y. pestis host cell invasion and bacterial virulence. Insertion site analysis of the library repeatedly identified a 9,042-bp chromosomal gene (YPO3944), intimin/invasin-like protein (Ilp), similar to the Gram-negative intimin/invasin family of surface proteins. Deletion mutants of ilp were generated in Y. pestis strains KIM5(pCD1(+)) Pgm(-) (pigmentation negative)/, KIM6(pCD1(-)) Pgm(+), and CO92. Comparative analyses were done with the deletions and the parental wild type for bacterial adhesion to and internalization by HEp-2 cells in vitro, infectivity and maintenance in the flea vector, and lethality in murine models of systemic and pneumonic plague. Deletion of ilp had no effect on bacterial blockage of flea blood feeding or colonization. The Y. pestis KIM5 Δilp strain had reduced adhesion to and internalization by HEp-2 cells compared to the parental wild-type strain (P < 0.05). Following intravenous challenge with Y. pestis KIM5 Δilp, mice had a delayed time to death and reduced dissemination to the lungs, livers, and kidneys as monitored by in vivo imaging using a lux reporter system (in vivo imaging system [IVIS]) and bacterial counts. Intranasal challenge in mice with Y. pestis CO92 Δilp had a 55-fold increase in the 50% lethal dose ([LD(50)] 1.64 × 10(4) CFU) compared to the parental wild-type strain LD(50) (2.98 × 10(2) CFU). These findings identified Ilp as a novel virulence factor of Y. pestis.

Escherichia coli O157:H7: animal reservoir and sources of human infection.

This review surveys the literature on carriage and transmission of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the context of virulence factors and sampling/culture technique. EHEC of the O157:H7 serotype are worldwide zoonotic pathogens responsible for the majority of severe cases of human EHEC disease. EHEC O157:H7 strains are carried primarily by healthy cattle and other ruminants, but most of the bovine strains are not transmitted to people, and do not exhibit virulence factors associated with human disease. Prevalence of EHEC O157:H7 is probably underestimated. Carriage of EHEC O157:H7 by individual animals is typically short-lived, but pen and farm prevalence of specific isolates may extend for months or years and some carriers, designated as supershedders, may harbor high intestinal numbers of the pathogen for extended periods. The prevalence of EHEC O157:H7 in cattle peaks in the summer and is higher in postweaned calves and heifers than in younger and older animals. Virulent strains of EHEC O157:H7 are rarely harbored by pigs or chickens, but are found in turkeys. The bacteria rarely occur in wildlife with the exception of deer and are only sporadically carried by domestic animals and synanthropic rodents and birds. EHEC O157:H7 occur in amphibian, fish, and invertebrate carriers, and can colonize plant surfaces and tissues via attachment mechanisms different from those mediating intestinal attachment. Strains of EHEC O157:H7 exhibit high genetic variability but typically a small number of genetic types predominate in groups of cattle and a farm environment. Transmission to people occurs primarily via ingestion of inadequately processed contaminated food or water and less frequently through contact with manure, animals, or infected people.