Temporal dynamics of SARS-CoV-2 genome and detection of variants of concern in wastewater influent from two metropolitan areas in Arkansas.

Although SARS-CoV-2 can cause severe illness and death, a percentage of the infected population is asymptomatic. This, along with other factors, such as insufficient diagnostic testing and underreporting due to self-testing, contributes to the silent transmission of SARS-CoV-2 and highlights the importance of implementing additional surveillance tools. The fecal shedding of the virus from infected individuals enables its detection in community wastewater, and this has become a valuable public health tool worldwide as it allows the monitoring of the disease on a populational scale. Here, we monitored the presence of SARS-CoV-2 and its dynamic genomic changes in wastewater sampled from two metropolitan areas in Arkansas during major surges of COVID-19 cases and assessed how the viral titers in these samples related to the clinical case counts between late April 2020 and January 2022. The levels of SARS-CoV-2 RNA were quantified by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) using a set of TaqMan assays targeting three different viral genes (encoding ORF1ab polyprotein, surface glycoprotein, and nucleocapsid phosphoprotein). An allele-specific RT-qPCR approach was used to screen the samples for SARS-CoV-2 mutations. The identity and genetic diversity of the virus were further investigated through amplicon-based RNA sequencing, and SARS-CoV-2 variants of concern were detected in wastewater samples throughout the duration of this study. Our data show how changes in the virus genome can affect the sensitivity of specific RT-qPCR assays used in COVID-19 testing with the surge of new variants. A significant association was observed between viral titers in wastewater and recorded number of COVID-19 cases in the areas studied, except when assays failed to detect targets due to the presence of particular variants. These findings support the use of wastewater surveillance as a reliable complementary tool for monitoring SARS-CoV-2 and its genetic variants at the community level.

Genomics Analyses of GIV and GVI Noroviruses Reveal the Distinct Clustering of Human and Animal Viruses.

Noroviruses are highly diverse viruses that are the major viral cause of acute gastroenteritis in humans. Although these viruses can infect multiple mammalian species, their potential for zoonosis is not well understood, especially within Genogroup IV (GIV), which contains viruses that infect humans, canines, and felines. The study of GIV viruses has been, in part, hindered by the limited number of complete genomes. Here, we developed a full-genome amplicon-based platform that facilitated the sequencing of canine noroviruses circulating in the United States. Eight novel nearly full-length canine norovirus genomes and two nearly complete VP1 sequences, including four GIV.2, three GVI.1, and three GVI.2 viruses, were successfully obtained. Only animal strains exhibited GVI/GIV chimeric viruses, demonstrating restrictions in norovirus recombination. Using genomic, phylogenetic, and structural analyses, we show that differences within the major capsid protein and the non-structural proteins of GIV and GVI noroviruses could potentially limit cross-species transmission between humans, canines, and felines.

Silicon Dioxide Impedes Antiviral Response and Causes Genotoxic Insult During Calicivirus Replication.

Noroviruses (NoV) are the leading cause of nonbacterial gastroenteritis in humans, and replicate extensively in the human gastrointestinal (GI) tract. Silica (also known as silicon dioxide, SiO2) nanoparticles (NPs) used in processed foods, dairy products, and beverages also accumulate in the GI tract. We investigated the effect of silica NPs on NoV replication and host cell response during virus infection, using murine norovirus (MNV-1) infection of RAW 264.7 murine macrophages. Pretreatment with 10 µg/ml silica significantly reduced the viability of macrophages, but no cumulative effects on viability of macrophages were observed with MNV-1 infection. No difference was observed between exposure to control or silica NPs on either the quantity of viral genome copies or the production of infectious virus in macrophages infected with MNV-1. Silica NPs reduced the ability of macrophages to upregulate genes encoding bone morphogenic proteins (BMPs), chemokine ligands and cytokines for which expression levels were otherwise found to be upregulated in response to MNV-1 infection. Furthermore, silica NPs reduced the levels of proinflammatory cytokines secreted by macrophages in response to MNV infection. Finally, silica NPs with MNV-1 infection produced a genotoxic insult to macrophages. Strikingly, this genotoxic insult was also found to occur as a synergistic effect of silica NPs and feline calicivirus infection in feline kidney epithelial cells. Taken together, our study suggests important safety considerations related to reducing exposure to silica NPs affecting the GI tract in individuals infected with NoVs and possibly other foodborne viruses.

Titanium Dioxide Nanoparticles Evoke Proinflammatory Response during Murine Norovirus Infection Despite Having Minimal Effects on Virus Replication.

Noroviruses (NoV) have enhanced tropism for the gastrointestinal (GI) tract and are the major cause of nonbacterial gastroenteritis in humans. Titanium dioxide (TiO2) nanoparticles (NPs) used as food additives, dietary supplements, and cosmetics accumulate in the GI tract. We investigated the effect anatase TiO2 NPs on NoV replication and host response during virus infection, using murine norovirus (MNV-1) infection of RAW 264.7 macrophages. Pretreatment with 20 µg/ml anatase NPs significantly reduced the viability of macrophages alone or during virus infection, but did not alter virus replication. In contrast, pre-incubation with 2 µg/ml anatase NPs reduced virus replication fivefold at 48 h. The presence of anatase NPs during MNV-1 infection evoked a pro-inflammatory response, as measured by a significant increase in expression of cytokines, including IL-6, IFN-γ, TNFα and the TGFß1. No genotoxic insults due to anatase TiO2 NPs alone or to their presence during MNV-1 infection were detected. This study highlights important safety considerations related to NP exposure of the GI tract in individuals infected with noroviruses or other foodborne viruses.

Infection of Murine Macrophages by Salmonella enterica Serovar Heidelberg Blocks Murine Norovirus Infectivity and Virus-induced Apoptosis.

Gastroenteritis caused by bacterial and viral pathogens constitutes a major public health threat in the United States accounting for 35% of hospitalizations. In particular, Salmonella enterica and noroviruses cause the majority of gastroenteritis infections, with emergence of sporadic outbreaks and incidence of increased infections. Although mechanisms underlying infections by these pathogens have been individually studied, little is known about the mechanisms regulating co-infection by these pathogens. In this study, we utilized RAW 264.7 murine macrophage cells to investigate the mechanisms governing co-infection with S. enterica serovar Heidelberg and murine norovirus (MNV). We demonstrate that infection of RAW 264.7 cells with S. enterica reduces the replication of MNV, in part by blocking virus entry early in the virus life cycle, and inducing antiviral cytokines later in the infection cycle. In particular, bacterial infection prior to, or during MNV infection affected virus entry, whereas MNV entry remained unaltered when the virus infection preceded bacterial invasion. This block in virus entry resulted in reduced virus replication, with the highest impact on replication observed during conditions of co-infection. In contrast, bacterial replication showed a threefold increase in MNV-infected cells, despite the presence of antibiotic in the medium. Most importantly, we present evidence that the infection of MNV-infected macrophages by S. enterica blocked MNV-induced apoptosis, despite allowing efficient virus replication. This apoptosis blockade was evidenced by reduction in DNA fragmentation and absence of poly-ADP ribose polymerase (PARP), caspase 3 and caspase 9 cleavage events. Our study suggests a novel mechanism of pathogenesis whereby initial co-infection with these pathogens could result in prolonged infection by either of these pathogens or both together.

Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) production and Lactobacillus species growth in a defined medium simulating vaginal secretions.

Lactobacillus species are commensal with the healthy vaginal environment and inhibit the growth of many pathogenic bacteria in the vaginal tract by a variety of mechanisms, such as the production of hydrogen peroxide, organic acids, and antimicrobial substances. Simulation of the vaginal environment is crucial for proper investigation of the effects of Lactobacillus species on pathogenic bacteria. In this study, we modified a medium used to simulate vaginal secretions to improve the growth of toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus clinical strains and Lactobacillus species so that interactions between these bacteria may be examined. A medium consisting of basal salts, vitamins, albumin, glycogen, mucin, urea, sodium bicarbonate, polyoxyethylene sorbitan monolaurate, and amino acids supported the growth of S. aureus and the production of TSST-1 as determined by Western analysis. Improved growth of the Lactobacillus species was seen when this same medium was supplemented with manganese chloride, sodium acetate, and an increase in glucose concentration. However, growth of S. aureus in the supplemented medium resulted in reduced levels of TSST-1. Production of TSST-1 was not detected in a medium routinely used for the growth of Lactobacillus species although S. aureus growth was not inhibited. The development of an improved genital tract secretion medium provides a more authentic environment in which to study the interactions of Lactobacillus species and vaginal pathogens, such as S. aureus.

A metallo-ß-lactamase is responsible for the degradation of ceftiofur by the bovine intestinal bacterium Bacillus cereus P41.

Ceftiofur is a highly effective veterinary cephalosporin, yet it is rapidly degraded by bacteria in the gut. The goal of this work was to directly determine the mechanism of ceftiofur degradation by the bovine intestinal isolate Bacillus cereus P41. B. cereus P41 was isolated from the feces of a cow that had not been treated with cephalosporins, and was found to rapidly degrade ceftiofur in culture. Analysis of spent culture media by HPLC/UV and HPLC/MS revealed one major metabolite of ceftiofur, with a negative ion m/z of 127. Comparison of ceftiofur, ceftriaxone, and cefpodoxime degradation suggested that the major stable ceftiofur metabolite was the thiofuroic acid group eliminated from the C-3 position of the drug after hydrolysis by ß-lactamase. Genomic DNA from B. cereus P41 was cloned into Escherichia coli, and the transformants were screened for growth in the presence of ceftiofur. DNA sequencing of the plasmid pHSG299-BC-3 insert revealed the presence of a gene encoding a metallo-ß-lactamase. Incubation of ceftiofur with either the E. coli transformant or a commercial B. cereus metallo-ß-lactamase showed degradation of the drug and formation of the same major metabolite produced by B. cereus P41. These data demonstrate that a metallo-ß-lactamase plays a major role in the degradation of ceftiofur by the bovine intestinal bacterium B. cereus P41.

Human Respiratory Coronaviruses Detected In Patients with Influenza-Like Illness in Arkansas, USA.

Acute respiratory viruses often result in significant morbidity and mortality. The potential impact of human respiratory coronavirus (CoV) infections was underestimated until the severe acute respiratory syndrome (SARS-CoV) outbreak in 2003, which showed that new, highly pathogenic coronaviruses could be introduced to humans, highlighting the importance of monitoring the circulating coronaviruses. The use of sensitive molecular methods has contributed to the differential diagnosis of viruses circulating in humans. Our study aim was to investigate the molecular epidemiology of human CoV strains circulating in Arkansas, their genetic variability and their association with reported influenza-like symptoms. We analyzed 200 nasal swab samples, collected by the Arkansas Department of Health in 2010, for influenza diagnosis. All samples were from patients showing acute respiratory symptoms while testing negative for influenza. Samples were pre-screened, using a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) multiprobe for coronavirus, and subjected to confirmatory pancoronavirus and/or strain-specific reverse transcriptase (RT)-PCR followed by sequence analysis. Seventy-nine samples (39.5%) were positive by qRT-PCR and 35 samples (17.5%) were confirmed by conventional RT-PCR. Twenty-three of the confirmed samples (59%) were sequenced. The most frequent strain detected was HCoV-OC43-like followed by NL63-like; only one sample was positive for HCoV-229E and one for HCoV-HKU1. Feline-like CoV strains were detected in three samples, representing possible evidence of interspecies transmission or a new human strain. Seventeen percent of the coronavirus positive samples were also positive for other respiratory viruses, such as Respiratory Syncytial Virus (RSV), Parainfluenza 2 and 3, and Rhinovirus. Thus, HCoV-OC43, NL63, HKU1 and new feline-like strains were circulating in Arkansas in 2010. HCoV was the sole respiratory virus detected in 16% of the patients who showed acute respiratory symptoms with negative diagnoses for influenza virus.

Stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions.

Fecal suspensions with an aerosol route of transmission were responsible for a cluster of severe acute respiratory syndrome (SARS) cases in 2003 in Hong Kong. Based on that event, the World Health Organization recommended that research be implemented to define modes of transmission of SARS coronavirus through sewage, feces, food and water. Environmental studies have shown that animal coronaviruses remain infectious in water and sewage for up to a year depending on the temperature and humidity. In this study, we examined coronavirus stability on lettuce surfaces. A cell culture adapted bovine coronavirus, diluted in growth media or in bovine fecal suspensions to simulate fecal contamination was used to spike romaine lettuce. qRT-PCR detected viral RNA copy number ranging from 6.6 × 104 to 1.7 × 106 throughout the experimental period of 30 days. Whereas infectious viruses were detected for at least 14 days, the amount of infectious virus varied, depending upon the diluent used for spiking the lettuce. UV and confocal microscopic observation indicated attachment of residual labeled virions to the lettuce surface after the elution procedure, suggesting that rates of inactivation or detection of the virus may be underestimated. Thus, it is possible that contaminated vegetables may be potential vehicles for coronavirus zoonotic transmission to humans.

Single nucleotide polymorphism analysis of the major tripartite multidrug efflux pump of Escherichia coli: functional conservation in disparate animal reservoirs despite exposure to antimicrobial chemotherapy.

AcrAB-TolC imparts a strong intrinsic resistance phenotype to many clinically significant molecules in Escherichia coli. This complex is composed of a pump, AcrB, and a periplasmic protein, AcrA, that exports substrates through a common outer membrane porin, TolC. A sequence survey of the pump-specific components, acrA and acrB, was conducted on three discrete animal reservoirs: rodents, bovines, and catfish. Although two of the reservoirs (bovine and catfish) were agrarian, and antibiotic use (ceftiofur and oxytetracycline/Romet 30, respectively) was reported for them, the vast majority of structural polymorphisms were silent except for T104A (AcrA) and Q733R (AcrB), found in certain bovine-derived strains. Overall, the genes were well conserved, with high ratios of synonymous to nonsynonymous substitutions (d(S)/d(N) ratios), consistent with or, in the case of acrB, better than those of standard multilocus sequence typing (MLST) loci. Furthermore, predicted recombination points from single nucleotide polymorphism (SNP) patterns in acrB support a modular evolution of transporter proteins, consistent with an ancient origin. However, functional studies with clones representing the major silent SNPs and the nonsilent mutation in acrB failed to generate significant differences in resistance to a range of common efflux pump substrates. Interestingly, a comparison between log-phase acrA and acrB expression profiles yielded inconsistent trends, with acrB expression increasing modestly (<1.8-fold) in many strains from the antibiotic-enriched pools. Our results suggest that structural polymorphisms in this major efflux pump system may not contribute significantly to adaptive resistance by altering function or substrate specificity but may have a potential use in improving phylogenetic relationships and/or source tracking.

Interaction of dietary resveratrol with animal-associated bacteria.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene), an antifungal phytoalexin produced by grapes, peanuts, and Japanese knotweeds, is thought to be a beneficial dietary phytochemical in red wine and grape juice. Information on its antibacterial properties and biotransformation, however, is limited. We surveyed the interactions of resveratrol with 43 strains of bacterial species that are often animal- or human-associated. Resveratrol at 50 mg L(-1) reduced the growth rates of most of the bacteria tested, but did not totally prevent growth even at much higher levels. Eleven of the 43 bacteria were capable of transforming at least 20% of the resveratrol. Three major metabolites were identified as resveratroloside, piceid, and dihydroresveratrol, and three other metabolites were partially characterized.

Lactobacillus-mediated inhibition of clinical toxic shock syndrome Staphylococcus aureus strains and its relation to acid and peroxide production.

The inhibitory activities of 39 strains representing 20 different species of Lactobacillus toward a menstrual toxic shock syndrome (TSS) Staphylococcus aureus archetype strain MN8 were investigated. Nearly every strain (38 of 39) produced an inhibitory effect under both aerobic and anaerobic conditions when assayed on agar medium. In addition, the MN8 inhibition was conserved against at least 10 other clinical TSS S. aureus isolates and, interestingly, required actively growing cultures of Lactobacillus (verified with a two-well co-culture system in broth medium). This general uniform inhibition could be ameliorated by organic buffer (PIPES) supplied in the growth medium and, with only one exception, MRS medium adjusted with non-organic acid (HCl) failed to support growth of TSS strains at or below pH 5.5. By comparison, the vast majority of lactobacilli in this study decreased culture pH to a range of 4-5. Hydrogen peroxide production by the lactobacilli was also assessed and verified by two different methodologies revealing a broad spectrum of phenotypes that, contrary to reports touting its effectiveness, did not seem to correspond with our inhibition studies. Furthermore, resistances to peroxide by MN8, other TSS strains, and a subset of lactobacilli used in this study were nearly identical whereas the S. aureus collection was slightly more sensitive to racemic lactic acid than the lactobacilli. Collectively, these data suggest that the underlying inhibition toward Staphylococcus is generally conserved in Lactobacillus sp. and is related to a common factor in this genus involving promotion of acidic conditions.

Substrate competition studies using whole-cell accumulation assays with the major tripartite multidrug efflux pumps of Escherichia coli.

AcrAB-TolC is the major, constitutively expressed tripartite multidrug efflux system in Escherichia coli that recognizes various structurally unrelated molecules, including many antibiotics, dyes, and steroids. The AcrB inner membrane pump portion of the efflux system has been shown in recent structural studies to bind substrates at multiple sites, suggesting that particular substrate "sets" may compete for efflux by interfering with a certain binding site(s). However, our data indicate that the general structural class does not appear to dictate a particular substrate binding site that can be competitively inhibited in whole cells. In our study, substrate competition failed to increase cell-associated levels of steroids or dyes to levels characteristic of AcrB- or AcrB/EmrAB-deficient genomic mutants or achieved with the pump inhibitor carbonyl cyanide m-chlorophenylhydrazone. In addition, this general observation was sustained even with (i) a cocktail containing seven-pump substrates supplied slightly below their respective wild-type MIC levels, (ii) competing drug substrates of the same structural class (steroids or macrolides), and (iii) hyper-MIC levels of the exogenously supplied agents. Thus, this pump system (and possibly EmrAB-TolC) may have an extraordinary capacity to simultaneously handle multiple-drug substrates that is not necessarily reflected in MIC analyses. In addition, our study has extended the range of substrates recognized by the AcrAB- and EmrAB-TolC systems.

Mammalian steroid hormones are substrates for the major RND- and MFS-type tripartite multidrug efflux pumps of Escherichia coli.

A steroid-hormone-dependent growth suppression was observed in Escherichia coli efflux-deficient backgrounds containing mutations in the major RND- and MFS-type tripartite multidrug efflux systems, AcrAB-TolC and EmrAB-TolC, respectively. In addition to their previously known natural steroid spectrum, which includes bile acids, both systems were shown to transport the hormones estradiol and progesterone, whereas hydrocortisone served as a substrate of only AcrAB-TolC. Furthermore, at least two other RND-type pumps, YhiV and AcrD, were capable of transporting such hormones when overexpressed on plasmid vectors (with some demonstrable specificity observed with AcrD). When this activity was examined in a wild-type background, cell-associated estradiol levels remained largely unaffected by competition with exogenous bile acids and hydrocortisone, in contrast to progesterone, which produced a significant modulation in estradiol uptake.

Bile-mediated aminoglycoside sensitivity in Lactobacillus species likely results from increased membrane permeability attributable to cholic acid.

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus plantarum WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 mug/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-3H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide m-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since L. plantarum, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.

Viral gene expression during acute simian varicella virus infection.

Simian varicella virus (SVV) causes a natural varicella-like disease in nonhuman primates. Outbreaks of simian varicella occur sporadically in primate facilities. Simian varicella is used as a model for investigation of varicella-zoster virus (VZV) pathogenesis and latency. In this study, SVV gene expression and histopathology were analysed in tissues of acutely infected vervet monkeys. RT-PCR analysis demonstrated expression of specific SVV immediate early, early and late genes in the skin, lung, liver and ganglia tissues of acutely infected monkeys. Viral antigen expression and histopathology, including necrosis and inflammation, were detected in the skin, lungs, liver and spleen of infected monkeys by immunohistochemical analysis. Viral antigen expression, but little or no histopathology, was evident in the neural ganglia, the eventual site of viral latency. The study provides a foundation for further investigation on the role of viral genes in varicella pathogenesis and latency.