The Long-Term Persistence of Borrelia burgdorferi Antigens and DNA in the Tissues of a Patient with Lyme Disease.

Whether Borrelia burgdorferi, the causative agent of Lyme disease, can persist for long periods in the human body has been a controversial question. The objective of this study was to see if we could find B. burgdorferi in a Lyme disease patient after a long clinical course and after long-term antibiotic treatment. Therefore, we investigated the potential presence of B. burgdorferi antigens and DNA in human autopsy tissues from a well-documented serum-, PCR-, and culture-positive Lyme disease patient, a 53-year-old female from northern Westchester County in the lower Hudson Valley Region of New York State, who had received extensive antibiotic treatments during extensive antibiotic treatments over the course of her 16-year-long illness. We also asked what form the organism might take, with special interest in the recently found antibiotic-resistant aggregate form, biofilm. We also examined the host tissues for the presence of inflammatory markers such as CD3+ T lymphocytes. Autopsy tissue sections of the brain, heart, kidney, and liver were analyzed by histological and immunohistochemical methods (IHC), confocal microscopy, fluorescent in situ hybridization (FISH), polymerase chain reaction (PCR), and whole-genome sequencing (WGS)/metagenomics. We found significant pathological changes, including borrelial spirochetal clusters, in all of the organs using IHC combined with confocal microscopy. The aggregates contained a well-established biofilm marker, alginate, on their surfaces, suggesting they are true biofilm. We found B. burgdorferi DNA by FISH, polymerase chain reaction (PCR), and an independent verification by WGS/metagenomics, which resulted in the detection of B. burgdorferi sensu stricto specific DNA sequences. IHC analyses showed significant numbers of infiltrating CD3+ T lymphocytes present next to B. burgdorferi biofilms. In summary, we provide several lines of evidence that suggest that B. burgdorferi can persist in the human body, not only in the spirochetal but also in the antibiotic-resistant biofilm form, even after long-term antibiotic treatment. The presence of infiltrating lymphocytes in the vicinity of B. burgdorferi biofilms suggests that the organism in biofilm form might trigger chronic inflammation.

Genomic Comparison Reveals Natural Occurrence of Clinically Relevant Multidrug-Resistant Extended-Spectrum-ß-Lactamase-Producing Escherichia coli Strains.

The effectiveness of antibiotics has been challenged by the increasing frequency of antimicrobial resistance (AMR), which has emerged as a major threat to global health. Despite its negative impact on the development of AMR, there are few effective strategies for reducing AMR in food-producing animals. Using whole-genome sequencing and comparative genomics of 36 multidrug-resistant (MDR) Escherichia coli strains isolated from beef cattle with no previous exposure to antibiotics, we obtained results suggesting that the occurrence of MDR E. coli also arises in animals with no antibiotic selective pressure. Extended-spectrum-ß-lactamase-producing E. coli strains with enhanced virulence capacities for toxin production and adherence have evolved, which implies important ramifications for animal and human health. Gene exchanges by conjugative plasmids and insertion elements have driven widespread antibiotic resistance in clinically relevant pathogens. Phylogenetic relatedness of E. coli strains from various geographic locations and hosts, such as animals, environmental sources, and humans, suggests that transmission of MDR E. coli strains occurs intercontinentally without host barriers.IMPORTANCE Multidrug-resistant (MDR) Escherichia coli isolates pose global threats to public health due to the decreasing availability of treatment options. To better understand the characteristics of MDR E. coli isolated from food-producing animals with no antibiotic exposure, we employed genomic comparison, high-resolution phylogenetics, and functional characterization. Our findings highlight the potential capacity of MDR E. coli to cause severe disease and suggest that these strains are widespread intercontinentally. This study underlines the occurrence of MDR E. coli in food-producing animals raised without antibiotic use, which has alarming, critical ramifications within animal and human medical practice.

High Prevalence of Cefotaxime Resistant Bacteria in Grazing Beef Cattle: A Cross Sectional Study.

Although the over-use of antibiotics during food animal production is a potential driver of antimicrobial resistant microorganisms (ARMs), a high prevalence of cefotaxime resistant bacteria (CRB) has been observed in grazing animals raised without antibiotic supplementation. In this cross-sectional study, the prevalence and concentration of CRB in beef cattle on grazing farms were investigated. Fecal samples from the recto-anal junction of cattle (n = 840) and environmental samples (n = 258) were collected from 17 farms in North and Central Florida in the United States, and a survey of farm characteristics, animal husbandry practices, and antibiotic usage was conducted. CRB were detected in fecal samples from 47.4% of all cattle, with the prevalence ranging from 21.1 to 87.5% on farms, and significantly higher (P < 0.001) in calves compared to adult cows (54.1 vs. 41.8%). Environmental samples had a higher prevalence than fecal samples (P < 0.001), with CRB detected in 88.6% of water, 98.7% of soil, and 95.7% of forage samples. Compared to the concentration (log CFU/g) of CRB in fecal samples (2.95, 95% CI: 2.89, 3.02), the concentration of CRB was higher (P < 0.001) in soil and forage samples (5.37, 95% CI: 5.16, 5.57) and lower (P < 0.001) in water samples (1.08, 95% CI: 0.82, 1.36). Soil microbiota from farms with high prevalence of CRB clustered closer together and the proportion of Phylum Proteobacteria was higher on farms with high prevalence of CRB resistance. Large farming operations were associated with a 58% higher likelihood of CRB detection in fecal samples. Regular cleaning of drinking troughs and the addition of ionophores to feed were associated with CRB reduction in fecal samples. Taken together, the widespread of CRB into both cattle seldom treated with cephalosporin antibiotics and the surrounding environment suggests the environment is a natural source of antimicrobial resistance in beef cattle.

An evaluation of the virulence and adherence properties of avian pathogenic Escherichia coli.

Avian pathogenic E. coli (APEC) cause disease primarly in poultry; however, the link between APEC and infections in humans is questionable. In this current study, a total of 100 APEC strains isolated from chickens in Delmarva were evaluated for the presence of virulence genes to investigate their zoonotic potential in humans. A total of 28 isolates possessed one Enterohaemorrhagic E. coli (EHEC) virulence factor each and 87 isolates possessed up to 5 extraintestinal pathogenic E. coli (ExPEC) virulence factors. Five APEC isolates exhibited stronger attachment to chicken breast than both human E. coli outbreak strains tested. Ten APEC isolates exhibited stronger attachment to human epithelial cells (HCT-8) than both E. coli outbreak strains. While the APEC isolates in this study were not found to possess all the virulence genes necessary to cause clinical illness in humans, their potential to acquire these genes in the environment as well as their ability to attach to food surfaces and human cells warrants further attention.

Human Norovirus and Its Surrogates Induce Plant Immune Response in Arabidopsis thaliana and Lactuca sativa.

Human norovirus is the leading cause of foodborne illness worldwide with the majority of outbreaks linked to fresh produce and leafy greens. It is essential that we thoroughly understand the type of relationship and interactions that take place between plants and human norovirus to better utilize control strategies to reduce transmission of norovirus in the field onto plants harvested for human consumption. In this study the expression of gene markers for the salicylic acid (SA) and jasmonic acid (JA) plant defense pathways was measured and compared in romaine lettuce (Lactuca sativa) and Arabidopsis thaliana Col-0 plants that were inoculated with Murine Norovirus-1, Tulane Virus, human norovirus GII.4, or Hank's Balanced Salt Solution (control). Genes involving both the SA and JA pathways were expressed in both romaine lettuce and A. thaliana for all three viruses, as well as controls. Studies, including gene expression of SA- and JA-deficient A. thaliana mutant lines, suggest that the JA pathway is more likely involved in the plant immune response to human norovirus. This research provides the first pieces of information regarding how foodborne viruses interact with plants in the preharvest environment.

Water for Agriculture: the Convergence of Sustainability and Safety.

Agricultural water is a precious and limited resource. Increasingly more water types and sources are being explored for use in irrigation within the United States and across the globe. As outlined in this chapter, the Produce Safety Rule (PSR) in the Food Safety and Modernization Act (FSMA) provide irrigation water standards for application of water to fruits and vegetables consumed raw. These rules for production and use of water will continue to develop and be required as the world experiences aspects of a changing climate including flooding as well as drought conditions. Research continues to assess the use of agricultural water types. The increased use of reclaimed water in the United States as well as for selected irrigation water needs for specific crops may provide increased water availability. The use of surface water can be used in irrigation as well, but several studies have shown the presence of some enteric bacterial pathogens (enterohemorrhagic E. coli, Salmonella spp. and Listeria monocytogenes) in these waters that may contaminate fruits and vegetables. There have been outbreaks of foodborne illness in the U.S., South America, Europe, and Australia related to the use of contaminated water in fruit and vegetable irrigation or washing. Unreliable water supplies, more stringent microbial water standards, mitigation technologies and expanded uses of reclaimed waters have all increased interest in agricultural water.

Application of Bacillus subtilis to the roots of leafy greens, in the presence of Listeria innocua and Salmonella Newport, induces closure of stomata.

Plant growth-promoting rhizobacterium Bacillus subtilis UD1022 has been shown to trigger an induced systemic response in Arabidopsis thaliana. This interaction causes plant stomata to close, protecting the plant from infection by plant pathogens and thereby increasing crop yield. The purpose of this study was to determine whether UD1022 applied to the roots of plants is able to induce stomata closure in leafy greens as well as influence the persistence of human pathogens (Listeria and Salmonella) on plants. UD1022 induced stomata closure in the presence of human pathogens on both lettuce and spinach 3 h post-inoculation (p<0.0001). Results were confirmed by root inoculation with heat-killed UD1022, which did not induce stomata closure. Presence of UD1022 on lettuce roots significantly reduced the persistence of Listeria on plants after 3 days post-inoculation (p=0.02) but had less of an effect on the persistence of Salmonella. The results of this study indicate that plant growth-promoting rhizobacterium B. subtilis UD1022 may be able to prevent contamination by some human pathogens. This is the first study to investigate the use of plant growth-promoting rhizobacteria to control the persistence of human pathogens on plants.

Inactivation of Human Norovirus and Its Surrogates on Alfalfa Seeds by Aqueous Ozone.

Alfalfa sprouts have been associated with numerous foodborne outbreaks. Previous studies investigated the effectiveness of aqueous ozone on bacterially contaminated seeds, yet little is known about the response of human norovirus (huNoV). This study assessed aqueous ozone for the disinfection of alfalfa seeds contaminated with huNoV and its surrogates. The inactivation of viruses without a food matrix was also investigated. Alfalfa seeds were inoculated with huNoV genogroup II, Tulane virus (TV), and murine norovirus (MNV); viruses alone or inoculated on seeds were treated in deionized water containing 6.25 ppm of aqueous ozone with agitation at 22°C for 0.5, 1, 5, 15, or 30 min. The data showed that aqueous ozone resulted in reductions of MNV and TV infectivity from 1.66 ± 1.11 to 5.60 ± 1.11 log PFU/g seeds; for all treatment times, significantly higher reductions were observed for MNV (P < 0.05). Viral genomes were relatively resistant, with a reduction of 1.50 ± 0.14 to 3.00 ± 0.14 log genomic copies/g seeds; the reduction of TV inoculated in water was similar to that of huNoV, whereas MNV had significantly greater reductions in genomic copies (P < 0.05). Similar trends were observed in ozone-treated viruses alone, with significantly higher levels of inactivation (P < 0.05), especially with reduced levels of infectivity for MNV and TV. The significant inactivation by aqueous ozone indicates that ozone may be a plausible substitute for chlorine as an alternative treatment for seeds. The behavior of TV was similar to that of huNoV, which makes it a promising surrogate for these types of scenarios.

Efficient reduction of pathogenic and spoilage microorganisms from apple cider by combining microfiltration with UV treatment.

Thermal pasteurization can achieve the U. S. Food and Drug Administration-required 5-log reduction of pathogenic Escherichia coli O157:H7 and Cryptosporidium parvum in apple juice and cider, but it can also negatively affect the nutritional and organoleptic properties of the treated products. In addition, thermal pasteurization is only marginally effective against the acidophilic, thermophilic, and spore-forming bacteria Alicyclobacillus spp., which is known to cause off-flavors in juice products. In this study, the efficiency of a combined microfiltration (MF) and UV process as a nonthermal treatment for the reduction of pathogenic and nonpathogenic E. coli, C. parvum, and Alicyclobacillus acidoterrestris from apple cider was investigated. MF was used to physically remove suspended solids and microorganisms from apple cider, thus enhancing the effectiveness of UV and allowing a lower UV dose to be used. MF, with ceramic membranes (pore sizes, 0.8 and 1.4 µm), was performed at a temperature of 10 °C and a transmembrane pressure of 155 kPa. The subsequent UV treatment was conducted using at a low UV dose of 1.75 mJ/cm(2). The combined MF and UV achieved more than a 5-log reduction of E. coli, C. parvum, and A. acidoterrestris. MF with the 0.8-µm pore size performed better than the 1.4-µm pore size on removal of E. coli and A. acidoterrestris. The developed nonthermal hurdle treatment has the potential to significantly reduce pathogens, as well as spores, yeasts, molds, and protozoa in apple cider, and thus help juice processors improve the safety and quality of their products.

Survival of murine norovirus, Tulane virus, and hepatitis A virus on alfalfa seeds and sprouts during storage and germination.

Human norovirus (huNoV) and hepatitis A virus (HAV) have been involved in several produce-associated outbreaks and identified as major food-borne viral etiologies. In this study, the survival of huNoV surrogates (murine norovirus [MNV] and Tulane virus [TV]) and HAV was investigated on alfalfa seeds during storage and postgermination. Alfalfa seeds were inoculated with MNV, TV, or HAV with titers of 6.46 ± 0.06 log PFU/g, 3.87 ± 0.38 log PFU/g, or 7.01 ± 0.07 log 50% tissue culture infectious doses (TCID50)/g, respectively. Inoculated seeds were stored for up to 50 days at 22°C and sampled during that storage period on days 0, 2, 5, 10, and 15. Following storage, virus presence was monitored over a 1-week germination period. Viruses remained infectious after 50 days, with titers of 1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID50/g for MNV, TV, and HAV, respectively. HAV demonstrated greater persistence than MNV and TV, without a statistically significant reduction over 20 days (<1 log TCID50/g); however, relatively high levels of genomic copies of all viruses persisted over the testing time period. Low titers of viruses were found on sprouts and were located in all tissues as well as in sprout-spent water sampled on days 1, 3, and 6 following seed planting. Results revealed the persistence of viruses in seeds for a prolonged period of time, and perhaps of greater importance these data suggest the ease of which virus may transfer from seeds to sprouts and spent water during germination. These findings highlight the importance of sanitation and prevention procedures before and during germination.

Pathogenic psychrotolerant sporeformers: an emerging challenge for low-temperature storage of minimally processed foods.

Sporeforming bacteria are a significant problem in the food industry as they are ubiquitous in nature and capable of resisting inactivation by heat and chemical treatments designed to inactivate them. Beyond spoilage issues, psychrotolerant sporeformers are becoming increasingly recognized as a potential hazard given the ever-expanding demand for refrigerated processed foods with extended shelf-life. In these products, the sporeforming pathogens of concern are Bacillus cereus, Bacillus weihenstephanensis, and Clostridium botulinum type E. This review article examines the foods, conditions, and organisms responsible for the food safety issue caused by the germination and outgrowth of psychrotolerant sporeforming pathogens in minimally processed refrigerated foods.