Survival of Salmonella and Shiga Toxin-Producing Escherichia coli and Changes in Indigenous Microbiota during Fermentation of Home-Brewed Kombucha.

ABSTRACT: Survival and growth of Salmonella and Shiga toxin-producing Escherichia coli (STEC) were investigated in kombucha prepared from four brands of commercially available kombucha kits intended for use by home brewers. Changes in populations of the indigenous microbiota responsible for fermentation of kombucha were also determined. An initial population of Salmonella (6.77 log CFU/mL) decreased to below the detection limit (0.30 log CFU/mL) within 10 days in kombucha prepared from two of the test brands. Populations of 1.85 and 1.20 log CFU/mL were detected in two brands fermented for 14 days. An initial STEC population of 7.02 log CFU/mL decreased to <0.30 log CFU/mL in two brands within 14 days; 0.77 and 0.87 log CFU/mL were detected in kombucha prepared from the other two brands. Salmonella and STEC increased within 1 day in three brands of base tea used to prepare kombucha and were stable throughout 14 days of incubation. Both pathogens steadily declined in base tea prepared from one brand of kombucha kit. Inactivation of the pathogens occurred as the pH of the kombucha decreased, but a clear correlation between rates of inactivation among different brands of kits and decrease in pH was not evident. Growth and peak populations of mesophilic aerobic microorganisms, yeasts, lactic acid bacteria, and acetic acid bacteria varied depending on the kombucha kit brand. No strong evidence was found of a correlation between the behavior of Salmonella or STEC and that of any of these groups of indigenous microbiota. Results of this study show that survival of Salmonella and STEC in kombucha and base tea used to prepare kombucha is dependent on inherent differences in commercially available kombucha kits intended for use in home settings. Strict application of hygienic practices is essential for preventing contamination with Salmonella or STEC and reducing the risk of illness associated the consumption of kombucha.

Survival and internalization of Salmonella and Escherichia coli O157:H7 sprayed onto different cabbage cultivars during cultivation in growth chambers.

BACKGROUND: Cabbage may become contaminated with enteric pathogens during cultivation. Using multiple cabbage cultivars at two maturity stages (small plants or plants with small heads) in growth chamber studies, the fate (internalization or surface survival) of Salmonella and Escherichia coli O157:H7 (0157) were examined in conjunction with any potential relationships to the plant's antimicrobial content. RESULTS: Internalized Salmonella was detected in cabbage within 24 h with prevalence ranging from 62% (16 of 26) for the 'Super Red 80' cultivar to 92% (24 of 26) for the 'Red Dynasty' cultivar. Surface survival of pathogens on small cabbage plants over nine days was significantly affected by cultivar with both pathogens surviving the most on the 'Farao' cultivar and Salmonella and O157 surviving the least on the 'Super Red 80' and 'Capture' cultivars, respectively (P < 0.05). Survival of O157 was slightly higher on cabbage heads for O157 than small plants suggesting that the maturity stage may affect this pathogen's fate. An inverse relationship existed between antimicrobial levels and the pathogen's surface survival on cabbage heads (P < 0.05). CONCLUSIONS: The fate of pathogens varied with the cabbage cultivar in growth chamber studies highlighting the potential to explore cultivar in field studies to reduce the risk of microbiological contamination in this crop. © 2019 Society of Chemical Industry.

Mitochondrial genome sequence variation as a useful marker for assessing genetic heterogeneity among Cyclospora cayetanensis isolates and source-tracking.

BACKGROUND: Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. For effective outbreak identification and investigation, it is essential to rapidly assess the genetic heterogeneity of C. cayetanensis specimens from cluster cases and identify the likely occurrence of outbreaks. METHODS: In this study, we developed a quantitative PCR (qPCR) targeting the polymorphic link region between copies of the mitochondrial genome of C. cayetanensis, and evaluated the genetic heterogeneity among 36 specimens from six countries using melt curve, gel electrophoresis, and sequence analyses of the qPCR products. RESULTS: All specimens were amplified successfully in the qPCR and produced melt peaks with different Tm values in the melt curve analysis. In gel electrophoresis of the qPCR products, the specimens yielded bands of variable sizes. Nine genotypes were identified by DNA sequencing of the qPCR products. Geographical segregation of genotypes was observed among specimens analyzed, which could be useful in geographical source-tracking. CONCLUSIONS: The length and nucleotide sequence variations in the mitochondrial genome marker allow rapid assessment of the genetic heterogeneity among C. cayetanensis specimens by melt curve, gel electrophoresis, or DNA sequence analysis of qPCR products. The sequence data generated could be helpful in the initial source-tracking of the pathogen.

Population genetic characterization of Cyclospora cayetanensis from discrete geographical regions.

Cyclospora cayetanensis is an emerging pathogen that is endemic in developing countries and responsible for many large foodborne cyclosporiasis outbreaks in North America since 1990s. Because of the lack of typing targets, the genetic diversity and population genetics of C. cayetanensis have not been investigated. In this study, we undertook a population genetic analysis of multilocus sequence typing data we recently collected from 64 C. cayetanensis specimens. Despite the extensive genetic heterogeneity in the overall C. cayetanensis population, there were significant intra- and inter-genic linkage disequilibria (LD). A disappearance of LD was observed when only multilocus genotypes were included in the population genetic analysis, indicative of an epidemic nature of C. cayetanensis. Geographical segregation-associated sub-structuring was observed between specimens from China and those from Peru and the United States. The two subpopulations had reduced LD, indicating the likely occurrence of genetic exchange among isolates in endemic areas. Further analyses of specimens from other geographical regions are necessary to fully understand the population genetics of C. cayetanensis.

Stray cats are more frequently infected with zoonotic protists than pet cats.

Faecal samples were collected from cats kept as pets (n = 120) and stray cats (n = 135) in Central Europe (Czech Republic, Poland and Slovakia) and screened for the presence of Cryptosporidium spp., Giardia intestinalis (Kunstler, 1882), Encephalitozoon spp. and Enterocytozoon bieneusi Desportes, Le Charpentier, Galian, Bernard, Cochand-Priollet, Lavergne, Ravisse et Modigliani, 1985 by PCR analysis of the small-subunit of rRNA (Cryptosporidium spp. and G. intestinalis) and ITS (microsporidia) genes. Sequence analysis of targeted genes revealed the presence of C. felis Iseki, 1979, G. intestinalis assemblage F, E. cuniculi Levaditi, Nicolau et Schoen, 1923 genotype II, and E. bieneusi genotype D. There was no correlation between the occurrence of detected parasites and sex, presence of diarrhoea or drug treatment (drug containing pyrantel and praziquantel). Compared to pet cats (7%), stray cats (30%) were statistically more frequently infected with protist parasites and overall may present a greater risk to human health.

Retention of Viability of Salmonella in Sucrose as Affected by Type of Inoculum, Water Activity, and Storage Temperature.

Outbreaks of salmonellosis have been associated with consumption of high-sugar, low-water activity (aw) foods. The study reported here was focused on determining the effect of storage temperature (5 and 25°C) on survival of initially high and low levels of Salmonella in dry-inoculated sucrose (aw 0.26 ± 0.01 to 0.54 ± 0.01) and wet-inoculated sucrose (aw 0.24 ± 0.01 to 0.44 ± 0.04) over a 52-week period. With the exception of dry-inoculated sucrose at aw 0.26, Salmonella survived for 52 weeks in dry- and wet-inoculated sucrose stored at 5 and 25°C. Retention of viability was clearly favored in sucrose stored at 5°C compared with 25°C, regardless of level or type of inoculum or aw. Survival at 5°C was not affected by aw. Initial high-inoculum counts of 5.18 and 5.25 log CFU/g of dry-inoculated sucrose (aw 0.26 and 0.54, respectively) stored for 52 weeks at 5°C decreased by 0.56 and 0.53 log CFU/g; counts decreased by >4.18 and >4.25 log CFU/g in samples stored at 25°C. Inactivation rates in wet-inoculated sucrose were similar to those in dry-inoculated sucrose; however, a trend toward higher persistence of Salmonella in dry- versus wet-inoculated sucrose suggests there was a higher proportion of cells in the wet inoculum with low tolerance to osmotic stress. Survival patterns were similar in sucrose initially containing a low level of Salmonella (2.26 to 2.91 log CFU/g). The pathogen was recovered from low-inoculated sucrose stored at 5°C for 52 weeks regardless of type of inoculum or aw and from dry-inoculated sucrose (aw 0.54) and wet-inoculated sucrose (aw 0.24) stored at 25°C for 12 and 26 weeks, respectively. Results emphasize the importance of preventing contamination of sucrose intended for use as an ingredient in foods not subjected to a treatment that would be lethal to Salmonella.

Multilocus Sequence Typing Tool for Cyclospora cayetanensis.

Because the lack of typing tools for Cyclospora cayetanensis has hampered outbreak investigations, we sequenced its genome and developed a genotyping tool. We observed 2 to 10 geographically segregated sequence types at each of 5 selected loci. This new tool could be useful for case linkage and infection/contamination source tracking.

In Vitro Study of Taenia solium Postoncospheral Form.

BACKGROUND: The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages. METHODOLOGY/PRINCIPAL FINDINGS: T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development--days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15-30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages. CONCLUSIONS/SIGNIFICANCE: This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite's immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations.

Cryptosporidium proliferans n. sp. (Apicomplexa: Cryptosporidiidae): Molecular and Biological Evidence of Cryptic Species within Gastric Cryptosporidium of Mammals.

The morphological, biological, and molecular characteristics of Cryptosporidium muris strain TS03 are described, and the species name Cryptosporidium proliferans n. sp. is proposed. Cryptosporidium proliferans obtained from a naturally infected East African mole rat (Tachyoryctes splendens) in Kenya was propagated under laboratory conditions in rodents (SCID mice and southern multimammate mice, Mastomys coucha) and used in experiments to examine oocyst morphology and transmission. DNA from the propagated C. proliferans isolate, and C. proliferans DNA isolated from the feces of an African buffalo (Syncerus caffer) in Central African Republic, a donkey (Equus africanus) in Algeria, and a domestic horse (Equus caballus) in the Czech Republic were used for phylogenetic analyses. Oocysts of C. proliferans are morphologically distinguishable from C. parvum and C. muris HZ206, measuring 6.8-8.8 (mean = 7.7 µm) × 4.8-6.2 µm (mean = 5.3) with a length to width ratio of 1.48 (n = 100). Experimental studies using an isolate originated from T. splendens have shown that the course of C. proliferans infection in rodent hosts differs from that of C. muris and C. andersoni. The prepatent period of 18-21 days post infection (DPI) for C. proliferans in southern multimammate mice (Mastomys coucha) was similar to that of C. andersoni and longer than the 6-8 DPI prepatent period for C. muris RN66 and HZ206 in the same host. Histopatologicaly, stomach glands of southern multimammate mice infected with C. proliferans were markedly dilated and filled with necrotic material, mucus, and numerous Cryptosporidium developmental stages. Epithelial cells of infected glands were atrophic, exhibited cuboidal or squamous metaplasia, and significantly proliferated into the lumen of the stomach, forming papillary structures. The epithelial height and stomach weight were six-fold greater than in non-infected controls. Phylogenetic analyses based on small subunit rRNA, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, heat shock protein 70, actin, heat shock protein 90 (MS2), MS1, MS3, and M16 gene sequences revealed that C. proliferans is genetically distinct from C. muris and other previously described Cryptosporidium species.

Contamination of knives and graters by bacterial foodborne pathogens during slicing and grating of produce.

Poor hygiene and improper food preparation practices in consumers' homes have previously been demonstrated as contributing to foodborne diseases. To address potential cross-contamination by kitchen utensils in the home, a series of studies was conducted to determine the extent to which the use of a knife or grater on fresh produce would lead to the utensil's contamination with Escherichia coli O157:H7 or Salmonella enterica. When shredding inoculated carrots (ca. 5.3 log CFU/carrot), all graters became contaminated and the number of E. coli O157:H7 present on the utensil was significantly greater than Salmonella (p < 0.05). Contamination of knives after slicing inoculated produce (4.9-5.4 log CFU/produce item) could only be detected by enrichment culture. After slicing tomatoes, honeydew melons, strawberries, cucumbers, and cantaloupes, the average prevalence of knife contamination by the two pathogens was 43%, 17%, 15%, 7%, and 3%, respectively. No significant increase in the incidence or level of contamination occurred on the utensils when residues were present (p > 0.05); however, subsequent contamination of 7 produce items processed with the contaminated utensils did occur. These results highlight the necessity of proper sanitization of these utensils when used in preparation of raw produce.

Role of Brushes and Peelers in Removal of Escherichia coli O157:H7 and Salmonella from Produce in Domestic Kitchens.

Consumers are being advised to increase their consumption of fruits and vegetables to reduce their risk of chronic disease. However, to achieve that goal, consumers must be able to implement protocols in their kitchens to reduce their risk of consuming contaminated produce. To address this issue, a study was conducted to monitor the fate of Escherichia coli O157:H7 and Salmonella on produce (cantaloupe, honeydew melon, carrots, and celery) that were subjected to brushing or peeling using common kitchen utensils. Removal of similar levels of Salmonella from carrots was accomplished by peeling and by brushing, but significantly greater removal of E. coli O157:H7 from carrots was accomplished by peeling than by brushing under running water (P < 0.05). Brushing removed significantly fewer pathogens from contaminated cantaloupes than from other produce items (P < 0.05), suggesting that the netted rind provided sites where the pathogen cells could evade the brush bristles. A Sparta polyester brush was less effective than a scouring pad for removing Salmonella from carrots (P < 0.05). In all cases, brushing and peeling failed to eliminate the pathogens from the produce items, which may be the result of contamination of the utensil during use. High incidences of contamination (77 to 92%) were found among peelers used on carrots or celery, the Sparta brush used on carrots, and the scouring pad used on carrots and cantaloupe. Of the utensils investigated, the nylon brush had the lowest incidence of pathogen transference from contaminated produce (0 to 12%). Transfer of pathogens from a potentially contaminated Sparta brush or peeler to uncontaminated carrots did not occur or occurred only on the first of seven carrots processed with the utensil. Therefore, risk of cross-contamination from contaminated utensils to uncontaminated produce may be limited.

Novel rat model for neurocysticercosis using Taenia solium.

Neurocysticercosis is caused by Taenia solium infecting the central nervous system and is the leading cause of acquired epilepsy and convulsive conditions worldwide. Research into the pathophysiology of the disease and appropriate treatment is hindered by lack of cost-effective and physiologically similar animal models. We generated a novel rat neurocysticercosis model using intracranial infection with activated T. solium oncospheres. Holtzman rats were infected in two separate groups: the first group was inoculated extraparenchymally and the second intraparenchymally, with different doses of activated oncospheres. The groups were evaluated at three different ages. Histologic examination of the tissue surrounding T. solium cysticerci was performed. Results indicate that generally infected rats developed cysticerci in the brain tissue after 4 months, and the cysticerci were observed in the parenchymal, ventricle, or submeningeal brain tissue. The route of infection did not have a statistically significant effect on the proportion of rats that developed cysticerci, and there was no dependence on infection dose. However, rat age was crucial to the success of the infection. Epilepsy was observed in 9% of rats with neurocysticercosis. In histologic examination, a layer of collagen tissue, inflammatory infiltrate cells, perivascular infiltrate, angiogenesis, spongy change, and mass effect were observed in the tissue surrounding the cysts. This study presents a suitable animal model for the study of human neurocysticercosis.

The food industry's current and future role in preventing microbial foodborne illness within the United States.

During the past century, the microbiological safety of the US food supply has improved; however, many foodborne illnesses and outbreaks occur annually. Hence, opportunities for the food industry to improve the safety of both domestic and imported food exist through the adoption of risk-based preventive measures. Challenging food safety issues that are on the horizon include demographic changes to a population whose immune system is more susceptible to foodborne and opportunistic pathogens, climate changes that will shift where food is produced, and consumers' preferences for raw and minimally processed foods. Increased environmental and product testing and anonymous data sharing by the food industry with the public health community would aid in identifying system weaknesses and enabling more targeted corrective and preventive actions. Clinicians will continue to play a major role in reducing foodborne illnesses by diagnosing and reporting cases and in helping to educate the consumer about food safety practices.

Efficacy of wash solutions in recovering Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from basil.

Parasitic diseases can be acquired by ingestion of contaminated raw or minimally processed fresh produce (herbs and fruits). The sensitivity of methods used to detect parasites on fresh produce depends in part on the efficacy of wash solutions in removing them from suspect samples. In this study, six wash solutions (sterile E-Pure water, 3% levulinic acid-3% sodium dodecyl sulfate, 1 M glycine, 0.1 M phosphate-buffered saline, 0.1% Alconox, and 1% HCl-pepsin) were evaluated for their effectiveness in removing Cyclospora cayetanensis, Cryptosporidium parvum, and Toxoplasma gondii from basil. One hundred or 1,000 oocysts of these parasites were inoculated onto the adaxial surfaces of 25 g of basil leaves, placed in stomacher bags, and stored for 1 h at 21°C or 24 h at 4°C. Leaves were hand washed in each wash solution for 1 min. DNA was extracted from the wash solutions and amplified using PCR for the detection of all parasites. Oocysts inoculated at a concentration of 1,000 oocysts per 25 g of basil were detected in all wash solutions. At an inoculum concentration of 100 oocysts per 25 g, oocysts were detected in 18.5 to 92.6% of the wash solutions. The lowest variability in recovering oocysts from basil inoculated with 100 oocysts was observed in 1% HCl-pepsin wash solution. Oocyst recovery rates were higher at 1 h than at 24 h postinoculation. Unlike most bacteria, parasites cannot be enriched; therefore, an optimal recovery process for oocysts from suspected foods is critical. The observations in this study provide guidance concerning the selection of wash solutions giving the highest retrieval of parasite oocysts.

Cyclospora cayetanensis in a pediatric hospital in Morelia, México.

Cyclospora cayetanensis, a coccidian parasite, can cause gastrointestinal illness in humans and is characterized by watery and persistent diarrhea and abdominal pain. Cyclosporiasis has been associated with traveler's diarrhea. The infection is acquired through food and waterborne transmission, particularly by consumption of contaminated fresh fruits and vegetables. In the present study, stool samples from 8,877 children were examined for ova and parasites at the Pediatric Hospital of Morelia in Michoacán, Mexico, during 2000-2009. Sixty children (0.67%) had Cyclospora in their stools. Diarrhea (45.8%), abdominal pain (39.6%), and vomiting (18.8%) were the most frequent symptoms of cases with cyclosporiasis. Most of the cases (93.3%) were observed during June-August, the rainy season. In 45 children, Cyclospora was the only parasitic pathogen detected (75%); 15 children were co-infected with commensal, pathogenic, or both groups of parasites. Our findings suggest that C. cayetanensis is endemic to Michoacán and shows characteristically temporal patterns.

Population genetics of Cryptosporidium meleagridis in humans and birds: evidence for cross-species transmission.

Population genetic studies have been used to understand the transmission of pathogens in humans and animals, especially the role of zoonotic infections and evolution and dispersal of virulent subtypes. In this study, we analysed the genetic diversity and population structure of Cryptosporidium meleagridis, the only known Cryptosporidium species that infects both avian and mammalian hosts and is responsible for approximately 10% of human cryptosporidiosis in some areas. A total of 62 C. meleagridis specimens from children, AIDS patients, and birds in Lima, Peru were characterised by sequence analysis of the ssrRNA gene and five minisatellite, microsatellite and polymorphic markers in chromosome 6, including the 60 kDa glycoprotein (gp60), 47 kDa glycoprotein (CP47), a serine repeat antigen (MSC6-5), retinitis pigmentosa GTPase regulator (RPGR) and thrombospondin protein 8 (TSP8). The multilocus sequence analysis identified concurrent infections with Cryptosporidium hominis in four AIDS patients and three children. Unique subtypes of C. meleagridis ranged from eight at the gp60 locus (gene diversity -Hd=0.651), three at the RPGR (Hd=0.556), three at the MSC6-5 locus (Hd=0.242), two at TSP8 (Hd=0.198), to one at CP47 (monomorphic), much lower than that of C. hominis in the same area. Intragenic linkage disequilibrium was strong and complete at all gene loci. Intergenic linkage disequilibrium was highly significant (P<0.001) for all pairs of polymorphic loci. Two major groups of subtypes were seen, with most subtypes belonging to group 1. Within group 1, there was no clear population segregation, and two of the 14 multilocus subtypes of C. meleagridis were found in both AIDS patients and birds. We believe that these results provide the first evidence of a clonal population structure of C. meleagridis and the likely occurrence of cross-species transmission of C. meleagridis between birds and humans.

Genetic characterization of human-pathogenic Cyclospora cayetanensis parasites from three endemic regions at the 18S ribosomal RNA locus.

Cyclospora cayetanensis is an apicocomplexan parasite that infects the gastrointestinal tract and causes acute diarrheal disease in humans. In recent years, this human-pathogenic parasite has led to several foodborne outbreaks in the United States and Canada, mostly associated with imported produce. Understanding the biology and epidemiology of C. cayetanensis is difficult because little is known about its origin, possible zoonotic reservoirs, and genetic relationships with other coccidian parasites. Recently, we developed a 70kDa heat shock protein (HSP70) gene based nested PCR protocol for detection of C. cayetanensis parasite and sequenced the PCR products of 16 human isolates from Nepal, Mexico, and Peru. In this study, we have characterized the regions of 18S ribosomal RNA (rRNA) gene of 17 human C. cayetanensis isolates for molecular detection, and also to ascertain the genetic diversity of this parasite. The 18S rRNA primer sets were further tested by PCR amplification followed by nucleotide sequencing of the PCR amplified products of previously characterized C. cayetanensis isolates from three endemic regions at HSP70 locus. Although no genetic polymorphism was observed at the regions of HSP70 locus characterized in our previous study, the data analysis of this study revealed a minor genetic diversity at the 18S rRNA locus among the C. cayetanensis isolates. The 18S rRNA gene-based nested PCR protocol provides a useful genetic marker for the detection of C. cayetanensis parasite and confirms it as a genetically distinct species in genus Cyclospora. The results also supported lack of geographic segregation and existence of genetically homogeneous population for the C. cayetanensis parasites both at the HSP70 as well as at the18S rRNA loci.

Impacts of globalisation on foodborne parasites.

Globalisation is a manmade phenomenon encompassing the spread and movement of everything, animate and inanimate, material and intangible, around the planet. The intentions of globalisation may be worthy--but may also have unintended consequences. Pathogens may also be spread, enabling their establishment in new niches and exposing new human and animal populations to infection. The plethora of foodborne parasites that could be distributed by globalisation has only recently been acknowledged and will provide challenges for clinicians, veterinarians, diagnosticians, and everyone concerned with food safety. Globalisation may also provide the resources to overcome some of these challenges. It will facilitate sharing of methods and approaches, and establishment of systems and databases that enable control of parasites entering the global food chain.