Chester Z. Sarreal

Wrinkled Alfalfa Seeds Harbor More Aerobic Bacteria and Are More Difficult To Sanitize than Smooth Seeds

At least 14 separate outbreaks of food poisoning attributed to either Salmonella enterica or Escherichia coli O157:H7 have been traced to sprouts in the past decade. Seeds contaminated with human pathogens caused most of these outbreaks, thus many sprout growers are now treating alfalfa seeds with the sanitizing agent, calcium hypochlorite (Ca[OCl]2), prior to sprouting. The efficacy of alfalfa seed sanitation varies between seed lots and between seeds within each lot. Alfalfa seeds from different seed lots were sorted by type in an effort to determine if certain seed types carry more aerobic bacteria than other seed types. Seeds with a wrinkled type, characteristic of lygus bug damage, had significantly higher levels of culturable aerobic bacteria and were more difficult to sanitize than smooth, healthy seeds. After sanitation, wrinkled alfalfa seeds that had been inoculated with S. enterica ser. Newport carried significantly higher levels of Salmonella Newport than smooth seeds. If S. enterica is present on wrinkled seeds in naturally contaminated seed lots, it may be difficult to chemically sanitize the seed lot. Removal of the wrinkled alfalfa seeds from the seed lots, perhaps by adapting color sorting equipment similar to that used to sort rice grains and other seeds, should reduce the level of aerobic bacteria in seed lots and may result in lower levels of human pathogens on contaminated alfalfa seeds.

Bacterial Communities in Aerosols and Manure Samples from Two Different Dairies in Central and Sonoma Valleys of California

Aerosols have been suspected to transport food pathogens and contaminate fruits and vegetables grown in close proximity to concentrated animal feeding operations, but studies are lacking that substantiate such transport. To monitor the potential transport of bacteria originated from fresh or dry manure through aerosols on a dairy, we identified by 16S rRNA sequencing, bacteria in aerosols collected within 2 to 3 meters from dairy cows at two dairies. Gram-positive Firmicutes were predominant in aerosols from a dairy in Sonoma, California, and surrounded by vineyards, in contrast to sequences of Gram-negative Proteobacteria predominant in aerosols from a dairy in Modesto, California, also surrounded by other dairies. Although Firmicutes represented approximately 50% of the 10 most abundant sequences, aerosols from the Sonoma dairy also contained sequences of Bacteriodetes and Actinobacteria, identified previously with animal feces. While none of the top 10 sequences from fresh or dry manure from Modesto dairy were detected in aerosols, two of the sequences from the phylum Bacteriodetes and one from class Clostridia from fresh manure were detected in aerosols from Sonoma. Interestingly, none of the sequences from dry manure were in the top 10 sequences in aerosols from both dairies. The 10 most abundant sequences in aerosols from the Modesto dairy were all from Proteobacteria and nearly half of them were from genus Massilia, which have been isolated previously from immune-compromised people and aerosols. We conclude that the predominant bacteria in aerosols are diverse among locations and that they do not reflect the predominant species of bacteria present in cow feces and/or in close proximity to cows. These results suggest that the aerosol sequences did not originate from manure. Large volumes of aerosols would be required to determine if bacterial sequences from aerosols could be used to track bacteria in manure to crops grown in proximity.

Effect of Molasses on Regrowth of E. coli O157:H7 And Salmonella in Compost Teas

Compost water extracts (compost teas) are gaining popularity among organic growers, largely because of their disease suppressive activity when applied to foliage or soil. Production methods often include addition of supplemental constituents, particularly molasses, to stimulate plant-beneficial microbial populations. We have found that molasses amendments also favor regrowth of human pathogenic bacteria, raising public health concerns about potential contamination of treated crops, particularly produce intended for fresh consumption. Using disease outbreak strains marked with green fluorescent protein (GFP) and spontaneous antibiotic-resistance, we found that regrowth of Salmonella enterica serovar Thompson and Escherichia coli O157:H7 was positively correlated with molasses concentration. For Salmonella, regrowth was also dependent on the type of starter compost material used. Salmonella populations increased from 1 at time 0 to over 1000 CFU ml−1 in dairy manure compost tea with 1% molasses, and from 1 at time 0 to over 350,000 CFU ml−1 in chicken manure compost tea by 72 h. E. coli populations increased from 1 at time 0 to approximately 1000 CFU ml−1 in both types of tea by 72 h. Pathogen regrowth did not occur when molasses was eliminated or kept to 0.2%.

Identification of Protozoa in Dairy Lagoon Wastewater that Consume Escherichia coli O157:H7 Preferentially

Escherichia coli O157:H7 (EcO157), an agent of life threatening hemolytic-uremic syndrome, resides in ruminants and is released in feces at numbers as high as 10 million cells/gram. EcO157 could survive in manure for as long as 21 months, but we observed a 90% decrease in cells of an outbreak strain of EcO157 within half a day in wastewater from dairy lagoons. Although chemical, environmental and biological factors may be responsible for this decrease, we observed an 11-fold increase in native protozoa when wastewater was re-inoculated with 2×107 cells of EcO157/mL. These protozoa engulfed the green fluorescent protein labeled EcO157 within 2 hours after inoculation, but expelled vacuoles filled with live EcO157 cells within 3 days into surrounding wastewater, whereas other protozoa retained the EcO157-filled vacuoles for 7 days. EcO157 was not detected by confocal microscopy either inside or outside protozoa after 7 days. Mixed cultures of protozoa enriched from wastewater consumed EcO157 preferentially as compared to native aerobic bacteria, but failed to eliminate them when EcO157 cells declined to 104/mL. We isolated three protozoa from mixed cultures and typed them by 18S sequencing as Vorticella microstoma, Platyophyra sp. and Colpoda aspera. While all three protozoa internalized EcO157, only Platyophyra and Colpoda acted as predators. Similar to mixed cultures, these protozoa failed to eliminate EcO157 from PBS containing no other supplemental nutrients or prey. However, spiking PBS with cereal grass medium as nutrients induced predation of EcO157 by Platyophyra sp. after 3 days or enhanced predation by Colpoda after 5 days. Therefore, attempts to enrich protozoa to decrease EcO157 from dairy lagoons, may correspond to an increase in protozoa similar to Vorticella and possibly facilitate transport of bacterial pathogens to food crops grown in proximity.

Altered protozoan and bacterial communities and survival of Escherichia coli O157:H7 in monensin-treated wastewater from a dairy lagoon.

Surviving predation is a fitness trait of Escherichia coli O157:H7 (EcO157) that provides ample time for the pathogen to be transported from reservoirs (e.g. dairies and feedlots) to farm produce grown in proximity. Ionophore dietary supplements that inhibit rumen protozoa may provide such a selective advantage for EcO157 to proliferate in lagoons as the pathogen is released along with the undigested supplement as manure washings. This study evaluated the fate of an outbreak strain of EcO157, protozoan and bacterial communities in wastewater treated with monensin. Although total protozoa and native bacteria were unaffected by monensin, the time for 90% decrease in EcO157 increased from 0.8 to 5.1 days. 18S and 16S rRNA gene sequencing of wastewater samples revealed that monensin eliminated almost all colpodean and oligohymenophorean ciliates, probably facilitating the extended survival of EcO157. Total protozoan numbers remained high in treated wastewater as monensin enriched 94% of protozoan sequences undetected with untreated wastewater. Monensin stimulated 30-fold increases in Cyrtohymena citrina, a spirotrichean ciliate, and also biflagellate bicosoecids and cercozoans. Sequences of gram-negative Proteobacteria increased from 1% to 46% with monensin, but gram-positive Firmicutes decreased from 93% to 46%. It is noteworthy that EcO157 numbers increased significantly (P<0.01) in Sonneborn medium containing monensin, probably due to monensin-inhibited growth of Vorticella microstoma (P<0.05), a ciliate isolated from wastewater. We conclude that dietary monensin inhibits ciliate protozoa that feed on EcO157. Feed supplements or other methods that enrich these protozoa in cattle manure could be a novel strategy to control the environmental dissemination of EcO157 from dairies to produce production environments.

Survival of Salmonella enterica in Aerated and Nonaerated Wastewaters from Dairy Lagoons

Salmonella is the most commonly identified foodborne pathogen in produce, meat and poultry. Cattle are known reservoirs of Salmonella and the pathogen excreted in feces ends up in manure flush lagoons. Salmonella enterica survival was monitored in wastewater from on-site holding lagoons equipped or not with circulating aerators at two dairies. All strains had poor survival rates and none proliferated in waters from aerated or settling lagoons. Populations of all three Salmonella serovars declined rapidly with decimal reduction times (D) of <2 days in aerated microcosms prepared from lagoon equipped with circulators. Populations of Salmonella decreased significantly in aerated microcosms (D = 4.2 d) compared to nonaerated waters (D = 7.4 d) and in summer (D = 3.4 d) compared to winter (D = 9.0 d). We propose holding the wastewater for sufficient decimal reduction cycles in lagoons to yield pathogen-free nutrient-rich water for crop irrigations and fertilization.

Strain differences in fitness of Escherichia coli O157:H7 to resist protozoan predation and survival in soil.

Escherichia coli O157:H7 (EcO157) associated with the 2006 spinach outbreak appears to have persisted as the organism was isolated, three months after the outbreak, from environmental samples in the produce production areas of the central coast of California. Survival in harsh environments may be linked to the inherent fitness characteristics of EcO157. This study evaluated the comparative fitness of outbreak-related clinical and environmental strains to resist protozoan predation and survive in soil from a spinach field in the general vicinity of isolation of strains genetically indistinguishable from the 2006 outbreak strains. Environmental strains from soil and feral pig feces survived longer (11 to 35 days for 90% decreases, D-value) with Vorticella microstoma and Colpoda aspera, isolated previously from dairy wastewater; these D-values correlated (P<0.05) negatively with protozoan growth. Similarly, strains from cow feces, feral pig feces, and bagged spinach survived significantly longer in soil compared to clinical isolates indistinguishable by 11-loci multi-locus variable-number tandem-repeat analysis. The curli-positive (C+) phenotype, a fitness trait linked with attachment in ruminant and human gut, decreased after exposure to protozoa, and in soils only C− cells remained after 7 days. The C+ phenotype correlated negatively with D-values of EcO157 exposed to soil (r s = −0.683; P = 0.036), Vorticella (r s = −0.465; P = 0.05) or Colpoda (r s = −0.750; P = 0.0001). In contrast, protozoan growth correlated positively with C+ phenotype (Vorticella, r s = 0.730, P = 0.0004; Colpoda, r s = 0.625, P = 0.006) suggesting a preference for consumption of C+ cells, although they grew on C− strains also. We speculate that the C− phenotype is a selective trait for survival and possibly transport of the pathogen in soil and water environments.

Persistence of F-Specific RNA Coliphages in Surface Waters from a Produce Production Region along the Central Coast of California

F+ RNA coliphages (FRNA) are used to source-track fecal contamination and as surrogates for enteric pathogen persistence in the environment. However, the environmental persistence of FRNA is not clearly understood and necessitates the evaluation of the survival of prototype and environmental isolates of FRNA representing all four genogroups in surface waters from the central coast of California. Water temperature played a significant role in persistence–all prototype and environmental strains survived significantly longer at 10°C compared to 25°C. Similarly, the availability of host bacterium was found to be critical in FRNA survival. In the absence of E. coli Famp, all prototypes of FRNA disappeared rapidly with a D-value (days for one log reduction) of <1.2 d from water samples incubated at 25°C; the longest surviving prototype was SP. However, in the presence of the host, the order of persistence at 25°C was QB>MS2>SP>GA and at 10°C it was QB = MS2>GA>SP. Significant differences in survival were observed between prototypes and environmental isolates of FRNA. While most environmental isolates disappeared rapidly at 25°C and in the absence of the host, members of genogroups GIII and GI persisted longer with the host compared to members of GII and GIV. Consequentially, FRNA based source tracking methods can be used to detect phages from recent fecal contamination along with those that persist longer in the environment as a result of cooler temperatures and increased host presence.

Fitness of Outbreak and Environmental Strains of Escherichia coli O157:H7 in Aerosolizable Soil and Association of Clonal Variation in Stress Gene Regulation.

Airborne dust from feedlots is a potential mechanism of contamination of nearby vegetable crops with Escherichia coli O157:H7 (EcO157). We compared the fitness of clinical and environmental strains of EcO157 in <45 µm soil from a spinach farm. Differences in survival were observed among the 35 strains with D-values (days for 90% decreases) ranging from 1–12 days. Strains that survived longer, generally, were from environmental sources and lacked expression of curli, a protein associated with attachment and virulence. Furthermore, the proportion of curli-positive (C+) variants of EcO157 strains decreased with repeated soil exposure and the strains that were curli-negative (C−) remained C− post-soil exposure. Soil exposure altered expression of stress-response genes linked to fitness of EcO157, but significant clonal variation in expression was measured. Mutations were detected in the stress-related sigma factor, rpoS, with a greater percentage occurring in parental strains of clinical origin prior to soil exposure. We speculate that these mutations in rpoS may confer a differential expression of genes, associated with mechanisms of survival and/or virulence, and thus may influence the fitness of EcO157.

Rapid detection of predation of Escherichia coli O157:H7 and sorting of bacterivorous Tetrahymena by flow cytometry.

Protozoa are known to harbor bacterial pathogens, alter their survival in the environment and make them hypervirulent. Rapid non-culture based detection methods are required to determine the environmental survival and transport of enteric pathogens from point sources such as dairies and feedlots to food crops grown in proximity. Grazing studies were performed on a soil isolate of Tetrahymena fed green fluorescent protein (GFP) expressing Escherichia coli O157:H7 to determine the suitability of the use of such fluorescent prey bacteria to locate and sort bacterivorous protozoa by flow cytometry. In order to overcome autofluorescence of the target organism and to clearly discern Tetrahymena with ingested prey vs. those without, a ratio of prey to host of at least 100:1 was determined to be preferable. Under these conditions, we successfully sorted the two populations using short 5–45 min exposures of the prey and verified the internalization of E. coli O157:H7 cells in protozoa by confocal microscopy. This technique can be easily adopted for environmental monitoring of rates of enteric pathogen destruction vs. protection in protozoa.