Short communication: Characterization of Salmonella phages from dairy calves on farms with history of diarrhea

Salmonella enterica can cause disease and mortality in calves. This pathogen is also a zoonosis that can be transmitted by animal contact or by food. The prevalence of Salmonella in dairy farms has been reported to range from 0 to 64%, and, due to the diversity of Salmonella serovars that can be circulating, Salmonella is an important concern for dairy production. Bacteriophages that infect Salmonella have been documented to be abundant and widely distributed in the dairy environment. The current study investigated the diversity of Salmonella serovars and Salmonella phages in 8 dairy farms with a history of diarrhea in southern Chile. A total of 160 samples from sick calves, healthy calves, and the environment were analyzed for Salmonella and phage. Isolated phages were characterized and classified by their host range using a panel of 26 Salmonella isolates representing 23 serovars. Host ranges were classified according to lysis profiles (LP) and their spatial distribution was mapped. Salmonella-infecting phages were identified, but none of the 160 samples were positive for Salmonella. A total of 45 phage isolates were obtained from sick calves (11), healthy calves (16), or the environment (18). According to their host range, 19 LP were identified, with LP1 being the most common on all 8 farms; LP1 represents phages that only lyse serogroup D Salmonella. The identification of Salmonella phages but not Salmonella in the same samples could suggest that these phages are controlling Salmonella in these farms.     

Short communication: Determination of Salmonella clustered regularly interspaced short palindromic repeats (CRISPR) diversity on dairy farms in Wisconsin and Minnesota

Salmonella enterica ssp. enterica is a foodborne pathogen able to cause disease in both humans and animals. Diverse serovars of this pathogen exist, some of which are host specific, causing a range of clinical symptoms from asymptomatic infection through morbidity and mortality. According to a 2007 survey by the USDA National Animal Health Monitoring System, fecal shedding of Salmonella from healthy cows occurs on 39.7% of dairy farms in the United States. Certain serovars are frequently isolated from dairy farms and the majority of isolates from the National Animal Health Monitoring System study were represented by 5 serovars; however, genotypic diversity was not examined. The objective of this study was to determine the diversity of clustered regularly interspaced short palindromic repeats (CRISPR) loci in Salmonella collected from 8 dairy farms with a previous history of salmonellosis. None of the cows or calves sampled on 2 of the 8 dairy farms were shedding Salmonella, although Salmonella was detected in a cow bedding sample on 1 of these farms. Salmonella populations were discrete on each farm, according to CRISPR typing, with the exception of an Anatum var. 15+ type on farms 5 and 6 and the Montevideo type on farms 1 and 2. One to 4 distinct CRISPR genotypes were identified per farm. The CRISPR typing differed within serovars, as Montevideo, Anatum var. 15+, and Muenster serovars had no overlap of spacer content, even on the same farm, reflecting between- and within-serovar genetic diversity. The dynamic nature of Salmonella populations was shown in a farm that was sampled longitudinally over 13.5 mo. Changes in serovar from 3,19:-:z27 to Montevideo was observed between the first sampling time and 8 mo later, with concomitant change in CRISPR alleles. The results indicate that Salmonella strains present in smaller dairy herds (<500 head) are specific to that farm and new Salmonella strains may emerge over time.     

Fecal prevalence and diversity of Salmonella species in lactating dairy cattle in four states

Salmonella is one of the most serious foodborne pathogenic bacteria in the United States, causing an estimated 1.3 million human illnesses each year. Dairy cows can be reservoirs of foodborne pathogenic bacteria, including Salmonella spp.; it is estimated that from 27 to 31% of dairy herds across the United States are colonized by Salmonella. The present study was designed to examine the occurrence of Salmonella spp. on dairies and to examine the serotypic diversity of Salmonella isolates on sampled dairies from across the United States. Fecal samples (n = 60 per dairy) were collected from 4 dairies in each of 4 states for a total of 960 fecal samples representing a total population of 13,200 dairy cattle. In the present study, 93 of 960 samples (9.96%) collected were culture-positive for Salmonella enterica. At least one Salmonella fecal-shedding cow was found in 9 of the 16 herds (56%) and the within-herd prevalence varied in our study from 0% in 7 herds to a maximum of 37% in 2 herds, with a mean prevalence among Salmonella-positive herds of 17%. Seventeen different serotypes were isolated, representing 7 different Salmonella serogroups. There were 2 or more different serogroups and serotypes present on 7 of the 9 Salmonella-positive farms. Serotypes Montevideo and Muenster were the most frequent and widespread. From our data, it appears that subclinical colonization with Salmonella enterica is relatively common on dairy farms and is represented by diverse serotypes on US dairy farms.     

Evaluation of a cocktail of three bacteriophages for the biocontrol of Salmonella of wastewater

Salmonella serovars are increasing in importance as significant pathogens of both human and animals. Although water and wastewater are treated to eliminate pathogenic microorganisms, they still play an important role in the transmission of Salmonella spp. In this study, bacteriophages infecting Salmonella spp. were isolated from wastewater and evaluated; for their potential to lyse environmental Salmonella strains in vitro at different MOIs and temperatures; and to control the wastewater bacterial community. Three distinct phages designated sww65, sww275, and sww297; as defined by plaque morphology, electron microscopy and host range; were obtained from wastewater. Challenge tests were performed at 37, and 30 °C with the infection of the Salmonella cultures with individual phage, a mixture of two phages, and cocktail of three phages at MOIs of 10⁰, 10², and 10⁴ PFU/CFU. At 30, and 37 °C, a cocktail of three phages reduced all of the Salmonella cultures tested. These results required a high multiplicity of infection. However, when infected with only one phage or a mixture of two phages at MOIs of 10⁰ or 10 ² PFU/CFU, an emergence of bacterial resistance was observed. The dynamic monitoring of wastewater enterobacterial community was conducted using Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). The number of bands decreased gradually with the use of individual phage or phage cocktails. Moreover, the dynamic monitoring of Salmonella community during wastewater treatment was performed using PCR detection of virulence gene invA. The results correlated with the ERIC-PCR fingerprints, and suggested that Salmonella community was affected by the phage treatment. Indeed, in wastewater, bacteriophages are reducing Salmonella and other members of the Enterobacteriaceae. These results indicated that dynamic changes are closely related with the process of treatment. The introduction of wide host range bacteriophages in wastewater can have a potential impact on the dynamics of the microbial communities, manifested by the reduction or the elimination of microbial species.     

Diversity of Streptococcus thermophilus phages in a large-production cheese factory in Argentina

Phage infections still represent a serious risk to the dairy industry, in which Streptococcus thermophilus is used in starter cultures for the manufacture of yogurt and cheese. The goal of the present study was to analyze the biodiversity of the virulent S. thermophilus phage population in one Argentinean cheese plant. Ten distinct S. thermophilus phages were isolated from cheese whey samples collected in a 2-mo survey. They were then characterized by their morphology, host range, and restriction patterns. These phages were also classified within the 2 main groups of S. thermophilus phages (cos- and pac-type) using a newly adapted multiplex PCR method. Six phages were classified as cos-type phages, whereas the 4 others belonged to the pac-type group. This study illustrates the phage diversity that can be found in one factory that rotates several cultures of S. thermophilus. Limiting the number of starter cultures is likely to reduce phage biodiversity within a fermentation facility.     

Molecular epidemiology and antimicrobial resistance profiles of Salmonella isolates from dairy heifer calves and adult lactating cows in a Mediterranean pasture-based system of Australia

Dairy cows can be reservoirs of foodborne pathogens such as Salmonella that pose serious public health risks to humans. The study was designed to examine the molecular epidemiology and antimicrobial resistance profiles of Salmonella isolates from dairy heifer calves and adult lactating cows in the pasture-based system of Australia. A total of 838 animals (328 heifer calves and 510 lactating cows) from 22 farms were sampled. Overall, 54 Salmonella isolates were recovered (calves 28/328 and cows 26/510). A herd-level Salmonella prevalence of 50% (95% confidence interval: 31%–69%) was recorded. Within-herd prevalence for Salmonella ranged between 4%–29% and 4%–45% among the heifer calves and adult lactating cows, respectively. Three different serovars were identified with Salmonella Infantis being the most common serovar (n = 33, 61%) followed by Salmonella Kiambu (n = 20, 37.0%) and one isolate of Salmonella Cerro (2%). The highest antimicrobial resistance prevalence of Salmonella isolates was found against streptomycin (n = 31, 57%), followed by cefoxitin (n = 12, 22%), ceftriaxone (n = 2, 4%), and chloramphenicol (n = 1, 2%). Multiple class resistance was observed on 4 isolates against cefoxitin, chloramphenicol, and streptomycin. Multilocus sequence types ST32 (61%), ST309 (37%), and ST367 (2%) were strongly linked to the serovars Salmonella Infantis, Salmonella Kiambu, and Salmonella Cerro, respectively. Whole genome sequencing of Salmonella isolates detected only 2 resistance genes: aac(6′) gene that confers resistance against aminoglycosides among 40.7% of the isolates, and a single isolate positive for the blaDHA-16 gene. Two distinct clusters among the serovars were observed suggesting 2 independent sources of spread. Despite the low prevalence of antimicrobial resistance among Salmonella from the dairy farms, our findings contribute to the regional and national understanding of antimicrobial resistance in dairy herds in Australia. There is need for continued antimicrobial resistance stewardship and surveillance programs to ensure the production of high-quality food products and the long-term protection of both animal and human health.     

Introduction of new multidrug-resistant Salmonella enterica strains into commercial dairy herds

A longitudinal observational study of 59 dairy herds was conducted in Washington State to estimate the rate of introduction of new multidrug-resistant (MDR) Salmonella enterica strains onto commercial dairy herds. Samples were collected on these herds over 7 visits separated by intervals of 2 to 4 mo over a period of 15 to 21 mo. Samples were cultured for Salmonella spp. and serogroup, serovar, and antimicrobial susceptibility patterns were identified for MDR Salmonella isolates. Fingerprinting generated by pulsed-field gel electrophoresis (PFGE) using XbaI restriction enzyme digestion generated genotyping profiles for all MDR isolates identified in the study. The rate of new MDR Salmonella strain introduction was 0.9 per herd-year (95% confidence interval: 0.6-1.4). The rates for the most commonly introduced MDR Salmonella serovars were 0.4/herd-year for Typhimurium, 1.2/herd-year for Newport, and 0.1/herd-year for Dublin. Thirty-three of 59 herds (56%) had at least one new MDR Salmonella introduction during the study period. The number of new MDR Salmonella strains acquired by dairy herds ranged from zero to 8. Thirteen of the 59 herds had a history of clinical salmonellosis. Among these 13 herds, 6 herds acquired new MDR Salmonella strains, although these strains were different than historical clinical strains. These data indicate that acquisition of new MDR Salmonella strains by dairy herds was a common event in participating herds, although the number of strains introduced varied greatly among herds.     

Molecular characterization of antibiotic resistant Salmonella isolates from Indian foods

Salmonella isolates belonging to five serovars, Salmonella enterica Ohio, S. Oslo, S. Tennessee, S. Weltevreden and S. Typhimurium, isolated during 2006-2008 from food samples like sprouts and different varieties of fresh water and marine fish were tested for antibiotic resistance. High percentages (97%) of the isolates were resistant to at least one antibiotic and 82% of the isolates were resistant to more than one antibiotic. S. Oslo was the most resistant serovar and it exhibited resistance to 13 out of 16 antibiotics tested. Integron 1, which has been shown to confer multidrug resistance to various Salmonella serovars, was detected in multidrug resistant S. Oslo. PFGE studies revealed that serovars showed very high genetic diversity. The multidrug resistant S. Oslo showed unique PFGE pattern, which could be used in epidemiological studies.     

Prevalence and antibiotic resistance of Salmonella serovars in ducks,duck rearing and processing environments in Penang,Malaysia

An investigation was carried out to determine the prevalence and antibiotic resistance of Salmonella serovars in ducks, their rearing and processing environments in Penang, Malaysia. A total of 531 samples collected from wet markets and duck farms, were examined from August 2009 to October 2010. The overall prevalence of Salmonella serovars was 23.5% (125/531). The 125 Salmonella isolates belong to 10 different serovars namely Typhimirium (29.6%), Enteritidis (12.0%), Gallinarum (2.4%), Braenderup (12.0%), Albany (11.2%), Hadar (20.8%), Derby (6.4%), Weltevreden (1.6%), Newbrunswick (3.4%) and London (0.8%). Salmonella serovars also showed various resistance patterns against 13 different antibiotics. All the serovars were resistant to erythromycin but susceptible to cephalothin, gentamicin and ceftriaxone. Plasmids were detected in 91 (72.8%) of the isolates with sizes ranging from 1.4 to 23.1 Kbp. Our findings provide baseline information on the distribution of Salmonella serovars in ducks, their rearing and processing environments, and indicate that ducks should be considered as an important source of food-borne pathogens.     

Antimicrobial susceptibility of Salmonella from organic and conventional dairy farms

The objective of this study was to compare antimicrobial susceptibility of Salmonella isolated from conventional and organic dairy farms in the Midwest and Northeast United States. Environmental and fecal samples were collected from organic (n = 26) and conventional (n = 69) farms in Michigan, Minnesota, New York, and Wisconsin every 2 mo from August 2000 to October 2001. Salmonella isolates (n = 1,243) were tested using a broth microdilution method for susceptibility to amoxicillin-clavulanic acid, ampicillin, ceftiofur, ceftriaxone, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim-sulfamethoxazole. Herd-level logistic regression and logistic proportional hazards multivariable models were used to examine the association between farm management type and susceptibility to antimicrobial agents. For most antimicrobial agents tested, susceptibility of Salmonella isolates was similar on organic and conventional herds when controlling for herd size and state. Conventional farms were more likely to have at least one Salmonella isolate resistant to streptomycin using logistic regression (odds ratio = 7.5; 95% confidence interval = 1.7-5.4). Conventional farms were more likely to have Salmonella isolates with greater resistance to streptomycin (odds ratio = 5.4; 95% confidence interval = 1.5-19.0) and sulfamethoxazole (odds ratio = 4.2; 95% confidence interval = 1.2-14.1) using logistic proportional hazards models. Although not statistically significant, conventional farms tended to be more likely to have at least one Salmonella isolate resistant to 5 or more antimicrobial agents when compared with organic farms.     

Molecular diversity of Staphylococcus aureus and the role of milking equipment adherences or biofilm as a source for bulk tank milk contamination

Staphylococcus aureus is one of the most frequent pathogens causing intramammary infections in dairy herds. Consequently, virulence factors, pathobiology, and epidemiology of Staphylococcus aureus strains have been widely assessed through the years. Nevertheless, not much has been described about the epidemiology of Staph. aureus strains from bulk tank milk (BTM) and adherences on milking equipment (AMES), even when these strains may play a role in the quality of milk that is intended for human consumption. The objective of this study was to assess the strain diversity of 166 Staph. aureus isolates collected from 3 consecutive BTM samples, and from AMES in contact with milk from 23 Chilean dairy farms. Isolates were analyzed and typed using pulsed-field gel electrophoresis. Diversity of strains, both within and among farms, was assessed using Simpson's index of diversity (SID). On farms where Staph. aureus was isolated from both AMES and BTM (n = 8), pulsotypes were further analyzed to evaluate the role of AMES as a potential source of Staph. aureus strains in BTM. Among all Staph. aureus analyzed by pulsed-field gel electrophoresis, a total of 42 pulsotypes (19 main pulsotypes and 23 subtypes) were identified. Among dairy farms, strain diversity was highly heterogeneous (SID = 0.99). Within dairy farms, Staph. aureus strain diversity was variable (SID = 0 to 1), and 18 dairy operations (81.8%) had one pulsotype that was shared between at least 2 successive BTM samples. In those farms where Staph. aureus was isolated in both AMES and BTM (n = 8), 7 (87.5%) showed a clonal distribution of Staph. aureus strains between these 2 types of samples. The overlapping of certain Staph. aureus strains among dairy farms may point out common sources of Staph. aureus among otherwise epidemiologically unrelated farms. Indistinguishable Staph. aureus strains between AMES and BTM across dairy farms suggest that Staph. aureus–containing AMES may represent a source for BTM contamination, thus affecting milk quality. Our study highlights the role of viable Staph. aureus in AMES as a source for BTM contamination on dairy farms, and also describes the overlapping and presence of specific BTM and AMES pulsotypes among farms.     

Prevalence of Salmonella infecting bacteriophages associated with Ontario pig farms and the holding area of a high capacity pork processing facility

BACKGROUND: There is interest in applying bacteriophages to control Salmonella in pig production and pork processing. The following reports on the prevalence of Salmonella infecting bacteriophages within Ontario pig farms and associated with the holding area of a pork slaughterhouse. RESULTS: Salmonella infecting bacteriophages were present in 30 and 28 of the effluent manure samples collected from 36 farms using S. Typhimurium DT104 or S. Heidelberg as host cell respectively. Bacteriophages were recovered in 95–100% of the 48 samples taken from holding pens within a high capacity slaughterhouse over a 12 month period. Bacteriophages isolated from farms exhibited similar host ranges which differed to that of slaughterhouse isolates. Salmonella (n = 21) from the slaughterhouse were susceptible to the endogenous bacteriophages. Despite being susceptible to the resident phages, the Salmonella populations were found to be genetically stable with the same genotypes being recovered over successive visits. Salmonella isolated from the farms were frequently resistant to the endogenous phages. CONCLUSIONS: Bacteriophages are prevalent in the pig slaughterhouse environment although they do not have a significant impact on the genetic structure of Salmonella populations. However, there was evidence that the Salmonella population structure on farms is influenced by the presence of infecting phages. Copyright © 2010 Society of Chemical Industry     

Prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples and their potential as biocontrol agents

The prevalence of bacteriophages infecting Staphylococcus aureus in dairy samples was assessed. Fourteen Staph. aureus strains were used in enrichment cultures of 75 dairy samples. All samples grew specific Staph. aureus bacteriophages. According to the host range, 8 different phages were isolated. Three of them, phages ΦH5, ΦG7, and ΦA72, were found in 89% of the samples; all the isolated phages were temperate. Phages ΦH5 and ΦA72 were used in preliminary bacterial challenge tests against Staph. aureus in milk. A phage mixture (1:1) was more effective than each single phage, most likely by preventing the survival of lysogenized cells. Phages inhibited Staph. aureus in UHT and pasteurized whole-fat milk. However, the phages were less active in semi-skimmed raw milk and little inhibition was achieved in whole, raw milk. Killing of Staph. aureus was observed at room temperature and at 37°C, but not at refrigeration temperature.     

Diversity of Salmonella isolates using serotyping and multilocus sequence typing

One hundred and twenty-one Salmonella isolates were obtained from food, feed, and live chicken samples derived from 13 countries or regions. In this study, their subtypes were evaluated by serotyping and multilocus sequence typing (MLST), and their genetic profiles were also characterized. It was demonstrated by serotyping on these isolates that 36 various serovars were obtained in this study, of which three serotypes S. Babelsberg, S. Fresno, and S. II were first found in mainland China. Based on Simpson’s index of diversity, the serotyping method had a 0.943 discriminatory power. Meanwhile, there were a total of 42 unique sequence types (STs) characterized by MLST, and the discriminatory power of MLST (D = 0.947) was close to that of the serotyping method. In MLST, hisD revealed the highest levels of nucleotide diversity. In addition, ST-92 was the most common ST represented by 16 Salmonella isolates, followed by ST-367 which was represented by 14 isolates. Seven new alleles were identified, which were associated with other alleles and resulted in the assignment of nine new STs. It was concluded from the results that MLST was generally associated with serotype, but not associated with the epidemiological source of the samples, and antimicrobial resistance patterns.     

Antibiogram and coagulase diversity in staphylococcal enterotoxin-producing Staphylococcus aureus from bovine mastitis

We investigated antibiogram and coagulase gene diversity in staphylococcal enterotoxin (StE)-producing Staphylococcus aureus isolated from raw milk samples of cows infected with mastitis from 140 dairy farms in Korea between 1997 and 2004. Of the 696 Staph. aureus isolates collected in this study, 164 isolates (23.6%) produced one or more staphylococcal enterotoxins (A to D), and 19 isolates (2.7%) were methicillin-resistant. The percentage of StE-producing Staph. aureus (SES) isolates resistant to methicillin, kanamycin, neomycin, amikacin, and tetracycline was greater than that of non-SES. Ten coagulase genotype patterns were observed, including 4 main types comprising I (25.4%), II (13.9%), VII (13.2%), and VIII (17.8%). More than 4 Staph. aureus types were isolated from each of 82 dairy farms in different geographic locations, and only 1 coagulase genotype pattern was observed in 39 of the herds (47.6%). There was no significant correlation between coagulase genotypes harbored by Staph. aureus and their specific StE type. The percentage of isolates producing major StE types (A, B, AC, and ABCD) and being resistant to cephalothin and methicillin was greater among the Staph. aureus isolates with the 4 predominant coagulase genotypes (I, II, VII, and VIII) than among the isolates harboring the 6 rare coagulase types (III, IV, V, VI, IX, and X). Based on coagulase gene polymorphisms, our data indicate that a broad distribution of identical or closely related enterotoxin-producing Staph. aureus strains seem to contribute to bovine mastitis in the Republic of Korea.     

Genetic diversity and molecular epidemiology of outbreaks of Klebsiella pneumoniae mastitis on two large Chinese dairy farms

Klebsiella pneumoniae is an opportunistic and environmental mastitis-causing pathogen, with potential for contagious transmission. Repetitive element sequence-based PCR was used to determine genetic diversity and explore potential transmission and reservoirs for mastitis caused by K. pneumoniae on 2 large Chinese dairy farms. A total of 1,354 samples was collected from the 2 dairy farms, including milk samples from cows with subclinical and clinical mastitis, bedding, feces, feed, teat skin, and milking liners. Environmental samples were collected from all barns and milking parlors and extramammary samples from randomly selected dairy cows on both farms. In total, 272 and 93 K. pneumoniae isolates were obtained from Farms A and B, respectively (with ~8K and 2K lactating cows, respectively). Isolation rates from clinical mastitis (CM), subclinical mastitis (SCM), and environmental or extramammary samples were 34, 23 and 37%, respectively for Farm A and 42, 3, and 34% for Farm B. The K. pneumoniae isolated from CM milk and extramammary or environmental sources had high genetic diversity (index of diversity >90%) on the 2 farms and from SCM on Farm A. However, on Farm B, 9 SCM isolates were classified as 2 genotypes, resulting in a relatively low index of diversity (Simpson's index of diversity = 0.39; 95% CI = 0.08–0.70). Genotypes of K. pneumoniae causing mastitis were commonly detected in feces, bedding, and milking liners (Farm A), or from teat skin, sawdust bedding, and feed (Farm B). Based on its high level of genetic diversity, we inferred K. pneumoniae was an opportunistic and environmental pathogen causing outbreaks of CM on these 2 large Chinese dairy farms. Nevertheless, that only a few genotypes caused SCM implied some strains had increased udder adaptability and a contagious nature or a common extramammary source. Finally, control of intramammary infections caused by K. pneumoniae on large Chinese dairy farms must consider farm-level predictors, as the 2 outbreaks had distinct potential environmental sources of infection.     

Biodiversity of bacteriophages infecting Lactococcus lactis starter cultures

In the current study, we characterized 137 Lactococcus lactis bacteriophages that had been isolated between 1997 and 2012 from whey samples obtained from industrial facilities located in 16 countries. Multiplex PCR grouping of these 137 phage isolates revealed that the majority (61.31%) belonged to the 936 group, with the remainder belonging to the P335 and c2 groups (23.36 and 15.33%, respectively). Restriction profile analysis of phage genomic DNA indicated a high degree of genetic diversity within this phage collection. Furthermore, based on a host-range survey of the phage collection using 113 dairy starter strains, we showed that the c2-group isolates exhibited a broader host range than isolates of the 936 and P335 groups.     

Determination of water quality variables, endotoxin concentration, and Enterobacteriaceae concentration and identification in southern High Plains dairy lagoons

The objectives of this study were to determine the concentration of endotoxin, determine 20 water quality variables, and identify and enumerate fungal and bacterial pathogens from United States southern High Plains dairy lagoons and control lakes during summer and winter. Water samples were collected in triplicate from the north, south, east, and west quadrants of each body of water. The mean (± SEM) winter dairy lagoon endotoxin concentration was significantly higher (9,678 ± 1,834 ng/mL) than the summer concentration (3,220 ± 810 ng/mL). The mean endotoxin concentration of the 2 control lakes (summer: 58.1 ± 8.8 ng/mL; winter: 38.6 ± 4.2 ng/mL) was significantly less than that of the dairy lagoons. Two hundred-one Salmonella enterica spp. isolates were identified, 7 serovars were recovered from the dairy lagoons, and 259 Salmonella ssp. were identified from 5 other dairy locations (milk barn, ditch effluent, settling basin, feed alley pad flush, and center pivots). Twenty-eight Salmonella spp. were identified from center pivot water. Escherichia coli O157:H7 pathogens were isolated from other dairy locations but not from lagoons. Neither Salmonella spp. nor E. coli O157:H7 were identified from control lakes. Enterobacteriaceae opportunistic pathogens were isolated from both dairies and control lakes. Important mesophilic and thermophilic catabolic (to manure biosolids) fungal isolates were identified from dairy effluent locations, but no thermophilic fungal isolates were cultured from the control lakes. Adequate curing of green forage following center pivot irrigation is important to kill lagoon water enteric pathogens, even though the lagoon water is mixed with fresh water. Recirculating lagoon water to flush the feed alley pad, where cows stand while eating, to remove manure and using lagoon water to abate dairy dust in loafing pens and unimproved dairy roads is inconsistent with good environmental practice management.     

Bacteriophages BCP1-1 and BCP8-2 require divalent cations for efficient control of Bacillus cereus in fermented foods

Bacillus cereus is a foodborne bacterial pathogen that causes diarrhea and vomiting. In this study, the usefulness of bacteriophages to eradicate B. cereus from fermented foods was investigated. A total of 13 phages were isolated from Korean fermented food products, and 2 (BCP1-1 and BCP8-2) were further characterized. Transmission electron microscopy (TEM), restriction enzyme digestion pattern analysis, and SDS-PAGE of the structural proteins suggest that both phages belong to the family Myoviridae, containing approximately 150 kbp-long genomes. The host ranges of both phages were limited to B. cereus group species (12/13), as they were not able to lyse other Gram-positive or negative strains including Bacillus subtilis. Purified phages were used to inhibit B. cereus growth in a model fermented food system, cheonggukjang, a fast-fermented soybean paste product. BCP1-1 and BCP8-2 were able to effectively eradicate B. cereus from the food only if divalent cations (Ca²⁺, Mg²⁺, or Mn²⁺) were added to the medium. Further studies reveal that divalent cations are essential for phage adsorption, while a monovalent cation (Na⁺) is required for the post-adsorption phase of phage infection. Taken together, our findings imply that a phage could be an ideal anti-bacterial agent for use in fermented food products that require the presence of beneficial microflora and, during phage application, optimization of phage reaction conditions is critical for the successful utilization of phage biocontrol.