Silage Collected from Dairy Farms Harbors an Abundance of Listeriaphages with Considerable Host Range and Genome Size Diversity

Since the food-borne pathogen Listeria monocytogenes is common in dairy farm environments, it is likely that phages infecting this bacterium (“listeriaphages”) are abundant on dairy farms. To better understand the ecology and diversity of listeriaphages on dairy farms and to develop a diverse phage collection for further studies, silage samples collected on two dairy farms were screened for L. monocytogenes and listeriaphages. While only 4.5% of silage samples tested positive for L. monocytogenes, 47.8% of samples were positive for listeriaphages, containing up to >1.5 × 10⁴ PFU/g. Host range characterization of the 114 phage isolates obtained, with a reference set of 13 L. monocytogenes strains representing the nine major serotypes and four lineages, revealed considerable host range diversity; phage isolates were classified into nine lysis groups. While one serotype 3c strain was not lysed by any phage isolates, serotype 4 strains were highly susceptible to phages and were lysed by 63.2 to 88.6% of phages tested. Overall, 12.3% of phage isolates showed a narrow host range (lysing 1 to 5 strains), while 28.9% of phages represented broad host range (lysing ≥11 strains). Genome sizes of the phage isolates were estimated to range from approximately 26 to 140 kb. The extensive host range and genomic diversity of phages observed here suggest an important role of phages in the ecology of L. monocytogenes on dairy farms. In addition, the phage collection developed here has the potential to facilitate further development of phage-based biocontrol strategies (e.g., in silage) and other phage-based tools.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Catalase and superoxide dismutase activities after heat injury of Listeria monocytogenes.

Four strains of Listeria monocytogenes were examined for catalase (CA) and superoxide dismutase (SOD) activities. The two strains having the highest CA activities (LCDC and Scott A) also possessed the highest SOD activities. The CA activity of heated cell extracts of all four strains examined decreased sharply between 55 and 60 degrees C. SOD was more heat labile than CA. Two L. monocytogenes strains demonstrated a decline in SOD activity after heat treatment at 45 degrees C, whereas the other two strains demonstrated a decline at 50 degrees C. Sublethal heating of the cells at 55 degrees C resulted in increased sensitivity to 5.5% NaCl. Exogenous hydrogen peroxide was added to suspensions of L. monocytogenes; strains producing the highest CA levels showed the greatest H2O2 resistance.     

Classification of virulent and temperate bacteriophages of Listeria spp. on the basis of morphology and protein analysis.

A set of 22 phages of Listeria species (listeriaphages) (21 temperate and 1 virulent) were compared on the basis of morphology and protein composition. All 22 phages had icosahedral heads and exhibited either contractile or noncontractile tails. They represented two different morphotypes. Twenty phages belonged to the Siphoviridae family and could be differentiated only on the basis of tail length. Accordingly, they could be assigned to previously defined listeriaphage species. Two other phages were classified as members of the Myoviridae family, one of which (A511) should be regarded as a new species. It was found to be substantially different from all other known listeriaphages. All phages exhibited typical protein profiles, which were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent laser densitometrical analysis of the gels. It was then possible to distinguish eight protein subgroups on the basis of unique protein patterns. This classification corresponds well to the previous groupings based on host range. Most of the phages revealed two or three major proteins ranging from 21 to 24 kDa and 30 to 36 kDa. In addition, at least 10 minor proteins could be observed for each phage. Our results indicate that the major proteins are structural proteins of the capsid and tail, and the protein band ranging from 30 to 35 kDa could clearly be assigned to the proteins of the phage capsid.     

Host ranges of Listeria-specific bacteriophages from the turkey processing plant environment in the United States

Even though at least 400 Listeria phages have been isolated from various sources, limited information is available on phages from the food processing plant environment. Phages in the processing plant environment may play critical roles in determining the Listeria population that becomes established in the plant. In this study, we pursued the isolation of Listeria-specific phages from environmental samples from four turkey processing plants in the United States. These environmental samples were also utilized to isolate Listeria spp. Twelve phages were isolated and classified into three groups in terms of their host range. Of these, nine (group 1) showed a wide host range, including multiple serotypes of Listeria monocytogenes, as well as other Listeria spp. (L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii). The remaining phages mostly infected L. monocytogenes serotype 4b as well as L. innocua, L. ivanovii, and/or L. welshimeri. All but one of the strains of the serotype 4b complex (4b, 4d, 4e) from the processing plant environment could be readily infected by the wide-host-range phages isolated from the environment of the processing plants. However, many strains of other serotypes (1/2a [or 3a] and 1/2b [or 3b]), which represented the majority of L. monocytogenes strains isolated from the environmental samples, were resistant to infection by these phages. Experiments with two phage-resistant strains showed reduced phage adsorption onto the host cells. These findings suggest that phage resistance may be an important component of the ecology of L. monocytogenes in the turkey processing plants.     

Inactivation of barotolerant strains of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by ultra high pressure and <Emphasis Type="Italic">tert</Emphasis>-butylhydroquinone combination

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23+/-2 degrees C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (> or = 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.     

AFFINITY CONCENTRATION OF LISTERIA MONOCYTOGENES CELLS ON CONCANAVALIN A-COATED POLYESTER CLOTH AND SUBSEQUENT DETECTION BY THE POLYMERASE CHAIN REACTION

The Canavalia ensiformis lectin concanavalin A (con A) was immobilized on macroporous polyester cloth to form an inexpensive high surface area adsorbent (con A-cloth) for the affinity concentration of bacterial cells in aqueous suspensions. When the con A-cloth was packed into a 1 ml pipette tip, the resulting mini-column could be used to concentrate large volumes of dilute L. monocytogenes cell suspensions, followed by the polymerase chain reaction (PCR) amplification of L. monocytogenes-specific hly A sequences from lysates of the captured cells. This improved the effective sensitivity of the PCR as compared to the assay of L. monocytogenes in unconcentrated suspensions. Several enrichment broths (Fraser Broth, Listeria Enrichment Broth and Brain Heart Infusion Broth) were found to be inhibitory to the PCR of L. monocytogenes when introduced directly into the reaction mixture. This inhibitory effect was completely eliminated when the L. monocytogenes cells were captured on the con A-cloth and washed to remove the enrichment broth components prior to performing the PCR. Since the lectin con A is reactive with a broad variety of Gram positive and Gram negative bacteria, this simple and inexpensive affinity concentration method should be applicable to the PCR detection of other pathogens in enrichment cultures.     

Thermal and chemical resistance of Lactobacillus casei and Lactobacillus paracasei bacteriophages

AIMS: The survival of two collection Lactobacillus casei and L. paracasei bacteriophages when subjected to thermal and chemical treatments was investigated. METHODS AND RESULTS: Thermal resistance was evaluated by heating phage suspensions at 63, 72 and 90 degrees C in three different media [Tris-magnesium gelatin (TMG) buffer: 10 mmol l(-1) Tris-Cl, 10 mmol l(-1) MgSO(4) and 0.1% w/v gelatin; Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)]. A marked heat sensitivity was evident in both phages, as 15 min at 72 degrees C was enough to completely inactivate (6 log(10) reduction) them. No clear influence was demonstrated by the suspension media. The phages also showed similar resistance to biocides. Peracetic acid and sodium hypochlorite (800 ppm) were the most effective ones, destroying the phages within 5 min. Concentrations of 75 and 100% ethanol were not suitable to inactivate phage particles even after 45 min. Isopropanol did not show an effect on phage viability. CONCLUSIONS: The data obtained in this work are important to design more effective control procedures in order to inactivate phages in dairy plants and laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute to enhance the background knowledge about phages of probiotic bacteria.     

Catalase, superoxide dismutase, and hemolysin activities and heat susceptibility of Listeria monocytogenes after growth in media containing sodium chloride.

The activities of catalase, superoxide dismutase, and a thiol-activated hemolysin produced by four strains of Listeria monocytogenes propagated in media containing various concentrations of sodium chloride were examined. L. monocytogenes 7644 showed an increase in catalase, superoxide dismutase, and thiol-activated hemolysin activities when grown in a medium containing 2.5% (wt/vol) NaCl followed by a decrease in activities when propagated in media containing salt concentrations higher than 2.5%. L. monocytogenes LCDC 81-861 demonstrated enhanced catalase activity when grown in media containing NaCl ranging from 1.5 to 4.6% and increased superoxide dismutase activity when propagated in media containing 1.5 to 3.5% NaCl. L. monocytogenes LCDC 81-861 did not exhibit any detectable hemolysin activity under the conditions tested. After growth in various NaCl-containing media, both strains were subjected to sublethal heat injury for 30 min at 55 degrees C. L. monocytogenes LCDC 81-861 showed increased sensitivity to the heat treatment when grown in media containing 4.6 and 6.5% NaCl, whereas L. monocytogenes 7644 did not exhibit enhanced heat lability.     

Catalase, superoxide dismutase, and hemolysin activities and heat susceptibility of Listeria monocytogenes after growth in media containing sodium chloride

The activities of catalase, superoxide dismutase, and a thiol-activated hemolysin produced by four strains of Listeria monocytogenes propagated in media containing various concentrations of sodium chloride were examined. L. monocytogenes 7644 showed an increase in catalase, superoxide dismutase, and thiol-activated hemolysin activities when grown in a medium containing 2.5% (wt/vol) NaCl followed by a decrease in activities when propagated in media containing salt concentrations higher than 2.5%. L. monocytogenes LCDC 81-861 demonstrated enhanced catalase activity when grown in media containing NaCl ranging from 1.5 to 4.6% and increased superoxide dismutase activity when propagated in media containing 1.5 to 3.5% NaCl. L. monocytogenes LCDC 81-861 did not exhibit any detectable hemolysin activity under the conditions tested. After growth in various NaCl-containing media, both strains were subjected to sublethal heat injury for 30 min at 55 degrees C. L. monocytogenes LCDC 81-861 showed increased sensitivity to the heat treatment when grown in media containing 4.6 and 6.5% NaCl, whereas L. monocytogenes 7644 did not exhibit enhanced heat lability.     

Two novel type II restriction-modification systems occupying genomically equivalent locations on the chromosomes of Listeria monocytogenes strains

Listeria monocytogenes is responsible for the potentially life-threatening food-borne disease listeriosis. One epidemic-associated clonal group of L. monocytogenes, epidemic clone I (ECI), harbors a Sau3AI-like restriction-modification (RM) system also present in the same genomic region in certain strains of other lineages. In this study, we identified and characterized two other, novel type II RM systems, LmoJ2 and LmoJ3, at this same locus. LmoJ2 and LmoJ3 appeared to recognize GCWGC (W = A or T) and GCNGC, respectively. Both RM systems consisted of genes with GC content below the genome average and were in the same genomic region in strains of different serotypes and lineages, suggesting site-specific horizontal gene transfer. Genomic DNA from the LmoJ2 and LmoJ3 strains grown at various temperatures (4 to 42°C) was resistant to digestion with restriction enzymes recognizing GCWGC or GCNGC, indicating that the methyltransferases were expressed under these conditions. Phages propagated in an LmoJ2-harboring strain exhibited moderately increased infectivity for this strain at 4 and 8°C but not at higher temperatures, while phages propagated in an LmoJ3 strain had dramatically increased infectivity for this strain at all temperatures. Among the sequenced Listeria phages, lytic phages possessed significantly fewer recognition sites for these RM systems than lysogenic phages, suggesting that in lytic phages sequence content evolved toward reduced susceptibility to such RM systems. The ability of LmoJ2 and LmoJ3 to protect against phages may affect the efficiency of phages as biocontrol agents for L. monocytogenes strains harboring these RM systems.     

Functional activity of mouse peritoneal macrophages contaminated with gram-positive bacteria

The analysis of the functional and enzymatic activity of mouse peritoneal macrophages contaminated with Staphylococcus aureus and Listeria monocytogenes virulent strains is presented. The low bactericidal and digestive activity of these cells with respect to the above-mentioned microorganisms was determined. In this study a decrease in the activity of plasmatic membrane enzymes (5'-nucleotidase and ATP-ase) of macrophages contaminated with S. aureus and L. monocytogenes was observed, which was indicative of the stimulation of phagocytes. A rise in the activity of the oxygen-dependent system of macrophages contaminated with S. aureus and L. monocytogenes was detected by means of the nitro blue tetrazolium test. At the same time a decrease in the intracellular content of nitrogen oxide end metabolites in macrophages was detected with a rise in content of nitrogen oxide in the supernatants.     

Expression of superoxide dismutase in Listeria monocytogenes.

The nature and expression of superoxide dismutase (SOD; EC 1.15.1.1) in the gram-positive food-borne pathogen Listeria monocytogenes were examined. Metal depletion and reconstitution studies and resistance to H2O2 and potassium cyanide inactivation indicated that L. monocytogenes has a single SOD which utilizes manganese as a metal cofactor. The specific activity of SOD was unchanged in cells exposed to a heat shock at 42 degrees C or grown in the presence of paraquat-generated superoxide anion or of metal chelators in the medium. SOD levels increased, however, as the cells progressed through the logarithmic phase of growth and into the stationary phase. Furthermore, SOD activity decreased with decreasing growth temperatures and declined concurrently with decreased growth when higher concentrations of sodium chloride were added to the medium. Cells grown anaerobically possessed relatively high levels of SOD, although these levels were about 10 to 30% lower than those of aerobically grown bacteria. Different isolates of L. monocytogenes were found to produce approximately equivalent levels of SOD, although greater differences in SOD expression were seen among other species of Listeria. When compared with L. monocytogenes, for example, Listeria welshimeri typically produced about 30% greater SOD activity, whereas Listeria murrayi produced about 60% less total SOD activity. Although all species of Listeria produced a single Mn-type SOD, differences in the relative electrophoretic mobility of the native enzymes were noted. These data suggest that the single L. monocytogenes SOD enzyme is constitutively produced in response to many environmental factors and may also be responsive to the cellular growth rate.     

Ultrastructural changes and diosgenin content in cell suspensions of <Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis> L. by ethylene treatment

This paper studies the effect of different concentrations of ethephon (an ethylene releasing compound) on the level of the steroidal sapogenin diosgenin in cell suspensions of Trigonella foenum-graecum L. The results reveal that diosgenin synthesis and/or accumulation was stimulated by 5 ppm ethephon treatment, an increase of 126% being observed, while concentrations of 25 ppm and 50 ppm reduced the levels of this secondary compound. Changes in the isopentenyl diphosphate isomerase activity, increased cell diameter, decreased cell packing (with all the ethephon concentrations assayed), increased cytoplasmic density (5 ppm ethephon treatment) and alterations in the membrane structures (25 and 50 ppm ethephon treatments) were also observed.     

Inhibitory effects of raw carrots on Listeria monocytogenes

The survival and growth of two strains of Listeria monocytogenes on raw and cooked carrots stored at 5 and 15 degrees C and in carrot juice media at 30 degrees C were investigated. The influence of shredding, chlorine treatment, and packaging under an atmosphere containing 3% O2 and 97% N2 on the behavior of L. monocytogenes and naturally occurring microflora was determined. Populations of viable L. monocytogenes decreased upon contact with whole and shredded raw carrots but not cooked carrots. Viable populations also decreased in cell suspensions in which raw carrots were dipped. Small populations of L. monocytogenes detected on whole carrots immediately after dipping were essentially nondetectable after 7 days of storage at 5 or 15 degrees C. After a lag of 7 days at 5 degrees C, significant (P less than or equal to 0.05) increases in populations were detected on shredded carrots after 24 days of storage. Carrots stored at 5 or 15 degrees C spoiled before L. monocytogenes grew. Populations of mesophilic aerobes, psychrophiles, and yeasts and molds increased throughout storage at 5 and 15 degrees C. Cutting treatment (whole or shredded carrots), chlorine treatment, and modified-atmosphere packaging had no effect on the survival or growth of L. monocytogenes or naturally occurring microflora. The presence of raw carrot juice in tryptic phosphate broth at a concentration as low as 1% substantially reduced the maximum population of L. monocytogenes reached after 24 h at 30 degrees C. The anti-Listeria effect of carrots was essentially eliminated when the carrots were cooked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10 degrees C but increased significantly on fresh-cut honeydew melons stored at 10 degrees C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.     

Inhibitory effects of raw carrots on Listeria monocytogenes.

The survival and growth of two strains of Listeria monocytogenes on raw and cooked carrots stored at 5 and 15 degrees C and in carrot juice media at 30 degrees C were investigated. The influence of shredding, chlorine treatment, and packaging under an atmosphere containing 3% O2 and 97% N2 on the behavior of L. monocytogenes and naturally occurring microflora was determined. Populations of viable L. monocytogenes decreased upon contact with whole and shredded raw carrots but not cooked carrots. Viable populations also decreased in cell suspensions in which raw carrots were dipped. Small populations of L. monocytogenes detected on whole carrots immediately after dipping were essentially nondetectable after 7 days of storage at 5 or 15 degrees C. After a lag of 7 days at 5 degrees C, significant (P less than or equal to 0.05) increases in populations were detected on shredded carrots after 24 days of storage. Carrots stored at 5 or 15 degrees C spoiled before L. monocytogenes grew. Populations of mesophilic aerobes, psychrophiles, and yeasts and molds increased throughout storage at 5 and 15 degrees C. Cutting treatment (whole or shredded carrots), chlorine treatment, and modified-atmosphere packaging had no effect on the survival or growth of L. monocytogenes or naturally occurring microflora. The presence of raw carrot juice in tryptic phosphate broth at a concentration as low as 1% substantially reduced the maximum population of L. monocytogenes reached after 24 h at 30 degrees C. The anti-Listeria effect of carrots was essentially eliminated when the carrots were cooked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.

High pH has been shown to rapidly destroy gram-negative food-borne pathogens; however, the mechanism of destruction has not yet been elucidated. Escherichia coli O157:H7, Salmonella enteritidis ATCC 13706, and Listeria monocytogenes F5069 were suspended in NaHCO3-NaOH buffer solutions at pH 9, 10, 11, or 12 to give a final cell concentration of approximately 5.2 x 10(8) CFU/ml and then held at 37 or 45 degrees C. At 0, 5, 10, and 15 min the suspensions were sterilely filtered and each filtrate was analyzed for material with A260. Viability of the cell suspensions was evaluated by enumeration on nonselective and selective agars. Cell morphology was evaluated by scanning electron microscopy and transmission electron microscopy. A260 increased dramatically with pH and temperature for both E. coli and S. enteritidis; however, with L. monocytogenes material with A260 was not detected at any of the pHs tested. At pH 12, numbers of E. coli and S. enteritidis decreased at least 8 logs within 15 s, whereas L. monocytogenes decreased by only 1 log in 10 min. There was a very strong correlation between the initial rate of release of material with A260 and death rate of the gram-negative pathogens (r = 0.997). At pH 12, gram-negative test cells appeared collapsed and showed evidence of lysis while gram-positive L. monocytogenes did not, when observed by scanning and transmission electron microscopy. It was concluded that destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.     

Evaluation of the international phage typing set and some experimental phages for typing of Listeria monocytogenes from poultry in Spain

AIMS: The validity of the international phage set and 13 experimental phages for subtyping Listeria monocytogenes strains isolated from poultry in Spain was investigated. METHODS AND RESULTS: Ninety-six L. monocytogenes strains (52 from serogroup 1/2 and 44 from serogroup 4) were phage-typed using the international phage set, 10 experimental phages for typing serogroup 1/2 strains (seven isolated in France: 1313, 9425, 1807, 351, 881, 717 and 586-, and three from Denmark: 5775, 12682 and 6223-) and three experimental phages isolated in France for typing serogroup 4 strains (2425 A, 4286 and 197). Percentages of serogroup 1/2, serogroup 4 and total phage-typeable strains were 57.7%, 52.3% and 55.2%, respectively. Important differences in the behaviour of the phages tested were found. The typeability rate, the specificity index and the percentage of strong reactions were greater in the phages of international set than in the experimental phages. The number of phage typeable strains and the number of phage types (42) were not modified by the use of experimental phages. CONCLUSIONS: The phage set used was not effective for typing L. monocytogenes strains from poultry in Spain, because a low typeability rate was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest the importance of the availability of new phages specific to a geographical area in order to improve the typeability of the system.