Abstract:
A non-selective and selective media and method are provided for the rapid enrichment of  Listeria  from samples. The method includes inoculating a sample that may contain  Listeria  into a non-selective or selective medium. Also, a sample that may contain  Listeria  may be provided by an environmental sponge or swab. More specifically, a small volume of the medium is added directly to an environmental sample for one-step enrichment in a short time period. This invention enables shorter enrichment time, as genetic detection methods become more rapid and sensitive.

Description:
[0001]     The invention provides selective and non-selective media and methods for the rapid enrichment of  Listeria  from samples. More specifically, a small volume of the medium is added directly to an environmental sample for one-step enrichment in a short time period. The selective medium includes antimicrobial agents that favor the growth of  Listeria  spp. while inhibiting the growth of other microflora.  
       BACKGROUND  
       [0002]      Listeria  is classified as gram-positive, rod-shaped bacteria and consists of the species  Listeria monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii , and  L. grayi . Among these,  L. monocytogenes  is responsible for the majority of human listeriosis cases and immunocompromised, pregnant women, elderly, and newborns have increased susceptibility to infection. The most common symptoms of listeriosis are septicemia, meningitis, and miscarriages.  
         [0003]     A large number of methods for detecting  Listeria  are known. Conventional detection methods for  L. monocytogenes  comprise of pre-enriching and subsequently isolating colonies on selection media (Lovett et al., J. Food Protection 50 (1987), 188-192; McClain &amp; Lee, J. Assoc. Off. Anal. Chem. 71 (1988), 660-664). Single colonies are examined for their morphology and biochemical properties. An analysis may take up to 6-8 days.  
         [0004]     Some current industry accepted  Listeria  enrichments require a 24 to 48 hour incubation time with one to two enrichment steps. Many of the media formulations are selective and can be inhibitory towards  Listeria  spp. Often, selective agents do not support the resuscitation of injured organisms. As genetic detection methods become more rapid and sensitive, shorter and simpler enrichment is needed. In order to achieve an improved enrichment method, an enrichment medium should sustain an increased growth rate, resuscitate injured cells, and either inhibit growth of competitor organisms or allow  Listeria  spp. to grow to detectable levels through a competitive environment in a short time period.  
         [0005]     Rapid, one-step enrichment for  Listeria  is needed. This invention provides such methods.  
       SUMMARY  
       [0006]     Selective and non-selective  Listeria  spp. enrichment media are provided. Both media contain sources of protein, carbohydrates, vitamins, minerals, buffer and essential ions. In an important aspect, both media contain pyruvate in amounts effective for stimulating the metabolism of stressed organisms. The selective medium further includes antimicrobial agents that favor the growth of  Listeria  spp. while inhibiting the growth of other microflora. The enrichment media are effective for maximizing the growth of  Listeria  spp. over a ten to twelve hour incubation time. In this aspect, both selective and non-selective media are effective for supporting minimally 2 logs of  Listeria  spp. growth after 10 hours at 30° C.  
         [0007]     The non-selective enrichment medium for  Listeria  includes the following components:  
                                                                         Component   Range g/L   Preferred g/L                                        Pyruvate    2.0-10.0   2.5           Carbohydrate   0.2-2.5   0.5           Yeast Extract    1.0-10.0   3.0           Sodium Chloride   1.0-5.0   5.0           Magnesium Source   0.1-0.5   0.25           Iron Source   0.1-2.5   0.1           Protein    1.0-20.0   10.0           Buffer    1.0-25.0   Sufficient amount to                   maintain pH 6.0-7.3 during                   incubation.                      
 
         [0008]     The selective enrichment medium for  Listeria  includes the following components:  
                                                                         Component   Range g/L   Preferred g/L                                        Pyruvate    2.0-10.0   2.5           Carbohydrate   0.2-2.5   0.5           Yeast Extract    1.0-10.0   3.0           Sodium Chloride   1.0-5.0   5.0           Magnesium Source   0.1-0.5   0.25           Iron Source   0.1-2.5   0.1           Polymyxin B   0.007-0.06    0.007-0.025           Acriflavine HCl   0.004-0.03    0.004-0.015           Ceftazidime   0.015-0.12    0.015-0.045           Protein    1.0-20.0   10.0           Buffer    1.0-25.0   Sufficient amount to                   maintain pH 6.0-7.3                   during incubation                      
 
         [0009]     Pyruvate is included in the media to enhance growth and recovery of stressed cells. The pyruvate is provided to the medium as a salt of pyruvic acid, preferably a sodium salt of pyruvic acid. In an important aspect, the medium includes from about 2.0 to about 10.0 g/L sodium pyruvate.  
         [0010]     The medium further includes carbohydrate such as dextrose, esculin, maltose, amygdalin, cellobiose, fructose, mannose, salicin, dextrin, (x-methyl-D-glucoside and mixtures thereof. In an important aspect, the medium includes from about 0.2 to about 2.5 g/L dextrose.  
         [0011]     The medium further includes Yeast Extract. In an important aspect, the medium includes from about 1.0 to about 10.0 g/L Yeast Extract.  
         [0012]     The medium further includes salts such as sodium, potassium, or calcium salts of chloride. In an important aspect, the medium includes from about 1.0 to about 5.0 g/L sodium chloride.  
         [0013]     Essential ions such as magnesium and iron are also included in the media. Magnesium which may be used including magnesium selected from the group consisting of magnesium sulfate, magnesium chloride, and mixtures thereof. Iron which may be used inludes iron selected from the group consisting of ferric ammonium citrate, ferrous sulfate, ferric sulfate, ferric citrate, ferrous ammonium sulfate, ferric chloride, and mixtures thereof. In an important aspect, the medium includes from about 0.1 to about 0.5 g/L magnesium sulfate heptahydrate and from about 0.1 to about 2.5 g/L ferric ammonium citrate.  
         [0014]     The medium further includes protein, which may be provided from a variety of sources. For example, the protein may be provided from sources such as Tryptone, Tryptose, Soytone, Peptone, Pantone, Bitone, Proteose Peptone, pancreatic digest of gelatin, and mixtures thereof. In an important aspect, the medium includes from about 1.0 to about 20.0 g/L protein.  
         [0015]     The medium further includes buffers, which are effective for maintaining the pH in a desired range. For example, buffers that may be used include buffers such as potassium phosphate monobasic, potassium phosphate dibasic, sodium phosphate dibasic, and mixtures thereof. In an important aspect, the medium includes from about 1.0 to about 25.0 g/L buffer.  
         [0016]     The selective medium further comprises of antimicrobial agents. Antimicrobials that may be used in the present invention include selective agents from the group consisting of Polymyxin B, Acriflavine HCl, Ceftazidime, and mixtures thereof. In an important aspect, the medium includes from about 0.007 to about 0.06 g/L Polymyxin B, from about 0.004 to about 0.03 g/L Acriflavine HCl, and from about 0.015 to about 0.12g/L Ceftazidime.  
         [0017]     An enrichment method for  Listeria  is also provided that includes inoculating a sample which may contain  Listeria  into a non-selective or selective medium as described. Enrichment in non-selective or selective medium may be practiced with a food sample, which may contain  Listeria . Also, a sample which may contain  Listeria  may be provided by an environmental sponge or swab. The environmental sponge or swab may include a medium selected from the group consisting of buffered peptone water. 
     
    
     BRIEF DESCRIPTION OF THE FIGURE  
       [0018]      FIG. 1  illustrates  Listeria  growth at 30° C. as compared to commercially available media. 
     
    
     DETAILED DESCRIPTION  
       [0000]     Definitions  
         [0019]     The term “Listeria” as used herein, refers to the bacteria classified as such in Bergey&#39;s Manual of Systematic Bacteriology (P. H. A. Sneath (ed), 1986, 1234-1245, Williams &amp; Wilkins). Therefore, the term “Listeria” as used herein includes  Listeria monocytogenes, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria ivanovii , and  Listeria grayi.    
         [0020]     “Non-selective” medium does not contain additives that intentionally inhibit the growth of any organisms picked up by a swab or sponge. Other organisms in addition to  Listeria  are capable of growing in this medium. The media components are optimized to achieve maximum resuscitation and growth of  Listeria  spp.  
         [0021]     “Selective” medium contains additives that inhibit many organisms&#39; growth other than  Listeria  spp. The “selective” medium is designed to maximize the growth of  Listeria  spp. and reduce background microflora. There are several microorganisms that are unaffected by the selective components so not all non- Listeria  organisms are inhibited. The medium is not selective for different  Listeria  species since the objective is to enrich for all  Listeria.    
         [0000]     Medium Preparation  
         [0022]     Both the selective and non-selective media may be prepared using known methods. Generally, all ingredients are weighed out in deionized water and filter sterilized using a 0.2 μm (Nalgene) filter or autoclaved at 121° C. for 15 minutes. Selective agents are reconstituted with sterile water and added just prior to use of the medium.  
         [0000]     Sample Preparation  
         [0023]     Samples to be tested for the presence of  Listeria  spp. may be added directly to selective and non-selective media or may be collected on swabs or sponges. Pre-hydrated sponges and swabs are used to collect microorganism according to manufacturer&#39;s instructions and placed back in original container. Selective or non-selective medium is added directly to the container, vortexed or stomached for 10 seconds, and incubated at 30-37° C. Small volumes of medium, such as about 30 ml or less, preferably about 5 ml, may be added to the swab or sponge in preparation for the enrichment.  
       EXAMPLES  
     Example 1  
     Media Formulations  
       [0024]     The g/L (total) column is the composition of the media and the Buffered Peptone Water (BPW) combined if 5 ml of medium is added to a sponge or swab containing 10 ml BPW. The right column is the composition for the medium considered independently from BPW. In this example, the buffer and protein source are supplied by the BPW, but if growth and buffering capacity are insufficient, the medium may be supplemented with additional protein and buffer.  
                                                                     g/L (total)   g/L                                        NON-SELECTIVE MEDIUM                   Sodium Chloride   1.667   5.00           Dextrose   0.500   1.50           Magnesium Sulfate 7H 2 0   0.250   0.75           Ferric Ammonium Citrate   0.100   0.30           Sodium Pyruvate   2.500   7.50           Yeast Extract   3.000   9.00           Deionized Water   333.333   1000.00           INGREDIENTS FROM BPW IN           SPONGE/SWAB           Pancreatic Digest of Gelatin   6.667   10.0           Sodium Chloride   3.333   5.0           Disodium Phosphate   2.333   3.5           Monopotassium Phosphate   1.000   1.5           Deionized Water   666.667   1000           SELECTIVE MEDIUM “A”           Sodium Chloride   1.667   5.14           Dextrose   0.500   1.54           Magnesium Sulfate 7H 2 0   0.250   0.77           Ferric Ammonium Citrate   0.100   0.31           Sodium Pyruvate   2.500   7.71           Yeast Extract   3.000   9.25           Deionized Water   324.333   1000.00           Polymyxin B   0.023   2.50           Acriflavine HCl   0.011   1.25           Ceftazidime   0.045   5.00           Sterile Deionized Water   9.000   1000.00           INGREDIENTS FROM BPW IN           SPONGE/SWAB           Pancreatic Digest of Gelatin   6.667   10.0           Sodium Chloride   3.333   5.0           Disodium Phosphate   2.333   3.5           Monopotassium Phosphate   1.000   1.5           Deionized Water   666.667   1000           SELECTIVE MEDIUM “B”           Sodium Chloride   1.667   5.05           Dextrose   0.500   1.51           Magnesium Sulfate 7H 2 0   0.250   0.76           Ferric Ammonium Citrate   0.100   0.30           Sodium Pyruvate   2.500   7.57           Yeast Extract   3.000   9.08           Deionized Water   330.333   1000.00           Polymyxin B   0.008   2.50           Acriflavine HCl   0.004   1.25           Ceftazidime   0.015   5.00           Sterile Deionized Water   3.000   1000.00           INGREDIENTS FROM BPW IN           SPONGE/SWAB           Pancreatic Digest of Gelatin   6.667   10.0           Sodium Chloride   3.333   5.0           Disodium Phosphate   2.333   3.5           Monopotassium Phosphate   1.000   1.5           Deionized Water   666.667   1000                      
 
       Example 2  
     Comparison of Growth of  Listeria monocytogenes  in Non-Selective and Selective Media Compared to Commercially Available Media in Environmental Sponges  
       [0025]     The slowest growing strain in a collection of  Listeria  was used.  Listeria monocytogenes  (DUP-1038, Serotype 4b) was inoculated into Brain Heart Infusion broth (BHI, Difco) and incubated for 24 h at 30° C. to reach stationary phase. The cultures were held at 4° C. for 5 days after reaching stationary phase. To determine the growth level after refrigeration, the culture was serially diluted in Butterfield&#39;s Buffer (International BioProducts), pour-plated with Oxford Medium Base (Difco), and incubated at 35° C. for 24 h. Based on the cell counts, the culture was diluted in Butterfield&#39;s Buffer and a volume of the diluted culture targeting an initial cell count of 10 CFU/sponge was inoculated into environmental sponges containing Buffered Peptone Water (International BioProducts). In addition, the inoculum was pour plated with Oxford Medium Base. The plates were incubated at 35° C. for 24 h and the cell counts were collected to confirm the CFU/sponge at T 0 .  
         [0026]     Spiked sponges were processed by adding 5 ml of a medium to the sponge in the original manufacturer&#39;s container, followed by stomaching for 10 seconds, and incubation for 10 hours at 30° C. The sponges were stomached again for 10 seconds and serial dilutions were pour-plated with Oxford Medium Base. The plates were incubated at 35° C. for 24 h and the cell counts were collected and compared to the To count at 8 h and 10 h.  
         [0027]     Commercial media were compared to Non-Selective and Selective media listed in Example 1. Commercial media tests consisted of Tryptic Soy broth (TSB, Difco), Universal Preenrichment broth (UPB, Difco), and Demi-Fraser Broth Base (DF, Difco)  
                                 TABLE 1                           Change in Log CFU/ml  Listeria monocytogenes  during       10-hour incubation at 30° C. in environmental sponges                Media   8 h   10 h                       TSB   1.36   1.70           UPB   1.39   2.30           Demi-Fraser   1.39   1.84           Non-Selective   1.75   2.64           Selective “A”   1.10   1.76           Selective “B”   1.65   2.42                      
 
         [0028]     Results show Non-Selective medium supports higher growth of  Listeria monocytogenes  than the commercially available media tested. Selective “A” medium contains the highest concentration of selective agents in the preferred range and performs similarly to Demi-Fraser, a commercially acceptable selective medium often used for the first step in a two-step  Listeria enrichment . Selective “B” medium contains the lowest concentration of the selective agents in the preferred range and performs slightly better than UPB.  
       Example 3  
     Comparison of Growth of  Listeria  and Environmental Isolates to Determine the Effect of Environmental Isolates on  Listeria  Growth and the Effect of the Polymyxin B, Acriflavine HCl, and Ceftazidime on Environmental Isolates  
       [0029]     Nineteen non- Listeria  organisms isolated from environmental sponges from meat and dairy manufacturing facilities were inoculated separately into Tryptic Soy broth (TSB, Difco) and incubated for 24 h at 30° C. The isolates were screened for growth in Selective and Non-Selective media by monitoring absorbance via a spectrophotometer. 900 μl of test media and either 100 μl of culture or Butterfield&#39;s Buffer (International BioProducts) were added to a cuvette and incubated at 30° C. Absorbance readings were taken at 660 nm on a spectrophotometer (Shimadzu UV160U) at To, 2 h, 4 h, and 6 h and compared to the corresponding blank cuvette with buffer. Growth was scored by absorbance readings.  
         [0030]     The Non-Selective medium consisted of Universal Preenrichment Broth (UPB, Difco) with 0.4% Yeast Extract (YE, Difco). The Selective medium consisted of Universal Preenrichment Broth (UPB, Difco) with 0.4% Yeast Extract (YE, Difco), 0.001% Polymymin B, 0.0005% Acriflavine HCl, and 0.002% Ceftazidime.  
                                                   TABLE 1                           Scoring of Environmental Isolates Based on Absorbance Readings       over 6 hours of Incubation at 30° C.                30° C.   35° C.                    Non-       Non-       Environmental Organism   Selective   Selective   Selective   Selective                 Serratia     ++   +++   ++   +++         Citrobacter     0   +++   0   +++         Enterobacter     ++   +++   0   +++         Pseudomonas     ++   +++   0   ++         Pantoea     +   ++   0   ++         Vibrio     −   +   0   0         Acinetobacter     −   +   0   +         Escherichia coli     0   +++   −   +++         Carnobacterium     ++   ++   ++   ++         Bacillus subtilis     0   +   0   +         Bacillus lichenformis     +   ++   0   ++         Lactococcus     ++   +++   ++   +++         Lactobacillus plantarum     −   +   +   +         Lactobacillus casei     −   +   +   +         Enterococcus     ++   ++   ++   ++         Staphylococcus     −   +++   −   +++         epedermis           Staphylococcus aureus     +   +++   +   +++         Leuconostoc     −   +   0   +         paramesenteroides           Streptococcus     −   0   0   0         thermophilus                   KEY            − negative absorbance            0 no change in absorbance            + low growth            ++ medium growth            +++ high growth             
 
         [0031]     Several environmental organisms screened via spectrophotometer were selected to test in mixed culture with  Listeria . Environmental isolates were inoculated separately into TSB and incubated for 24 h at 30° C. The growth level was determined by serial dilutions plated on Tryptic Soy agar (TSA, Difco) and incubated at 30° C. for 24 h. Based on the cell counts, the culture was diluted in Butterfield&#39;s Buffer and a volume of the diluted culture targeting an initial cell count of 1000 CFU/mL was inoculated into the test media.  
         [0032]      Listeria  monocytogenes (DUP-1038, Serotype 4b) and  Listeria welshermeri  (DUP-1073) were inoculated separately into Brain Heart Infusion broth (BHI, Difco) and incubated for 24 h at 30° C. to reach stationary phase. The cultures were held at 4° C. for 5 days after reaching stationary phase. To determine the growth level after refrigeration, the culture was serially diluted in Butterfield&#39;s Buffer, pour-plated with Oxford Medium Base (Difco), and incubated at 35° C. for 24 h. Based on the cell counts, the culture was diluted in Butterfield&#39;s Buffer and a volume of the diluted culture targeting an initial cell count of 100 CFU/mL was inoculated into the test media.  
         [0033]     30 mL of Non-Selective or Selective medium was added to 10 ml of Butterfield&#39;s Buffer to simulate the addition of media to environment sponge. A Non-Selective and Selective medium was inoculated with  Listeria  culture alone, environmental isolate alone, and  Listeria  and environment isolate combined. The media were incubated in a water bath at 30° C. Serial dilutions of inoculated media were collected at To and 8 h and plated on Oxford Media Base and a differential media particular to the environmental isolate being tested (see Table 2). The Oxford Media Base plates were incubated at 35° C. for 24 h and the cell counts were collected and compared to the T 0  count at 8 h.  
                             TABLE 2                           Differential Plating Medium                    Incubation Temperature       Isolate   Plating Medium   (° C.)                 Staphylococcus     Tryptic Soy Agar + 5.0%   30         aureus     Sheep Blood         Enterobacter     Tryptic Soy Agar + 5.0%   30           Sheep Blood         Bacillus subtilis     Tryptic Soy Agar + 5.0%   30           Sheep Blood         Enterococcus     Tryptic Soy Agar + 5.0%   45           Sheep Blood         Carnobacterium     Tryptic Soy Agar + 1.0%   30           Crystal Violet                  
 
         [0034]                                                                                                                                              TABLE 3                           Growth of  Listeria  and environmental isolates in Selective and Non-Selective Media                Change in               Log CFU/ml  Listeria             in 8 hours at 30° C.   Change in Log CFU/mL                Non-       Environmental Isolate                Environmental   Selective   Selective   Non-Selective   Selective              Listeria  strain   Isolate   Pure   Mixed   Pure   Mixed   Mixed                      L. welshermeri       Staphylococcus     2.28   1.78   1.85   1.81   3.05   −1.07             aureus  Strain 1         L. welshermeri       Enterobacter     2.46   2.31   2.00   2.53   4.64   −2.70         L. welshermeri       Bacillus subtilis     2.10   2.19   1.55   1.88   3.11   −0.78         L. welshermeri       Enterococcus     2.56   2.44   2.28   2.19   3.48   2.68         L. welshermeri       Carnobacterium     2.46   2.63   1.89   1.92   2.51   1.33         L. monocytogenes       Enterococcus     1.71   1.88   1.38   1.47   3.37   1.97             durans  cocktail         L. monocytogenes       Staphylococcus     1.92   1.94   1.51   1.49   3.45   −0.50             aureus  Strain 2                    
 Polymyxin B, Acriflavine HCl, and Ceftazidime are effective selective agents against many environmental isolates that have the potential to be included in an environmental sample. At 8 h,  Staphylococcus aureus, Enterobacter, Bacillus subtilis  CFU/mL were reduced from the CFU/mL at T 0   . Enterococcus  and  Carnobacterium  grew in Selective medium, but 0.8-1.2 log less growth than in Non-Selective medium. In most cases, the presence of environmental isolates had minimal effect on the growth of  Listeria . In the Non-Selective medium,  L. welshermeri  showed 0.5 log less growth in the presence of  S. aureus  Strain 1 than in a pure culture. In the Selective medium,  L. welshermeri  showed 0.53 and 0.33 log more growth in the presence of  Enterobacter  and  B. subtilis  respectively than in a pure culture.