# Patent

## Title
Selective growth medium for Listeria spp

## Abstract

The invention is a selective growth medium for investigating, isolating, counting and directly identifying pathogenic bacteria of the genus Listeria.The medium promotes the Listeria spp. while simultaneously inhibiting the growth of non-Listeria organisms. The medium does not produce a bacterial biomass contaminated with interfering fluorophores. The medium contains nitrofurantoin, esculin and lithium chloride and lacks acriflavin.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority under 35 USC §119(e) to U.S. Provisional Application Ser. No. 60/543,947, file Feb. 12, 2004, the entirety of which is incorporated herein.


REFERENCES AND INCORPORATION BY REFERENCE
Complete bibliographic citations for the references cited herein are contained in a section titled “REFERENCES,” immediately preceding the claims. All of the documents listed in the “REFERENCES” section are incorporated herein.
FIELD OF THE INVENTION
The invention is directed to a Listeria-selective growth medium. The preferred embodiment of the medium comprises nitrofurantoin, esculin and lithium chloride, and is substantially devoid of acriflavin.
BACKGROUND OF THE INVENTION
Considerable microbiological research has been devoted to understanding the nutritional requirements and environmental conditions that promote selective growth of Listeria spp. Dependable selective culturing of Listeria spp. is becoming increasingly important in the food industry because of evolving federal and state regulations requiring more frequent monitoring of food-processing equipment and environments. Listeria spp. is considered to be a critical indicator of the effectiveness of industrial sanitation practices for two principle reasons: 1) organisms of the genus Listeria are ubiquitous; and 2) the species Listeria monocytogenes is pathogenic and thus a cause of concern for public health officials.
Among the bacteria of the genus Listeria spp., only the species monocytogenes is known to be pathogenic to humans. Other species of Listeria such as L. ivanovii are not generally pathogenic or are pathogenic only for animals. L. monocytogenes is a gram-positive, motile, aerobic and facultatively anaerobic bacterium which is ubiquitous in nature. It can cause various diseases in man including meningoencephalitis, low-grade septicemia, infectious mononucleosis-like syndrome, pneumonia, endocarditis, bacterial aortic aneurysm, localized abscesses, papular or pustular cutaneous lesions, conjunctivitis and urethritis.
In the past decade, L. monocytogenes has been recognized as a major food-borne pathogen. Outbreaks of listeriosis have been linked to a number of contaminated foods such as coleslaw, Mexican-style soft cheese, pasteurized millk and turkey franks. It has been isolated from fresh produce, dairy products, processed meats and seafood products. About 500 people die each year in the United States from Listerial food poisoning; the victims are usually the immunocompromised, pregnant women and neonates.
The isolation and the identification of the bacterium L. monocytogenes is a major problem in the monitoring of food hygiene and of medical bacteriology. While a number of putative media for selective culture of Listeria spp. have been described in the literature, each have disadvantages. For example, Lovett et al. describe an enrichment broth for selective isolation of Listeria spp. and U.S. Pat. No. 6,228,606 describes a method for inhibiting L. monocytogenes using a synthetic chromogenic substrate. However, these media detect every species of the genus Listeria spp. Thus, supplementary identification tests, such as microscopic, biochemical, immunological, and/or genetic tests must be used to establish the presence of the pathogenic monocytogenes species. However, these supplementary manipulations increase the length of time and cost of the analyses, require a vast number of reagents and the use of qualified personnel, and are often a source of error or at least the cause of lower precision and reliability. This is especially true when there is a very small amount of L. monocytogenes present.
Other methods for the selective culture of Listeria spp. have been described, such as Fraser and Sprerber's medium exploiting the high salt tolerance of Listeria spp., and its ability to hydrolyze esculin. Esculin is a glucoside (6-(beta-D-glucopyranosyloxy)-7-hydroxy-2H-1-benzopyran-2-one, CAS No. 531-75-9) obtained from Aesculus hippocastanum (the horsechestnut) and is characterized by its fine blue fluorescent solutions. In this approach, the beta-glucosidase activity of Listeria hydrolyzes esculin. The hydrolysis products, in combination with iron salts present in the medium, yield a black pigment that is used as a colorimetric indicator of a positive sample. Donnelly & Baigent developed a modified medium similar to the Fraser & Sprerber broth but lacking the colorimetric indicator. This medium exploits the salt tolerance of Listeria spp. in conjunction with several antibiotics to yield a medium selective for the growth of Listeria. However, these media slow the overall growth rate of Listeria cells to achieve inhibition of competitive micro-flora in the sample being tested. Further, the combination of high salt concentration and antibiotics prevents the growth of certain strains of Listeria, most notably L. ivanovii and L. grayi. 
Another complicating aspect of conventional selective media is the presence of acriflavin. Acriflavin is an acridine dye that is an effective inhibitor of competitive gram-positive bacteria such as Bacillus spp. Unfortunately, acriflavin not only is a suspected carcinogen but is also a fluorophore that is incorporated into the DNA and proteins of growing cells. Thus, acriflavin causes unwanted fluorescent interference in many fluorescence-based assays, such as enzyme-linked immunosorbent assays (ELISA) and the polymerase chain reaction (PCR). Many commercially available Listeria detection products rely upon the use of fluorescent reagents for analyte detection.
Thus, there remains a long-felt and unmet need for a Listeria-selective medium that 1) does not appreciably interfere with the growth rate of Listeria spp.; 2) does not yield bacterial biomass contaminated with interfering fluorophores; and 3) strongly inhibits the growth of non-Listeria organisms.
SUMMARY OF THE INVENTION
The present invention is a culture medium for investigating, isolating, counting and directly identifying pathogenic bacteria of the genus Listeria. The medium promotes the growth of Listeria spp. while simultaneously inhibiting the growth of non-Listeria organisms. Further, the medium does not produce a bacterial biomass contaminated with interfering fluorophores.
The medium or the present invention comprises nitrofurantoin, esculin and lithium chloride and is substantially devoid of acriflavin. In a preferred embodiment, no acriflavin is present. In an alternative embodiment, acriflavin is present in concentrations of about 0.01 g/L or less. The medium also uses much lower concentrations of lithium chloride than the prior art. In a preferred embodiment, lithium chloride is present in concentrations of about 5 g/L or less.
The rapid and accurate identification of Listeria spp is just one of the advantages the medium of the present invention. For instance, the medium of the present invention does not require a secondary transfer to another medium. Further, the medium does not fluoresce, and therefore is compatible with ELISA-and PCR-based tests to identify Listeria spp. Further still, procedures using the medium of the present invention require no special enrichment procedures or secondary manipulations. Finally, the medium of the present invention can be used to detect L. monocytogenes in a host of foods, food products and environmental samples, even in the presence of large populations of other non-Listeria organisms.
The complete scope of the invention will appear more fully from the following detailed description of the preferred embodiment of the invention.


DETAILED DESCRIPTION OF THE INVENTION
The selective medium of the present invention promotes and enhances the growth of Listeria spp. while simultaneously inhibiting the growth of non-Listeria organisms. The medium may be used with any type of food product or environmental sample.
In a preferred embodiment (see PDX-1 in Table 1), the medium comprises tryptone, in a concentration ranging from about 15 to about 25 g/L, with a preferred concentration of about 16 to about 18 g/L, with a further preferred concentration of about 17 g/L; peptone, in a concentration ranging from about 1 to about 5 g/L, with a preferred concentration of about 2 to about 4 g/L, with a further preferred concentration of about 3 g/L; sodium chloride, in a concentration ranging from about 1 to about 10 g/L, with a preferred concentration of about 2.5 to about 7.5 g/L, with a further preferred concentration of about 5 g/L; anhydrous dibasic potassium phosphate, in a concentration ranging from about 1 to about 10 g/L, with a preferred concentration of about 2.5 to about 7.5 g/L, with a further preferred concentration of about 5 g/L; yeast extract, in a concentration ranging from about 1 to about 10 g/L, with a preferred concentration of about 2.5 to about 7.5 g/L, with a further preferred concentration of about 6 g/L; cycloheximide, in a concentration ranging from about 0.01 to 0.1 g/L, with a preferred concentration of about 0.025 to about 0.075 g/L, with a further preferred concentration of about 0.05 g/L; acriflavin, in a concentration ranging from no more than about 0.01; naladixic acid, in a concentration ranging from about 0.01 to about 0.1 g/L, with a preferred concentration of about 0.025 to about 0.075 g/L, with a further preferred concentration of about 0.04 g/L; and esculin, in a concentration ranging from about 0.5 to 5 g/L, with a preferred concentration of about 0.75 to about 2 g/L, with a further preferred concentration of about 1 g/L.
In an especially preferred embodiment (see PDX-2 in Table 1), the selective medium of the present invention comprises tryptone, peptone, sodium chloride, anhydrous dibasic potassium phosphate, yeast extract, cycloheximide, naladixic acid and esculin in the amounts described above but contains no acriflavin. Acriflavin consistently inhibits all of the Bacillus spp. but also inhibits the hemolytic activity of L. monocytogenes. 
The ingredients of the selective medium of the present invention are dissolved in distilled water and autoclaved at approximately 121 psig until sterile, usually about 15 min. After cooling, supplements are added. Preferred supplements include ceftazidime, phosphomycin, polymyxin E, ferric ammonium citrate, lithium chloride and nitrofurantoin (Table 2).
Ceftazidime, phosphomycin, polymyxin E and nitrofurantoin are all antibiotics. Ceftazidime is a third generation cephalosporin, and acts to inhibit cell wall synthesis. Other cephalosporins such as ceftriaxone, moxolactam, cefotaxime, cefpodoxime, ceftizoxime, cefoperazone may also be used. The medium of the present invention preferably contains ceftazidime in a concentration ranging from about 0.04 g/L.
Phosphomycin is an antibiotic principally excreted through the kidney. Several studies have shown its activity against gram-positive and gram-negative organisms (Kestle, Kwan), and its clinical efficiency in the treatment of infections of the respiratory (Bacardi), gastrointestinal (Taylor), and urinogenital (Allona) tracts. The medium of the present invention preferably contains phosphomycin in a concentration of about 0.04 g/L.
Polymyxin E, also known as colistin, (CAS No. 1066-17-7) is frequently used as an oral drug for flora suppression of the gastrointestinal canal. The suppression effect is dose dependent because polymyxin E is moderately inactivated by faecal and food compounds. Polymyxin compounds are derived from various species of the soil bacterium Bacillus, and are active against gram-negative bacteria. Polymyxin E acts by disrupting the cell membranes of bacteria, destroying their ability to function as osmotic barriers. The medium of the present invention preferably contains polymyxin E in a concentration of about 0.01 g/L.
Ferric ammonium citrate is an iron-containing salt that is often used in the treatment of some forms of anemia. The present medium uses ferric ammonium citrate as a growth enhancer for Listeria spp. A concentration ranging from about 0.1 to about 1.0 g/L of ferric ammonium citrate is used, with a preferred concentration ranging from about 0.025 g/L to about 0.075 g/L, and a further preferred concentration of 0.05 g/L.
Lithium chloride is a salt commonly used in selective growth media because high salinity was believed necessary to control bacterial competitors of Listeria spp., such as Enterococcus spp. and Bacillus spp. However, the medium of the present invention uses much lower concentrations of lithium chloride than conventional media. Surprisingly, the low levels of salinity remain effective at inhibiting bacterial competitors. For instance, conventional media often contain lithium chloride in concentrations ranging as high as 10 g/L to 15 g/L. However, the medium of the present invention preferably contains a concentration of lithium chloride ranging from about 1 to about 10 g/L, with a preferred concentration ranging from about 2.5 to about 7.5 g/L, with a further preferred concentration of about 5 g/L or less.
Nitrofurantoin is an antibiotic with signifiant anti-microbial activity against many potential gram-positive competitors. Nitrofurantoin has been shown to be effective at concentrations from about one to two orders of magnitude lower than the minimum inhibitory concentration for Listeria spp. (Soriano, Safdar). Further, because nitrofurantoin is non-fluorescent, the selective medium of the present invention does not interfere with ELISA-or PCR-based detection protocols. The medium of the present invention preferably contains a concentration of nitrofurantoin ranging from about 0.001 to about 0.01 g/L, with a preferred concentration of about 0.0025 to about 0.0075 g/L, with a further preferred concentration of about 0.006 g/L.
The following Examples illustrate the features of the novel selective medium disclosed and claimed herein. The Examples are included solely to provide a more complete disclosure of the invention and do not limit the scope of the medium disclosed and claimed herein in any fashion.
EXAMPLES
Example 1







TABLE 1







Medium Formulation, Versions PDX-1 and PDX-2.









Ingredient
PDX-1 (g/L)
PDX-2 (g/L)












Tryptone
17.0
17.0


Peptone
3.0
3.0


Sodium Chloride
5.0
5.0


Dibasic Potassium Phosphate (anhydrous)
6.0
6.0


Yeast extract
6.0
6.0


Cycloheximide
0.05
0.05


Acriflavin
0.01
—


Naladixic acid
0.04
0.04


Esculin
1.0
1.0









The solid ingredients were dissolved in distilled water and autoclaved at 121 psig for 15 min to sterilize. After cooling, the following supplements were added:







TABLE 2







Supplements.











Supplement name
PDX-1
PDX-2






Ceftazidime
0.04 g/L
0.04 g/L



Phosphomycin
0.04 g/L
0.04 g/L



Polymyxin E
0.01 g/L
0.01 g/L



Ferric Ammonium Citrate
 0.5 g/L
 0.5 g/L



Lithium Chloride*
 5.0 g/L
 5.0 g/L



Nitrofurantoin**
—
0.006 g/L 





*Lithium chloride is exothermic when dissolved in water. Appropriate care must be taken when adding it to the medium.


**Nitrofurantoin is insoluble in water. A 10 mg/mL stock solution was made in sterile DMSO. The nitrofurantoin/DMSO stock solution was then added to the rest of the medium (600 microliters of stock solution/L medium yields 0.006 g/L nitrofurantoin in the final medium). Solid-medium plates were made from the liquid medium by adding 15 g agar per liter of liquid medium, bringing the medium to a boil to dissolve the agar, cooling the solutions, and sterilizing the same.






Example 2
Comparison of Growth Rates of: PDX-1 vs. Fraser Broth
The purpose of this Example is to compare the growth rate of L. monocytogenes in Fraser broth versus the growth of L. monocytogenes in PDX-1 liquid medium.
Cultures of L. monocytogenes (100 microliters of 10-7 dilution; 1/10 serial dilutions on peptone from overnight L. monocytogenes culture in tryptone soy broth (TSB)) were added to 3 mL of Fraser broth and 3 mL of PDX-1. Every hour starting at the time of inoculation, 100 microliters of both the PDX-1 medium and Fraser medium were plated on PALCAM plates in duplicate and incubated at 37 C for the enumeration of colonies. (For data on PALCAM plates, see Van Netten). PALCAM plates are available commercially from a number of international suppliers.) The growth rates of Listeria spp. on PDX-1 and Fraser broth media are shown in Table 3.







TABLE 3







Growth Rates of PDX-1 compared to Fraser Broth (CFU/0.1 mL)










Hour















0
1
2
3
4
5



















PDX-1
65
73
69
72
131
273




67
72
76
100
149
257



Fraser
59
74
66
68
94
70




57
66
81
81
77
100










The data show that the Listeria spp. in the PDX-1 sample were able to recover from inoculation and start growth faster than the samples grown in Fraser broth. Also of interest is the fact that both sets of samples were inoculated from the same stock and had the same volume of inoculation. Thus the difference in initial cell counts between the two media (65 and 67 for PDX-1; 59 and 57, for Fraser broth) is significant, suggesting that the PDX-1 medium is less stressful to the cells at initial inoculation. In both runs, the Listeria displayed greater survivability in the PDX-1 medium as compared to the Fraser broth.
Example 3
Growth of ATCC Cultures on Solid PDX-1, PDX-2, and Modified Oxford Broth
The purpose of this Example was to plate out ATCC cultures of various bacteria, including Listeria spp., on solid PDX-1 and PDX-2 media to obtain a record of their respective colony morphologies, as well as to compare these morphologies to those of corresponding colonies grown on conventional media.
A loop of overnight Trypticase Soy Broth (TSB) culture was streaked out on PDX-1, PDX-2, and Oxford broth supplemented with moxalactam. The various primary cultures were obtained from the American Type Culture Collection, Manassas, Virginia. Plates were stored at 37 C and checked at 20 and 40 hours. The results after 20 hours incubation are shown in Table 4.







TABLE 4







Growth of Different Species on Various Solid media After 20 Hr Incubation at 37 C.










Species
PDX-1
PDX-2
Oxford + Mox





S. choleraesuis
−
−
−


M. luteus
−
−
−


S. aureus
−
−
Regular, round, off-white





colonies


L. welshimeri
+
+
+


L. ivanovii
+
+
+


L. grayi
−
−
Area of some





discoloration, without any





noticeable colonies where





the streak started


L. seelgreri
−
−
−


L. monocytogenes
+
+
+


L. innocua
+
+
+


E. faecalis
Discoloration without
Discoloration without visible
Discoloration without



visible colonies at
colonies at location of start of
visible colonies at



location of start of
streak
location of start of streak



streak









As can be seen from the data, the medium according to the present invention is highly selective for the growth of Listeria spp. and highly inhibitory of the growth of non-Listeria species.
It is understood that the invention is not confined to the particular construction and arrangement of parts illustrated and described herein, but embraces such modified forms thereof as come within the scope of the following claims.
REFERENCES


    Allona, A. et al. (1977) “Fosfomycin in chronic urinary infections,” Chemotherapy (Basel) 23(Suppl. 1):267-274.
    Al-Zoreky, N. et al, (1990) “Highly Selective Medium for Isolation of Listeria monocytogenes from Food” Appl. Environ. Microbiol. October:3154-3157.
    Bacardi, R. et al. (1977) “Treatment of respiratory infections with fosfomycin,” Chemotherapthy 23(Suppl. 1) :343-347.
    Bannerman, E. et al. (1998) “A New Selective Medium for Isolating Listeria spp. from Heavily Contaminated Material, ”Appl. Environ. Microbiol. 165-167.
    Blanco, M. et al. (1989) “A Technique for the Direct Identification of Haemolytic-pathogenic Listeria on Selective Plating Media,” Letters in Appl. Microbiol. 125-128.
    Cassiday, P. et al. (1989) “Evaluation of Ten Selective Direct Plating Media for Enumeration of L. monocytogenes in Hams and Oysters, ” Food Microbiol. 113-125.
    Donnelly, C. & G. Baigent (1986) “Method for flow cytometric detection of Listeria monocytogenes in milk,” Appl. Environ. Microbiol. 52:689-695.
    Fraser, J and W. Sprerber (1988) “Rapid detection of Listeria in food and environmental samples by Esculin hydrolysis,” J. Food Prot. 51:726-765.
    Kestle, D. and W. Kirby (1970) “Clinical pharmacology and in vitro activity of phosphonomycin,” Antimicrob. Agents Chemother. 332-337.
    Kwan, K. et al. (1971) “Pharmacokinetics of fosphomycin in man” I. Intravenous administration,” J. Pharm. Sci. 60:678-684.
    Lovett, J. D. et al. (1987) “Listeria monocytogenes in raw milk: detection, incidence, and pathogenicity,” J. Food Prot. 50:188-192.
    Peterson, M. et al. (1993) “Parameters for Control of Listeria monocytogenesin Smoked Fishery Products . . . ,” J. Food Prot. 56:11:938-943.
    Safdar, A. & D. Armstrong (2003) “Antimicrobial activities against 84 Listeria monocytogenes isolates from patients with systemic Listeriosis at a comprehensive cancer center (1955-1997),” J. Clin. Microbiol. 41:483-485.
    Soriano, F. et al. (1995) “Antimicrobial susceptibilities of Corynebacteriun species and other non-spore forming gram-positive bacilli to 18 antimicrobial agents,” Antimicrob. Agents Chemother. 39:208-214.
    Taylor, C. et al. (1977) “Enteropathogenic E. coli gastroenterocolitis in neonates treated with fosfomycin,” Chemotherapy (Basel) 23(Suppl. 1):310-5314.
    Van Netten, P. et al. (1989) “Liquid and Solid Selective Differential Media for the Detection and Enumeration of L. monocytogenes and other Listeria spp., ” Int. J. of Food Microbiol. 1215-1217.

## Claims

1. A Listeria spp.-selective medium comprising, in combination,
a. tryptone, in a concentration of about 17.0 g/L;
b. peptone, in a concentration of about 3.0 g/L;
c. sodium chloride, in a concentration of about 5.0 g/L,
d. anhydrous dibasic potassium phosphate, in a concentration of about 6.0 g/L;
e. yeast extract, in a concentration of about 6.0 g/L;
f. cycloheximide, in a concentration of about 0.05 g/L;
g. naladixic acid, in a concentration of about 0.04 g/L;
h. esculin, in a concentration of about 1.0 g/L;
i. ceftazidime, in a concentration of about 0.04 g/L;
j. phosphomycin, in a concentration of about 0.04 g/L;
k. polymyxin E, in a concentration of about 0.01 g/L;
l. ferric ammonium citrate, in a concentration of about 0.5 g/L;
m. lithium chloride, in a concentration of about 5.0 g/L; and
n. nitrofurantoin, in a concentration of about 0.006 g/L.

2. The medium of claim 1, wherein the medium is substantially devoid of acriflavin.