Europaisches Patentamt J European Patent Office © Publication number: 0 489 392 A2 Office europeen des brevets © EUROPEAN PATENT APPLICATION © Application number: 91120711.6 © Int. CI.5: C12Q 1/04, C12N 1/38, //(C12N1/38,C12R1:21,1:36) @ Date of filing: 03.12.91 ® Priority: 06.12.90 US 622874 © Applicant: Becton Dickinson and Company One Becton Drive @ Date of publication of application: Franklin Lakes New Jersey 0741 7-1 880(US) 10.06.92 Bulletin 92/24 @ Inventor: Goldenbaum, Paul E. © Designated Contracting States: 732 Silver Creek Road AT BE CH DE DK ES FR GB GR IT LI LU NL SE Baltimore MD 21208(US) Inventor: Graziosi, Ann M. 3619 Gibbons Avenue Baltimore, MD 21208(US) © Representative: von Kreisler, Alek, Dipl.-Chem. et al Patentanwalte Von Kreisler-Selting-Werner, Deichmannhaus am Hauptbahnhof W-5000 Koln 1(DE) © Culture medium supplement for fastidious organisms. © The present invention is a fastidious organism supplement which comprises growth factors and components to neutralize the toxicity of a polyanionic anticoagulant. The supplement is useful to isolate, grow, recover and/or enhance detection of fastidious organisms in culture media. CM < CM Oi CO at CO Rank Xerox (UK) Business Services EP 0 489 392 A2 1 . Field of the Invention This invention relates to a fastidious organism supplement, and more particularly to a neutralizing supplement, and to the use of the supplement in culture media. 5 2. Description of Related Art A polyanionic anticoagulant is an essential ingredient in all blood culture media. Sodium polyanetholesulfonate (SPS) and sodium amylosulfate (SAS) are examples of polyanionic anticoagulants io that may be added to blood culture media. SPS, in particular, is routinely added to commercial blood culture media. SPS has many beneficial effects when used in culture media, such as the inhibition of enzymes, proteins and processes that inactivate the growth of microorganisms. Such enzymes, proteins and processes include phagocytosis, complement, lysozymes and aminoglycoside antibodies. However, SPS is 75 toxic to many strains of organisms, including Neisseria meningitidis, Neisseria gonorrhoeae, Peptostrep- tococcus anaerobius, Streptobacillus moniliformis, Gardnerella vaginalis, and Mycoplasma sp. A discussion about SPS and the variables influencing the speed and recovery rate of microorganisms is incorporated by reference, C.L. Strand and J.A. Shulman, Bloodstream Infections, 21-44 (1988). Numerous attempts have been made to neutralize the toxic effect of polyanionic anticoagulants in 20 different media, especially in blood culture media, and to enhance the isolation, growth, recovery and/or detection of fastidious organisms. The neutralization of SPS by hemoglobin has been reported by S.C. Edberg, et al. in "Inactivation of the Polyanionic Detergent Sodium Polyanetholesulfonate by Hemoglobin." J. Clin. Microbiol. 18:1047-1050 (1983). It was reported that hemoglobin can inactivate SPS but since hemoglobin is one of the major 25 constituents inside blood culture bottles, and since by lysis its concentration can change over time, it is extremely difficult to predict whether the amount of free hemoglobin will be enough to inhibit a particular isolate of Neisseria in an individual blood culture bottle. The neutralization of SPS by gelatin (typically 1.0 to 1.2% w/v) has been reported by M. Weinstein, et. al. in "Controlled Evaluation of Modified Radiometric Blood Culture Medium Supplemented with Gelatin for 30 Detection of Bacteriemia and Fungemia." J. Clin. Microbiol. 25:1373-1375 (1987). The presence of gelatin in SPS-containing blood culture medium was shown to significantly inhibit the recovery of a variety of fastidious organisms, including staphylococci and members of the Enterbacteriaceae family. U.S. Patent No. 4,217,411 to Le Frock, et al. discloses enrichment for enhancing the detection and growth of the etiological agents of bacteriemia comprising Fildes peptic digest of blood and certain other 35 compounds. The enrichment comprises Fildes peptic digest, Vitamin B12, NAD, L-Glutamine, Co-enzyme A, Pyruvate, Catalase, Glutamate, Vitamin Ki , Co-Carboxylase and Cysteine hydrochloride. The enrichment supports growth of a number of organisms, but does not contain a neutralizing effect for polyanionic anticoagulants. The addition of gelatin to blood culture media to prevent inhibition of various organisms caused by SPS 40 has been suggested by Reimer, et al., in "Controlled Evaluation of Trypticase Soy Broth with and without Gelatin and Yeast Extract in the Detection of Bacteriemia and Fungemia." Diagn. Microbiol. Infect. Dis. 8:19- 24 (1987). However, the addition of gelatin without sufficient growth factors was found not to enhance the growth of fastidious organisms. It was concluded that blood culture media could not be used for non-blood samples by Abdou, et al., in 45 "Unsuitability of Blood Culture Media Containing Sodium Polyanetholesulfonate for the Detection of Fastidious Microorganisms in CSF and Other Blood-Free Body Fluids." Zbl. Bakt. Hyg., I. Abt. Orig. A 254, 109-117 (1983). It was found that SPS containing media could not be used for culturing cerebral spinal fluid and other blood-free body fluids and that media without anticoagulants could not be used to culture blood. Fastidious organisms in clinical microbiology, are difficult to isolate or cultivate on ordinary media 50 because of their need for special nutritional factors. In particular, factor V and factor X are nutritional factors which stimulate the growth of a number of fastidious bacteria including Haemophilus influenzae, Haemophilus parainfluenzae and Gardnerella vaginalis. Since factors X and V cannot be made by fastidious organisms in a culture media, they need to be supplied nutritionally to a culture media for proper growth of the fastidious organisms. 55 Factor X is haemin (hemin), a complex organic molecule which is present in hemoglobin in red blood cells. Several species of Haemophilus are examples of bacteria which require factor X or haemin for growth. Factor V is nicotinamide adenine dinucleotide, or NAD, a large complex organic molecule which is required for the growth of several fastidious bacteria, including some species of Haemophilus. NAD is 2 EP 0 489 392 A2 present in whole blood and serum, but its level in these fluids is variable and not stable when in aqueous solution. Therefore, blood and blood products cannot be relied upon to serve as good sources of factor V. Most blood culture media do not contain adequate levels of these growth factors. This is due to the fact that most blood culture media are sterilized in manufacturing by autoclaving, and since some growth 5 factors, such as factor V, are heat labile, factor V becomes inactivated. There are a number of available supplements for use in microbiological culture media which are intended to enhance the isolation of fastidious organisms. None of these supplements, however, provides growth factors and neutralizes the toxic effects of polyanionic anticoagulants. Available supplements for stimulating the growth and recovery of nutritionally fastidious microorganisms io include Fildes Enrichment (sold by Becton Dickinson Microbiology Systems, Cockeysville, Maryland), ISOVITALEX® Enrichment (trademark of Becton, Dickinson and Company) sold by Becton Dickinson Microbiology Systems, Cockeysville, Maryland, BACTO® Supplements (trademark of Difco) sold by Difco Laboratories, Detroit, Ml and Hemoglobin (sold by Becton Dickinson Microbiology Systems, Cockeysville, Maryland). is Fildes Enrichment is a peptic digest of defibrinated sheep blood containing factor X, and possibly factor V. It is not able to neutralize the toxic effects of polyanionic anticoagulants in culture media. ISOVITALEX Enrichment, contains factor V, but does not contain factor X and is not able to neutralize the toxic effects of polyanionic anticoagulants in culture media. The BACTO Supplements include BACTO Supplements A, B, C and VX. BACTO Supplement A is a 20 desiccated yeast concentrate which contains crystal violet, a dye which inhibits growth of many bacteria and is not acceptable as a blood culture medium supplement. BACTO Supplement A preserves the thermolabile and thermostable growth accessory factors, including glutamine, factor V, cocoarboxylase and factor X. BACTO Supplement A is unable to neutralize the toxic effects of polyanionic anticoagulants in culture media. 25 BACTO Supplement B is a desiccated yeast concentrate which provides growth factors required to support the growth of both Haemophilus and Neisseria species. BACTO Supplement B preserves the thermolabile and thermostable growth accessory factors of fresh yeast, and contains factor V and factor X. BACTO Supplement B is unable to neutralize the toxic effects of polyanionic anticoagulants in culture media. 30 BACTO Supplement C is a desiccated yeast concentrate which provides growth factors required to support the cultivation of Haemophilus influenzae and Neisseria gonorrhoeae. BACTO Supplement C contains the thermolabile and thermostable growth accessory factors of fresh yeast, including factor V, factor X and cocoarboxylase. BACTO Supplement C is unable to neutralize the toxic effects of polyanionic anticoagulants in culture media. 35 BACTO Supplement VX is a lyophilized concentrate of growth factors used as an enrichment for the cultivation of Neisseria gonorrhoeae, Haemophilus influenzae and other fastidious organisms. BACTO Supplement VX contains factor V, but it does not contain factor X and is unable to neutralize