Microorganism identification method

专利摘要:
METHOD OF DETECTING A SPECIFIC MICROORGANISM A method of detecting a specific microorganism comprising contacting said microorganism with bacteriocins from a microorganism of a genus which is taxonomically unrelated to said specific organism. The result of such contact may be utilized to detect the presence of a microorganism belonging to a taxonomically unrelated genus. Radio-labeled or fluorescein-labeled bacteriocins can be reacted with specific bacteria in a biological sample and the presence of such specific bacteria detected by removing excess bacteriocins and determining the presence of fluorescent or radioactive bacteria in the sample. Neisseria gonorrhoeae is identified by spotting bacteriocins on a plate of clinical material; or using a disk impregnated with bacteriocins placed on a plate inoculated with the clinical material; or the bacteriocins can be incorporated into one-half of a split agar plate, the identification being made on the basis of a zone of inhibition surrounding the spot where the bacteriocins were applied, or growth inhibition on the portion of the plate to which the bacteriocins were added.
公开号:SU731904A3
申请号:SU772466678
申请日:1977-03-31
公开日:1980-04-30
发明作者:Аллен Морс Стефен；Хотан Иглевски Барбара
申请人:За витель；
IPC主号:

专利说明:

The culture of mitomycin C aiducirovain C is centrifuged at 2400 times acceleration for 30 minutes to remove cellular debris and the upper layer, which is added after settling, is treated with chloroform (5% by volume). This surface (pa up fraction is raw niocin.
Crude piocin is further purified by freezing i.M MnCb (60 ml per 1 l of lysate), while admixing the mixture with crude neiocin. After reaching a pH of 7.5 seconds by: using 1M NaOH, the resulting precipitate is removed by price1) by refluxing. The supernatant phase is a purified pyocin.
Further purification is carried out by adding (NH4) 2SO4 to 70% saturation and cultivating overnight at
, 1 ° s
-I.
After centrifuging for 30 minutes at 4 ° C, the precipitate from the centrifugation, containing the component with pyocine activity, is dissolved in 50 ml of O.OIM tris (hydroxymethyl) aminomethane (tris) hydrochloride, pH 7.5, containing 0.0IM MgCls and 0.01 M MgSO, and dialyzed overnight at 4 ° C. against 2 liters of the same buffer. If necessary, the preparation is clarified by centrifugation for 90 minutes. The gelatinous precipitate from the centrifugation is carefully dissolved in 20 ml of buffer and chromatographed on DEAE cellulose, preliminarily shymy 1, and brought to equilibrium using the same buffer. 8 ml of a sample of niocin were added to a column (1.5 x 28 cm in size and adsorbed for an hour. The column was washed with 200 ml of buffer to remove substances not adsorbed on DEAE type cellulose. Then piocin was eluted with 800 mm in 0.01 M buffer. Collected 5 ml fractions and analysis are based on absorption at 280 nm and on theocyan activity.
The fractions exhibiting pyodinate activity were analyzed for 0.01 M tris-buffer, pH 7.5 for distance and NaCl, and then concentrated by ultracentrifugation for 90 minutes.
Pyocin typing is carried out using the fluid method described by John.
Strains of Neisseria gonorrhoeae, which are susceptible to piocin, are grown overnight in agar plates. A suspension of these microorganisms is prepared in a diluent consisting of 0.85% NaCl and 0.1% HCl (pH 6.4), and adjusted to 1% Clett, paiBiKbix 50-60. Cup with agar medium infect with a swab immersed in the cell suspension. Undiluted or serially diluted preparations of pyocin (5 µl are applied to the surface of the plates with agar medium. All stump thicknesses., ...;
, -,, .-. ,:, “,
 .....
731904
virus overnight at 37 ° C with a high content of COs.
Piocins were prepared for examination under an electron microscope using the negative staining technique. The preparations of diocin are centrifuged with an acceleration of 100,000 q for 1 h, and the precipitate from centrifuging of the agitation is resuspended in a small volume.
1M NH4C2H302 (pH 7.0). Copper grids coated with formvar are introduced into a drop of the sample over a period of 1-2 minutes, and then blot dry with filter paper. These nets are then introduced into a drop of 1.5% sodium phosphoforphate sodium diluent (pH 7.0) for 30 seconds. Excess fluid is removed with filter paper.
Interaction of pyocins with cells
Neisseria gonorrhoeae is detected by a similar technique. 30 minutes after the addition of piocin to Liquid N ulutron N. gonorrhoeae 72H870, the sample is removed and processed in the same manner as described. The preparation with negatively stained spots was studied using an RCA electron microscope (ori 50 kV)
Table 1 shows the results of the production of the gonococcal factor inhibition synthesized during the growth of Pseudomona.s aeruginosa ATCC 29260 in the indicated medium. The addition of mitomycin C (1 µg1 ml) causes a wide lysis of the culture within 3 hours and leads to a 16-fold increase in the concentration of the inhibition factor, determined by titrating Psendomonas aernginosa Pp7 and 1947. The table also shows the title strains of N. gonorrhoeae JW-31 and JW-31 The DGI torus is inhibited, changing with changes in different strains of Neisseria gonorrhoeae, and there is no difference with the colonial variants of the single strain.
Table I
Note: a) Lnolpolschina C (I μg per 1 ml of medium) prk vosm. Subjective dilution was not able to inhibit the growth of these organisms at a dilution of 1: 2 (0.5 μg / ml);
b) N, D - not defined.
The inhibition factor was partially purified from the upstream liquid phases of the cultures of Pseudomonas aeruginosa strain ATCC 29260, induced by mitomycin C, according to the procedure described above. The inhibition factor is eluted from DEAE cellulose using a NaCl gradient from 0 to 1, OM. Two peaks containing inhibitory factor are detected. The main peak (a) is zoned at a NaCl concentration of 0.06 M. And has more than 90% inhibitory activity. The weak peak (6) elutes at a NaCl concentration of 0.91M and has less than 10% activity. The fractions constituting peak a were pooled, dialyzed against 0.01 trichlorohydrate buffer (pH 7.5) to remove NaCl and concentrated by ultracentrifugation (100,000-fold acceleration for 90 minutes). This preparation was used in the tests described below.
An electron microscope study of a drug with negative stains from purified inhibition factor indicates that the particles resemble R-type pyocins in both unabridged and reduced states. In the uncontracted state, these particles have a length of 11 5 nj and a width of 15.3 nm. In the reduced state, the particles consist of an internal core (105 nanometers long and a width of 6.5 "lg) surrounded by a contracted shell (44.4 nm long and 18.6 nm wide.}. In this, the cropped state was 20- 30% of the particles observed in these preparations.
The type of piocin in both partially purified and II in purified preparations is determined by the above method, and the results show that the model, i.e., is variable ;; zs cleaning time. Piocin refers to model 611131.
The effect of pyocin 611131 on the growth of the clinical isolate of Neisseria gonorrhoeae (strain 72H870).
The effect of shiodin on the growth of microorganism cells was determined by adding different concentrations of the purified preparation to exponentially growing cultures.
At high concentrations of pyocin, complete inhibition of growth, occurring within 1 hour, is accompanied by a wide culture LYSIS. An electron microscope study shows that there is a direct interaction between the pyocin and the sensitive cells of Neisseria gonorrhoeae. The interaction of pyocin with cells leads to a reduction in pyocin.
Spectrum Inhibit) Irovani piocin type 611131.
Aliquots of the purified preparation of piocin are spotted on crops obtained from clinical isolates of N. gonorrhoeae. The zone of inhibition is clearly visible in all the studied strains. No 1 is the difference between colonies of T-1 and T-4 type from the same strain. Also included is the negative control test of the producing strain of P. aeruginosa ATCC 29260.
Table 3 shows the inhibition of various Neisseria species with this pyocin. 2. All Isolation N. gonorrhoeae feces
table 2
Both scattered and non-dispersed infections are inhibited. However, only 3 of 20 N. meningitidis strains and 5 of 16 N. lactamica strains are inhibited by 40. No correlation was observed between the serological group and the inhibition of N. meningitidis. None; -: for the other five species tested, this pyocin is not detected. Fast identification of Neisseria gonorrhoeae.
The method for the rapid identification of Neisseria goni rrhoeae can be carried out using any of the following methods: pyocin 611131 is applied as a spot into an agar plate containing a biological sample that is being tested, or a disk impregnated with piocin is applied to the plate infected with the sample or, piocin, applied in one half of a cup with cleaved agar medium, becomes infected with a sample. Following cultivation, the microorganism is identified as N. gonorrhoeae on the basis of the zone of inhibition, surrounding the spot where pyocin was applied, or inhibiting typing methods, is especially useful in epidemiological studies, when the prevalence or appearance of new strains is important, and also Whether the failure is processing the resistance of the microorganism or re-infection with a new type of microorganism. Additional embodiments of the invention are described below. Identification of N. gonorrhoeae using fluorescein labeled piocins. According to this method, piocins produced and purified as described in the wipt are marked with fluorescein according to the Johnson method. Thus labeled piocins react on suspected N. gonorrhoeae on a glass slide prepared from clinical material or from isolated colonies from an agar plate. Excess cyocytes are removed by washing the slide with 0.02 molar physiological (pH 7.2) phosphate-buffered buffer, and the slide is examined under a UV microscope. Cells with typical N. gonorrhoeae morphology and showing fluorescence are considered positive for N. gonorrhoeae.
one hundred on that part of the thicket into which piocin was introduced
This method can be used to identify other bacteria.
Typization of Neisseria gonorrhoeae.
The typing of N. gonorrhoeae and other Neisseria species involves the use of several types of pyocin and is based on the inhibition or inhibition of the microorganisms tested. The observed results are used to identify the type of isolate. By relating them to results obtained using a variety of known types (Table 3). This
Table 3 Identification of bacteria using radiolabeled piocins. Radioactive pyocins are obtained by iodizing them with isotope j using the chloramine T method. They can be obtained labeled with I or C by introducing specific labeled amino acids into the cultured medium before starting the synthesis of pyocin in Pseudomonas aeruginosa using mitomycin C. Piocins produced according to This method is radioactive. Specific bacteria are easily identified by reacting radioactive pyocins with a suspension of bacteria. This suspension is then subjected to membrane filtration or centrifugation to separate unbound pyocins from pyocins, which are specifically bound to the surface of the bacterial cells. The filter or washed cell pellet in the tube after centrifugation is then taken into account in order to determine the amount of bound pyocin that has been labeled. The data are compared with the degree of non-specific binding, any increase above the level of non-specific binding indicates the presence of an unknown microorganism.
Sal. kadack
Sal. miarembe
Sal. onderspoort
Sal. thompson
Sal. spp. Group C
Serratia mercescen
S. marcescens
Shigella flexneri
Sh. spp. Group d
Brucella abortus
B. bronchisepticus
B. suis
Neisserla gonorrho
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. gonorrhoeae
N. meningitidis
N. meningitidis
N. meningitidis
N. meningitidis
N. meningitidis
N. flava
N. flavescens
N. laciamia
N. lactamica
N. mucosa
N. ovis
N. peiflava
N. subfiava
Acinetobacler anitra
A. anitratus
A. calcoaceiicus
Aeromonrjs hydioph
Alcaligenes viscosib
Azotomanas iiisoliid
EnteroDactcr aeroge
E. cloaceae
E. cloaceae
E. cloaceae
E. cloaceae
Exhericliia coli
E. cnii
E. coli
E. ooH
E. coli
E coli
E. coli B.
E cnli iiidole CP
E, Cdli Se rs
E. coli 1. C 411
E. coli K. 1
E. coli K. 12
E. cuii
Haffni.i Spp
Herrt le; i Vaginocoi
H VagiII with .la
H. Vaguii4-, lH V; igirioi () bi
Hk-bbiclla oz I II.:l
K. pneu ioi; i; k
K. pneumonia
K. pneuinuniae
Mima polymorpha Proteus rnorganic Providencia s tuartie
Table 4
+
+
+
+ + + + + + + + + + 4- + +
+ +
+
/ - +
+ / +
+
+
+ / + 7 + / + + + + + / I +
+ / +
+
+
+
+
+
+ // +
+ -
+ / + / +
+ + + +
+
+ I -
+
+
+
  j

- i +/- i +
II
Orga:
Pse i: d from on; s .i er ug i ;: os l
P. dertiijir: c-T
Ps. aerugin s; i non p; f.T
F alkuligc,; ifs
Ps. irniolo.MC.
Ps. inaltopliiha
Ps. n; a1Op)
PS saccliarcphili.i
S;: ln; onell; i ariz.
Sa. bergen
Sal. bornura
. cholerssuis
Sal. dahlem
Sal. deversoir
Sal. djakarta
Sal. diigbe
Sal. enieriditis
Sal. florida
Bacteriocjn I Bacteriocin 2 3-acleriocin 3 Bacteriocin 4 Bacterjocin 5 Baceriocin 6 BfiCteriocin 7
The invention may be used to identify antigens that are characteristic of taxonomic unrelated bacteria. Bacteriocins are known to bind to specific receptor sites on the outer surface of bacteria. These receptor sites are also subject to the action of host protection mechanisms and can thus stimulate the production of antibodies, which can be either bactericidal or opsonous types. Bacteria can secrete intrinsic antigens. These intrinsic antigens can also be bacteriocin receptor sites, so the interaction of bacteriocins with different types of bacteria can be used as an indicator of separating antigens (Table 4). This, in turn, can be used in the isolation and purification of antigens for the production of vaccines. Thus, a minor antigen on one microorganism can be the main antigen on another, and therefore can facilitate purification and can be crawled in large quantities.
The method can also be used to demonstrate intrinsic antigens (or surface components) in mammalian cells. Due to the intrinsic specificity of their receptors, bacteriocins can be used interchangeably.
Continued table. four
B KTSrIOTSKR
: PS aeruginosa FS-5: PS aeruginosa FS-6: PS aeruginosa PA 108: PS aeruginosa PS-7: PS Vibriocholera Her Toz: PS Serratia marcesceus: Aeromonas hydro.philia
one hundred compounds, such as lectins, kl :. in techniques that include tissue typing.:;). The invention can also be used to identify other bakgee zy. Thus, it is shown in Table. 4, bacteriocins from Vibro cholerae, Aeroni s Hydrophila and Serratia marcescens usually cross-react with a taxonomic unbound microorganism. The proposed method allows to simplify the determination of the presence of microorganisms using any bacteriazin.
Formula I310
L A method for the identification of microorganisms in a biolopic 1M material is confusing: govev on a nutrient medium, followed by contacting the growing colonies with bacteriocin, incubating and taking into account the presence or absence of growth, so that, in order to simplify the method, grown colonies are in contact with R-type bacteriocins from microorganisms; y; oE taxonomically unrelated.
2. A method according to claim I, characterized in that as a bacteriocin isp. 1, piocin Pseudomonas aeruginosa ATCC 29260, asp 611131, is used.
3. A method according to claim I, characterized in that the microorganism Xeisseria gonorrhoeas is identified. 13 Source of information, understanding during examination: t. G. Volk and S. Kraus adopted in meningococcal urethritis. Possible protective “Asymptomatic cal Diseases, 1973, 49, p. 511-512. 731904 And value against gonococcal infection by bacteriocin production, "British J. of venerologi

权利要求:
Claims (3)
[1]
15 claims
1. A method for the identification of microorganisms in bioptic material; m material by plating on a nutrient medium followed by
[2]
2® by contacting the grown colonies with bacteriocin, incubation and taking into account the presence or absence of growth, which is due to the fact that, in order to simplify the method, the grown colonies are in contact with 25 R-type bacteriocins from microorganisms of a taxonomically unrelated kind.
2. The method according to π. 1, characterized in that. that the bacteriocin is used Pioudomonas aeruginosa ATCC pyocin
30 2 9 2 60, typed as 611131.
[3]
3. The method according to π. 1, characterized in that the microorganism Neisseria gonorrhoeae is identified.

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US4351761A|1978-05-15|1982-09-28|Research Corporation|Purified antigen to test for Neisseria gonorrhoeae antibodies|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US05/672,292|US4142939A|1976-03-31|1976-03-31|Method of producing an r-type bacteriocin and its use in the detection of specific microorganisms|

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