CN104651473A - Method for simultaneously determining agent resistance and agent tolerance of sublethal-dose staphylococcus aureus - Google Patents
Method for simultaneously determining agent resistance and agent tolerance of sublethal-dose staphylococcus aureus Download PDFInfo
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- CN104651473A CN104651473A CN201510046318.8A CN201510046318A CN104651473A CN 104651473 A CN104651473 A CN 104651473A CN 201510046318 A CN201510046318 A CN 201510046318A CN 104651473 A CN104651473 A CN 104651473A
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Abstract
The invention discloses a method for simultaneously determining the agent resistance and the agent tolerance of the sublethal-dose staphylococcus aureus. The method comprises the following steps: (1) preparing a bacterium suspension of the staphylococcus aureus containing an interference agent; (2) determining the agent resistance; (3) determining the agent tolerance; (4) determining the sensibility recovery; (5) finding out the change rules of the agent resistance and the agent tolerance of the sublethal-dose staphylococcus aureus to a disinfectant. According to the method for simultaneously determining the agent resistance and the agent tolerance of the sublethal-dose staphylococcus aureus, the determinations of the agent resistance, the agent tolerance and the agent sensitivity recovery of a bacterium are combined in an experiment, particularly the change rule and the relation between the agent resistance and the agent tolerance can be intuitively found so that the internal relation between the agent resistance and the agent tolerance can be accurately and directly found.
Description
Technical field
The present invention relates to a kind of Simultaneously test resistance and drug method, be specifically related to a kind of method of Simultaneously test sublethal dose streptococcus aureus resistance and resistance.
Background technology
First find MRSA from jevons in 1961 in Britain, middle 1960s extends to European many countries and Canada, and MRSA had and increased extend over the entire globe the end of the seventies, and resistance scope expanding day, drug-resistant intensity is day by day serious.The later stage eighties has become global pathogenic bacteria, occupies the first place of Nosocomial Infection Pathogens, accounts for the 60-80% that teaching hospital is separated S. aureus L-forms.There is again resistance to MRSA through the ages in the later stage nineties 20th century.Current MRSA is the important component part of superbacteria, and its resistance is strong, mortality ratio is high has become and HBV, AIDS worldwide 3 large scabrous infectious diseases side by side.Therefore inquire into source and the resistance mechanism thereof of MRSA, find effective control method, be containment its infect popular most effective way.
Sterilizing agent and microbiotic belong to the chemical substance with special suppression microbicide effect, and they also have a lot of same or similar part to the mechanism of action of microorganism, and the microorganism of some resistance may produce the resistance phenomenon to sterilizing agent equally.American scholar proposes microbiotic and sterilizing agent facilitates the selection of MRSA and the possibility of maintenance, and experiment proves that S. aureus L-forms contains the drug resistance plasmid of Multiple Classes of Antibiotics and sterilizing agent, and finds to there is certain contact between them.A large amount of epidemic datas also shows: the Resistant strain of streptococcus aureus raises the resistance of sterilizing agent.Structure gene research around streptococcus aureus family qac gene and beta-lactamase shows, the plasmid at qac gene place carries Drug-resistant genes simultaneously, and the transposon Tn552 of the structure gene blaz of staphylococcus beta-lactamase and two close-connected gene blai and blaR and Large plasmid are if pST6 is also on same plasmid.Also qac gene, beta-lactamase and latk gene coexist on many R-plasmids pMs62 to have report to point out, the latter also carries ermc, frA, acAaphD, produce to the antibiotics resistance such as erythromycin, Sulfamonomethoxine, these for streptococcus aureus resistance and sterilizing agent crossing drug resistant provide some prompting and references.
Summary of the invention
The invention provides a kind of sublethal dose streptococcus aureus resistance and drug method, the inventive method has recovers triplicity by the resistance of bacterium, resistance and susceptibility, particularly very intuitively can see the Changing Pattern between resistance, resistance and contact, have accurately, directly disclose both the advantage of internal connection.
A method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance, comprises the steps:
(1) preparation is containing the bacteria suspension of the streptococcus aureus of agent interfering;
(2) bacteria suspension of sterilizing agent to the described streptococcus aureus containing agent interfering preparing different sorts different concns carries out bactericidal assay and measures the resistance of every generation sublethal dose streptococcus aureus; Described in Simultaneously test, every generation sublethal dose streptococcus aureus is to antibiotic resistance; Did for 20 generations continuously;
(3) susceptibility recover experiment: choose through sterilizing agent sublethal dose screening the 20th cash equivalent staphylococcus aureus as primary bacterium, be inoculated in blood increasing bacterial context soup pipe to cultivate and be prepared into blood and increase bacterial context soup bacteria suspension, described blood is increased bacterial context soup bacteria suspension and carry out drug sensitivity test; Repeat above-mentioned steps to the 40th generation.
(4) according to the measurement result of described step (2), (3) and (4), described sublethal dose streptococcus aureus is found out to the resistance of described sterilizing agent and to the Changing Pattern of described antibiotic resistance and contact.
The method of Simultaneously test sublethal dose streptococcus aureus resistance of the present invention and resistance, wherein, described step (1) specifically comprises the steps:
By described streptococcus aureus through Zengjing Granule and separation and Culture, after separation and Culture, described streptococcus aureus is divided into single bacterium, and makes its independent fissiparity form independent bacterium colony; Be inoculated into nutrient agar slopes with the colonies typical that separation and Culture described in inoculating needle picking obtains, under 35-37 DEG C of condition, cultivate 24h form lawn; Be that 3% phosphate buffered saline buffer washes lower described lawn with massfraction, after sterile absorbent cotton is filtered, obtain filtrate through shaken well, dilute described filtrate with sterilized water and be prepared into the bacteria suspension that concentration is 0.5 Maxwell standard; Described bacteria suspension and agent interfering solution being done the concentration that two-fold dilution is made into 10ml is 7.5 × 10
7the bacteria suspension that cfu/ml contains agent interfering is for subsequent use, be prepared from 0.45 μm of filtering with microporous membrane is degerming after being mixed by finished product serum albumin 30g and distilled water 1000mL during described agent interfering solution, in described agent interfering solution, the massfraction of bovine serum albumin is 3%.
The method of Simultaneously test sublethal dose streptococcus aureus resistance of the present invention and resistance, wherein, described step (2) specifically comprises the steps:
The preparation of each concentration Iodophor: get 11 sterile test tube numbering A respectively
1~ A
11, with syringe holder numbering A
2~ A
11test tube respectively add 4 ml distilled waters, get 4 milliliters of the original iodine liquids and add A
1in number test tube, in described the original iodine liquid, the concentration of available iodine is 5000mg/l; Get 4 milliliters of described the original iodine liquids again and add A
2the mixing of number test tube obtains A
2iodophor liquid, the concentration of its available iodine is 2500mg/l; Then from A
24 milliliters of A got by number test tube
2iodophor liquid is added to A
3number test tube, the concentration of its available iodine is 1250mg/l; Single job is to A
10number test tube, the rest may be inferred A
4the concentration of number test tube available iodine is 625mg/l; A
5the concentration of number test tube available iodine is 312.5mg/l; A
6the concentration of number test tube available iodine is 156.25mg/l; A
7the concentration of number test tube available iodine is 78.13mg/l; A
8the concentration of number test tube available iodine is about 39.06mg/l; A
9the concentration of number test tube available iodine is about 19.53mg/l; A
10the concentration of number test tube available iodine is 9.77mg/l; A
11number test tube does not add thimerosal, and just 4 ml distilled waters are as positive control;
The preparation of each concentration staphylococcus lysozyme: get 9 sterile test tube numbering a respectively
1~ a
9, with syringe holder numbering a
2~ a
9test tube respectively add 4 ml distilled waters, get 4 milliliters of staphylococcus lysozyme stostes and add a
1in number test tube, in described staphylococcus proenzyme liquid, the concentration of staphylococcus lysozyme is 1000U/L; Get 4 milliliters of described staphylococcus lysozyme stostes again and add a
2the mixing of number test tube obtains a
2dissolving staphylococcal bacteria enzyme solution, its concentration is 500U/L; Then 4 milliliters of described a are got
2dissolving staphylococcal bacteria enzyme solution is added to a
3a is obtained in number test tube
3dissolving staphylococcal bacteria enzyme solution, the concentration of its staphylococcus lysozyme is 250U/L; Be operated to a successively
8number test tube, the rest may be inferred a
4the concentration of number test tube staphylococcus lysozyme is 125U/L; a
5the concentration of number test tube staphylococcus lysozyme is 62.50U/L; a
6the concentration of number test tube staphylococcus lysozyme is 31.25U/L; a
7number test tube dissolving staphylococcal bacteria enzyme concn is 15.63U/L; a
8the concentration of number test tube staphylococcus lysozyme is 7.80U/L; a
9number test tube only add 4 ml distilled waters containing thimerosal as positive control;
The preparation of each concentration chlorine-containing disinfection liquid: get 11 sterile test tube and numbering B
1~ B
11, first prepare available chlorine be 5000.0mg/l as stoste, use syringe holder B
2~ B
11test tube respectively add 4 ml distilled waters, getting 4 ml concns is that 5000.0mg/l available chlorine stoste adds B
1in number test tube, the concentration of available chlorine is 5000mg/l; Get 4 milliliters again and add B
2the mixing of number test tube obtains B
2chlorine-containing disinfection liquid, the concentration of its available chlorine is 2500mg/l; Then 4 milliliters of described B are got
2chlorine-containing disinfection liquid is added to described B
3the mixing of number test tube to B
3chlorine-containing disinfection liquid, the concentration of its available chlorine is 1250mg/l; Be operated to B successively
10number test tube, the rest may be inferred B
4in number test tube, the concentration of available chlorine is 625mg/l; B
5in number test tube, the concentration of available chlorine is 312.5mg/l; B
6in number test tube, the concentration of available chlorine is 156.25mg/l; B
7in number test tube, the concentration of available chlorine is 78.13mg/l; B
8in number test tube, the concentration of available chlorine is 39.06mg/l; B
9in number test tube, the concentration of available chlorine is 19.53mg/l; B
10in number test tube, the concentration of available chlorine is 9.77mg/l; No. 11 test tubes only add 4 ml distilled waters, do not contain thimerosal as positive control;
Be primary with the described bacteria suspension of streptococcus aureus containing agent interfering prepared, the required medium slant of sampling inoculation, then respectively with described in configure each concentration Iodophor, chlorine-containing disinfectant and composite lysostaphin effect, the action time of described Iodophor is 0.5min, the action time of described chlorine-containing disinfectant is 5min, and the action time of described composite lysostaphin is 6min; Then to proceed at once in the meat soup pipe containing corresponding neutralizing agent and 10 minutes, transferred species counting plate after neutralization, the streptococcus aureus of sublethal dose is got again from respective counting plate, repeat above-mentioned steps to 20 generation, more described 20 generation sublethal dose streptococcus aureus resistance obtain described sublethal dose streptococcus aureus the resistance of sterilizing agent changed;
While the described sublethal dose streptococcus aureus of mensuration is to the resistance change of sterilizing agent, adopt K-B scraps of paper test method(s), the respectively change of the golden yellow grape bacterial antibiotic susceptibility in the sublethal dose 1-20 generation of three kinds of thimerosals described in Continuous Observation record, described microbiotic is following one of several: penicillin, erythromycin, vancomycin, gentamicin, Rifampin, levofloxacin, cefoxitin, SMZ-CO.
The method of Simultaneously test sublethal dose streptococcus aureus resistance of the present invention and resistance, wherein, described step (4) specifically comprises the steps:
Choose the 20th cash equivalent staphylococcus aureus through the screening of sterilizing agent sublethal dose as primary bacterium, dip typical bacterium colony with transfering loop, be inoculated into blood and increase that to cultivate 24h in bacterial context soup pipe be a generation; Dip cultured blood with described transfering loop and increase bacterial context soup bacteria suspension, be inoculated into and be separated in plate, be placed in 35 DEG C-37 DEG C and cultivate 18-24h; Be inoculated on nutrient agar slopes with the colonies typical that described transfering loop dips in described separation plate, cultivate 18-24h for 35 DEG C-37 DEG C and obtain lawn; Massfraction be 3% phosphate buffered saline buffer wash lower lawn, shaken well also filters through sterile absorbent cotton and is prepared into described blood and increases bacterial context soup bacteria suspension; Carry out drug sensitivity test according to described step (3), record each drug sensitivity tests, repeat continuously to go down to posterity from the 20th generation to the 40th generation.
The method of Simultaneously test sublethal dose streptococcus aureus resistance of the present invention and resistance, wherein, the mensuration of described sublethal dose streptococcus aureus to antibiotic resistance specifically comprises the steps:
The bacteria suspension of described 1-20 for the streptococcus aureus of sublethal dose is dipped respectively with aseptic cotton carrier, in vitro wall ullage rotates and presses, unnecessary bacterium liquid is squeezed and goes, then on each M-H culture medium flat plate agar surface, uniform application inoculates 3 times, each Rotating Plates 60 DEG C, to guarantee to inoculate being uniformly distributed of bacterium, finally smear one week along dull and stereotyped inner edge, dry 5 minutes; Then on flat board, be placed with the diameter 5mm Antibiotic discs chosen, make it be close to planar surface, each plate pastes 4, build plate, be placed in 35-37 DEG C and cultivate 18-20h, measure inhibition zone with vernier callipers, record each drug sensitivity tests, result judges according to Clinical microorganism normalizing operation; Do the test of 20 generations continuously.
The method of Simultaneously test sublethal dose streptococcus aureus resistance of the present invention and resistance, wherein, increase bacterial context soup containing blood in described increasing bacterial context soup pipe, its component is: peptone 10.0g/L; Extracted beef powder 3.0g/L; Sodium-chlor 5.0g/L; Glucose 1.0g/L; Sodium Citrate 3.0g/L; Dipotassium hydrogen phosphate 2g g/L; Para-amino benzoic acid 0.05g/L; Magnesium sulfate 2.4g/L; Phenol red 0.024g/L; All the other are distilled water; It is 7.4 ± 0.1 that blood increases bacterial context soup pH value; For subsequent use through 121 DEG C of sterilizing 15min and 35 DEG C 24h sterility test.
The method of Simultaneously test sublethal dose streptococcus aureus resistance of the present invention and resistance, wherein, described step (3) specifically comprises the steps: it is adopt K-B scraps of paper test method(s), the change of the raw plain susceptibility of every generation antagonism of the yellow grape bacterium of the sublethal dose 1-20 cash equivalent of three kinds of thimerosals described in Continuous Observation record, described microbiotic is following one of several: penicillin, erythromycin, vancomycin, gentamicin, Rifampin, levofloxacin, cefoxitin, SMZ-CO.
In the inventive method, 0.5 Maxwell opacity tube compound method is: 0.48mol/L Bacl2 (1.17%w/v BaCl
22H
2o) solution 0.5ml; 0.18mol/L H
2sO
4solution 99.5ml; By two liquid mixing, split sealing in pipe, use front mixing; 0.5 Maxwell standard: bacteria containing amount 1.5 × 10
8cfu/ml
Sublethal dose streptococcus aureus resistance of the present invention and drug method, perform key problems in process of the present invention as follows:
The pollution discharging technology of various substratum: test nutrient broth pipe used, numeration plate, nutrient agar slopes, be placed in for first 2 days in incubator in experiment and cultivate 48h through 35-37 DEG C, do not have pollution to use;
The pollution discharging technology of experiment bacteria suspension: from the first smear gramstaining of the bacteria suspension slant medium is washed, whether be streptococcus aureus, after getting rid of the pollution of experiment bacteria suspension, then do sterilization experiment if observing;
The pollution discharging technology of environment: the i.e. utilization of aseptic technique;
Before experiment, experimental article is got all the ready: autoclaved barrier gown, use disposable breathing mask and cap, sterile gloves; The following article putting into clean bench are first used 75% alcohol wipe dedusting: blood increases various substratum (pre-incubated), spirit lamp, transfering loop, inoculating needle, disposable syringe, sterile test tube, Sterile Hard Water, three kinds of thimerosals, calf serums etc. such as bacterial context Tang Guan simultaneously;
The integrity of checking experiment equipment: 35-37 DEG C incubator etc.;
The preparation of laboratory overall situation: test and carry out at biocontainment laboratory, each experiment various surface of clean room the day before yesterday and ground, first experimental day opens the disinfection by ultraviolet light more than 40 minutes of room and super clean bench;
The preparation of operating environment: wash hands, wears sterilizing barrier gown, band disposable breathing mask, cap, band sterile gloves; With 75% alcohol disinfecting wiping Bechtop face before test;
Sterilization in the present invention and anti-drug sensitive test all operate according to disinfection technology standard.
Compared with prior art, the method for Simultaneously test sublethal dose streptococcus aureus resistance of the present invention and resistance has as follows a little:
The present invention is by the resistance of bacterium, resistance and drug susceptibility recover three ingenious combinations of experimental technique, can be accurate, clearly measure the dynamic relationship between three, measurement result is with a high credibility, disclose sublethal dose sterilizing agent (being equivalent to use sterilizing agent lack of standardization in real work), not only select the bacterium that sterilizing agent tolerance is increased, and the bacterial antibiotic selected also produces resistance, illustrate there is crossing drug resistant phenomenon between the two, therefore this experiment is that the use management of reinforcement sterilizing agent in the future (will as use antibacterials, want Normalization rule sterilizing agent, reduce the generation of bacterial drug resistance and resistance, delay sterilizing agent and antibiotic life-span) theoretical direction is provided.
Embodiment
Embodiment 1
A method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance, comprises the steps:
(1) preparation is containing the bacteria suspension of the streptococcus aureus of agent interfering;
(2) bacteria suspension of sterilizing agent to the described streptococcus aureus containing agent interfering preparing different sorts different concns carries out bactericidal assay and measures the resistance of every generation sublethal dose streptococcus aureus; Described in Simultaneously test, every generation sublethal dose streptococcus aureus is to antibiotic resistance; Did for 20 generations continuously;
(3) susceptibility recover experiment: choose through sterilizing agent sublethal dose screening the 20th cash equivalent staphylococcus aureus as primary bacterium, be inoculated in blood increasing bacterial context soup pipe to cultivate and be prepared into blood and increase bacterial context soup bacteria suspension, described blood is increased bacterial context soup bacteria suspension and carry out drug sensitivity test; Repeat above-mentioned steps to the 40th generation.
(4) according to the measurement result of described step (2), (3) and (4), described sublethal dose streptococcus aureus is found out to the resistance of described sterilizing agent and to the Changing Pattern of described antibiotic resistance and contact.
Embodiment 2
A method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance, comprises the steps:
(1) by described streptococcus aureus through Zengjing Granule and separation and Culture, after separation and Culture, described streptococcus aureus is divided into single bacterium, and makes its independent fissiparity form independent bacterium colony; Be inoculated into nutrient agar slopes with the colonies typical that separation and Culture described in inoculating needle picking obtains, under 35-37 DEG C of condition, cultivate 24h form lawn; Be that 3% phosphate buffered saline buffer washes lower described lawn with massfraction, after sterile absorbent cotton is filtered, obtain filtrate through shaken well, dilute described filtrate with sterilized water and be prepared into the bacteria suspension that concentration is 0.5 Maxwell standard; Described bacteria suspension and agent interfering solution being done the concentration that two-fold dilution is made into 10ml is 7.5 × 10
7the bacteria suspension that cfu/ml contains agent interfering is for subsequent use, be prepared from 0.45 μm of filtering with microporous membrane is degerming after being mixed by finished product serum albumin 30g and distilled water 1000mL during described agent interfering solution, in described agent interfering solution, the massfraction of bovine serum albumin is 3%;
(2) preparation of each concentration Iodophor: get 11 sterile test tube numbering A respectively
1~ A
11, with syringe holder numbering A
2~ A
11test tube respectively add 4 ml distilled waters, get 4 milliliters of the original iodine liquids and add A
1in number test tube, in described the original iodine liquid, the concentration of available iodine is 5000mg/l; Get 4 milliliters of described the original iodine liquids again and add A
2the mixing of number test tube obtains A
2iodophor liquid, the concentration of its available iodine is 2500mg/l; Then from A
24 milliliters of A got by number test tube
2iodophor liquid is added to A
3number test tube, the concentration of its available iodine is 1250mg/l; Single job is to A
10number test tube, the rest may be inferred A
4the concentration of number test tube available iodine is 625mg/l; A
5the concentration of number test tube available iodine is 312.5mg/l; A
6the concentration of number test tube available iodine is 156.25mg/l; A
7the concentration of number test tube available iodine is 78.13mg/l; A
8the concentration of number test tube available iodine is about 39.06mg/l; A
9the concentration of number test tube available iodine is about 19.53mg/l; A
10the concentration of number test tube available iodine is 9.77mg/l; A
11number test tube does not add thimerosal, and just 4 ml distilled waters are as positive control;
The preparation of each concentration staphylococcus lysozyme: get 9 sterile test tube numbering a respectively
1~ a
9, with syringe holder numbering a
2~ a
9test tube respectively add 4 ml distilled waters, get 4 milliliters of staphylococcus lysozyme stostes and add a
1in number test tube, in described staphylococcus proenzyme liquid, the concentration of staphylococcus lysozyme is 1000U/L; Get 4 milliliters of described staphylococcus lysozyme stostes again and add a
2the mixing of number test tube obtains a
2dissolving staphylococcal bacteria enzyme solution, its concentration is 500U/L; Then 4 milliliters of described a are got
2dissolving staphylococcal bacteria enzyme solution is added to a
3a is obtained in number test tube
3dissolving staphylococcal bacteria enzyme solution, the concentration of its staphylococcus lysozyme is 250U/L; Be operated to a successively
8number test tube, the rest may be inferred a
4the concentration of number test tube staphylococcus lysozyme is 125U/L; a
5the concentration of number test tube staphylococcus lysozyme is 62.50U/L; a
6the concentration of number test tube staphylococcus lysozyme is 31.25U/L; a
7number test tube dissolving staphylococcal bacteria enzyme concn is 15.63U/L; a
8the concentration of number test tube staphylococcus lysozyme is 7.80U/L; a
9number test tube only add 4 ml distilled waters containing thimerosal as positive control;
The preparation of each concentration chlorine-containing disinfection liquid: get 11 sterile test tube and numbering B
1~ B
11, first prepare available chlorine be 5000.0mg/l as stoste, use syringe holder B
2~ B
11test tube respectively add 4 ml distilled waters, getting 4 ml concns is that 5000.0mg/l available chlorine stoste adds B
1in number test tube, the concentration of available chlorine is 5000mg/l; Get 4 milliliters again and add B
2the mixing of number test tube obtains B
2chlorine-containing disinfection liquid, the concentration of its available chlorine is 2500mg/l; Then 4 milliliters of described B are got
2chlorine-containing disinfection liquid is added to described B
3the mixing of number test tube to B
3chlorine-containing disinfection liquid, the concentration of its available chlorine is 1250mg/l; Be operated to B successively
10number test tube, the rest may be inferred B
4in number test tube, the concentration of available chlorine is 625mg/l; B
5in number test tube, the concentration of available chlorine is 312.5mg/l; B
6in number test tube, the concentration of available chlorine is 156.25mg/l; B
7in number test tube, the concentration of available chlorine is 78.13mg/l; B
8in number test tube, the concentration of available chlorine is 39.06mg/l; B
9in number test tube, the concentration of available chlorine is 19.53mg/l; B
10in number test tube, the concentration of available chlorine is 9.77mg/l; No. 11 test tubes only add 4 ml distilled waters, do not contain thimerosal as positive control;
Be primary with the described bacteria suspension of streptococcus aureus containing agent interfering prepared, the required medium slant of sampling inoculation, then respectively with described in configure each concentration Iodophor, chlorine-containing disinfectant and composite lysostaphin effect, the action time of described Iodophor is 0.5min, the action time of described chlorine-containing disinfectant is 5min, and the action time of described composite lysostaphin is 6min; Then to proceed at once in the meat soup pipe containing corresponding neutralizing agent and 10 minutes, transferred species counting plate after neutralization, the streptococcus aureus of sublethal dose is got again from respective counting plate, repeat above-mentioned steps to 20 generation, more described 20 generation sublethal dose streptococcus aureus resistance obtain described sublethal dose streptococcus aureus the resistance of sterilizing agent changed.
While the described sublethal dose streptococcus aureus of mensuration is to the resistance change of sterilizing agent, adopt K-B scraps of paper test method(s), the respectively change of the golden yellow grape bacterial antibiotic susceptibility in the sublethal dose 1-20 generation of three kinds of thimerosals described in Continuous Observation record, described microbiotic is following one of several: penicillin, erythromycin, vancomycin, gentamicin, Rifampin, levofloxacin, cefoxitin, SMZ-CO;
The bacteria suspension of described 1-20 for the streptococcus aureus of sublethal dose is dipped respectively with aseptic cotton carrier, in vitro wall ullage rotates and presses, unnecessary bacterium liquid is squeezed and goes, then on each M-H culture medium flat plate agar surface, uniform application inoculates 3 times, each Rotating Plates 60 DEG C, to guarantee to inoculate being uniformly distributed of bacterium, finally smear one week along dull and stereotyped inner edge, dry 5 minutes; Then on flat board, be placed with the diameter 5mm Antibiotic discs chosen, make it be close to planar surface, each plate pastes 4, build plate, be placed in 35-37 DEG C and cultivate 18-20h, measure inhibition zone with vernier callipers, record each drug sensitivity tests, result judges according to Clinical microorganism normalizing operation; Do the test of 20 generations continuously;
(3) susceptibility recovers experiment: choose the 20th cash equivalent staphylococcus aureus through the screening of sterilizing agent sublethal dose as primary bacterium, dip typical bacterium colony with transfering loop, is inoculated into blood and increases that to cultivate 24h in bacterial context soup pipe be a generation; Dip cultured blood with described transfering loop and increase bacterial context soup bacteria suspension, be inoculated into and be separated in plate, be placed in 35 DEG C-37 DEG C and cultivate 18-24h; Be inoculated on nutrient agar slopes with the colonies typical that described transfering loop dips in described separation plate, cultivate 18-24h for 35 DEG C-37 DEG C and obtain lawn; Massfraction be 3% phosphate buffered saline buffer wash lower lawn, shaken well also filters through sterile absorbent cotton and is prepared into described blood and increases bacterial context soup bacteria suspension; Carry out drug sensitivity test according to described step (3), record each drug sensitivity tests, repeat continuously to go down to posterity from the 20th generation to the 40th generation;
Increase bacterial context soup containing blood in described increasing bacterial context soup pipe, its component is: peptone 10.0g/L; Extracted beef powder 3.0g/L; Sodium-chlor 5.0g/L; Glucose 1.0g/L; Sodium Citrate 3.0g/L; Dipotassium hydrogen phosphate 2g g/L; Para-amino benzoic acid 0.05g/L; Magnesium sulfate 2.4g/L; Phenol red 0.024g/L; All the other are distilled water; It is 7.4 ± 0.1 that blood increases bacterial context soup pH value; For subsequent use through 121 DEG C of sterilizing 15min and 35 DEG C 24h sterility test.
(4) according to the measurement result of described step (2), (3) and (4), described sublethal dose streptococcus aureus is found out to the resistance of described sterilizing agent and to the Changing Pattern of described antibiotic resistance and contact.
The result that the present embodiment records is as follows:
Sterilizing agent to difference go down to posterity killing bacteria effect change:
Through observing display to primary to 20 cash equivalent staphylococcus aureus killing effects, available iodine concentration, within the scope of 39.06mg/L ~ 9.77mg/L, all acts on 0.5min; Available chlorine, within the scope of 19.53mg/L ~ 9.77mg/L, all acts on 5min; Though have fluctuation to the streptococcus aureus killing rate of different passage number, do not reduce trend, concrete outcome is in table 1.
Table 1 two kinds of chemostefilants are to different passage number streptococcus aureus killing effect
Dissolving staphylococcal bacteria enzyme concn is within the scope of 1000U/L ~ 7.8U/L, all act on 6min, though have fluctuation to the streptococcus aureus killing rate of 20 times of going down to posterity, but there is no downtrending, result is pointed out, streptococcus aureus type strain is through 20 cultures, and it does not change to the drag of tested three kinds of sterilizing agents, and concrete outcome is in table 2.
Table 2 staphylococcus lysozyme is to different passage number streptococcus aureus killing effect
Three kinds of sterilizing agent induction streptococcus aureus drug sensitive tests and susceptibility recover experimental result
The go down to posterity result in 20 generations of three kinds of sterilizing agents shows to occur resistance through the go down to posterity streptococcus aureus of 10 times of Iodophor effect to penicillin and erythromycin, and goes down to posterity 20 times with hemotrophic nutrition, and susceptibility is not recovered; Bacterial strain after another two kinds of sterilizing agent effects is then to the basic irregularities change of penicillin, erythromycin and vancomycin 3 kinds of antibacterials drags, and concrete data are in table 3.
Table 3 sterilizing agent induction streptococcus aureus drug sensitive test result
Remarks: odd number stringer be 1-20 for sterilization experiment drug sensitivity tests, even numbers stringer is the drug sensitivity tests of 21-40 for blood Zengjing Granule
Resistant strain recovers test-results to thimerosal susceptibility
Result shows, through the streptococcus aureus in sterilizing agent inducing culture 20 generation, then menses culture medium culturing 20 generation, its susceptibility of sterilizing agent is not recovered or the sign that changes in table 4 and table 5; But the bacterial strain in lower concentration N,O-Diacetylmuramidase inducing culture 20 generation, blood meida continues go down to posterity after 20 times, the trend that drag strengthens on the contrary, in table 6.
Table 4 resistant strain is to iodophor disinfectant susceptibility recovery situation
Note: A40 on behalf of the S. aureus L-forms of inducing through Iodophor again hemoculture passed for 20 generations
Table 5 resistant strain is to chlorine-containing disinfectant susceptibility recovery situation
Note: B40 on behalf of the S. aureus L-forms of inducing through chlorine-containing disinfectant again hemoculture passed for 20 generations
Table 6 resistant strain is to staphylococcus lysozyme susceptibility recovery situation
Note: a40 on behalf of the S. aureus L-forms of inducing through N,O-Diacetylmuramidase again hemoculture passed for 20 generations
2.4 streptococcus aureuses are homophyletic drug sensitive test result not
Test shows, through the streptococcus aureus of above-mentioned sterilizing agent inducing culture and the maximum Secondary Culture of MRSA bacterial strain 45 times, its phenomenon all do not recovered the susceptibility of different antibacterials, concrete data are in table 7.
Table 7 is homophyletic streptococcus aureus drug sensitive test result not
Note: in table, A, B, a represent respectively through Iodophor, chlorine-containing disinfectant and N,O-Diacetylmuramidase induction streptococcus aureus
Can be drawn the following conclusions by above result:
The concentration of sterilizing agent is one of deciding factor of germicidal action, research finds, this tests three kinds of sterilizing agents used has good killing action to all experimental bacteria comprising multi-drug resistant bacteria under low concentration, but has test organisms survival in the thimerosal of very low (sub-lethal) concentration.In real work, the residual of sublethal dose sterilizing agent can exist everywhere, as the body surface etc. on the skin after skin degerming or after various hand-wrist bones and after sterilization.
This research finds, sublethal dose thimerosal can induce the bacterial strain that existing certain resistance has again resistance, and it is to Disinfectant resistance and have certain contacting to the resistance of antibacterials.Test proves, resistant strain had both made to go down to posterity for 30 generations on nutrient-reinforced substratum, the sign that its susceptibility is not also recovered.The reports such as Zhang, clinical nurse nasal cavity streptococcus aureus carrying rate is 20.5%, MRSA definite value rate is 3.21%, and nurse's nasal cavity is separated the recall rate of flora qacA/B gene up to 41.2%.Because nurse has the chance of more contact severe case and contaminated hospital environment, and the selective pressure of hospital disinfection agent causes formation and the propagation of resistant to disinfectants bacterial strain, the risk " 8 " of hospital infection can be formed by continuous low-level sterilizing agent residual region.Therefore, to when participating in transplanting contour risk operations, contact large-area burns or need the patient of Protective isolation, medical personnel should reduce resistant organism field planting or initiatively remove and kill the Resistant strain that human body carries.
Find in this research, experiment culture plate of being everlasting grows coarse and fried egg look bacterium colony, also has research to turn out similar bacterium colony from hospital's endoscope cleaning machine, and prompting test organisms can be different and change with nutritional status in culturing process.One of sterilizing agent resistance influence factor is microbial nutrition state, and nutrition limitations affect factor only has relevant with halogen resistance, is mediated by Special Proteins, and cAMP can promote that its resistance produces.The resistant strain transferred species blood Zengjing Granule pipe 2-3 of this theoretical reasonable dismissal this research bactericidal assay induction recovers to some extent for the rear susceptibility to vancomycin, but awaits further confirmation.
Have report to contact Iodophor frequently for a long time, the resistance of microorganism to Iodophor has increase in various degree, and generation tolerance and MRSA may have inherent contacting with it to the resistance of Iodophor to antibiotic tolerance.This research also finds that multi-drug resistant bacteria does not increase chlorine-containing disinfectant resistance, increases to some extent to the resistance of Iodophor.Staphylococcus lysozyme does not induce Resistant strain under sublethal dose condition as Iodophor, and front 3 concentration also 100.0% can kill MRSA.
Research discloses further, and there is crossed resistance between sterilizing agent and antibacterials, bacterium, after disengaging certain sterilizing agent for a long time, can not disappear at once to the resistance of this sterilizing agent.The continuous action of the sterilizing agent of sublethal dose may increase the selective action of thimerosal Resistant strain, makes it in hospital environment, obtain growth vigor, and antibiotic a large amount of use that vice versa also likely produces sterilizing agent resistant strain and selects.Therefore advise, the use range of sterilizing agent be limited, stop abuse; Low effect disinfectants is preferably used alternatingly; Resistance and patience and multi-drug resistant bacteria are produced to sublethal dose thimerosal bacterium and to increase in bacterial context soup the phenomenons such as susceptibility recovery at blood, it is desirable to further to be studied.
Above-described embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that those of ordinary skill in the art make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determines.
Claims (7)
1. a method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance, is characterized in that, comprises the steps:
(1) preparation is containing the bacteria suspension of the streptococcus aureus of agent interfering;
(2) bacteria suspension of sterilizing agent to the described streptococcus aureus containing agent interfering preparing different sorts different concns carries out bactericidal assay and measures the resistance of every generation sublethal dose streptococcus aureus; Described in Simultaneously test, every generation sublethal dose streptococcus aureus is to antibiotic resistance; Did for 20 generations continuously;
(3) susceptibility recover experiment: choose through sterilizing agent sublethal dose screening the 20th cash equivalent staphylococcus aureus as primary bacterium, be inoculated in blood increasing bacterial context soup pipe to cultivate and be prepared into blood and increase bacterial context soup bacteria suspension, described blood is increased bacterial context soup bacteria suspension and carry out drug sensitivity test; Repeat above-mentioned steps to the 40th generation.
(4) according to the measurement result of described step (2), (3) and (4), described sublethal dose streptococcus aureus is found out to the resistance of described sterilizing agent and to the Changing Pattern of described antibiotic resistance and contact.
2. the method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance according to claim 1, it is characterized in that, described step (1) specifically comprises the steps:
By described streptococcus aureus through Zengjing Granule and separation and Culture, after separation and Culture, described streptococcus aureus is divided into single bacterium, and makes its independent fissiparity form independent bacterium colony; Be inoculated into nutrient agar slopes with the colonies typical that separation and Culture described in inoculating needle picking obtains, under 35-37 DEG C of condition, cultivate 24h form lawn; Be that 3% phosphate buffered saline buffer washes lower described lawn with massfraction, after sterile absorbent cotton is filtered, obtain filtrate through shaken well, dilute described filtrate with sterilized water and be prepared into the bacteria suspension that concentration is 0.5 Maxwell standard; Described bacteria suspension and agent interfering solution being done the concentration that two-fold dilution is made into 10ml is 7.5 × 10
7the bacteria suspension that cfu/ml contains agent interfering is for subsequent use, be prepared from 0.45 μm of filtering with microporous membrane is degerming after being mixed by finished product serum albumin 30g and distilled water 1000mL during described agent interfering solution, in described agent interfering solution, the massfraction of bovine serum albumin is 3%.
3. the method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance according to claim 1, it is characterized in that, described step (2) specifically comprises the steps:
The preparation of each concentration Iodophor: get 11 sterile test tube numbering A respectively
1~ A
11, with syringe holder numbering A
2~ A
11test tube respectively add 4 ml distilled waters, get 4 milliliters of the original iodine liquids and add A
1in number test tube, in described the original iodine liquid, the concentration of available iodine is 5000mg/l; Get 4 milliliters of described the original iodine liquids again and add A
2the mixing of number test tube obtains A
2iodophor liquid, the concentration of its available iodine is 2500mg/l; Then from A
24 milliliters of A got by number test tube
2iodophor liquid is added to A
3number test tube, the concentration of its available iodine is 1250mg/l; Single job is to A
10number test tube, the rest may be inferred A
4the concentration of number test tube available iodine is 625mg/l; A
5the concentration of number test tube available iodine is 312.5mg/l; A
6the concentration of number test tube available iodine is 156.25mg/l; A
7the concentration of number test tube available iodine is 78.13mg/l; A
8the concentration of number test tube available iodine is about 39.06mg/l; A
9the concentration of number test tube available iodine is about 19.53mg/l; A
10the concentration of number test tube available iodine is 9.77mg/l; A
11number test tube does not add thimerosal, and just 4 ml distilled waters are as positive control;
The preparation of each concentration staphylococcus lysozyme: get 9 sterile test tube numbering a respectively
1~ a
9, with syringe holder numbering a
2~ a
9test tube respectively add 4 ml distilled waters, get 4 milliliters of staphylococcus lysozyme stostes and add a
1in number test tube, in described staphylococcus proenzyme liquid, the concentration of staphylococcus lysozyme is 1000U/L; Get 4 milliliters of described staphylococcus lysozyme stostes again and add a
2the mixing of number test tube obtains a
2dissolving staphylococcal bacteria enzyme solution, its concentration is 500U/L; Then 4 milliliters of described a are got
2dissolving staphylococcal bacteria enzyme solution is added to a
3a is obtained in number test tube
3dissolving staphylococcal bacteria enzyme solution, the concentration of its staphylococcus lysozyme is 250U/L; Be operated to a successively
8number test tube, the rest may be inferred a
4the concentration of number test tube staphylococcus lysozyme is 125U/L; a
5the concentration of number test tube staphylococcus lysozyme is 62.50U/L; a
6the concentration of number test tube staphylococcus lysozyme is 31.25U/L; a
7number test tube dissolving staphylococcal bacteria enzyme concn is 15.63U/L; a
8the concentration of number test tube staphylococcus lysozyme is 7.80U/L; a
9number test tube only add 4 ml distilled waters containing thimerosal as positive control;
The preparation of each concentration chlorine-containing disinfection liquid: get 11 sterile test tube and numbering B
1~ B
11, first prepare available chlorine be 5000.0mg/l as stoste, use syringe holder B
2~ B
11test tube respectively add 4 ml distilled waters, getting 4 ml concns is that 5000.0mg/l available chlorine stoste adds B
1in number test tube, the concentration of available chlorine is 5000mg/l; Get 4 milliliters again and add B
2the mixing of number test tube obtains B
2chlorine-containing disinfection liquid, the concentration of its available chlorine is 2500mg/l; Then 4 milliliters of described B are got
2chlorine-containing disinfection liquid is added to described B
3the mixing of number test tube to B
3chlorine-containing disinfection liquid, the concentration of its available chlorine is 1250mg/l; Be operated to B successively
10number test tube, the rest may be inferred B
4in number test tube, the concentration of available chlorine is 625mg/l; B
5in number test tube, the concentration of available chlorine is 312.5mg/l; B
6in number test tube, the concentration of available chlorine is 156.25mg/l; B
7in number test tube, the concentration of available chlorine is 78.13mg/l; B
8in number test tube, the concentration of available chlorine is 39.06mg/l; B
9in number test tube, the concentration of available chlorine is 19.53mg/l; B
10in number test tube, the concentration of available chlorine is 9.77mg/l; No. 11 test tubes only add 4 ml distilled waters, do not contain thimerosal as positive control;
Be primary with the described bacteria suspension of streptococcus aureus containing agent interfering prepared, the required medium slant of sampling inoculation, then respectively with described in configure each concentration Iodophor, chlorine-containing disinfectant and composite lysostaphin effect, the action time of described Iodophor is 0.5min, the action time of described chlorine-containing disinfectant is 5min, and the action time of described composite lysostaphin is 6min; Then to proceed at once in the meat soup pipe containing corresponding neutralizing agent and 10 minutes, transferred species counting plate after neutralization, the streptococcus aureus of sublethal dose is got again from respective counting plate, repeat above-mentioned steps to 20 generation, more described 20 generation sublethal dose streptococcus aureus resistance obtain described sublethal dose streptococcus aureus the resistance of sterilizing agent changed;
While the described sublethal dose streptococcus aureus of mensuration is to the resistance change of sterilizing agent, adopt K-B scraps of paper test method(s), the respectively change of the golden yellow grape bacterial antibiotic susceptibility in the sublethal dose 1-20 generation of three kinds of thimerosals described in Continuous Observation record, described microbiotic is following one of several: penicillin, erythromycin, vancomycin, gentamicin, Rifampin, levofloxacin, cefoxitin, SMZ-CO.
4. the method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance according to claim 1, it is characterized in that, described step (4) specifically comprises the steps:
Choose the 20th cash equivalent staphylococcus aureus through the screening of sterilizing agent sublethal dose as primary bacterium, dip typical bacterium colony with transfering loop, be inoculated into blood and increase that to cultivate 24h in bacterial context soup pipe be a generation; Dip cultured blood with described transfering loop and increase bacterial context soup bacteria suspension, be inoculated into and be separated in plate, be placed in 35 DEG C-37 DEG C and cultivate 18-24h; Be inoculated on nutrient agar slopes with the colonies typical that described transfering loop dips in described separation plate, cultivate 18-24h for 35 DEG C-37 DEG C and obtain lawn; Be that 3% phosphate buffered saline buffer washes lower lawn with massfraction, shaken well also filters through sterile absorbent cotton and is prepared into described blood and increases bacterial context soup bacteria suspension; Carry out drug sensitivity test according to described step (3), record each drug sensitivity tests, repeat continuously to go down to posterity from the 20th generation to the 40th generation.
5. the method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance according to claim 3, it is characterized in that, the mensuration of described sublethal dose streptococcus aureus to antibiotic resistance specifically comprises the steps:
The bacteria suspension of described 1-20 for the streptococcus aureus of sublethal dose is dipped respectively with aseptic cotton carrier, in vitro wall ullage rotates and presses, unnecessary bacterium liquid is squeezed and goes, then on each M-H culture medium flat plate agar surface, uniform application inoculates 3 times, each Rotating Plates 60 DEG C, to guarantee to inoculate being uniformly distributed of bacterium, finally smear one week along dull and stereotyped inner edge, dry 5 minutes; Then on flat board, be placed with the diameter 5mm Antibiotic discs chosen, make it be close to planar surface, each plate pastes 4, build plate, be placed in 35-37 DEG C and cultivate 18-20h, measure inhibition zone with vernier callipers, record each drug sensitivity tests, result judges according to Clinical microorganism normalizing operation; Do the test of 20 generations continuously.
6. the method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance according to claim 5, is characterized in that, increase bacterial context soup containing blood in described increasing bacterial context soup pipe, its component is: peptone 10.0g/L; Extracted beef powder 3.0g/L; Sodium-chlor 5.0g/L; Glucose 1.0g/L; Sodium Citrate 3.0g/L; Dipotassium hydrogen phosphate 2g g/L; Para-amino benzoic acid 0.05g/L; Magnesium sulfate 2.4g/L; Phenol red 0.024g/L; All the other are distilled water; It is 7.4 ± 0.1 that described blood increases bacterial context soup pH value; For subsequent use through 121 DEG C of sterilizing 15min and 35 DEG C 24h sterility test.
7. the method for Simultaneously test sublethal dose streptococcus aureus resistance and resistance according to claim 4, it is characterized in that, described step (3) specifically comprises the steps: it is adopt K-B scraps of paper test method(s), the change of the raw plain susceptibility of every generation antagonism of the yellow grape bacterium of sublethal dose 1-20 cash equivalent of three kinds of thimerosals described in Continuous Observation record.
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| CN110305933A (en) * | 2019-08-20 | 2019-10-08 | 郑州安图生物工程股份有限公司 | A kind of method of quick detection susceptibility |
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