CN104651473B - The method for determining the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance simultaneously - Google Patents
The method for determining the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance simultaneously Download PDFInfo
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Abstract
The invention discloses a kind of while the method for the measure sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, comprises the following steps：(1) bacteria suspension of the staphylococcus aureus containing agent interfering is prepared；(2) resistance to the action of a drug is determined；(3) drug resistance is determined；(4) sensitiveness is recovered to determine；(5) the sub-lethal dose staphylococcus aureus is found out to the resistance of the disinfectant and its changing rule of drug resistance.The present invention determines the sub-lethal dose staphylococcus aureus resistance to the action of a drug simultaneously and the resistance to the action of a drug, drug resistance and drug sensitivity the recovery measure of bacterium are combined by the method for drug resistance in an experiment, changing rule and the contact between the resistance to the action of a drug, drug resistance particularly can be can be visually seen very much, the internal connection with both accurate, direct announcements.
Description
Technical field
Resistance and drug method are determined simultaneously the present invention relates to a kind of, and in particular to a kind of to determine simultaneously
The method of the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance.
Background technology
Find MRSA first in Britain from jevons in 1961, middle 1960s extend to many countries in Europe and
Canada, the end of the seventies, great increase of MRSA extended over the entire globe, and resistance scope expands day by day, and drug-resistant intensity is increasingly serious.The eighties
Later stage turns into global pathogen, occupies the first place of Nosocomial Infection Pathogens, accounts for the 60- that teaching hospital separates S. aureus L-forms
80%.Later stage 1990s occurs in that resistance to MRSA through the ages again.Current MRSA is the important component of superbacteria, its
Drug resistance is strong, the death rate is high has turned into and HBV, AIDS worldwide 3 big scabrous infectious diseases arranged side by side.Therefore visit
MRSA source and its resistance mechanism is begged for, effective control method is found, is the most effective approach for containing that its infection is popular.
Disinfectant and antibiotic belong to the chemical substance acted on special suppression microbicide, their works to microorganism
Also there are many same or similar parts, the equally possible resistance phenomenon produced to disinfectant of microorganism of some resistances with mechanism.
American scholar proposes antibiotic and disinfectant facilitates MRSA selection and the possibility of maintenance, and it is many that experiment proves that S. aureus L-forms contain
The drug resistance plasmid of antibiotic and disinfectant is planted, and finds to there is certain contact between them.Substantial amounts of epidemiology number
According to displaying that：The antibody-resistant bacterium of staphylococcus aureus is raised to the resistance of disinfectant.Around staphylococcus aureus family qac
Plasmid where the structural gene studies have shown that qac genes of gene and beta-lactamase carries antibiotic resistance base simultaneously
Because, and the structural gene blaz of staphylococcus beta-lactamase and two close-connected gene blai and blaR transposons
Tn552 and Large plasmid such as pST6 are also on same plasmid.Also it is reported that qac genes, beta-lactamase and latk genes are same
On many R-plasmid pMs62, the latter also carries ermc, frA, acAaphD, produces to antibiotic such as erythromycin, Sulfamonomethoxines
Resistance, these are that the staphylococcus aureus resistance to the action of a drug and disinfectant crossing drug resistant provide some promptings and reference.
The content of the invention
The invention provides a kind of sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug method, present invention side
Method has recovers triplicity by the resistance to the action of a drug, drug resistance and sensitiveness of bacterium, particularly can be visually seen very much the resistance to the action of a drug, resistance
Changing rule and contact between property, have the advantages that the internal connection for accurately and directly disclosing the two.
It is a kind of while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, comprise the following steps：
(1) bacteria suspension of the staphylococcus aureus containing agent interfering is prepared；
(2) bacteria suspension of the disinfectant to the staphylococcus aureus containing agent interfering of variety classes various concentrations is prepared
Carry out sterilization test and determine the resistance to the action of a drug of every generation sub-lethal dose staphylococcus aureus；The Asia per a generation is determined simultaneously to cause
Drug resistance of the dead amount staphylococcus aureus to antibiotic；Continuously did for 20 generations；
(3) sensitiveness recovers experiment：Choose the 20th cash equivalent staphylococcus aureus conduct of sterilized dose of sub-lethal dose screening
Primary bacterium, is inoculated into and blood increasing bacterial context soup bacteria suspension is cultivated and be prepared into blood increasing bacterial context soup pipe, the blood is increased into bacterial context
Soup bacteria suspension carries out drug sensitivity test；Repeat the above steps to the 40th generation.
(4) according to the measurement result of the step (2), (3) and (4), the sub-lethal dose staphylococcus aureus is found out
The resistance to the action of a drug to the disinfectant and its changing rule to the drug resistance of the antibiotic and contact.
It is of the present invention while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, wherein, institute
State step (1) and specifically include following steps：
The staphylococcus aureus is passed through into Zengjing Granule and is separately cultured, by the golden yellow Portugal after being separately cultured
Grape coccus is divided into single bacterium, and its independent schizogamy is formed independent bacterium colony；It is separately cultured with described in transfer needle picking
To colonies typical be inoculated into nutrient agar slopes, under the conditions of 35-37 DEG C cultivate 24h formation lawn；It is 3% with mass fraction
Phosphate buffer washes the lower lawn, obtains filtrate after being filtered through shaken well and through sterile absorbent cotton, is diluted with sterilized water
The filtrate is prepared into the bacteria suspension that concentration is 0.5 Maxwell standard；The bacteria suspension is done into two-fold dilution with interference agent solution to match somebody with somebody
Concentration into 10ml is 7.5 × 107The bacteria suspension that cfu/ml contains agent interfering is standby, by finished product serum during the interference agent solution
Albumin 30g and distilled water 1000mL is prepared from after mixing with 0.45 μm of filtering with microporous membrane is degerming, the interference agent solution
The mass fraction of middle bovine serum albumin is 3%.
It is of the present invention while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, wherein, institute
State step (2) and specifically include following steps：
The preparation of each concentration Iodophor：Take 11 sterile test tubes numbering A respectively1~A11, with syringe holder numbering A2~A11's
Test tube respectively adds 4 milliliters of distilled water, takes 4 milliliters of the original iodine liquids to add A1In number test tube, available iodine is dense in the original iodine liquid
Spend for 5000mg/l；4 milliliters of the original iodine liquids are taken to add A again2Number test tube is uniformly mixed so as to obtain A2Iodophor liquid, the concentration of its available iodine
For 2500mg/l；Then from A2Number test tube takes 4 milliliters of A2Iodophor liquid is added to A3Number test tube, the concentration of its available iodine is 1250mg/l；
Once-through operation is to A10Number test tube, the rest may be inferred A4The concentration of number test tube available iodine is 625mg/l；A5The concentration of number test tube available iodine
For 312.5mg/l；A6The concentration of number test tube available iodine is 156.25mg/l；A7The concentration of number test tube available iodine is 78.13mg/l；
A8The concentration of number test tube available iodine is about 39.06mg/l；A9The concentration of number test tube available iodine is about 19.53mg/l；A10Number test tube
The concentration of available iodine is 9.77mg/l；A11Number test tube is not added with thimerosal, and simply 4 milliliters of distilled water are used as positive control；
The preparation of each concentration staphylococcus lysozyme：Take 9 sterile test tubes numbering a respectively1~a9, with syringe holder numbering a2
~a9Test tube respectively add 4 milliliters of distilled water, take 4 milliliters of staphylococcus lysozyme stostes to add a1In number test tube, the staphylococcus
The concentration of staphylococcus lysozyme is 1000U/L in proenzyme liquid；4 milliliters of staphylococcus lysozyme stostes are taken to add a again2Number test tube
It is uniformly mixed so as to obtain a2Dissolving staphylococcal bacteria enzyme solutions, its concentration is 500U/L；Then 4 milliliters of a are taken2Dissolving staphylococcal bacteria enzyme solutions add
To a3A is obtained in number test tube3Dissolving staphylococcal bacteria enzyme solutions, the concentration of its staphylococcus lysozyme is 250U/L；Operate successively to a8Number
Test tube, the rest may be inferred a4The concentration of number test tube staphylococcus lysozyme is 125U/L；a5The concentration of number test tube staphylococcus lysozyme is
62.50U/L；a6The concentration of number test tube staphylococcus lysozyme is 31.25U/L；a7Number test tube dissolving staphylococcal bacteria enzyme concentration is
15.63U/L；a8The concentration of number test tube staphylococcus lysozyme, is 7.80U/L；a9Number test tube only adds 4 milliliters of distilled water without disappearing
Venom is used as positive control；
The preparation of each concentration chlorine-containing disinfection liquid：Take 11 sterile test tubes and numbering B1~B11, effective chlorine is prepared first is
5000.0mg/l uses syringe holder B as stoste2~B11Test tube respectively add 4 milliliters of distilled water, take 4 milliliters of concentration to be
5000.0mg/l effective chlorine stoste adds B1In number test tube, the concentration of effective chlorine is 5000mg/l；4 milliliters of addition B are taken again2Number examination
Pipe is uniformly mixed so as to obtain B2Chlorine-containing disinfection liquid, the concentration of its effective chlorine is 2500mg/l；Then 4 milliliters of B are taken2Chlorine-containing disinfection liquid adds
To the B3What number test tube was mixed arrives B3Chlorine-containing disinfection liquid, the concentration of its effective chlorine is 1250mg/l；Operate successively to B10Number examination
Pipe, the rest may be inferred B4The concentration of effective chlorine is 625mg/l in number test tube；B5The concentration of effective chlorine is 312.5mg/l in number test tube；
B6The concentration of effective chlorine is 156.25mg/l in number test tube；B7The concentration of effective chlorine is 78.13mg/l in number test tube；B8Number test tube
The concentration of middle effective chlorine is 39.06mg/l；B9The concentration of effective chlorine is 19.53mg/l in number test tube；B10Effective chlorine in number test tube
Concentration be 9.77mg/l；No. 11 test tubes only add 4 milliliters of distilled water, and positive control is used as without thimerosal；
Bacteria suspension using the staphylococcus aureus containing agent interfering prepared is cultivated to be primary needed for sampling inoculation
Base inclined-plane, is then acted on each concentration Iodophor configured, chlorine-containing disinfectant and composite lysostaphin respectively, described
The action time of Iodophor is 0.5min, and the action time of the chlorine-containing disinfectant is 5min, the composite lysostaphin
Action time is 6min；Then it is transferred in the meat soup pipe containing corresponding nertralizer and neutralizes 10 minutes at once, transferred species meter after neutralization
Plate is counted, then the staphylococcus aureus of sub-lethal dose is taken from respective counting plate, repeats the above steps to 20 generations, compares
The resistance to the action of a drug of the 20 generation sub-lethal dose staphylococcus aureus obtains the sub-lethal dose staphylococcus aureus to disinfectant
The resistance to the action of a drug change；
While the measure sub-lethal dose staphylococcus aureus changes to the resistance to the action of a drug of disinfectant, using K-B paper
Piece test method(s), respectively Continuous Observation record the golden yellow grape bacterium in the sub-lethal dose 1-20 generations of three kinds of thimerosals to antibiosis
The change of plain susceptibility, the antibiotic is following one of several：Penicillin, erythromycin, vancomycin, gentamicin, rifampin,
Lavo-ofloxacin, Cefoxitin, SMZ-CO.
It is of the present invention while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, wherein, institute
State step (4) and specifically include following steps：
The 20th cash equivalent staphylococcus aureus of sterilized dose of sub-lethal dose screening are chosen as primary bacterium, are dipped with oese
Typical bacterium colony, it is a generation to be inoculated into blood to increase culture 24h in bacterial context soup pipe；Cultured blood is dipped with the oese to increase
Bacterial context soup bacteria suspension, is inoculated into separation plate, is placed in 35 DEG C of -37 DEG C of culture 18-24h；Described point is dipped with the oese
It is inoculated into from the colonies typical in plate on nutrient agar slopes, 35 DEG C of -37 DEG C of culture 18-24h obtain lawn；Mass fraction is
3% phosphate buffer washes lower lawn, and shaken well is simultaneously prepared into the blood increasing bacterial context soup bacterium through the filtering of sterile absorbent cotton
Suspension；According to the step (3) carry out drug sensitivity test, record each drug sensitivity tests, continuously repeat passage from the 20th generation to
40th generation.
It is of the present invention while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, wherein, institute
State sub-lethal dose staphylococcus aureus and following steps are specifically included to the measure of the drug resistance of antibiotic：
Bacteria suspensions of the 1-20 for the staphylococcus aureus of sub-lethal dose is dipped with aseptic cotton carrier respectively, in vitro
The rotation of wall ullage is pressed, and unnecessary bacterium solution is squeezed and gone, is then uniformly smeared on each M-H culture medium flat plates agar surface
Inoculation 3 times, each 60 DEG C of Rotating Plates to ensure to be inoculated with being uniformly distributed for bacterium, are smeared one week finally along flat board inner edge, dried
5 minutes；Then the diameter 5mm Antibiotic discs of selection are placed with flat board, it is close to planar surface, each plate patch 4
, plate is covered, 35-37 DEG C of culture 18-20h is placed in, measures inhibition zone with slide measure, record each drug sensitivity tests, as a result
Judge according to Clinical microorganism normalizing operation；It is continuous to do the experiment of 20 generations.
It is of the present invention while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, wherein, institute
State in increasing bacterial context soup pipe and increase bacterial context soup containing blood, its component is：Peptone 10.0g/L；Extracted beef powder 3.0g/L；Sodium chloride
5.0g/L；Glucose 1.0g/L；Sodium citrate 3.0g/L；Dipotassium hydrogen phosphate 2g g/L；P-aminobenzoic acid 0.05g/L；Sulfuric acid
Magnesium 2.4g/L；Phenol red 0.024g/L；Remaining is distilled water；It is 7.4 ± 0.1 that blood, which increases bacterial context soup pH value,；Through 121 DEG C of sterilizings
15min and 35 DEG C of 24h sterility test is standby.
It is of the present invention while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, wherein, institute
State step (3) and specifically include following steps：It is to use K-B scraps of paper test method(s)s, Continuous Observation records the Asia of three kinds of thimerosals
The change of the raw plain susceptibility of every generation confrontation of lethal dose 1-20 cash equivalent yellow grape bacteriums, the antibiotic for it is following it is several it
One：Penicillin, erythromycin, vancomycin, gentamicin, rifampin, lavo-ofloxacin, Cefoxitin, SMZ-CO.
0.5 Maxwell opacity tube compound method is in the inventive method：0.48mol/L Bacl2 (1.17%w/v BaCl2·
2H2O) solution 0.5ml；0.18mol/L H2SO4Solution 99.5ml；Two liquid are mixed, pipe interior sealing is split, are mixed with preceding；0.5
Maxwell standard：Bacteria containing amount 1.5 × 108cfu/ml
The sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug method of the present invention, perform process of the present invention
Middle key problems are as follows：
The blowdown dyeing technique of various culture mediums：Experiment nutrient broth pipe used, numeration plate, nutrient agar slopes, in
Experiment is placed in incubator through 35-37 DEG C of culture 48h for first 2 days, and not polluting to use；
The blowdown dyeing technique of experiment bacteria suspension：From slant medium wash under the first smear Gram's staining of bacteria suspension, see
Whether be staphylococcus aureus, after the pollution for excluding experiment bacteria suspension, then do sterilization experiment if examining；
The blowdown dyeing technique of environment：That is the utilization of aseptic technique；
Experimental article is got all the ready before experiment：Autoclaved barrier gown, uses disposable breathing mask and cap, sterile gloves；
Simultaneously the following article being put into clean bench first with 75% alcohol wipe dedusting：Blood increases the various culture mediums such as bacterial context soup pipe
It is (preculture), alcolhol burner, oese, transfer needle, disposable syringe, sterile test tube, Sterile Hard Water, three kinds of thimerosals, small
Cow's serum etc.；
The integrity of checking experiment equipment：35-37 DEG C of incubator etc.；
The preparation of laboratory overall situation：Experiment is carried out in biocontainment laboratory, and experiment the previous day clean room is each every time
Kind of surface and ground, experimental day are first turned on the disinfection by ultraviolet light more than 40 minutes of room and super-clean bench；
The preparation of operating environment：Wash hands, wear sterilizing barrier gown, band disposable breathing mask, cap, band sterile gloves；Examination
Before testing superclean bench face is wiped with 75% alcohol disinfecting；
Sterilization and anti-drug sensitive test in the present invention are operated according to disinfection technology standard.
Compared with prior art, the present invention determines the side of the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance simultaneously
Method has as follows a little：
The resistance to the action of a drug, drug resistance and drug sensitivity of bacterium are recovered three ingenious combinations of experimental method by the present invention, can accurately,
The dynamic relationship between three is clearly measured, measurement result is with a high credibility, disclose sub-lethal dose disinfectant (equivalent to reality
Use disinfectant lack of standardization in work), not only select to the increased bacterium of disinfectant tolerance, and the bacterium pair selected
Antibiotic also produces drug resistance, illustrates there is crossing drug resistant phenomenon between the two, therefore this experiment is the reinforcement disinfectant in future
(to be reduced the generation of bacterial drug resistance and resistance as using antibacterials, Normalization rule disinfectant, prolonged using management
The life-span of slow disinfectant and antibiotic) theoretical direction is provided.
Embodiment
Embodiment 1
It is a kind of while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, comprise the following steps：
(1) bacteria suspension of the staphylococcus aureus containing agent interfering is prepared；
(2) bacteria suspension of the disinfectant to the staphylococcus aureus containing agent interfering of variety classes various concentrations is prepared
Carry out sterilization test and determine the resistance to the action of a drug of every generation sub-lethal dose staphylococcus aureus；The Asia per a generation is determined simultaneously to cause
Drug resistance of the dead amount staphylococcus aureus to antibiotic；Continuously did for 20 generations；
(3) sensitiveness recovers experiment：Choose the 20th cash equivalent staphylococcus aureus conduct of sterilized dose of sub-lethal dose screening
Primary bacterium, is inoculated into and blood increasing bacterial context soup bacteria suspension is cultivated and be prepared into blood increasing bacterial context soup pipe, the blood is increased into bacterial context
Soup bacteria suspension carries out drug sensitivity test；Repeat the above steps to the 40th generation.
(4) according to the measurement result of the step (2), (3) and (4), the sub-lethal dose staphylococcus aureus is found out
The resistance to the action of a drug to the disinfectant and its changing rule to the drug resistance of the antibiotic and contact.
Embodiment 2
It is a kind of while determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, comprise the following steps：
(1) staphylococcus aureus is passed through into Zengjing Granule and be separately cultured, will be described golden yellow after being separately cultured
Color staphylococcus is divided into single bacterium, and its independent schizogamy is formed independent bacterium colony；Trained with being separated described in transfer needle picking
Support obtained colonies typical and be inoculated into nutrient agar slopes, 24h formation lawns are cultivated under the conditions of 35-37 DEG C；Use mass fraction
The lower lawn is washed for 3% phosphate buffer, filtrate is obtained after being filtered through shaken well and through sterile absorbent cotton, uses sterilized water
Dilute the filtrate and be prepared into the bacteria suspension that concentration is 0.5 Maxwell standard；The bacteria suspension is done to dilute again with interference agent solution
It is 7.5 × 10 to release and be made into 10ml concentration7The bacteria suspension that cfu/ml contains agent interfering is standby, by finished product during the interference agent solution
Seralbumin 30g and distilled water 1000mL is prepared from after mixing with 0.45 μm of filtering with microporous membrane is degerming, the agent interfering
The mass fraction of bovine serum albumin is 3% in solution；
(2) preparation of each concentration Iodophor：Take 11 sterile test tubes numbering A respectively1~A11, with syringe holder numbering A2~A11
Test tube respectively add 4 milliliters of distilled water, take 4 milliliters of the original iodine liquids to add A1In number test tube, available iodine in the original iodine liquid
Concentration is 5000mg/l；4 milliliters of the original iodine liquids are taken to add A again2Number test tube is uniformly mixed so as to obtain A2Iodophor liquid, its available iodine it is dense
Spend for 2500mg/l；Then from A2Number test tube takes 4 milliliters of A2Iodophor liquid is added to A3Number test tube, the concentration of its available iodine is 1250mg/
l；Once-through operation is to A10Number test tube, the rest may be inferred A4The concentration of number test tube available iodine is 625mg/l；A5Number test tube available iodine it is dense
Spend for 312.5mg/l；A6The concentration of number test tube available iodine is 156.25mg/l；A7The concentration of number test tube available iodine is 78.13mg/
l；A8The concentration of number test tube available iodine is about 39.06mg/l；A9The concentration of number test tube available iodine is about 19.53mg/l；A10Number examination
The concentration of pipe available iodine is 9.77mg/l；A11Number test tube is not added with thimerosal, and simply 4 milliliters of distilled water are used as positive control；
The preparation of each concentration staphylococcus lysozyme：Take 9 sterile test tubes numbering a respectively1~a9, with syringe holder numbering a2
~a9Test tube respectively add 4 milliliters of distilled water, take 4 milliliters of staphylococcus lysozyme stostes to add a1In number test tube, the staphylococcus
The concentration of staphylococcus lysozyme is 1000U/L in proenzyme liquid；4 milliliters of staphylococcus lysozyme stostes are taken to add a again2Number test tube
It is uniformly mixed so as to obtain a2Dissolving staphylococcal bacteria enzyme solutions, its concentration is 500U/L；Then 4 milliliters of a are taken2Dissolving staphylococcal bacteria enzyme solutions add
To a3A is obtained in number test tube3Dissolving staphylococcal bacteria enzyme solutions, the concentration of its staphylococcus lysozyme is 250U/L；Operate successively to a8Number
Test tube, the rest may be inferred a4The concentration of number test tube staphylococcus lysozyme is 125U/L；a5The concentration of number test tube staphylococcus lysozyme is
62.50U/L；a6The concentration of number test tube staphylococcus lysozyme is 31.25U/L；a7Number test tube dissolving staphylococcal bacteria enzyme concentration is
15.63U/L；a8The concentration of number test tube staphylococcus lysozyme, is 7.80U/L；a9Number test tube only adds 4 milliliters of distilled water without disappearing
Venom is used as positive control；
The preparation of each concentration chlorine-containing disinfection liquid：Take 11 sterile test tubes and numbering B1~B11, effective chlorine is prepared first is
5000.0mg/l uses syringe holder B as stoste2~B11Test tube respectively add 4 milliliters of distilled water, take 4 milliliters of concentration to be
5000.0mg/l effective chlorine stoste adds B1In number test tube, the concentration of effective chlorine is 5000mg/l；4 milliliters of addition B are taken again2Number examination
Pipe is uniformly mixed so as to obtain B2Chlorine-containing disinfection liquid, the concentration of its effective chlorine is 2500mg/l；Then 4 milliliters of B are taken2Chlorine-containing disinfection liquid adds
To the B3What number test tube was mixed arrives B3Chlorine-containing disinfection liquid, the concentration of its effective chlorine is 1250mg/l；Operate successively to B10Number examination
Pipe, the rest may be inferred B4The concentration of effective chlorine is 625mg/l in number test tube；B5The concentration of effective chlorine is 312.5mg/l in number test tube；
B6The concentration of effective chlorine is 156.25mg/l in number test tube；B7The concentration of effective chlorine is 78.13mg/l in number test tube；B8Number test tube
The concentration of middle effective chlorine is 39.06mg/l；B9The concentration of effective chlorine is 19.53mg/l in number test tube；B10Effective chlorine in number test tube
Concentration be 9.77mg/l；No. 11 test tubes only add 4 milliliters of distilled water, and positive control is used as without thimerosal；
Bacteria suspension using the staphylococcus aureus containing agent interfering prepared is cultivated to be primary needed for sampling inoculation
Base inclined-plane, is then acted on each concentration Iodophor configured, chlorine-containing disinfectant and composite lysostaphin respectively, described
The action time of Iodophor is 0.5min, and the action time of the chlorine-containing disinfectant is 5min, the composite lysostaphin
Action time is 6min；Then it is transferred in the meat soup pipe containing corresponding nertralizer and neutralizes 10 minutes at once, transferred species meter after neutralization
Plate is counted, then the staphylococcus aureus of sub-lethal dose is taken from respective counting plate, repeats the above steps to 20 generations, compares
The resistance to the action of a drug of the 20 generation sub-lethal dose staphylococcus aureus obtains the sub-lethal dose staphylococcus aureus to disinfectant
The resistance to the action of a drug change.
While the measure sub-lethal dose staphylococcus aureus changes to the resistance to the action of a drug of disinfectant, using K-B paper
Piece test method(s), respectively Continuous Observation record the golden yellow grape bacterium in the sub-lethal dose 1-20 generations of three kinds of thimerosals to antibiosis
The change of plain susceptibility, the antibiotic is following one of several：Penicillin, erythromycin, vancomycin, gentamicin, rifampin,
Lavo-ofloxacin, Cefoxitin, SMZ-CO；
Bacteria suspensions of the 1-20 for the staphylococcus aureus of sub-lethal dose is dipped with aseptic cotton carrier respectively, in vitro
The rotation of wall ullage is pressed, and unnecessary bacterium solution is squeezed and gone, is then uniformly smeared on each M-H culture medium flat plates agar surface
Inoculation 3 times, each 60 DEG C of Rotating Plates to ensure to be inoculated with being uniformly distributed for bacterium, are smeared one week finally along flat board inner edge, dried
5 minutes；Then the diameter 5mm Antibiotic discs of selection are placed with flat board, it is close to planar surface, each plate patch 4
, plate is covered, 35-37 DEG C of culture 18-20h is placed in, measures inhibition zone with slide measure, record each drug sensitivity tests, as a result
Judge according to Clinical microorganism normalizing operation；It is continuous to do the experiment of 20 generations；
(3) sensitiveness recovers experiment：Choose the 20th cash equivalent staphylococcus aureus conduct of sterilized dose of sub-lethal dose screening
Primary bacterium, typical bacterium colony is dipped with oese, and it is a generation to be inoculated into blood to increase culture 24h in bacterial context soup pipe；With the inoculation
Ring dips cultured blood and increases bacterial context soup bacteria suspension, is inoculated into separation plate, is placed in 35 DEG C of -37 DEG C of culture 18-24h；With
The colonies typical that the oese is dipped in the separation plate is inoculated on nutrient agar slopes, 35 DEG C of -37 DEG C of culture 18-
24h obtains lawn；Mass fraction washes lower lawn for 3% phosphate buffer, and shaken well simultaneously filters system through sterile absorbent cotton
It is standby to increase bacterial context soup bacteria suspension into the blood；Drug sensitivity test is carried out according to the step (3), each drug sensitivity tests are recorded,
Continuously repeat passage from the 20th generation to the 40th generation；
Increase bacterial context soup containing blood in the increasing bacterial context soup pipe, its component is：Peptone 10.0g/L；Extracted beef powder
3.0g/L；Sodium chloride 5.0g/L；Glucose 1.0g/L；Sodium citrate 3.0g/L；Dipotassium hydrogen phosphate 2g g/L；P-aminobenzoic acid
0.05g/L；Magnesium sulfate 2.4g/L；Phenol red 0.024g/L；Remaining is distilled water；It is 7.4 ± 0.1 that blood, which increases bacterial context soup pH value,；Through
15min and 35 DEG C of 24h sterility test of 121 DEG C of sterilizings is standby.
(4) according to the measurement result of the step (2), (3) and (4), the sub-lethal dose staphylococcus aureus is found out
The resistance to the action of a drug to the disinfectant and its changing rule to the drug resistance of the antibiotic and contact.
The result that the present embodiment is measured is as follows：
Disinfectant changes to different passage killing bacteria effects：
Through to it is primary to 20 cash equivalent staphylococcus aureus killing effects observe show, effective iodine concentration 39.06mg/L~
In the range of 9.77mg/L, 0.5min is acted on；Effective chlorine acts on 5min in the range of 19.53mg/L~9.77mg/L；To not
Though the staphylococcus aureus killing rate with passage number has fluctuation, there is no reduction trend, concrete outcome is shown in Table 1.
1 two kinds of chemosterilants of table are to different passage number staphylococcus aureus killing effects
Dissolving staphylococcal bacteria enzyme concentration acts on 6min in the range of 1000U/L~7.8U/L, to the passage golden yellow of 20 times
Though staphylococcus killing rate has fluctuation, without downward trend, as a result point out, staphylococcus aureus type strain is commissioned to train through 20
Support, its drag to three kinds of tested disinfectants does not change, and concrete outcome is shown in Table 2.
The staphylococcus lysozyme of table 2 is to different passage number staphylococcus aureus killing effects
Three kinds of disinfectant induction staphylococcus aureus drug sensitive tests and sensitiveness recover experimental result
The result that three kinds of disinfectants passed on for 20 generations shows to act on the staphylococcus aureus for passing on 10 times to mould through Iodophor
There is resistance in element and erythromycin, and are passed on 20 times with hemotrophic nutrition, and sensitiveness is not recovered；After the effect of other two disinfectant
Bacterial strain then changes to penicillin, erythromycin and the basic irregularities of 3 kinds of antibacterials drags of vancomycin, and specific data are shown in Table 3.
The disinfectant of table 3 induces staphylococcus aureus drug sensitive test result
Remarks：Odd number stringer is 1-20 for sterilization experiment drug sensitivity tests, and even numbers stringer is 21-40 for blood Zengjing Granule
Drug sensitivity tests
Resistant strain recovers result of the test to thimerosal sensitiveness
As a result show, the staphylococcus aureus in the sterilized dose of generation of Fiber differentiation 20, then the generation of menses medium culture 20, its
The sign that sensitiveness to disinfectant is not recovered or changed is shown in Table 4 and table 5；But the bacterium in the generation of low concentration lysozyme Fiber differentiation 20
Strain, continues after passing on 20 times on blood meida, and drag enhanced trend on the contrary is shown in Table 6.
The resistant strain of table 4 is to iodophor disinfectant sensitiveness recovery situation
Note：On behalf of the S. aureus L-forms induced through Iodophor, blood culture passed for 20 generations to A40 again
The resistant strain of table 5 is to chlorine-containing disinfectant sensitiveness recovery situation
Note：On behalf of the S. aureus L-forms induced through chlorine-containing disinfectant, blood culture passed for 20 generations to B40 again
The resistant strain of table 6 is to staphylococcus lysozyme sensitiveness recovery situation
Note：On behalf of the S. aureus L-forms through bacteriolyze enzyme induction, blood culture passed for 20 generations to a40 again
2.4 staphylococcus aureuses not homophyletic drug sensitive test result
Experiment shows, the most Secondary Cultures 45 of staphylococcus aureus and MRSA bacterial strains through above-mentioned disinfectant Fiber differentiation
Secondary, the phenomenon that its sensitiveness to different antibacterials is not recovered, specific data are shown in Table 7.
The not homophyletic staphylococcus aureus drug sensitive test result of table 7
Note：A, B, a are represented through Iodophor, chlorine-containing disinfectant and bacteriolyze enzyme induction staphylococcus aureus respectively in table
It can be drawn the following conclusions by result above：
The concentration of disinfectant is one of deciding factor of bactericidal action, and research is found, this experiment three kinds of sterilizations used
Agent has good killing action under low concentration to all experimental bacterias including multi-drug resistant bacteria, but very low (sub-
It is lethal) test organisms survival is had in the thimerosal of concentration.In real work, the residual of sub-lethal dose disinfectant can be deposited everywhere
In the body surface on the skin after such as skin degerming or after various hand-wrist bones and after sterilization.
The study find that, sub-lethal dose thimerosal can induce existing certain resistance the bacterial strain of drug resistance again, and its is right
Disinfectant resistance has certain contact with the drug resistance to antibacterials.Experiment proves that resistant strain had both made in enriched nutritive
Passed on for 30 generations on culture medium, the sign that its sensitiveness is not also recovered.The report such as Zhang, clinical nurse nasal cavity golden yellow grape
Coccus carrying rate is that 20.5%, MRSA definite values rate is 3.21%, and the recall rate of nurse's nasal cavity separation flora qacA/B genes is up to
41.2%.Because nurse has a chances of more contact severe cases and contaminated hospital environment, and hospital disinfection agent
Selection pressure causes the formation and propagation of resistant to disinfectants bacterial strain, can constitute hospital by continuous low-level disinfectant residual region
The risk " 8 " of infection.Therefore, to participating in the contour risk operations of transplanting, contacting large-area burns or needing the trouble of Protective isolation
During person, medical personnel should reduce drug-fast bacteria field planting or actively remove and kill the antibody-resistant bacterium that human body is carried.
Found in this research, often grow coarse and fried egg color bacterium colony in experiment culture plate, also have research from hospital
Similar bacterium colony is turned out on endoscope cleaning machine, points out test organisms to change in incubation with nutritional status difference
Become.One of disinfectant resistance influence factor is microbial nutrition state, and nutrition limitations affect factor is only relevant with halogen resistance,
Mediated by Special Proteins, cAMP can promote its resistance to produce.The induction of this theoretical reasonable dismissal this research sterilization test
Resistant strain transferred species blood Zengjing Granule pipe 2-3 generations after the sensitiveness of vancomycin has been recovered, but await further
Confirm.
Have been reported that and frequently contact Iodophor for a long time, microorganism has different degrees of increase to the resistance of Iodophor, produces resistance to
By and MRSA may have to the tolerance of antibiotic with it to the resistance of Iodophor inherence contact.This research is it has also been found that multiple resistance to
Medicine bacterium does not increase chlorine-containing disinfectant resistance, and the resistance to Iodophor increased.Staphylococcus lysozyme is in sub-lethal dose condition
It is lower that antibody-resistant bacterium is not induced as Iodophor, and preceding 3 concentration also can 100.0% killing MRSA.
Research is further disclosed, and cross tolerance is there is between disinfectant and antibacterials, and bacterium departs from long-time to be connect
Touch after certain disinfectant, the resistance to the disinfectant will not disappear at once.The continuous action of the disinfectant of sub-lethal dose may increase
The selection index system of thimerosal antibody-resistant bacterium, makes it obtain growth vigor in hospital environment, and vice versa, and a large amount of of antibiotic make
Selected with being also possible to produce disinfectant resistant strain.It is therefore proposed that to limit the use scope of disinfectant, prevent abuse；
Low effect disinfectants is preferably used alternatingly；Resistance and patience and multi-drug resistant bacteria are produced to sub-lethal dose thimerosal bacterium in blood
Liquid increases the phenomenon such as sensitiveness recovery in bacterial context soup, it would be desirable to accesses and further studies.
Embodiment described above is only that the preferred embodiment of the present invention is described, not to the model of the present invention
Enclose and be defined, on the premise of design spirit of the present invention is not departed from, technical side of the those of ordinary skill in the art to the present invention
In various modifications and improvement that case is made, the protection domain that claims of the present invention determination all should be fallen into.
Claims (5)
1. it is a kind of while determining the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance, it is characterised in that including
Following steps：
(1) bacteria suspension of the staphylococcus aureus containing agent interfering is prepared, bacteria suspension is done into two-fold dilution with interference agent solution and matched somebody with somebody
Concentration into 10ml is 7.5 × 107The bacteria suspension that cfu/ml contains agent interfering is standby, and the interference agent solution is by finished product serum
Albumin 30g and distilled water 1000mL is prepared from after mixing with 0.45 μm of filtering with microporous membrane is degerming；
(2) disinfectant for preparing variety classes various concentrations is carried out to the bacteria suspension of the staphylococcus aureus containing agent interfering
Sterilization test and the resistance to the action of a drug for determining every generation sub-lethal dose staphylococcus aureus；Determine described per generation sub-lethal dose simultaneously
Drug resistance of the staphylococcus aureus to antibiotic；Continuously did for 20 generations；
(3) sensitiveness recovers experiment：The 20th cash equivalent staphylococcus aureus of sterilized dose of sub-lethal dose screening are chosen as primary
Bacterium, is inoculated into and blood increasing bacterial context soup bacteria suspension is cultivated and be prepared into blood increasing bacterial context soup pipe, the blood is increased into bacterial context soup bacterium
Suspension carries out drug sensitivity test；Repeat the above steps to the 40th generation；
(4) according to the step (2) and the measurement result of (3), find out the sub-lethal dose staphylococcus aureus and disappear to described
The resistance to the action of a drug of toxic agent and its changing rule to the drug resistance of the antibiotic and contact；
The step (2) specifically includes following steps：
The preparation of each concentration Iodophor：Take 11 sterile test tubes numbering A respectively1~A11, with syringe holder numbering A2~A11Test tube
4 milliliters of distilled water are respectively added, take 4 milliliters of the original iodine liquids to add A1In number test tube, the concentration of available iodine is in the original iodine liquid
5000mg/l；4 milliliters of the original iodine liquids are taken to add A again2Number test tube is uniformly mixed so as to obtain A2Iodophor liquid, the concentration of its available iodine is
2500mg/l；Then from A2Number test tube takes 4 milliliters of A2Iodophor liquid is added to A3Number test tube, the concentration of its available iodine is 1250mg/l；One
It is secondary to operate to A10Number test tube, the rest may be inferred A4The concentration of number test tube available iodine is 625mg/l；A5The concentration of number test tube available iodine is
312.5mg/l；A6The concentration of number test tube available iodine is 156.25mg/l；A7The concentration of number test tube available iodine is 78.13mg/l；A8
The concentration of number test tube available iodine is about 39.06mg/l；A9The concentration of number test tube available iodine is about 19.53mg/l；A10Number test tube has
The concentration for imitating iodine is 9.77mg/l；A11Number test tube is not added with thimerosal, and simply 4 milliliters of distilled water are used as positive control；
The preparation of each concentration staphylococcus lysozyme：Take 9 sterile test tubes numbering a respectively1~a9, with syringe holder numbering a2~a9's
Test tube respectively adds 4 milliliters of distilled water, takes 4 milliliters of staphylococcus lysozyme stostes to add a1In number test tube, the staphylococcus proenzyme
The concentration of staphylococcus lysozyme is 1000U/L in liquid；4 milliliters of staphylococcus lysozyme stostes are taken to add a again2Number test tube is mixed
Obtain a2Dissolving staphylococcal bacteria enzyme solutions, its concentration is 500U/L；Then 4 milliliters of a are taken2Dissolving staphylococcal bacteria enzyme solutions are added to a3
A is obtained in number test tube3Dissolving staphylococcal bacteria enzyme solutions, the concentration of its staphylococcus lysozyme is 250U/L；Operate successively to a8Number examination
Pipe, the rest may be inferred a4The concentration of number test tube staphylococcus lysozyme is 125U/L；a5The concentration of number test tube staphylococcus lysozyme is
62.50U/L；a6The concentration of number test tube staphylococcus lysozyme is 31.25U/L；a7Number test tube dissolving staphylococcal bacteria enzyme concentration is
15.63U/L；a8The concentration of number test tube staphylococcus lysozyme, is 7.80U/L；a9Number test tube only adds 4 milliliters of distilled water without disappearing
Venom is used as positive control；
The preparation of each concentration chlorine-containing disinfection liquid：Take 11 sterile test tubes and numbering B1~B11, effective chlorine is prepared first is
5000.0mg/l uses syringe holder B as stoste2~B11Test tube respectively add 4 milliliters of distilled water, take 4 milliliters of concentration to be
5000.0mg/l effective chlorine stoste adds B1In number test tube, the concentration of effective chlorine is 5000mg/l；4 milliliters of addition B are taken again2Number examination
Pipe is uniformly mixed so as to obtain B2Chlorine-containing disinfection liquid, the concentration of its effective chlorine is 2500mg/l；Then 4 milliliters of B are taken2Chlorine-containing disinfection liquid adds
To the B3What number test tube was mixed arrives B3Chlorine-containing disinfection liquid, the concentration of its effective chlorine is 1250mg/l；Operate successively to B10Number examination
Pipe, the rest may be inferred B4The concentration of effective chlorine is 625mg/l in number test tube；B5The concentration of effective chlorine is 312.5mg/l in number test tube；
B6The concentration of effective chlorine is 156.25mg/l in number test tube；B7The concentration of effective chlorine is 78.13mg/l in number test tube；B8Number test tube
The concentration of middle effective chlorine is 39.06mg/l；B9The concentration of effective chlorine is 19.53mg/l in number test tube；B10Effective chlorine in number test tube
Concentration be 9.77mg/l；No. 11 test tubes only add 4 milliliters of distilled water, and positive control is used as without thimerosal；
Bacteria suspension using the staphylococcus aureus containing agent interfering prepared is primary, and culture medium needed for sampling inoculation is oblique
Face, is then acted on, the Iodophor with each concentration Iodophor configured, chlorine-containing disinfectant and composite lysostaphin respectively
Action time be 0.5min, action time of the chlorine-containing disinfectant is 5min, the effect of the composite lysostaphin
Time is 6min；Then it is transferred in the meat soup pipe containing corresponding nertralizer and neutralizes 10 minutes at once, transferred species counts flat after neutralization
Ware, then the staphylococcus aureus of sub-lethal dose is taken from respective counting plate, repeat the above steps to 20 generations, it is relatively more described
The resistance to the action of a drug of 20 generation sub-lethal dose staphylococcus aureuses obtains the sub-lethal dose staphylococcus aureus and disinfectant is resisted
The property of medicine changes；
While the measure sub-lethal dose staphylococcus aureus changes to the resistance to the action of a drug of disinfectant, tried using the K-B scraps of paper
Method is tested, Continuous Observation records the golden yellow grape bacterial antibiotic medicine in the sub-lethal dose 1-20 generations of three kinds of thimerosals respectively
Quick change, the antibiotic is following one of several：Penicillin, erythromycin, vancomycin, gentamicin, rifampin, left oxygen
Flucloxacillin, Cefoxitin, SMZ-CO.
2. determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance simultaneously according to claim 1, its
It is characterised by, the step (1) specifically includes following steps：
The staphylococcus aureus is passed through into Zengjing Granule and is separately cultured, by the Staphylococcus aureus after being separately cultured
Bacterium is divided into single bacterium, and its independent schizogamy is formed independent bacterium colony；It is separately cultured what is obtained with described in transfer needle picking
Colonies typical is inoculated into nutrient agar slopes, and 24h formation lawns are cultivated under the conditions of 35-37 DEG C；It is 3% phosphoric acid with mass fraction
Salt buffer washes the lower lawn, and filtrate is obtained after being filtered through shaken well and through sterile absorbent cotton, dilutes described with sterilized water
Filtrate is prepared into the bacteria suspension that concentration is 0.5 Maxwell standard；The mass fraction of bovine serum albumin is in the interference agent solution
3%.
3. determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance simultaneously according to claim 1, its
It is characterised by, the step (3) specifically includes following steps：
The 20th cash equivalent staphylococcus aureus of sterilized dose of sub-lethal dose screening are chosen as primary bacterium, typical case is dipped with oese
Bacterium colony, be inoculated into blood increase bacterial context soup pipe in culture 24h be a generation；Cultured blood is dipped with the oese and increases bacterial context
Soup bacteria suspension, is inoculated into separation plate, is placed in 35 DEG C of -37 DEG C of culture 18-24h；The separation is dipped with the oese flat
Colonies typical in ware is inoculated on nutrient agar slopes, and 35 DEG C of -37 DEG C of culture 18-24h obtain lawn；It is with mass fraction
3% phosphate buffer washes lower lawn, and shaken well is simultaneously prepared into the blood through the filtering of sterile absorbent cotton and increases bacterial context soup bacterium and hang
Liquid；Drug sensitivity test is carried out according to the step (3), each drug sensitivity tests are recorded, passage is continuously repeated from the 20th generation to the
40 generations.
4. determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance simultaneously according to claim 1, its
It is characterised by, the sub-lethal dose staphylococcus aureus specifically includes following steps to the measure of the drug resistance of antibiotic：
Bacteria suspensions of the 1-20 for the staphylococcus aureus of sub-lethal dose is dipped with aseptic cotton carrier respectively, in vitro wall liquid
Rotation is pressed above face, and unnecessary bacterium solution is squeezed and gone, then uniform on each M-H culture medium flat plates agar surface to smear inoculation
3 times, each 60 DEG C of Rotating Plates to ensure to be inoculated with being uniformly distributed for bacterium, are smeared one week finally along flat board inner edge, dry 5 points
Clock；Then the diameter 5mm Antibiotic discs of selection are placed with flat board, it is close to planar surface, each plate pastes 4,
Plate is covered, 35-37 DEG C of culture 18-20h is placed in, measures inhibition zone with slide measure, record each drug sensitivity tests, result judgement
According to Clinical microorganism normalizing operation；It is continuous to do the experiment of 20 generations.
5. determine the method for the sub-lethal dose staphylococcus aureus resistance to the action of a drug and drug resistance simultaneously according to claim 3, its
It is characterised by, increases bacterial context soup containing blood in the increasing bacterial context soup pipe, its component is：Peptone 10.0g/L；Extracted beef powder
3.0g/L；Sodium chloride 5.0g/L；Glucose 1.0g/L；Sodium citrate 3.0g/L；Dipotassium hydrogen phosphate 2g g/L；P-aminobenzoic acid
0.05g/L；Magnesium sulfate 2.4g/L；Phenol red 0.024g/L；Remaining is distilled water；The blood increase bacterial context soup pH value be 7.4 ±
0.1；It is standby through 15min and 35 DEG C of 24h sterility test of 121 DEG C of sterilizings.
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