CN110484595A - A kind of evaluation method being atomized bacteriophage bactericidal effect - Google Patents
A kind of evaluation method being atomized bacteriophage bactericidal effect Download PDFInfo
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Abstract
The application belongs to health care Prevention Technique field, and in particular to a kind of evaluation method for being atomized bacteriophage bactericidal effect.This method comprises: setting sample spot, bacteriophage preparation, bacteriophage atomization spray.In general, in existing bacteriophage sterilizing research, due to being mainly limited to experimental exploratory development, and for practical application, it then needs to consider the balance between bacteriophage application mode, application amount and application effect in detail, could be that good technique basis is established in application of the bacteriophage in drug-resistant bacteria prevention and treatment.In the application, the Quality Control of mode and the evaluation of spraying effect are sprayed by bacteriophage, tentatively establishes a kind of more comprehensive bacteriophage application effect appraisal method, and certain technology evaluation basis can be established for practical popularization and application of the bacteriophage in drug-resistant bacteria prevention and control.
Description
Technical field
The application belongs to health care Prevention Technique field, and in particular to a kind of evaluation side for being atomized bacteriophage bactericidal effect
Method.
Background technique
In recent years, superbacteria (drug-resistant bacteria) has attracted increasing attention.On 2 27th, 2017, the world
Therefore frequency that health organization is infected based on the drug resistance situation in clinic, the death rate caused by drug resistance, people and these feel
It contaminates and is born to health care system bring, issued 12 kinds of superbacteria lists for being listed in highest priority, and root for the first time
Urgent, high isopreference level and high medium priority three classes are divided them into according to degree of danger.It is wherein resistance to carbapenem antibiotic
The Acinetobacter baumannii of medicine, Pseudomonas aeruginosa and enterobacteriaceae lactobacteriaceae degree of danger highest, be put into emergency priority, these height cause
Germ can not only cause drug resistant infection, and such as pneumonia, wound or blood infection or even fatal, it is heavy to bring to existing medical system
Heavy burden.
Omnibus survey studies have shown that superbacteria recall rate of these emergency priorities in hospital environment is very high.They
Can by directly contacting, mediate contact, droplet and air, even by mosquito fly etc. quietly in hospital environment and in people and
Middle fast propagation between people, this will be fatal to the urgent patient of hospital's High Risk Department.And it is existing conventional use of to environment
With the physics and chemical method of sterilisation of objects, because having irritation, toxicity and corrosivity, in densely populated place, carry disease germs people
Group continues existing hospital environment, therefore sphere of action is restricted, it is impossible to reach thorough cleaning-sterilizing, block super thin
The function and effect that bacterium is propagated, this is that the propagation of superbacteria prevalence causes inside-hospital infection to continue existing basic reason.People need
Superbacteria in the scientific and effective prevention and control hospital of new formulation is wanted to infect.
Bacteriophage is the virus of a kind of bacterial infection, is the generally existing bacterium natural enemy of nature.They have very strong
Host specificity is strong, and sterilization precisely, does not destroy Tiny ecosystem, while will not more infect the mankind or animals and plants, therefore, precisely kills ring
The bacteriophage of target superbacteria will be a kind of following pure natural, colourless non-stimulated, nontoxic, noresidue, green height in border
The disinfecting product of effect.Therefore, the application of bacteriophage is for controlling the diffusion of superbacteria in medical environment and ensuring to cure
Therapeutic effect has highly important technological value.
But then, the evaluation method of broad spectrum activity sterilization effect is not particularly suited for narrow spectrum antimicrobial form in the prior art
The evaluation of bacteriophage sterilization effect, main cause and difference are: broad spectrum type Evaluation of Germicidal Efficacy method mainly passes through calculating
The total micro organism quantity difference in disinfection front and back (mainly including bacterium and fungi etc.) can calculate；And bacteriophage is due to the object that sterilizes
It is more single-minded, for existing sterilizing object (host strain) again with the presence of non-sterilizing object in the case of, be difficult through quantitative area
It is not evaluated directly.And in routine evaluations method, bacteriophage function and effect are evaluated, are generally mainly based upon again specific
The morphological indexs such as the phagocytosis spot diameter of host strain, amount of bacterial plaque, transparency are evaluated, and phagocytosis in actual environment is not particularly suited for
The evaluation of body sterilization effect.Therefore, a kind of more science and bacteriophage sterilization effect evaluation objective, suitable for practical application are designed
Method, for promoting bacteriophage sterilizing application, drug-resistant bacteria control all has highly important technological value.
Summary of the invention
The application is designed to provide a kind of evaluation method for being atomized bacteriophage bactericidal effect, to exist for related bacteriophage
Actual medical environment sterilizing in apply and superbacteria prevention and treatment etc. establish certain technical foundation.
Details are as follows for the technical solution that the application is taken.
A kind of evaluation method being atomized bacteriophage bactericidal effect, includes the following steps:
(1) sample spot is arranged
Sample for evaluation point is selected in environment to be evaluated, then lays Quality Control Quality Control sample and evaluation with commenting in counter sample point
Valence sample, is preferably also provided with control sample；
The Quality Control sample are as follows: the plate of sterile saline containing 10ml (or SM culture solution or other aseptic culture fluids)；
The evaluation sample are as follows: the plate of the object bacteria (host strain) containing bacteriophage effect target；
The control sample: for monitor microorganism storage situation in existing environment containing sterile LB or NB or other be suitble to cultivate mesh
Mark the plate of the culture medium of superbacteria；
(2) prepared by bacteriophage
Bacteriophage and host strain are added in LB culture medium according to optimal multiplicity of infection (MOI), shake condition in 37 DEG C, 120rpm
Lower proliferation becomes clarification by muddiness to bacterium solution, final to prepare high-purity and high-titer phage stock body, bites in final bacteriophage liquid
Cell concentration should reach 1011PFU/ml or more；
When specific preparation high-purity and high-titer phage stock body, can refer to " molecular cloning ", (J. Pehanorm Brooker, D.W. are drawn
Sai Er, Molecular Cloning:A Laboratory guide (third edition), Science Press, 2002) in λ bacteriophage large-scale culture, concentration and purifying
Method prepared；
(3) bacteriophage atomization sprays
Bacteriophage uniform atomizing prepared in step (2) is sprayed in the environment to be evaluated in step (1)；
Distributed effect after the ejection effect of bacteriophage is monitored and evaluated by Quality Control sample and is sprayed；
Bacteriophage is monitored to the direct killing effect of object bacteria by evaluation sample；
Before specific atomization sprays, prepared bacteriophage in step (2) is suitably diluted using physiological saline, in order to rear
Continuous atomization sprays, but liquid pnagus medius final concentration should be not less than 10 after dilution9 PFU/ml, to ensure post sterilization effect effect
Fruit；
When specific atomization sprays, Ying Caiyong household or industrial grade, with can control or surrounding oscillating fan and atomizing particle
It is sprayed for the atomizer progress bacteriophage atomization of nanoscale size (1 μm of <), to ensure to spray uniformly；
During atomization sprays, the room needs of atomization are closed the doors and windows, to ensure the accuracy of evaluation result；
It, can practical potency according to bacteriophage liquid, atomizer pair used to the total time of room atomization during atomization sprays
The nebulization efficiency of liquid, room area are estimated；When atomization reaches complete sterilization, the concentration of bacteriophage is higher, and/or is atomized
The efficiency of device is higher, and required nebulisation time is shorter, conversely, longer；
When practical operation, with reference to method provided by the present invention, it can first pass through and implement specific atomization test, measuring makes to monitor position
It is corresponding to monitor bacteriophage collected by site Quality Control plate when the object bacteria being artificially inoculated in point sterilization plate is killed completely
Minimum, in conjunction with room real area, obtains the estimation of pnagus minimus amount of application needed for sterilizing completely further according to this minimum
It is worth (PFU/cm2), using estimated value as the reference of practical amount of application；
The following calculation formula that can refer to total time of bacteriophage atomization is estimated:
Nebulisation time (min)=[bacteriophage amount of application estimated value (PFU/cm2) × room area (cm2)] ÷ [nebulization efficiency (ml/
Min) × phage titer (PFU/ml)]
The practical amount of application of bacteriophage can refer to following calculation formula and be calculated:
Bacteriophage amount of application (PFU/cm2)=nebulisation time (min) × nebulization efficiency (ml/min) × phage titer (PFU/ml)
÷ room area (cm2)；
For the calculated result of the amount of application of bacteriophage, it should be ensured that the amount of application of bacteriophage every square centimeter is super after spraying
Sterilization pnagus minimus amount of application completely is crossed (that is, the sterilizing rate of Target indication bacterium reaches in the sample that carries disease germs artificially placed to monitoring point
To 100%).
It needs to be explained further, since bacteriophage is one kind can be sterilized with the biological bactericide of self duplication
Process can increase with bacteriophage is exponential, therefore, amount and not absolute terms required for practical application bacteriophage.Above-mentioned technology
It is the phagocytosis that (high infection multiplicity sterilization) is estimated in the case where not considering bacteriophage multiplication that the numerical value obtained is calculated in scheme
The minimum amount of application of body sterilization easily uniformly divides with atomization process since phage particle only has tens nanosizeds
Cloth, the dragnet just as having planted bacteriophage " particle bullet " formation in the room, this amount of application can guarantee that bacteriophage is being proliferated
Or under all situations not being proliferated, the superbacteria that corresponding monitoring point is artificially inoculated with can thoroughly be killed by being attained by, therefore, theoretical
The bacteriophage " particle bullet " that upper such concentration is structured the formation, which can similarly be killed, sends out the super of popular bacteriophage typing of the same race in environment
Grade bacterium.
In general, in existing bacteriophage sterilizing research, due to being mainly limited to experimental exploratory development, and with regard to actually answering
For, then needs to consider the balance between bacteriophage application mode, application amount and application effect in detail, could be that bacteriophage exists
Good technique basis is established in application in drug-resistant bacteria prevention and treatment.In the application, the Quality Control of mode is sprayed by bacteriophage and is sprayed
The evaluation of effect tentatively establishes a kind of more comprehensive bacteriophage application effect appraisal method, can be thin in drug resistance for bacteriophage
Certain technology evaluation basis is established in practical popularization and application in bacterium prevention and control.
Detailed description of the invention
Fig. 1 is the bacteriophage collecting amount monitored in each sampled point Quality Control sample plate when bacteriophage is atomized 1min；
Fig. 2 is the bactericidal effect that bacteriophage is atomized that each sampled point when 1min evaluates object bacteria in plate；
Fig. 3 is the bacteriophage collecting amount monitored in each sampled point Quality Control sample plate when bacteriophage is atomized 10 min,
Fig. 4 is the bactericidal effect that bacteriophage is atomized that each sampled point when 10min evaluates object bacteria in plate；
Fig. 5 is the bacteriophage collecting amount monitored in each sampled point Quality Control sample plate when bacteriophage is atomized 40 min；
Fig. 6 is the bactericidal effect that bacteriophage is atomized that each sampled point when 40min evaluates object bacteria in plate；
Fig. 7 is the cultivation results that part monitors plate, and wherein left figure is control plate cultivation results, and right figure is bacteriophage atomization
The cultivation results of 40min sterilization plate.
Specific embodiment
Explanation is further explained to the application with reference to the accompanying drawings and examples.Before introducing specific embodiment, just
Involved biomaterial situation, which is briefly introduced, in following embodiments is described as follows.
Biomaterial:
Object bacteria employed in following embodiments is clinical bacteria strain Acinetobacter bauamnnii common in certain medical environment (life
Entitled A186), bacteriophage used is then also the phagocytosis that can efficiently kill this plant of pathogenic bacteria by acquisition separated in actual environment
Body (is named as PA186).It should be noted that bacterial strain and bacteriophage are that applicant is obtained in the collection of related medical unit
Common antibody-resistant bacterium and its corresponding bacteriophage, the technical solution for being merely for convenience and purposes of illustration of the application are illustrated card, be should not be understood
The specified microorganisms material is limited only to for the technical solution of the application.
Embodiment
For the objectivity of the objective evaluation method for investigating atomization bacteriophage bactericidal effect provided herein, accuracy and
Applicability, inventor is first using aseptic experiment room as environment to be evaluated, to investigate the feasibility of related concrete operations, specifically
Process is briefly discussed below.
(1) sample spot is arranged
With certain rectangle room, (long and width is 360cm and 300cm respectively, therefore the gross area is 10.8m2) it is space to be evaluated.
According to statistics needs, to ensure result accuracy, it is room quadrangle respectively that sample for evaluation point is selected in aseptic experiment room
The center and.Then counter sample point lays Quality Control Quality Control sample, evaluation use and evaluates sample and as the ground control of ground control
Sample, while the blank control sample of a blank control is set；
The Quality Control sample are as follows: the plate (Quality Control plate) of the sterile saline containing 10ml, for evaluating bacteriophage atomization effect；
The evaluation sample are as follows: the object bacteria (host strain uses Acinetobacter bauamnnii A186) containing bacteriophage effect target
Plate (sterilization plate), is atomized bacteriophage bactericidal effect for monitoring and evaluation；
The ground control sample: for monitor total microorganism storage (quantity) situation in existing environment containing LB aseptic culture medium
Plate (sterilized petri dishes)；
The plate is all made of the plate that common diameter is 90mm；
The blank control sample, for acted on containing bacteriophage target object bacteria (host strain) plate, but without subsequent mist
Change the control sample of bacteriophage processing；
It should be noted that each sample plate of each sample spot is no less than 3, to ensure the accuracy of testing result.
The evaluation sample can refer to following operation: by the target superbacteria bacterium solution being incubated overnight in nothing specifically when preparation
It is accordingly diluted in bacteria culture fluid, diluted final bacteria concentration is made to reach 103 CFU/ml~104Then CFU/ml takes
100ul is uniformly applied to solid culture primary surface with spreading rod.
(2) prepared by bacteriophage
By bacteriophage PA186 host strain A186 corresponding to its according to optimal multiplicity of infection (105 PFU/ml:107 CFU/ml it) is added
In LB culture medium, " molecular cloning " (J. Pehanorm Brooker, D.W. Russell, Molecular Cloning:A Laboratory guide (third edition), section is pressed
Learn publishing house, 2002) λ bacteriophage large-scale culture, concentration and the method for purifying prepare high-purity high-titer bacteriophage in
PA186, final products pnagus medius concentration are 1011PFU/ml or so.
(3) bacteriophage atomization sprays
Normal saline dilution is added to 10 in bacteriophage prepared in step (2)9PFU/ml or so, using certain brand household mist
Change device carry out atomization spray (atomizer with can surrounding oscillating fan, atomizing particle be nanoscale size (300nm ~
400nm)；It is very little, very light in general, because phage particle only has tens nanometers, easily uniformly broadcast in the environment with air
It dissipates, therefore is atomized when spraying and one of atomizer requirement is it should be ensured that air can be made to flow, so should have can
Oscillating fan).
During atomization sprays, the room of atomization need to close the doors and windows
It, can practical potency according to bacteriophage liquid, atomizer pair used to the total time of room atomization during atomization sprays
The nebulization efficiency of liquid, room area are estimated, using estimated value as the reference of practical amount of application；
The following calculation formula that can refer to total time of bacteriophage atomization is estimated:
Nebulisation time (min)=[bacteriophage amount of application estimated value (PFU/cm2) × room area (cm2)] ÷ [nebulization efficiency (ml/
Min) × phage titer (PFU/ml)]
The amount of application of bacteriophage can refer to following calculation formula and be calculated:
Amount of application (the PFU/cm of bacteriophage2)=nebulisation time (min) × nebulization efficiency (ml/min) × phage titer (PFU/
Ml) ÷ room area (cm2).
Calculated result shows, it is ensured that the amount of application of bacteriophage every square centimeter is more than that sterilization minimum completely is bitten after spraying
Thallus amount of application are as follows: 1 × 108 PFU/ plate.
Before phagocytosis spray body, it should be ensured that each plate sample is open, so that it is guaranteed that atomization bacteriophage can fall into plate；
It is to be understood that the ultimate density of the bacteriophage of atomization application in the environment is mainly by phagocytosis in atomization bacteriophage liquid
Bulk concentration determines.That is, as long as atomized liquid pnagus medius concentration is sufficiently high (such as more than 1011 PFU/ml), it is atomized very
Short time is the high concentration that can reach sterilization and need.Conversely, if atomization bacteriophage strength of fluid is very low, by extending nebulisation time
The way of environment bacteriophage quantity is improved, often efficiency is extremely low.It is illustrated below:
Such as the phagocytosis body fluid final concentration of 10 being atomized9 PFU/ml, atomization 1ml can into environment the theory of application of bacteriophage it is total
Number is 109 PFU, 10ml are 1010 PFU, 100ml are 1011 PFU, 1000 ml are 1012 PFU.If 1h can be atomized 100ml
Bacteriophage amount of liquid, atomization 1000ml must need 10 hours, the order of magnitude base for the bacteriophage applied within this 10 hours
Originally it will not become, all be 1011 It theoretically can just can reach 10 after PFU, 10h12 PFU。
If the phagocytosis body fluid final concentration of atomization is low, only 107 PFU/ml, atomization 1ml can into environment application of bacteriophage
Theory sum only 107 PFU, 10ml can be only achieved 108 PFU, 100ml just reach 109 PFU, 1000 ml also just just reach
1010 PFU.1h is atomized 100ml amount of liquid, and bacteriophage application sum can only achieve 109 It theoretically also can only just be reached after PFU, 10h
To 1010 PFU wants to reach 1012 PFU, must need 1000h, and this efficiency is unfavorable for the practical application of bacteriophage.
After atomization, distributed effect after the ejection effect of bacteriophage is monitored and evaluated by Quality Control sample and is sprayed；Tool
When gymnastics is made, appropriate collection liquid is taken first, and after doing appropriate dilution, phage titer (pfu/ml) is surveyed using double-layer agar technique；It needs
It should be noted that the sum of plate pnagus medius should reach 107PFU or more just can ensure that and artificially be inoculated in sterilization plate
Bacterium has obvious bactericidal effect；
And to evaluation sample and control sample, it should cover after lid is placed in 37 DEG C of culture 48h, be calculated and commented by clump count difference
Valence bacteriophage is to the direct killing effect of object bacteria, i.e. sterilizing rate；
Sterilizing rate (%)=(control plate clump count-sterilization plate clump count-sterilized petri dishes clump count) × 100%/control plate
Clump count.
Part of test results is as shown in Fig. 1 to Fig. 6.For concrete analysis:
It will be seen from figure 1 that the phagocytosis bulk concentration of each monitoring point monitoring is less than 10 when atomization 1min6PFU/ml, and it is each
The bacteriophage concentration difference of monitoring point monitoring is larger (p < 0.05)；This result shows that, be atomized 1min when, bacteriophage is in each monitoring
The distribution of point is simultaneously uneven；And Fig. 2 the result shows that, when being atomized 1min, each monitoring point average bactericidal rate is only 83.8%, and each prison
The sterilizing rate difference of measuring point is also larger (p < 0.05).
And from Fig. 3, Fig. 4 can be seen that atomization 10min after, the sterilizing rate of each monitoring point is more than 99%, average bactericidal rate
Up to 99.8%；The bacteriophage concentration difference of each monitoring point monitoring is not statistically significant (p > 0.05) at this time, illustrates that bacteriophage is distributed
It has reached uniformly, and monitor value is more than 1 × 106PFU/ml(i.e. 1 × 107PFU/ plate).
It can be seen that from Fig. 5, Fig. 6 after being atomized 40min, each monitoring point sterilizing rate is 100%, the practical sterilizing result in part
As shown in Figure 7.The phagocytosis bulk concentration of each monitoring point monitoring improves an order of magnitude again at this time, and the bacteriophage of each monitoring point monitoring is dense
It spends no significant difference (p > 0.05), more than 107PFU/ml.The physiological saline being added in plate is 10ml, according to
It is 1 × 10 that this result, which can estimate sterilization pnagus minimus amount of application completely,8 PFU/ plate, and 1m3Being equivalent to 157 diameters is
The plate area (10000 ÷ 63.6=157.2) of 9cm, it is possible to calculate every square metre and sterilize required bacteriophage completely
Minimum amount of application about should be 157 × 108 PFU(1.57 × 1010 PFU/m3).
Room area in the present embodiment is 10.8m3, the pnagus minimus amount of application that can calculate entire room accordingly is
1.7×1011 PFU(1.57 × 1010 PFU×10.8 m3=1.7×1011 PFU).And phagocytosis spray body used in the present embodiment
Practical potency be 1.2 × 1011 PFU/ml, can calculate the smallest bacteriophage amount of application accordingly is 1.4 ml.In view of atomization
Bacteriophage can be consumed loss (such as suspension of air pnagus medius；The absorption to bacteriophage such as wall, furniture and ceiling),
Therefore, it should be greater than calculated value in practical application, such as can be according to the closed effect of the height in room and indoor article and room
Fruit improves amount of application, and usually the tens of estimated value times arrive hundred times.
The nebulization efficiency (amount of liquid that atomization sprays per minute) of atomizer employed in the present embodiment is 1ml/min, mist
The practical final concentration of the bacteriophage liquid diluting of change is 1.2 × 1011 PFU/ml, therefore 40min is atomized 40ml phagocytosis body fluid altogether
Body.
In short, the bacteriophage amount of application that sum up result can be seen that atomization 40min is put down the evaluation sample that site is placed is monitored
The superbacteria that is inoculated in ware is all killed, and sterilizing rate is up to 100%；It, can basis accordingly as a result, when facing practical sterilizing and needing
This result sets reasonable bacteriophage amount of application.
Claims (2)
1. a kind of evaluation method for being atomized bacteriophage bactericidal effect, which is characterized in that this method comprises the following steps:
A kind of evaluation method being atomized bacteriophage bactericidal effect, which is characterized in that this method comprises the following steps:
(1) sample spot is arranged
Then selection, design evaluatio sample spot in environment to be evaluated are laid Quality Control Quality Control sample in counter sample point and are commented
Valence evaluation sample；
The Quality Control sample are as follows: the plate containing sterile saline or SM culture solution；
The evaluation sample are as follows: the plate of the object bacteria containing bacteriophage effect target；
(2) prepared by bacteriophage
Bacteriophage and host strain are added in culture medium according to optimal multiplicity of infection, proliferation to phagocytosis bulk concentration reaches 1011 PFU/
Ml or more；
(3) bacteriophage atomization sprays
Bacteriophage uniform atomizing prepared in step (2) is sprayed in the environment to be evaluated in step (1)；
Distributed effect after the ejection effect of bacteriophage is monitored and evaluated by Quality Control sample and is sprayed；
Bacteriophage is monitored to the direct killing effect of object bacteria by evaluation sample；
The total time calculation formula of bacteriophage atomization are as follows:
Nebulisation time (min)=[bacteriophage amount of application estimated value (PFU/cm2) × room area (cm2)] ÷ [nebulization efficiency (ml/
Min) × phage titer (PFU/ml)]
The amount of application calculation formula of bacteriophage are as follows:
The amount of application PFU/cm of bacteriophage2=nebulisation time min × nebulization efficiency ml/min × phage titer PFU/ml ÷ is to be evaluated
Valence environment area cm2；
For the calculated result of the amount of application of bacteriophage, it should be ensured that the amount of application of bacteriophage every square centimeter is super after spraying
Cross sterilization pnagus minimus amount of application completely.
2. the evaluation method of atomization bacteriophage bactericidal effect as described in claim 1, which is characterized in that in step (1), sample spot
It is provided with control sample；The control sample: contain sterile LB or NB culture medium for monitor microorganism storage situation in existing environment
Plate.
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