JP3163571B2 - Drug resistant staphylococcal measurement unit - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measurement unit for drug-resistant staphylococci suspended in the air, and more particularly, to a drug-resistant staphylococcus measuring device for environmental measurement, which is suspended in the air. A unit for collecting and culturing bacteria and a method for using the same to measure the number of drug-resistant staphylococci.

[0002]

2. Description of the Related Art Conventionally, in a hospital or the like, the number of bacteria floating in the air per unit volume has been measured for environmental management in a room. Staphylococci include those having no resistance to existing antibacterial drugs and staphylococci that have recently become resistant to methicillin. Recently, drug-resistant staphylococci have rapidly spread, are rapidly becoming resistant to various antibacterial drugs, and are rapidly becoming resistant to newly developed antibacterial drugs. It is even higher.

[0003] Methycillin Resistant Staphyrococcus Aure which is currently employed.
us, hereafter referred to as MRSA) is to grow colonies of all species by enrichment culture of samples, then isolate and culture each colony to identify staphylococci, and further culture for the presence or absence of methicillin resistance By confirming by MRSA
Have confirmed. Therefore, since a multi-stage culture test is required, it takes at least about two weeks to confirm and measure MRSA. Therefore, it has been extremely difficult to constantly perform measurements for environmental management in hospitals and the like and quickly manage the sanitary condition of indoor air based on the results.

[0004] Here, as a medium for selectively culturing staphylococci, a mannitol medium containing D-mannitol and salt as a main component and a Baird Parker medium containing sodium pyruvate, lithium chloride and glycine as main components are used. Is conventionally known.

[0005] The present applicant has previously developed a simple and quick method for measuring the number of bacteria floating in the air for use in environmental management (Japanese Patent Application No. 3-87697). Conventionally, put a sterilized medium in a Petri dish, leave the lid open for a certain period of time, and during that time, collect bacteria that have fallen from the air on the surface of the medium,
A method of displaying the environment based on the number of colonies after culture has been adopted. However, in this method, the operation is complicated and the measurement requires a long time, and the obtained measured values vary greatly. Therefore, the present applicant has focused on the bacteria collecting property of the membrane filter, simplified the collecting device and the culture method, and made it possible to measure the number of bacteria floating in the air in a short time.

[0006]

Recently, measurement of MRSA has become an important issue in environmental management in hospitals and the like. For this reason, the simplification of the MRSA measurement method and the measurement time, that is,
There is a strong demand for the development of efficient measurement methods and devices.

[0007]

Means for Solving the Problems The present inventors have developed a simple and easy-to-operate bacterium collecting apparatus capable of measuring the number of bacteria floating in the air in a short time and a culturing method which is easy to operate. The method applied to the measurement of was studied. As a result, in order to improve the reproducibility of the measured values, when collecting bacteria floating in the air with a membrane filter, consider that the bacteria may collide with the membrane and damage and kill the cell membrane, and air And the structure of the capsule were examined. Further, the composition of a highly selective culture solution capable of growing only MRSA colonies in only one culture was studied. In addition, a method for adjusting the culture medium with a simple operation in a short time was also studied, and based on those results, the present invention was reached.

That is, the present invention relates to a membrane filter.
And a filter capsule equipped with an absorbent pad and a drug
Capsule back lid with filling chamber for resistant staphylococcal broth?
And the membrane filter allows air to flow
With the function of collecting airborne bacteria on the surface, and after collecting the bacteria
Filling the culture solution by attaching the back cover to the capsule
An opening is formed in the chamber and the culture solution is injected into the absorbent pad.
Medium to selectively culture drug-resistant staphylococci.
To form colonies on the membrane surface.
Airborne drug-resistant Staphylococcus aureus measurement unit
It is .

[0009] Here, as a culture solution capable of selectively cultivating drug-resistant staphylococci, a mannitol medium containing D-mannitol and sodium chloride as main components or a sodium pyruvate, glycine and lithium chloride as main components. A medium obtained by adding methicillin and / or oxacillin to the prepared Baird Parker medium and adjusting the pH with sodium carbonate or hydrochloric acid can be used.

Hereinafter, the present invention will be described in detail.

The filter capsule of the drug-resistant staphylococcal measuring unit of the present invention is usually used by being attached to a floating bacteria measuring device made to flow a constant amount of air at a constant flow rate. The capsule has a structure in which a membrane filter and an absorbent pad are closely attached inside. In one embodiment, as shown in FIG. 1, a membrane filter and an absorbent pad are mounted on top of each other,
The air enters from the inlet and passes in the order of filter → pad. The membrane filter needs to have a function of collecting bacteria floating in the air when the air is circulated. Many types of bacteria are usually suspended in the air, but the most abundant are micrococcus and staphyrococcus.
It is. In general, bacteria are spherical with a diameter of 1 to 2 μm and are enveloped by a cell membrane mainly composed of peptidoglycan. The cytoplasm is contained inside the cell membrane.

The membrane filter is made of a film of nitrocellulose, cellulose acetate, polyether sulfone, polytetrafluoroethylene, or the like. Fine pores having a substantially uniform pore diameter and a nearly circular shape are densely formed on the entire surface of the thin film. Film filter. Since the conventional filter is formed by entanglement of a large number of fibers, the shape of the pores is irregular, and the separability when filtering bacteria is insufficient. However, the membrane filter is characterized in that it has very uniform pores, and thus exhibits a very sharp separation property. For collecting bacteria floating in the air, a membrane filter having a pore size of usually 0.3 to 0.8 μm, preferably 0.4 to 0.5 μm is used.

[0013] The measurement unit of the present invention is superior in principle to the conventional petri dish method in collecting suspended bacteria from a large amount of air. However, there was considerable variation in the measurement results at the beginning, and as a result of the examination, it was found that the influence of the flow velocity was large. As shown in FIG. 3, when the bacteria suspended in the air were collected by the membrane filter, the bacteria were collected on the membrane. It was clarified that the cell membrane was damaged by the collision and died. From this result, it has become clear that it is preferable that the air passing speed through the filter be 15 cm / sec or less in order to increase the measurement accuracy.

An absorbent pad is laminated on the membrane filter, and has a function of holding the filter when air is circulated at the time of collecting bacteria. Further, after collecting the suspended bacteria, the pad is impregnated with a culture solution. The medium can be adjusted as it is.

The culture solution of the drug-resistant staphylococcus in the measurement unit is filled in an ampoule or the like. The medium containing this culture solution has the property that only MRSA can be selectively cultured.
A medium capable of selectively culturing staphylococci as described above is already commercially available. However, in this medium, both staphylococci having drug resistance and staphylococci having no drug resistance existing in the past propagate and produce yellow colonies. The culture using this medium alone cannot confirm MRSA, which has recently become a problem.

Further, all of these media are commercially available in powder form, which are raw materials for agar media. Therefore, when used, it is necessary to dissolve it in a predetermined concentration with purified water, inject it into a petri dish or the like used for culture, and solidify the agar for adjustment. Therefore, when measuring MRSA, a considerably complicated operation is required only by adjusting the medium.

In order to simplify this operation, the present measuring unit uses an ampoule or capsule after sterilizing the culture solution.
The medium can be adjusted immediately by filling a filling chamber or the like provided inside the back cover and impregnating the pad at the time of use. Compared to a solid agar medium, the medium can be adjusted by a very simple operation, which is one of the major features of this measurement unit.

The composition of a culture solution filled in an ampoule or the like is based on a mannitol medium and a Baird Parker medium that can selectively culture staphylococci, and has been developed so as to be compatible with the present measurement unit. The medium is adjusted to a pH suitable for culture with sodium carbonate or diluted hydrochloric acid by removing agar from the components contained in the medium, and further adjusted by adding methicillin, oxacillin or other antibacterial agents in consideration of clinical use conditions.

That is, the composition of a culture solution containing mannitol and sodium chloride as main components is as follows: 2.5 g of meat extract, 10 g of peptone, 10 g of D-mannitol, 75 g of sodium chloride, 0.025 g of phenol red and methicillin per liter of purified water. And / or oxacillin and adjusted to pH 7.2 to 7.6 with sodium carbonate or hydrochloric acid.

The composition of a culture solution containing sodium pyruvate and lithium chloride as the main components is as follows: per liter of purified water, 5 g of meat extract, 10 g of casein peptone, 1 g of yeast extract, 10 g of sodium pyruvate, 12 g of glycine,
It is a liquid containing 5 g of lithium chloride and methicillin and / or oxacillin, and further adjusted to pH 6.6 to 7.0 with sodium carbonate or hydrochloric acid after adding 20 ml of a 1% potassium tellurite solution.

Antimicrobial agents, for example, methicillin, oxacillin and the like are added in advance to the ampule, but may be added with a syringe or the like when adjusting the culture medium. By culturing the thus obtained medium and a membrane filter that has been previously laminated and that has collected airborne bacteria in the air, only MRSA propagates and a yellow colony usually forms on the surface of the membrane.

The cultivation is usually carried out in an incubator, which is performed at a temperature of 35 to 38 ° C. for 24 to 48 hours in a culture solution containing mannitol and salt as a main component. When MRSA is present, yellow colonies are formed, and the number indicates the number of MRSA collected by the membrane.

The culture conditions of the culture solution containing sodium pyruvate and lithium chloride as main components are as follows.
26 hours. When MRSA is present, it produces shiny black colonies. The number of MRSA collected by the membrane can be determined from the number of generated colonies.

The fact that MRSA can be measured in a single culture only in a single culture is different from the conventional MRSA detection method in that MRSA can be measured only in a multi-stage culture test requiring a long time. It is far superior both in terms of time and time, which is the most significant feature of the measuring unit of the present invention.

As shown in FIG. 1, the inside of the filter capsule is equipped with a membrane filter and an absorbent pad, and the air inlet is sealed with a lid. Since the capsule is sterilized and stored after assembly, the capsule is kept in a sterilized state from the lid of the air inlet to the membrane filter. The capsule is preferably used by being attached to a device for measuring airborne bacteria in the air, which has a function of allowing a certain amount of air to pass through the membrane at a certain flow rate.

Many kinds of bacteria are always floating in the air. The most frequent are micrococcus and staphylococci, among them staphylococci including MRSA, which has recently become a particular problem. When collecting MRSA floating in the air, remove the cap of the capsule, switch on the floating bacteria measuring device, and let a fixed amount of air pass through the membrane filter at a constant flow rate. Air enters from the inlet and flows in the direction of the membrane filter → absorbent pad, and MRSA floating in the air is collected on the surface of the membrane together with other bacteria.

At this time, the air passage speed of the membrane is MRSA
It has a significant effect on the reproducibility of the measured values. This was found during the comparative study of the reproducibility of the measured value with the Petri dish method.The biggest cause of the dispersion of the measured value is that when the bacteria floating in the air are collected by the membrane, if the flow rate is high, the bacteria will It was found that the cell membrane was damaged and killed when colliding with the membrane. Air passage velocity of the membrane (cavity velocity)
As a result of examining the relationship between and the residual rate of the bacteria using the apparatus shown in FIG. 2, the results shown in FIG. 3 were obtained. Accordingly, it is necessary to maintain the air passage speed of the membrane at 15 cm / sec or less in order to ensure reproducibility of the measured values. After the collection of the suspended bacteria, the air inlet is closed by a lid, and the surface of the membrane is kept sealed and cultured.

When air is passed through the membrane filter to collect suspended bacteria, the absorbent pad has a function of keeping the membrane in close contact with the membrane. This is because the membrane is very thin and is deformed by a slight pressure of air or the like, and the pore diameter may change. After the collection of the suspended bacteria, the culture solution is cut from a drug-resistant staphylococcal culture solution ampule or the like and injected from the back side of the absorbent pad. The liquid uniformly penetrates into the inside of the pad by capillary action, and becomes the medium of MRSA as it is.

After injecting the culture solution into the absorbent pad, a back cover is attached to the capsule, and the capsule is usually placed in an incubator and cultured at a constant temperature for a predetermined time. The medium is
Since only MRSA has the property of selectively proliferating, when MRSA is collected on the membrane, culturing usually produces yellow colonies. From this number and the amount of air passing through the membrane, the presence or absence of MRSA and its density can be immediately determined.

[0030] In addition to this measurement unit as shown in FIG. 4,
A drug-resistant Staphylococcus aureus culture solution filling chamber is installed on the back of the capsule.
Those having only the form Ru Oh. After collecting the suspended bacteria with the capsule, when the back cover is attached, an opening is formed at the protrusion of the air outlet, the culture solution is injected into the absorbent pad, it becomes the medium, and it can be cultured in the incubator as it is It is possible to do it.

As described above, one of the major features of the measurement unit of the present invention is that the operation of adjusting the culture medium, culturing, detecting and measuring MRSA is extremely simple.

[0032]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 FIG. 1 is a sectional view and a perspective view of one embodiment of (a) a filter capsule and (b) a container filled with a drug-resistant staphylococcus culture solution, which constitutes the drug-resistant staphylococcus measuring unit of the present invention. The figure is shown. (C) is a perspective view of a dropper for injecting a culture solution.

The main body of the filter capsule 1 is a transparent plastic molded product. The main body is provided with an air inlet lid 2 as shown in (a), and a membrane filter 3 and an absorbent pad 4 are mounted inside the lid. It is mounted on the membrane and pad holding plate 5. A large number of concentric and radial shallow grooves are cut in the surface of the membrane and the pad holding plate 5 which comes into contact with the pad, so that air passes through each part of the membrane at a uniform speed. A cylindrical air outlet 6 is provided at the center of the holding plate. (b) The ampoule 7 of the drug-resistant staphylococcal culture solution is filled with a culture solution 17 having a property of selectively culturing MRSA.

The membrane filter 3 is a thin nitrocellulose film having an inner diameter of 37 mm in the air passage portion, and pores having a uniform diameter of about 0.45 μm, which are almost uniform, are formed densely on the entire surface. The absorbent pad 4 is made of a cellulosic nonwoven fabric having excellent gas permeability and moisture absorption.

After the filter capsule is assembled, the stopper 18 is attached to the opening 21 of the lid, and it is sealed and sterilized before being stored. Therefore, the space from the lid 2 at the air inlet to the membrane filter is kept in a sterilized state. Although the space between the absorbent pad 4 and the air outlet 6 is open, the air inlet is closed, so that no air flows through the membrane from the air outlet 6 and the membrane is stored during storage. No airborne bacteria adhere to the surface of the surface.

Next, using an apparatus as shown in FIG.
The residual density of MRSA was measured when the cavity velocity of air passing through the membrane was changed. The sample used was air from a highly contaminated hospital room, two filter capsules 1, 1 'were connected to the same air reservoir 8, and flow meters 9, 9' and a control valve were used.
The suction is performed by the same suction pump 11 via 10, 10 '. While keeping the cavity speed of the capsule 1 constant, 1 ′
The density of MRSA was measured by the measurement method of the present invention while changing the cavity velocity, and the relationship between the cavity velocity and the residual ratio of MRSA was examined. The result is shown in FIG. The measured values shown in the figure are average values of 20 measurements, and the range of variation of the measured values is indicated by a vertical bar.

Thus, it can be seen that when the cavity velocity becomes 15 cm / sec or more, the survival rate of the bacteria decreases as the cavity velocity increases, and the measurement results greatly fluctuate.
Therefore, the cavity velocity as air passes through the membrane
If it is kept at 15 cm / sec or less, there is almost no killing of bacteria,
It is also clear that the dispersion of the measured values is significantly reduced.

Next, the filter capsule has a function of allowing a fixed amount of air to pass through the membrane at a constant flow rate, and is a device for measuring airborne bacteria [Air Trend, manufactured by Kuraray Co., Ltd.] "]. Remove the lid 21 of the air inlet, put the switch of the floating bacteria measuring instrument, set the air passage time to 20 minutes to collect the floating bacteria,
After collection, the lid of the air inlet was closed. During this time, the air flow through the membrane was 4 ft ³ and the passing speed (cavity speed) was 8.7 c
m / sec.

Next, the ampoule 7 of the drug-resistant staphylococcal culture solution was cut, and 1 ml of the enclosed culture solution 17 was added to a dropper 19.
And injected into the absorbent pad 4 from the air outlet 6. The solution was quickly absorbed by the pad, and the medium was quickly adjusted.

The culture solution is a solution containing mannitol and sodium chloride as main components. The composition is 2.5 g of meat extract, 10 g of peptone, 10 g of D-mannitol, 75 g of sodium chloride, 0.025 g of phenol red per liter of purified water. And methicillin, and the pH is adjusted to 7.2 to 7.6 with sodium carbonate and diluted hydrochloric acid.
It is a liquid adjusted to.

Next, the capsule was put on the back cover, placed in an incubator, and cultured at 37 ° C. for 48 hours by a conventional method. As a result, four yellow colonies were confirmed from the outside of the capsule. Density of floating MRSA is the one / ft ^3.

[0043]

[0044]

[0045]

Example 2 FIG. 4 shows a perspective view of one embodiment of the drug-resistant staphylococcal measuring unit of the present invention. The filter capsule 13 has the same structure as that of the first embodiment, except that the air outlet has a protrusion 14. In addition, a drug-resistant staphylococcal culture solution filling chamber was provided
The back cover 15 of the capsule is a plastic molded product (polycarbonate), and has a structure in which a culture solution filling chamber is incorporated inside the inside of the back cover of the capsule. After injection of the culture liquid in the filling chamber, EVAL film 16 is heat-sealed and stored in sterilized at the tip of the filling chamber.

After collecting the suspended bacteria, the capsule back lid 15 in which the culture solution filling chamber is incorporated is pushed into the back side of the capsule, and the eval film 16 at the filling chamber entrance is formed by the projection 14 provided at the air outlet. The culture medium is ruptured, and the culture solution is poured into the absorbent pad to prepare the medium.

The culture solution sealed in the culture solution filling chamber is a solution containing sodium pyruvate and lithium chloride as main components.
g, casein peptone 10g, yeast extract 1g, sodium pyruvate 10g, glycine 12g, lithium chloride 5g
And 20 ml of a 1% potassium tellurite solution, and the pH was adjusted with sodium carbonate or hydrochloric acid.
It is a culture solution adjusted to 6.6 to 7.0.

The suspended bacteria were collected from the contaminated air different from that in Example 1, and the medium was adjusted as described above. Then, the capsule was placed in an incubator and cultured at 37 ° C. for 24 hours. As a result, six colonies were confirmed from the outside of the capsule. The density of floating MRSA is 1.5 / ft ³ .

[0050]

In recent years, staphylococci resistant to methicillin have spread in medical facilities such as hospitals, and attention has been paid to the intractability of diseases caused by the infection, and strict environmental management is required. The drug-resistant staphylococcus measurement unit of the present invention greatly simplifies the conventional complicated and time-consuming measurement method,
The use of the SA selective medium enables measurement with only one culture, which is extremely effective in environmental management for MRSA.

[Brief description of the drawings]

FIG. 1 shows one embodiment of a drug-resistant staphylococcal measuring unit of the present invention. (a) is a cross-sectional view of the filter capsule, (b)
Is a container filled with a drug-resistant staphylococcal culture solution. Also,
(c) is a perspective view of a dropper for injecting a culture solution.

FIG. 2 shows a system diagram of an apparatus for measuring a cavity velocity of air passing through a membrane and a residual ratio of MRSA.

FIG. 3 shows the relationship between the cavity velocity of air passing through the membrane and the residual ratio of MRSA.

FIG. 4 shows one of the drug-resistant staphylococcal measurement units of the present invention .
FIG. 2 shows a cross-sectional view of the embodiment .

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1, 1 'Filter capsule 2 Air inlet lid 3 Membrane filter 4 Absorbent pad 5 Membrane and pad holding plate 6 Air outlet 7 Drug-resistant staphylococcus culture solution filling container 8 Air reservoir 9, 9' Flow meter 10, 10 ′ Control valve 11 Suction pump 13 Filter capsule with protrusion at air outlet 14 Projection at air outlet 15 Capsule back with drug-resistant staphylococcus culture solution filling chamber 16 Eval film 17 Drug-resistant staphylococcus culture solution 18 Air inlet Lid lid 19 dropper 21 air inlet lid opening

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-4-218393 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12M 1/34

Claims (1)

(57) [Claims] (1) a membrane filter and an absorbent pad;
Filter capsule and drug-resistant staphylococcal culture
Consisting of a capsule back lid with a nutrient solution filling chamber, a membrane
Filter floats on the surface by allowing air to flow through it.
It has a function of collecting bacteria, and after collecting bacteria, put a back lid on the capsule.
By mounting, an opening is formed in the culture solution filling chamber
The culture solution is injected into the absorbent pad to become a medium,
Selective culture of drug-resistant staphylococci on the membrane surface
Airborne, capable of forming colonies
Drug resistant staphylococcal measurement unit.