WO1999021960A1

WO1999021960A1 - Method for culturing microbiological samples in a gas pressure gradient

Info

Publication number: WO1999021960A1
Application number: PCT/FI1998/000828
Authority: WO
Inventors: Olli-Pekka Lehtonen
Original assignee: Oy Aboatech Ab
Priority date: 1997-10-24
Filing date: 1998-10-23
Publication date: 1999-05-06
Also published as: FI105044B; EP1025202A1; FI974043A0; FI974043A

Abstract

The present invention relates to a method for culturing microbiological samples. The method comprises filtering a prepared sample and culturing the sample on the filter in the presence of a pressure gradient.

Description

METHOD FOR CULTURING MICROBIOLOGICAL SAMPLES IN A GAS PRESSURE GRADIENT

Field of the invention

Background of the invention

Culture is currently the basic method in clinical microbiological laboratories. Despite of the development of various techniques, especially the genetic engineering in the diagnosis of infectious diseases, clinical laboratories do need the culture methods also in the future. Culture is versatile for a wide spectrum of organisms being the only simple method to detect new and unexpected organisms. It is relatively cheap to perform and thus it has an established position in the diagnosis of the most common bacterial infections. Culture is also the only method to supply with live organisms for further reference and phenotypic testing of drug susceptibility. Environmental and food laboratories do also exploit the advantages of culture techniques.

Culture is said to be slow. However, a bacterial mass large enough for modern susceptibility testing and identification can be grown in less than 10 hours. Most of the clinically relevant bacteria yield visible colonies in 16 hours. Iden- tification by antigen detection or gene hybridization can be performed in 1 to 2 hours and modern susceptibility testing for rapidly growing bacteria can be done in 4 to 6 hours. Thus, the overall analysis time for rapidly growing bacteria is less than 24 hours. Considering assisting in the choice of antimicrobials this is quite a reasonable delivery time. However, in current microbiological laboratories the total delivery time for a sample submitted for culture tends to be substantially longer. In addition to transport of the sample and reporting of the results, there are long time periods when the sample is not in the process. Samples wait for culturing, cultures wait for interpretation, subcultures wait for identification, and susceptibility tests wait for reading. There are automatic apparatuses available for the follow-up of growth, identification and suscepti- bility testing (Jδrgensen, 1987). However, optimal use of these would necessarily require a laboratory with three working shifts because transfer of the material from one automate to another is still purely manual.

It has been pointed out that clinical bacteriology has only a limited impact on clinical decision-making ( Campo & Mylotte, 1988; Rintala et al . , 1991; Lehtonen et al . , 1993). On the other hand, there are some data indicating that a rapid and timely culture report can improve the clinical impact, achieve substantial savings in hospital costs and even decrease mortality (Doern et al., 1982; Trenholme et al., 1989; Doern et al., 1994). Thus, an optimal method for culture would be a totally automated system with a minimal delay in processing the samples all the way until reporting with no manual phases which interrupt the process.

The present status of bacteriological process automation can be described briefly as follows.

Automatic sample processing and culture inoculation are theoretically possible, at least for liquid or liquefied samples. Some devices for dilution of samples are available.

Culture follow-up automates are widely used for blood culture. Still, subcultures have to be done manually. Some attempts have been made for direct inoculation from the blood culture bottle to identification (Davis & Fuller, 1991), but even in this case mixed cultures cannot be handled. It seems that at least in blood culture plating of individual colonies is critical for proper susceptibility testing results (Travis & MacLowry, 1989).

There are no methods for automatic judging of the cultures and making subcultures from individual colonies.

Automated systems for identification are available. Most of those currently available require some preliminary classification of the bacteria (e.g. Gram-staining) , which has to be done manually. Analysis of membrane fatty acids (Eerola & Lehtonen, 1988) or sequencing of 16S ribosomal DNA (Jens- sen et al . , 1993) are methods which can be applied without such a previous classification.

Automated susceptibility testing systems are available. They, however, need a standardized inoculum, preferably from colonies grown on a solid medium.

The most critical steps for process automation are located in the beginning of the process, especially in the separation of the clones in the sample. Liquid cultures are cumbersome because they cannot cope with mixed cultures of different strains. Conventional agar plates are not very handy for automated processes. They have a limited shelf- life and are prone to contamination in automated handling. This is why the present inventor has attended to develop- ment of a new design for bacterial culture.

Description of the invention

The present invention provides a filter culture method which enables automated culturing of microbes. The method is called "Bactexpress" method. The method can be applied for various biological samples, e.g. body fluids, but also for water samples, or filterable food or environmental samples .

In the method a sample is first prepared to be filterable, if appropriate, i.e. in the case of a biological sample eukaryotic cells present in said sample are lysed. Then the sample obtained is filtered using a microbiological filter, and the filter is flushed with a liquid nutrient medium, which remains on the other side of the filter. The microbes which remain on the filter, are made to grow on it as if they grew on an agar plate, by establishing a gas pressure gradient using appropriate gases or gas mixtures. The medium should remain in contact with the filter. This can be accomplished e.g. by turning the filter around. The pressure gradient is used to prevent the medium from bleeding to the side of the filter where the microbes have been trapped. It is easy to change nutrient media and the gas mixtures during incubation. The method is suitable for automation as the culture method can be carried out using liquid flows, for which suitable technologies exist.

The culture method of the invention uses bacteriological filters (e.g. cellulose acetate) with pore sizes of 0.22 to 1.0 μm. Such filters are manufactured by e.g. Millipore. Most of the clinically relevant samples (blood, urine, ascites, pus, cerebrospinal fluid, synovial fluid) can be prepared for filtration by lysing the eukaryotic cells present therein. E.g. Zierdt (1982) has described a lysis process which can be used in the present method. Filtration of biological samples is described in various publications (e.g. Wallis & Melnick, 1985, Longoria et al . , 1987 and Bernhardt et al . , 1991), and any such filtration system can be used in the present method. After filtration of the sample, the filter is flushed with a liquid medium to remove growth inhibitors possibly present in a clinical sample. Then, a further amount of liquid medium is added and filtered until to the other side of the filter. After filtration the filter is turned around, and a gas pressure gradient is established leading a gas or a gas mixture to the filter from below upwards, thereby constructing a solid-liquid interface. In the appended Figure l pilot scale arrangement for this system is illustrated. This setup gives a growth environment which results in visible colonies similarly as on an agar plate. A similar type of idea of combining liquid and solid media was disclosed decades ago (Kanz, 1951) but without the filtration procedure for inoculation of the sample. It should also be noted that Kanz used cellophane which is quite inappropriate for filtration. Contamination of the liquid medium is easy to avoid because the liquid flows in one direction only through the filter. The liquid medium can easily be changed, even during incubation, if necessary. Different gas mixtures can be used for different atmospheric requirements of the microbes. The principle of the inventive method is illustrated in the appended Figure 2.

The now presented configuration would enable automation because control of the culture process is easy by regulating the flow of the liquid nutrient medium. Using solid culture media would necessitate very cumbersome robotics. The liquid flow serves for inoculation of the sample, con- struction of the "solid" culture medium as well as disinfection and removal of infectious material. Video technology could be used for the subsequent surveillance of the growth of the colonies, and gas chromatography or sequence analysis of ribosomal DNA would be useful for further auto- matic identification of the cultures.

Other applications for the culture method of the invention are e.g. construction of a microbiological culture method in field conditions from dry nutrient medium and water using the filter, as well as replacement of natural agar in microbiological culture research. Brief description of drawings

Fig. 1 Pilot scale arrangement for culture system according to the invention.

Fig. 2 The liquid nutrient broth and the test sample are first filtered through the filter; the bacteria remain on the side B. The filter is then turned around, and the nutrient broth on the side A acts as growth medium. Diffu- sion of the nutrient broth to side B and prevention of excessive moisture by a pressure gradient (0.01 to 0.5 bar) enable growth of visible and dispersed colonies as on agar plates.

Experimental

Filterability testing of blood samples

The experiments were carried out using blood culture samples . The blood samples were filterable in the given circumstances (see Wallis and Melnick, 1985) when detergents were added to a commercially available sample tube (Isolator™).

The microbes tested in pure cultures: Escherichia coll, Enterococcus faecalis , Haemophilus influ- enzae. Streptococcus pneυmoniae , Streptococcus miller 1, Streptococcus agalactiae, Candida albicans, Pseudomonas aeruginosa, Staphylococcus epidermidis, Pasteurella multo- cida, Bacteroides fragiliε

Example 1: Comparative tests - present system versus agar plate cultivation

Comparative tests using the present filter culture method and on the other hand transferring the filter onto a conventional agar plate were carried out with the above listed microbes. The agar plate method with filter is currently used e.g. in food laboratories. The parameters measured were 1) number of the colonies, 2) size of the colonies, and 3 ) existence of separate colonies. The variables were a) composition of the nutrient medium, b) incubation time and c) different types of membrane filters. The influence of antibiotics on the filters as well as changing of the nutrient medium during the incubation^' were also studied.

Results

Results of the experiments carried out with different nutrient medium compositions are given in Table 1.

Table legend:

+: a successful test, i.e. good growth (+): a faint growth

-: an unsuccessful test, i.e. no growth; empty field: no experiment so far.

Abbreviations in Table 1 : BHI = Brain Heart Infusion (Difco)

FBS = Fetal Bovine Serum 5% (Finnzymes Oy) BLOOD = heated sheep blood, 5% NAD = a growth factor in blood (7.5 μg/ml ) HEMIN = a growth factor in blood (7.5 μg/ml )

The results given in Table 1 show that various bacteria having clinical importance can be successfully cultivated with the present "Bactexpress" system. However, the most fastidious species need medium supplements. Table 1

Example 2 : Comparative tests with Escherichia coli and Enterococcus faecalis

Comparative tests using the present filter culture method and on the other hand transferring the filter onto a conventional agar plate were carried out using Escherichia coli (ATCC 25922) and Enterococcus faecalis (ATCC 29212).

Conditions: Nutrient media: Brain Heart Infusion (BHI) Broth (Dif- co)

Brain Heart Infusion Agar (1.5% Bacto

Agar)

Temperature: 37 °C

Filter: Cellulose acetate membrane 0.45 μm

Variables: Pressure (air) 0.5 bar

0.3 bar

0.1 bar

0.05 bar Incubation time 14 h

21 h

Quantities measured: number of colonies size of colonies number of separate colonies

Six replications were used in both methods.

The results are given in Table 2.

Table 2 Comparison of Bactexpress and agar methods

According to the results obtained it seems that the lower pressure, the bigger size of the colonies. The lowest pressures used, i.e. 0.05 to O.l bar, are sufficient and are functioning best. The number and size .of the colonies using the Bactexpress method are smaller than the average number and size of the colonies using the conventional agar plate system. The inventive method can, however, be considered quite functional . Example 3: Seeking the optimum conditions for Streptococcus agalactiae

The optima of Bactexpress-method for a clinical strain of Streptococcus agalactiae (TY 659) were studied by varying the pressure and concentration of fetal bovine serum (FBS) in tryptic soya broth. The yield of colonies was compared to the yield of colonies on tryptic soya agar with 0.5 per cent FBS. The mean diameter of the colonies was measured from 10 individual colonies on filter. The number of colonies not confluent to adjacent colonies was counted and expressed as the percentage of the total number of colonies. Each combination of pressure and serum concentration was studied in five parallel filters. The results are shown in Tables 3, 4 and 5.

Table 3. Effect of the pressure and concentration of FBS on the yield of colonies of Streptococcus agalactiae in Bactexpress-method, per cent out of the colonies grown on tryptic soya agar with 0.5 per cent FBS.

Table 4. Effect of the pressure and concentration of FBS on the mean diameter of colonies of Streptococcus agalactiae in Bactexpress-method.

Table 5. Effect of the pressure and concentration of FBS on the percentage of non-confluent colonies of Streptococcus agalactiae in Bactexpress-method.

The results show that Bactexpress-method needs more serum supplement than agar culture to yield the same number of colonies. However, using pressure of 0.2 bar, and FBS concentration of 10 per cent, a good yield of mostly non- confluent colonies is obtained.

References

Bernhardt M, Pennell DR, Aimer LS, Schell RF. Detection of bacteria in blood by centrifugation and filtration. J. Clin. Microbiol. 1991; 29: 422-425.

Campo L, Mylotte JM. Use of microbiology reports by physicians in prescribing antimicrobial agents. Am. J. Med. Sci. 1988; 296: 392-398.

Davis TB, Fuller DD. Direct identification of bacterial isolates in blood cultures by using a DNA probe. J. Clin. Microbiol. 1991; 29: 2193-2196.

Doern GV, Scott DR, Rashad AL. Clinical impact of rapid antimicrobial susceptibility testing of blood culture isolates. Antimicrob. Agents Chemother 1982; 21: 1023-1024.

Doern GV, Vautour R, Gaudet M, Levy B. Clinical impact of rapid in vitro susceptibility testing and bacterial identification. J. Clin. Microbiol. 1994; 32: 1757-1762.

Eerola E, Lehtonen 0-P. Optimal data processing procedure for automatic bacterial identification by gas-liquid chro- matography of cellular fatty acids. J. Clin. Microbiol. 1988; 26: 1745-53.

Jenssen MA, Webster JA, Straus N. Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl. Environ. Microbiol. 1993; 59: 945-952.

Jorgensen JH. Automation in clinical microbiology. CRC Press Inc. Boca Raton, Florida, 1987.

Kanz E. Das Flϋfe-Verfahren, eine neue Form von Membran- nahrbδden. Arch. Hyg. 1951; 140: 106-129.

Lehtonen 0-P, Rintala E, Eerola E. Poor value of routine susceptibility tests in guidance of antimicrobial therapy, (abstract) 7th Congress of European Society of Clinical Microbiology and Infectious Diseases, Seville, 1993.

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Rintala E, Kairisto V, Eerola E, Nikoskelainen J, Lehtonen 0-P. Antimicrobial therapy of septicemic patients in intensive care units before and after blood culture reporting. Scand. J. Infect. Dis. 1991; 23: 341-346.

Travis LB, MacLowry JD. Clinically significant differences in antibiograms of morphologic variants of blood culture isolates. Diagn. Microbiol. Infect. Dis. 1989; 12: 177-179. Trenholme GM, Kaplan RL, Karakusis PH, Stine T. Fuhrer J. Landau W, Levin S. Clinical impact of rapid identification and susceptibility testing of bacterial blood culture isolates. J. Clin. Microbiol. 1989; 27: 1342-1345.

Wallis C, Melnick JL. Rapid, colorimetric method for the detection of microorganisms in blood culture. J. Clin. Microbiol.1985; 221 : 505-508.

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Claims

1. A method for culturing microorganisms present in a sample, comprising a) filtering the sample through a microbiological filter, b) flushing the filter with a liquid nutrient medium, and c) establishing a gas pressure gradient for retaining contact between the filter and the liquid medium but enabling growth of separate colonies during the subsequent incubation.

2. The method according to claim 1, wherein, prior to step a) the sample is prepared to be filterable by lysing eukaryotic cells present in the sample.

3. The method according to claim 2, wherein the sample is a whole blood sample.

4. The method according to claim 2 , wherein the sample is synovial fluid, cerebrospinal fluid, ascites, or any other body liquid.

5. The method according to claim 1, wherein the pore size of the filter is 0.22 to 1.0 ╬╝m, and wherein a pressure of 0.01 to 0.5 bar is used.

6. The method according to claim 5 , wherein a pressure of 0.05 to 0.1 bar is used.