Method for recovering microbial cells

Abstract

The present invention provides a method of recovering viable microbial cells from a complex sample, said method comprising: a) providing a sample having a volume of at least 1 ml; b) contacting said sample with a buffer solution and one or more proteases, wherein said buffer solution has a pH of at least pH 6 and less than pH 11, wherein said buffer solution and said one more proteases do not comprise a detergent or a chaotrope, and wherein the buffer solution/protease/sample mixture is non-hypotonic; c) filtering the mixture obtained in step (b) through a filter suitable for retaining microbial cells; and d) recovering the microbial cells retained by the filter in step (c), wherein the recovered microbial cells are viable, and a microbial recovery device for the same.

Claims

1. A method of recovering viable microbial cells from a complex sample containing non-microbial cells, said method comprising: a) providing a sample having a volume of at least 1 ml, wherein where the sample has previously been contacted with a detergent or chaotrope, the sample is processed such that in subsequent steps (b) and (c), no chaotrope or detergent is present; b) contacting said sample with a buffer solution and one or more proteases, wherein said buffer solution has a pH of at least pH 6 and less than pH 11, wherein said buffer solution and said one more proteases do not comprise a detergent or a chaotrope, wherein the buffer solution/protease/sample mixture is non-hypotonic to the non-microbial cells, and wherein no detergent or chaotrope is present or used in step (b); c) subjecting the mixture produced in step (b), in which no detergent or chaotrope is present, to a filtration step, by filtering the mixture obtained in step (b) through a filter suitable for retaining microbial cells; and d) recovering the microbial cells retained by the filter in step (c), wherein the recovered microbial cells are viable.

2. The method of claim 1, wherein the sample is contacted with the buffer solution and the one or more proteases separately.

3. The method of claim 1, wherein the buffer solution and the one or more proteases are mixed prior to step (b).

4. The method of claim 1, wherein the sample is or comprises a clinical sample.

5. The method of claim 1, wherein the sample is a clinical, biological or environmental sample added to a medium.

6. The method of claim 5, wherein the medium is a culture medium.

7. The method of claim 4, wherein the clinical sample is a blood sample.

8. The method of claim 7, wherein said blood sample is collected in a blood culture flask.

9. The method of claim 1, wherein said protease is proteinase K.

10. The method of claim 1, wherein the pH of the buffer solution is between pH 7 and less than pH 10.7.

11. The method of claim 1, wherein the pH of the buffer solution is between pH 7 and pH 10.5.

12. The method of claim 1, wherein the pH of the buffer solution is between pH 9 and pH 10.5.

13. The method of claim 1, wherein the filter is a membrane filter.

14. The method of claim 1, wherein the filter is a polyamide filter.

15. The method of claim 1, wherein the filter has a pore size of less than 0.4 μm.

16. The method of claim 1, wherein the cells retained on the filter are recovered through back-flushing the filter using a liquid.

17. The method of claim 16, wherein the cells are back-flushed using culture medium.

18. The method of claim 1, wherein said method further comprises one or more wash steps between steps (c) and (d).

19. The method of claim 18, wherein said wash step selectively lyses non-microbial cells retained on the filter.

Description

EXAMPLES

(1) Samples consisting of BD Bactec BCF media and EDTA blood (25%) spiked with bacteria (E. coli or S. pyogenes) (approximately 10.sup.6 CFU/ml) were added to 0.3 M CAPS buffer containing protease in the absence of detergent. 5 ml of the blood/media mixture were added to 10 ml buffer and incubated before filtration by hand using a 0.2 μm filter, diameter 25 mm. After filtration, if the entire volume went through the filter, the filters were washed with 2 volumes of MH medium or PBS. Samples were resuspended to recover cells by back-flushing the filters with 5 ml MH-media or PBS. Filtration time and volume of sample it was possible to filter were measured for each sample. Viability of microbial cells recovered from the membrane was performed on TSA-plates and was recorded as CFU after overnight incubation. Recovery was calculated based on input number of bacteria an output number of bacteria corrected for the varying volume of in and out sample. pH of the buffer was as stated in the Examples below.

Example 1—Addition of a Buffer Containing a Protease to a Sample Enhance its Filterability

(2) An initial experiment was performed to determine the effect of the addition of a buffer containing a protease on the filterability of a sample. Samples were prepared using E. coli as outlined above and incubated with a buffer solution containing proteinase K, at a range of different pH values. As a comparison, a further sample was incubated with a buffer solution at pH 10.5 which did not contain proteinase K, Results indicating the filterability of each sample following treatment with the respective buffers are shown in Table 1.

(3) TABLE-US-00001 TABLE 1 Sample pH 9 pH 9.5 pH 10 pH 10.5 pH 11 pH 11.5 Buffer only — — — <1 ml — — Buffer + 15 ml 15 ml 15 ml 15 ml 15 ml 15 ml proteinase K Recovery of 5%  5% 20%  5% 0%  0% viable cells after backflush Viability of cells N.D 100% N.D >90% N.D <1% after incubation (without filtration)

(4) Samples incubated with buffer solutions containing proteinase K at each of the pH values tested showed enhanced filterability compared with the sample which did not contain a proteinase. This demonstrates that a proteinase may be of use in increasing the filterability of samples

(5) A further experiment was conducted to confirm the importance of Proteinase K in enhancing the filterability of a clinical sample. Samples were prepared using E. coli as outlined above, and the effect of the addition of buffer with or without proteinase K on the filterability of a sample was assessed using CAPS 0.3M buffer pH 10.5+proteinase K. Results are shown in Table 2.

(6) TABLE-US-00002 TABLE 2 Did volume pass filter? Approximate time to filter Sample 1 (+Prot K) All passed 1 min, 45 sec Sample 2 (−Prot K) 4.5 ml 1 min, 22 sec Sample 3 (+Prot K) All passed 1 min, 46 sec Sample 4 (−Prot K) 4.5. ml  59 sec

Example 2—Viability of Microbial Cells

(7) Samples were prepared using E. coli, a Gram-negative bacterium, and S. pyogenes, a Gram-positive bacterium, as outlined above. Samples were contacted with buffer at pH 10.0 containing Proteinase K, followed by a wash with PBS and subsequent treatment with DNaseI for 5 minutes, and cells on the filter were resuspended as outlined above. Viability of the microbial cells recovered from the samples were subsequently tested, and the results are shown in Table 3.

(8) TABLE-US-00003 TABLE 3 E. coli S. pyogenes Expt. 1 13-20% 31-32% Expt. 2 30-34% 47-56% Expt. 3 25-27% 28-39% Expt. 4 32-36% Average 27% (n = 12) 39% (n = 9)

(9) Both the Gram-negative E. coli and Gram-positive S. pyogenes were found to be viable at pH 10.0, with S. pyogenes appearing to have a greater degree of viability under these conditions.

(10) Recovered cells were also used for preparation of bacterial DNA using the DNA kit from Molzym (Molzym GmbH, Bremen, Germany) and results were quantified with real time PCR for presence of bacterial as well as residual human DNA.

(11) For E. coli approximately the total pre-efficiency from added bacteria measured as CFU to bacterial DNA measured as genomic copies present in eluate were about 7% and for S. pyogenes around 14%. Human DNA was found to be reduced by a factor of more than 99.9%.

Example 3—the Effect of Filter Material on Filterability

(12) Four filter materials: regenerated cellulose acetate (RegCA) (Ø 25 mm); polyamide (PA) (Ø 25 mm); polyethersulfone (PES) (Ø 30 mm); polyvinylidene (PVD) (Ø 30 mm); were tested and compared to the standard cellulose acetate (CA) (Ø 30 mm). All filters had a pore size of 0.2 μm. Samples were prepared as outlined above using a buffer at pH 10.5 and containing proteinase K. Results are shown in Table 4.

(13) TABLE-US-00004 TABLE 4 Did volume pass filter? Approximate time to filter PES 6.5 ml 3 min 40 sec PVD All passed 3 min 26 sec CA All passed 4 min 42 sec RegCA All passed 6 min 17 sec Polyamide All passed 2 min

(14) This demonstrates that although a range of different filter materials may be used in the methods of the present invention. Polyamide and cellulose acetate filters were selected for further testing to establish the efficiency or recovery of the microbial cells retained on the filter. Recovery of bacteria from different filters is shown in Table 5.

(15) TABLE-US-00005 TABLE 5 Did volume Approximate CFU Recovery pass filter time to filter recovered efficiency CA (1) All passed 2 min 6 sec 1E+06 69.1% PA (1) All passed 1 min 50 sec 1E+06 53.1% CA (2) All passed 3 min 1E+06 79.1% PA (2) All passed 1 min 44 sec 8E+05 45.6%

(16) A good efficiency of recovery of E. coli cells was observed for both the polyamide and cellulose acetate filters.

Example 4—the Effect of pH on Filterability and Viability

(17) Multiple CAPS buffers all at 0.3 M were prepared and adjusted to pH 7, 8, 9, 9.5, 10, 10.5 and 11. Samples were added to each CAPS buffer followed by incubation with Proteinase K. Samples were then filtered using polyamide filters and resuspended using phosphate-buffered saline (PBS) solution. Recovery was calculated based on colony formation (i.e. only viable cells were counted). Filtration and recovery of microbial cells at different pH values is shown in Table 6.

(18) TABLE-US-00006 TABLE 6 Did volume Approximate CFU Recovery pass filter? time to filter recovered efficiency BCF 2E6  — pH 9.0 All passed 1 min 21 sec 8E+4 4% pH 9.5 All passed 1 min 38 sec 2E+5 9% pH 10.3 All passed 1 min 58 sec 5E+5 24%  pH 10.7 All passed 1 min 13 sec 3E+3 0.1%.sup.  pH 11.0 All passed 46 sec 0 0 pH 11.5 All passed 32 sec 0

(19) An improvement was in filterability was seen for treatment with buffers at pH11-11.5, however, microbial viability at these high pH values was compromised. Following the finding that buffers having pH values of at least pH 9 could be used to enhance the filterability of a clinical sample, buffers having a wider range of pH values were tested for their effect on enhancing the filterability of a clinical sample. The results of this are shown in Table 7.

(20) TABLE-US-00007 TABLE 7 Volume filterable pH 7 7.5 8 8.5 9 9.5 10 10.5 11 Buffer only n/d n/d n/d n/d n/d <1.5 ml n/d Buffer + >15 ml >15 ml >15 ml >15 ml >15 ml  >15 ml >15 ml proteinase K Recovery of 22% 21% 19% 8% 6% 9% 20% 6% <1% viable bacteria

(21) These data indicate that the filterability of a clinical sample may be enhanced using a buffer solution containing Proteinase K at a wide range of pH values. Thus a method to allow filtration of large volumes of complex matrices, such as blood and blood+culture media at any pH between pH 7 and less than pH 11 using a protease to facilitate filtration is demonstrated and we believe that such a method may be used with buffers at any pH from pH 6 to less than pH 11. Viability of microbial cells between pH 7 and pH 10.5 was found to be close to 100% as evaluated after incubation with CAPS and proteinase K buffer and direct plating for counting viable count. At pH 7 between 92 to 120% survival was recorded after 5 to 15 minutes proteinase K treatment. At pH 10.5 between 95 to 120% survival was recorded after 10 minutes proteinase K treatment. The over 100% survival can be accounted for imprecision in the plating procedure that was done manually in these experiments. Accordingly, whilst the recovery of viable bacteria is reduced between pH 8.5 and 9.5 in the data in Table 7 above, this may be addressed by optimisation of the particular reaction conditions at each pH, e.g. reaction time, protease concentration etc.

Example 5—Conditions Used for Filtration are Less Harmful to Microbial Cells than Those Used in the Art

(22) A further experiment was performed to confirm that the pH of the filtration conditions identified herein are less harmful to microbial cells than those currently in the art. Selective lysis of non-microbial cells has been shown to be possible at very high pH values, however, the viability of the microbial cells under such conditions is poor.

(23) EDTA-blood samples spiked with E. coli were incubated with a buffer solution at pH 10.5 or a solution containing 8% NaOH. A comparison of these conditions with the filtration conditions described herein is provided in Table 8.

(24) TABLE-US-00008 TABLE 8 5 ml blood + 10 ml 5 ml blood + 5 ml Sample Before pH 10.5 buffer 8% NaOH, pH 14 CFU/ml 1E+6 5E+5 0 Recovery* — 123% 0% CFU/ml 6E+5 2E+5 0 Recovery* — 103% 0%

(25) Incubation of the sample with a solution containing 8% NaOH resulted in no viable microbial cells being recovered from the sample. In contrast, addition of the buffer solution at pH 10.5 did not substantially affect the viability of the microbial cells in the sample. Recovery of over 100% was observed for samples incubated with buffer at pH 10.5 due to viability experiments only being performed in duplicate.