Comparative evaluation of the MGIT and BACTEC systems for the culture of Mycobacterium avium subsp. paratuberculosis from milk

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1 Comparative evaluation of the MGIT and BACTEC systems for the culture of Mycobacterium avium subsp. paratuberculosis from milk Grant IR 1, Kirk RB 2, Hitchings EIJ 1 and Rowe MT 1,2 1 Department of Food Microbiology, Queen s University of Belfast. 2 Food Science Division, Department of Agriculture and Rural Development for N. Ireland, Agriculture and Food Science Centre, Newforge Lane, Belfast BT9 5PX, N. Ireland, UK. Corresponding author: Grant IR. Phone Fax i.grant@qub.ac.uk Keywords: Mycobacterium avium subsp. paratuberculosis (Map), BACTEC culture, MGIT culture, milk, decontamination. SUMMARY The non-radiometric Mycobacterium Growth Indicator Tube (MGIT TM ) culture system has not previously been evaluated for the culture of Mycobacterium avium subsp. paratuberculosis (Map) from milk. A study was undertaken to compare the detection capabilities and detection times of the MGIT system with those of the radiometric BACTEC 460 system currently used in our laboratory. UHT milk samples were spiked with a range of Map concentrations ( cells/ml). MGIT tubes supplemented with MGIT OADC, MGIT PANTA and mycobactin J, and BACTEC vials supplemented with PANTA and mycobactin J, were inoculated with 500 µl of triplicate milk samples at each Map concentration. MGIT tubes were read manually using a UV transilluminator and BACTEC vials on the BACTEC 460TB machine. Time to detection of growth in days was recorded for each system. A corresponding BACTEC count was determined from the BACTEC growth index readings using a published formula. A further experiment assessed the recovery of Map from milk by both culture systems following decontamination with 0.75 % (w/v) cetylpyridinium chloride for 5 h. In the absence of any decontamination step both the MGIT and BACTEC culture systems were capable of detecting growth from milk samples containing Map cells within d. The correlation coefficient between MGIT and BACTEC detection times was MGIT detection times tended to be shorter than BACTEC detection times when low numbers of Map were present in a milk sample and slightly longer when high numbers of Map were present. After decontamination only milk samples originally spiked with > Map exhibited growth in both culture systems. Decontamination caused a significant reduction (mean 1.44 log) in numbers of viable Map in all spiked milk samples. Overall, the MGIT system was shown to have similar detection capabilities to the BACTEC system for recovering Map from milk. INTRODUCTION Many laboratories worldwide, including our own, routinely use the BACTEC 460TB system for the culture and detection of mycobacteria. Becton Dickinson (BD) first introduced this technology over 20 years ago. In the mid 1990s the BBL MGIT TM Mycobacteria Growth Indicator Tube was introduced as a nonradiometric alternative to the BACTEC 460 system (8). In contrast to the BACTEC system where the amount of 14 C-labelled CO 2 released gives a measure of growth, the MGIT tubes utilize fluorometric technology to indicate that growth has occurred. When MGIT tubes are placed on a UV transilluminator (365 nm wavelength) growth positive tubes emit a vivid orange fluorescent glow at the tube base and at the meniscus, whereas growth negative tubes show negligible or no glow. The fluorescence indicates that dissolved oxygen in the MGIT broth medium has been consumed by respiring bacteria. The MGIT system is reported to have a number of clear advantages over the BACTEC 460TB system: (a) it is a non-radioactive system so the disposal of 14 C in used tubes does not present problems; (b) MGIT tubes take up less incubator space than BACTEC vials; (c) syringes and needles are not required for the addition of supplements or specimen; and (d) MGIT tubes can be read manually on a UV transilluminator so there is no need for an expensive automated reader (although such a machine is available, MGIT 960 system). To date the MGIT culture system has not been widely used, or properly evaluated, for the culture of Mycobacterium avium subsp. paratuberculosis (Map). Map is much slower growing than M. tuberculosis and it also has a growth requirement for mycobactin J, an iron- 208

2 chelating compound, which must be added to any solid or liquid medium used for its culture. In addition egg yolk emulsion has been added to BACTEC radiometric 12B medium in the past to encourage the growth of very small numbers of Map. These additional medium supplements have the potential to interfere with the fluorometric detection system in the MGIT tubes. Stitt et al. (8) reported a preliminary evaluation of methods for growing Map using MGIT tubes. Since then veterinary and clinical laboratories in New Zealand and USA respectively, have reported the successful culture of Map from animal tissue (2), human Crohn s disease tissue (7) and breast milk of Crohn s patients (6) using the MGIT culture system. Our particular interest in Map over recent years has been in determining whether milk could be a potential vehicle of transmission of this organism from cattle to humans, and both naturally infected and artificially spiked milk samples have been cultured using the BACTEC 460TB system in the course of our research. Generally culture of Map from naturally infected milk must be preceded by chemical decontamination of the milk samples with 0.75 % cetylpyridinium chloride for 5 h at room temperature (3). The objectives of the study reported here were twofold: (a) to compare the performance of the MGIT and BACTEC culture systems in terms of detection times and minimum detection limits when used to detect Map in milk; and (b) to determine if chemical decontamination of milk samples before culture affects the detection capabilities of either culture system. In order to exclude possible interference from other elements of the milk microflora spiked UHT whole milk rather than spiked raw milk was used throughout this study. MATERIALS AND METHODS Comparison of the minimum detection limits and detection times for BACTEC and MGIT culture. A 10 ml broth culture of Map NCTC 8578 was vortexed with five sterile glass beads (3 mm diameter) for 2 min in order to de-clump cells. Serial dilutions of this de-clumped Map cell suspension were prepared in UHT whole milk obtained from a local supermarket to yield spiked milk samples containing 10 1 to 10 7 Map cells/ml. Five hundred microlitres of each dilution were inoculated into three vials of BACTEC Middlebrook 12B medium (Becton Dickinson, Cowley, Oxford) supplemented with 100 µl of PANTA antibiotic supplement (Becton Dickinson) and 2 µg/ml mycobactin J (Synbiotics Europe SAS, Lyon, France). Three MGIT tubes (Becton Dickinson) supplemented with 100 µl of BBL MGIT PANTA antibiotic mixture and 0.5 ml BBL MGIT OADC enrichment (both Becton Dickinson) and 2 µg/ml mycobactin J were also inoculated in a similar manner. Egg yolk emulsion was not added to either medium because it has been reported to quench fluorescence in the MGIT system (8) and we have encountered inhibition problems with egg yolk in BACTEC cultures of milk samples previously (4). BACTEC vials and MGIT tubes were incubated at 37 ºC and read at intervals of 3-4 days for up to 50 days. BACTEC vials were read using the BACTEC 460TB instrument (Becton Dickinson) and MGIT tubes were examined on a 365 nm UV transilluminator (UltraViolet Products Ltd. Cambridge, U.K.). The time to a growth index (GI) reading > 50 was recorded in the case of each BACTEC culture and the number of Map added to each vial initially was estimated using the formula published by Lambrecht et al. (5) which relates detection time (in days) and the associated cumulative GI reading to Log 10 number of Map cells present. In the case of the MGIT tubes, the time (in days) until fluorescence was observed was noted for each milk sample. The whole experiment was repeated on three separate occasions using Map strain NCTC Comparison of the performance of BACTEC and MGIT culture systems when chemical decontamination was applied to milk before culture. A dilution series was prepared from a broth culture of Map NCTC 8578 and thirty-six 8 ml volumes of UHT whole milk were dispensed into 10 ml centrifuge tubes. Sets of six milk samples were spiked with six different Map dilutions to yield milk samples containing 10 1 to 10 7 Map cells. All 36 milk samples were then centrifuged at 2,500 x g for 15 min and the milk pellets in 18 of the tubes, three for each Map cell concentration, were resuspended in 1 ml phosphate buffered saline containing 0.05 % Tween 20 (PBS-T, Sigma Chemical Co. Ltd, Poole, Dorset). These 18 tubes represented the no decontamination treatment. The remaining 18 milk pellets were resuspended in 10 ml 0.75 % (w/v) cetylpyridinium chloride (CPC; Sigma) and incubated at room temperature (20 ºC) for 5 h. Following centrifugation (as above) the pellets were resuspended in 1 ml PBS-T. These 18 samples represented the After HPC decontamination treatment. The resuspended pellet from each milk sample, irrespective of treatment, was inoculated into one vial of BACTEC 12B medium and into one MGIT tube (both supplemented as described above), 500 µl per vial or tube. Both culture media were incubated at 37 ºC for up to 50 days and examined periodically as described above. Time to detection of growth (in days) in both culture systems was recorded for each milk sample, and 209

3 a BACTEC count determined for each milk sample using the published formula (5). The whole experiment was repeated twice using Map strain NCTC Statistical analysis of results. Detection times recorded for the BACTEC and MGIT culture systems, with and without prior HPC decontamination, were compared using the paired t-test and the correlation between BACTEC and MGIT detection times was determined. RESULTS The detection times recorded for the MGIT and BACTEC culture systems for all the milk samples tested during this study did not differ significantly, irrespective of whether decontamination had been applied to the milk samples before culture (t-test, p = ). There was shown to be a good correlation between MGIT and BACTEC detection times (r = , p < ). Typical detection times for the two culture systems are provided in Table 1. Both culture systems were shown to be capable of detecting 10 Map cells in a milk sample within days in the absence of prior decontamination. Chemical decontamination of the spiked milk samples with 0.75 % CPC for 5 h at room temperature prior to culture resulted in a significant (p < ) reduction in numbers of viable Map recovered (mean log 10 reduction of 1.44, 95 % confidence interval: log 10 cycles). Consequently, when decontamination was applied before culture, both the MGIT and BACTEC systems were only capable of detecting growth in milk samples originally spiked with Map (Figure 1). Detection sensitivity had decreased for both culture systems as a consequence of the adverse effect of HPC on Map viability. Table 1. Typical detection times observed for MGIT and BACTEC culture systems for the recovery of Map from milk. No. of Map present a Mean detection time (d) ± (cells/ml) BACTEC MGIT ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.0 a BACTEC count derived using the formula of Lambrecht et al. ( 5). DISCUSSION This study was carried out to compare the performance of the MGIT and BACTEC 460 culture systems for the isolation of Map from milk. Results indicate that both culture systems are capable of detecting low numbers of Map within similar timeframes. Detection times recorded for the two culture systems (Table 1) were not significantly different even when chemical decontamination was applied to the milk samples prior to culture. Consequently, MGIT culture could be used instead of BACTEC for the culture of Map from milk without loss of detection sensitivity. Currently, we have no plans to change from the BACTEC to the MGIT system because MGIT tubes and allied supplements are even more expensive than BACTEC 12B medium and PANTA supplement. However, should local restrictions regarding disposal of 14 C become prohibitive in the future we would then switch to using the nonradiometric MGIT system. Spiked UHT milk was used throughout the study in order to 7 6 No decontamination After HPC decontamination Mean Log10 no. of MAPrecovered No. of MAPcells spiked into milk sample originally Figure 1. Effect of chemical decontamination on the numbers of Map recovered by BACTEC culture from spiked milk samples. 210

4 exclude any possible interference from the normal background microflora of raw milk. The real test will come when the MGIT system is used to culture Map from naturally infected milk samples. Contamination rates have been reported by several groups to be slightly higher in the MGIT medium compared to the BACTEC medium. This is possibly due to the addition of MGIT OADC to MGIT medium making it a richer medium than BACTEC 12B (1). We have suspected for some time that HPC decontamination of milk containing Map resulted in loss of viability in some Map cells present. However, we had not previously been able to quantify this loss. BACTEC counts were obtained for each milk sample tested during this study and counts before and after HPC decontamination (Figure 1) were significantly different with a mean reduction in numbers of viable Map of 1.44 log being observed. HPC decontamination had an adverse effect on the detection sensitivity of both the MGIT and BACTEC systems, Map cells could be detected in the absence of decontamination but only cells after decontamination. This finding has implications for the detection of Map in naturally infected milk samples using either of the culture systems. Low numbers of Map are likely to be present in naturally infected raw milk and raw milk normally requires chemical decontamination in some shape or form to be applied before culture. The findings of this study suggest that if HPC decontamination is applied before culture only when Map is present at levels of 10 2 or above will culture be successful. HPC decontamination has previously been shown to be less harmful to Map than, for example, the Cornell method used by other groups to test milk (3). Consequently, even greater inactivation of Map may occur if decontamination protocols other than 0.75 % HPC for 5 h are used before culture in MGIT or BACTEC. This possibility needs to be borne in mind by researchers attempting culture of Map from naturally infected milk. During this study we manually examined MGIT tubes for evidence of fluorescence by placing them on a UV transilluminator at regular intervals during incubation. When three people independently read the tubes, the detection times recorded by each person were never significantly different. We, therefore, found manual reading of the tubes to be a satisfactory means of assessing growth of Map in the relatively small number of MGIT tubes involved in this study. An automated MGIT reader, the MGIT 960 system, is available from Becton Dickinson that will accommodate 960 MGIT tubes for the period of incubation and automatically indicate when growth has been detected in any of the tubes. However, this is an expensive piece of equipment and, in our view, the price would be prohibitive for many laboratories unless very large numbers of MGIT cultures were being processed, whereas most laboratories either already have, or can easily afford to buy, a UV transilluminator. CONCLUSIONS The MGIT culture system was shown to have similar detection capabilities to the BACTEC system when used to culture Map from spiked milk and so this non-radiometric culture system could be substituted for the radiometric BACTEC 460 system for milk testing purposes. Prior HPC decontamination of the milk samples prolonged detection times in both culture systems to a similar degree and minimum detection limits were, consequently, reduced. Manual reading of the MGIT tubes on a UV transilluminator proved a satisfactory means of assessing growth in MGIT cultures during this study. ACKNOWLEDGEMENTS This study forms part of ongoing Map research funded by the Department of Agriculture and Rural Development for N. Ireland. REFERENCES 1. Cornfield DB, Beavis KG, Greene JA, Bojak M and Bondi J Mycobacterial growth and bacterial contamination in the Mycobacteria Growth Indicator Tube and BACTEC 460 culture systems. J Clin Microbiol 35: De Lisle GW, Yates GF, Cavaignac S and Collins DM Evaluation of the MGIT system for culturing Mycobacterium avium subsp. paratuberculosis and characterization of strains by Polymerase Chain Reaction tests. Proceedings of the 6 th International Colloquium on Paratuberculosis, p Dundee L, Grant IR, Ball HJ and Rowe MT Comparative evaluation of four decontamination protocols for the isolation of Mycobacterium avium subsp. paratuberculosis from milk, Lett Appl Microbiol 33: Grant IR, Ball HJ and Rowe MT Incidence of Mycobacterium paratuberculosis in bulk raw and commercially pasteurised cows milk from approved dairy processing establishments in the United Kingdom. Appl Environ Microbiol 68: Lambrecht RS, Carriere JF and Collins MT A model for analyzing growth kinetics of a slowly growing Mycobacterium sp., Appl Environ Microbiol 54: Naser S, Shafran I and Schwartz D Isolation of Mycobacterium avium subsp. paratuberculosis from breast milk of Crohn s 211

5 disease patients. Amer J Gastroenterol 95: Schwartz D, Shafran I, Romero C, Piromalli C, Biggerstaff J, Naser N, Chamberlain W and Naser SA Use of short-term culture for identification of Mycobacterium avium subsp. paratuberculosis in tissue from Crohn s disease patients. Clin Microbiol Infect 6: Stitt DT, Sturn KM and Hagemann PA Preliminary methods for growing Mycobacterium paratuberculosis using the BBL MGIT TM Mycobacteria Growth Indicator Tube. Proceedings of the 5 th International Colloquium on Paratuberculosis, pp