Creating the right atmosphere

Technical developments have brought anaerobic culture systems within reach of all clinical microbiology laboratories, yet they vary in their capacities to isolate and identify anaerobes. Here Steve Robertson considers the clinical and financial benefits of improved anaerobic technique, and applications of modified atmosphere technology in groundbreaking new areas such as hypoxic research for cancer therapeutics

Anaerobic technology has moved on since the days of old brass jars

INTRODUCTION of anaerobic workstations in the 1970s provided microbiologists with the means to easily process, culture and examine samples without exposure to atmospheric oxygen. Despite their introduction, many laboratories still insisted on using anaerobic jars, although it had been well established that anaerobe isolation rates were significantly reduced. Wren (1977)1 showed an increase in isolation rate of anaerobes from 9.7% when using anaerobic jars opened at both 24 and 48 hours for examination, to 28.1% with jars left for 48 hours before being opened and to 35.7% when using an anaerobic workstation. Wren concluded that uninterrupted anaerobic incubation for 48 h substantially increases the yield of anaerobic isolates. Ten years later, a study by Sisson2 showed an isolation rate of 43.6% using anaerobic jars, which increased to 56.4% with an anaerobic workstation. The principal reason for these differences is straightforward: anaerobic jars are frequently not anaerobic, due to either a poisoned catalyst or poor technique when setting up the jar. Ensuring that samples are not exposed to oxygen following sampling is fundamental to good anaerobic microbiology as oxygen exposure reduces the ability to culture the most stringent of anaerobes in the sample.

In 1990, Downe3 compared the performance of two bag systems compared with that of the anaerobic chamber in their ability to support the growth of 25 anaerobic stock strains and to recover anaerobes present in clinical specimens. From the total of 171 anaerobic organisms isolated from 49 clinical specimens cultured, the anaerobic chamber proved to be superior for the cultivation of anaerobes, with all stock strains cultured and 169 (99%) of the total clinical isolates recovered. As expected for the more fastidious anaerobes, the poorest recovery was from the bag systems. This was thought to be due in part to the delay in obtaining anaerobic conditions in the bags resulting in longer oxygen exposure times, whereas plates were placed in an anaerobic atmosphere immediately when using the chamber.

A survey carried out by Goldstein (1990)4 investigated the methods in use for the isolation, identification, and susceptibility testing of anaerobic bacteria in relatively large non-teaching hospitals across the United States. Over half the laboratories using anaerobic chambers examined their primary plates after 24 hours of incubation, as opposed to 48 hours in laboratories using other systems such as jars or pouches. In identifying methods to improve clinical utility of anaerobic cultures, they proposed the use of selective media and an anaerobic chamber should lead to the availability of clinically relevant results of the presence of the B. fragilis group within 24 hours.

The first evidence-based study documenting the impact of using improved anaerobic techniques in the clinical microbiology laboratory was reported in 2002 by Barenfanger5 (Memorial Medical Center, Illinois USA). Her group compared parameters on 150 patients with anaerobic infections during two different time periods. The first when anaerobic cultures were done as previously (the control or ‘before’ group) and the second when anaerobic cultures were performed after a programme to improve anaerobic techniques (IAT). Matching of the two groups by diagnosis-related group (DRG) was done for the mortality, length of stay, and costs.

The IAT programme consisted of: converting to routine use of an anaerobe workstation and improved media; sending technologists to a workshop in anaerobic procedures; educating practitioners and nursing staff about anaerobic cultures; and strict adherence to improved guidelines for working with suspect anaerobes. For the group using improved techniques the average turnaround time for preliminary anaerobic data was 17 hours less than that from the control group. The crude mortality rate was 9.5% lower, the average length of hospital stay was 1.3 day less per patient, the average variable cost was $2,433 (£1,224) lower per patient and the average laboratory cost for all tests was $343 (£172) less per patient.

Based on the hospital admitting approximately 250 patients per year with positive anaerobic cultures, Barenfanger calculated that her hospital could expect to save $608,250 (£306,084 per year by using improved procedures for testing pathogens. The savings were offset by about $7,300 (£3,673) for the increased time taken for a skilled technician to carry out the additional work but importantly there were further savings of about $31,755 (£15,980) per year by using an anaerobic chamber instead of the previously used anaerobic culture systems. When all the figures were combined, a net annual cost saving through improved anaerobic testing of $632,705 (£318,403) was estimated. Not only had patient care been enhanced but significant savings were made. Anaerobic microbiology is clearly important with clear evidence that utilisation of best practice is consistent with improving patient care and providing significant cost savings.

Modular Atmosphere Controlled System (MACS) workstation

Research at the laboratories of Don Whitley Scientific has allowed the development of the Modular Atmosphere Controlled System (MACS) workstations for applications other than anaerobic culture.
Dr Roger Phillips at the University of Bradford’s Institute of Cancer Therapeutics, conducts research into the identification and evaluation of potential therapeutic compounds for treatment of hypoxic cells in tumours. Hypoxia or low oxygen tension is a state found in solid tumours, due to the combination of an inadequate blood supply and different physiological structure of blood vessels compared to that of normal vessels. In these poorly perfused regions, the environment is one of low oxygen tension, acidic pH, low nutrients and high catabolites, and contains cells with particular characteristics dictated by their main function of survival rather than proliferation.

Targeting these cells using conventional therapies has proved problematic, as they are very resistant to anti-cancer drugs and radiotherapy. As this environment is one unique to solid tumours, it is a potential target for therapeutic intervention using drugs designed to act selectively on these cells. Using a MACS VA variable atmosphere workstation, Phillips’ research has employed cellular assays to investigate changes over a closely controlled range of oxygen tensions, in order to demonstrate the required preferential activity against the hypoxic cells. The creation of this specialised hypoxic environment also allows the unique biochemical pathways involved to be accurately replicated, critically important for both biological and therapeutic understanding in the identification of new targets.

Essential to this research is the tight level of control over the gas mixture allowed by the variable atmosphere system, and its high degree of user friendliness. With CO2 required to maintain culture conditions as well as low O2 tension, the workstation can also function as a CO2 incubator to keep the cells under hypoxic conditions for longer periods. This is particularly important to ensure truly representative physiological conditions, rather than returning cells to their normal environment following treatment. Cells can be retained under hypoxic conditions during drug exposure and afterwards, allowing longer-term effects to be observed.

It can be seen that the flexibility in experimental design afforded by such close control over environmental conditions is playing a key role in the development of anti-cancer therapeutics and increased understanding of the role of hypoxia in tumour biology. As a result, research performed in this laboratory has led to two drugs entering clinical trials that show huge potential in ultimately improving patient care6, 7. From the two very different perspectives of clinical anaerobic microbiology and hypoxic research for cancer therapeutics, these advances have been made possible through the careful design and development of controlled atmospheric systems capable of accurately reproducing physiological conditions.

References
1. Wren M.W.D. (1977). The culture of clinical specimens for anaerobic bacteria: a comparison of three regimens.  Journal of Medical Microbiology, 10:195-201
2. Sisson P.R., Ingham H.R., Byrne P.O. (1987). Wise Anaerobic Work Station: an evaluation. Journal of Clinical Pathology, 40: 286-291
3. Downes J., Mangels J.I., Holden J., Ferraro M.J., Baron E.J. (1990). Evaluation of two single-plate incubation systems and the anaerobic chamber for the cultivation of anaerobic bacteria. Journal of Clinical Microbiology, 28 (2): 246-248
4. Goldstein E.J.C., Citron D.M., Goldman R.J. (1992). National hospital survey of anaerobic culture and susceptibility testing methods: results and recommendations for improvement. Journal of Clinical Microbiology, 30 (6): 1529-1534
5. Barenfanger J., Drake C.A., Lawhorn J., Kopec C., Killiam R. (1992). Outcomes of improved techniques in clinical microbiology. Clinical Infectious Diseases, 35 (Suppl 1): S78-83
6. Puri R., Palit V., Loadman P.M., Flannigan M., Shah T., Choudry G.A., Basu S., Double JA., Lenaz G., Chawla S., Beer M., Van Kalken C., de Boer R., Beijnen J.H, Twelves C.J., Phillips R.M. (2006) Phase I/II pilot study of intravesical Eoquin (EO9) against superficial bladder cancer. Journal of Urology, 176: 1344-1348
7. Harris P.A, Dunk C.R., Albertella M.R., Loadman P.M., Phillips R.M., Jones P., Twelves C.J. (2006) Tumor-specific activation of the hypoxic cell cytotoxin AQ4N: a phase I clinical study in solid tumors.  AACR Meeting Abstracts, Apr 2006: 571 – 572

By Steve Robertson. Steve is Sales Director at Don Whitley Scientific where his role includes researching new markets and products.