Within NCTC, great care is taken when authenticating all bacteria within the collection. Fastidious anaerobes pose a unique challenge. NCTC must ensure that strains are free from contamination from their aerobic counterparts and that there is enough biomass for the organism to survive in its freeze-dried state for a number of years. In addition, identification of anaerobic bacteria to species-level has been made easier in recent years through the improved databases of MALDI-TOF MS and VITEK2, although specialist reference laboratories play a large role in confirming the identification and specific characteristics.
Here are a few examples of the measures NCTC takes to provide the numerous anaerobic bacteria in the collection.
A potential issue with anaerobic bacteria is contamination from aerobic bacteria, particularly skin flora such as Staphylococcus and Micrococcus spp. Practices within NCTC when manufacturing the freeze-dried ampoules reduce the chances of this happening but when it does, the contamination can be hard to detect. The anaerobic conditions and the spreading colony morphology of some anaerobes such as Clostridium botulinum can suppress the appearance of contaminants present. To overcome this, the anaerobic NCTC strains are also grown aerobically for up to seven days to encourage the growth of any contaminants that may be present.
Increasing viability and ensuring longevity
Whilst some bacteria can survive the freeze-drying process for over 60 years, certain anaerobes do not fare so well. Porphyromonas and Actinomycete spp. can take over a week to grow and so prove tricky to cultivate. It was found that the viability of freeze-dried Clostridium novyi drops dramatically within one year; therefore NCTC monitors the viability of the anaerobes in the collection closely on a strain-by-strain basis to keep them alive in the collection.
Multiple approaches are used to confirm the identity of the anaerobes in the NCTC collection. Whilst MALDI-TOF and the VITEK2 are able to identify many of the clinically significant anaerobes, there are shortfalls in the databases available. In this instance 16S ribosomal RNA identification, derived from next-generation sequencing will be employed. Furthermore, detection of specific characteristics such as Clostridium difficile ribotype and C. perfringens toxin status is carried out by the specialist reference laboratories in Cardiff and Colindale respectively.