Inactivation of National Collection of Type Cultures (NCTC) bacteria for the NCTC 3000 sequencing project

Mohammed-Abbas Fazal1, Sarah Alexander1, Ana Deheer-Graham1, Nick Grayson2, Karen Oliver2, Julian Parkhill2, Julie E. Russell1
1Public Health England, London, United Kingdom. 2Wellcome Trust Sanger Institute, Cambridge, United Kingdom

INTRODUCTION RESULTS
Established in 1920, National Collection of Type Cultures (NCTC) was the first collection in the world to offer a supply service of authenticated Thus far the project has successfully inactivated and extracted DNA
bacteria of medical and veterinary interest to the scientific community. Today the collection contains over 5,000 bacterial cultures of known 23 lysates of E.coli and E.faecalis were incubated for lag time from over 2300 strains from 74 different families, and 533 bacterial
provenance. NCTC 3000 is a five-year collaborative project between Public Health England (PHE) Culture Collections and the Wellcome Trust (7:30 minutes) + 1 minute (half time in minutes required for total species using the methods developed. Out of 74 families, the majority
Sanger Institute (WTSI) funded by the Wellcome Trust with reagent support by Pacific Biosciences (PacBio). The project aims to sequence the inactivation, D-value). ‘No growth’ was observed in all 23 lysates were Enterobacteriaceae (35 %), 14% were Streptococcaceae and 9%
produced following heat inactivation at 80˚C for a total of 8:30 belonged to Staphylococcaceae (figure 2). The remaining 42% of the
genomes of 3,000 different containment level (CL2) 2 and CL3 type and reference strains from the collection using the PacBio Single Molecule,
minutes. Therefore, we can assume 1 minute inactivation time is strains sequenced thus far contain 949 strains representing 71
Real-Time (SMRT®) DNA Sequencing technology.
sufficient to reduce a starting culture of E.coli and E.faecalis different families
from 1010 to 100.
Heat treatment is the method of bacterial destruction most frequently used in food processing, accurate prediction of thermal death rates is
necessary to achieve desired safety margins whilst minimizing processing. In most studies, thermal inactivation has been described using first- To determine the quality of DNA produced following heat Clostridiaceae, 3%
order reaction kinetics and D-values. When exposed to lethal stresses, microbes are assumed to be inactivated with first order kinetic model. If inactivation. Suspensions of E. coli @~1010 in 300 ml of lysis Corynebacteriaceae, 2%
during one unit of time, 9 out of 10 organisms are inactivated then during an additional unit of time, 9 out of 10 of the survivors will be buffer were set up. Heat inactivation (800C) on lysates was Streptococcaceae, 14%

inactivated and so on. The unit of time will depend mainly on what the microbe is and what the lethal stress is, but within each combination, the performed for 9:30 minutes (lag time + 2 minutes) and DNA
unit of time for a decimal reduction will be constant. This unit of time is called a “D-value”. extracted and analysed (figure 1).

If a microbial suspension is subjected to a lethal stress with no survivors found on culture, it is possible that the log count in the suspension is Staphylococcaceae, 9%
around 10-1; a 1 in 10 chance of a survivor, which equates to one surviving microbe in every ten samples processed in that way.

Pseudomonadaceae, 3%
Enterobacteriaceae, 35%

Pasteurellaceae, 4%

METHODS
Neisseriaceae, 3%
NCTC bacterial strains were cultured from the earliest subculture available in order to avoid sequence mutations or loss of genetic
information (e.g. plasmids) caused by serial in vitro passage.
Figure 1. Electropherogram (EPG) from replicates of heat Legionellaceae, 3%
inactivated E.coli DNA showing HMW DNA with a peak at 60kbp
Inactivation of CL2 bacterial strains was performed by a combination of heat inactivation and enzymatic digestion with proteinase K, without shearing. Figure 2. Breakdown of the bacterial families sequenced thus far. The strains
highlighted above include Enterobacteriaceae (35%), Streptococcaceae (14%),
mutanolysin, lysostaphin, lysozyme was utilised. Bacteria such as Clostridia, Bacilli, Mycobacteria were incubated overnight at 85 ˚C Staphylococcaceae (9%), Pasteurellaceae (4%), Pseudomonadaceae (3%),
respectively to support cell lysis. To confirm inactivation, 10% of lysate volume was plated out onto appropriate medium and once ‘no Neisseriaceae (3%), Legionellaceae (3%), Clostridiaceae (3%),
Corynebacteriaceae (2%)
growth‘ was observed then DNA extracted.

Prior to heat inactivation of bacterial strains for CL3 bacteria, the lag time of heating block to reach 80 ˚C inside the tube was conducted by
setting up 24 water samples to determine the time taken in minutes for the tubes containing water to go from room temperature to 80 ˚C.
The different times were recorded on the heat block map. The longest time to reach 80 ˚C was determined was referred to as the “lag
time”. The lag time was determined to be 7 minutes and 30 seconds
D-values for NCTC 9001 Escherichia coli (E.coli) and NCTC 13379 Enterococcus faecalis (E.faecalis) were determined by conducting half
time experiment [“lag time” + 1 minute (8 minutes and 30 seconds)] to ensure inactivation of these strains using 23 replicates. E.faecalis
was a model organism for robust heat resistant vegetative bacteria to extrapolate data to CL3 Enterobacteriaceae. Therefore doubling the
inactivation time to 2 minutes to achieve double logarithmic inactivation of E.coli to one in 1010 chance of viable microbe present in the
lysate.
The optimum heat inactivation temperature was determined to be 80˚C for 9 minutes and 30 seconds without affecting the quality of DNA
extracted (figure 1).
CONCLUSIONS
Whole genome sequencing is set to revolutionise medical microbiology. Genomic characterisation of expertly-authenticated type and reference
Culture Collection strains will contribute significantly to a wide range of clinical and research applications as we progress further into this new
genomic era. SMRT sequencing delivers long read lengths with the highest consensus accuracy and uniform coverage, generating the most
comprehensive de novo assemblies. In order to deliver these long read sequence lengths, the quality of DNA is of utmost importance.
Subsequently DNA extraction methods have been developed and optimised to yield HMW DNA with an average fragment size of 60 kb or greater
from a range of Gram negative and positive bacteria.
Thus far over 2300 gram negative and positive bacteria were successfully inactivated from 74 different families, and 533 bacterial species. This
project will also sequence CL3 strains from the collection and it is of utmost importance to ensure bacteria are inactivated before proceeding with
DNA extraction and sequencing. Therefore methods to inactivate strains will be developed by heat inactivation and determining D-values for every
genus to ensure total lethality.

INTERACTIONS
Websites

 www.pheculturecollections.org.uk/collections/nctc-3000-project
ACKNOWLEDGEMENTS FUTURE WORK
 www.sanger.ac.uk/resources/downloads/bacteria/nctc

 www.ncbi.nlm.nih.gov/bioproject/251923  Public Health England As the project has now sequenced over two-thirds of total genomes, the next focus is to extract HWM
DNA from Advisory Committee on Dangerous Pathogens (ACDP) Containment Level 3 (CL3) pathogens
Social Media  Wellcome Trust Sanger Institute of which there are 181 in the collection. We anticipate that the some will be successfully extracted using
methodologies developed for CL2 organisms. However we also anticipate some challenging bacteria
Follow us on Twitter: @NCTC_3000  Pacific Biosciences such as spore forming bacteria which will be difficult to heat inactivate.
Currently the status of plasmids within NCTC strains is unknown. The project will also look into
If you would like to deposit strains with NCTC please contact: sarah.alexander@phe.gov.uk  Dr Peter Hoffmann investigating the loss of plasmids from the strains sequenced thus far. This would provide complete
genetic information of the strains deposited.