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Considerations for Authenticating Bacterial Strains Coming Into and Going Out of Service Culture Collections

Paper i proceeding
Författare Edward R.B. Moore
Elisabeth Inganäs
Susanne Jensie-Markopoulos
Maria Ohlén
Sofia Wernersson
Liselott A Svensson
Publicerad i Proceedings of the XXVII ECCO Annual Meeting, June 10-11, Ghent, Belgium
Sidor 22-24
Publiceringsår 2008
Publicerad vid Institutionen för biomedicin, avdelningen för infektionssjukdomar
Sidor 22-24
Språk en
Ämneskategorier Biologisk systematik


Culture collections effectively provide important depositories of prokaryotic diversity, to be available for academic, biotechnological and commercial exploitation. Given the rationale for maintaining reference collections of bacterial strains, it is essential that the collections insure four important points: 1) the viability of strains; 2) the purity of strains; 3) the “authenticity” of strains, i.e., strains are what have been described and represented by the depositors; and 4) the accessibility of strains. Indeed, Rule 27(3) of the Code of Nomenclature of Prokaryotes states, “in the case of species or subspecies the culture collection numbers of at least two publicly accessible service collections in different countries where a subculture of the type strain has been deposited must be indicated”. Since 2002, proof of deposit and availability from culture collections has been required. Implicit in these requirements is the responsibility for the collection to provide proof of deposit to confirm the authenticity of the strain. However, culture collections should not consider themselves obligated to repeat the complete experimental procedures of depositors. The numbers of new taxonomic names included in categories covered by the rules of the Code of Nomenclature of Prokaryotes and validly published have risen an average of 14% each year for the last five years, to more than 900 in 2007 (www.bacterio.cict.fr/). The designated type strain for each of these new species and new combinations must be confirmed as available within culture collections. In principle, it is the responsibility of the respective culture collections to confirm the authenticity of these additions. In 2007, the CCUG issued 118 Certificates of Deposit (CoD) for type strains of newly proposed species. This represents 13% of the total number of new taxa described for that year and was an increase of 34% more than the number of CoD issued by the CCUG in 2006. In light of such increasing numbers of new taxa, culture collections must also respect the time, effort and expense required for “confirming” the authenticities of new deposits. The CCUG has assumed the responsibility for confirming authenticities of deposited strains, i.e., with respect to the protocols used to analyse the strains, and including the qualification: with a high degree of confidence. To this end, culture collections must consider carefully the methods they employ for analysing the authenticity of deposits. The methods to be used should necessarily be reproducible and elucidate the resolution adequate for recognising incorrect strains or contaminations. The CCUG employs phenotypic and cellular fatty acid (CFA) profiling as first- and second-phase analyses, with subsequent 16S rRNA gene sequencing as a tertiary-phase tool in cases wherein the phenotyping and CFA analyses prove to be inadequate. Phenotyping. CCUG “phenotyping” employs an initial screening of Gram-reaction, oxidase, catalase and cultivation on differential media. As has been pointed out before, if the results of simple, initial screening do not conform to the description of the organism, there is no need to carry on with more extensive analyses. Assuming that initial screening agrees with the description received from the depositor, phenotyping proceeds, employing taxon-specific customised and commercial (API, bioMérieux) tests (described in “CCUG Databases, Worksheets and Statistics”, www.ccug.se/default.cfm?navID=160). The resulting data are compared with the differential characteristics described by the depositors (i.e., the CCUG strongly recommends that depositors send the manuscript describing the strains, to be held in confidence). The CCUG carries out “phenotyping” analyses on most deposits. Thus, phenotypic authentications or identifications were performed on most of 1,715 strains deposited with the CCUG in 2007. Such analyses are reliable and useful for some taxa. Unfortunately, phenotyping also possesses well-known inherent problems. Firstly, the protocols require cultivation periods to allow the reactions to develop within the test panels. This generally takes 4 – 48 hours for most commercial systems, and some tests can require as long as six days. Secondly, reproducing phenotypic profiles in the culture collection lab that match those produced in the labs of depositors can be a challenge. Many reasons are responsible for inter-laboratory variability noted in phenotyping results. Additionally, it is recognised that some bacteria do not respond well to the substrates in commercial test panels (e.g., Stenotrophomonas spp., Sphingomonas spp.,), resulting in profiles of limited diversity. Deposits of such bacteria can not be reliably assessed, using phenotyping protocols. Furthermore, some bacteria do not present profiles that distinguish them from other species (e.g., Burkholderia cepacia complex spp., Streptococcus mitis complex spp., etc.). Thus, although bacteria belonging to closely related “species complexes” may be easily differentiated from organisms outside the respective species complex, the culture collection should be aware that inadvertent switches or contaminations with closely related species will most likely not be detected. For this reason, it is recommended that handling multiple samples of closely related and similar organisms on the bench should be done at different times. Chemotyping. CCUG “chemotyping”, may be applied, using cellular fatty acid (CFA) profiling and the protocols described in the MIDI Technical Note #101 (www.midi-inc.com). In 2007, the CCUG determined CFA profiles of 743 deposits (43%). In many cases, such analyses are able to confirm the authenticity of a deposited bacterial strain. Unfortunately, chemotyping faces some of the same limitations observed for phenotyping, in that some bacteria will produce CFA profiles of minimal diversity (e.g., Methylobacterium spp., etc.) or profiles that are not distinguishing (e.g., species of Enterobacteriaceae, etc.). The same risks associated with switching samples or contamination with closely related organisms must be acknowledged by culture collections. Furthermore, a minimal amount (100 mg) of biomass is necessary, which may be problematic to obtain from some fastidious organisms. Genotyping. It is clear that, just as phenotypic and chemotypic profiling may not be adequate for identifications of some bacteria, they also will prove less than perfect for reliable authentications of some bacteria. In such cases, 16S rRNA gene sequence determinations and comparative analyses may provide evidence supporting the authenticity of deposits. The CCUG employs partial 16S rRNA gene sequences for analysing the authenticities of deposited bacteria. The 16S rRNA genes are amplified by PCR, using universal primers. Amplification reactions are set up in duplicate, in order to minimise potential sequence mistakes caused by PCR error. The duplicate amplification products are combined prior to setting up sequence reactions. For purposes of authenticating deposited strains, the CCUG depends upon a single sequence reaction, resulting in determination of approximately 500 nucleotide positions (one third of the 16S rRNA gene) at the 5’-terminus of the 16S rRNA gene. Throughout the length of the 16S rRNA gene, five “hypervariable” regions have been observed to encompass the majority of nucleotide positions exhibiting variation. Three of the regions, comprising 71% of the variable positions in the five “hypervariable” regions, are located within the range of 500 nucleotide positions at the 5’-terminus of the gene. Thus, the probability is high that any deviation in sequence identity between sequences determined for deposited strains and the sequences determined for the strains by the depositors will be detected in this region. In order to provide a systematic assessment of deposited strains, using 16S rRNA gene sequence analysis, the exact region of sequence comparison must be specified, preferably with reference to the E. coli or other species 16S rRNA gene sequence. An extremely high, or perfect, correlation of sequence identities would be expected in order to confirm the deposited strains as being the same as described by the depositors. Of course, the limitations of using 16S rRNA gene sequence analysis as a tool for authenticating deposited strains can be the same as those faced by phenotyping and chemotyping, i.e., the problem of resolution between very closely related organisms. However, again, the question is whether the deposited strains can be considered to be the same organisms described by the depositors – with a high degree of confidence. In point of fact, the authentication process for strains coming into the CCUG is overly complex because information obtained from new organisms is, at the same time, incorporated into the CCUG identification databases. If the only issue were to confirm the authenticity of new strains deposited in the collection, a partial, single-reaction sequence of 16S rRNA genes could be recommended as adequate, in most cases. Such an approach would provide the necessary confidence in the authenticity of newly deposited strains and would help minimise the time, effort and expense of analyses. It is important to note that the 16S rRNA gene sequence is the only characteristic that is required in all cases of new bacterial species descriptions. Whereas phenotypic differential characteristics are required, the specific analyses are not defined. Chemotypic data may be recommended, but are not necessarily required. G+C% content and DNA-DNA similarities are required only in specific conditions. Thus, it is reasonable to assume that information certain to be included in the descriptions of new organisms would be used also by culture collections for authenticating new deposits. Although the methods described above may be considered to be “adequate” for authentications – with a high degree of confidence, culture collections must always qualify their conclusions because of the limitations in the methods used. Methods affording higher resolution could be employed, e.g., MLSA, VNTR, etc. However, not all new organisms are described by such methods. Thus, we see that procedures for authenticating deposits in culture collections necessarily are defined by the methods used for describing new taxa. As information accumulates from multi-locus analyses, taxon-specific house-keeping gene sequence data could be employed directly, forgoing current phenotyping, chemotyping or genotyping protocols. Certainly, with the on-going effort of the Joint Genome Initiative to sequence the genomes of all type strains, genomic data will accumulate which will enable culture collections to confirm authenticities of deposits, without qualification, by assessing specific differential characteristics defined by the depositor. In the future, the CCUG will propose to institute a standard “Strain Character Authentication Form” to be filled out by depositors, in addition to the current Deposit Form, which will list specific relevant information for assessing the authenticity of the strain. The form will include the accession number for the determined 16S rRNA gene sequence of the strain to be deposited. The sequence similarities to the most closely related species will be listed, including the accession numbers of the respective 16S rRNA gene sequences. Additional information will include a listing of the differential characteristics for the strain to be deposited and the most closely related species. Listings of the specific phenotypic, chemotypic and genotypic differential data would “stream-line” the process for deciding which analyses are necessary for confirming that the deposited strain is what has been described. Costs should be reduced by eliminating the time, effort and materials that would be used in carrying out uninformative analyses. The overall authentication process would be greatly enhanced.

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