Bristol Centre for Antimicrobial Research and Evaluation, Bacterial Genomics Group, Department of Pathology & Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
Received 7 January 2004; returned 9 February 2004; revised 24 March 2004; accepted 30 March 2004
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Abstract |
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Materials and methods: Evolutionary mapping used a shortest-path analysis of aligned blaSHV variants, and other basic bioinformatic approaches, such as CLUSTAL W and Blast were employed.
Results: Two main branches of the blaSHV evolutionary tree were located; both are derived from variant blaSHV-1 alleles. Identical mutations, responsible for extended-spectrum SHV substrate profiles, have been selected independently in each branch. There is evidence for a pool of non-mobile blaSHV framework sequences. Analysis of the genome sequence of Klebsiella pneumoniae confirms the chromosomal origin of blaSHV, whose mobilization has occurred at least twice, once for each of the main evolutionary branches. Both these mobilization events have been catalysed by IS26. Evolution of blaSHV to give common extended-spectrum variants is most likely to have occurred following mobilization.
Conclusions: These data shed new light on the evolution and mobilization of blaSHV, and these observations may be useful in predicting what might happen in future, both for blaSHV, and for other ß-lactamase genes.
Keywords: evolutionary trees , K. pneumoniae , mobile DNA elements
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Introduction |
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There have been some attempts to map the evolution of SHV, but these have generally used only amino acid sequences (e.g. Ref. 4). Furthermore, the generally accepted archive of SHV variants, maintained by Drs Jacoby and Bush (http://www.lahey.org/studies/webt.asp?D=http://www.lahey.org/studies/webt.stm&C = 404) only quotes amino acid variants, and has no facility to report variants with silent nucleotide changes. Such silent changes are of considerable value when studying evolutionary relatedness between sequences, because they are most unlikely to occur convergently, in contrast to functionally important amino acid changes. Many blaSHV variants with silent changes are reported on the NCBI Entrez database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD = Search&DB = nucleotide), but are time-consuming to extract and interpret, particularly given the large number of sequences involved.
We have a particular interest in studying the evolution and mobilization of SHV ß-lactamase genes, because understanding the processes that have gone on in the past might inform us about how genes might mutate, or become mobilized in the future, not just for SHV, but for other, newly mobilized ß-lactamase genes, and even those that are so far exclusively chromosomal. In this report, we have combined a series of data-mining experiments designed to find out the most likely way that SHV has evolved and been mobilized.
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Materials and methods |
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EvolConnEval (Evolution Connectivity Evaluator) was written in the Java programming language specifically to analyse blaSHV evolution. It was designed for use on the UNIX platform, where a local copy of CLUSTAL W10 is required. The program is written to automate (and so speed up) the following procedure, which could be carried out by hand. Put simply, the program accesses the NCBI GenBank database via the Entrez protein portal and performs a search for database entries containing the words SHV and lactamase. These entries contain coding sequences (CDS). Some only have one, encoding SHV, but some contain several. The program then downloads every CDS sequence (in FASTA format) in each database entry. These DNA sequences are pre-processed to remove duplicate copies (for example, there will be several identical examples of some blaSHV alleles, but only one of each is required for the analysis), and the modal length of the collection of CDSs is determined, and used to remove sequences differing from this by more than 3 bp. This final step is required because a few database entries highlighted in the search will contain multiple CDS sequences, only one of which will be the blaSHV sequence required (e.g. if an entire plasmid sequence were in one database entry). Furthermore, it is sometimes the case that FASTA files of blaSHV sequences contain truncated sequences, which, whilst being suitable for rough evolutionary tree prediction, always reduce the probability that the tree is correct. Our aim was to obtain a tree as near to perfect as possible, so these truncated sequences were not mapped into the automatic analysis. Following filtering, the final set of blaSHV sequences is inputted to the CLUSTAL W alignment program.10
Phylogenetic analysis of the blaSHV alignment using a Dijkstra's approach
The output file produced by CLUSTAL W is read by EvolConnEval to obtain, for each blaSHV sequence, a gapped alignment appropriate for matching to all other blaSHV sequences in the set. This alignment could be read manually to determine how close (in terms of the number of mutations) each pair of blaSHV alleles are, and so produce an evolutionary tree. However, to save time, EvolConnEval does this automatically. All relationships between blaSHV sequence pairs that fall within a defined threshold of similarity (up to two mutations were allowed during our analysis, again to maximize the likelihood that the tree we predicted is correct) are recorded in an nxn distance matrix. To penalize the requirement for multiple mutations to link two sequences, and so favour evolutionary paths that involve single mutations as much as possible, the distance between two sequences is recorded as the square of the actual number of mutations (i.e. the maximum gap between two sequences is 4, because the maximum number of mutations allowed is 2). Relationships between the entire set of blaSHV sequences can be viewed from the point of view of an undirected, (possibly cyclic) graph, and using the above distance matrix, the most likely (i.e. shortest) evolutionary paths are identified using a Dijkstra's classic shortest-path search algorithm.11 Again, this could be done manually, using trial and error. Having found all of the inter-relationships amongst the sequences, EvolConnEval attempts to identify which of these sequences is most likely to be the ancestor of the others, by finding that which is closest to all other sequences. In this case, the distance between two directly linked sequences is the number of mutational events required to convert one to the other, and in the case of longer putative evolutionary paths used to indirectly link two sequences, the separation is the sum of the distances along each of the intermediary paths. In order to reward the overall proximity of related sequences to a candidate sequence, rather than the number of sequences to which a link can be made (which will be equal for all members of the analysis), the centrality of the sequence is calculated as the sum of the inverse of the distances between the candidate sequence and all linked sequences. This approach is only valid for a situation where the ancestor sequence is present in the group, but in the case of mobile blaSHV sequences, this was thought highly likely to be the case.
EvolConnEval will be made available by the authors upon request.
Estimating the date of evolution of blaSHV alleles
EvolConnEval chooses the first example of each blaSHV allele that it comes across in the Entrez search output [i.e. in order of when the database entry was updated (most recent first)] so to find the oldest example of each allele, and so allow estimation of when that allele evolved, a manual analysis had to be carried out. The sequence of each blaSHV allele chosen by EvolConnEval was downloaded by reference to the GenBank accession number associated with it. This sequence was used to carry out a Blastn12 search on the GenBank nucleotide sequence database. Accession(s) containing sequences 100% identical to the input were visited, and the isolation date for that oldest example of the allele was determined. The minimum estimated evolutionary date for each blaSHV was equated to the date of first isolation of the bacterium containing it (if known), otherwise, the date of the publication reporting it (if known) or the date of its appearance on the database, whichever occurred first.
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Results and discussion |
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The amino acid sequence of SHV-1 was used as bait to run Blast searches10 on the available K. pneumoniae strain MGH78578 genome sequence (http://genome.wustl.edu/projects/bacterial/kpneumoniae/). This revealed two high-scoring hits (one with an e value of 6.2 x 10147, and the other with an e value of 1.2 x 10145) meaning that strain MGH78578 actually possesses two blaSHV genes, something that is not uncommon in modern, clinical K. pneumoniae isolates.9 One of these genes encodes an amino acid sequence that is identical to the SHV-11 subtype, and the other gene encodes a protein identical to the extended-spectrum ß-lactamase (ESBL), SHV-12.13
The K. pneumoniae MGH78578 blaSHV-11v4 gene, which is a silent variant of the previously isolated blaSHV-11v1,14 appears to form an independent transcriptional unit (i.e. is not part of an operon), and is located within a cluster of genes (Figure 1). Some of these neighbouring genes are known to be chromosomal in K. pneumoniae (e.g. the lac operon),15 and others are homologues of chromosomal Escherichia coli genes (i.e. the ygb operon).16 Guanine/cytosine content analysis across this region confirmed that blaSHV-11v4 does not stand out from the background (data not shown). Accordingly, there is no evidence that blaSHV-11v4 was acquired at this locus in recent evolutionary time, and there is every probability that this gene evolved in this species. The location of the E. coli ampC ß-lactamase gene is also as a single transcriptional unit, surrounded by seemingly unrelated genes.17
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Development of an evolutionary tree for blaSHV
EvolConnEval was run on the NCBI Entrez protein search engine using the keywords SHV and lactamase. One hundred and sixty-one GenBank entries were revealed, and within them, all CDS links were followed and associated nucleotide sequence information downloaded. In all, 48 non-identical blaSHV sequences passed through the various filters (see Materials and methods). Information about each of these alleles is given in Table 1. All the sequences were locally aligned with CLUSTAL W and analysed using a Dijkstra's algorithm as set out in Materials and methods. The resultant evolutionary map is shown in Figure 2.
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Definition of the ancestor blaSHV allele and evidence for convergent evolution of extended spectrum variants
The computational analysis, encouragingly, puts the oldest known allele, blaSHV-1v1, as the probable ancestral blaSHV (Figure 2). Two main branches have apparently evolved from this starting point: one branch produced genes encoding the ESBLs, SHV-2 and SHV-5, and the other branch produced genes encoding the ESBLs, SHV-2a and SHV-12 (Figure 2). This is in agreement with a more limited phylogenetic analysis of blaSHV nucleotide sequences carried out previously using a traditional maximum likelihood approach.19 Indeed, this general correlation of our analysis (Figure 2) with this previous approach validates the novel algorithm used here. The previous analysis used fewer blaSHV sequences (because fewer were known at the time) and tried to determine an evolutionary scenario that included all the sequences.19 However, our analysis, whilst it included more sequences initially, was specifically designed to only compare very closely related sequences, and so produce a tree that is more likely to be correct. Accordingly, whilst the traditional approach gives a possible big picture for blaSHV evolution,19 our simpler approach gives a more likely picture, but one targeted to a few closely related sequences.
Convergent evolution is apparent in the evolution of ESBLs in both main branches of the blaSHV evolutionary tree. Both blaSHV-2 and blaSHV-2av1 have the same G700A mutation, producing the amino acid change Gly238Ser, yet they have been derived from different blaSHV backbones. blaSHV-2av1 is derived from blaSHV-11v1 and blaSHV-2 is derived from blaSHV-1v1 (Figure 2). Furthermore, the additional amino acid substitution (Glu240Lys) found in SHV-5 (derived from SHV-2) is also present in SHV-12 (derived from SHV-2a), because of an identical nucleotide substitution occurring in the two progenitor genes (G703A), meaning that convergent evolution has facilitated the same two active site changes in both main branches of the SHV evolutionary tree. Clearly, therefore, these active site changes provide a strong survival advantage. The first of these changes (in both branches) occurred during the mid-1980s, and probably reflected the widespread use of cefotaxime at this time, since ESBLs having this change are able to hydrolyse cefotaxime.4 The second change (again, in both branches) occurred during the early 1990s, and probably reflected a move to the clinical use of ceftazidime.4 Both changes are required to enable ceftazidime hydrolysis, and the second, G703A change is not found alone in any known blaSHV sequence.
Analysis of 5' proximal sequence associated with blaSHV alleles, and evidence of IS26-dependent mobilization
Further evidence for the accuracy of our evolutionary tree comes from analysis of sequences 5' proximal to the genes where that sequence is available (Table 1). blaSHV-1v1, 2 and 5v1, all have identical 5' flanking sequences. In contrast, blaSHV-1v2, 11v1, 2av1 and 12v1 all have a single C to T point mutation (compared to blaSHV-1v1) at 26 with respect to the first nucleotide of the gene (i.e. the A in ATG). This is further evidence for the two-domain prediction of blaSHV evolution, where the upstream mutation occurred after the separation of the two domains, but before the evolution of ESBL alleles.
Some of the 5' flanking sequences associated with blaSHV alleles are long enough to reveal the presence of an IS26 element. It should be noted that most blaSHV alleles are sequenced following PCR reactions, which use primers that only amplify the structural gene, so it is likely that other mobile blaSHV genes are also associated with IS26, but the information to confirm this is not present on the various nucleotide sequence databases (Table 1). Some of the sequences with enough 5' proximal sequence to tell, however, reveal blaSHV alleles that are associated with IS26, which is 75 bp from the start of the gene, as in the case for blaSHV-12v4 in the K. pneumoniae MGH78578 genome sequence (Figure 1). Linkage to IS26 at this distance is found with blaSHV-2av1, and blaSHV-12v1 (Table 1). Interestingly, these alleles are found in the first putative branch of blaSHV evolution (Figure 2). Accordingly, it can be said with some confidence that mutation of blaSHV-2av1 into blaSHV-12v1 occurred after blaSHV-2av1 existed in a mobile form. Furthermore, blaSHV-2av1 is most probably derived from blaSHV-11v1 (Figure 2) which is found in an identical IS26-linked context (Table 1)14 so conversion of the non-ESBL, SHV-11 into an ESBL is highly likely to have followed the mobilization of blaSHV-11v1. The gene from which blaSHV-11v1 is probably derived, blaSHV-1v2 (Figure 2), is chromosomal, as are several other blaSHV-11 alleles, e.g. blaSHV-11v2.13 Accordingly, this is strong evidence for random variation at an (ancient) chromosomal locus existing in K. pneumoniae (the population) as might be expected, with one such allele being mobilized (probably since the first use of ß-lactam antibiotics) and subsequently founding one branch of ESBLs (Figure 2 and Table 1).
The chromosomal form of blaSHV-1v12 is the likely ancestor of the chromosomal blaSHV-1v2,13 and so the blaSHV-11 variants (Figure 2). The mobile form of blaSHV-1v13 seems to have evolved into the second main branch of mobile blaSHV ESBLs, including blaSHV-2 and blaSHV-5 (Figure 2). An analysis of this group of sequences reveals that their mobilization is also likely to be IS26-dependent. In this case, IS26 insertions in the chromosomal blaSHV region resulted in the generation of a larger piece of mobile DNA than was generated during mobilization of blaSHV-11, the IS26 element being 2643 bp upstream from blaSHV (Figure 1). However, because of the large amount of 5' proximal sequence required to confirm linkage of blaSHV with IS26 at this distance, it is only possible to say with certainty that it has happened with blaSHV-5v1. However, enough 5' proximal sequence information is available for blaSHV-1 and blaSHV-2 alleles, which are known to be mobile,3,4 to confirm that they are not closely linked to IS26 (Table 1).
Interestingly, it has previously been noted that, in South East Asia, blaSHV-1, blaSHV-2 and blaSHV-5 (which are rare in this region) do not have a closely linked 5' proximal IS26 element, but that the more common blaSHV-2a and blaSHV-12 alleles do, leading the authors to believe that the two clusters of sequences have evolved separately in that part of the world. The data presented here provide a more detailed analysis of the links between these sequences, which supports and extends these earlier conclusions.20
The close 5' proximal linkage of IS26 with blaSHV-11v1, and its derivatives probably explains the previous observation that blaSHV-2a is expressed more strongly than is blaSHV-2 (without a closely associated IS26) because of promoter differences that, at the time the observation was made, were not understood.21
Evidence for novel blaSHV backbone alleles, and derivatives thereof
Currently, fifty-four sequences have been officially assigned SHV numbers (http://www.lahey.org/studies/webt.asp?D=http://www.lahey.org/studies/webt.stm&C = 404) . However, SHV-17 has since been withdrawn, sequences for SHV-30, 31, 47, 49, 52, 53 and 54 have not yet been released onto the databases and DNA sequences encoding SHV-3, 4 and SHV-10 were never placed there. Furthermore, EvolConnEval deliberately removes truncated sequences, primarily so that non-related sequences in the same database entry as a blaSHV gene are not included in the blaSHV alignment. There are a number of blaSHV sequences in the database that are truncated (encoding SHVs 6, 19, 20, 21, 22, 23 and 39) and so were ignored because their inclusion in phylogenetic trees can only be tentative, since there may be mutations in the remainder of the sequence that are not known about. So, of 50 available, full-length blaSHV gene sequences (Table 1) encoding variants of 37 SHV enzymes, 25 (50%) could not be placed within the blaSHV evolutionary tree by EvolConnEval (Figure 2 and Table 1), even when allowing for up to two mutations (deletions and additions count as one mutation, even if a group of bases are involved). There are two variants of blaSHV-28, so, in total, 24 of 37 SHV enzymes (65%) could not be mapped by the program. The reason for this is that we wanted to produce a highly accurate map. Inclusion of divergent sequences into an evolutionary tree might give the most likely possibility of evolutionary relationship, but the divergent sequences have the potential to skew the entire tree, making it statistically reasonable, but actually, incorrect. This is borne out by the less-defined tree produced previously, when all blaSHV available sequences were included in the analysis.19
Sequences 5' proximal to each of the unmapped blaSHV alleles (where available) were analysed (Table 1). Interestingly, none of the 25 orphan blaSHV genes carry the 5' flanking sequence defined for blaSHV-1v2-derived genes, above, so none can be conclusively assigned to this cluster. In all cases where a 5' flanking sequence is available on the database entry (11 genes) they are identical to the 5' flanking sequence of blaSHV-1v1 (Table 1) so may have been derived from blaSHV-1v1, or may have evolved from a common ancestor. Of the 11 blaSHV alleles where a 5' flanking sequence is available, but which were not fitted by EvolConnEval, a reasonable case for direct descendance from blaSHV-1v1 can be made for blaSHV-16. With respect to blaSHV-1v1, there are only two changes in blaSHV-16: one insertion and one 4 bp rearrangement, but EvolConnEval treated the rearrangement as four separate nucleotide changes, and so did not fit blaSHV-16 into the evolutionary tree. Also, a case for direct descendance from blaSHV-5v1 can be made for blaSHV-9 (one triplet deletion and two rearrangements). These two alleles have been included in the evolutionary map (Figure 2).
It is likely that the remaining 23 full-length unmapped blaSHV sequences, some of which have been used previously to produce a less-targeted blaSHV phylogenetic tree,19 are derivatives of blaSHV variants whose sequences have not yet been determined. Indeed, of the 25 sequences, 15 are chromosomal, suggestive of chance isolation of novel, chromosomal blaSHV backbones (Table 1). Of the remaining 10 unmapped, blaSHV genes that are known to be plasmid-mediated, blaSHV-7 is the oldest.22 There is some evidence that blaSHV-18 (also mobile) was derived from blaSHV-7,23 suggesting that blaSHV-7 is a mobile ancestor allele distinct from both blaSHV-1v1 and 11v1. Not enough sequence information is available to convincingly demonstrate what the mechanism used to mobilize blaSHV-7 is, but analysis of the sequence22 gives hints that it might involve a mechanism not associated with IS26. The first 88 bases upstream of blaSHV-7 are identical to those upstream of blaSHV-1v1, but the remaining 37 bp of available sequence22 is highly homologous (33/37 identities) with a region of sequence that flanks IS10224 but has no homology with any other known sequences. This raises the possibility that blaSHV-7 was mobilized via an IS102-type insertion sequence, representing at least the third separate mobilization of a chromosomal blaSHV gene.
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Conclusions |
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The sort of evolutionary tree determination presented here, which excludes alleles that differ from others by more than two nucleotides, requires a situation where the phylogenetic root is present, together with a considerable number of very closely related sequences. Such an analysis could not be carried out with many groups of resistance gene sequences other than blaSHV. However, as more variants of other mobile ß-lactamase genes are reported, particularly alleles containing silent variations, it might be possible to produce a similar analysis for them.
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Acknowledgements |
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Footnotes |
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References |
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