Bristol Centre for Antimicrobial Research and Evaluation, Department of Pathology & Microbiology, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
Received 25 February 2004; accepted 12 March 2004
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Abstract |
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Methods: ß-Lactamase and 16S rRNA genes were amplified by PCR and sequenced by standard methods. ß-Lactamase induction was attempted in liquid-grown cultures using cefoxitin. Nitrocefin hydrolysis assays were performed using a spectrophotometer.
Results: Analysis of 16S rRNA gene sequences showed that Citrobacter spp. isolates with an inducible ß-lactamase gene, cdiA, closely related to C. koseri NF85 and ULA27 are actually Citrobacter amalonaticus. C. koseri isolates, whose identities were confirmed by 16S rRNA sequencing, produce a class A ß-lactamase, Cko, constitutively at low levels. The cko and cdiA ß-lactamase genes share <45% identity.
Conclusions: We have confirmed that cko is a ß-lactamase gene carried by C. koseri, and that isolates previously identified as C. koseri , but carrying the cdiA ß-lactamase gene are C. amalonaticus. Thus, ß-lactamase-gene-specific PCR may provide a valuable tool to differentiate these biochemically homogeneous Citrobacter species.
Keywords: Citrobacter, ß-lactamases, phylogenetics, induction
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Introduction |
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Both C. freundii and C. diversus were shown to express single, chromosomally encoded, inducible ß-lactamases, but the two have quite different substrate profiles and come from different molecular classes. C. freundii produces a class C ß-lactamase, AmpC, which hydrolyses cephalosporins, including later generation compounds, and some penicillins.3 In contrast, C. diversus was shown to produce a class A ß-lactamase, CdiA, hydrolysing aminopenicillins and carboxypenicillins.4 ß-Lactamase genes from two C. diversus isolates, NF854 and ULA27,5 have been cloned and sequenced. The two cdiA genes are 98% identical.
In 1982, another species was introduced into the genus Citrobacter, named Citrobacter amalonaticus, which was only differentiated from C. diversus by the fact that the former is not able to utilize malonate as sole carbon and energy source.6 Isolates ULA27 and NF85 were identified as C. diversus using biochemical profiles set down in 1977, before the introduction of C. amalonaticus.7 At that time, it was acknowledged that C. diversus was variably positive for malonate utilization, with 12% of isolates being negative.2
In 1993, C. diversus was rejected as a species name in favour of C. koseri8 because of an error in the naming of C. diversus, and the previously (and correctly) proposed name, C. koseri, for isolates with the C. diversus/C. koseri/L. malonaticus biochemical profile took over.8 This helped clarify the taxonomy of Citrobacter spp. at a time when eight new genomospecies were being proposed.1 At this time, all nucleotide database entries originally designated containing sequences from C. diversus were automatically altered to state that the sequence came from C. koseri.
The C. diversus NF85 and ULA27 cdiA nucleotide sequence database entries (EMBL accession numbers CAA54738 and CAA44485) currently state that these isolates are C. koseri, and so CdiA ß-lactamase is now thought of as being a C. koseri enzyme. However, in the first report characterizing C. diversus ULA27 and NF85 ß-lactamases, differences from a ß-lactamase, said to be expressed by a strain identified as C. koseri, were reported.7 C. diversus ß-lactamases, typified by CdiA from NF85 and ULA27, are inducible upon ß-lactam challenge, or if they are constitutively expressed, this is at a high level, as is common for strains carrying inducible ß-lactamases that have mutated to overexpress them.4 In contrast, the previously reported C. koseri ß-lactamase was said to be constitutively expressed at low levels.7 An apparently less significant difference noted was that the pI of the C. diversus CdiA enzymes was determined experimentally as just under 7.0, but that of the C. koseri enzyme as around 5.0.7 Given that all these isolates are now known as C. koseri, such a pI difference implies, at the very least, sequence heterogeneity at the ß-lactamase locus in this species. More recently, a group of biochemically confirmed C. koseri isolates have been shown to have a low-level constitutively expressed, acidic ß-lactamase.9 Using DNA hybridization techniques, the ß-lactamase gene carried by these C. koseri isolates was shown to be significantly different from cdiA.9 The ß-lactamase gene carried by one of these isolates, CK4, has recently been cloned and sequenced (Petrella et al., unpublished data; EMBL accession number AF477396). This gene, named cko, is predicted to encode a class A ß-lactamase sequence, having a pI of 5.0, and is dramatically different (only 40% similarity) from cdiA. To find such a large amount of variation at one chromosomal locus within a species is most unusual. Another possibility, however, is that isolates ULA27 and NF85 are not actually C. koseri isolates. Since they were identified as C. diversus before the separation of C. amalonaticus from C. diversus, it is possible that they are in fact C. amalonaticus, and that the automatic renaming of these isolates as C. koseri by the NCBI database was in error.
Accordingly, the aims of this study were: first to definitively identify isolates NF85 and ULA27 using 16S rRNA sequencing; second, to definitively identify a set of isolates also originally identified as C. diversus by API 20E profiling, and to determine the sequence of the ß-lactamase gene carried by each, and its level of expression.
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Materials and methods |
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Test isolates (see Table 1) were collected from faecal samples of inpatients at Southmead Hospital, Bristol, during 19801984. The identity of each isolate was determined as C. diversus using API 20E test strips (bioMérieux, La Balme les Grottes, France) at the time of isolation, and they were stored as glycerol stocks at 70°C. Isolates ULA27 and NF857 were also used. Bacteria were grown at 37°C in air using nutrient broth and nutrient agar (Oxoid, Basingstoke, UK). Nitrocefin was purchased from Becton Dickinson (Cockeysville, MD, USA). PCR primers were purchased from Sigma-Genosys Ltd (Pampisford, UK). All other reagents were from Sigma Chemical Co. or BDH, both of Poole, UK.
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16S rRNA and ß-lactamase gene-specific PCRs were used, with genomic DNA from boiled colonies and the method described previously,10 with primers, CKO +ve (5'-ATGAGAAACGAGGAAGTCAT-3') and CKO ve (5'TTAATCATAGACTGCGAGTG-3') for amplification of the entire C. koseri CK4 cko ß-lactamase gene (903 bp) (EMBL accession number AF477396); primers CDIA +ve (5'-ACAGGTCCAACA- AAAGCTGG-3') and CDIA ve (5'-GTTTTATCGCCAACCACCCA-3') for amplification of a 650 bp internal fragment of the C. diversus NF85 cdiA ß-lactamase gene;4 primers rRNA +ve (5'-TCAGATTTG- AACGCTGGCGGCA-3') and rRNA ve (5'-CGTATTACCGCGGCTGCTGCCAC-3') for amplification of a 500 bp hypervariable region from the 16S rRNA gene. All PCR products were cleaned and sequenced as described previously.10 DNA sequence analysis, alignment and phylogenetic mapping was performed using the suite of programs, Lasergene (DNA-Star, Madison, WI, USA). For the phylogenetic analysis, an alignment was produced using the CLUSTAL W algorithm, applying a BLOSUM matrix with a gap-opening penalty of 10 and a gap-extension penalty of 0.1. The resultant alignment was analysed using a maximal likelihood method with the standard parameters in Lasergene.
Induction of ß-lactamase expression, isolation and assay of ß-lactamases
Induction of ß-lactamase expression was attempted using nutrient broth-grown cells by adding cefoxitin (10 mg/L) for 2 h to cultures at a starting density of 0.6 OD600. Crude cell extracts were then prepared and hydrolysis of nitrocefin was examined by spectrophotometric analysis, as previously described,10 measuring product formation at 482 nm and using an extinction coefficient of 17 400 AU/M/cm. The protein concentration of each bacterial extract was determined using the Bio-Rad protein assay reagent (Bio-Rad, Munich, Germany) according to the manufacturers instructions.
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Results |
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To investigate the hypothesis that cdiA is a C. amalonaticus-specific ß-lactamase gene, and that cko is a C. koseri-specific ß-lactamase gene, we used PCR with primers specific for an internal portion of cdiA, or for the entire cko gene, together with stringent annealing conditions. In fact, five isolates gave appropriately sized amplicons (650 bp) with the cdiA primers, and four gave amplicons of the correct size (903 bp) with the cko primers (not shown). All four C. amalonaticus isolates were positive for cdiA and the sequences were >99% identical across the region sequenced, and all four C. koseri isolates were positive for cko, with the sequences being >97% identical. The isolate that could not be identified by 16S rRNA sequencing, C37 (Figure 1a), gave an amplicon with the cdiA primers, but the sequence was only 85% identical to those of the other cdiA amplicons. These sequence homologies are represented in the phylogenetic tree drawn in Figure 1(b), which also includes the database Citrobacter spp. class A ß-lactamase sequences. This analysis confirms that the ß-lactamase genes carried by the test isolates fall into two major groups, with one group clustering around the C. koseri CK4 cko, and one clustering around the C. amalonaticus NF85 cdiA. There is only 40% identity between these two clusters. The variant cdiA sequence from isolate C37 does not cluster more closely with any other known Citrobacter spp. ß-lactamase sequence than with cdiA (Figure 1b).
Previously, it has been reported that CdiA-producing isolates like ULA27 and NF85 produce the enzyme inducibly, or, if the induction system is mutated, then at high levels4,7 and that Cko-producing isolates like CK4, do so constitutively at low levels.9 To test whether the level/inducibility of ß-lactamase production is generally consistent with the ß-lactamase gene carried (and so the actual Citrobacter species), ß-lactamase induction was attempted in all nine test clinical Citrobacter spp. isolates, and ß-lactamase production was quantified as the ability of cell extracts to hydrolyse nitrocefin (Table 1). All the isolates that have a cdiA-like ß-lactamase gene (including isolate C37) express an inducible enzyme, except isolate C13, which expresses the enzyme constitutively at high levels (Table 1). Of the four isolates that have a cko ß-lactamase gene, three produce a ß-lactamase enzyme constitutively at low levels, and one, C45, produces an enzyme constitutively at high levels (Table 1). This high-level nitrocefin hydrolysing activity in isolate C45, was found by PCR to be due to the presence of an acquired TEM-1 ß-lactamase, whereas no evidence of an acquired ß-lactamase was found for isolate C13 (data not shown).
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Discussion |
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The gene encoding the low-level constitutively expressed ß-lactamase from C. koseri isolate CK4, cko, was cloned and sequenced by Petrella et al., and the sequence has been deposited on the EMBL database (accession number AF477396). Using this unpublished sequence, we have designed PCR primers, and have amplified a homologue (<3% heterogeneity) from four definitively identified C. koseri isolates. The cko gene is only 40% identical to cdiA. We await with interest the publication of the full sequence of Cko, and its biochemical characterization.
The data presented here show that at least two nucleotide sequence database entries (cdiA from isolates NF85 and ULA27) have been erroneously altered via the NCBI taxonomy database as coming from C. koseri, because it was incorrectly assumed that all C. diversus isolates (the previous identification of NF85 and ULA27)7 have biochemical profiles comparable with C. koseri. The reason for this error is that some isolates were identified as C. diversus prior to the acceptance of C. amalonaticus as a species, at a time when malonate utilization was known to be variable for C. diversus isolates.2 Thus nothing about the malonate-utilization status of such isolates should be assumed from their prior classification as C. diversus. In fact, we have confirmed here that both NF85 and ULA27 are C. amalonaticus. Accordingly, all previous work using NF85 and ULA27, of which much has been published, should be reinterpreted in the light of this error in taxonomy.
Given the biochemical variability of the genus Citrobacter,1 and the difficulty that commercially available systems have in identifying different Citrobacter spp., simple molecular tests may be of considerable value in the diagnostic laboratory. Of course, it is already possible to employ rRNA gene sequencing to identify Citrobacter spp. definitively, as we have done here, but there would be a considerable cost and time advantage if a binary PCR could be developed, which was specific for each species. Data presented here suggest that ß-lactamase gene-specific PCR might be suitable for differentiating C. koseri and C. amalonaticus, which are difficult to differentiate biochemically.1 However, we do not propose that the specific PCR methodology employed here is suitable for such a test, not least because the cdiA PCR does not differentiate between C. amalonaticus cdiA and the homologue carried by isolate C37, which is 15% different.
Indeed, a final, unexpected point to come out of this work is the discovery of this novel partial ß-lactamase gene sequence. Fifteen percent divergence between Citrobacter ß-lactamase genes is a significant degree of heterogeneity given that Citrobacter sedlakii sedA is 20% different from C. amalonaticus cdiA.9 In terms of API 20E profile, isolate C37 is currently identified as Citrobacter youngae, but its 16S rRNA gene sequence does not agree with this classification, and does not allow definitive identification of the isolate. All these data point to the possibility that isolate C37 represents a new Citrobacter species, although this remains to be seen.
Acknowledgements
We thank Professor Alasdair MacGowan, Department of Microbiology, Southmead Hospital, Bristol, UK, for providing the clinical isolates, Jenny Douthwaite, Department of Biochemistry, University of Bristol, for DNA sequencing and Dr Amy Smith, bioMérieux, Inc., for help with analysis of API 20E profiles.
ß-Lactamase research at the Bristol Centre for Antimicrobial Research and Evaluation is funded by the British Society for Antimicrobial Chemotherapy and the Biotechnology and Biological Sciences Research Council.
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Footnotes |
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References |
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2 . Ewing, W. H. & Davis, B. R. (1972). Biochemical characterization of Citrobacter diversus (Burkey) Werkman and Gillen and designation of the neotype strain. International Journal of Systematic Bacteriology 22, 128.
3 . Lindberg, F. P. & Normark, S. (1986). Sequence of the Citrobacter freundii OS60 ampC ß-lactamase gene. European Journal of Biochemistry 156, 4415.[Abstract]
4 . Jones, M. E. & Bennett, P. M. (1995). Inducible expression of the chromosomal cdiA from Citrobacter diversus NF85, encoding an ambler class A ß-lactamase, is under similar genetic control to the chromosomal ampC, encoding an ambler class C enzyme, from Citrobacter freundii OS60. Microbial Drug Resistance 1, 28591.[ISI][Medline]
5 . Perilli, M., Franceschini, N., Segatore, B. et al. (1991). Cloning and nucleotide sequencing of the gene encoding the ß-lactamase from Citrobacter diversus. FEMS Microbiology Letters 67, 7984.[CrossRef][ISI][Medline]
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8 . Anonymous. (1993). Judicial opinion 67: rejection of the name Citrobacter diversus Werkman and Gillen 1932. International Journal of Systematic Bacteriology 43, 392.
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Petrella, S., Clermont, D., Casin, I. et al. (2001). Novel class A ß-lactamase Sed-1 from Citrobacter sedlakii: genetic diversity of ß-lactamases within the Citrobacter genus. Antimicrobial Agents and Chemotherapy 45, 228798.
10 . Avison, M. B., von Heldreich, C. J., Higgins, C. S. et al. (2001). A TEM-2 ß-lactamase encoded on an active Tn1-like transposon in the genome of a clinical isolate of Stenotrophomonas maltophilia. Journal of Antimicrobial Chemotherapy 46, 87984.[ISI]