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 5 September 2003; returned 16 November 2003; revised 18 December 2003; accepted 23 January 2004
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Abstract |
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Methods: ß-Lactamase gene-containing recombinant plasmids transformed into Escherichia coli were selected using ampicillin. PCR analysis was used to locate specific ampC and 16S rRNA genes, and the amplicons were sequenced. Random amplified polymorphic DNA PCR was used to group isolates and API 20E biochemical profiling was used to identify them putatively.
Results: Of 50 ceftazidime-resistant clinical Enterobacteriaceae isolates, 36 were identified (>95% confidence)using API 20E test stripsas being organisms known to express inducible class C ß-lactamases (Citrobacter freundii, Enterobacter cloacae, Morganella morganii or Hafnia alvei). The rest were biochemically atypical. Of these, isolate I113, putatively identified as E. coli, possesses a chromosomally encoded ampC which differs by 15% from C. freundii OS60 ampC and by >30% from E. coli ampC. A related ampC gene was found in another seven of the atypical isolates. The use of various identification methods, including ampC sequence analysis, revealed that these I113-like ampC-positive isolates represent Citrobacter murliniae and Citrobacter youngae.
Conclusions: We report sequences for two new Citrobacter spp. ampC genes, and provide evidence that ampC sequencing is a discriminatory method for identifying atypical Citrobacter spp. isolates.
Keywords: Citrobacter, lactamases, overexpression, ceftazidime resistance, phylogeny
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Introduction |
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The C. koseri LysR-type transcriptional regulator protein, CdiR, which is divergently expressed from cdiA, has also been characterized, and its mode of action is thought to be identical to the well studied C. freundii AmpC regulator, AmpR.3,5 Consistent with this same mode of action, AmpR and CdiR are substantially more homologous than are the ß-lactamases whose expression they regulate.5 More recently, CdiA homologues from Citrobacter sedlakii and Citrobacter rodentium have been characterized. There is up to 40% divergence between these three class A ß-lactamases.6 The variability of class C ß-lactamases in the genus Citrobacter has been examined by PCR/hybridization experiments, which suggested that there is a similar variability to that seen with class A enzymes,7 and a recent paper, describing the sequences of ampC genes from Citrobacter braakii, Citrobacter murliniae and Citrobacter werkmanii, confirmed this to be the case.8
Even though 11 Citrobacter species have been described, there is much heterogeneity among phenotypic and genotypic parameters within several of these species.1,2 This makes identifying Citrobacter species difficult, with many isolates being identified as atypical or being identified incorrectly.9 The aim of this study was to look at ß-lactamase gene sequences in biochemically atypical (mainly Citrobacter spp.) isolates collected during routine surveillance of members of the Enterobacteriaceae at a Bristol childrens hospital to determine whether, as has been previously suggested,7 ß-lactamase gene sequencing might be a more reliable method for identifying Citrobacter spp. than conventional methods.9
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Materials and methods |
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Isolates were collected from faecal samples of oncology patients undergoing chemotherapy at the Bristol Childrens Hospital during 19821991. The criteria for including isolates in this study were that they were identified as members of the Enterobacteriaceae, and were resistant, according to disc susceptibility testing,10 to ceftazidime. The identity of each isolate was determined using API 20E test strips (bioMérieux, La Balme les Grottes, France) at the time of isolation, and their identities were confirmed using the same method at intervals during the 20 years that this project has been running. Some isolates were also subjected to much more detailed biochemical analysis using standard methods.2 Bacteria were grown at 37°C in air using, unless otherwise stated, nutrient broth and nutrient agar (Oxoid, Basingstoke, UK). The recipient for recombinant plasmids containing cloned Citrobacter sp. I113 ampR-ampC was Escherichia coli SN03 (ampA1, ampC8, recA, pyrB, thi), an AmpC hypoproducer,11 which was also used as a control for a number of other studies, along with C. freundii OS60.4,12 Recombinant plasmid pNU305, carrying the inducible OS60 ß-lactamase with its associated ampR regulator gene, was also used in control experiments.12
Materials
Nitrocefin was purchased from Becton Dickinson (Cockeysville, MD, USA). PCR primers were purchased from Sigma-Genosys Ltd (Pampisford, UK). General reagents for DNA manipulation were obtained from GIBCO-BRL (Life Technology Ltd, Paisley, UK). All other general reagents were from Sigma Chemical Co., or BDH, both of Poole, Dorset, UK.
Susceptibility tests
All susceptibility data were determined using Iso-Sensitest agar (Oxoid). MIC values were quantified using Etest strips (AB Biodisk, Solna, Sweden) with an inoculum with a turbidity equivalent to that of a 0.5 McFarland standard following incubation at 37°C for 24 h.
Induction of ß-lactamase expression, isolation and assay of ß-lactamases and isoelectric focusing
Induction of ß-lactamase expression was attempted, crude cell extracts were prepared and hydrolysis of nitrocefin was examined by spectrophotometric analysis, as previously described.13 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. Isoelectric focusing of proteins in crude cell extracts and staining of ß-lactamase bands with nitrocefin was performed as described previously.14
Cloning the Citrobacter sp. I113 ampR-ampC locus
Preparation of chromosomal DNA from Citrobacter sp. I113 and general molecular biology techniques were performed as described previously.15 DNA was partially restricted with Sau3A, ligated into BamHI-linearized pK1816 and used to transform E. coli strain SN03,11 by electrotransformation15 with a Gene Pulser (Bio-Rad, Munich, Germany) set at 2.5 kV, 25 µF and 200 . Transformed bacteria were selected on agar containing kanamycin and ampicillin (both at 30 mg/L). Resistant transformants were recovered, plasmid DNA was isolated, and the insert was sequenced using an ABI PRISM 377 automated DNA sequencer. Sequences were determined on both strands using a custom primer walking strategy.15 Citrobacter spp. 16S rRNA gene sequences were downloaded from the EMBL nucleotide sequence database. Computer-assisted sequence manipulation, alignments and phylogenetic trees were made using the Lasergene software package (DNA star, Madison, WI, USA).
PCR protocols
16S rRNA gene-specific PCR was performed using the protocol and primers described previously.17 Colony PCR for the I113 and OS60 ampC genes was performed using the method and primers described previously.7 Random amplified polymorphic DNA (RAPD) PCR was performed using 0.1 µM universal primer 55884 (5'-GTCAATAACGTC AAAAAG-3') in a 50 µL total reaction volume, including 10 µL of clarified cell extracts (produced as described before)7 and a reaction mixture containing 10 mM TrisHCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl, 0.1% w/v gelatin, 0.1% w/v Triton X-100, 200 µM dNTPs and 0.1 units of Super-Taq DNA polymerase (Stratech Scientific, Luton, UK). The PCR program was 5 min 96°C, followed by four cycles of 1 min 96°C, 1 min 26°C and 1 min 72°C; 28 cycles of 1 min 96°C, 1 min 36°C and 1 min 72°C; four cycles of 1 min 96°C, 1 min 50°C and 1 min 72°C, then a 5 min extension step at 72°C.
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Results |
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In total, 50 ceftazidime-resistant Enterobacteriaceae isolates were collected and identified to species level using API 20E test strips immediately following their first culture. Twenty-two (44%) of the isolates were identified (>95% confidence value) as being Enterobacter cloacae, nine (18%) as being C. freundii, four (8%) as being Morganella morganii and one (2%) as being Hafnia alvei. In addition, tentative identifications were made for seven (14%) of the isolates as being Citrobacter spp., five (10%) as being Enterobacter spp. and two (4%) as being E. coli. The 36 (72%) isolates identified definitively come from species known to have the potential to become ceftazidime-resistant, usually following chromosomal mutations that lead to constitutive overproduction of their normally inducible AmpC ß-lactamases,3 so these isolates were not looked at further in this study. Table 1 sets out API 20E profiles for the remaining 14 isolates, which were biochemically atypical. In Table 1, species identifications are those derived from the 1986 issue of the API 20E code book, which was current at the time of their isolation. The significance of this point will be discussed later. MICs of various ß-lactams for these 14 atypical isolates are also reported in Table 1.
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Analysis of ß-lactamase activity produced by atypical Enterobacteriaceae isolates and cloning of a ß-lactamase gene from the putative E. coli isolate, I113
ß-Lactamase activities were measured in cell extracts of the putative E. coli isolates C376 and I113 following growth in the presence or absence of ß-lactam inducer (cefoxitin, 10 mg/L, 2 h) using the chromogenic ß-lactam, nitrocefin. In extracts of both isolates, ß-lactamase activity was readily detected in uninduced cells, and the production of enzyme was induced only a small amount following cefoxitin challenge (Table 2). A similarly high uninduced ß-lactamase activity with only a small induction ratio was seen with the putative C. freundii and Enterobacter agglomerans isolates (Table 2). This contrasts with the much higher induction ratio seen following challenge of the prototypical C. freundii isolate, OS60 with cefoxitin as an induction control. The OS60 induction ratio is higher than those of the clinical isolates, mainly because of a lower uninduced ß-lactamase activity; the induced activity is similar (Table 2). MICs of ß-lactams for OS60 are generally less than those for the clinical isolates, particularly for later generation cephalosporins (Table 1).
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Isoelectric focusing of cell extracts from the two putative E. coli isolates, I113 and C376 followed by staining the gel with nitrocefin, revealed a single ß-lactamase (pI = 8.6) in each extract (not shown). Plasmid preparations proved negative for both isolates, so it was concluded that the gene encoding the ß-lactamase was most likely to be chromosomal in each. Accordingly, chromosomal DNA was used to clone the ß-lactamase gene, initially from isolate I113, into E. coli SN03 (an AmpC hypoproducer). More than one ß-lactamase-encoding insert was identified (as is commonly the case when using Sau3A partial digests), one (carried on recombinant plasmid pUB6066) being 2.0 kb, and another (carried on recombinant plasmid pUB6074) being 3.4 kb. MICs of ß-lactams for E. coli SN03 recombinants carrying pUB6074 were found to be similar to those carrying the C. freundii OS60 ampC on plasmid pNU305 (Table 1).4 E. coli SN03 recombinants carrying pUB6074 expressed ß-lactamase activity that was inducible with cefoxitin, but recombinants carrying pUB6066 did not. In this case, the induction ratio was similar to that seen with the cloned OS60 ampC in the presence of the ampR regulatory gene on plasmid pNU305 (Table 2).4
Both I113 ß-lactamase-encoding inserts were sequenced to reveal a molecular class C ß-lactamase gene, ampC encoded immediately downstream of and oppositely oriented to an ampR gene, predicted to encode a regulator of AmpC expression because it is 89% identical to the C freundii OS60 AmpR.4 The insert of plasmid pUB6074 also carries genes either side of ampR-ampC, frdD and blc, which confirms the same genomic location as is found with ampC from E. coli and C. freundii OS60.4,12 The sequence of the large insert has been deposited in the EMBL database under accession number AJ607409. In the shorter cloned insert, in plasmid pUB6066, ampR is truncated after 238 nucleotides, whereas ampC is present in its full-length form. Given that the shorter construct encodes a ß-lactamase that is not inducible upon ß-lactam challenge (Table 2), this provides evidence for a role of I113 AmpR in AmpC induction.
Analysis of the distribution of I113-like ampC ß-lactamase genes among other atypical isolates collected during this study
Sequence alignment showed that I113 AmpC ß-lactamase is 85% identical to the C. freundii OS60 AmpC, but only 65% identical to E. coli AmpC, yet I113 had been putatively identified by API 20E as E. coli. Furthermore, E. coli AmpC is not inducible, and ampC is not linked to an ampR regulator gene in E. coli,10 as is clearly the case in I113. Accordingly, it was thought likely that isolate I113 had been misidentified as E. coli and probably represented a member of the genus Citrobacter.
I113 ampC-specific PCR primers were used to determine whether any of the other atypical isolates collected in this study (Table 1) carried an ampC gene similar to that from I113. Stringent PCR reactions (annealing temperature, 60°C; MgCl2 concentration, 1.5 mM)7 produced amplicons of the expected size (850 bp) using genomic DNA from isolate I113 as template (as expected), but also from isolates A184, B284, C376, C525, G557, I626 and I715 (Figure 1a). Of the remaining six atypical isolates, threeD571, D643 and G24all identified as atypical C. freundii (Table 1), produced a faint, incorrectly sized PCR amplicon (1 kb) using the I113 PCR primer set (Figure 1a), but produced a strong, correctly sized PCR amplicon (850 bp) using a primer set designed to target the C. freundii OS60 ampC sequence (Figure 1b).7 The remaining isolates, E413, E548 and I684, all putatively identified as E. cloacae (Table 1) were negative in PCR reactions using either Citrobacter spp. ampC-specific primer set (Figure 1). This was expected for true E. cloacae isolates. Control PCR reactions using C. freundii OS60 and E. coli SN03 gave expected results using the ampC-specific primer sets (Figure 1). To confirm the integrity of the templates, and so the validity of negative ampC-specific PCR results in some cases, 16S rRNA gene-specific PCR was performed for all isolates and controls. All yielded products, and sequencing of those from isolates E413, E548 and I684 confirmed them to be E. cloacae (not shown).
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A detailed biochemical profile of multiple atypical C. freundii isolates has led to the definition of several new species.2 Analysis of these data led to the conclusion that isolate I113, as representative of isolates giving RAPD-PCR pattern 1 (Figure 2), most closely matches with C. murliniae (Table 3). Isolate A184, as representative of the four I113 ß-lactamase PCR-positive isolates giving RAPD-PCR pattern 2 (Figure 2), most closely matches with Citrobacter youngae. However, in both cases, some tests were not as expected from published data,2 so identification was not definitive (Table 3). 16S rRNA gene sequencing (Figure 3a) was also not particularly discriminatory. Whereas isolates C376, I113, G557 and I626 (RAPD-PCR pattern 1) are identical to the C. murliniae type strain in the 500 bp (hypervariable) region of the 16S rRNA gene sequenced, agreeing with the biochemical data (Table 3), the EMBL nucleotide database entry for C. youngae 16S rRNA is also identical. In contrast, the four isolates, A184, B284, C525 and I715, of RAPD-PCR pattern 2, which were biochemically identified as C. youngae (Table 3), differ from C. youngae EMBL nucleotide database entry by one nucleotide in the 16S rRNA hypervariable region. The C. freundii type strain has the next closest 16S rRNA homologue to those of the RAPD-PCR pattern 2 isolates, but there are five nucleotide differences, so from this test, C. youngae/C. murliniae is the most appropriate identification for all eight I113-like ampC PCR-positive atypical Citrobacter spp. isolates. The three Citrobacter isolates that were positive for the C. freundii ampC PCR screen (Figure 1b) are also difficult to identify from 16S rRNA gene sequencing. Whereas isolate D571 has a 500 bp 16S rRNA hypervariable region that is identical to the C. freundii type strain, so is clearly C. freundii, isolate G24s 16S rRNA 500 bp hypervariable region differs by two nucleotides from the C. freundii type strain sequence. Even with this two nucleotide difference, however, isolate G24 is most closely related to C. freundii, but isolate D634, whose 16S rRNA 500 bp hypervariable region differs by two nucleotides from the C. freundii type strain sequence, also differs by this amount from C. murliniae and C. youngae, so it is not possible to identify isolate D634 using this test.
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Discussion
The sequence of ampC from Enterobacteriaceae isolate I113 was determined in 1992, and was deposited on the EMBL nucleotide database in 1993 (accession number X76636). We delayed publishing this sequence at the time because we were not able to rationalize the identification of I113 as E. coli using API 20E. In 1999, advances in the taxonomy of Citrobacter spp.2,9 led to this project being restarted, and, through targeted biochemical tests, we became aware that I113 may represent a C. murliniae isolate. At this time, however, there was still doubt of the C. murliniae identification. The reason for our reticence was that very few isolates have been identified as C. murliniae, and so levels of permitted variation in biochemical parameters have not been accurately determined for this species.2 So, whereas the biochemical data pointed to I113 being most closely related to C. murliniae, there are a number of discrepancies with the published biochemical profile, meaning that the isolate could be either C. murliniae, C. freundii or C. youngae. In this situation, DNA sequence is often turned to for true identification, but 16S rRNA sequencing was not entirely helpful because the C. murliniae type strain database entry for 16S rRNA and that from a C. youngae isolate are identical. However, this analysis did confirm that I113 is not C. freundii. It was not until the very recent publication of the C. murliniae type strain ampC sequence8 that we became convinced that this is the correct classification for isolate I113. A similar situation occurred with the other isolates identified as C. murliniae, and those identified as C. youngae, where ampC sequence was the deciding factor for us to definitively identify them. Interestingly, the 16S rRNA gene from the isolates identified by ampC sequencing as C. youngae is one nucleotide different from the C. youngae EMBL nucleotide database 16S rRNA sequence across the 500 bp region used in this analysis. Accordingly, it may be that the EMBL nucleotide database entry is not actually of a C. youngae isolate, but represents C. murliniae, since it is identical to the 16S rRNA gene from the C. murliniae type strain. This is the only 16S rRNA sequence for C. youngae in the EMBL nucleotide database, and is not from the type strain, adding weight to the idea that it has been misidentified. We were not able to find any other C. youngae 16S rRNA gene sequences. Widely used internet rRNA sequence databases, such as the Michigan State University Ribosomal Database Project (http://rdp.cme.msu.edu/html/last accessed by the authors 18 December 2003), only report the EMBL database entry for C. youngae. In contrast to the situation with the 16S rRNA gene, the C. youngae database ampC sequence is from the type strain, so we are confident of the ampC-directed identification of our clinical isolates. Indeed, the most up-to-date API 20E profile book would correctly identify two of the isolates, A184 and I715, as C. youngae with a >99% confidence value, although it would not allow identification of any of the others. Isolates B284, C525, G557 and I626 are all currently identified by API 20E as C. freundii, with confidence values of 69.5%, 94.1%, 86.2% and 86.2%, respectively. The other two isolates, I113 and C376, are currently best identified as C. braakii, but with a confidence value of only 47% for both. It has been documented, using a larger collection of isolates, that API 20E profiling does not distinguish well between these less common Citrobacter species.9
Our long-standing hypothesis, that ß-lactamase gene sequencing might be a useful method for identifying Citrobacter spp. isolates,7 has been given a boost by this study. The advantage of ampC sequencing over 16S rRNA gene sequencing in this case is that more variation is observed between different species at the ampC locus than at the 16S rRNA locus, allowing more definitive identification to species level. Furthermore, sequence differences are such that species-specific PCR primer sets can be made, which, with a simple binary PCR test, not requiring sequencing, can be used to identify isolates.7 Accordingly, we feel that identification protocols involving some aspect of ampC sequence differentiation are very useful for what remains a highly biochemically diverse genus of bacteria.
It would appear that the reason the biochemically atypical Citrobacter spp. and E. cloacae isolates described here appeared following a screen for ceftazidime-resistant Enterobacteriaceae is that they have acquired mutation(s) that led to overproduction of their chromosomal AmpC ß-lactamase. Normally, Citrobacter spp. and Enterobacter spp. having inducible AmpCs are not resistant to ceftazidime.3,8 The majority of mutations previously shown to achieve AmpC overexpression, and so ceftazidime resistance in these organisms, have been mapped to the ampD gene,3 although we have not confirmed that this is the case for the clinical isolates reported here. The dynamic nature of community carriage and environmental prevalence of C. murliniae and C. youngae is not known, but under the correct selective pressure, they can clearly be found in patients. Reported isolations of these species from clinical specimens are rare,2 but it is likely that their presence has been under-reported because of the identification difficulties discussed above. In our study, 8% of ceftazidime-resistant Enterobacteriaceae isolates were confirmed as C. murliniae and 8% as C. youngae.
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Acknowledgements |
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Footnotes |
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References |
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