Bristol Centre for Antimicrobial Research and Evaluation, Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
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Abstract |
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Introduction |
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Initially categorized as a member of the genus Achromobacter, DNADNA hybridization and 16S sequencing data led to the foundation of the genus Ochrobactrum in 1988 and its partitioning within the Rhizobiaceae group. This group includes plant and animal intracellular pathogens such as Agrobacterium spp., Bartonella spp. and Brucella spp.5,6 Among all these bacteria, O. anthropi is most closely related to the Brucellaceae, and recent reports have described immunological cross-reactivity between Brucella spp. and O. anthropi antigens.7,8 However, because O. anthropi constitutes a heterogeneous group of bacteria on the basis of phenotypic characterization and DNADNA hybridization studies, further subdivision of the genus into two species, O. anthropi and Ochrobactrum intermedium has recently been proposed, with O. intermedium being more closely related to Brucella spp.9
In general, O. anthropi strains are reported to be resistant to cephalosporins and penicillins, but susceptible to carbapenems, quinolones and tetracyclines.2,4 Nothing has been reported previously concerning the mechanism of ß-lactam resistance in O. anthropi. The O. anthropi isolates we have studied come from three oncology patients with bacteraemias that recurred following courses of piperacillin/tazobactam and ceftazidime therapy. The antimicrobial susceptibilities of the isolates were determined, and the production of ß-lactamase was investigated as a possible cause of ß-lactam resistance.
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Materials and methods |
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Clinical isolates of O. anthropi were recovered over a period of several years from blood cultures of bacteraemic oncology patients being treated at the Bristol Royal Infirmary (BRI). Isolates were plated on to nutrient agar (Oxoid, Basingstoke, UK) to check culture purity and the identity of each was confirmed using API 20NE test strips (bioMérieux, La Balme les Grottes, France). Bacteria were grown at 37°C in air, unless otherwise stated. Bacterial strains and plasmids used in this study are listed in Table I.
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Unless otherwise stated, media used were nutrient broth and nutrient agar (Oxoid). ß-Lactams used were nitrocefin (Beckton Dickinson, Cockeysville, MD, USA); clavulanic acid and BRL 42715 (SmithKline Beecham, Worthing, UK); ampicillin and cephaloridine (Sigma Chemical Co., St Louis, MO, USA); ceftazidime (Glaxo Laboratories Ltd, Greenford, UK); piperacillin (Lederle, Carolina, Puerto Rico); and meropenem (Zeneca Pharmaceuticals, Macclesfield, UK). 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
Antibiotic susceptibility was determined by Etest (AB Biodisk, Solna, Sweden) on IsoSensitest agar (Oxoid) with an inoculum of 0.5 McFarland. The MIC of the test antibiotic was defined as the lowest concentration of the antibiotic that prevented growth after incubation at 37°C for 24 h.
Induction of ß-lactamase expression, isolation and assay of ß-lactamases
Induction of ß-lactamase expression was attempted and crude cell extracts were prepared as described previously.10 Hydrolysis of ß-lactam antibiotics was examined by spectrophotometric analysis as described previously10 at the following wavelengths: 233, 265, 265, 299, 482 and 233 nm, and using the following extinction coefficients: 809, 6980, 9000, 2500, 17400 and 936 Au/M/cm, respectively, for ampicillin, cephaloridine, ceftazidime, meropenem, nitrocefin and piperacillin. The protein concentration of each bacterial extract was determined using the Bio-Rad protein assay reagent (Bio-Rad, München, Germany) according to the manufacturer's instructions. One unit of ß-lactamase activity was defined as that required to hydrolyse 1 nmol of substrate per min at 25°C. Specific activity was defined as the number of units of ß-lactamase per mg of protein in the cell extract.
Isoelectric focusing
Ten micrograms of total protein from each bacterial extract (above) were resolved by isoelectric focusing (IEF) gel electrophoresis, and ß-lactamase(s) was visualized as described previously.11
Cloning the O. anthropi ampRampC locus
Chromosomal DNA was prepared from O. anthropi N217I and general molecular biology techniques were performed as described previously.12 DNA was restricted with EcoRI, ligated into appropriately restricted pSU1813 and used to transform Escherichia coli strain UB5252,10 by electrotransformation with a Gene Pulser (Bio-Rad) set at 2.5 V, 25 µF and 200 . UB5252 is a permeability mutant of E. coli HfrH, which is hypo-susceptible to ß-lactams, allowing easier cloning of ß-lactamase genes that are poorly expressed in E. coli. Transformed bacteria were selected on agar containing chloramphenicol and cephaloridine (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.12
Polymerase chain reaction
Genomic polymerase chain reaction (PCR) was performed as described previously.14 The primers used to amplify O. anthropi ampC were (forward) 5'-GAGATGCGGTCGAACCAT-3' and (reverse) 5'-CTGACTGAACTGCTGCCG-3'. The primers were designed to span the entire coding sequence of the ampC gene. PCR products were purified using a QIAquick PCR purification kit (Qiagen Ltd, Crawley, UK) according to the manufacturer's instructions and sequenced on both strands using the PCR primers to initiate sequencing.
In silico sequence analysis
Computer-assisted sequence manipulation and basic alignments were done with the Lasergene software package (DNA star, Madison, WI). Interrogation of the NCBI microbial BLAST database with O. anthropi AmpC protein sequence was carried out using the tblastn search tool.15 Positive M13 random contigs (average size c. 600 bp) from the Sinorhizobium meliloti genome database were aligned in Lasergene using Seqman to produce a contiguous sequence for the ampR and ampC genes. Sequence data for S. meliloti were generated by S. R. Long and her colleagues at the Department of Biological Sciences, Howard Hughes Medical Institute and the Stanford DNA Sequencing and Technology Centre.
For the phylogenetic analysis, an alignment of published AmpC protein sequences was produced using the CLUSTAL W program16 applying the BLOSUM matrix with a gap-opening penalty of 10 and a gap-extension penalty of 0.1. The resulting alignment was analysed using a maximum likelihood method17 with the program protml18 and the Jones, Taylor, Thornton substitution model.19
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Results |
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Initially, the MICs of a variety of ß-lactams for the three O. anthropi clinical isolates were determined. The three isolates were resistant (according to BSAC breakpoints for the Enterobacteriaceae20) to all cephalosporins and penicillins tested (including four combinations of ß-lactam plus clavulanic acid or tazobactam), and aztreonam, but were susceptible to carbapenems (Table II).
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IEF using induced cell extracts revealed only one ß-lactamase band (pI = 9.0) following nitrocefin staining (data not shown). The enzyme appeared to be a strict cephalosporinase, as induced cell extracts hydrolysed nitrocefin, cephaloridine and ceftazidime, but not meropenem, ampicillin or piperacillin (Table III). The ß-lactamase activity (nitrocefin hydrolysis) was completely inhibited by the broad-spectrum serine ß-lactamase inhibitor, BRL 42715 (10 µM), but not by clavulanic acid (10 µM) or EDTA (50 mM) (Table III
). Almost identical results were obtained when analysing the other two clinical O. anthropi strains (data not shown).
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The ß-lactamase gene from O. anthropi isolate N217I was cloned into E. coli UB5252 following the construction of an EcoRI genebank of N217I genomic DNA using cloning vector pSU18. Recombinants were selected with chloramphenicol and cephaloridine (both at 30 mg/L) and all contained pSU18 carrying a 4.8 kb insert. One such plasmid recombinant was designated pUB6052. E. coli UB5252 containing pUB6052 was found to have decreased susceptibility to cefotaxime and ceftazidime, but not to ampicillin or meropenem, when compared with untransformed UB5252 (Table IV). Extracts of UB5252:pUB6052 hydrolysed cephaloridine, but not meropenem or ampicillin (Table IV
). Finally, IEF analysis showed the cloned ß- lactamase had a pI of 9.0, indistinguishable from that expressed by O. anthropi strain N217I (data not shown).
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The O. anthropi N217I ampC gene extends for 1173 nucleotides, encoding a polypeptide of 390 amino acids. A credible ribosome binding site is located 15 nucleotides upstream from the initiation codon, and 16 nucleotides downstream of the gene there is an inverted repeat followed by a poly(T) run, which is likely to be a Rho-independent transcription terminator.22 No obvious promoter with significant homology to the 70 promoter of E. coli has been identified for ampC, though there are a number of excellent putative 10 boxes (Figure 1
).23 A credible 25 amino acid secretion leader peptide24 in AmpC is defined by alanine residues at positions 25 and 26. The mature 365 amino acid protein has a predicted molecular weight of 39.6 kDa and a predicted pI of 9.0 (the same as that determined experimentally).
PCR primers were designed from the N217I ampC sequence and used to amplify the gene from the other two clinical O. anthropi isolates. The PCR amplicons were sequenced to assess the level of sequence heterogeneity amongst the O. anthropi ampC genes. In fact, all three sequences were identical. Importantly, this also demonstrates that the cloned ß-lactamase gene is from O. anthropi.
Analysis of O. anthropi AmpR and the mechanism of ampC induction
The 193 bp ampRampC intercistronic region contains a putative 10 box for ampR, but no recognizable 35 box (Figure 1).23 Having shown that the O. anthropi ampC is inducible, we measured the inducibility of an E. coli construct containing pUB6052 (ampRampC). Following cefoxitin challenge at sub-MIC levels, an increase in ß- lactamase activity was observed (Table IV
) in extracts of E. coli carrying pUB6052. In addition, cloning of pUB6052 into the ampD mutant, JRG582 (which has high basal levels of the enterobacterial AmpR signal25) resulted in significantly higher ß-lactamase activity than those in HfrH,26 the parent of JRG582,25 containing pUB6052 (Table IV
).
The presence of AmpC ß-lactamases in other members of the -subdivision
The O. anthropi AmpC represents the first AmpC ß- lactamase to be found outside of the -subdivision of the bacterial kingdom. We used the O. anthropi AmpC amino acid sequence to search for homologues in genome sequences from bacteria that are not part of the
-subdivision. Only one homologue was found, in S. meliloti. In this case, the ampC gene is also downstream of an ampR homologue (data not shown). We have not determined whether the S. meliloti ampC gene encodes an active ß-lactamase, or if its expression is inducible. The two novel AmpC amino acid sequences were aligned with all previously published examples of chromosomal AmpC-type ß-lactamases (Figure 2
). This information was used to construct a phylogenetic tree for chromosomal AmpC enzymes (Figure 3
).
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Discussion |
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The multiple alignment of class C AmpC ß-lactamases shown in Figure 2 reveals a large degree of sequence heterogeneity. This variation has come about through divergent evolution, and the extent of this variation implies that the ancestral ampC gene was acquired a considerable time ago. There are regions within the AmpC proteins with a high degree of sequence conservation, however, and upon mapping these conserved regions on to known AmpC crystal structures,28 all can be associated with recognizable structural motifs within the enzyme, so a high degree of homology is unsurprising. There is likely to be strong selective pressure on these proteins to maintain the integrity of the active site and hence the individual motifs stabilizing that site. Other, less important, regions change more freely, however.
The phylogenetic analysis (Figure 3) of the AmpC proteins clearly places O. anthropi AmpC within the same group as the predicted AmpC from another
-proteobacterium, S. meliloti. As this analysis produces an unrooted tree (Figure 3
), it is not possible to say with certainty what the ancestral AmpC sequence was. More AmpC sequence data from organisms outside the
-subdivision will help clarify this situation. However, our current study does indicate that AmpC enzymes evolved before the divergence of the
and
groups, which is more evidence that the acquisition of ampC is an ancient event, and occurred millennia before the first clinical use of ß-lactams.
Whilst the -subdivision of the bacterial kingdom contains most of the common animal pathogens, the
- subdivision is composed mainly of environmental organisms and plant symbionts such as S. meliloti. Clearly, in these latter organisms, the selective pressure of clinical ß-lactam use has not maintained the presence of AmpC. The true role of AmpC and other ß-lactamases is unknown, though they could have evolved to protect cells against ß-lactams in the environment. It is just as likely that AmpC enzymes serve a useful physiological purpose, however. That production of AmpC is inducible argues that the enzyme is useful to the cell during specific circumstances, rather than all the time. It was recently reported that the AmpC ß-lactamase of E. cloacae displays d,d-carboxypeptidase activity, suggestive of a role in peptidoglycan metabolism.29 This correlates well with the AmpC induction process being associated with peptidoglycan turnover.21
The greatest problem of clinical antibiotic resistance is caused by acquired ß-lactamases, usually because these are expressed at high levels.30 In contrast, inducible ß-lactamases rarely cause clinical resistance, though mutations that cause ß-lactamase overexpression can lead to therapeutic failure.30 It would seem strange to evolve a sophisticated induction mechanism to bring about ß-lactam resistance, if the only way resistance occurs is following mutation of the induction machinery. Clearly, however, the potential to produce any ß-lactamase is an advantage for a pathogen that comes into contact with ß-lactams. O. anthropi is such an organism, and it may be that its intrinsic antibiotic resistance mechanisms are part of the reason for its recent rise. O. anthropi is categorized amongst the Brucellaceae, and antigenic cross-reactivity between O. anthropi and Brucella abortus has been noted.79 Interestingly, a recent report showed that Brucella spp. are resistant to cephalosporins during growth in nutrient broth or on nutrient agar.31 It is therefore possible that these organisms also encode an ampC ß-lactamase.
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Acknowledgments |
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Notes |
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References |
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Received 18 December 2000; returned 22 February 2001; revised 1 March 2001; accepted 2 March 2001