Expression of the carbapenemase gene (cfiA) in Bacteroides fragilis

R. Edwards* and P. N. Read

Division of Microbiology and PHLS Laboratory, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteroides fragilis strains were studied to examine carbapenemase gene (cfiA) expression and insertion sequence (IS) element promoters. High-level resistance was associated with high meropenemase activity and IS elements upstream of cfiA. Sequencing revealed two element types; IS1187 and elements related to IS942 and IS1170. The latter was implicated in the conversion to carbapenem resistance in a cfiA-positive isolate during imipenem therapy. Two strains showing low-level resistance, and strains susceptible to meropenem, did not possess IS elements upstream of cfiA. The prevalence in Nottingham of clinical isolates of B. fragilis with cfiA and efficient IS element promoters was low (0.6%).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteroides fragilis is an important anaerobic pathogen commonly associated with polymicrobial infections. Carbapenems are normally highly active against B. fragilis because of the stability of these antibiotics to the typical ß-lactamases produced by these organisms. However, a minority of isolates are capable of producing metallo-ß- lactamases which can result in reduced susceptibility or resistance to carbapenems and other ß-lactam antibiotics.1 These carbapenemases are encoded by the cfiA gene.2 High cfiA expression is thought to be caused by insertion sequence (IS) elements, immediately upstream of the gene, carrying efficient promoters.3 Seven IS element types have been reported in B. fragilis; some of these (IS942, IS1186 and IS4351) are associated with cfiA.3,4

This study examined the prevalence, type and efficiency as promoters of IS elements upstream of cfiA among clinical isolates of B. fragilis from Nottingham and other centres.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Twenty-one B. fragilis isolates, previously found to be cfiA positive, were examined. Eighteen of these were obtained from screening clinical isolates from Nottingham Public Health Laboratory between 1986 and 1998,1,5–8 and three strains were isolated in Japan and Spain. The Nottingham isolates included 12 from a cfiA prevalence survey of 175 isolates7 and two obtained from a patient before and during therapy with imipenem8 (TableGo). B. fragilis NCTC9344 (carbapenemase sensitive) and B. fragilis TAL3636 (cfiA positive) were included as control organisms.


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Table. Expression of the cfiA gene and analysis of PCR product upstream of cfiA
 
Determination of MICs

Antibiotics were incorporated in brain–heart infusion agar supplemented with yeast extract (5 g/L), haemin (5 mg/L) and menadione (1 mg/L). Approximately 106 organisms were delivered on to the agar surface using a multipoint inoculator (Denley Instruments, Billingshurst, UK). The MIC was defined as the lowest antibiotic concentration that completely inhibited growth after 48 h of incubation at 37°C in an anaerobic cabinet.

Carbapenemase activity

Carbapenemase activity was detected by the change in absorbance of a mixture of crude cell extract sonicate (0.2 mL), meropenem (0.2 mL of 250 mg/L) and phosphate buffer pH 7.0 (0.6 mL). Measurements were made at 299 nm over 1 h at 37°C. Specific activities (nmol meropenem hydrolysed/min/mg protein) were calculated.

Detection of the cfiA gene

PCR with primers comprising nucleotides 557–582 and the complementary sequence of 1266–1285 were used.2 The reactions were incubated for 35 cycles in a programmable heating block for 1 min at 94°C, 2 min at 50°C and 2 min at 72°C. PCR products were visualized on agarose gels containing ethidium bromide under UV light. Their sizes were compared with those of DNA markers and of the product generated from B. fragilis TAL3636.

Amplification of DNA upstream of cfiA

PCR, using the conditions described above, was also used to amplify DNA upstream of the cfiA genes. One primer comprised the complementary sequence 565–598 within cfiA.2 This was used with one of two upstream primers derived from regions upstream of cfiA: oligonucleotide G9 and sequence 5'-GTTTTGACCGGGAGACCATCCTC-3'. The PCR products were visualized and their sizes estimated as above.

Detection of IS elements

DNA from the amplified region upstream of cfiA was purified using QIAquick PCR Purification Kit (Qiagen, Crawley, UK) and sequenced using the primer within cfiA. The presence and type of IS elements were determined by comparing partial sequences of the insertions with sequences on the GenBank database using the gapped-basic local alignment search tool (BLAST).

Randomly amplified polymorphic DNA (RAPD) analysis

The B. fragilis isolates obtained before and during imipenem therapy8 (isolates 7/5 and P16/16, respectively) were examined by RAPD using primer M13 to ascertain whether they represented the same strain.10 PCR products were visualized on agarose gels containing ethidium bromide and compared.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The presence of the cfiA gene was confirmed in all test strains by the detection of a PCR product of c. 720 bp, identical to that produced from B. fragilis TAL3636. The MICs of meropenem ranged from 0.03 to >256 mg/L. No meropenemase activity was detected with five strains. The remainder showed a range of specific meropenemase activities from 2.7 to 334 nmol meropenem hydrolysed/ min/mg protein (TableGo). A positive correlation was shown between these two sets of values.

Sixteen isolates produced small PCR products for the region upstream of cfiA (c. 300 bp) which did not contain IS elements. All these isolates showed meropenem MICs of <=8 mg/L, with the exception of B. fragilis isolates 57 and 119, for which the meropenem MIC was 16 mg/L. The remaining five strains showed high meropenem MICs (>32 mg/L) and gave larger PCR products upstream of cfiA of 1.5 or 1.9 kb (examples given in the FigureGo) which contained IS elements. B. fragilis isolates T2 and 288.89 possessed IS elements 99 and 74% identical to IS1187, respectively. B. fragilis P16/16, FS and GAI30144 contained sequences showing 67–81% identity with IS942 and IS1170 (TableGo). RAPD analysis showed that the profiles generated by PCR products of B. fragilis isolates 7/5 and P16/16 were indistinguishable.



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Figure. PCR products using primers for the region upstream of cfiA.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolates with cfiA that were highly resistant to meropenem (MIC >= 64 mg/L) all showed high levels of carbapenemase activity and possessed IS elements immediately upstream of the gene. This suggests the presence of efficient promoters on these elements. Two isolates possessed IS elements closely related to IS1187, an element submitted to GenBank (accession number Y18979) by I. Podglajen and E. Collatz, which is associated with B. fragilis. Three strains harboured IS elements upstream of cfiA that were 67–81% identical to the closely related IS942 and IS1170, elements previously reported in B. fragilis to control expression of cfiA and the nitroimidazole resistance gene nim, respectively.3,4 Multiple alignments of sequences revealed that insertions in these strains are more similar to each other than to any previously characterized insertion sequences, including IS942 and IS1170. This suggests that they may represent a novel IS element type.

Two isolates investigated in the study, B. fragilis 7/5 and P16/16, had previously shown evidence of in vivo conversion to carbapenem resistance.8 These were isolated from the same patient before and during imipenem therapy, and it was suggested that they may be two isolates of the same strain in which a switch from reduced sensitivity to clinical resistance had occurred. Evidence from RAPD analysis and sequencing upstream of cfiA suggested that the two isolates were indistinguishable. Therefore, the insertion of an IS942/1170-related element upstream of cfiA was responsible for the conversion to increased carbapenemase production and carbapenem resistance in the isolate obtained during therapy. The presence of this element in the genome of the pre-therapy isolate, B. fragilis 7/5, has yet to be investigated.

Isolates sensitive to meropenem (MIC < 8 mg/L) showed no or weak carbapenemase activity and did not possess IS elements upstream of cfiA. These findings were consistent with those of Podglajen et al.3 The cfiA expression was clearly low in these strains and the absence of IS elements with efficient promoters is to be expected. Some of these strains, however, showed higher meropenem MICs than typical sensitive B. fragilis strains. This, together with detectable carbapenemase activity, indicated the presence of a weak promoter upstream of cfiA in the absence of an IS element. Such expression of cfiA was also apparent in B. fragilis isolates 57 and 119; these strains showed low-level resistance to meropenem (MIC 16 mg/L) and moderate specific meropenemase activity.

The prevalence of IS promoters of cfiA among B. fragilis isolates in Nottingham was assessed from analysis of the 12 cfiA-positive strains that were part of a survey of 175 isolates.7 Only one isolate, B. fragilis T2, was shown to possess a cfiA-associated IS element. This element was 99% identical to IS1187 and associated with meropenem resistance. The prevalence of isolates with IS elements upstream of the cfiA gene was, therefore, low, at one in 175 (0.6%). The remaining 11 cfiA-positive isolates of the 175 survey strains comprise the important sub-group of sensitive strains capable of conversion to high-level carbapenem resistance by the insertion of an IS element upstream of cfiA.8,9

This study highlights the importance of IS elements upstream of cfiA in high-level expression of the cfiA gene. Despite the low prevalence of highly expressed cfiA genes, their significance should not be overlooked. The frequency of occurrence of isolates carrying the cfiA gene should be monitored and, with the development of oral carbapenems, the susceptibility of B. fragilis to carbapenems must not be taken for granted.


    Acknowledgments
 
B. fragilis GAI30144 was kindly donated by Professor K. Watanabe, Institute of Anaerobic Bacteriology, Gifu University School of Medicine, Japan. This study was supported in part by a grant from the British Society for Antimicrobial Chemotherapy. Preliminary findings were presented at the Third European Congress of Chemotherapy, Madrid, Spain, May, 2000.


    Notes
 
* Corresponding author. Tel: +44-115-9709162; Fax: +44-115-9709233; E-mail: richard.edwards{at}nottingham.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Edwards, R. & Greenwood, D. (1992). An investigation of ß- lactamases from clinical isolates of Bacteroides species. Journal of Medical Microbiology 36, 89–95.[Abstract]

2 . Thompson, J. S. & Malamy, M. H. (1990). Sequencing the gene for an imipenem-cefoxitin-hydrolyzing enzyme (CfiA) from Bacteroides fragilis TAL2480 reveals strong similarity between CfiA and Bacillus cereus ß-lactamase II. Journal of Bacteriology 172, 2584–93.[ISI][Medline]

3 . Podglajen, I., Breuil, J., Casin, I. & Collatz, E. (1995). Genotypic identification of two groups within the species Bacteroides fragilis by ribotyping and by analysis of PCR-generated fragment patterns and insertion sequence content. Journal of Bacteriology 177, 5270–5.[Abstract]

4 . Smith, C. J., Tribble, G. D. & Bayley, D. P. (1998). Genetic elements of Bacteroides species: a moving story. Plasmid 40, 12–29.[ISI][Medline]

5 . Eley, A. & Greenwood, D. (1986). Characterization of ß-lactamases in clinical isolates of Bacteroides. Journal of Antimicrobial Chemotherapy 18, 325–33.[Abstract]

6 . Eley, A. & Greenwood, D. (1986) Beta-lactamases of type culture strains of the Bacteroides fragilis group and of strains that hydrolyse cefoxitin, latamoxef and imipenem. Journal of Medical Microbiology 21, 49–57.[Abstract]

7 . Edwards, R., Hawkyard, C. V., Garvey, M. T. & Greenwood, D. (1999) Prevalence and degree of expression of the carbapenemase gene (cfiA) among clinical isolates of Bacteroides fragilis in Nottingham, UK. Journal of Antimicrobial Chemotherapy 43, 273–6.[Abstract/Free Full Text]

8 . Turner, P., Edwards, R., Weston, V., Gazis, A., Ispahani, P. & Greenwood, D. (1995). Simultaneous resistance to metronidazole, co-amoxiclav, and imipenem in clinical isolates of Bacteroides fragilis. Lancet 345, 1275–7.[ISI][Medline]

9 . Podglajen, I., Breuil, J. & Collatz, E. (1994). Insertion of a novel DNA sequence, IS1186, upstream of the silent carbapenemase gene cfiA, promotes expression of carbapenemase resistance in clinical isolates of Bacteroides fragilis. Molecular Microbiology 12, 105–14.[ISI][Medline]

10 . Welsh, J. & McClelland, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18, 7213–8.[Abstract]

Received 31 May 2000; returned 21 August 2000; revised 30 August 2000; accepted 8 September 2000