Division of Microbiology and PHLS Laboratory, University Hospital, Queen':s Medical Centre, Nottingham NG7 2UH, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cfiA gene may be `silent' or expressed to various degrees resulting in a wide range of levels of carbapenem resistance. 3 Most isolates appear sensitive to carbapenems on conventional testing, 4 but some can convert to high-level resistance following antibiotic pressure. 3,5 We have examined the prevalence, copy number and degree of expression of the cfiA gene among clinical isolates of B. fragilis in Nottingham.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One hundred and seventy-five randomly selected, clinically significant B. fragilis isolates were collected during 1996 and 1997 from Nottingham PHL. Isolates were identified by conventional testing or the Rapid ID 32 A system (bioMérieux, Basingstoke, UK). B. fragilis NCTC 9344 (carbapenem-sensitive) and B. fragilis TAL3636 (metallo-ß-lactamase-producer 6) were included as controls.
Antibiotic titrations
Antibiotics were incorporated into Brain Heart Infusion agar supplemented with yeast extract (5 g/L), haemin (5 mg/L) and menadione (1 mg/L). Inocula of 106 organisms were delivered on to the agar surface and the MIC was taken as the lowest antibiotic concentration to inhibit growth completely after 48 h of incubation at 37°C in an anaerobic cabinet.
Detection of the cfiA gene
PCR was used to screen isolates for the cfiA gene. Supernates of bacterial cells boiled in water were used as tem-plate DNA. Reaction mixtures each contained 5 µL of 10x reaction buffer (Boehringer Mannheim Lewis, UK), 1 µL (2.5 mM) each of dATP, dCTP, dGTP and dTTP (Boehringer Mannheim), 1 µL (35 pmoles) of each primer, 5 µL of DNA preparation, 33 µL of sterile water and 1 U Taq polymerase (Boehringer Mannheim). The primers were 5'-TCCATGCTTTTCCCTGTCGCAGTTAT (sequence of nucleotides 557582) and 5'-GGGCTATG G CTTTGAAGTGC (the complementary sequence of nucleotides 1266- 1285). 2 The reactions were incubated for 40 cycles in a programmable heating block (Techne, Duxford, UK) for 1 min at 92°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 and their sizes were compared with those of HaeIII digest markers (NBL Gene Sciences, Cramlington, UK) and the product generated from B. fragilis TAL3636.
Southern blotting and hybridization with a cfiA-specific probe were used to confirm the PCR results and to determine the copy number of the cfiA gene. The protocol described by Ausubel et al. 7 was used. Isolated genomic DNA was digested with the restriction enzymes EcoRI or AvaI (Boehringer Mannheim), electrophoresed and transferred to a Hybond N+ membrane (Boehringer Mannheim) and probed with a digoxigenin-labelled probe obtained by random priming of the 700 bp fragment from B. fragilis TAL3636. Hybridization was performed at 42°C overnight and bound probe was visualized with CDP star (Boehringer Mannheim) and exposure to a Kodak X-OMAT film (Sigma, Poole, UK).
Biological assay
Iso-Sensitest agar (Unipath, Basingstoke, UK) was inoculated with the indicator strain Escherichia coli NCTC 10418 to achieve semi-confluent growth. Cell extracts were prepared as previously described, 4 freeze dried and reconstituted in buffer to provide four-fold concentrates. Concentrated cell extracts (30 µL), imipenem 25 mg/L (10 µL, final concentration of 5 mg/L) and phosphate buffer pH 7.0 (10 µL) were dispensed into wells in the agar. After aerobic incubation at 37°C for 24 h, the diameters of zones of inhibition were compared with those of wells containing cell extract of B. fragilis NCTC 9344, and with the buffer replaced with EDTA (final concentration 2 mM).
Spectrophotometry
Metallo-ß-lactamase activity was detected by a change in absorbance at 299 nm of a mixture of concentrated cell extract (0.2 mL), imipenem (0.2 mL; 250 mg/L) and phosphate buffer pH 7.0 (0.6 mL) with and without EDTA (final concentration 2 mM) over 1 h at 37°C. Specific activities (nmol imipenem hydrolysed/min/mg protein) were calculated from changes in absorbance and the protein content of the cell extracts was measured with a Sigma protein assay kit.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The 700 bp cfiA gene was detected in 11 isolates by PCR; an additional strain, B. fragilis Q8, showed products of c. 500 bp (Figure 1a). Southern blotting revealed the presence of single copies of the gene in all these 12 strains (Figure 1b).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ß-Lactamase activity among cfiA-positive isolates did not appear to be dependent on the gene copy number as all the isolates tested possessed only one copy. Podglajen et al. 9 identified three strains with multiple cfiA gene copies among 11 cfiA-positive strains, although the copy number showed no correlation with the degree of resistance.
Metallo-ß-lactamase activity was not detected in one third of the cfiA-positive isolates, indicating that the cfiA status is a more reliable indicator of these problem strains. The prevalence of carbapenemase production (4.6%) has not risen markedly in Nottingham since 1985, when carbapenemases were detected in 3% of B. fragilis isolates. 1
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 258493.[ISI][Medline]
3 . Podglajen, I., Breuil, J., Bordon, F., Gutmann, L. & Collatz, E. (1992). A silent carbapenemase gene in strains of Bacteroides fragilis can be expressed after a one-step mutation. FEMS Microbiology Letters 91, 2130.[ISI]
4 . Edwards, R. & Greenwood, D. (1992). An investigation of ß-lactamases from clinical isolates of Bacteroides species. Journal of Medical Microbiology 36, 8995.[Abstract]
5 . 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, 12757.[ISI][Medline]
6 . Cuchural, G. J., Malamy, M. H. & Tally, F. P. (1986). ß-Lactamase-mediated imipenem resistance in Bacteroides fragilis. Antimicrobial Agents and Chemotherapy 30,645 8.[ISI][Medline]
7 . Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. et al. (1994). Current Protocols in Molecular Biology . Greene Publishing Associates and Wiley Interscience, New York.
8 . 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, 52705.[Abstract]
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, 10514.[ISI][Medline]
10 . Bandoh, K., Ueno, K., Watanabe, K. & Kato, N. (1993). Susceptibility patterns and resistance to imipenem in the Bacteroides fragilis group species in Japan: a 4-year study. Clinical Infectious Diseases 16 , Suppl. A, S3826.[ISI][Medline]
Received 29 May 1998; returned 6 July 1998; revised 28 July 1998; accepted 21 September 1998