Journal of Antimicrobial Chemotherapy (1999) 43, 273-276
© 1999 The British Society for Antimicrobial Chemotherapy
Brief reports |
Prevalence and degree of expression of the carbapenemase gene (cfiA) among clinical isolates of Bacteroides fragilis in Nottingham, UK
Division of Microbiology and PHLS Laboratory, University Hospital, Queen':s Medical Centre, Nottingham NG7 2UH, UK
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Abstract |
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The carbapenemase gene (cfiA) was detected in 12 (6.9%) out of 175 clinical isolates of Bacteroides fragilis from Nottingham, three times the frequency reported from France. Only one gene copy was present in all cases. Eleven (6.3%) of the cfiA-positive isolates were susceptible to imipenem or meropenem (MIC < 8 mg/L); strains of this type have the potential to convert to high-level resistance. One isolate (0.6%) displayed resistance to both carbapenem antibiotics and was associated with specific carbapenemase activity six times that of the sensitive isolates.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The carbapenem antibiotics are useful therapeutic agents for infections associated with Bacteroides fragilis owing to their stability to common ß-lactamases. However, a minority of clinically significant B. fragilis strains (c. 3%) produce carbapenemases. 1 These are typically metallo-ß-lactamases that are inhibited by ethylenediaminetetra-acetic acid (EDTA) and are products of the cfiA gene, which has been cloned and sequenced. 2
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.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Bacterial strains
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 557–582) 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.
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Results |
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MICs of imipenem for the isolates ranged from 0.03 to 16 mg/L; 21 isolates showed reduced susceptibility to imipenem (MICs 0.5- 2 mg/L), and one isolate displayed resistance (MIC 16 mg/L).
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).
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For ten of the cfiA-positive isolates, the MICs of the carbapenems were 0.03- 2 mg/L (Table). The MICs of imipenem and meropenem for B. fragilis Q8 were 2 mg/L and 16 mg/L, respectively. B. fragilis T2 was resistant to both carbapenem antibiotics (MIC of imipenem, 16 mg/L; MIC of meropenem, 64 mg/L).
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Metallo-ß-lactamase activity was detected in eight of the 12 cfiA-positive strains (Table). With B. fragilis J1, imipenem hydrolysis was detected only in the biological assay. The specific activities of six of the isolates (imipenem MIC, 1 or 2 mg/L) were between 1.9 and 8.2 nmol imipenem hydrolysed/min/mg protein; that of the resistant isolate was 49.4 nmol imipenem hydrolysed/min/mg protein. MICs of imipenem for the four carbapenemase-negative strains were 0.03–1 mg/L.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Twelve (6.9%) of the 175 clinical isolates of B. fragilis from Nottingham possessed the cfiA gene; substantially more than reported in France (2.4%). 8 All but one of the isolates would be classed as sensitive by conventional criteria (imipenem MIC <8 mg/L). However, for most of these isolates the MIC was at least ten times that of fully sensitive isolates. The proportion of cfiA-positive isolates that were carbapenem-sensitive (6.3% of total) was higher than that reported in France (1.6%). 8 These isolates have the potential to convert to a high level of metallo-ß-lactamase expression and resistance owing to the presence of an insertion element carrying an efficient promoter immediately upstream of the cfiA gene. 3,9 Only one isolate (0.6%) was resistant to both imipenem and meropenem; it exhibited a specific activity at least six times that of the more sensitive strains. Similar low rates of resistance have been found in USA and France, 8 although in Japan, where imipenem is widely used, rates of 5.9% have been reported. 10
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
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Acknowledgments |
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This work was supported by a grant from the British Society for Antimicrobial Chemotherapy. We thank Dr A. Cockayne for his advice and help with the molecular methods. Preliminary findings of this study were presented at the Eighth International Congress on Infectious Diseases, Boston, MA, USA, May 1998.
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Notes |
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* Tel: +44-115-9249924, ext. 42632; Fax: +44-115-9709233; E-mail: richard.edwards{at}nottingham.ac.uk
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References |
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1 . Edwards, R., Thirlwell, D. & Greenwood, D. (1996). Changes in ß-lactam antibiotic susceptibility and ß-lactamase production of clinical isolates of Bacteroides and Prevotella species over a 9 year period. Journal of Antimicrobial Chemotherapy 37, 636–8.
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Received 29 May 1998; returned 6 July 1998; revised 28 July 1998; accepted 21 September 1998
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