1 Area de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios, 51, 26006 Logroño, Spain; 2 Servicio de Microbiología, Hospital Universitario Central de Asturias, Oviedo, Spain
Received 4 April 2005; returned 20 August 2005; revised 5 September 2005; accepted 15 September 2005
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Abstract |
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Methods: Thirty-four of the 1700 E. coli isolates recovered from unrelated patients in a Spanish hospital showed expanded-spectrum cephalosporin resistance. The presence of genes encoding TEM, SHV, CTX-M, CMY-2-type or FOX ß-lactamases as well as the existence of mutations in the regulatory region of the chromosomal ampC gene were studied by PCR and sequencing in these 34 E. coli isolates.
Results: The following extended-spectrum ß-lactamases (ESBLs) or plasmidic class C ß-lactamase genes were detected (number of isolates): blaCTX-M-14 (14), blaCTX-M-9 (4), blaCTX-M-32 (1), blaTEM-52 (2), blaSHV-12 (3) and blaCMY-2 (2). The remaining eight isolates showed a mutation in the promoter/attenuator region of the ampC chromosomal gene at position 42, in combination with mutations at positions 18, 1 and +58. The blaTEM-1 gene was also detected in 12 of the ESBL-producing isolates, in both CMY-2-producing isolates and in four of the eight isolates that showed a mutation at position 42 of the ampC promoter. Other mutations in the promoter/attenuator region were detected in association with ESBL or CMY-2 genes, such as the combination 18, 1 and +58, 28 and +58, or +22, +26, +27 and +32. No clonal relationship was found among the CTX-M-producing E. coli isolates by PFGE with XbaI enzyme.
Conclusions: Approximately 1.5% of the E. coli isolates of our hospital harboured ESBL genes, those of the CTX-M-9 group being the most common ones.
Keywords: E. coli , ESBLs , class C ß-lactamases , Spain
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Introduction |
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Resistance to expanded-spectrum cephalosporins can also be associated in E. coli with the production of plasmidic class C ß-lactamases, such as CMY enzymes,8 or with the overproduction of the chromosomal AmpC ß-lactamase.7,9,10 The objective of this work was to characterize the ß-lactam resistance mechanisms in all the clinical E. coli isolates with reduced susceptibility or resistance to expanded-spectrum cephalosporins, recovered during a 1 year period in a Spanish hospital, and to analyse their clonal relationship.
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Materials and methods |
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The presence of genes encoding TEM (forward, 5'-ATTCTTGAAGACGAAAGGGC-3'; reverse, 5'-ACGCTCAGTGGAACGAAAAC-3'), SHV (forward, 5'-CACTCAAGGATGTATTGTG-3'; reverse, 5'-TTAGCGTTGCCAGTGCTCG-3'), CTX-M (forward, 5'-GTGACAAAGAGAGTGCAACGG-3'; reverse, 5'-ATGATTCTCGCCGCTGAAGCC-3'), CMY-2-type (forward, 5'-GATTCCTTGGACTCTTCAG3'; reverse, 5'-TAAAACCAGGTTCCCAGATAGC-3') and FOX (forward, 5'-CACCACGAGAATAACCAT-3'; reverse, 5'-ATGTGGACGCCTTGAACT-3') ß-lactamases was studied by specific PCRs7 and their identification was verified by sequencing and comparison with those sequences included in the EMBL database. The promoter and attenuator region of the chromosomal ampC gene was also amplified by PCR (forward, 5'-AATGGGTTTTCTACGGTCTG-3'; reverse, 5'-GGGCAGCAAATGTGGAGCAA-3'), sequenced and compared with the same region of the E. coli K12 ampC gene,10 in order to analyse the mutations in that region, associated with the overexpression of the ampC gene. Positive and negative controls were included in all PCR assays.
The clonal relationship among the strains was studied by PFGE, using XbaI as restriction enzyme. PFGE patterns were classified into four groups: indistinguishable (all the bands match), closely related (13 different bands), possibly related (46 different bands) and unrelated (>6 different bands).
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Results and discussion |
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A positive ESBL screening test was demonstrated in 24 of the 34 studied isolates, which represents 1.4% of the 1700 E. coli isolates recovered in the 1 year period. A higher MIC value of cefotaxime (4 to >256 mg/L) than of ceftazidime (0.12516 mg/L) was detected in 19 of them, and the MIC values of ceftazidime were higher than those of cefotaxime in three other isolates (28 and 32128 mg/L, respectively). Similar MIC values of cefotaxime and ceftazidime were found in the last two isolates (32 mg/L). Specific PCR and sequencing allowed identification of five different ESBL genes among these 24 ESBL-positive isolates: blaCTX-M-14 (14 isolates), blaCTX-M-9 (4 isolates), blaCTX-M-32 (1 isolate), blaSHV-12 (3 isolates) and blaTEM-52c (2 isolates). A blaTEM-1 gene (molecular variants blaTEM-1b or blaTEM-1c) was also detected in 12 of them that harboured blaCTX-M-14, blaCTX-M-9 or blaSHV-12 genes (Table 1).
The percentages of resistance to non-ß-lactam antibiotics detected in the series of 24 E. coli isolates harbouring ESBLs were as follows: nalidixic acid, 75%; ciprofloxacin, 50%; tetracycline, 71%; trimethoprim/sulfamethoxazole, 63%; streptomycin, 67%; kanamycin, 25%; gentamicin, 13%; tobramycin, 8%; amikacin, 4%; and chloramphenicol, 17%.
A CTX-M-32-harbouring E. coli strain, recovered from one urine sample, has been detected in this study. This ESBL was first described by Cartelle et al.4 from a human clinical E. coli strain and, very recently, has been also found in one animal E. coli strain,7 in both cases in Spain. CTX-M-32 differs from CTX-M-1 through a single amino acid substitution (Asp-240Gly), which confers by itself hydrolytic activity against ceftazidime.4 As a matter of fact, our blaCTX-M-32-containing E. coli strain showed a relatively high MIC value of ceftazidime (4 mg/L).
It is interesting to underline the extended dissemination of blaCTX-M genes, of different groups, among ESBL-containing E. coli strains of our hospital (19 of the 24 ESBL producers, 79%), blaCTX-M-14 being the most frequent one (14 of the 19 isolates, 74%). CTX-M-9-group ß-lactamases, such as CTX-M-14 or CTX-M-9, have been increasingly found in human clinical E. coli isolates in different countries13,5,6 and they begin to be the most frequently found ESBLs in Spain either in clinical or faecal human and animal E. coli isolates.3,6,7
Mechanisms of resistance in the ESBL-negative strains
Ten of the 34 E. coli isolates of our series showed a negative result for the ESBL screen test, being resistant to amoxicillin/clavulanic acid (MIC 32 mg/L) and cefoxitin (16256 mg/L). The blaCMY-2 gene was found in two of these isolates, associated with either the blaTEM-1a or blaTEM-1b genes. No ESBLs or plasmidic cephalosporinases were detected in the remaining eight isolates. Nevertheless, mutations in the promoter and attenuator region of the ampC gene (at positions 42, 18, 1 and +58) were identified together in all these eight isolates (Table 1). It is known that specific mutations in the ampC promoter region render an increase in the MIC values of expanded-spectrum cephalosporins as well as of cephamycins.9,10 In this sense, mutations at positions 42 (CT) and at 18 (G
A) create new 35 and 10 boxes separated by 17 bp, the optimal distance to enhance the expression, resulting in the formation of a strong promoter.9,10 Mutations at position 42 have not been detected in association with ESBLs either in this study or, to our knowledge, in others.
Mutations at positions +22, +26, +27 and +32 of the ampC attenuator region were found together in one isolate which harboured the blaCMY-2 gene and mutations at positions 18, 1 and +58 were found in the other CMY-2-producing isolate (Table 1). The high MIC of cefoxitin for these two isolates could be explained by the expression of the blaCMY-2 gene, although any effect due to the mutations in the promoter (position 18) or attenuator (positions +22, +26, +27 and +32) ampC region cannot be excluded.9
Mutations in the ampC promoter/attenuator region in the ESBL-producing strains
Mutations at positions 18, 1 and +58 were found together in three of our isolates that harboured ESBL genes (blaSHV-12, one isolate; blaTEM-52, two isolates). These isolates showed cefoxitin MIC values of 416 mg/L, lower than those presented by isolates harbouring a mutation at position 42 (1664 mg/L). Caroff et al.10 indicate that mutation at position 18, by creating a new 10 box, plays an important role in ampC expression. Our results suggest that further studies are necessary to determine the real role of this mutation.
Besides, mutations at positions 28 (GA) and +58 (C
T) were also detected together in three isolates harbouring blaCTX-M-14 or blaCTX-M-32 genes (Table 1), which showed cefoxitin MIC values of 416 mg/L. Any association between a mutation at position 28 and overexpression of the ampC gene has not been previously demonstrated. Nevertheless, the nucleotide at this position could interact with RNA polymerase, due to its localization in the spacer, as suggested by Mulvey et al.9
Clonal relationship of ESBL-producing strains
No clonal relationship was observed among the 19 CTX-M-producing or the two CMY-producing E. coli strains when their PFGE patterns were compared after XbaI digestion. Nevertheless, an indistinguishable PFGE pattern was obtained for the two TEM-52-producing E. coli strains, both of them recovered from urine samples (Figure 1). These results suggest the existence of horizontal transfer of blaCTX-M genes rather than the dissemination of specific clones, as was also suggested by other authors.1,3,6
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References |
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