Abstract
Genes encoding CTX-M-14, CTX-M-9, CTX-M-1, CTX-M-32, SHV-12, TEM-52, or CMY-2 β-lactamases were detected in 21 Escherichia coli strains recovered during 2003 from sick animals (11 of 459 [2.4%] strains) and healthy animals (10 of 158 [6.3%] strains) in Spain. Twelve of these strains harbored blaCTX-M genes and showed unrelated pulsed-field gel electrophoresis patterns.
Escherichia coli strains harboring extended-spectrum β-lactamases (ESBLs) or plasmidic class C β-lactamases have been arising as detected in humans (3, 5, 19) but have been rarely reported for animals (6, 7, 13, 22, 24-26). A new group of class A ESBLs, CTX-M enzymes, are increasingly being detected among human clinical E. coli strains in the last several years (3).
Since 1996, there has been a Veterinary-Antimicrobial-Resistance-Surveillance (VAV) Network in Spain focused on monitoring antibiotic resistance in bacteria of animal origin (16). The characterization of ESBLs and plasmidic class C β-lactamases in E. coli strains of sick and healthy animals recovered through the VAV Network during the periods 1997 to 2001 and 2000 to 2001, respectively, had been previously reported by our group (6, 7). The objective of the present work was to follow their evolution during the whole year of 2003. A total of 459 E. coli isolates from sick animals were submitted during 2003 to the VAV Network from eight public and private laboratories of animal health from all over Spain. No previous selection of the E. coli isolates according to their antimicrobial susceptibility was performed. Of the 459 E. coli isolates, 13 (2.8%) showed resistance or diminished susceptibility (drug MIC ≥ 2 μg/ml) to ceftazidime (CAZ) or cefotaxime (CTX), which were included in the study (Table 1). In addition, 160 fecal samples of healthy chickens (1 per farm) from nine Spanish slaughterhouses were obtained via the VAV Network during 2003. Fecal samples were seeded into non-antibiotic-supplemented MacConkey agar plates, and one E. coli isolate per sample was studied. A total of 158 E. coli isolates were recovered, and 13 of them (8.2%) showed resistance or diminished susceptibility to CAZ or CTX, which were also included in this study (Table 2).
TABLE 1.
MIC values of different β-lactams and β-lactamase genes detected in the 13 E. coli isolates obtained from sick animals with diminished susceptibility or resistance to broad-spectrum cephalosporins
| E. coli isolate | Animal (sample) | MIC (μg/ml)a
|
ESBL test resultb | Plasmidic β- lactamase genes detected | Mutations in the promoter-attenuator of the ampC gene | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AMP | TIC | AMC | CFZ | FOX | CAZ | CTX | CRO | IMP | ATM | |||||
| TER008 | Pig (gut) | >256 | >128 | 4 | >128 | 8 | 1 | 32 | 128 | 0.125 | 4 | + | blaTEM-1c + blaCTX-M-14 | −18, −1, +58 |
| BAR037 | Pig (liver) | >256 | >128 | 4 | >128 | 4 | 1 | 32 | 64 | 0.125 | 2 | + | blaTEM-1b + blaCTX-M-14 | No mutations |
| TER007 | Pig (gut) | >256 | >128 | 8 | >128 | 8 | 1 | 128 | >256 | 0.125 | 4 | + | blaTEM-1b + blaCTX-M-14 | −18, −1, +58 |
| AND014 | Poultry (lung) | >256 | >128 | 8 | >128 | 8 | 1 | 128 | 256 | ≤0.06 | 4 | + | blaTEM-le + blaCTX-M-14 | −18, −1, +58 |
| MAD156 | Poultry (yolk) | >256 | >128 | 8 | >128 | 4 | ≤0.5 | 8 | 32 | 0.125 | 2 | + | blaCTX-M-9 | −18, −1, +58 |
| BAD159 | Pig (lung) | >256 | >128 | 4 | >128 | 8 | 32 | 256 | >256 | 0.125 | >64 | + | blaCTX-M-32 | No mutations |
| BAR045 | Cattle (milk) | >256 | >128 | 8 | >128 | 8 | 2 | 128 | >256 | 0.125 | 16 | + | blaTEM-1b + blaCTX-M-I | No mutations |
| BUR048 | Rabbit (lung) | >256 | >128 | 8 | 32 | 8 | 4 | 32 | 32 | ≤0.06 | 4 | + | blaTEM-52b | −18, −1, +58 |
| COR029 | Pig (liver) | >256 | >128 | 8 | 32 | 8 | 16 | 2 | 2 | 0.125 | 16 | + | blaTEM-1b + blaSHV-12 | −18, −1, +58 |
| A32/03/Hg | Poultry (liver) | >256 | >128 | 2 | 32 | 4 | 16 | 4 | 2 | ≤0.06 | 32 | + | blaSHV-12 | −18, −1, +58 |
| TAR004 | Partridge (gut) | >256 | >128 | 64 | >128 | 128 | 64 | 64 | 64 | 0.06 | 16 | − | blaTEM-1b + blaCMY-2 | −18, −1, +58 |
| LUG699 | Cattle (feces) | >256 | >128 | 64 | 32 | 32 | 4 | 2 | 1 | 0.125 | 4 | − | No bla genes detected | −42, −18, −1, +58 |
| LUG737 | Pig (gut) | 128 | 64 | 32 | 128 | 64 | 4 | 4 | 4 | 0.06 | 4 | − | No bla genes detected | −42, −18, −1, +58 |
AMP, ampicillin; TIC, ticarcillin; AMC, amoxicillin-clavulanic acid; CFZ, cefazolin; FOX, cefoxitin; CAZ, ceftazidime; CTX, cefotaxime; CRO, ceftriaxone; IMP, imipenem; ATM, aztreonam.
+, positive; −, negative.
TABLE 2.
MIC values of different β-lactams and β-lactamase genes detected in the 13 E. coli strains obtained from fecal samples of healthy chickens with diminished susceptibility or resistance to broad-spectrum cephalosporins
| E. coli isolate | MIC (μg/ml)
|
ESBL test resultb | Plasmidic β-lactamase gene(s) detected | Mutations in the promoter-attenuator of the ampC gene | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AMP | TIC | AMC | CFZ | FOX | CAZ | CTX | CRO | IMP | ATM | ||||
| A3SAEC 269-271 | >256 | >128 | 4 | >128 | 8 | 1 | 64 | 128 | 0.125 | 2 | + | blaTEM-1b + blaCTX-M-14 | No mutations |
| A3SZEC 377-379 | >256 | >128 | 4 | >128 | 4 | 1 | 64 | 128 | 0.125 | 2 | + | blaCTX-M-14 | No mutations |
| A3SZEC 443-445 | >256 | >128 | 8 | >128 | 4 | ≤0.5 | 32 | 128 | ≤0.06 | 2 | + | blaCTX-M-14 | No mutations |
| A3YEEC 209-211 | >256 | >128 | 4 | >128 | 4 | 1 | 64 | 128 | ≤0.06 | 4 | + | blaCTX-M-14 | −18, −1, +58 |
| A3SZEC 431-433 | >256 | >128 | 8 | >128 | 4 | ≤0.5 | 8 | 16 | 0.125 | 2 | + | blaTEM-1b + blaCTX-M-9 | −28, +58 |
| A3AGEC 425-427 | >256 | >128 | 8 | 32 | 4 | 16 | 4 | 4 | 0.125 | 32 | + | blaTEM-1b + blaSHV-12 | No mutations |
| A3SZEC 368-370 | >256 | >128 | 8 | 32 | 8 | 16 | 4 | 4 | 0.125 | 32 | + | blaTEM-1b + blaSHV-12 | +22, +26, +27, +32 |
| A3LEEC 064-066 | >256 | >128 | 4 | 32 | 8 | 32 | 8 | 8 | 0.125 | 32 | + | blaSHV-12 | No mutations |
| A3YEEC 296-298 | >256 | >128 | 32 | >128 | 64 | 8 | 8 | 16 | 0.125 | 8 | − | blaTEM-1b + blaCMY-2 | No mutations |
| A3AAEC 031-033 | 256 | 64 | 64 | >128 | 64 | 2 | 8 | 16 | 0.125 | 4 | − | blaCMY-2 | No mutations |
| A3SAEC 239-241 | >256 | >128 | 32 | 32 | 16 | 2 | 1 | 0.5 | 0.125 | 2 | − | blaTEM-1b | −42, −18, −1, +58 |
| A3AVEC 404-406 | 256 | 32 | 64 | 32 | 16 | 2 | 2 | 0.5 | 0.125 | 2 | − | No bla genes detected | −42, −18, −1, +58 |
| A3COEC 551-553 | 128 | 32 | 32 | 16 | 16 | 2 | 1 | 0.5 | 0.125 | 2 | − | No bla genes detected | −42, −18, −1, +58 |
AMP: ampicillin; TIC: ticarcillin; AMC: amoxicillin-clavulanic acid; CFZ: cefazolin; FOX: cefoxitin; CAZ: ceftazidime; CTX: cefotaxime; CRO: ceftriaxone; IMP: imipenem; ATM: aztreonam.
+, positive; −, negative.
Determination of the MICs of 10 β-lactams was carried out by the agar dilution method. Disks of CAZ and CTX supplemented or not supplemented with clavulanic acid were used to screen the ESBL production (17). The detection and characterization of genes encoding TEM (2), SHV (20), FOX (1), CTX-M (12, 18), and CMY (23) β-lactamases was carried out by PCR and sequencing. The regulatory region of the chromosomal ampC gene was amplified by PCR (8), sequenced, and compared with the same region in the E. coli K12 ampC gene (9, 15).
Characterization of β-lactamase genes in E. coli strains from sick animals.
The characteristics of the 13 E. coli strains from sick animals with resistance or diminished susceptibility to broad-spectrum cephalosporins are shown in Table 1. ESBL production was detected by the screening method for 10 of these strains.
The presence of genes encoding ESBLs or plasmidic class C β-lactamases of the type TEM, SHV, CTX-M, or CMY in 11 of the 13 E. coli strains was demonstrated. The following combination of genes were found: blaCTX-M-14 + blaTEM-1b (two strains), blaCTX-M-14 + blaTEM-1c (one strain), blaCTX-M-14 + blaTEM-1e (one strain), blaCTX-M-1 + blaTEM-1b (one strain), blaCTX-M-9 (one strain), blaCTX-M-32 (one strain), blaSHV-12 + blaTEM-1b (one strain), blaSHV-12 (one strain), blaTEM-52b (one strain), blaCMY-2 + blaTEM-1b (one strain), and none (two strains).
As mentioned above, one of our ESBL-producing strains (BAD159), recovered from a lung sample of a sick pig, harbored the blaCTX-M-32 gene, whose sequence was very recently found in one human clinical E. coli strain in Spain (10). The CTX-M-32 β-lactamase shows an Asp240-Gly amino acid substitution with respect to CTX-M-1 that seems to be associated with the increase in the level of resistance to CAZ (10). As a matter of fact, the results obtained with our blaCTX-M-32-containing E. coli strain showed a high CAZ MIC (32 μg/ml) as well as high CTX and ceftriaxone (both ≥256 μg/ml) and aztreonam (>64 μg/ml) MICs.
The specific C→T mutation at position −42 of the promoter-attenuator region of ampC chromosomal gene was only detected in the two strains in which no plasmidic β-lactamase genes were found (both strains showed resistance to amoxicillin-clavulanic acid and cefoxitin) (Table 1). Mutations at positions −18, −1, and +58 were also detected in these two strains and in other eight of this series. According to previous reports (9), the mutations at position −42 or −32 are associated with overexpression of the ampC gene in E. coli. Nevertheless, other mutations such as those at positions −18, −1 (attenuator region), and +58 are frequently found in β-lactam-susceptible E. coli strains (8).
It is interesting to underline that 8 of the 10 ESBL-harboring E. coli strains were recovered from pigs or poultry. In addition, 11 of the 13 strains, especially those of the CTX-M type (7 of these strains), presented an ESBL or a plasmidic class C β-lactamase as the mechanism of resistance. In our previous work carried out with E. coli strains from sick animals recovered during the period 1997 to 2001, ESBL or plasmidic class C β-lactamases were detected in a small proportion of strains and mutations in the promoter of ampC gene were the main mechanism of broad-spectrum cephalosporin resistance (6).
Characterization of β-lactamase genes in E. coli strains of healthy animals.
The characteristics of the 13 E. coli strains from healthy animals with diminished susceptibility or resistance to broad-spectrum cephalosporins are shown in Table 2. ESBL production in eight of these strains was detected by the screening method.
ESBL or plasmidic class C β-lactamase genes were detected in 10 of these 13 E. coli strains and in 5 of them were combined with the blaTEM-1b gene (Table 2). The genes found were the following: blaCTX-M-14 (four strains, in one case associated with blaTEM-1b gene), blaSHV-12 (three strains, in two cases associated with blaTEM-1b gene), blaCTX-M-9 (one strain, associated with blaTEM-1b gene) and blaCMY-2 (two strains, in one case associated with blaTEM-1b gene).
The remaining three E. coli strains showed CTX and CAZ MICs in the range of 1 to 2 μg/ml and harbored the specific mutation (C→T) at position −42 in the promoter region of the ampC gene related to its overexpression (in one of the cases associated with the blaTEM-1b gene) (Table 2).
Genes encoding CMY-2, CTX-M-14, and SHV-12 β-lactamases were previously found by our group in fecal samples obtained during the period 2000 to 2001 from healthy chickens (7), and the presence of ESBL or plasmidic class C β-lactamase genes was demonstrated in 2.5% of those strains at that time. In the present work, the ratio of this type of genes was higher (6.3%) and the diversity of β-lactamase genes, including the blaCTX-M-9 gene, was also wider. This ratio is especially high when we consider that no antibiotic-supplemented medium was used for the analysis of the animal fecal samples.
Clonal diversity of E. coli strains.
All eight E. coli strains from sick and healthy animals which carried the blaCTX-M-14 gene showed unrelated pulsed-field gel electrophoresis patterns with the XbaI enzyme (14). Similarly, no clonal relationship was detected among strains containing blaCTX-M-9 (two strains), blaSHV-12 (four strains), or blaCMY-2 (three strains) genes.
It is interesting to underline the broad dissemination of blaCTX-M genes of different groups among E. coli strains of sick and healthy animals, with blaCTX-M-14 being the most frequently detected. This specific β-lactamase gene has been increasingly found in human clinical E. coli isolates in different countries, including Spain, in the last several years (3, 4, 11, 21). The factors that could have contributed to the dissemination of blaCTX-M genes among animal E. coli strains are unknown but could be associated with a potential coselection with other antibiotics due to the possible association of the resistance determinants in the same genetic mobile elements.
An increase in the percentage and variety of ESBL-producing E. coli strains in healthy and sick animals in this study (year 2003) compared to the results of previous studies (before 2002) carried out by our group has been observed. Monitoring ESBL-producing E. coli strains, including both clinical and commensal strains, should be continued in the human and animal fields to investigate their evolution and to analyze the factors that contribute to their selection and spread.
Acknowledgments
This work has been supported in part by grants FIS01/973 and AGL2002/02637. The VAV work was supported by the Ministery of Agriculture, Fish and Food of Spain.
We acknowledge Fernanda Ruiz-Larrea and Myriam Zarazaga for reviewing the manuscript.
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