In the past, CP was widely used in veterinary medicine because of its efficacy against broad-spectrum bacteria. However, it became evident that CP may cause toxic adverse effects, such as aplastic anemia in humans.1 Therefore, a ban on the use of CP in food-producing animals was instituted in 1998 in Japan and other countries.2-5 The percentage of CP-resistant Escherichia coli in apparently healthy cattle before the ban ranged from 9.6% to 16.6%, and this percentage in apparently healthy pigs ranged from 18.6% to 27.1%.6-8 Although 6 years have elapsed since the ban was imposed, CP-resistant E coli are still found in food-producing animals in Japan and other countries.2-5
In Japan, TP and FFC have been approved for use in the treatment of enteric and respiratory diseases in food-producing animals. Phenicols, including CP, TP, and FFC, inhibit bacterial protein synthesis by binding to the 50S ribosomal subunit and inhibiting the activity of peptidyl transferase on the 70S ribosome.9 It is generally believed that enzymatic and nonenzymatic resistance to CP is caused by CAT and the efflux pump (eg, CmlA and Flo proteins), respectively,10-12 with CAT and the CmlA protein involved in resistance to TP but not to FFC.9,13 In contrast, the Flo protein causes cross-resistance to both TP and FFC.2,14
Class 1 integrons, which integrate resistance genes and transfer them among bacteria, contribute to the emergence of multiple-drug resistance.15-17 Multiple-drug resistance induces selection for resistance to antimicrobials that have not been used; this phenomenon is called coresistance.18 The emergence of multiple-drug resistance by a transposon or plasmid carrying the integrons as well as that caused by the integron itself is an issue of major concern. However, limited information is currently available regarding the epidemiologic characterization of CP-resistance genes and integrons in E coli strains derived from food-producing animals.
It is believed that the ban on antimicrobial use will help prevent the development of antimicrobial resistance in bacteria isolated from food-producing animals. The purpose of the study reported here was to evaluate the effect of coresistance and cross-resistance on resistance to CP. We examined the susceptibility of E coli strains isolated from sick cattle and pigs in Japan to TP and FFC and the relationship between the prevalence of CP-resistance genes and the gene cassette within class 1 integrons.
Materials and Methods
Sample population—Bacterial strains used in the study reported here included 71 CP-resistant (MIC ≥ 32 μg/mL) and 104 CP-susceptible (MIC, 2 to 16 μg/mL) strains isolated from sick cattle and pigs in Japan between 2001 and 2004.4 The MICs of AMP, DSM, OTC, and TMP were determined in another study.4 All the strains were stored in 10% skim milk at –80°C until analyzed.
Antimicrobial susceptibility testing—The MICs of TP and FFC were determined by an agar dilution method in accordance with the guidelines of the National Committee for Clinical Laboratory Standards.19 Several bacterial isolates, namely E coli ATCC 25922, Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212, were used as quality-control samples for MIC determination. The breakpoints of TP and FFC were set at 256 and 64 μg/mL, respectively,20 on the basis of the midpoint between the MIC peaks in the study reported here.
PCR amplification of CP-resistance genes and the variable region of integrons—Bacterial DNA was prepared by boiling in water.16 Primer sets were used for detection of CP-resistance genes, namely the cat1, cat2, cmlA, and flo genes, and the variable region of the integrons (Appendix).14,15,17,21,22 Products amplified by a PCR assay were analyzed by gel electrophoresis by use of a 2% (wt:vol) agarose gel in 1X triphosphate EDTA buffer at 100 V. Gels were stained with ethidium bromide, developed with ultraviolet irradiation, and imaged by use of a fluorescent imaging system.a
PCR mapping—The PCR mapping for the strains that harbored the CP-resistance gene and the integrons was conducted as described in another study.23 Mapping was accomplished by use of the primers for the aadA1, aadA2, dhfrI, dhfrVII, dhfrXII, and dhfrXVII genes and the variable region of the integrons (Appendix). The strains containing dhfrVII and dhfrXVII were differentiated by use of PCR restriction fragment length polymorphism.21
Statistical analysis—The χ2 test was used for all statistical analyses in the study. Values of P < 0.05 were considered significant.
Results
Prevalence of CP-resistance genes—Of the 71 CP-resistant strains, 46 harbored cat1, 22 harbored cmlA, 4 harbored cat2, and 3 harbored flo genes (Table 1). Eight isolates harbored 2 or 3 CP-resistance genes. No CP-resistance genes were detected in the 6 CP-resistant and 101 CP-susceptible strains, and the remaining 3 CP-susceptible strains harbored only cat1. Of the 20 CP-resistant strains isolated from cattle, 18 (90%) harbored the cat1 gene, whereas 28 (54.9%) and 20 (39.2%) strains from among the 51 CP-resistant strains isolated from pigs harbored cat1 and cmlA, respectively.
Prevalence of the cat1, cat2, cmlA, and flo genes in CP-resistant and CP-susceptible Escherichia coliisolated from sick cattle and pigs in Japan.
| CP-resistance gene | No. of CP-resistant strains (n = 71) | No. of CP-susceptible strains (104) | ||||
|---|---|---|---|---|---|---|
| Cattle | Pigs | Total | Cattle | Pigs | Total | |
| cat1 | 16 | 24 | 40 | 1 | 2 | 3 |
| cmlA | 1 | 15 | 16 | 0 | 0 | 0 |
| cat1 and cmlA | 1 | 2 | 3 | 0 | 0 | 0 |
| cat1, cat2, and cmlA | 0 | 2 | 2 | 0 | 0 | 0 |
| cat1 and flo | 1 | 0 | 1 | 0 | 0 | 0 |
| cat2 and cmlA | 0 | 1 | 1 | 0 | 0 | 0 |
| cat2 and flo | 0 | 1 | 1 | 0 | 0 | 0 |
| flo | 1 | 0 | 1 | 0 | 0 | 0 |
| None | 0 | 6 | 6 | 36 | 65 | 101 |
| Total | 20 | 51 | 71 | 37 | 67 | 104 |
Comparison between the MIC distributions of phenicols in E coli harboring CP-resistance genes—The MIC distributions of the strains harboring each CP-resistance gene were compared (Table 2). The MIC90 for CP in the strains harboring cmlA was 128 μg/mL, whereas MIC90 for CP was 512 μg/mL for the strains harboring cat1. In contrast, the MIC90 for TP and FFC was > 512 and 16 μg/mL, respectively, in both of those strains.
Distribution of the MICs of various phenicols for strains of E coliwith and without CP-resistance genes.
| Antimicrobial | CP-resistance gene | No. of strains | MIC (μg/mL) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | 512 | >512 | |||
| CP | cat1 | 43 | 0 | 0 | 2 | 1 | 0 | 0 | 0 | 2 | 23 | 12 | 3 |
| cmlA | 16 | 0 | 0 | 0 | 0 | 0 | 4 | 8 | 4 | 0 | 0 | 0 | |
| cmlA and cat1 | |||||||||||||
| or cat2 | 6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 3 | 2 | |
| flo and cat1 or | |||||||||||||
| cat2 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 2 | 0 | |
| None | 107 | 0 | 14 | 42 | 40 | 5 | 5 | 1 | 0 | 0 | 0 | 0 | |
| Total | 175 | 0 | 14 | 44 | 41 | 5 | 9 | 9 | 6 | 25 | 17 | 5 | |
| TP | cat1 | 43 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 2 | 10 | 18 | 11 |
| cmlA | 16 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 13 | 2 | |
| cmlA and cat1 | |||||||||||||
| or cat2 | 6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 3 | |
| flo and cat1 | |||||||||||||
| or cat2 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | |
| None | 107 | 0 | 0 | 0 | 0 | 0 | 2 | 31 | 56 | 15 | 2 | 1 | |
| Total | 175 | 0 | 0 | 0 | 0 | 1 | 2 | 32 | 58 | 26 | 36 | 20 | |
| FFC | cat1 | 43 | 0 | 0 | 1 | 34 | 8 | 0 | 0 | 0 | 0 | 0 | 0 |
| cmlA | 16 | 0 | 0 | 1 | 4 | 10 | 1 | 0 | 0 | 0 | 0 | 0 | |
| cmlA and cat1 | |||||||||||||
| or cat2 | 6 | 0 | 0 | 0 | 2 | 1 | 2 | 1 | 0 | 0 | 0 | 0 | |
| flo and cat1 | |||||||||||||
| or cat2 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 1 | 0 | |
| None | 107 | 1 | 4 | 16 | 77 | 7 | 2 | 0 | 0 | 0 | 0 | 0 | |
| Total | 175 | 1 | 4 | 18 | 117 | 26 | 5 | 1 | 2 | 0 | 1 | 0 | |
Within each antimicrobial, the dashed vertical line indicates the mean breakpoint.
Association between CP-resistance genes and phenotypes for other classes of antimicrobial resistance—In bacteria isolated from cattle, strains that harbored cat1 had significantly higher rates of resistance to AMP, DSM, and TMP (P = 0.01) and OTC (P < 0.05), compared with strains that did not harbor any CP-resistance genes. In bacteria isolated from pigs, strains that harbored cat1 or cmlA had significantly higher rates of resistance to DSM (P < 0.05) and TMP (P = 0.01), compared with strains that did not harbor any CP-resistance genes (Table 3).
Rates of resistance (percentages) to AMP, DSM, OTC, and TMP in E colistrains with and without CP-resistance genes.
| Antimicrobial | Cattle (n = 57) | Pigs (118) | |||
|---|---|---|---|---|---|
| cat1 (19)* | None (36) | cat1 (30)* | cmlA (20) | None (71) | |
| AMP | 89.5a | 41.7b | 50.0 | 45.0 | 40.8 |
| DSM | 100a | 63.9b | 76.7c | 95.0a | 54.9b,d |
| OTC | 94.7c | 61.1d | 93.3 | 85.0 | 74.6 |
| TMP | 68.4a | 13.9b | 56.7c | 70.0a | 29.6b,d |
The strains that harbored cat1 or cml/A include the strains that harbored the CP-resistance genes.
Within a row within a species, value for strains with CP-resistance genes differed significantlya,b P = 0.01;c,d P <0.05) from the value for strains without CP-resistance genes.
Prevalence of class 1 integrons in strains that harbored CP-resistance genes—Class 1 integrons were detected in 14 of 17 (82.4%) strains that harbored cat1 and were isolated from cattle and in 18 of 26 (69.2%) and 12 of 15 (80.0%) strains that harbored cat1 and cmlA, respectively, and were isolated from pigs. Integrons were significantly (P = 0.01) more prevalent in strains with some CP-resistance genes (51/68) than in those without CP-resistance genes (25/107).
Amplicons of the integrons were 0.5, 1.0, 1.6, 1.9, 2.4, and 3.0 kb. As a result of PCR mapping, integrons in the CP-resistant strains were classified into 11 types of gene cassettes. There were 2 strains with no resistance gene in the 0.5-kb fragment; 17 strains with the aadA1 gene, 2 strains with the aadA2 gene, and 1 strain with the dhfrXII gene in the 1.0-kb fragment; 6 strains with the dhfrI and aadA1 genes, 1 strain with the dhfrVII and aadA1 genes, 2 strains with the dhfrXVII and aadA1 genes, and 2 strains with the dhfrXVII and aadA2 genes in the 1.6-kb fragment; 19 strains with the dhfrXII and aadA2 genes in the 1.9-kb fragment; 1 strain with the dhfrI and cat1 genes in the 2.4-kb fragment; and 1 strain with the dhfrI, aadA1, and cmlA genes in the 3.0-kb fragment (Tables 4 and 5).
Prevalence of the integrons in 21 E coli strains harboring the CP-resistance genes and isolated from cattle.
| CP-resistance genes | Variable region (kb) | Gene cassette* | Resistance pattern |
|---|---|---|---|
| cat1 (17) | 1.0 (4) | aadA1 (2) | AMP-DSM-OTC (2) |
| aadA2 (2) | AMP-DSM-OTC-TMP (2) | ||
| 1.6 (4) | dhfrI and aadA1 (3) | AMP-DSM-OTC-TMP (3) | |
| dhfrXVII and aadA2 (1) | DSM-OTC-TMP (1) | ||
| 1.9 (4) | dhfrXII and aadA2 (4) | AMP-DSM-OTC-TMP (4) | |
| 2.4 (1) | dhfrI and cat1 (1) | DSM-OTC-TMP (1) | |
| 1.6 and 1.9 (1) | dhfrI, aadA1, dhfrXII, and aadA2 (1) | AMP-DSM-OTC-TMP (1) | |
| None (3) | NA (3) | AMP-DSM (1) | |
| AMP-DSM-OTC (2) | |||
| cmlA (1) | 3.0 (1) | dhfrI, aadA1, and cmlA (1) | AMP-DSM-OTC-TMP (1) |
| cat1 and cmlA (1) | None (1) | NA (1) | AMP-DSM-OTC-TMP (1) |
| cat1 and flo (1) | 1.0 (1) | aadA1 (1) | AMP-DSM-OTC (1) |
| flo (1) | 1.0 (1) | aadA1 (1) | AMP-DSM-OTC (1) |
Values in parentheses are number of E coli strains.
The aadA1 and aadA2 genes are streptomycin-resistance genes, whereas dhfrI, dhfrVII, dhfrXII, and dhfrXVII are TMP-resistance genes.
Prevalence of the integrons in 47 strains harboring the CP-resistance genes and isolated from pigs.
| CP-resistance genes | Variable region (kb) | Gene cassette* | Resistance pattern |
|---|---|---|---|
| cat1 (26) | 1.0 (9) | aadA1 (9) | DSM (1) |
| OTC (1) | |||
| DSM-OTC (1) | |||
| DSM-OTC-TMP (2) | |||
| AMP-DSM-OTC (1) | |||
| AMP-TMP (1) | |||
| AMP-DSM-OTC-TMP (2) | |||
| 1.6 (3) | dhfrI and aadA1 (1) | AMP-DSM-OTC-TMP (1) | |
| dhfrVII and aadA1 (1) | AMP-DSM-OTC-TMP (1) | ||
| dhfrXVII and aadA2 (1) | AMP-DSM-OTC-TMP (1) | ||
| 1.9 (4) | dhfrXII and aadA2 (4) | OTC-TMP (1) | |
| AMP-OTC-TMP (1) | |||
| DSM-OTC-TMP (1) | |||
| AMP-DSM-OTC-TMP (1) | |||
| 1.0 and 1.6 (2) | aadA1, dhfrXVII, and aadA1 | (2) DSM-OTC-TMP (2) | |
| None (8) | NA (8) | OTC (3) | |
| DSM-OTC (1) | |||
| AMP-DSM-OTC (3) | |||
| AMP-DSM-OTC-TMP (1) | |||
| cmlA (15) | 0.5 (2) | No resistance gene (2) | AMP-DSM (1) |
| DSM-OTC (1) | |||
| 1.0 (3) | aadA1 (2) | DSM-OTC-TMP (1) | |
| AMP-DSM-OTC-TMP (1) | |||
| dhfrXII (1) | DSM-OTC-TMP (1) | ||
| 1.6 (1) | dhfrI and aadA1 (1) | AMP-DSM-OTC-TMP (1) | |
| 1.9 (6) | dhfrXII and aadA2 (6) | DSM-TMP (1) | |
| DSM-OTC-TMP (4) | |||
| AMP-DSM-OTC-TMP (1) | |||
| None (3) | NA (3) | AMP-DSM (1) | |
| DSM-OTC (1) | |||
| AMP-DSM-OTC-TMP (1) | |||
| cat1 and cmlA (2) | None (2) | NA (2) | OTC (1) |
| DSM-OTC (1) | |||
| cat1, cat2, and cmlA (2) | 1.9 (2) | dhfrXII and aadA2 (2) | AMP-DSM-OTC-TMP (2) |
| cat2 and cmlA (1) | 1.9 (1) | dhfrXII and aadA2 (1) | AMP-DSM-OTC-TMP (1) |
| cat2 and flo (1) | 1.9 (1) | dhfrXII and aadA2 (1) | AMP-DSM-OTC-TMP (1) |
See Table 4 for key.
Discussion
In the study reported here, populations of CP-resistance genes differed among E coli strains depending on the animal species from which these strains were isolated. For example, cat1 was predominant in strains isolated from sick cattle, whereas cat1 and cmlA were predominant in strains isolated from sick pigs. A difference in the gene population has also been observed in the United States, where the dominant CP-resistance gene (87.5%) in E coli isolated from diarrheic cattle24 was flo and cmlA was the dominant CP-resistance gene (97.9%) in pathogenic E coli isolated from diarrheic pigs.3 In these reports,3,24 it was suggested that cross-resistance as a result of the flo gene in bacteria isolated from cattle and coresistance as a result of mul-tiple-drug resistance in bacteria isolated from pigs can result in CP resistance in E coli strains. Because CP is not used in domestic food-producing animals in the United States and several other countries, this result raises the possibility that the use of the same or other classes of antimicrobials contributes to selection of CP-resistant E coli in sick animals.
The MIC90 for CP was higher in the strains with cat1 (512 μg/mL) than in those with cmlA (128 μg/mL). However, the MIC90 of TP and FFC for those strains was almost the same (>512 and 16 μg/mL, respectively). Whether the use of TP and FFC affects the prevalence of CP-resistance genes is still unclear.
Incidentally, the duration of the use of TP and FFC in cattle, which were approved in 1993 and 1995, respectively, is relatively shorter, compared with the duration of their use in pigs (approved in 1967 and 1992, respectively). Additionally, the volume of TP and FFC used in 2003 was less in cattle (900 kg) than in pigs (11,200 kg),25 and only the injectable formulation of both of these antimicrobials has been approved for use in cattle, whereas both the oral and injectable formulations have been approved for use in pigs in Japan. In fact, the frequency of CP resistance in bacteria isolated from pigs did not decrease much after the ban was initiated (from 27.1% to 22.3%), compared with the decrease in bacteria isolated from cattle (16.6% to 3.1%).6,26 Thus, the effect of cross-resistant selection attributable to use of the same class of antimicrobial may be larger for pigs than for cattle.
The breakpoint for FFC was set at 64 μg/mL on the basis of our results. Currently, National Committee for Clinical Laboratory Standards do not recommend breakpoints for FFC in E coli, whereas it is set at 8 μg/mL in bovine respiratory tract pathogens.19 There is a possibility of overestimating FFC resistance when the breakpoint of bovine respiratory tract pathogens is adapted for E coli.27 In the study reported here, the MICs of FFC were ≥ 128 μg/mL, whereas the MICs for FFC are at least 32 μg/mL in other reports.2,28-30
Class 1 integrons were contained in 17 of 21 (81.0%) of the isolates from cattle and 34 of 47 (72.3%) of the isolates from pigs with CP-resistance genes. In apparently healthy pigs in 1 study,16 approximately half of the tetracycline-resistant E coli contained class 1 integrons. Resistance to tetracycline is the most frequently observed type of resistance in E coli derived from apparently healthy pigs in Japan.26 In the sick animals of the study reported here, 22 of 42 (52.4%) and 47 of 95 (49.5%) tetracycline-resistant strains isolated from cattle and pigs, respectively, contained class 1 integrons (data not shown). It is suggested that CP-resistance genes are more closely linked to inclusion of integrons than to inclusion of tetracy-cline-resistance genes.
Prevalence of CP resistance in strains isolated from pigs was correlated with resistances to DSM and TMP, whereas that prevalence in strains isolated from cattle was correlated with resistances to DSM and TMP as well as AMP and OTC (Table 3). Antimicrobials such as DSM and TMP are frequently used for therapeutic purposes in the cattle and swine industries. Thus, the use of DSM and TMP in both species, in addition to the use of AMP and OTC in cattle, can lead to coselection of CP-resistant E coli in Japan. The strains with cat1 or cmlA frequently harbored class 1 integrons. Most of the resistance genes within the class 1 integrons encoded resistances to DSM or TMP. Class 1 integrons, which can participate in coresistance, play an important role in CP resistance in E coli isolated from sick cattle and pigs.
The study reported here revealed the genetic diversity of CP-resistant E coli strains isolated from cattle and pigs, which indicates that it is likely for characteristic antimicrobial resistance mechanisms, such as coresistance, to contribute considerably to the selection of CP-resistant E coli strains in sick animals. Furthermore, our results may lend authenticity to the possibility that bacterial resistance to CP in food-producing animals would persist despite the ban on CP use. To control antimicrobial resistance associated with coresistance, it may be necessary to regulate the use of all related antimicrobials. However, this decision would involve risks that could greatly affect animal health and welfare.
| CP | Chloramphenicol |
| TP | Thiamphenicol |
| FFC | Florfenicol |
| CAT | CP acetyltransferase |
| CmlA | CP efflux pump |
| Flo | FFC efflux pump |
| MIC | Minimum inhibitory concentration |
| AMP | Ampicillin |
| DSM | Dihydrostreptomycin |
| OTC | Oxytetracycline |
| TMP | Trimethoprim |
| ATCC | American tissue culture collection |
| cat | CAT gene |
| cmlA | Nonenzymatic CP resistance gene |
| flo | FFC resistance gene |
| aadA | Aminoglycoside-3″-adenylyltransferase gene |
| dhfr | Dihydrofolate reductase gene |
| MIC90 | Minimum inhibitory concentration at which 90% of isolates are inhibited |
GelDoc, BioRad Laboratories, Hercules, Calif.
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Appendix 1
Appendix 1
PCR primers used for detection of the resistance genes and variable region of the integrons.
| Antimicrobial | Target gene | Forward primer | Reverse primer | Reference |
|---|---|---|---|---|
| CP | cat1 | 5′–CTT GTC GCC TTG CGT ATA AT–3′ | 3′–GAA ATG CTA CGG TAA CCC TA–5′ | 15 |
| cat2 | 5′–AAC GGC ATG ATG AAC CTG AA–3′ | 3′–GAA ATG CTA CGG TAA CCC TA–5′ | 15 | |
| cmlA | 5′–CGC CAC GGT GTT GTT GTT AT–3′ | 3′–CAC TGT AAA TGC GTC CAG CG–5′ | 15 | |
| flo | 5′–AAT CAC GGG CCA CGC TGT ATC–3′ | 3′–CTT CCA CTT CTT ACT GCC GC–5′ | 14 | |
| DSM | aadA1 | 5′–TAT CAG AGG TAG TTG GCG TCA T–3′ | 3′–TTA CTT TGG AAT TGC GAT ACC TTG–5′ | 17 |
| aadA2 | 5′–TGT TGG TTA CTG TGG CCG TA–3′ | 3′–CGA AAC ACT TTC CGC TCT AG–5′ | 17 | |
| TP | dhfrI | 5′–CGG TCG TAA CAC GTT CAA GT–3′ | 3′–GTG AAC TTG CAC ACC GGA TTC–5′ | 15 |
| dhfrVII and | ||||
| dhfrXVII | 5′–GTC GCC CTA AAA CAA AGT TA–3′ | 3′–TGT AAA CTG AGA TAC CCG C–5′ | 21 | |
| dhfrXII | 5′–AAA TTC CGG GTG AGC AGA AG–3′ | 3′–GAT TGG TAA GGC AGT TGC CC–5′ | 15 | |
| NA | Variable region of integron | 5′–GGC ATC CAA GCA GCA AGC–3′ | 3′–TAG TCC AGT TCA GAC GAA–5′ | 22 |
NA = Not
