Article Figures & Data
Figures
- FIG. 1.
Plasmids with the cmy-2 gene in E. coli transferred by conjugation to serovar Newport in the intestinal tracts of poults. Lanes 1, E. coli MPS57 (O168:H7) with 72-MDa plasmid (containing cmy-2 gene) and 66-MDa plasmid; lanes 2, Salmonella serovar Newport 02-6203 before conjugation; lanes 3, serovar Newport 02-6203 after conjugation with E. coli MPS57 containing the 72-MDa plasmid; lanes 4 and 5, E. coli CE332-1 (O168:H7; 72- and 66-MDa plasmids) and serovar Newport CE332-1 (72-MDa plasmid), isolated from the cecal contents of the same poult; lanes 6, reference plasmids of 96, 62, 36, 23, 3.7, 3.4, 2.8, 2.0, and 1.4 MDa; lanes 7 and 8, E. coli CE3313-1 (O168:H7; 72- and 66-MDa plasmids) and serovar Newport CE3313-1 (72-MDa plasmid) isolated from the cecal contents of the same poult; lanes 9 and 10, E. coli CE331-2 (O101:H9; 80-, 72-, 3.6-, 3.0-, and 1.0-MDa plasmids) and serovar Newport CE331-2 (72-MDa plasmid) isolated from the cecal contents of the same poult; lanes 11 and 12, E. coli CE338-2 (O101:H9; 96-, 72-, 3.6-, 3.0-, and 1.0-MDa plasmids) and serovar Newport CE338-2 (72-MDa plasmid) isolated from the cecal contents of the same poult. The isolates with the suffix “-1” were isolated during the first trial; those with the suffix “-2” were isolated during the second trial.
Tables
- TABLE 1.
Antimicrobials and concentrations used to test susceptibility of E. coli and Salmonella isolates
Antimicrobial (abbreviation) Breakpoint concn (μg/ml)a Susceptibility Resistance Amikacin (AMK) 16 ND Ampicillin (AMP) ND 32 Amoxicillin-clavulanate (AMC) ND 64/16b Apramycin (APR)c ND 32d Carbadox (CRB)c ND 30e Cephalothin (CEF) ND 32 Ceftriaxone (CRO) 8 ND Ceftiofur (CTF) ND 8 Cefoxitin (FOX) ND 32 Chloramphenicol (CHL) ND 32 Ciprofloxacin (CIP) 0.125f ND Florfenicol (FLO)c ND 16g Gentamicin (GEN) ND 16 Kanamycin (KAN) ND 64 Nalidixic acid (NAL) ND 32 Neomycin (NEO)c ND 16d Nitrofurantoin (NIT) ND 64h Spectinomycin (SPT)c ND 64d Streptomycin (STR)c ND 32d Sulfisoxazole (SUL) ND 512 Sulfamethoxazole-trimethoprim (SXT) ND 76/4 Tetracycline (TET) ND 16 Tobramycin (TOB) ND 8 Trimethoprim (TMP) ND 16 ↵a The breakpoint concentrations to determine susceptibility and resistance are those specified by NCCLS standards M31-A (26) and M100-S12 (27). ND, not determined.
↵b The strains were considered resistant when growing on agar plates with amoxicillin-clavulanic acid at 64 and 16 μg/ml; respectively.
↵c There are no interpretive criteria specified by NCCLS standards M31-A (26) or M100-S12 (27) for these drugs.
↵d Strains were considered resistant to apramycin, neomycin, spectinomycin, and streptomycin at 32, 16, 64, and 32 μg/ml; respectively.
↵e Strains were considered resistant to carbadox, a veterinary growth promoter for pigs, at 30 μg/ml (12).
↵f A 0.125-μg/ml concentration of ciprofloxacin determines reduced sensitivity to ciprofloxacin (34).
↵g Strains were considered resistant to florfenicol at the level of 16 μg/ml (36).
↵h Strains were considered resistant to nitrofurantoin at 64 μg/ml; human urinary tract isolates are considered resistant at 128 μg/ml (27).
- TABLE 2.
Experimental design of study—dosing and treatment of poults
Day Dosing and treatment for poults in pena: 1 2 3 4 1 Dosing with antibiotic-susceptible serovar Newport Dosing with antibiotic-susceptible serovar Newport Dosing with antibiotic-susceptible serovar Newport and ESC-resistant E. coli No treatment 2 Administration of CTF (0.17 mg in 0.2 ml of saline) subcutaneouslyb Administration of saline (0.2 ml) subcutaneously No treatment with antimicrobials No treatment ↵a The pens were isolation units in an animal isolation building. Pens 1 and 4 contained 12 poults; pens 2 and 3 contained 13 poults. Poults given serovar Newport were dosed orally with 4.2 × 106 (trial 1) or 3.8 × 106 (trial 2) serovar Newport 02-6203 bacteria, an antimicrobial-susceptible strain that did not harbor any plasmids. The poults in pen 3 were also dosed with 2.5 × 106 (trial 1) or 1.4 × 106 (trial 2) bacteria of the ESC-resistant E. coli strain MPS57 that is resistant to AMP, AMC, CEF, CFT, CRO at 8 μg/ml; FOX; CHL, SPT, and SUL and that contains two plasmids of 66 and 72 MDa; the latter plasmid is self-transmissible, contains the cmy-2 gene, and encodes resistance to all the above-mentioned antimicrobials. Saline, phosphate-buffered saline.
↵b Administered subcutaneously at a dose and route indicated for the prevention of colibacillosis in 1-day-old poults (5).
- TABLE 3.
Experimental design of the study—sample collection
Day Trial(s) Sample type (no. of samples) and time of collectiona 1 1 and 2 Shavings (4), feed (1), water (1); before poults were placed in pens 1 1 Cloacal swabsb (12 or 13; 1/poult); when poults were placed in pens and before they were dosed with serovar Newport (pens 1 to 3) and E. coli (pen 3) 2 2 Cloacal swabs (12 or 13; 1/poult); after poults were dosed with serovar Newport (pens 1 to 3) and E. coli (pen 3) on day 1 but before CTF (pen 1) or saline (pen 2) treatment on day 2 4 1 and 2 Litter (4), feed with litter (1), water (1), cloacal swabs (12 or 13; 1/poult) 10 1 and 2 Litter (4), feed with litter (1), water (1), cecal contentsc (12 or 13; 1/poult) ↵a Samples were taken from 12 or 13 poults in each of four pens.
↵b Cloacal swabs were taken by inserting a cotton swab wetted with BPW into the cloaca.
↵c Upon necropsy; cecal contents were taken and examined for the presence of Salmonella and E. coli; one isolate each of E. coli and serovar Newport per sample, if present, was further characterized.
- TABLE 4.
Trial 1 antimicrobial resistance patterns and isolation rates of E. coli and Salmonella serovar Newport
Pen Day Resistance patterns (no. of isolates) ofa: E. coli Serovar Newport 1 1 AMP-STR (6), AMP-CIP-NAL-(STR)-TET (5), KAN-NEO-SPT-STR-SUL-TET (2) NIb 4 AMP-STR (1), AMP-CIP-NAL-(STR)-(SUL)-TET (15), KAN-NEO-SPT-STR-(SUL)-TET (2) Susc.c (18) 10 AMP-(AMC)-(CEF)-CIP-NAL-(NIT)-(STR)-TET (16), (AMP)-KAN-NEO-SPT-STR-SUL-TET (2) Susc. (16), SPT-STR-SUL (2) 2 1 AMP-STR (6), AMP-CIP-NAL-(STR)-TET (4), (AMP)-KAN-NEO-SPT-STR-SUL-TET (3) NI 4 AMP-STR (4), AMP-CIP-NAL-STR-(TET) (10), KAN-NEO-SPT-STR-SUL-TET (4) Susc. (18) 10 AMP-STR (4), AMP-CIP-NAL-(STR)-TET (12), KAN-NEO-STR-SUL-TET (2) Susc. (11), SPT-(STR)-SUL (6) 3 1 AMP-STR (5), AMP-CIP-NAL-(STR)-TET (6), KAN-NEO-SUL-TET (2) NI 4 AMP-STR (2), AMP-CIP-NAL-STR-TET (3), KAN-NEO-(STR)-SUL-TET (8), AMP-(AMC)-CEF-(CRO)-(CTF)-FOX-SPT-SUL (7) Susc. (18), AMP-CEF-CRO-CTF-FOX-CHL-SPT-SUL (1) 10 AMP-STR (2), AMP-CIP-NAL-(STR)-TET (3), KAN-NEO-(SPT)-(STR)-SUL-TET (6), AMP-(AMC)-CEF-CRO-(CTF)-FOX-SPT-SUL (8) Susc. (11), AMP-(AMC)-CEF-(CRO)-(CTF)-FOX-CHL-SPT-SUL (8) 4 1 AMP-STR (2), AMP-CIP-NAL-(STR)-TET (4), (AMP)-(GEN)-KAN-NEO-SPT-STR-SUL-TET (6) NI 4 AMP-STR (2), AMP-CIP-NAL-(STR)-TET (9), (AMP)-(CIP)-KAN-(NAL)-NEO-(SPT)-STR-SUL-TET (7) NI 10 AMP-STR (1), AMP-CIP-NAL-(SPT)-STR-TET (8), (AMP)-KAN-NEO-SPT-STR-SUL-TET (8) Susc. (1) ↵a See Table 1 for abbreviations for antimicrobials. Isolates were considered resistant to CIP when growth occurred on MH agar containing 0.125 μg of CIP/ml. Antimicrobials shown in parentheses are those to which the isolates may or may not be additionally resistant.
↵b NI, none isolated.
↵c Susc., susceptible.
- TABLE 5.
Trial 2 antimicrobial resistance patterns and isolation rates of E. coli and Salmonella serovar Newport
Pen Day Resistance pattern(s) (no. of isolates) ofa: E. coli Serovar Newport 1 2 NIb Susc.c(12) 4 NI Susc. (17) 10 Susc. (1), AMP-STR (1) Susc. (16), SUL (1) 2 2 NI Susc. (13) 4 NI Susc. (18) 10 SPT-SUL (18) Susc. (16), SUL (2) 3 2 AMP-AMC-CEF-CRO-(CTF)-FOX-SPT-SUL (13) Susc. (13) 4 Susc. (7), AMP-(AMC)-CEF-(CRO)-(CTF)-(FOX)-SPT-SUL-(TET) (11) Susc. (18) 10 Susc. (6), AMP-(AMC)-CEF-(CRO)-(CTF)-FOX-SPT-SUL (12) Susc. (5), AMP-(AMC)-CEF-(CRO)-(CTF)-(FOX)-CHL-SPT-SUL (13) 4 2 AMP-CIP-NAL-TET (2) NI 4 AMP-CIP-NAL-(STR)-TET (15), KAN-NEO-SUL-TET (2) NI 10 AMP-STR (4), AMP-CIP-NAL-(SPT)-STR-SUL-TET (10), KAN-NEO-(SPT)-STR-SUL-TET (3) NI - TABLE 6.
Relationships among serovars, antimicrobial resistances, and plasmid profiles of E. coli isolates
Trial Pen E.coli serovara Antimicrobial resistance patternb Plasmid profile (MDa)c 1 1 O8:H9 AMP-STR 2.8, 1.0 O77:H18 AMP-CIP-NAL-(STR)-(SUL)-TET 96, 60, 30, (1.0) O92:NM (AMP)-KAN-NEO-SPT-STR-SUL-TET 80, 66, 18, 4.8, 2.0 2 O8:H9 AMP-STR (80), (66), 2.8, 1.0 O77:H18 AMP-CIP-NAL-(STR)-(TET) 96, 60, 30 O92:NM (AMP)-KAN-NEO-SPT-STR-SUL-TET 80, 66, 18, 4.8, 2.0 3 O8:H9 AMP-STR 80, 66, 2.8, 1.0 O17:H18 KAN-NEO-SUL-TET NONE O77:H18 AMP-CIP-NAL-(STR)-TET 96, 60, 30 O168:H7 AMP-AMC-CEF-CRO-(CFT)-FOX-SPT-SUL 72, 66 4 O8:H9 AMP-STR 2.8, 1.0 O77:H18 AMP-CIP-NAL-STR-TET 96, 60, 30, (1.0) O92:NM (AMP)-(GEN)-KAN-NEO-(SPT)-STR-SUL-TET 80, 66, 18, 4.8, 2.0 2 1 O8:H9 AMP-STR 2.8, 1.0 O159:H28 Susc. None 2 O11:H11 SPT-SUL 90, 84 3 O101:H9 Susc. 96, 3.6, 3.0, 1.0 O101:H9 AMP-AMC-CEF-FOX-SPT-SUL 96 or 80, 72, 3.6, 3.0, 1.0 O168:H7 AMP-AMC-CEF-CRO-(CTF)-FOX-SPT-SUL 72, 66 4 O8:H9 AMP-STR 80, 66, 24, 2.8, 1.0 O17:H18 KAN-NEO-SUL-TET None O17:H18 AMP-CIP-NAL-TET 96, 60, 30 O92:NM KAN-NEO-(SPT)-STR-SUL-TET 80, 66, 18, 2.0 ↵a None of the E. coli isolates were verotoxigenic. NM, nonmotile.
↵b Antimicrobials shown in parentheses are those to which the isolates may or may not be additionally resistant.
↵c Those plasmids indicated in parentheses may or may not be present. The 72-MDa plasmid indicated in bold was the only one that hybridized with the cmy-2 probe.
