Characterization of multidrug-resistant Escherichia coli isolates associated with nosocomial infections in dogs

Abstract

Multidrug-resistant opportunistic pathogens have become endemic to the veterinary hospital environment. Escherichia coli isolates resistant to 12 antibiotics were isolated from two dogs that were housed in the intensive care unit at The University of Georgia Veterinary Teaching Hospital within 48 h of each other. Review of 21 retrospective and prospective hospital-acquired E. coli infections revealed that the isolates had similar antibiotic resistance profiles, characterized by resistance to most cephalosporins, beta-lactams, and the beta-lactamase inhibitor clavulanic acid as well as resistance to tetracycline, spectinomycin, sulfonamides, chloramphenicol, and gentamicin. E. coli isolates with similar resistance profiles were also isolated from the environment in the intensive care unit and surgery wards. Multiple E. coli genetic types were endemic to the hospital environment, with the pulsed-field gel electrophoresis fingerprint identified among E. coli isolates from diseased animals and the hospital environment matching. The extended-spectrum cephalosporin resistance in these nosocomial E. coli isolates was attributed to the cephamycinase-encoding gene, bla(CMY2). Chloramphenicol resistance was due in part to the dissemination of the florfenicol resistance gene, flo, among these isolates. Resistance encoded by both genes was self-transmissible. Although bla(CMY2) and flo were common to the polyclonal, nosocomial E. coli isolates, there was considerable diversity in the genetic compositions of class 1 integrons, especially among isolates belonging to the same genetic type. Two or more integrons were generally present in these isolates. The gene cassettes present within each integron ranged in size from 0.6 to 2.4 kb, although a 1.7-kb gene cassette was the most prevalent. The 1.7-kb gene cassette contained spectinomycin resistance gene aadA5 and trimethoprim resistance gene dfrA17.

FIG. 1.

E. coli genetic types identified by PFGE. (A) Patterns obtained by PFGE with XbaI. Lanes 1 and 14, S. cerevisiae DNA standards (BioWhittaker Molecular Applications, Rockland, Maine); lane 3, PFGE pattern H1 (isolate 29); lane 4, H1.2 (isolate 42614A); lane 5, H1.4 (isolate 38); lane 6, I (isolate 4517A); lane 7, J (isolate A1-72); lane 8, K (isolate 27315); lane 9, L (isolate 40362); lane 10, M (isolate 42614B); lane 11, N (isolate 1888); lane 12, O (isolate 29610); and lane 13, P (isolate 21). (B) Patterns obtained by PFGE with BlnI. Lanes 1 and 15, S. cerevisiae DNA standards (BioWhittaker Molecular Applications); lane 3, PFGE pattern A1 (isolate 29); lane 4, A2.1 (isolate 38); lane 5, A2.2 (isolate 4479); lane 6, A2.3 (isolate 4517A); lane 7, B1 (isolate 27315); lane 8, B1.1 (isolate 42614A); lane 9, B1.2 (isolate A1-72); lane 10, C (isolate 29610); lane 11, D (isolate 42614B); lane 12, E (isolate 22559); lane 13, F (isolate 1888); and lane 14, G (isolate 21). E. coli K-12 LE392 in panels A and B (lanes 2) served as an internal control for the reproducibility of every PFGE run. Alphabetical designations were assigned to PFGE patterns with seven or more band differences (56). The numbers represent four to six band differences within a PFGE pattern, and the numbers following decimal points designate slight genetic differences of one to three bands.

FIG. 1.

E. coli genetic types identified by PFGE. (A) Patterns obtained by PFGE with XbaI. Lanes 1 and 14, S. cerevisiae DNA standards (BioWhittaker Molecular Applications, Rockland, Maine); lane 3, PFGE pattern H1 (isolate 29); lane 4, H1.2 (isolate 42614A); lane 5, H1.4 (isolate 38); lane 6, I (isolate 4517A); lane 7, J (isolate A1-72); lane 8, K (isolate 27315); lane 9, L (isolate 40362); lane 10, M (isolate 42614B); lane 11, N (isolate 1888); lane 12, O (isolate 29610); and lane 13, P (isolate 21). (B) Patterns obtained by PFGE with BlnI. Lanes 1 and 15, S. cerevisiae DNA standards (BioWhittaker Molecular Applications); lane 3, PFGE pattern A1 (isolate 29); lane 4, A2.1 (isolate 38); lane 5, A2.2 (isolate 4479); lane 6, A2.3 (isolate 4517A); lane 7, B1 (isolate 27315); lane 8, B1.1 (isolate 42614A); lane 9, B1.2 (isolate A1-72); lane 10, C (isolate 29610); lane 11, D (isolate 42614B); lane 12, E (isolate 22559); lane 13, F (isolate 1888); and lane 14, G (isolate 21). E. coli K-12 LE392 in panels A and B (lanes 2) served as an internal control for the reproducibility of every PFGE run. Alphabetical designations were assigned to PFGE patterns with seven or more band differences (56). The numbers represent four to six band differences within a PFGE pattern, and the numbers following decimal points designate slight genetic differences of one to three bands.

FIG. 2.

E. coli genetic types identified by ERIC PCR. Lanes 1 and 20, 100-bp ladder (Roche Molecular Biochemicals, Indianapolis, Ind.); lane 3, ERIC pattern II (isolate 29); lane 4, RR (isolate 32); lane 5, LL (isolate 4479); lane 6, NN (isolate 4517A); lane 7, VV (isolate 37); lane 8, KK (isolate 56); lane 9, SS (isolate 42614B); lane 10, QQ (isolate 1745); lane 11, TT (isolate 12279); lane 12, OO (isolate 29610); lane 13, JJ (isolate 21); lane 14, UU (isolate 18397); lane 15, PP (isolate A1-72); lane 16, MM (isolate 38); lane 17, WW (isolate 55-1); lane 18, empty; and lane 19, no-DNA control. E. coli K-12 LE392 (lane 2) served as an internal control for the reproducibility of every ERIC PCR run. Since no interpretative criteria have been developed for analysis of the patterns obtained by ERIC PCR, DNA patterns with one or more band differences were given a different alphabetical designation.

FIG. 3.

E. coli genetic types identified by RAPD analysis with primer 1290. Lanes 1 and 22, 100-bp ladder (Roche Molecular Biochemicals); lane 2, RAPD 1290 pattern R (isolate 29); lane 3, FF (isolate 21411); lane 4, T (isolate 4479); lane 5, EE (isolate 23120); lane 6, U (isolate 22559); lane 7, Z (isolate 42614B); lane 8, Y (isolate 40362); lane 9, S (isolate A1-72); lane 10, GG (isolate 37); lane 11, HH (isolate 55-1); lane 12, V (isolate 4517A); lane 13, AA (isolate 1888); lane 14, Q (isolate 39737); lane 15, DD (isolate 18397); lane 16, X (isolate 27315); lane 17, BB (isolate 29610); lane 18, CC (isolate 12279); lane 19, W (isolate 1745); and lane 21, no-DNA control. E. coli K-12 LE392 (lane 20) served as an internal control for reproducibility of every RAPD analysis run. Since no interpretative criteria have been developed for analysis of RAPD PCR patterns, DNA patterns with one or more band differences were given a different alphabetical designation.

FIG. 4.

Plasmid profiles of canine E. coli isolates. Lanes 1 and 24, supercoiled DNA ladder; lane 2, V517, plasmid control strain; lane 3, isolate A1-72; lane 4, isolate 1745; lane 5, isolate 1888; lane 6, isolate 4479; lane 7, isolate 4517A; lane 8, isolate 12279; lane 9, isolate 18397; lane 10, isolate 18813; lane 11, 22559; lane 12, isolate 25055a; lane 13, isolate 25055b; lane 14, isolate 25055a; lane 15, isolate 27315; lane 16, isolate 29610; lane 17, isolate 39737; lane 18, isolate 40362, lane 19, isolate 42614A; lane 20, isolate 42614B; lane 21, isolate 22; lane 22, isolate 34; and lane 23, isolate 53. The asterisk indicates the position of chromosomal DNA on the gel.

FIG. 5.

Gene cassette(s) in class 1 integrons of nosocomial E. coli. The gene cassette(s) present within the class 1 integron integration site (attI) was amplified by PCR with oligonucleotide primers specific for conserved sequences 5′ and 3′ of the attI site (32). PCR amplifications were done with canine E. coli isolates positive for the intI1class 1 integrase gene, a marker for class 1 integrons. Lanes 2 to 10, canine E. coli isolates with representative PCR amplicons associated with class 1 integrons in nosocomial isolates; lane 1, 1-kb ladder (Roche Molecular Biochemicals); lane 2, E. coli isolate 1745; lane 3, isolate 1888; lane 4, isolate 4479; lane 5, isolate 4517A; lane 6, isolate 12279; lane 7, isolate 18397; lane 8, isolate 21411; lane 9, isolate 39737; lane 10, isolate 38; and lane 11, no-DNA control.