Characterization of Fluoroquinolone-Resistant Escherichia coli Causing Septicemic Colibacillosis in Calves in Italy: Emergence of a Multiresistant O78 Clonal Group

Bacterial strains

Forty-seven E. coli displaying reduced susceptibility to enrofloxacin (zone diameter ≤18 mm, enrofloxacin disc 5 μg) were selected from 137 strains causing septicemic colibacillosis in young calves [mean age, 9.87 days±9.88 (mean±standard deviation)]. All strains were isolated from blood and tissue samples (spleen, liver, and bone marrow) from septicemic animals bred in 45 widely dispersed large-scale farms in Piemonte, a region in the northwest of Italy. Strains were consecutively isolated during the years 2006–2008 at the Istituto Zooprofilattico Sperimentale in Turin, Italy, and then referred to the Microbiology Section (DISC Department) of the University of Genoa, Italy, where they were reidentified to the species level using the API 20E system (bioMèrieux, Marcy l'Etoile, France) and stored at −80°C until further analysis.

Susceptibility tests

Susceptibility to selected antimicrobial agents was determined by using the disk diffusion test and interpreted according to the Clinical Laboratory Standards Institute (CLSI) criteria for bacteria isolated from animals, or, when not available, according to the CLSI criteria for bacteria isolated from humans. ⁸ Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality-control strains.

β-Lactamase studies

To detect extended-spectrum β-lactamase (ESBL) production, the CLSI ESBL initial screen test was carried out. ⁷ To detect plasmid-mediated ampC β-lactamase, a phenotypic test (disk-based inhibition screening with cefoxitin and cloxacillin) and PCR amplification were carried out as previously described.^18,20 All strains were screened for the presence of bla _TEM , bla _SHV , and bla _CTX-M genes by PCR using primers and conditions as previously reported. ⁵

O serotyping

O serotyping was performed using E. coli O antisera purchased from Denka Seiken, Co Ltd (Tokyo, Japan), according to the manufacturers' instructions.

Virulence genotyping

All of the isolates were screened for the virulence markers astA, pap, iucD, afaD8/E8, eae, bfpA, cnf1/2, and stx1/2 using previously described primers and amplification conditions.^2,15,16

Phylotyping

E. coli phylogenetic group (A, B1, B2, or D) was determined by an established multiplex PCR assay described by other authors and as reported previously. ⁵

Screening for plasmid-mediated quinolone resistance genes

All isolates were screened for the presence of qnr (qnrA, qnrB and qnrS), aac(6′)-Ib-cr, and qepA genes using previously described primers and amplification conditions.^5,15 Plasmids pmG252 (qnrA 1), pmG298 (qnrB 1), pmG306 (qnr S 1), pPSTV28 (qepA), and pBCSK(+) (aac(6′)-Ib-cr) were used as positive controls.

Randomly amplified polymorphic DNA and pulsed-field gel electrophoresis

Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) analysis, and the interpretation of results (banding patterns) was carried out as previously described. ⁵

Outer membrane protein profiles

To ascertain if resistance to third-generation cephalosporins and cefoxitin (strain E. coli Eco 28) was due to impermeability, outer membrane protein (OMP) profiles analysis was carried out. OMP preparations were obtained after sonic disruption of bacterial cells grown in nutrient broth, followed by selective solubilization of cytoplasmic material with sodium N-lauroyl sarcosinate (2% w/v) and ultracentrifugation as previously described. ⁵ The preparations were run on sodium dodecyl sulfate–polyacrylamide gels (12%) and stained with Coomassie Blue. Escherichia coli strain HB 101 and E. coli ATCC 25922 were used as fluoroquinolone-susceptible controls.

PCR amplification of the E. coli Eco 28 ampC promoter

Primers AB1 (5′-GATCGTTCTGCCGCTGTG-3′) and AmpC2 (5′-GGGCAGCAAATGTGGAGCAA-3′) were used to amplify a 271-bp fragment from the E. coli ampC promoter, as described by Caroff et al. ⁶ The PCR products were purified and sequenced as previously described. ⁵

PCR amplification and DNA sequencing of gyrA and parC

The gyrA and parC gene fragments of four O78 E. coli strains, representing four nonidentical PFGE profiles and four different levels of fluoroquinolone resistance [ciprofloxacin minimum inhibitory concentration (MIC) of 0.5, 4, 32, and >128 mg/L, respectively] were amplified by using previously described PCR conditions and primers. ⁵ PCR amplimer purification and sequencing were carried out as described above.

Conjugation experiments

Conjugal transfer of ampicillin, tetracycline, co-trimoxazole, gentamicin, and thiamphenicol resistance among all strains and E. coli K12 J53 Rif ^R was attempted by filter mating. Transconjugants were selected on rifampicin (256 mg/L) and either ampicillin (100 mg/L) or tetracycline (10 mg/L), or chloramphenicol (20 mg/L) or gentamicin (2 mg/L) or trimethoprim (5 mg/L).

Plasmid extraction

Plasmid DNA was extracted by the alkaline lysis method. ¹⁹

Susceptibility tests

Table 1 shows the disk diffusion susceptibility patterns of E. coli strains with reduced susceptibility to enrofloxacin. Third-generation cephalosporins (cefotaxime and ceftazidime) emerged as the most active drugs tested (97.9% of susceptible strains). Only one strain (Eco 28) was resistant to ceftazidime and cefotaxime. Overall, >90% of the strains were susceptible to cefoxitin. In contrast, the activity of the remaining antimicrobial agents was dramatically reduced (<50% of susceptible strains).

All microorganisms studied carried resistance to two or more additional drugs, with the pattern fluoroquinolone–ampicillin–co-trimoxazole–tetracycline–gentamicin–thiamphenicol being the most represented (18 out of 47 strains; 38.3%), (Table 2). All strains belonging to the O78 clonal group were simultaneously resistant to enrofloxacin, ampicillin, co-trimoxazole, tetracycline, and thiamphenicol.

β-Lactamase studies

The only strain simultaneously resistant to cefotaxime, ceftazidime, and cefoxitin (Eco 28) was positive by the CLSI ESBL initial screen test, but displayed the AmpC phenotype. This strain was PCR negative for AmpC plasmid-mediated genes. Molecular characterization by PCR showed that none of the strains carried bla _SHV , bla _CTX-M , genes, whereas 41 strains carried a bla _TEM gene.

Serotyping

Thirty-nine out of the 47 strains were typeable, 6 were autoagglutinating, and 2 were untypeable. A high variability was found among our collection (18 different serotypes/39 typeable strains). Overall, the most common serogroup was O78 (11 strains), followed by O111 (4) and O63 (3) (Table 2).

Detection of virulence-associated genes

The isolates produced one or more of the following potential virulence factors: The astA gene was detected in 18 strains, 19 possessed the pap gene, 31 the iucD gene, and 14 the afaD8/afaE8 genes. No strain was found positive for the other virulence-associated genes tested.

Phylogenetic groups

Approximately 70% of the strains (72.3 %) belonged to the less-virulent commensal phylogenetic group A and the remaining 27.7% to virulent phylogenetic group D, as shown in Table 2.

Quinolone plasmid-mediated resistance

By PCR analysis, isolates carrying qnr-like or aac(6′)-Ib-cr and qepA genes were not detected.

RAPD PCR profiles

All 47 strains underwent RAPD PCR with two different primers. Overall, 37 different profiles could be distinguished by this method, The most-represented RAPD profile, designated A, was related to serogroup O78 and was found in seven strains. Three strains belonging to serogroup O63 had the same RAPD profile (Table 2).

PFGE profiles

Finer resolution of the clonal relationship among the 11 O78 E. coli strains and the 3 O63 E. coli strains was obtained by PFGE analysis. Seven O78 strains were related (differing from 0 to 6 bands). The remaining 4 O78 strains had a different unrelated profile. Of the 3 O63 E.coli strains, 2 had a related profile (Fig. 1).

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FIG. 1.

Pulsed-field gel electrophoresis (PFGE) patterns of O78 (A) and O63 (B) Escherichia coli studied. Lanes 1, 13, 14, and 18, molecular weight standards; lanes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, E. coli Eco 12, Eco 17, Eco 50, Eco 26, Eco 69, Eco 39, Eco 34, Eco 4, Eco 42, Eco 67, and Eco 23; lanes 15, 16, and 17, E. coli Eco 27, Eco 54, and Eco56.

OMP profiles

The only cefoxitin-resistant E. coli (Eco 28) isolate expressed both the OmpC and OmpF proteins. Because OMP loss was not observed in this strain, its phenotype was compatible with hyperproduction of chromosomal cephalosporinase.

PCR amplification of the E. coli Eco 28 ampC promoter

The promoter of E. coli Eco 28 differed from strain K 12 at bases −88, −82, −42, −18, −1, and +58. All of these mutations have been already described in E. coli strains characterized by increased ampC transcription and hyperproduction of chromosomal cephalosporinase. ⁶

PCR amplification and DNA sequencing of gyrA and parC

Compared to the amino acids in wild-type E. coli K12, one out of the four isolates analyzed (ciprofloxacin MIC 0.5 mg/L) had a single mutation in gyrA, resulting in alterations at positions Ser-83 (Ser→Leu). Two isolates with ciprofloxacin MIC 4 and 16 mg/L, respectively, possessed two mutations in gyrA and one mutation in parC, resulting in alterations at positions Ser-83 (Ser→Leu) and Asp-87 (Asp acid→Asn) in GyrA and positions Ser-80 (Ser→Ile) in ParC, respectively. The fourth strain (ciprofloxacin MIC >128 mg/L) had two mutations in gyrA (the same observed in the other strains studied) and two mutations in parC, resulting in alterations at position Ser-80 (the same observed in the other strains studied) and position Glu-84 (Glu→Val).

Plasmid analysis

Conjugation experiments and antimicrobial susceptibility testing of the transconjugants revealed that only 4 out of 47 (8.5%) strains were able to transfer ampicillin and tetracycline resistance to the recipient. Ampicillin resistance and tetracycline resistance were transferred simultaneously. No detectable transfer was observed for the other resistance markers tested (trimethoprim, gentamicin, and chloramphenicol). The plasmid profile analysis of the four transconjugants revealed three distinct profiles.