Abstract

The genus Citrobacter is an opportunistic pathogen causing infections in animals, and the published data for its resistance to florfenicol are scarce. In this study, we investigated the antimicrobial susceptibility and molecular characteristics of florfenicol resistance genes among Citrobacter isolates from animal and relevant environmental samples and conducted a comparative analysis of a multidrug-resistant Citrobacter freundii strain isolated from a rabbit. Among 20 Citrobacter strains isolated from animal samples, resistance was most commonly observed to ampicillin (100%), tetracycline (75%), streptomycin (65%), florfenicol (60%), chloramphenicol (60%), and aztreonam (50%), while all the strains found in environmental samples were resistant to few antibiotics. The florfenicol resistance gene floR was detected in 12 isolates (48%, 12/25) from animal samples, and all of the floR-positive isolates were resistant to florfenicol with minimum inhibitory concentration (MIC) values ≥256 μg/mL. Sequencing and comparative analysis of the plasmids from a multidrug-resistant C. freundii isolate named R47 showed that the floR-containing region in the plasmid pR47-54 was a truncated transposon-like structure and could be found on both plasmids and chromosomes of bacteria of either animal or human origin. Furthermore, a range of antimicrobial and metal resistance genes associated with mobile genetic elements could be identified in pR47-54 and the other plasmid pR47-309 of C. freundii R47. These results provide in-depth views into the phenotypic and molecular characteristics of Citrobacter isolates recovered from animal and relevant environmental samples, as well as highlight the role horizontal gene transfer plays in the dissemination of plasmid-encoded resistance genes.

Highlights

  • The genus Citrobacter is a Gram-negative, nonsporulating, and facultative anaerobic bacillus of the family Enterobacteriaceae [1], which at present is divided into 15 genetically distinct genomospecies: 11 previously recognized species, C. freundii, C. koseri, C. amalonaticus, C. farmeri, C. youngae, C. braakii, C. werkmanii, C. sedlakii, C. rodentium, C. gillenii, and C. murliniae; and four recently described species, C. pasteurii, C. europaeus, C. bitternis, and C. portucalensis [2,3,4,5,6,7], commonly found in surface waters, sewage, soil, and intestinal microflora of animals and humans

  • The complete sequence of floR flanked by two integrons was present in the chromosomes of several Salmonella serovars [19, 30]; the floR-containing region in the plasmid pMBSF1 of E. coli was composed of three parts homologous to Tn5393, the floR plasmid from E. coli 10660 and Tn1721 [31], and the floRflanking regions in some bacteria were associated with previously described ISCR2 transposable elements [32]

  • 64% (16/25) were isolated from fish, 16% (4/25) from rabbits, 12% (3/25) from soil, and 8% (2/25) from sewage. These isolates consisted of 4 species, including C. freundii (72%, 18/25), C. murliniae (16%, 4/25), C. farmeri (8%, 2/25), and C. gillenii (4%, 1/25)

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Summary

Introduction

The genus Citrobacter is a Gram-negative, nonsporulating, and facultative anaerobic bacillus of the family Enterobacteriaceae [1], which at present is divided into 15 genetically distinct genomospecies: 11 previously recognized species, C. freundii, C. koseri, C. amalonaticus, C. farmeri, C. youngae, C. braakii, C. werkmanii, C. sedlakii, C. rodentium, C. gillenii, and C. murliniae; and four recently described species, C. pasteurii, C. europaeus, C. bitternis, and C. portucalensis [2,3,4,5,6,7], commonly found in surface waters, sewage, soil, and intestinal microflora of animals and humans. Citrobacter strains are closely associated with disorders in fish, which were first described as newly emerged fish pathogens in sunfish (Mola mola) by Sato et al [10]. Similar to other antimicrobial resistance (AMR) genes, the floR gene has been identified on numerous bacterial plasmids and chromosomes with various MGEs. For example, the complete sequence of floR flanked by two integrons was present in the chromosomes of several Salmonella serovars [19, 30]; the floR-containing region in the plasmid pMBSF1 of E. coli was composed of three parts homologous to Tn5393, the floR plasmid from E. coli 10660 and Tn1721 [31], and the floRflanking regions in some bacteria were associated with previously described ISCR2 transposable elements [32]

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