Genetic Diversity, Multidrug Resistance, and Virulence of Citrobacter freundii From Diarrheal Patients and Healthy Individuals

Liyun Liu; Daoli Chen; Liqin Liu; Ruiting Lan; Shuai Hao; Wenjie Jin; Hui Sun; Yiting Wang; Ying Liang; Jianguo Xu

doi:10.3389/fcimb.2018.00233

Genetic Diversity, Multidrug Resistance, and Virulence of Citrobacter freundii From Diarrheal Patients and Healthy Individuals

Front Cell Infect Microbiol. 2018 Jul 10:8:233. doi: 10.3389/fcimb.2018.00233. eCollection 2018.

Authors

Liyun Liu^{1

2}, Daoli Chen³, Liqin Liu⁴, Ruiting Lan⁵, Shuai Hao⁶, Wenjie Jin⁴, Hui Sun¹, Yiting Wang¹, Ying Liang¹, Jianguo Xu^{1

2}

Affiliations

¹ State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
² Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang, China.
³ Maanshan Center for Disease Control and Prevention, Ma'anshan, China.
⁴ School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, China.
⁵ School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.
⁶ Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, China.

Abstract

Objectives:Citrobacter freundii is a frequent cause of nosocomial infections and a known cause of diarrheal infections, and has increasingly become multidrug resistant (MDR). In this study, we aimed to determine the genetic diversity, the antimicrobial resistance profiles and in vitro virulence properties of C. freundii from diarrheal patients and healthy individuals. Methods: 82 C. freundii isolates were obtained from human diarrheal outpatients and healthy individuals. Multilocus Sequence Typing (MLST) of seven housekeeping genes was performed. Antimicrobial susceptibility testing was carried out using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Adhesion and cytotoxicity to HEp-2 cells were assessed. PCR and sequencing were used to identify bla_CTX-M, bla_SHV, bla_TEM, qnrA, qnrB, qnrS, qnrC, qnrD, aac(6')-Ib-cr, and qepA genes. Results: The 82 C. freundii isolates were divided into 76 sequence types (STs) with 65 STs being novel, displaying high genetic diversity. Phylogenetic analysis divided the 82 isolates into 5 clusters. All 82 isolates were sensitive to imipenem (IPM), but resistant to one or more other 16 antibiotics tested. Twenty-six isolates (31.7%) were multidrug resistant to three or more antibiotic classes out of the 10 distinct antibiotic classes tested. Five MDR isolates, all of which were isolated from 2014, harbored one or more of the resistance genes, bla_TEM-1, bla_CTX-M-9, aac(6')-Ib-cr, qnrS1, qnrB9, and qnrB13. All 11 qnrB-carrying C. freundii isolates belonged to cluster 1, and one C. freundii isolate carried a new qnrB gene (qnrB92). Six isolates showed strong cytotoxicity to HEp-2 cells, one of which was multidrug resistant. Conclusions:C. freundii isolates from human diarrheal outpatients and healthy individuals were diverse with variation in sequence types, antibiotic resistance profiles and virulence properties.

Keywords: Citrobacter freundii; adhesion; cytotoxicity; multidrug resistance; multilocus sequence typing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Adhesion
Cell Line
Cell Survival
Citrobacter freundii / classification*
Citrobacter freundii / drug effects
Citrobacter freundii / isolation & purification*
Citrobacter freundii / pathogenicity
Diarrhea / microbiology*
Disk Diffusion Antimicrobial Tests
Drug Resistance, Multiple, Bacterial*
Enterobacteriaceae Infections / microbiology*
Genetic Variation*
Hepatocytes / microbiology
Humans
Multilocus Sequence Typing