Characterization of a carbapenem-resistant Citrobacter amalonaticus coharbouring bla
IMP-4 and qnrs1 genes
Authors: Zhao Yunan , Chen Xuefeng , Hu Xiaolei , Shi Yang , Zhao Xinmi , Xu Jianfen , Ding Hui , Wu Rongzhen , Huang Jiansheng , Zhao Zhigang 
Published: June 2021

Introduction. Members of the genus

Citrobacter

are facultative anaerobic Gram-negative bacilli belonging to the

Enterobacterales

[Janda J Clin Microbiol 1994; 32(8):1850–1854; Arens Clin Microbiol Infect 1997;3(1):53–57]. Formerly, Citrobacter species were occasionally reported as nosocomial pathogens with low virulence [Pepperell Antimicrob Agents Chemother 2002;46(11):3555–60]. Now, they are consistently reported to cause nosocomial infections of the urinary tract, respiratory tract, bone, peritoneum, endocardium, meninges, intestines, bloodstream and central nervous system. Among

Citrobacter

species, the most common isolates are

C. koseri

and

C. freundii

, while

C. amalonaticus

has seldom been isolated [Janda J Clin Microbiol 1994; 32(8):1850–1854; Marak Infect Dis (Lond) 2017;49(7):532–9]. Further,

Citrobacter

spp. are usually susceptible to carbapenems, aminoglycosides, tetracyclines and colistin [Marak Infect Dis (Lond) 2017;49(7):532–9].

Hypothesis/Gap Statement. As

C. amalonaticus

is rare, only one clinical isolate, coharbouring carbapenem resistance gene bla
IMP-4 and quinolone resistance gene qnrs1, has been reported.

Aim. To characterize a carbapenem-resistant

C. amalonaticus

strain from PR China coharbouring bla
IMP-4 and qnrs1.

Methodology. Three hundred and forty nonrepetitive carbapenem-resistant

Enterobacterales

(CRE) strains were collected during 2011–2018. A carbapenem-resistant

C. amalonaticus

strain was detected and confirmed using a VITEK mass spectrometry-based microbial identification system and 16S rRNA sequencing. Minimum inhibitory concentrations (MICs) for clinical antimicrobials were obtained by the broth microdilution method. Whole-genome sequencing (WGS) was performed for antibiotic resistance gene analysis, and a phylogenetic tree of

C. amalonaticus

strains was constructed using the Bacterial Pan Genome Analysis (BPGA) tool. The transferability of the resistance plasmid was verified by conjugal transfer.

Results. A rare carbapenem-resistant

C. amalonaticus

strain (CA71) was recovered from a patient with cerebral obstruction and the sequences of 16S rRNA gene shared more than 99 % similarity with

C. amalonaticus

CITRO86, FDAARGOS 165. CA71 is resistant to β-lactam, quinolone and aminoglycoside antibiotics, and even imipenem and meropenem (MICs of 2 and 4 mg l−1 respectively), and is only sensitive to polymyxin B and tigecycline. Six antibiotic resistance genes were detected via WGS, including the β-lactam genes bla
IMP-4, bla
CTX-M-18 and bla
Sed1, the quinolone gene qnrs1, and the aminoglycoside genes AAC(3)-VIIIa, AadA24. Interestingly, bla
IMP-4 and qnrs1 coexist on an IncN1-type plasmid (pCA71-IMP) and successfully transferred to

Escherichia coli

J53 via conjugal transfer. Phylogenetic analysis showed that CA71 is most similar to

C. amalonaticus

strain CJ25 and belongs to the same evolutionary cluster along with seven other strains.

Conclusion. To the best of our knowledge, this is the first report of a carbapenem-resistant

C. amalonaticus

isolate coharbouring bla
IMP-4 and qnrs1.