Characterization of a carbapenem-resistant Citrobacter amalonaticus coharbouring bla IMP-4 and qnrs1 genes

Abstract

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.

Keywords: Citrobacter amalonaticus; carbapenem resistance; whole-genome sequencing analysis.