Carbapenem-Resistant Citrobacter spp. as an Emerging Concern in the Hospital-Setting: Results From a Genome-Based Regional Surveillance Study

Abstract

The rise of Carbapenem-resistant Enterobacterales (CRE) represents an increasing threat to patient safety and healthcare systems worldwide. Citrobacter spp., long considered not to be a classical nosocomial pathogen, in contrast to Klebsiella pneumoniae and Escherichia coli, is fast gaining importance as a clinical multidrug-resistant pathogen. We analyzed the genomes of 512 isolates of 21 CRE species obtained from 61 hospitals within a three-year-period and found that Citrobacter spp. (C. freundii, C. portucalensis, C. europaeus, C. koseri and C. braakii) were increasingly detected (n=56) within the study period. The carbapenemase-groups detected in Citrobacter spp. were KPC, OXA-48/-like and MBL (VIM, NDM) accounting for 42%, 31% and 27% respectively, which is comparable to those of K. pneumoniae in the same study. They accounted for 10%, 17% and 14% of all carbapenemase-producing CRE detected in 2017, 2018 and 2019, respectively. The carbapenemase genes were almost exclusively located on plasmids. The high genomic diversity of C. freundii is represented by 22 ST-types. KPC-2 was the predominantly detected carbapenemase (n=19) and was located in 95% of cases on a highly-conserved multiple-drug-resistance-gene-carrying pMLST15 IncN plasmid. KPC-3 was rarely detected and was confined to a clonal outbreak of C. freundii ST18. OXA-48 carbapenemases were located on plasmids of the IncL/M (pOXA-48) type. About 50% of VIM-1 was located on different IncN plasmids (pMLST7, pMLST5). These results underline the increasing importance of the Citrobacter species as emerging carriers of carbapenemases and therefore as potential disseminators of Carbapenem- and multidrug-resistance in the hospital setting.

Keywords: ARGs; Carbapenemase; Citrobacter; Germany; IncN-plasmid; MLST; WGS.

Figure 1

Phylogenetic tree of the 56 sequenced Citrobacter spp. isolates based on SNPs-analysis with the C. freundii strain ATCC 8090 (Access No. BBMV01000001) as reference genome, generated and annotated with the iTOL tool (https://itol.embl.de/tree/) (Letunic and Bork, 2021). The bacterial species is displayed with different branch line colors: C. freundii, black, C. portucalensis, orange, C. europaeus, blue, C. braakii, green, and C. koseri, red. The hospitals, from which the isolates were obtained, are indicated with the 16 different colored circles on the tree nodes. The different identified carbapenemase types are marked with different colors according to the isolate labels. ST, C. freundii MLST types of the C. freundii isolates. IncN[pMLST15]: the bla _KPC-2 gene-carrying plasmid are represented by an asterisk in the KPC-2 carbapenemase-producing isolates. IncL/M(pOXA-48): the Inc type of plasmids identified in the OXA-48-like carbapenemase-positive isolates and likely to carry their bla _OXA-48 or bla _OXA-162.

Figure 2

Genetic map of the completed genomes of the bla _KPC-2-encoding IncN[pMLST15] (A) and the distribution of the IncN pMLST15 plasmids according to the time from 2016 to 2019 and hospitals as well as the genome types and species (B). The plasmid pCF37969-KPC2 was almost identical to the early identified pCF13066-KPC2, which has been isolated from a multi-species carbapenem-resistant outbreak in 2014 in Hesse (separate study), and differed by a ~11 kb insertion. Each box in (B) indicates one isolate. The numbers in the boxes indicate the MLST-types of the C. freundii isolates as well as the symbol (*) for the species C. koseri and the symbol (^#) for C. portucalensis.

Figure 3

Genetic Map of IncA/C2 plasmid encoding a Tn4401 associated bla _KPC-3- carbapenemase.

Figure 4

Two types of the genetic surroundings of the bla _OXA-48 and bla _OXA-162. Type A and B differ only in the presence or absence of a repetitive 15bp sequence (GGTGATGCTGCCAAC). Type A was detected in 9 bla _OXA-48 or bla _OXA-162-encoding IncL/M(pOXA-48) plasmids from 8 C. freundii isolates, i.e. Survcare050, Survcare150, Survcare247, Survcare311, Survcare315, Survcare316, Survcare396 and Survcare410 and one C. braakii Survcare336, as well as in the non-IncL/M(pOXA-48)-bearing Survcare252, which harbored a bla _OXA-48 gene. Type B was present in four bla _OXA-48-harboring isolates, whereas two isolates (Survcare163 and Survcare314) were associated with the IncL/M(pOXA-48) plasmid and two (Survcare162 and NRZ-45233) were not.

Figure 5

Genetic map of the complete genome of the bla _VIM-1-encoding IncN[pMLST7] plasmid (A) and Depiction of insertion elements/transposons present in this plasmid (B). The picture B was created using the Galileo AMR software (Partridge and Tsafnat, 2018).