Nationwide molecular epidemiology of carbapenemase-producing Citrobacter spp. in France in 2019 and 2020

ABSTRACT Carbapenemase-producing Enterobacterales have emerged in the last decades as one of the main threats in modern medicine. Enterobacter is a genus increasingly recognized as a key player in carbapenemase diffusion besides Escherichia coli and Klebsiella pneumoniae, but information regarding Citrobacter spp. remained scarce. A collection of 803 isolates of Citrobacter spp. recovered from the French National Center for carbapenem resistance over a 2-year period during 2019–2020 was analyzed. A total of 15 different carbapenemases were identified of which OXA-48 followed by NDM-1 and OXA-181 represented the main enzymes. Phylogenetic analysis of this collection revealed that Citrobacter freundii was the main species among which three main clones are circulating in France, being ST8, ST22, and ST91. ST22 and ST8 were distributed in all regions whereas ST91 demonstrated a regional spread. Analysis of the species diversity revealed that among the C. freundii complex, Citrobacter portucalensis and Citrobacter braakii were also disseminated. We revealed the dissemination of a clone carrying both blaDHA-15 (β-lactamase-encoding gene rarely reported in Enterobacterales) and blaOXA-48 in the north of France and allowed to identify an outbreak not previously investigated. This study analyzed the largest collection of carbapenemase-producing Citrobacter spp. and allowed deciphering of molecular epidemiology of Citrobacter spp. in France. IMPORTANCE The emergence of carbapenemase producers in Enterobacterales mostly involves Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex species. However, in France, we observed the emergence and the rapid dissemination of carbapenemase in Citrobacter spp. In this study, we demonstrated that a wide variety of carbapenemases is produced by many different species of Citrobacter spp. However, we clearly identify three high-risk clones of Citrobacter freundii, ST8, ST22, and ST91 that drive the spread of carbapenemase in France. This epidemiological study paves the way of further analysis that would aim to identify the virulence factors involved in this pellicular ability of these three clones to disseminate at the hospital.

Inside this genus, C. koseri is genetically distant from C. freundii and the other subspecies (5).Moreover, C. koseri belongs to the group II Enterobacterales, as it produces a class A chromosomally encoded penicillinase that confers resistance to amino-, carboxy-, and ureido-penicillins (amoxicillin) and is inhibited by clavulanate or tazobactam (6).C. freundii and the other Citrobacter spp.belong to the group III Enterobacterales as they possess an Ambler class C chromosomally encoded cephalo sporinase (bla CMY-like ) conferring an intrinsic resistance to aminopenicillins, to classical β-lactamase inhibitors such as clavulanate or tazobactam, and first-generation cephalo sporins (7).Overexpression of bla CMY-like genes may lead to resistance to expanded-spec trum cephalosporins and even carbapenems, if associated with decreased permeability (7).
The aim of this study was to characterize the epidemiology of carbapenemase-pro ducing Citrobacter spp. received at the French National Reference Center (F-NRC) for CPEs in 2019-2020.

Strain collection
A total of 1,121 carbapenem non-susceptible Citrobacter spp.were received at the F-NRC for CPEs between January 1st, 2019, and December 31st, 2020.Clinical isolates were firstly identified by MALDI-TOF mass spectrometry (MALDI Biotyper, Wissembourg, France).
The carbapenemase activity was assessed using the updated Carba NP test as described (10), and the type of carbapenemase was identified by the lateral flow immunoassay (LFIA) NG-Test Carba5 (NG Biotech, Guipry, France) (11).

Whole-genome phylogeny
To assess the genetic diversity of Citrobacter spp., a phylogenetic tree was constructed based on the whole genome of the 803 carbapenemase-producing isolates (Fig. 1).As expected, it revealed that the main species corresponded to C. freundii sensu stricto.
However, WGS results revealed that several isolates sent to the F-NRC were incorrectly identified as C. freundii using MALDI-TOF, including isolates of C. europaeus, C. portucalen sis, C. cronae, and C. werkmanii (Fig. 1) This is likely due the inability of MALDI-TOF to distinguish between these C. freundii complex species.The more distant species, and correctly identified by MALDI-TOF, included C. koseri, C. farmeri, C. amalonaticus, and C. pasteurii.Among the two C. pasteurii identified, one was very distant and could be considered as C. pasteurii-like.Further characterizations are needed to decipher if it truly belongs to C. pasteurii or to a novel species.
C. freundii isolates of ST22 were the most prevalent (Fig. 1).They were mostly recovered from three regions being Paris area (Ile-de-France, n = 45), north-east of France (Hauts-de-France, n = 22), and south-east (Auvergne-Rhone-Alpes, n = 22).However, isolates belonging to ST22 were also from French island such as Réunion Island (Indian Ocean) (Fig. 2).The in-depth analysis of the phylogenetic tree of ST22 revealed a wide genetic diversity inside this ST.Indeed, the size of the branches demonstrated a broad genetic diversity (Fig. 2A).As an example, despite being part of the ST22, some isolates seemed more distantly related (e.g., 262H6 and 254F8 isolates).This genetic diversity might also be highlighted by the presence in the same branch of clearly unrelated isolates recovered from different regions.Inside this genetic diversity, few outbreaks can be evidenced, such as 9 OXA-48-producing isolates recovered from an outbreak in Hauts-De-France or 14 clonally related strains in the region of Lyon (highlighted in red in Fig. 2A).Regarding this last outbreak which involved 14 isolates in Lyon, it is worthy to note that 3 isolates produced VIM-1, 10 isolates produced OXA-48, and the remaining isolate co-produced both carbapenemases.It suggests a wide spread of this particular clone that independently or successively acquired VIM-1 and OXA-48 carbapenemases in a restricted area (same hospital).
The remaining main clone, ST8, has been identified in different regions of France and associated with different carbapenemase (Fig. 2C).It might indicate a wide spread of this clone rather than a recent dissemination as observed for ST91.

Antimicrobial susceptibility and carbapenemase production
Among the 1,121 carbapenem non-susceptible Citrobacter spp. received at the F-NRC for CPEs over the study period, 1,023 isolates produced a carbapenemase as revealed by Carba NP.The OXA-48-like carbapenemases were the most frequent, followed by NDM, VIM, KPC, and IMP as revealed by LFIA and further confirmed by WGS (Table 1).
Fosfomycin, tigecycline, and ceftazidime/avibactam remained in the susceptibility range for 90.7%, 92.4%, and 82.6%, respectively.Diameter distribution of ceftazidime/avi bactam revealed a bimodal distribution with a highly resistant population likely indicating a production of metal-β-lactamase.
Besides carbapenemase genes, numerous broad-spectrum β-lactamase-encoding genes were identified including ESBL-encoding genes such as eight different bla CTX-M alleles (bla CTX-M-1 , bla CTX-M-3 , bla CTX-M-9 , bla CTX-M-14 , bla CTX-M-15 , bla CTX-M-32 , bla CTX-  By contrast with bla DHA-1 gene identified in multiple STs, bla DHA-15 gene have only been identified in ST500.Epidemiological data revealed that all these ST500 C. freundii isolates were from the same city and thus revealed a loco-regional spread of a single clone.As described in Morganella, the natural progenitor of bla DHA -like genes, bla DHA-15 is associated to its transcriptional regulator LysR.Analysis of the genetic of the bla DHA-15 environment revealed the presence of a quinolone resistance protein qnrB4 close to this gene. Among ESBLs identified in this collection, bla GES-1 was present in 10 isolates including 8 C. freundii, 1 C. koseri, and 1 C. amalonaticus.This gene was identified in four different STs in the C. freundii complex being ST116 (n = 4), ST216 (n = 2), ST22 (n = 1), and ST118 (n = 1).Noticeably, the extended-spectrum class D bla OXA-35-like was identified in 22 isolates including 20 C. freundii and 2 C. portucalensis.Analysis of the genetic background identified a wide genetic diversity of STs (n = 10) carrying this gene.

Outbreak of DHA-15 and OXA-48-producing isolates in northern France
During the analysis of resistomes, some β-lactamase-encoding genes rarely reported in Enterobacterales, but commonly identified in Pseudomonas spp., caught our interest such as bla VIM-2 , bla GES-1 , and a rare plasmid-encoded cephalosporinase bla DHA-15 gene.The bla DHA-15 gene is present in GenBank database in only two sequences correspond ing to the initial description (GenBank accession number NG_049060) and a plasmidencoded sequence identified in C. freundii (GenBank accession number OW969688).In our collection, DHA-15 was present in nine isolates of C. freundii belonging to ST500.This ST500 was reported only once in PubMLST database (https://pubmlst.org/) in Poland in 2017.

DISCUSSION
This study characterized the epidemiology of carbapenemase-producing Citrobacter spp. at a genomic level and identified the most prevalent clones that circulated in France in 2019 and 2020.A total of 803 phenotypically characterized isolates were whole-genome sequenced.
Analysis of the resistome revealed that besides the wide dissemination of the bla OXA-48 gene and into a lesser extent bla NDM-1 , an unexpected high diversity of other beta-lactamases was identified in this collection.This includes the production of VIM-2 rarely described in Enterobacterales ( 16) and commonly identified in Pseudomonas spp.(17).Surprisingly, analysis of the genetic context surrounding the bla VIM-2 gene revealed a close genetic context with a plasmid identified in P. aeruginosa suggesting a genetic transfer between these two genus.Another particularity is the presence of the bla GES-1 gene in 10 isolates belonging to different Citrobacter species and the dissemination of the bla DHA-15 gene that seems to be correlated to the spread of C. freundii ST500 and responsible of several outbreaks in the north of France.
Analysis of the clones circulating in France revealed that three main clones were disseminating carbapenemase being ST8, ST22, and ST91.Despite that these clones are more prevalent, the drivers for their dissemination differ.Indeed, ST91 dissemination corresponded to a clonal expansion mainly within a single region whereas ST8 and ST22 seem to have disseminated in the entire country with a more genetic diversity.In addition, the dissemination of these two STs (ST8 and ST22) is uncorrelated to the production of a given carbapenemase suggesting that these STs can be defined as "high-risk" clones of multidrug resistance.This is the main caveat of this study.We should acknowledge that our collection included only carbapenemase-producing Citrobacter spp.isolates.To really define ST8 and ST22 as a high-risk clone, it will be needed to study antibiotic-susceptible Citrobacter spp. or ESBL producers, too.This additional investiga tion might help differentiate clones involved in carbapenemase dissemination from those that are intrinsically highly prevalent.Our results pave the way of the analysis of core genomes of these highly prevalent STs to identify features that might be associated with their success.
Finally, as previously observed for other Enterobacterales such as the Enterobacter cloacae complex (18)(19)(20), we demonstrated that MALDI-TOF is not precise enough to accurately identify C. freundii complex isolates at the species level.It might be of interest to use our collection of deeply characterized Citrobacter spp.isolates to increase the MALDI-TOF database to see whether specific markers may help separate these species.

FIG 1
FIG 1 Global phylogeny of carbapenemase-producing Citrobacter spp.isolated in France in 2019 and 2020.

TABLE 1
Distribution of carbapenemases by Citrobacter species

TABLE 2
Resistance profiles of Citrobacter spp.included in this study a S, susceptible; I, susceptible at high exposure; R, resistant.b N/A, not available.