The increase in infections caused by carbapenem-resistant Enterobacterales (CRE) represents a worrying threat to patient safety and healthcare systems worldwide and patients with CRE infections oftentimes cannot be treated effectively with available antibiotics (Grundmann et al., 2017). In contrast to Carbapenem-resistant Klebsiella pneumoniae and other carbapenem-resistant species, such as Escherichia coli and Enterobacter spp. which are commonly detected in nosocomial settings (Brolund et al., 2019; Pitout et al., 2020), Citrobacter was not considered to be a classical nosocomial pathogen. Citrobacter species belong to a group of facultative, anaerobic, Gram-negative bacilli within the Enterobacteriaceae family. They are frequently found in water, soil, food, and intestines of animals and humans and are mostly recognized as environmental contaminants or harmless colonizers. However, outbreaks in hospital settings are described, mostly occurring in neonates, young children or immunocompromised patients (Lipsky et al., 1980; Liu et al., 2017). They are able to cause a wide spectrum of infections involving the urinary tract, liver, biliary tract, peritoneum, intestines, bone, respiratory tract, endocardium, meninges, and the bloodstream. In addition, Citrobacter spp. are now fast gaining importance as a clinical multidrug-resistant pathogen causing opportunistic nosocomial and community-acquired infections (Mohanty et al., 2007; Villa et al., 2013; Wang et al., 2015; Arana et al., 2017a; Oyeka and Antony, 2017; Yao et al., 2017; Babiker et al., 2020). The carriage of antibiotic-resistant C. freundii was a mortality risk factor for immunocompromised patients with bacteremia (Liu et al., 2018). Recruitment of mobile colistin resistance, e.g. of the mcr-1 gene, could further limit antibiotic therapeutic options (Hu et al., 2017; Giani et al., 2018).

In a regionally performed three-year-CRE-genomic surveillance study in the state of Hesse, Germany, 512 CRE isolates from 21 species, of which 368 were carbapenemase-producing Enterobacterales (CPE) underwent whole-genome analysis. Here we could demonstrate that the carbapenemase-gene-harbouring Citrobacter spp. contributed a high proportion among the CPE and the carbapenemase-producing Citrobacter freundii (CPC) was the third most frequently (~14%) identified species, following Klebsiella pneumoniae and Escherichia coli. CPC has increased in both absolute abundance and proportion in 2018 and 2019 compared to 2017, representing an emerging infection control and public health challenge (Yao et al. unpublished data). In the present study, the characteristics of carbapenem-resistant Citrobacter spp. are discussed in detail based on the results of the performed genomic analysis.

In this study, we conducted a retrospective review of genomic typing and epidemiology of CR Citrobacter spp. from hospital settings in a medium sized State with 6.2 Mio habitants in Germany over a period of more than three years. We analyzed the genomes of 56 Citrobacter spp. in the context of a CRGN project consisting of 512 Enterobacterales isolates. We found that rates of carbapenemase-producing Citrobacter spp. (CPCs) did increase over the study period, with 10.1%, 16.7% and 14.3% in 2017, 2018 and 2019, respectively, and that 27% of patients, from whom the CPCs were isolated, suffered from a clinical infection in contrast to a colonization. Our data revealed a large genetic diversity of Citrobacter spp. in the study region suggesting that the emergence was not only due to clonal transfer. We also found that the carbapenem resistance phenotype was mostly mediated by the acquisition of carbapenemases and interestingly, the carbapenemase-producing fraction in the Citrobacter spp. (CPCs), was the highest among all CR Enterobacterales including species Klebsiella pneumoniae, Escherichia coli and Enterobacter spp. in the same study period and region (Yao et al., unpublished data).

The current increase in CPCs, is in line with studies in Spain and the US, which indicates towards a sustained global trend (Arana et al., 2017a; Babiker et al., 2020). The patients´ carbapenem application was considered an associated risk factor in a long-term-study (Babiker et al., 2020). In our data, we found Citrobacter freundii as the predominant carbapenemase-producing species, which accounted for 83% of isolates, with the remaining 17% comprising of the species C. portucalensis, C. koseri, C. europaeus, C. braakii. The predominance of C. freundii and the distribution of other Citrobacter species were similar to previous studies (Arana et al., 2017b; Babiker et al., 2020). In our study, the C. freundii STs 19 and 22 seem to be emerging over the study period.

OXA-48 and VIM-1 are the most frequent carbapenemase types detected in Germany and K. pneumoniae, E. coli and Enterobacter spp. are the most affected species (Grundmann et al., 2017; Becker et al., 2018; Pfennigwerth, 2020). However, very little information is available about carbapenemase-producing Citrobacter freundii (Peter et al., 2014; Yao et al., 2014; Schweizer et al., 2019). Nine different carbapenemase types were detected among Citrobacter spp. isolates under study Their distribution, with a predominance of KPC-2 and OXA-48, differed substantially from studies in other countries or regions. While 59% VIM, 32% OXA-48 and 20% KPC-2 were found in the Citrobacter spp. in Spain, more than two-thirds KPC-3 were found in the isolates of Citrobacter species in a US study (Arana et al., 2017a; Babiker et al., 2020). This demonstrates that carbapenemase diversity varies according to the geographic location, as previously stated (Nordmann et al., 2011). We describe for the first time two C. freundii co-producing KPC-2 and either VIM-1 or VIM-4 carbapenemases in Germany. Some earlier studies have reported the co-production of two carbapenemases such as KPC-2 plus NDM-1 or VIM-1 plus OXA-48 in C. freundii and KPC-2 plus VIM-2 in K. pneumoniae (Feng et al., 2015; Falco et al., 2016; Arana et al., 2017a), but no Citrobacter spp. with KPC-2 and VIM. As acquisition of mcr-1 resistance gene to the last-line antibiotic colistin was reported in Citrobacter europaeus from a healthy child, the presence of this gene represented a potential for expanding the ARGs profile (Giani et al., 2018). However, mcr-1 could not be detected in the studied isolates.

In this study, 95% of the carbapenem-resistant Citrobacter spp. isolates were found to have carbapenemase-encoding genes, a rate higher than that reported in prior studies, which was roughly two-thirds (Arana et al., 2017a; Babiker et al., 2020). This was also higher than that of other Enterobacterales species e.g. E. coli, Klebsiella spp. and Enterobacter spp. in the same study period. This could be explained by the fact that in the studied isolates a high diversity of plasmids and a relatively high proportion of carbapenemase-encoding plasmids was found. In addition, plasmid-mediated carbapenemase transmission between Citrobacter spp. and other bacterial species could be demonstrated when the patients carried multiple bacteria with the same carbapenemase-encoding plasmids, such as the four cases mentioned above.

The analysed Citrobacter spp. isolates carried up to 22 ARGs, exclusive the carbapenemase genes reflecting their MDR phenotypes. This impressing accumulation of resistance traits is exceptional, compared to other Enterobacterales and might point towards these Citrobacter becoming a relevant reservoir of potentially transmissible resistance in the healthcare-setting. We predicted a few known-virulence genes such as an astA-like gene in one of the two OXA-162 positive C. freundii isolates as well as the cloacin-encoding gene ccl and the plasmid-encoded enterotoxin- encoding gene senB in the both C. koseri isolates.

All CR Citrobacter isolates, including the Non-carbapenemase-producing isolates harboured alternation/disruption of at least one gene of the outer membrane porine-encoding genes ompC and ompF. The absence of mutations or the presence of both intact genes ompC and ompF in all carbapenem-susceptible isolates (Arana et al., 2017a) conversely hints that these mutations could indirectly contribute to reduced susceptibility to carbapenems.

The analyses of the surrounding genetic structure of the carbapenemase-encoding genes and the identified respective Inc types of the carbapenemase-carrying plasmids in the Citrobacter spp. isolates suggests that about 70% of the identified carbapenemases could be characterized into three Inc groups, IncN, IncL/M and IncA/C2. We identified a KPC-3-encoding IncA/C2 plasmid for all five KPC-3 positives isolates, a KPC-2- encoding IncN[pMLST15] plasmid for 18 of 19 KPC-2-producing isolates and pMLST-7 IncN plasmids for two of six VIM-1-producing isolates. Furthermore, we predicted the IncL/M[pOXA-48] plasmid type for 12 of the 16 OXA-48 and OXA-162- carrying isolates.

The KPC-2-encoding IncN[pMLST15]-carrying isolates originated from eight different hospitals during a time period from 2016 to 2019 ( Figure 2B ). The whole-genome types of these isolates were heterogeneous. However, the mutually present KPC-2 IncN[pMLST15] plasmids were almost identical and occurred across the entire study period, independently of hospital-settings and bacterial genome types. The plasmid sequences were also highly homolog to a plasmid which had mediated a previous KPC-2 multi-species nosocomial outbreak in Hesse in 2014. In this context, we could observe that a patient who was involved in this previous outbreak was colonized with KPC-2 positive C. freundii harboring the same plasmid for a long period of time after the outbreak (first sample May 2014, followed Dec. 2014, June and Sep. 2015, Feb and June 2016) in the follow-up screening. It is particularly noteworthy that KPC-2 Citrobacter isolates with the same plasmid were consistently identified in this hospital during this study period. Furthermore, in a separate study, this plasmid was repetitively identified in different species (Yao et al., 2021 unpublished data). The Citrobacter spp. especially C. freundii seem to be frequent carriers of this plasmid and might even represent a specific reservoir. At least in our setting this plasmid seems to be both endemic and emerging and might be classified as a high-risk plasmid in terms of CR dissemination across different bacterial species. The reasons for this are difficult to determine and this impression might be biased by the study parameters. Therefore, further studies are needed to determine whether carbapenemase-producing Citrobacter spp. act as an intermediate reservoir to spread antibiotic resistance determinants to more virulent bacterial species or represent a more permanent reservoir for drug resistance accumulation with the risk of becoming pan-drug resistant pathogens themselves, and whether this multi-resistant KPC-2-bearing plasmid provides additional fitness advantage to the host bacteria.

Our study provides a molecular-epidemiological overview on CR Citrobacter spp. originating from hospitals in Hesse, a midrange federal state of Germany according to population size and geographical area, over a three-year time period, based on whole-genome analyses. To the best of our knowledge, this study was the first of its kind in Germany. We detected a moderate increase in CR Citrobacter spp. numbers between 2016 and 2019 with carbapenemase production being the most prevalent mechanism for carbapenem resistance (95%). Our data revealed several additional findings: (i). clonal and plasmid-mediated polyclonal and interspecies spread of carbapenemase-producing Citrobacter spp. across the region in different hospitals; (ii). Carriage of nine different carbapenemase enzymes of the groups KPC, OXA-48, NDM and VIM by five species of Citrobacter spp., especially C. freundii that comprised a large genetic heterogeneity; however, emerging sequence types could be demonstrated (iii). Genetic locations of almost carbapenemases on plasmids and other genetic mobile elements evidencing that the studied Citrobacter spp. acquired carbapenem resistance via horizontal gene transfer; (iv). Detection of two potential high-risk C. freundii clones ST19 and ST22; and (v). Several specific plasmid types for carbapenemase-carriage, e.g. the IncN[pMLST15] for KPC-2, have been determined. In conclusion, the points towards Citrobacter spp. being an emerging bacterial species in terms of Carbapenemase carriage, resistance accumulation and potential transmission of resistance traits within the hospital setting. The Citrobacter spp. isolates, while usually not regarded as a typical human colonizer or nosocomial pathogen, display an above-average propensity for resistance accumulation of mobile and transferable resistance traits, thereby potentially representing a still underestimated reservoir for CR dissemination. Thus, the carbapenem-resistant Citrobacter spp. are an emerging health care and public health challenge which must be studied further to determine their role in resistance accumulation, dissemination and persistence in particular concerning carbapenem resistance in the hospital but also in the general population one health context. Further specific genomic surveillance studies in the mentioned settings seem to be mandatory.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, BioProject PRJNA692829.

Ethical approval was sought at the Ethics Committee of the State Medical Association of Hesse in Frankfurt/Main. The Committee decided on the 24th of January 2018, that an ethical approval for the project was not necessary, as for this study patient data were rendered anonymous.

YY, TC, and CI designed the study. AH, PH, YY, CI, TC, JF, LF, and ED collected the data and samples. AH, PH, JF, LF, CI, TC, and ED performed phenotyping and WGS of the isolates. YY, AH, PH, CI, TC, JF, LF, and ED analyzed the data. YY and JF performed the Genomic analyses. YY, CI, and TC wrote the manuscript that was critically reviewed and approved by all authors. All authors contributed to the article and approved the submitted version.

This work was supported by the Bundesministerium fuer Bildung und Forschung (BMBF, Germany) within the German Center for Infection Research (DZIF/grant numbers, 8032808811, 8032808818, 8032808820 to TC/CI). Support was also obtained from the Hessian State Ministry for Social Affairs and Integration (HMSI) within the project SurvCARE Hessen and the Hessian Ministry of Higher Education, Research and Arts within the project HuKKH (Hessisches universitaeres Kompetenzzentrum Krankenhaushygiene). The funders of the study had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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