Citrobacter spp. are facultative anaerobic Gram negative bacteria within the family Enterobacteriaceae. Citrobacter spp. have been associated with nosocomial infections involving the urinary tract, liver, biliary tract, peritoneum, intestines, bone, respiratory tract, endocardium, wounds, soft tissue, meninges, and the bloodstream (Khorasani et al., 2008; Kumar et al., 2013; Liu et al., 2018b). C. freundii is the most frequently isolated Citrobacter species from a range of infections (Khorasani et al., 2008) and has also caused small outbreaks in healthcare settings (Nada et al., 2004; Mohanty et al., 2007; Samonis et al., 2009; Bai et al., 2012). C. koseri can cause meningitis and brain abscesses in neonates and central nervous system (CNS) infections in head trauma, facial fractures, post neurosurgical procedures, or immunocompromised individuals (Vaz Marecos et al., 2012; Chao et al., 2013; Lechowicz et al., 2017; Reyes et al., 2017). C. braakii has been reported to cause bacteriemia (Lai et al., 2010; Hirai et al., 2016; Oyeka and Antony, 2017).

Emergence of multidrug-resistant (MDR) Citrobacter strains is an increasing concern (Khorasani et al., 2008). MDR C. freundii strains have been associated with a higher rate of in-hospital mortality compared to susceptible strains (Deal et al., 2007). MDR Citrobacter spp. with production of β-lactamase (Amp-C), broad-spectrum β-lactamase, extended-spectrum β-lactamase (ESBL), or even carbapenemase has been reported by several international surveillance programs (Wang et al., 2000; Mohanty et al., 2007; Zhang et al., 2008; Samonis et al., 2009; Kanamori et al., 2011; Lee et al., 2015; Liu et al., 2018b). It has been reported that 39–48% of C. freundii isolates were resistant to broad-spectrum cephalosporins (ceftriaxone, ceftazidime), piperacillin, and piperacillin/tazobactam (Khorasani et al., 2008). Moreover, a few studies have reported C. freundii harboring carbapenemases, particularly metallo-β-lactamases (MBLs) or Klebsiella pneumoniae carbapenemase (KPC) types (Weile et al., 2007; Protonotariou et al., 2008; Zhang et al., 2008; Gaibani et al., 2013; Schweizer et al., 2019; Gobeille Paré et al., 2020; Räisänen et al., 2021). Quinolone resistance determinant including qnr and aac(6’)-Ib-cr genes have been reported in Citrobacter spp. (Park et al., 2007; Zhang et al., 2012). Numerous qnrB alleles have been detected, and about 40 qnrB variants are located on the chromosome of Citrobacter spp., especially C. freundii (Jacoby et al., 2014, Liao et al., 2015). Fluoroquinolone resistance is associated with mutations in gyrA and parC genes (Minarini and Darini, 2012). Mutations in gyrA were found in fluoroquinolone resistant C. freundii isolates (Weigel et al., 1998; Minarini and Darini, 2012).

Studies on Citrobacter spp. from extraintestinal infections have been mostly focused on antibiotic resistance, and little is known about their genetic diversity and virulence properties. Citrobacter spp. can be isolated from fecal samples of healthy individuals and can also cause food-borne infections (Tassew et al., 2010; Bai et al., 2012; Ifeadike et al., 2012; Minarini and Darini, 2012; Liu et al., 2017; Liu et al., 2018a; Liu et al., 2020). The possible source of strains causing extraintestinal infections and their relationships to strains from other infections and other sources have not been well studied. In our previous studies, we analyzed Citrobacter isolates from diarrheal patients, foods, and environment in China (Bai et al., 2012; Liu et al., 2017; Liu et al., 2018a; Liu et al., 2020). We found high diversity of Citrobacter strains from these sources in sequence types (STs), antibiotic resistance profiles, and virulence properties (Liu et al., 2017; Liu et al., 2018a; Liu et al., 2020). In this study, we collected Citrobacter spp. isolates from extraintestinal infections of inpatients in Maanshan people's hospital, Anhui Province, China and examined these isolates by multilocus sequence typing (MLST), antibiotic resistance profiling, and in vitro virulence testing to obtain an insight into their genetic diversity, antibiotic resistance, and virulence.

In this study, we surveyed Citrobacter extraintestinal infections in a hospital and found that Citrobacter isolates were mostly isolated from urine with 54.3% of the 46 isolates, followed by sputum samples (30.4%). Our findings were similar to those previously reported in India (Mohanty et al., 2007) and the USA (Hodges et al., 1978).

The 46 isolates were separated into 38 STs. The 38 STs from this study were compared with 566 STs from the Citrobacer MLST database, including isolates from all countries and sources. No ST in this study was shared with isolates from the database. In our previous study, 11 STs were shared with isolates from the database from other countries or regions or from different sources, and isolates of the same ST may be widely present in human fecal, food, and human extra intestinal samples (Liu et al., 2018a). We further analyzed the STs by CCs to ascertain any sharing of CCs and to determine whether there are any widely distributed CCs. The four CCs found in this study did not share any ST or CCs with other countries or other Chinese isolates. Fifteen CCs were found to be globally distributed. Some CCs contained human clinical isolates, including from diarrheal patient fecal samples and human extra-intestinal samples, which suggests that some CCs are more likely to cause diseases in humans.

A key strength of this study was that the population diversity and relationships of the isolates were assessed by MLST. The use of a standardized MLST scheme allowed comparison of data from this study with local and international MLST data from different sources. The combined MLST data from isolates from this study, isolates from human fecal samples and food samples from our previous studies, and other international isolates have revealed that there was no prevalent strains or clones, unlike many other bacterial pathogens such as UTI causing MDR E. coli ST131 with global distribution (Petty et al., 2014). However it is much needed of more studies using MLST or genome sequencing to better understand the genetic diversity and virulence of Citrobacter populations.

Clinical Citrobacter spp. strains are often resistant to multiple classes of antibiotics (Leski et al., 2016). Infections by MDR Citrobacter strains have been associated with a higher rate of in-hospital mortality compared to susceptible strains (Leski et al., 2016). Similarly, our study found that 54.3% of the isolates from extraintestinal infections were MDR with resistance to penicillin (84.8%), cephalosporins (67.4%), and azithromycin (65.2%), but susceptible to carbapenems (87.0%).

Carbapenem resistant Enterobacteriaceae (CRE) has become a major public health threat that requires urgent attention (Ramsamy et al., 2020). Carbapenem-resistant Citrobacter spp. isolates have been reported due to the acquisition of worldwide disseminated carbapenemases, such as New Delhi Metallo-β-lactamase (NDM), VIM-1, OXA-48, KPC-2, and VIM-2 (Hammerum et al., 2016; Arana et al., 2017; Faccone et al., 2019; Schweizer et al., 2019; Gobeille Paré et al., 2020; Räisänen et al., 2021). bla _NDM-1-positive C. freundii has been increasingly reported in China, India, Denmark, and South Africa (Yang et al., 2018) and VIM-1- and VIM-2-positive C. freundii have also been reported in Europe (Gaibani et al., 2013; Porres-Osante et al., 2014; Santos et al., 2017). In this study, four isolates were resistant to IMP or MEM. We did not determine the molecular mechanisms of carbapenem resistance of these four isolates which will be done in future studies. In our previous study, all isolates which were isolated from food, diarrheal patient fecal, healthy individual fecal, and environmental samples were susceptible to carbapenems (Liu et al., 2017; Liu et al., 2018a; Liu et al., 2020).

The prevalence of quinolone resistance and mutations of quinolone resistance genes varied among Citrobacter isolates. In our previous study, C. braakii had the highest proportion of quinolone resistant isolates (52.6%), followed by C. freundii with 23.7% (Liu et al., 2020). In this study, C. freundii had the highest proportion of quinolone resistant isolates. Citrobacter isolates with mutations in the quinolone resistance determining region of gyrA, including Thr83Ile and Asp87Asn, have shown reduced susceptibility to fluoroquinolones (Weigel et al., 1998; Minarini and Darini, 2012). In our previous studies (Liu et al., 2018a; Liu et al., 2020), four quinolone resistant C. freundii isolates had mutations in Thr59Ile, Gln111Arg, and Ile134Val. Twenty-seven quinolone resistant isolates carried mutations in Thr59Ile and one having three mutations in Thr59Ile, Gln111Arg, and Ile134Val. In this study, among eight quinolone resistant C. freundii isolates, all had the Thr59Ile mutation in the gyrA gene.

Cytotoxicity and adhesive ability in vitro were assessed for all isolates, which varied widely. Among the 50 isolates, only five and one were shown to be highly adhesive and highly cytotoxic, respectively. We did not find any association of cytotoxicity and adhesive ability with the source of the isolates (urine or sputum samples). Since all isolates were from clinical infections, it seems that in vitro cytotoxicity and adhesive ability of an isolate may not be indicative of their disease causing ability. However, the numbers of isolates were small and there were no patient data to determine whether any of these parameters is suggestive of more severe disease outcomes.

We analyzed 46 extraintestinal clinical Citrobacter isolates (26 C. freundii, 6 C. braakii, and 14 C. koseri isolates) from 2014 to 2018 in Maanshan people’s hospital of Anhui Province, China. The isolates showed high diversity with 38 STs, all of which were novel STs. Nine of the 38 STs belonged to four CCs, but no isolates or STs from this study shared the same CCs with isolates from other countries or other Chinese isolates reported. MDR was prevalent among the isolates causing extraintestinal infections at 54.3%, and four isolates (8.7%) were carbapenem resistant (IMP or MEM). All eight quinolone resistant C. freundii isolates carried the Thr59Ile mutation in the gyrA gene. Only a small proportion of the isolates were found to be highly cytotoxic and adhesive with no correlation to sample sources. This study has shed more light on the genetic diversity and antibiotic resistance of extraintestinal infection causing Citrobacter in China.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethical Committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, China (No. ICDC-2016007). The patients/participants provided their written informed consent to participate in this study.

LL and JX designed the project. YW and LZ carried out the sampling work. HZ, MY and HS carried out the experiments. LL, RL, and DH analyzed data. LL and RL drafted the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the National Key Research and Development Program of China (2019YFC1200505 and 2019YFC1200500) and grants from National Natural Science Foundation of China (No. 81301401).

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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