FosA3 emerging in clinical carbapenemase-producing C. freundii

Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales, but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii. The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3, blaKPC-2, and blaVIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3-harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C. freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales, affecting even low-virulence pathogens, as C. freundii.


Introduction
Carbapenem-resistant Enterobacteriaceae (CRE) infections, which present a considerable challenge for clinicians, are an increasing global threat.Currently, combination therapy based on carbapenem and fosfomycin is a valid option for the treatment of CRE infections (Bakthavatchalam et al., 2020).
Fosfomycin (FOS) is a phosphoric acid derivate, active against both Gram-negative and Gram-positive bacteria, which regained clinical interest in the last 20 years as a valid candidate in the treatment of multidrug-resistant (MDR) infections (Dijkmans et al., 2017).Currently, FOS is indicated for the treatment of uncomplicated UTIs, whereas the parenteral FOS has been used in case of systemic infections caused by MDR organisms (Falagas et al., 2009).FOS used in combination therapy is usually associated with good clinical outcome and bacteriological cure (Shiju et al., 2020).However, in recent years, an increased rate of resistant bacteria has been reported globally, mainly due to FOS-modifying enzymes (such as FosA).
As of February 2024, 10 fosA variants have been reported in the members of Enterobacterales.Plasmid-mediated dissemination of fosA-like genes is recognized as a worrying new challenge for the public health; fosA3 is the most widespread variant, with endemic cases reported from both veterinary and clinical settings in China (Singkham-In et al., 2020;Zhang et al., 2020;Wang et al., 2021;Zou et al., 2021;Mattioni Marchetti et al., 2023a).
Citrobacter spp.are considered as low-risk pathogens, yet can act as silent reservoirs for relevant resistance genes, especially in case of Citrobacter freundii (Bitar et al., 2019).Recent evidence has suggested that the rate of infections caused by carbapenemase-producing Citrobacter spp. is increasing, with relevant reports among Mediterranean countries (Yao et al., 2021;Nobrega et al., 2023).In this scenario, high FOS MICs may further impair antibiotic effectiveness.The co-occurrence of carbapenemases and FosA in Citrobacter spp. is scarcely reported in the literature, with the sole clinical case from the Czech Republic (Mattioni Marchetti et al., 2023b).
Therefore, the aim of our study is to molecularly characterize an XDR carbapenemase-producing C. freundii isolate with high FOS MIC.

Identification of the bacterial isolate, susceptibility determination, and detection of enzymes
Identification of the C. freundii strain (AK867) was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) with MALDI Biotyper software (Bruker Daltonics, Bremen, Germany).The production of carbapenemases (metallo-b-lactamase, OXA-48, and KPC) was assessed with the ROSCO test and with the NG-Test CARBA 5 immunochromatographic assay (NG Biotech Laboratories) (Chudejova et al., 2021).FOS MICs were evaluated using ADM and interpreted according to EUCAST clinical breakpoints v 13.0 and the new v 14.0, whereas the production of FosA-like and FosC2 enzymes was detected by the PPF test (Nakamura et al., 2014).In accordance with Nakamura in-house protocol, the PPF test requires MH agar plates added with 25 mg/L glucose-6-phosphate (G6P), confluence growth of 0.5 MacFarland solution of the isolate to investigate, one disk of FOS (50 mg), and one of FOS (50 mg) plus PPF (1 mg).The cutoff was set to a 5-mm enlargement in the inhibition zone of FOS plus PPF disk compared with the FOS disk alone (Nakamura et al., 2014;Mattioni Marchetti et al., 2023a).

Long-read sequencing
For genomic characterization, genomic DNA was extracted using the NucleoSpin Microbial DNA kit (Macherey-Nagel, Duren, Germany) and sheared using the Hydropore-long on Megaruptor 2 (Diagenode).Microbial multiplexing library preparation was performed without size selection according to the manufacturer's instructions.The multiplexed library was sequenced using long-read sequencing technology using the Sequel I platform (Pacific Biosciences, Menlo Park, CA, USA) for a 10-h movie run.Assembly was performed using the "Microbial Assembly" pipeline offered by the SMRT Link v10.0.with the default settings (minimum seed coverage of 30×).Assembled sequences were annotated using the RAST (Rapid Annotation using Subsystems Technology) server (Aziz et al., 2008).In-silico multilocus sequence typing (MLST) of the strains and of the plasmids (pMLST) were performed when applicable.Reconstruction of the resistome, plasmidome, and virulome of the isolates was accomplished using ResFinder, PlasmidFinder, and the Virulence Factors Database (VFDB) via ABRicate (github.com/tseemann/ABRicate).BRIG v.0.95 was used to produce figures of comparison of the circular plasmids' sequences.A linear map of chromosomal environments was created by using Easyfig (Sullivan et al., 2011) and the graphic editor Procreate (Savage Interactive, Tasmania, Australia).

Phylogenetic analysis
Phylogenetic relationships between AK867 and 117 global genomes, downloaded from the NCBI assembly database, including complete and draft genomes, were investigated.SNP-based phylogeny was depicted using parsnp v1.2 (Treangen et al., 2014) and using randomly GCF_029840125.1 as the reference genome.A graphic illustration of the trees was built with the Interactive Tree Of Life (iTOL) (https://itol.embl.de/).The clustering of genetic sequence was performed on the total pool of C. freundii ST116 by the FastBaps algorithm (Tonkin-Hill et al., 2019).

Data access
The plasmid sequence of pfosA3_CFR867 has been uploaded to GenBank under the accession number CP151860-CP151866.

Isolation and antimicrobial susceptibility profile
On 31/01/2023, a C. freundii (AK867) from rectal swab was collected.The sample was part of ongoing 3-year surveillance on carbapenemase-producing Enterobacterales (CPE) conducted locally at Modena Hospital in Italy.AK867 was isolated from a 41-year-old patient admitted in Modena Hospital, suffering from fever due to inguinal abscess by anaerobic bacteria.AK867 showed an extensively drug-resistant (XDR) phenotype, being susceptible only to colistin.FOS MIC was evaluated by ADM (FOS MIC >128 mg/L).The high FOS MIC was corroborated by the production of FosA-like enzymes, as suggested by a positive phenotypic PPF test.

Phylogenetic analysis
The SNP-based phylogeny on the 118 C. freundii ST116 genomes downloaded from the NCBI pointed out the presence of three clades (CL1, CL2, and CL3), confirmed by the FastBaps algorithm (Figure 3).AK867 falls into CL3 and clustered together with GCF_032192735.1, collected in 2017 from a German patient, and GCA_028404165.2, collected in 2023 from an American patient.According to the available metadata, the three clades are circulating worldwide since 2012, with CL3 as the predominant clade.Referring to the antimicrobial resistance content, C. freundii ST116 revealed cluster-related resistomes, with carbapenemase KPC-2 common in the three clades.Moreover, except for AK867, the occurrence of VIM-1 carbapenemase is rare in ST116.Interestingly, the presence of fosA3 already occurred in C. freundii S T 1 1 6 , b u t i n C L 1 f r o m s e w a g e s a m p l e i n C h i n a (GCF_032747095.1).Moreover, another fosA-like gene, fosA5, fits in C. freundii ST116 CL1 as reported by three human samples collected in China (GCF_029104415.1,GCF_029104385.1, GCF_029104405.1) (Figure 3).Additionally, the three clusters revealed different plasmidome, with a predominance of pKPC-CAV1312 plasmids in CL2 (Supplementary Figure S1).pKPC-CAV1312 seemed to not easily fit in CL1 and CL3, where there is a higher incidence of IncFIB, IncFII, and IncA/C2 (Supplementary Figure S1).Thus, the entry and stabilization of pKPC-CAV1312 that harbor fosA-like genes in ST116 provide further knowledge on the real incidence of FOS resistance within C. freundii ST116.The presence of multireplicon IncFIB: HI1B was not reported in ST116 except for AK867.Concerning the virulome, all the three clusters shared similar virulence gene content, including adhesions (csg locus), metabolism (chuX, entB, entE, fepC), invasion (ompA), and chemotaxis (fliG) genes (Figure 3).Interestingly, AK867 showed a wider virulome, carrying the adhesion genes fyuA, irp-1 and -2, and the locus ybt for siderophores (Figure 3).

Discussion
FosA3 is a subtype of the FosA enzyme family that, since its first report in 2010, is currently worldwide disseminated, especially in China (Hou et al., 2012;Mattioni Marchetti et al., 2023a).The major vehicles involved in dissemination are IncFII (Hou et al., 2012), followed by IncI1 (Sato et al., 2013), IncN (Liu et al., 2022), IncHI2 (Chen et al., 2021), and IncP (Hameed et al., 2022).Furthermore, it has been documented that IS26 plays a fundamental role in antimicrobial resistance genes (AMR) transposition and dissemination among Enterobacterales (Lv et al., 2020).Here, we depicted the genomic organization of a novel fosA3-harboring transposon on a pKPC-CAV1312 plasmids.As suggested by the available genomes on NCBI, pKPC-CAV1312 plasmids are likely to promote the entry and the subsequent fitting of fosA3 in Citrobacter spp.Interestingly, pfosA3_CFR867 did not show a perfect identity with others fosA3-mediated pKPC-CAV1312 plasmids, suggesting consistent rearrangements in the plasmid backbone structure.The plasmid-mediated fosA3 is generally organized in a composite transposon of 4 kb in size, consisting in two IS26 elements with the same orientation, flanking the cassette fosA3-orf1-orf2-Dorf3 (Wachino et al., 2010).In the present study, fosA3 was included in a large composite transposon of >12 kb that contained several orfs.The occurrence of fosA3 in large transposon, with the ability to carry different genes, and combined with the transposition potential of IS26, poses a further challenge in containing the spread of such emergent antibioticresistant strains at a global level.
strand DNA and allowing the acquisition of functional attributes to the host cell, such as AMR genes (Edgell, 2009).The association between fosA-like genes and HNH endonucleases has already been pointed out in literature, speculating an undefined role of HNH in the dissemination of fosA genes, in absence of any surrounding insertion elements, within Citrobacter spp (Li et al., 2020;Mattioni Marchetti et al., 2023b).The pfosA3_CFR867 contained an HNH endonuclease, at a 6,530-bp distance from the fosA3 transposon.
In the present study, AK867 showed an XDR profile due to the co-presence of clinically relevant carbapenemases KPC-2 and VIM-1.In this prospective, the resistance to carbapenems and high FOS MIC in C. freundii could represent a novel menace and reduce the current antimicrobial armamentarium.This possibility is also strengthened by the easy transfer of such gene into the E. coli recipient, inducing an increase in MIC values beyond the current EUCAST breakpoints (resistance category with MIC >8 mg/L).
EUCAST clinical breakpoints for FOS underwent on a recent revision, with FOS cutoffs applicable on E. coli only and not recommending the use of FOS for other Enterobacterales than E. coli.However, FOS still represents a valid option in the treatment of urinary tract infection by ESBL-producing E. coli and C. freundii (Bielen and Likic, 2019).Moreover, FOS is also recognized as a valid option in combination therapy against several MDR Gram-negative infections, due to its relevant synergistic effect (Antonello et al., 2020).Based on the previous EUCAST breakpoints, C. freundii strains maintain high susceptibility levels to FOS (average MIC = 4 mg/L), but an eventual stabilization of transferable FosA enzymes may mark a turning point in the evolution of antibiotic resistance in C. freundii (Bielen et al., 2018).
In fact, cases of FosA-like enzymes in uncommon pathogens, as C. freundii, are slowly emerging in the literature (Chen et al., 2021;Mattioni Marchetti et al., 2023a;Mattioni Marchetti et al., 2023b).For this reason, despite the EUCAST revision, FosA enzyme detection and related transferability should be assessed even in low-risk pathogens, in order to track the transmission routes from these pathogens to clinically relevant clones, such as E. coli ST131 (Chudejova et al., 2024).Doubtless, largescale surveillance on FOS-resistance profiles among Enterobacterales are demanding due to the lack of rapid kit as reliable as the reference ADM method.However, the coexistence of carbapenemases and FosA-like enzymes requires additional effort in clinical surveillance programs (Nakamura et al., 2014).
In conclusion, the incidence of FOS resistance is increasing globally among Enterobacterales, reaching and fitting even in low-