Identification of IncA Plasmid, Harboring blaVIM-1 Gene, in S. enterica Goldcoast ST358 and C. freundii ST62 Isolated in a Hospitalized Patient

In the present study, we analyzed the genome of two S. enterica strains TS1 and TS2 from stool and blood cultures, respectively, and one strain of C. freundii TS3, isolated from a single hospitalized patient with acute myeloid leukemia. The S. enterica Goldcoast ST358 (O:8 (C2-C3) serogroup), sequenced by the MiSeq Illumina system, showed the presence of β-lactamase genes (blaVIM-1, blaSHV-12 and blaOXA-10), aadA1, ant(2″)-Ia, aac(6′)-Iaa, aac(6′)-Ib3, aac(6′)-Ib-cr, qnrVC6, parC(T57S), and several incompatibility plasmids. A wide variety of insertion sequences (ISs) and transposon elements were identified. In C. freundii TS3, these were the blaVIM-1, blaCMY-150, and blaSHV-12, aadA1, aac(6′)-Ib3, aac(6′)-Ib-cr, mph(A), sul1, dfrA14, ARR-2, qnrVC6, and qnrB38. IncA plasmid isolated from E.coli/K12 transconjugant and C. freundii exhibited a sequence identity >99.9%. The transfer of IncA plasmid was evaluated by conjugation experiments.


Introduction
Salmonella enterica is an intracellular facultative anaerobe Gram-negative bacterium, belonging to the family of Enterobacterales.This common zoonotic pathogen is responsible for infections in both humans and animals [1].Salmonella infection poses a major public health concern worldwide as a foodborne illness.Salmonellosis is the second most commonly reported gastrointestinal infection in the EU/EEA, and an important cause of food-borne outbreaks [2,3].There are more than 2600 serovars of Salmonella on the basis of the lipopolysaccharide (LPS) O antigen and the flagellar protein (H) antigen [4].From the clinical point of view, S. enterica subspecies enterica is the primary cause of human infections.S. enterica species can be subdivided into two big groups including the invasive typhoidal group (i.e., S. Typhy, S. Paratyphy A, B and C) and non-typhoidal Salmonella.Several hundred serovar of the S. enterica subspecies enterica are identified in different animal species (i.e., cattle, pig, poultry, human, etc.) [5,6].Invasive typhoidal Salmonella causes enteric fever with clinical and severe complications if not treated [7].Nontyphoidal Salmonella (NTS) strains include the S. enterica serovar Goldcoast, a rare zoonotic pathogen for human infection.The infection is usually acquired through contaminated food of animal origin [8].S. enterica Goldcoast is a rare serovar for humans in the United States but it is more common in European countries.To date, a few epidemic outbreaks involving S. enterica Goldcoast have been described in Germany, Hungary, England, and Italy [9][10][11][12].Recently, the increasing resistance to antimicrobials in Salmonella has posed a significant threat to public health.Salmonella strains resistant to ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol, extended-spectrum cephalosporins, fluoroquinolones, tetracyclines and even carbapenems have emerged worldwide [1,13].B-Lactams are the most common antibiotics employed in clinical practice and their extensive use has led to the emergence of resistance.The World Health Organization (WHO) has estimated a high risk of the dissemination of antibiotic resistant bacteria (ARB) and an urgent worldwide action plan is required [14].Carbapenems represent the "last-line agents" of defense for serious clinical infections caused by multidrug-resistant (MDR) Gram-negative bacteria and β-lactamases resistant to carbapenems (i.e., "carbapenemases") are disseminated, mainly, among Enterobacterales.Currently, carbapenem-resistant Enterobacterales (CPEs) represent a serious problem in healthcare settings [14].Resistance to carbapenems is due to the cooperation of different mechanisms including (i) production of carbapenemases, extended-spectrum-β-lactamases and AmpC, (ii) mutations that alter the expression and function of PBPs and porins, and (iii) efflux pump alteration [15].The most worrisome carbapenemases found in Enterobacterales are represented by subclass B1 metallo-β-lactamases (MBLs) (i.e., NDM, VIM, and IMP), class A (i.e., KPC), and class D (i.e., OXA-48) enzymes of the Ambler classification.MBLs utilize one or two zinc ions for their catalysis while class A and D carbapenemases are serineβ-lactamases able to hydrolyze the β-lactams substrates by forming a transient or stable acyl enzyme through an active site serine.The dissemination of carbapenemase genes in bacterial isolates is due to their localization in mobile genetic elements which play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of resistance genes.The most widespread CPEs worldwide in clinical settings are Klebsiella pneumoniae and Escherichia coli [16].However, other CPEs causing nosocomial infections such as Citrobacter spp.and Salmonella spp.have, recently, been emerging [17,18].Molecular epidemiology data on carbapenem-resistant Citrobacter spp.indicate the presence of class A and D carbapenemases (i.e., KPC-types and OXA-48, respectively) and MBLs (i.e., NDM-, VIM-, and IMP-variants) [17].Even though carbapenems are not the drugs of choice for Salmonella infections, the presence of carbapenemases has been detected in NTS S. enterica identified in humans, animals, and food [19][20][21][22].In particular, the first member of MBLs, the IMP-4 enzyme, was identified in a S. waycross isolate recovered in 2007 in Australia from an 87-year-old woman with a urinary tract infection followed by diarrhea [23].However, the most common MBL reported worldwide in S. enterica is VIM-2, isolated for the first time in 2010 from the blood and urine of hospitalized patient in Morocco [24].To date, the bla VIM-1 gene has mainly been identified in S. enterica strains isolated from livestock farms and food [25][26][27].
In the present study, we have reported a detailed characterization of a genome of two S. enterica (TS1 and TS2) strains and C. freundii TS3 isolated, from the stool and blood cultures of a single patient admitted to the University Hospital of Trieste (Northeastern Italy) with acute myeloid leukemia who underwent allogeneic stem cell transplantation complicated by graft-versus-host disease (GVHD).The IncA plasmid identified in both S. enterica and C. freundii was sequenced by Illumina and Nanopore systems in order to verify sequence similarity.Conjugation experiments were performed to support the theory of a possible "jump" in IncA plasmid or genetic determinants between S. enterica and C. freundii.Antibiotic resistance genes (ARGs), mobile genetic elements, and virulence factors were identified in both strains.

Clinical Aspects and Strains Selection
We describe a patient with acute myeloid leukemia who underwent allogeneic stem cell transplantation (March 2022) complicated by graft-versus-host disease (GVHD).In late November 2022, the patient was hospitalized for acute diarrhea.Stool and blood cultures were positive for the third-generation cephalosporin-susceptible group C S. enterica.The strain was susceptible to carbapenems, cephalosporins, and aminoglycosides.In addition, Clostridioides difficile infection (CDI) was diagnosed.The patient was treated with fidaxomicin (10 days) and ceftriaxone (14 days) with clinical improvement and was discharged.Rectal colonization by KPC-producing K. pneumoniae and VIM-producing C. freundii was also detected.In late December 2022, the patient was readmitted for CDI recurrence that was treated with fidaxomicin (a tapered regimen for 20 days) and bezlotoxumab.The S. enterica group C was again identified from blood and stool cultures.Indeed, a second targeted antibiotic therapy with high-dose meropenem plus fosfomycin (14 days both) was administered.The Xpert Carba-R assay was carried out on S. enterica TS1, S. enterica TS2, and C. freundii TS3, and the presence of the bla VIM gene was detected in these strains.The patient experienced a gradual improvement with symptoms resolution but remained hospitalized for GVHD management.A PET scan ruled out a deep-seated infection and bloodstream clearance was documented.In late January 2023, hospitalacquired pneumonia (HAP) was diagnosed.High-dose meropenem and colistin were started.Due to clinical deterioration and the recurrence of bloodstream infection (BSI) due to VIM-producing group C S. enterica, the antibiotic regimen was modified in highdose tigecycline, cefiderocol, and colistin.The samples underwent genomic sequencing.Recurrent BSI due to infected gallstones and secondary HAP were hypothesized.After targeted therapy and removal of all intravascular devices, the blood cultures collected were negative.Unfortunately, the patient died of causes unrelated to the infection ten days later.

Antimicrobial Susceptibility of S. enterica and C. freundii
The S. enterica TS1 and S. enterica TS2 isolated from stool and blood cultures, respectively, were analyzed for their susceptibility profile against different antibiotics.Both strains showed resistance to cephalosporins and ciprofloxacin, susceptibility at increased exposure to imipenem, and susceptibility to meropenem, aminoglycosides, and tigecycline (Table 1).The C. freundii strain showed resistance to cephalosporins, susceptibility at increased exposure to imipenem, susceptibility to meropenem, aminoglycosides, ciprofloxacin, and tigecycline.

IncA Plasmid Mapping
In order to verify the sequence similarity of the IncA plasmid identified in S. enterica and C. freundii, the IncA plasmid was extracted from C. freundii TS3 and from transconjugant E. coli K12/S.enterica (see conjugation experiments in the materials and methods section).A total of 1.699.858.150bp paired-end raw reads (510 Mb) were obtained by Illumina, and 126.920 raw reads with a mean length of 909.8 bp (115 Mb) by ONT for the C. freundii plasmid.The hybrid assembly produced 278 contigs with a largest contig of 150,373 bp.All contigs were used as inputs for MOB-suite to define their molecular type, only three were marked as plasmid sequences including the largest contig that results as a complete and circular plasmid.The IncA plasmids from transconjugant E. coli K12/S.enterica and C. freundii showed a sequence identity, by BLAST analysis, higher than 99.9%.The main genetic elements identified in the IncA plasmid were represented by plasmid replication module (repA), ARGs, IS family transposases, and genetic elements related to a conjugal transfer function.IncA harbored bla VIM-1 and bla SHV-12 which confer resistance to carbapenems and extended-spectrum β-lactams; aacA4 is an aminoglycoside N-acetyltransferase that confers resistance to broad-spectrum aminoglycosides; qnrVC6 confers resistance to fluoroquinolones; ant1 is a streptomycin adenyltransferase; and the mph(A) gene is a macrolide phosphotransferase, which, mainly, inactivates erythromycin.A similarity search in a public sequence database indicated the presence, in the IncA plasmid, of 12 conjugation genes (traB to traW).The traF, traG, and traH genetic elements, identified as components of outer membrane complex, seem to be involved in conjugation transfer (Figure 1).

IncA Plasmid Mapping
In order to verify the sequence similarity of the IncA plasmid identified in S. enterica and C. freundii, the IncA plasmid was extracted from C. freundii TS3 and from transconjugant E. coli K12/S.enterica (see conjugation experiments in the materials and methods section).A total of 1.699.858.150bp paired-end raw reads (510 Mb) were obtained by Illumina, and 126.920 raw reads with a mean length of 909.8 bp (115 Mb) by ONT for the C. freundii plasmid.The hybrid assembly produced 278 contigs with a largest contig of 150,373 bp.All contigs were used as inputs for MOB-suite to define their molecular type, only three were marked as plasmid sequences including the largest contig that results as a complete and circular plasmid.The IncA plasmids from transconjugant E. coli K12/S.enterica and C. freundii showed a sequence identity, by BLAST analysis, higher than 99.9%.The main genetic elements identified in the IncA plasmid were represented by plasmid replication module (repA), ARGs, IS family transposases, and genetic elements related to a conjugal transfer function.IncA harbored blaVIM-1 and blaSHV-12 which confer resistance to carbapenems and extended-spectrum β-lactams; aacA4 is an aminoglycoside N-acetyltransferase that confers resistance to broad-spectrum aminoglycosides; qnrVC6 confers resistance to fluoroquinolones; ant1 is a streptomycin adenyltransferase; and the mph(A) gene is a macrolide phosphotransferase, which, mainly, inactivates erythromycin.A similarity search in a public sequence database indicated the presence, in the IncA plasmid, of 12 conjugation genes (traB to traW).The traF, traG, and traH genetic elements, identified as components of outer membrane complex, seem to be involved in conjugation transfer (Figure 1).The IncA plasmid harbored a class 1 integron which includes (i) an integrase 1 gene from 117,171 bp to 116,158 bp, (ii) a variable region of about 4000 bp including five gene cassettes (bla VIM-1 , aacA4, qnrVC6, arr2, and ant1), and (iii) a 3 CS region including qacEdelta1 and sul1 genes (Figure 2).Upstream and downstream of integron element, there are typical elements of transposon.
The IncA plasmid harbored a class 1 integron which includes (i) an integrase 1 gene from 117,171 bp to 116,158 bp, (ii) a variable region of about 4000 bp including five gene cassettes (blaVIM-1, aacA4, qnrVC6, arr2, and ant1), and (iii) a 3 CS region including qacEdelta1 and sul1 genes (Figure 2).Upstream and downstream of integron element, there are typical elements of transposon.

IncA Transfer by Conjugation Assays
The transfer of IncA plasmid was evaluated by conjugation experiments.The selection of transconjugants was performed in plates supplemented with ceftazidime having ascertained the presence, in the IncA plasmid, of blaVIM-1 and blaSHV-12.Resistance to ceftazidime in E. coli K12 transconjugants was surely conferred by blaSHV-12.The correct transfer of the IncA plasmid was confirmed by amplification of the blaVIM-1 and blaSHV-12 genes in the E. coli K12 transconjugant of S. enterica and the E. coli K12 transconjugant of C. freundii.

Discussion
Herein we describe the first identification in Italy of the VIM-1-producing S. enterica serovar Goldcoast ST358 isolated from the blood and stool cultures of a single patient colonized by multiple germs including K. pneumoniae, C. difficile, and C. freundii during a hospitalization for acute myeloid leukemia.The genome DNA of S. enterica (TS1 and TS2 strains) and C. freundii was sequenced and various ARGs were identified in the chromosome and plasmids of these clinical isolates.IncA was identified in both the S. enterica TS2 and C. freundii TS3 strains.In addition to IncA, S. enterica also possessed IncHI and IncFII plasmids.The complete sequence of incompatibility of the IncA plasmid, carrying blaVIM-1, recovered by both S. enterica TS2 and C. freundii TS3, showed a nucleotide sequence similarity higher than 99%, suggesting a possible transfer of IncA between the two isolates.The theory that C. freundii TS3 exchanged IncA plasmid with S. enterica TS2 or vice versa, during the patient's hospitalization, was supported by the positive results of "in vitro" conjugation experiments and the presence of a genetic complex system subordinated to the conjugal function [28].The IncA/C plasmids have an extremely broad host range coupled with a high ability to spread via conjugative transfer [29][30][31].Conjugation is the most effective mechanism for horizontal gene transfer (HGT).In this context, various incompatibility plasmids have a huge impact on the global emergence of MDR bacteria in clinical, animal, and environmental settings [32,33].Recently, a highly conjugative IncA plasmid has been characterized in several VIM-1-producing Enterobacterales [34].Arcari and coworkers identified, in VIM-IncA, several genetic regions (IS6000, dfrA14, mph(A), blaSHV-12, aac(6′)-Ib3, and aadA1) which were also encountered in our strains [34].Despite the presence of blaVIM-1 in the IncA plasmid, both S. enterica TS2 and C. freundii TS3 were found to be susceptible to imipenem and meropenem.This is not unusual because several

IncA Transfer by Conjugation Assays
The transfer of IncA plasmid was evaluated by conjugation experiments.The selection of transconjugants was performed in plates supplemented with ceftazidime having ascertained the presence, in the IncA plasmid, of bla VIM-1 and bla SHV-12 .Resistance to ceftazidime in E. coli K12 transconjugants was surely conferred by bla SHV-12 .The correct transfer of the IncA plasmid was confirmed by amplification of the bla VIM-1 and bla SHV-12 genes in the E. coli K12 transconjugant of S. enterica and the E. coli K12 transconjugant of C. freundii.

Discussion
Herein we describe the first identification in Italy of the VIM-1-producing S. enterica serovar Goldcoast ST358 isolated from the blood and stool cultures of a single patient colonized by multiple germs including K. pneumoniae, C. difficile, and C. freundii during a hospitalization for acute myeloid leukemia.The genome DNA of S. enterica (TS1 and TS2 strains) and C. freundii was sequenced and various ARGs were identified in the chromosome and plasmids of these clinical isolates.IncA was identified in both the S. enterica TS2 and C. freundii TS3 strains.In addition to IncA, S. enterica also possessed IncHI and IncFII plasmids.The complete sequence of incompatibility of the IncA plasmid, carrying bla VIM-1 , recovered by both S. enterica TS2 and C. freundii TS3, showed a nucleotide sequence similarity higher than 99%, suggesting a possible transfer of IncA between the two isolates.The theory that C. freundii TS3 exchanged IncA plasmid with S. enterica TS2 or vice versa, during the patient's hospitalization, was supported by the positive results of "in vitro" conjugation experiments and the presence of a genetic complex system subordinated to the conjugal function [28].The IncA/C plasmids have an extremely broad host range coupled with a high ability to spread via conjugative transfer [29][30][31].Conjugation is the most effective mechanism for horizontal gene transfer (HGT).In this context, various incompatibility plasmids have a huge impact on the global emergence of MDR bacteria in clinical, animal, and environmental settings [32,33].Recently, a highly conjugative IncA plasmid has been characterized in several VIM-1-producing Enterobacterales [34].Arcari and coworkers identified, in VIM-IncA, several genetic regions (IS6000, dfrA14, mph(A), bla SHV-12 , aac(6 )-Ib3, and aadA1) which were also encountered in our strains [34].Despite the presence of bla VIM-1 in the IncA plasmid, both S. enterica TS2 and C. freundii TS3 were found to be susceptible to imipenem and meropenem.This is not unusual because several studies have reported low-carbapenem minimum inhibitory concentration values (MICs) in VIM-1-producing Enterobacterales [35,36].In the chromosome of S. enterica, we have identified OXA-10, a class D oxacillinase commonly produced by Pseudomonas aeruginosa, which has been described as possessing weak activity against carbapenems [37].The presence, in the IncA plasmid, of a class 1 integron with five gene cassettes has led to its rapid dissemination among bacteria, in particular when they are exposed to antibiotics.The IncA plasmid harbored the bla SHV-12 gene, whose presence also confers resistance to extended-spectrum β-lactams, and the fluoroquinolones and aminoglycoside resistance genes (qnrVC6 and aacA4, respectively).qnrVC6 is an emerging quinolone-resistance determinant in the qnr family which has mainly been identified in non-Enterobacterales species such as Acinetobacter baumannii, Pseudomonas spp., and C. freundii or, occasionally, in Vibrio parahaemolyticus [38][39][40][41].In S. enterica TS2, resistance to fluoroquinolones is not only due to qnrVC6 but also to aac(6 )Ib-cr and mutation in T57S amino acid substitution in parC.Mutation in parC, identified in the QRDR of S. enterica reduces susceptibility to ciprofloxacin even if ciprofloxacin is a drug of choice for treating S. enterica infections in humans [42].In S. enterica TS2, the situation is exacerbated

Genomic and Plasmid Extraction
Total nucleic acid was extracted from liquid cultures of S. enterica (TS1 and TS2 strains) and C. freundii TS3 using a modified protocol of the MagMAX Microbiome Ultra Nucleic Acid Isolation kit (Applied Biosystems, ThermoFisher Scientific, Monza, Italy) as previously reported [54].The IncA plasmid was extracted from S. enterica TS2 and C. freundii TS3 using a Large-Construct Kit, which allows a purified plasmid free of genomic DNA contamination to be obtained.

WGS-Based AMR Identification of C. freundii TS3
ARGs, plasmids, mobile genetic elements, MLST and pMLST were detected as described in the above section, using services provided by Center for Genomic Epidemiology (https://www.genomicepidemiology.org/, accessed on 25 January 2023).

Conjugation Experiments
S. enterica and C. freundii and E. coli K12 strains were used as donors and recipient for conjugation experiments, respectively.Transconjugants were selected on Luria-Bertani (LB) agar plates supplemented with 250 mg/L of streptomycin and 32 mg/L ceftazidime.The detection sensitivity of the assay was ≥5 × 10 −7 transconjugants per recipient.

Conclusions
The increase in resistance to β-lactams is a result of the high plasticity of β-lactamases genes and the genetic adaptability of the bacterial population in a short period of time.The presence, in the IncA plasmid, of genetic components involved in conjugation (i.e., traF and traH) facilitates the genetic transfer of ARGs [28].Carbapenem-resistant S. enterica Goldcoast and C. freundii are not considered to be a classical nosocomial pathogen but they possessed many genetic elements which conferred high resistance to several antibiotic families.Bloodstream S. enterica infections are very common and represent a real risk for public health, in particular when multiple bacterial species colonize the same patient [62].The therapeutic options are very limited and the patient risks their own life.

Figure 1 .
Figure 1.Circular map of IncA plasmid.The circles, from the outermost to the innermost, show: (i) coding DNA sequences (CDSs) encoded on the plus and minus DNA strands (annotation is reported for known genes only); and (ii) the G+C content, shown as deviation from the average G+C content of the entire molecule.Genes encoding protein of known functions are in different colors, as detailed in the legend.

Figure 2 .
Figure 2. Structure of the class 1 integron carried on the analyzed plasmid.The intI1 integrase gene is indicated by a violet arrow; the variable region includes five gene cassettes (red arrows).

Figure 2 .
Figure 2. Structure of the class 1 integron carried on the analyzed plasmid.The intI1 integrase gene is indicated by a violet arrow; the variable region includes five gene cassettes (red arrows).

Table 1 .
Antimicrobial susceptibility pattern of S. enterica and C. freundii clinical strains.

Table 2 .
ARGs and mobile genetic elements in the S. enterica Goldcoast TS2 strain.

Table 4 .
ARGs and mobile genetic elements of the C. freundii TS3 strain.