Genomic Insights into Pluralibacter gergoviae Sheds Light on Emergence of a Multidrug-Resistant Species Circulating between Clinical and Environmental Settings

Pluralibacter gergoviae is a member of the Enterobacteriaceae family that has been reported sporadically. Although P. gergoviae strains exhibiting multidrug-resistant profiles have been identified an in-depth genomic analysis focusing on antimicrobial resistance (AMR) has been lacking, and was therefore performed in this study. Forty-eight P. gergoviae strains, isolated from humans, animals, foods, and the environment during 1970–2023, were analyzed. A large number of single-nucleotide polymorphisms were found, indicating a highly diverse population. Whilst P. gergoviae strains were found to be circulating at the One Health interface, only human and environmental strains exhibited multidrug resistance genotypes. Sixty-one different antimicrobial resistance genes (ARGs) were identified, highlighting genes encoding mobile colistin resistance, carbapenemases, and extended-spectrum β-lactamases. Worryingly, the co-occurrence of mcr-9.1, blaKPC-2, blaCTX-M-9, and blaSHV-12, as well as mcr-10.1, blaNDM-5, and blaSHV-7, was detected. Plasmid sequences were identified as carrying clinically important ARGs, evidencing IncX3 plasmids harboring blaKPC-2, blaNDM-5, or blaSHV-12 genes. Virulence genotyping underlined P. gergoviae as being a low-virulence species. In this regard, P. gergoviae is emerging as a new multidrug-resistant species belonging to the Enterobacteriaceae family. Therefore, continuous epidemiological genomic surveillance of P. gergoviae is required.


Introduction
Antimicrobial resistance (AMR) is a growing global health concern that affects several sectors.In clinical settings, AMR reduces the effectiveness of antimicrobials and leads to longer hospital stays.Consequently, there is an increase in mortality rates [1].Furthermore, healthcare-associated infections caused by MDR pathogens are on the rise, which is concerning [2].In the environment, antimicrobial-resistant strains are circulating especially in water bodies and agricultural soils, impacting human and animal populations [3].These strains may be reaching food products and leading to difficult-to-treat foodborne infections [4].In this context, AMR is a multifactorial problem that requires a One Health approach [5].
Pluralibacter gergoviae, formerly Enterobacter gergoviae, was first described in the 1980s in human clinical specimens and the environment in France and the United States [6,7].Subsequently, this species was identified as contaminating cosmetic formulations, colonizing the human oral cavity, and causing nosocomial outbreaks [8][9][10][11].From the 2010s, multidrug-resistant (MDR) P. gergoviae strains, harboring clinically important antimicrobial resistance genes (ARGs) and highlighting carbapenemases, began to emerge in clinical settings [11][12][13].Whilst MDR P. gergoviae strains have been described, an in-depth genomic analysis is lacking.Therefore, this study aimed to perform a genomic characterization of P. gergoviae strains circulating at the One Health interface and provide genomic insights into AMR.

Distribution of P. gergoviae Strains
Forty-eight P. gergoviae strains were included in this study.These strains were isolated from humans (abscesses, blood, bronchoalveolar lavage, dentine caries, drainage, endotracheal and tracheal aspirate, feces, gastrojejunostomy tubes, nasopharyngeal swabs, peri-bile duct tissue, right shin wound cultures, tracheostomy sites, and urine), an animal (dog, urine), foods (fish balls and ground beef), or the environment (wastewater), evidencing their circulation at the One Health interface.These strains were obtained from 1970 onwards in countries belonging to the Australian (Australia), American (Brazil, Peru, and the United States), Asian (Bangladesh and Malaysia), and European (Bulgaria, Greece, France, and the United Kingdom) continents (Figure 1).

SNPs Data
SNP differences among all P. gergoviae genomes ranged from 27 to 33,993, whereas among human and environmental strains SNP differences ranged from 80 to 33,993 and 27 to 1073, respectively.Between food strains (FB2 and H11), differences of 22,871 SNPs were found.Overall, P. gergoviae genomes were grouped according to country or host, but divergences in these characteristics were also observed.The largest clade grouped human and environmental strains (214632, 1951017-11 to -15, and 1952417-11 to -15) from the United Kingdom.Interestingly, human and animal strains (FDAARGOS-386, PG1351, and CCBH27438) from the American continent (Brazil, Peru, and the United States) were clustered.In this regard, the finding of a large number of SNPs indicates a highly diverse population with a range of genetic adaptations that may be related to genetic exchanges or recombination events.The SNP-based phylogenetic tree of P. gergoviae strains distributed worldwide.Strain, host, country, year, and BioSample data were retrieved from the GenBank ® database.Red, blue, yellow, and green painted circles represent positive antimicrobial resistance genes, biocide resistance genes, virulence genes, and plasmid replicons, respectively.The tree was rooted at the midpoint.
Most strains (58%) carried at least four ARGs.Among them, strains isolated from humans and the environment in Greece, the United Kingdom, and the United States harbored ten or more ARGs related mainly to polymyxins, β-lactams, fluoroquinolones, aminoglycosides, tetracyclines, and folate pathway antagonists.The presence of genes that encode mobile colistin resistance (mcr-9.1 and mcr-10.1),carbapenemases (bla KPC , bla NDM , and bla VIM ), extended-spectrum β-lactamases (ESBLs; bla CTX-M and bla SHV ), and plasmidmediated quinolone resistance [PMQR; qnr and aac(6 )-Ib-cr] is highlighted.Interestingly, the coexistence of the aforementioned clinically relevant ARGs was identified in human and environmental strains from the United States and the United Kingdom, respectively.In addition, three human strains (2022DK-00159, 2022DK-00154, and 2021DK-00143) presented more than 20 ARGs (Figure 1).These characteristics contribute to the emergence of multidrug-resistant strains and the spread of clinically important ARGs worldwide.

WGS-Predicted Antimicrobial Susceptibility
Corroborating with the resistome, it was found that most strains (58%) were predicted to be MDR since they presented resistance to one or more antimicrobial agents in three or more antimicrobial categories.Although MDR strains presented resistance to critically important antimicrobials [e.g., polymyxins, penicillins + β-lactamase inhibitors, extended-spectrum cephalosporins (3rd and 4th generations), monobactams, carbapenems, fluoroquinolones, and/or aminoglycosides], they were susceptible mainly to minocycline, tigecycline, and/or fosfomycin.MDR strains were obtained exclusively from humans and the environment, while pan-susceptible strains were recovered from humans, animals, and foods (Table 1).

Virulence Genotyping
Virulence genes that encode a heat shock survival AAA family ATPase (clpK1), a lipoprotein NlpI precursor (nlpI), an outer-membrane protein complement resistance (traT), and a tellurium ion resistance protein (terC) were identified among the P. gergoviae genomes.Most strains presented only one of the virulence genes found; however, some of them harbored two genes.Strain C3 from Australia was isolated from a patient with cystic fibrosis and presented clpK1 and terC genes, while environmental strains from the United Kingdom carried terC and traT genes (Figure 1).

Discussion
In this study, we present a first comparative genomic analysis of P. gergoviae distributed worldwide.Overall, P. gergoviae strains were recovered from humans, animals, foods, and the environment, but MDR strains were isolated only from humans and the environment, which may be related to the limited number of food and animal strains analyzed.In this context, it can be said that this species has been circulating at the One Health interface and that MDR strains may also emerge within it.Therefore, AMR is a multifaceted challenge that requires coordinated global activities to reduce the spread of MDR bacteria [17].Following the emergence of P. gergoviae strains as MDR, high-resolution molecular typing methods, including multilocus sequence typing, should be encouraged in order to improve population structure analysis, strain differentiation, international comparisons, and longitudinal studies.
Species belonging to the Enterobacteriaceae family, including E. coli and K. pneumoniae, are known to be potentially pathogenic and MDR [18].In general, AMR studies are focused on them, while in other species, mainly newer ones, little is known about the AMR's genetic background.Recently, Raoultella species have gained notoriety for being MDR and shedding clinically important ARGs [19,20].In this regard, P. gergoviae emerges as a new species with these traits since it harbors an arsenal of plasmid-mediated ARGs.As enterobacteria are known for their ability to exchange genetic materials [21], P. gergoviae also demonstrates the potential to act as a disseminator of ARG-bearing plasmids.Furthermore, P. gergoviae strains also carried biocide resistance genes, contributing to the co-selection of AMR and their persistence in clinical settings [22].
Among known AMR-related plasmid families in Enterobacteriaceae, IncA, IncF, IncH, and IncN stand out for harboring various ARGs.These conjugative plasmids have been reported especially in medically important bacteria [23], and now also in P. gergoviae.Curiously, the first report of AMR-related plasmids in P. gergoviae was published in the United States in 2022.In this report, large IncA and IncHI1B/FIB plasmids carrying bla KPC-4 and bla NDM-1 , respectively, were identified as referring to BWH-P-GER-1 and BWH-P-GER-6 strains [24].Furthermore, IncX3 plasmids have great conjugation ability and high stability, and have been identified as carrying ESBL-or carbapenemase-encoding genes in P. gergoviae strains, reinforcing the importance of P. gergoviae in the dissemination of ARGs within Enterobacteriaceae [25].
Recently, single-and multireplicon plasmids carrying mcr-9 or mcr-10 have been identified in different sources.Consequently, mcr-positive and carbapenem-resistant Enterobacteriaceae strains have emerged worldwide [26,27] and were also described in this study.In addition, the co-occurrence of ESBL-and carbapenemase-encoding genes in P. gergoviae strains has been detected, demonstrating a similar genotype to other medically important enterobacteria [28].Based on the P. gergoviae resistome, it is possible to predict antimicrobial susceptibility and highlight the multidrug resistance phenotype.Although some mcr genes do not confer resistance to polymyxins in certain bacterial species [29], good correlation between the genotypic and phenotypic resistance for other antimicrobials was documented [30], including in clinical P. gergoviae strains [24].Furthermore, antimicrobial susceptibility may vary substantially from predictions and may even be underestimated since mutational mechanisms were not investigated in this study, suggesting that MDR strains could still be classified as extensively drug-resistant or pandrug-resistant.
Previous studies have reported that P. gergoviae causes opportunistic infections and even nosocomial outbreaks [11,31,32]; however, virulence genotyping strengthened that P. gergoviae is a low-virulence bacterium [24], although virulence databases are still limited in the case of this species.Indeed, the role of virulence genes in the pathogenicity of this species needs to be further investigated.Worryingly, P. gergoviae-derived infections, especially those caused by carbapenem-resistant strains, can be difficult to treat, representing a significant healthcare challenge and serious public health concern [33].As described in high-risk clones of E. coli and K. pneumoniae [34], the convergence of virulence and AMR did not occur in P. gergoviae.In addition, no species-and host-specific genes related to AMR or virulence were identified, evidencing an acquired and diverse genetic background.

Conclusions
In summary, this study provides the first genomic overview of P. gergoviae strains circulating at the One Health interface.Comparative analysis revealed a highly diverse population that harbored an arsenal of ARGs.Conjugative plasmids were identified as carrying clinically important ARGs embedded in conserved genetic contexts, evidencing their capture of other medically important bacteria.In this sense, P. gergoviae is emerging as a new multidrug-resistant species belonging to the Enterobacteriaceae family.Therefore, more studies investigating the genetic background of AMR in non-clinically relevant enterobacteria should be encouraged and continuous epidemiological genomic surveillance of P. gergoviae should be required.

Figure 1 .
Figure1.The SNP-based phylogenetic tree of P. gergoviae strains distributed worldwide.Strain, host, country, year, and BioSample data were retrieved from the GenBank ® database.Red, blue, yellow, and green painted circles represent positive antimicrobial resistance genes, biocide resistance genes, virulence genes, and plasmid replicons, respectively.The tree was rooted at the midpoint.