Genomic landscape of NDM-1 producing multidrug-resistant Providencia stuartii causing burn wound infections in ...

Abstract

The increasing antimicrobial resistance in Providencia stuartii (P. stuartii) worldwide, particularly concerning for immunocompromised and burn patients, has raised concern in Bangladesh, where the significance of this infectious opportunistic pathogen had been previously overlooked, prompting a need for investigation. The two strains of P. stuartii (P. stuartii SHNIBPS63 and P. stuartii SHNIBPS71) isolated from wound swab of two critically injured burn patients were found to be multidrug-resistant and P. stuartii SHNIBPS63 showed resistance to all the 22 antibiotics tested as well as revealed the co-existence of bla_VEB-6 (Class A), bla_NDM-1 (Class B), bla_OXA-10 (Class D) beta lactamase genes. Complete resistance to carbapenems through the production of NDM-1, is indicative of an alarming situation as carbapenems are considered to be the last line antibiotic to combat this pathogen. Both isolates displayed strong biofilm-forming abilities and exhibited resistance to copper, zinc, and iron, in addition to carrying multiple genes associated with metal resistance and the formation of biofilms. The study also encompassed a pangenome analysis utilizing a dataset of eighty-six publicly available P. stuartii genomes (n = 86), revealing evidence of an open or expanding pangenome for P. stuartii. Also, an extensive genome-wide analysis of all the P. stuartii genomes revealed a concerning global prevalence of diverse antimicrobial resistance genes, with a particular alarm raised over the abundance of carbapenem resistance gene bla_NDM-1. Additionally, this study highlighted the notable genetic diversity within P. stuartii, significant informations about phylogenomic relationships and ancestry, as well as potential for cross-species transmission, raising important implications for public health and microbial adaptation across different environments.

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Introduction

Antimicrobial resistance of bacteria is a phenomenon that had been at the peak of utmost concern, notably in the field of infectious diseases since last two decades¹. Many of the advances in the design and development of antibiotics and antimicrobial drugs have resulted from the extensive effort and meticulous action to combat the ever-evolving mechanisms of resistance that render most of the existing antibiotics obsolete, thus prompting the investigation for introducing new-found or modified molecules having potentiality to be more effective and more resilient². Burns are one of the most common and devastating form of trauma and injury³. On a global scale, burns constitute highly severe injuries, contributing to over 265,000 fatalities worldwide⁴. In the specific context of Bangladesh, an annual toll of approximately 3000 individuals succumb to injuries attributable to burns⁵. A cross-sectional survey, conducted from January to December 2003 on a population basis in Bangladesh, revealed an elevated burn mortality rate among females. The majority of fatalities resulted from accidental incidents, with a mere 5% attributed to self-inflicted burns. Furthermore, the mortality rate was notably higher in rural areas as opposed to urban areas⁴. A research investigation conducted at the National Institute of Burn and Plastic Surgery in Dhaka, Bangladesh, unveiled that for non-lethal burn incidents, demographic groups at elevated risk encompassed young adult males in their early thirties belonging to lower socioeconomic strata⁵. Within the pediatric population, children under eight years old emerged as the most susceptible subgroup. Prevalent non-lethal burn categories included flame-induced injuries (35%), electrical burns (31%), and scald injuries (24%). Regarding mortality cases, high-risk demographics comprised young adult males and children approximately eight years of age. The average extent of total body surface area (TBSA) affected in these instances stood at 46.4%, with complications arising from flame burns constituting the cause of death in 65% of cases⁵. Microbial infection in severe burn wounds by Multidrug-Resistant (MDR) organisms is a serious matter of concern and is a leading cause of morbidity and mortality in hospitalized burn patients⁶. A number of organisms are responsible for causing infections in the wounds of burn patients including MDR Enterobacteriaceae being a major concern. Among the members of Enterobacteriaceae family causing severe infections in burn patients, Providencia stuartii (P. stuartii) is one of the most alarming and matter of growing concern because of its intrinsic resistance to several antibiotics including colistin and gradual occurrence to complete resistance toward carbapenems, considered to be the last line antibiotics against this particular infectious agent⁷. To date, there has been limited documentation of P. stuartii in Bangladesh, particularly in the context of burn wound infections. Some previously published reports from Bangladesh consistently highlighted Pseudomonas aeruginosa (P. aeruginosa) as the predominant microorganism associated with infections in burn patients^8,9,10. Subsequent to the prevalence of P. aeruginosa, other identified pathogens in burn wound infections included Staphylococcus aureus, Proteus spp., Klebsiella spp., Acinetobacter spp., and Escherichia coli, as documented in the scientific literatures^8,9,10,11. Providencia spp. belong to the Morganellaceae family of the order Enterobacterales¹². P. stuartii is a gram-negative rod-shaped bacterium commonly found in soil, water and sewage. It is the most common Providencia species capable of causing human infections and is an opportunistic pathogen. P. stuartii is well known and characterized for causing nosocomial infections which puts immune compromised elderly individuals at a greater risk¹³. Occurrence of sepsis because of P. stuartii infection is considered to be a new challenge in the treatment of thermal injury. In addition to that, many studies have established that, P. stuartii has the ability to migrate to several organs causing endocarditis, peritonitis, pericarditis and meningitis¹³. The extensive and alarming antimicrobial resistance pattern and its severe invasive properties along with uprising infection and mortality rate, makes P. stuartii indisputably a potential threat and to be considered a matter of great concern as well as a significant, cabalistic and interesting subject to research in order to investigate its pathogenicity, antimicrobial resistance mechanisms, alternative and effective treatment methods.

Carbapenems, the most potent class of β-lactams which are the efficacious addition in our antibiotic armamentarium because of its broad spectrum activity against both Gram-positive organisms and Gram negative especially multidrug-resistant Gram-negative bacilli¹⁴. Association of mobile genetic elements (MGEs) with carbapenemases even crossed the species barrier resulting in the worldwide rapid transmission of carbapenemases in nonclonally related isolates and species and the most important types based on rapid and extensive epidemiological dissemination and clinical relevance are bla_KPC-type, bla_OXA-type, bla_VIM-type, bla_NDM-type of cabapenemases¹⁵. As P. stuartii is intrinsically resistant to many antibiotics such as Gentamycin, Tobramycin, Ampicillin, Cefazolin including Colistin; the carbapenems are considered to be the last line drug to treat infections caused by this organism¹⁶. Carbapenem resistant P. stuartii are increasingly reported mainly due to bla_NDM-1¹⁷. So, emerging resistance of this organism to carbapenems could be undoubtedly alarming also in the perspective of Bangladesh as worldwide. Whole genome sequencing (WGS), also known as complete genome sequencing, is the process of determining the entire or nearly the entire DNA sequence of an organism's genome at a single time which reveals almost every detail encoded about that particular organism¹⁸. The recent advancement in bacterial whole genome sequencing approach made it a widely used technique in research, clinical diagnostic and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence¹⁹.

In the perspect of Bangladesh, P. stuartii is not advertently reported and no report has been drawn up or published yet regarding the occurrence of this potential threat in burn patients where its role is certainly serious. In addition to the lack of report about the investigation of P. stuartii in Bangladesh, no whole genome sequencing data of this particular potential threatening organism has been published yet from Bangladesh. The unaware inattention toward this infectious opportunistic pathogen which is being reported as highly dangerous for burn patients worldwide, roused the importance and concernment to investigate about this pathogen in Bangladesh, most importantly in burn and immunocompromised patients. In this study, we have reported about the cultural and genomic characteristics of two MDR isolate Providencia stuartii SHNIBPS63 (Carbapenem resistant) and Providencia stuartii SHNIBPS71 (Carbapenem sensitive), isolated from infected burn wound of two critically injured burn patient.

Results and discussion

Whole genome sequence analysis, identification and mapping

The identification through KmerFinder predicted the two isolates to be Providencia stuartii (P. stuartii). Besides, the identification through Average Nucleotide Identity (ANI) approach validated the identification and revealed the test isolates to be Providencia stuartii as the test isolates showed significant average nucleotide identity of more than 99% with the references (Table 1). Along with that, the isolates were identified as P. stuartii by 16S rDNA-based identification as well. Following the identification, the two test isolates were designated as Providencia stuartii SHNIBPS63 and Providencia stuartii SHNIBPS71. The draft genome analysis revealed that the genomes were of quite quality having around 100 percent completeness and less than 0.6% contamination which indicated toward the good quality and consistency of the genome. The genome size and GC contents of the sequenced isolates were consistent with the published genomes of P. stuartii. Detailed annotation of the two isolates can be found in Table 2. The genome maps revealed constructive and diverse features of both genome (Fig. 1). The sources of each contigs were listed in Table 3.

Table 1 Identification of the test isolates.

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Table 2 General features of the annotated genomes.

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Table 3 Source of each node detected in the draft genomes.

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The draft genome sequences of the two isolate Providencia stuartii SHNIBPS63 and Providencia stuartii SHNIBPS71 were deposited in DDBJ/ENA/GenBank of NCBI under the accession number JASDEX000000000 and JASDEW000000000 respectively. From the annotated data, it was found that both strains contained > 2000 genes in common. Around 35% hypothetical proteins (HP) are indicative toward the functional diversity, evolutionary divergence along with the possibility to contain novel genes or proteins having potential significance. By definition, an HP is a predicted product derived from an open reading frame (ORF) whose translation has not been experimentally demonstrated, and its functional significance remains to be characterized²⁰. From extensive analysis of these hypothetical proteins from P. stuartii, it might be possible to find out proteins having novel functions, new diagnostic markers for P. stuartii or drug targets to combat this pathogenic bacterium. The genome size and GC contents of the sequenced isolates were consistent with the published reference genomes of P. stuartii.

Antimicrobial susceptibility testing and cultural characterization

The two targeted isolates showed multidrug resistance pattern which is depicted in Supplementary Fig. 1. The P. stuartii SHNIBPS63 showed resistance to all the 22 antibiotics tested against it. The other isolate P. stuartii SHNIBPS71 exerted sensitivity to meropenem, doripenem and ciprofloxacin. It showed intermediate sensitivity to imipenem, tigecycline, aztreonam and also showed tolerance against imipenem after the tolerance assay performed against carbapenems. As carbapenems, polymyxins and according to some report tigecycline is considered to be the last line or resort of antibiotics²¹, CRE (Carbapenem Resistant Enterobacterales) like P. stuartii SHNIBPS63 is a matter of concern as P. stuartii is known to be naturally resistant in colistin and tigecycline. Only P. stuartii SHNIBPS63 gave positive result in Modified Hodge test which indicated toward the production of carbapenemase. Along with that, both isolates were predicted to be strong biofilm former. The ability of bacteria to form biofilm is one the mechanism of resistance to survive in the presence of antibiotics²². Metal sensitivity test revealed both isolates to be resistant in 0.01 M iron, copper and zinc solution. The isolates gave characteristic yellow/orange colonies on MacConkey agar and pinpoint colorless colonies on Cetrimide agar media. They were able to ferment glucose, but not lactose and sucrose. Additionally, they showed characteristics such as indole production, nitrate reduction, a positive methyl red reaction, and were oxidase negative but catalase positive. These traits matched the cultural characteristics typically observed in P. stuartii.

Antimicrobial resistance genes and mobile genetic elements investigation

The antimicrobial resistance genes investigation revealed that P. stuartii SHNIBPS63 harboured plethora of genes which are responsible for conferring resistance to many antibiotics. This particular isolate is co-harboured with bla_VEB-6 (Class A), bla_NDM-1 (Class B) and bla_OXA-10 (Class D) which confer resistance to beta lactams. The localization and source investigation of the antimicrobial resistance genes revealed that bla_NDM-1 and bla_VEB-6 genes were predicted to be located in the chromosome. NDM-1, the product of bla_NDM-1, which is a metallo beta lactamase enzyme (carbapenemase), has the capability to confer resistance against carbapenems. According to plasmid mapping, it was determined that 11 antimicrobial resistance genes, including bla_OXA-10, were likely carried by plasmids (Fig. 2). Conversely, in the case of P. stuartii SHNIBPS71, it possessed the plasmid-mediated class C beta-lactamase gene, bla_CMY-16. The presence of bla_CMY-16 gene in P. stuartii SHNIBPS71 conferred resistance to certain beta-lactams, notably cephalosporins, but not able to protect the bacteria from carbapenems. No other beta lactam resistance genes were present in this isolate which indicated toward the sensitivity of this isolate to carbapenems. All the antimicrobial resistance genes carried by the two isolates are listed in Table 4. Total nine antimicrobial resistance genes were anticipated to be plasmid mediated in this isolate. In case of P. stuartii SHNIBPS63, the presence of bla_NDM-1 is responsible for conferring complete resistance to carbapenems like meropenem, imipenem, ertapenem and doripenem. Usually, bla_NDM-1 gene is mediated by plasmids and reported to be carried by plasmids²³. But, in case of P. stuartii SHNIBPS63 it was carried in the chromosome which was verified through plasmid and genome analysis. In depth analysis revealed that the bla_NDM-1 gene was surrounded by several insertion sequences, transposase, integrase genes which denoted toward the possibility of the gene to be acquired or inserted into the chromosome through horizontal gene transfer (Fig. 3).

Table 4 Investigation of antimicrobial resistance genes.

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Investigation on colistin resistance in both test isolates revealed the presence of arnBCADTEF operon (Fig. 4) which was predicted to be responsible for conferring intrinsic resistance to colistin. The arnBCADTEF operon encodes for a set of enzymes that modify lipopolysaccharides (LPS), which are a major component of the outer membrane of gram-negative bacteria. The 4-amino-4-deoxy-l-arabinose (l-Ara4N) chemical groups are specifically added to the lipid A portion of LPS by the arnBCADTEF operon²⁴. This can inhibit the binding of colistin to the bacterial membrane and hence reduce the drug's effectiveness. Additionally, sapABCDEF operon, phoPQ (A two component system), acrAB-TolC, lpxA/C/D, emrA/B/D/E, ramA and ugd gene sequences were detected having potential role in conferring colistin resistance.

Integron investigation revealed the presence of class I integron in P. stuartii SHNIBPS71 and 2 CALIN (Class 3) type and 1 In0 type integron in P. stuartii SHNIBPS63. Although studies have indicated that CALIN integrons can contribute to the transmission of resistance to a number of kinds of antibiotics, including aminoglycosides and beta-lactams, their involvement in the dissemination of antibiotic resistance genes is not yet entirely understood²⁵. On the other hand, In0-type integrons are of interest because they are thought to represent an intermediate stage in the evolution of active integrons. Some researchers have hypothesized that In0-type integrons might be the precursors of active integrons, and they have the ablility or potentiality to evolve into active integrons by acquiring an integrase gene and associated recombination site²⁶. Additionally, several mobile genetic elements were detected in the genomes and especially around the antimicrobial resistance gene cassettes in P. stuartii SHNIBPS63 which indicated toward the horizontal transfer of antimicrobial resistance genes (Fig. 3).

Metal resistance genes and virulence factor genes analysis

Metal resistance genes analysis through BacMet revealed the presence of >100 metal and biocide resistance genes (Supplementary Table 1). The analysis disclosed the presence of genes capable of conferring resistance to several metals such as copper, zinc, iron, cadmium, arsenic, antimony, tungsten and manganese. Both isolates revealed metal and biocide resistance genes such as acrA, acrB, envC, acrF, acrR, actP, arsA, arsB, copA, mdtA, mdtB, mdtC, sitA and many more. The presence of such genes can have important implications in healthcare. For example, in a clinical context, it may signify that the bacterium has developed resistance to metals that are sometimes used in antimicrobial agents. This resistance can make it more challenging to treat infections caused by these bacteria, as traditional antimicrobial treatments may be less effective. The presence of several metal resistance genes in P. stuartii infecting the wound of burn patients can limit the use of metal-based therapeutics and medications which are applied in wound treatment and repair. Additionally, the presence of metal resistance genes may suggest that the bacterium has developed a degree of adaptability and resilience, making it potentially more difficult to eradicate. Understanding the presence of these genes can be important for choosing appropriate treatment strategies and managing the spread of drug-resistant bacteria. In a comparative analysis, it was observed that P. stuartii SHNIBPS63 possessed merA, merD, merE, merP, and merT genes, which were conspicuously absent in P. stuartii SHNIBPS71. These genes play a crucial role in providing resistance against mercury.

PathogenFinder tool predicted the two isolates of P. stuartii to be highly pathogenic. Virulence factor genes investigation revealed the presence of a plethora of virulence genes in the two test isolates. Several genes were found having role in adherence and biofilm formation such as fliA, fliC, fliD, fliE, fliG, fliH, fliI, fliJ, fliK, fliL, fliM, fliN, fliP, fliQ, fliR, fliS, fliT, fliZ, flgK, flgH, flgF, flgD, flgB, flgM, flgN, rhlE, rhlB, fimA, fimD, pilJ, pilM, pilN, pilQ, papC and papD. The abundance of genes related with biofilm formation correlated with the phenotypic trait of being strong biofilm former of the two isolates. Also, virulence genes prediction revealed the presence of yijD gene, which has a putative function of invading the brain endothelial cells, is indicative toward the capacity of P. stuartii to cause meningitis²⁷. Besides, type I, type II, type III, type VI secretion systems and relevant genes were also found in the two isolates. Bacterial secretion systems are critical for the virulence and pathogenicity of various pathogens and in the dynamics of burn wound infection. These systems enable bacteria to deliver toxins, effector proteins, and other molecules into host cells, manipulate host processes, evade the immune system, and acquire essential nutrients²⁸. By using secretion systems, bacteria can establish infections, form biofilms, and communicate with each other. Understanding these mechanisms is crucial for combating bacterial infections and developing effective treatments. On the otherhand, chaperone-usher fimbriae and fimbrial clusters were also investigated and numbers of proteins associated with fimbriae formation were detected in our test isolates of P. stuartii (Supplementary Table 2). In case of burn wound infection by P. stuartii, the role of fimbriae can play pivotal role in case of adherence to host tissues. This adherence is often a crucial initial step in the establishment of infection. Fimbriae facilitate the colonization of host surfaces, allowing pathogens to resist mechanical clearance and evade host immune responses²⁹. Some toxins such as actin cross linking toxin VgrG-1, persistence and stress-resistance toxin PasT, toxin-antitoxin system YoeB/YefM were found to be associated with the isolates. Actin cross linking toxins are reported to specifically target phagocytic cells to promote survival of bacteria after the onset of innate immune defenses³⁰. Previous investigations have demonstrated the essential role of VgrG-1 in facilitating host cell cytotoxicity through the Type VI Secretion System (T6SS) and in hindering phagocytosis mediated by actin cytoskeleton, thereby preventing bacterial engulfment by macrophages³¹. These mechanisms could potentially exert a substantial influence on the initiation and advancement of burn wound infections caused by P. stuartii. Several other virulence factor genes such as rfaD, rfaC, wecA, galE, galU, bioA, bioB, luxS, luxR and many more were present in the two test isolates of P. stuartii which were predicted to be responsible for the pathogenicity and had putative role in burn wound infection dynamics.

Subsystems, metabolic pathways, prophage and CRISPR/Cas system investigation

The subsystems coverage and feature count analysis through RAST server showed that both isolates contained 344 subsystems. The subsystem feature counts and category distribution are not highly different in the two isolates (Supplementary Fig. 2). The metabolic pathway analysis revealed a plethora of pathways having prominent functional impacts. Several pathways were detected having potentiality of different xenobiotic/toxic pollutants biodegradations such as bisphenol A, DDT, toluene, naphthalene, tetrachloroethene and 2, 4-dichlorobenzoate which denoted the probable capacity of the isolates to grow on such harsh environmental conditions indicating toward the potency of the rigidness of the isolates. The isolates also harbor pathways involved in different secondary metabolite biosynthesis such as zeatin, anthocyanin, carotenoid, tropane, flavonoid, biterpenoid, phenylpropanoid and many more. The detailed pathways of both isolates are listed in Supplementary Table 3 and 4. For both isolates, multiple regions of phage integration were identified. P. stuartii SHNIBPS63 contained 8 regions of phage integration and P. stuartii SHNIBPS71 contained 6 regions (Supplementary Fig. 4). The prophage regions detected belonged to bacteriophage family Myoviridae and Siphoviridae. On the other hand, in our study, P. stuartii SHNIBPS63 was found to harbor class 1 I-F type CRISPR/Cas system in its genome (Supplementary Fig. 3). This CRISPR/Cas system could possibly function toward the resistance against phage as previously described in case of Enterobacteriaceae.

Genome wide antimicrobial resistance gene pool investigation

The genome wide analysis of 86 whole genome sequences of P. stuartii available in public repositories (NCBI and BV-BRC) including the two isolates of this study, revealed the notable presence of 47 different categories of antimicrobial resistance genes (Fig. 5A). The abundance investigation revealed antibiotic resistance gene variant or mutant (Elfamycin resistance gene), antibiotic inactivation enzyme (Aminoglycoside resistance genes) and efflux pump genes to be the most abundant categories of antimicrobial resistance genes in case of P. stuartii. The noteworthy prevalence of diverse categories of antimicrobial resistance genes in P. stuartii highlighted a concerning global issue, with a growing pattern of antimicrobial resistance that is becoming increasingly alarming. Further, the abundance analysis of beta lactam resistance genes revealed high abundance of bla_OXA, bla_TEM, bla_CTX-M and bla_NDM genes respectively (Fig. 5B). All the beta lactamase genes harbored by the 86 isolates of P. stuartii are listed in Supplementary Table 5. An in-depth analysis brought to light the presence of various classes and variants of antimicrobial resistance genes, particularly beta-lactamases (Fig. 5C). Class D beta lactamase gene bla_OXA-10 was found to be most abundant and was followed by bla_NDM-1 and bla_TEM-1. The high occurrence of bla_NDM-1 is highly alarming as this gene can confer resistance to almost all the beta lactam antibiotics including carbapenems. As, P. stuartii is intrinsically resistant to many antibiotics including colistin, carbapenems are considered to be the last resort of antibiotics to fight this pathogen. So, high abundance of carbapenem resistance genes like bla_NDM-1 in P. stuartii is highly alarming. Genome wide analysis also disclosed the presence of bla_NDM-1 in P. stuartii F3W which was isolated from a goose in China. The potential for the transmission of carbapenem-resistant P. stuartii with carbapenemase genes between different species poses a substantial threat to public health. This underscores the need for One Health approach to address this issue.

Pangenome analysis revealed an open pangenome of Providencia stuartii

For pangenome analysis, total 86 genomes including the two isolates analyzed in this study were selected. The pangenome analysis revealed the core genome being significantly short having only 195 core genes. The too short core genome which is indicative toward the high genomic diversity of P. stuartii worldwide (Fig. 6). Total number of genes predicted through pangenome analysis were 27,818 including 165 soft core genes, 4392 shell genes and 23,066 cloud genes (Supplementary Table 6). From pangenome analysis, a too small core genome and large accessory genome clearly depicted that the pangenome of P. stuartii is still open which implies ongoing gene acquisition and diversity. An open pangenome arises when a particular taxonomic lineage continues to expand its repertoire of new gene families, and this augmentation does not exhibit asymptotic behavior, irrespective of the number of newly added genomes to the pangenome. The continuous acquisition of genes is a characteristic that could lead to heightened virulence and alterations in the antimicrobial resistance profile of P. stuartii. Moreover, the sequencing of additional strains is anticipated to reveal novel genes, potentially giving rise to the adaptation of new mechanisms pertaining to pathogenicity, virulence, and antimicrobial resistance. The pangenome based phylogenetic analysis revealed that the isolates having non-human sources such as P. stuartii strain F3W (Isolated from Goose in China), P. stuartii strain M2, M4 and M5 (Isolated from Mouse in China), P. stuartii TYL-Y13 (Isolated from fermentation dreg in China) formed a clade which were distantly related strains from our two test isolates and most other isolates collected from human source. Another isolate collected from non-human source, P. stuartii strain Crippen (Isolated from Lucilia sericata in USA, North America) was predicted to be closely related with P. stuartii strain 3-1238-2 (Isolated from China) and isolates collected from bathing water in Algeria (P. stuartii strain M147 and strain M97). The SNP based phylogenetic analysis also validated the relationship between these strains which are clearly depicted in the rooted tree (Fig. 7A). The SNP based phylogenomic analysis likely provided a more comprehensive representation of the evolutionary lineage and relatedness of the isolates and established a comparative linkage with the genomic characteristics derived from pangenome analysis. From Fig. 7A, it can be clearly observed that P. stuartii strain F3W (Isolated from Goose in China) and P. stuartii strain M2 (Isolated from Mouse in China) are the isolates which can be denoted as basal taxa from evolutionary aspect. These two isolates shared a common ancestry and through the analysis, they are predicted to be diverged early in the evolutionary history. Following that, the evolution of P. stuartii strains M2 and M4 was observed, both of which were isolated from mice in China. Subsequently, another strain, P. stuartii strain TYL-Y13, isolated from fermentation dregs in China, was deduced to have undergone evolutionary processes. Despite some ambiguity, based on the assessment of evolutionary relationships through pangenomic and phylogenomic analyses, it can tentatively be inferred that the potential origin of the ancestral lineage of P. stuartii may be associated with geographical regions proximate to China. Regarding P. stuartii SHNIBPS63, analyses based on both the pangenome and SNP-derived phylogenetic assessments indicated a close relationship between this isolate and P. stuartii MRSN2154, substantiating this connection in both genomic and evolutionary contexts. On the other hand, regarding P. stuartii SHNIBPS71, a close genomic association was observed with P. stuartii AS012498 which was isolated from USA (Fig. 6A). However, in terms of evolutionary relationships, the isolate showed a close relationship with P. stuartii PRV00005 and P. stuartii 2020CK-00448 which were isolated from USA (Fig. 7A). Phylogenomic analysis also pointed out the divergence of P. stuartii of non-human sources with the clinical isolates of P. stuartii collected from human sources. From the comparative genomic feature diversity analysis (Fig. 7B, C), it could clearly be observed that the genomes of P. stuartii from different continents and countries were significantly diversed from each other having a small (Including the isolates from non-human sources) and moderate (considering only the strains isolated from human sources) number of genes within the core genome.

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