Citrobacter freundii is a zoonotic and opportunistic pathogen that has been linked to occurrences of gastroenteritis and food poisoning. Antimicrobial-resistant and virulent C. freundii has the potential to be transferred from poultry to humans via direct contact and the food chain, suggesting a serious public health threat. But the occurrence of C. freundii in poultry, especially in ducks is still not studied well in Bangladesh. As far as we know, there is no data on the identification and characterization of C. freundii in ducks in Bangladesh. Moreover, the deleterious consequences of C. freundii could be minimized if their evolutionary history, metabolic capability, and VFG and ARG profiles were well understood. Therefore, in the present study, we detected a C. freundii BAU_TM8 isolate in a domesticated duck in Bangladesh and characterized the isolate using WGS to determine its antimicrobial resistance and virulence profiles.
Citrobacter freundii can cause opportunistic infections in people due to its versatile genetic make-up, which allows it to colonize a wide range of habitats [17]. In this study, WGS analysis revealed that the C. freundii BAU_TM8 strain had a diverse and dynamic genetic makeup. The genomic characteristics of the C. freundii BAU_TM8 genome that have been characterized in the current study are similar to the genome properties of a variety of C. freundii strains that have been reported in the past from other countries [18, 19]. Moreover, this strain was 100% similar to a reference genome, C. freundii MRSN 12115 isolated from a human sample in the USA. Complete genome sequencing (using WGS) for phylogenetic analysis allows for a more accurate assessment of the evolutionary relationships between sampled species. Epidemiological surveillance should be automated and strengthened by using this type of analysis.
Citrobacter freundii has developed a higher level of resistance to a variety of antimicrobial classes throughout time [20]. Moreover, AMR and/or ARGs have been exacerbated by the widespread use of various antimicrobials for poultry disease control, particularly in lower-middle-income countries, including Bangladesh [21]. In this study, the C. freundii BAU_TM8 strain exhibited a high level of antimicrobial resistance in both phenotypic and genomic assays. It appears that the genome of C. freundii BAU_TM8 contains genes for MDR efflux systems with similar specificities. Among these are the putative MFS and RND family gene sets (emrB and emrR and acrA and acrB) which may both transport the cation fluoroquinolone and ansamycin groups of antibiotics, as reported by Yap et al. [1]. However, it is not clear why cells have many coping mechanisms against the same type of toxic materials. These overlapping specificities of MDR efflux systems are thought to improve bacterial fitness by allowing cells to maximize the removal of target substances [1, 22]. In addition, this study revealed that the present strain harbored two plasmids, including IncR, exhibiting numerous transposable elements that carried multifarious resistance genes [23]. This finding demonstrates that the plasmid's transmissibility raises the danger of antibiotic-resistant bacterial pathogen and presents a formidable obstacle to disease treatment [2]. This also suggests that C. freundii BAU TM8 may have developed widespread resistance to almost all antibiotics used for disease treatment and may have emerged as a pandrug-resistant bacterium in Bangladesh due to the presence of ARGs, plasmids, and similar resistance mechanisms. WGS was also used to learn more about the mechanisms behind AMR, such as the MDR efflux systems, transcriptional activator, probable aminoglycoside efflux pump, intrinsic MDR mechanism, MDR efflux pump outer membrane protein, and MDR proteins. This agreement is in line with previous research and lends credence to the data already available on WGS's reliability in predicting AMR phenotypes [1, 6, 24, 25]. Moreover, this strain contained resistance genes under the cephalosporin and cephamycin classes of antimicrobial agents (blaTEM−1 and blaCMY−51). Resistance genes conferring cephalosporin and cephamycin resistance were also detected in the previous studies conducted in Bangladesh, China, Malaysia, and Taiwan [1, 6, 9, 20]. The presence of resistance genes against a high number of antibiotic classes in this strain suggests quick action to curb the overuse of antimicrobials for the treatment of poultry diseases.
Moreover, phylogenetic analysis between the present strain and selected 41 global strains revealed that this strain was closely related to six strains of the USA (n = 3), China (n = 2), and Denmark (n = 1), isolated from human and environmental samples, respectively. Since a few caveats, for example, the phylogeny analysis was conducted on a random assortment of archived C. freundii isolates from the NCBI database, we cannot draw the conclusion that the ratios of ARGs and phylogeny revealed in the study are significant. However, in this study, we showed the possible utility of whole genome sequencing to suggest the spread of strains, which might be connected with data from adjacent ARGs and many other details about the strains.
Virulence determinants recognized in Citrobacter species were determined in the present strain, including fimbriae, toxin, acid resistance, flagellar motility, iron uptake, antiphagocytosis, and others. Although the virulence genes of the C. freundii BAU_TM8 strain show a high degree of conservation, the strain also demonstrates a number of distinctive characteristics, such as the ability to form biofilms, that may affect its ecological adaptations [1]. In the current study, a total of 66 VFGs were detected in this strain using the VFDB database. We found that this strain is particularly abundant in virulent-strain-specific proteins, as evidenced by the existence of distinct genes encoding for flagellar hook protein (flg genes) and flagellar filament structural or regulatory protein (fli genes) [1]. These VFGs play an essential role in the development of the pathogenesis of a wide variety of infectious consequences brought on by a variety of pathotypes of Enterobacterales, including species of Citrobacter [9]. This strain harbored several fimbrial adherence determinant and dispensary virulence genes, such as fimA, fimC, fimF, fimH, fimD, etc., which have a significant role in the invasion of human epithelial cells in the gastrointestinal tract, the urinary tract, and the lungs. In fact, the suppression of the fimD gene in C. freundii led to a reduction in the organism’s ability to adhere to and invade epithelial cells, as demonstrated by an expression analysis [26]. Moreover, it is possible that bacteria with these genes have a higher potential for causing disease and adapting to human environments. Moreover, PathogenFinder exhibited that this strain had a very high pathogenicity index (0.919), indicating the isolate as a very high pathogenic for humans. The present study revealed that the C. freundii BAU TM8 strain isolated from a duck expresses multiple VFGs, suggesting that it may act as a zoonotic pathogen.