K. pneumoniae is an important opportunistic pathogen that causes a wide range of infections in both community and healthcare settings (Keynan and Rubinstein, 2007). The World Health Organization has identified K. pneumoniae as an important threat to humanity because it can acquire and express resistance to various antimicrobial classes. This species is also listed as one of the pathogens in the Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) group. It is a major cause of neonatal infection, urinary tract infection, septicemia, and soft tissue infection in hospitals. Community-acquired infections such as pyogenic liver abscesses, pneumonia, and meningitis are also serious health concerns caused by K. pneumoniae (Holt et al., 2015). Although humans are considered major reservoirs of these organisms, especially in the gastrointestinal tract, Klebsiella species have been identified in different habitats, including surface waters, sewage, industrial effluents, soil, and vegetation (Bagley, 1985). Moreover, they have also been associated with clinical mastitis in dairy cattle (Munoz et al., 2006), pneumonia in horses (Estell et al., 2016), and septicemia in pigs (Bidewell et al., 2018).

Despite the prevalence of K. pneumoniae, the role of foods in the prevalence and dissemination of this bacterium remains relatively underexplored. A prevalence of K. pneumoniae of approximately 21% was recently reported in raw meat and ready-to-eat food items from Singapore (Hartantyo et al., 2020). Several researchers have also documented food-borne outbreaks associated with K. pneumoniae (Calbo et al., 2011; Tambekar et al., 2011; Zhang et al., 2018). The selling of raw meat was cited as one of the transmission modes of virulence and especially antimicrobial resistance (AMR) genes from food to humans (Davis et al., 2015). In this context, exploring the molecular epidemiology and resistance development of isolates originating from food holds great significance in understanding the risk of Klebsiella infection and its transmission efficacy to humans (Wareth and Neubauer, 2021).

K. pneumoniae harbors a wide array of virulence factors, including capsules, fimbriae siderophores, lipopolysaccharides, aerobactin, salmochelin, yersiniabactin, colibactin, and various secretion systems (Lam et al., 2018). In addition, K. pneumoniae reportedly possesses multiple AMR genes, which severely limits therapeutic options for infections. The available data estimated that extended-spectrum beta-lactamase-producing and carbapenem-resistant strains of K. pneumoniae account for more than 90,000 infections and 7,000 deaths, respectively, in Europe alone (Wyres et al., 2020). With the increasing affordability and availability of next-generation sequencing (NGS), whole-genome sequencing (WGS) has emerged as a powerful tool to complement traditional microbiological characterization of foodborne pathogens. WGS provides the opportunity for in-depth analysis of organisms at the genomic scale by enabling rapid identification and detection of virulence traits and antimicrobial resistance by establishing phylogenetic relationships with related strains to infer epidemiology. Although many previous studies have examined the genomic features of K. pneumoniae, there is a paucity of data on the genomic features of K. pneumoniae of food origin. Therefore, in the present study, we reported the results of a comparative genomic analysis of K. pneumoniae isolates from fish to bridge the knowledge gaps in the genomic features of K. pneumoniae of food origin and to explore its virulence and antimicrobial resistance traits. Furthermore, the genomes of all available Indian K. pneumoniae isolates of food/environmental origin were compared for phylogenomic relatedness. To our understanding, this report constitutes the first such study from the Indian subcontinent.

Kerala is one of the leading fish-eating states, with almost 85% of the population consuming a variety of freshwater and seawater fishes. The local retail fish markets in Cochin are influenced by the tastes and preferences of consumers (Shyam, 2020). Small and medium fishes are the mainstays of daily cuisine. Varieties such as sardines, mackerel, tilapia, shrimp, crab, clam, mullet, and ertoplus are the most common fishes sold on the market. As fish are among the prominent foods in Kerala cuisine, tracing, identifying, and characterizing the biological contaminants that may affect human health are highly important.

Klebsiella species are ubiquitous in the natural environment and are widely distributed in the gastrointestinal tracts of humans and animals (Gundogan, 2014). It is an important opportunistic pathogen of humans and has acquired international heterogeneity due to the increasing occurrence of AMR. In general, three distinct species of Klebsiella are classified into different phylogroups: K. pneumoniae as KpI, Klebsiella quasipneumoniae as KpII, and Klebsiella varicola as KpIII (Dworkin et al., 2006). K. pneumoniae is associated with a wide range of hosts, from humans to livestock, and different environmental niches (Brisse et al., 2014). In the present study, we reported the genomic features of K. pneumoniae of fish origin and evaluated them in comparison with other food/environmentally sourced genomes of K. pneumoniae available in public databases from the Indian subcontinent for the first time.

A total of 6 K.pneumoniae, 15 E.coli, 3 Pseudomonas aeruginosa, 2 Acinetobacter baumanni/calcoacetius complex, 1 Pseudomonas putida, 1 Aeromonas veronii bv veronii, and 1 Aeromons caviae were identified by the BD Phoenix automated identification and AST system. Almost all of the identified isolates were multidrug resistant (MDR) since we had selectively isolated the ESBL-producing Enterobacteriaceae from the fish samples with 1 µg/ml of cefotaxime in the MaConkey agar plate and selected the suspected colonies. Furthermore, we only selected MDR strains for further characterization by PCR for AGRs and then WGS. The Klebsiella isolates studied were susceptible to the majority of the antibiotics tested, while an intermediate MIC was observed for cephalosporins and fluoroquinolones; these isolates were classified as non-ESL strains. These non-ESBL strains are pathogenic bacteria and are devoid of extended-spectrum beta-lactamases (ESBLs). Compared to non-ESBL K. pneumoniae, ESBL-producing K. pneumoniae is more pathogenic and virulent. However, in most instances, this does not reduce the risk associated with K. pneumoniae. It was shown in one study that the potential risk factors linked to both forms of Enterobacteriaceae are quite comparable when comparing ESBL-producing and non-ESBL-producing Enterobacteriaceae in the clinical context. Only the treatment regimen differed in the usage of antibiotics for ESBL-producing and non-ESBL-producing patients (Bhavnani et al., 2006).

The presence of 30 different AMR genes, including genes associated with reduced permeability, antibiotic efflux, 16S rRNA methylases, and aminoglycoside-modifying enzymes, in the genomes studied, indicated potential hazards, especially when considering the abundance of plasmids that are known to carry AMR genes horizontally. The efflux pump systems, which include the AcrAB and mdtK systems, are also considered to be responsible for MDR in K. pneumoniae (Padilla et al., 2010; Meletis et al., 2012), and they belong to the resistance nodulation division (RND) class and multi-antimicrobial extrusion (MATE) family of efflux pumps, respectively (Wasfi et al., 2016). In all the genomes studied, the universal presence of these genes reflects the inherent transmissibility of the viruses. Unique cases of SHV variants have been reported in clinical settings globally (Liakopoulos et al., 2016). Similarly, in this particular study, different variants, such as bla _SHV-1, bla _SHV-110, and bla _SHV-60, were detected in the fish-sourced isolates, while bla _SHV-1, bla _SHV-106, bla _SHV-120, bla _SHV-134, bla _SHV-182 and bla _SHV-187 were detected in other reference genomes from food/environmental sources. The presence of different variants of bla _SHV genes in all the isolates is attributed to the organism’s ability to adapt to selective environmental pressure (Effah et al., 2020).

Several factors contribute to the virulence and pathogenicity of K. pneumoniae. Among these, the capsular serotype, lipopolysaccharide, iron-acquisition siderophores, and fimbrial and non-fimbrial adhesions are significant (Fuursted et al., 2012). Fimbrial adhesions play a major role in biofilm formation and are classified as virulence factors. The presence of type I and III fimbriae genes in the genome points to the ability of these isolates to adhere to host cells and form biofilms. The presence of the fim operon and mrkA gene in all the isolates contributed to the association of these genes with communities that induce pathogenicity (Alcántar-Curiel et al., 2013). The mrkD gene is a type 3 fimbrial adhesion gene that mediates agglutination and was found in all four genomes of fish origin. It is known to be rare in K. pneumoniae strains but highly conserved in K. oxytoca (Schurtz et al., 1994; Sebghati et al., 1998). This confirms the ability of the isolates to adhere to human renal/pulmonary epithelial cells or tissues, thus rendering them pathogenic. The absence of rmpA is a key feature of all the studied genomes, along with other reference genomes. Since rmpA is associated with virulence in Klebsiella, the absence of this particular virulence factor is intriguing and might be due to the draft nature of the genome. Of the present genomes from the food studied, 50% were O3b and the other 50% were O1v2. O1 is the most common serotype, followed by O3, while the O3b locus in particular is considered to be very rare according to studies of the global genome dataset (n=573) of K. pneumoniae (Follador et al., 2016).

Although our isolates did not show any carbapenemase or oxacillin resistance, the clonal type ST37 is indicative of its ability to transform into a highly epidemic clone. ST37 is a known high-risk international clone of K. pneumoniae with a greater ability to confer multidrug resistance to extended beta-lactamase, AmpC, or carbapenemase producers (Roe et al., 2019; Huynh et al., 2020). According to the definition by Huynh et al. (2020), “high-risk clones” are those that are represented at least 10 times in NCBI genomes, although reports of ST37 clones in the Indian context are very limited. There is a single report of an ST37 K. pneumoniae clinical isolate (either a urine or respiratory tract sample) harboring bla _NDM-1 from Chennai Hospital, India (Giske et al., 2012). Archived data on sequence type 37 of Klebsiella spp. from fish as food origin is not available. The molecular characterization of NDM-1 K. pneumoniae isolates belonging to other STs has been reported from the neonatal ward of a tertiary care hospital in Agartala, Northeast India (Mukherjee et al., 2019). NDM-1-producing K. pneumoniae with ST37 clones were isolated from neonates at a Chinese children’s hospital (Zhu et al., 2016). The incidence of epidemic clone ST37 has been associated with nosocomial infections and has also been reported in canines in Japan (Taniguchi et al., 2017). A high prevalence of ESBL-associated ST37 clones of K. pneumoniae was reported in companion animals in China (Xia et al., 2017). Strains with high-risk clonal lineages can colonize people outside hospital settings (Holt et al., 2015). The present findings highlight the possibility of producing high-risk clones even from food sources, and to the best of our knowledge, this is the first Indian report on ST37 K. pneumoniae isolated from a non-human source, i.e., fish. ST37 and ST192 clones of K. pneumoniae have been reported from the fecal samples of healthy individuals by Lepuschitz et al. (2020), who cited food as a possible source of these pathogens. Our study emphasizes the finding that food can act as a potential reservoir of high-risk clones, creating an ideal niche for the colonization of K. pneumoniae in the community. Guo et al. (2016) reported the presence of ST515 in Klebsiella pneumoniae isolated from ventilator-associated pneumonia patients in mainland China. Sequence type 515 was associated with non-hypermucoviscous strains of K. pneumoniae in this study. The IncR incompatibility group plasmid has been widely reported in clinical isolates worldwide. This particular plasmid is generally associated with MDR genes (Guo et al., 2016). In our case, the IncR plasmid was associated with ST515 clones along with the bla _SHV-1 variant. The phylogenetic analysis indicated a distinct lineage for K. pneumoniae from fishes, although proximity was observed to one of the isolates and the reference genome Kp_HS11286. This study highlights the significance of the circulation of new STs in this fishery niche and its adaptation. However, the small number of samples (n=37) will not represent the complete picture of K.pneumoniae prevalence and the genetic diversity of its AMR. This preliminary study could shed light on the presence of AMR K.pneumoniae and more detailed studies with more AMR pathogens are warranted.

In summary, epidemiological risk factors such as the demography of a place, environment, food, livestock, and companion animals are known sources of K. pneumoniae infection. The present study revealed that food can be a potential reservoir of pathogenic K. pneumoniae. This is the first study to report various STs, namely, ST37, ST515, and ST192, and rare serotype O3b K. pneumoniae isolates from fish. Whole-genome sequencing is a technique that provides greater insights into the clonal complexity of pathogenic bacteria. However, a large number of samples should be used so as to obtain the true occurrences of MDR K. pneumoniae in fish samples. The elimination of virulent or antimicrobial-resistant strains of Klebsiella in food must be enforced at all stages of the food chain.