Survival and Genome Evolution Signatures of Klebsiella pneumoniae Isolates Originated in Seven Species of Aquatic Animals

Abstract

Klebsiella pneumoniae can cause life-threatening pneumonia in humans. The bacterium is also the causative agent of nosocomial infection diseases. In our recent research, we reported, for the first time, the presence of K. pneumoniae in fourteen species of aquatic animals sampled in Shanghai, China. Here, we further investigated the bacterial survival and genome evolution traits. The results revealed that K. pneumoniae isolates (n = 7), recovered from 7 species of commonly consumed aquatic animals, had multiple antibiotic and heavy metal resistance profiles. The isolates were capable of growing vigorously at pH 4.5−7.5 and 0.5−1.0% NaCl in TSB medium at 37 °C. Draft genome sequences of the K. pneumoniae isolates were determined (5,256,522−5,857,823 bp, 56.35–57.81% GC contents), which carried many mobile genetic elements, including genomic islands (n = 87), prophages (n = 14), integrons (n = 4), and insertion sequences (n = 22), indicating possible active horizontal gene transfer during the genome evolution. Meanwhile, numerous strain-specific (n = 199−605) genes, antibiotic resistance (n = 20−35, e.g., β-lactamase) genes, and virulence (n = 43−59, e.g., enterobactin)-related genes, were also identified, demonstrating considerable genome variation in the K. pneumoniae isolates. Overall, the results of this study fill prior gaps in understanding the K. pneumoniae genomes derived from aquatic animals.

1. Introduction

Klebsiella pneumoniae was first described by Carl Friedlander in 1882, which was isolated from the lungs of patients who died from pneumonia [1]. Since then, the Gram-negative bacterium has also been reported to cause bloodstream infections (BSIs), meningitis, osteomyelitis, thrombophlebitis, urinary tract infections (UTIs), and invasive pyogenic liver abscess syndrome [2,3,4]. To date, more than 79 capsular (K antigen) serotypes have been reported in K. pneumoniae isolates, of which K1, K2, K5, K20, K54, and K57 serotypes are strongly associated with the bacterial pathogenesis [5,6].

Antibiotics are widely used in clinics for prevention and therapy of bacterial infections [3]. Nevertheless, the overuse and/or misuse of antibiotics have accelerated the spread of antibiotic-resistant pathogens, particularly in developing countries [2,7,8]. For instance, in China, more than 40% (267/666) of clinical K. pneumoniae isolates, collected from 30 medical centers across the country, were identified as carbapenem-resistant K. pneumoniae (CRKP) in 2017 [7]. The mortality of patients with CRKP infection (main BSIs) was up to 70% [8]. The rising incidence of multiple drug resistant (MDR) K. pneumoniae creates serious threats to public health.

K. pneumoniae are found growing in hospital wastewaters, urban rivers, and even in shrimp farms [9,10,11,12]. Intensive farming in aquaculture drives indiscriminate use of antibiotics, which results in antibiotic residues in aquatic products and MDR pathogens [13]. For example, recently, Luo et al. [14] detected chloramphenicol (CHL) residue in shrimp, shellfish, and fish samples (n = 291) sampled in Shenzhen, China. They found that positive detection rates of CHL were 13.6% (3/22), 37.2% (64/172), and 16.5% (16/97) in shrimp, shellfish, and fish, respectively [14]. Xu et al. [15] recently reported K. pneumoniae isolates (n = 94) present in 14 species of aquatic animals sampled in Shanghai, China, which showed higher resistance rates to sulfamethoxazole–trimethoprim (SXT, 52.1%) and CHL (31.9%). On the other hand, heavy metal concentrations in marine environments, rivers, and soils have been increasing because of industrialization and environmental pollution, which has given rise to heavy-metal-tolerant bacteria [16]. Moreover, heavy metals could trigger the proliferation of antibiotic resistance by increasing mobile genetic element (MGE) abundance or by influencing bacterial communities [17]. It has been reported that the majority of the K. pneumoniae isolates were also tolerant to heavy metals e.g., Cr³⁺ (96.8%), Pb²⁺ (89.4%), and Hg²⁺ (81.9%) [15]. The co-selection between antibiotics and heavy metals, leading to MDR K. pneumoniae, threatens population health [18,19].

Aquatic environments, considered as a pool of antibiotic resistance genes (ARGs), contain diverse microbial communities, where the dissemination of ARGs could partially be attributed to horizontal gene transfer (HGT) mediated via MGEs in bacterial genomes [20,21]. To date, complete genome sequences of over 1757 K. pneumoniae isolates are available in the GenBank database (https://www.ncbi.nlm.nih.gov/, accessed on 14 March 2023). Of these, the majority of the K. pneumoniae strains (n = 1659) were isolated from humans, followed by animals (n = 87), water environments (n = 4), and others (n = 7). To the best of our knowledge, current literature on the genomes of K. pneumoniae isolates originating in aquatic animals is rare.

In our recent research, we reported, for the first time, the presence of K. pneumoniae in 14 species of aquatic animals [15]. Based on the finding, in this study, we further investigated the survival and genome evolution traits of the K. pneumoniae isolates recovered from seven species of commonly consumed aquatic animals. The major objectives of this study were (1) to examine phenotypes, genotypes, and growth traits of the K. pneumoniae isolates; (2) to determine draft genome sequences of the K. pneumoniae isolates using the Illumina Hiseq × Ten sequencing technique and to identify MGEs, such as genomic islands (GIs), prophages, integrons (INs), and insertion sequences (ISs) in the K. pneumoniae genomes; (3) to identify virulence- and resistance-related genes in the K. pneumoniae genomes; and (4) to analyze phylogenetic relatedness of the K. pneumoniae isolates. The results of this study will enrich genome data and fill prior gaps in understanding K. pneumoniae genomes derived from aquatic animals.

2. Materials and Methods

2.1. K. pneumoniae Isolates and Cultural Conditions

K. pneumoniae strains (Table S1) were isolated from three species of shellfish: Maoctra veneriformis, Cipangopaludina cahayensis and Tegillarca granosa; two species of crustaceans: Eriocheir sinensis and Procambarus clarkii; and two species of fish: Epinephelus fuscoguttatus and Misgurnus anguillicaudatus, which were sampled in Shanghai, China in July−September of 2018−2019 [15]. The K. pneumoniae isolates were identified by biochemical and molecular biological methods [15] and stored at −80 °C in a freezer in our laboratory at Shanghai Ocean University, in Shanghai, China. The isolates were routinely incubated in Tryptic Soy Broth (TSB) medium (pH 7.2, 0.5% NaCl) (Beijing Land Bridge Technology, Beijing, China) at 37 °C aerobically, with shaking at 175 rpm [15]. K. pneumoniae ATCC13883 was used as a positive control strain.

2.2. Antibiotic Susceptibility and Heavy Metal Tolerance Assays

Antibiotic susceptibility of the K. pneumoniae isolates were tested according to the disc diffusion method approved by the Clinical and Laboratory Standards Institute in the United State (CLSI, M100-S28, 2018). The Mueller–Hinton (MH) medium and antibiotic discs were purchased from OXOID, Basingstoke, UK, as described in our recent reports [15,22]. Heavy metal tolerance of the K. pneumoniae isolates was performed according to the broth dilution testing (microdilution, CLSI) [15,22]. The HgCl₂, NiCl₂, CrCl₃, CdCl₂, PbCl₂, CuCl₂, ZnCl₂, and MnCl₂ (Analytical Reagent, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) were applied in the range from 3200 to 3.125 μg/mL. The results were interpreted by minimal inhibitory concentrations (MICs) that completely inhibited the growth of the bacteria. Escherichia coli ATCC25922 and K12 strains (Institute of Industrial Microbiology, Shanghai, China) were used as quality control strains [15,22].

2.3. Growth Curve Assay

The TSB was adjusted to different pH (3.5, 4.5, 5.5, 6.5, and 7.5) and NaCl concentrations (0.5%, 1%, 2%, 3%, and 4%) as described in our previous studies [23,24]. Growth curves of the K. pneumoniae isolates under different pH (3.5–7.5) and NaCl (0.5–4%) conditions were individually determined at 37 °C for 25 h using Multimode Microplate Reader (BioTek Instruments, Winooski, VT, USA).

2.4. Polymerase Chain Reaction (PCR) Assay

The 16S rRNA gene, virulence-associated genes (aerobactin, magA, tarT, wcaG, iroN, rmpA, entB, fimH, mrkD, and ybtA), and capsule serotypes (K1, K2, K5, K20, K54, and K57) were detected using the PCR assay as described in our recent report [15]. The primers (Table S2) were synthesized by the Sangon (Shanghai, China).

2.5. Genome Sequencing, Assembly, and Annotation

The K. pneumoniae isolates were individually incubated in the TSB (pH 7.2, 0.5% NaCl) to logarithmic growth stage (LGS). Bacterial cells were harvested by centrifugation, and genomic DNA was extracted using TIANamp Bacteria DNA Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China) according to the manufacture’s instruction. Three separately produced DNA samples were used for each of the K. pneumoniae isolates. DNA samples were analyzed, as described in our previous report [21,24], and only high quality samples were subjected to the genome sequencing.

Whole-genome sequencing of the K. pneumoniae isolates was conducted by Shanghai Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China, using Illumina Hiseq × Ten (Illumina, San Diego, CA, USA) platform [21]. High-quality sequence assembly was performed using SOAPdenovo (version 2.04) software [25]. Coding sequences (CDSs), rRNA genes, tRNA genes, and Clusters of Orthologous Groups (COG) of proteins were predicted using the same software Glimmer (version 3.02) [26], Barrnap tool (https://github.com/tseemann/barrnap, accessed on 31 July 2022), tRNAscan-SE (version 2.0) [27], and Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST, accessed on 31 July 2022) with default parameters as described in our recent report [18]. The virulence factor database (http://www.mgc.ac.cn/VFs, accessed on 31 July 2022) and the ARGs database (http://arpcard.Mcmaster.ca, accessed on 31 July 2022) were used to detect virulence- and antibiotic resistance-related genes, respectively [18].

2.6. Comparative Genome Analysis

GIs, prophages, INs, ISs, and CRISPR-Cas repeats in the K. pneumoniae genomes were predicted using the same software IslandViewer (version 1.2) [28], Phage_Finder [29], Integron_Finder (version 2.0) [30], ISEScan (version 1.7.2.1) [31], as well as Mined software (version 3) and CRISPRtyper [32] with default parameters as described in our recent report [21].

Core genes were the set of genes encoding orthologous proteins in all the genomes tested, while pan–genes were the set of all genes present in all the tested genomes. Only proteins with ≥60% amino acid similarity and ≥80% sequence coverage were designated as direct relatives, while those with ≤30% or no hits were assigned as strain-specific genes at E ≤ 1 × 10⁻⁵ [21].

A phylogenetic tree was constructed on the basis of amino acid data sets of single-copy orthologs that were present in all the analyzed genomes of 72 K. pneumoniae isolates, of which complete genome sequences of 65 K. pneumoniae isolates were downloaded from the GenBank database (Table S3). The maximum likelihood method was used to build a tree by RAxML (version 8) software [33], with 1000 bootstrap replications and a cut-off threshold of ≥50% bootstrap values.

2.7. Statistical Analysis

The SPSS statistical analysis software (version 17.0, SPSS Inc., Chicago, IL, USA) was used to analyze the data. All tests were conducted in triplicate.

3. Results

3.1. Phenotypes and Genotypes of the K. pneumoniae Isolates

K. pneumoniae 7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, and 8-2-10-5 strains (Table S1) were isolated from 7 species of aquatic animals, including M. anguillicaudatus, M. veneriformis, E. sinensis, C. cahayensis, P. clarkii, T. granosa, and E. fuscoguttatus, respectively [15]. The isolates were confirmed by 16S rRNA gene sequencing and analysis, and the obtained 16S rDNA sequences were deposited in the National Center for Biotechnology Information (NCBI) database under the accession numbers shown in Table S1.

The K. pneumoniae isolates had different antibiotic resistance profiles (Table S1). For example, K. pneumoniae 7-5-4 from M. anguillicaudatus displayed resistance to CHL, ciprofloxacin (CIP), kanamycin (KAN), norfloxacin (NOR), SXT, and tetracycline (TET), while K. pneumoniae 7-10-14 from M. veneriformis was resistant to ampicillin (AMP), CHL, gentamicin (GEN), KAN, SXT, and TET. Conversely, K. pneumoniae 7-13-2, 7-17-8, and 8-1-12-1 isolates were solely resistant to AMP.

Meanwhile, the K. pneumoniae isolates tolerated 3−7 heavy metals, showing different tolerance patterns. For example, K. pneumoniae 8-1-12-1 from P. clarkii was tolerant to Zn²⁺/Pb²⁺/Mn²⁺/Hg²⁺/Cu²⁺/Cr³⁺/Cd²⁺, while K. pneumoniae 8-2-10-5 from E. fuscoguttatus was tolerant to Zn²⁺/Pb²⁺/Hg²⁺/Cu²⁺/Cr³⁺/Cd²⁺. Conversely, K. pneumoniae 7-17-8 from C. cahayensis tolerated the minimum number of heavy metals (Zn²⁺/Cu²⁺/Cr³⁺) tested.

In addition, all the K. pneumoniae isolates tested negative for the virulence-related genes, including the aerobactin, magA, tarT, wcaG, iroN, and rmpA. However, some virulence-related genes tested positive in the K. pneumoniae isolates. For instance, K. pneumoniae 7-10-14 carried the entB gene; K. pneumoniae 7-5-4, 7-13-2, 8-2-5-4, and 8-2-10-5 isolates tested positive for the entB/fimH/mrkD genes; and K. pneumoniae 7-17-8, and 8-1-12-1 isolates had the entB/fimH/mrkD/ybtA gene profile.

3.2. Survival of the K. pneumoniae Isolates at Different pH and NaCl Conditions

The pH of the human stomach normally ranges pH 1−3, but can rise above 6.0 after food consumption [23]. Therefore, we examined survival of the K. pneumoniae isolates at different pH conditions (pH 3.5–7.5) when incubated in the TSB (0.5% NaCl) at 37 °C. As shown in Figure 1A–G, the growth of all K. pneumoniae isolates was severely inhibited at the acidic pH 3.5. However, notably, the acidic pH 4.5 condition strongly promoted the bacterial growth, and all the isolates were capable of growing vigorously at pH 4.5−7.5. The highest biomass was observed at pH 7.5, with the maximum OD₆₀₀ at stationary growth phase (SGP) in the range of 1.37−1.51 (Figure 1).

Given that the K. pneumoniae isolates were recovered from different aquatic animals in water environments, we further determined growth curves of the K. pneumoniae isolates at different salinity concentrations (0.5−4% NaCl) when incubated in the TSB (pH 7.5) at 37 °C. As shown in Figure 2A−G, the growth of all K. pneumoniae isolates was severely inhibited at 4% NaCl. The biomass of the K. pneumoniae isolates gradually increased with the decreased NaCl concentrations (3–0.5%), and the highest biomass was observed at 0.5% NaCl, showing the maximum OD₆₀₀ values ranging from 1.26 to 1.44 at SGP.

For example, K. pneumoniae 7-5-4 from M. anguillicaudatus appeared the most susceptible to the higher salinity concentrations, as this isolate was strongly repressed at 3% NaCl as well. Conversely, this condition stimulated the growth of the other 6 isolates, but showed a longer retarded growth phase (RGP) (11−19 h) with lower biomass (OD₆₀₀ = 0.68−0.97). The decreased NaCl concentration (2%) promoted the growth of K. pneumoniae 7-5-4, although it had an RGP for 8 h. K. pneumoniae 7-5-4 was able to grow vigorously at 1.0−0.5%, the same case as the other isolates.

Taken together, the results demonstrated that the K. pneumoniae isolates of aquatic animal origins were able to grow vigorously at pH 4.5−7.5, 0.5−1.0% NaCl in the TSB at 37 °C. K. pneumoniae 7-5-4 was the most susceptible to higher salinity concentrations (4−3 NaCl) among the isolates tested in this study.

3.3. Genome Features of the K. pneumoniae Isolates of Aquatic Animal Origins

Based on the obtained results, we further determined draft genome sequences of the 7 K. pneumoniae isolates using the Illumina Hiseq × Ten sequencing platform, which generated approximately 76,382−107,851 clean single reads. The final assembly yielded 45−113 scaffolds with sequencing depth (on average) of 188.3–fold to 271.9−fold. The obtained genome sizes ranged from 5,256,522 to 5,857,823 bp with GC contents of 56.35–57.81% (

Table 1. Genome features of the K. pneumoniae isolates of aquatic animal origins.

Genome Feature	K. pneumoniae Isolate
	7-5-4	7-10-14	7-13-2	7-17-8	8-1-12-1	8-2-5-4	8-2-10-5
Genome size (bp)	5,857,823	5,376,532	5,412,275	5,438,640	5,593,530	5,432,731	5,256,522
G + C (%)	56.35	57.28	57.29	57.12	57.21	57.32	57.81
DNA Scaffold	81	113	79	50	74	64	45
Total predicted gene	5662	5142	5165	5189	5369	5143	4986
Protein-coding gene	5558	5044	5060	5089	5260	5042	4885
RNA gene	240	231	238	234	249	224	235
Genes assigned to COG	4986	4761	4757	4770	4916	4777	4639
Genes with unknown function	1363	1230	1288	1271	1357	1230	1188
GI	17	8	8	15	16	11	12
Prophage	2	2	2	2	3	1	2
IN	1	2	0	0	0	0	1
IS	2	2	6	2	4	4	2
CRISPR-Cas repeat	14	9	6	2	6	6	11
Source	This study	This study	This study	This study	This study	[15]	This study

, Figure S2). A total of 4885–5558 protein-coding genes were predicted, of which approximately 4639–4986 genes were classified into 22 functional catalogs in the COG database. Remarkably, the K. pneumoniae genomes carried many MGEs, including GIs (n = 87), prophages (n = 14), INs (n = 4), and ISs (n = 22), suggesting the HGT during the K. pneumoniae genome evolution via these MGEs.

The draft genomes of the K. pneumoniae 7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4 and 8-2-10-5 isolates were deposited in the GenBank database under the accession numbers JALJQY000000000, JALJQX000000000, JALJQW000000000, JALJQV000000000, JALJQT000000000, JALJQQ000000000, and JALJQR00000000, respectively.

3.4. MGEs in the K. pneumoniae Genomes of Aquatic Animal Origins

3.4.1. GIs

GIs can carry large foreign DNA fragments (~200 Kb) and endorse the host’s diverse biological functions [21]. In this study, remarkably, a total of 87 GIs were identified in the 7 K. pneumoniae genomes (Table S4), each of which contained 8−17 GIs, ranging from 3228 to 44,595 bp and encoding 5−47 genes (Figure 3). Interestingly, various functions carried by the GIs were found, e.g., virulence, resistance, substrate hydrolysis, transporting and utilization, restriction and modification, as well as phage and stress regulation.

The K. pneumoniae 7-5-4 genome contained the maximum number of the GIs (n = 17, GIs 1−17), whereas K. pneumoniae 7-10-14 had the fewest GIs (n = 8, GIs 1−8). Of note, virulence-related genes were found in 10 GIs in the 7 K. pneumoniae genomes (Table S5). For example, the hcp gene was present in the GI 12, GI 1, GI 3, GI 16, and GI 3 in K. pneumoniae 7-5-4, 7-10-14, 7-17-8, 8-1-12-1, and 8-2-5-4 genomes, respectively. It is a core gene of the T6SS in Acinetobacter baumannii, causing respiratory tract infection [34]. Antibiotic resistance and heavy-metal-tolerance-related genes were identified in some GIs, e.g., the GI 3 (17,350 bp) and GI 8 (6626 bp) in the K. pneumoniae 7-13-2 genome, respectively. Interestingly, the conjugative transfer-related genes were found in the GI 11 in K. pneumoniae 7-17-8. Additionally, there were some identified GIs carrying phage regulator genes in the K. pneumoniae genomes. For example, the GI 5 in K. pneumoniae 7-5-4, and the GI 11 in K. pneumoniae 8-1-12-1 contained the gene (Kp 7-5-4_1988; Kp 8-1-12-1_3134) encoding a lysB family phage lysis regulatory protein.

3.4.2. Prophages

Prophages are viruses that infect bacteria. They can transfer important biological characteristics to their bacterial hosts [35]. In this study, a total of 14 prophage gene clusters were identified in the 7 K. pneumoniae genomes (Table S6), each of which carried 1−3 prophages, ranging from 21,338 to 108,967 bp and encoding 34−115 genes (Figure 4).

The K. pneumoniae 8-1-12-1 genome contained the maximum number of prophage gene clusters (n = 3), which had sequence similarity to Ralstonia_phage_RSA1 (38,760 bp, NCBI accession number: NC_009382), Enterobacteria_phage_186 (30,624 bp, NCBI accession number: NC_001317), and Klebsiella_phage_phiKO2 (51,601 bp, NCBI accession number: NC_005857). Conversely, K. pneumoniae 8-2-5-4 carried only 1 prophage gene cluster similar to Enterobacteria_phage_HK022 (15,456 bp, NCBI accession number: NC_002161).

The identified 14 prophages in the 7 K. pneumoniae genomes were derived from 4 different genera, including Enterobacteria, Klebsiella, Pseudomonas, and Ralstonia, indicating extensive phage transmission across the genera boundaries. Moreover, the Enterobacteria_phage_186 homologue was present in the K. pneumoniae 7-5-4 and 8-1-12-1 genomes, but in different lengths, encoding different numbers of genes. The same was the case for the Pseudomonas_phage_D3 homologue, which was present in the K. pneumoniae 7-5-4 and 7-10-14 genomes. Likewise, the Klebsiella_phage_phiKO2 homologue was found in the K. pneumonia 7-17-8, 8-1-12-1 and 8-2-10-5 genomes carrying a similar set of phage structure genes but different accessory genes. These results also provided evidence of extensive genome rearrangement during the K. pneumoniae genome evolution.

3.4.3. INs

INs are considered as determinants in acquisition and evolution of virulence and antibiotic resistance [36]. They are classified into type I, type II, type III, and super integrons based on integrase genes (intI1, intI2, intI3, and intI4) [21]. In this study, INs were identified in the K. pneumoniae 7-5-4 (n = 1), 7-10-14 (n = 2), and 8-2-10-5 (n = 1) genomes, but absent from K. pneumoniae 7-13-2, 7-17-8, 8-1-12-1, and 8-2-5-4 genomes (Figure 5, Table S7).

The K. pneumoniae 7-5-4 genome contained only 1 complete IN (2737 bp), encoding a NAD (+)-rifampin ADP-ribosyltransferase Arr-2 (Kp 7_5_4_5567), an ANT(3″)-Ia family aminoglycoside nucleotidyltransferase AadA3 (Kp 7_5_4_5566), an Orf3/QacEdelta1 fusion protein (Kp 7_5_4_5565), and an intl1 (Kp 7_5_4_5568). The type 1 INs are strongly associated with the dissemination of antibiotic resistance in bacteria [37].

The K. pneumoniae 7-10-14 genome contained 2 incomplete INs (INs 1−2) with gene cassettes. The IN 1 encoded an aac(6′)-Ib family aminoglycoside 6′-N-acetyltransferase (Kp 7_10_14_5082), a NAD(+)-rifampin ADP-ribosyltransferase (Kp 7_10_14_5083), and a dihydrofolate reductase type 15 (Kp 7_10_14_5084), while the IN 2 encoded a quaternary ammonium compound efflux SMR (Kp 7_10_14_5103) and an aminoglycoside resistance protein (Kp 7_10_14_5104).

The K. pneumoniae 8-2-10-5 genome contained 1 incomplete IN, encoding a glycoside hydrolase family 31 protein (Kp 8_2_10_5_0283) and an ABC-F family ATPase (Kp 8_2_10_5_0284).

3.4.4. ISs

ISs are short discrete DNA fragments that can move themselves to a new position in DNA almost randomly in a single cell [38]. In this study, all the 7 K. pneumoniae genomes contained ISs (n = 2 to 6), ranging from 741 to 1588 bp (Table S8).

For instance, the K. pneumoniae 7-13-2 genome contained the maximum numbers of ISs (n = 6, IS001−IS006). The IS001 (807 bp) coded for a tyrosine-type recombinase/integrase (Kp 7_13_2_0367); IS002 (1265 bp) for a transposase IS116/IS110/IS902 family (Kp 7_13_2_4341); IS003 (1161 bp) for transposases (Kp 7_13_2_4675, Kp 7_13_2_4676); IS004 (1173 bp) for a IS5 family transposase (Kp 7_13_2_4820); IS005 (1257 bp) for a IS3 family transposase (Kp 7_13_2_5141, Kp 7_13_2_5142); and IS006 (819 bp) for a IS6-like element IS26 family transposase (Kp 7_13_2_5153).

3.5. CRISPR-Cas Repeats

The CRISPR-Cas systems provide adaptive immunity to prokaryotes from invasion by foreign nucleic acids in their hosts [39]. The systems are associated with drug resistance in K. pneumoniae [40,41]. In this study, a number of CRISPR-Cas repeats (n = 54) were identified in the 7 K. pneumoniae genomes, each of which contained 2−14 such gene clusters, ranging from 75 to 2649 bp. However, none of these systems contained the Cas protein, suggesting partial or inactive CRISPR-Cas systems in the K. pneumoniae isolates (Figure 6).

For instance, the K. pneumoniae 7-5-4 genome contained the maximum number of the CRISPR-Cas repeats (n = 14, CRISPRs 1−14), ranging from 77 to 2649 bp. The CRISPR 4 was the longest in size (2649 bp), with the maximum number of repetitive sequences (n = 44), whereas the CRISPR 3 was the shortest (77 bp), with the fewest repeats (n = 2). Conversely, the K. pneumoniae 7-17-8 genome had the fewest CRISPR-Cas repeats (n = 2, CRISPRs 1−2). The CRISPR 1 (201 bp) had 4 repeats, while the CRISPR 2 (188 bp) had 3 repeats.

3.6. Putative Virulence-Associated Genes in the K. pneumoniae Genomes

Many putative virulence-related genes (n = 43−59) were identified in the 7 K. pneumoniae genomes (Table 2). K. pneumoniae 8-1-12-1 form P. clarkii contained the maximum number of such genes (n = 59), whereas K. pneumoniae 7-10-14 from M. veneriformis carried relatively fewer (n = 43).

For instance, the entABCDEFS and fepABCDG gene clusters were identified in all the K. pneumoniae genomes, both of which are related to enterobactin in K. pneumoniae. The former is responsible for enterobactin biosynthesis, while the latter mediates enterobactin transport. Specifically, the fepA gene encodes a receptor for enterobactin uptake [3]. Of note, the iucABCD gene cluster was found in the K. pneumoniae 8-2-5-4 genome, which is involved in the biosynthesis of aerobactin, commonly detected in hypervirulent K. pneumoniae [42].

The other virulence-related genes involved in the bacterial persistence in the host were also identified in the seven K. pneumoniae genomes. For example, the fimH gene encoding a fimbrial protein was found in the K. pneumoniae 7-5-4, 7-17-8, 8-1-12-1, 8-2-5-4, and 8-2-10-5 genomes, which can bind to highly mannosylated UPIa to ensure stable adhesion of bacteria to tissues in the host [43].

Table 2. The putative virulence-related genes identified in the K. pneumoniae genomes.

Virulence-Related Gene	K. pneumoniae Genome	Reference
ecpABCDER	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[44]
entABCDEFS	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[45]
fepABCDG	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[46]
fes	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[46]
gnd	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[47]
kdsA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[48]
rcsAB	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[45]
tuf	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[49]
wza	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[50]
galU	7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[51]
hcp	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4	[52]
impBCGHJKL	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4	[52,53]
vasDG	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4	[53]
yhjH	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[54]
fimABCDEFGI	7-5-4, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[55]
fimH	7-5-4, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[43]
manBC	7-5-4, 7-10-14, 7-17-8, 8-1-12-1, 8-2-5-4	[56]
vgrG	7-5-4, 7-10-14, 7-13-2, 8-1-12-1, 8-2-5-4	[57]
iroE	7-13-2, 7-17-8, 8-2-5-4, 8-2-10-5	[46]
glf	7-10-14, 7-13-2, 8-2-5-4	[58]
vasJ	7-10-14, 8-2-5-4	[59]
fyuA	7-17-8, 8-1-12-1	[60]
irp12345	7-17-8, 8-1-12-1	[55,61]
mbtI	7-17-8, 8-1-12-1	[62]
ybtAX	7-17-8, 8-1-12-1	[63,64]
allABCDRS	8-2-10-5	[65]
iucABCD	8-2-5-4	[45]

Table 2. The putative virulence-related genes identified in the K. pneumoniae genomes.

Virulence-Related Gene	K. pneumoniae Genome	Reference
ecpABCDER	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[44]
entABCDEFS	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[45]
fepABCDG	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[46]
fes	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[46]
gnd	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[47]
kdsA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[48]
rcsAB	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[45]
tuf	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[49]
wza	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[50]
galU	7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[51]
hcp	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4	[52]
impBCGHJKL	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4	[52,53]
vasDG	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4	[53]
yhjH	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[54]
fimABCDEFGI	7-5-4, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[55]
fimH	7-5-4, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[43]
manBC	7-5-4, 7-10-14, 7-17-8, 8-1-12-1, 8-2-5-4	[56]
vgrG	7-5-4, 7-10-14, 7-13-2, 8-1-12-1, 8-2-5-4	[57]
iroE	7-13-2, 7-17-8, 8-2-5-4, 8-2-10-5	[46]
glf	7-10-14, 7-13-2, 8-2-5-4	[58]
vasJ	7-10-14, 8-2-5-4	[59]
fyuA	7-17-8, 8-1-12-1	[60]
irp12345	7-17-8, 8-1-12-1	[55,61]
mbtI	7-17-8, 8-1-12-1	[62]
ybtAX	7-17-8, 8-1-12-1	[63,64]
allABCDRS	8-2-10-5	[65]
iucABCD	8-2-5-4	[45]

3.7. Antibiotic and Heavy Metal Resistance-Associated Genes in the K. pneumoniae Genomes

Antimicrobial resistance-related genes (n = 20−35) were also identified in the 7 K. pneumoniae genomes (Table 3). K. pneumoniae 7-5-4 from M. anguillicaudatus contained the maximum number of such genes (n = 35), whereas K. pneumoniae 7-13-2, 7-17-8, and 8-1-12-1 isolates had the least (n = 20).

All the K. pneumoniae genomes contained the genes for MDR, e.g., a multidrug efflux SMR transporter subunit (KpnE), a spermidine export protein mdtJ (KpnF), a multidrug efflux MFS transporter periplasmic adaptor subunit emrA (KpnG), a multidrug resistance protein (KpnH), a multidrug efflux MFS transporter (mdtG), a multidrug efflux RND transporter permease subunit (acrB), a multidrug efflux RND transporter periplasmic adaptor subunit (oqxA), a multidrug efflux RND transporter permease subunit (oqxB), and a multidrug ABC transporter permease/ATP-binding protein (yojI).

The qnrs1 gene, encoding a quinolone resistance pentapeptide repeat protein, was present in the K. pneumoniae 7-5-4 and 8-2-10-5 genomes, while the floR, mphA, and sul1 genes were found in the K. pneumoniae 7-5-4, 7-10-14, and 8-2-10-5 genomes, which encoded a chloramphenicol/florfenicol efflux MFS transporter (floR), a mph(A) family macrolide 2-phosphotransferase (mphA), and a sulfonamide-resistant dihydropteroate synthase (sul1), respectively. Additionally, the genes involved in the resistance to β-lactam antibiotics (penicillins, cephalosporins, carbapenems, and monobactams) and aminoglycosides were also found in some of the K. pneumoniae genomes (Table 3). These results provided genome-wide evidence for antibiotic resistance phenotypes of the seven K. pneumoniae isolates.

Several genes involved in heavy metal tolerance were identified in the K. pneumoniae genomes as well (Table 3). For example, the copA gene, which plays a key role in the export of excess copper [66], was present in the seven K. pneumoniae genomes. Moreover, the cusARS genes, which are involved in the heavy metal efflux RND transporter [66,67], were also found in the seven K. pneumoniae genomes. Additionally, the K. pneumoniae 7-10-14 genome carried the zntA gene as well, which encodes a Zn/Cd/Hg/Pb-transporting ATPase, while K. pneumoniae 7-13-2 had the arsABCR genes, which are essential for heavy metal As resistance [68].

Table 3. The antibiotic and heavy metal resistance-related genes identified in the K. pneumoniae genomes.

Antibiotic/Heavy Metal	Gene	K. pneumoniae Genome	Reference
Cephalosporin	acrB	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[69]
Fluoroquinolone	hns	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[70]
	emrR	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[71]
	marA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[72]
	ramA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[72]
	crp	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[73]
	qnrS1	7-5-4, 8-2-10-5	[74]
	qnrB2	7-10-14, 8-2-10-5	[75]
Tetracycline	oqxAB	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[44]
	tet(A)	7-5-4, 7-10-14, 8-2-10-5, 8-2-5-4	[44]
Aminoglycoside	KpnEFGH	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[76,77]
	ompK37	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[78]
	aph(3′)-Ia	7-5-4, 7-10-14, 8-2-10-5	[79]
	aph(3″)-Ib	7-5-4, 7-10-14, 8-2-10-5	[79]
	aph(6)-Id	7-5-4, 7-10-14, 8-2-10-5	[79]
	aac(3)-IId	7-10-14, 8-2-10-5	[79]
	aac(6′)-Ib-cr	7-10-14, 8-2-10-5	[80]
	aadA16	7-10-14, 8-2-10-5	[79]
	aadA8	7-5-4	[81]
Diaminopyrimidine	dfrA27	7-10-14, 8-2-10-5	[82]
Beta-lactam	SHV-11	7-5-4, 7-17-8	[83]
	SHV-1	7-10-14, 7-13-2	[84]
	SHV-38	8-1-12-1, 8-2-5-4	[85]
	TEM-1	7-5-4	[85]
	OKP-B-7	8-2-10-5	[86]
	TEM-116	8-2-5-4	[85]
Macrolide	mphA	7-5-4, 7-10-14, 8-2-10-5	[44]
	ermB	7-5-4	[44]
	mphE	7-5-4	[87]
	msrE	7-5-4	[87]
Nitroimidazole	msbA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[88]
Phenicol	floR	7-5-4, 7-10-14, 8-2-10-5	[89]
Peptide	pmrF	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[90]
	ugd	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[91]
	yojI	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[92]
Sulfonamide	sul1	7-5-4, 7-10-14, 8-2-10-5	[44]
	sul2	7-5-4, 8-2-10-5	[44]
Rifamycin	arr-2	7-5-4	[80]
	arr-3	7-10-14	[46]
Fosfomycin	FosA5	7-5-4, 7-10-14	[80]
	FosA6	7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[80]
	mdtG	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[93]
Heavy metal	cusARS	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[66,67]
Heavy metal	copA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[66]
Heavy metal	zntA	7-10-14	[94]
Heavy metal	arsABCR	7-13-2	[68]

Table 3. The antibiotic and heavy metal resistance-related genes identified in the K. pneumoniae genomes.

Antibiotic/Heavy Metal	Gene	K. pneumoniae Genome	Reference
Cephalosporin	acrB	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[69]
Fluoroquinolone	hns	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[70]
	emrR	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[71]
	marA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[72]
	ramA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[72]
	crp	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[73]
	qnrS1	7-5-4, 8-2-10-5	[74]
	qnrB2	7-10-14, 8-2-10-5	[75]
Tetracycline	oqxAB	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[44]
	tet(A)	7-5-4, 7-10-14, 8-2-10-5, 8-2-5-4	[44]
Aminoglycoside	KpnEFGH	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[76,77]
	ompK37	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[78]
	aph(3′)-Ia	7-5-4, 7-10-14, 8-2-10-5	[79]
	aph(3″)-Ib	7-5-4, 7-10-14, 8-2-10-5	[79]
	aph(6)-Id	7-5-4, 7-10-14, 8-2-10-5	[79]
	aac(3)-IId	7-10-14, 8-2-10-5	[79]
	aac(6′)-Ib-cr	7-10-14, 8-2-10-5	[80]
	aadA16	7-10-14, 8-2-10-5	[79]
	aadA8	7-5-4	[81]
Diaminopyrimidine	dfrA27	7-10-14, 8-2-10-5	[82]
Beta-lactam	SHV-11	7-5-4, 7-17-8	[83]
	SHV-1	7-10-14, 7-13-2	[84]
	SHV-38	8-1-12-1, 8-2-5-4	[85]
	TEM-1	7-5-4	[85]
	OKP-B-7	8-2-10-5	[86]
	TEM-116	8-2-5-4	[85]
Macrolide	mphA	7-5-4, 7-10-14, 8-2-10-5	[44]
	ermB	7-5-4	[44]
	mphE	7-5-4	[87]
	msrE	7-5-4	[87]
Nitroimidazole	msbA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[88]
Phenicol	floR	7-5-4, 7-10-14, 8-2-10-5	[89]
Peptide	pmrF	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[90]
	ugd	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[91]
	yojI	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[92]
Sulfonamide	sul1	7-5-4, 7-10-14, 8-2-10-5	[44]
	sul2	7-5-4, 8-2-10-5	[44]
Rifamycin	arr-2	7-5-4	[80]
	arr-3	7-10-14	[46]
Fosfomycin	FosA5	7-5-4, 7-10-14	[80]
	FosA6	7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[80]
	mdtG	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[93]
Heavy metal	cusARS	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[66,67]
Heavy metal	copA	7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, 8-2-5-4, 8-2-10-5	[66]
Heavy metal	zntA	7-10-14	[94]
Heavy metal	arsABCR	7-13-2	[68]

3.8. Strain-Specific Genes of the K. pneumoniae Isolates of Aquatic Animal Origins

Comparative genomic analyses revealed approximately 4111 core genes shared by the K. pneumoniae genomes, which accounted for 65.7% of pan genes (n = 6255). Meanwhile, many strain-specific genes (n = 199−605) were found in the K. pneumoniae isolates (Figure 7). Interestingly, K. pneumoniae 7-5-4 from M. anguillicaudatus contained the highest number of strain-specific genes (n = 605), whereas K. pneumoniae 7-10-14 from T. veneriformis had the fewest (n = 199). Remarkably, higher percentages of the strain-specific genes (30.2−54.4%) encoded unknown proteins. These results also provided the evidence of the considerable genome variation in the K. pneumoniae isolates of aquatic animal origins.

3.9. Phylogenetic Relatedness of the K. pneumoniae Isolates of Aquatic Animal Origins

To address phylogenetic relatedness of the K. pneumoniae isolates of aquatic animal origins, we constructed a phylogenetic tree on the basis of 72 K. pneumoniae genomes. Of these, complete genomes of 65 K. pneumoniae strains were derived from the GenBank database, the majority of which (n = 57) were isolated from human samples, followed by animals (n = 5, bovine, cat, chicken, dog, pig, and rabbit), and water environment (n = 3) in 1999−2021. The 7 K. pneumoniae isolates of aquatic animal origins tested negative for the toxic K1, K2, K5, K20, K54, and K57 serotypes, except K. pneumoniae 8-2-5-4 of serotype K2 [15]. The phylogenetic tree analysis revealed seven distinct clusters, designated as Clusters A−F (Figure 8).

K. pneumoniae 8-1-12-1 from P. clarkii fell into Cluster B, together with K. pneumoniae JX-CR-hvKP (GenBank accession no. NZ_CP064208), which was isolated from human blood in 2019 in China.

Both K. pneumoniae 7-10-14 from M. Veneriformis and K. pneumoniae 7-17-8 from C. cahayensis were classified into Cluster F, together with K. pneumoniae 49,210 (GenBank accession no. NZ_CP089024) isolated from humans in 2016 in China.

Both K. pneumoniae 7-5-4 from M. anguillicaudatus and K. pneumoniae 8-2-10-5 from E. fuscoguttatus were grouped into Cluster E, together with K. pneumoniae SWHE3 (GenBank accession no. NZ_CP055061) isolated from humans in 2018 in China, and K. pneumoniae SB617 (GenBank accession no. NZ_CP084825), which was isolated from water in 2000 in the Netherlands.

K. pneumoniae 7-13-2 from E. sinensis was classified into a single Cluster A, phylogenetically distant from all the other genomes tested, suggesting its unique genome trait.

K. pneumoniae 8-2-5-4 of serotype K2 from T. granosa was classified into Cluster D, showing the closest phylogenetic distance with K. pneumoniae BcKp067 (GenBank accession no. NZ_CP084829), which was isolated from the water environment in 1999 in the Netherlands.

In addition, 8 K. pneumoniae isolates belonging to the capsule serotypes K1, K2, K5, K54 and K57 were classified into Clusters D−F.

Taken together, these results demonstrated the genome diversity of the K. pneumoniae isolates of the clinical and environmental origins.

3.10. Sequence Types (ST) of the K. pneumoniae Isolates of Aquatic Animal Origins

Based on the 7 conserved core genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) in K. pneumoniae [15], the multilocus sequence typing (MLST) analysis against the MLST database (https://cge.food.dtu.dk/services/MLST/ (accessed on 23 November 2022)) revealed that K. pneumoniae 7-5-4, 7-10-14, 7-13-2, 7-17-8, 8-1-12-1, and 8-2-5-4 isolates belonged to the ST-273, ST-1310, ST-101, ST-353, ST-6289, and ST-2026, respectively, whereas the K. pneumoniae 8-2-10-5 isolate was not classified into any known STs.

4. Discussion

The inappropriate use of antibiotics may result in increasing levels of bacterial resistance [8,95]. For example, Fatima et al. [96] examined the resistance of K. pneumoniae isolates from urine (n = 72) and sputum (n = 35) isolated in Balochistan to 17 antimicrobial agents. They found that the majority of the K. pneumoniae isolates were resistant to GEN (76.2%), followed by SXT (66.7%), and NOR (42.9%) [96]. Marques et al. [97] determined the resistance of K. pneumoniae isolates from companion animals (n = 27) and humans (n = 77), isolated in Lisbon during 2002 to 2015, to 29 antimicrobial agents. Their results showed that most K. pneumoniae isolates were resistant to AMP (95.2%), followed by CHL (31.7%), KAN (27.9%), TET (28.8%), and CIP (25%) [97]. In this study, the K. pneumoniae isolates had different antibiotic resistance profiles. For example, K. pneumoniae 7-5-4 from M. anguillicaudatus and K. pneumoniae 7-10-14 from M. veneriformis displayed resistance to CHL/CIP/KAN/NOR/SXT/TET and AMP/CHL/GEN/KAN/SXT/TET, respectively. K. pneumoniae 8-2-10-5 from E. fuscoguttatus was resistant to CHL/CIP/KAN/SXT/TET, while K. pneumoniae 8-2-5-4 from T. granosa was resistant to AMP/CHL/TET, suggesting different antibiotic exposure levels or pollution sources of aquaculture environments.

Previous studies have reported heavy metal residues in various aquatic environments and aquatic products sampled around the world, especially in developing countries [98,99]. For example, Varol and Sunbul et al. [98] examined the residues of five heavy metals in biota samples, including one species of mussel, crayfish, and farmed fish, respectively, and six species of wild fish, collected from the Euphrates River in Turkey. The highest concentrations of As, Cd, and Pb were detected in mussels, while the highest concentrations of Cu and Zn were detected in crayfish [98]. Recently, Ni et al. [99] reported Cu, Hg, Pb, and Cd residues in 41 species of aquatic animals, sampled in Shanghai, China in July−September of 2018−2019, with positive sample rates of 100%, 100%, 77.4%, and 34.0%, respectively, but none of which exceeded their maximum residue limits [99]. In this study, the K. pneumoniae isolates had different heavy metal tolerance patterns. For example, K. pneumoniae 8-1-12-1 from P. clarkii was tolerant to Cu²⁺/Cd²⁺/Cr³⁺/Hg²⁺/Mn²⁺/Pb²⁺/Zn²⁺, while K. pneumoniae 8-2-10-5 from E. fuscoguttatus was tolerant to Zn²⁺/Pb²⁺/Hg²⁺/Cu²⁺/Cr³⁺/Cd²⁺. These results suggested a potential risk of consuming these aquatic animals.

The human acidic stomach environment challenges survival and infection of K. pneumoniae. Therefore, we examined survival of the K. pneumoniae isolates at pH 3.5–7.5 when incubated in the TSB (0.5% NaCl) at 37 °C. Unexpectedly, all the isolates were capable of growing vigorously at pH 4.5–7.5, although the highest biomass was observed at pH 7.5. These results provided evidence of the acidic tolerance of the K. pneumoniae isolates of animal origins, which may have been attributed to the bacterial survival across the acidic stomach boundary in the host.

The K. pneumoniae isolates were recovered from different aquatic animals in water environments. M. anguillicaudatus, E. sinensis, C. cahayensis, and P. clarkii were derived from freshwater, while M. Veneriformis, T. granosa, and E. fuscoguttatus from seawater. Therefore, we determined growth curves of the K. pneumoniae isolates at different salinity concentrations (0.5−4% NaCl) when incubated in the TSB (pH 7.5) at 37 °C. Our results indicated that all the K. pneumoniae isolates were able to grow vigorously at 0.5−1.0% NaCl in the TSB (pH 7.5) at 37 °C. Of these, K. pneumoniae 7-5-4 from M. anguillicaudatus was the most susceptible to higher salinity concentrations (4−3% NaCl), consistent with its freshwater culture environment.

Draft genomes of the seven K. pneumoniae isolates were determined using the Illumina Hiseq × Ten sequencing platform. The typical Poisson distribution, with a clear single peak at the only 17–mers frequency, was observed in the sequencing data, indicating less repetitive DNA in the K. pneumoniae genomes (Figure S1, Table S9). The assembled genomes were 5,256,522−5,857,823 bp with GC contents of 56.35−57.81%, similar to the other K. pneumoniae genomes [42,100]. For example, Yu et al. [100] used MiSeq short-read sequencing and Oxford nanopore long-read sequencing to determine whole genomes of 7 CRKP strains, and obtained genome sizes ranging from 5.4 to 5.8 Mb with an average GC content of 57.2% [100]. Du et al. [42] sequenced and de novo assembled the genomes of 6 hvKP strains, which ranged 5.34−5.58 Mb and GC percentages ranging from 57.22 to 57.46% [42]. The genome sizes of 1757 K. pneumoniae strains, which were available in the GenBank database (accessed on 14 March, 2023), ranged from 4.76 Mb to 6.37 Mb, most of which contained 3107 to 5973 predicted genes. In this study, interestingly, a total of 4885–5558 protein-coding genes were predicted, of which 1188−1363 protein-coding genes encoded unknown proteins. Moreover, about 7.1−41.0% of the strain-specific (n = 199−605) genes encoded unknown proteins as well. These results highlighted specific genome traits of the K. pneumoniae isolates of aquatic animal origins, which may result from the high numbers of identified MGEs.

Remarkably, the K. pneumoniae genomes carried many MGEs, including GIs (n = 87), prophages (n = 14), INs (n = 4), and ISs (n = 22). The identified MGEs carrying a large number of genes may constitute an important driving force in K. pneumoniae genome evolution and speciation. For instance, the identified 87 GIs endowed the bacterium with a variety of biological functions for fitness into niches, such as virulence, resistance, substrate hydrolysis, transporting and utilization, and restriction and modification, as well as phage and stress regulation.

There are approximately 10³¹ bacteriophages on earth, which play a critical role in virulence and evolution of bacterial genomes [101]. Bleriot et al. [102] reported 40 prophages (11.454−84.199 kb) in 16 clinical CRKP strains, 27 of which belonged to the family Myoviridae, 10 to Siphoviridae, and 3 to Podoviridae [102]. In this study, we found 14 prophages (12,633−109,928 bp) in the 7 K. pneumoniae genomes, which were derived from different genera, including Enterobacteria, Klebsiella, Pseudomonas, and Ralstonia. The results in this study, coupled with previous report [102], indicated extensive phage transmission between Klebsiella and the other bacterial genera. In this study, the identified prophage homologues, e.g., Enterobacteria_phage_186, Klebsiella_phage_phiKO2, and Pseudomonas_phage_D3, were present in different K. pneumoniae genomes, but in different lengths encoding different numbers of genes, which provided evidence of extensive genome rearrangement during the K. pneumoniae evolution.

It has been reported that the Type 1 IN is the most prevalent and common in clinical bacteria [103]. For example, Firoozeh et al. [104] isolated K. pneumoniae strains (n = 181) from clinical specimens and found 82.9% (n = 150) of the isolates with MDR phenotypes. Of the MDR isolates, 100% (n = 150) and 36.7% (n = 55) carried intI1 and intI2 genes, respectively, but none had the intI3 gene by PCR amplification [104]. In this study, 4 INs (1229−4332 bp) were identified in the K. pneumoniae 7-5-4, 7-10-14, and 8-2-10-5 genomes. Of these, K. pneumoniae 7-5-4 carried the Type 1 IN. Moreover, this IN contained ARGs, encoding a NAD(+)-rifampin ADP-ribosyltransferase arr-2 (Kp 7_5_4_5567) and an ANT(3″)-Ia family aminoglycoside nucleotidyltransferase aadA3 (Kp 7_5_4_5566), suggesting possible transmission of ARGs mediated by the IN.

ISs consist of two inverted repeat sequences and one or two genes encoding transposases [105]. In this study, all the 7 K. pneumoniae genomes contained ISs (n = 2 to 6), ranging from 741 to 1588 bp. They belonged to the IS3 family, IS5 family, IS6 family, IS91 family, and IS110 family.

The CRISPR-Cas systems defend the prokaryotes from invasion by MGEs [106]. In this study, 54 CRISPR-Cas gene clusters (75−2649 bp) were identified in the 7 K. pneumoniae genomes. However, all the predicted clusters lacked the Cas protein, which plays an essential role in the function of the CRISPR-Cas systems [107]. These results provided indirect evidence for inactive CRISPR-Cas repeats and possible active HGT in the 7 K. pneumoniae isolates.

Many virulence-related genes have been identified in K. pneumoniae isolates [108,109]. For example, Remya et al. [108] detected 9 virulence genes in K. pneumoniae isolates (n = 370) by PCR amplification, including the magA, allS, kfu, K2A, rmpA, entB, ybtS, fimH, and uge genes. They found that 93.2% (345/370) of the isolates carried multiple virulence genes, 4.0% (15/370) carried 1, and 2.7% (10/370) had none [108]. Kuş et al. [109] detected 16 virulence genes in K. pneumoniae strains (n = 53) isolated from nosocomial infections in Turkey by PCR amplification, including the fimH-1, mrkD, kpn, iutA, ycfM, entB, irp-1, irp-2, ybtS, fyuA, iroN, rmpA, magA, traT, hlyA, and cnf-1 genes. Their results showed that the entB gene was the most predominant (96.2%), followed by the ycfM (86.8%), mrkD (83.0%), fimH-1 (64.2%), fyuA (54.7%), and kpn (49.1%). The detection rates of the ybtS, irp-1, irp-2, traT, and iutA genes ranged from 41.5 to 5.7%, whereas the other genes (iroN, rmpA, magA, hlyA, and cnf-1) tested negative in the isolates [109]. In this study, based on the obtained genome sequences, we also identified many virulence-related genes (n = 43−59) in the 7 K. pneumoniae isolates of aquatic animal origins, e.g., ecpABCDE, entABCDEF, fimABCDEFGHI, iucABCD, fepABCDG, fyuA, vgrG, galU, gnd, vgrG, fimH, entB, mrkd, ybta, and T6SS-associated genes, which were involved in adhesion, antiphagocytosis, secretion system, and gene regulation of K. pneumoniae. Of note, K. pneumoniae 8-1-12-1 from P. clarkii contained the maximum number of the virulence-associated genes (n = 59), whereas K. pneumoniae 7-10-14 from M. veneriformis had relatively fewer (n = 43). These virulence-related genes may be candidate targets for the development of new diagnostics, vaccines, and treatments to control K. pneumoniae infection.

The emergence and spread of MDR pathogens poses a serious threat to public safety [110]. For example, Marques et al. [97] reported 15 antibiotic resistance-related genes in K. pneumoniae isolates, causing UTIs from companion animals (n = 27) and humans (n = 77), e.g., qnrB, qnrS, sul1, sul2, sul3, dfrA12, dfrIa, tet(A), tet(B), and floR [97]. In this study, many antibiotic resistance-related genes (n = 20−35) were identified in the 7 K. pneumoniae genomes, e.g., tetA, acrB, hns, oqxA, aac (6′)-Ib-cr, ermB, msbA, floR, pmrF, sul1, arr-2, and fosA5, which are involved in the resistance to cephalosporin, fluoroquinolone, tetracycline, aminoglycoside, macrolide, phenicol, sulfonamide, rifamycin, and fosfomycin. Moreover, several genes in heavy metal tolerance were also identified in the seven K. pneumoniae genomes, such as the cusASR, copA, zntA, and arsABCR genes. These results provided genome-wide evidence for the resistance phenotypes of the K. pneumoniae isolates of aquatic animal origins.

5. Conclusions

The K. pneumoniae 7-10-14, 7-17-8, 8-2-5-4, 7-13-2, 8-1-12-1, 8-2-10-5, and 7-5-4 strains of aquatic animal origins had multiple antibiotic resistance and heavy metal tolerance profiles, and were capable of growing vigorously at pH 4.5−7.5 and 0.5−1.0% NaCl in the TSB medium at 37 °C.

Remarkably, the K. pneumoniae genomes carried many MGEs, including GIs (n = 87), prophages (n = 14), INs (n = 4), and ISs (n = 22), as well as partial or inactive CRISPR-Cas systems (n = 54), indicating possible active HGT during the K. pneumoniae genome evolution. Many antibiotic resistance (n = 20−35) and virulence (n = 43−59)-related genes were found in the K. pneumoniae genomes. K. pneumoniae 7-5-4 from M. anguillicaudatus contained the maximum number of ARGs (n = 35), while K. pneumoniae 8-1-12-1 from P. clarkii carried the most virulence-related genes (n = 59). Additionally, numerous strain-specific (n = 199−605) genes were present in the K. pneumoniae isolates, approximately 30.2−54.4% of which encoded unknown proteins. These results, coupled with the phylogenetic tree analysis, demonstrated considerable genome variation and high genome plasticity of the K. pneumoniae isolates.

Overall, the results of this study enrich genome data and fill prior gaps in understanding the K. pneumoniae genomes derived from aquatic animals.