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Article

Genomics of Klebsiella pneumoniae Species Complex Reveals the Circulation of High-Risk Multidrug-Resistant Pandemic Clones in Human, Animal, and Environmental Sources

by
Sergio Morgado
,
Erica Fonseca
and
Ana Carolina Vicente
*
Laboratory of Molecular Genetics of Microorganisms, Oswaldo Cruz Institute, Av. Brasil, 4365—Manguinhos, Rio de Janeiro 21040-900, Brazil
*
Author to whom correspondence should be addressed.
Microorganisms 2022, 10(11), 2281; https://doi.org/10.3390/microorganisms10112281
Submission received: 18 October 2022 / Revised: 11 November 2022 / Accepted: 14 November 2022 / Published: 17 November 2022
(This article belongs to the Special Issue 10th Anniversary of Microorganisms: Past, Present and Future)
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Abstract

:
The Klebsiella species present a remarkable genetic and ecological diversity, being ubiquitous in nature. In particular, the Klebsiella pneumoniae species complex (KpSC) has emerged as a major public health threat in the world, being an interesting model to assess the risk posed by strains recovered from animals and the environment to humans. We therefore performed a genomic surveillance analysis of the KpSC using every public genome in Brazil, aiming to show their local and global relationships, and the connectivity of antibiotic resistance and virulence considering human, animal, and environmental sources. The 390 genomes from distinct sources encompassed the K. pneumoniae, Klebsiella quasipneumoniae subsp. quasipneumoniae, Klebsiella quasipneumoniae subsp. similipneumoniae, Klebsiella variicola subsp. variicola, Klebsiella variicola subsp. tropica, and Klebsiella grimontii species and subspecies. K. pneumoniae harbored dozens of antibiotic resistance genes, while most of the genomes belong to the high-risk pandemic CC258 occurring in humans, animals, and the environment. In K. pneumoniae ST11, a high prevalence of the virulence determinants yersiniabactin, colibactin, and T6SS was revealed in association with multi-drug resistance (MDR), including carbapenem resistance. A diversity of resistance genes is carried by plasmids, some shared between strains from different STs, regions, and sources. Therefore, here were revealed some factors driving the success of KpSC as a pathogen.

1. Introduction

The Klebsiella species (spp.) are ubiquitous, dispersed in natural environments such as soil, water, plants, and livestock, and may eventually be associated with both human and animal infections. This bacteria group presents a remarkable genetic and ecological heterogeneity, representing, therefore, a pertinent taxon for assessing the risk to public health posed by animal and environmental reservoirs [1]. Among the Klebsiella genus, the Klebsiella pneumoniae species is, so far, the best-studied species, mainly due to its frequent association with community-acquired and nosocomial outbreaks. Moreover, K. pneumoniae is included in the ESKAPE group due to its association with the spread and development of multi-drug resistance (MDR), being considered one of the major public health challenges worldwide [2].
All members of the K. pneumoniae species complex (KpSC), which includes K. pneumoniae, Klebsiella quasipneumoniae subsp. quasipneumoniae, Klebsiella quasipneumoniae subsp. similipneumoniae, Klebsiella variicola subsp. variicola, and Klebsiella variicola subsp. tropica, can be recovered from the environment, as well as from human and animal infections, showing resistance to multiple drugs and virulent phenotypes worldwide. [3,4,5,6,7,8,9]. These species, especially K. pneumoniae, can develop multidrug resistance to all antibiotic classes, including carbapenems, through gene acquisition via horizontal gene transfer [2]. β-lactams are antibiotics widely used around the world in the treatment of Klebsiella infections, such as penicillins, cephalosporins, and carbapenems. However, some strains develop resistance to these antibiotics by acquiring plasmid-mediated extended-spectrum β-lactamases (ESBL), AmpC, and carbapenemase genes, such as blaCTX-M-15, blaSHV, blaTEM, blaDHA, blaCMY, blaKPC, blaOXA-48, blaVIM, blaIMP, and blaNDM, being considered one of the greatest threats to global health [2]. The acquisition of mobile genetic elements has also led to the development of hypervirulent Kp (hvKp) that carry plasmids and integrative conjugative elements (ICEs) encoding several important virulence determinants, such as siderophores (salmochelin (iro), aerobactin (iuc), and yersiniabactin (ybt)), the colibactin toxin (clb) and/or genes responsible for a hypermucoviscosity phenotype (rmpA/rmpA2) [10]. Therefore, genomic studies could provide insights into the dynamics of dissemination of this bacterial complex, as well as the genes associated with antibiotic resistance and virulence.
Brazil is a continental country bordering most South American countries, and it is characterized by six major biomes, namely Amazon, Cerrado, Caatinga, Pantanal, Atlantic Forest, and Pampa, which is supposed to have the greatest biodiversity in the world [11]. In this country, KpSC has already been incriminated in nosocomial infections, including high-risk clones presenting multidrug resistance mainly due to the presence of carbapenemase genes, such as blaNDM and blaKPC [4,12,13,14,15,16,17]. Moreover, virulent and multidrug-resistant strains from natural environments and animals have also been reported [18,19,20]. Therefore, based on this scenario, we performed a genomic surveillance analysis on hundreds of KpSC genomes from Brazil, considering the three axes of the One Health concept (environment—animal—human), to reveal their resistome, virulome and genetic relationships in the context of this diverse scenario.

2. Materials and Methods

2.1. Public Data Set

Complete and draft genomes of Klebsiella isolated from Brazil (n = 378) were obtained from the National Center for Biotechnology Information (NCBI) on 29 October 2021: (https://www.ncbi.nlm.nih.gov/genome/browse/#!/prokaryotes/klebsiella). The accession numbers are supplied in Table S1.

2.2. Genome Sequencing and Assembly

In this study, we generated 21 Klebsiella spp. genomes, in which 14/21 were from the Brazilian Amazon (RR—Roraima), including animal and human/clinical isolates. The remaining genomes (n = 7) were human clinical ones from Rio de Janeiro (RJ). In this way, we were able to contribute genomic information from underrepresented Brazilian regions (Table S1).
The genomic DNA extraction was done using a NucleoSpin Microbial DNA kit (Macherey-Nagel), and the genome libraries were constructed using Nextera paired-end libraries. The sequencing was performed by Illumina Hiseq 2500, generating reads of 250 bp length. The raw reads were filtered and trimmed using a NGS QC Toolkit v.2.3.3 [21], considering a Phred quality score ≥ 20. The genomes were de novo assembled with a SPAdes assembler v3.14.1 [22] or a A5-miseq pipeline v20160825 (IDBA-UD assembler) [23] and improved using a Pilon v1.23 [24].

2.3. Characterization of Klebsiella Genomes

The Kleborate v2.1.0 [25] pipeline was used for the genetic characterization of the Klebsiella genomes considering species designation, sequence typing, and identification of acquired virulence and antibiotic resistance genes. In addition, the Kleborate scored the virulence and antibiotic resistance profiles of the genomes based on the presence of clinically relevant gene markers. Virulence scores range from 0 to 5, based on the presence of three loci (yersiniabactin, colibactin, and aerobactin): yersiniabactin only (score 1), colibactin without aerobactin (score 2), aerobactin only (score 3), aerobactin and yersiniabactin without colibactin (score 4), and all three present (score 5). The resistance scores range from 0 to 3, based on the presence of genes associated with ESBL (score 1), carbapenemases (score 2), and carbapenemase plus colistin resistance (score 3).
In addition, ABRicate v1.0.1 (https://github.com/tseemann/abricate accessed on 1 January 2022) was used for plasmid replicon typing (PlasmidFinder database) [26], and the identification of virulence (VFDB database) [27] and antibiotic resistance genes (CARD database) [28] in the putative plasmids.

2.4. Phylogeny

The genomes were annotated by Prokka v1.12 [29] and their core genomes were estimated using Roary v3.13 [30]. Next, single nucleotide polymorphisms (SNPs) were extracted from the concatenated core genes by snp-sites v2.5.1 [31] and submitted to phylogenetic analysis using an IQTree v1.6.12 [32] to obtain a maximum likelihood tree with 1000 ultrafast bootstrap replicates [33]. The tree was generated using the iTOL web platform (https://itol.embl.de) accessed on 1 January 2022) [34].

3. Results

3.1. K. pneumoniae Species Complex Epidemiology and Sequence Typing

To establish the epidemiological links of the KpSC concerning the One Health axes, we performed a genomic analysis with new genomes as well as genomes available in the NCBI database. In total, 399 Klebsiella genomes from Brazil were analyzed, of which 21 were obtained in the present study and 378 from the NCBI (Table S1). These genomes belonged to Klebsiella isolated from the environment, animals, and humans in every Brazilian region (CO, midwest; N, north; NE, northeast; S, south; and SE, southeast) (Figure 1) between 2003 and 2020 (Figure 2).
Based on Kleborate taxonomic criteria, five Klebsiella species were identified, of which two had two subspecies: Klebsiella pneumoniae (n = 350), Klebsiella quasipneumoniae subsp. quasipneumoniae (n = 2), Klebsiella quasipneumoniae subsp. similipneumoniae (n = 16), Klebsiella variicola subsp. variicola (n = 20), Klebsiella variicola subsp. tropica (n = 1), Klebsiella aerogenes (n = 9), and Klebsiella grimontii (n = 1) (Table S2). Most of the isolates (n = 349) were recovered from humans, while the others were from animals (n = 30; including vertebrates and invertebrates), and natural environments (n = 18; water, sewage, soil, plants) (Table S1). All K. aerogenes (n = 9) genomes were from human/clinics and, therefore, were not included in the further analyses.
Most of the remaining 390 genomes belonged to K. pneumoniae (n = 350), encompassing many STs, some persisting for several years; most of them have been reported globally and, therefore, are pandemics, such as ST11 (n = 98), ST437 (n = 52), ST258 (n = 37), and ST340 (n = 27) (Figure 3, Tables S1 and S2). These four prevalent STs are part of the same clonal complex (CC258) and represent 61% (214/350) of the K. pneumoniae genomes from Brazil. Considering the Brazilian regions, ST11 was the most prevalent, except in the South (S) region, in which ST437 prevailed. However, as most of the regions are underrepresented (S, 15; NE, 27; N, 35; CO, 19) compared with the SE region (n = 288), this ST prevalence could be biased (Table S1). Importantly, some STs, including pandemic ones, occurred in different sources (and sometimes in different regions): ST340, ST15, and ST11 in humans, animals (e.g., crabs and dogs), and environments (e.g., mangroves and water); ST307 in humans and animals (e.g., mussels, dogs, horses); ST437 in humans and environments (e.g., water); ST198 in humans and the environment (e.g., lettuce) (Table S1). About the other species, K. variicola subsp. variicola was represented by multiple STs in humans (n = 9), animals (n = 8), and environments (n = 3), occurring in all Brazilian regions. In particular, ST355 was observed both in animals (e.g., Bos indicus) and in the environment (e.g., plants). In the same way, K. quasipneumoniae subsp. similipneumoniae was represented by several STs in the country’s regions and, interestingly, the ST1308 was observed in both humans and mosquitoes (Figure 3 and Table S1).

3.2. K. pneumoniae Species Complex Antibiotic Resistance and Virulence Determinants

The antibiotic resistance and virulence determinants of the KpSC genomes were surveyed using the Kleborate tool, which determines score values based on loci that contribute to clinically relevant antibiotic resistance (score from 1 to 3) or hypervirulence (score from 1 to 5) phenotypes.
Regarding antibiotic resistance for K. pneumoniae, most genomes were classified in score 2, followed by 3, 1, and 0; for K. variicola subsp. variicola, most genomes were classified in score 0, followed by 2; for K. quasipneumoniae subsp. similipneumoniae, the genomes were classified in scores 0 or 2 (Figure 3 and Figure 4A, and Table S2).
In total, 336 genomes presented positive scores for β-lactamases, where few of them only harbor ESBLs (score 1; n = 59), while the others presented carbapenemases (score 2; n = 165), and carbapenemases with colistin resistance determinants (score 3; n = 112). Score 3 was only observed in K. pneumoniae species, including clinical and environmental species. Although genomes harboring only ESBLs (score 1) were outnumbered (n = 59), ESBLs also co-occurred in genomes carrying carbapenemases (53%; n = 213), where 128/165 genomes with score 2 had carbapenemase and ESBL (77%), and 85/112 genomes with score 3 had carbapenemase with colistin resistance determinants and ESBL (75%). Thus, most Klebsiella genomes co-harbor carbapenemase and ESBL genes.
The median number of antibiotic resistance genes contained in the genomes varied depending on the source, being 0, 5, and 10 genes in the animal, environmental and human genomes, respectively. In general, genomes of the Klebsiella species of the three sources presented heterogeneity of antibiotic resistance genes (Table 1) and resistance profiles (Table 2).
Some genomes from the same ST but occurring in different sources shared several antibiotic resistance genes, such as catA1, catB3, sul1, blaCTX-M-2, blaCTX-M-15, blaOXA-1, blaOXA-2, blaKPC-2, aac(3), tet(A), tet(D) (Table S2). For instance: in ST11, GCA_013303005.1 genome (dog/2019) and GCA_011037475.1 genome (human/2016) shared qnrS1, sul1, dfrA12, blaLAP-2, blaOXA-1, blaCTX-M-15, blaKPC-2; in ST307, GCA_003194695.1 genome (horse/2017) and JALJAB000000000 genome (human/2017) shared qnrB1, sul2, tet(A), dfrA14, blaTEM-1, blaOXA-1, blaCTX-M-15. Most genomes analyzed (n = 342; 87%) were classified as MDR, since they harbored antibiotic resistance genes conferring resistance to ≥3 drug classes (Table S2). Considering the three bacterial sources, differences in the proportion of MDR genomes were observed: human, 92% (314/340); environment, 66% (12/18); and animal, 46% (14/30). Concerning enzymatic resistance, among the carbapenemases, blaKPC-2 was the most prevalent, and the main alleles of ESBLs were blaCTX-M-14 and blaCTX-M-15 (Table S2). The sul (sulfonamide resistance) and dfrA (trimethoprim resistance) genes were observed in 73% and 70% of the genomes, respectively, while to a lesser extent, qnr (quinolone resistance) and tet (tetracycline resistance) genes were found in 30% and 45% of the genomes, respectively (Table S2). The mcr gene, involved in colistin resistance, was only observed in four clinical genomes: three of K. pneumoniae from different STs, and one from K. quasipneumoniae subsp. similipneumoniae. However, 110 genomes presented mutations in the mgrB and/or pmrB genes (genomes with score 3), also associated with colistin resistance (Table S2). In addition, this in silico resistome investigation revealed the presence of the rare blaSCO-1 in five clinical genomes of K. pneumoniae ST11, ST307, ST392 (n = 2), and ST442, recovered from different regions (Table S2). Moreover, based on class C beta-lactamase gene family sequences from the National Database of Antibiotic Resistant Organisms (NDARO), we searched ampC genes in all Klebsiella genomes, and none of them were detected.
The virulence loci analysis revealed that 146 genomes had score 1 (n = 75), score 2 (n = 68), score 3 (n = 2), and score 5 (n = 1), while 244 genomes presented the virulence score 0 (Figure 4B and Table S2). Regarding Klebsiella species, there was only a high prevalence of genes associated with yersiniabactin and colibactin virulence loci in K. pneumoniae (Table 3), particularly in genomes belonging to the high-risk clone ST11 (Table 4). Interestingly, T6SS occurred in all species, as well as in all K. pneumoniae STs (Table 3 and Table 4).
The virulence loci yersiniabactin and colibactin identified in animal, environmental, and human K. pneumoniae genomes were associated with different ICEKp (ICEKp3, 4, 10, 12). For instance, the ICEKp4, which carries the yersiniabactin 10 (ybt 10) loci, occurred in genomes from dogs and water (ST15), and mangroves and humans (ST11); and the ICEKp10, which carries the yersiniabactin 0 or 17 (ybt 0 and ybt 17) loci and the colibactin loci 3 (clb 3), occurred within ST11 genomes from crabs, water and humans (Table S2). Most of K. pneumoniae belonging to ST11 had score 2, therefore harboring colibactin loci or colibactin plus yersiniabactin loci, the latter being the prevalent composition (Figure 3 and Table S2). However, a deep analysis revealed that some of them had their loci annotated as truncated and/or incomplete (yersiniabactin, 17/144; colibactin, 20/69). The same occurred with other virulence loci (aerobactin, 2/3; salmochelin, 3/4; rmpADC, 1/3) (Table S2), hence were probably nonfunctional. Interestingly, some animal and environmental genomes were identified with positive virulence scores, such as 2 or 3 (Table S1).

3.3. K. pneumoniae Species Complex Plasmids

Using the ABRicate tool with the Plasmidfinder database, we predicted thousands of putative plasmid sequences with dozens of plasmid replicon types (n = 52) in 384 Klebsiella genomes of all species. It was not always possible to recover the entire plasmid sequence, as many genomes were highly fragmented, but it was possible to infer that some plasmid sequences were shared (considering coverage ≥ 70% and identity ≥ 80%) by strains from different STs, regions, and sources (Table 5).
In addition, similar plasmids (also considering coverage ≥ 70% and identity ≥ 80%) could be observed being shared among different species, such as a group of small plasmids (~7.3–9.2 kb) present in K. pneumoniae (WERP01000003; water source), K. quasipneumoniae (VDFZ01000103; human source), and K. variicola (SZND01000045; human source). Additionally, a putative plasmid from K. quasipneumoniae (JAGTYC010000025; ~76 kb), isolated from a mosquito, presented an identity (82%) with a putative plasmid from K. pneumoniae (NTHU02000076; ~104 kb), isolated from a human. Curiously, the latter plasmid carried the aph(3), aph(6), and sul2 genes, while the K. quasipneumoniae plasmid did not. All putative plasmid sequences were screened for virulence and antibiotic resistance genes; however, due to the genome fragmentation, only sequences presenting these genes with any plasmid replicon gene in the same contig were considered. Among these 384 genomes, only five (four K. pneumoniae and one K. grimontii) presented putative plasmids with virulence genes (Table S3). Interestingly, one of these plasmids (LYZC01000019.1; 148 kb) harbored seven core genes of the Type VI secretion system (tssB/tssC/tssK/tssL/tssD/tssH/tssI). In addition, it was possible to observe that two K. pneumoniae genomes (SPSP01000000.1 and SPSO01000000.1), both isolated from animals (Callithrix penicillata), harbored the pLVPK virulence plasmid (~220 kb; ~100% coverage), in fragmented contigs, encoding the aerobactin operon (iucABCD-iutA) together with the tellurium (ter) resistance operon, and rmpA2, a regulator gene of the mucoid phenotype of HvKP. Thus, this plasmid was responsible for the virulence score 3 of these genomes, since they do not encode either yersiniabactin or colibactin. Regarding antibiotic resistance genes, 224 plasmid sequences carried from one to 13 genes (Table S4), where blaKPC was the most common (n = 93). Interestingly, despite most of the KPC genes belonging to the same allele, several of them were in different putative plasmids and genomes of K. pneumoniae from different Brazilian regions. Other beta-lactamases were also found, such as blaCTX-M (−2, −8, −9, and −15), blaGES (−14), and blaOXA (−1, −9, and −543), in addition to genes associated with resistance to aminoglycoside (aac, aph), fluoroquinolone (qnr), macrolide (ermB), chloramphenicol (cat), rifamycin (arr), sulfonamide (sul), colistin (mcr) (Table S4). Some putative plasmids carrying antibiotic resistance genes were shared by different genomes from different sources and STs (Table 5). For instance, the putative plasmids LYMZ01000021 (animal, ST340) and JABBZB010000030 (human, ST11) harbored sequences encoding blaLAP-2 and qnrS1; JABSUB010000003 (animal, ST11) and JAEVGJ010000028 (human, ST437) harbored sequences encoding blaKPC-2 (Table 5). Thus, similar plasmids, carrying the same antibiotic resistance genes, are dispersed in strains from human, environmental, and animal sources. Importantly, there is a set of plasmids harbored by environmental genomes carrying enzymatic genes related to resistance to several antibiotics (Table 6).

4. Discussion

In 2019, the WHO established a group of critical priority pathogens associated with the worldwide spread of antimicrobial resistance [35]. This group of bacteria includes the remarkable pathogen K. pneumoniae, which together with other Klebsiella species constitutes the K. pneumoniae species complex (KpSC), which is strongly associated with the spread of antimicrobial resistance due to its high susceptibility to horizontal gene transfer [36], thus favoring the emergence of high-risk clones that can evolve into dominant clones and cause outbreaks. Furthermore, Klebsiella species are ubiquitous and can be found in a wide range of sources, such as humans, animals, natural environments, and foods [37]. In fact, we observed in the present analysis the same STs in different sources (e.g., K. pneumoniae: ST340, ST15, and ST11 in humans, animals, and the environment; K. variicola: ST355 in animals and the environment; K. quasipneumoniae: ST1308 in humans and animals), characterizing the zoonotic profile of these species. Furthermore, genomes from different sources were dispersed in the phylogeny, not forming groups by source. Therefore, there are no specific lineage associations with the source. Most environmental genomes were obtained from aquatic environments, which can be directly affected by the industrial, hospital, and sewage effluents, being potential “hot spots” for resistant bacteria and the exchange of genetic material between species [38]. In fact, we were able to identify some putative plasmids that were shared between different Klebsiella species, albeit from different sources. This suggests that the environment is not a barrier for Klebsiella species to exchange genetic material with each other.
Heterogeneity of K. pneumoniae STs has been reported in distinct Brazilian regions, mainly STs from the high-risk pandemic CC258 [16,39,40,41,42]. Based on the available Klebsiella spp. genomes, we observed that CC258 is prevalent in Brazil, considering the national level (61% of the K. pneumoniae genomes), mainly through the presence of ST11 (28% of the K. pneumoniae genomes). So far, ST11 has been mostly associated with outbreaks in Asia, mainly in China [43], while ST258 and ST512 are epidemics in Latin America and Europe [44]. However, in the present study, it was noticed that ST258 and ST512, together, represented only ~10% of the K. pneumoniae genomes (observed in three of the five Brazilian regions). Therefore, it is possible that ST11 outbreaks in Brazil are being underreported. Interestingly, this scenario can also be seen considering the animal and environmental K. pneumoniae genomes belonging to STs from CC258.
An in-silico analysis, such as the one performed here, can also reveal information about genes that are not usually tracked, as occurred with blaSCO-1, which is supposedly rare in K. pneumoniae [45]; here, it was identified in some clinical genomes of K. pneumoniae of ST11, ST307, ST392, and ST442 that occur worldwide. Furthermore, several antibiotic resistance genes, identified in similar putative plasmids, were shared between genomes from different sources, suggesting, in general, a connection between these three One Health axes. Interestingly, a clear prevalence of yersiniabactin, colibactin, and T6SS was observed in ST11 genomes, including environmental and animal genomes, which may favor their success in causing outbreaks and invasive infections [46]. In addition to ST11, ST16 was another ST with a prevalence of the yersiniabactin loci, where about half of the analyzed ST16 genomes presented this locus. These genomes belong to a study that used a larval model to show the association of ST16 with high mortality rates when compared to ST11 [47].
In terms of South America, some comparisons can be made between K. pneumoniae data from Brazil and Colombia, a blaKPC-endemic country in South America [48]. In both countries, strains of CC258 prevail (mainly ST258/512 in Colombia and ST11 in Brazil) with high levels of MDR (~93%), but with differences in the rate of carbapenemase-producing strains, 91.5% in Colombia [48], and 75% in Brazil. Furthermore, in Colombia, there is no single prevalence of carbapenemases (blaKPC-3, 39%; blaKPC-2, 30%; blaNDM-1, 14%) [48], while blaKPC-2 is practically the only circulating allele (92%) in Brazil. Virulence was low among CC258 strains in both countries, with a similar prevalence of the clb 3 lineage (25.2% in Colombia and 28% in Brazil). On the other hand, the prevalence of yersiniabactin differed, with 76.9% of the CC258 isolates carrying this locus in Colombia (mainly ybt 17 and ybt 10) [48], and 44% in Brazil (mainly ybt 0 and ybt 9).
Our analysis revealed a K. quasipneumoniae genome from the human/SE region belonging to ST334. This ST has been reported as a potential emerging outbreak-associated MDR clone [44], having already been identified in Pakistan, Cambodia, and Singapore [49]. Indeed, this Brazilian genome displayed a broad resistome, being in vitro confirmed as an XDR (data not shown). Two ST334 genomes from Singapore present a similar MDR profile but they carry distinct carbapenemases (blaNDM-5 and blaOXA-181) [49] about the Brazilian genome that carries blaKPC-2. These findings reveal the diversity of carbapenemase genes in this Klebsiella species.
Although K. variicola is commonly associated with plant ecosystems, infections have been reported in humans and less frequently in animals [50]. Here, we observed a wide range of sources, including soil, different animals, and humans, with most genomes presenting a score of 0, both for virulence and antibiotic resistance. Few genomes harbored carbapenemases (blaGES-5 and blaKPC-2), and ESBLs (blaCTX) (some in plasmids), showing that this Klebsiella species has the potential to develop the MDR profile [17].
Recently, several reports from Asia, Europe, and North America described the emergence of Klebsiella hypervirulent clones, in which there was a convergence of virulence and antibiotic resistance traits [9,10,51]. However, among Brazilian genomes, we could not observe this convergence (i.e., genomes presenting a virulence score ≥ 3 and an antibiotic resistance score ≥ 1; [25]). The exception was only one clinical genome (GCA_003326465.1) belonging to a new ST, but close to ST23 (it had a variant locus), and presenting a virulence score of 5 and antibiotic resistance score of 3. Indeed, ST23 is commonly associated with hypervirulent strains, as are other STs, such as ST37 [25,52]. The ST37 was observed here; however, it did not show any virulence trait, although it was predicted as MDR. The lack of convergence of virulence and antibiotic resistance was also observed in other Klebsiella species, as well as in genomes from animal and environmental sources.
As we have seen here that most of the population of KpSC in Brazil would be MDR, there could be a tendency for the appearance of hypervirulent clones since MDR clones circulating in clinical settings would be more likely to acquire virulence genes [10]. Indeed, the virulent plasmid pLVPK was identified here in two genomes of K. pneumoniae isolated from Callithrix penicillata, a commensal animal in urban areas [53]. Interestingly, these strains showed a virulent phenotype, but with an antibiotic susceptibility profile [54]. However, as K. pneumoniae is highly susceptible to horizontal gene transfer, these strains could show a convergent phenotype of virulence and resistance.
This study therefore contributes to the understanding of some underlying factors, resistome and virulome, driving the success of the Klebsiella pneumoniae species complex as a pathogen.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms10112281/s1, Table S1: List of analyzed genomes and metadata; Table S2: Kleborate output; Table S3: Virulence genes in Klebsiella plasmids; Table S4: Antibiotic resistance genes in Klebsiella plasmids.

Author Contributions

Conceptualization, A.C.V.; Formal analysis, S.M.; Methodology, A.C.V. and S.M.; Writing—original draft, A.C.V., E.F. and S.M.; Writing—review & editing, A.C.V. and S.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Inova Fiocruz/VPPCB post-doctoral fellowship, and Oswaldo Cruz Institute grants.

Institutional Review Board Statement

Not Applicable.

Data Availability Statement

Genome information is detailed in supplementary materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Brazilian regions and distribution of Klebsiella genomes. The values represent the amount of Klebsiella genomes obtained from each Brazilian state. The Brazilian regions are colored in green (North), red (Northeast), yellow (Midwest), lilac (Southeast), and blue (South).
Figure 1. Brazilian regions and distribution of Klebsiella genomes. The values represent the amount of Klebsiella genomes obtained from each Brazilian state. The Brazilian regions are colored in green (North), red (Northeast), yellow (Midwest), lilac (Southeast), and blue (South).
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Microorganisms 10 02281 g002 550
Figure 2. Number of sequenced and available Klebsiella genomes (y axis) by year of isolation (x axis).
Figure 2. Number of sequenced and available Klebsiella genomes (y axis) by year of isolation (x axis).
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Microorganisms 10 02281 g003 550
Figure 3. Maximum-likelihood tree based on the core genome of Klebsiella spp. The different species are indicated by the background colors of the accession numbers. The ST number of each genome is next to the accession number. There are two orbits of colored blocks, where the innermost represents the Brazilian regions from where the genome was obtained, and the outermost represents the source of isolation. More external to these blocks, the different sizes of the thin bars (0 to 5) indicate the antibiotic resistance (red) and virulence (blue) scores of the genomes. Outer-colored circles indicate genomes with complete operons of yersiniabactin (green), and colibactin (beige) loci; and the presence of Type VI secretion system gene clusters (red).
Figure 3. Maximum-likelihood tree based on the core genome of Klebsiella spp. The different species are indicated by the background colors of the accession numbers. The ST number of each genome is next to the accession number. There are two orbits of colored blocks, where the innermost represents the Brazilian regions from where the genome was obtained, and the outermost represents the source of isolation. More external to these blocks, the different sizes of the thin bars (0 to 5) indicate the antibiotic resistance (red) and virulence (blue) scores of the genomes. Outer-colored circles indicate genomes with complete operons of yersiniabactin (green), and colibactin (beige) loci; and the presence of Type VI secretion system gene clusters (red).
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Microorganisms 10 02281 g004 550
Figure 4. Antibiotic resistance (A) and virulence (B) scores of KpSC.
Figure 4. Antibiotic resistance (A) and virulence (B) scores of KpSC.
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Table
Table 1. Antibiotic resistance genes identified in the genomes of Klebsiella species.
Table 1. Antibiotic resistance genes identified in the genomes of Klebsiella species.
SpeciesAGlyColFlqMLSPheRifSulTetTmtBlaBla_ESBLBla_Carb
K. quasipneumoniaeaac aadA aph rmtD strA strBqnrA qnrB qnrEereAcatB sultetDdfrAoxa temctxges kpc ndm
K. variicolaaac aadA sat2 strA strBqnrB catA sul dfrAoxa temctxges kpc
K. pneumoniaeaac aadA ant aph armA rmtB
rmtB rmtC rmtG sat2 strA strB		mcrqnrA qnrB qnrE
qnrS qnrVC		ereA ermB mphA
mphE		catA catB catII
cmlA floR		arrsultetA tetB tetD tetGdfrAlap oxa shv
sco tem		ctximp kpc ndm
AGly, aminoglycosides; Col, colistin; Flq, fluoroquinolone; MLS, macrolides; Phe, phenicols; Rif, rifampin; Sul, sulfonamides; Tet, tetracyclines; Tmt, trimethoprim; Bla, β-lactamases; Bla_ESBL, ESBL extended-spectrum β-lactamases; Bla_Carb, carbapenemases.
Table
Table 2. Number of Klebsiella genomes carrying antibiotic resistance genes to different classes of antibiotics per species.
Table 2. Number of Klebsiella genomes carrying antibiotic resistance genes to different classes of antibiotics per species.
Species#AGlyColFlqMLSPheRifSulTetTmtBlaBla_ESBLBla_Carb# MDR
K. quasipneumoniae1811 (61%)1 (5%)6 (33%)1 (5%)7 (38%)09 (50%)1 (5%)8 (44%)7 (38%)7 (38%)9 (50%)10 (55%)
K. variicola213 (14%)01 (4%)01 (4%)02 (9%)02 (9%)2 (9%)2 (9%)2 (9%)3 (14%)
K. pneumoniae350113 (32%)3 (<1%)113 (32%)194 (55%)225 (64%)20 (5%)277 (79%)177 (50%)267 (76%)278 (79%)262 (74%)265 (75%)328 (93%)
AGly, aminoglycosides; Col, colistin; Flq, fluoroquinolone; MLS, macrolides; Phe, phenicols; Rif, rifampin; Sul, sulfonamides; Tet, tetracyclines; Tmt, trimethoprim; Bla, β-lactamases; Bla_ESBL, ESBL extended-spectrum β-lactamases; Bla_Carb, carbapenemases; #, number.
Table
Table 3. Prevalence of virulence loci in Klebsiella species in Brazil.
Table 3. Prevalence of virulence loci in Klebsiella species in Brazil.
#ybtclbiucirormpADCT6SS
pneumoniae350142 (40%)69 (19%)3 (<1%)4 (<1%)3 (<1%)268 (76%)
K. quasipneumoniae18000004 (22%)
K. variicola211 (<1%)000018 (85%)
Ybt, yersiniabactin; Clb, colibactin; Iuc, aerobactin; Iro, salmochelin; rmpADC, hypermucoidy loci; #, number.
Table
Table 4. Prevalence of virulence loci in the main STs of Klebsiella pneumoniae in Brazil.
Table 4. Prevalence of virulence loci in the main STs of Klebsiella pneumoniae in Brazil.
ST#ybtclbiucirormpADCT6SS
119890 (91%)59 (60%)00067 (68%)
437522 (<1%)2 (<1%)00048 (92%)
258370000021 (56%)
340273 (11%)100012 (44%)
162512 (48%)000025 (100%)
15183 (16%)000018 (100%)
Ybt, yersiniabactin; Clb, colibactin; Iuc, aerobactin; Iro, salmochelin; rmpADC, hypermucoidy loci; #, number.
Table
Table 5. Putative plasmids shared between Klebsiella genomes.
Table 5. Putative plasmids shared between Klebsiella genomes.
Accession NumberSize (bp)SourceRegionYearST
NCVW01000066140.347AnimalSE/SP2016307
PPHO01000091140.347ArugulaSE/SP20162739
LZCY0100005194.582RiverSE/SP2011437
QXMY01000007119.474HumanSE/SP201011
JABBZB01000003064.338HumanSE/SP201811
LYMZ0100002188.051AnimalSE/MG2012340
JAAQON01000002966.917MeatN/PA20193827
JABBZB01000003548.305HumanSE/SP201811
JABUOR01000003947.331LakeSE/SP201715
JABSUB01000000354.609AnimalSE/SP201911
JAEVGJ01000002853.207HumanSE/SP2014437
JABBZC01000003912.764HumanSE/SP2014340
JABEPG01000006912.143AnimalSE/SP201711
MJBQ0100003012.096LakeSE/SP2011340
JAEVGO01000002254.438HumanSE/SP2014437
KX06209152.536Urban riverSE/SP2011437
QXMZ0100000252.364HumanSE/SP2009437
Table
Table 6. Antibiotic resistance genes identified in putative plasmids from Klebsiella environmental genomes.
Table 6. Antibiotic resistance genes identified in putative plasmids from Klebsiella environmental genomes.
Putative PlasmidsSourceRegionSpeciesSTaac(6′)-Ib8aac(6′)-Ib9ant(3″)-IIages-5kpc-2oxa-9shv-134shv-5tem-181dfr22dfrA30qacErmtDsul1
CP066860.1Sewer effluentSK. quasipneumoniaeST5527 XXX X XXX XX
CP076869.1Urban lakeCOK. pneumoniaeST5236 X
WERP01000062.1WaterSEK. pneumoniaeST4416 X
CP067435.1Sewer effluentSK. grimontiiNA X X
CP067436.1 X
CP067439.1X
CP067440.1 X
LZCZ01000029.1Urban riverSEK. pneumoniaeST437 XX
NSLG01000092.1Urban lakeSEK. pneumoniaeST11 XX
WERN01000017.1WaterSEK. pneumoniaeST661 X
WERO01000020.1WaterSEK. pneumoniaeST4415 X
(X): gene presence.
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Morgado, S.; Fonseca, E.; Vicente, A.C. Genomics of Klebsiella pneumoniae Species Complex Reveals the Circulation of High-Risk Multidrug-Resistant Pandemic Clones in Human, Animal, and Environmental Sources. Microorganisms 2022, 10, 2281. https://doi.org/10.3390/microorganisms10112281

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Morgado S, Fonseca E, Vicente AC. Genomics of Klebsiella pneumoniae Species Complex Reveals the Circulation of High-Risk Multidrug-Resistant Pandemic Clones in Human, Animal, and Environmental Sources. Microorganisms. 2022; 10(11):2281. https://doi.org/10.3390/microorganisms10112281

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Morgado, Sergio, Erica Fonseca, and Ana Carolina Vicente. 2022. "Genomics of Klebsiella pneumoniae Species Complex Reveals the Circulation of High-Risk Multidrug-Resistant Pandemic Clones in Human, Animal, and Environmental Sources" Microorganisms 10, no. 11: 2281. https://doi.org/10.3390/microorganisms10112281

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