
==== Front
101084138
22395
Infect Genet Evol
Infect Genet Evol
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases
1567-1348
1567-7257

39002874
10.1016/j.meegid.2024.105640
nihpa2021110
Article
Comprehensive genomics reveals novel sequence types of multidrug resistant Klebsiella oxytoca with uncharacterized capsular polysaccharide K- and lipopolysaccharide O-antigen loci from the National Hospital of Uganda
Maghembe Reuben S. abcd*
Magulye Maximilian A.K. bd
Makaranga Abdalah c
Moto Edward e
Sekyanzi Simon f
Mwesigwa Savannah b
Katagirya Eric b
a Department of Microbiology and Immunology, Faculty of Biomedical Sciences, Kampala International University-Western Campus (KIU-WC), Ishaka, Uganda
b Department of Immunology and Molecular Biology, School of Biomedical Sciences, Makerere University, P. O. Box 7072, Kampala, Uganda
c Biological and Marine Sciences Unit, Faculty of Science, Marian University College, P. O. Box 47, Bagamoyo, Tanzania
d Department of Biomedial Sciences, Didia Education and Health Organization (DEHO), P. O. Box 113, Shinyanga, Tanzania
e Department of Biology, College of Natural and Mathematical Sciences, University of Dodoma, Dodoma, Tanzania
f Department of Medical Microbiology, 2nd Floor Pathology BLDG, College of Health Sciences, Makerere University, Upper Mulago Hill Road, P.O. Box 7072, Kampala, Uganda
* Corresponding author at: Department of Microbiology and Immunology, Faculty of Biomedical Sciences, Kampala International University-Western Campus (KIU-WC), Ishaka, Uganda., rmaghembe@gmail.com (R.S. Maghembe).
15 9 2024
9 2024
11 7 2024
21 9 2024
123 105640105640
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The Klebsiella oxytoca complex comprises diverse opportunistic bacterial pathogens associated with hospital and community-acquired infections with growing alarming antimicrobial resistance. We aimed to uncover the genomic features underlying the virulence and antimicrobial resistance of isolates from Mulago National Hospital in Uganda. We coupled whole genome sequencing with Pathogenwatch multilocus sequence typing (MLST) and downstream bioinformatic analysis to delineate sequence types (STs) capsular polysaccharide K- and O-antigen loci, along with antimicrobial resistance (AMR) profiles of eight clinical isolates from the National Referral Hospital of Uganda. Our findings revealed that only two isolates (RSM6774 and RSM7756) possess a known capsular polysaccharide K-locus (KL74). The rest carry various unknown K-loci (KL115, KL128, KLI52, KL161 and KLI63). We also found that two isolates possess unknown loci for the lipopolysaccharide O-antigen (O1/O2v1 type OL104 and unknown O1). The rest possess known O1 and O3 serotypes. From MLST, we found four novel sequence types (STs), carrying novel alleles for the housekeeping genes glyceraldehyde-6-phosphate dehydrogenase A (gapA), glucose-6-phosphate isomerase (pgi), and RNA polymerase subunit beta (rpoB). Our AMR analysis revealed that all the isolates are resistant to ampicillin and ceftriaxone, with varied resistance to other antibiotics, but all carry genes for extended-spectrum beta-lactamases (ESBLs). Notably, one strain (RSM7756) possesses outstanding chromosomal and plasmid-encoded AMR to beta-lactams, cephalosporins, fluoroquinolones and methoprims. Conclusively, clinical samples from Mulago National Referral Hospital harbor novel STs and multidrug resistant K. oxytoca strains, with significant public health importance, which could have been underrated.

Genomics
Klebsiella oxytoca
Virulence factors
Antimicrobial resistance
Capsular polysaccharides
O-antigens
Multilocus sequence typing (MLST)
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pmc1. Introduction

The Klebsiella oxytoca complex comprises nine species, including K. oxytoca, K. michiganensis, K. grimontii, K. huaxiensis, K. pasteurii, K. spallanzanii and Raoultella ornithinolytica, which are better delineated along with two that are yet to accurately named (Jing et al., 2021). Organisms under this group are emerging as opportunistic pathogens clinically implicated in nosocomical infections including urinary tract infection (UTI), respiratory tract infection, bloodstream infection, gastrointestinal tract inflammatory disease including, and most commonly, antibiotic-associated hemorrhagic colitis (Long Haiyan et al., 2022). Globally, more attention has been given to K. pneumoniae compared to K. oxytoca. However, existing clinical isolates from various studies have associated species of the K. oxytoca complex with infections and resistance to various antibiotic classes, including beta-lactams, carbapenems, fluoroquinolones and tetracyclines (Long Haiyan et al., 2022; Wan et al., 2023). In addition, evidence shows that a significant number of virulence factors and AMR genotypes are shared between the K.pneumoniae complex and K. oxytoca complex (Moradigaravand et al., 2017; Serena et al., 2022). With our current understanding, some of the virulence factors carred by Klebsiella spp. fall under antiphagocytosis (e. capsule) adherence and biofilm (e.g. fimbria), iron acquision (eg. enterobactin, kleboxymycin), and secretion systems (e.g. T6SS) (Moradigaravand et al., 2017; King et al., 2021). The capsular polysaccharide K and lipopolysaccharide O-antigen comprise a vital virulence feature, which has been used as an important criterion to characterize various Klebsiella serotypes (Long Haiyan et al., 2022). However, since the vast majority of the K. oxytoca complex is unepxplored, our understating on strains and serotypes inhabiting most clinical settings is still limited. In sub-Saharan Africa., most genomic studies have emphasized the clinical importance of K. pneumoniae with accidental encounters of K. oxytoca species (Muraya et al., 2022; Sengeruan et al., 2022). This justifies the need to explore more strains from this group, for better and more relevant intervention options. In the current study, we sought to elucidate the pathogenic and antimicrobial resistance profiles of the K. oxytoca complex using clinical isolates from the National Referral Hospital of Uganda. Here, we report genome sequences with uncharacterized capsular polysaccharides, O-antigens, and novel alleles for housekeeping genes. Furhter, we report the genotypes underlying the virulence and antimicrobial resistance profiles of clinical K. oxytoca. Finally we characterize the genomic structural elemetns underlying the the most drug resistant isolate, for the first time from Uganda.

2. Materials and methods

2.1. Species identification and antimicrobial susceptibility testing (AST)

Samples from different clinical specimens were isolated stored back in 2022 and stored under − 80 °C, then retrieved in 2023 and subcultured for 24 h to MacConkey and Mueller Hinton to check their purity. Then, species re-identification was performed based on colony morphology and conventional biochemical methods. Antimicrobial susceptibility tests (AST) were performed using the Kirby-Bauer disc diffusion method and the results were interpreted as peracccroding the Clinical and Laboratory Standards Institute (CLSI, 2022). The antibiotic panel included trimethoprim-sulfamethoxazole (SXT), clindamycin (CN), ciprofloxacin (CIP), tetracycline (TE), chloramphenicol (C), imipenem (IPM), cefuroxime (CXM), ampicillin (AMP), ceftriaxone (CRO), cefepime (FEP), and amoxicillin-clavulanate (AMC). Genome sequencing, quality control, assembly and annotation

DNA was extracted, sequenced with Illumina Novaseq 6000, followed by read quality control and assembly as described in our recent work (Maghembe et al., 2024). Based on assembly parameters such N50, L50, number of contigs, and genome completeness, each assembly was assessed with the latest algorithm of QUAST (https://quast.sourceforge.net/quast.html). Contigs from each assembly were mapped to the closest relative genome using the Medusa genome finishing tool with default parameters (Bosi et al., 2015) to generate chromosomes. Plasmid replicons were assembled with plasmidSPAdes (v3.15.5) (Antipov et al., 2016). To predict the type of each plasmid, the contigs were subjected to Pathogenwatch web service analysis- (Argimón et al., 2021), which fetched the corresponding Inc. types from PlasmidFinder v2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/). Finally, we assessed the completness of each plasmid with Ori-Finder (https://tubic.tju.edu.cn/Ori-Finder3). Then, annotation was performed using the Prokaryotic Genome Annotation Pipeline (PGAP, https://www.ncbi.nlm.nih.gov/refseq/annotation_prok/). Appropriate BLASTn/p was performed against the GenBank databases within the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) to confirm specific nucleotide and peptide sequences.

2.2. Genomic strain identification and phylogenetic analysis

The closest relative strains were determined based on average nucleotide identity (ANI) values computed using the Microbial Genomes Atlas (MiGA) webserver (http://microbial-genomes.org/). Whole genome-based phylogeny and strain confirmation were accomplished using the Type Strain Genome Server (TYGS) algorithms (Meier-Kolthoff and Göker, 2019).

2.3. Analysis of virulence and antimicrobial resistance genotypes

Virulence factors were predicted using the Pathogenwatch platform (Argimón et al., 2021), which integrates Kleborate multilocus sequence typing and Kaptive v2.0 (Lam et al., 2022). On the other hand, resistance genotypes with respect to specific drugs were analyzed using ResFinder v 4.5.0 (http://genepi.food.dtu.dk/resfinder), in which a threshold of 85% identity was for both chromosomal mutations and acquired resistance genes, at a minimum sequence lenght of 60% For detailed genome-based mechanisms of resistance covering a wide range of genes, each genome assembly was analyzed using the comprehensive antimicrobial resistance database (CARD, https://card.mcmaster.ca/). Selected resistance genotypes encoded by plasmids were annotated and mapped with PlasMapper v3.0 (Wishart et al., 2023). The overall analysis of mobile genetic elements (MGEs) was accomplished with mobileOG-db integrated in Proksee (Grant et al., 2023). Additionally, ISFinder (Kichenaradja et al., 2010) was used to locate insertion sequences of interest.

3. Results

3.1. Sample description

Samples were retrieved from the −80 °C storage at the Clinical Microbiology Laboratory, Department of Medical Microbiology, School of Biomedical Sciences of Makerere University in Uganda. The samples were of different specimens, as indicated in Table 1.

3.2. Phenotypic antimicrobial susceptibility profile

A differential pattern of susceptibility and resistance was observed across the complex. While all the isolates were susceptible to imipenem and ciprofloxacin, they all exhibited resistance to ampicillin (Table 2). The isolates RSM5033, RSM5034 and RSM6895 have comparatively equal resistance rate of 36.4%. These were resistant to ampicillin, cefuroxime, ceftriaxone and cefepime. Exclusively, the most resistant isolate was RSM7756 (54.5%). The most susceptible K. oxytoca isolates were RSM6774 (18.2%) and RSM9152 (9.1%). The isolate RSM7756 appeared phenotypically the most resistant of all the isolates, expressing resistance to six of the eleven drugs (Table 2). This resistance rate is approximately 54.5%. The two isolates, RSM5061 and RSM7096, did not sufficiently grow on our subculture and, therefore to avoid errors, were not included in the AST experiment. However, we were curious to find if we could still obtain some DNA from the retrieved samples. Thus, we successfully extracted tand sequenced their DNA samples, and their genomic information will therefore be presented in a nutshell.

3.3. Genome assembly features and phylogrouping of each isolate

The number of contigs ranged from 43 (isolate RSM5034) to 89 (isolate RSM6895). Details are shown in Supplementary Table 1. The strains in the K. oxytoca complex were highly recovered. Four of the genomes (K. oxytoca sensu stricto) contained no plasmids but isolates RSM5061, RSM7756 and RSM9152 each contained two plasmids. The rest of the isolates contained two to three plasmids. Generally, all the assemblies showed completeness of >98%. Further details of completeness can be viewed through the BioProject and/or Biosample link showing the assembly features of each genome deposited in the GenBank (https://www.ncbi.nlm.nih.gov/biosample/?term=Reuben%20Maghembe). Combining ANI with Pathogenwatch analysis, each isolate was unambiguously placed into the appropriate species and the closest relative was established. Shown in Table 3, isolates RSM5033, RSM5034, RSM6774 and RSM6895 fall under Klebsiella oxytoca sensu stricto, while RSM5061, RSM7756 and RSM9152 are placed into Klebsiella michiganensis. Generaaly, the isolates fall under three phylotypes, i. e., Klebsiella oxytoca sensu stricto, Klebsiella michiganensis and Raoultella ornithinolytica. The rest of the strains presented in Fig. 1A are reference samples whose genomes were retrieved from the GenBank database, guided by published clinical studies of Klebsiella spp. (Argimón et al., 2021; Hussain et al., 2023; Sonda et al., 2018) and used for comparison in this study.

3.4. Findings from multilocus sequence typing

To predict sequence types, PubMLST used seven established housekeeping genes: gapA, infB, mdh, pgi, phoE, rpoB, and tonB. Six of the eight isolates fall into three categories of two sequence types (ST) each. i.e. ST*c1ed (RSM5033 and RSM5034), ST*1b23 (RSM5061 and RSM9152), ST*eac0 (RSM6895 and RSM7096), one isolate belongs to ST2 (RSM6774) and another one ST330 (RSM7756). As shown in Fig. 1B, Four isolates carried novel alleles for given marker genes. While the isolates RSM5061 and RSM9152 carry a novel allele for the gene pgi, both RSM6895 and RSM7096 carry novel alleles for gapA and rpoB. Details, including these alleles’ genomic position and protein sequence identities, are presented in Supplementary Table 3.

3.5. Genomic virulence factors

3.5.1. Klebprate-Kaptive-based typing of apsular polysaccharide K and lipopolysaccharide O-antigen loci

Capsular polysaccharides K-antigen and O-antigen play an important role in the pathogenesis of Klebsiella as antigphagocytic and inflammatory agents (Rendueles, 2020; Shibu et al., 2021). From Kleborate analysis, different capsule types were predicted for each isolate (Fig. 2A). The best match for isolates RSM5033 and RSM5034 was type KL128, known to be carried by clinical isolates of K. oxytoca and K. quasapneunoniae (Li et al., 2024; Wesley et al., 2017). The best match capsule locus for isolate RSM6895 is KL163, while that of RSM6774 and RSM7756 is KL74. The isolate RSM9152 matched KL152, whereas RSM7096 was KL115. While Kaptive analysis revealed that only the locus KL74 was found to be a known capsule type, the rest are yet to be characterized. The characterized genes encoding capsules are wza, wzb, wzc, wzi, wzx, and wzy (Lam et al., 2022; Lam et al., 2021). From Kleborate analysis, only the isolate RSM6774 possesses a characterized version of wzi, i.e., wzi531. Alternative annotation with PGAP revealed that strains RSM5033 and RSM5034 carry wzb, wzc, wzi, wzx and wzy, which are related to the role in capsule polysaccharide assembly. On the other hand, the isolate RSM7756 carries a wzxE, wzyE and wzzE, all related to capsular polysaccharide assembly.

Furhter findings from Kleborate analysis revealed that among the eight isolates, the isolates RSM5061, RSM7756, and RSM9152, all of which are classified as K.michiganensis, exhibit subtly distinctive K and O loci characteristics (Supplementary Table 2). The K (KL74, gene wzi531) and O loci (OL101, type O1/O2v1 unknown type) for isolate RSM7756 exhibit high identity (up to 98%) against the standard serologically characterized strain from all the genes comprising the operons for these virulence factor clusters. Findings from Kleborate revealed differential but significantluy higher AMR profiles (Fig. 2B), with the strains RSM7756 and RSM7096 portraying the highest AMR, accounting for extended spectrum beta lactamases (ESBL).

3.6. Antimicrobial resistance genotypes

The analysis from all methods revealed that all the isolates possess significant resistance to amplicin, with a differential pattern of genotypic resistance to other beta-lactam agents. While all the isolates belonging to K. oxytoca (RSM5033, RSM5034, RSM6774, RSM6895) carry no resistance genotypes to aminoglycosides, they exhibited OXY-2–6 and OXY-2–11 genes, confering resistance to cephalosporins and other beta-lactams. On the other hand, all the K. michiganensis isolates RSM5061 and RSM9152 (novel STs) have acquired the aminoglycoside resistance gene aph3-Ia.v1^, in addition to the OXY-1–1. From ResFinder analysis, only the isolate RSM9152 harbors the transposon Tn903, related to kanamycin resistance. Generally, all the isolates showed resistance to piperacillin-tazobactam marked by the genotypes blaOXY-2–6 and blaOXY-2–8, which are also known to confer resistance to ampicillin and amoxicillin (Long Haiyan et al., 2022).

Of all the recovered isolates, RSM7756 exhibited the highest phenotypic and genotypic AMR profile with increasingly high resistance to beta-lactams including cephalosporins. The predominant ESBL gene in the RSM7756 genome is OXY-2–8, carried by both plasmids (Fig. 3) and the chromosome (Fig. 4). Both Kleborate and ResFinder results (Table 4) revealed that the isolate RSM7756 harbors a plasmid carrying the gene qnrS1 (13). Other acquired AMR genes for isolate RSM7756 are tet(A).v1 (tetracycline), sul1^ (sulfonamides), dfrA14.v2* and dfrA15.v2 (trimethoprim resistance), encoded by the plasmids pRSM7756_p1 and pRSM7756_p2 as well as the chromosome (Figs. 3 and 4).

Mobile genetic elements have been increasingly associated with high AMR rates (Johansson et al., 2021). Here we compared the number and types of MGEs among the isolates to find out their relevance to observed AMR profiles. The overall observation is that phages contribute to the largest percent of all the mobile genetic elements across the K. oxytoca complex in this work (Fig. 5A). On average, K. oxytoca isolates contain a relatively smaller number as compared to K. michiganesnsis and Raoultella ornithinolytica strains included in this study. The pattern is K. oxytoca (316) - R. ornithinolyrica (413) – K. michiganensis (476). Comprehensive analysis from CARD revealed that efflux pumps, especially the OmpA along wih Oqx and AdeF (Fig. 5B), account for the most resistance mechanisms. In addition, the most common genes for AMR in these isolates include beta-lactamases associated with resistance to penicillins and cephalosporins, followed by acquired single nucleotide polymorphisms (SNPs) mediating quinolone and cephalosporintarget modifications.

While all the novel STs did not show any outstanding pattern of AMR, our observation shows that the mobile genetic element trend from low to high is K. oxytoca, – R. ornithinolytica, – K. michiganensis, except for isolate RSM7096. Since isolate RSM7756 was was adequately recovered and characterized from AST demonstrating exclusively higher AMR than any of the isolates tested phenotypically (Table 2), we were interested in understanding the potential role of mobile genetic elements at the molecular level. Fig. 4 shows the distribution of some of the mobile genetic elements related to the acquisition of the dfrA14 gene. We found that both the plasmid pRSM7756_p1 (CP132017.1) and the chromosome (CP132016.1) possess the dfrA14 gene (Fig. 4). The chromosome region harboring the dfrA14 trimethoprim/sulfamethoxazole resistance cassette spans from 5,576,986–5,584,453 bp in the RSM7756 chromosome.

The cassette comprises the gene dfrA14 flanked by class 1 integron integrase IntI1(WLP16674.1) and plasmid mobilization relaxosome protein MobC (WLP19370.1), which is downstream to the IS6-like element IS6100 family transposase (WLP16673.1). Upstream of IS6100 is the DDE-type integrase/transposase/recombinase (WLP16672.1) along with type II site-specific deoxyribonuclease (DNase, “WLP16671.1), DNA cytosine methyltransferase (DCMT, WLP16670.1), IS1 family transposase (WP_001288433.1) and restriction endonuclease (WLP16668.1).

4. Discussion

The clinical importance of the K. oxytoca complex is defined by blood, gastrointestinal, respiratory, and urinary tract infections (Moradigaravand et al., 2017; Seng et al., 2016). However, interest in the K. oxytoca complex is subtle, creating a paucity in our understanding of the diversity of strains in various clinical settings. With the integration of multiple genome analysis approaches, we successfully divulged genomes of eight isolates of the K. oxytoca complex. The use of the housekeeping genes gapA, infB, mdh, pgi, phoE, rpoB, and tonB has been widely applied as a gold standard approach to discriminate clinical bacterial pathogens in Klebsiella spp. (Abdollahi et al., 2018; Stephen et al., 2022; Saxenborn et al., 2021; Silago and Mshana, 2022). Interestingly, we were able to discriminate the isolates through the discovery of novel alleles for the genes gapA, pgi, and rpoB. These findings constitute a strong criterion that could be considered for the diagnostic identification of the K.oxytoca complex strains. The recovery of these novel alleles is critical for future surveillance and diagnostic strategies to delineate the pathogens prevailing in our clinical settings. It is also fascinating that the isolates possess a spectrum of virulence factors entailing unique signatures as well as those exhibiting an overlap with some counterparts in the K. pneumoniae group.Among the virulence factors recently reported to be shared with K. pneumoniae include some of the capsular polysaccharide K and lipopolysaccharide O-antigen types (Galiot et al., 2023). In this study, Kleborate analysis revealed that all the K. oxytoca isolates (RSM5033, RSM5034 and RSM6895) harbor K-loci with a lower identity of 75–78% (Supplementary Table 2) of the characterized K-loci. The loci also miss a number of other genes, including KL128_02_cpsACP, KL128_05_wzb, KL128_06_wzc, KL128_08 and KL163_02, among others. However, although these genes were not successfully characterized with the use of Kleborate, PGAP annotation revealed that all the isolates possess these genes, including wzb, wzc, wzi, wzx and wzy, all of whose functions are related to capsule assembly and trafficking (Kenyon et al., 2021). This suggests that these genes are, by far, less characterized, and their involvement in the K. oxytoca pathogenesis is far from understood. To better improve our understanding of the complex, experimental characterization of their capsule biosynthetic operons is crucial to precise clinical diagnostics and management of K. oxytoca infections. Comparison with selected isolates from other studies on the K. oxytoca complex revealed similar findings, i.e., most of the capsular polysaccharide K-antigens are unknown. This suggests that the K-antigens for most of the K. oxytoca strains remain uncharacterized, which hampers clinical serological profiling. Our findings align with other recent studies (Stewart et al., 2022), strongly suggesting an urgent need for large-scale serotyping of the K. oxytoca complex based on the capsular antigen sequence types. Rarely occurring in K. pneumoniae (Vasaikar and Obi, 2018), the locus KL74 is also considerably found in strains of the K. oxytoca complex (Long Haiyan et al., 2022; Moradigaravand et al., 2017). Although the role of this KL74 in the virulence of K. oxytoca is yet to be clearly substantiated, its detection in only two strains (RSM6774 and RSM7756) in our study accounts for a potentially unique determinant, which could be utilized for differential diagnostics against other strains.

Along with our isolates, we comparatively analyzed strains from other studies to find out how they could be related to the strains in our current study. We found that K. oxytoca strain KCRI-259C (NCBI Bio-Sample ID accession SAMEA4646304) from Kilimanjaro, Tanzania (Sonda et al., 2018) also carries the locus KL74 and the gene wzi531, similar to our isolate RSM6774 as well as those from the United States K. oxytoca strain 2020CK-00213 (CP118212.1) and the UK K. oxytoca strains NCTC13727 (LR134333.1) and 2880STDY5682532 and 2880STDY5682681 (NCBI BioProject PRJEB5065). However, one important finding from our analysis is that although the KL74 locus could be shared between K. oxytoca sensu stricto and K. michiganensis, only the former possesses the gene wizi531. This observation also includes strains from other studies reanalyzed in our study (Figs. 1 and 3). Therefore, a comprehensive analysis of large datasets would further our understanding of the KL74 and the wizi531 gene for differential diagnostic purposes.

In the case of O-antigen serotyping, our findings demonstrated that except for isolate RSM6774 (unknown (OL104) and isolate RSM 5061 (novel ST, unknown O1), the rest possess known O-antigen loci. Evidence shows that the OL104 has been reported to be more prevalent in the K. oxytoca complex (Long Haiyan et al., 2022; Cuénod et al., 2021) and shares sequence homology with O1, O2 and O2ac, found in K. pneumoniae (Patro et al., 2020), which makes it a potentially shared virulence factor across the genus Klebsiella. Although the OL104 antigen could not be useful as a differential diagnostic marker for K. oxytoca, it expands our understanding as a shared virulence factor, which could account for some of the mechanisms of Klebsiella infection, pathogenesis and potential crossreactivity from serology. Serotypes carrying the penta-mannose O3 antigen are common in K. pneumoniae (Guachalla et al., 2017). While the tetra-mannose 3a version is rare, it is also found among K. pneumoniae and is known to bind cross-reacting antibodies with the prevalent O3 (Guachalla et al., 2017). Moreover, the recently described O1/O2v1 serotype is also prevalent in K. pneumoniae (Pajand et al., 2023), which extends the evidence of shared virulence between K. oxytoca and K. pneumoniae. Therefore, from these findings, the characteristics of these O-antigens from the K. oxytoca complex provides strong evidence of shared pathogenic signatures with K. pneumoniae, concurring with recent reports (Long Haiyan et al., 2022; Singh et al., 2016).

Apart from the K- and O-loci, members of the family Enterobacteriaceae have been historically characterized for pathogenicity related to expression of the siderophore yersiniabactin, associated with gut and pulmonary infection, among other effects (Sonda et al., 2018). Except for RSM7756, all the isolates in the current study were found to possess unknown yersiniabactin biosynthetic gene clusters. In most studies, yersiniabactin gene clusters are better characterized in K. pneumoniae but only detected in K. oxytoca strains in the pursuit of K. pneumoniae (Sonda et al., 2018). This suggests that the yersiniabactin gene cluster is insufficiently studied and, hence, yet to be clearly substantiated in the K. oxytoca complex, which creates a need for further studies to establish its diversity and potential implication in pathogenicity, diagnostics, therapeutic and genome mining-based vaccine design and development.

Rapid evolution of drug-resistant strains is especially prominent and substantially elucidated in the Klebsiella pneumoniae complex but insufficiently delineated across the K. oxytoca complex. In Europe and Asia, significant efforts have been devoted to understanding the potential risk of K. oxytoca species in antibiotic resistance (Stephen et al., 2022; Stewart et al., 2022; Vasaikar and Obi, 2018). However, in Africa, the detection of K. oxytoca has been accidental from studies focusing on the isolation and characterization of K. pneumoniae (Sonda et al., 2018; Vasaikar and Obi, 2018), giving little attention to the potential health risks of K. oxytoca isolates. In this study, we demonstrate that strains within the complex have the potential to cause infections and yet resist multiple antibiotic agents. From our phenotypic AST results, all the isolates were resistant to ampicillin. Integrating Kleborate analysis with ResFinder and CARD pangenome screening, we note that all the isolates possess multidrug resistance to beta-lactams, chloramphenicol and quinolones. The most predominant resistance genotype for fluoroquinolones is the qnrS, encoded by K. michiganensis isolate RSM7756. The latter is unique in that it harbors resistance genes in both the chromosome as well as the two plasmids with recurring genotypes for multidrug resistance (Figs. 3 and 6). Two chromosomal encoded beta-lactamase genes include blaOXY-5–1, blaOXY-5–2 (spanning from 829,344–1,830,216 bp) associated with resistance to amoxicillin, amoxicillin+clavulanic acid, ampicillin, ampicillin+clavulanic, cefotaxime, cefoxitin, ceftazidime, piperacillin, piperacillin+tazobactam, ticarcillin, and ticarcillin+clavulanic acid, among other antibiotics. Although the dfrA14 gene was detected in the plasmid pRSM7756_p1 as it is mostly a plasmid-born gene (Miranda et al., 2016), it was also present on the chromosome (between genome position 582,711 to 5,583,184) (Fig. 4), suggesting that this isolate has significant potential to withstand trimethoprim-sulfamethoxazole. Most of the genotypes carried by this isolate are observed in clinical studies from K. pneumoniae (Moradigaravand et al., 2017; Stephen et al., 2022). Therefore, these findings strongly demonstrate the multidrug resistance propensity for isolate RSM7756. The same isolate (RSM7756) was also found to carry two AMR-confering single nucleotide polymorphisms (SNPs), ompK36: p.N49S and ompK36:p.T184P, which could explain why the strain was also found to be resistant to cefuroxime (CXM) from AST and ResFinder (Table 4). Among the established ESBL genotypes in the family Enterobacteriaceae include the CTX-M-15 and OXY-5–1* genes (Muraya et al., 2022; Stephen et al., 2022), both revealed in isolate RSM7756. These findings identify this isolate as the outstanding AMR isolate of this study, portraying ESBL with unnoticed existence.

The evolution of complex mechanisms of antimicrobial resistance has integrated the role of mobile genetic elements (Galiot et al., 2023). Mobile genetic elements can insert into chromosomal and extrachromosomal replicons, leading to alterations in the functions of a wide range of target genes, including virulence and antimicrobial resistance genes in bacteria (Galiot et al., 2023). In various experimental and analytical studies, mobile genetic elements of different families and groups have been implicated in the alterations of gene expression among Gram-positive and Gram-negative bacteria (Johansson et al., 2021). The insertion sequences of the family IS1, IS3, IS6/26, as well as the transposon Tn3, have been strongly associated with drug resistance (Johansson et al., 2021). Here, we demonstrate that the genome of isolate RSM7756 (K. michiganensis) carries multiple copies of the most notorious mobile genetic elements (Fig. 5). It is known that the trimethoprim resistance gene, dfrA14, is frequently plasmid-encoded and carried by integrons (Miranda et al., 2016). Intriguingly, the isolate RSM7756 exceptionally carries this entire cassette of mobile genetic elements and the integron unit with the dfrA14 gene in the chromosome. These findings are alarming about the existence of isolates with a unique set of antimicrobial resistance integrating plasmid and chromosomal mechanisms yet to be deciphered.

Furthermore, the pRSM7756_p1 (Fig. 2) possesses several insertion sequences upstream and downstream of the antimicrobial resistance genes. This could potentially result in genetic instability and, thus, SNPs for some of the drug targets, including chloramphenicol, tetracycline, kanamycin and multiple beta-lactam antibiotics (Wan et al., 2023; Stephen et al., 2022). Interestingly, although this isolate does not appear unique in terms of virulence, its pattern of AMR is comparable to those of K. pneumoniae, suggesting the existence of unnoticed multidrug resisant K. oxytoca superbags in the clinics.

Although the K. oxytoca complex is considered to be less virulent and drug resistant relative to the K. pneumoniae complex, the scarcity of experimental and genomic datasets limits rigorous justification for this understanding. The vast majority of studies have been directed towards K. pneumonae, which is why databases are better enriched with more K. pneumoniae STs, virulence factor and AMR information, which could limit our understating of the K. oxytoca complex under the same parameters. This study for example, is purely based on samples from different encounters not focused on the K. oxytoca complex. This could limit our rigorous association of the isolates with clinical cases and establishment of the causal relation with the inherent infections.

5. Conclusions

The K. oxytoca complex is both biologically and clinically diverse. Our study presents novel strains carrying novel alleles for the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase A (gapA), glucose-6-phosphate isomerase (gpi) and ribosomal polymerase subunit beta (rpoB). Through analysis of virulence factors based on the capsular polysaccharide K and lipopolysaccharide O-antigens, we infer that K. oxytoca complex isolates from Uganda portray similar signatures with those of K. pneumoniae. Therefore, other virulence factors, such as those related to the enterotoxin family, could potentially discriminate the pathogens if well studied. Most importantly, we demonstrate, for the first time from Uganda and East Africa, that the K. oxytoca complex poses a significant antimicrobial resistance threat due to the carriage of both plasmid and chromosome-encoded antimicrobial resistance markers. Furthermore, we show that ampicillin and ceftriaxone are ineffective in all the isolatrs. Therefore, we strongly recommend a dedicated surveillance of the K. oxytoca complex from clinical and environmental niches within East Africa.

Supplementary Material

1

Acknowledgments

The authors would like to thank Mr. Paul Mabala of St. John’s University of Tanzania for his assistance during antimicrobial susceptibility testing experiments.

Funding

This study was partially funded by Case Western Reserve University through the US-NIH-Fogarty International Centre, Grant #D43TW010319.

Data availability

The data supporting this study’s findings are available from the NCBI under the Bioproject accession link: https://www.ncbi.nlm.nih.gov/bioproject/992249.

Fig. 1. A) A phylogenomic tree inferred with FastME 2.1.6.1 (Lefort et al., 2015) from GBDP distances calculated from proteome sequences. The branch lengths are scaled in terms of the GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 78.4%. The tree was rooted at the midpoint (Farris, 1972). Species clusters are denoted by the two letters of the binomial scientific name: K. pneumoniae (Kp), R. ornithinolytica (Ro), K. michiganensis (Km) and K. oxytoca (Ko). B) A MLST-based heatmap indicating known STs as well as novel STs based on novel alleles (0) for corresponding housekeeping genes.

Fig. 2. (A) A heatmap representing capsular polysaccharide and O-antigent loci types for each isolate against selected known reference strains. (B) AMR gene count for selected groups of antimicrobials retrieved from ResFinder via integrative Kleborate annotation. Legend bars besides each heatmap represent capsule/O-antigen presence (A) and resistance degree (B) for each strain.

Fig. 3. Circular representations of the plasmids pRSM7756_p1(A) and pRSM7756 (B) showing positions of AMR genotypes and insertion sequences. The gene for ESBL AMR is represented as blacTX-M-15. The resistance for quinolone is represented by qnrS1, while that of trimethoprim is indicated with the dfr14. Insertion sequences of interest are also shown. The replication origins for this plasmid are shown as OriC01–3.

Fig. 4. Genome map of K. michiganensis strain RSM7756 showing the distribution of mobile genetic elements across the entire chromosome. Regions harboring phage-related insertions are indicated in blue, integration/excision and transfer in green, while excision/repair are indicated in purple. The regions harboring the CARD-annotated AMR are indicated as CARD-AMR. The region carrying the dfrA14 trimethoprim-sulfamethoxazole-resistance gene is shown by the bar cassette of genes spanning between 5,576,986–5,584,453 bp.

Fig. 5. A) Distribution of mobile genetic elements among the Klebsiella oxytoca complex strains, including selected species from reference studies. The column represents the type of GEs: IS = Insertion (integration/excision), R = Replication (recombination/repair), P = Phage, and S = Stability (transfer/defense transfer). The legend on the right denotes the number of the GEs detected for each isolate: The number increases with ball size and color intensity. B) AMR genotypes detected from CARD prediction (strict-perfect hit method) indicating SNPs and mechanisms of resistance.

Table 1 A summary of sample description.

SN	Sample ID	Species	Specimen	Infection	
1	RSM5033	K. oxytoca	Feces	GIT infection	
2	RSM5034	K. oxytoca	Feces	GIT infection	
3	RSM5061	K. oxytoca	Blood	Blood stream infection	
4	RSM6774	K. oxytoca	Blood	Blood stream infection	
5	RSM6895	K. oxytoca	Rectal swab	GIT infection	
6	RSM7756	K. oxytoca	Blood	Blood stream infection	
7	RSM9152	K. oxytoca	Blood	Blood stream infection	
8	RSM7096	K. oxytoca	Blood	Blood stream infection	
Sample sources are indicated as specimens with corresponding clinical information.

Table 2 Phenotypic antimicrobial susceptibility profile of each isolate sequenced from the current study.

	Antibiotic resistance		
Strains	SXT	CN	CIP	TE	C	IPM	CXM	AMP	CRO	FEP	AMC	
RSM5033	26	16	32	30	30	32	6	6	15	15	14	
I	S	S	S	S	S	S	R	R	R	R	I	
RSM5034	26	16	30	28	28	26	6	6	14	22	12	
I	S	S	S	S	S	S	R	R	R	I	R	
RSM6895	30	17	32	28	30	30	6	6	16	24	10	
I	S	S	S	S	S	S	R	R	R	I	R	
RSM9152	26	16	32	30	29	34	24	6	26	32	24	
I	S	S	S	S	S	S	S	R	S	S	S	
RSM7756	6	20	30	6	6	35	6	6	10	22	26	
I	R	S	S	R	R	S	R	R	R	I	S	
RSM6774	25	10	30	26	28	30	22	10	28	32	24	
I	S	R	S	S	S	S	S	R	S	S	S	
Teste-dantibiotics include trimethoprim-sulfamethoxazole (SXT), clindamycin (CN), ciprofloxacin (CIP), tetracycline (TE), chloramphenicol (C), imipenem (IPM), cefuroxime (CXM), ampicillin (AMP), ceftriaxone (CRO), cefepime (FEP), and amoxicillin-clavulanate (AMC). Colored boxes represent resistant (R), while unshaded boxes represent susceptible (S) and intermediate response (I).

Table 3 Taxonomic placement of each isolate based on ANI and other classification methods.

Isolate	Species	Strain	NCBI Accession	Closest relative	Accession number	ANI (%)	
RSM5033	Klebsiella oxytoca	RSM5033	CP132967.1	K. oxytoca strain NCTC13727	LR134333.1	99.42	
RSM5034	Klebsiella oxytoca	RSM5034	CP130582.1	K. oxytoca strain NCTC13727	LR134333.1	99.42	
RSM5061	Klebsiella michiganensis	RSM5061	GCA_030490345	K. michiganensis	GCA_009173485	98.85	
RSM6774	Klebsiella oxytoca	RSM6774	CP130256.1	K. oxytoca NBRC 105695	GCA 001598695	99.35	
RSM6895	Klebsiella oxytoca	RSM6895	CP130257.1	K. oxytoca strain NCTC13727	LR134333.1	99.12	
RSM7096	Raoultella ornithinolytica	RSM7096	CP132011- CP132014	R. ornithinolytica NBRC 105727	GCA_001598295	99.37	
RSM7756	Klebsiella michiganensis	RSM7756	CP132016- CP132018	K.michiganensis	GCA_009173485T	97.77	
RSM9152	Klebsiella michiganensis	RSM9152	CP129895- CP129897	K. michiganensis	6095 GCA_009173485	98.85	
Each isolate is renamed into a species based on the closest relative. The accession numbers of each assembly and/or nucleotide sequence are deposited in the NCBI GenBank database. The average nucleotide identity (ANI) values are also included, representing similarity with one of the closest strains retrievable from NCBI.

Table 4 Comparative AMR profile from AST and genome analysis from ResFinder (RF).

	RSM5033	RSM5034	RSM9152	RSM7756	RSM6774	RSM6895	
	AST	RF	AST	RF	AST	RF	AST	RF	AST	RF	AST	RF	
SXT	0	0	0	0	0	0	1	1	0	0	0	0	
CN	0	0	0	0	0	0	0	0	1	0	0	0	
CIP	0	0	0	0	0	0	0	1	0	0	0	0	
TE	0	0	0	0	0	0	1	1	0	0	0	0	
C	0	0	0	0	0	0	1	0	0	0	0	0	
PM	0	0	0	0	0	0	0	0	0	0	0	0	
CXM	1	0	1	0	0	0	1	0	0	0	1	0	
AMP	1	1	1	1	1	1	1	1	1	1	1	1	
CRO	1	1	1	0	0	0	1	1	0	1	1	0	
FEP	1	0	0	0	0	0	0	1	0	0	0	0	
AMC	0	1	1	1	0	1	0	1	0	1	1	1	
Susceptibility is denoted by 0, while resistance is denoted by 1. AST = Antimicrobial susceptibility test, RF = ResFinder. Antibiotics include trimethoprim-sulfamethoxazole (SXT), clindamycin (CN), ciprofloxacin (CIP), tetracycline (TE), chloramphenicol (C), imipenem (IPM), cefuroxime (CXM), ampicillin (AMP), ceftriaxone (CRO), cefepime (FEP), and amoxicillin-clavulanate (AMC).

Ethical statement

This study was approved by the Institutional Review Board (IRB), Makerere University, School of Biomedical Sciences Research and Ethics Committee (SBS-REC), approval number SBS-2023–380.

CRediT authorship contribution statement

Reuben S. Maghembe: Writing – original draft, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Maximilian A.K. Magulye: Writing – review & editing, Validation, Software, Methodology, Formal analysis, Data curation. Abdalah Makaranga: Writing – review & editing, Validation, Software, Data curation. Edward Moto: Writing – review & editing, Visualization, Formal analysis. Simon Sekyanzi: Writing – review & editing, Resources, Investigation, Funding acquisition, Conceptualization. Savannah Mwesigwa: Writing – review & editing, Supervision, Resources, Project administration, Data curation, Conceptualization. Eric Katagirya: Writing – review & editing, Validation, Supervision, Project administration, Data curation, Conceptualization.

Declaration of competing interest

The authors have no conflict of interest to declare.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.meegid.2024.105640.
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