Molecular Characterization of Klebsiella pneumoniae Clinical Isolates Through Whole-Genome Sequencing: A Comprehensive Analysis of Serotypes, Sequence Types, and Antimicrobial and Virulence Genes

Introduction Antimicrobial resistance (AMR) has become a menace, spreading among bacterial species globally. AMR is now recognized as a silent pandemic responsible for treatment failures. Therefore, an effective surveillance mechanism is warranted to understand the bacterial species isolated from human clinical specimens. The present study employed next-generation sequencing (NGS) or whole-genome sequencing (WGS) to identify the resistance and virulence genes, sequence type, and serotypes. Methods This study included 18 multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) isolates obtained from patients suffering from different infections attending the Prathima Institute of Medical Sciences, Karimnagar, India. All isolates were identified, and antimicrobial susceptibility profiles were determined through conventional microbiological techniques and confirmed by automated systems. All the isolates were investigated using NGS or WGS to identify the genes coding for resistance, such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases, and virulence genes. Multilocus sequence typing (MLST) was conducted to identify the sequence types, and Kleborate analysis was performed to confirm the species, genes for AMR, and virulence and evaluate the capsular polysaccharide (KL) and cell wall/lipopolysaccharide (O) serotypes carried by the isolates. Results The mean age of the patients was 46.11±20.35 years. Among the patients included, 12 (66.66%) were males and 6 (33.33%) were females. A high percentage (>50%) of hypervirulent K. pneumoniae (hvKp) strains that had genes coding for AMR and plasmids having the potential to carry blaNDM and resistance genes were observed. Among the isolates, 16 (88.88%) revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance. There was a high prevalence of blaSHV (17/18; 94.44%) and blaCTX-M-15 (16/18; 88.88%) AMR genes. Other AMR genes identified included blaTEM (83.33%; 15/18) and blaOXA (14/18; 77.77%). Two (11.11%) strains each showed the presence of blaNDM-1 and blaNDM-5 genes. The virulence genes identified included gapA, infB, mdh, pgi, phoE, rpoB, tonB, and ybt. The most frequent K. pneumoniae serotypes found were KL51:O1v2 (3/18, 16.66%), KL17:O1v1 (3/18, 16.66%), and KL64:O2v1 (3/18, 16.66%). KL64 (4/18; 22.22%) was the most common capsular serotype identified among the isolates. The most frequent MLST-based sequence type (ST) identified included ST-147 (5/18, 27.77%), followed by ST-231 (3/18, 16.66%) and ST-101 (2/18, 11.11%). Conclusions The molecular analysis of K. pneumoniae isolates revealed multiple AMR, plasmid, and virulence genes. Additionally, many global STs were noticed by MLST. The results noted a high prevalence of hvKp strains. Molecular characterization of bacterial strains using NGS/WGS is important to understand the epidemiology of bacterial strains and the antibiotic resistance and virulence genes they are potentially carrying. The data obtained from this study may be utilized to devise careful antibiotic-prescribing approaches and improve patient management practices.


Introduction
Klebsiella pneumoniae (K.pneumoniae) is one of the pathogenic bacteria listed under the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens [1].These bacteria utilize mechanisms such as antibiotic resistance and virulence determinants to counteract human defenses and cause invasive diseases.K. pneumoniae is a versatile gram-negative capsule-forming bacillus associated with several human infections that range from mild to deep-seated invasive infections.Some of the infections frequently associated with K. pneumoniae include respiratory tract infections (RTIs), urinary tract infections (UTIs), abscesses, and septicemia [2].
Interestingly, K. pneumoniae can cause community-acquired infections (CAIs) and hospital-acquired infections (HAIs) [3].CAIs spread among people while moving socially, in places such as classrooms, movie theaters, parks, recreation centers, and other crowded areas.HAIs are contracted by people after 48 hours of hospital admission, wherein they develop an infection different from the condition for which they were hospitalized.Patients admitted to the intensive care units (ICUs) are predisposed to HAIs.
Extended-spectrum β-lactamase (ESBL) and carbapenem-resistant K. pneumoniae (CR-Kp) strains have been spreading globally and contributing to treatment failures [4,5].The antimicrobial resistance (AMR) gene bla OXA is one of several genes identified in K. pneumoniae isolates that contribute to carbapenemase activity and resistance to the carbapenem group of antibiotics, which are generally employed as last resort antimicrobial agents to treat infections with multidrug-resistant (MDR) bacteria [6].Other genes that confer MDR identified among K. pneumoniae clinical isolates include bla CTX-M , bla SHV , bla TEM , ompK36, and ompK37 [7].
A few strains, characterized as hypervirulent K. pneumoniae (hvKp), have been associated with severe and invasive infections among healthy and immunocompetent individuals.These strains are known to harbor virulence genes such as iro (salmochelin biosynthesis), iuc (aerobactin synthesis), and rmpA (regulator of mucoid phenotype) [8].
Therefore, it is important to improve our understanding of the sequence types (STs) and serotypes of K. pneumoniae, the AMR genes, plasmids, and the virulence genes they are potentially carrying.

Materials And Methods
An observational, analytical, and cross-sectional study was conducted among 18 MDR K. pneumoniae isolates acquired from patients attending Prathima Institute of Medical Sciences, Karimnagar, India.The study period was between April 2018 and April 2020.All isolates were identified, and antimicrobial susceptibility profiles were determined through conventional microbiological techniques and confirmed by automated systems [9][10][11][12].Additionally, all the isolates were investigated using next-generation sequencing (NGS) or whole-genome sequencing (WGS) to identify the genes coding for resistance, such as ESBLs, metallo-betalactamases (MBLs), and virulence genes.Multilocus sequence typing (MLST) was carried out to determine the sequence types, and serotyping was carried out to evaluate the capsular polysaccharide (K) and cell wall/lipopolysaccharide (O) serotypes carried by the isolates.

Whole-genome sequencing and genomic characterization for resistance and virulence genes and sequence types
The deoxyribonucleic acid (DNA) was extracted from K. pneumoniae isolates using the Qiagen QIAamp DNA Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.Double-stranded DNA libraries with 450 base pairs (bp) insert size were prepared and sequenced on the Illumina platform with 150 bp paired-end chemistry.The genomes that passed sequence quality control were assembled using Spades v3.14 [13] to generate contigs and annotated with Prokka v1.5 [14].Species identification was carried out using a bactinspector, and contamination was assessed using confindr.All the quality metrics were combined using MultiQC and Qualifyr to generate web-based reports.MLST, AMR, and virulence factors were identified using the ARIBA tool v2.14.4 [15] with BIGSdb-Pasteur MLST database, National Center for Biotechnological Information (NCBI) AMR-acquired gene, PointFinder databases, and VFDB, respectively [16][17][18].All the bioinformatic analyses were conducted using Nextflow pipelines developed as a part of the Global Health Research Unit (GHRU), United Kingdom, for AMR surveillance.

Kleborate analysis
The Kleborate tool was used to confirm the species, genes for AMR, and virulence and evaluate the capsular polysaccharide (KL) and cell wall/lipopolysaccharide (O) serotypes [19].

Results
The mean age of the patients was 46.11±20.35years.Among the patients included, 12 (66.66%)were males and 6 (33.33%) were females.Among the samples included were blood (2/18; 11.11%), sputum/respiratory secretions (3/18; 16.66%), pus/wound (4/18; 22.22%), and urine (9/18; 50%).A high percentage (>50%) of hvKp strains that had genes coding for AMR and plasmids having the potential to carry bla NDM and resistance genes were observed.Of the 18 isolates, 16 (88.88%)revealed the presence of multiple AMR and virulence genes, with evidence of at least one gene coding for beta-lactamase resistance.The study identified the presence of aerobactin (AbST) and yersiniabactin (YbST) STs based on the occurrence of virulence genes such as iuc (A-D) and iutA for AbST and ybt (A, E, P, Q, S, T, U X, 9, 10, 14, 15, 16), and irp (1-2) for YbST.The genes coding for AMR and virulence identified in this study are detailed in Table 1.The MLST was performed using selected/functional virulence genes called housekeeping genes.The details of the plasmid replicons and virulence genes and functions along with their usefulness to the bacteria are shown in Table 4.

Discussion
Among many public health problems encountered in the present time is AMR, which is responsible for treatment failures and results in severe morbidity and mortality.There is an increased concern about the emergence and spread of MDR K. pneumoniae (MDR-Kp) strains carrying genes for AMR, including the genes coding for carbapenemase that confer resistance to carbapenem groups of antibiotics, such as imipenem and meropenem, that are considered as last resort antimicrobial agents [20].
In this study, ST-147 was the most frequent ST with a serotype combination of KL64: O2v1 (three strains), KL128: O3b (one strain), and KL17:O1v1 (one strain).The ST-147, in combination with KL64, was previously identified among K. pneumoniae isolated from patients admitted to ICUs [21].A high percentage (>50%) of hvKp strains that had genes coding for AMR and plasmids having the potential to carry bla NDM and resistance genes were observed.

Hypervirulent K. pneumoniae strains
There are no recommended criteria for classifying K. pneumoniae isolates into hypervirulent (hvKp) types.In the clinical setting, the string test can help in the provisional diagnosis of hvKP infection.The test was considered positive if a viscous string measuring more than 5 mm long was obtained by pulling bacterial colonies grown on an agar plate with a bacteriology inoculation wire loop or needle [22].The K. pneumoniae strains that show hyperviscosity in the texture of colonies on primary isolation from the clinical specimens are preliminarily identified as hvKp strains (Figure 1).

FIGURE 1: Klebsiella pneumoniae colony textures after being isolated from patient specimens
Image credit: Venkataramana Kandi The hvKp strains are known to secrete increased quantities of capsular polysaccharides due to the rmpA and rmpA2 genes.Further, hvKp strains carry specific plasmids such as pPMK-NDM, which carry AMR genes, and virulence genes such as iuc, iro, and iut.K. pneumoniae strains having genetic markers for the biosynthesis of salmochelin (iro), aerobactin (iuc), yersiniabactin (ybt), and colibactin (col) have been considered as hvKp strains.Besides, the presence of Integrative and conjugative elements of K. pneumoniae (ICEKp) indicates hypervirulence [8].The present study demonstrated a high percentage (>50%) of hvKp strains, which was considerably high compared to a recent study from Italy that noted a hvKP prevalence of 3.7%.This study observed that hypervirulence was associated with ST-23 and ST-86, and ST-147 carried a bla NDM gene [23].NDM carriage was noticed among ST-147, ST-15, and ST-101 in our study.
Our study noted one (1/18; 5.55%) aerobactin ST (AbST) in one isolate (ST-231), which revealed iuc and iut genes.Conversely, a study from Morocco showed only 1.5% of isolates belonging to AbST [24].Our investigation revealed 11 distinct STs, with ST-147, ST-231, and ST-101 being the predominant ones.A study from South India revealed ST-2096 with hypervirulent markers, including AMR genes (aadA2, armA, bla OXA- In a study from North India, 11.6% of strains were identified as hvKp.This study noted K64 as the most frequent capsular serotype, similar to our results.ST-2096 was the most common ST, along with others such as ST-231 and ST-43, which were also found [28].Although ST-231 and ST-2096 were detected in our study, ST-43 was not documented.
A study from Iran that analyzed more than 400 strains of K. pneumoniae revealed an hvKp prevalence of 4% based on the presence of the rmpA gene.This study observed the predominance of K1 and K2 capsular types, and 7.8% of strains had bla NDM genes [29].A higher percentage of hvKp in our study may be attributed to the comprehensive evaluation of the isolates using WGS/NGS for plasmid, AMR, and virulence genes.
A recent study from Turkey that investigated virulence and resistance genes among K. pneumoniae isolates found that 45% of the isolates were hvKp.The study found bla KPC and bla OXA as predominant resistance genes; none of the isolates had bla NDM [30].In contrast, our study found 11.11% of strains carrying bla NDM genes, and none of the isolates revealed bla KPC .

Study limitations
This study was conducted among limited numbers of K. pneumoniae isolates acquired from different clinical specimens.The major limitation of this study is it did not compare the genomic evidence with the phenotypic resistance patterns.Also, this study did not evaluate the chromosomal and plasmid origins of the resistance and virulence genes.Based on the clinical condition, the study did not try to establish any relationship between the serotype or ST and the bacterium's virulence.

Conclusions
The results from the genomic analysis of K. pneumoniae clinical isolates indicate a very high percentage of them carrying multiple genes conferring AMR and virulence.Multiple AMR genes coding for carbapenemase resistance and ESBLs were identified in the isolates.The study also recognized plasmids carrying AMR and virulence genes in most isolates that can be potentially transmissible between strains and other bacterial species.Besides, more than half of the isolates included in this study were identified as hypervirulent (hvKP) strains.Screening the clinical isolates from hospitalized patients for the presence of AMR genes, virulence genes, and plasmids through NGS/WGS could improve the understanding of the epidemiological characteristics and invasive disease-causing potential of the bacteria prevalent in the hospital environment.

TABLE 3 : Klebsiella pneumoniae serotypes identified in this study
KP: Klebsiella pneumoniae, KL: capsule type based on Kleborate analysis